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Sample records for phytopathogen verticillium longisporum

  1. Identification and Differentiation of Verticillium Species and V. longisporum Lineages by Simplex and Multiplex PCR Assays.

    PubMed

    Inderbitzin, Patrik; Davis, R Michael; Bostock, Richard M; Subbarao, Krishna V

    2013-01-01

    Accurate species identification is essential for effective plant disease management, but is challenging in fungi including Verticillium sensu stricto (Ascomycota, Sordariomycetes, Plectosphaerellaceae), a small genus of ten species that includes important plant pathogens. Here we present fifteen PCR assays for the identification of all recognized Verticillium species and the three lineages of the diploid hybrid V. longisporum. The assays were based on DNA sequence data from the ribosomal internal transcribed spacer region, and coding and non-coding regions of actin, elongation factor 1-alpha, glyceraldehyde-3-phosphate dehydrogenase and tryptophan synthase genes. The eleven single target (simplex) PCR assays resulted in amplicons of diagnostic size for V. alfalfae, V. albo-atrum, V. dahliae including V. longisporum lineage A1/D3, V. isaacii, V. klebahnii, V. nonalfalfae, V. nubilum, V. tricorpus, V. zaregamsianum, and Species A1 and Species D1, the two undescribed ancestors of V. longisporum. The four multiple target (multiplex) PCR assays simultaneously differentiated the species or lineages within the following four groups: Verticillium albo-atrum, V. alfalfae and V. nonalfalfae; Verticillium dahliae and V. longisporum lineages A1/D1, A1/D2 and A1/D3; Verticillium dahliae including V. longisporum lineage A1/D3, V. isaacii, V. klebahnii and V. tricorpus; Verticillium isaacii, V. klebahnii and V. tricorpus. Since V. dahliae is a parent of two of the three lineages of the diploid hybrid V. longisporum, no simplex PCR assay is able to differentiate V. dahliae from all V. longisporum lineages. PCR assays were tested with fungal DNA extracts from pure cultures, and were not evaluated for detection and quantification of Verticillium species from plant or soil samples. The DNA sequence alignments are provided and can be used for the design of additional primers.

  2. Identification and Differentiation of Verticillium Species and V. longisporum Lineages by Simplex and Multiplex PCR Assays

    PubMed Central

    Inderbitzin, Patrik; Davis, R. Michael; Bostock, Richard M.; Subbarao, Krishna V.

    2013-01-01

    Accurate species identification is essential for effective plant disease management, but is challenging in fungi including Verticillium sensu stricto (Ascomycota, Sordariomycetes, Plectosphaerellaceae), a small genus of ten species that includes important plant pathogens. Here we present fifteen PCR assays for the identification of all recognized Verticillium species and the three lineages of the diploid hybrid V. longisporum. The assays were based on DNA sequence data from the ribosomal internal transcribed spacer region, and coding and non-coding regions of actin, elongation factor 1-alpha, glyceraldehyde-3-phosphate dehydrogenase and tryptophan synthase genes. The eleven single target (simplex) PCR assays resulted in amplicons of diagnostic size for V. alfalfae, V. albo-atrum, V. dahliae including V. longisporum lineage A1/D3, V. isaacii, V. klebahnii, V. nonalfalfae, V. nubilum, V. tricorpus, V. zaregamsianum, and Species A1 and Species D1, the two undescribed ancestors of V. longisporum. The four multiple target (multiplex) PCR assays simultaneously differentiated the species or lineages within the following four groups: Verticillium albo-atrum, V. alfalfae and V. nonalfalfae; Verticillium dahliae and V. longisporum lineages A1/D1, A1/D2 and A1/D3; Verticillium dahliae including V. longisporum lineage A1/D3, V. isaacii, V. klebahnii and V. tricorpus; Verticillium isaacii, V. klebahnii and V. tricorpus. Since V. dahliae is a parent of two of the three lineages of the diploid hybrid V. longisporum, no simplex PCR assay is able to differentiate V. dahliae from all V. longisporum lineages. PCR assays were tested with fungal DNA extracts from pure cultures, and were not evaluated for detection and quantification of Verticillium species from plant or soil samples. The DNA sequence alignments are provided and can be used for the design of additional primers. PMID:23823707

  3. Broadening the Genetic Basis of Verticillium longisporum Resistance in Brassica napus by Interspecific Hybridization.

    PubMed

    Rygulla, W; Snowdon, R J; Eynck, C; Koopmann, B; von Tiedemann, A; Lühs, W; Friedt, W

    2007-11-01

    ABSTRACT Verticillium wilt caused by the vascular fungal pathogen Verticillium longisporum is one of the most important pathogens of oilseed rape (Brassica napus sp. oleifera) in northern Europe. Because production of this major oilseed crop is expanding rapidly and no approved fungicides are available for V. longisporum, long-term control of the disease can only be achieved with cultivars carrying effective quantitative resistance. However, very little resistance to V. longisporum is available within the gene pool of oilseed rape, meaning that interspecific gene transfer from related species is the only possibility for broadening levels of resistance in current varieties. The amphidiploid species B. napus can be resynthesized by crossing the two progenitor species Brassica oleracea and Brassica rapa, hence resistant accessions of these two diploid species can be used as resistance donors. In this study a total of 43 potential B. rapa and B. oleracea resistance donors were tested with regard to their reaction to a mixture of two aggressive V. longisporum isolates, and resistances from diverse lines were combined by embryo rescue-assisted interspecific hybridization in resynthesized rapeseed lines. Progenies from crosses of the two B. rapa gene bank accessions 13444 and 56515 to the B. oleracea gene bank accessions BRA1008, CGN14044, 8207, BRA1398, and 7518 showed a broad spectrum of resistance in pathogenicity tests. Of 45 tested resynthesized lines, 41 lines exhibited a significantly higher level of resistance than the moderately V. longisporum-tolerant oilseed rape cultivar Express. These lines represent a promising basis for the combination of different resistance resources in new varieties.

  4. Soluble phenylpropanoids are involved in the defense response of Arabidopsis against Verticillium longisporum.

    PubMed

    König, Stefanie; Feussner, Kirstin; Kaever, Alexander; Landesfeind, Manuel; Thurow, Corinna; Karlovsky, Petr; Gatz, Christiane; Polle, Andrea; Feussner, Ivo

    2014-05-01

    Verticillium longisporum is a soil-borne vascular pathogen causing economic loss in rape. Using the model plant Arabidopsis this study analyzed metabolic changes upon fungal infection in order to identify possible defense strategies of Brassicaceae against this fungus. Metabolite fingerprinting identified infection-induced metabolites derived from the phenylpropanoid pathway. Targeted analysis confirmed the accumulation of sinapoyl glucosides, coniferin, syringin and lignans in leaves from early stages of infection on. At later stages, the amounts of amino acids increased. To test the contribution of the phenylpropanoid pathway, mutants in the pathway were analyzed. The sinapate-deficient mutant fah1-2 showed stronger infection symptoms than wild-type plants, which is most likely due to the lack of sinapoyl esters. Moreover, the coniferin accumulating transgenic plant UGT72E2-OE was less susceptible. Consistently, sinapoyl glucose, coniferyl alcohol and coniferin inhibited fungal growth and melanization in vitro, whereas sinapyl alcohol and syringin did not. The amount of lignin was not significantly altered supporting the notion that soluble derivatives of the phenylpropanoid pathway contribute to defense. These data show that soluble phenylpropanoids are important for the defense response of Arabidopsis against V. longisporum and that metabolite fingerprinting is a valuable tool to identify infection-relevant metabolic markers.

  5. ERECTA, salicylic acid, abscisic acid, and jasmonic acid modulate quantitative disease resistance of Arabidopsis thaliana to Verticillium longisporum

    PubMed Central

    2014-01-01

    Background Verticillium longisporum is a soil-borne vascular pathogen infecting cruciferous hosts such as oilseed rape. Quantitative disease resistance (QDR) is the major control means, but its molecular basis is poorly understood so far. Quantitative trait locus (QTL) mapping was performed using a new (Bur×Ler) recombinant inbred line (RIL) population of Arabidopsis thaliana. Phytohormone measurements and analyses in defined mutants and near-isogenic lines (NILs) were used to identify genes and signalling pathways that underlie different resistance QTL. Results QTL for resistance to V. longisporum-induced stunting, systemic colonization by the fungus and for V. longisporum-induced chlorosis were identified. Stunting resistance QTL were contributed by both parents. The strongest stunting resistance QTL was shown to be identical with Erecta. A functional Erecta pathway, which was present in Bur, conferred partial resistance to V. longisporum-induced stunting. Bur showed severe stunting susceptibility in winter. Three stunting resistance QTL of Ler origin, two co-localising with wall-associated kinase-like (Wakl)-genes, were detected in winter. Furthermore, Bur showed a much stronger induction of salicylic acid (SA) by V. longisporum than Ler. Systemic colonization was controlled independently of stunting. The vec1 QTL on chromosome 2 had the strongest effect on systemic colonization. The same chromosomal region controlled the level of abscisic acid (ABA) and jasmonic acid (JA) in response to V. longisporum: The level of ABA was higher in colonization-susceptible Ler than in colonization-resistant Bur after V. longisporum infection. JA was down-regulated in Bur after infection, but not in Ler. These differences were also demonstrated in NILs, varying only in the region containing vec1. All phytohormone responses were shown to be independent of Erecta. Conclusions Signalling systems with a hitherto unknown role in the QDR of A. thaliana against V. longisporum were

  6. Verticillium longisporum Infection Affects the Leaf Apoplastic Proteome, Metabolome, and Cell Wall Properties in Arabidopsis thaliana

    PubMed Central

    Floerl, Saskia; Majcherczyk, Andrzej; Possienke, Mareike; Feussner, Kirstin; Tappe, Hella; Gatz, Christiane; Feussner, Ivo; Kües, Ursula; Polle, Andrea

    2012-01-01

    Verticillium longisporum (VL) is one of the most devastating diseases in important oil crops from the family of Brassicaceae. The fungus resides for much time of its life cycle in the extracellular fluid of the vascular system, where it cannot be controlled by conventional fungicides. To obtain insights into the biology of VL-plant interaction in the apoplast, the secretome consisting of the extracellular proteome and metabolome as well as cell wall properties were studied in the model Brassicaceae, Arabidopsis thaliana. VL infection resulted in increased production of cell wall material with an altered composition of carbohydrate polymers and increased lignification. The abundance of several hundred soluble metabolites changed in the apoplast of VL-infected plants including signalling and defence compounds such as glycosides of salicylic acid, lignans and dihydroxybenzoic acid as well as oxylipins. The extracellular proteome of healthy leaves was enriched in antifungal proteins. VL caused specific increases in six apoplast proteins (three peroxidases PRX52, PRX34, P37, serine carboxypeptidase SCPL20, α-galactosidase AGAL2 and a germin-like protein GLP3), which have functions in defence and cell wall modification. The abundance of a lectin-like, chitin-inducible protein (CILLP) was reduced. Since the transcript levels of most of the induced proteins were not elevated until late infection time points (>20 dpi), whereas those of CILLP and GLP3 were reduced at earlier time points, our results may suggest that VL enhances its virulence by rapid down-regulation and delay of induction of plant defence genes. PMID:22363647

  7. The plant host Brassica napus induces in the pathogen Verticillium longisporum the expression of functional catalase peroxidase which is required for the late phase of disease.

    PubMed

    Singh, Seema; Braus-Stromeyer, Susanna A; Timpner, Christian; Valerius, Oliver; von Tiedemann, Andreas; Karlovsky, Petr; Druebert, Christine; Polle, Andrea; Braus, Gerhard H

    2012-04-01

    The devastating soilborne fungal pathogen Verticillium longisporum is host specific to members of the family Brassicaceae, including oilseed rape (Brassica napus) as the economically most important crop. The fungus infects through the roots and causes stunting and early senescence of susceptible host plants and a marked decrease in crop yield. We show here that V. longisporum reacts to the presence of B. napus xylem sap with the production of six distinct upregulated and eight downregulated proteins visualized by two-dimensional gel electrophoresis. Identification of 10 proteins by mass spectrometry revealed that all upregulated proteins are involved in oxidative stress response. The V. longisporum catalase peroxidase (VlCPEA) was the most upregulated protein and is encoded by two isogenes, VlcpeA-1 and VlcpeA-2. Both genes are 98% identical, corroborating the diploid or "amphihaploid" status of the fungus. Knock downs of both VlcpeA genes reduced protein expression by 80% and resulted in sensitivity against reactive oxygen species. Whereas saprophytic growth and the initial phase of the plant infection were phenotypically unaffected, the mutants were not able to perform the late phases of disease. We propose that the catalase peroxidase plays a role in protecting the fungus from the oxidative stress generated by the host plant at an advanced phase of the disease. PMID:22112218

  8. Identification and characterization of microRNAs in oilseed rape (Brassica napus) responsive to infection with the pathogenic fungus Verticillium longisporum using Brassica AA (Brassica rapa) and CC (Brassica oleracea) as reference genomes.

    PubMed

    Shen, Dan; Suhrkamp, Ina; Wang, Yu; Liu, Shenyi; Menkhaus, Jan; Verreet, Joseph-Alexander; Fan, Longjiang; Cai, Daguang

    2014-11-01

    Verticillium longisporum, a soil-borne pathogenic fungus, causes vascular disease in oilseed rape (Brassica napus). We proposed that plant microRNAs (miRNAs) are involved in the plant-V. longisporum interaction. To identify oilseed rape miRNAs, we deep-sequenced two small RNA libraries made from V. longisporum infected/noninfected roots and employed Brassica rapa and Brassica oleracea genomes as references for miRNA prediction and characterization. We identified 893 B. napus miRNAs representing 360 conserved and 533 novel miRNAs, and mapped 429 and 464 miRNAs to the AA and CC genomes, respectively. Microsynteny analysis with the conserved miRNAs and their flanking protein coding sequences revealed 137 AA-CC genome syntenic miRNA pairs and 61 AA and 42 CC genome-unique miRNAs. Sixty-two miRNAs were responsive to the V. longisporum infection. We present data for specific interactions and simultaneously reciprocal changes in the expression levels of the miRNAs and their targets in the infected roots. We demonstrate that miRNAs are involved in the plant-fungus interaction and that miRNA168-Argonaute 1 (AGO1) expression modulation might act as a key regulatory module in a compatible plant-V. longisporum interaction. Our results suggest that V. longisporum may have evolved a virulence mechanism by interference with plant miRNAs to reprogram plant gene expression and achieve infection.

  9. Identification and characterization of microRNAs in oilseed rape (Brassica napus) responsive to infection with the pathogenic fungus Verticillium longisporum using Brassica AA (Brassica rapa) and CC (Brassica oleracea) as reference genomes.

    PubMed

    Shen, Dan; Suhrkamp, Ina; Wang, Yu; Liu, Shenyi; Menkhaus, Jan; Verreet, Joseph-Alexander; Fan, Longjiang; Cai, Daguang

    2014-11-01

    Verticillium longisporum, a soil-borne pathogenic fungus, causes vascular disease in oilseed rape (Brassica napus). We proposed that plant microRNAs (miRNAs) are involved in the plant-V. longisporum interaction. To identify oilseed rape miRNAs, we deep-sequenced two small RNA libraries made from V. longisporum infected/noninfected roots and employed Brassica rapa and Brassica oleracea genomes as references for miRNA prediction and characterization. We identified 893 B. napus miRNAs representing 360 conserved and 533 novel miRNAs, and mapped 429 and 464 miRNAs to the AA and CC genomes, respectively. Microsynteny analysis with the conserved miRNAs and their flanking protein coding sequences revealed 137 AA-CC genome syntenic miRNA pairs and 61 AA and 42 CC genome-unique miRNAs. Sixty-two miRNAs were responsive to the V. longisporum infection. We present data for specific interactions and simultaneously reciprocal changes in the expression levels of the miRNAs and their targets in the infected roots. We demonstrate that miRNAs are involved in the plant-fungus interaction and that miRNA168-Argonaute 1 (AGO1) expression modulation might act as a key regulatory module in a compatible plant-V. longisporum interaction. Our results suggest that V. longisporum may have evolved a virulence mechanism by interference with plant miRNAs to reprogram plant gene expression and achieve infection. PMID:25132374

  10. The Vascular Pathogen Verticillium longisporum Requires a Jasmonic Acid-Independent COI1 Function in Roots to Elicit Disease Symptoms in Arabidopsis Shoots1[W

    PubMed Central

    Ralhan, Anjali; Schöttle, Sonja; Thurow, Corinna; Iven, Tim; Feussner, Ivo; Polle, Andrea; Gatz, Christiane

    2012-01-01

    Verticillium longisporum is a soil-borne vascular pathogen that causes reduced shoot growth and early senescence in Arabidopsis (Arabidopsis thaliana). Here, we report that these disease symptoms are less pronounced in plants that lack the receptor of the plant defense hormone jasmonic acid (JA), CORONATINE INSENSITIVE1 (COI1). Initial colonization of the roots was comparable in wild-type and coi1 plants, and fungal DNA accumulated to almost similar levels in petioles of wild-type and coi1 plants at 10 d post infection. Completion of the fungal life cycle was impaired in coi1, as indicated by the reduced number of plants with microsclerotia, which are detected on dead plant material at late stages of the disease. Contrary to the expectation that the hormone receptor mutant coi1 should display the same phenotype as the corresponding hormone biosynthesis mutant delayed dehiscence2 (dde2), dde2 plants developed wild-type-like disease symptoms. Marker genes of the JA and the JA/ethylene defense pathway were induced in petioles of wild-type plants but not in petioles of dde2 plants, indicating that fungal compounds that would activate the known COI1-dependent signal transduction chain were absent. Grafting experiments revealed that the susceptibility-enhancing COI1 function acts in the roots. Moreover, we show that the coi1-mediated tolerance is not due to the hyperactivation of the salicylic acid pathway. Together, our results have unraveled a novel COI1 function in the roots that acts independently from JA-isoleucine or any JA-isoleucine mimic. This COI1 activity is required for a yet unknown root-to-shoot signaling process that enables V. longisporum to elicit disease symptoms in Arabidopsis. PMID:22635114

  11. The vascular pathogen Verticillium longisporum requires a jasmonic acid-independent COI1 function in roots to elicit disease symptoms in Arabidopsis shoots.

    PubMed

    Ralhan, Anjali; Schöttle, Sonja; Thurow, Corinna; Iven, Tim; Feussner, Ivo; Polle, Andrea; Gatz, Christiane

    2012-07-01

    Verticillium longisporum is a soil-borne vascular pathogen that causes reduced shoot growth and early senescence in Arabidopsis (Arabidopsis thaliana). Here, we report that these disease symptoms are less pronounced in plants that lack the receptor of the plant defense hormone jasmonic acid (JA), CORONATINE INSENSITIVE1 (COI1). Initial colonization of the roots was comparable in wild-type and coi1 plants, and fungal DNA accumulated to almost similar levels in petioles of wild-type and coi1 plants at 10 d post infection. Completion of the fungal life cycle was impaired in coi1, as indicated by the reduced number of plants with microsclerotia, which are detected on dead plant material at late stages of the disease. Contrary to the expectation that the hormone receptor mutant coi1 should display the same phenotype as the corresponding hormone biosynthesis mutant delayed dehiscence2 (dde2), dde2 plants developed wild-type-like disease symptoms. Marker genes of the JA and the JA/ethylene defense pathway were induced in petioles of wild-type plants but not in petioles of dde2 plants, indicating that fungal compounds that would activate the known COI1-dependent signal transduction chain were absent. Grafting experiments revealed that the susceptibility-enhancing COI1 function acts in the roots. Moreover, we show that the coi1-mediated tolerance is not due to the hyperactivation of the salicylic acid pathway. Together, our results have unraveled a novel COI1 function in the roots that acts independently from JA-isoleucine or any JA-isoleucine mimic. This COI1 activity is required for a yet unknown root-to-shoot signaling process that enables V. longisporum to elicit disease symptoms in Arabidopsis.

  12. The tolerance of the Arabidopsis defense hormone receptor mutant coi1 against the vascular pathogen Verticillium longisporum is not due to increased levels of the active hormone jasmonoyl-isoleucine.

    PubMed

    Ralhan, Anjali; Thurow, Corinna; Gatz, Christiane

    2013-11-01

    Verticillium longisporum is a soil-borne vascular pathogen found primarily on oilseed rape in Northern Europe. Infection of the model plant Arabidopsis thaliana can be achieved under laboratory conditions. In the article related to this addendum, we have shown that Arabidopsis dde2-2 mutants that are compromised in their ability to synthesize the defense hormone jasmonoyl-isoleucine (JA-Ile) are slightly more susceptible than wild-type. Contrary to the expectation that hormone biosynthesis mutants and their respective receptor mutants should have the same phenotype, we found that plants that lack the JA-Ile receptor CORONATINE INSENSITIVE1 (COI1) are more tolerant to the disease. This addendum addressed the question whether the increased JA-Ile levels found in coi1 are responsible for its tolerance phenotype. Based on the evidence that the JA-Ile-deficient dde2-2 coi1-t double mutant is as tolerant as coi1-t, we conclude that increased JA-Ile levels do not protect Arabidopsis against the fungus in the absence of COI1.

  13. The tolerance of the Arabidopsis defense hormone receptor mutant coi1 against the vascular pathogen Verticillium longisporum is not due to increased levels of the active hormone jasmonoyl-isoleucine

    PubMed Central

    Ralhan, Anjali; Thurow, Corinna; Gatz, Christiane

    2013-01-01

    Verticillium longisporum is a soil-borne vascular pathogen found primarily on oilseed rape in Northern Europe. Infection of the model plant Arabidopsis thaliana can be achieved under laboratory conditions. In the article related to this addendum, we have shown that Arabidopsis dde2–2 mutants that are compromised in their ability to synthesize the defense hormone jasmonoyl-isoleucine (JA-Ile) are slightly more susceptible than wild-type. Contrary to the expectation that hormone biosynthesis mutants and their respective receptor mutants should have the same phenotype, we found that plants that lack the JA-Ile receptor CORONATINE INSENSITIVE1 (COI1) are more tolerant to the disease. This addendum addressed the question whether the increased JA-Ile levels found in coi1 are responsible for its tolerance phenotype. Based on the evidence that the JA-Ile-deficient dde2–2 coi1-t double mutant is as tolerant as coi1-t, we conclude that increased JA-Ile levels do not protect Arabidopsis against the fungus in the absence of COI1. PMID:24300304

  14. Transposable elements in phytopathogenic Verticillium spp.: insights into genome evolution and inter- and intra-specific diversification

    PubMed Central

    2012-01-01

    Background Verticillium dahliae (Vd) and Verticillium albo-atrum (Va) are cosmopolitan soil fungi causing very disruptive vascular diseases on a wide range of crop plants. To date, no sexual stage has been identified in either microorganism suggesting that somatic mutation is a major force in generating genetic diversity. Whole genome comparative analysis of the recently sequenced strains VdLs.17 and VaMs.102 revealed that non-random insertions of transposable elements (TEs) have contributed to the generation of four lineage-specific (LS) regions in VdLs.17. Results We present here a detailed analysis of Class I retrotransposons and Class II “cut-and-paste” DNA elements detected in the sequenced Verticillium genomes. We report also of their distribution in other Vd and Va isolates from various geographic origins. In VdLs.17, we identified and characterized 56 complete retrotransposons of the Gypsy-, Copia- and LINE-like types, as well as 34 full-length elements of the “cut-and-paste” superfamilies Tc1/mariner, Activator and Mutator. While Copia and Tc1/mariner were present in multiple identical copies, Activator and Mutator sequences were highly divergent. Most elements comprised complete ORFs, had matching ESTs and showed active transcription in response to stress treatment. Noticeably, we found evidences of repeat-induced point mutation (RIP) only in some of the Gypsy retroelements. While Copia-, Gypsy- and Tc1/mariner-like transposons were prominent, a large variation in presence of the other types of mobile elements was detected in the other Verticillium spp. strains surveyed. In particular, neither complete nor defective “cut-and-paste” TEs were found in VaMs.102. Conclusions Copia-, Gypsy- and Tc1/mariner-like transposons are the most wide-spread TEs in the phytopathogens V. dahliae and V. albo-atrum. In VdLs.17, we identified several retroelements and “cut-and-paste” transposons still potentially active. Some of these elements have undergone

  15. A non-cyclic baboon θ-defensin derivative exhibiting antimicrobial activity against the phytopathogen Verticillium dahliae.

    PubMed

    Ni, Mi; Zhao, Yijing; Bibi, Noreen; Shao, Mingyan; Yuan, Shuna; Fan, Kai; Zhang, Gaixia; Li, Feng; Wang, Xuede

    2013-03-01

    θ-Defensins are the only natural cyclic proteins found in primates. They have strong antimicrobial activity related to their trisulfide ladders and macrocyclic conformation. A non-cyclic baboon θ-defensin (BTD) was synthesized by substituting valine with phenylalanine at position 17, at the C-terminal end of the BTD; this was termed "BTD-S." The antimicrobial activities of this synthetic peptide were investigated against Escherichia coli and two cotton phytopathogens: Verticillium dahliae and Fusarium oxysporum. The minimum inhibitory concentration (MIC) of BTD-S for E. coli was 10 μg/mL and for V. dahliae was 5 μg/mL, significantly lower than that for F. oxysporum (40.0 μg/mL). A time course analysis of fungal cultures indicated that the growth of V. dahliae was completely inhibited after 96 h of BTD-S treatment. Furthermore, hemolysis assays revealed that BTD-S was not toxic to mammalian cells as it could not induce lysis of sheep red blood cells even at ten times the MIC (50 μg/mL). Scanning electron microscopy and double-stained (calcofluor white and propidium iodide binding) fluorescence microscopy showed that exposure of spores of V. dahliae to BTD-S either disabled normal germination or disintegrated the spores. The size of cells exposed to BTD-S was significantly reduced compared with controls, and their number increased in a dose-dependent curve when measured by flow cytometry. These findings suggest that BTD-S has great potential to inhibit the growth of V. dahliae and can be utilized as an effective remedy to control economic losses caused by Verticillium wilt in the development of wilt-resistant cotton.

  16. Comparative analyses of secreted proteins from the phytopathogenic fungus Verticillium dahliae in response to nitrogen starvation.

    PubMed

    Chu, Jun; Li, Wei-Fang; Cheng, Wang; Lu, Mo; Zhou, Ke-Hai; Zhu, He-Qin; Li, Fu-Guang; Zhou, Cong-Zhao

    2015-05-01

    The soilborne fungus Verticillium dahliae is the major pathogen that causes the verticillium wilt disease of plants, which leads to huge economic loss worldwide. At the early stage of infection, growth of the pathogen is subject to the nutrition stress of limited nitrogen. To investigate the secreted pathogenic proteins that play indispensable roles during invasion at this stage, we compared the profiles of secreted proteins of V. dahliae under nitrogen starvation and normal conditions by using in-gel and in-solution digestion combined with liquid chromatography-nano-electrospray ionization tandem mass spectrometry (LC-nanoESI-MS). In total, we identified 212 proteins from the supernatant of liquid medium, including 109 putative secreted proteins. Comparative analysis indicated that the expression of 76 proteins was induced, whereas that of 9 proteins was suppressed under nitrogen starvation. Notably, 24 proteins are constitutively expressed. Further bioinformatic exploration enabled us to classify the stress-induced proteins into seven functional groups: cell wall degradation (10.5%), reactive oxygen species (ROS) scavenging and stress response (11.8%), lipid effectors (5.3%), protein metabolism (21.1%), carbohydrate metabolism (15.8%), electron-proton transport and energy metabolism (14.5%), and other (21.0%). In addition, most stress-suppressed proteins are involved in the cell-wall remodeling. Taken together, our analyses provide insights into the pathogenesis of V. dahliae and might give hints for the development of novel strategy against the verticillium wilt disease.

  17. Transposable elements in phytopathogenic Verticillium spp.: insights into genome evolution and inter- and intra-specific diversification

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Verticillium dahliae (Vd) and Verticillium albo-atrum (Va) are cosmopolitan soil fungi causing very disruptive vascular diseases on a wide range of crop plants. To date, no sexual stage has been identified in either microorganism suggesting that somatic mutation is a major force in generating geneti...

  18. "Cryptic" group-I introns in the nuclear SSU-rRNA gene of Verticillium dahliae.

    PubMed

    Papaioannou, Ioannis A; Dimopoulou, Chrysoula D; Typas, Milton A

    2014-08-01

    Group-I introns are widespread--though irregularly distributed--in eukaryotic organisms, and they have been extensively used for discrimination and phylogenetic analyses. Within the Verticillium genus, which comprises important phytopathogenic fungi, a group-I intron was previously identified in the SSU-rRNA (18S) gene of only V. longisporum. In this work, we aimed at elucidating the SSU-located intron distribution in V. dahliae and other Verticillium species, and the assessment of heterogeneity regarding intron content among rDNA repeats of fungal strains. Using conserved PCR primers for the amplification of the SSU gene, a structurally similar novel intron (sub-group IC1) was detected in only a few V. dahliae isolates. However, when intron-specific primers were used for the screening of a diverse collection of Verticillium isolates that originally failed to produce intron-containing SSU amplicons, most were found to contain one or both intron types, at variable rDNA repeat numbers. This marked heterogeneity was confirmed with qRT-PCR by testing rDNA copy numbers (varying from 39 to 70 copies per haploid genome) and intron copy ratios in selected isolates. Our results demonstrate that (a) IC1 group-I introns are not specific to V. longisporum within the Verticillium genus, (b) V. dahliae isolates of vegetative compatibility groups (VCGs) 4A and 6, which bear the novel intron at most of their rDNA repeats, are closely related, and (c) there is considerable intra-genomic heterogeneity for the presence or absence of introns among the ribosomal repeats. These findings underline that distributions of introns in the highly heterogeneous repetitive rDNA complex should always be verified with sensitive methods to avoid misleading conclusions for the phylogeny of fungi and other organisms.

  19. Complete genome sequence of a novel dsRNA mycovirus isolated from the phytopathogenic fungus Verticillium dahliae Kleb.

    PubMed

    Feng, Zili; Zhu, Heqin; Li, Zhifang; Shi, Yongqiang; Zhao, Lihong; Liu, Lijiang; Jiang, Daohong

    2013-12-01

    A novel double-stranded RNA (dsRNA) mycovirus, designated Verticillium dahliae partitivirus 1 (VdPV1), was isolated from a strain of the fungus Verticillium dahliae. The VdPV1 genome has two dsRNA genome segments. The larger segment (1768 bp) has a single open reading frame (ORF) with a conserved RNA-dependent RNA polymerase (RdRP) domain. The smaller segment (1587 bp) contains a single ORF encoding a putative coat protein. Analysis of its genomic structure indicated that VdPV1 is a new member of the genus Partitivirus. We report the full-length sequence of this partitivirus that infects Verticillium dahliae, the causal agent of verticillium wilt of cotton.

  20. Genomic characterization of a novel dsRNA virus detected in the phytopathogenic fungus Verticillium dahliae Kleb.

    PubMed

    Cao, Yue-Fen; Zhu, Xi-Wu; Xiang, Yu; Li, De-Qiang; Yang, Jia-Rong; Mao, Qian-Zhuo; Chen, Ji-Shuang

    2011-07-01

    Four novel double-stranded RNA segments were detected in a Verticillium dahliae Kleb. strain (V. dahliae isolate 0-21), a causal fungal agent of Verticillium wilt disease of cotton. Each dsRNA genome segment contains a single large open reading frame (ORF) that encodes a distinctive protein with modest levels of sequence similarities to the corresponding putative proteins in the genus Chrysovirus. These include an RNA-dependent RNA polymerase (RdRp), a coat protein, an undefined replication-related protein and an ovarian tumor domain peptidase. Phylogenetic analysis of the four putative proteins unanimously indicated that they are evolutionarily related to viruses in Chrysovirus. The 5'- and 3'-untranslated regions of the four dsRNAs share highly similar internal sequence and contain conserved sequence stretches of UGAUAAAAAA(/U)UG(/U)AAAAA- (in the 5'-UTR) and -UUUACUACU (in the 3'-UTR), indicating that they have a common virus origin. Indeed, isometric virus-like particles (VLPs) with a diameter of approximately 34nm were extracted from the fungal mycelia, and the four dsRNA segments were also detected in the virus-like particle (VLP) fraction. These results suggest that the mycovirus with four different dsRNA genome segments from the fungal isolate 0-21 is a new member of the genus Chrysovirus. We named the virus Verticillium dahliae chrysovirus 1 (VdCV1).

  1. Verticillium infection triggers VASCULAR-RELATED NAC DOMAIN7-dependent de novo xylem formation and enhances drought tolerance in Arabidopsis.

    PubMed

    Reusche, Michael; Thole, Karin; Janz, Dennis; Truskina, Jekaterina; Rindfleisch, Sören; Drübert, Christine; Polle, Andrea; Lipka, Volker; Teichmann, Thomas

    2012-09-01

    The soilborne fungal plant pathogen Verticillium longisporum invades the roots of its Brassicaceae hosts and proliferates in the plant vascular system. Typical aboveground symptoms of Verticillium infection on Brassica napus and Arabidopsis thaliana are stunted growth, vein clearing, and leaf chloroses. Here, we provide evidence that vein clearing is caused by pathogen-induced transdifferentiation of chloroplast-containing bundle sheath cells to functional xylem elements. In addition, our findings suggest that reinitiation of cambial activity and transdifferentiation of xylem parenchyma cells results in xylem hyperplasia within the vasculature of Arabidopsis leaves, hypocotyls, and roots. The observed de novo xylem formation correlates with Verticillium-induced expression of the VASCULAR-RELATED NAC DOMAIN (VND) transcription factor gene VND7. Transgenic Arabidopsis plants expressing the chimeric repressor VND7-SRDX under control of a Verticillium infection-responsive promoter exhibit reduced de novo xylem formation. Interestingly, infected Arabidopsis wild-type plants show higher drought stress tolerance compared with noninfected plants, whereas this effect is attenuated by suppression of VND7 activity. Together, our results suggest that V. longisporum triggers a tissue-specific developmental plant program that compensates for compromised water transport and enhances the water storage capacity of infected Brassicaceae host plants. In conclusion, we provide evidence that this natural plant-fungus pathosystem has conditionally mutualistic features.

  2. Verticillium infection triggers VASCULAR-RELATED NAC DOMAIN7-dependent de novo xylem formation and enhances drought tolerance in Arabidopsis.

    PubMed

    Reusche, Michael; Thole, Karin; Janz, Dennis; Truskina, Jekaterina; Rindfleisch, Sören; Drübert, Christine; Polle, Andrea; Lipka, Volker; Teichmann, Thomas

    2012-09-01

    The soilborne fungal plant pathogen Verticillium longisporum invades the roots of its Brassicaceae hosts and proliferates in the plant vascular system. Typical aboveground symptoms of Verticillium infection on Brassica napus and Arabidopsis thaliana are stunted growth, vein clearing, and leaf chloroses. Here, we provide evidence that vein clearing is caused by pathogen-induced transdifferentiation of chloroplast-containing bundle sheath cells to functional xylem elements. In addition, our findings suggest that reinitiation of cambial activity and transdifferentiation of xylem parenchyma cells results in xylem hyperplasia within the vasculature of Arabidopsis leaves, hypocotyls, and roots. The observed de novo xylem formation correlates with Verticillium-induced expression of the VASCULAR-RELATED NAC DOMAIN (VND) transcription factor gene VND7. Transgenic Arabidopsis plants expressing the chimeric repressor VND7-SRDX under control of a Verticillium infection-responsive promoter exhibit reduced de novo xylem formation. Interestingly, infected Arabidopsis wild-type plants show higher drought stress tolerance compared with noninfected plants, whereas this effect is attenuated by suppression of VND7 activity. Together, our results suggest that V. longisporum triggers a tissue-specific developmental plant program that compensates for compromised water transport and enhances the water storage capacity of infected Brassicaceae host plants. In conclusion, we provide evidence that this natural plant-fungus pathosystem has conditionally mutualistic features. PMID:23023171

  3. The Arabidopsis thaliana DNA-binding protein AHL19 mediates verticillium wilt resistance.

    PubMed

    Yadeta, Koste A; Hanemian, Mathieu; Smit, Patrick; Hiemstra, Jelle A; Pereira, Andy; Marco, Yves; Thomma, Bart P H J

    2011-12-01

    Verticillium spp. are destructive soilborne fungal pathogens that cause vascular wilt diseases in a wide range of plant species. Verticillium wilts are particularly notorious, and genetic resistance in crop plants is the most favorable means of disease control. In a gain-of-function screen using an activation-tagged Arabidopsis mutant collection, we identified four mutants, A1 to A4, which displayed enhanced resistance toward the vascular wilt species Verticillium dahliae, V. albo-atrum and V. longisporum but not to Fusarium oxysporum f. sp. raphani. Further testing revealed that mutant A2 displayed enhanced Ralstonia solanacearum resistance, while mutants A1 and A3 were more susceptible toward Pseudomonas syringae pv. tomato. Identification of the activation tag insertion site in the A1 mutant revealed an insertion in close proximity to the gene encoding AHL19, which was constitutively expressed in the mutant. AHL19 knock-out alleles were found to display enhanced Verticillium susceptibility whereas overexpression of AHL19 resulted in enhanced Verticillium resistance, showing that AHL19 acts as a positive regulator of plant defense. PMID:21864046

  4. DNA sequence analysis of conserved genes reveals hybridization events that increase genetic diversity in Verticillium dahliae.

    PubMed

    Collado-Romero, Melania; Jiménez-Díaz, Rafael M; Mercado-Blanco, Jesús

    2010-01-01

    The hybrid origin of a Verticillium dahliae isolate belonging to the vegetative compatibility group (VCG) 3 is reported in this work. Moreover, new data supporting the hybrid origin of two V. dahliae var. longisporum (VDLSP) isolates are provided as well as information about putative parentals. Thus, isolates of VDLSP and V. dahliae VCG3 were found harboring multiple sequences of actin (Act), β-tubulin (β-tub), calmodulin (Cal) and histone 3 (H3) genes. Phylogenetic analysis of these sequences, the internal transcribed sequences (ITS-1 and ITS-2) of the rRNA genes and of a V. dahliae-specific sequence provided molecular evidences for the interspecific hybrid origin of those isolates. Sequence analysis suggests that some of VDLSP isolates may have resulted from hybridization events between a V. dahliae isolate of VCG1 and/or VCG4A and, probably, a closely related taxon to Verticillium alboatrum but not this one. Similarly, phylogenetic analysis and PCR markers indicated that a V. dahliae VCG3 isolate might have arisen from a hybridization event between a V. dahliae VCG1B isolate and as yet unidentified parent. This second parental probably does not belong to the Verticillium genus according to the gene sequences dissimilarities found between the VCG3 isolate and Verticillium spp. These results suggest an important role of parasexuality in diversity and evolution in the genus Verticillium and show that interspecific hybrids within this genus may not be rare in nature.

  5. Verticillium Infection Triggers VASCULAR-RELATED NAC DOMAIN7–Dependent de Novo Xylem Formation and Enhances Drought Tolerance in Arabidopsis[W

    PubMed Central

    Reusche, Michael; Thole, Karin; Janz, Dennis; Truskina, Jekaterina; Rindfleisch, Sören; Drübert, Christine; Polle, Andrea; Lipka, Volker; Teichmann, Thomas

    2012-01-01

    The soilborne fungal plant pathogen Verticillium longisporum invades the roots of its Brassicaceae hosts and proliferates in the plant vascular system. Typical aboveground symptoms of Verticillium infection on Brassica napus and Arabidopsis thaliana are stunted growth, vein clearing, and leaf chloroses. Here, we provide evidence that vein clearing is caused by pathogen-induced transdifferentiation of chloroplast-containing bundle sheath cells to functional xylem elements. In addition, our findings suggest that reinitiation of cambial activity and transdifferentiation of xylem parenchyma cells results in xylem hyperplasia within the vasculature of Arabidopsis leaves, hypocotyls, and roots. The observed de novo xylem formation correlates with Verticillium-induced expression of the VASCULAR-RELATED NAC DOMAIN (VND) transcription factor gene VND7. Transgenic Arabidopsis plants expressing the chimeric repressor VND7-SRDX under control of a Verticillium infection-responsive promoter exhibit reduced de novo xylem formation. Interestingly, infected Arabidopsis wild-type plants show higher drought stress tolerance compared with noninfected plants, whereas this effect is attenuated by suppression of VND7 activity. Together, our results suggest that V. longisporum triggers a tissue-specific developmental plant program that compensates for compromised water transport and enhances the water storage capacity of infected Brassicaceae host plants. In conclusion, we provide evidence that this natural plant–fungus pathosystem has conditionally mutualistic features. PMID:23023171

  6. Analysis of a MULE-cyanide hydratase gene fusion in Verticillium dahliae

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The genome of the phytopathogenic fungus Verticillium dahliae encodes numerous Class II “cut-and-paste” transposable elements, including those of a small group of MULE transposons. We have previously identified a fusion event between a MULE transposon sequence and sequence encoding a cyanide hydrata...

  7. Phylogenetics and taxonomy of the fungal vascular wilt pathogen Verticillium, with the descriptions of five new species.

    PubMed

    Inderbitzin, Patrik; Bostock, Richard M; Davis, R Michael; Usami, Toshiyuki; Platt, Harold W; Subbarao, Krishna V

    2011-01-01

    Knowledge of pathogen biology and genetic diversity is a cornerstone of effective disease management, and accurate identification of the pathogen is a foundation of pathogen biology. Species names provide an ideal framework for storage and retrieval of relevant information, a system that is contingent on a clear understanding of species boundaries and consistent species identification. Verticillium, a genus of ascomycete fungi, contains important plant pathogens whose species boundaries have been ill defined. Using phylogenetic analyses, morphological investigations and comparisons to herbarium material and the literature, we established a taxonomic framework for Verticillium comprising ten species, five of which are new to science. We used a collection of 74 isolates representing much of the diversity of Verticillium, and phylogenetic analyses based on the ribosomal internal transcribed spacer region (ITS), partial sequences of the protein coding genes actin (ACT), elongation factor 1-alpha (EF), glyceraldehyde-3-phosphate dehydrogenase (GPD) and tryptophan synthase (TS). Combined analyses of the ACT, EF, GPD and TS datasets recognized two major groups within Verticillium, Clade Flavexudans and Clade Flavnonexudans, reflecting the respective production and absence of yellow hyphal pigments. Clade Flavexudans comprised V. albo-atrum and V. tricorpus as well as the new species V. zaregamsianum, V. isaacii and V. klebahnii, of which the latter two were morphologically indistinguishable from V. tricorpus but may differ in pathogenicity. Clade Flavnonexudans comprised V. nubilum, V. dahliae and V. longisporum, as well as the two new species V. alfalfae and V. nonalfalfae, which resembled the distantly related V. albo-atrum in morphology. Apart from the diploid hybrid V. longisporum, each of the ten species corresponded to a single clade in the phylogenetic tree comprising just one ex-type strain, thereby establishing a direct link to a name tied to a herbarium specimen

  8. Phylogenetics and Taxonomy of the Fungal Vascular Wilt Pathogen Verticillium, with the Descriptions of Five New Species

    PubMed Central

    Inderbitzin, Patrik; Bostock, Richard M.; Davis, R. Michael; Usami, Toshiyuki; Platt, Harold W.; Subbarao, Krishna V.

    2011-01-01

    Knowledge of pathogen biology and genetic diversity is a cornerstone of effective disease management, and accurate identification of the pathogen is a foundation of pathogen biology. Species names provide an ideal framework for storage and retrieval of relevant information, a system that is contingent on a clear understanding of species boundaries and consistent species identification. Verticillium, a genus of ascomycete fungi, contains important plant pathogens whose species boundaries have been ill defined. Using phylogenetic analyses, morphological investigations and comparisons to herbarium material and the literature, we established a taxonomic framework for Verticillium comprising ten species, five of which are new to science. We used a collection of 74 isolates representing much of the diversity of Verticillium, and phylogenetic analyses based on the ribosomal internal transcribed spacer region (ITS), partial sequences of the protein coding genes actin (ACT), elongation factor 1-alpha (EF), glyceraldehyde-3-phosphate dehydrogenase (GPD) and tryptophan synthase (TS). Combined analyses of the ACT, EF, GPD and TS datasets recognized two major groups within Verticillium, Clade Flavexudans and Clade Flavnonexudans, reflecting the respective production and absence of yellow hyphal pigments. Clade Flavexudans comprised V. albo-atrum and V. tricorpus as well as the new species V. zaregamsianum, V. isaacii and V. klebahnii, of which the latter two were morphologically indistinguishable from V. tricorpus but may differ in pathogenicity. Clade Flavnonexudans comprised V. nubilum, V. dahliae and V. longisporum, as well as the two new species V. alfalfae and V. nonalfalfae, which resembled the distantly related V. albo-atrum in morphology. Apart from the diploid hybrid V. longisporum, each of the ten species corresponded to a single clade in the phylogenetic tree comprising just one ex-type strain, thereby establishing a direct link to a name tied to a herbarium specimen

  9. Phylogenetics and taxonomy of the fungal vascular wilt pathogen Verticillium, with the descriptions of five new species.

    PubMed

    Inderbitzin, Patrik; Bostock, Richard M; Davis, R Michael; Usami, Toshiyuki; Platt, Harold W; Subbarao, Krishna V

    2011-01-01

    Knowledge of pathogen biology and genetic diversity is a cornerstone of effective disease management, and accurate identification of the pathogen is a foundation of pathogen biology. Species names provide an ideal framework for storage and retrieval of relevant information, a system that is contingent on a clear understanding of species boundaries and consistent species identification. Verticillium, a genus of ascomycete fungi, contains important plant pathogens whose species boundaries have been ill defined. Using phylogenetic analyses, morphological investigations and comparisons to herbarium material and the literature, we established a taxonomic framework for Verticillium comprising ten species, five of which are new to science. We used a collection of 74 isolates representing much of the diversity of Verticillium, and phylogenetic analyses based on the ribosomal internal transcribed spacer region (ITS), partial sequences of the protein coding genes actin (ACT), elongation factor 1-alpha (EF), glyceraldehyde-3-phosphate dehydrogenase (GPD) and tryptophan synthase (TS). Combined analyses of the ACT, EF, GPD and TS datasets recognized two major groups within Verticillium, Clade Flavexudans and Clade Flavnonexudans, reflecting the respective production and absence of yellow hyphal pigments. Clade Flavexudans comprised V. albo-atrum and V. tricorpus as well as the new species V. zaregamsianum, V. isaacii and V. klebahnii, of which the latter two were morphologically indistinguishable from V. tricorpus but may differ in pathogenicity. Clade Flavnonexudans comprised V. nubilum, V. dahliae and V. longisporum, as well as the two new species V. alfalfae and V. nonalfalfae, which resembled the distantly related V. albo-atrum in morphology. Apart from the diploid hybrid V. longisporum, each of the ten species corresponded to a single clade in the phylogenetic tree comprising just one ex-type strain, thereby establishing a direct link to a name tied to a herbarium specimen

  10. Verticillium alfalfae and V. dahliae, agents of Verticillium wilt diseases

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Verticillium wilts are vascular wilt diseases caused by species of Verticillium, and are among the most devastating fungal diseases worldwide. Over 400 different plant hosts, including major agricultural crops and ornamentals, are susceptible to Verticillium wilt mainly in temperate, less frequently...

  11. Biosynthetic arginine decarboxylase in phytopathogenic fungi.

    PubMed

    Khan, A J; Minocha, S C

    1989-01-01

    It has been reported that while bacteria and higher plants possess two different pathways for the biosynthesis of putrescine, via ornithine decarboxylase (ODC) and arginine decarboxylase (ADC); the fungi, like animals, only use the former pathway. We found that contrary to the earlier reports, two of the phytopathogenic fungi (Ceratocystis minor and Verticillium dahliae) contain significant levels of ADC activity with very little ODC. The ADC in these fungi has high pH optimum (8.4) and low Km (0.237 mM for C. minor, 0.103 mM for V. dahliae), and is strongly inhibited by alpha-difluoromethylarginine (DFMA), putrescine and spermidine, further showing that this enzyme is probably involved in the biosynthesis of polyamines and not in the catabolism of arginine as in Escherichia coli. The growth of these fungi is strongly inhibited by DFMA while alpha-difluoromethylornithine (DFMO) has little effect.

  12. Dynamics of verticillium species microsclerotia in field soils in response to fumigation, cropping patterns, and flooding.

    PubMed

    Short, Dylan P G; Sandoya, German; Vallad, Gary E; Koike, Steven T; Xiao, Chang-Lin; Wu, Bo-Ming; Gurung, Suraj; Hayes, Ryan J; Subbarao, Krishna V

    2015-05-01

    Verticillium dahliae is a soilborne, economically significant fungal plant pathogen that persists in the soil for up to 14 years as melanized microsclerotia (ms). Similarly, V. longisporum is a very significant production constraint on members of the family Brassicaceae. Management of Verticillium wilt has relied on methods that reduce ms below crop-specific thresholds at which little or no disease develops. Methyl bromide, a broad-spectrum biocide, has been used as a preplant soil fumigant for over 50 years to reduce V. dahliae ms. However, reductions in the number of ms in the vertical and horizontal soil profiles and the rate at which soil recolonization occurs has not been studied. The dynamics of ms in soil before and after methyl bromide+chloropicrin fumigation were followed over 3 years in six 8-by-8-m sites in two fields. In separate fields, the dynamics of ms in the 60-cm-deep vertical soil profile pre- and postfumigation with methyl bromide+chloropicrin followed by various cropping patterns were studied over 4 years. Finally, ms densities were assessed in six 8-by-8-m sites in a separate field prior to and following a natural 6-week flood. Methyl bromide+chloripicrin significantly reduced but did not eliminate V. dahliae ms in either the vertical or horizontal soil profiles. In field studies, increases in ms were highly dependent upon the crop rotation pattern followed postfumigation. In the vertical soil profile, densities of ms were highest in the top 5 to 20 cm of soil but were consistently detected at 60-cm depths. Six weeks of natural flooding significantly reduced (on average, approximately 65% in the total viable counts of ms) but did not eliminate viable ms of V. longisporum. PMID:25626074

  13. Autophagy in plants and phytopathogens.

    PubMed

    Yoshimoto, Kohki; Takano, Yoshitaka; Sakai, Yasuyoshi

    2010-04-01

    Plants and plant-associated microorganisms including phytopathogens have to adapt to drastic changes in environmental conditions. Because of their immobility, plants must cope with various types of environmental stresses such as starvation, oxidative stress, drought stress, and invasion by phytopathogens during their differentiation, development, and aging processes. Here we briefly describe the early studies of plant autophagy, summarize recent studies on the molecular functions of ATG genes, and speculate on the role of autophagy in plants and phytopathogens. Autophagy regulates senescence and pathogen-induced cell death in plants, and autophagy and pexophagy play critical roles in differentiation and the invasion of host cells by phytopathogenic fungi. PMID:20079356

  14. Interactions between Verticillium dahliae and its host: vegetative growth, pathogenicity, plant immunity.

    PubMed

    Luo, Xiumei; Xie, Chengjian; Dong, Jinyan; Yang, Xingyong; Sui, Anping

    2014-08-01

    Verticillium dahliae is a soil-borne phytopathogenic fungus that causes vascular wilt diseases in a wide variety of crop plants, resulting in extensive economic losses. In the past 5 years, progress has been made in elaborating the interaction between this hemibiotrophic fungus and its host plants. Some genes responsible for the vegetative growth and/or pathogenicity in V. dahliae have been identified. Plants have accrued a series of defense mechanisms, including inducible defense signaling pathways and some resistant genes to combat V. dahliae infection. Here, we have reviewed the progress in V. dahliae-plant interaction research.

  15. Structural and phylogenetic analysis of the rDNA intergenic spacer region of Verticillium dahliae.

    PubMed

    Papaioannou, Ioannis A; Dimopoulou, Chrysoula D; Typas, Milton A

    2013-10-01

    The nuclear ribosomal intergenic spacer (IGS) region was structurally analyzed and exploited for molecular discrimination and phylogenetic analysis of vegetative compatibility groups (VCGs) of Verticillium dahliae. A structural study of 201 available IGS sequences of the fungus was performed, and four classes of ubiquitous repetitive elements, organized in higher-order repetitive structures or composite blocks, were detected in a variable IGS subregion. This subregion was amplified from an international collection of 59 V. dahliae isolates covering all VCGs, together with nine representative V. albo-atrum and V. longisporum isolates, and sequenced. Structural and phylogenetic analyses of the sequences of this polymorphic IGS subregion were consistently informative and allowed the identification of two main lineages in V. dahliae, that is, clade I including VCGs 1A, 1B, 2A, 4B, and 3 and clade II containing VCGs 2B, 4A, and 6. Analysis of IGS sequences proved a highly suitable molecular tool for (a) rapid interspecific differentiation, (b) intraspecific discrimination among VCGs of V. dahliae, facilitating high-throughput VCG confirmation and prediction/profiling, and (c) phylogenetic analysis within and among V. dahliae VCGs.

  16. A novel method for rapidly isolating microbes that suppress soil-borne phytopathogens

    NASA Astrophysics Data System (ADS)

    Cooper, Sarah; Agnew, Linda; Pereg, Lily

    2016-04-01

    Seedling establishment faces a large number of challenges related to soil physical properties as well as to fungal root diseases. It is extremely difficult to eliminate fungal pathogens from soils where their populations are established due to the persistent nature of their spores and since fumigation of resident fungi is very ineffective in clay-containing soils. Therefore it is necessary to find ways to overcome disease in areas where the soils are infected with fungal phytopathogens. The phenomenon of disease suppressive soils, where the pathogen is present but no disease observed, suggests that microbial antagonism in the soil may lead to the suppression of the growth of fungal pathogens. There are also cases in the literature where soil microorganisms were isolated that suppress the growth of phytopathogens. Antibiosis is one of the most important mechanisms responsible for fungal antagonism, with some significant antifungal compounds involved including antibiotics, volatile organic compounds, hydrogen cyanide and lytic enzymes. Isolation of pathogen-suppressive microorganisms from the soil is time consuming and tedious. We established a simple method for direct isolation of soil microbes (bacteria and fungi) that suppress fungal phytopathogens as well as procedures for confirmation of disease suppression. We will discuss such methods, which were so far tested with the cotton fungal pathogens Thielaviopsis basicola, Verticillium dahliae and Fusarium oxysporum and Verticillium fungicola. We have isolated a diversity of T. basicola-suppressive fungi and bacteria from two vastly different soil types. Identification of the antagonistic isolates revealed that they are a diverse lot, some belong to groups known to be suppressive of a wide range of fungal pathogens, endorsing the power of this technique to rapidly and directly isolate soil-borne microbes antagonistic to a wide variety of fungal pathogens.

  17. SNP association analysis of resistance to Verticillium wilt (Verticillium dahliae Kleb.) in spinach

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Verticillium wilt, caused by Verticillium dahliae Kleb. is one of important fungus diseases in spinach (Spinacia oleracea L.) and the most economical method of control this disease is through the use of genetic resistance, especially for organic growers. The objective of this research is to evaluate...

  18. Detection and quantification of Verticillium dahliae in spinach seed

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Verticillium dahliae is a soilborne fungus that causes Verticillium wilt on multiple crops in central coastal California. Although spinach crops grown in this region for fresh and processing commercial production do not display Verticillium wilt symptoms, spinach seeds produced in the United States ...

  19. Expression of BvGLP-1 encoding a germin-like protein from sugar beet in Arabidopsis thaliana leads to resistance against phytopathogenic fungi.

    PubMed

    Knecht, Katrin; Seyffarth, Monique; Desel, Christine; Thurau, Tim; Sherameti, Irena; Lou, Binggan; Oelmüller, Ralf; Cai, Daguang

    2010-04-01

    Nematode (Heterodera schachtii) resistance in sugar beet (Beta vulgaris) is controlled by a single dominant resistance gene, Hs1(pro-1). BvGLP-1 was cloned from resistant sugar beet. The BvGLP-1 messenger (m)RNA is highly upregulated in the resistant plants after nematode infection, suggesting its role in the Hs1(pro-1) mediated resistance. BvGLP-1 exhibits sequence homology to a set of plant germin-like proteins (GLP), from which several have proved to be functional in plant basal or defense resistance against fungal pathogens. To test whether BvGLP-1 is also involved in the plant-fungus interaction, we transferred BvGLP-1 into Arabidopsis and challenged the transgenic plants with the pathogenic fungi Verticillium longisporum and Rhizoctonia solani as well as with the beneficial endophytic fungus Piriformospora indica. The expression of BvGLP-1 in Arabidopsis elevated the H(2)O(2) content and conferred significant resistance to V. longisporum and R. solani but did not affect the beneficial interaction with P. indica in seedlings. Microscopic observations revealed a dramatic reduction in the amount of hyphae of the pathogenic fungi on the root surface as well as of fungal mycelium developed inside the roots of transgenic Arabidopsis compared with wild-type plants. Molecular analysis demonstrated that the BvGLP-1 expression in Arabidopsis constitutively activates the expression of a subset of plant defense-related proteins such as PR-1 to PR-4 and PDF1.2 but not PDF2.1 and PDF2.3. In contrast, the PDF2.1 mRNA level was downregulated. These data suggest an important role of BvGLP-1 in establishment of plant defense responses, which follow specific signaling routes that diverge from those induced by the beneficial fungus. PMID:20192832

  20. Expression of BvGLP-1 encoding a germin-like protein from sugar beet in Arabidopsis thaliana leads to resistance against phytopathogenic fungi.

    PubMed

    Knecht, Katrin; Seyffarth, Monique; Desel, Christine; Thurau, Tim; Sherameti, Irena; Lou, Binggan; Oelmüller, Ralf; Cai, Daguang

    2010-04-01

    Nematode (Heterodera schachtii) resistance in sugar beet (Beta vulgaris) is controlled by a single dominant resistance gene, Hs1(pro-1). BvGLP-1 was cloned from resistant sugar beet. The BvGLP-1 messenger (m)RNA is highly upregulated in the resistant plants after nematode infection, suggesting its role in the Hs1(pro-1) mediated resistance. BvGLP-1 exhibits sequence homology to a set of plant germin-like proteins (GLP), from which several have proved to be functional in plant basal or defense resistance against fungal pathogens. To test whether BvGLP-1 is also involved in the plant-fungus interaction, we transferred BvGLP-1 into Arabidopsis and challenged the transgenic plants with the pathogenic fungi Verticillium longisporum and Rhizoctonia solani as well as with the beneficial endophytic fungus Piriformospora indica. The expression of BvGLP-1 in Arabidopsis elevated the H(2)O(2) content and conferred significant resistance to V. longisporum and R. solani but did not affect the beneficial interaction with P. indica in seedlings. Microscopic observations revealed a dramatic reduction in the amount of hyphae of the pathogenic fungi on the root surface as well as of fungal mycelium developed inside the roots of transgenic Arabidopsis compared with wild-type plants. Molecular analysis demonstrated that the BvGLP-1 expression in Arabidopsis constitutively activates the expression of a subset of plant defense-related proteins such as PR-1 to PR-4 and PDF1.2 but not PDF2.1 and PDF2.3. In contrast, the PDF2.1 mRNA level was downregulated. These data suggest an important role of BvGLP-1 in establishment of plant defense responses, which follow specific signaling routes that diverge from those induced by the beneficial fungus.

  1. Data on morphological features of mycosis induced by Colletotrichum nymphaeae and Lecanicillium longisporum on citrus orthezia scale.

    PubMed

    Mascarin, Gabriel Moura; Guarín-Molina, Juan Humberto; Arthurs, Steven Paul; Humber, Richard Alan; de Andrade Moral, Rafael; Demétrio, Clarice Garcia Borges; Delalibera, Ítalo

    2016-09-01

    We describe symptoms of mycosis induced by two native fungal entomopathogens of the citrus orthezia scale, Praelongorthezia praelonga (Hemiptera: Ortheziidae), an important pest of citrus orchards. The data presented in this article are related to the article entitled "Seasonal prevalence of the insect pathogenic fungus Colletotrichum nymphaeae in Brazilian citrus groves under different chemical pesticide regimes" [1]. The endemic fungal pathogen, C. nymphaeae, emerges through the thin cuticular intersegmental regions of the citrus orthezia scale body revealing orange salmon-pigmented conidiophores bearing conidial masses, as well as producing rhizoid-like hyphae that extend over the citrus leaf. By contrast, nymphs or adult females of this scale insect infected with Lecanicillium longisporum exhibit profuse outgrowth of bright white-pigmented conidiophores with clusters of conidia emerging from the insect intersegmental membranes, and mycosed cadavers are commonly observed attached to the leaf surface by hyphal extensions. These morphological differences are important features to discriminate these fungal entomopathogens in citrus orthezia scales. PMID:27274531

  2. Data on morphological features of mycosis induced by Colletotrichum nymphaeae and Lecanicillium longisporum on citrus orthezia scale.

    PubMed

    Mascarin, Gabriel Moura; Guarín-Molina, Juan Humberto; Arthurs, Steven Paul; Humber, Richard Alan; de Andrade Moral, Rafael; Demétrio, Clarice Garcia Borges; Delalibera, Ítalo

    2016-09-01

    We describe symptoms of mycosis induced by two native fungal entomopathogens of the citrus orthezia scale, Praelongorthezia praelonga (Hemiptera: Ortheziidae), an important pest of citrus orchards. The data presented in this article are related to the article entitled "Seasonal prevalence of the insect pathogenic fungus Colletotrichum nymphaeae in Brazilian citrus groves under different chemical pesticide regimes" [1]. The endemic fungal pathogen, C. nymphaeae, emerges through the thin cuticular intersegmental regions of the citrus orthezia scale body revealing orange salmon-pigmented conidiophores bearing conidial masses, as well as producing rhizoid-like hyphae that extend over the citrus leaf. By contrast, nymphs or adult females of this scale insect infected with Lecanicillium longisporum exhibit profuse outgrowth of bright white-pigmented conidiophores with clusters of conidia emerging from the insect intersegmental membranes, and mycosed cadavers are commonly observed attached to the leaf surface by hyphal extensions. These morphological differences are important features to discriminate these fungal entomopathogens in citrus orthezia scales.

  3. Distribution of phytopathogenic bacteria in infested seeds

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Populations of phytopathogenic bacteria representing five host-pathogen combinations were assessed to determine if there was a mathematical relationship common across seedborne bacterial diseases. Bacterial populations were estimated from naturally-infested seeds of cowpea (Vigna unguiculata), peppe...

  4. MADS-Box Transcription Factor VdMcm1 Regulates Conidiation, Microsclerotia Formation, Pathogenicity, and Secondary Metabolism of Verticillium dahliae.

    PubMed

    Xiong, Dianguang; Wang, Yonglin; Tian, Longyan; Tian, Chengming

    2016-01-01

    Verticillium dahliae, a notorious phytopathogenic fungus, causes vascular wilt diseases in many plant species resulting in devastating yield losses worldwide. Due to its ability to colonize plant xylem and form microsclerotia, V. dahliae is highly persistent and difficult to control. In this study, we show that the MADS-box transcription factor VdMcm1 is a key regulator of conidiation, microsclerotia formation, virulence, and secondary metabolism of V. dahliae. In addition, our findings suggest that VdMcm1 is involved in cell wall integrity. Finally, comparative RNA-Seq analysis reveals 823 significantly downregulated genes in the VdMcm1 deletion mutant, with diverse biological functions in transcriptional regulation, plant infection, cell adhesion, secondary metabolism, transmembrane transport activity, and cell secretion. When taken together, these data suggest that VdMcm1 performs pleiotropic functions in V. dahliae. PMID:27536281

  5. MADS-Box Transcription Factor VdMcm1 Regulates Conidiation, Microsclerotia Formation, Pathogenicity, and Secondary Metabolism of Verticillium dahliae

    PubMed Central

    Xiong, Dianguang; Wang, Yonglin; Tian, Longyan; Tian, Chengming

    2016-01-01

    Verticillium dahliae, a notorious phytopathogenic fungus, causes vascular wilt diseases in many plant species resulting in devastating yield losses worldwide. Due to its ability to colonize plant xylem and form microsclerotia, V. dahliae is highly persistent and difficult to control. In this study, we show that the MADS-box transcription factor VdMcm1 is a key regulator of conidiation, microsclerotia formation, virulence, and secondary metabolism of V. dahliae. In addition, our findings suggest that VdMcm1 is involved in cell wall integrity. Finally, comparative RNA-Seq analysis reveals 823 significantly downregulated genes in the VdMcm1 deletion mutant, with diverse biological functions in transcriptional regulation, plant infection, cell adhesion, secondary metabolism, transmembrane transport activity, and cell secretion. When taken together, these data suggest that VdMcm1 performs pleiotropic functions in V. dahliae. PMID:27536281

  6. Verticillium comparative genomics--understanding pathogenicity and diversity.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Verticillium dahliae is the primary causal agent of Verticillium wilt that causes billions of dollars in annual losses worldwide. This soil-borne fungal pathogen exhibits extraordinary genetic plasticity, capable of colonizing a broad range of hosts in diverse ecological niches. Moreover, V. dahlia...

  7. A soilless Verticillium wilt assay using an early flowering lettuce

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A soilless growth chamber assay was evaluated for rapid assessment of Verticillium wilt symptoms on lettuce. Seedlings of the early flowering plant introduction (PI) 251246 were inoculated in tubes with conidial suspensions of Verticillium spp. isolates from lettuce or cauliflower. PI 251246 devel...

  8. VdCrz1 is involved in microsclerotia formation and required for full virulence in Verticillium dahliae.

    PubMed

    Xiong, Dianguang; Wang, Yonglin; Tang, Chen; Fang, Yulin; Zou, Jingyi; Tian, Chengming

    2015-09-01

    Calcium signaling plays crucial roles in ion stress tolerance, sporulation and pathogenicity in fungi. Although the signaling pathway mediated by calcineurin and the calcineurin-responsive zinc finger transcription factor Crz1 is well characterized in other fungi, this pathway is not well characterized in the phytopathogenic fungus, Verticillium dahliae. To better understand the role of this calcineurin-dependent transcription factor in V. dahliae, an ortholog of CRZ1, VdCrz1, was identified and characterized functionally. Transcriptional analysis of VdCrz1 and GFP expression driven by the VdCrz1 promoter indicated that VdCrz1 was involved in microsclerotia development. After targeted deletion of VdCrz1, microsclerotia formation and melanin accumulation were impaired. Furthermore, the ΔVdCrz1 mutants were hypersensitive to high concentrations of Ca(2+) and cell wall-perturbing agents, such as sodium dodecyl sulfate. The addition of Mg(2+) to the medium restores the microsclerotia formation in ΔVdCrz1 mutants. The ΔVdCrz1 mutants exhibited delayed Verticillium wilt symptoms on smoke tree. These results suggest that VdCrz1 plays important roles in Ca(2+) signaling, cell wall integrity, microsclerotia development and full virulence in V. dahliae.

  9. Complete mitochondrial genome of the Verticillium-wilt causing plant pathogen Verticillium nonalfalfae

    PubMed Central

    Jelen, Vid; de Jonge, Ronnie; Van de Peer, Yves; Javornik, Branka; Jakše, Jernej

    2016-01-01

    Verticillium nonalfalfae is a fungal plant pathogen that causes wilt disease by colonizing the vascular tissues of host plants. The disease induced by hop isolates of V. nonalfalfae manifests in two different forms, ranging from mild symptoms to complete plant dieback, caused by mild and lethal pathotypes, respectively. Pathogenicity variations between the causal strains have been attributed to differences in genomic sequences and perhaps also to differences in their mitochondrial genomes. We used data from our recent Illumina NGS-based project of genome sequencing V. nonalfalfae to study the mitochondrial genomes of its different strains. The aim of the research was to prepare a V. nonalfalfae reference mitochondrial genome and to determine its phylogenetic placement in the fungal kingdom. The resulting 26,139 bp circular DNA molecule contains a full complement of the 14 "standard" fungal mitochondrial protein-coding genes of the electron transport chain and ATP synthase subunits, together with a small rRNA subunit, a large rRNA subunit, which contains ribosomal protein S3 encoded within a type IA-intron and 26 tRNAs. Phylogenetic analysis of this mitochondrial genome placed it in the Verticillium spp. lineage in the Glomerellales group, which is also supported by previous phylogenetic studies based on nuclear markers. The clustering with the closely related Verticillium dahliae mitochondrial genome showed a very conserved synteny and a high sequence similarity. Two distinguishing mitochondrial genome features were also found—a potential long non-coding RNA (orf414) contained only in the Verticillium spp. of the fungal kingdom, and a specific fragment length polymorphism observed only in V. dahliae and V. nubilum of all the Verticillium spp., thus showing potential as a species specific biomarker. PMID:26839950

  10. Identification of fungal phytopathogens using Fourier transform infrared-attenuated total reflection spectroscopy and advanced statistical methods

    NASA Astrophysics Data System (ADS)

    Salman, Ahmad; Lapidot, Itshak; Pomerantz, Ami; Tsror, Leah; Shufan, Elad; Moreh, Raymond; Mordechai, Shaul; Huleihel, Mahmoud

    2012-01-01

    The early diagnosis of phytopathogens is of a great importance; it could save large economical losses due to crops damaged by fungal diseases, and prevent unnecessary soil fumigation or the use of fungicides and bactericides and thus prevent considerable environmental pollution. In this study, 18 isolates of three different fungi genera were investigated; six isolates of Colletotrichum coccodes, six isolates of Verticillium dahliae and six isolates of Fusarium oxysporum. Our main goal was to differentiate these fungi samples on the level of isolates, based on their infrared absorption spectra obtained using the Fourier transform infrared-attenuated total reflection (FTIR-ATR) sampling technique. Advanced statistical and mathematical methods: principal component analysis (PCA), linear discriminant analysis (LDA), and k-means were applied to the spectra after manipulation. Our results showed significant spectral differences between the various fungi genera examined. The use of k-means enabled classification between the genera with a 94.5% accuracy, whereas the use of PCA [3 principal components (PCs)] and LDA has achieved a 99.7% success rate. However, on the level of isolates, the best differentiation results were obtained using PCA (9 PCs) and LDA for the lower wavenumber region (800-1775 cm-1), with identification success rates of 87%, 85.5%, and 94.5% for Colletotrichum, Fusarium, and Verticillium strains, respectively.

  11. Sources of Verticillium dahliae affecting lettuce.

    PubMed

    Atallah, Zahi K; Maruthachalam, Karunakaran; Subbarao, Krishna V

    2012-11-01

    ABSTRACT Since 1995, lettuce in coastal California, where more than half of the crop in North America is grown, has consistently suffered from severe outbreaks of Verticillium wilt. The disease is confined to this region, although the pathogen (Verticillium dahliae) and the host are present in other crop production regions in California. Migration of the pathogen with infested spinach seed was previously documented, but the geographic sources of the pathogen, as well as the impact of lettuce seed sparsely infested with V. dahliae produced outside coastal California on the pathogen population in coastal California remain unclear. Population analyses of V. dahliae were completed using 16 microsatellite markers on isolates from lettuce plants in coastal California, infested lettuce seed produced in the neighboring Santa Clara Valley of California, and spinach seed produced in four major spinach seed production regions: Chile, Denmark, the Netherlands, and the United States (Washington State). California produces 80% of spinach in the United States and all seed planted with the majority infested by V. dahliae comes from the above four sources. Three globally distributed genetic populations were identified, indicating sustained migration among these distinct geographic regions with multiple spinach crops produced each year and repeated every year in coastal California. The population structure of V. dahliae from coastal California lettuce plants was heavily influenced by migration from spinach seed imported from Denmark and Washington. Conversely, the sparsely infested lettuce seed had limited or no contribution to the Verticillium wilt epidemic in coastal California. The global trade in plant and seed material is likely contributing to sustained shifts in the population structure of V. dahliae, affecting the equilibrium of native populations, and likely affecting disease epidemiology.

  12. Identification and characterization of a pathogenicity-related gene VdCYP1 from Verticillium dahliae.

    PubMed

    Zhang, Dan-Dan; Wang, Xin-Yan; Chen, Jie-Yin; Kong, Zhi-Qiang; Gui, Yue-Jing; Li, Nan-Yang; Bao, Yu-Ming; Dai, Xiao-Feng

    2016-01-01

    Verticillium dahliae is a phytopathogenic fungus that causes vascular wilt disease in a wide variety of crop plants, thereby causing extensive economic loss. In present study, one V. dahliae T-DNA mutant M01C06 showed the pathogenicity loss on cotton, and the expression of a flanking gene encoding cytochrome P450 monooxygenase (P450, VdCYP1) was strongly repressed. P450s of fungi could affect the fungal pathogenicity by involving in the synthesis of secondary metabolites. However, there was no report about the pathogenic function of P450s in V. dahliae. VdCYP1 gene deletion and complementation experiments confirmed that VdCYP1 was the pathogenicity-related gene in V. dahliae. A comparison of culture supernatants of the VdCYP1 deletion mutants and wild-type strains indicates that at least 14 kinds of secondary metabolites syntheses were affected due to VdCYP1 gene deletion. One of these compounds, sulfacetamide, had the ability to induce the necrosis and wilting symptoms in cotton. Above results indicate that VdCYP1 could participate in pathogenesis by involving the secondary metabolism in V. dahliae, such as the compound sulfacetamide. In conclusion, VdCYP1 acts as an important pathogenicity-related factor to involve in secondary metabolism that likely contributes to the pathogenic process in V. dahliae. PMID:27329129

  13. Identification and characterization of a pathogenicity-related gene VdCYP1 from Verticillium dahliae

    PubMed Central

    Zhang, Dan-Dan; Wang, Xin-Yan; Chen, Jie-Yin; Kong, Zhi-Qiang; Gui, Yue-Jing; Li, Nan-Yang; Bao, Yu-Ming; Dai, Xiao-Feng

    2016-01-01

    Verticillium dahliae is a phytopathogenic fungus that causes vascular wilt disease in a wide variety of crop plants, thereby causing extensive economic loss. In present study, one V. dahliae T-DNA mutant M01C06 showed the pathogenicity loss on cotton, and the expression of a flanking gene encoding cytochrome P450 monooxygenase (P450, VdCYP1) was strongly repressed. P450s of fungi could affect the fungal pathogenicity by involving in the synthesis of secondary metabolites. However, there was no report about the pathogenic function of P450s in V. dahliae. VdCYP1 gene deletion and complementation experiments confirmed that VdCYP1 was the pathogenicity-related gene in V. dahliae. A comparison of culture supernatants of the VdCYP1 deletion mutants and wild-type strains indicates that at least 14 kinds of secondary metabolites syntheses were affected due to VdCYP1 gene deletion. One of these compounds, sulfacetamide, had the ability to induce the necrosis and wilting symptoms in cotton. Above results indicate that VdCYP1 could participate in pathogenesis by involving the secondary metabolism in V. dahliae, such as the compound sulfacetamide. In conclusion, VdCYP1 acts as an important pathogenicity-related factor to involve in secondary metabolism that likely contributes to the pathogenic process in V. dahliae. PMID:27329129

  14. Molecular characterization and functional analysis of a specific secreted protein from highly virulent defoliating Verticillium dahliae.

    PubMed

    Liu, Shao-Yan; Chen, Jie-Yin; Wang, Jin-Long; Li, Lei; Xiao, Hong-Li; Adam, Sami M; Dai, Xiao-Feng

    2013-10-25

    Verticillium dahliae Kleb. is a phytopathogenic fungus that causes wilt diseases in hundreds of dicotyledonous plant species. Previous research has demonstrated that the secretome plays an important role in the pathogenicity of V. dahliae. In this study, the specific secreted protein gene (VdSSP1) in highly virulent defoliating V. dahliae strain VDG1 was cloned, and considered to be a secreted protein by signal peptide activity assay. VdSSP1 deletion mutants in VDG1 significantly compromised virulence, and the fungal growth decreased in media with pectin and starch as carbon sources. Pathogenicity and carbon utilization were restored upon complementation of the VdSSP1 deletion strains or low virulence non-defoliating strain VDG2, which lacks VdSSP1. It is indicated that the virulence role of VdSSP1 is associated with plant cell wall degradation. In conclusion, our data suggested that VdSSP1 is a secreted protein that is engaged in the pathogenicity of the highly virulent defoliating V. dahliae.

  15. Insects as alternative hosts for phytopathogenic bacteria.

    PubMed

    Nadarasah, Geetanchaly; Stavrinides, John

    2011-05-01

    Phytopathogens have evolved specialized pathogenicity determinants that enable them to colonize their specific plant hosts and cause disease, but their intimate associations with plants also predispose them to frequent encounters with herbivorous insects, providing these phytopathogens with ample opportunity to colonize and eventually evolve alternative associations with insects. Decades of research have revealed that these associations have resulted in the formation of bacterial-vector relationships, in which the insect mediates dissemination of the plant pathogen. Emerging research, however, has highlighted the ability of plant pathogenic bacteria to use insects as alternative hosts, exploiting them as they would their primary plant host. The identification of specific bacterial genetic determinants that mediate the interaction between bacterium and insect suggests that these interactions are not incidental, but have likely arisen following the repeated association of microorganisms with particular insects over evolutionary time. This review will address the biology and ecology of phytopathogenic bacteria that interact with insects, including the traditional role of insects as vectors, as well as the newly emerging paradigm of insects serving as alternative primary hosts. Also discussed is one case where an insect serves as both host and vector, which may represent a transitionary stage in the evolution of insect-phytopathogen associations.

  16. Insects as alternative hosts for phytopathogenic bacteria.

    PubMed

    Nadarasah, Geetanchaly; Stavrinides, John

    2011-05-01

    Phytopathogens have evolved specialized pathogenicity determinants that enable them to colonize their specific plant hosts and cause disease, but their intimate associations with plants also predispose them to frequent encounters with herbivorous insects, providing these phytopathogens with ample opportunity to colonize and eventually evolve alternative associations with insects. Decades of research have revealed that these associations have resulted in the formation of bacterial-vector relationships, in which the insect mediates dissemination of the plant pathogen. Emerging research, however, has highlighted the ability of plant pathogenic bacteria to use insects as alternative hosts, exploiting them as they would their primary plant host. The identification of specific bacterial genetic determinants that mediate the interaction between bacterium and insect suggests that these interactions are not incidental, but have likely arisen following the repeated association of microorganisms with particular insects over evolutionary time. This review will address the biology and ecology of phytopathogenic bacteria that interact with insects, including the traditional role of insects as vectors, as well as the newly emerging paradigm of insects serving as alternative primary hosts. Also discussed is one case where an insect serves as both host and vector, which may represent a transitionary stage in the evolution of insect-phytopathogen associations. PMID:21251027

  17. Breeding and genetics of lettuce against race 2 of Verticillium wilt

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Verticillium wilt, caused by the soil-borne fungus Verticillium dahliae, is a destructive disease of lettuce in California. Verticillium resistance 1 (Vr1) confers resistance to race 1 isolates but is defeated by race 2 isolates, which are predicted to increase in prevalence. Three Plant Introductio...

  18. The Mitogen-Activated Protein Kinase Kinase VdPbs2 of Verticillium dahliae Regulates Microsclerotia Formation, Stress Response, and Plant Infection

    PubMed Central

    Tian, Longyan; Wang, Yonglin; Yu, Jun; Xiong, Dianguang; Zhao, Hengjun; Tian, Chengming

    2016-01-01

    Verticillium dahliae, a ubiquitous phytopathogenic fungus, forms resting structures, known as microsclerotia that play crucial roles in Verticillium wilt diseases. VdHog1, a mitogen-activated protein kinase (MAPK), controls microsclerotia formation, virulence, and stress response in V. dahliae. In this study, we present detailed evidence that the conserved upstream component of VdHog1, VdPbs2, is a key regulator of microsclerotia formation, oxidative stress and fungicide response and plant virulence in V. dahliae. We identified VdPbs2, homologous to the yeast MAPK kinase Pbs2. Similar to the VdHog1 deletion mutant, VdPbs2 deletion strains exhibited delayed melanin synthesis and reduced formation of microsclerotia. When exposed to stresses, VdPbs2 mutants were more sensitive than the wild type to osmotic agents and peroxide, but more resistant to inhibitors of cell wall synthesis and some fungicides. Finally, VdPbs2 deletion mutants exhibited reduced virulence on smoke tree and tobacco seedlings. When taken together, we implicate that VdPbs2 and VdHog1 function in a cascade that regulates microsclerotia formation and virulence, but not all VdHog1 dependent functions are VdPbs2 regulated. This study thus provides novel insights into the signal transduction mechanisms that regulate microsclerotia formation and pathogenesis in this fungus. PMID:27729908

  19. A synthetic antimicrobial peptide BTD-S expressed in Arabidopsis thaliana confers enhanced resistance to Verticillium dahliae.

    PubMed

    Li, Feng; Shen, Hao; Wang, Ming; Fan, Kai; Bibi, Noreen; Ni, Mi; Yuan, Shuna; Wang, Xuede

    2016-08-01

    BTD-S is a synthetic non-cyclic θ-defensin derivative which was previously designed in our laboratory based on baboon θ-defensins (BTDs). It shows robust antimicrobial activity against economically important phytopathogen, Verticillium dahliae. Here, we deduced the coding nucleotide sequence of BTD-S and introduced the gene into wild-type (ecotype Columbia-0) Arabidopsis thaliana plants. Results demonstrated that BTD-S-transgenic lines displayed in bioassays inhibitory effects on the growth of V. dahliae in vivo and in vitro. Based on symptom severity, enhanced resistance was found in a survey of BTD-S-transgenic lines. Besides, crude protein extracts from root tissues of BTD-S-transformed plants significantly restricted the growth of fungal hyphae and the germination of conidia. Also, fungal biomass over time determined by real-time PCR demonstrated the overgrowth of V. dahliae in wild-type plants 2-3 weeks after inoculation, while almost no fungal DNA was detected in aerial tissues of their transgenic progenitors. The result suggested that fungus failed to invade and progress acropetally up to establish a systemic infection in BTD-S-transgenic plants. Moreover, the assessment of basal defense responses was performed in the leaves of WT and BTD-S-transgenic plants. The mitigated oxidative stress and low antioxidase level in BTD-S-transgenic plants revealed that BTD-S acts via permeabilizing target microbial membranes, which is in a category different from hypersensitive response-dependent defense. Taken together, our results demonstrate that BTD-S is a promising gene to be explored for transgenic engineering for plant protection against Verticillium wilt.

  20. Clonal expansion of Verticillium dahliae in lettuce.

    PubMed

    Gurung, S; Short, D P G; Atallah, Z K; Subbarao, K V

    2014-06-01

    Few studies in population biology have documented how structure and diversity of pathogens evolve over time at local scales. With the historical samples of Verticillium dahliae available from lettuce, we investigated the structure and diversity of this pathogen in time and space. Three hundred twenty-nine V. dahliae isolates from lettuce fields collected over 18 years were characterized with polymorphic microsatellite markers and polymerase chain reaction tests for race and mating type. Genetic variation within and among commercial lettuce fields in a single season was also investigated using an additional 146 isolates. Sixty-two haplotypes (HTs) were observed among the 329 isolates. A single HT was frequently observed over multiple years and locations (61.40%). Genetic diversity, allelic richness, and private allelic richness suggested a relatively recent clonal expansion. Race 1 (93.63%) and MAT1-2-1 (99.69%) were overwhelmingly represented among the isolates. Linkage disequilibrium was significant (P < 0.001) for all populations, suggesting limited sexual recombination in the sampled populations from lettuce. Populations from 2006, 2009, and 2010 had higher numbers of unique HTs, implying a recent introduction of novel HTs. We conclude that V. dahliae population from lettuce evaluated in this study is expanding clonally, consistent with an asexually reproducing pathogen, and the movement of clonal genotypes locally occurs over time.

  1. Caffeine fostering of mycoparasitic fungi against phytopathogens.

    PubMed

    Sugiyama, Akifumi; Sano, Cecile M; Yazaki, Kazufumi; Sano, Hiroshi

    2016-01-01

    Caffeine (1,3,7-trimethixanthine) is a typical purine alkaloid produced in more than 80 plant species. Its biological role is considered to strengthen plant's defense capabilities, directly as a toxicant to biotic attackers (allelopathy) and indirectly as an activator of defense system (priming). Caffeine is actively secreted into rhizosphere through primary root, and possibly affects the structure of microbe community nearby. The fungal community in coffee plant rhizosphere is enriched with particular species, including Trichoderma family, a mycoparasite that attacks and kills phytopathogens by coiling and destroying their hyphae. In the present study, the caffeine response of 8 filamentous fungi, 4 mycoparasitic Trichoderma, and 4 prey phytopathogens, was examined. Results showed that allelopathic effect of caffeine on fungal growth and development was differential, being stronger on pathogens than on Trichoderma species. Upon confronting, the prey immediately ceased the growth, whereas the predator continued to grow, indicating active mycoparasitism to have occurred. Caffeine enhanced mycoparasitism up to 1.7-fold. Caffeine thus functions in a double-track manner against fungal pathogens: first by direct suppression of growth and development, and second by assisting their natural enemy. These observations suggest that caffeine is a powerful weapon in the arms race between plants and pathogens by fostering enemy's enemy, and we propose the idea of "caffeine fostering" as the third role of caffeine.

  2. Caffeine fostering of mycoparasitic fungi against phytopathogens.

    PubMed

    Sugiyama, Akifumi; Sano, Cecile M; Yazaki, Kazufumi; Sano, Hiroshi

    2016-01-01

    Caffeine (1,3,7-trimethixanthine) is a typical purine alkaloid produced in more than 80 plant species. Its biological role is considered to strengthen plant's defense capabilities, directly as a toxicant to biotic attackers (allelopathy) and indirectly as an activator of defense system (priming). Caffeine is actively secreted into rhizosphere through primary root, and possibly affects the structure of microbe community nearby. The fungal community in coffee plant rhizosphere is enriched with particular species, including Trichoderma family, a mycoparasite that attacks and kills phytopathogens by coiling and destroying their hyphae. In the present study, the caffeine response of 8 filamentous fungi, 4 mycoparasitic Trichoderma, and 4 prey phytopathogens, was examined. Results showed that allelopathic effect of caffeine on fungal growth and development was differential, being stronger on pathogens than on Trichoderma species. Upon confronting, the prey immediately ceased the growth, whereas the predator continued to grow, indicating active mycoparasitism to have occurred. Caffeine enhanced mycoparasitism up to 1.7-fold. Caffeine thus functions in a double-track manner against fungal pathogens: first by direct suppression of growth and development, and second by assisting their natural enemy. These observations suggest that caffeine is a powerful weapon in the arms race between plants and pathogens by fostering enemy's enemy, and we propose the idea of "caffeine fostering" as the third role of caffeine. PMID:26529400

  3. Caffeine fostering of mycoparasitic fungi against phytopathogens

    PubMed Central

    Sugiyama, Akifumi; Sano, Cecile M.; Yazaki, Kazufumi; Sano, Hiroshi

    2016-01-01

    Caffeine (1,3,7-trimethixanthine) is a typical purine alkaloid produced in more than 80 plant species. Its biological role is considered to strengthen plant's defense capabilities, directly as a toxicant to biotic attackers (allelopathy) and indirectly as an activator of defense system (priming). Caffeine is actively secreted into rhizosphere through primary root, and possibly affects the structure of microbe community nearby. The fungal community in coffee plant rhizosphere is enriched with particular species, including Trichoderma family, a mycoparasite that attacks and kills phytopathogens by coiling and destroying their hyphae. In the present study, the caffeine response of 8 filamentous fungi, 4 mycoparasitic Trichoderma, and 4 prey phytopathogens, was examined. Results showed that allelopathic effect of caffeine on fungal growth and development was differential, being stronger on pathogens than on Trichoderma species. Upon confronting, the prey immediately ceased the growth, whereas the predator continued to grow, indicating active mycoparasitism to have occurred. Caffeine enhanced mycoparasitism up to 1.7-fold. Caffeine thus functions in a double-track manner against fungal pathogens: first by direct suppression of growth and development, and second by assisting their natural enemy. These observations suggest that caffeine is a powerful weapon in the arms race between plants and pathogens by fostering enemy's enemy, and we propose the idea of "caffeine fostering" as the third role of caffeine. PMID:26529400

  4. Lipofuscins and sclerotial differentiation in phytopathogenic fungi.

    PubMed

    Georgiou, Christos D; Zees, Athanasios

    2002-01-01

    Lipofuscins of lipidic and proteinaceous origin were identified by their excitation and emission spectra in phytopathogenic fungal representatives of different sclerotial differentiation types. Lipofuscin pigments in Sclerotium rolfsii, Rhizoctonia solani, Sclerotinia minor and Sclerotinia sclerotiorum showed similar excitation and emission maxima (ex-em 330-450, 330-450, 330-470 and 330-470 nm, respectively). Sclerotial differentiation of these fungi was proceeded by a 4.2, 2.5, 2.7, 2.5 and 6, 2.9, 3.8, 3.1 fold increase of lipofuscin accumulation (per lipid and protein content), per respective fungus, as compared to their undifferentiated stage. Lipofuscin levels were higher in older than in younger mycelia and this phenomenon was more profound in S. rolfsii. Since lipofuscins are considered as indicators of oxidative stress, these data are in accordance with the hypothesis that suggests oxidative stress to be a common underlying factor in sclerotial differentiation of sclerotia-forming filamentous phytopathogenic fungi. PMID:12014481

  5. Entomopathogenic potential of Verticillium and Acremonium species (Deuteromycotina: Hyphomycetes).

    PubMed

    Steenberg, T; Humber, R A

    1999-05-01

    Hyphomycetes with conidia formed in slimy heads from hyaline mycelium were isolated from a range of insect hosts. Isolation on artificial medium and microscopic examination revealed that these fungi in many cases were not Verticillium lecanii despite a superficial resemblance to this common entomopathogen. The fungi were identified as Verticillium fusisporum, Verticillium psalliotae, Verticillium lamellicola, and species of Acremonium. Isolates of these fungi were bioassayed against the sweet-potato whitefly (Bemisia tabaci) and against the housefly (Musca domestica) to examine their entomopathogenicity. A test was also conducted with a coleopteran (lesser mealworm, Alphitobius diaperinus) to further evaluate the host range for some of the fungi. V. lamellicola was not pathogenic to the two species of insects treated, while varying levels of pathogenicity were shown for the other species. In general, V. lecanii was the most pathogenic species. Immature whiteflies appeared to be more susceptible to fungal infection than adult houseflies, and the host range for several of the fungi also included lesser mealworm. PMID:10222186

  6. Phytopathogenic fungal inhibitors from celery seeds.

    PubMed

    Liu, Tao; Liu, Fu-Guang; Xie, Hui-Qin; Mu, Qing

    2012-07-01

    Extract of celery (Apium graveolens L.) seeds was investigated against phytopathogenic fungi. The light petroleum extract showed promising inhibition activities in the tests against Rhizoctonia solani and Fusarium oxysporum f. sp. vasinfecum. Chromatographic separation of the extract gave 19 fractions, one of which, QCZ-4, possessed significant inhibitory rates of 64.6%, 88.4% and 54.7% at a concentration of 100 ppm against R. solani, F. oxysporium f. sp. vasinfecum and Alternaria alternata, respectively. Major components in the active fraction were identified by GC-MS as p-(2-aminoethyl)phenol (39.7%), 3-(3,4-dimethybenzoyl) propionic acid (32.6%) and p-heptylphenol (26.9%).

  7. Evaluation of verticillium wilt resistance in commercial cultivars and advanced breeding lines of cotton

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Verticillium wilt (VW), caused by Verticillium dahliae Kleb, is one of the most destructive diseases in cotton (Gossypium spp.). The most efficient and cost-effective method of controlling the disease is the use of resistant cotton cultivars. Most commercial cultivars and elite breeding lines are de...

  8. Using comparative genomics to develop a new model of understanding Verticillium wilt resistance in potato

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Verticillium wilt (VW) is a persistent and costly disease affecting potato production. VW is caused by the soilborne fungi Verticillium dahliae and V. albo-atrum. Because of the difficulty in dealing with this disease, host resistance offers a low input, effective method of control. Most potato cult...

  9. Genetics of the partial resistance against race 2 of Verticillium dahliae in lettuce

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Lettuce (Lactuca sativa L.) production on the Coastal California is threatened by Verticillium wilt, a soil borne fungal disease caused by Verticillium dahliae that diminishes yield and quality. Two races of V. dahliae were identified on lettuce, race 1 and race 2. Complete resistance to race 1 is c...

  10. Pathogenicity, vegetative compatibility, and genetic diversity in verticillium dahliae from sugar beet and historical strains

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Verticillium wilt of sugar beet is a disease problem that has received very little attention in the literature, but has been reported to reduce sucrose production and purity. To improve our understanding of Verticillium wilt, a survey of sugar beet plants with wilt symptoms (leaves with yellow or n...

  11. Screening of wild and cultivated Capsicum germplasm reveals new sources of Verticillium wilt resistance

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Verticillium wilt caused by Verticillium dahliae is an important soilborne disease of pepper (Capsicum species) worldwide. Most commercial pepper cultivars lack resistance to this pathogen. Our objective was to identify resistance to multiple V. dahliae isolates in wild and cultivated Capsicum acces...

  12. Decreased defense gene expression in tolerance versus resistance to Verticillium dahliae in potato

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Verticillium dahliae Kleb. is a soil-borne fungi that colonizes vascular tissues and induces early senescence in potato in a disease called Verticillium wilt. A diploid potato clone, 07506-01, was infected at high levels with V. dahliae but did not develop symptoms, indicating tolerance to the patho...

  13. Verticillium comparative genomics yields insights into niche adaptation by plant vascular wilt pathogens

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The vascular wilt fungi Verticillium dahliae and V. albo-atrum infect over 200 plant species worldwide, causing recurring crop losses estimated in the billions of dollars annually. Plant pathogenic Verticillium species are soilborne, and produce dormant structures that enable survival for years in ...

  14. The transcriptome of Verticillium dahliae-infected Nicotiana benthamiana determined by deep RNA sequencing.

    PubMed

    Faino, Luigi; de Jonge, Ronnie; Thomma, Bart P H J

    2012-09-01

    Verticillium wilt disease is caused by fungi of the Verticillium genus that occur on a wide range of host plants, including Solanaceous species such as tomato and tobacco. Currently, the well characterized Ve1 gene of tomato is the only Verticillium wilt resistance gene cloned. During experiments to identify the Verticillium molecule that activates Ve1 resistance in tomato, RNA sequencing (RNA-Seq) of Verticillium-infected Nicotiana benthamiana was performed. In total, over 99% of the obtained reads were derived from N. benthamiana. Here, we report the assembly and annotation of the N. benthamiana transcriptome. In total, 142,738 transcripts > 100 bp were obtained, amounting to a total transcriptome size of 38.7 Mbp, which is comparable to the Arabidopsis transcriptome. About 30,282 transcripts could be annotated based on homology to Arabidopsis genes. By assembly of the N. benthamiana transcriptome, we provide a catalogue of transcripts of a Solanaceous model plant under pathogen stress.

  15. Bidirectional promoter trapping T-DNA for insertional mutagenesis in Verticillium dahliae.

    PubMed

    Deng, Sheng; Wang, Cai-yue; Zhang, Xin; Lin, Ling

    2014-07-01

    Transfer DNA (T-DNA)-based random insertional mutagenesis is a universal forward genetic approach for gene identification and cloning in many phytopathogenic fungi. In a large number of randomly selected transformants, screening for mutants with a specific phenotype is laborious, especially for pathogenicity-defective mutants. To accelerate mutant screening and gene identification, a bidirectional promoter-trapping Ti binary vector, 1300-bisGFP-hyg, was constructed and deployed in this study. More than 6000 Verticillium dahliae transformants were obtained by the mediation of Agrobacterium tumefaciens carrying the vector. One thousand randomly selected transformants were cultured on Czapek-Dox and on Czapek-Dox plus cotton root extract media plates. The cultured transformants with green fluorescent protein (GFP) expression or changes in phenotype were selected and used in virulence or promoter-trapping assays. Based on the virulence assay of 60 transformants, the pathogenicity of 17 of these mutants was compromised. Ten pathogenicity-defective mutants were found with GFP expression, and 6 with expression in Czapek-Dox plus cotton root extract media specifically. Using TAIL-PCR (thermal asymmetric interlaced polymerase chain reaction), the T-DNA insertion sites were identified in 8 GFP-expressing transformants, including 5 pathogenicity-defective mutants and 3 unaffected transformants. Promoters of 6 genes were successfully trapped using the T-DNA method in this study. The nonpathogenic transformant 24C9 was the subject of additional investigation. It displayed strong GFP expression on water agar medium supplemented with cotton root extracts and on cotton seedling stems. The results obtained by Southern blot and quantitative real-time PCR confirmed that the transcription level of VdUGPU (encoding UTP-glucose-1-phosphate uridylyltransferase) was significantly reduced owing to T-DNA insertion in the gene promoter region. These results indicate that the bidirectional

  16. Bidirectional promoter trapping T-DNA for insertional mutagenesis in Verticillium dahliae.

    PubMed

    Deng, Sheng; Wang, Cai-yue; Zhang, Xin; Lin, Ling

    2014-07-01

    Transfer DNA (T-DNA)-based random insertional mutagenesis is a universal forward genetic approach for gene identification and cloning in many phytopathogenic fungi. In a large number of randomly selected transformants, screening for mutants with a specific phenotype is laborious, especially for pathogenicity-defective mutants. To accelerate mutant screening and gene identification, a bidirectional promoter-trapping Ti binary vector, 1300-bisGFP-hyg, was constructed and deployed in this study. More than 6000 Verticillium dahliae transformants were obtained by the mediation of Agrobacterium tumefaciens carrying the vector. One thousand randomly selected transformants were cultured on Czapek-Dox and on Czapek-Dox plus cotton root extract media plates. The cultured transformants with green fluorescent protein (GFP) expression or changes in phenotype were selected and used in virulence or promoter-trapping assays. Based on the virulence assay of 60 transformants, the pathogenicity of 17 of these mutants was compromised. Ten pathogenicity-defective mutants were found with GFP expression, and 6 with expression in Czapek-Dox plus cotton root extract media specifically. Using TAIL-PCR (thermal asymmetric interlaced polymerase chain reaction), the T-DNA insertion sites were identified in 8 GFP-expressing transformants, including 5 pathogenicity-defective mutants and 3 unaffected transformants. Promoters of 6 genes were successfully trapped using the T-DNA method in this study. The nonpathogenic transformant 24C9 was the subject of additional investigation. It displayed strong GFP expression on water agar medium supplemented with cotton root extracts and on cotton seedling stems. The results obtained by Southern blot and quantitative real-time PCR confirmed that the transcription level of VdUGPU (encoding UTP-glucose-1-phosphate uridylyltransferase) was significantly reduced owing to T-DNA insertion in the gene promoter region. These results indicate that the bidirectional

  17. VdNUC-2, the Key Regulator of Phosphate Responsive Signaling Pathway, Is Required for Verticillium dahliae Infection.

    PubMed

    Deng, Sheng; Wang, Cai-yue; Zhang, Xin; Wang, Qing; Lin, Ling

    2015-01-01

    In fungal cells, a phosphate (Pi) responsive signaling and metabolism (PHO) pathway regulates Pi-homeostasis. NUC-2/PHO81 and its homologs are one of the most important components in the regulation pathway. In soil-borne phytopathogenic fungus Verticillium dahliae, we identified a Neurospora crassa nuc-2 homolog gene VdNUC-2. VdNUC-2 is composed of 1,018 amino acids, and is highly conserved in tested filamentous fungi. Under conditions of Pi-starvation, compared with the wild-type strain and ectopic complementation strains, the VdNUC-2 knocked out mutants exhibited reduced radial growth, decreased production of conidia and microsclerotia, and were more sensitive to hydrogen peroxide stress. The virulence of VdNUC-2 defective mutants was significantly compromised, and that was unable to be restored by exogenous application of extra Pi. Additionally, the deletion mutants of VdNUC-1, a key transcription factor gene positively controlled by VdNUC-2 in the PHO pathway, showed the similar cultural phenotypes as VdNUC-2 mutants when both of them grew in Pi-limited conditions. However, the virulence of VdNUC-1 mutants was comparable to the wild-type strain. These evidences indicated that the virulence reduction in VdNUC-2 mutants is not due to the interruptions in the PHO pathway or the disturbance of Pi-homeostasis in V. dahliae cytoplasm. VdNUC-2 is not only a crucial gene in the PHO pathway in V. dahliae, but also is required for the full virulence during host-infection.

  18. Molecular characterization and functional analysis of a necrosis- and ethylene-inducing, protein-encoding gene family from Verticillium dahliae.

    PubMed

    Zhou, Bang-Jun; Jia, Pei-Song; Gao, Feng; Guo, Hui-Shan

    2012-07-01

    Verticillium dahliae Kleb. is a hemibiotrophic, phytopathogenic fungus that causes wilt disease in a wide range of crops, including cotton. Successful host colonization by hemibiotrophic pathogens requires the induction of plant cell death to provide the saprophytic nutrition for the transition from the biotrophic to the necrotrophic stage. In this study, we identified a necrosis-inducing Phytophthora protein (NPP1) domain-containing protein family containing nine genes in a virulent, defoliating isolate of V. dahliae (V592), named the VdNLP genes. Functional analysis demonstrated that only two of these VdNLP genes, VdNLP1 and VdNLP2, encoded proteins that were capable of inducing necrotic lesions and triggering defense responses in Nicotiana benthamiana, Arabidopsis, and cotton plants. Both VdNLP1 and VdNLP2 induced the wilting of cotton seedling cotyledons. However, gene-deletion mutants targeted by VdNLP1, VdNLP2, or both did not affect the pathogenicity of V. dahliae V592 in cotton infection. Similar expression and induction patterns were found for seven of the nine VdNLP transcripts. Through a comparison of the conserved amino acid residues of VdNLP with different necrosis-inducing activities, combined with mutagenesis-based analyses, we identified several novel conserved amino acid residues, in addition to the known conserved heptapeptide GHRHDWE motif and the cysteine residues of the NPP domain-containing protein, that are indispensable for the necrosis-inducing activity of the VdNLP2 protein.

  19. Maintenance of sex-related genes and the co-occurrence of both mating types in Verticillium dahliae.

    PubMed

    Short, Dylan P G; Gurung, Suraj; Hu, Xiaoping; Inderbitzin, Patrik; Subbarao, Krishna V

    2014-01-01

    Verticillium dahliae is a cosmopolitan, soilborne fungus that causes a significant wilt disease on a wide variety of plant hosts including economically important crops, ornamentals, and timber species. Clonal expansion through asexual reproduction plays a vital role in recurring plant epidemics caused by this pathogen. The recent discovery of recombination between clonal lineages and preliminary investigations of the meiotic gene inventory of V. dahliae suggest that cryptic sex appears to be rare in this species. Here we expanded on previous findings on the sexual nature of V. dahliae. Only 1% of isolates in a global collection of 1120 phytopathogenic V. dahliae isolates contained the MAT1-1 idiomorph, whereas 99% contained MAT1-2. Nine unique multilocus microsatellite types comprised isolates of both mating types, eight of which were collected from the same substrate at the same time. Orthologs of 88 previously characterized sex-related genes from fungal model systems in the Ascoymycota were identified in the genome of V. dahliae, out of 93 genes investigated. Results of RT-PCR experiments using both mating types revealed that 10 arbitrarily chosen sex-related genes, including MAT1-1-1 and MAT1-2-1, were constitutively expressed in V. dahliae cultures grown under laboratory conditions. Ratios of non-synonymous (amino-acid altering) to synonymous (silent) substitutions in V. dahliae MAT1-1-1 and MAT1-2-1 sequences were indistinguishable from the ratios observed in the MAT genes of sexual fungi in the Pezizomycotina. Patterns consistent with strong purifying selection were also observed in 18 other arbitrarily chosen V. dahliae sex-related genes, relative to the patterns in orthologs from fungi with known sexual stages. This study builds upon recent findings from other laboratories and mounts further evidence for an ancestral or cryptic sexual stage in V. dahliae.

  20. VdNUC-2, the Key Regulator of Phosphate Responsive Signaling Pathway, Is Required for Verticillium dahliae Infection

    PubMed Central

    Deng, Sheng; Wang, Cai-yue; Zhang, Xin; Wang, Qing; Lin, Ling

    2015-01-01

    In fungal cells, a phosphate (Pi) responsive signaling and metabolism (PHO) pathway regulates Pi-homeostasis. NUC-2/PHO81 and its homologs are one of the most important components in the regulation pathway. In soil-borne phytopathogenic fungus Verticillium dahliae, we identified a Neurospora crassa nuc-2 homolog gene VdNUC-2. VdNUC-2 is composed of 1,018 amino acids, and is highly conserved in tested filamentous fungi. Under conditions of Pi-starvation, compared with the wild-type strain and ectopic complementation strains, the VdNUC-2 knocked out mutants exhibited reduced radial growth, decreased production of conidia and microsclerotia, and were more sensitive to hydrogen peroxide stress. The virulence of VdNUC-2 defective mutants was significantly compromised, and that was unable to be restored by exogenous application of extra Pi. Additionally, the deletion mutants of VdNUC-1, a key transcription factor gene positively controlled by VdNUC-2 in the PHO pathway, showed the similar cultural phenotypes as VdNUC-2 mutants when both of them grew in Pi-limited conditions. However, the virulence of VdNUC-1 mutants was comparable to the wild-type strain. These evidences indicated that the virulence reduction in VdNUC-2 mutants is not due to the interruptions in the PHO pathway or the disturbance of Pi-homeostasis in V. dahliae cytoplasm. VdNUC-2 is not only a crucial gene in the PHO pathway in V. dahliae, but also is required for the full virulence during host-infection. PMID:26670613

  1. VdNUC-2, the Key Regulator of Phosphate Responsive Signaling Pathway, Is Required for Verticillium dahliae Infection.

    PubMed

    Deng, Sheng; Wang, Cai-yue; Zhang, Xin; Wang, Qing; Lin, Ling

    2015-01-01

    In fungal cells, a phosphate (Pi) responsive signaling and metabolism (PHO) pathway regulates Pi-homeostasis. NUC-2/PHO81 and its homologs are one of the most important components in the regulation pathway. In soil-borne phytopathogenic fungus Verticillium dahliae, we identified a Neurospora crassa nuc-2 homolog gene VdNUC-2. VdNUC-2 is composed of 1,018 amino acids, and is highly conserved in tested filamentous fungi. Under conditions of Pi-starvation, compared with the wild-type strain and ectopic complementation strains, the VdNUC-2 knocked out mutants exhibited reduced radial growth, decreased production of conidia and microsclerotia, and were more sensitive to hydrogen peroxide stress. The virulence of VdNUC-2 defective mutants was significantly compromised, and that was unable to be restored by exogenous application of extra Pi. Additionally, the deletion mutants of VdNUC-1, a key transcription factor gene positively controlled by VdNUC-2 in the PHO pathway, showed the similar cultural phenotypes as VdNUC-2 mutants when both of them grew in Pi-limited conditions. However, the virulence of VdNUC-1 mutants was comparable to the wild-type strain. These evidences indicated that the virulence reduction in VdNUC-2 mutants is not due to the interruptions in the PHO pathway or the disturbance of Pi-homeostasis in V. dahliae cytoplasm. VdNUC-2 is not only a crucial gene in the PHO pathway in V. dahliae, but also is required for the full virulence during host-infection. PMID:26670613

  2. A model for multiseasonal spread of verticillium wilt of lettuce.

    PubMed

    Wu, B M; Subbarao, K V

    2014-09-01

    Verticillium wilt, caused by Verticillium dahliae, is a destructive disease in lettuce, and the pathogen is seedborne. Even though maximum seed infestation rates of <5% have been detected in commercial lettuce seed lots, it is necessary to establish acceptable contamination thresholds to prevent introduction and establishment of the pathogen in lettuce production fields. However, introduction of inoculum into lettuce fields for experimental purposes to determine its long term effects is undesirable. Therefore, we constructed a simulation model to study the spread of Verticillium wilt following pathogen introduction from seed. The model consists of four components: the first for simulating infection of host plants, the second for simulating reproduction of microsclerotia on diseased plants, the third for simulating the survival of microsclerotia, and the fourth for simulating the dispersal of microsclerotia. The simulation results demonstrated that the inoculum density-disease incidence curve parameters and the dispersal gradients affect disease spread in the field. Although a steep dispersal gradient facilitated the establishment of the disease in a new field with a low inoculum density, a long-tail gradient allowed microsclerotia to be dispersed over greater distances, promoting the disease spread in fields with high inoculum density. The simulation results also revealed the importance of avoiding successive lettuce crops in the same field, reducing survival rate of microsclerotia between crops, and the need for breeding resistance against V. dahliae in lettuce cultivars to lower the number of microsclerotia formed on each diseased plant. The simulation results, however, suggested that, even with a low seed infestation rate, the pathogen would eventually become established if susceptible lettuce cultivars were grown consecutively in the same field for many years. A threshold for seed infestation can be established only when two of the three drivers of the disease

  3. A model for multiseasonal spread of verticillium wilt of lettuce.

    PubMed

    Wu, B M; Subbarao, K V

    2014-09-01

    Verticillium wilt, caused by Verticillium dahliae, is a destructive disease in lettuce, and the pathogen is seedborne. Even though maximum seed infestation rates of <5% have been detected in commercial lettuce seed lots, it is necessary to establish acceptable contamination thresholds to prevent introduction and establishment of the pathogen in lettuce production fields. However, introduction of inoculum into lettuce fields for experimental purposes to determine its long term effects is undesirable. Therefore, we constructed a simulation model to study the spread of Verticillium wilt following pathogen introduction from seed. The model consists of four components: the first for simulating infection of host plants, the second for simulating reproduction of microsclerotia on diseased plants, the third for simulating the survival of microsclerotia, and the fourth for simulating the dispersal of microsclerotia. The simulation results demonstrated that the inoculum density-disease incidence curve parameters and the dispersal gradients affect disease spread in the field. Although a steep dispersal gradient facilitated the establishment of the disease in a new field with a low inoculum density, a long-tail gradient allowed microsclerotia to be dispersed over greater distances, promoting the disease spread in fields with high inoculum density. The simulation results also revealed the importance of avoiding successive lettuce crops in the same field, reducing survival rate of microsclerotia between crops, and the need for breeding resistance against V. dahliae in lettuce cultivars to lower the number of microsclerotia formed on each diseased plant. The simulation results, however, suggested that, even with a low seed infestation rate, the pathogen would eventually become established if susceptible lettuce cultivars were grown consecutively in the same field for many years. A threshold for seed infestation can be established only when two of the three drivers of the disease

  4. Use of the phytopathogenic effect for studies of Burkholderia virulence.

    PubMed

    Molchanova, E V; Ageeva, N P

    2015-02-01

    The phytopathogenic effect of the pseudomallei group Burkholderia is demonstrated on the Peireskia aculeata model. A method for evaluation of the effect is suggested. The effect correlates with the levels of Burkholderia pseudomallei, Burkholderia mallei, and Burkholderia thailandensis virulence for laboratory animals. P. aculeata can be used as a model for preliminary studies of the virulence of the above species.

  5. Systematics of key phytopathogenic fusaria: Current status and future challenges

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This brief review is intended to provide plant pathologists and other scientists, especially ones in Japan, with a current overview of the most important fusarial phytopathogens and mycotoxin producers. Knowledge of Fusarium species diversity and their evolutionary relationships has increased dramat...

  6. To what extent are soil amendments useful to control Verticillium wilt?

    PubMed

    Goicoechea, Nieves

    2009-08-01

    The genus Verticillium includes several species that attack economically important crops throughout the world. The control of Verticillium spp. becomes especially difficult when they form microsclerotia that can survive in the field soil for several years. It has been common practice to fumigate soil with chemicals such as methyl bromide and/or chloropicrin to control soil-borne fungal pathogens. Other chemicals that are used against Verticillium spp. are the antifungal antibiotic aureofungin, the fungicides benomyl, captan, carbendazim, thiram, azoxystrobin and trifloxystrobin and the plant defence activator acibenzolar-S-methyl. However, the potential risks involved in applying phytochemicals to crop plants for both the environment and human health, together with their limited efficacy for controlling Verticillium-induced diseases, support the need to find alternatives to replace their use or improve their efficacy. Soil amendment with animal or plant organic debris is a cultural practice that has long been used to control Verticillium spp. However, today the organic farming industry is becoming a significant player in the global agricultural production scene. In this review, some of the main results concerning the efficacy of several soil amendments as plant protectors against Verticillium spp. are covered, and the limitations and future perspectives of such products are discussed in terms of the control of plant diseases.

  7. [Secretion of proteolytic enzymes by three phytopathogenic microorganisms].

    PubMed

    2013-01-01

    Serine proteinases from three phytopathogenic microorganisms that belong to different fungal families and cause diseases in potatoes were studied and characterized. The oomycete Phytophthora infestans (Mont.) de Bary and the fungi Rhizoctonia solani and Fusarium culmorum were shown to secrete serine proteinases. An analysis of the substrate specificity of these enzymes and their sensitivity to synthetic and protein inhibitors allowed us to refer them to trypsin- and subtilisin-like proteinases. The correlation between the trypsin- and subtilisin-like proteinases depended on the composition of the culture medium, particularly on the form of the nitrogen source. A phylogenetic analysis was carried out. In contrast to basidiomycetes R. solani, ascomycetes F. culmorum and oomycetes P. infestans produced a similar set of exoproteinases, although they had more distant phylogenetic positions. This indicated that the secretion of serine proteinases by various phytopathogenic microorganisms also depended on their phylogenetic position. These results allowed us to suggest that exoproteinases from phytopathogenic fungi play a different role in pathogenesis. They may promote the adaptation of fungi if the range of hosts is enlarged. On the other hand, they may play an important role in the survival of microorganisms in hostile environements outside their hosts. PMID:25508654

  8. [Secretion of proteolytic enzymes by three phytopathogenic microorganisms].

    PubMed

    Kudriavtseva, N N; Sof'in, A V; Revina, T A; Gvozdeva, E L; Ievleva, E V; Valueva, T A

    2013-01-01

    Serine proteinases from three phytopathogenic microorganisms that belong to different fungal families and cause diseases in potatoes were studied and characterized. The oomycete Phytophthora infestans (Mont.) de Bary and the fungi Rhizoctonia solani and Fusarium culmorum were shown to secrete serine proteinases. An analysis of the substrate specificity of these enzymes and their sensitivity to synthetic and protein inhibitors allowed us to refer them to trypsin- and subtilisin-like proteinases. The correlation between the trypsin- and subtilisin-like proteinases depended on the composition of the culture medium, particularly on the form of the nitrogen source. A phylogenetic analysis was carried out. In contrast to basidiomycetes R. solani, ascomycetes F. culmorum and oomycetes P. infestans produced a similar set of exoproteinases, although they had more distant phylogenetic positions. This indicated that the secretion of serine proteinases by various phytopathogenic microorganisms also depended on their phylogenetic position. These results allowed us to suggest that exoproteinases from phytopathogenic fungi play a different role in pathogenesis. They may promote the adaptation of fungi if the range of hosts is enlarged. On the other hand, they may play an important role in the survival of microorganisms in hostile environements outside their hosts. PMID:25474875

  9. A study of interaction between Verticillium wilt Verticillium dahliae and root-knot nematode Meloidogyne javanica in olive cultivars.

    PubMed

    Saeedizadeh, Ayatollah; Kheiri, Ahmad; Zad, Javad; Etebarian, Hasan Reza; Bandani, Ali Reza; Nasiri, Mohammad Bagher

    2009-01-01

    Second stage juvenile (J2) of root-knot nematode, Meloidogyne javanica, and microsclerotia of verticillium wilt, Verticillium dahliae, were used as the source of inoculum for nematode and fungus respectively. One-year-old seedlings of olive cultivars, Zard, Roghani, Koroneiki and Manzanilla, were transplanted to pots containing 2000g of sterilized sandy loam soil. Experiment was conducted in completely randomized design with 32 treatments and five replications. Treatments were as follows: control, nematode alone, fungus alone and fungus + nematode. Pots were inoculated with (0, 2000, 3000, 4000) J2 of nematode and/or (10 no/g soil) microsclerotia of fungus according to the treatments. Experiment was terminated after 10 months and fallowing parameters were determined i.e., fresh weight of root and stem, number of galls and egg masses per root system, and percentage of incidence of symptom on aerial parts, browning of vascular tissue, decrease of seedling height and stem/root tissue colonization by fungus. Results showed that presence of nematode caused reduction on colonization of the fungus in the root and stem and vice versa i.e. presence of fungus caused reduction on number of galls and egg masses produced by the nematode. Severe fungus wilt on aerial parts of Manzanilla cultivar was observed when both pathogens were inoculated and mild fungus wilt was observed in fungus alone treatments of Koroneiki cultivar. Galling and egg mass production in root system were reduced in cvs Manzanilla, Zard, Roghani and Koroneiki, respectively (p < or = 0/05). Based on the results obtained in this study, verticilliosis symptoms and galling of nematode in olive seedlings were be less on cvs Koroneiki, Roghani, Zard and Manzanilla, respectively (p < or = 0/05). PMID:20222619

  10. Segregation of unknown signaling components in potato complicates marker-assisted selection for Ve-mediated Verticillium resistance

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Verticillium wilt (VW), caused primarily by soil-borne fungi Verticillium dahliae Kleb and V. albo-atrum Reinke and Berthold is characterized by unilateral wilting of leaves, chlorosis, and premature senescence. VW is mainly controlled by fumigating fields with metam sodium, which has negative econo...

  11. Non-defoliating and defoliating strains from cotton correlate with races 1 and 2 of Verticillium dahliae

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Verticillium wilt caused by Verticillium dahliae is an important disease of cotton worldwide. Isolates of V. dahliae can be characterized as race 1 or race 2 based on the responses of differential cultivars of tomato and lettuce or as defoliating or non-defoliating based on symptom expression in cot...

  12. Identification of molecular markers associated with verticillium wilt resistance in alfalfa (medicago sativa l.) using high-resolution melting

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Verticillium wilt (VW), caused by the soilborne fungus, Verticillium alfalfae, is one of the most serious diseases of alfalfa (Medicago sativa L.) worldwide. To identify loci associated with resistance to VW, an association study was conducted using autotetraploid alfalfa populations composed of 352...

  13. Fifteen years of verticillium wilt of lettuce in america’s salad bowl: a tale of immigration, subjugation and abatement.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Lettuce is a popular leafy vegetable that is globally cultivated. The US ranks second in production, with coastal California producing half of the US supply. In 1995, Verticillium wilt caused by the soil borne fungus Verticillium dahliae was identified as a disease of lettuce in coastal California, ...

  14. Frequency of Verticillium species in commercial spinach fields and transmission of V. dahliae from spinach to subsequent lettuce crops

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Verticillium wilt, caused by Verticillium dahlia,e is a devastating disease of lettuce in California. The disease on lettuce is currently restricted to a small geographic area in the central coastal California, even though cropping patterns in other coastal lettuce production regions in the state ar...

  15. Quantitative trait locus analysis of Verticillium wilt resistance in an introgressed recombinant inbred population of Upland cotton

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Verticillium wilt (VW) of Upland cotton (Gossypium hirsutum L.) is caused by the soil-borne fungal pathogen Verticillium dahlia Kleb. The availability of VW-resistant cultivars is vital for control of this economically important disease, but there is a paucity of Upland cotton breeding lines and cul...

  16. Proteomic identification of differentially expressed proteins in Gossypium thurberi inoculated with cotton Verticillium dahliae.

    PubMed

    Zhao, Fu'an; Fang, Weiping; Xie, Deyi; Zhao, Yuanming; Tang, Zhongjie; Li, Wu; Nie, Lihong; Lv, Shuping

    2012-04-01

    Thurber's cotton (Gossypium thurberi) is the wild relative of cultivated cotton. It is highly resistant to cotton Verticillium wilt, a disease that significantly affects cotton yield and quality. To reveal the mechanism of disease resistance in G. thurberi and to clone resistance-related genes, we used two-dimensional electrophoresis (2-DE) and tandem time-of-flight mass spectrometry (MALDI-TOF-MS) to identify differentially expressed proteins in Thurber's cotton after inoculation with Verticillium dahliae. A total of 57 different protein spots were upregulated, including 52 known proteins representing 11% of the total protein spots. These proteins are involved in resistance to stress and disease, transcriptional regulation, signal transduction, protein processing and degradation, photosynthesis, production capacity, basic metabolism, and other processes. In addition, five disease resistance proteins showed intense upregulation, indicating that resistance genes (R genes) may play a critical role in resistance to Verticillium wilt in Thurber's cotton. Our results suggest that disease and stress resistance are the combined effects of multiple co-expressed genes. This provides a basis for further, detailed investigation into the mechanisms underlying Verticillium wilt resistance of G. thurberi and for cloning essential genes into cotton cultivars to produce Verticillium wilt resistant plants.

  17. Characterization of Two ABC Transporters from Biocontrol and Phytopathogenic Fusarium oxysporus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    ABC transporter genes from four strains of Fusarium oxysporum [two biocontrol and two phytopathogenic (f. sp. lycopersici Race 1) isolates] indicated that this gene is well conserved. However, sequences of promoter regions of FoABC1 differed between 8 phytopathogenic and 11 biocontrol strains of F....

  18. Identification of molecular markers associated with Verticillium wilt resistance in alfalfa (Medicago sativa L.) using high-resolution melting.

    PubMed

    Zhang, Tiejun; Yu, Long-Xi; McCord, Per; Miller, David; Bhamidimarri, Suresh; Johnson, David; Monteros, Maria J; Ho, Julie; Reisen, Peter; Samac, Deborah A

    2014-01-01

    Verticillium wilt, caused by the soilborne fungus, Verticillium alfalfae, is one of the most serious diseases of alfalfa (Medicago sativa L.) worldwide. To identify loci associated with resistance to Verticillium wilt, a bulk segregant analysis was conducted in susceptible or resistant pools constructed from 13 synthetic alfalfa populations, followed by association mapping in two F1 populations consisted of 352 individuals. Simple sequence repeat (SSR) and single nucleotide polymorphism (SNP) markers were used for genotyping. Phenotyping was done by manual inoculation of the pathogen to replicated cloned plants of each individual and disease severity was scored using a standard scale. Marker-trait association was analyzed by TASSEL. Seventeen SNP markers significantly associated with Verticillium wilt resistance were identified and they were located on chromosomes 1, 2, 4, 7 and 8. SNP markers identified on chromosomes 2, 4 and 7 co-locate with regions of Verticillium wilt resistance loci reported in M. truncatula. Additional markers identified on chromosomes 1 and 8 located the regions where no Verticillium resistance locus has been reported. This study highlights the value of SNP genotyping by high resolution melting to identify the disease resistance loci in tetraploid alfalfa. With further validation, the markers identified in this study could be used for improving resistance to Verticillium wilt in alfalfa breeding programs.

  19. Identification of Molecular Markers Associated with Verticillium Wilt Resistance in Alfalfa (Medicago Sativa L.) Using High-Resolution Melting

    PubMed Central

    Zhang, Tiejun; Yu, Long-Xi; McCord, Per; Miller, David; Bhamidimarri, Suresh; Johnson, David; Monteros, Maria J.; Ho, Julie; Reisen, Peter; Samac, Deborah A.

    2014-01-01

    Verticillium wilt, caused by the soilborne fungus, Verticillium alfalfae, is one of the most serious diseases of alfalfa (Medicago sativa L.) worldwide. To identify loci associated with resistance to Verticillium wilt, a bulk segregant analysis was conducted in susceptible or resistant pools constructed from 13 synthetic alfalfa populations, followed by association mapping in two F1 populations consisted of 352 individuals. Simple sequence repeat (SSR) and single nucleotide polymorphism (SNP) markers were used for genotyping. Phenotyping was done by manual inoculation of the pathogen to replicated cloned plants of each individual and disease severity was scored using a standard scale. Marker-trait association was analyzed by TASSEL. Seventeen SNP markers significantly associated with Verticillium wilt resistance were identified and they were located on chromosomes 1, 2, 4, 7 and 8. SNP markers identified on chromosomes 2, 4 and 7 co-locate with regions of Verticillium wilt resistance loci reported in M. truncatula. Additional markers identified on chromosomes 1 and 8 located the regions where no Verticillium resistance locus has been reported. This study highlights the value of SNP genotyping by high resolution melting to identify the disease resistance loci in tetraploid alfalfa. With further validation, the markers identified in this study could be used for improving resistance to Verticillium wilt in alfalfa breeding programs. PMID:25536106

  20. Antifungal activity of Artemisia annua endophyte cultures against phytopathogenic fungi.

    PubMed

    Liu, C H; Zou, W X; Lu, H; Tan, R X

    2001-07-12

    Artemisia annua, well recognized for its production of antimalarial drug artemisinin, is seldom attacked by any of phytopathogenic fungi, which could be partially associated with the presence of endophytes. Present investigation is aiming at disclosing whether the endophytes inside A. annua produce antifungal substances. A total of 39 endophytes were isolated and fermented, and the ferment broth was evaluated in vitro for the antifungal activity against crop-threatening fungi Gaeumannomyces graminis var. tritici, Rhizoctonia cerealis, Helminthosporium sativum, Fusarium graminearum, Gerlachia nivalis and Phytophthora capsici. These plant pathogens are still causing wheat take-all, sharp eyespot, common rot, scab, snow mould, and pepper phytophthora blight, respectively. Out of 39 endophytes investigated, 21 can produce in vitro substances that are inhibitory to all or a few of the tested phytopathogens whereas the rest yielded nothing active. Moreover, the most active broth of endophyte IV403 was extracted with EtOAc and n-butanol, and comparisons of the antifungal activity of the extracts indicated that the major active metabolites were EtOAc-extractable.

  1. Verticillium dahliae Infects, Alters Plant Biomass, and Produces Inoculum on Rotation Crops.

    PubMed

    Wheeler, D L; Johnson, D A

    2016-06-01

    Verticillium wilt, caused by Verticillium dahliae, reduces yields of potato and mint. Crop rotation is a potential management tactic for Verticillium wilt; however, the wide host range of V. dahliae may limit the effectiveness of this tactic. The hypothesis that rotation crops are infected by V. dahliae inoculum originating from potato and mint was tested by inoculation of mustards, grasses, and Austrian winter pea with eight isolates of V. dahliae. Inoculum density was estimated from plants and soil. Typical wilt symptoms were not observed in any rotation crop but plant biomass of some crops was reduced, not affected, or increased by infection of specific isolates. Each isolate was host-specific and infected a subset of the rotation crops tested but microsclerotia from at least one isolate were observed on each rotation crop. Some isolates were host-adapted and differentially altered plant biomass or produced differential amounts of inoculum on rotation crops like arugula and Austrian winter pea, which supported more inoculum of specific isolates than potato. Evidence of asymptomatic and symptomatic infection and differential inoculum formation of V. dahliae on rotation crops presented here will be useful in designing rotations for management of Verticillium wilt. PMID:26828231

  2. Verticillium dahliae Infects, Alters Plant Biomass, and Produces Inoculum on Rotation Crops.

    PubMed

    Wheeler, D L; Johnson, D A

    2016-06-01

    Verticillium wilt, caused by Verticillium dahliae, reduces yields of potato and mint. Crop rotation is a potential management tactic for Verticillium wilt; however, the wide host range of V. dahliae may limit the effectiveness of this tactic. The hypothesis that rotation crops are infected by V. dahliae inoculum originating from potato and mint was tested by inoculation of mustards, grasses, and Austrian winter pea with eight isolates of V. dahliae. Inoculum density was estimated from plants and soil. Typical wilt symptoms were not observed in any rotation crop but plant biomass of some crops was reduced, not affected, or increased by infection of specific isolates. Each isolate was host-specific and infected a subset of the rotation crops tested but microsclerotia from at least one isolate were observed on each rotation crop. Some isolates were host-adapted and differentially altered plant biomass or produced differential amounts of inoculum on rotation crops like arugula and Austrian winter pea, which supported more inoculum of specific isolates than potato. Evidence of asymptomatic and symptomatic infection and differential inoculum formation of V. dahliae on rotation crops presented here will be useful in designing rotations for management of Verticillium wilt.

  3. Dynamics of Verticillium species microsclerotia in field soils in response to fumigation, cropping patterns, and flooding

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Many soil-inhabiting fungi are capable of surviving the dynamic soil microenvironment through the formation of resilient resting structures, such as thick-walled spores, melanized hyphae, and sclerotia. Verticillium dahliae is a soil-inhabiting, economically significant plant pathogenic fungus that ...

  4. Draft Genome Sequence and Gene Annotation of the Entomopathogenic Fungus Verticillium hemipterigenum

    PubMed Central

    Horn, Fabian; Habel, Andreas; Scharf, Daniel H.; Dworschak, Jan; Brakhage, Axel A.; Guthke, Reinhard

    2015-01-01

    Verticillium hemipterigenum (anamorph Torrubiella hemipterigena) is an entomopathogenic fungus and produces a broad range of secondary metabolites. Here, we present the draft genome sequence of the fungus, including gene structure and functional annotation. Genes were predicted incorporating RNA-Seq data and functionally annotated to provide the basis for further genome studies. PMID:25614560

  5. Colonization of spinach (Spinacia oleracea L.) by GFP-tagged verticillium dahliae.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The soilborne fungus, Verticillium dahliae, causes wilt in a wide range of hosts, including spinach (Spinacia oleracea L.). The interaction between a green fluorescent protein (GFP)-tagged V. dahliae strain and spinach was studied by confocal laser scanning microscopy. The roots of spinach seedlings...

  6. Verticillium comparative genomics yields insights into niche adaptation by plant vascular wilt pathogens.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The vascular wilt fungi Verticillium dahliae and V. albo-atrum infect over 200 plant species, causing billions of dollars in annual losses. The characteristic vascular wilt symptoms are a result of colonization and proliferation of the pathogens in the xylem vessels. To gain insights into the mechan...

  7. Analysis of returns above variable costs for management of Verticillium wilt in cotton.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A large plot study located in Halfway, TX, was conducted from 2007 to 2013 in an irrigated field infested with Verticillium wilt. Management options (crop rotation, irrigation amount, variety election) and combinations of options that can reduce this disease were compared using returns above variabl...

  8. The inheritance of resistance to Verticillium wilt caused by race 1 isolates of Verticillium dahliae in the lettuce cultivar La Brillante.

    PubMed

    Hayes, Ryan J; McHale, Leah K; Vallad, Gary E; Truco, Maria Jose; Michelmore, Richard W; Klosterman, Steve J; Maruthachalam, Karunakaran; Subbarao, Krishna V

    2011-08-01

    Verticillium wilt of lettuce caused by Verticillium dahliae can cause severe economic damage to lettuce producers. Complete resistance to race 1 isolates is available in Lactuca sativa cultivar (cv.) La Brillante and understanding the genetic basis of this resistance will aid development of new resistant cultivars. F(1) and F(2) families from crosses between La Brillante and three iceberg cultivars as well as a recombinant inbred line population derived from L. sativa cv. Salinas 88 × La Brillante were evaluated for disease incidence and disease severity in replicated greenhouse and field experiments. One hundred and six molecular markers were used to generate a genetic map from Salinas 88 × La Brillante and for detection of quantitative trait loci. Segregation was consistent with a single dominant gene of major effect which we are naming Verticillium resistance 1 (Vr1). The gene described large portions of the phenotypic variance (R(2) = 0.49-0.68) and was mapped to linkage group 9 coincident with an expressed sequence tag marker (QGD8I16.yg.ab1) that has sequence similarity with the Ve gene that confers resistance to V. dahliae race 1 in tomato. The simple inheritance of resistance indicates that breeding procedures designed for single genes will be applicable for developing resistant cultivars. QGD8I16.yg.ab1 is a good candidate for functional analysis and development of markers suitable for marker-assisted selection.

  9. [THE SENSITIVITY OF PHYTOPATHOGENIC BACTERIA TO STREPTOMYCIN UNDER THE INFLUENCE OF PESTICIDES].

    PubMed

    Buletsa, N M; Butsenko, L M; Pasichnyk, L A; Patyka, V P

    2015-01-01

    The results of the streptomycin sensitivity changes of phytopathogenic Pseudomonas syringae and Xanthomonas translucens bacteria under the action of pesticides are pre- sented. It is demonstrated that phytopathogenic strains show greater changes of strepto- mycin sensitivity compared to epiphytic Pantoea agglomerans strain under the pesticides influence. Granstar herbicide, Tviks and Alpha Super insecticides increase the number of streptomycin resistant cells of Xanthomonas translucens 3164, P syringae pv. syringae YKM B-1027 and P syringae pv. atrofaciens YKM B-1011. This fact indicates mutagenic action of these pesticides against researched phytopathogenic bacteria. PMID:26829841

  10. Phytopathogen type III effector weaponry and their plant targets

    PubMed Central

    Block, Anna; Li, Guangyong; Fu, Zheng Qing; Alfano, James R.

    2008-01-01

    Summary Phytopathogenic bacteria suppress plant innate immunity and promote pathogenesis by injecting proteins called type III effectors into plant cells using a type III protein secretion system. These type III effectors use at least three major strategies to alter host responses. One strategy is to alter host protein turnover, either by direct cleavage or by modulating ubiquitination and targeting to the 26S proteasome. Another strategy involves alteration of RNA metabolism by transcriptional activation or ADP-ribosylation of RNA-binding proteins. A third major strategy is to inhibit the kinases involved in plant defence signalling, either by removing phosphates or by direct inhibition. The wide array of strategies bacterial pathogens employ to suppress innate immunity suggest that circumvention of innate immunity is critical for bacterial pathogenicity of plants. PMID:18657470

  11. [Extracellular proteinases from the phytopathogenic fungus Fusarium culmorum].

    PubMed

    Ievleva, E V; Revina, T A; Kudriavtseva, N N; Sof'in, A V; Valueva, T A

    2006-01-01

    The growth of Fusarium culmorum fungus on a medium containing thermostable proteins from potato tubers was accompanied by the production of proteinases, exhibiting activity over a broad pH range (from 6.0-10.0). When studied by SDS-PAGE in the presence of beta-mercaptoethanol, extracellular proteinases were represented by at least five species with a molecular weight of 30-60 kDa. Inhibitor analysis and studies of enzyme activities with synthetic substrates demonstrated that the culture liquid of Fusarium culmorum contained serine proteinases of various classes. The amount of subtilisin-like proteinases was the highest. A near-complete inhibition of the enzymes was caused by proteinaceous proteinase inhibitors from potato tubers. These data suggest that proteinases of the phytopathogen Fusarium culmorum serve as a metabolic target for natural inhibitors of potato proteinases.

  12. Growth rate inhibition of phytopathogenic fungi by characterized chitosans

    PubMed Central

    Oliveira Junior, Enio N.; Gueddari, Nour E. El; Moerschbacher, Bruno. M.; Franco, Telma T.

    2012-01-01

    The inhibitory effects of fifteen chitosans with different degrees of polymerization (DP) and different degrees of acetylation (FA) on the growth rates (GR) of four phytopathogenic fungi (Alternaria alternata, Botrytis cinerea, Penicillium expansum, and Rhizopus stolonifer) were examined using a 96-well microtiter plate and a microplate reader. The minimum inhibitory concentrations (MICs) of the chitosans ranged from 100 μg ×mL-1 to 1,000 μg ×mL-1 depending on the fungus tested and the DP and FA of the chitosan. The antifungal activity of the chitosans increased with decreasing FA. Chitosans with low FA and high DP showed the highest inhibitory activity against all four fungi. P. expansum and B. cinerea were relatively less susceptible while A. alternata and R. stolonifer were relatively more sensitive to the chitosan polymers. Scanning electron microscopy of fungi grown on culture media amended with chitosan revealed morphological changes. PMID:24031893

  13. Growth rate inhibition of phytopathogenic fungi by characterized chitosans.

    PubMed

    Oliveira Junior, Enio N; Gueddari, Nour E El; Moerschbacher, Bruno M; Franco, Telma T

    2012-04-01

    The inhibitory effects of fifteen chitosans with different degrees of polymerization (DP) and different degrees of acetylation (FA) on the growth rates (GR) of four phytopathogenic fungi (Alternaria alternata, Botrytis cinerea, Penicillium expansum, and Rhizopus stolonifer) were examined using a 96-well microtiter plate and a microplate reader. The minimum inhibitory concentrations (MICs) of the chitosans ranged from 100 μg ×mL(-1) to 1,000 μg ×mL(-1) depending on the fungus tested and the DP and FA of the chitosan. The antifungal activity of the chitosans increased with decreasing FA. Chitosans with low FA and high DP showed the highest inhibitory activity against all four fungi. P. expansum and B. cinerea were relatively less susceptible while A. alternata and R. stolonifer were relatively more sensitive to the chitosan polymers. Scanning electron microscopy of fungi grown on culture media amended with chitosan revealed morphological changes.

  14. Diversity of endophytic fungi from different Verticillium-wilt-resistant Gossypium hirsutum and evaluation of antifungal activity against Verticillium dahliae in vitro.

    PubMed

    Li, Zhi-Fang; Wang, Ling-Fei; Feng, Zi-Li; Zhao, Li-Hong; Shi, Yong-Qiang; Zhu, He-Qin

    2014-09-01

    Cotton plants were sampled and ranked according to their resistance to Verticillium wilt. In total, 642 endophytic fungi isolates representing 27 genera were recovered from Gossypium hirsutum root, stem, and leaf tissues, but were not uniformly distributed. More endophytic fungi appeared in the leaf (391) compared with the root (140) and stem (111) sections. However, no significant difference in the abundance of isolated endophytes was found among resistant cotton varieties. Alternaria exhibited the highest colonization frequency (7.9%), followed by Acremonium (6.6%) and Penicillium (4.8%). Unlike tolerant varieties, resistant and susceptible ones had similar endophytic fungal population compositions. In three Verticillium-wilt-resistant cotton varieties, fungal endophytes from the genus Alternaria were most frequently isolated, followed by Gibberella and Penicillium. The maximum concentration of dominant endophytic fungi was observed in leaf tissues (0.1797). The evenness of stem tissue endophytic communities (0.702) was comparatively more uniform than the other two tissues. Eighty endophytic fungi selected from 27 genera were evaluated for their inhibition activity against highly virulent Verticillium dahliae isolate Vd080 in vitro. Thirty-nine isolates exhibited fungistasis against the pathogen at varying degrees. Seven species, having high growth inhibition rates (≥75%), exhibited strong antifungal activity against V. dahliae. The antifungal activity of both volatile and nonvolatile metabolites was also investigated. The nonvolatile substances produced by CEF-818 (Penicillium simplicissimum), CEF-325 (Fusarium solani), CEF-714 (Leptosphaeria sp.), and CEF-642 (Talaromyces flavus) completely inhibited V. dahliae growth. These findings deepen our understanding of cotton-endophyte interactions and provide a platform for screening G. hirsutum endophytes with biocontrol potential. PMID:24836187

  15. Diversity of endophytic fungi from different Verticillium-wilt-resistant Gossypium hirsutum and evaluation of antifungal activity against Verticillium dahliae in vitro.

    PubMed

    Li, Zhi-Fang; Wang, Ling-Fei; Feng, Zi-Li; Zhao, Li-Hong; Shi, Yong-Qiang; Zhu, He-Qin

    2014-09-01

    Cotton plants were sampled and ranked according to their resistance to Verticillium wilt. In total, 642 endophytic fungi isolates representing 27 genera were recovered from Gossypium hirsutum root, stem, and leaf tissues, but were not uniformly distributed. More endophytic fungi appeared in the leaf (391) compared with the root (140) and stem (111) sections. However, no significant difference in the abundance of isolated endophytes was found among resistant cotton varieties. Alternaria exhibited the highest colonization frequency (7.9%), followed by Acremonium (6.6%) and Penicillium (4.8%). Unlike tolerant varieties, resistant and susceptible ones had similar endophytic fungal population compositions. In three Verticillium-wilt-resistant cotton varieties, fungal endophytes from the genus Alternaria were most frequently isolated, followed by Gibberella and Penicillium. The maximum concentration of dominant endophytic fungi was observed in leaf tissues (0.1797). The evenness of stem tissue endophytic communities (0.702) was comparatively more uniform than the other two tissues. Eighty endophytic fungi selected from 27 genera were evaluated for their inhibition activity against highly virulent Verticillium dahliae isolate Vd080 in vitro. Thirty-nine isolates exhibited fungistasis against the pathogen at varying degrees. Seven species, having high growth inhibition rates (≥75%), exhibited strong antifungal activity against V. dahliae. The antifungal activity of both volatile and nonvolatile metabolites was also investigated. The nonvolatile substances produced by CEF-818 (Penicillium simplicissimum), CEF-325 (Fusarium solani), CEF-714 (Leptosphaeria sp.), and CEF-642 (Talaromyces flavus) completely inhibited V. dahliae growth. These findings deepen our understanding of cotton-endophyte interactions and provide a platform for screening G. hirsutum endophytes with biocontrol potential.

  16. Mapping QTL associated with Verticillium dahliae resistance in the cultivated strawberry (Fragaria × ananassa).

    PubMed

    Antanaviciute, L; Šurbanovski, N; Harrison, N; McLeary, K J; Simpson, D W; Wilson, F; Sargent, D J; Harrison, R J

    2015-01-01

    A biparental cross of octoploid strawberry segregating for resistance to Verticillium dahliae, the causative agent of Verticillium wilt, was screened under field conditions for three seasons. Average wilt scores were significantly associated with multiple QTL, which were mostly significant across all years. Markers significantly associated with the traits were used to screen material with known wilt resistance and susceptibility phenotypes. A clear and statistically significant relationship was observed between resistant, tolerant and susceptible material and the total number of markers present in the different resistance classes. In field situations resistance QTL appear to behave in an additive manner. These markers are abundant in the cultivated strawberry germplasm indicating that, despite the large number of markers, clear genetic gain is possible through marker-assisted breeding. PMID:26504565

  17. Mapping QTL associated with Verticillium dahliae resistance in the cultivated strawberry (Fragaria × ananassa).

    PubMed

    Antanaviciute, L; Šurbanovski, N; Harrison, N; McLeary, K J; Simpson, D W; Wilson, F; Sargent, D J; Harrison, R J

    2015-01-01

    A biparental cross of octoploid strawberry segregating for resistance to Verticillium dahliae, the causative agent of Verticillium wilt, was screened under field conditions for three seasons. Average wilt scores were significantly associated with multiple QTL, which were mostly significant across all years. Markers significantly associated with the traits were used to screen material with known wilt resistance and susceptibility phenotypes. A clear and statistically significant relationship was observed between resistant, tolerant and susceptible material and the total number of markers present in the different resistance classes. In field situations resistance QTL appear to behave in an additive manner. These markers are abundant in the cultivated strawberry germplasm indicating that, despite the large number of markers, clear genetic gain is possible through marker-assisted breeding.

  18. Frequency of Verticillium Species in Commercial Spinach Fields and Transmission of V. dahliae from Spinach to Subsequent Lettuce Crops.

    PubMed

    Short, D P G; Gurung, S; Koike, S T; Klosterman, S J; Subbarao, K V

    2015-01-01

    Verticillium wilt caused by V. dahliae is a devastating disease of lettuce in California (CA). The disease is currently restricted to a small geographic area in central coastal CA, even though cropping patterns in other coastal lettuce production regions in the state are similar. Infested spinach seed has been implicated in the introduction of V. dahliae into lettuce fields but direct evidence linking this inoculum to wilt epidemics in lettuce is lacking. In this study, 100 commercial spinach fields in four coastal CA counties were surveyed to evaluate the frequency of Verticillium species recovered from spinach seedlings and the area under spinach production in each county was assessed. Regardless of the county, V. isaacii was the most frequently isolated species from spinach followed by V. dahliae and, less frequently, V. klebahnii. The frequency of recovery of Verticillium species was unrelated to the occurrence of Verticillium wilt on lettuce in the four counties but was related to the area under spinach production in individual counties. The transmission of V. dahliae from infested spinach seeds to lettuce was investigated in microplots. Verticillium wilt developed on lettuce following two or three plantings of Verticillium-infested spinach, in independent experiments. The pathogen recovered from the infected lettuce from microplots was confirmed as V. dahliae by polymerase chain reaction assays. In a greenhouse study, transmission of a green fluorescence protein-tagged mutant strain of V. dahliae from spinach to lettuce roots was demonstrated, after two cycles of incorporation of infected spinach residue into the soil. This study presents conclusive evidence that V. dahliae introduced via spinach seed can cause Verticillium wilt in lettuce.

  19. Frequency of Verticillium Species in Commercial Spinach Fields and Transmission of V. dahliae from Spinach to Subsequent Lettuce Crops.

    PubMed

    Short, D P G; Gurung, S; Koike, S T; Klosterman, S J; Subbarao, K V

    2015-01-01

    Verticillium wilt caused by V. dahliae is a devastating disease of lettuce in California (CA). The disease is currently restricted to a small geographic area in central coastal CA, even though cropping patterns in other coastal lettuce production regions in the state are similar. Infested spinach seed has been implicated in the introduction of V. dahliae into lettuce fields but direct evidence linking this inoculum to wilt epidemics in lettuce is lacking. In this study, 100 commercial spinach fields in four coastal CA counties were surveyed to evaluate the frequency of Verticillium species recovered from spinach seedlings and the area under spinach production in each county was assessed. Regardless of the county, V. isaacii was the most frequently isolated species from spinach followed by V. dahliae and, less frequently, V. klebahnii. The frequency of recovery of Verticillium species was unrelated to the occurrence of Verticillium wilt on lettuce in the four counties but was related to the area under spinach production in individual counties. The transmission of V. dahliae from infested spinach seeds to lettuce was investigated in microplots. Verticillium wilt developed on lettuce following two or three plantings of Verticillium-infested spinach, in independent experiments. The pathogen recovered from the infected lettuce from microplots was confirmed as V. dahliae by polymerase chain reaction assays. In a greenhouse study, transmission of a green fluorescence protein-tagged mutant strain of V. dahliae from spinach to lettuce roots was demonstrated, after two cycles of incorporation of infected spinach residue into the soil. This study presents conclusive evidence that V. dahliae introduced via spinach seed can cause Verticillium wilt in lettuce. PMID:25098494

  20. Temperature Modulates the Secretome of the Phytopathogenic Fungus Lasiodiplodia theobromae

    PubMed Central

    Félix, Carina; Duarte, Ana S.; Vitorino, Rui; Guerreiro, Ana C. L.; Domingues, Pedro; Correia, António C. M.; Alves, Artur; Esteves, Ana C.

    2016-01-01

    Environmental alterations modulate host–microorganism interactions. Little is known about how climate changes can trigger pathogenic features on symbiont or mutualistic microorganisms. Current climate models predict increased environmental temperatures. The exposing of phytopathogens to these changing conditions can have particularly relevant consequences for economically important species and for humans. The impact on pathogen/host interaction and the shift on their biogeographical range can induce different levels of virulence in new hosts, allowing massive losses in agricultural and health fields. Lasiodiplodia theobromae is a phytopathogenic fungus responsible for a number of diseases in various plants. It has also been described as an opportunist pathogen in humans, causing infections with different levels of severity. L. theobromae has a high capacity of adaptation to different environments, such as woody plants, moist argillaceous soils, or even humans, being able to grow and infect hosts in a wide range of temperatures (9–39°C). Nonetheless, the effect of an increase of temperature, as predicted in climate change models, on L. theobromae is unknown. Here we explore the effect of temperature on two strains of L. theobromae – an environmental strain, CAA019, and a clinical strain, CBS339.90. We show that both strains are cytotoxic to mammalian cells but while the environmental strain is cytotoxic mainly at 25°C, the clinical strain is cytotoxic mainly at 30 and 37°C. Extracellular gelatinolytic, xylanolytic, amylolytic, and cellulolytic activities at 25 and 37°C were characterized by zymography and the secretome of both strains grown at 25, 30, and 37°C were characterized by electrophoresis and by Orbitrap LC-MS/MS. More than 75% of the proteins were identified, mostly enzymes (glycosyl hydrolases and proteases). The strains showed different protein profiles, which were affected by growth temperature. Also, strain specific proteins were identified

  1. Temperature Modulates the Secretome of the Phytopathogenic Fungus Lasiodiplodia theobromae.

    PubMed

    Félix, Carina; Duarte, Ana S; Vitorino, Rui; Guerreiro, Ana C L; Domingues, Pedro; Correia, António C M; Alves, Artur; Esteves, Ana C

    2016-01-01

    Environmental alterations modulate host-microorganism interactions. Little is known about how climate changes can trigger pathogenic features on symbiont or mutualistic microorganisms. Current climate models predict increased environmental temperatures. The exposing of phytopathogens to these changing conditions can have particularly relevant consequences for economically important species and for humans. The impact on pathogen/host interaction and the shift on their biogeographical range can induce different levels of virulence in new hosts, allowing massive losses in agricultural and health fields. Lasiodiplodia theobromae is a phytopathogenic fungus responsible for a number of diseases in various plants. It has also been described as an opportunist pathogen in humans, causing infections with different levels of severity. L. theobromae has a high capacity of adaptation to different environments, such as woody plants, moist argillaceous soils, or even humans, being able to grow and infect hosts in a wide range of temperatures (9-39°C). Nonetheless, the effect of an increase of temperature, as predicted in climate change models, on L. theobromae is unknown. Here we explore the effect of temperature on two strains of L. theobromae - an environmental strain, CAA019, and a clinical strain, CBS339.90. We show that both strains are cytotoxic to mammalian cells but while the environmental strain is cytotoxic mainly at 25°C, the clinical strain is cytotoxic mainly at 30 and 37°C. Extracellular gelatinolytic, xylanolytic, amylolytic, and cellulolytic activities at 25 and 37°C were characterized by zymography and the secretome of both strains grown at 25, 30, and 37°C were characterized by electrophoresis and by Orbitrap LC-MS/MS. More than 75% of the proteins were identified, mostly enzymes (glycosyl hydrolases and proteases). The strains showed different protein profiles, which were affected by growth temperature. Also, strain specific proteins were identified, such

  2. A real-time PCR assay for detection and quantification of Verticillium dahliae in spinach seed.

    PubMed

    Duressa, Dechassa; Rauscher, Gilda; Koike, Steven T; Mou, Beiquan; Hayes, Ryan J; Maruthachalam, Karunakaran; Subbarao, Krishna V; Klosterman, Steven J

    2012-04-01

    Verticillium dahliae is a soilborne fungus that causes Verticillium wilt on multiple crops in central coastal California. Although spinach crops grown in this region for fresh and processing commercial production do not display Verticillium wilt symptoms, spinach seeds produced in the United States or Europe are commonly infected with V. dahliae. Planting of the infected seed increases the soil inoculum density and may introduce exotic strains that contribute to Verticillium wilt epidemics on lettuce and other crops grown in rotation with spinach. A sensitive, rapid, and reliable method for quantification of V. dahliae in spinach seed may help identify highly infected lots, curtail their planting, and minimize the spread of exotic strains via spinach seed. In this study, a quantitative real-time polymerase chain reaction (qPCR) assay was optimized and employed for detection and quantification of V. dahliae in spinach germplasm and 15 commercial spinach seed lots. The assay used a previously reported V. dahliae-specific primer pair (VertBt-F and VertBt-R) and an analytical mill for grinding tough spinach seed for DNA extraction. The assay enabled reliable quantification of V. dahliae in spinach seed, with a sensitivity limit of ≈1 infected seed per 100 (1.3% infection in a seed lot). The quantification was highly reproducible between replicate samples of a seed lot and in different real-time PCR instruments. When tested on commercial seed lots, a pathogen DNA content corresponding to a quantification cycle value of ≥31 corresponded with a percent seed infection of ≤1.3%. The assay is useful in qualitatively assessing seed lots for V. dahliae infection levels, and the results of the assay can be helpful to guide decisions on whether to apply seed treatments.

  3. Proteomic analysis of the sea-island cotton roots infected by wilt pathogen Verticillium dahliae.

    PubMed

    Wang, Fu-Xin; Ma, Yin-Ping; Yang, Chun-Lin; Zhao, Pi-Ming; Yao, Yuan; Jian, Gui-Liang; Luo, Yuan-Ming; Xia, Gui-Xian

    2011-11-01

    Verticillium wilt of cotton is a vascular disease mainly caused by the soil-born filamentous fungus Verticillium dahliae. To study the mechanisms associated with defense responses in wilt-resistant sea-island cotton (Gossypium barbadense) upon V. dahliae infection, a comparative proteomic analysis between infected and mock-inoculated roots of G. barbadense var. Hai 7124 (a cultivar showing resistance against V. dahliae) was performed by 2-DE combined with local EST database-assisted PMF and MS/MS analysis. A total of 51 upregulated and 17 downregulated proteins were identified, and these proteins are mainly involved in defense and stress responses, primary and secondary metabolisms, lipid transport, and cytoskeleton organization. Three novel clues regarding wilt resistance of G. barbadense are gained from this study. First, ethylene signaling was significantly activated in the cotton roots attacked by V. dahliae as shown by the elevated expression of ethylene biosynthesis and signaling components. Second, the Bet v 1 family proteins may play an important role in the defense reaction against Verticillium wilt. Third, wilt resistance may implicate the redirection of carbohydrate flux from glycolysis to pentose phosphate pathway (PPP). To our knowledge, this study is the first root proteomic analysis on cotton wilt resistance and provides important insights for establishing strategies to control this disease.

  4. Upward movement of Verticillium dahliae from soil to olive plants detected by qPCR.

    PubMed

    Ceccherini, Maria Teresa; Luchi, Nicola; Pantani, Ottorino-Luca; Ascher, Judith; Capretti, Paolo; Pietramellara, Giacomo

    2013-10-01

    Olive trees play an important role in cultural, ecological, environmental and social fields, constituting in large part the Mediterranean landscape. In Tuscany, an important economic activity is based on olive. Unfortunately, the Verticillium wilt affects this species and causes vascular disease. In the present study, a real-time quantitative PCR approach has been used to detect and quantify Verticillium dahliae in soil and in olive tree tissues both in micropropagated and in seedling olives. The minimum amounts of V. dahliae DNA sequences detected in soil were 11.4 fg which is equivalent to less than one fungal haploid genome. In micropropagated olive the pathogen was detected in the leaves after 43 days, showing a vertical upward movement of the fungus from the culture medium to stem and leaves. A similar fungal behaviour was observed in inoculated olive stem where after 15 days the fungal DNA was detected from symptomless stem tissue above 8 cm the inoculation site. The described molecular approach is expected to provide a more sensitive and less time-consuming alternative detection method for V. dahliae than plating assay procedures, which were traditionally proposed as an early diagnosis method for Verticillium wilt to farmers and tree nursery growers.

  5. VdCYC8, encoding CYC8 glucose repression mediator protein, is required for microsclerotia formation and full virulence in Verticillium dahliae

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Verticillium dahliae is the primary causal agent for Verticillium wilt disease on a diverse array of economically important crops, including cotton. In previous research, we screened a T-DNA insertional mutant library of the highly virulent isolate Vd080 derived from cotton. In this study, the targ...

  6. Quantitative trait locus mapping for Verticillium wilt resistance in a backcross inbred line population of cotton (Gossypium hirsutum × Gossypium barbadense) based on RGA-AFLP analysis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Verticillium wilt (VW), caused by Verticillium dahliae, is one of the most important diseases in cotton. Development and growing of VW resistant cultivars is the most effective and economic strategy in controlling the disease. However, little is currently known on the genetic basis of VW resistanc...

  7. Molecular Basis of Arabinobio-hydrolase Activity in Phytopathogenic Fungi

    PubMed Central

    Carapito, Raphaël; Imberty, Anne; Jeltsch, Jean-Marc; Byrns, Simon C.; Tam, Pui-Hang; Lowary, Todd L.; Varrot, Annabelle; Phalip, Vincent

    2009-01-01

    The phytopathogenic fungus Fusarium graminearum secretes a very diverse pool of glycoside hydrolases (GHs) aimed at degrading plant cell walls. α-l-Arabinanases are essential GHs participating in the complete hydrolysis of hemicellulose, a natural resource for various industrial processes, such as bioethanol or pharmaceuticals production. Arb93A, the exo-1,5-α-l-arabinanase of F. graminearum encoded by the gene fg03054.1, belongs to the GH93 family, for which no structural data exists. The enzyme is highly active (1065 units/mg) and displays a strict substrate specificity for linear α-1,5-l-arabinan. Biochemical assays and NMR experiments demonstrated that the enzyme releases α-1,5-l-arabinobiose from the nonreducing end of the polysaccharide. We determined the crystal structure of the native enzyme and its complex with α-1,5-l-arabinobiose, a degradation product of α-Me-1,5-l-arabinotetraose, at 1.85 and 2.05Å resolution, respectively. Arb93A is a monomeric enzyme, which presents the six-bladed β-propeller fold characteristic of sialidases of clan GHE. The configuration of the bound arabinobiose is consistent with the retaining mechanism proposed for the GH93 family. Catalytic residues were proposed from the structural analysis, and site-directed mutagenesis was used to validate their role. They are significantly different from those observed for GHE sialidases. PMID:19269961

  8. Streptomycetes and micromycetes as perspective antagonists of fungal phytopathogens.

    PubMed

    Postolaky, O; Syrbu, T; Poiras, N; Baltsat, K; Maslobrod, S; Boortseva, S

    2012-01-01

    Among natural factors that permanently influence on the plants, the soil microorganisms play a special role for the growing of plants as habitants of their rhizosphere. Mainly they are the representatives of actinomycetes genus Streptomyces and fungal genus Penicillium and their metabolic products stimulate plant growth and inhibit the growth of pathogenic fungi and bacteria. The aim of our study was to determine the antagonism of actinomycetes and micromycetes isolated from soils of R. Moldova against the fungal pathogens of agricultural plants. The strains were isolated from 5 types of chernozem (black soil) from central zone of R. Moldova, with different concentration of humus. Most of micromycetes and streptomycetes were isolated from soil sample 1 (monoculture of maize) and soil sample 2 (Poltava road border) with similar humus content (2.4-2.6%). The antifungal activity of micromycetes strains was occurring mostly against Fusarium solani and Thelaviopsis basicola, at streptomycetes against Alternaria alternata and Botrytis cinerea. It was revealed the strains completely inhibit the growth of Alt. alternata (streptomycetes strains 23, 33, 37), B. cinerea (Streptomyces sp. 17), and F. solani (Penicillium sp. 104). Our results allow to consider the actinomycetes Streptomyces sp.9, Streptomyces sp. 12, Streptomyces sp. 17, Streptomyces sp. 37 Streptomyces sp. 66 and micromycetes Penicillium sp. 5, Penicillium sp. 65, Penicillium sp. 104 isolated from soils of R. Moldova, as prospective strains-antagonists against the phytopathogenic fungus, the causative agents of agricultural plants deseasis. PMID:23878981

  9. Susceptibility of Phytopathogenic Bacteria to Wheat Purothionins In Vitro

    PubMed Central

    De Caleya, R. Fernandez; Gonzalez-Pascual, Blanca; García-Olmedo, F.; Carbonero, Pilar

    1972-01-01

    Purothionins are basic polypeptides with antimicrobial properties that are present in the endosperm of wheat and other cereal species. Susceptibility to wheat purothionins among phytopathogenic bacteria of the genera Pseudomonas, Xanthomonas, Agrobacterium, Erwinia, and Corynebacterium has been investigated. Sensitive strains have been found in all of these genera except Agrobacterium (the only strain of A. tumefaciens available proved to be resistant). Minimal inhibitory concentrations (MIC) with partially purified crude purothionins ranged from 1 μg/ml for C. sepedonicum (C.5) to 540 μg/ml for E. amylovora (E.3). Minimal bactericidal concentrations (MBC) were not higher than twice the MIC value, except for C. poinsettiae (C.4) (MBC/MIC = 8). Purothionins α and β, obtained by carboxymethyl-cellulose column chromatography, were tested against P. solanacearum (P.2) and X. phaseoli (X.2); α purothionin was more active than β against X.2, and β more active than α against P.2. This suggests a relationship between polypeptide sequence and specificity of action. PMID:5031563

  10. Polyamine Metabolism in Fungi with Emphasis on Phytopathogenic Species

    PubMed Central

    Valdés-Santiago, Laura; Cervantes-Chávez, José Antonio; León-Ramírez, Claudia Geraldine; Ruiz-Herrera, José

    2012-01-01

    Polyamines are essential metabolites present in all living organisms, and this subject has attracted the attention of researchers worldwide interested in defining their mode of action in the variable cell functions in which they are involved, from growth to development and differentiation. Although the mechanism of polyamine synthesis is almost universal, different biological groups show interesting differences in this aspect that require to be further analyzed. For these studies, fungi represent interesting models because of their characteristics and facility of analysis. During the last decades fungi have contributed to the understanding of polyamine metabolism. The use of specific inhibitors and the isolation of mutants have allowed the manipulation of the pathway providing information on its regulation. During host-fungus interaction polyamine metabolism suffers striking changes in response to infection, which requires examination. Additionally the role of polyamine transporter is getting importance because of its role in polyamine regulation. In this paper we analyze the metabolism of polyamines in fungi, and the difference of this process with other biological groups. Of particular importance is the difference of polyamine biosynthesis between fungi and plants, which makes this process an attractive target for the control of phytopathogenic fungi. PMID:22957208

  11. Genetic transformation of cotton with a harpin-encoding gene hpaXoo confers an enhanced defense response against Verticillium dahliae Kleb.

    PubMed

    Miao, Weiguo; Wang, Jingsheng

    2013-01-01

    The soil-borne fungal pathogen Verticillium dahliae Kleb causes Verticillium wilt in a wide range of crops including cotton (Gossypium hirsutum). To date, most upland cotton varieties are susceptible to V. dahliae and the breeding for cotton varieties with the resistance to Verticillium wilt has not been successful. Hpa1Xoo is a harpin protein from Xanthomonas oryzae pv. oryzae which induces the hypersensitive cell death in plants. When hpa1Xoo was transformed into the susceptible cotton line Z35 through Agrobacterium-mediated transformation, the transgenic cotton line (T-34) with an improved resistance to Verticillium dahliae was obtained. Here, we describe the related research approach, such as Western blot, Southern blot, immuno-gold labeling, evaluation of resistance to Verticillium dahliae, and how to detect the micro-hypersensitive response and oxidative burst elicited by harpin(Xoo) in plant tissue.

  12. Genetic transformation of cotton with a harpin-encoding gene hpaXoo confers an enhanced defense response against Verticillium dahliae Kleb.

    PubMed

    Miao, Weiguo; Wang, Jingsheng

    2013-01-01

    The soil-borne fungal pathogen Verticillium dahliae Kleb causes Verticillium wilt in a wide range of crops including cotton (Gossypium hirsutum). To date, most upland cotton varieties are susceptible to V. dahliae and the breeding for cotton varieties with the resistance to Verticillium wilt has not been successful. Hpa1Xoo is a harpin protein from Xanthomonas oryzae pv. oryzae which induces the hypersensitive cell death in plants. When hpa1Xoo was transformed into the susceptible cotton line Z35 through Agrobacterium-mediated transformation, the transgenic cotton line (T-34) with an improved resistance to Verticillium dahliae was obtained. Here, we describe the related research approach, such as Western blot, Southern blot, immuno-gold labeling, evaluation of resistance to Verticillium dahliae, and how to detect the micro-hypersensitive response and oxidative burst elicited by harpin(Xoo) in plant tissue. PMID:23143497

  13. Verticillium transcription activator of adhesion Vta2 suppresses microsclerotia formation and is required for systemic infection of plant roots.

    PubMed

    Tran, Van-Tuan; Braus-Stromeyer, Susanna A; Kusch, Harald; Reusche, Michael; Kaever, Alexander; Kühn, Anika; Valerius, Oliver; Landesfeind, Manuel; Aßhauer, Kathrin; Tech, Maike; Hoff, Katharina; Pena-Centeno, Tonatiuh; Stanke, Mario; Lipka, Volker; Braus, Gerhard H

    2014-04-01

    Six transcription regulatory genes of the Verticillium plant pathogen, which reprogrammed nonadherent budding yeasts for adhesion, were isolated by a genetic screen to identify control elements for early plant infection. Verticillium transcription activator of adhesion Vta2 is highly conserved in filamentous fungi but not present in yeasts. The Magnaporthe grisea ortholog conidiation regulator Con7 controls the formation of appressoria which are absent in Verticillium species. Vta2 was analyzed by using genetics, cell biology, transcriptomics, secretome proteomics and plant pathogenicity assays. Nuclear Vta2 activates the expression of the adhesin-encoding yeast flocculin genes FLO1 and FLO11. Vta2 is required for fungal growth of Verticillium where it is a positive regulator of conidiation. Vta2 is mandatory for accurate timing and suppression of microsclerotia as resting structures. Vta2 controls expression of 270 transcripts, including 10 putative genes for adhesins and 57 for secreted proteins. Vta2 controls the level of 125 secreted proteins, including putative adhesins or effector molecules and a secreted catalase-peroxidase. Vta2 is a major regulator of fungal pathogenesis, and controls host-plant root infection and H2 O2 detoxification. Verticillium impaired in Vta2 is unable to colonize plants and induce disease symptoms. Vta2 represents an interesting target for controlling the growth and development of these vascular pathogens.

  14. Verticillium transcription activator of adhesion Vta2 suppresses microsclerotia formation and is required for systemic infection of plant roots.

    PubMed

    Tran, Van-Tuan; Braus-Stromeyer, Susanna A; Kusch, Harald; Reusche, Michael; Kaever, Alexander; Kühn, Anika; Valerius, Oliver; Landesfeind, Manuel; Aßhauer, Kathrin; Tech, Maike; Hoff, Katharina; Pena-Centeno, Tonatiuh; Stanke, Mario; Lipka, Volker; Braus, Gerhard H

    2014-04-01

    Six transcription regulatory genes of the Verticillium plant pathogen, which reprogrammed nonadherent budding yeasts for adhesion, were isolated by a genetic screen to identify control elements for early plant infection. Verticillium transcription activator of adhesion Vta2 is highly conserved in filamentous fungi but not present in yeasts. The Magnaporthe grisea ortholog conidiation regulator Con7 controls the formation of appressoria which are absent in Verticillium species. Vta2 was analyzed by using genetics, cell biology, transcriptomics, secretome proteomics and plant pathogenicity assays. Nuclear Vta2 activates the expression of the adhesin-encoding yeast flocculin genes FLO1 and FLO11. Vta2 is required for fungal growth of Verticillium where it is a positive regulator of conidiation. Vta2 is mandatory for accurate timing and suppression of microsclerotia as resting structures. Vta2 controls expression of 270 transcripts, including 10 putative genes for adhesins and 57 for secreted proteins. Vta2 controls the level of 125 secreted proteins, including putative adhesins or effector molecules and a secreted catalase-peroxidase. Vta2 is a major regulator of fungal pathogenesis, and controls host-plant root infection and H2 O2 detoxification. Verticillium impaired in Vta2 is unable to colonize plants and induce disease symptoms. Vta2 represents an interesting target for controlling the growth and development of these vascular pathogens. PMID:24433459

  15. Overexpression of GbRLK, a putative receptor-like kinase gene, improved cotton tolerance to Verticillium wilt

    PubMed Central

    Jun, Zhao; Zhang, Zhiyuan; Gao, Yulong; Zhou, Lei; Fang, Lei; Chen, Xiangdong; Ning, Zhiyuan; Chen, Tianzi; Guo, Wangzhen; Zhang, Tianzhen

    2015-01-01

    Verticillium dahliae is a causative fungal pathogen and only a few genes have been identified that exhibit critical roles in disease resistance and few has shown positive effects on the resistance to Verticillium wilt in transgenic cotton. We cloned a receptor-like kinase gene (GbRLK) induced by Verticillium dahliae (VD) in the disease-resistant cotton Gossypium barbadense cv. Hai7124. Northern blotting revealed that the GbRLK was induced by VD at 96 h after inoculation. The functional GbRLK is from D subgenome since a single base deletion results in a frameshift or dysfunctional homologue in the A subgenome in tetraploid cotton. To verify the function of GbRLK, we developed the overexpression transgenic GbRLK cotton and Arabidopsis lines, and found that they all showed the higher resistance to Verticillium in the greenhouse and field trial. The results of the expression profile using transgenic and non-transgenic Arabidopsis thaliana revealed that the GbRLK regulated expressions of a series genes associated with biotic and abiotic stresses. Therefore, we propose that the increased resistance to Verticillium dahliae infection in transgnic plants could result from reduction in the damage of water loss and regulation of defense gene expression. PMID:26446555

  16. Hyphopodium-Specific VdNoxB/VdPls1-Dependent ROS-Ca2+ Signaling Is Required for Plant Infection by Verticillium dahliae

    PubMed Central

    Zhao, Yun-Long; Zhou, Ting-Ting; Guo, Hui-Shan

    2016-01-01

    Verticillium dahliae is a phytopathogenic fungus obligate in root infection. A few hyphopodia differentiate from large numbers of hyphae after conidia germination on the root surface for further infection. However, the molecular features and role of hyphopodia in the pathogenicity of V. dahliae remain elusive. In this study, we found that the VdPls1, a tetraspanin, and the VdNoxB, a catalytic subunit of membrane-bound NADPH oxidases for reactive oxygen species (ROS) production, were specifically expressed in hyphopodia. VdPls1 and VdNoxB highly co-localize with the plasma membrane at the base of hyphopodia, where ROS and penetration pegs are generated. Mutant strains, VdΔnoxb and VdΔpls1, in which VdPls1 and VdNoxB were deleted, respectively, developed defective hyphpodia incapable of producing ROS and penetration pegs. Defective plasma membrane localization of VdNoxB in VdΔpls1 demonstrates that VdPls1 functions as an adaptor protein for the recruitment and activation of the VdNoxB. Furthermore, in VdΔnoxb and VdΔpls1, tip-high Ca2+ accumulation was impaired in hyphopodia, but not in vegetative hyphal tips. Moreover, nuclear targeting of VdCrz1 and activation of calcineurin-Crz1 signaling upon hyphopodium induction in wild-type V. dahliae was impaired in both knockout mutants, indicating that VdPls1/VdNoxB-dependent ROS was specifically required for tip-high Ca2+ elevation in hyphopodia to activate the transcription factor VdCrz1 in the regulation of penetration peg formation. Together with the loss of virulence of VdΔnoxb and VdΔpls1, which are unable to initiate colonization in cotton plants, our data demonstrate that VdNoxB/VdPls1-mediated ROS production activates VdCrz1 signaling through Ca2+ elevation in hyphopodia, infectious structures of V. dahliae, to regulate penetration peg formation during the initial colonization of cotton roots. PMID:27463643

  17. Functional characterization of cotton genes responsive to Verticillium dahliae through bioinformatics and reverse genetics strategies

    PubMed Central

    Xu, Lian; Zhang, Wenwen; He, Xin; Liu, Min; Zhang, Kun; Shaban, Muhammad; Sun, Longqing; Zhu, Jiachen; Luo, Yijing; Yuan, Daojun; Zhang, Xianlong; Zhu, Longfu

    2014-01-01

    Verticillium wilt causes dramatic cotton yield loss in China. Although some genes or biological processes involved in the interaction between cotton and Verticillium dahliae have been identified, the molecular mechanism of cotton resistance to this disease is still poorly understood. The basic innate immune response for defence is somewhat conserved among plant species to defend themselves in complex environments, which makes it possible to characterize genes involved in cotton immunity based on information from model plants. With the availability of Arabidopsis databases, a data-mining strategy accompanied by virus-induced gene silencing (VIGS) and heterologous expression were adopted in cotton and tobacco, respectively, for global screening and gene function characterization. A total of 232 Arabidopsis genes putatively involved in basic innate immunity were screened as candidate genes, and bioinformatic analysis suggested a role of these genes in the immune response. In total, 38 homologous genes from cotton were singled out to characterize their response to V. dahliae and methyl jasmonate treatment through quantitative real-time PCR. The results revealed that 24 genes were differentially regulated by pathogen inoculation, and most of these genes responded to both Verticillium infection and jasmonic acid stimuli. Furthermore, the efficiency of the strategy was illustrated by the functional identification of six candidate genes via heterologous expression in tobacco or a knock-down approach using VIGS in cotton. Functional categorization of these 24 differentially expressed genes as well as functional analysis suggest that reactive oxygen species, salicylic acid- and jasmonic acid-signalling pathways are involved in the cotton disease resistance response to V. dahliae. Our data demonstrate how information from model plants can allow the rapid translation of information into non-model species without complete genome sequencing, via high-throughput screening and

  18. RNA-seq analyses of gene expression in the microsclerotia of Verticillium dahliae

    PubMed Central

    2013-01-01

    Background The soilborne fungus, Verticillium dahliae, causes Verticillium wilt disease in plants. Verticillium wilt is difficult to control since V. dahliae is capable of persisting in the soil for 10 to 15 years as melanized microsclerotia, rendering crop rotation strategies for disease control ineffective. Microsclerotia of V. dahliae overwinter and germinate to produce infectious hyphae that give rise to primary infections. Consequently, microsclerotia formation, maintenance, and germination are critically important processes in the disease cycle of V. dahliae. Results To shed additional light on the molecular processes that contribute to microsclerotia biogenesis and melanin synthesis in V. dahliae, three replicate RNA-seq libraries were prepared from 10 day-old microsclerotia (MS)-producing cultures of V. dahliae, strain VdLs.17 (average = 52.23 million reads), and those not producing microsclerotia (NoMS, average = 50.58 million reads). Analyses of these libraries for differential gene expression revealed over 200 differentially expressed genes, including up-regulation of melanogenesis-associated genes tetrahydroxynaphthalene reductase (344-fold increase) and scytalone dehydratase (231-fold increase), and additional genes located in a 48.8 kilobase melanin biosynthetic gene cluster of strain VdLs.17. Nearly 50% of the genes identified as differentially expressed in the MS library encode hypothetical proteins. Additional comparative analyses of gene expression in V. dahliae, under growth conditions that promote or preclude microsclerotial development, were conducted using a microarray approach with RNA derived from V. dahliae strain Dvd-T5, and from the amicrosclerotial vdh1 strain. Differential expression of selected genes observed by RNA-seq or microarray analysis was confirmed using RT-qPCR or Northern hybridizations. Conclusion Collectively, the data acquired from these investigations provide additional insight into gene expression and molecular

  19. Companion cropping with potato onion enhances the disease resistance of tomato against Verticillium dahliae.

    PubMed

    Fu, Xuepeng; Wu, Xia; Zhou, Xingang; Liu, Shouwei; Shen, Yanhui; Wu, Fengzhi

    2015-01-01

    Intercropping could alleviate soil-borne diseases, however, few studies focused on the immunity of the host plant induced by the interspecific interactions. To test whether or not intercropping could enhance the disease resistance of host plant, we investigated the effect of companion cropping with potato onion on tomato Verticillium wilt caused by Verticillium dahliae (V. dahliae). To investigate the mechanisms, the root exudates were collected from tomato and potato onion which were grown together or separately, and were used to examine the antifungal activities against V. dahliae in vitro, respectively. Furthermore, RNA-seq was used to examine the expression pattern of genes related to disease resistance in tomato companied with potato onion compared to that in tomato grown alone, under the condition of infection with V. dahliae. The results showed that companion cropping with potato onion could alleviate the incidence and severity of tomato Verticillium wilt. The further studies revealed that the root exudates from tomato companied with potato onion significantly inhibited the mycelia growth and spore germination of V. dahliae. However, there were no significant effects on these two measurements for the root exudates from potato onion grown alone or from potato onion grown with tomato. RNA-seq data analysis showed the disease defense genes associated with pathogenesis-related proteins, biosynthesis of lignin, hormone metabolism and signal transduction were expressed much higher in the tomato companied with potato onion than those in the tomato grown alone, which indicated that these defense genes play important roles in tomato against V. dahliae infection, and meant that the disease resistance of tomato against V. dahliae was enhanced in the companion copping with potato onion. We proposed that companion cropping with potato onion could enhance the disease resistance of tomato against V. dahliae by regulating the expression of genes related to disease

  20. Functional characterization of cotton genes responsive to Verticillium dahliae through bioinformatics and reverse genetics strategies.

    PubMed

    Xu, Lian; Zhang, Wenwen; He, Xin; Liu, Min; Zhang, Kun; Shaban, Muhammad; Sun, Longqing; Zhu, Jiachen; Luo, Yijing; Yuan, Daojun; Zhang, Xianlong; Zhu, Longfu

    2014-12-01

    Verticillium wilt causes dramatic cotton yield loss in China. Although some genes or biological processes involved in the interaction between cotton and Verticillium dahliae have been identified, the molecular mechanism of cotton resistance to this disease is still poorly understood. The basic innate immune response for defence is somewhat conserved among plant species to defend themselves in complex environments, which makes it possible to characterize genes involved in cotton immunity based on information from model plants. With the availability of Arabidopsis databases, a data-mining strategy accompanied by virus-induced gene silencing (VIGS) and heterologous expression were adopted in cotton and tobacco, respectively, for global screening and gene function characterization. A total of 232 Arabidopsis genes putatively involved in basic innate immunity were screened as candidate genes, and bioinformatic analysis suggested a role of these genes in the immune response. In total, 38 homologous genes from cotton were singled out to characterize their response to V. dahliae and methyl jasmonate treatment through quantitative real-time PCR. The results revealed that 24 genes were differentially regulated by pathogen inoculation, and most of these genes responded to both Verticillium infection and jasmonic acid stimuli. Furthermore, the efficiency of the strategy was illustrated by the functional identification of six candidate genes via heterologous expression in tobacco or a knock-down approach using VIGS in cotton. Functional categorization of these 24 differentially expressed genes as well as functional analysis suggest that reactive oxygen species, salicylic acid- and jasmonic acid-signalling pathways are involved in the cotton disease resistance response to V. dahliae. Our data demonstrate how information from model plants can allow the rapid translation of information into non-model species without complete genome sequencing, via high-throughput screening and

  1. Overexpression of potato miR482e enhanced plant sensitivity to Verticillium dahliae infection.

    PubMed

    Yang, Liu; Mu, Xiaoying; Liu, Chao; Cai, Jinghui; Shi, Ke; Zhu, Wenjiao; Yang, Qing

    2015-12-01

    Verticillium wilt of potato is caused by the fungus pathogen Verticillium dahliae. Present sRNA sequencing data revealed that miR482 was in response to V. dahliae infection, but the function in potato is elusive. Here, we characterized potato miR482 family and its putative role resistance to Verticillium wilt. Members of the potato miR482 superfamily are variable in sequence, but all variants target a class of disease-resistance proteins with nucleotide binding site (NBS) and leucine-rich repeat (LRR) motifs. When potato plantlets were infected with V. dahliae, the expression level of miR482e was downregulated, and that of several NBS-LRR targets of miR482e were upregulated. Transgenic potato plantlets overexpressing miR482e showed hypersensitivity to V. dahliae infection. Using sRNA and degradome datasets, we validated that miR482e targets mRNAs of NBS-LRR disease-resistance proteins and triggers the production of trans-acting (ta)-siRNAs, most of which target mRNAs of defense-related proteins. Thus, the hypersensitivity of transgenic potato could be explained by enhanced miR482e and miR482e-derived ta-siRNA-mediated silencing on NBS-LRR-disease-resistance proteins. It is speculated that a miR482-mediated silencing cascade mechanism is involved in regulating potato resistance against V. dahliae infection and could be a counter defense action of plant in response to pathogen infection.

  2. Companion cropping with potato onion enhances the disease resistance of tomato against Verticillium dahliae

    PubMed Central

    Fu, Xuepeng; Wu, Xia; Zhou, Xingang; Liu, Shouwei; Shen, Yanhui; Wu, Fengzhi

    2015-01-01

    Intercropping could alleviate soil-borne diseases, however, few studies focused on the immunity of the host plant induced by the interspecific interactions. To test whether or not intercropping could enhance the disease resistance of host plant, we investigated the effect of companion cropping with potato onion on tomato Verticillium wilt caused by Verticillium dahliae (V. dahliae). To investigate the mechanisms, the root exudates were collected from tomato and potato onion which were grown together or separately, and were used to examine the antifungal activities against V. dahliae in vitro, respectively. Furthermore, RNA-seq was used to examine the expression pattern of genes related to disease resistance in tomato companied with potato onion compared to that in tomato grown alone, under the condition of infection with V. dahliae. The results showed that companion cropping with potato onion could alleviate the incidence and severity of tomato Verticillium wilt. The further studies revealed that the root exudates from tomato companied with potato onion significantly inhibited the mycelia growth and spore germination of V. dahliae. However, there were no significant effects on these two measurements for the root exudates from potato onion grown alone or from potato onion grown with tomato. RNA-seq data analysis showed the disease defense genes associated with pathogenesis-related proteins, biosynthesis of lignin, hormone metabolism and signal transduction were expressed much higher in the tomato companied with potato onion than those in the tomato grown alone, which indicated that these defense genes play important roles in tomato against V. dahliae infection, and meant that the disease resistance of tomato against V. dahliae was enhanced in the companion copping with potato onion. We proposed that companion cropping with potato onion could enhance the disease resistance of tomato against V. dahliae by regulating the expression of genes related to disease

  3. Guanacastane-type diterpenoids from the insect-associated fungus Verticillium dahliae.

    PubMed

    Wu, Fa-Biao; Li, Tian-Xiao; Yang, Ming-Hua; Kong, Ling-Yi

    2016-01-01

    Four new guanacastane-type diterpenoids, namely dahlianes A (1), B (2), C (3), and D (4), were isolated from cultures of Verticillium dahliae. Their structures were elucidated on the basis of extensive spectroscopic data analysis. Their absolute configurations were determined by a combination of Mo2(OAc)4-induced electronic circular dichroism experiment and Mosher ester method. In cytotoxicity evaluation against human tumor cell lines, compounds 2 and 3 exhibited significant cytotoxicities against MCF-7 cell lines with IC50 values of 3.35 and 4.72 μM, respectively.

  4. Horizontal transfer in the phytopathogenic fungal genus Leptosphaeria and host-range expansion.

    PubMed

    Taylor, J L; Pedras, M S; Morales, V M

    1995-05-01

    All isolates examined of the phytopathogenic fungus Leptosphaeria maculans that are aggressive to Brassica napus and Brassica rapa have a repetitive element, LMR1. Horizontal transfer of LMR1 to an isolate of a closely related species of Leptosphaeria correlates with the expansion of the host range of this isolate to include Brassica juncea.

  5. Transcriptional profile of P. syringae pv. phaseolicola NPS3121 at low temperature: Physiology of phytopathogenic bacteria

    PubMed Central

    2013-01-01

    Background Low temperatures play key roles in the development of most plant diseases, mainly because of their influence on the expression of various virulence factors in phytopathogenic bacteria. Thus far, studies regarding this environmental parameter have focused on specific themes and little is known about phytopathogenic bacteria physiology under these conditions. To obtain a global view regarding phytopathogenic bacteria strategies in response to physiologically relevant temperature changes, we used DNA microarray technology to compare the gene expression profile of the model bacterial pathogen P. syringae pv. phaseolicola NPS3121 grown at 18°C and 28°C. Results A total of 236 differentially regulated genes were identified, of which 133 were up-regulated and 103 were down-regulated at 18°C compared to 28°C. The majority of these genes are involved in pathogenicity and virulence processes. In general, the results of this study suggest that the expression profile obtained may be related to the fact that low temperatures induce oxidative stress in bacterial cells, which in turn influences the expression of iron metabolism genes. The expression also appears to be correlated with the profile expression obtained in genes related to motility, biofilm production, and the type III secretion system. Conclusions From the data obtained in this study, we can begin to understand the strategies used by this phytopathogen during low temperature growth, which can occur in host interactions and disease development. PMID:23587016

  6. The genome of the saprophytic fungus Verticillium tricorpus reveals a complex effector repertoire largely resembling its pathogenic relatives

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Vascular wilts belong to the most destructive plant diseases, affecting hundreds of economically and ecologically important plant species. The fungal genus Verticillium presently comprises ten species, of which only few are causal agents of vascular wilt diseases. Of these, V. dahliae is the most no...

  7. Constitutive expression of a novel antimicrobial protein, Hcm1, confers resistance to both Verticillium and Fusarium wilts in cotton

    PubMed Central

    Zhang, Zhiyuan; Zhao, Jun; Ding, Lingyun; Zou, Lifang; Li, Yurong; Chen, Gongyou; Zhang, Tianzhen

    2016-01-01

    Fusarium and Verticillium wilts, two of the most important diseases in cotton, pose serious threats to cotton production. Here we introduced a novel antimicrobial protein Hcm1, which comprised harpin protein from Xanthomonas oryzae pv. oryzicola (Xoc), and the chimeric protein, cecropin A-melittin, into cotton. The transgenic cotton lines with stable Hcm1 expression showed a higher resistance to Verticillium and Fusarium wilts both in greenhouse and field trials compared to controls. Hcm1 enabled the transgenic cotton to produced a microscopic hypersensitive response (micro-HR), reactive oxygen species (ROS) burst, and caused the activation of pathogenesis-related (PR) genes in response to biotic stress, indicating that the transgenic cotton was in a primed state and ready to protect the host from pathogenic infection. Simultaneously, Hcm1 protein inhibited the growth of Verticillium dahliae (V. dahliae) and Fusarium oxysporum (F. oxysporum) in vitro. The spread of fungal biomass was also inhibited in vivo since the V. dahliae biomass was decreased dramatically in transgenic cotton plants after inoculation with V. dahliae. Together, these results demonstrate that Hcm1 could activate innate immunity and inhibit the growth of V. dahliae and F. oxysporum to protect cotton against Verticillium and Fusarium wilts. PMID:26856318

  8. Antibacterial activities of Ligaria cuneifolia and Jodina rhombifolia leaf extracts against phytopathogenic and clinical bacteria.

    PubMed

    Soberón, José R; Sgariglia, Melina A; Dip Maderuelo, María R; Andina, María L; Sampietro, Diego A; Vattuone, Marta A

    2014-11-01

    Six plant extracts prepared from Ligaria cuneifolia and Jodina rhombifolia were screened for their potential antimicrobial activities against phytopathogens and clinically standard reference bacterial strains. Bioautography and broth microdilution methods were used to study samples antibacterial activities against 7 bacterial strains. The minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) of samples were attained. An antibacterial activity guided isolation and identification of active compounds was carried out for L. cuneifolia methanolic extract (LCME). Both methanolic and aqueous extracts from L. cuneifolia showed inhibitory activities against phytopathogenic bacteria, with MICs ranging from 2.5 to 156 μg mL(-1) for LCME and 5 mg mL(-1) for the aqueous extract. None of the three J. rhombifolia extracts showed significant antibacterial activities against phytopathogenic strains (MIC > 5 mg mL(-1)), except for the aqueous extracts against Pseudomonas syringae (MIC = 312 μg mL(-1)). Only LCME showed bactericidal activities against phytopathogenic strains (MBCs = 78 μg mL(-1)). The LCME exhibited significant inhibitory activity against reference clinical strains: Escherichia coli (MIC = 156 μg mL(-1)) and Staphylococcus aureus (MIC = 78 μg mL(-1), MBC = 312 μg mL(-1)). LCME active compounds were identified as flavonol mono and diglycosides, and gallic acid. The antibacterial activity of purified compounds was also evaluated. A synergistic effect against S. aureus was found between gallic acid and a quercetin glycoside. Hence, anti-phytopathogenic bacteria potential compounds isolated from L. cuneifolia could be used as an effective source against bacterial diseases in plants. PMID:24894684

  9. Comparative Proteomic Analysis of Gossypium thurberi in Response to Verticillium dahliae Inoculation

    PubMed Central

    Fang, Weiping; Xie, Deyi; Zhu, Heqin; Li, Wu; Xu, Zhenzhen; Yang, Lirong; Li, Zhifang; Sun, Li; Wang, Jinxia; Nie, Lihong; Tang, Zhongjie; Lv, Shuping; Zhao, Fu’an; Sun, Yao; Zhao, Yuanming; Hou, Jianan; Yang, Xiaojie

    2015-01-01

    Verticillium wilt is threatening cotton productivity globally. This disease is caused by soil-borne Verticillium dahliae which directly infects cotton roots, and exclusively colonizes and occludes xylem vessels, finally resulting in necrosis, defoliation, and most severely, plant death. For the first time, iTRAQ (isobaric tags for relative and absolute quantification) was applied to screen the differentially expressed proteins of Gossypium thurberi inoculated with V. dahliae. A total of 6533 proteins were identified from the roots of G. thurberi after inoculation with V. dahliae, and 396 showed up- and 279 down-regulated in comparison to a mock-inoculated roots. Of these identified proteins, the main functional groups were those involved in cell wall organization and reinforcement, disease-resistant chemicals of secondary metabolism, phytohormone signaling, pathogenesis-related proteins, and disease-resistant proteins. Physiological and biochemical analysis showed that peroxidase activity, which promotes the biosynthesis and accumulation of lignin, was induced early in the hypocotyl after inoculation with V. dahliae. Similarly, salicylic acid also accumulated significantly in hypocotyl of the seedlings after inoculation. These findings provide an important knowledge of the molecular events and regulatory networks occurring during G. thurberi-V. dahliae interaction, which may provide a foundation for breeding disease-resistance in cotton. PMID:26506344

  10. Analysis of upland cotton (Gossypium hirsutum) response to Verticillium dahliae inoculation by transcriptome sequencing.

    PubMed

    Shao, B X; Zhao, Y L; Chen, W; Wang, H M; Guo, Z J; Gong, H Y; Sang, X H; Cui, Y L; Wang, C H

    2015-10-27

    Verticillium wilt is one of the main diseases in cotton (Gossypium hirsutum), severely reduces yield and fiber quality, and is difficult to be con-trolled effectively. At present, the molecular mechanism that confers resistance to this disease is unclear. Transcriptome sequencing is an important method to detect resistance genes, explore metabolic pathways, and study resistance mechanisms. In this study, the transcriptome of a disease-resistant inbred cot-ton line inoculated with Verticillium dahliae was sequenced. A total of 126,402 unigenes were obtained using de novo assembly and data analysis, 99,712 (78.88%) of which were annotated into the Nr, Nt, Swiss-Prot, KEGG, COG, and GO databases. The expression patterns of 16 candidate disease-resis-tance genes showed that some genes were upregulated soon after V. dahliae inoculation and others were upregulated later, which may indicate instanta-neous basal defense and lagged specific defense, respectively. We conducted a preliminary analysis of the transcriptome database, which will contribute to further research regarding the cloning of disease-resistance genes.

  11. Weathering of chrysotile asbestos by the serpentine rock-inhabiting fungus Verticillium leptobactrum.

    PubMed

    Daghino, Stefania; Turci, Francesco; Tomatis, Maura; Girlanda, Mariangela; Fubini, Bice; Perotto, Silvia

    2009-07-01

    Verticillium leptobactrum, a rare fungal species, has repeatedly been isolated from serpentinic rocks in the Western Alps, thus suggesting that it adapts easily to this selective mineral substrate. The rRNA internal transcribed spacer region of several isolates has been sequenced to confirm their identity and taxonomic position within Verticillium, a recently revised polyphyletic entity. Isolates of V. leptobactrum have also been investigated to establish their ability to weather asbestos chrysotile, the most common mineral in the isolation sites. The results of solubilization assays on magnesium and silicon, as well as measurement of the Mg/Si ratio in the asbestos fibres after exposure to fungal mycelia, indicate a high bioweathering activity of V. leptobactrum towards chrysotile. Comparison with data on Fusarium oxysporum shows differences among species, with V. leptobactrum being more active than F. oxysporum in removing structural ions from chrysotile. Asbestos weathering by fungi could be envisaged as a bioremediation strategy for hazardous asbestos-rich soils (e.g. abandoned mines). Fungi that have adapted to live in serpentine sites could be good candidates for this purpose.

  12. Incidence and impact of Verticillium dahliae in soil associated with certified potato seed lots.

    PubMed

    Dung, Jeremiah K S; Hamm, Philip B; Eggers, Jordan E; Johnson, Dennis A

    2013-01-01

    Verticillium dahliae causes Verticillium wilt of potato and can be found in soil associated with potato seed tubers. The purpose of this research was to quantify V. dahliae in soil associated with certified seed tubers and determine if this potential inoculum source is related to disease development in the field. Approximately 68% of seed lots assayed contained V. dahliae-infested soil on seed tuber surfaces (seed tuber soil). Over 82% of seed lots contained V. dahliae in loose seed lot soil obtained from bags and trucks used to transport seed tubers. Most samples contained ≤50 CFU/g but some contained >500 CFU/g. Most isolates (93%) were vegetative compatibility group 4A. Populations of V. dahliae in stem sap increased with increasing inoculum densities in field soils only when V. dahliae concentrations in seed tuber soil were low. High concentrations of V. dahliae in seed tuber soil resulted in greater stem sap colonization when V. dahliae inoculum densities in field soil were low (P < 0.01) and resulted in greater pathogen inoculum densities in postharvest field soils (P = 0.04). Seed tubers contaminated with V. dahliae-infested soils may introduce the pathogen into fields not previously cropped to potato or recontaminate those which have received preplant management practices. Long-term management of V. dahliae requires reducing propagules in soil associated with seed lots.

  13. A Ve homologous gene from Gossypium barbadense, Gbvdr3, enhances the defense response against Verticillium dahliae.

    PubMed

    Chen, Tianzi; Kan, Jialiang; Yang, Yuwen; Ling, Xitie; Chang, Youhong; Zhang, Baolong

    2016-01-01

    The tomato Ve1 gene and several Ve1 homologues are involved in the resistance to Verticillium dahliae. Here, we report on another Ve homologous gene, Gbvdr3, from a Verticillium wilt-resistant cotton cultivar, Gossypium barbadense Hai7124, which has a 3207-bp region that encodes a predicted receptor-like protein. Transient expression analyses indicated that Gbvdr3 is localized in the plasma membrane, and virus-induced gene silencing of Gbvdr3 compromised the resistance of Hai7124 cotton to a defoliating strain of V. dahliae, V991, but not to a non-defoliating strain, BP2. This resistance pattern was further confirmed by over-expression of Gbvdr3 in transgenic Arabidopsis, which significantly elevated the expression of the ethylene-regulated gene GST2, the ethylene- and jasmonic acid-regulated defense-related genes PR3 and PDF1.2, and the salicylic acid-regulated genes PR1 and PR5, but not the PR2 gene. It also triggered the accumulation of hydrogen peroxide and callose at early time points during infection by the V991 defoliating strain. In contrast, elevated accumulation of hydrogen peroxide or callose in Gbvdr3-expressed Arabidopsis leaves was not apparent under infection by the non-defoliating strain, BP2. These results suggested that Gbvdr3 is involved in the resistance to a unique spectrum of defoliating V. dahliae strains.

  14. Comparative Proteomic Analysis of Gossypium thurberi in Response to Verticillium dahliae Inoculation.

    PubMed

    Fang, Weiping; Xie, Deyi; Zhu, Heqin; Li, Wu; Xu, Zhenzhen; Yang, Lirong; Li, Zhifang; Sun, Li; Wang, Jinxia; Nie, Lihong; Tang, Zhongjie; Lv, Shuping; Zhao, Fu'an; Sun, Yao; Zhao, Yuanming; Hou, Jianan; Yang, Xiaojie

    2015-10-22

    Verticillium wilt is threatening cotton productivity globally. This disease is caused by soil-borne Verticillium dahliae which directly infects cotton roots, and exclusively colonizes and occludes xylem vessels, finally resulting in necrosis, defoliation, and most severely, plant death. For the first time, iTRAQ (isobaric tags for relative and absolute quantification) was applied to screen the differentially expressed proteins of Gossypium thurberi inoculated with V. dahliae. A total of 6533 proteins were identified from the roots of G. thurberi after inoculation with V. dahliae, and 396 showed up- and 279 down-regulated in comparison to a mock-inoculated roots. Of these identified proteins, the main functional groups were those involved in cell wall organization and reinforcement, disease-resistant chemicals of secondary metabolism, phytohormone signaling, pathogenesis-related proteins, and disease-resistant proteins. Physiological and biochemical analysis showed that peroxidase activity, which promotes the biosynthesis and accumulation of lignin, was induced early in the hypocotyl after inoculation with V. dahliae. Similarly, salicylic acid also accumulated significantly in hypocotyl of the seedlings after inoculation. These findings provide an important knowledge of the molecular events and regulatory networks occurring during G. thurberi-V. dahliae interaction, which may provide a foundation for breeding disease-resistance in cotton.

  15. Genome-wide profiling of miRNAs and other small non-coding RNAs in the Verticillium dahliae-inoculated cotton roots.

    PubMed

    Yin, Zujun; Li, Yan; Han, Xiulan; Shen, Fafu

    2012-01-01

    MicroRNAs (miRNAs) and small interfering RNAs (siRNAs) are short (19-25 nucleotides) non-coding RNA molecules that have large-scale regulatory effects on development and stress responses in plants. Verticillium wilt is a vascular disease in plants caused by the fungal pathogen Verticillium dahliae. The objective of this study is to investigate the transcriptional profile of miRNAs and other small non-coding RNAs in Verticillium-inoculated cotton roots. Four small RNA libraries were constructed from mocked and infected roots of two cotton cultured species which are with different Verticillium wilt tolerance ('Hai-7124', Gossypium barbadense L., a Verticillium-tolerant cultivar, and 'Yi-11', Gossypium hirsutum L. a Verticillium-sensitive cultivar). The length distribution of obtained small RNAs was significantly different between libraries. There were a total of 215 miRNA families identified in the two cotton species. Of them 14 were novel miRNAs. There were >65 families with different expression between libraries. We also identified two trans-acting siRNAs and thousands of endogenous siRNA candidates, and hundred of them exhibited altered expression after inoculation of Verticillium. Interesting, many siRNAs were found with a perfect match with retrotransposon sequences, suggested that retrotransposons maybe one of sources for the generation of plant endogenous siRNAs. The profiling of these miRNAs and other small non-coding RNAs lay the foundation for further understanding of small RNAs function in the regulation of Verticillium defence responses in cotton roots.

  16. Biological preparation of chitosan nanoparticles and its in vitro antifungal efficacy against some phytopathogenic fungi.

    PubMed

    Sathiyabama, M; Parthasarathy, R

    2016-10-20

    The aim of the present study was to prepare Chitosan nanoparticles through biological method with high antifungal activities. Chitosan nanoparticles were prepared by the addition of anionic proteins isolated from Penicillium oxalicum culture to chitosan solutions. The formation of chitosan nanoparticles was preliminary confirmed by UV-vis spectrophotometric analysis. The physico-chemical properties of the chitosan nanoparticles were determined by size and zeta potential analysis, FTIR analysis, HRTEM and XRD pattern. The chitosan nanoparticles were evaluated for its potential to inhibit the growth of phytopathogens viz., Pyricularia grisea, Alternaria solani, Fusarium oxysporum. It is evident from our results that chitosan nanoparticles inhibit the growth of phytopathogens tested. Chitosan nanoparticle treated chickpea seeds showed positive morphological effects such as enhanced germination%, seed vigor index and vegetative biomass of seedlings. All these results indicate that chitosan nanoparticle can be used further under field condition to protect various crops from the devastating fungal pathogens as well as growth promoters.

  17. Chemical Composition and Antibacterial Activity of Essential Oils of Two Species of Lamiaceae against Phytopathogenic Bacteria.

    PubMed

    Gormez, Arzu; Bozari, Sedat; Yanmis, Derya; Gulluce, Medine; Sahin, Fikrettin; Agar, Guleray

    2015-01-01

    In this study, we aimed to determine chemical composition and antibacterial activities of Satureja hortensis and Calamintha nepeta against to 20 phytopathogenic bacteria causing serious crop loss. The essential oils of S. hortensis and C. nepeta were isolated by the hydrodistillation method and the chemical composition of the essential oils were analyzed by GC-MS. The antibacterial properties of the essential oils were evaluated against 20 phytopathogenic bacteria through Disc diffusion assay and micro dilution assay. The results revealed that the essential oils of S. hortensis and C. nepeta have significant antibacterial activity. Furthermore, the findings of the study are valuable for future investigations focusing on the alternative natural compounds to control plant diseases. PMID:26373171

  18. [Cellulase and xylanase activity of phytopathogenic and endophytic fungal strains of Alternaria alternata (Fr.) Keissler].

    PubMed

    Kurchenko, I M; Sokolova, O V; Zhdanova, N M; Iarynchyn, A M; Iovenko, O M

    2008-01-01

    A comparative analysis of cellulase and xylanase activity of 25 fungal strains of phytopathogenic and endophytic Alternaria alternata had been realized for the first time using the qualitative reactions. The rate of their linear growth on the media with carboxymethylcellulose or xylane had been studied. The cellulase and xylanase activities clearly depended on the distinct strain. The absence of distinct dependence of cellulase and xylanase activities on the species and organs of host plants was demonstrated. The majority of investigated strains of A. alternata did not possess a cellulase activity or the latter was low, but as a whole the phytopathogenic strains were more active than endophytic ones. Xylanase activity was considerable for the fungal strains of all trophyc groups. It was shown that the level of xylanase activity cannot become a biochemical marker of the A. alternata isolate pathogenicity.

  19. [Effect of proteinaceous proteinase inhibitors from potato tubers on the growth and development of phytopathogenic microorganisms].

    PubMed

    Revina, T A; Gerasimova, N G; Kladnitskaia, G V; Chalenko, G I; Valueva, T A

    2008-01-01

    We studied the effect of two proteins, PSPI-21 and PKSI, on the growth and development of phytopathogenic microorganisms (Phytophthora infestans oomycete and Fusarium culmorum fungus). Both proteins were isolated from potato tubers (Solanum tuberosum L., cv. Istrinskii) and served as inhibitors of serine proteinases. These proteins differed in the ability to inhibit growth of Phytophthora infestans oomycete and Fusarium culmorum fungus. PSPI-21 was the most potent in modulating the growth of oomycete mycelium. PKSI primarily affected the growth of the fungal mycelium. The proteins under study induced complete destruction of oomycete zoospores and partial destruction of fungal macroconidia. Our results suggest that these proteins are involved in the protection of potato plants from phytopathogenic microorganisms.

  20. Biological preparation of chitosan nanoparticles and its in vitro antifungal efficacy against some phytopathogenic fungi.

    PubMed

    Sathiyabama, M; Parthasarathy, R

    2016-10-20

    The aim of the present study was to prepare Chitosan nanoparticles through biological method with high antifungal activities. Chitosan nanoparticles were prepared by the addition of anionic proteins isolated from Penicillium oxalicum culture to chitosan solutions. The formation of chitosan nanoparticles was preliminary confirmed by UV-vis spectrophotometric analysis. The physico-chemical properties of the chitosan nanoparticles were determined by size and zeta potential analysis, FTIR analysis, HRTEM and XRD pattern. The chitosan nanoparticles were evaluated for its potential to inhibit the growth of phytopathogens viz., Pyricularia grisea, Alternaria solani, Fusarium oxysporum. It is evident from our results that chitosan nanoparticles inhibit the growth of phytopathogens tested. Chitosan nanoparticle treated chickpea seeds showed positive morphological effects such as enhanced germination%, seed vigor index and vegetative biomass of seedlings. All these results indicate that chitosan nanoparticle can be used further under field condition to protect various crops from the devastating fungal pathogens as well as growth promoters. PMID:27474573

  1. Superoxide radical induces sclerotial differentiation in filamentous phytopathogenic fungi: a superoxide dismutase mimetics study.

    PubMed

    Papapostolou, Ioannis; Georgiou, Christos D

    2010-03-01

    This study shows that the superoxide radical (O(2) *( -)), a direct indicator of oxidative stress, is involved in the differentiation of the phytopathogenic filamentous fungi Rhizoctonia solani, Sclerotinia sclerotiorum, Sclerotium rolfsii and Sclerotinia minor, shown by using superoxide dismutase (SOD) mimetics to decrease their sclerotial differentiation. The production rate of O(2) *(-) and SOD levels in these fungi, as expected, were significantly lowered by the SOD mimetics, with concomitant decrease of the indirect indicator of oxidative stress, lipid peroxidation. PMID:20007647

  2. VdMsb regulates virulence and microsclerotia production in the fungal plant pathogen Verticillium dahliae.

    PubMed

    Tian, Liangliang; Xu, Jun; Zhou, Lei; Guo, Wangzhen

    2014-10-25

    The vascular wilt fungus Verticillium dahliae infects the roots of cotton plants and can seriously diminish the yield and quality of this and other dicotyledons. However, the key genes involved in V. dahliae infection and pathogenesis in cotton remain unclear. Msb encodes a transmembrane mucin that is highly conserved in the MAPK signal pathway. Msb has been implicated previously in pathogenicity in various aerial plant fungi. In this study, V. dahliae Msb (VdMsb) was found to be required for fungal virulence and microsclerotia production. Strains lacking VdMsb exhibited reduced conidiation and microsclerotia formation. Compared with wild-type and gene-complemented strains, the invasive growth and adhesive capacity of VdMsb deletion mutants were significantly decreased. These results suggest that VdMsb plays a role in development and virulence in V. dahliae.

  3. Isolation and characterization of a chitinase gene from entomopathogenic fungus Verticillium lecanii

    PubMed Central

    Zhu, Yanping; Pan, Jieru; Qiu, Junzhi; Guan, Xiong

    2008-01-01

    Entomopathogenic fungus Verticillium lecanii is a promising whitefly and aphid control agent. Chitinases secreted by this insect pathogen have considerable importance in the biological control of some insect pests. An endochitinase gene Vlchit1 from the fungus was cloned and overexpressed in Escherichia coli. The Vlchit1 gene not only contains an open reading frame (ORF) which encodes a protein of 423 amino acids (aa), but also is interrupted by three short introns. Vlchit1 protein showed that the chitinase Vlchit1 has a (a/b)8 TIM barrel structure. Overexpression test and Enzymatic activity assay indicated that the Vlchit1 is a functional enzyme that can hydrolyze the chitin substrate, so the Vlchit1 gene can service as a useful gene source for genetic manipulation leading to strain improvement of entomopathogenic fungi or constructing new transgenic plants with resistance to various fungal and insects pests. PMID:24031223

  4. [Screening probiotic endophytic bacteria from medicinal plant flex cornuta and the phytopathogen-inhibiting effect].

    PubMed

    Zhao, Long-fei; Xu, Ya-jun; Lai, Xin-he; Kou, Tian-chao; Yan, Jun-li; Zhou, Pei-pei; Fan, Shan-shan; Yan, Yong-feng

    2015-05-01

    Culturable endophytic bacteria were isolated from medicinal plant Ilex cornuta by plate-spreading method, strains with strong inhibitory effect on phytopathogen were screened by confrontation culture and fermentation filtrate culture methods, and the morphological changes of phytopathogen hyphae treated with endophytic bacteria were examined by microscopy and micrograph. Their phylogenetic relationships were determined by homology analysis of the 16S rDNA sequences of PCR products and the taxonomic status of the selected strains was determined based on their morphology, physiology, biochemical test results and 16S rDNA sequence analysis. A total of 85 endophytic bacteria were isolated from the healthy roots, stems, leaves and fruits of I. cornuta, and 10 strains of them showed strong inhibitory effect on Alternaria alternata, Magnaporthe grisea, Fusarium oxysporum, and were preliminarily identified belonging to four genera and seven species. Three strains with the strongest inhibitory effect, GG78 (60.3%), GG31 (48.1%) and GG13 (61.0%) belonged to Enterobacter cloacae, Enterobacter ludwigii and Bacillus cereus, respectively. Microscopic analyses showed that the inhibited phytopathogen hyphae became deformed, distorted, and partially expanded forming plasma concentration and hair-like branch on the hyphae base. These morphological changes could be caused by the extracellular metabolic substances secreted by the endophytic bacteria, such as antibiotics, hydrolytic enzymes, alkaloids and so on. PMID:26571677

  5. [Screening probiotic endophytic bacteria from medicinal plant flex cornuta and the phytopathogen-inhibiting effect].

    PubMed

    Zhao, Long-fei; Xu, Ya-jun; Lai, Xin-he; Kou, Tian-chao; Yan, Jun-li; Zhou, Pei-pei; Fan, Shan-shan; Yan, Yong-feng

    2015-05-01

    Culturable endophytic bacteria were isolated from medicinal plant Ilex cornuta by plate-spreading method, strains with strong inhibitory effect on phytopathogen were screened by confrontation culture and fermentation filtrate culture methods, and the morphological changes of phytopathogen hyphae treated with endophytic bacteria were examined by microscopy and micrograph. Their phylogenetic relationships were determined by homology analysis of the 16S rDNA sequences of PCR products and the taxonomic status of the selected strains was determined based on their morphology, physiology, biochemical test results and 16S rDNA sequence analysis. A total of 85 endophytic bacteria were isolated from the healthy roots, stems, leaves and fruits of I. cornuta, and 10 strains of them showed strong inhibitory effect on Alternaria alternata, Magnaporthe grisea, Fusarium oxysporum, and were preliminarily identified belonging to four genera and seven species. Three strains with the strongest inhibitory effect, GG78 (60.3%), GG31 (48.1%) and GG13 (61.0%) belonged to Enterobacter cloacae, Enterobacter ludwigii and Bacillus cereus, respectively. Microscopic analyses showed that the inhibited phytopathogen hyphae became deformed, distorted, and partially expanded forming plasma concentration and hair-like branch on the hyphae base. These morphological changes could be caused by the extracellular metabolic substances secreted by the endophytic bacteria, such as antibiotics, hydrolytic enzymes, alkaloids and so on.

  6. Investigation of mycoviruses in endophytic and phytopathogenic strains of Colletotrichum from different hosts.

    PubMed

    Rosseto, P; Costa, A T; Polonio, J C; da Silva, A A; Pamphile, J A; Azevedo, J L

    2016-01-01

    Fungi belonging to the Colletotrichum genus can be categorized as endophytic or phytopathogenic. These fungi can be infected by viruses, termed mycoviruses, which are know to promote hypovirulence in infected fungi. However, there are few studies that have described mycoviral infections of endophytes. The production of secondary metabolites by endophytes with antimicrobial potential in inhibiting numerous pathogens has gained increasing attention. The aim of the current study was to investigate the presence of mycoviruses in endophytic and phytopathogenic fungi of the Colletotrichum genus, as well as to analyze the antimicrobial activity of crude extracts obtained from these samples. To detect the presence of mycoviruses in the samples, dsRNA was extracted, treated with enzymes, and analyzed following electrophoresis in agarose gel. Furthermore, isometric mycoviral particles were observed by transmission electron microscopy. Serial microdilution methodology was used to test crude extracts of Colletotrichum spp for antibacterial activity against Escherichia coli and Staphylococcus aureus, and antifungal activity against Fusarium solani. The results of the molecular and microscopic analyses indicated that a phytopathogenic strain presented infection by mycovirus. The antibacterial activity analysis revealed that the minimum inhibitory concentrations and minimum bactericidal concentrations were low for the fungal extracts of the two endophytes, indicating that these extracts were effective antibacterial agents. However, their antifungal activity against F. solani was not statistically different compared to that of the negative control. PMID:26985921

  7. Characterization of Race-Specific Interactions Among Isolates of Verticillium dahliae Pathogenic on Lettuce.

    PubMed

    Vallad, Gary E; Qin, Qing-Ming; Grube, Rebecca; Hayes, Ryan J; Subbarao, Krishna V

    2006-12-01

    ABSTRACT Verticillium wilt, caused by Verticillium dahliae, poses a major threat to lettuce (Lactuca sativa) production in California. Incorporation of resistance into commercial lettuce cultivars offers the least expensive technique of sustaining production in infested areas. To test the breadth of the resistance identified in field experiments, a pair of susceptible ('Salinas' and 'Sniper') and resistant ('La Brillante' and 'Little Gem') lettuce cultivars were used as differentials and individually inoculated with 29 isolates of V. dahliae and two isolates of V. albo-atrum from several hosts, including lettuce, in replicated greenhouse experiments. The reactions of the four cultivars were determined based on the disease severity at maturity. None of the V. albo-atrum isolates or V. dahliae isolates from cruciferous hosts caused significant disease on lettuce. Both Salinas and Sniper were susceptible to many isolates of V. dahliae (21 of 23) from noncruciferous hosts, and the isolates varied in their overall virulence. However, of these, only three isolates caused significant disease on the resistant cvs. La Brillante and Little Gem. These three isolates also were distinct from the other V. dahliae isolates based on sequence data from the intergenic spacer (IGS) region of the nuclear ribosomal RNA gene, suggesting that they form a phylogenetically distinct subgroup that differs in virulence toward specific lettuce genotypes. Accordingly, isolates of V. dahliae virulent on all tested cultivars, including the resistant La Brillante and Little Gem, were designated as race 2, whereas those virulent only on the susceptible Salinas and Sniper were designated as race 1. Although a range of virulence among isolates has been described in other hosts, this is the first description of distinct virulence phenotypes in V. dahliae since a similar race structure was described in tomato in the 1960s.

  8. Transcriptome analysis of the compatible interaction of tomato with Verticillium dahliae using RNA-sequencing

    PubMed Central

    Tan, Guangxuan; Liu, Kun; Kang, Jingmin; Xu, Kedong; Zhang, Yi; Hu, Lizong; Zhang, Ju; Li, Chengwei

    2015-01-01

    Tomato Verticillium wilt is a soil-borne vascular disease caused by the necrotrophic fungus Verticillium dahliae. Although some understanding of plant defense mechanisms against V. dahliae infection has been gained for incompatible interactions, including identification of inducible resistant genes and defense signaling pathways, the genes and signaling pathways involved in the compatible interaction remain unclear. To investigate the molecular basis of the compatible interaction between tomato and V. dahliae, transcriptomes of V. dahliae infected tomatoes were compared to those of a control group. A total of approximately 25 million high-quality reads were generated by means of the RNA sequencing (RNA-seq) method. The sequence reads were aligned to the tomato reference genome and analyzed to measure gene expression levels, and to identify alternative splicing events. Comparative analysis between the two samples revealed 1,953 significantly differentially expressed genes (DEGs), including 1,281 up-regulated and 672 down-regulated genes. The RNA-Seq output was confirmed using RT-qPCR for 10 selected genes. The Nr, Swiss-Prot, Gene Ontology (GO), Clusters of Orthologous Groups (COG), and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases were used to annotate DEG functions. Of the 1,953 DEGs identified, 1,953, 1,579, 1,739, 862, and 380 were assigned by Nr, Swiss-Prot, GO, COG, and KEGG, respectively. The important functional groups identified via GO and COG enrichment were those responsible for fundamental biological regulation, secondary metabolism, and signal transduction. Of DEGs assigned to 87 KEGG pathways, most were associated with phenylpropanoid metabolism and plant–pathogen interaction pathways. Most of the DEGs involved in these two pathways were up-regulated, and may be involved in regulating the tomato-V. dahliae compatible interaction. The results will help to identify key susceptible genes and contribute to a better understanding of the

  9. Biocontrol treatments confer protection against Verticillium dahliae infection of potato by inducing antimicrobial metabolites.

    PubMed

    El Hadrami, A; Adam, L R; Daayf, F

    2011-03-01

    Verticillium wilt, caused by Verticillium dahliae Kleb., is a serious potato (Solanum tuberosum L.) disease worldwide, and biocontrol represents a promising eco-friendly strategy to reduce its impact. We used extracts from Canada milk vetch (CMV) and a set of four V. dahliae-antagonistic bacterial strains to coat potato seeds at planting and examined the degree of protection provided against V. dahliae as well as accumulation of soluble phenolics as markers for induced resistance. All tested treatments were effective in reducing disease severity, and CMV showed the highest level of protection. In this treatment, flavonol-glycoside rutin was a highly abundant compound induced in potato tissues, with levels two to three times higher than those detected in noninoculated controls and V. dahliae-inoculated plants. We investigated dose-dependent effects of rutin on V. dahliae growth and sporulation in vitro and in planta. The effect of rutin on mycelial growth was inconsistent between disk assay and amended medium experiments. On the other hand, significant reduction of V. dahliae sporulation in vitro was consistently observed starting at 300 and 100 μM for isolates Vd-9 and Vd-21, respectively. We successfully detected 2-protocatechuoylphloroglucinolcarboxylic acid (2-PCPGCA) using ultra-performance liquid chromatography tandem mass spectrometry, indicating that V. dahliae dioxygenally oxidizes quercetin. Quercetin, as an aglycone, is freed from the sugar moiety by glucosidases and rhamnosidases produced by the fungus and is a substrate for quercetinases. The occurrence of quercetinases in V. dahliae provides a background to formulate a hypothesis about how by-product 2-PCPGCA may be interfering with potato defenses.

  10. Whole genome wide expression profiles on germination of Verticillium dahliae microsclerotia.

    PubMed

    Hu, Dongfang; Wang, Chunsheng; Tao, Fei; Cui, Qian; Xu, Xiangming; Shang, Wenjing; Hu, Xiaoping

    2014-01-01

    Verticillium dahliae is a fungal pathogen causing Verticillium wilt on a range of economically important crops. Microsclerotia are its main survival and dormancy structures and serve as the primary inoculum on many hosts. Studies were conducted to determine the effect of temperature (5 to 50°C), pH (2 to 12) and nutrient regimes on microsclerotia germination. The optimal condition for microsclerotium germination was 20°C with pH 8.0 whereas nutrient regimes had no significant effect on its germination. The whole genome wide expression profiles during microsclerotium germination were characterized using the Illumina sequencing technology. Approximately 7.4 million of 21-nt cDNA tags were sequenced in the cDNA libraries derived from germinated and non-germinated microsclerotia. About 3.9% and 2.3% of the unique tags were up-regulated and down-regulated at least five-fold, respectively, in the germinated microsclerotia compared with the non-germinated microsclerotia. A total of 1654 genes showing differential expression were identified. Genes that are likely to have played important roles in microsclerotium germination include those encoding G-protein coupled receptor, lipase/esterase, cyclopentanone 1,2-monooxygenase, H(+)/hexose cotransporter 1, fungal Zn(2)-Cys(6) binuclear cluster domain, thymus-specific serine protease, glucan 1,3-beta-glucosidase, and alcohol dehydrogenase. These genes were mainly up-regulated or down-regulated only in germinated microsclerotia, compared with non-germinated microsclerotia. The differential expression of genes was confirmed by qRT-PCR analysis of 20 randomly selected genes from the 40 most differentially expressed genes.

  11. Transcriptome analysis of the compatible interaction of tomato with Verticillium dahliae using RNA-sequencing.

    PubMed

    Tan, Guangxuan; Liu, Kun; Kang, Jingmin; Xu, Kedong; Zhang, Yi; Hu, Lizong; Zhang, Ju; Li, Chengwei

    2015-01-01

    Tomato Verticillium wilt is a soil-borne vascular disease caused by the necrotrophic fungus Verticillium dahliae. Although some understanding of plant defense mechanisms against V. dahliae infection has been gained for incompatible interactions, including identification of inducible resistant genes and defense signaling pathways, the genes and signaling pathways involved in the compatible interaction remain unclear. To investigate the molecular basis of the compatible interaction between tomato and V. dahliae, transcriptomes of V. dahliae infected tomatoes were compared to those of a control group. A total of approximately 25 million high-quality reads were generated by means of the RNA sequencing (RNA-seq) method. The sequence reads were aligned to the tomato reference genome and analyzed to measure gene expression levels, and to identify alternative splicing events. Comparative analysis between the two samples revealed 1,953 significantly differentially expressed genes (DEGs), including 1,281 up-regulated and 672 down-regulated genes. The RNA-Seq output was confirmed using RT-qPCR for 10 selected genes. The Nr, Swiss-Prot, Gene Ontology (GO), Clusters of Orthologous Groups (COG), and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases were used to annotate DEG functions. Of the 1,953 DEGs identified, 1,953, 1,579, 1,739, 862, and 380 were assigned by Nr, Swiss-Prot, GO, COG, and KEGG, respectively. The important functional groups identified via GO and COG enrichment were those responsible for fundamental biological regulation, secondary metabolism, and signal transduction. Of DEGs assigned to 87 KEGG pathways, most were associated with phenylpropanoid metabolism and plant-pathogen interaction pathways. Most of the DEGs involved in these two pathways were up-regulated, and may be involved in regulating the tomato-V. dahliae compatible interaction. The results will help to identify key susceptible genes and contribute to a better understanding of the

  12. Development of an assay for rapid detection and quantification of Verticillium dahliae in soil.

    PubMed

    Bilodeau, Guillaume J; Koike, Steven T; Uribe, Pedro; Martin, Frank N

    2012-03-01

    ABSTRACT Verticillium dahliae is responsible for Verticillium wilt on a wide range of hosts, including strawberry, on which low soil inoculum densities can cause significant crop loss. Determination of inoculum density is currently done by soil plating but this can take 6 to 8 weeks to complete and delay the grower's ability to make planting decisions. To provide a faster means for estimating pathogen populations in the soil, a multiplexed TaqMan real-time polymerase chain reaction (PCR) assay based on the ribosomal DNA (rDNA) intergenic spacer (IGS) was developed for V. dahliae. The assay was specific for V. dahliae and included an internal control for evaluation of inhibition due to the presence of PCR inhibitors in DNA extracted from soil samples. An excellent correlation was observed in regression analysis (R(2) = 0.96) between real-time PCR results and inoculum densities determined by soil plating in a range of field soils with pathogen densities as low as 1 to 2 microsclerotia/g of soil. Variation in copy number of the rDNA was also evaluated among isolates by SYBR Green real-time PCR amplification of the V. dahliae-specific amplicon compared with amplification of several single-copy genes and was estimated to range from ≈24 to 73 copies per haploid genome, which translated into possible differences in results among isolates of ≈1.8 cycle thresholds. Analysis of the variation in results of V. dahliae quantification among extractions of the same soil sample indicated that assaying four replicate DNA extractions for each field sample would provide accurate results. A TaqMan assay also was developed to help identify colonies of V. tricorpus on soil plates.

  13. Development of an assay for rapid detection and quantification of Verticillium dahliae in soil.

    PubMed

    Bilodeau, Guillaume J; Koike, Steven T; Uribe, Pedro; Martin, Frank N

    2012-03-01

    ABSTRACT Verticillium dahliae is responsible for Verticillium wilt on a wide range of hosts, including strawberry, on which low soil inoculum densities can cause significant crop loss. Determination of inoculum density is currently done by soil plating but this can take 6 to 8 weeks to complete and delay the grower's ability to make planting decisions. To provide a faster means for estimating pathogen populations in the soil, a multiplexed TaqMan real-time polymerase chain reaction (PCR) assay based on the ribosomal DNA (rDNA) intergenic spacer (IGS) was developed for V. dahliae. The assay was specific for V. dahliae and included an internal control for evaluation of inhibition due to the presence of PCR inhibitors in DNA extracted from soil samples. An excellent correlation was observed in regression analysis (R(2) = 0.96) between real-time PCR results and inoculum densities determined by soil plating in a range of field soils with pathogen densities as low as 1 to 2 microsclerotia/g of soil. Variation in copy number of the rDNA was also evaluated among isolates by SYBR Green real-time PCR amplification of the V. dahliae-specific amplicon compared with amplification of several single-copy genes and was estimated to range from ≈24 to 73 copies per haploid genome, which translated into possible differences in results among isolates of ≈1.8 cycle thresholds. Analysis of the variation in results of V. dahliae quantification among extractions of the same soil sample indicated that assaying four replicate DNA extractions for each field sample would provide accurate results. A TaqMan assay also was developed to help identify colonies of V. tricorpus on soil plates. PMID:22066673

  14. Identification of miRNAs and Their Targets in Cotton Inoculated with Verticillium dahliae by High-Throughput Sequencing and Degradome Analysis.

    PubMed

    Zhang, Yujuan; Wang, Wei; Chen, Jie; Liu, Jubo; Xia, Minxuan; Shen, Fafu

    2015-06-30

    MicroRNAs (miRNAs) are a group of endogenous small non-coding RNAs that play important roles in plant growth, development, and stress response processes. Verticillium wilt is a vascular disease in plants mainly caused by Verticillium dahliae Kleb., the soil-borne fungal pathogen. However, the role of miRNAs in the regulation of Verticillium defense responses is mostly unknown. This study aimed to identify new miRNAs and their potential targets that are involved in the regulation of Verticillium defense responses. Four small RNA libraries and two degradome libraries from mock-infected and infected roots of cotton (both Gossypium hirsutum L. and Gossypium barbadense L.) were constructed for deep sequencing. A total of 140 known miRNAs and 58 novel miRNAs were identified. Among the identified miRNAs, many were differentially expressed between libraries. Degradome analysis showed that a total of 83 and 24 genes were the targets of 31 known and 14 novel miRNA families, respectively. Gene Ontology analysis indicated that many of the identified miRNA targets may function in controlling root development and the regulation of Verticillium defense responses in cotton. Our findings provide an overview of potential miRNAs involved in the regulation of Verticillium defense responses in cotton and the interactions between miRNAs and their corresponding targets. The profiling of these miRNAs lays the foundation for further understanding of the function of small RNAs in regulating plant response to fungal infection and Verticillium wilt in particular.

  15. Identification of novel microRNAs in the Verticillium wilt-resistant upland cotton variety KV-1 by high-throughput sequencing.

    PubMed

    He, Xiaohong; Sun, Quan; Jiang, Huaizhong; Zhu, Xiaoyan; Mo, Jianchuan; Long, Lu; Xiang, Liuxin; Xie, Yongfang; Shi, Yuzhen; Yuan, Youlu; Cai, Yingfan

    2014-01-01

    Plant microRNAs (miRNAs) play essential roles in the post-transcriptional regulation of gene expression during development, flowering, plant growth, metabolism, and stress responses. Verticillium wilt is one of the vascular disease in plants, which is caused by the Verticillium dahlia and leads to yellowing, wilting, lodging, damage to the vascular tissue, and death in cotton plants. Upland cotton varieties KV-1 have shown resistance to Verticillium wilt in multiple levels. However, the knowledge regarding the post-transcriptional regulation of the resistance is limited. Here two novel small RNA (sRNA) libraries were constructed from the seedlings of upland cotton variety KV-1, which is highly resistant to Verticillium wilts and inoculated with the V991 and D07038 Verticillium dahliae (V. dahliae) of different virulence strains. Thirty-seven novel miRNAs were identified after sequencing these two libraries by the Illumina Solexa system. According to sequence homology analysis, potential target genes of these miRNAs were predicted. With no more than three sequence mismatches between the novel miRNAs and the potential target mRNAs, we predicted 49 target mRNAs for 24 of the novel miRNAs. These target mRNAs corresponded to genes were found to be involved in plant-pathogen interactions, endocytosis, the mitogen-activated protein kinase (MAPK) signaling pathway, and the biosynthesis of isoquinoline alkaloid, terpenoid backbone, primary bile acid and secondary metabolites. Our results showed that some of these miRNAs and their relative gene are involved in resistance to Verticillium wilts. The identification and characterization of miRNAs from upland cotton could help further studies on the miRNA regulatory mechanisms of resistance to Verticillium wilt. PMID:25332864

  16. Genetic structure, linkage disequilibrium and association mapping of Verticillium wilt resistance in elite cotton (Gossypium hirsutum L.) germplasm population.

    PubMed

    Zhao, Yunlei; Wang, Hongmei; Chen, Wei; Li, Yunhai

    2014-01-01

    Understanding the population structure and linkage disequilibrium in an association panel can effectively avoid spurious associations and improve the accuracy in association mapping. In this study, one hundred and fifty eight elite cotton (Gossypium hirsutum L.) germplasm from all over the world, which were genotyped with 212 whole genome-wide marker loci and phenotyped with an disease nursery and greenhouse screening method, were assayed for population structure, linkage disequilibrium, and association mapping of Verticillium wilt resistance. A total of 480 alleles ranging from 2 to 4 per locus were identified from all collections. Model-based analysis identified two groups (G1 and G2) and seven subgroups (G1a-c, G2a-d), and differentiation analysis showed that subgroup having a single origin or pedigree was apt to differentiate with those having a mixed origin. Only 8.12% linked marker pairs showed significant LD (P<0.001) in this association panel. The LD level for linked markers is significantly higher than that for unlinked markers, suggesting that physical linkage strongly influences LD in this panel, and LD level was elevated when the panel was classified into groups and subgroups. The LD decay analysis for several chromosomes showed that different chromosomes showed a notable change in LD decay distances for the same gene pool. Based on the disease nursery and greenhouse environment, 42 marker loci associated with Verticillium wilt resistance were identified through association mapping, which widely were distributed among 15 chromosomes. Among which 10 marker loci were found to be consistent with previously identified QTLs and 32 were new unreported marker loci, and QTL clusters for Verticillium wilt resistanc on Chr.16 were also proved in our study, which was consistent with the strong linkage in this chromosome. Our results would contribute to association mapping and supply the marker candidates for marker-assisted selection of Verticillium wilt

  17. Cotton gene expression profiles in resistant Gossypium hirsutum cv. Zhongzhimian KV1 responding to Verticillium dahliae strain V991 infection.

    PubMed

    Zhang, Wen-Wei; Jian, Gui-Liang; Jiang, Teng-Fei; Wang, Sheng-Zheng; Qi, Fang-Jun; Xu, Shi-Chang

    2012-10-01

    Verticillium wilt of cotton (Gossypium hirsutum) is a widespread and destructive disease that is caused by the soil-borne fungus pathogen Verticillium dahliae (V. dahliae). To study the molecular mechanism in wilt tolerance, suppression subtractive hybridization (SSH) and dot blot techniques were used to identify the specifically expressed genes in a superior wilt-resistant cotton cultivar (G. hirsutum cv. Zhongzhimian KV1) after inoculation with pathogen. cDNAs from the root tissues of Zhongzhimian KV1 inoculated with V. dahliae strain V991 or water mock were used to construct the libraries that contain 4800 clones. Based on the results from dot blot analysis, 147 clones were clearly induced by V. dahliae and selected from the SSH libraries for sequencing. A total of 92 up-regulated and 7 down-regulated non-redundant expressed sequences tags (ESTs) were identified as disease responsive genes and classified into 9 functional groups. Two important clues regarding wilt-resistant G. hirsutum were obtained from this study. One was Bet v 1 family; the other was UbI gene family that may play an important role in the defense reaction against Verticillium wilt. The result from real-time quantitative reverse transcription polymerase chain reaction showed that these genes were activated quickly and transiently after inoculation with V. dahliae.

  18. Island cotton Gbve1 gene encoding a receptor-like protein confers resistance to both defoliating and non-defoliating isolates of Verticillium dahliae.

    PubMed

    Zhang, Baolong; Yang, Yuwen; Chen, Tianzi; Yu, Wengui; Liu, Tingli; Li, Hongjuan; Fan, Xiaohui; Ren, Yongzhe; Shen, Danyu; Liu, Li; Dou, Daolong; Chang, Youhong

    2012-01-01

    Verticillium wilt caused by soilborne fungus Verticillium dahliae could significantly reduce cotton yield. Here, we cloned a tomato Ve homologous gene, Gbve1, from an island cotton cultivar that is resistant to Verticillium wilt. We found that the Gbve1 gene was induced by V. dahliae and by phytohormones salicylic acid, jasmonic acid, and ethylene, but not by abscisic acid. The induction of Gbve1 in resistant cotton was quicker and stronger than in Verticillium-susceptible upland cotton following V. dahliae inoculation. Gbve1 promoter-driving GUS activity was found exclusively in the vascular bundles of roots and stems of transgenic Arabidopsis. Virus-induced silencing of endogenous genes in resistant cotton via targeting a fragment of the Gbve1 gene compromised cotton resistance to V. dahliae. Furthermore, we transformed the Gbve1 gene into Arabidopsis and upland cotton through Agrobacterium-mediated transformation. Overexpression of the Gbve1 gene endowed transgenic Arabidopsis and upland cotton with resistance to high aggressive defoliating and non-defoliating isolates of V. dahliae. And HR-mimic cell death was observed in the transgenic Arabidopsis. Our results demonstrate that the Gbve1 gene is responsible for resistance to V. dahliae in island cotton and can be used for breeding cotton varieties that are resistant to Verticillium wilt. PMID:23251427

  19. Island cotton Gbve1 gene encoding a receptor-like protein confers resistance to both defoliating and non-defoliating isolates of Verticillium dahliae.

    PubMed

    Zhang, Baolong; Yang, Yuwen; Chen, Tianzi; Yu, Wengui; Liu, Tingli; Li, Hongjuan; Fan, Xiaohui; Ren, Yongzhe; Shen, Danyu; Liu, Li; Dou, Daolong; Chang, Youhong

    2012-01-01

    Verticillium wilt caused by soilborne fungus Verticillium dahliae could significantly reduce cotton yield. Here, we cloned a tomato Ve homologous gene, Gbve1, from an island cotton cultivar that is resistant to Verticillium wilt. We found that the Gbve1 gene was induced by V. dahliae and by phytohormones salicylic acid, jasmonic acid, and ethylene, but not by abscisic acid. The induction of Gbve1 in resistant cotton was quicker and stronger than in Verticillium-susceptible upland cotton following V. dahliae inoculation. Gbve1 promoter-driving GUS activity was found exclusively in the vascular bundles of roots and stems of transgenic Arabidopsis. Virus-induced silencing of endogenous genes in resistant cotton via targeting a fragment of the Gbve1 gene compromised cotton resistance to V. dahliae. Furthermore, we transformed the Gbve1 gene into Arabidopsis and upland cotton through Agrobacterium-mediated transformation. Overexpression of the Gbve1 gene endowed transgenic Arabidopsis and upland cotton with resistance to high aggressive defoliating and non-defoliating isolates of V. dahliae. And HR-mimic cell death was observed in the transgenic Arabidopsis. Our results demonstrate that the Gbve1 gene is responsible for resistance to V. dahliae in island cotton and can be used for breeding cotton varieties that are resistant to Verticillium wilt.

  20. Antibacterial activity of Artemisia nilagirica leaf extracts against clinical and phytopathogenic bacteria

    PubMed Central

    2010-01-01

    Background The six organic solvent extracts of Artemisia nilagirica were screened for the potential antimicrobial activity against phytopathogens and clinically important standard reference bacterial strains. Methods The agar disk diffusion method was used to study the antibacterial activity of A. nilagirica extracts against 15 bacterial strains. The Minimum Inhibitory Concentration (MIC) of the plant extracts were tested using two fold agar dilution method at concentrations ranging from 32 to 512 μg/ml. The phytochemical screening of extracts was carried out for major phytochemical derivatives in A. nilagirica. Results All the extracts showed inhibitory activity for gram-positive and gram-negative bacteria except for Klebsiella pneumoniae, Enterococcus faecalis and Staphylococcus aureus. The hexane extract was found to be effective against all phytopathogens with low MIC of 32 μg/ml and the methanol extract exhibited a higher inhibition activity against Escherichia coli, Yersinia enterocolitica, Salmonella typhi, Enterobacter aerogenes, Proteus vulgaris, Pseudomonas aeruginosa (32 μg/ml), Bacillus subtilis (64 μg/ml) and Shigella flaxneri (128 μg/ml). The phytochemical screening of extracts answered for the major derivative of alkaloids, amino acids, flavonoids, phenol, quinines, tannins and terpenoids. Conclusion All the extracts showed antibacterial activity against the tested strains. Of all, methanol and hexane extracts showed high inhibition against clinical and phytopathogens, respectively. The results also indicate the presence of major phytochemical derivatives in the A. nilagirica extracts. Hence, the isolation and purification of therapeutic potential compounds from A. nilagirica could be used as an effective source against bacterial diseases in human and plants. PMID:20109237

  1. [Effect of biopreparations on dynamics of the number of bacteria and phytopathogenic fungi in potato agroecosystem].

    PubMed

    Patyka, N V; Borodaĭ, V V; Zhitkevich, N V; Khomenko, E V; Gnatiuk, T T; Koltunov, V A; Patyka, V F

    2012-01-01

    Application of biological preparations such as Phytotsid and Planryz favoures the growth of the general number of soil's bacteria population compared with control by 13.0-36.1% in the variant of potato variety Scarbnytsya and by 4.5-24.6% of potato variety Oberig. It also decreases 1.2-1.8 times the number of soil phytopathogens--Fusarium and Alternaria. During the application of Rovral Akvaflo the Shenon's ecological index of species biodiversity was lower than during the bioprepation application. One could observe a decrease of species biodiversity and dominance of dark pigmentation in fungi--Alternaria sp., Cladosporium sp., Phoma sp., Doratomyces sp., and pigmented bacteria.

  2. Complete genome sequence of a filamentous bacteriophage, RS611, that infects the phytopathogen Ralstonia solanacearum.

    PubMed

    Van, Truong Thi Bich; Yoshida, Shohei; Miki, Kaito; Kondo, Akihiro; Kamei, Kaeko

    2015-03-01

    Filamentous bacteriophage RS611 (ϕRS611), which infects the phytopathogen Ralstonia solanacearum, had a circular single-stranded DNA genome that was characterized as an Ff-type phage belonging to the family Inoviridae. The ϕRS611 genome was composed of 6386 bases with a G + C content of 62.1 % and contained 11 putative open reading frames. The ϕRS611 genome showed high similarity to those of Ralstonia phages RSS0 and RSS1. However, approximately 900-nucleotide deletions were found in the region corresponding to open reading frames 10 and 11 of ϕRSS0 and ϕRSS1.

  3. Chaperones of the endoplasmic reticulum are required for Ve1-mediated resistance to Verticillium.

    PubMed

    Liebrand, Thomas W H; Kombrink, Anja; Zhang, Zhao; Sklenar, Jan; Jones, Alexandra M E; Robatzek, Silke; Thomma, Bart P H J; Joosten, Matthieu H A J

    2014-01-01

    The tomato receptor-like protein (RLP) Ve1 mediates resistance to the vascular fungal pathogen Verticillium dahliae. To identify the proteins required for Ve1 function, we transiently expressed and immunopurified functional Ve1-enhanced green fluorescent protein (eGFP) from Nicotiana benthamiana leaves, followed by mass spectrometry. This resulted in the identification of peptides originating from the endoplasmic reticulum (ER)-resident chaperones HSP70 binding proteins (BiPs) and a lectin-type calreticulin (CRT). Knock-down of the different BiPs and CRTs in tomato resulted in compromised Ve1-mediated resistance to V. dahliae in most cases, showing that these chaperones play an important role in Ve1 functionality. Recently, it has been shown that one particular CRT is required for the biogenesis of the RLP-type Cladosporium fulvum resistance protein Cf-4 of tomato, as silencing of CRT3a resulted in a reduced pool of complex glycosylated Cf-4 protein. In contrast, knock-down of the various CRTs in N. benthamiana or N. tabacum did not result in reduced accumulation of mature complex glycosylated Ve1 protein. Together, this study shows that the BiP and CRT ER chaperones differentially contribute to Cf-4- and Ve1-mediated immunity. PMID:24015989

  4. Characterization of dusts collected from swine confinement buildings. [Verticillium sp. ; Actinomycetes

    SciTech Connect

    Donham, K.J.; Scallon, L.J.; Popendorf, W.; Treauhaft, M.W.; Roberts, R.D.

    1986-07-01

    The air in 21 different swine confinement buildings was sampled with 37 mm cassette filters with and without cyclone preselectors and with cascade impactors. Filter results yielded a mean total aerosol of 6.3 mg/m/sup 3/, a mean respirable aerosol of 0.5 mg/m/sup 3/; the geometric mean diameter was 2.9 microns. Cascade impactor measurements revealed a mean total aerosol of 7.6 mg/m/sup 3/, a respirable aerosol of 2.5 mg/m/sup 3/ and a mass median diameter of 9.6 microns. The two major constituents in these aerosols were grain particles and dried fecal matter. The grain particles were larger than fecal particles and proportionately more abundant in finishing buildings where 50 kg-100 kg animals are housed. Therefore the respirable fraction was less in finishing buildings than in farrowing and nursery buildings. Culturing of settled dusts yielded six different mold species, with the highest counts for Verticillium sp. (5 x 10/sup 2/ cfu/mg dry dust) grown at 37/sup 0/C. Thermophilic Actinomycetes and both gram negative and gram positive bacteria were isolated.

  5. Evaluation of Fungicides on Growth and Conidial Germination of Verticillium theobromae Isolated from Plantain

    NASA Astrophysics Data System (ADS)

    Igeleke, C. L.; Ayanru, D. K. G.

    An evaluation of three fungicides, namely, benomyl [methyl l-(butyl-carbamoyl)-2-benzimidazole carbamate], calixin (tridomorph) and dithane M-45 (mancozeb), was carried out for their inhibitory effects on Verticillium theobromae, the causal organism of cigar-end rot disease of plantain (Musa paradisiaca). The LD50 of calixin, benomyl and dithane M-45 in inhibiting mycelial growth, were 0.14, 0.98 and 535.17 µg mL-1, respectively. Calixin was also the most effective in conidial germination inhibition with an LD50 of 21.78 µg mL-1, as compared with dithane M-45 86.39 µg mL-1 and benomyl 275.50 µg mL-1. Germ tubes of the test organism were distorted by the fungicides at varying concentrations. Calixin had greater distortion effect at a lower concentration of 10 µg mL-1, dithane M-45 (100 µg mL-1) and benomyl (1000 µg mL-1). The relevance of these results in relation to the control and management of cigar-end rot disease of plantain is discussed.

  6. Verticillium dahliae populations from mint and potato are genetically divergent with predominant haplotypes.

    PubMed

    Dung, Jeremiah K S; Peever, Tobin L; Johnson, Dennis A

    2013-05-01

    In total, 286 Verticillium dahliae isolates from mint, potato, and other hosts and substrates were characterized for mating type, vegetative compatibility group (VCG), and multilocus microsatellite haplotype to determine population genetic structure among populations infecting mint and potato. Populations from mint and potato fit a clonal reproductive model, with all isolates a single mating type (MAT1-2) and multiple occurrences of the same haplotypes. Haplotype H02 represented 88% of mint isolates and was primarily VCG2B, while haplotype H04 represented 70% of potato isolates and was primarily VCG4A. Haplotypes H02 and H04 typically caused severe disease on mint and potato, respectively, in greenhouse assays regardless of host origin. Principal coordinate analysis and analysis of molecular variance indicated that mint and potato populations were significantly genetically diverged (P = 0.02), and identification of private alleles and estimation of migration rates suggested restricted gene flow. Migration was detected between infected potato plants and seed tubers, infested tare soil, and field soils. Genetic differentiation of V. dahliae from mint and potato may be due to the occurrence of a single mating type and differences in VCG. Populations of V. dahliae in potato and mint were characterized by the presence of aggressive, clonally reproducing haplotypes which are widely distributed in commercial mint and potato production.

  7. A ku70 null mutant improves gene targeting frequency in the fungal pathogen Verticillium dahliae.

    PubMed

    Qi, Xiliang; Su, Xiaofeng; Guo, Huiming; Qi, Juncang; Cheng, Hongmei

    2015-12-01

    To overcome the challenges met with gene deletion in the plant pathogen Verticillium dahliae, a mutant strain with impaired non-homologous end joining DNA repair was generated to improve targeted gene replacement frequencies. A V. dahliae 991 ΔVdku70 null mutant strain was generated using Agrobacterium tumefaciens-mediated transformation. Despite having impaired non-homologous end joining DNA repair function, the ΔVdku70 strain exhibited normal growth, reproduction capability, and pathogenicity when compared with the wild-type strain. When the ΔVdku70 strain was used to delete 2-oxoglutarate dehydrogenase E2, ferric reductase transmembrane component 3 precursor, and ferric reductase transmembrane component 6 genes, gene replacement frequencies ranged between 22.8 and 34.7% compared with 0.3 and 0.5 % in the wild-type strain. The ΔVdku70 strain will be a valuable tool to generate deletion strains when studying factors that underlie virulence and pathogenesis in this filamentous fungus.

  8. Decreased defense gene expression in tolerance versus resistance to Verticillium dahliae in potato.

    PubMed

    Tai, Helen H; Goyer, Claudia; Bud Platt, H W; De Koeyer, David; Murphy, Agnes; Uribe, Pedro; Halterman, Dennis

    2013-08-01

    Verticillium dahliae Kleb., a soil-borne fungus that colonizes vascular tissues, induces wilting, chlorosis and early senescence in potato. Difference in senescence timing found in two diploid potato clones, 07506-01 and 12120-03, was studied and genetic variation in response to V. dahliae infection was identified as a causal factor. The clone, 07506-01, was infected with V. dahliae but did not develop symptoms, indicating tolerance to the pathogen. The other diploid clone, 12120-03 had low levels of pathogen with infection and moderate symptoms indicating partial resistance. 07506-01 was found to carry two susceptible alleles of the Ve2 gene and 12120-03 carried one Ve2 resistant and one susceptible allele. Infected leaves of the two clones were compared using gene expression profiling with the Potato Oligonucleotide Chip Initiative (POCI) microrarray. The results provide further evidence for differences in response of the two clones to infection with V. dahliae. Chlorophyll biosynthesis was higher in the tolerant 07506-01 compared to partially resistant 12120-03. On the other hand, expression of fungal defense genes, Ve resistance genes and defense phytohormone biosynthetic enzyme genes was decreased in 07506-01 compared to 12120-03 suggesting defense responses were suppressed in tolerance compared to resistance. Transcription factor gene expression differences pointed to the WRKY family as potential regulators of V. dahliae responses in potato.

  9. Transcriptomic profiles of the smoke tree wilt fungus Verticillium dahliae under nutrient starvation stresses.

    PubMed

    Xiong, Dianguang; Wang, Yonglin; Tian, Chengming

    2015-10-01

    Verticillium dahliae is a notorious plant pathogen that causes vascular wilt on more than 200 plant species. During plant infection, efficient pathogen nutrition during the interaction with the host is a requisite for successful infection. However, little attention has been focused on nutrient uptake and starvation responses in this fungus. Here, we used RNA-Seq to analyze the response of V. dahliae to nutrient starvation, including carbon and nitrogen depletion. Gene expression profile analysis showed that 1854 genes were differentially expressed under carbon starvation (852 upregulated and 539 downregulated genes) and nitrogen starvation (487 upregulated and 291 downregulated genes). Among the differentially expressed genes, genes involved in utilization or production acetyl-CoA, including glycolysis, fatty acid biosynthesis or metabolism, and melanin biosynthesis, were repressed under carbon starvation, whereas melanin biosynthesis genes were strongly induced under nitrogen starvation. These results, combined with VDH1 expression data, suggested that melanin biosynthesis and microsclerotia development were induced under nitrogen starvation, but microsclerotia development was suppressed under carbon starvation. Furthermore, many genes encoding carbohydrate-active enzymes and secreted proteins were induced under carbon starvation. Overall, the results improve our understanding of the response of V. dahliae to nutrient starvation and help to identify potential virulence factors for the development of novel disease control strategies.

  10. Verticillium dahliae alters Pseudomonas spp. populations and HCN gene expression in the rhizosphere of strawberry.

    PubMed

    DeCoste, Nadine J; Gadkar, Vijay J; Filion, Martin

    2010-11-01

    The production of hydrogen cyanide (HCN) by beneficial root-associated bacteria is an important mechanism for the biological control of plant pathogens. However, little is known about the biotic factors affecting HCN gene expression in the rhizosphere of plants. In this study, real-time reverse transcription PCR (qRT-PCR) assays were developed to investigate the effect of the plant pathogen Verticillium dahliae on hcnC (encoding for HCN biosynthesis) gene expression in Pseudomonas sp. LBUM300. Strawberry plants were inoculated with Pseudomonas sp. LBUM300 and (or) V. dahliae and grown in pots filled with nonsterilized field soil. RNA was extracted from rhizosphere soil sampled at 0, 15, 30, and 45 days following inoculation with V. dahliae and used for qRT-PCR analyses. Populations of V. dahliae and Pseudomonas sp. LBUM300 were also monitored using a culture-independent qPCR approach. hcnC expression was detected at all sampling dates. The presence of V. dahliae had a significant stimulation effect on hcnC gene expression and also increased the population of Pseudomonas sp. LBUM300. However, the V. dahliae population was not altered by the presence of Pseudomonas sp. LBUM300. To our knowledge, this study is the first to evaluate the effect of a plant pathogen on HCN gene expression in the rhizosphere soil.

  11. Verticillium dahliae Sge1 differentially regulates expression of candidate effector genes.

    PubMed

    Santhanam, Parthasarathy; Thomma, Bart P H J

    2013-02-01

    The ascomycete fungus Verticillium dahliae causes vascular wilt diseases in hundreds of dicotyledonous plant species. However, thus far, only few V. dahliae effectors have been identified, and regulators of pathogenicity remain unknown. In this study, we investigated the role of the V. dahliae homolog of Sge1, a transcriptional regulator that was previously implicated in pathogenicity and effector gene expression in Fusarium oxysporum. We show that V. dahliae Sge1 (VdSge1) is required for radial growth and production of asexual conidiospores, because VdSge1 deletion strains display reduced radial growth and reduced conidia production. Furthermore, we show that VdSge1 deletion strains have lost pathogenicity on tomato. Remarkably, VdSge1 is not required for induction of Ave1, the recently identified V. dahliae effector that activates resistance mediated by the Ve1 immune receptor in tomato. Further assessment of the role of VdSge1 in the induction of the nine most highly in-planta-induced genes that encode putative effectors revealed differential activity. Although the expression of one putative effector gene in addition to Ave1 was not affected by VdSge1 deletion, VdSge1 appeared to be required for the expression of six putative effector genes, whereas two of the putative effectors genes were found to be negatively regulated by VdSge1. In conclusion, our data suggest that VdSge1 differentially regulates V. dahliae effector gene expression.

  12. Isolation and characterization of putative endophytic bacteria antagonistic to Phoma tracheiphila and Verticillium albo-atrum.

    PubMed

    Kalai-Grami, Leila; Saidi, Sabrine; Bachkouel, Sarra; Ben Slimene, Imen; Mnari-Hattab, Monia; Hajlaoui, Mohamed Rebah; Limam, Ferid

    2014-09-01

    A collection of 200 bacterial isolates recovered from citrus plants (Citrus limon, Citrus sinensis, and Citrus reticulata), Medicago truncatula and Laurus nobilis, was established. In vitro screening indicated that 28 isolates exhibited an inhibitory activity against the vascular pathogens Phoma tracheiphila and Verticillium albo-atrum. Isolates were screened according to their hydrolytic activities, plant growth-promoting bacteria (PGPB) abilities, as well as for the presence of nonribosomal peptide synthetase (NRPS) genes responsible of the lipopeptide biosynthesis. The results were positive for 16 isolates which exhibited at least two PGPB activities and a single NRPS gene. Genetic diversity of the selected isolates was studied using random amplified polymorphic DNA (RAPD) and repetitive element PCR (REP) tools that showed clustering of strains into three major groups (I, II, and III) (i, ii, and iii), respectively. Clustering was further confirmed by the 16S rDNA sequencing that assigned nine isolates to Bacillus velezensis, four isolates to Bacillus methyltrophicus, one isolate to Bacillus amyloliquefaciens, and two isolates to Bacillus mojavensis. Organ-bacterial genotype interaction as well as positive correlation with NRPS genes are discussed. PMID:25074353

  13. Verticillium dahliae alters Pseudomonas spp. populations and HCN gene expression in the rhizosphere of strawberry.

    PubMed

    DeCoste, Nadine J; Gadkar, Vijay J; Filion, Martin

    2010-11-01

    The production of hydrogen cyanide (HCN) by beneficial root-associated bacteria is an important mechanism for the biological control of plant pathogens. However, little is known about the biotic factors affecting HCN gene expression in the rhizosphere of plants. In this study, real-time reverse transcription PCR (qRT-PCR) assays were developed to investigate the effect of the plant pathogen Verticillium dahliae on hcnC (encoding for HCN biosynthesis) gene expression in Pseudomonas sp. LBUM300. Strawberry plants were inoculated with Pseudomonas sp. LBUM300 and (or) V. dahliae and grown in pots filled with nonsterilized field soil. RNA was extracted from rhizosphere soil sampled at 0, 15, 30, and 45 days following inoculation with V. dahliae and used for qRT-PCR analyses. Populations of V. dahliae and Pseudomonas sp. LBUM300 were also monitored using a culture-independent qPCR approach. hcnC expression was detected at all sampling dates. The presence of V. dahliae had a significant stimulation effect on hcnC gene expression and also increased the population of Pseudomonas sp. LBUM300. However, the V. dahliae population was not altered by the presence of Pseudomonas sp. LBUM300. To our knowledge, this study is the first to evaluate the effect of a plant pathogen on HCN gene expression in the rhizosphere soil. PMID:21076481

  14. Relative Activities and Characteristics of Some Oxidative Respiratory Enzymes from Conidia of Verticillium albo-atrum

    PubMed Central

    Throneberry, G. O.

    1967-01-01

    Conidia of Verticillium albo-atrum Reinke and Berthold, collected from shake cultures grown in Czapek broth, were sonified for 4 or 8 minutes or ground frozen in a mortar to obtain cell-free homogenates. These were assayed for certain enzymes associated with respiratory pathways. Malic dehydrogenase was the most active, glucose-6-P and NADH dehydrogenase were less active, NADH-cytochrome c reductase, NADPH dehydrogenase, and cytochrome oxidase were low in activity, and succinic dehydrogenase and succinic cytochrome c reductase were very low to negligible in activity. No NADH oxidase activity was detected. With the exception of NADH-cytochrome c reductase and possibly succinic dehydrogenase and cytochrome c reductase, there was no evident increase in specific activity of the enzymes during germination. Some NADH-cytochrome c reductase and a small amount of succinic-dehydrogenase and cytochrome c reductase were associated with the particulate fraction from 105,000 × g centrifugation. The other enzymes, including cytochrome oxidase, almost completely remained in the supernatant fraction. Menadione and vitamin K-S(II) markedly stimulated NADH-cytochrome c reductase activity in the supernatant fraction but had much less effect on NADPH-cytochrome c reductase in this fraction or on either of these enzyme systems in the particulate fraction. Electron transport inhibitors affected particulate NADH- and NADPH-cytochrome c reductase activity but had no effect on these in the supernatant fraction. PMID:16656681

  15. A ku70 null mutant improves gene targeting frequency in the fungal pathogen Verticillium dahliae.

    PubMed

    Qi, Xiliang; Su, Xiaofeng; Guo, Huiming; Qi, Juncang; Cheng, Hongmei

    2015-12-01

    To overcome the challenges met with gene deletion in the plant pathogen Verticillium dahliae, a mutant strain with impaired non-homologous end joining DNA repair was generated to improve targeted gene replacement frequencies. A V. dahliae 991 ΔVdku70 null mutant strain was generated using Agrobacterium tumefaciens-mediated transformation. Despite having impaired non-homologous end joining DNA repair function, the ΔVdku70 strain exhibited normal growth, reproduction capability, and pathogenicity when compared with the wild-type strain. When the ΔVdku70 strain was used to delete 2-oxoglutarate dehydrogenase E2, ferric reductase transmembrane component 3 precursor, and ferric reductase transmembrane component 6 genes, gene replacement frequencies ranged between 22.8 and 34.7% compared with 0.3 and 0.5 % in the wild-type strain. The ΔVdku70 strain will be a valuable tool to generate deletion strains when studying factors that underlie virulence and pathogenesis in this filamentous fungus. PMID:26475327

  16. Phytopathogenic bacteria phenotype conversion as a result of their lysogenisation by coliphage P1.

    PubMed

    Faidiuk, I V; Tovkach, E I

    2014-01-01

    A set of lysogenic strains of phytopathogenic bacteria Erwinia "horticola" and Erwinia amylovora associated with woody plants was obtained using bacteriophage P1 Cmc1ts100. The phenotype conversion from Cm(S) to Cm(R) was shown to be connected with introducing of authentic prophage DNA of 94.8 kb as a single-copy plasmid into the cells. Prophage state is unstable: P1 plasmid is spontaneously lost with high frequency by the cells. In lysogenic cells the prophage genes of type III restriction-modification complex EcoP1I are actively expressed. The system formed by E. "horticola" 450 and 60 as well as their lysogenic derivatives and specific bacteriophages provides an opportunity to divide the latter into three groups according to the level of restriction in the course of their interaction with the enzyme EcoP1I. The difference in phage responses to the endonuclease presence in a lysogenized host presumably correlates with the number of enzyme recognition sequences and the adsorption sites availability. After the prophage plasmid DNA curing the characteristic value of phage sensitivity of cells is changed. The lysogenic strains obtained in this work allow for the exploration of EcoP1I restriction-modification gene complex interaction with polyvalent phages able to grow not only on E. coli, but also on such phytopathogens as E. "horticola" and E. amylovora. PMID:25000732

  17. Gene silencing and gene expression in phytopathogenic fungi using a plant virus vector

    PubMed Central

    Mascia, Tiziana; Nigro, Franco; Abdallah, Alì; Ferrara, Massimo; De Stradis, Angelo; Faedda, Roberto; Palukaitis, Peter; Gallitelli, Donato

    2014-01-01

    RNA interference (RNAi) is a powerful approach for elucidating gene functions in a variety of organisms, including phytopathogenic fungi. In such fungi, RNAi has been induced by expressing hairpin RNAs delivered through plasmids, sequences integrated in fungal or plant genomes, or by RNAi generated in planta by a plant virus infection. All these approaches have some drawbacks ranging from instability of hairpin constructs in fungal cells to difficulties in preparing and handling transgenic plants to silence homologous sequences in fungi grown on these plants. Here we show that RNAi can be expressed in the phytopathogenic fungus Colletotrichum acutatum (strain C71) by virus-induced gene silencing (VIGS) without a plant intermediate, but by using the direct infection of a recombinant virus vector based on the plant virus, tobacco mosaic virus (TMV). We provide evidence that a wild-type isolate of TMV is able to enter C71 cells grown in liquid medium, replicate, and persist therein. With a similar approach, a recombinant TMV vector carrying a gene for the ectopic expression of the green fluorescent protein (GFP) induced the stable silencing of the GFP in the C. acutatum transformant line 10 expressing GFP derived from C71. The TMV-based vector also enabled C. acutatum to transiently express exogenous GFP up to six subcultures and for at least 2 mo after infection, without the need to develop transformation technology. With these characteristics, we anticipate this approach will find wider application as a tool in functional genomics of filamentous fungi. PMID:24594602

  18. Lipopeptides as main ingredients for inhibition of fungal phytopathogens by Bacillus subtilis/amyloliquefaciens

    PubMed Central

    Cawoy, Hélène; Debois, Delphine; Franzil, Laurent; De Pauw, Edwin; Thonart, Philippe; Ongena, Marc

    2015-01-01

    Some isolates of the Bacillus subtilis/amyloliquefaciens species are known for their plant protective activity against fungal phytopathogens. It is notably due to their genetic potential to form an impressive array of antibiotics including non-ribosomal lipopeptides (LPs). In the work presented here, we wanted to gain further insights into the relative role of these LPs in the global antifungal activity of B. subtilis/amyloliquefaciens. To that end, a comparative study was conducted involving multiple strains that were tested against four different phytopathogens. We combined various approaches to further exemplify that secretion of those LPs is a crucial trait in direct pathogen ward off and this can actually be generalized to all members of these species. Our data illustrate that for each LP family, the fungitoxic activity varies in function of the target species and that the production of iturins and fengycins is modulated by the presence of pathogens. Our data on the relative involvement of these LPs in the biocontrol activity and modulation of their production are discussed in the context of natural conditions in the rhizosphere. PMID:25529983

  19. The DNA damage response signaling cascade regulates proliferation of the phytopathogenic fungus Ustilago maydis in planta.

    PubMed

    de Sena-Tomás, Carmen; Fernández-Álvarez, Alfonso; Holloman, William K; Pérez-Martín, José

    2011-04-01

    In the phytopathogenic fungus Ustilago maydis, the dikaryotic state dominates the period of growth occurring during the infectious phase. Dikaryons are cells in which two nuclei, one from each parent cell, share a single cytoplasm for a period of time without undergoing nuclear fusion. In fungal cells, maintenance of the dikaryotic state requires an intricate cell division process that often involves the formation of a structure known as the clamp connection as well as the sorting of one of the nuclei to this structure to ensure that each daughter dikaryon inherits a balance of each parental genome. Here, we describe an atypical role of the DNA damage checkpoint kinases Chk1 and Atr1 during pathogenic growth of U. maydis. We found that Chk1 and Atr1 collaborate to control cell cycle arrest during the induction of the virulence program in U. maydis and that Chk1 and Atr1 work together to control the dikaryon formation. These findings uncover a link between a widely conserved signaling cascade and the virulence program in a phytopathogen. We propose a model in which adjustment of the cell cycle by the Atr1-Chk1 axis controls fidelity in dikaryon formation. Therefore, Chk1 and Atr1 emerge as critical cell type regulators in addition to their roles in the DNA damage response.

  20. Identification of Novel Virulence-Associated Proteins Secreted to Xylem by Verticillium nonalfalfae During Colonization of Hop Plants.

    PubMed

    Flajsman, Marko; Mandelc, Stanislav; Radisek, Sebastjan; Stajner, Natasa; Jakse, Jernej; Kosmelj, Katarina; Javornik, Branka

    2016-05-01

    Plant pathogens employ various secreted proteins to suppress host immunity for their successful host colonization. Identification and characterization of pathogen-secreted proteins can contribute to an understanding of the pathogenicity mechanism and help in disease control. We used proteomics to search for proteins secreted to xylem by the vascular pathogen Verticillium nonalfalfae during colonization of hop plants. Three highly abundant fungal proteins were identified: two enzymes, α-N-arabinofuranosidase (VnaAbf4.216) and peroxidase (VnaPRX1.1277), and one small secreted hypothetical protein (VnaSSP4.2). These are the first secreted proteins so far identified in xylem sap following infection with Verticillium spp. VnaPRX1.1277, classified as a heme-containing peroxidase from Class II, similar to other Verticillium spp. lignin-degrading peroxidases, and VnaSSP4.2, a 14-kDa cysteine-containing protein with unknown function and with a close homolog in related V. alfalfae strains, were further examined. The in planta expression of VnaPRX1.1277 and VnaSSP4.2 genes increased with the progression of colonization, implicating their role in fungal virulence. Indeed, V. nonalfalfae deletion mutants of both genes exhibited attenuated virulence on hop plants, which returned to the level of the wild-type pathogenicity in the knockout complementation lines, supporting VnaPRX1.1277 and VnaSSP4.2 as virulence factors required to promote V. nonalfalfae colonization of hop plants. PMID:26883488

  1. Identification of Novel Virulence-Associated Proteins Secreted to Xylem by Verticillium nonalfalfae During Colonization of Hop Plants.

    PubMed

    Flajsman, Marko; Mandelc, Stanislav; Radisek, Sebastjan; Stajner, Natasa; Jakse, Jernej; Kosmelj, Katarina; Javornik, Branka

    2016-05-01

    Plant pathogens employ various secreted proteins to suppress host immunity for their successful host colonization. Identification and characterization of pathogen-secreted proteins can contribute to an understanding of the pathogenicity mechanism and help in disease control. We used proteomics to search for proteins secreted to xylem by the vascular pathogen Verticillium nonalfalfae during colonization of hop plants. Three highly abundant fungal proteins were identified: two enzymes, α-N-arabinofuranosidase (VnaAbf4.216) and peroxidase (VnaPRX1.1277), and one small secreted hypothetical protein (VnaSSP4.2). These are the first secreted proteins so far identified in xylem sap following infection with Verticillium spp. VnaPRX1.1277, classified as a heme-containing peroxidase from Class II, similar to other Verticillium spp. lignin-degrading peroxidases, and VnaSSP4.2, a 14-kDa cysteine-containing protein with unknown function and with a close homolog in related V. alfalfae strains, were further examined. The in planta expression of VnaPRX1.1277 and VnaSSP4.2 genes increased with the progression of colonization, implicating their role in fungal virulence. Indeed, V. nonalfalfae deletion mutants of both genes exhibited attenuated virulence on hop plants, which returned to the level of the wild-type pathogenicity in the knockout complementation lines, supporting VnaPRX1.1277 and VnaSSP4.2 as virulence factors required to promote V. nonalfalfae colonization of hop plants.

  2. Killer yeasts inhibit the growth of the phytopathogen Moniliophthora perniciosa, the causal agent of Witches' Broom disease.

    PubMed

    de Souza Cabral, Anderson; de Carvalho, Patricia Maria Barroso; Pinotti, Tatiana; Hagler, Allen Norton; Mendonça-Hagler, Leda Cristina Santana; Macrae, Andrew

    2009-01-01

    Fruit and soil yeasts isolated from the Amazon, Atlantic Rainforests and an organic farm were screened for killer activity against yeasts. Killer yeasts were then tested against the phytopathogen Moniliophthora perniciosa (syn. Crinipellis perniciosa) and a Dipodascus capitatus strain and a Candida sp strain inhibited its growth.

  3. Antimicrobial activity of essential oils of Thymus vulgaris and Origanum vulgare on phytopathogenic strains isolated from soybean.

    PubMed

    Oliva, M de las M; Carezzano, M E; Giuliano, M; Daghero, J; Zygadlo, J; Bogino, P; Giordano, W; Demo, M

    2015-05-01

    The aim of this work was to study the antimicrobial activity of essential oils obtained from Thymus vulgaris (thyme) and Origanum vulgare (oregano) on phytopathogenic Pseudomonas species isolated from soybean. Strains with characteristics of P. syringae were isolated from leaves of soybean plants with blight symptoms. Ten of these could be identified in Group Ia of LOPAT as P. syringae. Six of these were confirmed as P. syringae using 16S rRNA, indicating the presence of these phytopathogenic bacteria in east and central Argentina. All the phytopathogenic bacteria were re-isolated and identified from the infected plants. MIC values for thyme were 11.5 and 5.7 mg·ml(-1) on P. syringae strains, while oregano showed variability in the inhibitory activity. Both essential oils inhibited all P. syringae strains, with better inhibitory activity than the antibiotic streptomycin. The oils were not bactericidal for all pseudomonads. Both oils contained high carvacrol (29.5% and 19.7%, respectively) and low thymol (1.5%). Natural products obtained from aromatic plants represent potential sources of molecules with biological activity that could be used as new alternatives for the treatment of phytopathogenic bacteria infections. PMID:25359697

  4. Effect of anaerobic soil disinfestation and vermicompost on soilborne phytopathogenic agents under tree-crop nursery conditions

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Anaerobic soil disinfestation (ASD) is a fumigation-independent management strategy for controlling soilborne pathogens. Walnut nurseries currently employ preplant fumigation to control soilborne phytopathogens and weeds, and may be amenable to use ASD instead. We investigated the potential of ASD a...

  5. Antimicrobial activity of essential oils of Thymus vulgaris and Origanum vulgare on phytopathogenic strains isolated from soybean.

    PubMed

    Oliva, M de las M; Carezzano, M E; Giuliano, M; Daghero, J; Zygadlo, J; Bogino, P; Giordano, W; Demo, M

    2015-05-01

    The aim of this work was to study the antimicrobial activity of essential oils obtained from Thymus vulgaris (thyme) and Origanum vulgare (oregano) on phytopathogenic Pseudomonas species isolated from soybean. Strains with characteristics of P. syringae were isolated from leaves of soybean plants with blight symptoms. Ten of these could be identified in Group Ia of LOPAT as P. syringae. Six of these were confirmed as P. syringae using 16S rRNA, indicating the presence of these phytopathogenic bacteria in east and central Argentina. All the phytopathogenic bacteria were re-isolated and identified from the infected plants. MIC values for thyme were 11.5 and 5.7 mg·ml(-1) on P. syringae strains, while oregano showed variability in the inhibitory activity. Both essential oils inhibited all P. syringae strains, with better inhibitory activity than the antibiotic streptomycin. The oils were not bactericidal for all pseudomonads. Both oils contained high carvacrol (29.5% and 19.7%, respectively) and low thymol (1.5%). Natural products obtained from aromatic plants represent potential sources of molecules with biological activity that could be used as new alternatives for the treatment of phytopathogenic bacteria infections.

  6. Characterization of a Novel Cotton Subtilase Gene GbSBT1 in Response to Extracellular Stimulations and Its Role in Verticillium Resistance.

    PubMed

    Duan, Xingpeng; Zhang, Zhidong; Wang, Jin; Zuo, Kaijing

    2016-01-01

    Verticillium wilt is a disastrous vascular disease in plants caused by Verticillium dahliae. Verticillium pathogens secrete various disease-causing effectors in cotton. This study identified a subtilase gene GbSBT1 from Gossypium babardense and investigated the roles against V. dahliae infection. GbSBT1 gene expression is responsive to V. dahliae defense signals, jasmonic acid, and ethylene treatments. Moreover, the GbSBT1 protein is mainly localized in the cell membrane and moves into the cytoplasm following jasmonic acid and ethylene treatments. Silencing GbSBT1 gene expression through virus-induced GbSBT1 gene silencing reduced the tolerance of Pima-90 (resistant genotype), but not facilitated the infection process of V. dahliae in Coker-312 (sensitive genotype). Moreover, the ectopically expressed GbSBT1 gene enhanced the resistance of Arabidopsis to Fusarium oxysporum and V. dahliae infection and activated the expression levels of defense-related genes. Furthermore, pull-down, yeast two-hybrid assay, and BiFC analysis revealed that GbSBT1 interacts with a prohibitin (PHB)-like protein expressed in V. dahliae pathogens during infection. In summary, GbSBT1 recognizes the effector PHB protein secreted from V. dahliae and is involved in Verticillium-induced resistance in cotton. PMID:27088499

  7. Deep mRNA sequencing reveals stage-specific transcriptome alterations during microsclerotia development in the smoke tree vascular wilt pathogen, Verticillium dahliae

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Verticillium dahliae is a soil-borne fungus that causes vascular wilt diseases in a wide range of plant hosts. V. dahliae produces multicelled, melanized resting bodies, also known as microsclerotia (MS) that can survive for years in the soil. Thus, MS formation marks an important event in the disea...

  8. Characterization of a Novel Cotton Subtilase Gene GbSBT1 in Response to Extracellular Stimulations and Its Role in Verticillium Resistance

    PubMed Central

    Duan, Xingpeng; Zhang, Zhidong; Wang, Jin; Zuo, Kaijing

    2016-01-01

    Verticillium wilt is a disastrous vascular disease in plants caused by Verticillium dahliae. Verticillium pathogens secrete various disease-causing effectors in cotton. This study identified a subtilase gene GbSBT1 from Gossypium babardense and investigated the roles against V. dahliae infection. GbSBT1 gene expression is responsive to V. dahliae defense signals, jasmonic acid, and ethylene treatments. Moreover, the GbSBT1 protein is mainly localized in the cell membrane and moves into the cytoplasm following jasmonic acid and ethylene treatments. Silencing GbSBT1 gene expression through virus-induced GbSBT1 gene silencing reduced the tolerance of Pima-90 (resistant genotype), but not facilitated the infection process of V. dahliae in Coker-312 (sensitive genotype). Moreover, the ectopically expressed GbSBT1 gene enhanced the resistance of Arabidopsis to Fusarium oxysporum and V. dahliae infection and activated the expression levels of defense-related genes. Furthermore, pull-down, yeast two-hybrid assay, and BiFC analysis revealed that GbSBT1 interacts with a prohibitin (PHB)-like protein expressed in V. dahliae pathogens during infection. In summary, GbSBT1 recognizes the effector PHB protein secreted from V. dahliae and is involved in Verticillium-induced resistance in cotton. PMID:27088499

  9. The mitogen-activated protein kinase gene, VdHog1, regulates osmotic stress response, microsclerotia formation and virulence in Verticillium dahliae

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The fungus Verticillium dahliae has gained worldwide notoriety as a destructive plant pathogen, causing vascular wilt diseases on diverse plant species. The fungus produces melanized resting bodies, known as microsclerotia that can survive for 15 years in the soil, and are thus critically important ...

  10. Spatial Pattern of Verticillium dahliae Microsclerotia and Cotton Plants with Wilt Symptoms in Commercial Plantations

    PubMed Central

    Yang, Jiarong; Hu, Xiaoping; Xu, Xiangming

    2015-01-01

    Spatial patterns of pathogen inoculum in field soils and the resulting patterns of disease may reflect the underlying mechanisms of pathogen dispersal. This knowledge can be used to design more efficient sampling schemes for assessing diseases. Spatial patterns of Verticillium dahliae microsclerotia were characterized in commercial cotton fields through quadrat and point sampling in 1994 and 2013, respectively. Furthermore, cotton plants with wilt symptoms, caused by V. dahliae, were assessed in six commercial cotton fields in 2013. Soil samples were assayed for the density of microsclerotia (expressed as CFU g-1 of soil) using a wet-sieving plating method and a real-time quantitative PCR method for the 1994 and 2013 study, respectively. The estimated inoculum threshold for causing wilt development on individual plants varied with the three fields: ca. 1.6 CFU g-1 of soil for one field, and 7.2 CFU g-1 of soil for the other two. Both quadrat and point sampling spatial analyses showed that aggregation of V. dahliae inoculum in soils was usually not detected beyond 1.0 m. Similarly, the spatial patterns of wilted cotton plants indicated that spatial aggregation of diseased plants were only observed below the scale of 1.0 m in six commercial cotton plantations. Therefore, spatial aggregation of both V. dahliae inoculum and cotton plants with wilt symptoms is not likely to be detected above the scale of 1.0 m for most commercial cotton plantations. When designing schemes for assessing wilt inoculum and wilt development, this scale needs to be taken into consideration. PMID:26167868

  11. Genome-wide identification, phylogeny and expression profile of vesicle fusion components in Verticillium dahliae.

    PubMed

    Yang, Xue; Ben, Siqi; Sun, Yingjiao; Fan, Xinlei; Tian, Chengming; Wang, Yonglin

    2013-01-01

    Vesicular trafficking plays a crucial role in protein localization and movement, signal transduction, and multiple developmental processes in eukaryotic cells. Vesicle fusion is the final and key step in vesicle-mediated trafficking and mainly relies on SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors), the regulators including SM (Sec1/Munc18) family proteins, Rab GTPases and exocyst subunits. Verticillium dahliae is a widespread soil fungus that causes disruptive vascular diseases on a wide range of plants. To date, no genes involved in vesicular fusion process have been identified and characterized in V. dahliae. The recent publication of the draft genome sequence of V. dahliae allowed us to conduct a genome-wide identification, phylogeny and expression profile of genes encoding vesicular fusion components. Using compared genomics and phylogenetic methods, we identified 44 genes encoding vesicle fusion components in the V. dahliae genome. According to the structural features of their encoded proteins, the 44 V. dahliae genes were classified into 22 SNAREs (6 Qa-, 4 Qb-, 6 Qc-, 1 Qbc- and 5 R-types), 4 SM family proteins, 10 Rab GTPases and 8 exocyst proteins. Based on phylogeny and motif constitution analysis, orthologs of vesicle fusion component in filamentous fungi were generally clustered together into the same subclasses with well-supported bootstrap values. Analysis of the expression profiles of these genes indicated that many of them are significantly differentially expressed during vegetative growth and microsclerotia formation in V. dahliae. The analysis show that many components of vesicle fusion are well conserved in filamentous fungi and indicate that vesicle fusion plays a critical role in microsclerotia formation of smoke tree wilt fungus V. dahliae. The genome-wide identification and expression analysis of components involved in vesicle fusion should facilitate research in this gene family and give new insights toward

  12. Recombination between clonal lineages of the asexual fungus Verticillium dahliae detected by genotyping by sequencing.

    PubMed

    Milgroom, Michael G; Jiménez-Gasco, María del Mar; Olivares García, Concepción; Drott, Milton T; Jiménez-Díaz, Rafael M

    2014-01-01

    Most asexual species of fungi have either lost sexuality recently, or they experience recombination by cryptic sexual reproduction. Verticillium dahliae is a plant-pathogenic, ascomycete fungus with no known sexual stage, even though related genera have well-described sexual reproduction. V. dahliae reproduces mitotically and its population structure is highly clonal. However, previously described discrepancies in phylogenetic relationships among clonal lineages may be explained more parsimoniously by recombination than mutation; therefore, we looked for evidence of recombination within and between clonal lineages. Genotyping by sequencing was performed on 141 V. dahliae isolates from diverse geographic and host origins, resulting in 26,748 single-nucleotide polymorphisms (SNPs). We found a strongly clonal population structure with the same lineages as described previously by vegetative compatibility groups (VCGs) and molecular markers. We detected 443 recombination events, evenly distributed throughout the genome. Most recombination events detected were between clonal lineages, with relatively few recombinant haplotypes detected within lineages. The only three isolates with mating type MAT1-1 had recombinant SNP haplotypes; all other isolates had mating type MAT1-2. We found homologs of eight meiosis-specific genes in the V. dahliae genome, all with conserved or partially conserved protein domains. The extent of recombination and molecular signs of sex in (mating-type and meiosis-specific genes) suggest that V. dahliae clonal lineages arose by recombination, even though the current population structure is markedly clonal. Moreover, the detection of new lineages may be evidence that sexual reproduction has occurred recently and may potentially occur under some circumstances. We speculate that the current clonal population structure, despite the sexual origin of lineages, has arisen, in part, as a consequence of agriculture and selection for adaptation to

  13. Spatial Pattern of Verticillium dahliae Microsclerotia and Cotton Plants with Wilt Symptoms in Commercial Plantations.

    PubMed

    Wei, Feng; Shang, Wenjing; Yang, Jiarong; Hu, Xiaoping; Xu, Xiangming

    2015-01-01

    Spatial patterns of pathogen inoculum in field soils and the resulting patterns of disease may reflect the underlying mechanisms of pathogen dispersal. This knowledge can be used to design more efficient sampling schemes for assessing diseases. Spatial patterns of Verticillium dahliae microsclerotia were characterized in commercial cotton fields through quadrat and point sampling in 1994 and 2013, respectively. Furthermore, cotton plants with wilt symptoms, caused by V. dahliae, were assessed in six commercial cotton fields in 2013. Soil samples were assayed for the density of microsclerotia (expressed as CFU g-1 of soil) using a wet-sieving plating method and a real-time quantitative PCR method for the 1994 and 2013 study, respectively. The estimated inoculum threshold for causing wilt development on individual plants varied with the three fields: ca. 1.6 CFU g-1 of soil for one field, and 7.2 CFU g-1 of soil for the other two. Both quadrat and point sampling spatial analyses showed that aggregation of V. dahliae inoculum in soils was usually not detected beyond 1.0 m. Similarly, the spatial patterns of wilted cotton plants indicated that spatial aggregation of diseased plants were only observed below the scale of 1.0 m in six commercial cotton plantations. Therefore, spatial aggregation of both V. dahliae inoculum and cotton plants with wilt symptoms is not likely to be detected above the scale of 1.0 m for most commercial cotton plantations. When designing schemes for assessing wilt inoculum and wilt development, this scale needs to be taken into consideration.

  14. Globally invading populations of the fungal plant pathogen Verticillium dahliae are dominated by multiple divergent lineages.

    PubMed

    Short, Dylan P G; Gurung, Suraj; Gladieux, Pierre; Inderbitzin, Patrik; Atallah, Zahi K; Nigro, Franco; Li, Guoqing; Benlioglu, Seher; Subbarao, Krishna V

    2015-08-01

    The spread of aggressive fungal pathogens into previously non-endemic regions is a major threat to plant health and food security. Analyses of the spatial and genetic structure of plant pathogens offer valuable insights into their origin, dispersal mechanisms and evolution, and have been useful to develop successful disease management strategies. Here, we elucidated the genetic diversity, population structure and demographic history of worldwide invasion of the ascomycete Verticillium dahliae, a soil-borne pathogen, using a global collection of 1100 isolates from multiple plant hosts and countries. Seven well-differentiated genetic clusters were revealed through discriminant analysis of principal components (DAPC), but no strong associations between these clusters and host/geographic origin of isolates were found. Analyses of clonal evolutionary relationships among multilocus genotypes with the eBURST algorithm and analyses of genetic distances revealed that genetic clusters represented several ancient evolutionary lineages with broad geographic distribution and wide host range. Comparison of different scenarios of demographic history using approximate Bayesian computations revealed the branching order among the different genetic clusters and lineages. The different lineages may represent incipient species, and this raises questions with respect to their evolutionary origin and the factors allowing their maintenance in the same areas and same hosts without evidence of admixture between them. Based on the above findings and the biology of V. dahliae, we conclude that anthropogenic movement has played an important role in spreading V. dahliae lineages. Our findings have implications for the development of management strategies such as quarantine measures and crop resistance breeding.

  15. Recombination between Clonal Lineages of the Asexual Fungus Verticillium dahliae Detected by Genotyping by Sequencing

    PubMed Central

    Milgroom, Michael G.; Jiménez-Gasco, María del Mar; Olivares García, Concepción; Drott, Milton T.; Jiménez-Díaz, Rafael M.

    2014-01-01

    Most asexual species of fungi have either lost sexuality recently, or they experience recombination by cryptic sexual reproduction. Verticillium dahliae is a plant-pathogenic, ascomycete fungus with no known sexual stage, even though related genera have well-described sexual reproduction. V. dahliae reproduces mitotically and its population structure is highly clonal. However, previously described discrepancies in phylogenetic relationships among clonal lineages may be explained more parsimoniously by recombination than mutation; therefore, we looked for evidence of recombination within and between clonal lineages. Genotyping by sequencing was performed on 141 V. dahliae isolates from diverse geographic and host origins, resulting in 26,748 single-nucleotide polymorphisms (SNPs). We found a strongly clonal population structure with the same lineages as described previously by vegetative compatibility groups (VCGs) and molecular markers. We detected 443 recombination events, evenly distributed throughout the genome. Most recombination events detected were between clonal lineages, with relatively few recombinant haplotypes detected within lineages. The only three isolates with mating type MAT1-1 had recombinant SNP haplotypes; all other isolates had mating type MAT1-2. We found homologs of eight meiosis-specific genes in the V. dahliae genome, all with conserved or partially conserved protein domains. The extent of recombination and molecular signs of sex in (mating-type and meiosis-specific genes) suggest that V. dahliae clonal lineages arose by recombination, even though the current population structure is markedly clonal. Moreover, the detection of new lineages may be evidence that sexual reproduction has occurred recently and may potentially occur under some circumstances. We speculate that the current clonal population structure, despite the sexual origin of lineages, has arisen, in part, as a consequence of agriculture and selection for adaptation to

  16. Field resistance to Fusarium oxysporum and Verticillium dahliae in transgenic cotton expressing the plant defensin NaD1

    PubMed Central

    Anderson, Marilyn A.

    2014-01-01

    The plant defensin NaD1, from Nicotiana alata, has potent antifungal activity against a range of filamentous fungi including the two important cotton pathogens, Fusarium oxysporum f. sp. vasinfectum (Fov) and Verticillium dahliae. Transgenic cotton plants expressing NaD1 were produced and plants from three events were selected for further characterization. Homozygous plants were assessed in greenhouse bioassays for resistance to Fov. One line (D1) was selected for field trial testing over three growing seasons in soils naturally infested with Fov and over two seasons in soils naturally infested with V. dahliae. In the field trials with Fov-infested soil, line D1 had 2–3-times the survival rate, a higher tolerance to Fov (higher disease rank), and a 2–4-fold increase in lint yield compared to the non-transgenic Coker control. When transgenic line D1 was planted in V. dahliae-infested soil, plants had a higher tolerance to Verticillium wilt and up to a 2-fold increase in lint yield compared to the non-transgenic Coker control. Line D1 did not exhibit any detrimental agronomic features compared to the parent Coker control when plants were grown in non-diseased soil. This study demonstrated that the expression of NaD1 in transgenic cotton plants can provide substantial resistance to two economically important fungal pathogens. PMID:24502957

  17. Systemic responses in a tolerant olive (Olea europaea L.) cultivar upon root colonization by the vascular pathogen Verticillium dahliae

    PubMed Central

    Gómez-Lama Cabanás, Carmen; Schilirò, Elisabetta; Valverde-Corredor, Antonio; Mercado-Blanco, Jesús

    2015-01-01

    Verticillium wilt of olive (VWO) is caused by the vascular pathogen Verticillium dahliae. One of the best VWO management measures is the use of tolerant cultivars; however, our knowledge on VWO tolerance/resistance genetics is very limited. A transcriptomic analysis was conducted to (i) identify systemic defense responses induced/repressed in aerial tissues of the tolerant cultivar Frantoio upon root colonization by V. dahliae, and (ii) determine the expression pattern of selected defense genes in olive cultivars showing differential susceptibility to VWO. Two suppression subtractive hybridization cDNA libraries, enriched in up-regulated (FU) and down-regulated (FD) genes respectively, were generated from “Frantoio” aerial tissues. Results showed that broad systemic transcriptomic changes are taking place during V. dahliae-“Frantoio” interaction. A total of 585 FU and 381 FD unigenes were identified, many of them involved in defense response to (a)biotic stresses. Selected genes were then used to validate libraries and evaluate their temporal expression pattern in “Frantoio.” Four defense genes were analyzed in cultivars Changlot Real (tolerant) and Picual (susceptible). An association between GRAS1 and DRR2 gene expression patterns and susceptibility to VWO was observed, suggesting that these transcripts could be further evaluated as markers of the tolerance level of olive cultivars to V. dahliae. PMID:26441865

  18. Field resistance to Fusarium oxysporum and Verticillium dahliae in transgenic cotton expressing the plant defensin NaD1.

    PubMed

    Gaspar, Yolanda M; McKenna, James A; McGinness, Bruce S; Hinch, Jillian; Poon, Simon; Connelly, Angela A; Anderson, Marilyn A; Heath, Robyn L

    2014-04-01

    The plant defensin NaD1, from Nicotiana alata, has potent antifungal activity against a range of filamentous fungi including the two important cotton pathogens, Fusarium oxysporum f. sp. vasinfectum (Fov) and Verticillium dahliae. Transgenic cotton plants expressing NaD1 were produced and plants from three events were selected for further characterization. Homozygous plants were assessed in greenhouse bioassays for resistance to Fov. One line (D1) was selected for field trial testing over three growing seasons in soils naturally infested with Fov and over two seasons in soils naturally infested with V. dahliae. In the field trials with Fov-infested soil, line D1 had 2-3-times the survival rate, a higher tolerance to Fov (higher disease rank), and a 2-4-fold increase in lint yield compared to the non-transgenic Coker control. When transgenic line D1 was planted in V. dahliae-infested soil, plants had a higher tolerance to Verticillium wilt and up to a 2-fold increase in lint yield compared to the non-transgenic Coker control. Line D1 did not exhibit any detrimental agronomic features compared to the parent Coker control when plants were grown in non-diseased soil. This study demonstrated that the expression of NaD1 in transgenic cotton plants can provide substantial resistance to two economically important fungal pathogens.

  19. Characterization and localization of prodiginines from Streptomyces lividans suppressing Verticillium dahliae in the absence or presence of Arabidopsis thaliana.

    PubMed

    Meschke, Holger; Walter, Stefan; Schrempf, Hildgund

    2012-04-01

    The ascomycete Verticillium dahliae causes worldwide vascular wilt of many field and horticultural plants. The melanized resting structures of this fungus, so-called microsclerotia, survive for many years in soils and continuously re-infect plants. Due to the absence of known fungicides, Verticillium wilt causes immense crop losses. We discovered that the Gram-positive, spore-forming soil bacterium Streptomyces lividans expresses members of the prodiginine family during co-cultivation with V. dahliae. Using HPLC and LC-MS analysis of cultures containing S. lividans alone or grown together with V. dahliae, we found that undecylprodigiosin [394.4 M+H](+) is highly abundant, and streptorubin B [392.4 M+H](+) is present in smaller amounts. Within co-cultures, the quantity of undecylprodigiosin increased considerably and pigment concentrated at and within fungal hyphae. The addition of purified undecylprodigiosin to growing V. dahliae hyphae strongly reduced microsclerotia formation. Undecylprodigiosin was also produced when S. lividans grew on the roots of developing Arabidopsis thaliana plants. Furthermore, the presence of the undecylprodigiosin producer led to an efficient reduction of V. dahliae hyphae and microsclerotia on plant-roots. Based on these novel findings and previous knowledge, we deduce that the prodiginine investigated leads to multiple cellular effects, which ultimately impair specific pathways for signal transduction and apoptosis of the fungal plant pathogen.

  20. Identification of quantitative trait loci for resistance to Verticillium wilt and yield parameters in hop (Humulus lupulus L.).

    PubMed

    Jakse, Jernej; Cerenak, Andreja; Radisek, Sebastjan; Satovic, Zlatko; Luthar, Zlata; Javornik, Branka

    2013-06-01

    Verticillium wilt (VW) can cause substantial yield loss in hop particularly with the outbreaks of the lethal strain of Verticillium albo-atrum. To elucidate genetic control of VW resistance in hop, an F1 mapping population derived from a cross of cultivar Wye Target, with the predicted genetic basis of resistance, and susceptible male breeding line BL2/1 was developed to assess wilting symptoms and to perform QTL mapping. The genetic linkage map, constructed with 203 markers of various types using a pseudo-testcross strategy, formed ten major linkage groups (LG) of the maternal and paternal maps, covering 552.98 and 441.1 cM, respectively. A significant QTL for VW resistance was detected at LOD 7 on a single chromosomal region on LG03 of both parental maps, accounting for 24.2-26.0 % of the phenotypic variance. QTL analysis for alpha-acid content and yield parameters was also performed on this map. QTLs for these traits were also detected and confirmed our previously detected QTLs in a different pedigree and environment. The work provides the basis for exploration of QTL flanking markers for possible use in marker-assisted selection. PMID:23423654

  1. Genome sequence of a novel endornavirus from the phytopathogenic fungus Alternaria brassicicola.

    PubMed

    Shang, Hong-Hong; Zhong, Jie; Zhang, Ru-Jia; Chen, Chuan-Yuan; Gao, Bi-Da; Zhu, Hong-Jian

    2015-07-01

    In an effort to discover new mycoviruses from phytopathogenic fungi, a dsRNA molecule of 10,290 nt, resembling those associated with the viruses belonging to the family Endornaviridae, was isolated from Alternaria brassicicola, one of the causal agents of rapeseed black spot disease. Genome analysis revealed the presence of a single open reading frame coding for a polyprotein of 3400 aa containing conserved viral methyltransferase (MTR), viral RNA helicase 1 (Hel-1), and RNA-dependent RNA polymerase (RdRp) domains. In addition, a cysteine-rich region (CRR) with conserved CXCC motifs, shared among several endornaviruses, was also identified between the MTR and Hel-1 domains. Phylogenetic analysis based on the RdRp sequence strongly suggested that the virus infecting A. brassicicola should be considered a representative of a novel endornavirus species, and this virus was designated as Alternaria brassicicola endornavirus 1 (AbEV1).

  2. Antifungal Activity of Narceine Methyl Ester and Narceine Isolated from Corydalis longipes Against Some Phytopathogenic Fungi

    PubMed Central

    Chowdhury, Dibyendu; Maurya, S.; Pandey, M. B.; Pandey, V. B.; Sarma, B. K.

    2005-01-01

    Narceine methyl ester and narceine are potent alkaloids which were isolated from Corydalis longipes were found effective in vitro at very low concentration, i.e., 100~500 ppm against spore germination of some test plant pathogenic fungi (Alternaria solani, A. tagetica, Cercospora abelmoschi, Curvularia maculans, Erysiphe cichoracearum, E. pisi, Fusarium udum, Helminthosporium oryzae, H. penniseti, Ustilago cynodontis). Among the test, phytopathogens the spores of F. udum, C. maculans and H. penniseti were highly sensitive at 200 ppm. However, spores of E. pisi, A. solani and A. tagetica were less sensitive at low concentration followed by other test fungi. Most of the fungi showed zero or nearly zero percent spore germination at 400 and 500 ppm. PMID:24049502

  3. Anthelmintic Efficacy of Gold Nanoparticles Derived from a Phytopathogenic Fungus, Nigrospora oryzae

    PubMed Central

    Kar, Pradip Kumar; Murmu, Sanatan; Saha, Saswati; Tandon, Veena; Acharya, Krishnendu

    2014-01-01

    Exploring a green chemistry approach, this study brings to the fore, the anthelmintic efficacy of gold nanoparticles, highlighting the plausible usage of myconanotechnology. Gold nanoparticles of ∼6 to ∼18 nm diameter were synthesized by treating the mycelia-free culture filtrate of the phytopathogenic fungus with gold chloride. Their size and morphology were confirmed by UV-Vis spectroscopy, DLS data, AFM and TEM images. The XRD studies reveal a crystalline nature of the nanoparticles, which are in cubic phase. The FTIR spectroscopic studies before and after the formation of nanoparticles show the presence of possible functional groups responsible for the bio-reduction and capping of the synthesized gold nanoparticles. The latter were tested as vermifugal agents against a model cestode Raillietina sp., an intestinal parasite of domestic fowl. Further, ultrastructural and biochemical parameters were used to corroborate the efficacy study. PMID:24465424

  4. Plant lectin-like antibacterial proteins from phytopathogens Pseudomonas syringae and Xanthomonas citri.

    PubMed

    Ghequire, Maarten G K; Li, Wen; Proost, Paul; Loris, Remy; De Mot, René

    2012-08-01

    The genomes of Pseudomonas syringae pv. syringae 642 and Xanthomonas citri pv. malvacearum LMG 761 each carry a putative homologue of the plant lectin-like bacteriocin (llpA) genes previously identified in the rhizosphere isolate Pseudomonas putida BW11M1 and the biocontrol strain Pseudomonas fluorescens Pf-5. The respective purified recombinant proteins, LlpAPss642 and LlpAXcm761 , display genus-specific antibacterial activity across species boundaries. The inhibitory spectrum of the P. syringae bacteriocin overlaps partially with those of the P. putida and P. fluorescens LlpAs. Notably, Xanthomonas axonopodis pv. citri str. 306 secretes a protein identical to LlpAXcm761 . The functional characterization of LlpA proteins from two different phytopathogenic γ-proteobacterial species expands the lectin-like bacteriocin family beyond the Pseudomonas genus and suggests its involvement in competition among closely related plant-associated bacteria with different lifestyles. PMID:23760822

  5. "Light-tagged" bacteriophage as a diagnostic tool for the detection of phytopathogens.

    PubMed

    Schofield, David; Bull, Carolee T; Rubio, Isael; Wechter, W Patrick; Westwater, Caroline; Molineux, Ian J

    2013-01-01

    Detection of the phytopathogen Pseudomonas cannabina pv alisalensis, the causal agent of bacterial blight of crucifers is essential for managing this disease. A phage-based diagnostic assay was developed that detects and identifies P. cannabina pv alisalensis from cultures and diseased plant specimens. A recombinant "light-tagged" reporter phage was generated by integrating the luxAB genes into the P. cannabina pv alisalensis phage PBSPCA1 genome. PBSPCA1::luxAB is viable, stable and detects P. cannabina pv alisalensis within minutes and with high sensitivity by conferring a bioluminescent signal. Detection is dependent on cell viability since cells treated with a bactericidal disinfectant are unable to elicit a signal. Importantly, the reporter phage detects P. cannabina pv alisalensis from diseased plant specimens indicating the potential of the diagnostic for disease identification. The reporter phage displays promise for the rapid and specific diagnostic detection of cultivated isolates, and infected plant specimens.

  6. In vitro inhibition activity of essential oils from some Lamiaceae species against phytopathogenic fungi.

    PubMed

    Kumar, Vinod; Mathela, C S; Tewari, A K; Bisht, K S

    2014-09-01

    Natural products have been in focus as alternative, effective and safe materials against the phytopathogens. Investigations show Nepeta oils as effective in controlling the food crops decay. The inhibitory effects of essential oils derived from Nepeta leucophylla, Nepeta ciliaris, Nepeta clarkei and Calamintha umbrosa against five phytopthogenic fungi have been determined. In vitro antifungal activity varied with their constituents and target species. More active being the oils containing oxygenated terpenoids. Helminthosporium maydis was sensitive to the all oils, IC50 values have 43.6-109.3 μg mL(-1). The N. leucophylla oil possessing oxygenated iridoids was more effective against H. maydis (IC50 value of 43.6 μg mL(-1)) while N. ciliaris was more active against Fusarium oxysporum (IC50 value of 219.2 μg mL(-1)). The oils were effective against the spore germination of all the tested plant pathogens. PMID:25175652

  7. "Light-tagged" bacteriophage as a diagnostic tool for the detection of phytopathogens.

    PubMed

    Schofield, David; Bull, Carolee T; Rubio, Isael; Wechter, W Patrick; Westwater, Caroline; Molineux, Ian J

    2013-01-01

    Detection of the phytopathogen Pseudomonas cannabina pv alisalensis, the causal agent of bacterial blight of crucifers is essential for managing this disease. A phage-based diagnostic assay was developed that detects and identifies P. cannabina pv alisalensis from cultures and diseased plant specimens. A recombinant "light-tagged" reporter phage was generated by integrating the luxAB genes into the P. cannabina pv alisalensis phage PBSPCA1 genome. PBSPCA1::luxAB is viable, stable and detects P. cannabina pv alisalensis within minutes and with high sensitivity by conferring a bioluminescent signal. Detection is dependent on cell viability since cells treated with a bactericidal disinfectant are unable to elicit a signal. Importantly, the reporter phage detects P. cannabina pv alisalensis from diseased plant specimens indicating the potential of the diagnostic for disease identification. The reporter phage displays promise for the rapid and specific diagnostic detection of cultivated isolates, and infected plant specimens. PMID:22990589

  8. Antifungal activity of essential oil from Asteriscus graveolens against postharvest phytopathogenic fungi in apples.

    PubMed

    Znini, Mohamed; Cristofari, Gregory; Majidi, Lhou; Mazouz, Hamid; Tomi, Pierre; Paolini, Julien; Costa, Jean

    2011-11-01

    The essential oils of the aerial parts of Asteriscus graveolens have been studied using GC and GC-MS. Twenty-eight compounds were identified in the essential oil amounting to 94.9% of the total oil. The aerial part oils showed similar chromatographic profiles and were characterized by having a high content of oxygenated sesquiterpenes with 6-oxocyclonerolidol (66.7% +/- 5.5) and 6-hydroxycyclonerolidol (8.8% +/-1.2) as the major components. The antifungal effect of the essential oil from A. graveolens leaves was evaluated in vitro against three phytopathogenic fungi of apples (Alternaria sp., Penicillium expansum, and Rhizopus stolonifer). The results suggest that this essential oil has fungicidal properties towards Alternaria sp. from direct contact assay at 0.2% (v/v) and to P. expansum from vapor assay tests at 80 microL.

  9. Inhibition of the phytopathogenic fungus Fusarium proliferatum by volatile compounds produced by Pseudomonas.

    PubMed

    Cordero, Paula; Príncipe, Analía; Jofré, Edgardo; Mori, Gladys; Fischer, Sonia

    2014-11-01

    The Fusarium head blight of grain cereals is a significant disease worldwide. In Argentina, high levels of contamination with Fusarium proliferatum have been found in crops. Many strains of the Pseudomonas genus antagonize the growth of fungi by different mechanisms, such as the production of antibiotics, siderophores, volatiles, and extracellular enzymes. In this work, we have designed a new system for studying the growth inhibition of F. proliferatum-namely by volatile compounds produced by Pseudomonas fluorescens MGR12. In both rich and minimal media, the bacterium released volatiles that negatively affected the mycelial growth of that phytopathogenic fungus. These bacterial compounds were analyzed by gas chromatography-mass spectrometry, but only a few could be identified by comparing their mass spectra with the libraries of the National Institutes of Standards and Technology MS search. PMID:25085617

  10. Purification and characterization of beta-N-acetylhexosaminidase from the phytopathogenic fungus Bipolaris sorokiniana.

    PubMed

    Geimba, M P; Riffel, A; Brandelli, A

    1998-10-01

    N-acetylhexosaminidase (HEX) from the phytopathogenic fungus Bipolaris sorokiniana was isolated and characterized. The production of HEX by B. sorokiniana was not altered by growing on different carbon sources. Enzyme purification was carried out by sequential liquid chromatography on Sephacryl S-200 HR, and p-aminobenzyl-2-acetamido-2-deoxy-beta-D-thioglucopyranoside agarose. The purification was about 70-fold, with a yield of 41%, determined with p-nitrophenyl-N-acetylglucosaminide as substrate. The enzyme had pH and temperature optima of 4.5 and 55 degrees C, respectively. The molecular weight of non-denatured enzyme was estimated as 120,000 Da by gel filtration chromatography, and about 55,000 Da by SDS-PAGE. The fungal HEX had glycosylated residues as evidenced by binding to Concanavalin-A. Bipolaris sorokiniana enzyme was also active with p-nitrophenyl-chitobioside and p-nitrophenyl-N-acetylgalactosaminide as substrates.

  11. Genome sequence of a novel endornavirus from the phytopathogenic fungus Alternaria brassicicola.

    PubMed

    Shang, Hong-Hong; Zhong, Jie; Zhang, Ru-Jia; Chen, Chuan-Yuan; Gao, Bi-Da; Zhu, Hong-Jian

    2015-07-01

    In an effort to discover new mycoviruses from phytopathogenic fungi, a dsRNA molecule of 10,290 nt, resembling those associated with the viruses belonging to the family Endornaviridae, was isolated from Alternaria brassicicola, one of the causal agents of rapeseed black spot disease. Genome analysis revealed the presence of a single open reading frame coding for a polyprotein of 3400 aa containing conserved viral methyltransferase (MTR), viral RNA helicase 1 (Hel-1), and RNA-dependent RNA polymerase (RdRp) domains. In addition, a cysteine-rich region (CRR) with conserved CXCC motifs, shared among several endornaviruses, was also identified between the MTR and Hel-1 domains. Phylogenetic analysis based on the RdRp sequence strongly suggested that the virus infecting A. brassicicola should be considered a representative of a novel endornavirus species, and this virus was designated as Alternaria brassicicola endornavirus 1 (AbEV1). PMID:25951967

  12. Thiol redox state and related enzymes in sclerotium-forming filamentous phytopathogenic fungi.

    PubMed

    Patsoukis, Nikolaos; Georgiou, D Christos

    2008-05-01

    Thiol redox state (TRS) reduced and oxidized components form profiles characteristic of each of the four main types of differentiation in the sclerotiogenic phytopathogenic fungi: loose, terminal, lateral-chained, and lateral-simple, represented by Rhizoctonia solani, Sclerotinia sclerotiorum, Sclerotium rolfsii, and Sclerotinia minor, respectively. A common feature of these fungi is that as their undifferentiated mycelium enters the differentiated state, it is accompanied by a decrease in the low oxidative stress-associated total reduced thiols and/or by an increase of the high oxidative stress-associated total oxidized thiols either in the sclerotial mycelial substrate or in its corresponding sclerotium, indicating a relationship between TRS-related oxidative stress and sclerotial differentiation. Moreover, the four studied sclerotium types exhibit high activities of TRS-related antioxidant enzymes, indicating the existence of antioxidant protection of the hyphae of the sclerotium medulla until conditions become appropriate for sclerotium germination. PMID:18400483

  13. Postharvest Survival of Porcine Sapovirus, a Human Norovirus Surrogate, on Phytopathogen-Infected Leafy Greens.

    PubMed

    Esseili, Malak A; Chin, Ashlina; Saif, Linda; Miller, Sally A; Qu, Feng; Lewis Ivey, Melanie L; Wang, Qiuhong

    2015-08-01

    Leafy greens are increasingly being recognized as an important vehicle for human noroviruses (HuNoV), which cause recurring gastroenteritis outbreaks. Leafy greens often become infected by phytopathogens in the field, which may cause symptoms on the edible parts. Whether plant pathogen infections enhance the survival of HuNoV on leafy greens is unknown. Lettuce and spinach plants were infected with a bacterium, Xanthomonas campestris pv. vitians strain 701a, and with Cucumber mosaic virus strain Fny, respectively. The survival rate of porcine sapovirus (SaV), a HuNoV surrogate, on infected and noninfected postharvest leaves was then assessed. In addition, acibenzolar-S-methyl, a commercial chemical elicitor of plant systemic defense, was used to assess whether stimulating the plant host defense affects the postharvest survival of SaV. Leaves harvested from control and treated plants were inoculated with SaV and incubated for 7 days at 4°C. The infectivity (tissue culture infectious dose affecting 50% of the culture [TCID50]/ml) and RNA (genomic equivalent/ml) titers of SaV were assayed using immunohistochemistry staining and SaV-specific TaqMan real-time reverse transcription PCR. Our results showed that cucumber mosaic virus Fny induced mild, nonnecrotic symptoms on spinach leaves and had no effect on SaV survival. In contrast, X. campestris pv. vitians 701a induced small localized necrotic lesions and significantly enhanced SaV survival on lettuce leaves. Treatment with acibenzolar-S-methyl was effective in reducing X. campestris pv. vitians 701a-induced lesions on infected lettuce plants but had no direct effect on SaV survival when used on healthy lettuce plants. These findings indicate that phytopathogen-induced necrotic lesions may enhance the postharvest survival of HuNoV on lettuce leaves. Therefore, preventive measures aiming to maintain healthy plants and minimize preharvest biological damage are expected to improve the safety of leafy greens.

  14. Diversity of Bacterial Endosymbionts Associated with Macrosteles Leafhoppers Vectoring Phytopathogenic Phytoplasmas

    PubMed Central

    Ishii, Yoshiko; Matsuura, Yu; Kakizawa, Shigeyuki; Nikoh, Naruo

    2013-01-01

    Here, we investigate the endosymbiotic microbiota of the Macrosteles leafhoppers M. striifrons and M. sexnotatus, known as vectors of phytopathogenic phytoplasmas. PCR, cloning, sequencing, and phylogenetic analyses of bacterial 16S rRNA genes identified two obligate endosymbionts, “Candidatus Sulcia muelleri” and “Candidatus Nasuia deltocephalinicola,” and five facultative endosymbionts, Wolbachia, Rickettsia, Burkholderia, Diplorickettsia, and a novel bacterium belonging to the Rickettsiaceae, from the leafhoppers. “Ca. Sulcia muelleri” and “Ca. Nasuia deltocephalinicola” exhibited 100% infection frequencies in the host species and populations and were separately harbored within different bacteriocytes that constituted a pair of coherent bacteriomes in the abdomen of the host insects, as in other deltocephaline leafhoppers. Wolbachia, Rickettsia, Burkholderia, Diplorickettsia, and the novel Rickettsiaceae bacterium exhibited infection frequencies at 7%, 31%, 12%, 0%, and 24% in M. striifrons and at 20%, 0%, 0%, 20%, and 0% in M. sexnotatus, respectively. Although undetected in the above analyses, phytoplasma infections were detected in 16% of M. striifrons and 60% of M. sexnotatus insects by nested PCR of 16S rRNA genes. Two genetically distinct phytoplasmas, namely, “Candidatus Phytoplasma asteris,” associated with aster yellows and related plant diseases, and “Candidatus Phytoplasma oryzae,” associated with rice yellow dwarf disease, were identified from the leafhoppers. These results highlight strikingly complex endosymbiotic microbiota of the Macrosteles leafhoppers and suggest ecological interactions between the obligate endosymbionts, the facultative endosymbionts, and the phytopathogenic phytoplasmas within the same host insects, which may affect vector competence of the leafhoppers. PMID:23770905

  15. The Phytopathogen Dickeya dadantii (Erwinia chrysanthemi 3937) Is a Pathogen of the Pea Aphid†

    PubMed Central

    Grenier, Anne-Marie; Duport, Gabrielle; Pagès, Sylvie; Condemine, Guy; Rahbé, Yvan

    2006-01-01

    Dickeya dadantii (Erwinia chrysanthemi) is a phytopathogenic bacterium causing soft rot diseases on many crops. The sequencing of its genome identified four genes encoding homologues of the Cyt family of insecticidal toxins from Bacillus thuringiensis, which are not present in the close relative Pectobacterium carotovorum subsp. atrosepticum. The pathogenicity of D. dadantii was tested on the pea aphid Acyrthosiphon pisum, and the bacterium was shown to be highly virulent for this insect, either by septic injury or by oral infection. The lethal inoculum dose was calculated to be as low as 10 ingested bacterial cells. A D. dadantii mutant with the four cytotoxin genes deleted showed a reduced per os virulence for A. pisum, highlighting the potential role of at least one of these genes in pathogenicity. Since only one bacterial pathogen of aphids has been previously described (Erwinia aphidicola), other species from the same bacterial group were tested. The pathogenic trait for aphids was shown to be widespread, albeit variable, within the phytopathogens, with no link to phylogenetic positioning in the Enterobacteriaceae. Previously characterized gut symbionts from thrips (Erwinia/Pantoea group) were also highly pathogenic to the aphid, whereas the potent entomopathogen Photorhabdus luminescens was not. D. dadantii is not a generalist insect pathogen, since it has low pathogenicity for three other insect species (Drosophila melanogaster, Sitophilus oryzae, and Spodoptera littoralis). D. dadantii was one of the most virulent aphid pathogens in our screening, and it was active on most aphid instars, except for the first one, probably due to anatomical filtering. The observed difference in virulence toward apterous and winged aphids may have an ecological impact, and this deserves specific attention in future research. PMID:16517643

  16. In vitro photodynamic inactivation of conidia of the phytopathogenic fungus Colletotrichum graminicola with cationic porphyrins.

    PubMed

    Vandresen, Camila Chevonica; Gonçalves, Alan Guilherme; Ducatti, Diogo Ricardo Bazan; Murakami, Fabio Seigi; Noseda, Miguel Daniel; Duarte, Maria Eugenia Rabello; Barreira, Sandra Mara Woranovicz

    2016-05-11

    Photodynamic inactivation (PDI) is an efficient approach for the elimination of a series of microorganisms; however, PDI involving phytopathogenic filamentous fungi is scarce in the literature. In the present study, we have demonstrated the photoinactivating properties of five cationic meso-(1-methyl-4-pyridinio)porphyrins on conidia of the phytopathogen Colletotrichum graminicola. For this purpose, photophysical properties (photostability and (1)O2 singlet production) of the porphyrins under study were first evaluated. PDI assays were then performed with a fluence of 30, 60, 90 and 120 J cm(-2) and varying the porphyrin concentration from 1 to 25 μmol L(-1). Considering the lowest concentration that enabled the best photoinactivation, with the respective lowest effective irradiation time, the meso-(1-methyl-4-pyridinio)porphyrins herein studied could be ranked as follows: triple-charged 4 (1 μmol L(-1) with a fluence of 30 J cm(-2)) > double-charged-trans2 (1 μmol L(-1) with 60 J cm(-2)) > tetra-charged 5 (15 μmol L(-1) with 90 J cm(-2)) > mono-charged 1 (25 μmol L(-1) with 120 J cm(-2)). Double-charged-cis-porphyrin 3 inactivated C. graminicola conidia in the absence of light. Evaluation of the porphyrin binding to the conidia and fluorescence microscopic analysis were also performed, which were in agreement with the PDI results. In conclusion, the cationic porphyrins herein studied were considered efficient photosensitizers to inactivate C. graminicola conidia. The amount and position of positive charges are related to the compounds' amphiphilicity and therefore to their photodynamic activity. PMID:27109559

  17. Structure Analysis Uncovers a Highly Diverse but Structurally Conserved Effector Family in Phytopathogenic Fungi

    PubMed Central

    Gracy, Jérome; Fournier, Elisabeth; Kroj, Thomas; Padilla, André

    2015-01-01

    Phytopathogenic ascomycete fungi possess huge effector repertoires that are dominated by hundreds of sequence-unrelated small secreted proteins. The molecular function of these effectors and the evolutionary mechanisms that generate this tremendous number of singleton genes are largely unknown. To get a deeper understanding of fungal effectors, we determined by NMR spectroscopy the 3-dimensional structures of the Magnaporthe oryzae effectors AVR1-CO39 and AVR-Pia. Despite a lack of sequence similarity, both proteins have very similar 6 β-sandwich structures that are stabilized in both cases by a disulfide bridge between 2 conserved cysteins located in similar positions of the proteins. Structural similarity searches revealed that AvrPiz-t, another effector from M. oryzae, and ToxB, an effector of the wheat tan spot pathogen Pyrenophora tritici-repentis have the same structures suggesting the existence of a family of sequence-unrelated but structurally conserved fungal effectors that we named MAX-effectors (Magnaporthe Avrs and ToxB like). Structure-informed pattern searches strengthened this hypothesis by identifying MAX-effector candidates in a broad range of ascomycete phytopathogens. Strong expansion of the MAX-effector family was detected in M. oryzae and M. grisea where they seem to be particularly important since they account for 5–10% of the effector repertoire and 50% of the cloned avirulence effectors. Expression analysis indicated that the majority of M. oryzae MAX-effectors are expressed specifically during early infection suggesting important functions during biotrophic host colonization. We hypothesize that the scenario observed for MAX-effectors can serve as a paradigm for ascomycete effector diversity and that the enormous number of sequence-unrelated ascomycete effectors may in fact belong to a restricted set of structurally conserved effector families. PMID:26506000

  18. [Effect of surface-active substances of Acinetobacter calcoaceticus IMV B-7241, Rhodococcus erythropolis IMV Ac-5017, and Nocardia vaccinii K-8 on phytopathogenic bacteria].

    PubMed

    Pirog, T P; Konon, A D; Sofilkanich, A P; Iutinskaia, G A

    2013-01-01

    The effect of surface-active substances (SAS's) of Acinetobacter calcoaceticus IMV B-7241, Rhodococcus erythropolis IMV Ac-5017, and Nocardia vaccinii K-8 on phytopathogenic bacteria has been studied. It was shown that the survival of cells (10(5)-10(7) in a milliliter) of the Pseudomonas and Xanthomonas phytopathogenic bacteria was found to be 0-33% after treatment with SAS preparations of the IMV Ac-5017 and IMV B-7241 strains for 2 h (0.15-0.4 mg/mL). In the presence of N. vaccinii K-8 SAS preparations (0.085-0.85 mg/mL), the number of cells of the majority of the studied phytopathogenic bacteria decreased by 95-100%. These data show prospects for using microbial SAS's for the construction of ecologically friendly drugs for regulating the number of phytopathogenic bacteria.

  19. Graphene oxide exhibits broad-spectrum antimicrobial activity against bacterial phytopathogens and fungal conidia by intertwining and membrane perturbation

    NASA Astrophysics Data System (ADS)

    Chen, Juanni; Peng, Hui; Wang, Xiuping; Shao, Feng; Yuan, Zhaodong; Han, Heyou

    2014-01-01

    To understand the interaction mechanism between graphene oxide (GO) and typical phytopathogens, a particular investigation was conducted about the antimicrobial activity of GO against two bacterial pathogens (P. syringae and X. campestris pv. undulosa) and two fungal pathogens (F. graminearum and F. oxysporum). The results showed that GO had a powerful effect on the reproduction of all four pathogens (killed nearly 90% of the bacteria and repressed 80% macroconidia germination along with partial cell swelling and lysis at 500 μg mL-1). A mutual mechanism is proposed in this work that GO intertwinds the bacteria and fungal spores with a wide range of aggregated graphene oxide sheets, resulting in the local perturbation of their cell membrane and inducing the decrease of the bacterial membrane potential and the leakage of electrolytes of fungal spores. It is likely that GO interacts with the pathogens by mechanically wrapping and locally damaging the cell membrane and finally causing cell lysis, which may be one of the major toxicity actions of GO against phytopathogens. The antibacterial mode proposed in this study suggests that the GO may possess antibacterial activity against more multi-resistant bacterial and fungal phytopathogens, and provides useful information about the application of GO in resisting crop diseases.To understand the interaction mechanism between graphene oxide (GO) and typical phytopathogens, a particular investigation was conducted about the antimicrobial activity of GO against two bacterial pathogens (P. syringae and X. campestris pv. undulosa) and two fungal pathogens (F. graminearum and F. oxysporum). The results showed that GO had a powerful effect on the reproduction of all four pathogens (killed nearly 90% of the bacteria and repressed 80% macroconidia germination along with partial cell swelling and lysis at 500 μg mL-1). A mutual mechanism is proposed in this work that GO intertwinds the bacteria and fungal spores with a wide range

  20. Phenological and phytochemical changes correlate with differential interactions of Verticillium dahliae with broccoli and cauliflower.

    PubMed

    Njoroge, S M C; Vallad, G E; Park, S-Y; Kang, S; Koike, S T; Bolda, M; Burman, P; Polonik, W; Subbarao, K V

    2011-05-01

    Cauliflower (Brassica oleracea var. botrytis subvar. cauliflora) is susceptible to wilt caused by Verticillium dahliae but broccoli (B. oleracea var. italica subvar. cyamosa) is not. Infection of broccoli and cauliflower by a green fluorescent protein-expressing isolate of V. dahliae was examined using epifluorescence and confocal laser-scanning microscopy to follow infection and colonization in relation to plant phenology. Plant glucosinolate, phenolic, and lignin contents were also assayed at 0, 4, 14, and 28 days postinoculation. V. dahliae consistently infected and colonized the vascular tissues of all cauliflower plants regardless of age at inoculation, with the pathogen ultimately appearing in the developing seed; however, colonization decreased with plant age. In broccoli, V. dahliae infected and colonized root and stem xylem tissues of plants inoculated at 1, 2, or 3 weeks postemergence. However, V. dahliae colonized only the root xylem and the epidermal and cortical tissues of broccoli plants inoculated at 4, 5, and 6 weeks postemergence. The frequency of reisolation of V. dahliae from the stems (4 to 22%) and roots (10 to 40%) of mature broccoli plants was lower than for cauliflower stems (25 to 64%) and roots (31 to 71%). The mean level of aliphatic glucosinolates in broccoli roots was 6.18 times higher than in the shoots and did not vary with age, whereas it was 3.65 times higher in cauliflower shoots than in the roots and there was a proportional increase with age. Indole glucosinolate content was identical in both cauliflower and broccoli, and both indole and aromatic glucosinolates did not vary with plant age in either crop. Qualitative differences in characterized glucosinolates were observed between broccoli and cauliflower but no differences were observed between inoculated and noninoculated plants for either broccoli or cauliflower. However, the phenolic and lignin contents were significantly higher in broccoli following inoculation than in

  1. Clonal Expansion and Migration of a Highly Virulent, Defoliating Lineage of Verticillium dahliae.

    PubMed

    Milgroom, Michael G; Del Mar Jiménez-Gasco, María; Olivares-García, Concepción; Jiménez-Díaz, Rafael M

    2016-09-01

    We used a population genomics approach to test the hypothesis of clonal expansion of a highly fit genotype in populations of Verticillium dahliae. This fungal pathogen has a broad host range and can be dispersed in contaminated seed or other plant material. It has a highly clonal population structure, with several lineages having nearly worldwide distributions in agricultural crops. Isolates in lineage 1A are highly virulent and cause defoliation in cotton, okra, and olive (denoted 1A/D), whereas those in other lineages cause wilting but not defoliation (ND). We tested whether the highly virulent lineage 1A/D could have spread from the southwestern United States to the Mediterranean basin, as predicted from historical records. We found 187 single-nucleotide polymorphisms (SNPs), determined by genotyping by sequencing, among 91 isolates of lineage 1A/D and 5 isolates in the closely related lineage 1B/ND. Neighbor-joining and maximum-likelihood analyses on the 187 SNPs showed a clear divergence between 1A/D and 1B/ND haplotypes. Data for only 77 SNPs were obtained for all 96 isolates (no missing data); lineages 1A/D and 1B/ND differed by 27 of these 77 SNPs, confirming a clear divergence between the two lineages. No evidence of recombination was detected within or between these two lineages. Phylogenetic and genealogical analyses resulted in five distinct subclades of 1A/D isolates that correlated closely with geographic origins in the Mediterranean basin, consistent with the hypothesis that the D pathotype was introduced at least five times in independent founder events into this region from a relatively diverse source population. The inferred ancestral haplotype was found in two isolates sampled before 1983 from the southwestern United States, which is consistent with historical records that 1A/D originated in North America. The five subclades coalesce with the ancestral haplotype at the same time, consistent with a hypothesis of rapid population expansion in the

  2. A comparison of real-time PCR protocols for the quantitative monitoring of asymptomatic olive infections by Verticillium dahliae pathotypes.

    PubMed

    Gramaje, D; Pérez-Serrano, V; Montes-Borrego, M; Navas-Cortés, J A; Jiménez-Díaz, R M; Landa, B B

    2013-10-01

    Early, specific, and accurate in planta detection and quantification of Verticillium dahliae are essential to prevent the spread of Verticillium wilt in olive using certified pathogen-free planting material and development of resistance. We comparatively assessed the accuracy, specificity, and efficiency of eight real-time quantitative polymerase chain reaction protocols published since 2002 for the specific detection and quantification of V. dahliae in various host plant species and in soil, using a background of DNAs extracted from olive roots, stems, and leaves. Results showed that some of those protocols were not specific for V. dahliae or were inhibited when using backgrounds other than water. Ranking of protocols according to a weighted score system placed protocols TAQ (based on intergenic spacer ribosomal DNA target gene) and SYBR-4 (based on the β-tubulin 2 target gene) first in sensitivity and efficiency for the quantification of V. dahliae DNA in small amounts and different types of olive tissues (root and stem) tested. Use of TAQ and SYBR-4 protocols allowed accurate quantification of V. dahliae DNA regardless of the background DNA, with a detection limit being fixed at a cycle threshold of 36 (≈18 fg for SYBR-4 and 15 fg for TAQ) of V. dahliae. The amount of DNA from defoliating (D) and nondefoliating (ND) V. dahliae pathotypes was monitored in Verticillium wilt-resistant 'Frantoio' olive using the TAQ and SYBR-4 protocols. In the infection bioassay, higher amounts of D V. dahliae DNA were measured in olive stems, whereas the average amount of fungal DNA in roots was higher for ND-infected plants than D-infected ones. Overall, V. dahliae DNA amounts in all olive tissues tested tended to slightly decrease or remain stable by the end of the experiment (35 days after inoculation). The SYBR-4 and TAQ protocols further enabled detection of V. dahliae in tissues of symptomless plants, suggesting that both techniques can be useful for implementing

  3. The type III secretion system of biocontrol Pseudomonas fluorescens KD targets the phytopathogenic Chromista Pythium ultimum and promotes cucumber protection.

    PubMed

    Rezzonico, Fabio; Binder, Christian; Défago, Geneviève; Moënne-Loccoz, Yvan

    2005-09-01

    The type III secretion system (TTSS) is used by Proteobacteria for pathogenic or symbiotic interaction with plant and animal hosts. Recently, TTSS genes thought to originate from the phytopathogen Pseudomonas syringae were evidenced in Pseudomonas fluorescens KD, which protects cucumber from the oomycete Pythium ultimum (kingdom Chromista/Stramenopila). However, it is not known whether the TTSS contributes to plant protection by the bacterium and, if so, whether it targets the plant or the phytopathogen. Inactivation of TTSS gene hrcV following the insertion of an omega cassette strongly reduced the biocontrol activity of the pseudomonad against P. ultimum on cucumber when compared with the wild type, but had no effect on its root-colonization ability. Analysis of a plasmid-based transcriptional hrpJ'-inaZ reporter fusion revealed that expression in strain KD of the operon containing hrcV was strongly stimulated in vitro and in situ by the oomycete and not by the plant. In vitro, both strain KD and its hrcV mutant reduced the activity level of the pectinase polygalacturonase (a key pathogenicity factor) from P. ultimum, but the reduction was much stronger with the wild type. Together, these results show that the target range of bacterial TTSS is not restricted to plants and animals but also can include members of Chromista/Stramenopila, and suggest that virulence genes acquired horizontally from phytopathogenic bacteria were functionally recycled in biocontrol saprophytic Pseudomonas spp., resulting in enhanced plant protection by the latter.

  4. Effect of foliar application of antibiotics and gibberellic acid on the rhizosphre microflora of pea, infected with Verticillium dahliae.

    PubMed

    Ramarao, P; Isaac, I

    1980-01-01

    A study was made of the effects of foliar spray of bacitracin, chloramphenicol and gibberellic acid on the rhizosphere microflora of pea seedlings (Pisum sativum L.) infected with Verticillium dahliae. The antibiotics increased fungus and actinomycete counts and reduced the bacterial populations in the rhizosphere. Gibberellic acid at 10 ppm concentration reduced all three groups of microorganisms while at 100 ppm fungi and actinomycetes increased slightly. Invariably the rhizosphere effect was as follows: bacteria leads to fungi leads to actinomycetes. Foliar sprays also affected percentage occurrence of particular genera of fungi in the rhizosphere; for example, Trichoderma spp. were stimulated by all the treatments, the maximum being with 10 ppm gibberellic acid, even though the total fungus count was reduced. The disease severity was markedly reduced by foliar sprays. PMID:7191387

  5. [Relationship of Sa, No and H2O2 signals in the reponses of Arabidopsis to toxin of Verticillium dahliae].

    PubMed

    Li, Shuang Shi; Li, Ying Zhang

    2004-08-01

    The accumulation of hydrogen peroxide (H2O2) in Arabidopsis was induced by toxin of Verticillium dahliae (VD-toxin), exogenous salicylic acid (SA) and nitric oxide donor (SNP). The effect of SNP was the most intense. H2O2 level was not increased in Arabidopsis treated with nitric oxide synthase inhibitor (NNA). It was identified with the results of DAB staining of H2O2 accumulates in the leaves of Arabidopsis. H2O2 was cytochemically detected in the cells of epidermal hair and the cell wall of vascular parenchyma cell. The results suggest that H2O2 acts as a second messenger to involve the defenses in Arabidopsis induced by SA and NO signal transduction. The relationship of NO and H2O2 signals was likely closer.

  6. Cytoplasmic- and extracellular-proteome analysis of Diplodia seriata: a phytopathogenic fungus involved in grapevine decline

    PubMed Central

    2010-01-01

    Background The phytopathogenic fungus Diplodia seriata, whose genome remains unsequenced, produces severe infections in fruit trees (fruit blight) and grapevines. In this crop is recognized as one of the most prominent pathogens involved in grapevine trunk disease (or grapevine decline). This pathology can result in the death of adult plants and therefore it produces severe economical losses all around the world. To date no genes or proteins have been characterized in D. seriata that are involved in the pathogenicity process. In an effort to help identify potential gene products associated with pathogenicity and to gain a better understanding of the biology of D. seriata, we initiated a proteome-level study of the fungal mycelia and secretome. Results Intracellular and secreted proteins from D. seriata collected from liquid cultures were separated using two-dimensional gel electrophoresis. About 550 cytoplasmic proteins were reproducibly present in 3 independent extractions, being 53 identified by peptide mass fingerprinting and tandem mass spectrometry. The secretome analysis showed 75 secreted proteins reproducibly present in 3 biological replicates, being 16 identified. Several of the proteins had been previously identified as virulence factors in other fungal strains, although their contribution to pathogenicity in D. seriata remained to be analyzed. When D. seriata was grown in a medium supplemented with carboxymethylcellulose, 3 proteins were up-regulated and 30 down-regulated. Within the up-regulated proteins, two were identified as alcohol dehydrogenase and mitochondrial peroxyrredoxin-1, suggesting that they could play a significant role in the pathogenicity process. As for the 30 down-regulated proteins, 9 were identified being several of them involved in carbohydrate metabolism. Conclusions This study is the first report on proteomics on D. seriata. The proteomic data obtained will be important to understand the pathogenicity process. In fact, several of

  7. Complex molecular relationship between vegetative compatibility groups (VCGs) in Verticillium dahliae: VCGs do not always align with clonal lineages.

    PubMed

    Jiménez-Gasco, María Del Mar; Malcolm, Glenna M; Berbegal, Mónica; Armengol, Josep; Jiménez-Díaz, Rafael M

    2014-06-01

    Verticillium wilts caused by the soilborne fungus Verticillium dahliae are among the most challenging diseases to control. Populations of this pathogen have been traditionally studied by means of vegetative compatibility groups (VCGs) under the assumption that VCGs comprise genetically related isolates that correlate with clonal lineages. We aimed to resolve the phylogenetic relationships among VCGs and their subgroups based on sequences of the intergenic spacer region (IGS) of the ribosomal DNA and six anonymous polymorphic sequences containing single-nucleotide polymorphisms (VdSNPs). A collection of 68 V. dahliae isolates representing the main VCGs and subgroups (VCGs 1A, 1B, 2A, 2B, 3, 4A, 4B, and 6) from different geographic origins and hosts was analyzed using the seven DNA regions. Maximum parsimony (MP) phylogenies inferred from IGS and VdSNP sequences showed five and six distinct clades, respectively. Phylogenetic analyses of individual and combined data sets indicated that certain VCG subgroups (e.g., VCGs 1A and 1B) are closely related and share a common ancestor; however, other subgroups (e.g., VCG 4B) are more closely related to members of a different VCG (e.g., VCG 2A) than to subgroups of the same VCG (VCG 4B). Furthermore, MP analyses indicated that VCG 2B is polyphyletic, with isolates placed in at least three distinct phylogenetic lineages based on IGS sequences and two lineages based on VdSNP sequences. Results from our study suggest the existence of main VCG lineages that contain VCGs 1A and 1B; VCGs 2A and 4B; and VCG 4A, for which both phylogenies agree; and the existence of other VCGs or VCG subgroups that seem to be genetically heterogeneous or show discrepancies in their phylogenetic placement: VCG 2B, VCG 3, and VCG 6. These results raise important caveats regarding the interpretation of VCG analyses: genetic homogeneity and close evolutionary relationship between members of a VCG should not be assumed.

  8. Cotton polyamine oxidase is required for spermine and camalexin signalling in the defence response to Verticillium dahliae.

    PubMed

    Mo, Huijuan; Wang, Xingfen; Zhang, Yan; Zhang, Guiyin; Zhang, Jinfa; Ma, Zhiying

    2015-09-01

    Verticillium dahliae is a destructive, soil-borne fungal pathogen that causes vascular wilt disease in many economically important crops worldwide. A polyamine oxidase (PAO) gene was identified and cloned by screening suppression subtractive hybridisation and cDNA libraries of cotton genotypes tolerant to Verticillium wilt and was induced early and strongly by inoculation with V. dahliae and application of plant hormone. Recombinant cotton polyamine oxidase (GhPAO) was found to catalyse the conversion of spermine (Spm) to spermidine (Spd) in vitro. Constitutive expression of GhPAO in Arabidopsis thaliana produced improved resistance to V. dahliae and maintained putrescine, Spd and Spm at high levels. Hydrogen peroxide (H2 O2 ), salicylic acid and camalexin (a phytoalexin) levels were distinctly increased in GhPAO-overexpressing Arabidopsis plants during V. dahliae infection when compared with wild-type plants, and Spm and camalexin efficiently inhibited growth of V. dahliae in vitro. Spermine promoted the accumulation of camalexin by inducing the expression of mitogen-activated protein kinases and cytochrome P450 proteins in Arabidopsis and cotton plants. The three polyamines all showed higher accumulation in tolerant cotton cultivars than in susceptible cotton cultivars after inoculation with V. dahliae. GhPAO silencing in cotton significantly reduced the Spd level and increased the Spm level, leading to enhanced susceptibility to infection by V. dahliae, and the levels of H2 O2 and camalexin were distinctly lower in GhPAO-silenced cotton plants after V. dahliae infection. Together, these results suggest that GhPAO contributes to resistance of the plant against V. dahliae through the mediation of Spm and camalexin signalling.

  9. Colonization of spinach by Verticillium dahliae and effects of pathogen localization on the efficacy of seed treatments.

    PubMed

    Maruthachalam, Karunakaran; Klosterman, Steven J; Anchieta, Amy; Mou, Beiquan; Subbarao, Krishna V

    2013-03-01

    Verticillium wilt on spinach (Spinacia oleracea) is caused by the soilborne fungus Verticillium dahliae. The pathogen is seedborne and transmission through seed is a major concern because of the dispersal of the pathogen to areas where fresh and processing spinach crops are grown in rotation with susceptible crops. Reduction in seedborne inoculum minimizes pathogen spread; therefore, knowledge of pathogen localization in seed is critical to develop methods to reduce seedborne inoculum. Spinach seedlings were inoculated with conidial suspensions of a green fluorescent protein-tagged strain of V. dahliae and colonization events were followed through seed production by confocal laser-scanning microscopy. Between 24 to 96 h postinoculation (PI), conidia germinated and formed hyphal colonies on root tips and in root elongation zones. Hyphae colonized root cortical tissues both intra and intercellularly by 2 weeks, and colonized the taproot xylem with abundant mycelia and conidia that led to vascular discoloration coincident with foliar symptom expression by 8 weeks PI. At 10 weeks PI, the xylem of the upper stem, inflorescence, and spinach seed parts, including the pericarp, seed coat, cotyledons, and radicle, had been colonized by the pathogen but not the perisperm (the diploid maternal tissue). Maximum concentration of the fungus was in the seed coat, the outermost layer of the vasculature. Infection of V. dahliae in spinach seed was systemic and transmissible to developing seedlings. Additional analyses indicated that fungicide and steam seed treatments reduced detectable levels of the pathogen but did not eliminate the pathogen from the seed. This information will assist in the development of seed treatments that will reduce the seedborne inoculum transmission to crop production fields.

  10. Comparative pathogenicity, biocontrol efficacy, and multilocus sequence typing of Verticillium nonalfalfae from the invasive Ailanthus altissima and other hosts.

    PubMed

    Kasson, M T; Short, D P G; O'Neal, E S; Subbarao, K V; Davis, D D

    2014-03-01

    Verticillium wilt, caused by Verticillium nonalfalfae, is currently killing tens of thousands of highly invasive Ailanthus altissima trees within the forests in Pennsylvania, Ohio, and Virginia and is being considered as a biological control agent of Ailanthus. However, little is known about the pathogenicity and virulence of V. nonalfalfae isolates from other hosts on Ailanthus, or the genetic diversity among V. nonalfalfae from confirmed Ailanthus wilt epicenters and from locations and hosts not associated with Ailanthus wilt. Here, we compared the pathogenicity and virulence of several V. nonalfalfae and V. alfalfae isolates, evaluated the efficacy of the virulent V. nonalfalfae isolate VnAa140 as a biocontrol agent of Ailanthus in Pennsylvania, and performed multilocus sequence typing of V. nonalfalfae and V. alfalfae. Inoculations of seven V. nonalfalfae and V. alfalfae isolates from six plant hosts on healthy Ailanthus seedlings revealed that V. nonalfalfae isolates from hosts other than Ailanthus were not pathogenic on Ailanthus. In the field, 100 canopy Ailanthus trees were inoculated across 12 stands with VnAa140 from 2006 to 2009. By 2011, natural spread of the fungus had resulted in the mortality of >14,000 additional canopy Ailanthus trees, 10,000 to 15,000 Ailanthus sprouts, and nearly complete eradication of Ailanthus from several smaller inoculated stands, with the exception of a few scattered vegetative sprouts that persisted in the understory for several years before succumbing. All V. nonalfalfae isolates associated with the lethal wilt of Ailanthus, along with 18 additional isolates from 10 hosts, shared the same multilocus sequence type (MLST), MLST 1, whereas three V. nonalfalfae isolates from kiwifruit shared a second sequence type, MLST 2. All V. alfalfae isolates included in the study shared the same MLST and included the first example of V. alfalfae infecting a non-lucerne host. Our results indicate that V. nonalfalfae is host adapted and

  11. Use of Arabidopsis thaliana to study mechanisms of control of Verticillium wilt by Pseudomonas fluorescens PICF7.

    PubMed

    Maldonado-González, M M; Bakker, P A H M; Mercado-Blanco, J

    2012-01-01

    Verticillium wilt (VW), caused by Verticillium dahliae Kleb., is an important disease in many crops and its effective management has proven difficult. Among the various disease control measures to be implemented, the use of microbial antagonists (biological control agents, BCAs) constitutes an environmentally-friendly approach fitting criteria of modern sustainable agriculture. Pseudomonas fluorescens PICF7 was isolated from root tissues of nursery--propagated olive plants. Selection of this strain was based on in vitro growth inhibition of V. dahliae, colonizing ability of olive roots, endophytic lifestyle, and control of the highly-virulent defoliating (D) pathotype of V. dahliae in olive planting stocks. The mode of action by which PICF7 controls VW in olive is as yet unknown; moreover, to uncover potential biocontrol mechanisms poses additional difficulties in this pathosystem because the target is a tree. Therefore we used the model plant Arabidopsis thaliana to study: i) if PICF7 colonizes the rhizosphere of A. thaliana; ii) disease symptoms caused by V. dahliae in A. thaliana; iii) control of VW by PICF7 in different accessions and mutants of A. thaliana; and iv) if motility, antibiosis and/or siderophores are involved in control of V. dahliae by PICF7. Diverse bioassays were conducted and in all of them both the BCA and the pathogen were introduced in the rhizosphere of A. thaliana. Both D and non-defoliating isolates of V. dahliae caused disease symptoms in A. thaliana. PICF7 colonized and persisted in the rhizosphere of different Arabidopsis accessions and could control the D pathotype in some of them. PICF7 mutants affected in antibiosis significantly lost their ability to control VW in A. thaliana. We conclude that the model plant A. thaliana is useful to unravel interactions between this BCA and V. dahliae.

  12. Streptomyces lividans inhibits the proliferation of the fungus Verticillium dahliae on seeds and roots of Arabidopsis thaliana.

    PubMed

    Meschke, Holger; Schrempf, Hildgund

    2010-07-01

    Verticillium wilt, a vascular disease in more than 200 dicotyledonous plants, is due to the ascomycete fungus Verticillium dahliae. As documented by video-microscopy, the soil bacterium Streptomyces lividans strongly reduces the germination of V. dahliae conidia, and the subsequent growth of hyphae. Quantification by the use of DNA-intercalating dyes and Calcofluor-staining revealed that during prolonged co-cultivation, bacterial hyphae proliferate to a dense network, provoke a poor development of V. dahliae vegetative hyphae and lead to an enormous reduction of conidia and microsclerotia. Upon individual application to seeds of the model plant Arabidopsis thaliana, either the bacterial spores or the fungal conidia germinate at or within the mucilage, including its volcano-shaped structures. The extension of hyphae from each individual strain correlates with the reduction of the pectin-containing mucilage-layer. Proliferating hyphae then spread to roots of the emerging seedlings. Plants, which arise in the presence of V. dahliae within agar or soil, have damaged root cells, an atrophied stem and root, as well as poorly developed leaves with chlorosis symptoms. In contrast, S. lividans hyphae settle in bunches preferentially at the outer layer near tips and alongside roots. Resulting plants have a healthy appearance including an intact root system. Arabidopsis thaliana seeds, which are co-inoculated with V. dahliae and S. lividans, have preferentially proliferating bacterial hyphae within the mucilage, and at roots of the outgrowing seedlings. As a result, plants have considerably reduced disease symptoms. As spores of the beneficial S. lividans strain are obtainable in large quantity, its application is highly attractive.

  13. Use of Arabidopsis thaliana to study mechanisms of control of Verticillium wilt by Pseudomonas fluorescens PICF7.

    PubMed

    Maldonado-González, M M; Bakker, P A H M; Mercado-Blanco, J

    2012-01-01

    Verticillium wilt (VW), caused by Verticillium dahliae Kleb., is an important disease in many crops and its effective management has proven difficult. Among the various disease control measures to be implemented, the use of microbial antagonists (biological control agents, BCAs) constitutes an environmentally-friendly approach fitting criteria of modern sustainable agriculture. Pseudomonas fluorescens PICF7 was isolated from root tissues of nursery--propagated olive plants. Selection of this strain was based on in vitro growth inhibition of V. dahliae, colonizing ability of olive roots, endophytic lifestyle, and control of the highly-virulent defoliating (D) pathotype of V. dahliae in olive planting stocks. The mode of action by which PICF7 controls VW in olive is as yet unknown; moreover, to uncover potential biocontrol mechanisms poses additional difficulties in this pathosystem because the target is a tree. Therefore we used the model plant Arabidopsis thaliana to study: i) if PICF7 colonizes the rhizosphere of A. thaliana; ii) disease symptoms caused by V. dahliae in A. thaliana; iii) control of VW by PICF7 in different accessions and mutants of A. thaliana; and iv) if motility, antibiosis and/or siderophores are involved in control of V. dahliae by PICF7. Diverse bioassays were conducted and in all of them both the BCA and the pathogen were introduced in the rhizosphere of A. thaliana. Both D and non-defoliating isolates of V. dahliae caused disease symptoms in A. thaliana. PICF7 colonized and persisted in the rhizosphere of different Arabidopsis accessions and could control the D pathotype in some of them. PICF7 mutants affected in antibiosis significantly lost their ability to control VW in A. thaliana. We conclude that the model plant A. thaliana is useful to unravel interactions between this BCA and V. dahliae. PMID:23878957

  14. Potential of Lecanicillium spp. for management of insects, nematodes and plant diseases.

    PubMed

    Goettel, Mark S; Koike, Masanori; Kim, Jeong Jun; Aiuchi, Daigo; Shinya, Ryoji; Brodeur, Jacques

    2008-07-01

    Fungi in the genus Lecanicillium (formerly classified as the single species Verticillium lecanii) are important pathogens of insects and some have been developed as commercial biopesticides. Some isolates are also active against phytoparasitic nematodes or fungi. Lecanicillium spp. use both mechanical forces and hydrolytic enzymes to directly penetrate the insect integument and the cell wall of the fungal plant pathogen. In addition to mycoparasitism of the plant pathogen, the mode of action is linked to colonization of host plant tissues, triggering an induced systemic resistance. Recently it was demonstrated that development of Lecanicillium hybrids through protoplast fusion may result in strains that inherit parental attributes, thereby allowing development of hybrid strains with broader host range and other increased benefits, such as increased viability. Such hybrids have demonstrated increased virulence against aphids, whiteflies and the soybean cyst nematode. Three naturally occurring species of Lecanicillium, L. attenuatum, L. longisporum, and an isolate that could not be linked to any presently described species based on rDNA sequences have been shown to have potential to control aphids as well as suppress the growth and spore production of Sphaerotheca fuliginea, the causal agent of cucumber powdery mildew. These results suggest that strains of Lecanicillium spp. may have potential for development as a single microbial control agent effective against several plant diseases, pest insects and plant parasitic nematodes due to its antagonistic, parasitic and disease resistance inducing characteristics. However, to our knowledge, no Lecanicillium spp. have been developed for control of phytopathogens or phytoparasitic nematodes.

  15. Dysfunctionality of the xylem in Olea europaea L. Plants associated with the infection process by Verticillium dahliae Kleb. Role of phenolic compounds in plant defense mechanism.

    PubMed

    Báidez, Ana G; Gómez, Pedro; Del Río, José A; Ortuño, Ana

    2007-05-01

    Xylem ultrastructural modification and the possible participation of phenolic compounds in the natural defense or resistance mechanisms of olive plants infected with Verticillium dahliae Kleb. were studied. Microscopic study showed that the mycelium propagated and passed from one element to another through the pit. The formation of tyloses and aggregates contributed to obstruction of the xylem lumen. In vivo changes in the levels of these phenolic compounds in infected olive plants and their antifungal activity against Verticillium dahliae Kleb., as revealed by in vitro study, strongly suggest that they are involved in natural defense or resistance mechanisms in this plant material, the most active being quercetin and luteolin aglycons, followed by rutin, oleuropein, luteolin-7-glucoside, tyrosol, p-coumaric acid, and catechin. . PMID:17394331

  16. The effects of some polyamine biosynthetic inhibitors on growth and morphology of phytopathogenic fungi

    NASA Technical Reports Server (NTRS)

    Rajam, M. V.; Galston, A. W.

    1985-01-01

    We have studied the effects of two polyamine biosynthetic inhibitors, alpha-difluoromethylornithine (DFMO) and alpha-difluoromethylarginine (DFMA), and of polyamines (PAs), alone and in combination, on mycelial growth and morphology of four phytopathogenic fungi: Botrytis sp, B. cinerea, Rhizoctonia solani and Monilinia fructicola. The inhibitors were added to a Czapek agar medium to get final concentrations of 0.1, 0.5 and 1.0 mM. DFMO and DFMA, suicide inhibitors of ornithine decarboxylase (ODC) and arginine decarboxylase (ADC) respectively, inhibited mycelial growth strongly; the effect was generally more pronounced with DFMA than with DFMO, but each fungus had its own response pattern. The addition of the PAs putrescine (Put) and spermidine (Spd) to the culture medium resulted in a promotion of growth. In Botrytis sp and Monilinia fructicola exposed to inhibitors plus PAs, mycelial growth was actually increased above control values. Mycelial morphology was altered and cell size dramatically reduced in plates containing inhibitors alone, whereas with PAs alone, or in combination with inhibitors, morphology was normal, but cell length and diameters increased considerably. These results suggest that PAs are essential for growth in fungal mycelia. The inhibition caused by DFMA may be due to its arginase-mediated conversion to DFMO.

  17. Differences in stability of seed-associated microbial assemblages in response to invasion by phytopathogenic microorganisms.

    PubMed

    Rezki, Samir; Campion, Claire; Iacomi-Vasilescu, Beatrice; Preveaux, Anne; Toualbia, Youness; Bonneau, Sophie; Briand, Martial; Laurent, Emmanuelle; Hunault, Gilles; Simoneau, Philippe; Jacques, Marie-Agnès; Barret, Matthieu

    2016-01-01

    Seeds are involved in the vertical transmission of microorganisms from one plant generation to another and consequently act as reservoirs for the plant microbiota. However, little is known about the structure of seed-associated microbial assemblages and the regulators of assemblage structure. In this work, we have assessed the response of seed-associated microbial assemblages of Raphanus sativus to invading phytopathogenic agents, the bacterial strain Xanthomonas campestris pv. campestris (Xcc) 8004 and the fungal strain Alternaria brassicicola Abra43. According to the indicators of bacterial (16S rRNA gene and gyrB sequences) and fungal (ITS1) diversity employed in this study, seed transmission of the bacterial strain Xcc 8004 did not change the overall composition of resident microbial assemblages. In contrast seed transmission of Abra43 strongly modified the richness and structure of fungal assemblages without affecting bacterial assemblages. The sensitivity of seed-associated fungal assemblage to Abra43 is mostly related to changes in relative abundance of closely related fungal species that belong to the Alternaria genus. Variation in stability of the seed microbiota in response to Xcc and Abra43 invasions could be explained by differences in seed transmission pathways employed by these micro-organisms, which ultimately results in divergence in spatio-temporal colonization of the seed habitat. PMID:27077013

  18. Screening for Antimicrobial Activity of Wood Rotting Higher Basidiomycetes Mushrooms from Uruguay against Phytopathogens.

    PubMed

    Barneche, Stephanie; Jorcin, Gabriela; Cecchetto, Gianna; Cerdeiras, María Pía; Vázquez, Alvaro; Alborés, Silvana

    2016-01-01

    In this work, the antimicrobial activity of extracts of wood rotting higher Basidiomycetes mushrooms isolated from Eucalyptus plantations in Uruguay was studied using bacterial and fungal phytopathogens as targets. Fifty-one extracts from mycelia and growth broth were prepared from higher Basidiomycetes mushrooms, from which eight extracts (from Ganoderma resinaceum, Laetiporus sulphureus, Dictyopanus pusillus, and Bjerkandera adusta) showed antimicrobial activity against Xanthomonas vesicatoria, Aspergillus oryzae, Penicillium expansum, Botrytis cinerea, and Rhizopus stolonifer as assayed in the qualitative test. The minimum inhibitory concentration (MIC) for those fungal extracts was determined and the results showed that L. sulphureus deserved further study, with low MIC values against X. vesicatoria. The antimicrobial activity of L. sulphureus culture broth extracts grown under different culture conditions was evaluated against X. vesicatoria. From the results of these assays, larger-scale cultures for the production of the compound(s) with antimicrobial activity should be performed using malt extract broth, at pH 5, at 20°C and static culture conditions. PMID:27481160

  19. Identification of novel bioactive hexapeptides against phytopathogenic bacteria through rapid screening of a synthetic combinatorial library.

    PubMed

    Choi, Jaehyuk; Moon, Eunpyo

    2009-08-01

    Antimicrobial peptides (AMPs) are considered to be a promising alternative to conventional antibiotics for future generations. We identified four novel hexapeptides with antimicrobial activity: KCM11 (TWWRWW-NH(2)), KCM12 (KWRWIW-NH(2)), KCM21 (KWWWRW-NH(2)), and KRS22 (WRWFIH-NH(2)), through positional scanning of a synthetic peptide combinatorial library (PS-SCL). The ability of these peptides to inhibit the growth of a variety of bacteria and unicellular fungi was evaluated. KCM11 and KRS22 preferentially inhibited the normal growth of fungal strains, whereas KCM12 and KCM21 were more active against bacterial strains. Bactericidal activity was addressed in a clear zone assay against phytopathogenic bacteria, including Pectobacterium spp., Xanthomonas spp., Pseudomonas spp., etc. KCM21 showed the highest activity and was effective against a wide range of target organisms. Application of KCM21 with inoculation of Pectobacterium carotovorum subsp. carotovorum on detached cabbage leaves resulted in an immune phenotype or a significant reduction in symptom development, depending on the peptide concentration. Cytotoxicity of the four hexapeptides was evaluated in mouse and human epithelial cell lines using an MTT test. The results revealed a lack of cytotoxic effects.

  20. Possibilities of the use of vinasses in the control of fungi phytopathogens.

    PubMed

    Santos, M; Diánez, F; de Cara, M; Tello, J C

    2008-12-01

    The purpose of this research was to study the biocide effect of three agroindustrial subproducts, concretely sugar beet, sugar cane and wine vinasse. Results from in vitro testing determined that wine vinasse is what shows a 100% capacity to suppress fungal growth with concentrations between 5% and 7% for Fusarium oxysporum f.sp. melonis race 0 and 1, Sclerotinia sclerotiorum, Pythium aphanidermatum and Phytophthora parasitica and 10-15% for F. oxysporum f.sp. radicis-cucumerinum. On the other hand, sugar cane vinasse produced an increase at high concentrations and sugar beet vinasse showed an approximate 100% suppressor effect on fungal growth for only some of the phytopathogens tested: S. sclerotiorum (15%), P. aphanidermatum (7%), P. parasitica (15%) and F. oxysporum f.sp. radicis-cucumerinum (15%). In the soil samples analyzed none of the three vinasse extracts decreased fusaric microbiota, producing an increase in the three samples tested. This would implicitly convey an improvement in soil quality by producing a potential increase in bacterial and fungal microbiota. PMID:18519163

  1. Synthesis of chitosan based nanoparticles and their in vitro evaluation against phytopathogenic fungi.

    PubMed

    Saharan, Vinod; Mehrotra, Akanksha; Khatik, Rajesh; Rawal, Pokhar; Sharma, S S; Pal, Ajay

    2013-11-01

    The main aim of present study was to prepare chitosan, chitosan-saponin and Cu-chitosan nanoparticles to evaluate their in vitro antifungal activities. Various nanoparticles were prepared using ionic gelation method by interaction of chitosan, sodium tripolyphosphate, saponin and Cu ions. Their particle size, polydispersity index, zeta potential and structures were confirmed by DLS, FTIR, TEM and SEM. The antifungal properties of nanoparticles against phytopathogenic fungi namely Alternaria alternata, Macrophomina phaseolina and Rhizoctonia solani were investigated at various concentrations ranging from 0.001 to 0.1%. Among the various formulations of nanoparticles, Cu-chitosan nanoparticles were found most effective at 0.1% concentration and showed 89.5, 63.0 and 60.1% growth inhibition of A. alternata, M. phaseolina and R. solani, respectively in in vitro model. At the same concentration, Cu-chitosan nanoparticles also showed maximum of 87.4% inhibition rate of spore germination of A. alternata. Chitosan nanoparticles showed the maximum growth inhibitory effects (87.6%) on in vitro mycelial growth of M. phaseolina at 0.1% concentration. From our study it is evident that chitosan based nanoparticles particularly chitosan and Cu-chitosan nanoparticles have tremendous potential for further field screening towards crop protection. PMID:24141067

  2. Isolation of bacteria with antifungal activity against the phytopathogenic fungi Stenocarpella maydis and Stenocarpella macrospora.

    PubMed

    Petatán-Sagahón, Iván; Anducho-Reyes, Miguel Angel; Silva-Rojas, Hilda Victoria; Arana-Cuenca, Ainhoa; Tellez-Jurado, Alejandro; Cárdenas-Álvarez, Isabel Oyuki; Mercado-Flores, Yuridia

    2011-01-01

    Stenocarpella maydis and Stenocarpella macrospora are the causal agents of ear rot in corn, which is one of the most destructive diseases in this crop worldwide. These fungi are important mycotoxin producers that cause different pathologies in farmed animals and represent an important risk for humans. In this work, 160 strains were isolated from soil of corn crops of which 10 showed antifungal activity against these phytopathogens, which, were identified as: Bacillus subtilis, Pseudomonas spp., Pseudomonas fluorescens, and Pantoea agglomerans by sequencing of 16S rRNA gene and the phylogenetic analysis. From cultures of each strain, extracellular filtrates were obtained and assayed to determine antifungal activity. The best filtrates were obtained in the stationary phase of B. subtilis cultures that were stable to the temperature and extreme pH values; in addition they did not show a cytotoxicity effect against brine shrimp and inhibited germination of conidia. The bacteria described in this work have the potential to be used in the control of white ear rot disease. PMID:22016606

  3. Lactic acid bacteria from fresh fruit and vegetables as biocontrol agents of phytopathogenic bacteria and fungi.

    PubMed

    Trias, Rosalia; Bañeras, Lluís; Montesinos, Emilio; Badosa, Esther

    2008-12-01

    This study evaluated the efficacy of lactic acid bacteria (LAB) isolated from fresh fruits and vegetables as biocontrol agents against the phytopathogenic and spoilage bacteria and fungi, Xanthomonas campestris, Erwinia carotovora, Penicillium expansum, Monilinia laxa, and Botrytis cinerea. The antagonistic activity of 496 LAB strains was tested in vitro and all tested microorganisms except P. expansum were inhibited by at least one isolate. The 496 isolates were also analyzed for the inhibition of P. expansum infection in wounds of Golden Delicious apples. Four strains (TC97, AC318, TM319, and FF441) reduced the fungal rot diameter of the apples by 20%; only Weissella cibaria strain TM128 decreased infection levels by 50%. Cell-free supernatants of selected antagonistic bacteria were studied to determine the nature of the antimicrobial compounds produced. Organic acids were the preferred mediators of inhibition but hydrogen peroxide was also detected when strains BC48, TM128, PM141 and FF441 were tested against E. carotovora. While previous reports of antifungal activity by LAB are scarce, our results support the potential of LAB as biocontrol agents against postharvest rot.

  4. Growth of phytopathogenic fungi in the presence of partially acetylated chitooligosaccharides.

    PubMed

    Oliveira, E N; El Gueddari, Nour E; Moerschbacher, Bruno M; Peter, Martin G; Franco, Telma T

    2008-09-01

    Four phytopathogenic fungi were cultivated up to six days in media containing chitooligosaccharide mixtures differing in average DP and FA. The three different mixtures were named Q3 (which contained oligosaccharides of DP2-DP10, with DP2-DP7 as main components), Q2 (which contained oligosaccharides of DP2-DP12, with DP2-DP10 as main components) and Q1 (which derived from Q2 and contained oligomers of DP5-DP8 with hexamer and a heptamer as the main components). The novel aspect of this work is the description of the effect of mixtures of oligosaccharides with different and known composition on fungal growth rates. The growth rate of Alternaria alternata and Rhizopus stolonifer was initially inhibited by Q3 and Q2 at higher concentrations. Q1 had a growth stimulating effect on these two fungi. Growth of Botrytis cinerea was inhibited by Q3 and Q2, while Q1 had no effect on the growth of this fungus. Growth of Penicillium expansum was only slightly inhibited by higher concentrations of sample Q3, while Q2 and Q1 had no effect. The inhibition of growth rates or their resistance toward chitooligosaccharides correlated with the absence or presence of chitinolytic enzymes in the culture media, respectively.

  5. Genome sequence of a novel mitovirus identified in the phytopathogenic fungus Alternaria arborescens.

    PubMed

    Komatsu, Ken; Katayama, Yukie; Omatsu, Tsutomu; Mizutani, Tetsuya; Fukuhara, Toshiyuki; Kodama, Motoichiro; Arie, Tsutomu; Teraoka, Tohru; Moriyama, Hiromitsu

    2016-09-01

    The phytopathogenic fungus Alternaria spp. contains a variety of double-stranded RNA (dsRNA) elements of different sizes. Detailed analysis of next-generation sequencing data obtained using dsRNA purified from Alternaria arborescens, from which we had previously found Alternaria arborescens victorivirus 1, revealed the presence of another mycoviral-like dsRNA of approximately 2.5 kbp in length. When using the fungal mitochondrial genetic code, this dsRNA has a single open reading frame that potentially encodes an RNA-dependent RNA polymerase (RdRp) with significant to sequence similarity to those of viruses of the genus Mitovirus. Moreover, both the 5'- and 3'-untranslated regions have the potential to fold into stable stem-loop structures, which is characteristic of mitoviruses. Pairwise comparisons and phylogenetic analysis of the deduced amino acid sequences of RdRp indicated that the virus we identified in A. arborescens is a distinct member of the genus Mitovirus in the family Narnaviridae, designated as "Alternaria arborescens mitovirus 1" (AaMV1). PMID:27368994

  6. Genome sequence of the enterobacterial phytopathogen Erwinia carotovora subsp. atroseptica and characterization of virulence factors

    PubMed Central

    Bell, K. S.; Sebaihia, M.; Pritchard, L.; Holden, M. T. G.; Hyman, L. J.; Holeva, M. C.; Thomson, N. R.; Bentley, S. D.; Churcher, L. J. C.; Mungall, K.; Atkin, R.; Bason, N.; Brooks, K.; Chillingworth, T.; Clark, K.; Doggett, J.; Fraser, A.; Hance, Z.; Hauser, H.; Jagels, K.; Moule, S.; Norbertczak, H.; Ormond, D.; Price, C.; Quail, M. A.; Sanders, M.; Walker, D.; Whitehead, S.; Salmond, G. P. C.; Birch, P. R. J.; Parkhill, J.; Toth, I. K.

    2004-01-01

    The bacterial family Enterobacteriaceae is notable for its well studied human pathogens, including Salmonella, Yersinia, Shigella, and Escherichia spp. However, it also contains several plant pathogens. We report the genome sequence of a plant pathogenic enterobacterium, Erwinia carotovora subsp. atroseptica (Eca) strain SCRI1043, the causative agent of soft rot and blackleg potato diseases. Approximately 33% of Eca genes are not shared with sequenced enterobacterial human pathogens, including some predicted to facilitate unexpected metabolic traits, such as nitrogen fixation and opine catabolism. This proportion of genes also contains an overrepresentation of pathogenicity determinants, including possible horizontally acquired gene clusters for putative type IV secretion and polyketide phytotoxin synthesis. To investigate whether these gene clusters play a role in the disease process, an arrayed set of insertional mutants was generated, and mutations were identified. Plant bioassays showed that these mutants were significantly reduced in virulence, demonstrating both the presence of novel pathogenicity determinants in Eca, and the impact of functional genomics in expanding our understanding of phytopathogenicity in the Enterobacteriaceae. PMID:15263089

  7. Lactic acid bacteria from fresh fruit and vegetables as biocontrol agents of phytopathogenic bacteria and fungi.

    PubMed

    Trias, Rosalia; Bañeras, Lluís; Montesinos, Emilio; Badosa, Esther

    2008-12-01

    This study evaluated the efficacy of lactic acid bacteria (LAB) isolated from fresh fruits and vegetables as biocontrol agents against the phytopathogenic and spoilage bacteria and fungi, Xanthomonas campestris, Erwinia carotovora, Penicillium expansum, Monilinia laxa, and Botrytis cinerea. The antagonistic activity of 496 LAB strains was tested in vitro and all tested microorganisms except P. expansum were inhibited by at least one isolate. The 496 isolates were also analyzed for the inhibition of P. expansum infection in wounds of Golden Delicious apples. Four strains (TC97, AC318, TM319, and FF441) reduced the fungal rot diameter of the apples by 20%; only Weissella cibaria strain TM128 decreased infection levels by 50%. Cell-free supernatants of selected antagonistic bacteria were studied to determine the nature of the antimicrobial compounds produced. Organic acids were the preferred mediators of inhibition but hydrogen peroxide was also detected when strains BC48, TM128, PM141 and FF441 were tested against E. carotovora. While previous reports of antifungal activity by LAB are scarce, our results support the potential of LAB as biocontrol agents against postharvest rot. PMID:19204894

  8. Potential application of Northern Argentine propolis to control some phytopathogenic bacteria.

    PubMed

    Ordóñez, R M; Zampini, I C; Moreno, M I Nieva; Isla, M I

    2011-10-20

    The antimicrobial activity of samples of Northern Argentine propolis (Tucumán, Santiago del Estero and Chaco) against phytopathogenic bacteria was assessed and the most active samples were identified. Minimal inhibitory concentration (MIC) values were determined by agar macrodilution and broth microdilution assays. Strong antibacterial activity was detected against Erwinia carotovora spp carotovora CECT 225, Pseudomonas syringae pvar tomato CECT 126, Pseudomonas corrugata CECT 124 and Xanthomonas campestris pvar vesicatoria CECT 792. The most active propolis extract (Tucumán, T1) was selected to bioguide isolation and identified for antimicrobial compound (2',4'-dihydroxychalcone). The antibacterial chalcone was more active than the propolis ethanolic extract (MIC values of 0.5-1 μg ml(-1) and 9.5-15 μg ml(-1), respectively). Phytotoxicity assays were realized and the propolis extracts did not retard germination of lettuce seeds or the growth of onion roots. Propolis solutions applied as sprays on tomato fruits infected with P. syringae reduced the severity of disease. Application of the Argentine propolis extracts diluted with water may be promising for the management of post harvest diseases of fruits.

  9. Trichoderma species mediated differential tolerance against biotic stress of phytopathogens in Cicer arietinum L.

    PubMed

    Saxena, Amrita; Raghuwanshi, Richa; Singh, Harikesh Bahadur

    2015-02-01

    Trichoderma spp. have been reported to aid in imparting biotic as well as abiotic tolerance to plants. However, there are only few reports unfolding the differential ability of separate species of Trichoderma genera generally exploited for their biocontrol potential in this framework. A study was undertaken to evaluate the biocontrol potential of different Trichoderma species namely T. harzianum, T. asperellum, T. koningiopsis, T. longibrachiatum, and T. aureoviride as identified in the group of indigenous isolates from the agricultural soils of Eastern Uttar Pradesh, India. Their biocontrol potential against three major soilborne phytopathogens, i.e., Sclerotium rolfsii, Sclerotinia sclerotiorum, and Colletotrichum capsici was confirmed by dual culture plate technique. Efficient mycoparasitic ability was further assessed in all the isolates in relation to chitinase, β-1,3 glucanase, pectinase, lipase, amylase, and cellulase production while equally consistent results were obtained for their probable phosphate solubilization and indole acetic acid (IAA) production abilities. The selected isolates were further subjected to test their ability to promote plant growth, to reduce disease incidence and to tolerate biotic stress in terms of lignification pattern against S. rolfsii in chickpea plants. Among the identified Trichoderma species, excellent results were observed for T. harzianum and T. koningiopsis indicating better biocontrol potential of these species in the group and thus exhibiting perspective for their commercial exploitation.

  10. Structure determination of a sugar-binding protein from the phytopathogenic bacterium Xanthomonas citri

    PubMed Central

    Medrano, Francisco Javier; de Souza, Cristiane Santos; Romero, Antonio; Balan, Andrea

    2014-01-01

    The uptake of maltose and related sugars in Gram-negative bacteria is mediated by an ABC transporter encompassing a periplasmic component (the maltose-binding protein or MalE), a pore-forming membrane protein (MalF and MalG) and a membrane-associated ATPase (MalK). In the present study, the structure determination of the apo form of the putative maltose/trehalose-binding protein (Xac-MalE) from the citrus pathogen Xanthomonas citri in space group P6522 is described. The crystals contained two protein molecules in the asymmetric unit and diffracted to 2.8 Å resolution. Xac-MalE conserves the structural and functional features of sugar-binding proteins and a ligand-binding pocket with similar characteristics to eight different orthologues, including the residues for maltose and trehalose interaction. This is the first structure of a sugar-binding protein from a phytopathogenic bacterium, which is highly conserved in all species from the Xanthomonas genus. PMID:24817711

  11. Characters of compost teas from different sources and their suppressive effect on fungal phytopathogens.

    PubMed

    Marín, Francisco; Santos, Mila; Diánez, Fernando; Carretero, Francisco; Gea, Francisco J; Yau, José A; Navarro, María J

    2013-08-01

    Compost teas (CT) are fermented watery extracts of composted materials that are used to control plant diseases and on crop fertilization. In this work, aerated (ACT) and non-aerated compost teas (NCT) were obtained from four different composts: spent mushroom substrate compost, grape marc compost, greenhouse horticultural crop residues compost, and vermicompost. Physico-chemical and microbiological analysis were carried out to determine their properties. In vitro assays were performed to assess their suppressive effect on the mycelial growth of eight fungal phytopathogens. In vivo trials aimed to assess their effect on gummy stem blight (Didymella bryonae) and powdery mildew (Podosphaera fusca) in melon plants. Results showed that ACT and NCT filtrates inhibited the in vitro growth of all tested pathogens while autoclaved CT did not completely lose their inhibitory effect, and CT sterilized by microfiltration had no effect on the pathogen growth. The severity of powdery mildew was highly reduced by ACT and NCT from all sources, though in gummy stem blight assay only a delay in disease development was observed. In general, all compost teas showed a high level of microbial populations and nutrients. Results suggest that the efficacy of ACT and NCT firstly depend on the microbiota present in them. We consider compost teas from the four tested sources as a viable way to manage plant diseases and crop fertilization, throughout its integration in pest management programs and fertirrigation systems under different dilution rates.

  12. Differences in stability of seed-associated microbial assemblages in response to invasion by phytopathogenic microorganisms

    PubMed Central

    Rezki, Samir; Campion, Claire; Iacomi-Vasilescu, Beatrice; Preveaux, Anne; Toualbia, Youness; Bonneau, Sophie; Briand, Martial; Laurent, Emmanuelle; Hunault, Gilles; Simoneau, Philippe; Jacques, Marie-Agnès

    2016-01-01

    Seeds are involved in the vertical transmission of microorganisms from one plant generation to another and consequently act as reservoirs for the plant microbiota. However, little is known about the structure of seed-associated microbial assemblages and the regulators of assemblage structure. In this work, we have assessed the response of seed-associated microbial assemblages of Raphanus sativus to invading phytopathogenic agents, the bacterial strain Xanthomonas campestris pv. campestris (Xcc) 8004 and the fungal strain Alternaria brassicicola Abra43. According to the indicators of bacterial (16S rRNA gene and gyrB sequences) and fungal (ITS1) diversity employed in this study, seed transmission of the bacterial strain Xcc 8004 did not change the overall composition of resident microbial assemblages. In contrast seed transmission of Abra43 strongly modified the richness and structure of fungal assemblages without affecting bacterial assemblages. The sensitivity of seed-associated fungal assemblage to Abra43 is mostly related to changes in relative abundance of closely related fungal species that belong to the Alternaria genus. Variation in stability of the seed microbiota in response to Xcc and Abra43 invasions could be explained by differences in seed transmission pathways employed by these micro-organisms, which ultimately results in divergence in spatio-temporal colonization of the seed habitat. PMID:27077013

  13. Potential application of Northern Argentine propolis to control some phytopathogenic bacteria.

    PubMed

    Ordóñez, R M; Zampini, I C; Moreno, M I Nieva; Isla, M I

    2011-10-20

    The antimicrobial activity of samples of Northern Argentine propolis (Tucumán, Santiago del Estero and Chaco) against phytopathogenic bacteria was assessed and the most active samples were identified. Minimal inhibitory concentration (MIC) values were determined by agar macrodilution and broth microdilution assays. Strong antibacterial activity was detected against Erwinia carotovora spp carotovora CECT 225, Pseudomonas syringae pvar tomato CECT 126, Pseudomonas corrugata CECT 124 and Xanthomonas campestris pvar vesicatoria CECT 792. The most active propolis extract (Tucumán, T1) was selected to bioguide isolation and identified for antimicrobial compound (2',4'-dihydroxychalcone). The antibacterial chalcone was more active than the propolis ethanolic extract (MIC values of 0.5-1 μg ml(-1) and 9.5-15 μg ml(-1), respectively). Phytotoxicity assays were realized and the propolis extracts did not retard germination of lettuce seeds or the growth of onion roots. Propolis solutions applied as sprays on tomato fruits infected with P. syringae reduced the severity of disease. Application of the Argentine propolis extracts diluted with water may be promising for the management of post harvest diseases of fruits. PMID:21237629

  14. Secondary metabolites from nonhost plants affect the motility and viability of phytopathogenic Aphanomyces cochlioides zoospores.

    PubMed

    Islam, Md Tofazzal

    2008-01-01

    The motile zoospores of the damping-off pathogen Aphanomyces cochlioides aggregate on host plants (e.g., sugar beet, spinach) guided by the host-specific plant signal cochliophilin A before infection. To assess the potential role of secondary metabolites in nonhost resistance, acetone extracts of 200 nonhost traditional medicinal plants from Chinese and Bangladeshi origins were tested for the motility behaviour of A. cochlioides zoospores using a particle bioassay method. Nearly one third of the tested plant extracts exhibited diverse deleterious activities such as repellent, stimulant, motility halting and lysis against A. cochlioides zoospores. Among these active plants, an extract of the Chinese medicinal plant Dalbergia odorifera displayed potent repellent activity toward zoospores. Chromatographic separation of D. odorifera constituents revealed that the repellent activity was regulated by the cumulative effect of three motility-affecting isoflavonoids, viz. (+/-)-medicarpin (repellent at 150 microg/ml), (-)-claussequinone (stimulant at 100 microg/ml) and formononetin (stimulant and attractant at 50 microg/ml). A mixture (1:1:1, w/w/w) of these three compounds exhibited only repellent activity toward zoospores at a concentration lower than 50 microg/ml. These results suggest that nonhost plants might possess potential bioactive secondary metabolites to ward off zoosporic phytopathogens. PMID:18533468

  15. The effects of some polyamine biosynthetic inhibitors on growth and morphology of phytopathogenic fungi.

    PubMed

    Rajam, M V; Galston, A W

    1985-01-01

    We have studied the effects of two polyamine biosynthetic inhibitors, alpha-difluoromethylornithine (DFMO) and alpha-difluoromethylarginine (DFMA), and of polyamines (PAs), alone and in combination, on mycelial growth and morphology of four phytopathogenic fungi: Botrytis sp, B. cinerea, Rhizoctonia solani and Monilinia fructicola. The inhibitors were added to a Czapek agar medium to get final concentrations of 0.1, 0.5 and 1.0 mM. DFMO and DFMA, suicide inhibitors of ornithine decarboxylase (ODC) and arginine decarboxylase (ADC) respectively, inhibited mycelial growth strongly; the effect was generally more pronounced with DFMA than with DFMO, but each fungus had its own response pattern. The addition of the PAs putrescine (Put) and spermidine (Spd) to the culture medium resulted in a promotion of growth. In Botrytis sp and Monilinia fructicola exposed to inhibitors plus PAs, mycelial growth was actually increased above control values. Mycelial morphology was altered and cell size dramatically reduced in plates containing inhibitors alone, whereas with PAs alone, or in combination with inhibitors, morphology was normal, but cell length and diameters increased considerably. These results suggest that PAs are essential for growth in fungal mycelia. The inhibition caused by DFMA may be due to its arginase-mediated conversion to DFMO.

  16. Synthesis of chitosan based nanoparticles and their in vitro evaluation against phytopathogenic fungi.

    PubMed

    Saharan, Vinod; Mehrotra, Akanksha; Khatik, Rajesh; Rawal, Pokhar; Sharma, S S; Pal, Ajay

    2013-11-01

    The main aim of present study was to prepare chitosan, chitosan-saponin and Cu-chitosan nanoparticles to evaluate their in vitro antifungal activities. Various nanoparticles were prepared using ionic gelation method by interaction of chitosan, sodium tripolyphosphate, saponin and Cu ions. Their particle size, polydispersity index, zeta potential and structures were confirmed by DLS, FTIR, TEM and SEM. The antifungal properties of nanoparticles against phytopathogenic fungi namely Alternaria alternata, Macrophomina phaseolina and Rhizoctonia solani were investigated at various concentrations ranging from 0.001 to 0.1%. Among the various formulations of nanoparticles, Cu-chitosan nanoparticles were found most effective at 0.1% concentration and showed 89.5, 63.0 and 60.1% growth inhibition of A. alternata, M. phaseolina and R. solani, respectively in in vitro model. At the same concentration, Cu-chitosan nanoparticles also showed maximum of 87.4% inhibition rate of spore germination of A. alternata. Chitosan nanoparticles showed the maximum growth inhibitory effects (87.6%) on in vitro mycelial growth of M. phaseolina at 0.1% concentration. From our study it is evident that chitosan based nanoparticles particularly chitosan and Cu-chitosan nanoparticles have tremendous potential for further field screening towards crop protection.

  17. Production of lipopeptides among Bacillus strains showing growth inhibition of phytopathogenic fungi.

    PubMed

    Velho, R V; Medina, L F C; Segalin, J; Brandelli, A

    2011-07-01

    The biological activity and the presence of genes sfp and ituD (surfactin and iturin A) among Bacillus strains isolated from the Amazon basin were determined. Bacillus spp. were tested for hemolytic activity and inhibition of fungal growth by agar plate assays in parallel with PCR for identification of sfp and ituD genes. All strains tested produced surface-active compounds, giving evidence by lysis of erythrocytes and emulsifying activity on mineral oil and soybean oil. These strains of Bacillus caused growth inhibition of several phytopathogenic fungi, including Fusarium spp., Aspergillus spp., and Bipolaris sorokiniana. The presence of genes ituD and sfp was confirmed by PCR and sequence analysis. The only exception was Bacillus sp. P34 that lacks sfp gene. Lipopeptides were isolated from culture supernatants and analyzed by mass spectrometry. Characteristic m/z peaks for surfactin and iturin were observed, and some strains also produced fengycin and bacillomycin. The remarkable antifungal activity showed by the strains could be associated with the co-production of three or more lipopeptide antibiotics. Screening for novel bacteria producing useful biosurfactants or biocontrol agents for agriculture is a topic of greatest importance to eliminate chemical pollutants.

  18. Isolation of Bacteria with Antifungal Activity against the Phytopathogenic Fungi Stenocarpella maydis and Stenocarpella macrospora

    PubMed Central

    Petatán-Sagahón, Iván; Anducho-Reyes, Miguel Angel; Silva-Rojas, Hilda Victoria; Arana-Cuenca, Ainhoa; Tellez-Jurado, Alejandro; Cárdenas-Álvarez, Isabel Oyuki; Mercado-Flores, Yuridia

    2011-01-01

    Stenocarpella maydis and Stenocarpella macrospora are the causal agents of ear rot in corn, which is one of the most destructive diseases in this crop worldwide. These fungi are important mycotoxin producers that cause different pathologies in farmed animals and represent an important risk for humans. In this work, 160 strains were isolated from soil of corn crops of which 10 showed antifungal activity against these phytopathogens, which, were identified as: Bacillus subtilis, Pseudomonas spp., Pseudomonas fluorescens, and Pantoea agglomerans by sequencing of 16S rRNA gene and the phylogenetic analysis. From cultures of each strain, extracellular filtrates were obtained and assayed to determine antifungal activity. The best filtrates were obtained in the stationary phase of B. subtilis cultures that were stable to the temperature and extreme pH values; in addition they did not show a cytotoxicity effect against brine shrimp and inhibited germination of conidia. The bacteria described in this work have the potential to be used in the control of white ear rot disease. PMID:22016606

  19. Differential Response of Extracellular Proteases of Trichoderma Harzianum Against Fungal Phytopathogens.

    PubMed

    Sharma, Vivek; Salwan, Richa; Sharma, Prem N

    2016-09-01

    In the present study, production of extracellular proteases by Trichoderma harzianum was evaluated based on the relative gene expression and spectrophotometric assay. The fungal isolates were grown in Czapek Dox Broth medium supplemented with deactivated mycelium of plant fungal pathogens such as Fusarium oxysporum, Colletotrichum capsici, Gloeocercospora sorghi, and Colletotrichum truncatum. The maximum protease activity was detected after 48 h of incubation against Colletotrichum spp. Similarly in qRT-PCR, the relative gene expression of four proteases varied from 48 to 96 h against host pathogens in a time-independent manner. Among proteases, statistically significant upregulation of asp, asp, and srp was observed against Colletotrichum spp., followed by F. oxysporum. But in the case of pepM22, maximum upregulation was observed against F. oxysporum. The variation in enzyme assay and qRT-PCR of proteases at different time intervals against various fungal phytopathogens could be due to the limitation of using casein as a substrate for all types of proteases or protease-encoding transcripts selected for qRT-PCR, which may not be true representative of total protease activity. PMID:27278806

  20. Laccase is upregulated via stress pathways in the phytopathogenic fungus Sclerotinia sclerotiorum.

    PubMed

    Coman, Cristina; Moţ, Augustin C; Gal, Emese; Pârvu, Marcel; Silaghi-Dumitrescu, Radu

    2013-01-01

    We report on the factors affecting the production of the newly characterized laccase from the phytopathogenic fungus Sclerotinia sclerotiorum (Lib.) de Bary. The carbon/nitrogen ratio appears to be of great importance. Rather than a simple nutrient-rich nitrogen source, yeast extract (YE) behaves as a true laccase upregulator, apparently acting via a stress pathway. Chelidonium majus extract, a known antifungal agent, acts in a similar manner. The compound(s) in the YE responsible for enhancing laccase synthesis are suggested to be hydrolysable choline derivatives. Both extracts reduce biomass and sclerotia development and enhance laccase production, leading to an increase in laccase activity by one order of magnitude compared to controls. The pH of the medium, a well-known virulence regulator for this fungus, also acts as a true laccase regulator, though via a different mechanism. The effect of pH appeared to be linked to the acidification kinetics of the extracellular medium during fungal development. A number of other known laccase inducers were found to enhance laccase production at most twofold. PMID:23931118

  1. Trichoderma species mediated differential tolerance against biotic stress of phytopathogens in Cicer arietinum L.

    PubMed

    Saxena, Amrita; Raghuwanshi, Richa; Singh, Harikesh Bahadur

    2015-02-01

    Trichoderma spp. have been reported to aid in imparting biotic as well as abiotic tolerance to plants. However, there are only few reports unfolding the differential ability of separate species of Trichoderma genera generally exploited for their biocontrol potential in this framework. A study was undertaken to evaluate the biocontrol potential of different Trichoderma species namely T. harzianum, T. asperellum, T. koningiopsis, T. longibrachiatum, and T. aureoviride as identified in the group of indigenous isolates from the agricultural soils of Eastern Uttar Pradesh, India. Their biocontrol potential against three major soilborne phytopathogens, i.e., Sclerotium rolfsii, Sclerotinia sclerotiorum, and Colletotrichum capsici was confirmed by dual culture plate technique. Efficient mycoparasitic ability was further assessed in all the isolates in relation to chitinase, β-1,3 glucanase, pectinase, lipase, amylase, and cellulase production while equally consistent results were obtained for their probable phosphate solubilization and indole acetic acid (IAA) production abilities. The selected isolates were further subjected to test their ability to promote plant growth, to reduce disease incidence and to tolerate biotic stress in terms of lignification pattern against S. rolfsii in chickpea plants. Among the identified Trichoderma species, excellent results were observed for T. harzianum and T. koningiopsis indicating better biocontrol potential of these species in the group and thus exhibiting perspective for their commercial exploitation. PMID:25205162

  2. Histopathological studies of sclerotia of phytopathogenic fungi parasitized by a GFP transformed Trichoderma virens antagonistic strain.

    PubMed

    Sarrocco, Sabrina; Mikkelsen, Lisbeth; Vergara, Mariarosaria; Jensen, Dan Funck; Lübeck, Mette; Vannacci, Giovanni

    2006-02-01

    The gfp gene from the jellyfish Aequorea victoria, coding for the Green Fluorescent Protein (GFP), was used as a reporter gene to transform a Trichoderma virens strain I10, characterized as having a promising biocontrol activity against a large number of phytopathogenic fungi. On the basis of molecular and biological results, a stable GFP transformant was selected for further experiments. In order to evaluate the effects of GFP transformation on mycoparasitic ability of T. virens I10, sclerotia of Sclerotium rolfsii, Sclerotinia sclerotiorum and S. minor were inoculated with the T. virens strain I10 GFP transformant or the wild type strain. Statistical analysis of percentages of decayed sclerotia showed that the transformation of the antagonistic isolate with the GFP reporter gene did not modify mycoparasitic activity against sclerotia. Sclerotium colonization was followed by fluorescent microscopy revealing intracellular growth of the antagonist in the cortex (S. rolfsii) and inter-cellular growth in the medulla (S. rolfsii, and S. sclerotiorum). The uniformly distributed mycelium of T. virens just beneath the rind of sclerotia of both S. rolfsii and S. sclerotiorum suggests that the sclerotia became infected at numerous randomly distributed locations without any preferential point of entry. PMID:16388938

  3. Propiconazole inhibits the sterol 14α-demethylase in Glomus irregulare like in phytopathogenic fungi.

    PubMed

    Calonne, Maryline; Sahraoui, Anissa Lounès-Hadj; Campagnac, Estelle; Debiane, Djouher; Laruelle, Frédéric; Grandmougin-Ferjani, Anne; Fontaine, Joël

    2012-04-01

    The increasing concentrations impact (0.02, 0.2 and 2 mg L(-1)) of a Sterol Biosynthesis Inhibitor (SBI) fungicide, propiconazole, was evaluated on development and sterol metabolism of two non-target organisms: mycorrhizal or non-mycorrhizal transformed chicory roots and the arbuscular mycorrhizal fungus (AMF) Glomus irregulare using monoxenic cultures. In this work, we provide the first evidence of a direct impact of propiconazole on the AMF by disturbing its sterol metabolism. A significant decrease in end-products sterols contents (24-methylcholesterol and in 24-ethylcholesterol) was observed concomitantly to a 24-methylenedihydrolanosterol accumulation indicating the inhibition of a key enzyme in sterol biosynthesis pathway, the sterol 14α-demethylase like in phytopathogenic fungi. A decrease in end-product sterol contents in propiconazole-treated roots was also observed suggesting a slowing down of the sterol metabolism in plant. Taken together, our findings suggest that the inhibition of the both AM symbiotic partners development by propiconazole results from their sterol metabolism alterations.

  4. An Improved Single-Step Cloning Strategy Simplifies the Agrobacterium tumefaciens-Mediated Transformation (ATMT)-Based Gene-Disruption Method for Verticillium dahliae.

    PubMed

    Wang, Sheng; Xing, Haiying; Hua, Chenlei; Guo, Hui-Shan; Zhang, Jie

    2016-06-01

    The soilborne fungal pathogen Verticillium dahliae infects a broad range of plant species to cause severe diseases. The availability of Verticillium genome sequences has provided opportunities for large-scale investigations of individual gene function in Verticillium strains using Agrobacterium tumefaciens-mediated transformation (ATMT)-based gene-disruption strategies. Traditional ATMT vectors require multiple cloning steps and elaborate characterization procedures to achieve successful gene replacement; thus, these vectors are not suitable for high-throughput ATMT-based gene deletion. Several advancements have been made that either involve simplification of the steps required for gene-deletion vector construction or increase the efficiency of the technique for rapid recombinant characterization. However, an ATMT binary vector that is both simple and efficient is still lacking. Here, we generated a USER-ATMT dual-selection (DS) binary vector, which combines both the advantages of the USER single-step cloning technique and the efficiency of the herpes simplex virus thymidine kinase negative-selection marker. Highly efficient deletion of three different genes in V. dahliae using the USER-ATMT-DS vector enabled verification that this newly-generated vector not only facilitates the cloning process but also simplifies the subsequent identification of fungal homologous recombinants. The results suggest that the USER-ATMT-DS vector is applicable for efficient gene deletion and suitable for large-scale gene deletion in V. dahliae. PMID:26780432

  5. Isolation of a Ve homolog, mVe1, and its relationship to Verticillium wilt resistance in Mentha longifolia (L.) Huds.

    PubMed

    Vining, Kelly; Davis, Thomas

    2009-08-01

    As a step toward greater understanding of the genetics of verticillium wilt resistance in plants, we report the sequencing of a candidate wilt resistance gene, mVe1, from the mint diploid model species, Mentha longifolia (Lamiaceae). mVe1 is a putative homolog of tomato (Solanum lycopersicum L.) verticillium wilt (Ve) resistance genes. The mVe1 gene has a coding region of 3,051 bp. The predicted mVe1 protein contains a leucine-rich repeat domain, a common feature of plant disease resistance proteins. We compared 13 mVe1 alleles from three mint species. These alleles shared 96.2-99.6% nucleotide identity. We analyzed four M. longifolia populations segregating with respect to mVe1 alleles and wilt resistance versus susceptibility and found one association between mVe1 genotype and wilt phenotype. We conclude that mVe1 may play a role in mint verticillium wilt resistance, but variation for resistance in our segregating progenies is likely polygenic. Therefore, further investigations of mVe1 and identification of additional candidate genes are both warranted.

  6. Arabidopsis thaliana as a tool to identify traits involved in Verticillium dahliae biocontrol by the olive root endophyte Pseudomonas fluorescens PICF7

    PubMed Central

    Maldonado-González, M. Mercedes; Bakker, Peter A. H. M.; Prieto, Pilar; Mercado-Blanco, Jesús

    2015-01-01

    The effective management of Verticillium wilts (VW), diseases affecting many crops and caused by some species of the soil-borne fungus Verticillium, is problematic. The use of microbial antagonists to control these pathologies fits modern sustainable agriculture criteria. Pseudomonas fluorescens PICF7 is an endophytic bacterium isolated from olive roots with demonstrated ability to control VW of olive caused by the highly virulent, defoliating (D) pathotype of Verticillium dahliae Kleb. However, the study of the PICF7-V. dahliae-olive tripartite interaction poses difficulties because of the inherent characteristics of woody, long-living plants. To overcome these problems we explored the use of the model plant Arabidopsis thaliana. Results obtained in this study showed that: (i) olive D and non-defoliating V. dahliae pathotypes produce differential disease severity in A. thaliana plants; (ii) strain PICF7 is able to colonize and persist in the A. thaliana rhizosphere but is not endophytic in Arabidopsis; and (iii) strain PICF7 controls VW in Arabidopsis. Additionally, as previously observed in olive, neither swimming motility nor siderophore production by PICF7 are required for VW control in A. thaliana, whilst cysteine auxotrophy decreased the effectiveness of PICF7. Moreover, when applied to the roots PICF7 controlled Botrytis cinerea infection in the leaves of Arabidopsis, suggesting that this strain is able to induce systemic resistance. A. thaliana is therefore a suitable alternative to olive bioassays to unravel biocontrol traits involved in biological control of V. dahliae by P. fluorescens PICF7. PMID:25904904

  7. Arabidopsis thaliana as a tool to identify traits involved in Verticillium dahliae biocontrol by the olive root endophyte Pseudomonas fluorescens PICF7.

    PubMed

    Maldonado-González, M Mercedes; Bakker, Peter A H M; Prieto, Pilar; Mercado-Blanco, Jesús

    2015-01-01

    The effective management of Verticillium wilts (VW), diseases affecting many crops and caused by some species of the soil-borne fungus Verticillium, is problematic. The use of microbial antagonists to control these pathologies fits modern sustainable agriculture criteria. Pseudomonas fluorescens PICF7 is an endophytic bacterium isolated from olive roots with demonstrated ability to control VW of olive caused by the highly virulent, defoliating (D) pathotype of Verticillium dahliae Kleb. However, the study of the PICF7-V. dahliae-olive tripartite interaction poses difficulties because of the inherent characteristics of woody, long-living plants. To overcome these problems we explored the use of the model plant Arabidopsis thaliana. Results obtained in this study showed that: (i) olive D and non-defoliating V. dahliae pathotypes produce differential disease severity in A. thaliana plants; (ii) strain PICF7 is able to colonize and persist in the A. thaliana rhizosphere but is not endophytic in Arabidopsis; and (iii) strain PICF7 controls VW in Arabidopsis. Additionally, as previously observed in olive, neither swimming motility nor siderophore production by PICF7 are required for VW control in A. thaliana, whilst cysteine auxotrophy decreased the effectiveness of PICF7. Moreover, when applied to the roots PICF7 controlled Botrytis cinerea infection in the leaves of Arabidopsis, suggesting that this strain is able to induce systemic resistance. A. thaliana is therefore a suitable alternative to olive bioassays to unravel biocontrol traits involved in biological control of V. dahliae by P. fluorescens PICF7.

  8. An Improved Single-Step Cloning Strategy Simplifies the Agrobacterium tumefaciens-Mediated Transformation (ATMT)-Based Gene-Disruption Method for Verticillium dahliae.

    PubMed

    Wang, Sheng; Xing, Haiying; Hua, Chenlei; Guo, Hui-Shan; Zhang, Jie

    2016-06-01

    The soilborne fungal pathogen Verticillium dahliae infects a broad range of plant species to cause severe diseases. The availability of Verticillium genome sequences has provided opportunities for large-scale investigations of individual gene function in Verticillium strains using Agrobacterium tumefaciens-mediated transformation (ATMT)-based gene-disruption strategies. Traditional ATMT vectors require multiple cloning steps and elaborate characterization procedures to achieve successful gene replacement; thus, these vectors are not suitable for high-throughput ATMT-based gene deletion. Several advancements have been made that either involve simplification of the steps required for gene-deletion vector construction or increase the efficiency of the technique for rapid recombinant characterization. However, an ATMT binary vector that is both simple and efficient is still lacking. Here, we generated a USER-ATMT dual-selection (DS) binary vector, which combines both the advantages of the USER single-step cloning technique and the efficiency of the herpes simplex virus thymidine kinase negative-selection marker. Highly efficient deletion of three different genes in V. dahliae using the USER-ATMT-DS vector enabled verification that this newly-generated vector not only facilitates the cloning process but also simplifies the subsequent identification of fungal homologous recombinants. The results suggest that the USER-ATMT-DS vector is applicable for efficient gene deletion and suitable for large-scale gene deletion in V. dahliae.

  9. Detection and identification of phytopathogenic Xanthomonas strains by amplification of DNA sequences related to the hrp genes of Xanthomonas campestris pv. vesicatoria.

    PubMed Central

    Leite, R P; Minsavage, G V; Bonas, U; Stall, R E

    1994-01-01

    Three pairs of oligonucleotide primers specific for different regions of the hrp gene (hypersensitive reaction and pathogenicity) cluster of Xanthomonas campestris pv. vesicatoria were designed and tested for amplification of DNA isolated from a large number of different bacteria. DNA sequences related to the hrp genes were successfully amplified from X. fragariae and from 28 pathovars of X. campestris. No DNA amplification occurred with genomic DNA from phytopathogenic strains of X. campestris pv. secalis, X. campestris pv. translucens, and X. albilineans or from nonpathogenic opportunistic xanthomonads and phytopathogenic strains of the genera Acidovorax, Agrobacterium, Clavibacter, Erwinia, Pseudomonas, and Xylella. The DNA from those bacteria also failed to hybridize to hrp-specific fragments in Southern blot analysis. DNA fragments amplified with a particular primer pair were of identical size from each of the different phytopathogenic xanthomonads. However, restriction analysis of these fragments by using frequently cutting endonucleases revealed variation in the pattern for these hrp-related fragments amplified from the different Xanthomonas strains. The restriction patterns generated for the different fragments allowed distinction of the strains representing a pathovar or species of phytopathogenic xanthomonads. We believe that DNA amplification with hrp-specific oligonucleotide primers is a highly sensitive and specific method that can be applied for detection and identification of phytopathogenic xanthomonads. Images PMID:8017904

  10. Impact of phytopathogen infection and extreme weather stress on internalization of Salmonella Typhimurium in lettuce.

    PubMed

    Ge, Chongtao; Lee, Cheonghoon; Nangle, Ed; Li, Jianrong; Gardner, David; Kleinhenz, Matthew; Lee, Jiyoung

    2014-01-01

    Internalization of human pathogens, common in many types of fresh produce, is a threat to human health since the internalized pathogens cannot be fully inactivated/removed by washing with water or sanitizers. Given that pathogen internalization can be affected by many environmental factors, this study was conducted to investigate the influence of two types of plant stress on the internalization of Salmonella Typhimurium in iceberg lettuce during pre-harvest. The stresses were: abiotic (water stress induced by extreme weather events) and biotic (phytopathogen infection by lettuce mosaic virus [LMV]). Lettuce with and without LMV infection were purposefully contaminated with green fluorescence protein-labeled S. Typhimurium on the leaf surfaces. Lettuce was also subjected to water stress conditions (drought and storm) which were simulated by irrigating with different amounts of water. The internalized S. Typhimurium in the different parts of the lettuce were quantified by plate count and real-time quantitative PCR and confirmed with a laser scanning confocal microscope. Salmonella internalization occurred under the conditions outlined above; however internalization levels were not significantly affected by water stress alone. In contrast, the extent of culturable S. Typhimurium internalized in the leafy part of the lettuce decreased when infected with LMV under water stress conditions and contaminated with high levels of S. Typhimurium. On the other hand, LMV-infected lettuce showed a significant increase in the levels of culturable bacteria in the roots. In conclusion, internalization was observed under all experimental conditions when the lettuce surface was contaminated with S. Typhimurium. However, the extent of internalization was only affected by water stress when lettuce was infected with LMV. PMID:24220663

  11. Rhizospheric fungi of Panax notoginseng: diversity and antagonism to host phytopathogens

    PubMed Central

    Miao, Cui-Ping; Mi, Qi-Li; Qiao, Xin-Guo; Zheng, You-Kun; Chen, You-Wei; Xu, Li-Hua; Guan, Hui-Lin; Zhao, Li-Xing

    2015-01-01

    Background Rhizospheric fungi play an essential role in the plant–soil ecosystem, affecting plant growth and health. In this study, we evaluated the fungal diversity in the rhizosphere soil of 2-yr-old healthy Panax notoginseng cultivated in Wenshan, China. Methods Culture-independent Illumina MiSeq and culture-dependent techniques, combining molecular and morphological characteristics, were used to analyze the rhizospheric fungal diversity. A diffusion test was used to challenge the phytopathogens of P. notoginseng. Results A total of 16,130 paired-end reads of the nuclear ribosomal internal transcribed spacer 2 were generated and clustered into 860 operational taxonomic units at 97% sequence similarity. All the operational taxonomic units were assigned to five phyla and 79 genera. Zygomycota (46.2%) and Ascomycota (37.8%) were the dominant taxa; Mortierella and unclassified Mortierellales accounted for a large proportion (44.9%) at genus level. The relative abundance of Fusarium and Phoma sequences was high, accounting for 12.9% and 5.5%, respectively. In total, 113 fungal isolates were isolated from rhizosphere soil. They were assigned to five classes, eight orders (except for an Incertae sedis), 26 genera, and 43 species based on morphological characteristics and phylogenetic analysis of the internal transcribed spacer. Fusarium was the most isolated genus with six species (24 isolates, 21.2%). The abundance of Phoma was also relatively high (8.0%). Thirteen isolates displayed antimicrobial activity against at least one test fungus. Conclusion Our results suggest that diverse fungi including potential pathogenic ones exist in the rhizosphere soil of 2-yr-old P. notoginseng and that antagonistic isolates may be useful for biological control of pathogens. PMID:27158233

  12. Potential non-phytopathogenic filamentous fungi for bioconversion of domestic wastewater sludge.

    PubMed

    Molla, Abul Hossain; Fakhru'l-Razi, Ahmadun; Hanafi, Mohamed Musa; Abd-Aziz, Suraini; Alam, Md Zahangir

    2002-09-01

    Ten filamentous fungi adapted to domestic wastewater sludge (DWS) were further studied to evaluate their potential in terms of adaptation to higher sludge supplemented growing media and phytopathogenicity (induction of diseases to plants) to three germinating crop (Corn: Zea mays, Mung bean: Phaseolus aureus and Mustard: Brassica napus) seeds. The performances of the fungi in seed germination were evaluated based on percent germination index (GI) and infected/spotted seeds on direct fungal biomass (FBM) and fungal metabolite (FM). Significantly the highest biomass production was achieved with RW-P1 512 and Penicillium corylophilum (WW-P1003) at the highest (25%) sludge supplemented growing media that implied its excellent potentiality of adaptation and multiplication to domestic wastewater sludge. Significantly encouraging results of percent GI and spotted/infected seedlings were observed in FM than FBM by all fungi except the strain Aspergillus niger. A. niger gave the poorest percent of GI (24.30, 26.98 and 00.00%) and the highest percent of infected/spotted seeds (70, 100, and 100%) using FBM for corn, mung bean and mustard, respectively. On the other hand, comparatively the highest percent of GI (107.99, 106.25 and 117.67%) and the lowest percent of spotted/infected seedlings (3.3, 3.3 and 3.3%) were achieved with the isolate RW-P1 512 using FM. In FBM, the superior results of percent GI (86.61, 95.92 and 83.87%) and spotted/infected seedlings (3.3, 63.3 and 43.3%) were obtained by A. versicolor. Several crop seeds were responded differently for different fungal treatments. Hundred percent infected/spotted seeds in FM were recorded only for mustard with Trichoderma family that implied its strong sensitiveness to its metabolites.

  13. Methylated Cytokinins from the Phytopathogen Rhodococcus fascians Mimic Plant Hormone Activity1[OPEN

    PubMed Central

    Radhika, Venkatesan; Ueda, Nanae; Tsuboi, Yuuri; Kojima, Mikiko; Kikuchi, Jun; Kudo, Takuji; Sakakibara, Hitoshi

    2015-01-01

    Cytokinins (CKs), a class of phytohormones that regulate plant growth and development, are also synthesized by some phytopathogens to disrupt the hormonal balance and to facilitate niche establishment in their hosts. Rhodococcus fascians harbors the fasciation (fas) locus, an operon encoding several genes homologous to CK biosynthesis and metabolism. This pathogen causes unique leafy gall symptoms reminiscent of CK overproduction; however, bacterial CKs have not been clearly correlated with the severe symptoms, and no virulence-associated unique CKs or analogs have been identified. Here, we report the identification of monomethylated N6-(∆2-isopentenyl)adenine and dimethylated N6-(∆2-isopentenyl)adenine (collectively, methylated cytokinins [MeCKs]) from R. fascians. MeCKs were recognized by a CK receptor and up-regulated type-A ARABIDOPSIS THALIANA RESPONSE REGULATOR genes. Treatment with MeCKs inhibited root growth, a hallmark of CK action, whereas the receptor mutant was insensitive. MeCKs were retained longer in planta than canonical CKs and were poor substrates for a CK oxidase/dehydrogenase, suggesting enhanced biological stability. MeCKs were synthesized by S-adenosyl methionine-dependent methyltransferases (MT1 and MT2) that are present upstream of the fas genes. The best substrate for methylation was isopentenyl diphosphate. MT1 and MT2 catalyzed distinct methylation reactions; only the MT2 product was used by FAS4 to synthesize monomethylated N6-(∆2-isopentenyl)adenine. The MT1 product was dimethylated by MT2 and used as a substrate by FAS4 to produce dimethylated N6-(∆2-isopentenyl)adenine. Chemically synthesized MeCKs were comparable in activity. Our results strongly suggest that MeCKs function as CK mimics and play a role in this plant-pathogen interaction. PMID:26251309

  14. Impact of phytopathogen infection and extreme weather stress on internalization of Salmonella Typhimurium in lettuce.

    PubMed

    Ge, Chongtao; Lee, Cheonghoon; Nangle, Ed; Li, Jianrong; Gardner, David; Kleinhenz, Matthew; Lee, Jiyoung

    2014-01-01

    Internalization of human pathogens, common in many types of fresh produce, is a threat to human health since the internalized pathogens cannot be fully inactivated/removed by washing with water or sanitizers. Given that pathogen internalization can be affected by many environmental factors, this study was conducted to investigate the influence of two types of plant stress on the internalization of Salmonella Typhimurium in iceberg lettuce during pre-harvest. The stresses were: abiotic (water stress induced by extreme weather events) and biotic (phytopathogen infection by lettuce mosaic virus [LMV]). Lettuce with and without LMV infection were purposefully contaminated with green fluorescence protein-labeled S. Typhimurium on the leaf surfaces. Lettuce was also subjected to water stress conditions (drought and storm) which were simulated by irrigating with different amounts of water. The internalized S. Typhimurium in the different parts of the lettuce were quantified by plate count and real-time quantitative PCR and confirmed with a laser scanning confocal microscope. Salmonella internalization occurred under the conditions outlined above; however internalization levels were not significantly affected by water stress alone. In contrast, the extent of culturable S. Typhimurium internalized in the leafy part of the lettuce decreased when infected with LMV under water stress conditions and contaminated with high levels of S. Typhimurium. On the other hand, LMV-infected lettuce showed a significant increase in the levels of culturable bacteria in the roots. In conclusion, internalization was observed under all experimental conditions when the lettuce surface was contaminated with S. Typhimurium. However, the extent of internalization was only affected by water stress when lettuce was infected with LMV.

  15. Ag doped hollow TiO2 nanoparticles as an effective green fungicide against Fusarium solani and Venturia inaequalis phytopathogens

    NASA Astrophysics Data System (ADS)

    Sankar Boxi, Siddhartha; Mukherjee, Khushi; Paria, Santanu

    2016-02-01

    Chemical-based pesticides are widely used in agriculture to protect crops from insect infestation and diseases. However, the excessive use of highly toxic pesticides causes several human health (neurological, tumor, cancer) and environmental problems. Therefore nanoparticle-based green pesticides have become of special importance in recent years. The antifungal activities of pure and Ag doped (solid and hollow) TiO2 nanoparticles are studied against two potent phytopathogens, Fusarium solani (which causes Fusarium wilt disease in potato, tomato, etc) and Venturia inaequalis (which causes apple scab disease) and it is found that hollow nanoparticles are more effective than the other two. The antifungal activities of the nanoparticles were further enhanced against these two phytopathogens under visible light exposure. The fungicidal effect of the nanoparticles depends on different parameters, such as particle concentration and the intensity of visible light. The minimum inhibitory dose of the nanoparticles for V. inaequalis and F. solani are 0.75 and 0.43 mg/plate. The presence of Ag as a dopant helps in the formation of stable Ag-S and disulfide bonds (R-S-S-R) in cellular protein, which leads to cell damage. During photocatalysis generated •OH radicals loosen the cell wall structure and this finally leads to cell death. The mechanisms of the fungicidal effect of nanoparticles against these two phytopathogens are supported by biuret and triphenyl tetrazolium chloride analyses and field emission electron microscopy. Apart from the fungicidal effect, at a very low dose (0.015 mg/plate) the nanoparticles are successful in arresting production of toxic napthoquinone pigment for F. solani which is related to the fungal pathogenecity. The nanoparticles are found to be effective in protecting potatoes affected by F. solani or other fungi from spoiling.

  16. Ag doped hollow TiO2 nanoparticles as an effective green fungicide against Fusarium solani and Venturia inaequalis phytopathogens.

    PubMed

    Boxi, Siddhartha Sankar; Mukherjee, Khushi; Paria, Santanu

    2016-02-26

    Chemical-based pesticides are widely used in agriculture to protect crops from insect infestation and diseases. However, the excessive use of highly toxic pesticides causes several human health (neurological, tumor, cancer) and environmental problems. Therefore nanoparticle-based green pesticides have become of special importance in recent years. The antifungal activities of pure and Ag doped (solid and hollow) TiO2 nanoparticles are studied against two potent phytopathogens, Fusarium solani (which causes Fusarium wilt disease in potato, tomato, etc) and Venturia inaequalis (which causes apple scab disease) and it is found that hollow nanoparticles are more effective than the other two. The antifungal activities of the nanoparticles were further enhanced against these two phytopathogens under visible light exposure. The fungicidal effect of the nanoparticles depends on different parameters, such as particle concentration and the intensity of visible light. The minimum inhibitory dose of the nanoparticles for V. inaequalis and F. solani are 0.75 and 0.43 mg/plate. The presence of Ag as a dopant helps in the formation of stable Ag-S and disulfide bonds (R-S-S-R) in cellular protein, which leads to cell damage. During photocatalysis generated (•)OH radicals loosen the cell wall structure and this finally leads to cell death. The mechanisms of the fungicidal effect of nanoparticles against these two phytopathogens are supported by biuret and triphenyl tetrazolium chloride analyses and field emission electron microscopy. Apart from the fungicidal effect, at a very low dose (0.015 mg/plate) the nanoparticles are successful in arresting production of toxic napthoquinone pigment for F. solani which is related to the fungal pathogenecity. The nanoparticles are found to be effective in protecting potatoes affected by F. solani or other fungi from spoiling. PMID:26808118

  17. Ag doped hollow TiO2 nanoparticles as an effective green fungicide against Fusarium solani and Venturia inaequalis phytopathogens.

    PubMed

    Boxi, Siddhartha Sankar; Mukherjee, Khushi; Paria, Santanu

    2016-02-26

    Chemical-based pesticides are widely used in agriculture to protect crops from insect infestation and diseases. However, the excessive use of highly toxic pesticides causes several human health (neurological, tumor, cancer) and environmental problems. Therefore nanoparticle-based green pesticides have become of special importance in recent years. The antifungal activities of pure and Ag doped (solid and hollow) TiO2 nanoparticles are studied against two potent phytopathogens, Fusarium solani (which causes Fusarium wilt disease in potato, tomato, etc) and Venturia inaequalis (which causes apple scab disease) and it is found that hollow nanoparticles are more effective than the other two. The antifungal activities of the nanoparticles were further enhanced against these two phytopathogens under visible light exposure. The fungicidal effect of the nanoparticles depends on different parameters, such as particle concentration and the intensity of visible light. The minimum inhibitory dose of the nanoparticles for V. inaequalis and F. solani are 0.75 and 0.43 mg/plate. The presence of Ag as a dopant helps in the formation of stable Ag-S and disulfide bonds (R-S-S-R) in cellular protein, which leads to cell damage. During photocatalysis generated (•)OH radicals loosen the cell wall structure and this finally leads to cell death. The mechanisms of the fungicidal effect of nanoparticles against these two phytopathogens are supported by biuret and triphenyl tetrazolium chloride analyses and field emission electron microscopy. Apart from the fungicidal effect, at a very low dose (0.015 mg/plate) the nanoparticles are successful in arresting production of toxic napthoquinone pigment for F. solani which is related to the fungal pathogenecity. The nanoparticles are found to be effective in protecting potatoes affected by F. solani or other fungi from spoiling.

  18. 5,7-Diamino-5,7,9-trideoxynon-2-ulosonic acid: a novel sugar from a phytopathogenic Pseudomonas lipopolysaccharide.

    PubMed

    Corsaro, Maria Michela; Evidente, Antonio; Lanzetta, Rosa; Lavermicocca, Paola; Parrilli, Michelangelo; Ummarino, Salvatore

    2002-05-13

    Preliminary results on the structure of a novel sugar from a lipopolysaccharide from Pseudomonas corrugata, a plant pathogenic bacterium whose several aspects of phytopathogenic mechanism are under investigation, are described. This is a 5,7-diamino-5,7,9-trideoxynon-2-ulosonic acid, isolated as an O-glycoside from the Smith degradation of the O-chain. The structure was obtained both with NMR and MS methodologies. To the best of our knowledge, this is the first example of 3-hydroxylated non-2-ulosonic acid.

  19. Detection of Verticillium wilt of olive trees and downy mildew of opium poppy using hyperspectral and thermal UAV imagery

    NASA Astrophysics Data System (ADS)

    Calderón Madrid, Rocío; Navas Cortés, Juan Antonio; Montes Borrego, Miguel; Landa del Castillo, Blanca Beatriz; Lucena León, Carlos; Jesús Zarco Tejada, Pablo

    2014-05-01

    The present study explored the use of high-resolution thermal, multispectral and hyperspectral imagery as indicators of the infections caused by Verticillium wilt (VW) in olive trees and downy mildew (DM) in opium poppy fields. VW, caused by the soil-borne fungus Verticillium dahliae, and DM, caused by the biotrophic obligate oomycete Peronospora arborescens, are the most economically limiting diseases of olive trees and opium poppy, respectively, worldwide. V. dahliae infects the plant by the roots and colonizes its vascular system, blocking water flow and eventually inducing water stress. P. arborescens colonizes the mesophyll, appearing the first symptoms as small chlorotic leaf lesions, which can evolve to curled and thickened tissues and systemic infections that become deformed and necrotic as the disease develops. The work conducted to detect VW and DM infection consisted on the acquisition of time series of airborne thermal, multispectral and hyperspectral imagery using 2-m and 5-m wingspan electric Unmanned Aerial Vehicles (UAVs) in spring and summer of three consecutive years (2009 to 2011) for VW detection and on three dates in spring of 2009 for DM detection. Two 7-ha commercial olive orchards naturally infected with V. dahliae and two opium poppy field plots artificially infected by P. arborescens were flown. Concurrently to the airborne campaigns, olive orchards and opium poppy fields were assessed "in situ" to assess actual VW severity and DM incidence. Furthermore, field measurements were conducted at leaf and crown level. The field results related to VW detection showed a significant increase in crown temperature (Tc) minus air temperature (Ta) and a decrease in leaf stomatal conductance (G) as VW severity increased. This reduction in G was associated with a significant increase in the Photochemical Reflectance Index (PRI570) and a decrease in chlorophyll fluorescence. DM asymptomatic leaves showed significantly higher NDVI and lower green/red index

  20. Selection of endophytic fungi from comfrey (Symphytum officinale L.) for in vitro biological control of the phytopathogen Sclerotinia sclerotiorum (Lib.).

    PubMed

    Rocha, Rafaeli; da Luz, Daniela Eleutério; Engels, Cibelle; Pileggi, Sônia Alvim Veiga; de Souza Jaccoud Filho, David; Matiello, Rodrigo Rodrigues; Pileggi, Marcos

    2009-01-01

    Biological control consists of using one organism to attack another that may cause economic damage to crops. Integrated Pest Management (IPM) is a very common strategy. The white mold produced by Sclerotinia sclerotiorum (Lib.) causes considerable damage to bean crops. This fungus is a soil inhabitant, the symptoms of which are characterized by water-soaked lesions covered by a white cottony fungal growth on the soil surface and/or the host plant. Possible biological control agents taken from plants are being investigated as phytopathogen inhibitors. These are endophytic microorganisms that inhabit the intercellular spaces of vegetal tissues and are often responsible for antimicrobial production. The objective of the present study was to select endophytic fungi isolated from comfrey (Symphytum officinale L.) leaves with in vitro antagonist potential against the phytopathogenic fungus S. sclerotiorum. Twelve isolates of endophytic fungi and a pathogenic strain of S. sclerotiorum were used in the challenge method. With the aid of this method, four endophytes with the best antagonistic activity against S. sclerotiorum were selected. Pathogen growth inhibition zones were considered indicative of antibiosis. The percentages of pathogenic mycelia growth were measured both with and without the antagonist, resulting in growth reductions of 46.7% to 50.0% for S. sclerotiorum. These analyses were performed by evaluating the endophytic/pathogenic mycelia growth in mm/day over an eight-day period of antagonistic tests. PMID:24031320

  1. Anti-phytopathogenic activity of sporothriolide, a metabolite from endophyte Nodulisporium sp. A21 in Ginkgo biloba.

    PubMed

    Cao, Ling-Ling; Zhang, Ying-Ying; Liu, Ying-Jie; Yang, Ting-Ting; Zhang, Jin-Long; Zhang, Zheng-Guang; Shen, Li; Liu, Jun-Yan; Ye, Yong-Hao

    2016-05-01

    Phytopathogenic fungi such as Rhizoctonia solani and Sclerotinia sclerotiorum caused multiple plant diseases resulting in severe loss of crop production. Increasing documents endorsed that endophytes are a striking resource pool for numerous metabolites with various bioactivities such as anti-fungal. Here we reported the characterization and anti-phytopathogenic activity of sporothriolide, a metabolite produced by Nodulisporium sp. A21-an endophytic fungus in the leaves of Ginkgo biloba. Among the total twenty-five endophytic fungi isolated from the healthy leaves of G. biloba, the fermentation broth (FB) of the strain A21 was found potently inhibitory activity against R. solani and S. sclerotiorum using mycelia growth inhibition method. A21 was then identified as Nodulisporium sp., the asexual stage of Hypoxylon sp., by microscopic examination and ITS rDNA sequence data comparison. Under the bioassay-guided fractionation, sporothriolide was isolated from the petroleum ether extract of the FB of A21, whose structure was established by integrated interpretation of HR-ESI-MS and (1)H- and (13)C-NMR. Furthermore, the crystal structure of sporothriolide was first reported. In addition, sporothriolide was validated to be potently antifungal against R. solani, S. sclerotiorum and inhibit conidium germination of Magnaporthe oryzae in vitro and in vivo, indicating that it could be used as a lead compound for new fungicide development. PMID:27017876

  2. Fasciation induction by the phytopathogen Rhodococcus fascians depends upon a linear plasmid encoding a cytokinin synthase gene.

    PubMed Central

    Crespi, M; Messens, E; Caplan, A B; van Montagu, M; Desomer, J

    1992-01-01

    Rhodococcus fascians is a nocardiform bacteria that induces leafy galls (fasciation) on dicotyledonous and several monocotyledonous plants. The wild-type strain D188 contained a conjugative, 200 kb linear extrachromosomal element, pFiD188. Linear plasmid-cured strains were avirulent and reintroduction of this linear element restored virulence. Pulsed field electrophoresis indicated that the chromosome might also be a linear molecule of 4 megabases. Three loci involved in phytopathogenicity have been identified by insertion mutagenesis of this Fi plasmid. Inactivation of the fas locus resulted in avirulent strains, whereas insertions in the two other loci affected the degree of virulence, yielding attenuated (att) and hypervirulent (hyp) bacteria. One of the genes within the fas locus encoded an isopentenyltranferase (IPT) with low homology to analogous proteins from Gram-negative phytopathogenic bacteria. IPT activity was detected after expression of this protein in Escherichia coli cells. In R.fascians, ipt expression could only be detected in bacteria induced with extracts from fasciated tissue. R.fascians strains without the linear plasmid but containing this fas locus alone could not provoke any phenotype on plants, indicating additional genes from the linear plasmid were also essential for virulence. These studies, the first genetic analysis of the interaction of a Gram-positive bacterium with plants, suggest that a novel mechanism for plant tumour induction has evolved in R.fascians independently from the other branches of the eubacteria. Images PMID:1547783

  3. Selection of endophytic fungi from comfrey (Symphytum officinale L.) for in vitro biological control of the phytopathogen Sclerotinia sclerotiorum (Lib.)

    PubMed Central

    Rocha, Rafaeli; da Luz, Daniela Eleutério; Engels, Cibelle; Pileggi, Sônia Alvim Veiga; de Souza Jaccoud Filho, David; Matiello, Rodrigo Rodrigues; Pileggi, Marcos

    2009-01-01

    Biological control consists of using one organism to attack another that may cause economic damage to crops. Integrated Pest Management (IPM) is a very common strategy. The white mold produced by Sclerotinia sclerotiorum (Lib.) causes considerable damage to bean crops. This fungus is a soil inhabitant, the symptoms of which are characterized by water-soaked lesions covered by a white cottony fungal growth on the soil surface and/or the host plant. Possible biological control agents taken from plants are being investigated as phytopathogen inhibitors. These are endophytic microorganisms that inhabit the intercellular spaces of vegetal tissues and are often responsible for antimicrobial production. The objective of the present study was to select endophytic fungi isolated from comfrey (Symphytum officinale L.) leaves with in vitro antagonist potential against the phytopathogenic fungus S. sclerotiorum. Twelve isolates of endophytic fungi and a pathogenic strain of S. sclerotiorum were used in the challenge method. With the aid of this method, four endophytes with the best antagonistic activity against S. sclerotiorum were selected. Pathogen growth inhibition zones were considered indicative of antibiosis. The percentages of pathogenic mycelia growth were measured both with and without the antagonist, resulting in growth reductions of 46.7% to 50.0% for S. sclerotiorum. These analyses were performed by evaluating the endophytic/pathogenic mycelia growth in mm/day over an eight-day period of antagonistic tests. PMID:24031320

  4. The two-component system CpxAR is Essential for Virulence in the phytopathogen bacteria Dickeya dadantii EC3937

    PubMed Central

    Bontemps-Gallo, Sébastien; Madec, Edwige; Lacroix, Jean-Marie

    2015-01-01

    The CpxAR two-component system is present in many Proteobacteria. It controls expression of genes required to maintain envelope integrity in response to environmental injury. Consequently, this two-component system was shown to be required for virulence of several zoo-pathogens but it has never been investigated in phyto-pathogens. In this paper, we investigate the role of the CpxAR two-component system in vitro and in vivo in Dickeya dadantii, an enterobacterial phytopathogen that causes soft-rot disease in a large variety of plant species. cpxA null mutant displays a constitutively phosphorylated CpxR phenotype as shown by direct analysis of phosphorylation of CpxR by a Phos-Tag retardation gel approach. Virulence in plants is completely abolished in cpxA or cpxR mutants of D. dadantii. In planta, CpxAR is only activated at an early stage of the infection process as shown by Phos-Tag and gene fusion analyses. To our knowledge, this is the first time that the timing of CpxAR phosphorelay activation has been investigated during the infection process by direct monitoring of response regulator phosphorylation. PMID:25856505

  5. Superoxide radical is involved in the sclerotial differentiation of filamentous phytopathogenic fungi: identification of a fungal xanthine oxidase.

    PubMed

    Papapostolou, Ioannis; Georgiou, Christos D

    2010-01-01

    This study shows that the direct indicator of oxidative stress superoxide radical (O·₂⁻) is involved in the sclerotial differentiation of the phytopathogenic filamentous fungi Rhizoctonia solani, Sclerotinia sclerotiorum, Sclerotium rolfsii, and Sclerotinia minor. The production rate of O·₂⁻ and the antioxidant enzyme superoxide dismutase (SOD) levels in the sclerotiogenic fungi were significantly higher and lower, respectively, than those of their non-differentiating counterpart strains, which strongly suggests that the oxidative stress of the sclerotium differentiating fungi is higher than that of the non-differentiating ones. Xanthine oxidase (XO), which was detected for the first time in fungi in general, was localized in the cytoplasmic membrane. The contribution of XO in the overall O·₂⁻production was very significant, reaching 30-70% among the strains, especially in the transition developmental stage between the undifferentiated and the differentiated state, suggesting a sclerotium triggering and a phytopathogenic role of XO during plant infection. The additional finding that these fungi secrete extracellular SOD can be related to their protection from the response of plants to produce O·₂⁻ at infection sites. PMID:20943149

  6. The Innate Immune Signaling System as a Regulator of Disease Resistance and Induced Systemic Resistance Activity Against Verticillium dahliae.

    PubMed

    Gkizi, Danai; Lehmann, Silke; L'Haridon, Floriane; Serrano, Mario; Paplomatas, Epaminondas J; Métraux, Jean-Pierre; Tjamos, Sotirios E

    2016-04-01

    In the last decades, the plant innate immune responses against pathogens have been extensively studied, while biocontrol interactions between soilborne fungal pathogens and their hosts have received much less attention. Treatment of Arabidopsis thaliana with the nonpathogenic bacterium Paenibacillus alvei K165 was shown previously to protect against Verticillium dahliae by triggering induced systemic resistance (ISR). In the present study, we evaluated the involvement of the innate immune response in the K165-mediated protection of Arabidopsis against V. dahliae. Tests with Arabidopsis mutants impaired in several regulators of the early steps of the innate immune responses, including fls2, efr-1, bak1-4, mpk3, mpk6, wrky22, and wrky29 showed that FLS2 and WRKY22 have a central role in the K165-triggered ISR, while EFR1, MPK3, and MPK6 are possible susceptibility factors for V. dahliae and bak1 shows a tolerance phenomenon. The resistance induced by strain K165 is dependent on both salicylate and jasmonate-dependent defense pathways, as evidenced by an increased transient accumulation of PR1 and PDF1.2 transcripts in the aerial parts of infected plants treated with strain K165. PMID:26780421

  7. Isolation and functional analysis of the pathogenicity-related gene VdPR3 from Verticillium dahliae on cotton.

    PubMed

    Zhang, Ya-Lin; Li, Zhi-Fang; Feng, Zi-Li; Feng, Hong-Jie; Zhao, Li-Hong; Shi, Yong-Qiang; Hu, Xiao-Ping; Zhu, He-Qin

    2015-11-01

    The fungal plant pathogen Verticillium dahliae is the causal agent of vascular wilt, a disease that can seriously diminish cotton fiber yield. The pathogenicity mechanism and the identity of the genes that interact with cotton during the infection process still remain unclear. In this study, we investigated the low-pathogenic, non-microsclerotium-producing mutant vdpr3 obtained in a previous study from the screening of a T-DNA insertional library of the highly virulent isolate Vd080; the pathogenicity-related gene (VdPR3) in wild-type strain Vd080 was cloned. Knockout mutants (ΔVdPR3) showed lower mycelium growth and obvious reduction in sporulation ability without microsclerotium formation. An evaluation of carbon utilization in mutants and wild-type isolate Vd080 demonstrated that mutants-lacking VdPR3 exhibited decreased cellulase and amylase activities, which was restored in the complementary mutants (ΔVdPR3-C) to levels similar to those of Vd080. ΔVdPR3 postponed infectious events in cotton and showed a significant reduction in pathogenicity. Reintroduction of a functional VdPR3 copy into ΔVdPR3-C restored the ability to infect cotton plants. These results suggest that VdPR3 is a multifunctional gene involved in growth development, extracellular enzyme activity, and virulence of V. dahliae on cotton. PMID:25652159

  8. Perturbations in the Primary Metabolism of Tomato and Arabidopsis thaliana Plants Infected with the Soil-Borne Fungus Verticillium dahliae.

    PubMed

    Buhtz, Anja; Witzel, Katja; Strehmel, Nadine; Ziegler, Jörg; Abel, Steffen; Grosch, Rita

    2015-01-01

    The hemibiotrophic soil-borne fungus Verticillium dahliae is a major pathogen of a number of economically important crop species. Here, the metabolic response of both tomato and Arabidopsis thaliana to V. dahliae infection was analysed by first using non-targeted GC-MS profiling. The leaf content of both major cell wall components glucuronic acid and xylose was reduced in the presence of the pathogen in tomato but enhanced in A. thaliana. The leaf content of the two tricarboxylic acid cycle intermediates fumaric acid and succinic acid was increased in the leaf of both species, reflecting a likely higher demand for reducing equivalents required for defence responses. A prominent group of affected compounds was amino acids and based on the targeted analysis in the root, it was shown that the level of 12 and four free amino acids was enhanced by the infection in, respectively, tomato and A. thaliana, with leucine and histidine being represented in both host species. The leaf content of six free amino acids was reduced in the leaf tissue of diseased A. thaliana plants, while that of two free amino acids was raised in the tomato plants. This study emphasizes the role of primary plant metabolites in adaptive responses when the fungus has colonized the plant. PMID:26381754

  9. The effect of pH on the production of chitinolytic enzymes of Verticillium fungicola in submerged cultures.

    PubMed

    Ramirez-Coutiño, Laura; Espinosa-Marquez, Jesús; Peter, Martin G; Shirai, Keiko

    2010-12-01

    Chitinase and N-acetylhexosaminidase activities in submerged cultures of Verticillium fungicola increased up to 5-fold and 2.5-fold, respectively when the pH of the culture medium was raised from 5 to 8. SDS-PAGE and zymograms of the freeze-dried crude enzyme obtained from the cultures indicated four chitin degrading proteins of M(w) 24, 40, 55 and 63 kDa, whereas isoelectric focusing displayed the separation of three chitin degrading enzymes with isoelectric points of 4.7, 6.8 and 10, as well as two N-acetylhexosaminidases having isoelectric points of 3.2 and 13. Freeze-dried crude enzyme was characterized for its ability to produce chito-oligosaccharides from chitosans. Matrix-assisted laser desorption ionization time of flight mass spectrometry analyses revealed that monomers as well as hetero-oligomers with degree of polymerization 4 were initially the main products, whereas oligomers with degree of polymerization 2-11 were detected after extended reaction times.

  10. Perturbations in the Primary Metabolism of Tomato and Arabidopsis thaliana Plants Infected with the Soil-Borne Fungus Verticillium dahliae

    PubMed Central

    Buhtz, Anja; Witzel, Katja; Strehmel, Nadine; Ziegler, Jörg; Abel, Steffen; Grosch, Rita

    2015-01-01

    The hemibiotrophic soil-borne fungus Verticillium dahliae is a major pathogen of a number of economically important crop species. Here, the metabolic response of both tomato and Arabidopsis thaliana to V. dahliae infection was analysed by first using non-targeted GC-MS profiling. The leaf content of both major cell wall components glucuronic acid and xylose was reduced in the presence of the pathogen in tomato but enhanced in A. thaliana. The leaf content of the two tricarboxylic acid cycle intermediates fumaric acid and succinic acid was increased in the leaf of both species, reflecting a likely higher demand for reducing equivalents required for defence responses. A prominent group of affected compounds was amino acids and based on the targeted analysis in the root, it was shown that the level of 12 and four free amino acids was enhanced by the infection in, respectively, tomato and A. thaliana, with leucine and histidine being represented in both host species. The leaf content of six free amino acids was reduced in the leaf tissue of diseased A. thaliana plants, while that of two free amino acids was raised in the tomato plants. This study emphasizes the role of primary plant metabolites in adaptive responses when the fungus has colonized the plant. PMID:26381754

  11. Manipulating inoculum densities of Verticillium dahliae and Pratylenchus penetrans with green manure amendments and solarization influence potato yield.

    PubMed

    Macguidwin, A E; Knuteson, D L; Connell, T; Bland, W L; Bartelt, K D

    2012-05-01

    We used cover crops with demonstrated efficacy against Verticillium dahliae and Pratylenchus penetrans in combination with the biocidal practice of solarization to determine the importance of targeting both organisms for managing potato early dying, an issue relevant to the search for alternatives to soil fumigation. Two experiments were conducted in commercial fields using a split-plot design with cover crop treatments of rapeseed, marigold, forage pearl millet, sorghum-sudangrass, and corn as the main plot factor and solarization as the subplot factor. Cover crops were grown and solarization applied in year one, followed by potato in year two. The main effect of solarization was significant for reduced inoculum levels of both organisms in year two and increased tuber yields. The main effect of cover crop was also significant with lower population densities of P. penetrans following the marigold and millet treatments and of V. dahliae following rape and sorghum-sudangrass. The cover crop treatments influenced yield in only one of the experiments in the absence of solarization. The combinatorial effect of cover crops and solarization resulted in a wide range of pathogen population densities. Mean soil inoculum levels were negatively related to yield for V. dahliae in experiment 1, and for P. penetrans and the P. penetrans × V. dahliae interaction in both experiments. PMID:22494249

  12. Discovery and identification of candidate genes from the chitinase gene family for Verticillium dahliae resistance in cotton

    PubMed Central

    Xu, Jun; Xu, Xiaoyang; Tian, Liangliang; Wang, Guilin; Zhang, Xueying; Wang, Xinyu; Guo, Wangzhen

    2016-01-01

    Verticillium dahliae, a destructive and soil-borne fungal pathogen, causes massive losses in cotton yields. However, the resistance mechanism to V. dahilae in cotton is still poorly understood. Accumulating evidence indicates that chitinases are crucial hydrolytic enzymes, which attack fungal pathogens by catalyzing the fungal cell wall degradation. As a large gene family, to date, the chitinase genes (Chis) have not been systematically analyzed and effectively utilized in cotton. Here, we identified 47, 49, 92, and 116 Chis from four sequenced cotton species, diploid Gossypium raimondii (D5), G. arboreum (A2), tetraploid G. hirsutum acc. TM-1 (AD1), and G. barbadense acc. 3–79 (AD2), respectively. The orthologous genes were not one-to-one correspondence in the diploid and tetraploid cotton species, implying changes in the number of Chis in different cotton species during the evolution of Gossypium. Phylogenetic classification indicated that these Chis could be classified into six groups, with distinguishable structural characteristics. The expression patterns of Chis indicated their various expressions in different organs and tissues, and in the V. dahliae response. Silencing of Chi23, Chi32, or Chi47 in cotton significantly impaired the resistance to V. dahliae, suggesting these genes might act as positive regulators in disease resistance to V. dahliae. PMID:27354165

  13. Overexpression of cotton (Gossypium hirsutum) dirigent1 gene enhances lignification that blocks the spread of Verticillium dahliae.

    PubMed

    Shi, Haiyan; Liu, Zhihao; Zhu, Li; Zhang, Chaojun; Chen, Yun; Zhou, Ying; Li, Fuguang; Li, Xuebao

    2012-07-01

    Dirigent super-family abounds throughout the plant kingdom, especially vascular plants. To elucidate the function of cotton (Gossypium hirsutum) DIR genes in lignification, two cDNAs (designated GhDIR1 and GhDIR2) encoding putative dirigent proteins were isolated from cotton cDNA libraries. Real-time quantitative reverse transcription-polymerase chain reaction analysis revealed that GhDIR1 transcript was preferentially accumulated in cotton hypocotyls, whereas GhDIR2 was predominantly expressed in cotton fibers. Overexpression of GhDIR1 gene resulted in an increase in lignin content in transgenic cotton plants, compared with that of wild type. Histochemical assay revealed that the transgenic plants displayed more widespread lignification than that of wild type in epidermis and vascular bundle. Furthermore, the transgenic cotton plants displayed more tolerance to the infection of Verticillium dahliae. Our data suggest that GhDIR1 may be involved in cotton lignification which can block the spread of fungal pathogen V. dahliae.

  14. Discovery and identification of candidate genes from the chitinase gene family for Verticillium dahliae resistance in cotton.

    PubMed

    Xu, Jun; Xu, Xiaoyang; Tian, Liangliang; Wang, Guilin; Zhang, Xueying; Wang, Xinyu; Guo, Wangzhen

    2016-06-29

    Verticillium dahliae, a destructive and soil-borne fungal pathogen, causes massive losses in cotton yields. However, the resistance mechanism to V. dahilae in cotton is still poorly understood. Accumulating evidence indicates that chitinases are crucial hydrolytic enzymes, which attack fungal pathogens by catalyzing the fungal cell wall degradation. As a large gene family, to date, the chitinase genes (Chis) have not been systematically analyzed and effectively utilized in cotton. Here, we identified 47, 49, 92, and 116 Chis from four sequenced cotton species, diploid Gossypium raimondii (D5), G. arboreum (A2), tetraploid G. hirsutum acc. TM-1 (AD1), and G. barbadense acc. 3-79 (AD2), respectively. The orthologous genes were not one-to-one correspondence in the diploid and tetraploid cotton species, implying changes in the number of Chis in different cotton species during the evolution of Gossypium. Phylogenetic classification indicated that these Chis could be classified into six groups, with distinguishable structural characteristics. The expression patterns of Chis indicated their various expressions in different organs and tissues, and in the V. dahliae response. Silencing of Chi23, Chi32, or Chi47 in cotton significantly impaired the resistance to V. dahliae, suggesting these genes might act as positive regulators in disease resistance to V. dahliae.

  15. Isolation and functional analysis of the pathogenicity-related gene VdPR3 from Verticillium dahliae on cotton.

    PubMed

    Zhang, Ya-Lin; Li, Zhi-Fang; Feng, Zi-Li; Feng, Hong-Jie; Zhao, Li-Hong; Shi, Yong-Qiang; Hu, Xiao-Ping; Zhu, He-Qin

    2015-11-01

    The fungal plant pathogen Verticillium dahliae is the causal agent of vascular wilt, a disease that can seriously diminish cotton fiber yield. The pathogenicity mechanism and the identity of the genes that interact with cotton during the infection process still remain unclear. In this study, we investigated the low-pathogenic, non-microsclerotium-producing mutant vdpr3 obtained in a previous study from the screening of a T-DNA insertional library of the highly virulent isolate Vd080; the pathogenicity-related gene (VdPR3) in wild-type strain Vd080 was cloned. Knockout mutants (ΔVdPR3) showed lower mycelium growth and obvious reduction in sporulation ability without microsclerotium formation. An evaluation of carbon utilization in mutants and wild-type isolate Vd080 demonstrated that mutants-lacking VdPR3 exhibited decreased cellulase and amylase activities, which was restored in the complementary mutants (ΔVdPR3-C) to levels similar to those of Vd080. ΔVdPR3 postponed infectious events in cotton and showed a significant reduction in pathogenicity. Reintroduction of a functional VdPR3 copy into ΔVdPR3-C restored the ability to infect cotton plants. These results suggest that VdPR3 is a multifunctional gene involved in growth development, extracellular enzyme activity, and virulence of V. dahliae on cotton.

  16. Functional analysis of autophagy genes via Agrobacterium-mediated transformation in the vascular Wilt fungus Verticillium dahliae.

    PubMed

    Zhou, Lei; Zhao, Jun; Guo, Wangzhen; Zhang, Tianzhen

    2013-08-20

    Autophagy is a widely conserved intracellular process for degradation and recycling of proteins, organelles and cytoplasm in eukaryotic organisms and is now emerging as an important process in foliar infection by many plant pathogenic fungi. However, the role of autophagy in soil-borne fungal physiology and infection biology is poorly understood. Here, we report the establishment of an Agrobacterium tumefaciens-mediated transformation (ATMT) system and its application to investigate two autophagy genes, VdATG8 and VdATG12, by means of targeted gene replacement and complementation. Transformation of a cotton-infecting Verticillium dahliae strain Vd8 with a novel binary vector pCOM led to the production of 384 geneticin-resistant transformants per 1 × 10(6) conidia. V. dahliae mutants lacking either VdATG8 or VdATG12 exhibited reduced conidiation and impaired aerial hyphae production. Disease development on Arabidopsis plants was slightly delayed when inoculated with VdATG8 or VdATG12 gene deletion mutants, compared with the wild-type and gene complemented strains. Surprisingly, in vitro inoculation with unimpaired roots revealed that the abilities of root invasion were not affected in gene deletion mutants. These results indicate that autophagy is necessary for aerial hyphae development and plant colonization but not for root infection in V. dahliae.

  17. Manipulating inoculum densities of Verticillium dahliae and Pratylenchus penetrans with green manure amendments and solarization influence potato yield.

    PubMed

    Macguidwin, A E; Knuteson, D L; Connell, T; Bland, W L; Bartelt, K D

    2012-05-01

    We used cover crops with demonstrated efficacy against Verticillium dahliae and Pratylenchus penetrans in combination with the biocidal practice of solarization to determine the importance of targeting both organisms for managing potato early dying, an issue relevant to the search for alternatives to soil fumigation. Two experiments were conducted in commercial fields using a split-plot design with cover crop treatments of rapeseed, marigold, forage pearl millet, sorghum-sudangrass, and corn as the main plot factor and solarization as the subplot factor. Cover crops were grown and solarization applied in year one, followed by potato in year two. The main effect of solarization was significant for reduced inoculum levels of both organisms in year two and increased tuber yields. The main effect of cover crop was also significant with lower population densities of P. penetrans following the marigold and millet treatments and of V. dahliae following rape and sorghum-sudangrass. The cover crop treatments influenced yield in only one of the experiments in the absence of solarization. The combinatorial effect of cover crops and solarization resulted in a wide range of pathogen population densities. Mean soil inoculum levels were negatively related to yield for V. dahliae in experiment 1, and for P. penetrans and the P. penetrans × V. dahliae interaction in both experiments.

  18. VdThit, a Thiamine Transport Protein, Is Required for Pathogenicity of the Vascular Pathogen Verticillium dahliae.

    PubMed

    Qi, Xiliang; Su, Xiaofeng; Guo, Huiming; Qi, Juncang; Cheng, Hongmei

    2016-07-01

    Verticillium dahliae causes a serious wilt disease of important crops and is difficult to control. Few plasma-membrane transport proteins for nutrient acquisition have been identified for this fungus, and their involvement in the disease process is unknown. Here, a plasma-membrane protein, the V. dahliae thiamine transporter protein VdThit, was characterized functionally by deletion of the VdThit gene in V. dahliae. Disruption strains were viable, but growth and conidial germination and production were reduced and virulence was impaired. Interestingly, by supplementing exogenous thiamine, growth, conidiation, and virulence of the VdΔThit mutants were partially restored. Stress-tolerance assays showed that the VdΔThit mutant strains were markedly more susceptible to oxidative stress and UV damage. High-pressure liquid chromatography-mass spectrometry (HPLC-MS) and gas chromatography-mass spectrometry (GC-MS) analyses showed low levels of pyruvate metabolism intermediates acetoin and acetyl coenzyme A (acetyl-CoA) in the VdΔThit mutant strains, suggesting that pyruvate metabolism was suppressed. Expression analysis of VdThit confirmed the importance of VdThit in vegetative growth, reproduction, and invasive hyphal growth. Furthermore, a green fluorescent protein (GFP)-labeled VdΔThit mutant (VdΔThit-7-GFP) was suppressed in initial infection and root colonization, as viewed with light microscopy. Together, these results showed that VdThit plays an indispensable role in the pathogenicity of V. dahliae.

  19. The Innate Immune Signaling System as a Regulator of Disease Resistance and Induced Systemic Resistance Activity Against Verticillium dahliae.

    PubMed

    Gkizi, Danai; Lehmann, Silke; L'Haridon, Floriane; Serrano, Mario; Paplomatas, Epaminondas J; Métraux, Jean-Pierre; Tjamos, Sotirios E

    2016-04-01

    In the last decades, the plant innate immune responses against pathogens have been extensively studied, while biocontrol interactions between soilborne fungal pathogens and their hosts have received much less attention. Treatment of Arabidopsis thaliana with the nonpathogenic bacterium Paenibacillus alvei K165 was shown previously to protect against Verticillium dahliae by triggering induced systemic resistance (ISR). In the present study, we evaluated the involvement of the innate immune response in the K165-mediated protection of Arabidopsis against V. dahliae. Tests with Arabidopsis mutants impaired in several regulators of the early steps of the innate immune responses, including fls2, efr-1, bak1-4, mpk3, mpk6, wrky22, and wrky29 showed that FLS2 and WRKY22 have a central role in the K165-triggered ISR, while EFR1, MPK3, and MPK6 are possible susceptibility factors for V. dahliae and bak1 shows a tolerance phenomenon. The resistance induced by strain K165 is dependent on both salicylate and jasmonate-dependent defense pathways, as evidenced by an increased transient accumulation of PR1 and PDF1.2 transcripts in the aerial parts of infected plants treated with strain K165.

  20. Perturbations in the Primary Metabolism of Tomato and Arabidopsis thaliana Plants Infected with the Soil-Borne Fungus Verticillium dahliae.

    PubMed

    Buhtz, Anja; Witzel, Katja; Strehmel, Nadine; Ziegler, Jörg; Abel, Steffen; Grosch, Rita

    2015-01-01

    The hemibiotrophic soil-borne fungus Verticillium dahliae is a major pathogen of a number of economically important crop species. Here, the metabolic response of both tomato and Arabidopsis thaliana to V. dahliae infection was analysed by first using non-targeted GC-MS profiling. The leaf content of both major cell wall components glucuronic acid and xylose was reduced in the presence of the pathogen in tomato but enhanced in A. thaliana. The leaf content of the two tricarboxylic acid cycle intermediates fumaric acid and succinic acid was increased in the leaf of both species, reflecting a likely higher demand for reducing equivalents required for defence responses. A prominent group of affected compounds was amino acids and based on the targeted analysis in the root, it was shown that the level of 12 and four free amino acids was enhanced by the infection in, respectively, tomato and A. thaliana, with leucine and histidine being represented in both host species. The leaf content of six free amino acids was reduced in the leaf tissue of diseased A. thaliana plants, while that of two free amino acids was raised in the tomato plants. This study emphasizes the role of primary plant metabolites in adaptive responses when the fungus has colonized the plant.

  1. The G protein β subunit controls virulence and multiple growth- and development-related traits in Verticillium dahliae.

    PubMed

    Tzima, Aliki K; Paplomatas, Epaminondas J; Tsitsigiannis, Dimitrios I; Kang, Seogchan

    2012-04-01

    To gain insight into the role of G protein-mediated signaling in virulence and development of the soilborne, wilt causing fungus Verticillium dahliae, the G protein β subunit gene (named as VGB) was disrupted in tomato race 1 strain of V. dahliae. A resulting mutant strain, 70ΔGb15, displayed drastic reduction in virulence, increased microsclerotia formation and conidiation, and decreased ethylene production compared to the corresponding wild type (wt) strain 70wt-r1. Moreover, 70ΔGb15 exhibited an elongated rather than radial growth pattern on agar media. A transformant of 70ΔGb15 (named as 70ΔGbPKAC1) that carries an extra copy of VdPKAC1, a V. dahliae gene encoding the catalytic subunit of the cAMP-dependent protein kinase A, exhibited wt growth pattern and conidiation, was unable to form microsclerotia, produced high amounts of ethylene, and exhibited virulence between that of 70ΔGb15 and 70wt-r1 on tomato plants. Phenotypical changes observed in 70ΔGb15 and 70ΔGbPKAC1 correlated with transcriptional changes in several genes involved in signaling (MAP kinase VMK1) and development (hydrophobin VDH1 and ACC synthase ACS1) of V. dahliae. Results from the present work suggest a linkage between VGB and VdPKAC1 signaling pathways in regulating virulence, hormone production and development in V. dahliae.

  2. VdThit, a Thiamine Transport Protein, Is Required for Pathogenicity of the Vascular Pathogen Verticillium dahliae.

    PubMed

    Qi, Xiliang; Su, Xiaofeng; Guo, Huiming; Qi, Juncang; Cheng, Hongmei

    2016-07-01

    Verticillium dahliae causes a serious wilt disease of important crops and is difficult to control. Few plasma-membrane transport proteins for nutrient acquisition have been identified for this fungus, and their involvement in the disease process is unknown. Here, a plasma-membrane protein, the V. dahliae thiamine transporter protein VdThit, was characterized functionally by deletion of the VdThit gene in V. dahliae. Disruption strains were viable, but growth and conidial germination and production were reduced and virulence was impaired. Interestingly, by supplementing exogenous thiamine, growth, conidiation, and virulence of the VdΔThit mutants were partially restored. Stress-tolerance assays showed that the VdΔThit mutant strains were markedly more susceptible to oxidative stress and UV damage. High-pressure liquid chromatography-mass spectrometry (HPLC-MS) and gas chromatography-mass spectrometry (GC-MS) analyses showed low levels of pyruvate metabolism intermediates acetoin and acetyl coenzyme A (acetyl-CoA) in the VdΔThit mutant strains, suggesting that pyruvate metabolism was suppressed. Expression analysis of VdThit confirmed the importance of VdThit in vegetative growth, reproduction, and invasive hyphal growth. Furthermore, a green fluorescent protein (GFP)-labeled VdΔThit mutant (VdΔThit-7-GFP) was suppressed in initial infection and root colonization, as viewed with light microscopy. Together, these results showed that VdThit plays an indispensable role in the pathogenicity of V. dahliae. PMID:27089469

  3. Threshold microsclerotial inoculum for cotton verticillium wilt determined through wet-sieving and real-time quantitative PCR.

    PubMed

    Wei, Feng; Fan, Rong; Dong, Haitao; Shang, Wenjing; Xu, Xiangming; Zhu, Heqin; Yang, Jiarong; Hu, Xiaoping

    2015-02-01

    Quantification of Verticillium dahliae microsclerotia is an important component of wilt management on a range of crops. Estimation of microsclerotia by dry or wet sieving and plating of soil samples on semiselective medium is a commonly used technique but this method is resource-intensive. We developed a new molecular quantification method based on Synergy Brands (SYBR) Green real-time quantitative polymerase chain reaction of wet-sieving samples (wet-sieving qPCR). This method can detect V. dahliae microsclerotia as low as 0.5 CFU g(-1) of soil. There was a high correlation (r=0.98) between the estimates of conventional plating analysis and the new wet-sieving qPCR method for 40 soil samples. To estimate the inoculum threshold for cotton wilt, >400 soil samples were taken from the rhizosphere of individual plants with or without visual wilt symptoms in experimental and commercial cotton fields at the boll-forming stage. Wilt inoculum was estimated using the wet-sieving qPCR method and related to wilt development. The estimated inoculum threshold varied with cultivar, ranging from 4.0 and 7.0 CFU g(-1) of soil for susceptible and resistant cultivars, respectively. In addition, there was an overall relationship of wilt incidence with inoculum density across 31 commercial fields where a single composite soil sample was taken at each field, with an estimated inoculum threshold of 11 CFU g(-1) of soil. These results suggest that wilt risk can be predicted from the estimated soil inoculum density using the new wet-sieving qPCR method. We recommend the use of 4.0 and 7.0 CFU g(-1) as an inoculum threshold on susceptible and resistant cultivars, respectively, in practical risk prediction schemes.

  4. Identification of Molecular Variants in Mitochondrial DNAs of Members of the Genera Beauveria, Verticillium, Paecilomyces, Tolypocladium, and Metarhizium

    PubMed Central

    Hegedus, Dwayne D.; Khachatourians, George G.

    1993-01-01

    A set of five mitochondrial (mt) probes derived from a strain of Beauveria bassiana was used to evaluate the similarity of mtDNAs from 15 additional isolates of this fungus and five genera of other entomopathogenic fungi. The probes and genes encoded for (shown in parentheses) were pBbmtE2 (NADI, ATP6), pBbmtE3 (ATP6, small rRNA [srRNA]), pBbmtE4 (srRNA, CO3, NAD6), pBbSE1 (NAD6, tRNAVal, Ile, Ser, Trp, Pro, large rRNA [lrRNA]), and pBbXS1 (lrRNA). The probes produced identical hybridization patterns in EcoRI-digested DNA from nearly all isolates of B. bassiana and Beauveria caledonica. Similar patterns were also observed with Beauveria densa. The isolates of B. caledonica and B. densa DNAs could be differentiated from each other and from B. bassiana on the basis of a HindIII digestion and probing with pBbmtE3. Probe pBbmtE2 produced either a 5.0-kb or a 4.1-kb band in all of the B. bassiana isolates. This observation was used to categorize the mtDNA of B. bassiana into two types, designated A and B. Hybridization of the five probes produced distinct banding patterns in Beauveria brongniartii, Tolypocladium cylindrosporum, Tolypocladium nivea, Metarhizium anisopliae, Verticillium lecanii, and Paecilomyces farinosus. Hybridizations carried out with multiple probes simultaneously present produced unique patterns which characterized the B. bassiana group from all other fungi tested. These results are discussed in terms of how mtDNA polymorphisms in B. bassiana may relate to natural population structures, mt transmission in deuteromycetes, and the use of mtDNA polymorphisms in structural analysis of mtDNA. Images PMID:16349124

  5. Small GTPase Rac1 and its interaction partner Cla4 regulate polarized growth and pathogenicity in Verticillium dahliae.

    PubMed

    Tian, Hui; Zhou, Lei; Guo, Wangzhen; Wang, Xinyu

    2015-01-01

    Rac1 is a small GTPase coordinating diverse cellular functions such as cell polarity, vesicular trafficking, the cell cycle and transcriptional dynamics in many organisms. In this study, we investigate the biological functions of VdRac1, a Rac1 homolog in the soil-borne, wilt-causing fungus Verticillium dahliae. The VdRac1 gene was deleted in a V. dahliae virulence strain Vd8 isolated from a local cotton cultivar. ΔVdrac1 mutants display drastic reduction in colony expansion and form compact, convoluted colonies, show hyper-branching, loss of polarity and ability to penetrate, leading to severely reduced virulence. The p21-activated kinase Cla4 (named as VdCla4 in V. dahliae) null mutants ΔVdcla4 share identical phenotypes with ΔVdrac1. Yeast two-hybrid studies prove that VdCla4 is an effector of VdRac1. Localizations of actin and reactive oxygen species (ROS) in ΔVdrac1 and ΔVdcla4 compared with the corresponding wild-type strain reveal that VdRac1 and VdCla4 play a primary role in polarized hyphal growth via organization of ROS and play only a minor role in the organization of actin. The Vdrac1 and Vdcla4 null mutants are defective in conidiation and trace elements can partially compensate for the defect. Our data demonstrate that VdRac1 regulates polarized growth and pathogenicity by interacting with its effector VdCla4 in V. dahliae.

  6. [Elaboration of the in vitro model system to study the interaction of phytopathogenic mollicutes with plant cells].

    PubMed

    Korobkova, K S; Onyshchenko, A M; Panchenko, L P; Mamchur, O Ie; Dmytruk, O O; Red'ko, V I

    2009-01-01

    The model system based on the sugar beet calluses infected by mycoplasms (mollicutes) was elaborated, and changes in the callus cells morphology under the effect of these microorganisms were also studied. The calluses of sugar beet 3K51 cultivated on the Gamborg medium were infected by phytopathogenic mollicute Acholaplasma laidlawii var. granulum str.118. Under the effect of mollicute infection one could observe changes in the cell morphology of sugar beet calluses: the plant cells were transformed from round to lengthened, the intensity of polyploids forming was increased, their grouping and their total destruction were observed. Data of electron microscopy confirm the presence of the mollicute in the sugar beet calluses: acholeplasma cells were localized between and within undifferentiated plant cells. PMID:19938618

  7. Acyl-homoserine lactones from Erwinia psidii R. IBSBF 435T, a guava phytopathogen (Psidium guajava L.).

    PubMed

    Pomini, Armando M; Manfio, Gilson P; Araújo, Welington L; Marsaioli, Anita J

    2005-08-10

    The phytopathogen Erwinia psidii R. IBSBF 435T causes rot in branches, flowers, and fruits of guava (Psidium guajava L.), being responsible for crop losses, and has no effective control. It was demonstrated that this strain produces two compounds [S-(-)-N-hexanoyl and N-heptanoyl-homoserine lactone], both belonging to the class of quorum-sensing signaling substances. A protocol using gas chromatography-flame ionization detection with chiral stationary phase is described for the absolute configuration determination of a natural acyl-homoserine lactone. Biological assays with specific reporter and synthesis of identified substances are also described. This is the first report on the N-heptanoyl-homoserine lactone occurrence in the Erwinia genus.

  8. [Enhanced resistance to phytopathogenic bacteria in transgenic tobacco plants with synthetic gene of antimicrobial peptide cecropin P1].

    PubMed

    Zakharchenko, N S; Rukavtsova, E B; Gudkov, A T; Bur'ianov, Ia I

    2005-11-01

    Plasmids with a synthetic gene of the mammalian antimicrobial peptide cecropin P1 (cecP1) controlled by the constitutive promoter 35S RNA of cauliflower mosaic virus were constructed. Agrobacterial transformation of tobacco plants was conducted using the obtained recombinant binary vector. The presence of gene cecP1 in the plant genome was confirmed by PCR. The expression of gene cecP1 in transgenic plants was shown by Northern blot analysis. The obtained transgenic plants exhibit enhanced resistance to phytopathogenic bacteria Pseudomonas syringae, P. marginata, and Erwinia carotovora. The ability of transgenic plants to express cecropin P1 was transmitted to the progeny. F1 and F2 plants had the normal phenotype (except for a changed coloration of flowers) and retained the ability to produce normal viable seeds upon self-pollination. Lines of F1 plants with Mendelian segregation of transgenic traits were selected.

  9. Genomic characterization of ϕRS603, a filamentous bacteriophage that is infectious to the phytopathogen Ralstonia solanacearum.

    PubMed

    Van, Truong Thi Bich; Yoshida, Shohei; Miki, Kaito; Kondo, Akihiro; Kamei, Kaeko

    2014-12-01

    A filamentous bacteriophage (ϕ), ϕRS603, which is infectious to the phytopathogen Ralstonia solanacearum was isolated. ϕRS603 was found to have a circular single-stranded DNA genome composed of 7679 nucleotides and to contain 13 putative open reading frames (ORFs). The ϕRS603 genome showed strong similarity with those of Ralstonia phages ϕRSM1 and ϕRSM3, as reported by Askora et al. The ϕRS603 genome had no ORFs corresponding to ORFs 2, 3, 13 and 14 (integrase) of ϕRSM3. ϕRS603 had an ORF that was homologous to other Ralstonia phages ϕRSS0 and ϕRSS1; however, ϕRSM1 and ϕRSM3 did not.

  10. Highly selective biotransformation of ginsenoside Rb1 to Rd by the phytopathogenic fungus Cladosporium fulvum (syn. Fulvia fulva).

    PubMed

    Zhao, Xuesong; Wang, Juan; Li, Jie; Fu, Ling; Gao, Juan; Du, Xiuli; Bi, Hongtao; Zhou, Yifa; Tai, Guihua

    2009-05-01

    Fourteen phytopathogenic fungi were tested for their ability to transform the major ginsenosides to the active minor ginsenoside Rd. The transformation products were identified by TLC and HPLC, and their structures were assigned by NMR analysis. Cladosporium fulvum, a tomato pathogen, was found to transform major ginsenoside Rb(1) to Rd as the sole product. The following optimum conditions for transforming Rd by C. fulvum were determined: the time of substrate addition, 24 h; substrate concentration, 0.25 mg ml(-1); temperature, 37 degrees C; pH 5.0; and biotransformation period, 8 days. At these optimum conditions, the maximum yield was 86% (molar ratio). Further, a preparative scale transformation with C. fulvum was performed at a dose of 100 mg of Rb(1) by a yield of 80%. This fungus has potential to be applied on the preparation for Rd in pharmaceutical industry.

  11. The Thioredoxin GbNRX1 Plays a Crucial Role in Homeostasis of Apoplastic Reactive Oxygen Species in Response to Verticillium dahliae Infection in Cotton1[OPEN

    PubMed Central

    Zhang, Jie; Sun, Shu-Tao; Feng, De-Qin; Yang, Chun-Lin; Sun, Yong-Duo; Zhong, Nai-Qin

    2016-01-01

    Examining the proteins that plants secrete into the apoplast in response to pathogen attack provides crucial information for understanding the molecular mechanisms underlying plant innate immunity. In this study, we analyzed the changes in the root apoplast secretome of the Verticillium wilt-resistant island cotton cv Hai 7124 (Gossypium barbadense) upon infection with Verticillium dahliae. Two-dimensional differential gel electrophoresis and matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry analysis identified 68 significantly altered spots, corresponding to 49 different proteins. Gene ontology annotation indicated that most of these proteins function in reactive oxygen species (ROS) metabolism and defense response. Of the ROS-related proteins identified, we further characterized a thioredoxin, GbNRX1, which increased in abundance in response to V. dahliae challenge, finding that GbNRX1 functions in apoplastic ROS scavenging after the ROS burst that occurs upon recognition of V. dahliae. Silencing of GbNRX1 resulted in defective dissipation of apoplastic ROS, which led to higher ROS accumulation in protoplasts. As a result, the GbNRX1-silenced plants showed reduced wilt resistance, indicating that the initial defense response in the root apoplast requires the antioxidant activity of GbNRX1. Together, our results demonstrate that apoplastic ROS generation and scavenging occur in tandem in response to pathogen attack; also, the rapid balancing of redox to maintain homeostasis after the ROS burst, which involves GbNRX1, is critical for the apoplastic immune response. PMID:26869704

  12. Identification of MiRNA from Eggplant (Solanum melongena L.) by Small RNA Deep Sequencing and Their Response to Verticillium dahliae Infection

    PubMed Central

    Yang, Liu; Jue, Dengwei; Li, Wang; Zhang, Ruijie; Chen, Min; Yang, Qing

    2013-01-01

    MiRNAs are a class of non-coding small RNAs that play important roles in the regulation of gene expression. Although plant miRNAs have been extensively studied in model systems, less is known in other plants with limited genome sequence data, including eggplant (Solanum melongena L.). To identify miRNAs in eggplant and their response to Verticillium dahliae infection, a fungal pathogen for which clear understanding of infection mechanisms and effective cure methods are currently lacking, we deep-sequenced two small RNA (sRNA) libraries prepared from mock-infected and infected seedlings of eggplants. Specifically, 30,830,792 reads produced 7,716,328 unique miRNAs representing 99 known miRNA families that have been identified in other plant species. Two novel putative miRNAs were predicted with eggplant ESTs. The potential targets of the identified known and novel miRNAs were also predicted based on sequence homology search. It was observed that the length distribution of obtained sRNAs and the expression of 6 miRNA families were obviously different between the two libraries. These results provide a framework for further analysis of miRNAs and their role in regulating plant response to fungal infection and Verticillium wilt in particular. PMID:24015279

  13. Colonization process of olive tissues by Verticillium dahliae and its in planta interaction with the biocontrol root endophyte Pseudomonas fluorescens PICF7

    PubMed Central

    Prieto, Pilar; Navarro‐Raya, Carmen; Valverde‐Corredor, Antonio; Amyotte, Stefan G.; Dobinson, Katherine F.; Mercado‐Blanco, Jesús

    2009-01-01

    Summary The colonization process of Olea europaea by the defoliating pathotype of Verticillium dahliae, and the in planta interaction with the endophytic, biocontrol strain Pseudomonas fluorescens PICF7 were determined. Differential fluorescent protein tagging was used for the simultaneous visualization of P. fluorescens PICF7 and V. dahliae in olive tissues. Olive plants were bacterized with PICF7 and then transferred to V. dahliae‐infested soil. Monitoring olive colonization events by V. dahliae and its interaction with PICF7 was conducted using a non‐gnotobiotic system, confocal laser scanner microscopy and tissue vibratoming sections. A yellow fluorescently tagged V. dahliae derivative (VDAT‐36I) was obtained by Agrobacterium tumefaciens‐mediated transformation. Isolate VDAT‐36I quickly colonized olive root surface, successfully invaded root cortex and vascular tissues via macro‐ and micro‐breakages, and progressed to the aerial parts of the plant through xylem vessel cells. Strain PICF7 used root hairs as preferred penetration site, and once established on/in root tissues, hindered pathogen colonization. For the first time using this approach, the entire colonization process of a woody plant by V. dahliae is reported. Early and localized root surface and root endophytic colonization by P. fluorescens PICF7 is needed to impair full progress of verticillium wilt epidemics in olive. PMID:21255281

  14. The cotton MYB108 forms a positive feedback regulation loop with CML11 and participates in the defense response against Verticillium dahliae infection.

    PubMed

    Cheng, Huan-Qing; Han, Li-Bo; Yang, Chun-Lin; Wu, Xiao-Min; Zhong, Nai-Qin; Wu, Jia-He; Wang, Fu-Xin; Wang, Hai-Yun; Xia, Gui-Xian

    2016-03-01

    Accumulating evidence indicates that plant MYB transcription factors participate in defense against pathogen attack, but their regulatory targets and related signaling processes remain largely unknown. Here, we identified a defense-related MYB gene (GhMYB108) from upland cotton (Gossypium hirsutum) and characterized its functional mechanism. Expression of GhMYB108 in cotton plants was induced by Verticillium dahliae infection and responded to the application of defense signaling molecules, including salicylic acid, jasmonic acid, and ethylene. Knockdown of GhMYB108 expression led to increased susceptibility of cotton plants to V. dahliae, while ecotopic overexpression of GhMYB108 in Arabidopsis thaliana conferred enhanced tolerance to the pathogen. Further analysis demonstrated that GhMYB108 interacted with the calmodulin-like protein GhCML11, and the two proteins form a positive feedback loop to enhance the transcription of GhCML11 in a calcium-dependent manner. Verticillium dahliae infection stimulated Ca(2+) influx into the cytosol in cotton root cells, but this response was disrupted in both GhCML11-silenced plants and GhMYB108-silenced plants in which expression of several calcium signaling-related genes was down-regulated. Taken together, these results indicate that GhMYB108 acts as a positive regulator in defense against V. dahliae infection by interacting with GhCML11. Furthermore, the data also revealed the important roles and synergetic regulation of MYB transcription factor, Ca(2+), and calmodulin in plant immune responses.

  15. Ectopic expression of a Ve homolog VvVe gene from Vitis vinifera enhances defense response to Verticillium dahliae infection in tobacco.

    PubMed

    Tang, Juan; Lin, Jing; Yang, Yuwen; Chen, Tianzi; Ling, Xitie; Zhang, Baolong; Chang, Youhong

    2016-01-15

    Verticillium wilt is a soil borne disease that can cause devastating losses to the production of many economically important crops. A Ve1 homologous gene responding to Verticillium dahliae infection was identified in Vitis vinifera cv. "HeiFeng" by semi-quantitative reverse transcription polymerase chain reaction and was designated as VvVe. The overexpression of VvVe in transgenic Nicotiana benthamiana plants significantly enhanced the resistance to isolate V991 of V. dahliae when compared with the wild type plants. The expressions of defense-related genes including the salicylic acid regulated gene pathogen-related 1 (PR1) but not PR2, the ethylene- and jasmonic acid-regulated genes ethylene response factor 1 (ERF1) and lipoxygenase (LOX) were significantly increased due to over expression of VvVe. And greater accumulation of active oxygen, callose and phenylalanine-ammonia lyase were observed in the leaves of transgenic VvVe tobacco plants than the wild type when under infection by V. dahliae. Moreover, the hypersensitive response mimicking cell death was exclusively occurred in the transgenic VvVe tobacco plants but not in the wild type. Taken together, the VvVe gene is a Ve1 like gene which involves in the signal cascade of salicylic acid, jasmonate, and ethylene defense pathways and enhances defense response to V. dahliae infection in the transgenic tobacco.

  16. VdCYC8, Encoding CYC8 Glucose Repression Mediator Protein, Is Required for Microsclerotia Formation and Full Virulence in Verticillium dahliae.

    PubMed

    Li, Zhi-Fang; Liu, Yi-Jie; Feng, Zi-Li; Feng, Hong-Jie; Klosterman, Steven J; Zhou, Fang-Fang; Zhao, Li-Hong; Shi, Yong-Qiang; Zhu, He-Qin

    2015-01-01

    Verticillium dahliae is the primary causal agent for Verticillium wilt disease on a diverse array of economically important crops, including cotton. In previous research, we obtained the low-pathogenicity mutant T286 from the T-DNA insertional mutant library of the highly virulent isolate Vd080 derived from cotton. In this study, the target disrupted gene VdCYC8 was identified by TAIL-PCR, encoding a homolog of CYC8 proteins involved in glucose repression. The deletion mutant ΔCYC8 exhibited several developmental deficiencies, including reduced microsclerotia formation, reduced sporulation, and slower growth. Moreover, compared with the wild type strain Vd080, the pathogenicity of strain ΔCYC8 was significantly decreased on cotton seedlings. However, the complementary mutants ΔCYC8-C led to restoration of the wild type phenotype or near wild type levels of virulence on cotton. Interestingly, pathogenicity of the strains was correlated with VdCYC8 gene expression levels in complemented mutants. Gene expression analyses in the wild type strain Vd080, the ΔCYC8-45 strain, and complemented strain ΔCYC8-C26 indicated that VdCYC8 regulates the transcription levels of several genes in V. dahliae that have roles in melanin and production.

  17. Colonization process of olive tissues by Verticillium dahliae and its in planta interaction with the biocontrol root endophyte Pseudomonas fluorescens PICF7.

    PubMed

    Prieto, Pilar; Navarro-Raya, Carmen; Valverde-Corredor, Antonio; Amyotte, Stefan G; Dobinson, Katherine F; Mercado-Blanco, Jesús

    2009-07-01

    The colonization process of Olea europaea by the defoliating pathotype of Verticillium dahliae, and the in planta interaction with the endophytic, biocontrol strain Pseudomonas fluorescens PICF7 were determined. Differential fluorescent protein tagging was used for the simultaneous visualization of P. fluorescens PICF7 and V. dahliae in olive tissues. Olive plants were bacterized with PICF7 and then transferred to V. dahliae-infested soil. Monitoring olive colonization events by V. dahliae and its interaction with PICF7 was conducted using a non-gnotobiotic system, confocal laser scanner microscopy and tissue vibratoming sections. A yellow fluorescently tagged V. dahliae derivative (VDAT-36I) was obtained by Agrobacterium tumefaciens-mediated transformation. Isolate VDAT-36I quickly colonized olive root surface, successfully invaded root cortex and vascular tissues via macro- and micro-breakages, and progressed to the aerial parts of the plant through xylem vessel cells. Strain PICF7 used root hairs as preferred penetration site, and once established on/in root tissues, hindered pathogen colonization. For the first time using this approach, the entire colonization process of a woody plant by V. dahliae is reported. Early and localized root surface and root endophytic colonization by P. fluorescens PICF7 is needed to impair full progress of verticillium wilt epidemics in olive.

  18. VdCYC8, Encoding CYC8 Glucose Repression Mediator Protein, Is Required for Microsclerotia Formation and Full Virulence in Verticillium dahliae

    PubMed Central

    Li, Zhi-Fang; Liu, Yi-Jie; Feng, Zi-Li; Feng, Hong-Jie; Klosterman, Steven J.; Zhou, Fang-Fang; Zhao, Li-Hong; Shi, Yong-Qiang; Zhu, He-Qin

    2015-01-01

    Verticillium dahliae is the primary causal agent for Verticillium wilt disease on a diverse array of economically important crops, including cotton. In previous research, we obtained the low-pathogenicity mutant T286 from the T-DNA insertional mutant library of the highly virulent isolate Vd080 derived from cotton. In this study, the target disrupted gene VdCYC8 was identified by TAIL-PCR, encoding a homolog of CYC8 proteins involved in glucose repression. The deletion mutant ΔCYC8 exhibited several developmental deficiencies, including reduced microsclerotia formation, reduced sporulation, and slower growth. Moreover, compared with the wild type strain Vd080, the pathogenicity of strain ΔCYC8 was significantly decreased on cotton seedlings. However, the complementary mutants ΔCYC8-C led to restoration of the wild type phenotype or near wild type levels of virulence on cotton. Interestingly, pathogenicity of the strains was correlated with VdCYC8 gene expression levels in complemented mutants. Gene expression analyses in the wild type strain Vd080, the ΔCYC8-45 strain, and complemented strain ΔCYC8-C26 indicated that VdCYC8 regulates the transcription levels of several genes in V. dahliae that have roles in melanin and production. PMID:26633180

  19. Identification of MiRNA from eggplant (Solanum melongena L.) by small RNA deep sequencing and their response to Verticillium dahliae infection.

    PubMed

    Yang, Liu; Jue, Dengwei; Li, Wang; Zhang, Ruijie; Chen, Min; Yang, Qing

    2013-01-01

    MiRNAs are a class of non-coding small RNAs that play important roles in the regulation of gene expression. Although plant miRNAs have been extensively studied in model systems, less is known in other plants with limited genome sequence data, including eggplant (Solanum melongena L.). To identify miRNAs in eggplant and their response to Verticillium dahliae infection, a fungal pathogen for which clear understanding of infection mechanisms and effective cure methods are currently lacking, we deep-sequenced two small RNA (sRNA) libraries prepared from mock-infected and infected seedlings of eggplants. Specifically, 30,830,792 reads produced 7,716,328 unique miRNAs representing 99 known miRNA families that have been identified in other plant species. Two novel putative miRNAs were predicted with eggplant ESTs. The potential targets of the identified known and novel miRNAs were also predicted based on sequence homology search. It was observed that the length distribution of obtained sRNAs and the expression of 6 miRNA families were obviously different between the two libraries. These results provide a framework for further analysis of miRNAs and their role in regulating plant response to fungal infection and Verticillium wilt in particular.

  20. Plant Carbohydrate Scavenging through TonB-Dependent Receptors: A Feature Shared by Phytopathogenic and Aquatic Bacteria

    PubMed Central

    Boulanger, Alice; Lautier, Martine; Guynet, Catherine; Denancé, Nicolas; Vasse, Jacques

    2007-01-01

    TonB-dependent receptors (TBDRs) are outer membrane proteins mainly known for the active transport of iron siderophore complexes in Gram-negative bacteria. Analysis of the genome of the phytopathogenic bacterium Xanthomonas campestris pv. campestris (Xcc), predicts 72 TBDRs. Such an overrepresentation is common in Xanthomonas species but is limited to only a small number of bacteria. Here, we show that one Xcc TBDR transports sucrose with a very high affinity, suggesting that it might be a sucrose scavenger. This TBDR acts with an inner membrane transporter, an amylosucrase and a regulator to utilize sucrose, thus defining a new type of carbohydrate utilization locus, named CUT locus, involving a TBDR for the transport of substrate across the outer membrane. This sucrose CUT locus is required for full pathogenicity on Arabidopsis, showing its importance for the adaptation to host plants. A systematic analysis of Xcc TBDR genes and a genome context survey suggested that several Xcc TBDRs belong to other CUT loci involved in the utilization of various plant carbohydrates. Interestingly, several Xcc TBDRs and CUT loci are conserved in aquatic bacteria such as Caulobacter crescentus, Colwellia psychrerythraea, Saccharophagus degradans, Shewanella spp., Sphingomonas spp. or Pseudoalteromonas spp., which share the ability to degrade a wide variety of complex carbohydrates and display TBDR overrepresentation. We therefore propose that TBDR overrepresentation and the presence of CUT loci designate the ability to scavenge carbohydrates. Thus CUT loci, which seem to participate to the adaptation of phytopathogenic bacteria to their host plants, might also play a very important role in the biogeochemical cycling of plant-derived nutrients in marine environments. Moreover, the TBDRs and CUT loci identified in this study are clearly different from those characterized in the human gut symbiont Bacteroides thetaiotaomicron, which allow glycan foraging, suggesting a convergent

  1. Glucanases and chitinases as causal agents in the protection of Acacia extrafloral nectar from infestation by phytopathogens.

    PubMed

    González-Teuber, Marcia; Pozo, María J; Muck, Alexander; Svatos, Ales; Adame-Alvarez, Rosa M; Heil, Martin

    2010-03-01

    Nectars are rich in primary metabolites and attract mutualistic animals, which serve as pollinators or as an indirect defense against herbivores. Their chemical composition makes nectars prone to microbial infestation. As protective strategy, floral nectar of ornamental tobacco (Nicotiana langsdorffii x Nicotiana sanderae) contains "nectarins," proteins producing reactive oxygen species such as hydrogen peroxide. By contrast, pathogenesis-related (PR) proteins were detected in Acacia extrafloral nectar (EFN), which is secreted in the context of defensive ant-plant mutualisms. We investigated whether these PR proteins protect EFN from phytopathogens. Five sympatric species (Acacia cornigera, A. hindsii, A. collinsii, A. farnesiana, and Prosopis juliflora) were compared that differ in their ant-plant mutualism. EFN of myrmecophytes, which are obligate ant-plants that secrete EFN constitutively to nourish specialized ant inhabitants, significantly inhibited the growth of four out of six tested phytopathogenic microorganisms. By contrast, EFN of nonmyrmecophytes, which is secreted only transiently in response to herbivory, did not exhibit a detectable inhibitory activity. Combining two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis with nanoflow liquid chromatography-tandem mass spectrometry analysis confirmed that PR proteins represented over 90% of all proteins in myrmecophyte EFN. The inhibition of microbial growth was exerted by the protein fraction, but not the small metabolites of this EFN, and disappeared when nectar was heated. In-gel assays demonstrated the activity of acidic and basic chitinases in all EFNs, whereas glucanases were detected only in EFN of myrmecophytes. Our results demonstrate that PR proteins causally underlie the protection of Acacia EFN from microorganisms and that acidic and basic glucanases likely represent the most important prerequisite in this defensive function. PMID:20023149

  2. Do induced responses mediate the ecological interactions between the specialist herbivores and phytopathogens of an alpine plant?

    PubMed

    Röder, Gregory; Rahier, Martine; Naisbit, Russell E

    2011-01-01

    Plants are not passive victims of the myriad attackers that rely on them for nutrition. They have a suite of physical and chemical defences, and are even able to take advantage of the enemies of their enemies. These strategies are often only deployed upon attack, so may lead to indirect interactions between herbivores and phytopathogens. In this study we test for induced responses in wild populations of an alpine plant (Adenostyles alliariae) that possesses constitutive chemical defence (pyrrolizidine alkaloids) and specialist natural enemies (two species of leaf beetle, Oreina elongata and Oreina cacaliae, and the phytopathogenic rust Uromyces cacaliae). Plants were induced in the field using chemical elicitors of the jasmonic acid (JA) and salicylic acid (SA) pathways and monitored for one month under natural conditions. There was evidence for induced resistance, with lower probability and later incidence of attack by beetles in JA-induced plants and of rust infection in SA-induced plants. We also demonstrate ecological cross-effects, with reduced fungal attack following JA-induction, and a cost of SA-induction arising from increased beetle attack. As a result, there is the potential for negative indirect effects of the beetles on the rust, while in the field the positive indirect effect of the rust on the beetles appears to be over-ridden by direct effects on plant nutritional quality. Such interactions resulting from induced susceptibility and resistance must be considered if we are to exploit plant defences for crop protection using hormone elicitors or constitutive expression. More generally, the fact that induced defences are even found in species that possess constitutively-expressed chemical defence suggests that they may be ubiquitous in higher plants.

  3. Study on interaction between root-knot nematode Meloidogyne javanica and wilt fungus Verticillium dahliae on olive seedlings in greenhouse.

    PubMed

    Saeedizadeh, A; Kheiri, A; Okhovat, M; Hoseininejad, A

    2003-01-01

    Verticillium dahliae has been reported as a limiting factor in cotton, olive, potato and tomato fields from several countries in the world. Root-knot nematodes Meloidogyne javanica causes considerable damage to olive groves in olive growing areas. Since the presence of these two pathogens in olive trees and seedlings were confirmed in Golestan Province, this study was proposed to find the mode of their action and interaction with olive seedlings in greenhouse. The non-defoliant strain of the fungus (SS-4) was isolated from olive groves showing symptom in Golestan Province. M. javanica was also recovered from the infested olive seedlings. After species identification, it was reared on tomato seedlings var. Rutgers. The larvae were used as a source of inoculum. Conidia and microsclerotia of V. dahliae were used as a source of inoculum for pathogenesis in this study. Stem cuttings of olive cultivar Zard were transplanted in different sets of pots containing 720 ml. of sterilized loamy soil and sandy soil. Experiment was conducted in Completely Randomized Design with 6 treatments and 8 replicates including control, nematode alone, fungus alone, nematode and fungus simultaneously, nematode and fungus concomitantly, fungus two weeks prior to nematode, nematode and fungus concomitantly, nematode two weeks prior to fungus. Pots were inoculated with 1500 larvae of nematodes and 7200 microsclerotia of V. dahliae. Experiment was terminated after 9 months and following parameters were determined i.e. fresh weight of roots, number of galls and females, per root system and discoloration of leaf and root tissues. Presence of nematode prior to fungus caused reduction in colonization of fungus in the roots and the stems and vis presence of fungus prior to nematode caused reduction in number of galls produced by nematode. Sever symptom on aerial parts of plant was observed when both pathogens were inoculated simultaneously. However fresh weight of roots was reduced in all treatments

  4. Study on interaction between root-knot nematode Meloidogyne javanica and wilt fungus Verticillium dahliae on olive seedlings in greenhouse.

    PubMed

    Saeedizadeh, A; Kheiri, A; Okhovat, M; Hoseininejad, A

    2003-01-01

    Verticillium dahliae has been reported as a limiting factor in cotton, olive, potato and tomato fields from several countries in the world. Root-knot nematodes Meloidogyne javanica causes considerable damage to olive groves in olive growing areas. Since the presence of these two pathogens in olive trees and seedlings were confirmed in Golestan Province, this study was proposed to find the mode of their action and interaction with olive seedlings in greenhouse. The non-defoliant strain of the fungus (SS-4) was isolated from olive groves showing symptom in Golestan Province. M. javanica was also recovered from the infested olive seedlings. After species identification, it was reared on tomato seedlings var. Rutgers. The larvae were used as a source of inoculum. Conidia and microsclerotia of V. dahliae were used as a source of inoculum for pathogenesis in this study. Stem cuttings of olive cultivar Zard were transplanted in different sets of pots containing 720 ml. of sterilized loamy soil and sandy soil. Experiment was conducted in Completely Randomized Design with 6 treatments and 8 replicates including control, nematode alone, fungus alone, nematode and fungus simultaneously, nematode and fungus concomitantly, fungus two weeks prior to nematode, nematode and fungus concomitantly, nematode two weeks prior to fungus. Pots were inoculated with 1500 larvae of nematodes and 7200 microsclerotia of V. dahliae. Experiment was terminated after 9 months and following parameters were determined i.e. fresh weight of roots, number of galls and females, per root system and discoloration of leaf and root tissues. Presence of nematode prior to fungus caused reduction in colonization of fungus in the roots and the stems and vis presence of fungus prior to nematode caused reduction in number of galls produced by nematode. Sever symptom on aerial parts of plant was observed when both pathogens were inoculated simultaneously. However fresh weight of roots was reduced in all treatments

  5. Expression of antimicrobial peptides thanatin(S) in transgenic Arabidopsis enhanced resistance to phytopathogenic fungi and bacteria.

    PubMed

    Wu, Tingquan; Tang, Dingzhong; Chen, Weida; Huang, Hexun; Wang, Rui; Chen, Yongfang

    2013-09-15

    Thanatin(S) is an analog of thanatin, an insect antimicrobial peptide possessing strong and broad spectrum of antimicrobial activity. In order to investigate if the thanatin could be used in engineering transgenic plants for increased resistance against phytopathogens, the synthetic thanatin(S) was introduced into Arabidopsis thaliana plants. To increase the expression level of thanatin(S) in plants, the coding sequence was optimized by plant-preference codon. To avoid cellular protease degradation, signal peptide of rice Cht1 was fused to N terminal of thanatin(S) for secreting the expressed thanatin(S) into intercellular spaces. To evaluate the application value of thanatin(S) in plant disease control, the synthesized coding sequence of Cht1 signal peptide (Cht1SP)-thanatin(S) was ligated to plant gateway destination binary vectors pGWB11 (with FLAG tag). Meanwhile, in order to observe the subcellular localization of Cht1SP-thanatin(S)-GFP and thanatin(S)-GFP, the sequences of Cht1SP-thanatin(S) and thanatin(S) were respectively linked to pGWB5 (with GFP tag). The constructs were transformed into Arabidopsis ecotype Col-0 and mutant pad4-1 via Agrobacterium-mediated transformation. The transformants with Cht1SP-thanatin(S)-FLAG fusion gene were analyzed by genomic PCR, real-time PCR, and western blots and the transgenic Arabidopsis plants introduced respectively Cht1SP-thanatin(S)-GFP and thanatin(S)-GFP were observed by confocal microscopy. Transgenic plants expressing Cht1SP-thanatin(S)-FLAG fusion protein showed antifungal activity against Botrytis cinerea and powdery mildew, as well as antibacterial activity against Pseudomonas syringae pv. tomato. And the results from confocal observation showed that the GFP signal from Cht1SP-thanatin(S)-GFP transgenic Arabidopsis plants occurred mainly in intercellular space, while that from thanatin(S)-GFP transgenic plants was mainly detected in the cytoplasm and that from empty vector transgenic plants was distributed

  6. Complete genome sequence of Pseudomonas fluorescens strain PICF7, an indigenous root endophyte from olive (Olea europaea L.) and effective biocontrol agent against Verticillium dahliae.

    PubMed

    Martínez-García, Pedro Manuel; Ruano-Rosa, David; Schilirò, Elisabetta; Prieto, Pilar; Ramos, Cayo; Rodríguez-Palenzuela, Pablo; Mercado-Blanco, Jesús

    2015-01-01

    Pseudomonas fluorescens strain PICF7 is a native endophyte of olive roots. Previous studies have shown this motile, Gram-negative, non-sporulating bacterium is an effective biocontrol agent against the soil-borne fungus Verticillium dahliae, the causal agent of one of the most devastating diseases for olive (Olea europaea L.) cultivation. Here, we announce and describe the complete genome sequence of Pseudomonas fluorescens strain PICF7 consisting of a circular chromosome of 6,136,735 bp that encodes 5,567 protein-coding genes and 88 RNA-only encoding genes. Genome analysis revealed genes predicting factors such as secretion systems, siderophores, detoxifying compounds or volatile components. Further analysis of the genome sequence of PICF7 will help in gaining insights into biocontrol and endophytism.

  7. Complete genome sequence of Pseudomonas fluorescens strain PICF7, an indigenous root endophyte from olive (Olea europaea L.) and effective biocontrol agent against Verticillium dahliae

    PubMed Central

    2015-01-01

    Pseudomonas fluorescens strain PICF7 is a native endophyte of olive roots. Previous studies have shown this motile, Gram-negative, non-sporulating bacterium is an effective biocontrol agent against the soil-borne fungus Verticillium dahliae, the causal agent of one of the most devastating diseases for olive (Olea europaea L.) cultivation. Here, we announce and describe the complete genome sequence of Pseudomonas fluorescens strain PICF7 consisting of a circular chromosome of 6,136,735 bp that encodes 5,567 protein-coding genes and 88 RNA-only encoding genes. Genome analysis revealed genes predicting factors such as secretion systems, siderophores, detoxifying compounds or volatile components. Further analysis of the genome sequence of PICF7 will help in gaining insights into biocontrol and endophytism. PMID:25685259

  8. Complete genome sequence of Pseudomonas fluorescens strain PICF7, an indigenous root endophyte from olive (Olea europaea L.) and effective biocontrol agent against Verticillium dahliae.

    PubMed

    Martínez-García, Pedro Manuel; Ruano-Rosa, David; Schilirò, Elisabetta; Prieto, Pilar; Ramos, Cayo; Rodríguez-Palenzuela, Pablo; Mercado-Blanco, Jesús

    2015-01-01

    Pseudomonas fluorescens strain PICF7 is a native endophyte of olive roots. Previous studies have shown this motile, Gram-negative, non-sporulating bacterium is an effective biocontrol agent against the soil-borne fungus Verticillium dahliae, the causal agent of one of the most devastating diseases for olive (Olea europaea L.) cultivation. Here, we announce and describe the complete genome sequence of Pseudomonas fluorescens strain PICF7 consisting of a circular chromosome of 6,136,735 bp that encodes 5,567 protein-coding genes and 88 RNA-only encoding genes. Genome analysis revealed genes predicting factors such as secretion systems, siderophores, detoxifying compounds or volatile components. Further analysis of the genome sequence of PICF7 will help in gaining insights into biocontrol and endophytism. PMID:25685259

  9. The cotton MYB108 forms a positive feedback regulation loop with CML11 and participates in the defense response against Verticillium dahliae infection

    PubMed Central

    Cheng, Huan-Qing; Han, Li-Bo; Yang, Chun-Lin; Wu, Xiao-Min; Zhong, Nai-Qin; Wu, Jia-He; Wang, Fu-Xin; Xia, Gui-Xian

    2016-01-01

    Accumulating evidence indicates that plant MYB transcription factors participate in defense against pathogen attack, but their regulatory targets and related signaling processes remain largely unknown. Here, we identified a defense-related MYB gene (GhMYB108) from upland cotton (Gossypium hirsutum) and characterized its functional mechanism. Expression of GhMYB108 in cotton plants was induced by Verticillium dahliae infection and responded to the application of defense signaling molecules, including salicylic acid, jasmonic acid, and ethylene. Knockdown of GhMYB108 expression led to increased susceptibility of cotton plants to V. dahliae, while ecotopic overexpression of GhMYB108 in Arabidopsis thaliana conferred enhanced tolerance to the pathogen. Further analysis demonstrated that GhMYB108 interacted with the calmodulin-like protein GhCML11, and the two proteins form a positive feedback loop to enhance the transcription of GhCML11 in a calcium-dependent manner. Verticillium dahliae infection stimulated Ca2+ influx into the cytosol in cotton root cells, but this response was disrupted in both GhCML11-silenced plants and GhMYB108-silenced plants in which expression of several calcium signaling-related genes was down-regulated. Taken together, these results indicate that GhMYB108 acts as a positive regulator in defense against V. dahliae infection by interacting with GhCML11. Furthermore, the data also revealed the important roles and synergetic regulation of MYB transcription factor, Ca2+, and calmodulin in plant immune responses. PMID:26873979

  10. Evaluation of Reference Genes for RT-qPCR Expression Studies in Hop (Humulus lupulus L.) during Infection with Vascular Pathogen Verticillium albo-atrum

    PubMed Central

    Štajner, Nataša; Cregeen, Sara; Javornik, Branka

    2013-01-01

    Hop plant (Humulus lupulus L.), cultivated primarily for its use in the brewing industry, is faced with a variety of diseases, including severe vascular diseases, such as Verticillium wilt, against which no effective protection is available. The understanding of disease resistance with tools such as differentially expressed gene studies is an important objective of plant defense mechanisms. In this study, we evaluated twenty-three reference genes for RT-qPCR expression studies on hop under biotic stress conditions. The candidate genes were validated on susceptible and resistant hop cultivars sampled at three different time points after infection with Verticillium albo-atrum. The stability of expression and the number of genes required for accurate normalization were assessed by three different Excel-based approaches (geNorm v.3.5 software, NormFinder, and RefFinder). High consistency was found among them, identifying the same six best reference genes (YLS8, DRH1, TIP41, CAC, POAC and SAND) and five least stably expressed genes (CYCL, UBQ11, POACT, GAPDH and NADH). The candidate genes in different experimental subsets/conditions resulted in different rankings. A combination of the two best reference genes, YLS8 and DRH1, was used for normalization of RT-qPCR data of the gene of interest (PR-1) implicated in biotic stress of hop. We outlined the differences between normalized and non-normalized values and the importance of RT-qPCR data normalization. The high correlation obtained among data standardized with different sets of reference genes confirms the suitability of the reference genes selected for normalization. Lower correlations between normalized and non-normalized data may reflect different quantity and/or quality of RNA samples used in RT-qPCR analyses. PMID:23874551

  11. Proteomic and virus-induced gene silencing (VIGS) Analyses reveal that gossypol, brassinosteroids, and jasmonic acid contribute to the resistance of cotton to Verticillium dahliae.

    PubMed

    Gao, Wei; Long, Lu; Zhu, Long-Fu; Xu, Li; Gao, Wen-Hui; Sun, Long-Qing; Liu, Lin-Lin; Zhang, Xian-Long

    2013-12-01

    Verticillium wilt causes massive annual losses of cotton yield, but the mechanism of cotton resistance to Verticillium dahliae is complex and poorly understood. In this study, a comparative proteomic analysis was performed in resistant cotton (Gossypium barbadense cv7124) on infection with V. dahliae. A total of 188 differentially expressed proteins were identified by mass spectrometry (MALDI-TOF/TOF) analysis and could be classified into 17 biological processes based on Gene Ontology annotation. Most of these proteins were implicated in stimulus response, cellular processes and metabolic processes. Based on the proteomic analysis, several genes involved in secondary metabolism, reactive oxygen burst and phytohormone signaling pathways were identified for further physiological and molecular analysis. The roles of the corresponding genes were further characterized by employing virus-induced gene silencing (VIGS). Based on the results, we suggest that the production of gossypol is sufficient to affect the cotton resistance to V. dahliae. Silencing of GbCAD1, a key enzyme involving in gossypol biosynthesis, compromised cotton resistance to V. dahliae. Reactive oxygen species and salicylic acid signaling may be also implicated as regulators in cotton responsive to V. dahliae according to the analysis of GbSSI2, an important regulator in the crosstalk between salicylic acid and jasmonic acid signal pathways. Moreover, brassinosteroids and jasmonic acid signaling may play essential roles in the cotton disease resistance to V. dahliae. The brassinosteroids signaling was activated in cotton on inoculation with V. dahliae and the disease resistance of cotton was enhanced after exogenous application of brassinolide. Meanwhile, jasmonic acid signaling was also activated in cotton after inoculation with V. dahliae and brassinolide application. These data provide highlights in the molecular basis of cotton resistance to V. dahliae.

  12. Draft genome sequencing and secretome analysis of fungal phytopathogen Ascochyta rabiei provides insight into the necrotrophic effector repertoire

    PubMed Central

    Verma, Sandhya; Gazara, Rajesh Kumar; Nizam, Shadab; Parween, Sabiha; Chattopadhyay, Debasis; Verma, Praveen Kumar

    2016-01-01

    Constant evolutionary pressure acting on pathogens refines their molecular strategies to attain successful pathogenesis. Recent studies have shown that pathogenicity mechanisms of necrotrophic fungi are far more intricate than earlier evaluated. However, only a few studies have explored necrotrophic fungal pathogens. Ascochyta rabiei is a necrotrophic fungus that causes devastating blight disease of chickpea (Cicer arietinum). Here, we report a 34.6 megabase draft genome assembly of A. rabiei. The genome assembly covered more than 99% of the gene space and 4,259 simple sequence repeats were identified in the assembly. A total of 10,596 high confidence protein-coding genes were predicted which includes a large and diverse inventory of secretory proteins, transporters and primary and secondary metabolism enzymes reflecting the necrotrophic lifestyle of A. rabiei. A wide range of genes encoding carbohydrate-active enzymes capable for degradation of complex polysaccharides were also identified. Comprehensive analysis predicted a set of 758 secretory proteins including both classical and non-classical secreted proteins. Several of these predicted secretory proteins showed high cysteine content and numerous tandem repeats. Together, our analyses would broadly expand our knowledge and offer insights into the pathogenesis and necrotrophic lifestyle of fungal phytopathogens. PMID:27091329

  13. Synthesis and biological evaluation of novel fluorine-containing stilbene derivatives as fungicidal agents against phytopathogenic fungi.

    PubMed

    Jian, Weilin; He, Daohang; Xi, Pinggen; Li, Xinwei

    2015-11-18

    The rising development of resistance to conventional fungicides is driving the search for new alternative candidates to control plant diseases. In this study, a series of new fluorine-containing stilbene derivatives was synthesized on the basis of our previous quantitative structure-activity relationship analysis results. Bioassays in vivo revealed that the title compounds exhibited potent fungicidal activities against phytopathogenic fungi (Colletotrichum lagenarium and Pseudoperonospora cubensis) from cucumber plants. In comparison to the previous results, the introduction of a fluorine moiety showed improved activities of some compounds against those fungi. Notably, compound 9 exhibited a control efficacy against C. lagenarium (83.4 ± 1.3%) comparable to that of commercial fungicide (82.7 ± 1.7%). For further understanding the possible mode of action of the stilbene against C. lagenarium, the effects on hyphal morphology, electrolyte leakage, and respiration of mycelial cell suspension were studied. Microscopic observation showed considerably deformed mycelial morphology. The conductivity of mycelial suspension increased in the presence of compound 9, whereas no significantly inhibitory effect on respiration was observed. Taken together, the fungicidal mechanism of this stilbene is associated with its membrane disruption effect, resulting in increased membrane permeability. These results provide important clues for mechanistic study and derivatization of stilbenes as alternative sources of fungicidal agents for plant disease control.

  14. Complete sequence of a double-stranded RNA from the phytopathogenic fungus Erysiphe cichoracearum that might represent a novel endornavirus.

    PubMed

    Du, Zhenguo; Lin, Wenzhong; Qiu, Ping; Liu, Xiaojuan; Guo, Lingfang; Wu, Kangcheng; Zhang, Songbai; Wu, Zujian

    2016-08-01

    A double-stranded RNA (dsRNA) HBJZ1506 recovered from the phytopathogenic fungus Erysiphe cichoracearum infecting Calendula officinalis in Jingzhou, Hubei Province, China, was sequenced. HBJZ1506 comprises 11,908 nucleotides (nt) and contains a 11,859-nt-long open reading frame (ORF) coding for a polypeptide that is 61 % identical to that of a putative endornavirus named grapevine endophyte endornavirus (GeEV). The putative polyprotein has an RNA-dependent RNA polymerase (RdRp) domain and an RNA helicase domain, which show homology to and have an arrangement that is similar to that of their counterparts in approved or putative endornaviruses. In a phylogenetic tree constructed using amino acid sequences of the RdRp region of HBJZ1506 and selected endornaviruses, HBJZ1506 clustered with endornaviruses and formed a well-supported monophyletic branch with GeEV. These results suggest that HBJZ1506 might represent a novel endornavirus, for which the name Erysiphe cichoracearum endornavirus (EcEV) is proposed. PMID:27255746

  15. Insights into the diversity of φRSM phages infecting strains of the phytopathogen Ralstonia solanacearum complex: regulation and evolution.

    PubMed

    Askora, Ahmed; Kawasaki, Takeru; Fujie, Makoto; Yamada, Takashi

    2014-08-01

    The filamentous φRSM phages (φRSM1 and φRSM3) have integration/excision capabilities in the phytopathogenic bacterium Ralstonia solanacearum. In the present study, we further investigated φRSM-like sequences present in the genomes of R. solanacearum strains belonging to the four major evolutionary lineages (phylotypes I-IV). Based on bioinformatics and comparative genomic analyses, we found that φRSM homologs are highly diverse in R. solanacearum complex strains. We detected an open reading frame (ORF)15 located upstream of the gene for φRSM integrase, which exhibited amino acid sequence similarity to phage repressor proteins. ORF15-encoded protein (a putative repressor) was found to encode a 104-residue polypeptide containing a DNA-binding (helix-turn-helix) domain and was expressed in R. solanacearum lysogenic strains. This suggested that φRSM3-ORF15 might be involved in the establishment and maintenance of a lysogenic state, as well as in phage immunity. Comparison of the putative repressor proteins and their binding sites within φRSM-related prophages provides insights into how these regulatory systems of filamentous phages have evolved and diverged in the R. solanacearum complex. In conclusion, φRSM phages represent a unique group of filamentous phages that are equipped with innate integration/excision (ORF14) and regulatory systems (ORF15). PMID:24619102

  16. Genome, Proteome and Structure of a T7-Like Bacteriophage of the Kiwifruit Canker Phytopathogen Pseudomonas syringae pv. actinidiae

    PubMed Central

    Frampton, Rebekah A.; Lopez Acedo, Elena; Young, Vivienne L.; Chen, Danni; Tong, Brian; Taylor, Corinda; Easingwood, Richard A.; Pitman, Andrew R.; Kleffmann, Torsten; Bostina, Mihnea; Fineran, Peter C.

    2015-01-01

    Pseudomonas syringae pv. actinidiae is an economically significant pathogen responsible for severe bacterial canker of kiwifruit (Actinidia sp.). Bacteriophages infecting this phytopathogen have potential as biocontrol agents as part of an integrated approach to the management of bacterial canker, and for use as molecular tools to study this bacterium. A variety of bacteriophages were previously isolated that infect P. syringae pv. actinidiae, and their basic properties were characterized to provide a framework for formulation of these phages as biocontrol agents. Here, we have examined in more detail φPsa17, a phage with the capacity to infect a broad range of P. syringae pv. actinidiae strains and the only member of the Podoviridae in this collection. Particle morphology was visualized using cryo-electron microscopy, the genome was sequenced, and its structural proteins were analysed using shotgun proteomics. These studies demonstrated that φPsa17 has a 40,525 bp genome, is a member of the T7likevirus genus and is closely related to the pseudomonad phages φPSA2 and gh-1. Eleven structural proteins (one scaffolding) were detected by proteomics and φPsa17 has a capsid of approximately 60 nm in diameter. No genes indicative of a lysogenic lifecycle were identified, suggesting the phage is obligately lytic. These features indicate that φPsa17 may be suitable for formulation as a biocontrol agent of P. syringae pv. actinidiae. PMID:26114474

  17. Chitinase genes revealed and compared in bacterial isolates, DNA extracts and a metagenomic library from a phytopathogen suppressive soil

    SciTech Connect

    Hjort, K.; Bergstrom, M.; Adesina, M.F.; Jansson, J.K.; Smalla, K.; Sjoling, S.

    2009-09-01

    Soil that is suppressive to disease caused by fungal pathogens is an interesting source to target for novel chitinases that might be contributing towards disease suppression. In this study we screened for chitinase genes, in a phytopathogen-suppressive soil in three ways: (1) from a metagenomic library constructed from microbial cells extracted from soil, (2) from directly extracted DNA and (3) from bacterial isolates with antifungal and chitinase activities. Terminal-restriction fragment length polymorphism (T-RFLP) of chitinase genes revealed differences in amplified chitinase genes from the metagenomic library and the directly extracted DNA, but approximately 40% of the identified chitinase terminal-restriction fragments (TRFs) were found in both sources. All of the chitinase TRFs from the isolates were matched to TRFs in the directly extracted DNA and the metagenomic library. The most abundant chitinase TRF in the soil DNA and the metagenomic library corresponded to the TRF{sup 103} of the isolate, Streptomyces mutomycini and/or Streptomyces clavifer. There were good matches between T-RFLP profiles of chitinase gene fragments obtained from different sources of DNA. However, there were also differences in both the chitinase and the 16S rRNA gene T-RFLP patterns depending on the source of DNA, emphasizing the lack of complete coverage of the gene diversity by any of the approaches used.

  18. Characterization of an oxidative stress response regulator, homologous to Escherichia coli OxyR, from the phytopathogen Xylella fastidiosa.

    PubMed

    Toledo, M A S; Schneider, D R; Azzoni, A R; Favaro, M T P; Pelloso, A C; Santos, C A; Saraiva, A M; Souza, A P

    2011-02-01

    The OxyR oxidative stress transcriptional regulator is a DNA-binding protein that belongs to the LysR-type transcriptional regulators (LTTR) family. It has the ability to sense oxidative species inside the cell and to trigger the cell's response, activating the transcription of genes involved in scavenging oxidative species. In the present study, we have overexpressed, purified and characterized the predicted OxyR homologue (orf xf1273) of the phytopathogen Xylella fastidiosa. This bacterium is the causal agent of citrus variegated chlorosis (CVC) disease caused by the 9a5c strain, resulting in economic and social losses. The secondary structure of the recombinant protein was analyzed by circular dichroism. Gel filtration showed that XfoxyR is a dimer in solution. Gel shift assays indicated that it does bind to its own predicted promoter under in vitro conditions. However, considering our control experiment we cannot state that this interaction occurs in vivo. Functional complementation assays indicated that xfoxyR is able to restore the oxidative stress response in an oxyr knockout Escherichia coli strain. These results show that the predicted orfxf1273 codes for a transcriptional regulator, homologous to E. coli OxyR, involved in the oxidative stress response. This may be important for X. fastidiosa to overcome the defense mechanisms of its host during the infection and colonization processes. PMID:20951212

  19. Genome, Proteome and Structure of a T7-Like Bacteriophage of the Kiwifruit Canker Phytopathogen Pseudomonas syringae pv. actinidiae.

    PubMed

    Frampton, Rebekah A; Acedo, Elena Lopez; Young, Vivienne L; Chen, Danni; Tong, Brian; Taylor, Corinda; Easingwood, Richard A; Pitman, Andrew R; Kleffmann, Torsten; Bostina, Mihnea; Fineran, Peter C

    2015-07-01

    Pseudomonas syringae pv. actinidiae is an economically significant pathogen responsible for severe bacterial canker of kiwifruit (Actinidia sp.). Bacteriophages infecting this phytopathogen have potential as biocontrol agents as part of an integrated approach to the management of bacterial canker, and for use as molecular tools to study this bacterium. A variety of bacteriophages were previously isolated that infect P. syringae pv. actinidiae, and their basic properties were characterized to provide a framework for formulation of these phages as biocontrol agents. Here, we have examined in more detail φPsa17, a phage with the capacity to infect a broad range of P. syringae pv. actinidiae strains and the only member of the Podoviridae in this collection. Particle morphology was visualized using cryo-electron microscopy, the genome was sequenced, and its structural proteins were analysed using shotgun proteomics. These studies demonstrated that φPsa17 has a 40,525 bp genome, is a member of the T7likevirus genus and is closely related to the pseudomonad phages φPSA2 and gh-1. Eleven structural proteins (one scaffolding) were detected by proteomics and φPsa17 has a capsid of approximately 60 nm in diameter. No genes indicative of a lysogenic lifecycle were identified, suggesting the phage is obligately lytic. These features indicate that φPsa17 may be suitable for formulation as a biocontrol agent of P. syringae pv. actinidiae.

  20. Differential in vitro and in vivo effect of barley cysteine and serine protease inhibitors on phytopathogenic microorganisms.

    PubMed

    Carrillo, Laura; Herrero, Ignacio; Cambra, Inés; Sánchez-Monge, Rosa; Diaz, Isabel; Martinez, Manuel

    2011-10-01

    Protease inhibitors from plants have been involved in defence mechanisms against pests and pathogens. Phytocystatins and trypsin/α-amylase inhibitors are two of the best characterized protease inhibitor families in plants. In barley, thirteen cystatins (HvCPI-1 to 13) and the BTI-CMe trypsin inhibitor have been previously studied. Their capacity to inhibit pest digestive proteases, and the negative in vivo effect caused by plants expressing these inhibitors on pests support the defence function of these proteins. Barley cystatins are also able to inhibit in vitro fungal growth. However, the antifungal effect of these inhibitors in vivo had not been previously tested. Moreover, their in vitro and in vivo effect on plant pathogenous bacteria is still unknown. In order to obtain new insights on this feature, in vitro assays were made against different bacterial and fungal pathogens of plants using the trypsin inhibitor BTI-CMe and the thirteen barley cystatins. Most barley cystatins and the BTI-CMe inhibitor were able to inhibit mycelial growth but no bacterial growth. Transgenic Arabidopsis plants independently expressing the BTI-CMe inhibitor and the cystatin HvCPI-6 were tested against the same bacterial and fungal pathogens. Neither the HvCPI-6 expressing transgenic plants nor the BTI-CMe ones were more resistant to plant pathogen fungi and bacteria than control Arabidopsis plants. The differences observed between the in vitro and in planta assays against phytopathogenic fungi are discussed. PMID:21482127

  1. The Comprehensive Phytopathogen Genomics Resource: a web-based resource for data-mining plant pathogen genomes.

    PubMed

    Hamilton, John P; Neeno-Eckwall, Eric C; Adhikari, Bishwo N; Perna, Nicole T; Tisserat, Ned; Leach, Jan E; Lévesque, C André; Buell, C Robin

    2011-01-01

    The Comprehensive Phytopathogen Genomics Resource (CPGR) provides a web-based portal for plant pathologists and diagnosticians to view the genome and trancriptome sequence status of 806 bacterial, fungal, oomycete, nematode, viral and viroid plant pathogens. Tools are available to search and analyze annotated genome sequences of 74 bacterial, fungal and oomycete pathogens. Oomycete and fungal genomes are obtained directly from GenBank, whereas bacterial genome sequences are downloaded from the A Systematic Annotation Package (ASAP) database that provides curation of genomes using comparative approaches. Curated lists of bacterial genes relevant to pathogenicity and avirulence are also provided. The Plant Pathogen Transcript Assemblies Database provides annotated assemblies of the transcribed regions of 82 eukaryotic genomes from publicly available single pass Expressed Sequence Tags. Data-mining tools are provided along with tools to create candidate diagnostic markers, an emerging use for genomic sequence data in plant pathology. The Plant Pathogen Ribosomal DNA (rDNA) database is a resource for pathogens that lack genome or transcriptome data sets and contains 131 755 rDNA sequences from GenBank for 17 613 species identified as plant pathogens and related genera. Database URL: http://cpgr.plantbiology.msu.edu.

  2. Opportunistic, human-pathogenic species in the Herpotrichiellaceae are phenotypically similar to saprobic or phytopathogenic species in the Venturiaceae

    PubMed Central

    Crous, P.W.; Schubert, K.; Braun, U.; de Hoog, G.S.; Hocking, A.D.; Shin, H.-D.; Groenewald, J.Z.

    2007-01-01

    Although morphologically similar, species of Cladophialophora (Herpotrichiellaceae) were shown to be phylogenetically distinct from Pseudocladosporium (Venturiaceae), which was revealed to be synonymous with the older genus, Fusicladium. Other than being associated with human disorders, species of Cladophialophora were found to also be phytopathogenic, or to occur as saprobes on organic material, or in water, fruit juices, or sports drinks, along with species of Exophiala. Caproventuria and Metacoleroa were confirmed to be synonyms of Venturia, which has Fusicladium (= Pseudocladosporium) anamorphs. Apiosporina, based on A. collinsii, clustered basal to the Venturia clade, and appears to represent a further synonym. Several species with a pseudocladosporium-like morphology in vitro represent a sister clade to the Venturia clade, and are unrelated to Polyscytalum. These taxa are newly described in Fusicladium, which is morphologically close to Anungitea, a heterogeneous genus with unknown phylogenetic affinity. In contrast to the Herpotrichiellaceae, which were shown to produce numerous synanamorphs in culture, species of the Venturiaceae were morphologically and phylogenetically more uniform. Several new species and new combinations were introduced in Cladophialophora, Cyphellophora (Herpotrichiellaceae), Exophiala, Fusicladium, Venturia (Venturiaceae), and Cylindrosympodium (incertae sedis). PMID:18491000

  3. Sequence analysis and structural characterization of a glyceraldehyde-3-phosphate dehydrogenase gene from the phytopathogenic fungus Eremothecium ashbyi.

    PubMed

    Sengupta, Sudeshna; Chandra, T S

    2011-02-01

    Eremothecium ashbyi is a phytopathogenic fungus infesting cotton, soybeans and several other plants. This highly flavinogenic fungus has been phylogenetically characterized, but the genetic aspects of its central metabolic and riboflavin biosynthetic pathways are unknown. An ORF of 996 bp was obtained from E. ashbyi by using degenerate primers for glyceraldehyde-3-phosphate dehydrogenase (GPD) through reverse transcriptase polymerase chain reaction (RT-PCR) and 5'-3' rapid amplification of cDNA ends (RACE-PCR). This nucleotide sequence had a high similarity of 88% with GPD sequence of Ashbya gossypii. The putative GPD peptide of 331-aa had a high similarity of 85% with the GPD sequence from other ascomycetes. The ORF had an unusually strong codon bias with 5 amino acids showing strict preference of a single codon. The theoretical molecular weight for the putative peptide was 35.58 kDa with an estimated pI of 5.7. A neighbor-joining tree showed that the putative peptide from E. ashbyi displayed the highest similarity to GPD of A. gossypii. The gene sequence is available at the GenBank, accession number EU717696. Homology modeling done with Kluyveromyces marxianus GPD (PDB: 2I5P) as template indicated high structural similarity. PMID:20820924

  4. Insights into the diversity of φRSM phages infecting strains of the phytopathogen Ralstonia solanacearum complex: regulation and evolution.

    PubMed

    Askora, Ahmed; Kawasaki, Takeru; Fujie, Makoto; Yamada, Takashi

    2014-08-01

    The filamentous φRSM phages (φRSM1 and φRSM3) have integration/excision capabilities in the phytopathogenic bacterium Ralstonia solanacearum. In the present study, we further investigated φRSM-like sequences present in the genomes of R. solanacearum strains belonging to the four major evolutionary lineages (phylotypes I-IV). Based on bioinformatics and comparative genomic analyses, we found that φRSM homologs are highly diverse in R. solanacearum complex strains. We detected an open reading frame (ORF)15 located upstream of the gene for φRSM integrase, which exhibited amino acid sequence similarity to phage repressor proteins. ORF15-encoded protein (a putative repressor) was found to encode a 104-residue polypeptide containing a DNA-binding (helix-turn-helix) domain and was expressed in R. solanacearum lysogenic strains. This suggested that φRSM3-ORF15 might be involved in the establishment and maintenance of a lysogenic state, as well as in phage immunity. Comparison of the putative repressor proteins and their binding sites within φRSM-related prophages provides insights into how these regulatory systems of filamentous phages have evolved and diverged in the R. solanacearum complex. In conclusion, φRSM phages represent a unique group of filamentous phages that are equipped with innate integration/excision (ORF14) and regulatory systems (ORF15).

  5. Genome, Proteome and Structure of a T7-Like Bacteriophage of the Kiwifruit Canker Phytopathogen Pseudomonas syringae pv. actinidiae.

    PubMed

    Frampton, Rebekah A; Acedo, Elena Lopez; Young, Vivienne L; Chen, Danni; Tong, Brian; Taylor, Corinda; Easingwood, Richard A; Pitman, Andrew R; Kleffmann, Torsten; Bostina, Mihnea; Fineran, Peter C

    2015-07-01

    Pseudomonas syringae pv. actinidiae is an economically significant pathogen responsible for severe bacterial canker of kiwifruit (Actinidia sp.). Bacteriophages infecting this phytopathogen have potential as biocontrol agents as part of an integrated approach to the management of bacterial canker, and for use as molecular tools to study this bacterium. A variety of bacteriophages were previously isolated that infect P. syringae pv. actinidiae, and their basic properties were characterized to provide a framework for formulation of these phages as biocontrol agents. Here, we have examined in more detail φPsa17, a phage with the capacity to infect a broad range of P. syringae pv. actinidiae strains and the only member of the Podoviridae in this collection. Particle morphology was visualized using cryo-electron microscopy, the genome was sequenced, and its structural proteins were analysed using shotgun proteomics. These studies demonstrated that φPsa17 has a 40,525 bp genome, is a member of the T7likevirus genus and is closely related to the pseudomonad phages φPSA2 and gh-1. Eleven structural proteins (one scaffolding) were detected by proteomics and φPsa17 has a capsid of approximately 60 nm in diameter. No genes indicative of a lysogenic lifecycle were identified, suggesting the phage is obligately lytic. These features indicate that φPsa17 may be suitable for formulation as a biocontrol agent of P. syringae pv. actinidiae. PMID:26114474

  6. Identification of the central quorum sensing regulator of virulence in the enteric phytopathogen, Erwinia carotovora: the VirR repressor.

    PubMed

    Burr, Tom; Barnard, Anne M L; Corbett, Mark J; Pemberton, Clare L; Simpson, Natalie J L; Salmond, George P C

    2006-01-01

    In the Gram-negative phytopathogen, Erwinia carotovora ssp. atroseptica (Eca) virulence depends on the production of a N-(3-oxohexanoyl)-L-homoserine lactone (OHHL) quorum sensing (QS) signal. This work identifies the elusive 'missing link' between QS and virulence in Erwinia. We have identified and characterized a novel regulator of virulence, VirR, in Eca and show that a virR mutation completely restores virulence factor production to an Eca mutant unable to synthesize OHHL. This effect of the virR mutation translates to a restoration of virulence to wild-type levels and thus provides evidence that VirR acts to prevent the production of virulence factors at low cell density. We also show that, in Eca, transcription of virulence genes is controlled by OHHL and that this control is effected through the action of VirR. We also demonstrate that the VirR regulatory pathway is present and functional in both blackleg and soft rotting species of Erwinia.

  7. Draft genome sequencing and secretome analysis of fungal phytopathogen Ascochyta rabiei provides insight into the necrotrophic effector repertoire.

    PubMed

    Verma, Sandhya; Gazara, Rajesh Kumar; Nizam, Shadab; Parween, Sabiha; Chattopadhyay, Debasis; Verma, Praveen Kumar

    2016-01-01

    Constant evolutionary pressure acting on pathogens refines their molecular strategies to attain successful pathogenesis. Recent studies have shown that pathogenicity mechanisms of necrotrophic fungi are far more intricate than earlier evaluated. However, only a few studies have explored necrotrophic fungal pathogens. Ascochyta rabiei is a necrotrophic fungus that causes devastating blight disease of chickpea (Cicer arietinum). Here, we report a 34.6 megabase draft genome assembly of A. rabiei. The genome assembly covered more than 99% of the gene space and 4,259 simple sequence repeats were identified in the assembly. A total of 10,596 high confidence protein-coding genes were predicted which includes a large and diverse inventory of secretory proteins, transporters and primary and secondary metabolism enzymes reflecting the necrotrophic lifestyle of A. rabiei. A wide range of genes encoding carbohydrate-active enzymes capable for degradation of complex polysaccharides were also identified. Comprehensive analysis predicted a set of 758 secretory proteins including both classical and non-classical secreted proteins. Several of these predicted secretory proteins showed high cysteine content and numerous tandem repeats. Together, our analyses would broadly expand our knowledge and offer insights into the pathogenesis and necrotrophic lifestyle of fungal phytopathogens. PMID:27091329

  8. Transgenic Brassica juncea plants expressing MsrA1, a synthetic cationic antimicrobial peptide, exhibit resistance to fungal phytopathogens.

    PubMed

    Rustagi, Anjana; Kumar, Deepak; Shekhar, Shashi; Yusuf, Mohd Aslam; Misra, Santosh; Sarin, Neera Bhalla

    2014-06-01

    Cationic antimicrobial peptides (CAPs) have shown potential against broad spectrum of phytopathogens. Synthetic versions with desirable properties have been modeled on these natural peptides. MsrA1 is a synthetic chimera of cecropin A and melittin CAPs with antimicrobial properties. We generated transgenic Brassica juncea plants expressing the msrA1 gene aimed at conferring fungal resistance. Five independent transgenic lines were evaluated for resistance to Alternaria brassicae and Sclerotinia sclerotiorum, two of the most devastating pathogens of B. juncea crops. In vitro assays showed inhibition by MsrA1 of Alternaria hyphae growth by 44-62 %. As assessed by the number and size of lesions and time taken for complete leaf necrosis, the Alternaria infection was delayed and restricted in the transgenic plants with the protection varying from 69 to 85 % in different transgenic lines. In case of S. sclerotiorum infection, the lesions were more severe and spread profusely in untransformed control compared with transgenic plants. The sclerotia formed in the stem of untransformed control plants were significantly more in number and larger in size than those present in the transgenic plants where disease protection of 56-71.5 % was obtained. We discuss the potential of engineering broad spectrum biotic stress tolerance by transgenic expression of CAPs in crop plants.

  9. Identification of a Polyketide Synthase Gene in the Synthesis of Phleichrome of the Phytopathogenic Fungus Cladosporium phlei.

    PubMed

    So, Kum-Kang; Chung, Yun-Jo; Kim, Jung-Mi; Kim, Beom-Tae; Park, Seung-Moon; Kim, Dae-Hyuk

    2015-12-01

    Phleichrome, a pigment produced by the phytopathogenic fungus Cladosporium phlei, is a fungal perylenequinone whose photodynamic activity has been studied intensively. To determine the biological function of phleichrome and to engineer a strain with enhanced production of phleichrome, we identified the gene responsible for the synthesis of phleichrome. Structural comparison of phleichrome with other fungal perylenequinones suggested that phleichrome is synthesized via polyketide pathway. We recently identified four different polyketide synthase (PKS) genes encompassing three major clades of fungal PKSs that differ with respect to reducing conditions for the polyketide product. Based on in silico analysis of cloned genes, we hypothesized that the non-reducing PKS gene, Cppks1, is involved in phleichrome biosynthesis. Increased accumulation of Cppks1 transcript was observed in response to supplementation with the application of synthetic inducer cyclo-(l-Pro-l-Phe). In addition, heterologous expression of the Cppks1 gene in Cryphonectria parasitica resulted in the production of phleichrome. These results provide convincing evidence that the Cppks1 gene is responsible for the biosynthesis of phleichrome.

  10. Transgenic Brassica juncea plants expressing MsrA1, a synthetic cationic antimicrobial peptide, exhibit resistance to fungal phytopathogens.

    PubMed

    Rustagi, Anjana; Kumar, Deepak; Shekhar, Shashi; Yusuf, Mohd Aslam; Misra, Santosh; Sarin, Neera Bhalla

    2014-06-01

    Cationic antimicrobial peptides (CAPs) have shown potential against broad spectrum of phytopathogens. Synthetic versions with desirable properties have been modeled on these natural peptides. MsrA1 is a synthetic chimera of cecropin A and melittin CAPs with antimicrobial properties. We generated transgenic Brassica juncea plants expressing the msrA1 gene aimed at conferring fungal resistance. Five independent transgenic lines were evaluated for resistance to Alternaria brassicae and Sclerotinia sclerotiorum, two of the most devastating pathogens of B. juncea crops. In vitro assays showed inhibition by MsrA1 of Alternaria hyphae growth by 44-62 %. As assessed by the number and size of lesions and time taken for complete leaf necrosis, the Alternaria infection was delayed and restricted in the transgenic plants with the protection varying from 69 to 85 % in different transgenic lines. In case of S. sclerotiorum infection, the lesions were more severe and spread profusely in untransformed control compared with transgenic plants. The sclerotia formed in the stem of untransformed control plants were significantly more in number and larger in size than those present in the transgenic plants where disease protection of 56-71.5 % was obtained. We discuss the potential of engineering broad spectrum biotic stress tolerance by transgenic expression of CAPs in crop plants. PMID:24452332

  11. Specific genomic fingerprints of phytopathogenic Xanthomonas and Pseudomonas pathovars and strains generated with repetitive sequences and PCR

    SciTech Connect

    Louws, F.J.; Stephens, C.T.; Fulbright, D.W.

    1994-07-01

    DNA primers corresponding to conserved motifs in bacterial repetitive (REP, ERIC, and BOX) elements and PCR were used to show that REP-, ERIC-, and BOX-like DNA sequences are widely distributed in phytopathogenic Xanthomonas and Pseudomonas strains. REP-, ERIC-, and BOX-PCR (collectively known as rep-PCR) were used to generate genomic fingerprints of a variety of Xanthomonas and Pseudomonas isolates and to to identify pathovars and strains that were previously not distinguishable by other classification methods. Analogous rep-PCR-derived genomic fingerprints were generated from purified genomic DNA, colonies on agar plates, liquid cultures, and directly from lesions on infected plants. REP-, ERIC-, and BOX-PCR-generated fingerprints of specific Xanthomonas and Pseudomonas strains were found to yield similar conclusions with regard to the identity of and relationship between these strains. This suggests that the distribution of REP-, ERIC-, and BOX-like sequences in these strains is a reflection of their genomic structure. Thus, the rep-PCR technique appears to be a rapid, simple, and reproducible method to identify and classify Xanthomonas and Pseudomonas strains, and it may be a useful diagnostic tool for these important plant pathogens. 70 refs., 5 figs., 1 tab.

  12. Antimicrobial activities of novel mannosyl lipids isolated from the biocontrol fungus Simplicillium lamellicola BCP against phytopathogenic bacteria.

    PubMed

    Le Dang, Quang; Shin, Teak Soo; Park, Myung Soo; Choi, Yong Ho; Choi, Gyung Ja; Jang, Kyoung Soo; Kim, In Seon; Kim, Jin-Cheol

    2014-04-16

    The antagonistic fungus Simplicillium lamellicola BCP has been developed as a microbial biopesticide that effectively controls the development of various plant diseases caused by both pathogenic bacteria and pathogenic fungi. Antibacterial bioassay-directed fractionation was used to isolate mannosyl lipids from S. lamellicola BCP, and the structures of these compounds were elucidated using spectral analysis and chemical degradation. Three novel mannosyl lipids were characterized and identified as halymecins F and G and (3R,5R)-3-O-β-D-mannosyl-3,5-dihydrodecanoic acid. Massoia lactone and (3R, 5R)-3-hydroxydecan-5-olide were also isolated from S. lamellicola BCP. The three novel compounds inhibited the growth of the majority of phytopathogenic bacteria that were tested, and halymecin F displayed the strongest antibacterial activity. Agrobacterium tumefaciens was the most sensitive to the three novel compounds, with IC₅₀ values ranging from 1.58 to 24.8 μg/mL. The ethyl acetate extract of the fermentation broth from the antagonistic fungus effectively reduced the bacterial wilt caused by Ralstonia solanacearum on tomato seedlings. These results indicate that S. lamellicola BCP suppresses the development of plant bacterial diseases through the production of antibacterial metabolites. PMID:24660753

  13. Production, partial purification and characterization of protease from a phytopathogenic fungi Alternaria solani (Ell. and Mart.) Sorauer.

    PubMed

    Chandrasekaran, Murugesan; Sathiyabama, Muthukrishnan

    2014-08-01

    An alkaline serine protease producing strain Alternaria solani was optimized for its enzyme production under submerged conditions. The maximum production of protease by A. solani was achieved by using sodium nitrate at the optimum concentration of 0.2% w/v. A. solani produced higher quantities (3.75 [unit/mg of protein]) of an inducible extracellular proteases on day 9 after incubation in czapek's dox broth medium amended with 1% casein as an inducer at pH 8.5, temperature 27 °C and 3% sucrose as carbon source. Extracellular proteases were precipitated by ammonium sulphate saturation (80%) method and purified on Sephadex G-100 column chromatography. The molecular mass of SDS-PAGE and Sephadex G-100 Column Gel permeation chromatography purified protease was estimated to 42 kDa. In addition, trypsin digestion of 42 kDa protein band was carried out and analyzed by MALDI-TOF for the identification of protease. The sequence IKELATNGVVTNVK (378-391) segment of the alkaline serine protease was found by using MS/MS spectrum at 1485 m/z from the purified fraction. It showed optimal activity at 50 °C and pH 9-10 and broad pH stability between pH 6-12. The protease activity was inhibited by phenyl methyl sulfonyl fluoride (PMSF), all the results indicated that the presence of a serine residue in the active site and is thus most likely a member of the serine protease family. This may function as a virulence protein during pathogenesis by A. solani. The results suggested that the presence of appreciable extracellular proteolytic activity in filamentous fungi may serve as a marker of their phytopathogenicity.

  14. Deep-sea Rhodococcus sp. BS-15, lacking the phytopathogenic fas genes, produces a novel glucotriose lipid biosurfactant.

    PubMed

    Konishi, Masaaki; Nishi, Shinro; Fukuoka, Tokuma; Kitamoto, Dai; Watsuji, Tomo-O; Nagano, Yuriko; Yabuki, Akinori; Nakagawa, Satoshi; Hatada, Yuji; Horiuchi, Jun-Ichi

    2014-08-01

    Glycolipid biosurfactant-producing bacteria were isolated from deep-sea sediment collected from the Okinawa Trough. Isolate BS15 produced the largest amount of the glycolipid, generating up to 6.31 ± 1.15 g l(-1) after 4 days at 20 °C. Glucose was identified in the hydrolysate of the purified major component of the biosurfactant glycolipid. According to gas chromatography/mass spectrometry analysis, the hydrophobic moieties in the major component were hexadecanoate, octadecanoate, 3-hydroxyhexadecanoate, 2-hydroxyoctanoate, and succinate. The molecular weight of the purified major glycolipid was calculated to be 1,211, while (1)H and (13)C nuclear magnetic resonance spectra confirmed that the major component consisted of 2 mol of α-glucoside and 1 mol of β-glucoside. The molecular structure was assigned as novel trisaccharide-type glycolipid biosurfactant, glucotriose lipids. The critical micelle concentration of the purified major glycolipid was 2.3 × 10(-6) M, with a surface tension of 29.5 mN m(-1). Phylogenetic analysis showed isolate BS15 was closely related to a Rhodococcus strains isolated from Antarctica, and to Rhodococcus fascians, a phytopathogen. PCR analysis showed that the fasA, fasB, fasC, fasD, fasE, and fasF genes, which are involved in phytohormone-like cytokinin production, were not present in the genome of BS15; however, analysis of a draft genome sequence of BS15 (5.5 Mb) identified regions with 31 %, 53 %, 46 %, 30 %, and 31 % DNA sequence identity to the fasA, fasB, fasC, and fasD genes, respectively.

  15. Quantitative Resistance to Verticillium Wilt in Medicago truncatula Involves Eradication of the Fungus from Roots and Is Associated with Transcriptional Responses Related to Innate Immunity

    PubMed Central

    Toueni, Maoulida; Ben, Cécile; Le Ru, Aurélie; Gentzbittel, Laurent; Rickauer, Martina

    2016-01-01

    Resistance mechanisms to Verticillium wilt are well-studied in tomato, cotton, and Arabidopsis, but much less in legume plants. Because legume plants establish nitrogen-fixing symbioses in their roots, resistance to root-attacking pathogens merits particular attention. The interaction between the soil-borne pathogen Verticillium alfalfae and the model legume Medicago truncatula was investigated using a resistant (A17) and a susceptible (F83005.5) line. As shown by histological analyses, colonization by the pathogen was initiated similarly in both lines. Later on, the resistant line A17 eliminated the fungus, whereas the susceptible F83005.5 became heavily colonized. Resistance in line A17 does not involve homologs of the well-characterized tomato Ve1 and V. dahliae Ave1 genes. A transcriptomic study of early root responses during initial colonization (i.e., until 24 h post-inoculation) similarly was performed. Compared to the susceptible line, line A17 displayed already a significantly higher basal expression of defense-related genes prior to inoculation, and responded to infection with up-regulation of only a small number of genes. Although fungal colonization was still low at this stage, the susceptible line F83005.5 exhibited a disorganized response involving a large number of genes from different functional classes. The involvement of distinct phytohormone signaling pathways in resistance as suggested by gene expression patterns was supported by experiments with plant hormone pretreatment before fungal inoculation. Gene co-expression network analysis highlighted five main modules in the resistant line, whereas no structured gene expression was found in the susceptible line. One module was particularly associated to the inoculation response in A17. It contains the majority of differentially expressed genes, genes associated with PAMP perception and hormone signaling, and transcription factors. An in silico analysis showed that a high number of these genes also

  16. Chemical composition and antimicrobial activity of essential oils from Acantholippia deserticola, Artemisia proceriformis, Achillea micrantha and Libanotis buchtormensis against phytopathogenic bacteria and fungi.

    PubMed

    Sampietro, Diego A; Lizarraga, Emilio F; Ibatayev, Zharkyn A; Omarova, Akerke B; Suleimen, Yerlan M; Catalán, Cesar A N

    2016-09-01

    Essential oils from aerial parts of Acantholippia deserticola, Artemisia proceriformis, Achillea micrantha and Libanotis buchtormensis were analysed by GC-MS. The major compounds identified were β-thujone (66.5 ± 0.2%), and trans-sabinyl acetate (12.1 ± 0.2%) in A. deserticola; α-thujone (66.9 ± 0.4%) in A. proceriformis; 1,8-cineole (26.9 ± 0.5%), and camphor (17.7 ± 0.3%) in A. micrantha and cis-β-ocimene (23.3 ± 0.3%), and trans-β-ocimene (18.4 ± 0.2%) in L. buchtormensis. The oils showed a weak antimicrobial effect (MIC100 > 1.5 mg/ml) on most phytopathogens tested. A moderate antimicrobial activity (MIC100 between 0.5 and 1.5 mg/ml) was displayed by the oils of A. deserticola, A. micrantha and L. buchtormensis on Septoria tritici and by the oil of A. deserticola on Septoria glycine. The antimicrobial activity was associated to the contents of β-thujone, trans-sabinyl acetate and trans-sabinol. Our results indicate that the tested essential oils have little inhibitory potency not suitable for use as plant protection products against the phytopathogens assayed. PMID:26404704

  17. Analysis of Phaseolus vulgaris Response to Its Association with Trichoderma harzianum (ALL-42) in the Presence or Absence of the Phytopathogenic Fungi Rhizoctonia solani and Fusarium solani

    PubMed Central

    Pereira, Jackeline L.; Queiroz, Rayner M. L.; Charneau, Sébastien O.; Felix, Carlos R.; Ricart, Carlos A. O.; da Silva, Francilene Lopes; Steindorff, Andrei Stecca; Ulhoa, Cirano J.; Noronha, Eliane F.

    2014-01-01

    The present study was carried out to evaluate the ability of Trichoderma harzianum (ALL 42-isolated from Brazilian Cerrado soil) to promote common bean growth and to modulate its metabolism and defense response in the presence or absence of the phytopathogenic fungi Rhizoctonia solani and Fusarium solani using a proteomic approach. T. harzianum was able to promote common bean plants growth as shown by the increase in root/foliar areas and by size in comparison to plants grown in its absence. The interaction was shown to modulate the expression of defense-related genes (Glu1, pod3 and lox1) in roots of P. vulgaris. Proteomic maps constructed using roots and leaves of plants challenged or unchallenged by T. harzianum and phytopathogenic fungi showed differences. Reference gels presented differences in spot distribution (absence/presence) and relative volumes of common spots (up or down-regulation). Differential spots were identified by peptide fingerprinting MALDI-TOF mass spectrometry. A total of 48 identified spots (19 for leaves and 29 for roots) were grouped into protein functional classes. For leaves, 33%, 22% and 11% of the identified proteins were categorized as pertaining to the groups: metabolism, defense response and oxidative stress response, respectively. For roots, 17.2%, 24.1% and 10.3% of the identified proteins were categorized as pertaining to the groups: metabolism, defense response and oxidative stress response, respectively. PMID:24878929

  18. Analysis of Phaseolus vulgaris response to its association with Trichoderma harzianum (ALL-42) in the presence or absence of the phytopathogenic fungi Rhizoctonia solani and Fusarium solani.

    PubMed

    Pereira, Jackeline L; Queiroz, Rayner M L; Charneau, Sébastien O; Felix, Carlos R; Ricart, Carlos A O; da Silva, Francilene Lopes; Steindorff, Andrei Stecca; Ulhoa, Cirano J; Noronha, Eliane F

    2014-01-01

    The present study was carried out to evaluate the ability of Trichoderma harzianum (ALL 42-isolated from Brazilian Cerrado soil) to promote common bean growth and to modulate its metabolism and defense response in the presence or absence of the phytopathogenic fungi Rhizoctonia solani and Fusarium solani using a proteomic approach. T. harzianum was able to promote common bean plants growth as shown by the increase in root/foliar areas and by size in comparison to plants grown in its absence. The interaction was shown to modulate the expression of defense-related genes (Glu1, pod3 and lox1) in roots of P. vulgaris. Proteomic maps constructed using roots and leaves of plants challenged or unchallenged by T. harzianum and phytopathogenic fungi showed differences. Reference gels presented differences in spot distribution (absence/presence) and relative volumes of common spots (up or down-regulation). Differential spots were identified by peptide fingerprinting MALDI-TOF mass spectrometry. A total of 48 identified spots (19 for leaves and 29 for roots) were grouped into protein functional classes. For leaves, 33%, 22% and 11% of the identified proteins were categorized as pertaining to the groups: metabolism, defense response and oxidative stress response, respectively. For roots, 17.2%, 24.1% and 10.3% of the identified proteins were categorized as pertaining to the groups: metabolism, defense response and oxidative stress response, respectively.

  19. The Lysobacter capsici AZ78 Genome Has a Gene Pool Enabling it to Interact Successfully with Phytopathogenic Microorganisms and Environmental Factors.

    PubMed

    Puopolo, Gerardo; Tomada, Selena; Sonego, Paolo; Moretto, Marco; Engelen, Kristof; Perazzolli, Michele; Pertot, Ilaria

    2016-01-01

    Lysobacter capsici AZ78 has considerable potential for biocontrol of phytopathogenic microorganisms. However, lack of information about genetic cues regarding its biological characteristics may slow down its exploitation as a biofungicide. In order to obtain a comprehensive overview of genetic features, the L. capsici AZ78 genome was sequenced, annotated and compared with the phylogenetically related pathogens Stenotrophomonas malthophilia K729a and Xanthomonas campestris pv. campestris ATCC 33913. Whole genome comparison, supported by functional analysis, indicated that L. capsici AZ78 has a larger number of genes responsible for interaction with phytopathogens and environmental stress than S. malthophilia K729a and X. c. pv. campestris ATCC 33913. Genes involved in the production of antibiotics, lytic enzymes and siderophores were specific for L. capsici AZ78, as well as genes involved in resistance to antibiotics, environmental stressors, fungicides and heavy metals. The L. capsici AZ78 genome did not encompass genes involved in infection of humans and plants included in the S. malthophilia K729a and X. c. pv. campestris ATCC 33913 genomes, respectively. The L. capsici AZ78 genome provides a genetic framework for detailed analysis of other L. capsici members and the development of novel biofungicides based on this bacterial strain. PMID:26903975

  20. The opgC gene is required for OPGs succinylation and is osmoregulated through RcsCDB and EnvZ/OmpR in the phytopathogen Dickeya dadantii

    PubMed Central

    Bontemps-Gallo, Sébastien; Madec, Edwige; Robbe-Masselot, Catherine; Souche, Erika; Dondeyne, Jacqueline; Lacroix, Jean-Marie

    2016-01-01

    Osmoregulated periplasmic glucans (OPGs) are a family of periplasmic oligosaccharides found in the envelope of most Proteobacteria. They are required for virulence of zoo- and phyto-pathogens. The glucose backbone of OPGs is substituted by various kinds of molecules depending on the species, O-succinyl residues being the most widely distributed. In our model, Dickeya dadantii, a phytopathogenic bacteria causing soft rot disease in a wide range of plant species, the backbone of OPGs is substituted by O-succinyl residues in media of high osmolarity and by O-acetyl residues whatever the osmolarity. The opgC gene encoding a transmembrane protein required for the succinylation of the OPGs in D. dadantii was found after an in silico search of a gene encoding a protein with the main characteristics recovered in the two previously characterized OpgC of E. coli and R. sphaeroides, i.e. 10 transmembrane segments and one acyl-transferase domain. Characterization of the opgC gene revealed that high osmolarity expression of the succinyl transferase is controlled by both the EnvZ-OmpR and RcsCDB phosphorelay systems. The loss of O-succinyl residue did not affect the virulence of D. dadantii, suggesting that only the glucose backbone of OPGs is required for virulence. PMID:26790533

  1. The Lysobacter capsici AZ78 Genome Has a Gene Pool Enabling it to Interact Successfully with Phytopathogenic Microorganisms and Environmental Factors

    PubMed Central

    Puopolo, Gerardo; Tomada, Selena; Sonego, Paolo; Moretto, Marco; Engelen, Kristof; Perazzolli, Michele; Pertot, Ilaria

    2016-01-01

    Lysobacter capsici AZ78 has considerable potential for biocontrol of phytopathogenic microorganisms. However, lack of information about genetic cues regarding its biological characteristics may slow down its exploitation as a biofungicide. In order to obtain a comprehensive overview of genetic features, the L. capsici AZ78 genome was sequenced, annotated and compared with the phylogenetically related pathogens Stenotrophomonas malthophilia K729a and Xanthomonas campestris pv. campestris ATCC 33913. Whole genome comparison, supported by functional analysis, indicated that L. capsici AZ78 has a larger number of genes responsible for interaction with phytopathogens and environmental stress than S. malthophilia K729a and X. c. pv. campestris ATCC 33913. Genes involved in the production of antibiotics, lytic enzymes and siderophores were specific for L. capsici AZ78, as well as genes involved in resistance to antibiotics, environmental stressors, fungicides and heavy metals. The L. capsici AZ78 genome did not encompass genes involved in infection of humans and plants included in the S. malthophilia K729a and X. c. pv. campestris ATCC 33913 genomes, respectively. The L. capsici AZ78 genome provides a genetic framework for detailed analysis of other L. capsici members and the development of novel biofungicides based on this bacterial strain. PMID:26903975

  2. The attack of the phytopathogens and the trumpet solo: Identification of a novel plant antifungal peptide with distinct fold and disulfide bond pattern.

    PubMed

    Mandal, Santi M; Porto, William F; Dey, Prabuddha; Maiti, Mrinal K; Ghosh, Ananta K; Franco, Octavio L

    2013-10-01

    Phytopathogens cause economic losses in agribusiness. Plant-derived compounds have been proposed to overcome this problem, including the antimicrobial peptides (AMPs). This paper reports the identification of Ps-AFP1, a novel AMP isolated from the Pisum sativum radicle. Ps-AFP1 was purified and evaluated against phytopathogenic fungi, showing clear effectiveness. In silico analyses were performed, suggesting an unusual fold and disulfide bond pattern. A novel fold and a novel AMP class were here proposed, the αβ-trumpet fold and αβ-trumpet peptides, respectively. The name αβ-trumpet was created due to the peptide's fold, which resembles the musical instrument. The Ps-AFP1 mechanism of action was also proposed. Microscopic analyses revealed that Ps-AFP1 could affect the fungus during the hyphal elongation from spore germination. Furthermore, confocal microscopy performed with Ps-AFP1 labeled with FITC shows that the peptide was localized at high concentration along the fungal cell surface. Due to low cellular disruption rates, it seems that the main target is the fungal cell wall. The binding thermogram and isothermal titration, molecular dynamics and docking analyses were also performed, showing that Ps-AFP1 could bind to chitin producing a stable complex. Data here reported provided novel structural-functional insights into the αβ-trumpet peptide fold.

  3. Serine protease identification (in vitro) and molecular structure predictions (in silico) from a phytopathogenic fungus, Alternaria solani.

    PubMed

    Chandrasekaran, Murugesan; Chandrasekar, Raman; Sa, Tongmin; Sathiyabama, Muthukrishnan

    2014-07-01

    Serine proteases are involved in an enormous number of biological processes. The present study aims at characterizing three-dimensional (3D) molecular architecture of serine proteases from early blight pathogen, Alternaria solani that are hypothesized to be markers of phytopathogenicity. A serine protease was purified to homogeneity and MALDI-TOF-MS/MS analysis revealed that protease produced by A. solani belongs to alkaline serine proteases (AsP). AsP is made up of 403 amino acid residues with molecular weight of 42.1 kDa (Isoelectric point - 6.51) and its molecular formula was C1859 H2930 N516 O595 S4 . AsP structure model was built based on its comparative homology with serine protease using the program, MODELER. AsP had 16 β-sheets and 10 α-helices, with Ser(350) (G347-G357), Asp(158) (D158-H169), and His(193) (H193-G203) in separate turn/coil structures. Biological metal binding region situated near 6th-helix and His(193) residue is responsible for metal binding site. Also, calcium ion (Ca(2+)) is coordinated by the carboxyl groups of Lys(84), Ile(85), Lys(86), Asp(87), Phe(88), Ala(89), Ala(90) (K84-A90) for first Ca(2+) binding site and carbonyl oxygen atom of Lys(244), Gly(245), Arg(246), Thr(247), Lys(248), Lys(249), and Ala(250) (K244-A250), for second Ca(2+) binding site. Moreover, Ramachandran plot analysis of protein residues falling into most favored secondary structures were determined (83.3%). The predicted molecular 3D structural model was further verified using PROCHECK, ERRAT, and VADAR servers to confirm the geometry and stereo-chemical parameters of the molecular structural design. The functional analysis of AsP 3D molecular structure predictions familiar in the current study may provide a new perspective in the understanding and identification of antifungal protease inhibitor designing. PMID:24122785

  4. Identification of mycoparasitism-related genes against the phytopathogen Sclerotinia sclerotiorum through transcriptome and expression profile analysis in Trichoderma harzianum

    PubMed Central

    2014-01-01

    Background The species of T. harzianum are well known for their biocontrol activity against plant pathogens. However, few studies have been conducted to further our understanding of its role as a biological control agent against S. sclerotiorum, a pathogen involved in several crop diseases around the world. In this study, we have used RNA-seq and quantitative real-time PCR (RT-qPCR) techniques in order to explore changes in T. harzianum gene expression during growth on cell wall of S. sclerotiorum (SSCW) or glucose. RT-qPCR was also used to examine genes potentially involved in biocontrol, during confrontation between T. harzianum and S. sclerotiorum. Results Data obtained from six RNA-seq libraries were aligned onto the T. harzianum CBS 226.95 reference genome and compared after annotation using the Blast2GO suite. A total of 297 differentially expressed genes were found in mycelia grown for 12, 24 and 36 h under the two different conditions: supplemented with glucose or SSCW. Functional annotation of these genes identified diverse biological processes and molecular functions required during T. harzianum growth on SSCW or glucose. We identified various genes of biotechnological value encoding proteins with functions such as transporters, hydrolytic activity, adherence, appressorium development and pathogenesis. To validate the expression profile, RT-qPCR was performed using 20 randomly chosen genes. RT-qPCR expression profiles were in complete agreement with the RNA-Seq data for 17 of the genes evaluated. The other three showed differences at one or two growth times. During the confrontation assay, some genes were up-regulated during and after contact, as shown in the presence of SSCW which is commonly used as a model to mimic this interaction. Conclusions The present study is the first initiative to use RNA-seq for identification of differentially expressed genes in T. harzianum strain TR274, in response to the phytopathogenic fungus S. sclerotiorum. It provides

  5. Serine protease identification (in vitro) and molecular structure predictions (in silico) from a phytopathogenic fungus, Alternaria solani.

    PubMed

    Chandrasekaran, Murugesan; Chandrasekar, Raman; Sa, Tongmin; Sathiyabama, Muthukrishnan

    2014-07-01

    Serine proteases are involved in an enormous number of biological processes. The present study aims at characterizing three-dimensional (3D) molecular architecture of serine proteases from early blight pathogen, Alternaria solani that are hypothesized to be markers of phytopathogenicity. A serine protease was purified to homogeneity and MALDI-TOF-MS/MS analysis revealed that protease produced by A. solani belongs to alkaline serine proteases (AsP). AsP is made up of 403 amino acid residues with molecular weight of 42.1 kDa (Isoelectric point - 6.51) and its molecular formula was C1859 H2930 N516 O595 S4 . AsP structure model was built based on its comparative homology with serine protease using the program, MODELER. AsP had 16 β-sheets and 10 α-helices, with Ser(350) (G347-G357), Asp(158) (D158-H169), and His(193) (H193-G203) in separate turn/coil structures. Biological metal binding region situated near 6th-helix and His(193) residue is responsible for metal binding site. Also, calcium ion (Ca(2+)) is coordinated by the carboxyl groups of Lys(84), Ile(85), Lys(86), Asp(87), Phe(88), Ala(89), Ala(90) (K84-A90) for first Ca(2+) binding site and carbonyl oxygen atom of Lys(244), Gly(245), Arg(246), Thr(247), Lys(248), Lys(249), and Ala(250) (K244-A250), for second Ca(2+) binding site. Moreover, Ramachandran plot analysis of protein residues falling into most favored secondary structures were determined (83.3%). The predicted molecular 3D structural model was further verified using PROCHECK, ERRAT, and VADAR servers to confirm the geometry and stereo-chemical parameters of the molecular structural design. The functional analysis of AsP 3D molecular structure predictions familiar in the current study may provide a new perspective in the understanding and identification of antifungal protease inhibitor designing.

  6. Cultivation-independent analysis of Pseudomonas species in soil and in the rhizosphere of field-grown Verticillium dahliae host plants.

    PubMed

    Costa, Rodrigo; Salles, Joana Falcão; Berg, Gabriele; Smalla, Kornelia

    2006-12-01

    Despite their importance for rhizosphere functioning, rhizobacterial Pseudomonas spp. have been mainly studied in a cultivation-based manner. In this study a cultivation-independent method was used to determine to what extent the factors plant species, sampling site and year-to-year variation influence Pseudomonas community structure in bulk soil and in the rhizosphere of two Verticillium dahliae host plants, oilseed rape and strawberry. Community DNA was extracted from bulk and rhizosphere soil samples of flowering plants collected at three different sites in Germany in two consecutive years. Pseudomonas community structure and diversity were assessed using a polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE) system to fingerprint Pseudomonas-specific 16S rRNA gene fragments amplified from community DNA. Dominant and differentiating DGGE bands were excised from the gels, cloned and sequenced. The factors sampling site, plant species and year-to-year variation were shown to significantly influence the community structure of Pseudomonas in rhizosphere soils. The composition of Pseudomonas 16S rRNA gene fragments in the rhizosphere differed from that in the adjacent bulk soil and the rhizosphere effect tended to be plant-specific. The clone sequences of most dominant bands analysed belonged to the Pseudomonas fluorescens lineage and showed closest similarity to culturable Pseudomonas known for displaying antifungal properties. This report provides a better understanding of how different factors drive Pseudomonas community structure and diversity in bulk and rhizosphere soils.

  7. Influence of soil type, cultivar and Verticillium dahliae on the structure of the root and rhizosphere soil fungal microbiome of strawberry.

    PubMed

    Nallanchakravarthula, Srivathsa; Mahmood, Shahid; Alström, Sadhna; Finlay, Roger D

    2014-01-01

    Sustainable management of crop productivity and health necessitates improved understanding of the ways in which rhizosphere microbial populations interact with each other, with plant roots and their abiotic environment. In this study we examined the effects of different soils and cultivars, and the presence of a soil-borne fungal pathogen, Verticillium dahliae, on the fungal microbiome of the rhizosphere soil and roots of strawberry plants, using high-throughput pyrosequencing. Fungal communities of the roots of two cultivars, Honeoye and Florence, were statistically distinct from those in the rhizosphere soil of the same plants, with little overlap. Roots of plants growing in two contrasting field soils had high relative abundance of Leptodontidium sp. C2 BESC 319 g whereas rhizosphere soil was characterised by high relative abundance of Trichosporon dulcitum or Cryptococcus terreus, depending upon the soil type. Differences between different cultivars were not as clear. Inoculation with the pathogen V. dahliae had a significant influence on community structure, generally decreasing the number of rhizosphere soil- and root-inhabiting fungi. Leptodontidium sp. C2 BESC 319 g was the dominant fungus responding positively to inoculation with V. dahliae. The results suggest that 1) plant roots select microorganisms from the wider rhizosphere pool, 2) that both rhizosphere soil and root inhabiting fungal communities are influenced by V. dahliae and 3) that soil type has a stronger influence on both of these communities than cultivar. PMID:25347069

  8. Identification of pathogenicity-related genes in the vascular wilt fungus Verticillium dahliae by Agrobacterium tumefaciens-mediated T-DNA insertional mutagenesis.

    PubMed

    Maruthachalam, K; Klosterman, S J; Kang, S; Hayes, R J; Subbarao, K V

    2011-11-01

    Verticillium dahliae is the causal agent of vascular wilt in many economically important crops worldwide. Identification of genes that control pathogenicity or virulence may suggest targets for alternative control methods for this fungus. In this study, Agrobacterium tumefaciens-mediated transformation (ATMT) was applied for insertional mutagenesis of V. dahliae conidia. Southern blot analysis indicated that T-DNAs were inserted randomly into the V. dahliae genome and that 69% of the transformants were the result of single copy T-DNA insertion. DNA sequences flanking T-DNA insertion were isolated through inverse PCR (iPCR), and these sequences were aligned to the genome sequence to identify the genomic position of insertion. V. dahliae mutants of particular interest selected based on culture phenotypes included those that had lost the ability to form microsclerotia and subsequently used for virulence assay. Based on the virulence assay of 181 transformants, we identified several mutant strains of V. dahliae that did not cause symptoms on lettuce plants. Among these mutants, T-DNA was inserted in genes encoding an endoglucanase 1 (VdEg-1), a hydroxyl-methyl glutaryl-CoA synthase (VdHMGS), a major facilitator superfamily 1 (VdMFS1), and a glycosylphosphatidylinositol (GPI) mannosyltransferase 3 (VdGPIM3). These results suggest that ATMT can effectively be used to identify genes associated with pathogenicity and other functions in V. dahliae.

  9. Phylogenic analysis revealed an expanded C₂H₂-homeobox subfamily and expression profiles of C₂H₂ zinc finger gene family in Verticillium dahliae.

    PubMed

    Xiong, Dianguang; Wang, Yonglin; Deng, Chenglin; Hu, Ruowen; Tian, Chengming

    2015-05-15

    C2H2 zinc finger (CZF) proteins are a major class of transcription factors that play crucial roles in fungal growth, development, various stress responses, and virulence. Little genome-wide data is available regarding the roles of CZF proteins in Verticillium dahliae, a destructive pathogen that causes vascular wilt disease in more than 200 plant species. We identified a total of 79 typical CZF genes in V. dahliae. Comparative analysis revealed that four plant pathogenic fungi, V. dahliae, Fusarium oxysporum, Magnaporthe oryzae, and Botrytis cinerea, have comparable numbers of predicted CZF genes with similar characteristics. Phylogenetic analysis identified a C2H2-homeobox subfamily in V. dahliae containing seven genes with similar gene structures. V. dahliae and F. oxysporum (Hypocreomycetidae) have more genes of this subfamily than M. oryzae (Sordariomycetidae) and B. cinerea (Leotiomycetes). Furthermore, gene-expression analysis of the smoke tree wilt fungus V. dahliae strain XS11 using digital gene-expression profiling and RT-qPCR revealed that a number of CZF genes were differentially expressed during microsclerotia formation, nutritional starvation, and simulated in planta conditions. Furthermore, the expression profiles revealed that some CZF genes were overrepresented during multiple stages, indicating that they might play diverse roles. Our results provide useful information concerning the functions of CZF genes in microsclerotia formation, nutritional stress responses, and pathogenicity in V. dahliae, and form a basis for future functional studies of these genes.

  10. Influence of soil type, cultivar and Verticillium dahliae on the structure of the root and rhizosphere soil fungal microbiome of strawberry.

    PubMed

    Nallanchakravarthula, Srivathsa; Mahmood, Shahid; Alström, Sadhna; Finlay, Roger D

    2014-01-01

    Sustainable management of crop productivity and health necessitates improved understanding of the ways in which rhizosphere microbial populations interact with each other, with plant roots and their abiotic environment. In this study we examined the effects of different soils and cultivars, and the presence of a soil-borne fungal pathogen, Verticillium dahliae, on the fungal microbiome of the rhizosphere soil and roots of strawberry plants, using high-throughput pyrosequencing. Fungal communities of the roots of two cultivars, Honeoye and Florence, were statistically distinct from those in the rhizosphere soil of the same plants, with little overlap. Roots of plants growing in two contrasting field soils had high relative abundance of Leptodontidium sp. C2 BESC 319 g whereas rhizosphere soil was characterised by high relative abundance of Trichosporon dulcitum or Cryptococcus terreus, depending upon the soil type. Differences between different cultivars were not as clear. Inoculation with the pathogen V. dahliae had a significant influence on community structure, generally decreasing the number of rhizosphere soil- and root-inhabiting fungi. Leptodontidium sp. C2 BESC 319 g was the dominant fungus responding positively to inoculation with V. dahliae. The results suggest that 1) plant roots select microorganisms from the wider rhizosphere pool, 2) that both rhizosphere soil and root inhabiting fungal communities are influenced by V. dahliae and 3) that soil type has a stronger influence on both of these communities than cultivar.

  11. Mutational analysis of the Verticillium dahliae protein elicitor PevD1 identifies distinctive regions responsible for hypersensitive response and systemic acquired resistance in tobacco.

    PubMed

    Liu, Wenxian; Zeng, Hongmei; Liu, Zhipeng; Yang, Xiufen; Guo, Lihua; Qiu, Dewen

    2014-01-01

    In our previous study, PevD1 was characterized as a novel protein elicitor produced by Verticillium dahliae inducing hypersensitive response (HR) and systemic acquired resistance (SAR) in tobacco plants; however, the detailed mechanisms of PevD1's elicitor activity remain unclear. In this study, five mutant fragments of PevD1 were generated by polymerase chain reaction-based mutagenesis and the truncated proteins expressed in Escherichia coli were used to test their elicitor activities. Biological activity analysis showed that the N-terminal and C-terminal of PevD1 had distinct influence on HR and SAR elicitation. Fragment PevD1ΔN98, which spans the C-terminal 57 amino acids of PevD1, was critical for the induction of HR in tobacco plants. In contrast, fragment PevD1ΔC57, the N-terminal of 98 amino acids of PevD1, retained the ability to induce SAR against tobacco mosaic virus (TMV) but not induction of HR, suggesting that the induction of HR is not essential for SAR mediated by PevD1. Our results indicated that fragment PevD1ΔC57 could be a candidate peptide for plant protection against pathogens without causing negative effects.

  12. Cotton major latex protein 28 functions as a positive regulator of the ethylene responsive factor 6 in defense against Verticillium dahliae.

    PubMed

    Yang, Chun-Lin; Liang, Shan; Wang, Hai-Yun; Han, Li-Bo; Wang, Fu-Xin; Cheng, Huan-Qing; Wu, Xiao-Min; Qu, Zhan-Liang; Wu, Jia-He; Xia, Gui-Xian

    2015-03-01

    In this study, we identified a defense-related major latex protein (MLP) from upland cotton (designated GhMLP28) and investigated its functional mechanism. GhMLP28 transcripts were ubiquitously present in cotton plants, with higher accumulation in the root. Expression of the GhMLP28 gene was induced by Verticillium dahliae inoculation and was responsive to defense signaling molecules, including ethylene, jasmonic acid, and salicylic acid. Knockdown of GhMLP28 expression by virus-induced gene silencing resulted in increased susceptibility of cotton plants to V. dahliae infection, while ectopic overexpression of GhMLP28 in tobacco improved the disease tolerance of the transgenic plants. Further analysis revealed that GhMLP28 interacted with cotton ethylene response factor 6 (GhERF6) and facilitated the binding of GhERF6 to GCC-box element. Transient expression assay demonstrated that GhMLP28 enhanced the transcription factor activity of GhERF6, which led to the augmented expression of some GCC-box genes. GhMLP28 proteins were located in both the nucleus and cytoplasm and their nuclear distribution was dependent on the presence of GhERF6. Collectively, these results demonstrate that GhMLP28 acts as a positive regulator of GhERF6, and synergetic actions of the two proteins may contribute substantially to protection against V. dahliae infection in cotton plants.

  13. Influence of Soil Type, Cultivar and Verticillium dahliae on the Structure of the Root and Rhizosphere Soil Fungal Microbiome of Strawberry

    PubMed Central

    Nallanchakravarthula, Srivathsa; Mahmood, Shahid; Alström, Sadhna; Finlay, Roger D.

    2014-01-01

    Sustainable management of crop productivity and health necessitates improved understanding of the ways in which rhizosphere microbial populations interact with each other, with plant roots and their abiotic environment. In this study we examined the effects of different soils and cultivars, and the presence of a soil-borne fungal pathogen, Verticillium dahliae, on the fungal microbiome of the rhizosphere soil and roots of strawberry plants, using high-throughput pyrosequencing. Fungal communities of the roots of two cultivars, Honeoye and Florence, were statistically distinct from those in the rhizosphere soil of the same plants, with little overlap. Roots of plants growing in two contrasting field soils had high relative abundance of Leptodontidium sp. C2 BESC 319 g whereas rhizosphere soil was characterised by high relative abundance of Trichosporon dulcitum or Cryptococcus terreus, depending upon the soil type. Differences between different cultivars were not as clear. Inoculation with the pathogen V. dahliae had a significant influence on community structure, generally decreasing the number of rhizosphere soil- and root-inhabiting fungi. Leptodontidium sp. C2 BESC 319 g was the dominant fungus responding positively to inoculation with V. dahliae. The results suggest that 1) plant roots select microorganisms from the wider rhizosphere pool, 2) that both rhizosphere soil and root inhabiting fungal communities are influenced by V. dahliae and 3) that soil type has a stronger influence on both of these communities than cultivar. PMID:25347069

  14. Draft Genome Sequences of Pseudomonas fluorescens Strains PA4C2 and PA3G8 and Pseudomonas putida PA14H7, Three Biocontrol Bacteria against Dickeya Phytopathogens

    PubMed Central

    Cigna, Jérémy; Raoul des Essarts, Yannick; Mondy, Samuel; Hélias, Valérie; Beury-Cirou, Amélie

    2015-01-01

    Pseudomonas fluorescens strains PA4C2 and PA3G8 and Pseudomonas putida strain PA14H7 were isolated from potato rhizosphere and show an ability to inhibit the growth of Dickeya phytopathogens. Here, we report their draft genome sequences, which provide a basis for understanding the molecular mechanisms involved in antibiosis against Dickeya. PMID:25635023

  15. Anti-phytopathogen potential of endophytic actinobacteria isolated from tomato plants (Lycopersicon esculentum) in southern Brazil, and characterization of Streptomyces sp. R18(6), a potential biocontrol agent.

    PubMed

    de Oliveira, Margaroni Fialho; da Silva, Mariana Germano; Van Der Sand, Sueli T

    2010-09-01

    Tomato plants (Lycopersicon esculentum) are highly susceptible to phytopathogen attack. The resulting intensive application of pesticides on tomato crops can affect the environment and health of humans and animals. The objective of this study was to select potential biocontrol agents among actinobacteria from tomato plants, in a search for alternative phytopathogen control. We evaluated 70 endophytic actinobacteria isolated from tomato plants in southern Brazil, testing their antimicrobial activity, siderophore production, indoleacetic acid production, and phosphate solubility. The actinomycete isolate with the highest antimicrobial potential was selected using the agar-well diffusion method, in order to optimize conditions for the production of compounds with antimicrobial activity. For this study, six growth media (starch casein-SC, ISP2, Bennett's, Sahin, Czapek-Dox, and TSB), three temperatures (25 degrees C, 30 degrees C, and 35 degrees C) and different pH were tested. Of the actinobacteria tested, 88.6% showed antimicrobial activity against at least one phytopathogen, 72.1% showed a positive reaction for indoleacetic acid production, 86.8% produced siderophores and 16.2% showed a positive reaction for phosphate solubility. Isolate R18(6) was selected due to its antagonistic activity against all phytopathogenic microorganisms tested in this study. The best conditions for production were observed in the SC medium, at 30 degrees C and pH 7.0. The isolate R18(6) showed close biochemical and genetic similarity to Streptomyces pluricolorescens. PMID:20542109

  16. Anti-phytopathogen potential of endophytic actinobacteria isolated from tomato plants (Lycopersicon esculentum) in southern Brazil, and characterization of Streptomyces sp. R18(6), a potential biocontrol agent.

    PubMed

    de Oliveira, Margaroni Fialho; da Silva, Mariana Germano; Van Der Sand, Sueli T

    2010-09-01

    Tomato plants (Lycopersicon esculentum) are highly susceptible to phytopathogen attack. The resulting intensive application of pesticides on tomato crops can affect the environment and health of humans and animals. The objective of this study was to select potential biocontrol agents among actinobacteria from tomato plants, in a search for alternative phytopathogen control. We evaluated 70 endophytic actinobacteria isolated from tomato plants in southern Brazil, testing their antimicrobial activity, siderophore production, indoleacetic acid production, and phosphate solubility. The actinomycete isolate with the highest antimicrobial potential was selected using the agar-well diffusion method, in order to optimize conditions for the production of compounds with antimicrobial activity. For this study, six growth media (starch casein-SC, ISP2, Bennett's, Sahin, Czapek-Dox, and TSB), three temperatures (25 degrees C, 30 degrees C, and 35 degrees C) and different pH were tested. Of the actinobacteria tested, 88.6% showed antimicrobial activity against at least one phytopathogen, 72.1% showed a positive reaction for indoleacetic acid production, 86.8% produced siderophores and 16.2% showed a positive reaction for phosphate solubility. Isolate R18(6) was selected due to its antagonistic activity against all phytopathogenic microorganisms tested in this study. The best conditions for production were observed in the SC medium, at 30 degrees C and pH 7.0. The isolate R18(6) showed close biochemical and genetic similarity to Streptomyces pluricolorescens.

  17. Draft Genome Sequences of Pseudomonas fluorescens Strains PA4C2 and PA3G8 and Pseudomonas putida PA14H7, Three Biocontrol Bacteria against Dickeya Phytopathogens.

    PubMed

    Cigna, Jérémy; Raoul des Essarts, Yannick; Mondy, Samuel; Hélias, Valérie; Beury-Cirou, Amélie; Faure, Denis

    2015-01-29

    Pseudomonas fluorescens strains PA4C2 and PA3G8 and Pseudomonas putida strain PA14H7 were isolated from potato rhizosphere and show an ability to inhibit the growth of Dickeya phytopathogens. Here, we report their draft genome sequences, which provide a basis for understanding the molecular mechanisms involved in antibiosis against Dickeya.

  18. Effect of crude plant extracts from some Oaxacan flora on two deleterious fungal phytopathogens and extract compatibility with a biofertilizer strain.

    PubMed

    Lira-De León, Karla I; Ramírez-Mares, Marco V; Sánchez-López, Vladimir; Ramírez-Lepe, Mario; Salas-Coronado, Raúl; Santos-Sánchez, Norma F; Valadez-Blanco, Rogelio; Hernández-Carlos, Beatriz

    2014-01-01

    The antimicrobial activity of 12 plant extracts was tested against the phytopathogens Alternaria alternata and Fusarium solani. In addition, the compatibility of the extracts toward Bacillus liqueniformis, a biofertilizer and a non-target microorganism, was assessed. Plants tested belong to the Euphorbiaceae, Asteraceae, Crassulaceae, Rubiaceae, Convolvulaceae, Verbenaceae, Orchidaceae, Nyctaginaceae, Boraginaceae, and Tiliaceae families and were collected in the State of Oaxaca. The antifungal activity of the plant extracts (50-100 mg/mL) against A. alternata and F. solani, was determined by measuring the mycelium radial growth and obtaining the minimum inhibitory concentration (MIC) of fungal growth. In addition, with the aim of finding plant extracts which are compatible with a B. licheniformis biofertilizer strain and to test the non-toxic nature of the treatments, the toxicity of the extracts toward this strain was evaluated using the agar diffusion method. Azoxystrobin (12 μg) and chloramphenicol (30 μg) were used as positive controls for the pathogens and for the non-target bacteria, respectively. Plant extracts inhibited fungal growth in the ranges of 0.76-56.17% against F. solani and 2.02-69.07% against A. alternata. The extracts of Acalypha subviscida, Ipomoea murucoides, Tournefortia densiflora and Lantana achyranthifolia showed MIC values between 5.77-12.5 mg/mL for at least one of the fungal species. The best treatment, Adenophyllum aurantium, exhibited a maximum inhibition for both F. solani (56.17%, MIC = 7.78 mg/mL) and A. alternata (68.64% MIC = 7.78 mg/mL), and resulted innocuous toward B. licheniformis. Therefore, this plant has an outstanding potential for the agroecological control of fungal phytopathogens in industrial crops. PMID:25147544

  19. Effect of crude plant extracts from some Oaxacan flora on two deleterious fungal phytopathogens and extract compatibility with a biofertilizer strain.

    PubMed

    Lira-De León, Karla I; Ramírez-Mares, Marco V; Sánchez-López, Vladimir; Ramírez-Lepe, Mario; Salas-Coronado, Raúl; Santos-Sánchez, Norma F; Valadez-Blanco, Rogelio; Hernández-Carlos, Beatriz

    2014-01-01

    The antimicrobial activity of 12 plant extracts was tested against the phytopathogens Alternaria alternata and Fusarium solani. In addition, the compatibility of the extracts toward Bacillus liqueniformis, a biofertilizer and a non-target microorganism, was assessed. Plants tested belong to the Euphorbiaceae, Asteraceae, Crassulaceae, Rubiaceae, Convolvulaceae, Verbenaceae, Orchidaceae, Nyctaginaceae, Boraginaceae, and Tiliaceae families and were collected in the State of Oaxaca. The antifungal activity of the plant extracts (50-100 mg/mL) against A. alternata and F. solani, was determined by measuring the mycelium radial growth and obtaining the minimum inhibitory concentration (MIC) of fungal growth. In addition, with the aim of finding plant extracts which are compatible with a B. licheniformis biofertilizer strain and to test the non-toxic nature of the treatments, the toxicity of the extracts toward this strain was evaluated using the agar diffusion method. Azoxystrobin (12 μg) and chloramphenicol (30 μg) were used as positive controls for the pathogens and for the non-target bacteria, respectively. Plant extracts inhibited fungal growth in the ranges of 0.76-56.17% against F. solani and 2.02-69.07% against A. alternata. The extracts of Acalypha subviscida, Ipomoea murucoides, Tournefortia densiflora and Lantana achyranthifolia showed MIC values between 5.77-12.5 mg/mL for at least one of the fungal species. The best treatment, Adenophyllum aurantium, exhibited a maximum inhibition for both F. solani (56.17%, MIC = 7.78 mg/mL) and A. alternata (68.64% MIC = 7.78 mg/mL), and resulted innocuous toward B. licheniformis. Therefore, this plant has an outstanding potential for the agroecological control of fungal phytopathogens in industrial crops.

  20. lpxC and yafS are the Most Suitable Internal Controls to Normalize Real Time RT-qPCR Expression in the Phytopathogenic Bacteria Dickeya dadantii

    PubMed Central

    Oger-Desfeux, Christine; Pineau-Chapelle, Emilie; Van Gijsegem, Frederique; Nasser, William; Reverchon, Sylvie

    2011-01-01

    Background Quantitative RT-PCR is the method of choice for studying, with both sensitivity and accuracy, the expression of genes. A reliable normalization of the data, using several reference genes, is critical for an accurate quantification of gene expression. Here, we propose a set of reference genes, of the phytopathogenic bacteria Dickeya dadantii and Pectobacterium atrosepticum, which are stable in a wide range of growth conditions. Results We extracted, from a D. dadantii micro-array transcript profile dataset comprising thirty-two different growth conditions, an initial set of 49 expressed genes with very low variation in gene expression. Out of these, we retained 10 genes representing different functional categories, different levels of expression (low, medium, and high) and with no systematic variation in expression correlating with growth conditions. We measured the expression of these reference gene candidates using quantitative RT-PCR in 50 different experimental conditions, mimicking the environment encountered by the bacteria in their host and directly during the infection process in planta. The two most stable genes (ABF-0017965 (lpxC) and ABF-0020529 (yafS) were successfully used for normalization of RT-qPCR data. Finally, we demonstrated that the ortholog of lpxC and yafS in Pectobacterium atrosepticum also showed stable expression in diverse growth conditions. Conclusions We have identified at least two genes, lpxC (ABF-0017965) and yafS (ABF-0020509), whose expressions are stable in a wide range of growth conditions and during infection. Thus, these genes are considered suitable for use as reference genes for the normalization of real-time RT-qPCR data of the two main pectinolytic phytopathogenic bacteria D. dadantii and P. atrosepticum and, probably, of other Enterobacteriaceae. Moreover, we defined general criteria to select good reference genes in bacteria. PMID:21637857

  1. Effect of crude plant extracts from some Oaxacan flora on two deleterious fungal phytopathogens and extract compatibility with a biofertilizer strain

    PubMed Central

    Lira-De León, Karla I.; Ramírez-Mares, Marco V.; Sánchez-López, Vladimir; Ramírez-Lepe, Mario; Salas-Coronado, Raúl; Santos-Sánchez, Norma F.; Valadez-Blanco, Rogelio; Hernández-Carlos, Beatriz

    2014-01-01

    The antimicrobial activity of 12 plant extracts was tested against the phytopathogens Alternaria alternata and Fusarium solani. In addition, the compatibility of the extracts toward Bacillus liqueniformis, a biofertilizer and a non-target microorganism, was assessed. Plants tested belong to the Euphorbiaceae, Asteraceae, Crassulaceae, Rubiaceae, Convolvulaceae, Verbenaceae, Orchidaceae, Nyctaginaceae, Boraginaceae, and Tiliaceae families and were collected in the State of Oaxaca. The antifungal activity of the plant extracts (50–100 mg/mL) against A. alternata and F. solani, was determined by measuring the mycelium radial growth and obtaining the minimum inhibitory concentration (MIC) of fungal growth. In addition, with the aim of finding plant extracts which are compatible with a B. licheniformis biofertilizer strain and to test the non-toxic nature of the treatments, the toxicity of the extracts toward this strain was evaluated using the agar diffusion method. Azoxystrobin (12 μg) and chloramphenicol (30 μg) were used as positive controls for the pathogens and for the non-target bacteria, respectively. Plant extracts inhibited fungal growth in the ranges of 0.76–56.17% against F. solani and 2.02–69.07% against A. alternata. The extracts of Acalypha subviscida, Ipomoea murucoides, Tournefortia densiflora and Lantana achyranthifolia showed MIC values between 5.77–12.5 mg/mL for at least one of the fungal species. The best treatment, Adenophyllum aurantium, exhibited a maximum inhibition for both F. solani (56.17%, MIC = 7.78 mg/mL) and A. alternata (68.64% MIC = 7.78 mg/mL), and resulted innocuous toward B. licheniformis. Therefore, this plant has an outstanding potential for the agroecological control of fungal phytopathogens in industrial crops. PMID:25147544

  2. Glucanases and Chitinases as Causal Agents in the Protection of Acacia Extrafloral Nectar from Infestation by Phytopathogens1[W][OA

    PubMed Central

    González-Teuber, Marcia; Pozo, María J.; Muck, Alexander; Svatos, Ales; Adame-Álvarez, Rosa M.; Heil, Martin

    2010-01-01

    Nectars are rich in primary metabolites and attract mutualistic animals, which serve as pollinators or as an indirect defense against herbivores. Their chemical composition makes nectars prone to microbial infestation. As protective strategy, floral nectar of ornamental tobacco (Nicotiana langsdorffii × Nicotiana sanderae) contains “nectarins,” proteins producing reactive oxygen species such as hydrogen peroxide. By contrast, pathogenesis-related (PR) proteins were detected in Acacia extrafloral nectar (EFN), which is secreted in the context of defensive ant-plant mutualisms. We investigated whether these PR proteins protect EFN from phytopathogens. Five sympatric species (Acacia cornigera, A. hindsii, A. collinsii, A. farnesiana, and Prosopis juliflora) were compared that differ in their ant-plant mutualism. EFN of myrmecophytes, which are obligate ant-plants that secrete EFN constitutively to nourish specialized ant inhabitants, significantly inhibited the growth of four out of six tested phytopathogenic microorganisms. By contrast, EFN of nonmyrmecophytes, which is secreted only transiently in response to herbivory, did not exhibit a detectable inhibitory activity. Combining two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis with nanoflow liquid chromatography-tandem mass spectrometry analysis confirmed that PR proteins represented over 90% of all proteins in myrmecophyte EFN. The inhibition of microbial growth was exerted by the protein fraction, but not the small metabolites of this EFN, and disappeared when nectar was heated. In-gel assays demonstrated the activity of acidic and basic chitinases in all EFNs, whereas glucanases were detected only in EFN of myrmecophytes. Our results demonstrate that PR proteins causally underlie the protection of Acacia EFN from microorganisms and that acidic and basic glucanases likely represent the most important prerequisite in this defensive function. PMID:20023149

  3. Impact of Multi-year Cropping Regimes on Solanum tuberosum Tuber Yields in the Presence of Pratylenchus penetrans and Verticillium dahliae.

    PubMed

    Chen, J; Bird, G W; Mather, R L

    1995-12-01

    Five cropping regimes involving combinations of 2 legumes, alfalfa (Medicago sativa) and yellow sweet dover (Melilotus officinalis), 2 monocots, corn (Zea mays) and sudax (Sorghum halupeuse x Sorghum sudanese), and potato (Solanum tuberosum cv. Superior) were tested for their impact on potato yields in a field infested with Pratylenchus penetrans and Verticillium dahliae. No differences in 1990 tuber yields were observed among the five cropping regimes (P < 0.05). In 1991, yields following 1 year of corn, sudax, sweet clover, or alfalfa and 2 years of potato were not different from that of 3 years of continuous potato (P < 0.05). Two years of sweet clover or alfalfa followed by potato resulted in significantly increased potato tuber yields compared with 3 years of potato (P < 0.05). The 2-year legume and 2-year grain rotations resulted in lower P. penetrans population densities at the end of the 3-year rotation compared with 3 years of continuous potato (P < 0.01). The highest preplant V. dahliae population density (34 cfu/g soil), together with a P. penetrans density of 12/100 cm(3) of soil was in the sudax-sudax-potato cropping regime and resulted in the lowest potato tuber yield. The highest preplant P. penetrans population density (54/100 cm(3) soil), together with a V. dahliae population density of 19.5 cfu/g soil was observed in the corn-corn-potato cropping regime and resulted in the second lowest potato tuber yield in 1991. After 3 years, potato tuber yields were negatively related to preplant densities of V. dahliae (r(2) = 0.237), P. penetrans (r(2) = 0.175), and both pathogens (r(2) = 0.380). A comprehensive regression model was developed to isolate pathogen effects on potato yields from cropping regime effects encompassing all 10 cropping regimes (r(2) = 0.915).

  4. A New Synthetic Amphiploid (AADDAA) between Gossypium hirsutum and G. arboreum Lays the Foundation for Transferring Resistances to Verticillium and Drought.

    PubMed

    Chen, Yu; Wang, Yingying; Zhao, Ting; Yang, Jianwei; Feng, Shouli; Nazeer, Wajad; Zhang, Tianzhen; Zhou, Baoliang

    2015-01-01

    Gossypium arboreum, a cultivated cotton species (2n = 26, AA) native to Asia, possesses invaluable characteristics unavailable in the tetraploid cultivated cotton gene pool, such as resistance to pests and diseases and tolerance to abiotic stresses. However, it is quite difficult to transfer favorable traits into Upland cotton through conventional methods due to the cross-incompatibility of G. hirsutum (2n = 52, AADD) and G. arboreum. Here, we improved an embryo rescue technique to overcome the cross-incompatibility between these two parents for transferring favorable genes from G. arboreum into G. hirsutum. Our results indicate that MSB2K supplemented with 0.5 mg l(-1) kinetin and 250 mg(-1) casein hydrolysate is an efficient initial medium for rescuing early (3 d after pollination) hybrid embryos. Eight putative hybrids were successfully obtained, which were further verified and characterized by cytology, molecular markers and morphological analysis. The putative hybrids were subsequently treated with different concentrations of colchicine solution to double their chromosomes. The results demonstrate that four putative hybrid plants were successfully chromosome-doubled by treatment with 0.1% colchicine for 24 h and become amphiploid, which were confirmed by cytological observation, self-fertilization and backcrossing. Preliminary assessments of resistance at seedling stage indicate that the synthetic amphiploid showed highly resistant to Verticillium and drought. The synthetic amphiploid between G. hirsutum × G. arboreum would lay the foundation for developing G. arboreum-introgressed lines with the uniform genetic background of G. hirsutum acc TM-1, which would greatly enhance and simplify the mining, isolation, characterization, cloning and use of G. arboreum-specific desirable genes in future cotton breeding programs. PMID:26061996

  5. A New Synthetic Amphiploid (AADDAA) between Gossypium hirsutum and G. arboreum Lays the Foundation for Transferring Resistances to Verticillium and Drought

    PubMed Central

    Chen, Yu; Wang, Yingying; Zhao, Ting; Yang, Jianwei; Feng, Shouli; Nazeer, Wajad; Zhang, Tianzhen; Zhou, Baoliang

    2015-01-01

    Gossypium arboreum, a cultivated cotton species (2n = 26, AA) native to Asia, possesses invaluable characteristics unavailable in the tetraploid cultivated cotton gene pool, such as resistance to pests and diseases and tolerance to abiotic stresses. However, it is quite difficult to transfer favorable traits into Upland cotton through conventional methods due to the cross-incompatibility of G. hirsutum (2n = 52, AADD) and G. arboreum. Here, we improved an embryo rescue technique to overcome the cross-incompatibility between these two parents for transferring favorable genes from G. arboreum into G. hirsutum. Our results indicate that MSB2K supplemented with 0.5 mgl-1 kinetin and 250 mg-1 casein hydrolysate is an efficient initial medium for rescuing early (3 d after pollination) hybrid embryos. Eight putative hybrids were successfully obtained, which were further verified and characterized by cytology, molecular markers and morphological analysis. The putative hybrids were subsequently treated with different concentrations of colchicine solution to double their chromosomes. The results demonstrate that four putative hybrid plants were successfully chromosome-doubled by treatment with 0.1% colchicine for 24 h and become amphiploid, which were confirmed by cytological observation, self-fertilization and backcrossing. Preliminary assessments of resistance at seedling stage indicate that the synthetic amphiploid showed highly resistant to Verticillium and drought. The synthetic amphiploid between G. hirsutum × G. arboreum would lay the foundation for developing G. arboreum-introgressed lines with the uniform genetic background of G. hirsutum acc TM-1, which would greatly enhance and simplify the mining, isolation, characterization, cloning and use of G. arboreum-specific desirable genes in future cotton breeding programs. PMID:26061996

  6. A cupin domain-containing protein with a quercetinase activity (VdQase) regulates Verticillium dahliae's pathogenicity and contributes to counteracting host defenses

    PubMed Central

    El Hadrami, Abdelbasset; Islam, Md. Rashidul; Adam, Lorne R.; Daayf, Fouad

    2015-01-01

    We previously identified rutin as part of potato root responses to its pathogen Verticillium dahliae. Rutin was directly toxic to the pathogen at doses greater than 160 μM, a threshold below which many V. dahliae pathogenicity-related genes were up-regulated. We identified and characterized a cupin domain-containing protein (VdQase) with a dioxygenase activity and a potential role in V. dahliae-potato interactions. The pathogenicity of VdQase knock-out mutants generated through Agrobacterium tumefasciens-mediated transformation was significantly reduced on susceptible potato cultivar Kennebec compared to wild type isolates. Fluorescence microscopy revealed a higher accumulation of flavonols in the stems of infected potatoes and a higher concentration of rutin in the leaves in response to the VdQase mutants as compared to wild type isolates. This, along with the HPLC characterization of high residual and non-utilized quercetin in presence of the knockout mutants, indicates the involvement of VdQase in the catabolism of quercetin and possibly other flavonols in planta. Quantification of Salicylic and Jasmonic Acids (SA, JA) in response to the mutants vs. wild type isolates revealed involvement of VdQase in the interference with signaling, suggesting a role in pathogenicity. It is hypothesized that the by-product of dioxygenation 2-protocatechuoylphloroglucinolcarboxylic acid, after dissociating into phloroglucinol and protocatechuoyl moieties, becomes a starting point for benzoic acid and SA, thereby interfering with the JA pathway and affecting the interaction outcome. These events may be key factors for V. dahliae in countering potato defenses and becoming notorious in the rhizosphere. PMID:26113857

  7. A New Synthetic Amphiploid (AADDAA) between Gossypium hirsutum and G. arboreum Lays the Foundation for Transferring Resistances to Verticillium and Drought.

    PubMed

    Chen, Yu; Wang, Yingying; Zhao, Ting; Yang, Jianwei; Feng, Shouli; Nazeer, Wajad; Zhang, Tianzhen; Zhou, Baoliang

    2015-01-01

    Gossypium arboreum, a cultivated cotton species (2n = 26, AA) native to Asia, possesses invaluable characteristics unavailable in the tetraploid cultivated cotton gene pool, such as resistance to pests and diseases and tolerance to abiotic stresses. However, it is quite difficult to transfer favorable traits into Upland cotton through conventional methods due to the cross-incompatibility of G. hirsutum (2n = 52, AADD) and G. arboreum. Here, we improved an embryo rescue technique to overcome the cross-incompatibility between these two parents for transferring favorable genes from G. arboreum into G. hirsutum. Our results indicate that MSB2K supplemented with 0.5 mg l(-1) kinetin and 250 mg(-1) casein hydrolysate is an efficient initial medium for rescuing early (3 d after pollination) hybrid embryos. Eight putative hybrids were successfully obtained, which were further verified and characterized by cytology, molecular markers and morphological analysis. The putative hybrids were subsequently treated with different concentrations of colchicine solution to double their chromosomes. The results demonstrate that four putative hybrid plants were successfully chromosome-doubled by treatment with 0.1% colchicine for 24 h and become amphiploid, which were confirmed by cytological observation, self-fertilization and backcrossing. Preliminary assessments of resistance at seedling stage indicate that the synthetic amphiploid showed highly resistant to Verticillium and drought. The synthetic amphiploid between G. hirsutum × G. arboreum would lay the foundation for developing G. arboreum-introgressed lines with the uniform genetic background of G. hirsutum acc TM-1, which would greatly enhance and simplify the mining, isolation, characterization, cloning and use of G. arboreum-specific desirable genes in future cotton breeding programs.

  8. Roles of the catalytic subunit of cAMP-dependent protein kinase A in virulence and development of the soilborne plant pathogen Verticillium dahliae.

    PubMed

    Tzima, Aliki; Paplomatas, Epaminondas J; Rauyaree, Payungsak; Kang, Seogchan

    2010-05-01

    Verticillium dahliae is a soilborne fungus that causes vascular wilt disease in a broad range of hosts and survives for many years in the soil in the form of microsclerotia. Although the role of cAMP-dependent protein kinase A (PKA) has been extensively studied in foliar pathogens, there is limited information about its role in soilborne fungal pathogens that infect through the root system. Genome database search revealed the presence of two PKA catalytic subunit genes in V. dahliae, named VdPKAC1 and VdPKAC2. A phylogenetic analysis showed that VdPKAC2 groups with fungal PKA catalytic subunits that appear to play a minor role in PKA activity. This gene was expressed considerably lower than that of VdPKAC1. Although disruption of VdPKAC1 did not affect the ability of V. dahliae to infect through the roots of tomato and eggplant, disease severity was significantly reduced. Since pathogen-derived ethylene is presumed to play a major role in symptom induction in vascular wilt diseases, ethylene generation was measured in fungal culture. The mutants defective in VdPKAC1 produced less ethylene than the corresponding wild type strains, suggesting a regulatory role of PKA in ethylene biosynthesis. Growth rates of these mutants were similar to those of wild type strains, while the rate of spore germination was slightly elevated and conidia production was significantly reduced. When grown on minimal media, the mutants showed greater microsclerotia production compared with the wild type strains. These results suggest multiple roles of VdPKAC1, including virulence, conidiation, microsclerotia formation, and ethylene biosynthesis, in the soilborne fungus V. dahliae.

  9. Cotton WRKY1 mediates the plant defense-to-development transition during infection of cotton by Verticillium dahliae by activating JASMONATE ZIM-DOMAIN1 expression.

    PubMed

    Li, Chao; He, Xin; Luo, Xiangyin; Xu, Li; Liu, Linlin; Min, Ling; Jin, Li; Zhu, Longfu; Zhang, Xianlong

    2014-12-01

    Plants have evolved an elaborate signaling network to ensure an appropriate level of immune response to meet the differing demands of developmental processes. Previous research has demonstrated that DELLA proteins physically interact with JASMONATE ZIM-DOMAIN1 (JAZ1) and dynamically regulate the interaction of the gibberellin (GA) and jasmonate (JA) signaling pathways. However, whether and how the JAZ1-DELLA regulatory node is regulated at the transcriptional level in plants under normal growth conditions or during pathogen infection is not known. Here, we demonstrate multiple functions of cotton (Gossypium barbadense) GbWRKY1 in the plant defense response and during development. Although GbWRKY1 expression is induced rapidly by methyl jasmonate and infection by Verticillium dahliae, our results show that GbWRKY1 is a negative regulator of the JA-mediated defense response and plant resistance to the pathogens Botrytis cinerea and V. dahliae. Under normal growth conditions, GbWRKY1-overexpressing lines displayed GA-associated phenotypes, including organ elongation and early flowering, coupled with the down-regulation of the putative targets of DELLA. We show that the GA-related phenotypes of GbWRKY1-overexpressing plants depend on the constitutive expression of Gossypium hirsutum GhJAZ1. We also show that GhJAZ1 can be transactivated by GbWRKY1 through TGAC core sequences, and the adjacent sequences of this binding site are essential for binding specificity and affinity to GbWRKY1, as revealed by dual-luciferase reporter assays and electrophoretic mobility shift assays. In summary, our data suggest that GbWRKY1 is a critical regulator mediating the plant defense-to-development transition during V. dahliae infection by activating JAZ1 expression.

  10. Spatial gene expression analysis in tomato hypocotyls suggests cysteine as key precursor of vascular sulfur accumulation implicated in Verticillium dahliae defense.

    PubMed

    Klug, Katharina; Hogekamp, Claudia; Specht, André; Myint, San Shwe; Blöink, Dominik; Küster, Helge; Horst, Walter J

    2015-02-01

    Verticillium dahliae is a prominent generator of plant vascular wilting disease and sulfur (S)-enhanced defense (SED) mechanisms contribute to its in-planta elimination. The accumulation of S-containing defense compounds (SDCs) including elemental S (S(0) ) has been described based on the comparison of two near-isogenic tomato (Solanum lycopersicum) lines differing in fungal susceptibility. To better understand the effect of S nutrition on V. dahliae resistance both lines were supplied with low, optimal or supraoptimal sulfate-S. An absolute quantification demonstrated a most effective fungal elimination due to luxury plant S nutrition. High-pressure liquid chromatography (HPLC) showed a strong regulation of Cys levels and an S-responsive GSH pool rise in the bulk hypocotyl. High-frequency S peak accumulations were detected in vascular bundles of resistant tomato plants after fungal colonization by laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS). Global transcriptomic analysis suggested that early steps of the primary S metabolism did not promote the SDCs synthesis in the whole hypocotyl as gene expression was downregulated after infection. Enhanced S fertilization mostly alleviated the repressive fungal effect but did not reverse it. Upregulation of glutathione (GSH)-associated genes in bulk hypocotyls but not in vascular bundles indicated a global antioxidative role of GSH. To finally assign the contribution of S metabolism-associated genes to high S(0) accumulations exclusively found in the resistant tomato line, a spatial gene expression approach was applied. Laser microdissection of infected vascular bundles revealed a switch toward transcription of genes connected with cysteine (Cys) synthesis. The upregulation of LeOASTLp1 suggests a role for Cys as key precursor for local S accumulations (possibly S(0) ) in the vascular bundles of the V. dahliae-resistant tomato line.

  11. A study of the changes in quantitative activity of some enzymes in olive cultivars during the interaction between Verticillium wilt and root-knot nematode.

    PubMed

    Saeedizadeh, Ayatollah; Kheiri, Ahmad; Zad, Javad; Etebarian, Hasan Reza; Bandani, Ali Reza; Sharifi, Rouhollah

    2009-01-01

    Second stage juvenile (J2) of root-knot nematode, Meloidogyne javanica, and microsclerotia of verticilliosis, Verticillium dahliae, were used as the source of inoculum for nematode and fungus respectively. One-year-old seedlings of olive cultivar, Zard, Roghani, Koroneiki and Manzanilla, were transplanted to pots containing 2000g of sterilized sandy loam soil. Experiment was conducted in completely randomized design with 32 Treatments and five replications. Treatments were as follows: control, nematode alone, fungus alone and fungus+nematode. Pots were inoculated with (0, 2000, 3000, 4000) J2 of nematode and/or (10 no/g soil) microsclerotia of fungus according to the treatments. Quantitative activity of soluble proxidase, cell wall bounded proxidase, beta-1,3-glucanase and B-1,4-glucanase were determined by the pirocatechol, hydrogen proxide, guaiacol and laminarin-dinitrosalicylilate methods, respectively, on 1,10, 20 and 30 days after inoculation. Results showed that these enzymes in leaves and roots of seedlings inoculated with pathogens increased (p < or = 0/05). In this study changes of these enzymes as compared with nematode alone and fungus alone treatments were increased in fungus+nematode treatments in seedlings (p < or = 0/05). Maximum of quantitative changes of these enzymes were observed in fungus+nematode (4000J2) treatment on cv. Koroneiki. Based on the results obtained in this study, quantitative activity of these enzymes in olive roots and leaves were be more on cvs. Koroneiki, Roghani, Zard and Manzanilla, respectively (p < or = 0/05). PMID:20222618

  12. Purification, characterization of a CkChn134 protein from Cynanchum komarovii seeds and synergistic effect with CkTLP against Verticillium dahliae.

    PubMed

    Wang, Qinghua; Zhang, Yongan; Hou, Yuxia; Wang, Ping; Zhou, Sihong; Ma, Xiaowen; Zhang, Ning

    2012-06-01

    Cynanchum komarovii Al Iljinski is a desert plant that has been used as analgesic, anthelminthic, and antidiarrheal, but also as herbal medicine to treat cholecystitis in people. In this work, an antifungal protein with sequence homology to chitinase was isolated from C. komarovii seeds and named CkChn134. The three-dimensional structure prediction of CkChn134 indicated that the protein has a loop domain formed a thin cleft, which is able to bind molecules and substrates. The protein and CkTLP synergistically inhibited the fungal growth of Verticillium dahliae, Fusarium oxysporum, Rhizoctonia solani, Botrytis cinerea, and Valsa mali in vitro. The full-length cDNA was cloned by RT-PCR and RACE-PCR according to the partial protein sequences obtained by nanoESI-MS/MS. The real-time PCR showed that the transcription level of CkChn134 had a significant increase under the stress of ethylene, NaCl, low temperature, drought, and pathogen infection, which indicates that CkChn134 may play an important role in response to abiotic and biotic stresses. The CkChn134 protein was located in the extracellular space/cell wall by CkChn134::GFP fusion protein in transgenic Arabidopsis. Furthermore, overexpression of CkChn134 significantly enhanced the resistance of transgenic Arabidopsis against V. dahliae. Interestingly, the coexpression of CkChn134 and CkTLP showed substantially greater protection against the fungal pathogen V. dahliae than either transgene alone. The results suggest that the CkChn134 is a good candidate protein or gene, and it had a potential synergistic effect with CkTLP for contributing to the development of disease-resistant crops.

  13. The bacterial lipopeptide iturins induce Verticillium dahliae cell death by affecting fungal signalling pathways and mediate plant defence responses involved in pathogen-associated molecular pattern-triggered immunity.

    PubMed

    Han, Qin; Wu, Fengli; Wang, Xiaonan; Qi, Hong; Shi, Liang; Ren, Ang; Liu, Qinghai; Zhao, Mingwen; Tang, Canming

    2015-04-01

    Verticillium wilt in cotton caused by Verticillium dahliae is one of the most serious plant diseases worldwide. Because no known fungicides or cotton cultivars provide sufficient protection against this pathogen, V. dahliae causes major crop yield losses. Here, an isolated cotton endophytic bacterium, designated Bacillus amyloliquefaciens 41B-1, exhibited greater than 50% biocontrol efficacy against V. dahliae in cotton plants under greenhouse conditions. Through high-performance liquid chromatography and mass analysis of the filtrate, we found that the antifungal compounds present in the strain 41B-1 culture filtrate were a series of isoforms of iturins. The purified iturins suppressed V. dahliae microsclerotial germination in the absence or presence of cotton. Treatment with the iturins induced reactive oxygen species bursts, Hog1 mitogen-activated protein kinase (MAPK) activation and defects in cell wall integrity. The oxidative stress response and high-osmolarity glycerol pathway contribute to iturins resistance in V. dahliae. In contrast, the Slt2 MAPK pathway may be involved in iturins sensitivity in this fungus. In addition to antagonism, iturins could induce plant defence responses as activators and mediate pathogen-associated molecular pattern-triggered immunity. These findings suggest that iturins may affect fungal signalling pathways and mediate plant defence responses against V. dahliae.

  14. Verticillium dahliae race 2-specific PCR reveals a high frequency of race 2 strains in commercial spinach seed lots and delineates race structure.

    PubMed

    Short, Dylan P G; Gurung, Suraj; Maruthachalam, Karunakaran; Atallah, Zahi K; Subbarao, Krishna V

    2014-07-01

    Two pathogenic races of Verticillium dahliae have been described on lettuce and tomato. Host resistance to race 1 is governed by plant immune receptors that recognize the race 1-specific fungal effector Ave1. Only partial resistance to race 2 exists in lettuce. Although polymerase chain reaction (PCR) assays are available to identify race 1, no complementary test exists to positively identify race 2, except for lengthy pathogenicity assays on host differentials. Using the genome sequences of two isolates of V. dahliae, one each from races 1 and 2, we identified potential markers and PCR primers to distinguish the two races. Several primer pairs based on polymorphisms between the races were designed and tested on reference isolates of known race. One primer pair, VdR2F-VdR2R, consistently yielded a 256-bp amplicon in all race 2 isolates exclusively. We screened DNA from 677 V. dahliae isolates, including 340 from spinach seedlots, with the above primer pair and a previously published race 1-specific primer pair. DNA from isolates that did not amplify with race 1-specific PCRs amplified with the race 2-specific primers. To validate this, two differential lines of lettuce were inoculated with 53 arbitrarily selected isolates from spinach seed and their pathogenicity and virulence were assessed in a greenhouse. The reactions of the differential cultivars strongly supported the PCR data. V. dahliae race structure was investigated in crops in coastal California and elsewhere using primers specific to the two races. All artichoke isolates from California were race 1, whereas nearly all tomato isolates were race 2. Isolates from lettuce, pepper, and strawberry from California as well as isolates from spinach seed from two of four countries comprised both races, whereas only race 2 was observed in cotton, mint, olive, and potato. This highlights the importance of identifying resistance against race 2 in different hosts. The technique developed in this study will benefit

  15. An ethylene response-related factor, GbERF1-like, from Gossypium barbadense improves resistance to Verticillium dahliae via activating lignin synthesis.

    PubMed

    Guo, Weifeng; Jin, Li; Miao, Yuhuan; He, Xin; Hu, Qin; Guo, Kai; Zhu, Longfu; Zhang, Xianlong

    2016-06-01

    An ethylene response-related factor, GbERF1-like, from Gossypium barbadense cv. '7124' involved in the defence response to Verticillium dahliae was characterized. GbERF1-like transcripts present ubiquitously in various tissues, with higher accumulation in flower organs. GbERF1-like was also responsive to defence-related phytohormones and V. dahliae infection. The downregulation of GbERF1-like increased the susceptibility of cotton plants to V. dahliae infection, while overexpression of this gene improved disease resistance in both cotton and Arabidopsis, coupled with activation of the pathogenesis-related proteins. Further analysis revealed that genes involved in lignin synthesis, such as PAL, C4H, C3H, HCT, CCoAOMT, CCR and F5H, showed higher expression levels in the overexpressing cotton and Arabidopsis lines and lower expression levels in the RNAi cotton lines. The expression levels of these genes increased obviously when the GbERF1-like-overexpressing plants were inoculated with V. dahliae. Meanwhile, significant differences in the content of whole lignin could be found in the stems of transgenic and wild-type plants after inoculation with V. dahliae, as revealed by metabolic and histochemical analysis. More lignin could be detected in GbERF1-like-overexpressing cotton and Arabidopsis but less in GbERF1-like-silencing cotton compared with wild-type plants. The ratio of S and G monomers in GbERF1-like-overexpressing cotton and Arabidopsis increased significantly after infection by V. dahliae. Moreover, our results showed that the promoters of GhHCT1 and AtPAL3 could be transactivated by GbERF1-like in vivo based on yeast one-hybrid assays and dual-luciferase reporter assays. Knockdown of GhHCT1 in GbERF1-like over-expressing cotton decreases resistance to V. dahliae. Collectively, our results suggest that GbERF1-like acts as a positive regulator in lignin synthesis and contributes substantially to resistance to V. dahliae in plants.

  16. Verticillium dahliae race 2-specific PCR reveals a high frequency of race 2 strains in commercial spinach seed lots and delineates race structure.

    PubMed

    Short, Dylan P G; Gurung, Suraj; Maruthachalam, Karunakaran; Atallah, Zahi K; Subbarao, Krishna V

    2014-07-01

    Two pathogenic races of Verticillium dahliae have been described on lettuce and tomato. Host resistance to race 1 is governed by plant immune receptors that recognize the race 1-specific fungal effector Ave1. Only partial resistance to race 2 exists in lettuce. Although polymerase chain reaction (PCR) assays are available to identify race 1, no complementary test exists to positively identify race 2, except for lengthy pathogenicity assays on host differentials. Using the genome sequences of two isolates of V. dahliae, one each from races 1 and 2, we identified potential markers and PCR primers to distinguish the two races. Several primer pairs based on polymorphisms between the races were designed and tested on reference isolates of known race. One primer pair, VdR2F-VdR2R, consistently yielded a 256-bp amplicon in all race 2 isolates exclusively. We screened DNA from 677 V. dahliae isolates, including 340 from spinach seedlots, with the above primer pair and a previously published race 1-specific primer pair. DNA from isolates that did not amplify with race 1-specific PCRs amplified with the race 2-specific primers. To validate this, two differential lines of lettuce were inoculated with 53 arbitrarily selected isolates from spinach seed and their pathogenicity and virulence were assessed in a greenhouse. The reactions of the differential cultivars strongly supported the PCR data. V. dahliae race structure was investigated in crops in coastal California and elsewhere using primers specific to the two races. All artichoke isolates from California were race 1, whereas nearly all tomato isolates were race 2. Isolates from lettuce, pepper, and strawberry from California as well as isolates from spinach seed from two of four countries comprised both races, whereas only race 2 was observed in cotton, mint, olive, and potato. This highlights the importance of identifying resistance against race 2 in different hosts. The technique developed in this study will benefit

  17. FTIR spectroscopic evaluation of changes in the cellular biochemical composition of the phytopathogenic fungus Alternaria alternata induced by extracts of some Greek medicinal and aromatic plants

    NASA Astrophysics Data System (ADS)

    Skotti, Efstathia; Kountouri, Sophia; Bouchagier, Pavlos; Tsitsigiannis, Dimitrios I.; Polissiou, Moschos; Tarantilis, Petros A.

    2014-06-01

    In this study, the biological activity of aquatic extracts of selected Greek medicinal and aromatic plants to the phytopathogenic fungus Alternaria alternata was investigated. Lamiaceae species (Hyssopus officinalis L., Melissa officinalis L., Origanum dictamnus L., Origanum vulgare L. and Salvia officinalis L.) were found to enhance significantly the mycelium growth whereas Crocus sativus appears to inhibit it slightly. M. officinalis and S. officinalis caused the highest stimulation in mycelium growth (+97%) and conidia production (+65%) respectively. In order to further investigate the bioactivity of plant extracts to A. alternata, we employed Fourier Transform Infrared Spectroscopy (FTIR). Differences of original spectra were assigned mainly to amides of proteins. The second derivative transformation of spectra revealed changes in spectral regions corresponding to absorptions of the major cellular constituents such as cell membrane and proteins. Principal component analysis of the second derivative transformed spectra confirmed that fatty acids of the cell membranes, amides of proteins and polysaccharides of the cell wall had the major contribution to data variation. FTIR band area ratios were found to correlate with fungal mycelium growth.

  18. FTIR spectroscopic evaluation of changes in the cellular biochemical composition of the phytopathogenic fungus Alternaria alternata induced by extracts of some Greek medicinal and aromatic plants.

    PubMed

    Skotti, Efstathia; Kountouri, Sophia; Bouchagier, Pavlos; Tsitsigiannis, Dimitrios I; Polissiou, Moschos; Tarantilis, Petros A

    2014-06-01

    In this study, the biological activity of aquatic extracts of selected Greek medicinal and aromatic plants to the phytopathogenic fungus Alternaria alternata was investigated. Lamiaceae species (Hyssopus officinalis L., Melissa officinalis L., Origanum dictamnus L., Origanum vulgare L. and Salvia officinalis L.) were found to enhance significantly the mycelium growth whereas Crocus sativus appears to inhibit it slightly. M. officinalis and S. officinalis caused the highest stimulation in mycelium growth (+97%) and conidia production (+65%) respectively. In order to further investigate the bioactivity of plant extracts to A. alternata, we employed Fourier Transform Infrared Spectroscopy (FTIR). Differences of original spectra were assigned mainly to amides of proteins. The second derivative transformation of spectra revealed changes in spectral regions corresponding to absorptions of the major cellular constituents such as cell membrane and proteins. Principal component analysis of the second derivative transformed spectra confirmed that fatty acids of the cell membranes, amides of proteins and polysaccharides of the cell wall had the major contribution to data variation. FTIR band area ratios were found to correlate with fungal mycelium growth.

  19. Tallow amphopolycarboxyglycinate-stabilized silver nanoparticles: new frontiers in development of plant protection products with a broad spectrum of action against phytopathogens

    NASA Astrophysics Data System (ADS)

    Krutyakov, Yurii A.; Kudrinskiy, Alexey A.; Zherebin, Pavel M.; Yapryntsev, Alexey D.; Pobedinskaya, Marina A.; Elansky, Sergey N.; Denisov, Albert N.; Mikhaylov, Dmitry M.; Lisichkin, Georgii V.

    2016-07-01

    Sustainable agriculture calls for minimal use of agrochemicals in order to protect the environment. It has caused an increase in the rate of nanoparticles use, in particular silver nanoparticles (AgNPs) due to their safety for mammals, unique biological activity and a broad spectrum of action against fungal and bacterial pathogens. Until now the use of AgNPs dispersions in the agricultural sector has been essentially limited due to many factors decreased their stability (mixing with other pesticides, presence of electrolytes). We present a versatile synthesis of polyampholyte surfactant (tallow amphopolycarboxyglycinate) stabilized AgNPs. We took a close look at unique aggregation behavior (via dynamic light scattering and UV-vis spectroscopy) and biocidal activity of obtained silver colloids. AgNPs are characterized by exclusively high aggregative stability in the presence of coagulating agents NaNO3 and NaSO4 (up to 1 M), during drying/redispergation, and frost/defrost cycles. The dispersion of AgNPs shows high biocidal activity (EC50 is ten times lower than commercial species ones) with respect to Phytophthora infestans and phytopathogenic fungi. This points to the possibility of successful application of silver preparations within agriculture with the goal of partial reduction of the use of toxic and expensive synthetic antibiotics and pesticides.

  20. FTIR spectroscopic evaluation of changes in the cellular biochemical composition of the phytopathogenic fungus Alternaria alternata induced by extracts of some Greek medicinal and aromatic plants.

    PubMed

    Skotti, Efstathia; Kountouri, Sophia; Bouchagier, Pavlos; Tsitsigiannis, Dimitrios I; Polissiou, Moschos; Tarantilis, Petros A

    2014-06-01

    In this study, the biological activity of aquatic extracts of selected Greek medicinal and aromatic plants to the phytopathogenic fungus Alternaria alternata was investigated. Lamiaceae species (Hyssopus officinalis L., Melissa officinalis L., Origanum dictamnus L., Origanum vulgare L. and Salvia officinalis L.) were found to enhance significantly the mycelium growth whereas Crocus sativus appears to inhibit it slightly. M. officinalis and S. officinalis caused the highest stimulation in mycelium growth (+97%) and conidia production (+65%) respectively. In order to further investigate the bioactivity of plant extracts to A. alternata, we employed Fourier Transform Infrared Spectroscopy (FTIR). Differences of original spectra were assigned mainly to amides of proteins. The second derivative transformation of spectra revealed changes in spectral regions corresponding to absorptions of the major cellular constituents such as cell membrane and proteins. Principal component analysis of the second derivative transformed spectra confirmed that fatty acids of the cell membranes, amides of proteins and polysaccharides of the cell wall had the major contribution to data variation. FTIR band area ratios were found to correlate with fungal mycelium growth. PMID:24657421

  1. Tallow amphopolycarboxyglycinate-stabilized silver nanoparticles: new frontiers in development of plant protection products with a broad spectrum of action against phytopathogens

    NASA Astrophysics Data System (ADS)

    Krutyakov, Yurii A.; Kudrinskiy, Alexey A.; Zherebin, Pavel M.; Yapryntsev, Alexey D.; Pobedinskaya, Marina A.; Elansky, Sergey N.; Denisov, Albert N.; Mikhaylov, Dmitry M.; Lisichkin, Georgii V.

    2016-07-01

    Sustainable agriculture calls for minimal use of agrochemicals in order to protect the environment. It has caused an increase in the rate of nanoparticles use, in particular silver nanoparticles (AgNPs) due to their safety for mammals, unique biological activity and a broad spectrum of action against fungal and bacterial pathogens. Until now the use of AgNPs dispersions in the agricultural sector has been essentially limited due to many factors decreased their stability (mixing with other pesticides, presence of electrolytes). We present a versatile synthesis of polyampholyte surfactant (tallow amphopolycarboxyglycinate) stabilized AgNPs. We took a close look at unique aggregation behavior (via dynamic light scattering and UV–vis spectroscopy) and biocidal activity of obtained silver colloids. AgNPs are characterized by exclusively high aggregative stability in the presence of coagulating agents NaNO3 and NaSO4 (up to 1 M), during drying/redispergation, and frost/defrost cycles. The dispersion of AgNPs shows high biocidal activity (EC50 is ten times lower than commercial species ones) with respect to Phytophthora infestans and phytopathogenic fungi. This points to the possibility of successful application of silver preparations within agriculture with the goal of partial reduction of the use of toxic and expensive synthetic antibiotics and pesticides.

  2. Green synthesis of protein capped silver nanoparticles from phytopathogenic fungus Macrophomina phaseolina (Tassi) Goid with antimicrobial properties against multidrug-resistant bacteria

    PubMed Central

    2014-01-01

    In recent years, green synthesis of nanoparticles, i.e., synthesizing nanoparticles using biological sources like bacteria, algae, fungus, or plant extracts have attracted much attention due to its environment-friendly and economic aspects. The present study demonstrates an eco-friendly and low-cost method of biosynthesis of silver nanoparticles using cell-free filtrate of phytopathogenic fungus Macrophomina phaseolina. UV-visible spectrum showed a peak at 450 nm corresponding to the plasmon absorbance of silver nanoparticles. Scanning electron microscopy (SEM), atomic force microscopy (AFM), and transmission electron microscopy (TEM) revealed the presence of spherical silver nanoparticles of the size range 5 to 40 nm, most of these being 16 to 20 nm in diameter. X-ray diffraction (XRD) spectrum of the nanoparticles exhibited 2θ values corresponding to silver nanoparticles. These nanoparticles were found to be naturally protein coated. SDS-PAGE analysis showed the presence of an 85-kDa protein band responsible for capping and stabilization of the silver nanoparticles. Antimicrobial activities of the silver nanoparticles against human as well as plant pathogenic multidrug-resistant bacteria were assayed. The particles showed inhibitory effect on the growth kinetics of human and plant bacteria. Furthermore, the genotoxic potential of the silver nanoparticles with increasing concentrations was evaluated by DNA fragmentation studies using plasmid DNA. PMID:25114655

  3. Mobilization of horizontally acquired island 2 is induced in planta in the phytopathogen Pectobacterium atrosepticum SCRI1043 and involves the putative relaxase ECA0613 and quorum sensing.

    PubMed

    Vanga, Bhanupratap R; Ramakrishnan, Pavithra; Butler, Ruth C; Toth, Ian K; Ronson, Clive W; Jacobs, Jeanne M E; Pitman, Andrew R

    2015-11-01

    Integrative and conjugative elements (ICEs) contribute to the rapid evolution of bacterial pathogens via horizontal gene transfer of virulence determinants. ICEs have common mechanisms for transmission, yet the cues triggering this process under natural environmental or physiological conditions are largely unknown. In this study, mobilization of the putative ICE horizontally acquired island 2 (HAI2), present in the chromosome of the phytopathogen Pectobacterium atrosepticum SCRI1043, was examined during infection of the host plant potato. Under these conditions, mobilization of HAI2 increased markedly compared with in vitro cultures. In planta-induced mobilization of HAI2 was regulated by quorum sensing and involved the putative ICE-encoded relaxase ECA0613. Disruption of ECA0613 also reduced transcription of genes involved in production of coronafacic acid (Cfa), the major virulence factor harboured on HAI2, whereas their expression was unaffected in the quorum-sensing (expI) mutant. Thus, suppression of cfa gene expression was not regulated by the mobilization of the ICE per se, but was due directly to inactivation of the relaxase. The identification of genetic factors associated solely with in planta mobilization of an ICE demonstrates that this process is highly adapted to the natural environment of the bacterial host and can influence the expression of virulence determinants.

  4. Green synthesis of protein capped silver nanoparticles from phytopathogenic fungus Macrophomina phaseolina (Tassi) Goid with antimicrobial properties against multidrug-resistant bacteria

    NASA Astrophysics Data System (ADS)

    Chowdhury, Supriyo; Basu, Arpita; Kundu, Surekha

    2014-07-01

    In recent years, green synthesis of nanoparticles, i.e., synthesizing nanoparticles using biological sources like bacteria, algae, fungus, or plant extracts have attracted much attention due to its environment-friendly and economic aspects. The present study demonstrates an eco-friendly and low-cost method of biosynthesis of silver nanoparticles using cell-free filtrate of phytopathogenic fungus Macrophomina phaseolina. UV-visible spectrum showed a peak at 450 nm corresponding to the plasmon absorbance of silver nanoparticles. Scanning electron microscopy (SEM), atomic force microscopy (AFM), and transmission electron microscopy (TEM) revealed the presence of spherical silver nanoparticles of the size range 5 to 40 nm, most of these being 16 to 20 nm in diameter. X-ray diffraction (XRD) spectrum of the nanoparticles exhibited 2 θ values corresponding to silver nanoparticles. These nanoparticles were found to be naturally protein coated. SDS-PAGE analysis showed the presence of an 85-kDa protein band responsible for capping and stabilization of the silver nanoparticles. Antimicrobial activities of the silver nanoparticles against human as well as plant pathogenic multidrug-resistant bacteria were assayed. The particles showed inhibitory effect on the growth kinetics of human and plant bacteria. Furthermore, the genotoxic potential of the silver nanoparticles with increasing concentrations was evaluated by DNA fragmentation studies using plasmid DNA.

  5. Functional Annotation of the Ophiostoma novo-ulmi Genome: Insights into the Phytopathogenicity of the Fungal Agent of Dutch Elm Disease

    PubMed Central

    Comeau, André M.; Dufour, Josée; Bouvet, Guillaume F.; Jacobi, Volker; Nigg, Martha; Henrissat, Bernard; Laroche, Jérôme; Levesque, Roger C.; Bernier, Louis

    2015-01-01

    The ascomycete fungus Ophiostoma novo-ulmi is responsible for the pandemic of Dutch elm disease that has been ravaging Europe and North America for 50 years. We proceeded to annotate the genome of the O. novo-ulmi strain H327 that was sequenced in 2012. The 31.784-Mb nuclear genome (50.1% GC) is organized into 8 chromosomes containing a total of 8,640 protein-coding genes that we validated with RNA sequencing analysis. Approximately 53% of these genes have their closest match to Grosmannia clavigera kw1407, followed by 36% in other close Sordariomycetes, 5% in other Pezizomycotina, and surprisingly few (5%) orphans. A relatively small portion (∼3.4%) of the genome is occupied by repeat sequences; however, the mechanism of repeat-induced point mutation appears active in this genome. Approximately 76% of the proteins could be assigned functions using Gene Ontology analysis; we identified 311 carbohydrate-active enzymes, 48 cytochrome P450s, and 1,731 proteins potentially involved in pathogen–host interaction, along with 7 clusters of fungal secondary metabolites. Complementary mating-type locus sequencing, mating tests, and culturing in the presence of elm terpenes were conducted. Our analysis identified a specific genetic arsenal impacting the sexual and vegetative growth, phytopathogenicity, and signaling/plant–defense–degradation relationship between O. novo-ulmi and its elm host and insect vectors. PMID:25539722

  6. MRN- and 9-1-1-Independent Activation of the ATR-Chk1 Pathway during the Induction of the Virulence Program in the Phytopathogen Ustilago maydis.

    PubMed

    Tenorio-Gómez, María; de Sena-Tomás, Carmen; Pérez-Martín, Jose

    2015-01-01

    DNA damage response (DDR) leads to DNA repair, and depending on the extent of the damage, to further events, including cell death. Evidence suggests that cell differentiation may also be a consequence of the DDR. During the formation of the infective hypha in the phytopathogenic fungus Ustilago maydis, two DDR kinases, Atr1 and Chk1, are required to induce a G2 cell cycle arrest, which in turn is essential to display the virulence program. However, the triggering factor of DDR in this process has remained elusive. In this report we provide data suggesting that no DNA damage is associated with the activation of the DDR during the formation of the infective filament in U. maydis. We have analyzed bulk DNA replication during the formation of the infective filament, and we found no signs of impaired DNA replication. Furthermore, using RPA-GFP fusion as a surrogate marker of the presence of DNA damage, we were unable to detect any sign of DNA damage at the cellular level. In addition, neither MRN nor 9-1-1 complexes, both instrumental to transmit the DNA damage signal, are required for the induction of the above mentioned cell cycle arrest, as well as for virulence. In contrast, we have found that the claspin-like protein Mrc1, which in other systems serves as scaffold for Atr1 and Chk1, was required for both processes. We discuss possible alternative ways to trigger the DDR, independent of DNA damage, in U. maydis during virulence program activation.

  7. Isolation, characterization and molecular three-dimensional structural predictions of metalloprotease from a phytopathogenic fungus, Alternaria solani (Ell. and Mart.) Sor.

    PubMed

    Chandrasekaran, Murugesan; Chandrasekar, Raman; Chun, Se-Chul; Sathiyabama, Muthukrishnan

    2016-08-01

    The present study aims at isolation, identification, characterization and prediction of three-dimensional molecular architecture of a proteolytic enzyme from the early blight pathogen, Alternaria solani which are hypothesized to be a marker of phytopathogenicity. Maximum enzyme production by A. solani was observed in Czapex's Dox broth amended with 2% (w/v) casein than other inducer amendments. Results indicate that the enzyme remained highly active in a pH range of 7.0-10.0 and a temperature range of 45-50°C. The enzyme was strongly inhibited by EDTA, whereas phenylmethylsulfonyl fluoride and monovalent cations (Na(+), K(+)) had little effect. Metal ions such as MgSO4, CaCl2, KCl at 10 mM concentration showed a stimulatory effect (>85%) on protease activity. Matrix-assisted laser desorption and ionization time of flight/mass spectrometry analysis of partially purified enzyme revealed the presence of protease belonging to a keratinolytic protein (metalloprotease) of exopeptidase nature. Putative A. solani keratinolytic enzyme (AsK) is made up of 216 amino acid residues with molecular weight (MW) 24.5 kDa, having a molecular formula of C1094H1704N290O342S4. Ramachandran plot analysis of the protein residues falling into the most favored secondary structures was observed at 84.2%. The major protein structural blocks, 2-β-sheets, and 9-α-helices have a greater tendency to be conserved during the evolutionary process than do mere sequences of amino acids. Besides, AsK, model prediction showed the presence of a Zinc atom at helix regions (Helix 3, 6, 7: His(57), His(130), His(169), and Cys(123)). Thus, it can be concluded that the major proteinases of AsK are divalent cation-requiring metalloproteinases and make them potential targets of protease inhibitors designing. PMID:26924427

  8. Incidence of Genome Structure, DNA Asymmetry, and Cell Physiology on T-DNA Integration in Chromosomes of the Phytopathogenic Fungus Leptosphaeria maculans

    PubMed Central

    Bourras, Salim; Meyer, Michel; Grandaubert, Jonathan; Lapalu, Nicolas; Fudal, Isabelle; Linglin, Juliette; Ollivier, Benedicte; Blaise, Françoise; Balesdent, Marie-Hélène; Rouxel, Thierry

    2012-01-01

    The ever-increasing generation of sequence data is accompanied by unsatisfactory functional annotation, and complex genomes, such as those of plants and filamentous fungi, show a large number of genes with no predicted or known function. For functional annotation of unknown or hypothetical genes, the production of collections of mutants using Agrobacterium tumefaciens–mediated transformation (ATMT) associated with genotyping and phenotyping has gained wide acceptance. ATMT is also widely used to identify pathogenicity determinants in pathogenic fungi. A systematic analysis of T-DNA borders was performed in an ATMT-mutagenized collection of the phytopathogenic fungus Leptosphaeria maculans to evaluate the features of T-DNA integration in its particular transposable element-rich compartmentalized genome. A total of 318 T-DNA tags were recovered and analyzed for biases in chromosome and genic compartments, existence of CG/AT skews at the insertion site, and occurrence of microhomologies between the T-DNA left border (LB) and the target sequence. Functional annotation of targeted genes was done using the Gene Ontology annotation. The T-DNA integration mainly targeted gene-rich, transcriptionally active regions, and it favored biological processes consistent with the physiological status of a germinating spore. T-DNA integration was strongly biased toward regulatory regions, and mainly promoters. Consistent with the T-DNA intranuclear-targeting model, the density of T-DNA insertion correlated with CG skew near the transcription initiation site. The existence of microhomologies between promoter sequences and the T-DNA LB flanking sequence was also consistent with T-DNA integration to host DNA mediated by homologous recombination based on the microhomology-mediated end-joining pathway. PMID:22908038

  9. Antioxidant and Antifungal Activity of Extracts of the Aerial Parts of Thymus capitatus (L.) Hoffmanns Against Four Phytopathogenic Fungi of Citrus sinensis

    PubMed Central

    Tabti, Leila; Dib, Mohammed El Amine; Gaouar, Nassira; Samira, Bouayad; Tabti, Boufeldja

    2014-01-01

    Background: Many medicinal plants from the Lamiaceae family can be easily found in Algeria. These plants have been used as traditional medicines by local ethnic groups. Thymus capitatus is known in Algeria as "Zaatar" and has been commonly used as a spice, and reported to have many biological effects. Objectives: This paper focused on the assessment of the antioxidant potential and antifungal activity of essential oil and solvent extracts of T. capitatus against the growth of certain fungi. Materials and Methods: Essential oil, ethanol and hexane extracts of T. capitatus were tested for their antioxidant and antifungal activities. The 2, 2-diphenyl-1-picrylhydrazyl (DPPH) assay was used to determine the free radical quenching capacity. Antifungal activity was assessed using the radial growth technique. Results: DPPH free radical scavenging effect of the extracts was compared with standard antioxidant ascorbic acid and showed significant results. The ethanol extract showed high activity at the concentration of 80 g/mL, but less than the standard ascorbic acid. The essential oil was effective against all the fungi used in the experiment. The highest inhibitory effect on the growth of Aspergillus niger, Aspergillus oryzae, Penicillium digitatum, and Fusarium solani was exhibited by the essential oil at concentrations between 0.1 and 0.5 μg/mL. Conclusions: These findings demonstrated that ethanol extract obtained from T. capitatus is a potential source of natural antioxidant, while the essential oil extract can be exploited as an ideal alternative to synthetic fungicides for use in the treatment of many fungal phytopathogens. PMID:24644439

  10. Eukaryotic extracellular catalase–peroxidase from Magnaporthe grisea – Biophysical/chemical characterization of the first representative from a novel phytopathogenic KatG group

    PubMed Central

    Zámocký, Marcel; Droghetti, Enrica; Bellei, Marzia; Gasselhuber, Bernhard; Pabst, Martin; Furtmüller, Paul G.; Battistuzzi, Gianantonio; Smulevich, Giulietta; Obinger, Christian

    2012-01-01

    All phytopathogenic fungi have two catalase–peroxidase paralogues located either intracellularly (KatG1) or extracellularly (KatG2). Here, for the first time a secreted bifunctional, homodimeric catalase–peroxidase (KatG2 from the rice blast fungus Magnaporthe grisea) has been produced heterologously with almost 100% heme occupancy and comprehensively investigated by using a broad set of methods including UV–Vis, ECD and resonance Raman spectroscopy (RR), thin-layer spectroelectrochemistry, mass spectrometry, steady-state & presteady-state spectroscopy. RR spectroscopy reveals that MagKatG2 shows a unique mixed-spin state, non-planar heme b, and a proximal histidine with pronounced imidazolate character. At pH 7.0 and 25 °C, the standard reduction potential E°′ of the Fe(III)/Fe(II) couple for the high-spin native protein was found to fall in the range typical for the KatG family. Binding of cyanide was relatively slow at pH 7.0 and 25 °C and with a Kd value significantly higher than for the intracellular counterpart. Demonstrated by mass spectrometry MagKatG2 has the typical Trp118-Tyr251-Met277 adduct that is essential for its predominantly catalase activity at the unique acidic pH optimum. In addition, MagKatG2 acts as a versatile peroxidase using both one- and two-electron donors. Based on these data, structure–function relationships of extracellular eukaryotic KatGs are discussed with respect to intracellular KatGs and possible role(s) in host–pathogen interaction. PMID:21971530

  11. A novel single-stranded RNA virus isolated from a phytopathogenic filamentous fungus, Rosellinia necatrix, with similarity to hypo-like viruses

    PubMed Central

    Zhang, Rui; Liu, Shengxue; Chiba, Sotaro; Kondo, Hideki; Kanematsu, Satoko; Suzuki, Nobuhiro

    2014-01-01

    Here we report a biological and molecular characterization of a novel positive-sense RNA virus isolated from a field isolate (NW10) of a filamentous phytopathogenic fungus, the white root rot fungus that is designated as Rosellinia necatrix fusarivirus 1 (RnFV1). A recently developed technology using zinc ions allowed us to transfer RnFV1 to two mycelially incompatible Rosellinia necatrix strains. A biological comparison of the virus-free and -recipient isogenic fungal strains suggested that RnFV1 infects latently and thus has no potential as a virocontrol agent. The virus has an undivided positive-sense RNA genome of 6286 nucleotides excluding a poly (A) tail. The genome possesses two non-overlapping open reading frames (ORFs): a large ORF1 that encodes polypeptides with RNA replication functions and a smaller ORF2 that encodes polypeptides of unknown function. A lack of coat protein genes was suggested by the failure of virus particles from infected mycelia. No evidence was obtained by Northern analysis or classical 5′-RACE for the presence of subgenomic RNA for the downstream ORF. Sequence similarities were found in amino-acid sequence between RnFV1 putative proteins and counterparts of a previously reported mycovirus, Fusarium graminearum virus 1 (FgV1). Interestingly, several related sequences were detected by BLAST searches of independent transcriptome assembly databases one of which probably represents an entire virus genome. Phylogenetic analysis based on the conserved RNA-dependent RNA polymerase showed that RnFV1, FgV1, and these similar sequences are grouped in a cluster distinct from distantly related hypoviruses. It is proposed that a new taxonomic family termed Fusariviridae be created to include RnFV1 and FgV1. PMID:25101066

  12. A filamentous hemagglutinin-like protein of Xanthomonas axonopodis pv. citri, the phytopathogen responsible for citrus canker, is involved in bacterial virulence.

    PubMed

    Gottig, Natalia; Garavaglia, Betiana S; Garofalo, Cecilia G; Orellano, Elena G; Ottado, Jorgelina

    2009-01-01

    Xanthomonas axonopodis pv. citri, the phytopathogen responsible for citrus canker has a number of protein secretion systems and among them, at least one type V protein secretion system belonging to the two-partner secretion pathway. This system is mainly associated to the translocation of large proteins such as adhesins to the outer membrane of several pathogens. Xanthomonas axonopodis pv. citri possess a filamentous hemagglutinin-like protein in close vicinity to its putative transporter protein, XacFhaB and XacFhaC, respectively. Expression analysis indicated that XacFhaB was induced in planta during plant-pathogen interaction. By mutation analysis of XacFhaB and XacFhaC genes we determined that XacFhaB is involved in virulence both in epiphytic and wound inoculations, displaying more dispersed and fewer canker lesions. Unexpectedly, the XacFhaC mutant in the transporter protein produced an intermediate virulence phenotype resembling wild type infection, suggesting that XacFhaB could be secreted by another partner different from XacFhaC. Moreover, XacFhaB mutants showed a general lack of adhesion and were affected in leaf surface attachment and biofilm formation. In agreement with the in planta phenotype, adhesin lacking cells moved faster in swarming plates. Since no hyperflagellation phenotype was observed in this bacteria, the faster movement may be attributed to the lack of cell-to-cell aggregation. Moreover, XacFhaB mutants secreted more exopolysaccharide that in turn may facilitate its motility. Our results suggest that this hemagglutinin-like protein is required for tissue colonization being mainly involved in surface attachment and biofilm formation, and that plant tissue attachment and cell-to-cell aggregation are dependent on the coordinated action of adhesin molecules and exopolysaccharides.

  13. Region-wide analysis of genetic diversity in Verticillium dahliae populations infecting olive in southern Spain and agricultural factors influencing the distribution and prevalence of vegetative compatibility groups and pathotypes.

    PubMed

    Jiménez-Díaz, Rafael M; Olivares-García, Concepción; Landa, Blanca B; del Mar Jiménez-Gasco, María; Navas-Cortés, Juan A

    2011-03-01

    Severity of Verticillium wilt in olive trees in Andalusia, southern Spain is associated with the spread of a highly virulent, defoliating (D) Verticillium dahliae pathotype of vegetative compatibility group 1A (VCG1A) but the extent of this spread and the diversity of the pathogen population have never been documented. VCG typing of 637 V. dahliae isolates from 433 trees in 65 orchards from five olive-growing provinces in Andalusia indicated that 78.1% were of VCG1A, 19.8% of VCG2A, 0.6% of VCG2B, 1.4% of VCG4B, and one isolate was heterokaryon self-incompatible. A single VCG prevailed among isolates within most orchards but two and three VCGs were identified in 12 and 3 orchards, respectively, with VCG1A+VCG2A occurring in 10 orchards. VCG1A was the predominant VCG in the three most important olive-growing provinces, and was almost as prevalent as VCG2A in another one. Molecular pathotyping of the 637 isolates using specific polymerase chain reaction assays indicated that VCG1A isolates were of the D pathotype whereas isolates of VCG2A, -2B, and -4B were of the less virulent nondefoliating (ND) pathotype. The pathotype of isolates correlated with the disease syndrome affecting sampled trees. Only three (seq1, seq2, and seq4) of the seven known sequences of the V. dahliae-specific 539- or 523-bp amplicon were identified among the 637 isolates. Distribution and prevalence of VCGs and seq sequences among orchards indicated that genetic diversity within olive V. dahliae in Andalusia is higher in provinces where VCG1A is not prevalent. Log-linear analysis revealed that irrigation management, source of irrigation water, source of planting stock, and cropping history of soil were significantly associated with the prevalence of VCG1A compared with that of VCG2A. Multivariate analyses using a selected set of agricultural factors as variables allowed development of a discriminant model for predicting the occurrence of D and ND pathotypes in the area of the study. Blind

  14. Cloning and characterization of a pectin lyase gene from Colletotrichum lindemuthianum and comparative phylogenetic/structural analyses with genes from phytopathogenic and saprophytic/opportunistic microorganisms

    PubMed Central

    2011-01-01

    Background Microorganisms produce cell-wall-degrading enzymes as part of their strategies for plant invasion/nutrition. Among these, pectin lyases (PNLs) catalyze the depolymerization of esterified pectin by a β-elimination mechanism. PNLs are grouped together with pectate lyases (PL) in Family 1 of the polysaccharide lyases, as they share a conserved structure in a parallel β-helix. The best-characterized fungal pectin lyases are obtained from saprophytic/opportunistic fungi in the genera Aspergillus and Penicillium and from some pathogens such as Colletotrichum gloeosporioides. The organism used in the present study, Colletotrichum lindemuthianum, is a phytopathogenic fungus that can be subdivided into different physiological races with different capacities to infect its host, Phaseolus vulgaris. These include the non-pathogenic and pathogenic strains known as races 0 and 1472, respectively. Results Here we report the isolation and sequence analysis of the Clpnl2 gene, which encodes the pectin lyase 2 of C. lindemuthianum, and its expression in pathogenic and non-pathogenic races of C. lindemuthianum grown on different carbon sources. In addition, we performed a phylogenetic analysis of the deduced amino acid sequence of Clpnl2 based on reported sequences of PNLs from other sources and compared the three-dimensional structure of Clpnl2, as predicted by homology modeling, with those of other organisms. Both analyses revealed an early separation of bacterial pectin lyases from those found in fungi and oomycetes. Furthermore, two groups could be distinguished among the enzymes from fungi and oomycetes: one comprising enzymes from mostly saprophytic/opportunistic fungi and the other formed mainly by enzymes from pathogenic fungi and oomycetes. Clpnl2 was found in the latter group and was grouped together with the pectin lyase from C. gloeosporioides. Conclusions The Clpnl2 gene of C. lindemuthianum shares the characteristic elements of genes coding for pectin

  15. An Oxalyl-CoA Dependent Pathway of Oxalate Catabolism Plays a Role in Regulating Calcium Oxalate Crystal Accumulation and Defending against Oxalate-Secreting Phytopathogens in Medicago truncatula

    PubMed Central

    Foster, Justin; Luo, Bin; Nakata, Paul A.

    2016-01-01

    Considering the widespread occurrence of oxalate in nature and its broad impact on a host of organisms, it is surprising that so little is known about the turnover of this important acid. In plants, oxalate oxidase is the most well studied enzyme capable of degrading oxalate, but not all plants possess this activity. Recently, an Acyl Activating Enzyme 3 (AAE3), encoding an oxalyl-CoA synthetase, was identified in Arabidopsis. AAE3 has been proposed to catalyze the first step in an alternative pathway of oxalate degradation. Whether this enzyme and proposed pathway is important to other plants is unknown. Here, we identify the Medicago truncatula AAE3 (MtAAE3) and show that it encodes an oxalyl-CoA synthetase activity exhibiting high activity against oxalate with a Km = 81 ± 9 μM and Vmax = 19 ± 0.9 μmoles min-1mg protein-1. GFP-MtAAE3 localization suggested that this enzyme functions within the cytosol of the cell. Mtaae3 knock-down line showed a reduction in its ability to degrade oxalate into CO2. This reduction in the capacity to degrade oxalate resulted in the accumulation of druse crystals of calcium oxalate in the Mtaae3 knock-down line and an increased susceptibility to oxalate-secreting phytopathogens such as Sclerotinia sclerotiorum. Taken together, these results suggest that AAE3 dependent turnover of oxalate is important to different plants and functions in the regulation of tissue calcium oxalate crystal accumulation and in defense against oxalate-secreting phytopathogens. PMID:26900946

  16. Complete genome sequences of the Serratia plymuthica strains 3Rp8 and 3Re4-18, two rhizosphere bacteria with antagonistic activity towards fungal phytopathogens and plant growth promoting abilities.

    PubMed

    Adam, Eveline; Müller, Henry; Erlacher, Armin; Berg, Gabriele

    2016-01-01

    The Serratia plymuthica strains 3Rp8 and 3Re4-18 are motile, Gram-negative, non-sporulating bacteria. Strain 3Rp8 was isolated from the rhizosphere of Brassica napus L. and strain 3Re4-18 from the endorhiza of Solanum tuberosum L. Studies have shown in vitro activity against the soil-borne fungi Verticillium dahliae Kleb., Rhizoctonia solani Kühn, and Sclerotinia sclerotiorum. Here, we announce and describe the complete genome sequence of S. plymuthica 3Rp8 consisting of a single circular chromosome of 5.5 Mb that encodes 4954 protein-coding and 108 RNA-only encoding genes and of S. plymuthica 3Re4-18 consisting of a single circular chromosome of 5.4 Mb that encodes 4845 protein-coding and 109 RNA-only encoding genes. The whole genome sequences and annotations are available in NCBI under the locus numbers CP012096 and CP012097, respectively. The genome analyses revealed genes putatively responsible for the promising plant growth promoting and biocontrol properties including predicting factors such as secretion systems, iron scavenging siderophores, chitinases, secreted proteases, glucanases and non-ribosomal peptide synthetases, as well as unique genomic islands. PMID:27602183

  17. Cotton WRKY1 Mediates the Plant Defense-to-Development Transition during Infection of Cotton by Verticillium dahliae by Activating JASMONATE ZIM-DOMAIN1 Expression1[C][W

    PubMed Central

    Li, Chao; He, Xin; Luo, Xiangyin; Xu, Li; Liu, Linlin; Min, Ling; Jin, Li; Zhu, Longfu; Zhang, Xianlong

    2014-01-01

    Plants have evolved an elaborate signaling network to ensure an appropriate level of immune response to meet the differing demands of developmental processes. Previous research has demonstrated that DELLA proteins physically interact with JASMONATE ZIM-DOMAIN1 (JAZ1) and dynamically regulate the interaction of the gibberellin (GA) and jasmonate (JA) signaling pathways. However, whether and how the JAZ1-DELLA regulatory node is regulated at the transcriptional level in plants under normal growth conditions or during pathogen infection is not known. Here, we demonstrate multiple functions of cotton (Gossypium barbadense) GbWRKY1 in the plant defense response and during development. Although GbWRKY1 expression is induced rapidly by methyl jasmonate and infection by Verticillium dahliae, our results show that GbWRKY1 is a negative regulator of the JA-mediated defense response and plant resistance to the pathogens Botrytis cinerea and V. dahliae. Under normal growth conditions, GbWRKY1-overexpressing lines displayed GA-associated phenotypes, including organ elongation and early flowering, coupled with the down-regulation of the putative targets of DELLA. We show that the GA-related phenotypes of GbWRKY1-overexpressing plants depend on the constitutive expression of Gossypium hirsutum GhJAZ1. We also show that GhJAZ1 can be transactivated by GbWRKY1 through TGAC core sequences, and the adjacent sequences of this binding site are essential for binding specificity and affinity to GbWRKY1, as revealed by dual-luciferase reporter assays and electrophoretic mobility shift assays. In summary, our data suggest that GbWRKY1 is a critical regulator mediating the plant defense-to-development transition during V. dahliae infection by activating JAZ1 expression. PMID:25301887

  18. Effects of experimental warming on fungal disease progress in oilseed rape.

    PubMed

    Siebold, Magdalena; von Tiedemann, Andreas

    2013-06-01

    Global warming will influence the growth and development of both crops and pathogens. The aims of this study were to investigate potential effects of future warming on oilseed rape growth and the epidemiology of the three economically important pathogens Verticillium longisporum, Sclerotinia sclerotiorum, and Leptosphaeria maculans (anamorph: Phoma lingam). We utilized climate chambers and a soil warming facility, where treatments represented regional warming scenarios for Lower Saxony, Germany, by 2050 and 2100, and compared results of both approaches on a thermal time scale by calculating degree-days (dd) from day of sowing, December 1st and March 1st until sampling, the latter correlating best with disease progress. Regression analysis showed that plant growth and growth stages in spring responded almost linearly to increasing thermal time until 1000-1500 dd. Colonization of plant tissue by V. longisporum showed an exponential increase when exceeding 1300-1500 dd and reaching plant growth stage BBCH 74/75 (pod development). V. longisporum colonization of plants may be advanced, potentially leading to higher inoculum densities after harvest and increased economic importance of this pathogen under future warming. Sclerotia germination of S. sclerotiorum reached its maximum at 600-900 dd. Advance of these critical degree-days may lead to earlier apothecia production, potentially advancing the infection window, whereas the future importance of S. sclerotiorum may remain constant. Severity of phoma crown canker increased linearly with increasing thermal time, but showed also large variation in response to the warming scenarios, suggesting that factors such as canopy microclimate in fall or leaf shedding over winter may play a bigger role for L. maculans infection and disease severity than higher soil temperatures. Thermal time was a suitable tool to combine and integrate data on biological responses to soil and air temperature increases from climate chamber and field

  19. Effects of experimental warming on fungal disease progress in oilseed rape.

    PubMed

    Siebold, Magdalena; von Tiedemann, Andreas

    2013-06-01

    Global warming will influence the growth and development of both crops and pathogens. The aims of this study were to investigate potential effects of future warming on oilseed rape growth and the epidemiology of the three economically important pathogens Verticillium longisporum, Sclerotinia sclerotiorum, and Leptosphaeria maculans (anamorph: Phoma lingam). We utilized climate chambers and a soil warming facility, where treatments represented regional warming scenarios for Lower Saxony, Germany, by 2050 and 2100, and compared results of both approaches on a thermal time scale by calculating degree-days (dd) from day of sowing, December 1st and March 1st until sampling, the latter correlating best with disease progress. Regression analysis showed that plant growth and growth stages in spring responded almost linearly to increasing thermal time until 1000-1500 dd. Colonization of plant tissue by V. longisporum showed an exponential increase when exceeding 1300-1500 dd and reaching plant growth stage BBCH 74/75 (pod development). V. longisporum colonization of plants may be advanced, potentially leading to higher inoculum densities after harvest and increased economic importance of this pathogen under future warming. Sclerotia germination of S. sclerotiorum reached its maximum at 600-900 dd. Advance of these critical degree-days may lead to earlier apothecia production, potentially advancing the infection window, whereas the future importance of S. sclerotiorum may remain constant. Severity of phoma crown canker increased linearly with increasing thermal time, but showed also large variation in response to the warming scenarios, suggesting that factors such as canopy microclimate in fall or leaf shedding over winter may play a bigger role for L. maculans infection and disease severity than higher soil temperatures. Thermal time was a suitable tool to combine and integrate data on biological responses to soil and air temperature increases from climate chamber and field

  20. Generalized Transduction in the Phytopathogen Pseudomonas syringae.

    PubMed

    Nordeen, R O; Currier, T C

    1983-06-01

    Bacteriophages isolated from culture supernatants of Pseudomonas syringae pv. syringae and from sewage transferred various chromosomal genes to P. syringae PS224. Linkage between arginine and tryptophan loci was demonstrated. The number of transductants recovered per milliliter was not altered appreciably by UV irradiation of selected phage isolates. In addition, the presence of the IncP2 plasmid R38 in a P. syringae PS224 arginine auxotroph did not increase the transduction frequency as it does in Pseudomonas aeruginosa. Increasing the multiplicity of infection of transducing phage Pssy15 from 1 to 10 resulted in up to a 10-fold increase in the number of transductants recovered, although the actual transductional frequency remained about the same. Treatment of transduction mixtures with DNase did not affect transductional frequency.

  1. Enzymatic response of cotton plants to the pathogen, Verticillium dahliae

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Pathogen infection initiates a set of local and systemic responses in plants. These responses include local oxidative burst, which may lead to death of infected cells, and changes of cell walls composition in neighbouring tissues, and de novo synthesis of antimicrobial compounds (phytoalexins) and P...

  2. Phomalactone from a phytopathogenic fungus infecting Zinnia elegans (Asteraceae) leaves

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Zinnia elegans plants are infected by a fungus that causes necrosis with dark red spots particularly in late spring to the middle of summer in the Mid-South part of the United States. This fungal disease when untreated causes the leaves to wilt and eventually kills the plant. The fungus was isolated...

  3. New Avirulence Genes in the Phytopathogenic Fungus Leptosphaeria maculans.

    PubMed

    Balesdent, M H; Attard, A; Kühn, M L; Rouxel, T

    2002-10-01

    ABSTRACT Leptosphaeria maculans, the causal agent of stem canker of oilseed rape (Brassica napus), develops gene-for-gene interactions with oilseed rape, and four L. maculans avirulence (AVR) genes (AvrLm1, AvrLm2, AvrLm4, and alm1) were previously genetically characterized. Based on the analysis of progeny of numerous in vitro crosses between L. maculans isolates showing either already characterized or new differential interactions, this work aims to provide an overview of the AVR genes that may specify incompatibility toward B. napus and the related species B. juncea and B. rapa. Two novel differential interactions were thus identified between L. maculans and B. napus genotypes, one of them corresponding to a complete resistance to European races of L. maculans. In both cases, a single gene control of avirulence was established (genes AvrLm3 and AvrLm7). Similarly, a single gene control of avirulence toward a B. rapa genotype, also resistant to European L. maculans isolates, was demonstrated (gene AvrLm8). Finally, a digenic control of avirulence toward B. juncea was established (genes AvrLm5 and AvrLm6). Linkage analyses demonstrated that at least four unlinked L. maculans genomic regions, including at least one AVR gene cluster (AvrLm1-AvrLm2-AvrLm6), are involved in host specificity. The AvrLm3-AvrLm4-AvrLm7 region may correspond either to a second AVR gene cluster or to a multiallelic AVR gene.

  4. Anti-Phytopathogenic Activities of Macro-Algae Extracts

    PubMed Central

    Jiménez, Edra; Dorta, Fernando; Medina, Cristian; Ramírez, Alberto; Ramírez, Ingrid; Peña-Cortés, Hugo

    2011-01-01

    Aqueous and ethanolic extracts obtained from nine Chilean marine macro-algae collected at different seasons were examined in vitro and in vivo for properties that reduce the growth of plant pathogens or decrease the injury severity of plant foliar tissues following pathogen infection. Particular crude aqueous or organic extracts showed effects on the growth of pathogenic bacteria whereas others displayed important effects against pathogenic fungi or viruses, either by inhibiting fungal mycelia growth or by reducing the disease symptoms in leaves caused by pathogen challenge. Organic extracts obtained from the brown-alga Lessonia trabeculata inhibited bacterial growth and reduced both the number and size of the necrotic lesion in tomato leaves following infection with Botrytis cinerea. Aqueous and ethanolic extracts from the red-alga Gracillaria chilensis prevent the growth of Phytophthora cinnamomi, showing a response which depends on doses and collecting-time. Similarly, aqueous and ethanolic extracts from the brown-alga Durvillaea antarctica were able to diminish the damage caused by tobacco mosaic virus (TMV) in tobacco leaves, and the aqueous procedure is, in addition, more effective and seasonally independent. These results suggest that macro-algae contain compounds with different chemical properties which could be considered for controlling specific plant pathogens. PMID:21673886

  5. Myconanoparticles: synthesis and their role in phytopathogens management

    PubMed Central

    Alghuthaymi, Mousa A.; Almoammar, Hassan; Rai, Mahindra; Said-Galiev, Ernest; Abd-Elsalam, Kamel A.

    2015-01-01

    Nanotechnology can offer green and eco-friendly alternatives for plant disease management. Apart from being eco-friendly, fungi are used as bio-manufacturing units, which will provide an added benefit in being easy to use, as compared to other microbes. The non-pathogenic nature of some fungal species in combination with the simplicity of production and handling will improve the mass production of silver nanoparticles. Recently, a diverse range of fungi have been screened for their ability to create silver nanoparticles. Mycosynthesis of gold, silver, gold–silver alloy, selenium, tellurium, platinum, palladium, silica, titania, zirconia, quantum dots, usnic acid, magnetite, cadmium telluride and uraninite nanoparticles has also been reported by various researchers. Nanotechnological application in plant pathology is still in the early stages. For example, nanofungicides, nanopesticides and nanoherbicides are being used extensively in agriculture practices. Remote activation and monitoring of intelligent nano-delivery systems can assist agricultural growers of the future to minimize fungicides and pesticides use. Nanoparticle-mediated gene transfer would be useful for improvement of crops resistant to pathogens and pest. This review critically assesses the role of fungi in the synthesis of nanoparticles, the mechanism involved in the synthesis, the effect of different factors on the reduction of metal ions in developing low-cost techniques for the synthesis and recovery of nanoparticles. Moreover, the application of nanoparticles in plant disease control, antimicrobial mechanisms, and nanotoxicity on plant ecosystem and soil microbial communities has also been discussed in detail. PMID:26019636

  6. [Current concepts on the pathogenicity of phytopathogenic bacteria].

    PubMed

    Boucher, C; Genin, S; Arlat, M

    2001-10-01

    What are the molecular determinants that make a bacterium a plant pathogen? In the last 10-20 years, important progress has been made in answering this question. In the early 20th century soon after the discovery of infectious diseases, the first studies of pathogenicity were undertaken. These early studies relied mostly on biochemistry and led to the discovery of several major pathogenicity determinants, such as toxins and hydrolytic enzymes which govern the production of major disease symptoms. From these pioneering studies, a simplistic view of pathogenicity arose. It was thought that only a few functions were sufficient to transform a bacterium into a pathogen. This view rapidly changed when modern techniques of molecular genetics were applied to analyse pathogenicity. Modern analyses of pathogenicity determinants took advantage of the relatively simple organization of the haploid genome of pathogenic bacteria. By creating non-pathogenic mutants, a large number of genes governing bacterium-host interactions were identified. These genes are required either for host colonization or for the production of symptoms. Even though the role of motility and chemotaxis in these processes is still unclear, it is clear that a strong attachment of Agrobacterium to plant cells is a prerequisite for efficient plant transformation and disease. Other important pathogenicity factors identified with a molecular genetic approach include hydrolytic enzymes such as pectinases and cellulases which not only provide nutrients to the bacteria but also facilitate pathogen invasion into host tissues. The precise role of exopolysaccharide in pathogenicity is still under discussion, however it is has been established that it is crucial for the induction of wilt symptoms caused by Ralstonia solanacearum. Trafficking of effector proteins from the invading bacterium into the host cell emerged recently as a new central concept. In plant pathogenic bacteria, protein translocation takes place through the so-called 'type II secretion machinery' encoded by hrp genes in the bacterium. These genes are present in representatives of all the major groups of Gram negative plant pathogenic bacteria except Agrobacterium. Most of these genes have counterparts in pathogens of mammals (including those of human) and they also play a central role in pathogenicity. Additionally, recent evidence suggests that a 'type IV secretion machinery' injects bacterial proteins into host cells. This machinery, originally found to be involved in the transfer of t-DNA from Agrobacterium into plant cells, was recently shown to translocate pathogenicity proteins in pathogens of mammals such as Helicobacter pylori and Brucella. Discovery of the trafficking of proteins from the pathogen into host cells revolutionized our conception of pathogenicity. First, it rather unexpectedly established the conservation of basic pathogenicity strategies in plant and animal pathogens. Second, this discovery changes our ideas about the overall strategy (or mechanism) of pathogenicity, although we still think the end result is exploitation of host cell nutritive components. Rather than killing the host cell from outside, we envision a more subtle approach in which pathogens inject effector proteins into the host cell to effect a change in host cell biology advantageous to the pathogen. Identification of the effector proteins, of their function and of the corresponding molecular targets in the host is a new challenge which will contribute to the conception of new strategies to control diseases. PMID:11570279

  7. Comparative Analyses of Exoproteinases Produced by Three Phytopathogenic Microorganisms

    PubMed Central

    Valueva, Tatiana A.; Kudryavtseva, Natalia N.; Sof'in, Alexis V.; Revina, Tatiana A.; Gvozdeva, Ekaterina L.; Ievleva, Elena V.

    2011-01-01

    Proteinases secreted by the oomycete Phytophthora infestans (Mont.) de Bary, Rhizoctonia solani, and Fusarium culmorum belonging to different families of fungi have been studied to determine if the exoenzyme secretion depends on the environmental conditions and the phylogenetic position of the pathogen. The substrate specificity of the extracellular proteinases of F. culmorum, R. solani, and P. infestans and their sensitivity to the action of synthetic and protein inhibitors suggest that they contain trypsin-like and subtilisin-like enzymes regardless of culture medium composition. The relation of trypsin-like and subtilisin-like enzymes is dependent on the culture medium composition, especially on the form of nitrogen nutrition, particularly in the case of the exoenzymes secreted by R. solani. Phylogenetic analyses have shown that the exoproteinase set of ascomycetes and oomycetes has more similarities than basidiomycetes although they are more distant relatives. Our data suggests that the multiple proteinases secreted by pathogenic fungi could play different roles in pathogenesis, increasing the adaptability and host range, or could have different functions in survival in various ecological habitats outside the host. PMID:22567343

  8. Comparative analyses of exoproteinases produced by three phytopathogenic microorganisms.

    PubMed

    Valueva, Tatiana A; Kudryavtseva, Natalia N; Sof'in, Alexis V; Revina, Tatiana A; Gvozdeva, Ekaterina L; Ievleva, Elena V

    2011-01-01

    Proteinases secreted by the oomycete Phytophthora infestans (Mont.) de Bary, Rhizoctonia solani, and Fusarium culmorum belonging to different families of fungi have been studied to determine if the exoenzyme secretion depends on the environmental conditions and the phylogenetic position of the pathogen. The substrate specificity of the extracellular proteinases of F. culmorum, R. solani, and P. infestans and their sensitivity to the action of synthetic and protein inhibitors suggest that they contain trypsin-like and subtilisin-like enzymes regardless of culture medium composition. The relation of trypsin-like and subtilisin-like enzymes is dependent on the culture medium composition, especially on the form of nitrogen nutrition, particularly in the case of the exoenzymes secreted by R. solani. Phylogenetic analyses have shown that the exoproteinase set of ascomycetes and oomycetes has more similarities than basidiomycetes although they are more distant relatives. Our data suggests that the multiple proteinases secreted by pathogenic fungi could play different roles in pathogenesis, increasing the adaptability and host range, or could have different functions in survival in various ecological habitats outside the host. PMID:22567343

  9. ACTIVITY OF NATURAL PRODUCTS AGAINST SOME PHYTOPATHOGENIC FUNGI.

    PubMed

    La Torre, A; Caradonia, F; Gianferro, M; Molinu, M G; Battaglia, V

    2014-01-01

    The requirement of environmental protection and food safety is perceived with always major interest by public opinion and it is consistent with European Union legislation on the sustainable use of pesticides (Directive 2009/128/EC). This directive requires member states to promote low pesticide-input, giving priority to non-chemical methods and low risk plant protection products. In order to contribute to the achievement of these objectives antifungal activity of natural substances, characterized by a good toxicological and ecotoxicological profile, was tested. Essential oil of Melaleuca alternifolia, essential oil of Syzygium aromaticum and extract from Mimosa tenuiflora were tested against Alternaria alternata, Botrytis cinerea and Fusarium oxysporum f. sp. lycopersici (races 1 and 2). In vitro tests involved determination of radial growth of the colonies of fungi in the presence of varying concentrations of tested products in agar media and determination of germination percentage in the presence of tested product at various concentrations. The products based on essential oil of M. alternifolia were also tested in vivo on tomato fruits wounded and artificially inoculated with A. alternata or with B. cinerea. The in vitro tests showed the antifungal activity of both essential oils instead the extract from M. tenuiflora exhibited poor antifungal activity and only against A. alternata and B. cinerea. The results on tomato fruits showed inhibition of grey mould and black mould by essential oil of M. alternifolia. The antifungal activity increased with increasing concentrations. In conclusion, the obtained results in the present study showed promising prospects for the utilisation of investigated products to reduce the using of antifungal chemicals and to achieve a more sustainable use of pesticides. PMID:26080478

  10. Phytopathogenic bacteria in the system of modern agriculture.

    PubMed

    Patyka, V P; Pasichnyk, L A

    2014-01-01

    The stages of studying bacterial diseases of crops and weeds at various farming systems have been characterized, biological properties have been investigated and pathogens identified using traditional and modern molecular genetic methods of research.

  11. SaxA-Mediated Isothiocyanate Metabolism in Phytopathogenic Pectobacteria

    PubMed Central

    Rosengarten, Jamila F.; de Graaf, Rob M.; Jetten, Mike S. M.

    2016-01-01

    Pectobacteria are devastating plant pathogens that infect a large variety of crops, including members of the family Brassicaceae. To infect cabbage crops, these plant pathogens need to overcome the plant's antibacterial defense mechanisms, where isothiocyanates are liberated by hydrolysis of glucosinolates. Here, we found that a Pectobacterium isolate from the gut of cabbage root fly larvae was particularly resistant to isothiocyanate and even seemed to benefit from the abundant Brassica root metabolite 2-phenylethyl isothiocyanate as a nitrogen source in an ecosystem where nitrogen is scarce. The Pectobacterium isolate harbored a naturally occurring mobile plasmid that contained a sax operon. We hypothesized that SaxA was the enzyme responsible for the breakdown of 2-phenylethyl isothiocyanate. Subsequently, we heterologously produced and purified the SaxA protein and characterized the recombinant enzyme. It hydrolyzed 2-phenylethyl isothiocyanate to yield the products carbonyl sulfide and phenylethylamine. It was also active toward another aromatic isothiocyanate but hardly toward aliphatic isothiocyanates. It belongs to the class B metal-dependent beta-lactamase fold protein family but was not, however, able to hydrolyze beta-lactam antibiotics. We discovered that several copies of the saxA gene are widespread in full and draft Pectobacterium genomes and therefore hypothesize that SaxA might be a new pathogenicity factor of the genus Pectobacterium, possibly compromising food preservation strategies using isothiocyanates. PMID:26873319

  12. Activity of Vitis vinifera Tendrils Extract Against Phytopathogenic Fungi.

    PubMed

    Fraternale, Daniele; Ricci, Donata; Verardo, Giancarlo; Gorassini, Andrea; Stocchia, Vilberto; Sestili, Piero

    2015-06-01

    The in vitro antifungal activity was determined of an ethanolic extract of Vitis vinifera L. tendrils (TVV) against ten plant pathogenic fungi, using the agar dilution method; activity was shown against all tested fungi. Fusarium species were the most sensitive with MIC values ranging from 250 to 300 ppm, while the basidiomycete fungus Rhizoctonia solani was the most resistant, with a MIC value of 500 ppm. Electrospray ionization tandem mass spectrometry (ESI-MS(n)) was used to obtain qualitative information on the main components of TVV. The high amount of polyphenolic compounds contained in TVV is likely to contribute significantly to its antifungal activity.

  13. Generalized transduction in the enterobacterial phytopathogen Erwinia chrysanthemi.

    PubMed

    Chatterjee, A K; Brown, M A

    1980-09-01

    Bacteriophages induced by mitomycin treatment of Erwinia chrysanthemi KS612 produced plaques on lawns of E. chrysanthemi EC183 and KS605. Bacteriophage Erch-12, purified from one such plaque, transferred an array of chromosomal genes (arg, leu, his, ser, thr, trp, ura) to appropriate recipient strains derived from E. chrysanthemi EC 183. Recombinants were formed in the absence of cellular contact between donor and recipient bacteria and in the presence of deoxyribonuclease. Ultraviolet irradiation of the bacteriophage stimulated transductional frequency. Linkage was detected in two-factor crosses between the loci thr and ser and between rif and ade; several closely linked mutations in ser were mapped with respect to thr.

  14. SaxA-Mediated Isothiocyanate Metabolism in Phytopathogenic Pectobacteria.

    PubMed

    Welte, Cornelia U; Rosengarten, Jamila F; de Graaf, Rob M; Jetten, Mike S M

    2016-04-01

    Pectobacteria are devastating plant pathogens that infect a large variety of crops, including members of the family Brassicaceae. To infect cabbage crops, these plant pathogens need to overcome the plant's antibacterial defense mechanisms, where isothiocyanates are liberated by hydrolysis of glucosinolates. Here, we found that a Pectobacterium isolate from the gut of cabbage root fly larvae was particularly resistant to isothiocyanate and even seemed to benefit from the abundant Brassica root metabolite 2-phenylethyl isothiocyanate as a nitrogen source in an ecosystem where nitrogen is scarce. The Pectobacterium isolate harbored a naturally occurring mobile plasmid that contained a sax operon. We hypothesized that SaxA was the enzyme responsible for the breakdown of 2-phenylethyl isothiocyanate. Subsequently, we heterologously produced and purified the SaxA protein and characterized the recombinant enzyme. It hydrolyzed 2-phenylethyl isothiocyanate to yield the products carbonyl sulfide and phenylethylamine. It was also active toward another aromatic isothiocyanate but hardly toward aliphatic isothiocyanates. It belongs to the class B metal-dependent beta-lactamase fold protein family but was not, however, able to hydrolyze beta-lactam antibiotics. We discovered that several copies of the saxA gene are widespread in full and draft Pectobacterium genomes and therefore hypothesize that SaxA might be a new pathogenicity factor of the genus Pectobacterium, possibly compromising food preservation strategies using isothiocyanates. PMID:26873319

  15. Brassica napus L. cultivars show a broad variability in their morphology, physiology and metabolite levels in response to sulfur limitations and to pathogen attack

    PubMed Central

    Weese, Annekathrin; Pallmann, Philip; Papenbrock, Jutta; Riemenschneider, Anja

    2015-01-01

    Under adequate sulfur supply, plants accumulate sulfate in the vacuoles and use sulfur-containing metabolites as storage compounds. Under sulfur-limiting conditions, these pools of stored sulfur-compounds are depleted in order to balance the nitrogen to sulfur ratio for protein synthesis. Stress conditions like sulfur limitation and/or pathogen attack induce changes in the sulfate pool and the levels of sulfur-containing metabolites, which often depend on the ecotypes or cultivars. We are interested in investigating the influence of the genetic background of canola (Brassica napus) cultivars in sulfur-limiting conditions on the resistance against Verticillium longisporum. Therefore, four commercially available B. napus cultivars were analyzed. These high-performing cultivars differ in some characteristics described in their cultivar pass, such as several agronomic traits, differences in the size of the root system, and resistance to certain pathogens, such as Phoma and Verticillium. The objectives of the study were to examine and explore the patterns of morphological, physiological and metabolic diversity in these B. napus cultivars at different sulfur concentrations and in the context of plant defense. Results indicate that the root systems are influenced differently by sulfur deficiency in the cultivars. Total root dry mass and length of root hairs differ not only among the cultivars but also vary in their reaction to sulfur limitation and pathogen attack. As a sensitive indicator of stress, several parameters of photosynthetic activity determined by PAM imaging showed a broad variability among the treatments. These results were supported by thermographic analysis. Levels of sulfur-containing metabolites also showed large variations. The data were interrelated to predict the specific behavior during sulfur limitation and/or pathogen attack. Advice for farming are discussed. PMID:25699060

  16. Brassica napus L. cultivars show a broad variability in their morphology, physiology and metabolite levels in response to sulfur limitations and to pathogen attack.

    PubMed

    Weese, Annekathrin; Pallmann, Philip; Papenbrock, Jutta; Riemenschneider, Anja

    2015-01-01

    Under adequate sulfur supply, plants accumulate sulfate in the vacuoles and use sulfur-containing metabolites as storage compounds. Under sulfur-limiting conditions, these pools of stored sulfur-compounds are depleted in order to balance the nitrogen to sulfur ratio for protein synthesis. Stress conditions like sulfur limitation and/or pathogen attack induce changes in the sulfate pool and the levels of sulfur-containing metabolites, which often depend on the ecotypes or cultivars. We are interested in investigating the influence of the genetic background of canola (Brassica napus) cultivars in sulfur-limiting conditions on the resistance against Verticillium longisporum. Therefore, four commercially available B. napus cultivars were analyzed. These high-performing cultivars differ in some characteristics described in their cultivar pass, such as several agronomic traits, differences in the size of the root system, and resistance to certain pathogens, such as Phoma and Verticillium. The objectives of the study were to examine and explore the patterns of morphological, physiological and metabolic diversity in these B. napus cultivars at different sulfur concentrations and in the context of plant defense. Results indicate that the root systems are influenced differently by sulfur deficiency in the cultivars. Total root dry mass and length of root hairs differ not only among the cultivars but also vary in their reaction to sulfur limitation and pathogen attack. As a sensitive indicator of stress, several parameters of photosynthetic activity determined by PAM imaging showed a broad variability among the treatments. These results were supported by thermographic analysis. Levels of sulfur-containing metabolites also showed large variations. The data were interrelated to predict the specific behavior during sulfur limitation and/or pathogen attack. Advice for farming are discussed. PMID:25699060

  17. Keeping Control: The Role of Senescence and Development in Plant Pathogenesis and Defense

    PubMed Central

    Häffner, Eva; Konietzki, Sandra; Diederichsen, Elke

    2015-01-01

    Many plant pathogens show interactions with host development. Pathogens may modify plant development according to their nutritional demands. Conversely, plant development influences pathogen growth. Biotrophic pathogens often delay senescence to keep host cells alive, and resistance is achieved by senescence-like processes in the host. Necrotrophic pathogens promote senescence in the host, and preventing early senescence is a resistance strategy of plants. For hemibiotrophic pathogens both patterns may apply. Most signaling pathways are involved in both developmental and defense reactions. Increasing knowledge about the molecular components allows to distinguish signaling branches, cross-talk and regulatory nodes that may influence the outcome of an infection. In this review, recent reports on major molecular players and their role in senescence and in pathogen response are reviewed. Examples of pathosystems with strong developmental implications illustrate the molecular basis of selected control strategies. A study of gene expression in the interaction between the hemibiotrophic vascular pathogen Verticillium longisporum and its cruciferous hosts shows processes that are fine-tuned to counteract early senescence and to achieve resistance. The complexity of the processes involved reflects the complex genetic control of quantitative disease resistance, and understanding the relationship between disease, development and resistance will support resistance breeding. PMID:27135337

  18. Genetic modification of potato against microbial diseases: in vitro and in planta activity of a dermaseptin B1 derivative, MsrA2.

    PubMed

    Osusky, Milan; Osuska, Lubica; Kay, William; Misra, Santosh

    2005-08-01

    Dermaseptin B1 is a potent cationic antimicrobial peptide found in skin secretions of the arboreal frog Phyllomedusa bicolor. A synthetic derivative of dermaseptin B1, MsrA2 (N-Met-dermaseptin B1), elicited strong antimicrobial activities against various phytopathogenic fungi and bacteria in vitro. To assess its potential for plant protection, MsrA2 was expressed at low levels (1-5 microg/g of fresh tissue) in the transgenic potato (Solanum tuberosum L.) cv. Desiree. Stringent challenges of these transgenic potato plants with a variety of highly virulent fungal phytopathogens--Alternaria, Cercospora, Fusarium, Phytophthora, Pythium, Rhizoctonia and Verticillium species--and with the bacterial pathogen Erwinia carotovora demonstrated that the plants had an unusually broad-spectrum and powerful resistance to infection. MsrA2 profoundly protected both plants and tubers from diseases such as late blight, dry rot and pink rot and markedly extended the storage life of tubers. Due to these properties in planta, MsrA2 is proposed as an ideal antimicrobial peptide candidate to significantly increase resistance to phytopathogens and improve quality in a variety of crops worldwide with the potential to obviate fungicides and facilitate storage under difficult conditions.

  19. Diversity and biotransformative potential of endophytic fungi associated with the medicinal plant Kadsura angustifolia.

    PubMed

    Huang, Qian; An, Hongmei; Song, Hongchuan; Mao, Hongqiang; Shen, Weiyun; Dong, Jinyan

    2015-01-01

    This study investigated the diversity and host component-transforming activity of endophytic fungi in medicinal plant Kadsura angustifolia. A total of 426 isolates obtained were grouped into 42 taxa belonging to Fungi Imperfecti (65.96%), Ascomycota (27.00%), Zygomycota (1.64%), Basidiomycota (0.47%) and Mycelia Sterilia (4.93%). The abundance, richness, and species composition of endophytic assemblages were significantly dependent on the tissue and the sampling site. Many phytopathogenic species associated with healthy K. angustifolia were found prevalent. Among them, Verticillium dahliae was dominant with 16.43% abundance. From 134 morphospecies selected, 39 showed remarkable biocatalytic activity and were further identified as species belonging to the genera Colletotrichum, Eupenicillium, Fusarium, Hypoxylon, Penicillium, Phomopsis, Trametes, Trichoderma, Umbelopsis, Verticillium and Xylaria on the basis of the sequence analysis of the internal transcribed spacer (ITS1-5.8S-ITS2). The results obtained in this work show that K. angustifolia is an interesting reservoir of pathogenic fungal species, and could be a community model for further ecological and evolutionary studies. Additionally, the converting potency screening of some endophytic fungi from this specific medicinal plant may provide an interesting niche on the search for novel biocatalysts.

  20. High throughput analysis of gene expression in microsclerotia of Verticillium dahliae

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The processes of microsclerotia formation, maintenance, and germination are critically important in the disease cycle of V. dahliae. To shed additional light on the molecular processes involved in microsclerotia biogenesis and melanin synthesis in V. dahliae, three replicate RNA-seq libraries were p...

  1. Genomic Analysis of Verticillium Wilt Resistance and Drought Tolerance in Alfalfa

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Alfalfa (Medicago sativa L.) is the fourth largest crop in the United States. Changing trends to multipurpose uses increases demand for alfalfa. However, the production of alfalfa is challenged by endemic and emerging diseases and adverse environmental factors. Identification of genes/loci controlli...

  2. Characterization of spinach germplasm for resistance against two races of Verticillium dahliae

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Historically, wilt disease caused by V. dahliae has not presented a problem in California spinach production because the crop is harvested well before the symptoms develop during the post-stem elongation (bolting) stage. However, infested spinach seeds introduce or increase inoculum in the soil for...

  3. Fluorescent Pseudomonads in the Phyllosphere of Wheat: Potential Antagonists Against Fungal Phytopathogens.

    PubMed

    Müller, Thomas; Behrendt, Undine; Ruppel, Silke; von der Waydbrink, Grit; Müller, Marina E H

    2016-04-01

    Fluorescent pseudomonads isolated from wheat leaves were characterized regarding their antagonistic potential and taxonomy in relation to protect crop plants from infestation by Fusarium and Alternaria fungi causing diseases in wheat. Using a dual culture assay, inhibition of fungal growth was found for 40 isolates of 175 fluorescent pseudomonads. Twenty-two of the antagonists were able to suppress strains of Fusarium as well as Alternaria. By means of real-time qPCR, the phlD gene encoding the antibiotic 2,4-diacetylphloroglucinol was detected in 20 isolates. On the basis of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry spectral patterns, the isolates with antagonistic activity were assigned to the phylogenetic subgroup Pseudomonas fluorescens and the closely related Pseudomonas gessardii subgroup. The results of the study suggest that pseudomonads in the phyllosphere of crop plants may possibly contribute to natural plant protection. PMID:26687461

  4. Synthesis, Biological Evaluation, and Molecular Modeling Studies of New Oxadiazole-Stilbene Hybrids against Phytopathogenic Fungi

    PubMed Central

    Jian, Weilin; He, Daohang; Song, Shaoyun

    2016-01-01

    Natural stilbenes (especially resveratrol) play important roles in plant protection by acting as both constitutive and inducible defenses. However, their exogenous applications on crops as fungicidal agents are challenged by their oxidative degradation and limited availability. In this study, a new class of resveratrol-inspired oxadiazole-stilbene hybrids was synthesized via Wittig-Horner reaction. Bioassay results indicated that some of the compounds exhibited potent fungicidal activity against Botrytis cinerea in vitro. Among these stilbene hybrids, compounds 11 showed promising inhibitory activity with the EC50 value of 144.6 μg/mL, which was superior to that of resveratrol (315.6 μg/mL). Remarkably, the considerably abnormal mycelial morphology was observed in the presence of compound 11. The inhibitory profile was further proposed by homology modeling and molecular docking studies, which showed the possible interaction of resveratrol and oxadiazole-stilbene hybrids with the cytochrome P450-dependent sterol 14α-demethylase from B. cinerea (BcCYP51) for the first time. Taken together, these results would provide new insights into the fungicidal mechanism of stilbenes, as well as an important clue for biology-oriented synthesis of stilbene hybrids with improved bioactivity against plant pathogenic fungi in crop protection. PMID:27530962

  5. Population genetic analysis reveals cryptic sex in the phytopathogenic fungus Alternaria alternata.

    PubMed

    Meng, Jing-Wen; Zhu, Wen; He, Meng-Han; Wu, E-Jiao; Duan, Guo-Hua; Xie, Ye-Kun; Jin, Yu-Jia; Yang, Li-Na; Shang, Li-Ping; Zhan, Jiasui

    2015-12-15

    Reproductive mode can impact population genetic dynamics and evolutionary landscape of plant pathogens as well as on disease epidemiology and management. In this study, we monitored the spatial dynamics and mating type idiomorphs in ~700 Alternaria alternata isolates sampled from the main potato production areas in China to infer the mating system of potato early blight. Consistent with the expectation of asexual species, identical genotypes were recovered from different locations separated by hundreds of kilometers of geographic distance and spanned across many years. However, high genotype diversity, equal MAT1-1 and MAT1-2 frequencies within and among populations, no genetic differentiation and phylogenetic association between two mating types, combined with random association amongst neutral markers in some field populations, suggested that sexual reproduction may also play an important role in the epidemics and evolution of the pathogen in at least half of the populations assayed despite the fact that no teleomorphs have been observed yet naturally or artificially. Our results indicated that A. alternata may adopt an epidemic mode of reproduction by combining many cycles of asexual propagation with fewer cycles of sexual reproduction, facilitating its adaptation to changing environments and making the disease management on potato fields even more difficult.

  6. Detection and characterization of a novel Gammapartitivirus in the phytopathogenic fungus Colletotrichum acutatum strain HNZJ001.

    PubMed

    Zhong, Jie; Chen, Dan; Lei, Xiang Hua; Zhu, Hong Jian; Zhu, Jun Zi; Da Gao, Bi

    2014-09-22

    Spherical virus-like particles about 40nm in diameter were observed under transmission electron microscope (TEM) and two dsRNA bands (dsRNA-1 and dsRNA-2) were detected on agarose gel after extraction from the mycelial preparation of a Colletotrichum acutatum strain HNZJ001 that isolated from an anthracnose lesion on immature pepper fruit. The complete nucleotide sequences of the dsRNAs were determined. DsRNA-1 (1762 nt) and dsRNA-2 (1381 nt) each contained a single open reading frame and potentially encoded 62 kDa and 40 kDa proteins, respectively. The 62 kDa protein showed similarity to the RNA-dependent RNA polymerase (RdRp) of partitiviruses, while the 40 kDa product had no significant similarity to any published capsid protein throughout all databases, besides of low homology with the hypothetical "capsid" protein of a few partitiviruses in fungus Ustilaginoidea virens. Genome comparison and phylogenetic analysis indicated that the virus is closely related to the mycovirus in the family Partitiviridae. The results suggested a novel two-segment dsRNA virus be detected. We name it Colletotrichum acutatum partitivirus 1 (CaPV1). RT-PCR detection, using a primer pair based on the RdRp of the dsRNA-1 showed very high efficiency of CaPV1 transmission into the progenies of the fungus. Virus curing and fungal phenotype observation for evaluation of the impact of CaPV1 in host fungus were also carried out.

  7. Isolation and fusion of protoplasts from the phytopathogenic fungus sclerotium rolfsii(sacc.)

    PubMed Central

    Cai, Yun; Christias, Christos

    2010-01-01

    Sclerotium rolfsii (Sacc.) is a serious plant pathogenic fungus and lacks perfect (basidial) stage in production. Protoplast fusion technology was employed to reconstruct fusants from this fungus. Two strains designated as A and R were used. Maximum protoplast yields of 3.8x105/g mycelia and 2.8x105/g mycelia were formed in strains A and R respectively. Osmotic stabilizer sucrose 1M gave maximum yield. Lysing enzyme at the rate of 15mg/ml was found best for yield. Fusion of protoplasts from strains A and R was carried out in fusion media containing PEG 4000 30% (w/v) with 0.2mM CaCl2. Four fusants F1, F2, F3 and F4 were recovered. Morphological, physiological and pathogenic characters of fusants were compared with parent strains on carrots, beans and tomato. PMID:24031488

  8. Identification and characterization of polymorphic minisatellites in the phytopathogenic ascomycete Leptosphaeria maculans.

    PubMed

    Eckert, Maria; Gout, Lilian; Rouxel, Thierry; Blaise, Françoise; Jedryczka, Malgorzata; Fitt, Bruce; Balesdent, Marie-Hélène

    2005-01-01

    Leptosphaeria maculans causes phoma stem canker, the most serious disease of oilseed rape world-wide. Sexual recombination is important in the pathogen life cycle and increases the risk of plant resistance genes being overcome rapidly. Thus, there is a need to develop easy-to-use molecular markers suitable for large-scale population genetic studies. The minisatellite MinLm1, showing six alleles in natural populations, has previously been used as a marker to survey populations. Here, we report the characterization of five new minisatellites (MinLm2-MinLm6), of which four were identified by a systematic search for tandemly repeated polymorphic regions in BAC-end sequencing data from L. maculans. Of 782 BAC-end sequences analysed, 43 possessed putative minisatellite-type repeats and four of these (MinLm3-MinLm6) displayed both consistent PCR amplification and size polymorphism in a collection of L. maculans isolates of diverse origins. Cloning and sequencing of each allele confirmed that polymorphism was due to variation in the repeat number of a core motif ranging from 11 bp (MinLm3) to 51 bp (MinLm4). The number of alleles found for each minisatellite ranged from three (MinLm4) to nine (MinLm2), with eight, five and six for MinLm3, MinLm5 and MinLm6, respectively. MinLm2-MinLm6 are all single locus markers specific to L. maculans and share some common features, such as conservation of core motifs and incomplete direct repeats in the flanking regions. To our knowledge, L. maculans is the first fungal species for which six polymorphic single locus minisatellite markers have been reported.

  9. Phytotoxin production and phytoalexin elicitation by the phytopathogenic fungus Sclerotinia sclerotiorum.

    PubMed

    Pedras, M Soledade C; Ahiahonu, Pearson W K

    2004-11-01

    The fungus Sclerotinia sclerotiorum (Lib.) de Bary causes rot disease in a vast range of plant families, including Cruciferae (Brassicaceae). We investigated the production of phytotoxins by S. sclerotiorum by using a bioassay-guided isolation, as well as the phytoalexins produced by the resistant wild crucifer Erucastrum gallicum under elicitation by S. sclerotiorum and other agents. We established for the first time that S. sclerotiorum produces a somewhat selective phytotoxin, sclerin, which is phytotoxic to three cruciferous species (Brassica napus, B. juncea, and Sinapis alba) susceptible to Sclerotinia stem rot disease, causing severe necrosis and chlorosis, but not to a resistant species (Erucastrum gallicum). In addition, we have shown that oleic acid, the major fatty acid isolated from sclerotia of S. sclerotiorum is responsible for the toxic activity of extracts of sclerotia to brine shrimp larvae (Artemia salina). Phytoalexin elicitation in leaves of E. gallicum led to the isolation of three known phytoalexins: indole-3-acetonitrile, arvelexin, and 1-methoxyspirobrassinin. Considering that resistance of E. gallicum to S. sclerotiorum is potentially transferable to B. rapa, a susceptible canola species, and that arvelexin, and 1-methoxyspirobrassinin are not produced by B. rapa, these phytoalexins may become useful markers for resistance against S. sclerotiorum.

  10. Optimization for rapid synthesis of silver nanoparticles and its effect on phytopathogenic fungi

    NASA Astrophysics Data System (ADS)

    Krishnaraj, C.; Ramachandran, R.; Mohan, K.; Kalaichelvan, P. T.

    In this present study, silver nanoparticles were synthesized by green chemistry approach using Acalypha indica leaf extract as reducing agents. The reaction medium employed in the synthesis process was optimized to attain better yield, controlled size and stability. Further, the biosynthesized silver nanoparticles were conformed through UV-vis spectrum, XRD and HR-TEM analyses. Different concentration of silver nanoparticles were tested to know the inhibitory effect of fungal plant pathogens namely Alternaria alternata, Sclerotinia sclerotiorum, Macrophomina phaseolina, Rhizoctonia solani, Botrytis cinerea and Curvularia lunata. Interestingly, 15 mg concentration of silver nanoparticles showed excellent inhibitory activity against all the tested pathogens. Thus, the obtained results clearly suggest that silver nanoparticles may have important applications in controlling various plant diseases caused by fungi.

  11. Complex regulation of secondary metabolism controlling pathogenicity in the phytopathogenic fungus Alternaria alternata.

    PubMed

    Takaoka, Shinya; Kurata, Mariko; Harimoto, Yoshiaki; Hatta, Rieko; Yamamoto, Mikihiro; Akimitsu, Kazuya; Tsuge, Takashi

    2014-06-01

    The filamentous fungus Alternaria alternata includes seven pathogenic variants (pathotypes), which produce different host-selective toxins and cause disease on different plants. The Japanese pear, strawberry and tangerine pathotypes produce AK-toxin, AF-toxin and ACT-toxin, respectively, which have a common structural moiety, 9,10-epoxy-8-hydroxy-9-methyl-decatrienoic acid (EDA). Here, we identified a new gene, AKT7 (AK-toxin biosynthetic gene 7), from the Japanese pear pathotype, which encodes a cytochrome P450 monooxygenase and functions to limit AK-toxin production. AKT7 homologs were found in the strawberry pathotype, but not the tangerine pathotype. However, the strawberry pathotype homolog appeared to include a premature stop codon. Although the Japanese pear pathotype strain has multiple copies of AKT7, a single-copy disruption resulted in mutants with increased production of AK-toxin and EDA. AKT7 overexpression in the three pathotypes caused marked reductions of toxin and EDA production, suggesting that Akt7 catalyzes a side reaction of EDA or its precursor. AKT7 overexpression caused reduced virulence in these pathotypes. We also found that AKT7 transcripts predominantly include misspliced mRNAs, which have premature stop codons. Our observations suggest that the AK-toxin production required for full virulence is regulated in a complex way by the copy number and intron information content of AKT7.

  12. Genomic diversity of large-plaque-forming podoviruses infecting the phytopathogen Ralstonia solanacearum.

    PubMed

    Kawasaki, Takeru; Narulita, Erlia; Matsunami, Minaho; Ishikawa, Hiroki; Shimizu, Mio; Fujie, Makoto; Bhunchoth, Anjana; Phironrit, Namthip; Chatchawankanphanich, Orawan; Yamada, Takashi

    2016-05-01

    The genome organization, gene structure, and host range of five podoviruses that infect Ralstonia solanacearum, the causative agent of bacterial wilt disease were characterized. The phages fell into two distinctive groups based on the genome position of the RNA polymerase gene (i.e., T7-type and ϕKMV-type). One-step growth experiments revealed that ϕRSB2 (a T7-like phage) lysed host cells more efficiently with a shorter infection cycle (ca. 60 min corresponding to half the doubling time of the host) than ϕKMV-like phages such as ϕRSB1 (with an infection cycle of ca. 180 min). Co-infection experiments with ϕRSB1 and ϕRSB2 showed that ϕRSB2 always predominated in the phage progeny independent of host strains. Most phages had wide host-ranges and the phage particles usually did not attach to the resistant strains; when occasionally some did, the phage genome was injected into the resistant strain's cytoplasm, as revealed by fluorescence microscopy with SYBR Gold-labeled phage particles. PMID:26901487

  13. Population genetic analysis reveals cryptic sex in the phytopathogenic fungus Alternaria alternata

    PubMed Central

    Meng, Jing-Wen; Zhu, Wen; He, Meng-Han; Wu, E-Jiao; Duan, Guo-Hua; Xie, Ye-Kun; Jin, Yu-Jia; Yang, Li-Na; Shang, Li-Ping; Zhan, Jiasui

    2015-01-01

    Reproductive mode can impact population genetic dynamics and evolutionary landscape of plant pathogens as well as on disease epidemiology and management. In this study, we monitored the spatial dynamics and mating type idiomorphs in ~700 Alternaria alternata isolates sampled from the main potato production areas in China to infer the mating system of potato early blight. Consistent with the expectation of asexual species, identical genotypes were recovered from different locations separated by hundreds of kilometers of geographic distance and spanned across many years. However, high genotype diversity, equal MAT1-1 and MAT1-2 frequencies within and among populations, no genetic differentiation and phylogenetic association between two mating types, combined with random association amongst neutral markers in some field populations, suggested that sexual reproduction may also play an important role in the epidemics and evolution of the pathogen in at least half of the populations assayed despite the fact that no teleomorphs have been observed yet naturally or artificially. Our results indicated that A. alternata may adopt an epidemic mode of reproduction by combining many cycles of asexual propagation with fewer cycles of sexual reproduction, facilitating its adaptation to changing environments and making the disease management on potato fields even more difficult. PMID:26666175

  14. Biosynthesized silver nanoparticles as a nanoweapon against phytopathogens: exploring their scope and potential in agriculture.

    PubMed

    Mishra, Sandhya; Singh, H B

    2015-02-01

    The beneficial use of silver nanoparticles (AgNPs) in agroecosystems is not fully explored with partial information available, of which most of the studies are limited to laboratory conditions and only few involve natural ecosystems. AgNPs, being the most popular metallic nanoparticles exhibiting antimicrobial property, are predominantly used for plant disease management. Owing to the ill hazards of chemically synthesized AgNPs, their biosynthesis using environment-friendly biomolecules is gaining noteworthy attention. In addition, considering the advantages of nanoformulations over biopesticides, there is no doubt that biosynthesized AgNP-based biopesticides could revolutionize the agricultural sector in the future. Though enhanced commercial use of AgNPs has generated biosafety issues in modern scenario but expecting their significant contribution towards agricultural sector, it is too early to predict the risk factor associated with their usage. To unveil the toxicity factor of AgNPs, we need to focus and understand the major interactions of AgNPs in agroecosytems. Hence, the present review highlights (i) the potential application of AgNPs in the agricultural sector particularly for plant disease management, (ii) significance of biosynthesized AgNPs using microbes and plants over their chemical synthesis, (iii) major interactions of AgNPs in agroecosystems (with soil, soil biota, and plants) with emphasis to deal with toxicity-determining factors, and (iv) identifying future research work holding promising applications of biosynthesized AgNPs in agroecosystems.

  15. A functional 4-hydroxybenzoate degradation pathway in the phytopathogen Xanthomonas campestris is required for full pathogenicity

    PubMed Central

    Wang, Jia-Yuan; Zhou, Lian; Chen, Bo; Sun, Shuang; Zhang, Wei; Li, Ming; Tang, Hongzhi; Jiang, Bo-Le; Tang, Ji-Liang; He, Ya-Wen

    2015-01-01

    Plants contain significant levels of natural phenolic compounds essential for reproduction and growth, as well as defense mechanisms against pathogens. Xanthomonas campestris pv. campestris (Xcc) is the causal agent of crucifers black rot. Here we showed that genes required for the synthesis, utilization, transportation, and degradation of 4-hydroxybenzoate (4-HBA) are present in Xcc. Xcc rapidly degrades 4-HBA, but has no effect on 2-hydroxybenzoate and 3-hydroxybenzoate when grown in XOLN medium. The genes for 4-HBA degradation are organized in a superoperonic cluster. Bioinformatics, biochemical, and genetic data showed that 4-HBA is hydroxylated by 4-HBA 3-hydroxylase (PobA), which is encoded by Xcc0356, to yield PCA. The resulting PCA is further metabolized via the PCA branches of the β-ketoadipate pathway, including Xcc0364, Xcc0365, and PcaFHGBDCR. Xcc0364 and Xcc0365 encode a new form of β-ketoadipate succinyl-coenzyme A transferase that is required for 4-HBA degradation. pobA expression was induced by 4-HBA via the transcriptional activator, PobR. Radish and cabbage hydrolysates contain 2-HBA, 3-HBA, 4-HBA, and other phenolic compounds. Addition of radish and cabbage hydrolysates to Xcc culture significantly induced the expression of pobA via PobR. The 4-HBA degradation pathway is required for full pathogenicity of Xcc in radish. PMID:26672484

  16. Genome Sequences of Three Phytopathogenic Species of the Magnaporthaceae Family of Fungi

    PubMed Central

    Okagaki, Laura H.; Nunes, Cristiano C.; Sailsbery, Joshua; Clay, Brent; Brown, Doug; John, Titus; Oh, Yeonyee; Young, Nelson; Fitzgerald, Michael; Haas, Brian J.; Zeng, Qiandong; Young, Sarah; Adiconis, Xian; Fan, Lin; Levin, Joshua Z.; Mitchell, Thomas K.; Okubara, Patricia A.; Farman, Mark L.; Kohn, Linda M.; Birren, Bruce; Ma, Li-Jun; Dean, Ralph A.

    2015-01-01

    Magnaporthaceae is a family of ascomycetes that includes three fungi of great economic importance: Magnaporthe oryzae, Gaeumannomyces graminis var. tritici, and Magnaporthe poae. These three fungi cause widespread disease and loss in cereal and grass crops, including rice blast disease (M. oryzae), take-all disease in wheat and other grasses (G. graminis), and summer patch disease in turf grasses (M. poae). Here, we present the finished genome sequence for M. oryzae and draft sequences for M. poae and G. graminis var. tritici. We used multiple technologies to sequence and annotate the genomes of M. oryzae, M. poae, and G. graminis var. tritici. The M. oryzae genome is now finished to seven chromosomes whereas M. poae and G. graminis var. tritici are sequenced to 40.0× and 25.0× coverage respectively. Gene models were developed by the use of multiple computational techniques and further supported by RNAseq data. In addition, we performed preliminary analysis of genome architecture and repetitive element DNA. PMID:26416668

  17. Gall-ID: Tools for genotyping gall-causing phytopathogenic bacteria

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Understanding the population structure and genetic diversity of plant pathogens, as well as the effect of agricultural practices on pathogen evolution, are important for disease management. Developments in molecular methods have contributed to increasing the resolution for accurate pathogen identifi...

  18. Blue mold to genomics and beyond: Insights into the biology and virulence of phytopathogenic Penicillium species

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Pomes, mainly apples and pears, are economically important fruits produced and consumed worldwide. The United States is the second largest producer of pome fruit in the world behind China. Penicillium expansum and other Penicillium spp. are the most common fungal plant pathogens that cause blue mold...

  19. Whole genome sequences and annotation of Micrococcus luteus SUBG006, a novel phytopathogen of mango.

    PubMed

    Rakhashiya, Purvi M; Patel, Pooja P; Thaker, Vrinda S

    2015-12-01

    Actinobaceria, Micrococcus luteus SUBG006 was isolated from infected leaves of Mangifera indica L. vr. Nylon in Rajkot, (22.30°N, 70.78°E), Gujarat, India. The genome size is 3.86 Mb with G + C content of 69.80% and contains 112 rRNA sequences (5S, 16S and 23S). The whole genome sequencing has been deposited in DDBJ/EMBL/GenBank under the accession number JOKP00000000. PMID:26697318

  20. Population genetic analysis reveals cryptic sex in the phytopathogenic fungus Alternaria alternata.

    PubMed

    Meng, Jing-Wen; Zhu, Wen; He, Meng-Han; Wu, E-Jiao; Duan, Guo-Hua; Xie, Ye-Kun; Jin, Yu-Jia; Yang, Li-Na; Shang, Li-Ping; Zhan, Jiasui

    2015-01-01

    Reproductive mode can impact population genetic dynamics and evolutionary landscape of plant pathogens as well as on disease epidemiology and management. In this study, we monitored the spatial dynamics and mating type idiomorphs in ~700 Alternaria alternata isolates sampled from the main potato production areas in China to infer the mating system of potato early blight. Consistent with the expectation of asexual species, identical genotypes were recovered from different locations separated by hundreds of kilometers of geographic distance and spanned across many years. However, high genotype diversity, equal MAT1-1 and MAT1-2 frequencies within and among populations, no genetic differentiation and phylogenetic association between two mating types, combined with random association amongst neutral markers in some field populations, suggested that sexual reproduction may also play an important role in the epidemics and evolution of the pathogen in at least half of the populations assayed despite the fact that no teleomorphs have been observed yet naturally or artificially. Our results indicated that A. alternata may adopt an epidemic mode of reproduction by combining many cycles of asexual propagation with fewer cycles of sexual reproduction, facilitating its adaptation to changing environments and making the disease management on potato fields even more difficult. PMID:26666175

  1. Ancestral state reconstruction infers phytopathogenic origins of sooty blotch and flyspeck fungi on apple.

    PubMed

    Ismail, Siti Izera; Batzer, Jean Carlson; Harrington, Thomas C; Crous, Pedro W; Lavrov, Dennis V; Li, Huanyu; Gleason, Mark L

    2016-01-01

    Members of the sooty blotch and flyspeck (SBFS) complex are epiphytic fungi in the Ascomycota that cause economically damaging blemishes of apples worldwide. SBFS fungi are polyphyletic, but approx. 96% of SBFS species are in the Capnodiales. Evolutionary origins of SBFS fungi remain unclear, so we attempted to infer their origins by means of ancestral state reconstruction on a phylogenetic tree built utilizing genes for the nuc 28S rDNA (approx. 830 bp from near the 59 end) and the second largest subunit of RNA polymerase II (RPB2). The analyzed taxa included the well-known genera of SBFS as well as non-SBFS fungi from seven families within the Capnodiales. The non-SBFS taxa were selected based on their distinct ecological niches, including plant-parasitic and saprophytic species. The phylogenetic analyses revealed that most SBFS species in the Capnodiales are closely related to plant-parasitic fungi. Ancestral state reconstruction provided strong evidence that plant-parasitic fungi were the ancestors of the major SBFS lineages. Knowledge gained from this study may help to better understand the ecology and evolution of epiphytic fungi.

  2. Genome structure and reproductive behaviour influence the evolutionary potential of a fungal phytopathogen.

    PubMed

    Daverdin, Guillaume; Rouxel, Thierry; Gout, Lilian; Aubertot, Jean-Noël; Fudal, Isabelle; Meyer, Michel; Parlange, Francis; Carpezat, Julien; Balesdent, Marie-Hélène

    2012-01-01

    Modern agriculture favours the selection and spread of novel plant diseases. Furthermore, crop genetic resistance against pathogens is often rendered ineffective within a few years of its commercial deployment. Leptosphaeria maculans, the cause of phoma stem canker of oilseed rape, develops gene-for-gene interactions with its host plant, and has a high evolutionary potential to render ineffective novel sources of resistance in crops. Here, we established a four-year field experiment to monitor the evolution of populations confronted with the newly released Rlm7 resistance and to investigate the nature of the mutations responsible for virulence against Rlm7. A total of 2551 fungal isolates were collected from experimental crops of a Rlm7 cultivar or a cultivar without Rlm7. All isolates were phenotyped for virulence and a subset was genotyped with neutral genetic markers. Virulent isolates were investigated for molecular events at the AvrLm4-7 locus. Whilst virulent isolates were not found in neighbouring crops, their frequency had reached 36% in the experimental field after four years. An extreme diversity of independent molecular events leading to virulence was identified in populations, with large-scale Repeat Induced Point mutations or complete deletion of AvrLm4-7 being the most frequent. Our data suggest that increased mutability of fungal genes involved in the interactions with plants is directly related to their genomic environment and reproductive system. Thus, rapid allelic diversification of avirulence genes can be generated in L. maculans populations in a single field provided that large population sizes and sexual reproduction are favoured by agricultural practices. PMID:23144620

  3. Comparative and functional genomic analyses of the pathogenicity of phytopathogen Xanthomonas campestris pv. campestris

    PubMed Central

    Qian, Wei; Jia, Yantao; Ren, Shuang-Xi; He, Yong-Qiang; Feng, Jia-Xun; Lu, Ling-Feng; Sun, Qihong; Ying, Ge; Tang, Dong-Jie; Tang, Hua; Wu, Wei; Hao, Pei; Wang, Lifeng; Jiang, Bo-Le; Zeng, Shenyan; Gu, Wen-Yi; Lu, Gang; Rong, Li; Tian, Yingchuan; Yao, Zhijian; Fu, Gang; Chen, Baoshan; Fang, Rongxiang; Qiang, Boqin; Chen, Zhu; Zhao, Guo-Ping; Tang, Ji-Liang; He, Chaozu

    2005-01-01

    Xanthomonas campestris pathovar campestris (Xcc) is the causative agent of crucifer black rot disease, which causes severe losses in agricultural yield world-wide. This bacterium is a model organism for studying plant-bacteria interactions. We sequenced the complete genome of Xcc 8004 (5,148,708 bp), which is highly conserved relative to that of Xcc ATCC 33913. Comparative genomics analysis indicated that, in addition to a significant genomic-scale rearrangement cross the replication axis between two IS1478 elements, loss and acquisition of blocks of genes, rather than point mutations, constitute the main genetic variation between the two Xcc strains. Screening of a high-density transposon insertional mutant library (16,512 clones) of Xcc 8004 against a host plant (Brassica oleraceae) identified 75 nonredundant, single-copy insertions in protein-coding sequences (CDSs) and intergenic regions. In addition to known virulence factors, full virulence was found to require several additional metabolic pathways and regulatory systems, such as fatty acid degradation, type IV secretion system, cell signaling, and amino acids and nucleotide metabolism. Among the identified pathogenicity-related genes, three of unknown function were found in Xcc 8004-specific chromosomal segments, revealing a direct correlation between genomic dynamics and Xcc virulence. The present combination of comparative and functional genomic analyses provides valuable information about the genetic basis of Xcc pathogenicity, which may offer novel insight toward the development of efficient methods for prevention of this important plant disease. PMID:15899963

  4. The Influence of Learning on Host Plant Preference in a Significant Phytopathogen Vector, Diaphorina citri.

    PubMed

    Stockton, Dara G; Martini, Xavier; Patt, Joseph M; Stelinski, Lukasz L

    2016-01-01

    Although specialist herbivorous insects are guided by innate responses to host plant cues, host plant preference may be influenced by experience and is not dictated by instinct alone. The effect of learning on host plant preference was examined in the Asian citrus psyllid, Diaphorina citri; vector of the causal agent of citrus greening disease or huanglongbing. We investigated: a) whether development on specific host plant species influenced host plant preference in mature D. citri; and b) the extent of associative learning in D. citri in the form of simple and compound conditioning. Learning was measured by cue selection in a 2-choice behavioral assay and compared to naïve controls. Our results showed that learned responses in D. citri are complex and diverse. The developmental host plant species influenced adult host plant preference, with female psyllids preferring the species on which they were reared. However, such preferences were subject to change with the introduction of an alternative host plant within 24-48 hrs, indicating a large degree of experience-dependent response plasticity. Additionally, learning occurred for multiple sensory modalities where novel olfactory and visual environmental cues were associated with the host plant. However, males and females displayed differing discriminatory abilities. In compound conditioning tasks, males exhibited recognition of a compound stimulus alone while females were capable of learning the individual components. These findings suggest D. citri are dynamic animals that demonstrate host plant preference based on developmental and adult experience and can learn to recognize olfactory and visual host plant stimuli in ways that may be sex specific. These experience-based associations are likely used by adults to locate and select suitable host plants for feeding and reproduction and may suggest the need for more tailored lures and traps, which reflect region-specific cultivars or predominate Rutaceae in the area being monitored.

  5. Transcriptional responses of the bacterial antagonist Serratia plymuthica to the fungal phytopathogen Rhizoctonia solani.

    PubMed

    Neupane, Saraswoti; Finlay, Roger D; Alström, Sadhna; Elfstrand, Malin; Högberg, Nils

    2015-02-01

    Rhizobacteria with biocontrol ability exploit a range of mechanisms to compete successfully with other microorganisms and to ensure their growth and survival in the rhizosphere, ultimately promoting plant growth. The rhizobacterium Serratia plymuthica AS13 is able to promote oilseed rape growth and improve seedling survival in the presence of the fungal pathogen, Rhizoctonia solani AG 2-1; however, our understanding of the mechanisms underlying the antagonism of Serratia is limited. To elucidate possible mechanisms, genome-wide gene expression profiling of S. plymuthica AS13 was carried out in the presence or absence of R. solani. We used RNA sequencing methodology to obtain a comprehensive overview of Serratia gene expression in response to R. solani. The differential gene expression profiles of S. plymuthica AS13 revealed significantly increased expression of genes related to the biosynthesis of the antibiotic pyrrolnitrin (prnABCD), protease production and transporters. The results presented here provide evidence that antibiosis is a major functional mechanism underlying the antagonistic behaviour of S. plymuthica AS13.

  6. Gall-ID: tools for genotyping gall-causing phytopathogenic bacteria.

    PubMed

    Davis Ii, Edward W; Weisberg, Alexandra J; Tabima, Javier F; Grunwald, Niklaus J; Chang, Jeff H

    2016-01-01

    Understanding the population structure and genetic diversity of plant pathogens, as well as the effect of agricultural practices on pathogen evolution, is important for disease management. Developments in molecular methods have contributed to increase the resolution for accurate pathogen identification, but those based on analysis of DNA sequences can be less straightforward to use. To address this, we developed Gall-ID, a web-based platform that uses DNA sequence information from 16S rDNA, multilocus sequence analysis and whole genome sequences to group disease-associated bacteria to their taxonomic units. Gall-ID was developed with a particular focus on gall-forming bacteria belonging to Agrobacterium, Pseudomonas savastanoi, Pantoea agglomerans, and Rhodococcus. Members of these groups of bacteria cause growth deformation of plants, and some are capable of infecting many species of field, orchard, and nursery crops. Gall-ID also enables the use of high-throughput sequencing reads to search for evidence for homologs of characterized virulence genes, and provides downloadable software pipelines for automating multilocus sequence analysis, analyzing genome sequences for average nucleotide identity, and constructing core genome phylogenies. Lastly, additional databases were included in Gall-ID to help determine the identity of other plant pathogenic bacteria that may be in microbial communities associated with galls or causative agents in other diseased tissues of plants. The URL for Gall-ID is http://gall-id.cgrb.oregonstate.edu/. PMID:27547538

  7. Genome sequences of three phytopathogenic species of the Magnaporthaceae family of fungi

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Magnaporthaceae is a family of ascomycetes that includes three fungi of great economic importance: Magnaporthe oryzae, Gaeumannomyces graminis var. tritici, and Magnaporthe poae. These three fungi cause widespread disease and loss in cereal and grass crops, including rice blast disease (M. oryzae), ...

  8. Cell wall chitosaccharides are essential components and exposed patterns of the phytopathogenic oomycete Aphanomyces euteiches.

    PubMed

    Badreddine, Ilham; Lafitte, Claude; Heux, Laurent; Skandalis, Nicholas; Spanou, Zacharoula; Martinez, Yves; Esquerré-Tugayé, Marie-Thérèse; Bulone, Vincent; Dumas, Bernard; Bottin, Arnaud

    2008-11-01

    Chitin is an essential component of fungal cell walls, where it forms a crystalline scaffold, and chitooligosaccharides derived from it are signaling molecules recognized by the hosts of pathogenic fungi. Oomycetes are cellulosic fungus-like microorganisms which most often lack chitin in their cell walls. Here we present the first study of the cell wall of the oomycete Aphanomyces euteiches, a major parasite of legume plants. Biochemical analyses demonstrated the presence of ca. 10% N-acetyl-D-glucosamine (GlcNAc) in the cell wall. Further characterization of the GlcNAc-containing material revealed that it corresponds to noncrystalline chitosaccharides associated with glucans, rather than to chitin per se. Two putative chitin synthase (CHS) genes were identified by data mining of an A. euteiches expressed sequence tag collection and Southern blot analysis, and full-length cDNA sequences of both genes were obtained. Phylogeny analysis indicated that oomycete CHS diversification occurred before the divergence of the major oomycete lineages. Remarkably, lectin labeling showed that the Aphanomyces euteiches chitosaccharides are exposed at the cell wall surface, and study of the effect of the CHS inhibitor nikkomycin Z demonstrated that they are involved in cell wall function. These data open new perspectives for the development of antioomycete drugs and further studies of the molecular mechanisms involved in the recognition of pathogenic oomycetes by the host plants. PMID:18806214

  9. The Influence of Learning on Host Plant Preference in a Significant Phytopathogen Vector, Diaphorina citri