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Sample records for ralstonia solanacearum species

  1. Polyphasic taxonomic revision of the Ralstonia solanacearum species complex: proposal to emend the descriptions of Ralstonia solanacearum and Ralstonia syzygii and reclassify current R. syzygii strains as Ralstonia syzygii subsp. syzygii subsp. nov., R. solanacearum phylotype IV strains as Ralstonia syzygii subsp. indonesiensis subsp. nov., banana blood disease bacterium strains as Ralstonia syzygii subsp. celebesensis subsp. nov. and R. solanacearum phylotype I and III strains as Ralstonia pseudosolanacearum sp. nov.

    PubMed

    Safni, Irda; Cleenwerck, Ilse; De Vos, Paul; Fegan, Mark; Sly, Lindsay; Kappler, Ulrike

    2014-09-01

    The Ralstonia solanacearum species complex has long been recognized as a group of phenotypically diverse strains that can be subdivided into four phylotypes. Using a polyphasic taxonomic approach on an extensive set of strains, this study provides evidence for a taxonomic and nomenclatural revision of members of this complex. Data obtained from phylogenetic analysis of 16S-23S rRNA ITS gene sequences, 16S-23S rRNA intergenic spacer (ITS) region sequences and partial endoglucanase (egl) gene sequences and DNA-DNA hybridizations demonstrate that the R. solanacearum species complex comprises three genospecies. One of these includes the type strain of Ralstonia solanacearum and consists of strains of R. solanacearum phylotype II only. The second genospecies includes the type strain of Ralstonia syzygii and contains only phylotype IV strains. This genospecies is subdivided into three distinct groups, namely R. syzygii, the causal agent of Sumatra disease on clove trees in Indonesia, R. solanacearum phylotype IV strains isolated from different host plants mostly from Indonesia, and strains of the blood disease bacterium (BDB), the causal agent of the banana blood disease, a bacterial wilt disease in Indonesia that affects bananas and plantains. The last genospecies is composed of R. solanacearum strains that belong to phylotypes I and III. As these genospecies are also supported by phenotypic data that allow the differentiation of the three genospecies, the following taxonomic proposals are made: emendation of the descriptions of Ralstonia solanacearum and Ralstonia syzygii and descriptions of Ralstonia syzygii subsp. nov. (type strain R 001(T) = LMG 10661(T) = DSM 7385(T)) for the current R. syzygii strains, Ralstonia syzygii subsp. indonesiensis subsp. nov. (type strain UQRS 464(T) = LMG 27703(T) = DSM 27478(T)) for the current R. solanacearum phylotype IV strains, Ralstonia syzygii subsp. celebesensis subsp. nov. (type strain UQRS 627(T

  2. Genomes of three tomato pathogens within the Ralstonia solanacearum species complex reveal significant evolutionary divergence

    PubMed Central

    2010-01-01

    Background The Ralstonia solanacearum species complex includes thousands of strains pathogenic to an unusually wide range of plant species. These globally dispersed and heterogeneous strains cause bacterial wilt diseases, which have major socio-economic impacts. Pathogenicity is an ancestral trait in R. solanacearum and strains with high genetic variation can be subdivided into four phylotypes, correlating to isolates from Asia (phylotype I), the Americas (phylotype IIA and IIB), Africa (phylotype III) and Indonesia (phylotype IV). Comparison of genome sequences strains representative of this phylogenetic diversity can help determine which traits allow this bacterium to be such a pathogen of so many different plant species and how the bacteria survive in many different habitats. Results The genomes of three tomato bacterial wilt pathogens, CFBP2957 (phy. IIA), CMR15 (phy. III) and PSI07 (phy. IV) were sequenced and manually annotated. These genomes were compared with those of three previously sequenced R. solanacearum strains: GMI1000 (tomato, phy. I), IPO1609 (potato, phy. IIB), and Molk2 (banana, phy. IIB). The major genomic features (size, G+C content, number of genes) were conserved across all of the six sequenced strains. Despite relatively high genetic distances (calculated from average nucleotide identity) and many genomic rearrangements, more than 60% of the genes of the megaplasmid and 70% of those on the chromosome are syntenic. The three new genomic sequences revealed the presence of several previously unknown traits, probably acquired by horizontal transfers, within the genomes of R. solanacearum, including a type IV secretion system, a rhi-type anti-mitotic toxin and two small plasmids. Genes involved in virulence appear to be evolving at a faster rate than the genome as a whole. Conclusions Comparative analysis of genome sequences and gene content confirmed the differentiation of R. solanacearum species complex strains into four phylotypes. Genetic

  3. Ralstonia syzygii, the Blood Disease Bacterium and Some Asian R. solanacearum Strains Form a Single Genomic Species Despite Divergent Lifestyles

    PubMed Central

    Cellier, Gilles; Jacobs, Jonathan M.; Mangenot, Sophie; Barbe, Valérie; Lajus, Aurélie; Vallenet, David; Medigue, Claudine; Fegan, Mark; Allen, Caitilyn; Prior, Philippe

    2011-01-01

    The Ralstonia solanacearum species complex includes R. solanacearum, R. syzygii, and the Blood Disease Bacterium (BDB). All colonize plant xylem vessels and cause wilt diseases, but with significant biological differences. R. solanacearum is a soilborne bacterium that infects the roots of a broad range of plants. R. syzygii causes Sumatra disease of clove trees and is actively transmitted by cercopoid insects. BDB is also pathogenic to a single host, banana, and is transmitted by pollinating insects. Sequencing and DNA-DNA hybridization studies indicated that despite their phenotypic differences, these three plant pathogens are actually very closely related, falling into the Phylotype IV subgroup of the R. solanacearum species complex. To better understand the relationships among these bacteria, we sequenced and annotated the genomes of R. syzygii strain R24 and BDB strain R229. These genomes were compared to strain PSI07, a closely related Phylotype IV tomato isolate of R. solanacearum, and to five additional R. solanacearum genomes. Whole-genome comparisons confirmed previous phylogenetic results: the three phylotype IV strains share more and larger syntenic regions with each other than with other R. solanacearum strains. Furthermore, the genetic distances between strains, assessed by an in-silico equivalent of DNA-DNA hybridization, unambiguously showed that phylotype IV strains of BDB, R. syzygii and R. solanacearum form one genomic species. Based on these comprehensive data we propose a revision of the taxonomy of the R. solanacearum species complex. The BDB and R. syzygii genomes encoded no obvious unique metabolic capacities and contained no evidence of horizontal gene transfer from bacteria occupying similar niches. Genes specific to R. syzygii and BDB were almost all of unknown function or extrachromosomal origin. Thus, the pathogenic life-styles of these organisms are more probably due to ecological adaptation and genomic convergence during vertical

  4. A computer program for fast and easy typing of a partial endoglucanase gene sequence into genospecies and sequevars 1&2 of the Ralstonia solanacearum species complex.

    PubMed

    Stulberg, Michael J; Huang, Qi

    2016-04-01

    The phytopathogen Ralstonia solanacearum is a species complex that contains race 3 biovar 2 strains belonging to phylotype IIB sequevars 1 and 2 that are quarantined or select agent pathogens. Recently, the R. solanacearum species complex strains have been reclassified into three genospecies: R. solanacearum, Ralstonia pseudosolanacearum and Ralstonia syzygii. An unidentified R. solanacearum strain is considered a select agent in the US until proven to be a non-race 3 biovar 2 (non-phylotype IIB sequevars 1&2). Currently, sequevars of R. solanacearum species complex strains can only be determined by phylogenetic analysis of a partial endoglucanase (egl) sequence of approximately 700-bp in length. Such analysis, however, requires expert knowledge to properly trim the sequence, to include the correct reference strains, and to interpret the results. By comparing GenBank egl sequences of representative R. solanacearum species-complex strains, we identified genospecies- and sequevar 1 and 2-specific single nucleotide polymorphisms (SNPs). We also designed primers to amplify a shorter, 526-bp, egl fragment from R. solanacearum species complex strains for easy sequencing of the amplicon, and to facilitate direct and specific amplification of egl from R. solanacearum-infected plant samples without the need of bacterial isolation. We wrote a computer program (Ralstonia solanacearum typing program) that analyzes a minimum 400-bp user-input egl sequence from a R. solanacearum strain for egl homology and SNP content to determine 1) whether it belongs to the R. solanacearum species complex, 2) if so, to which genospecies, and 3) whether it is of the sequevar type (sequevars 1 and 2) associated with the select agent/quarantined R. solanacearum strain. The program correctly typed all 371 tested egl sequences with known sequevars, obtained either from GenBank or through personal communication. Additionally, the program successfully typed 25 R. solanacearum strains in our

  5. A computer program for fast and easy typing of a partial endoglucanase gene sequence into genospecies and sequevars 1&2 of the Ralstonia solanacearum species complex.

    PubMed

    Stulberg, Michael J; Huang, Qi

    2016-04-01

    The phytopathogen Ralstonia solanacearum is a species complex that contains race 3 biovar 2 strains belonging to phylotype IIB sequevars 1 and 2 that are quarantined or select agent pathogens. Recently, the R. solanacearum species complex strains have been reclassified into three genospecies: R. solanacearum, Ralstonia pseudosolanacearum and Ralstonia syzygii. An unidentified R. solanacearum strain is considered a select agent in the US until proven to be a non-race 3 biovar 2 (non-phylotype IIB sequevars 1&2). Currently, sequevars of R. solanacearum species complex strains can only be determined by phylogenetic analysis of a partial endoglucanase (egl) sequence of approximately 700-bp in length. Such analysis, however, requires expert knowledge to properly trim the sequence, to include the correct reference strains, and to interpret the results. By comparing GenBank egl sequences of representative R. solanacearum species-complex strains, we identified genospecies- and sequevar 1 and 2-specific single nucleotide polymorphisms (SNPs). We also designed primers to amplify a shorter, 526-bp, egl fragment from R. solanacearum species complex strains for easy sequencing of the amplicon, and to facilitate direct and specific amplification of egl from R. solanacearum-infected plant samples without the need of bacterial isolation. We wrote a computer program (Ralstonia solanacearum typing program) that analyzes a minimum 400-bp user-input egl sequence from a R. solanacearum strain for egl homology and SNP content to determine 1) whether it belongs to the R. solanacearum species complex, 2) if so, to which genospecies, and 3) whether it is of the sequevar type (sequevars 1 and 2) associated with the select agent/quarantined R. solanacearum strain. The program correctly typed all 371 tested egl sequences with known sequevars, obtained either from GenBank or through personal communication. Additionally, the program successfully typed 25 R. solanacearum strains in our

  6. Improved biovar test for Ralstonia solanacearum

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Race 3, biovar 2 strains of Ralstonia solanacearum are quarantined pathogens in Europe and Canada and Select Agent pathogens in the United States. The biovar classification of R. solanacearum strains is based on their biochemical abilities to utilize a carbohydrate panel. The standard biovar test us...

  7. Bacterial wilt resistance in tomato, pepper, and eggplant: genetic resources respond to diverse strains in the Ralstonia solanacearum species complex.

    PubMed

    Lebeau, A; Daunay, M-C; Frary, A; Palloix, A; Wang, J-F; Dintinger, J; Chiroleu, F; Wicker, E; Prior, P

    2011-01-01

    Bacterial wilt, caused by strains belonging to the Ralstonia solanacearum species complex, inflicts severe economic losses in many crops worldwide. Host resistance remains the most effective control strategy against this disease. However, wilt resistance is often overcome due to the considerable variation among pathogen strains. To help breeders circumvent this problem, we assembled a worldwide collection of 30 accessions of tomato, eggplant and pepper (Core-TEP), most of which are commonly used as sources of resistance to R. solanacearum or for mapping quantitative trait loci. The Core-TEP lines were challenged with a core collection of 12 pathogen strains (Core-Rs2) representing the phylogenetic diversity of R. solanacearum. We observed six interaction phenotypes, from highly susceptible to highly resistant. Intermediate phenotypes resulted from the plants' ability to tolerate latent infections (i.e., bacterial colonization of vascular elements with limited or no wilting). The Core-Rs2 strains partitioned into three pathotypes on pepper accessions, five on tomato, and six on eggplant. A "pathoprofile" concept was developed to characterize the strain clusters, which displayed six virulence patterns on the whole set of Core-TEP host accessions. Neither pathotypes nor pathoprofiles were phylotype specific. Pathoprofiles with high aggressiveness were mainly found in strains from phylotypes I, IIB, and III. One pathoprofile included a strain that overcame almost all resistance sources. PMID:20795852

  8. TALE-Like Effectors Are an Ancestral Feature of the Ralstonia solanacearum Species Complex and Converge in DNA Targeting Specificity.

    PubMed

    Schandry, Niklas; de Lange, Orlando; Prior, Philippe; Lahaye, Thomas

    2016-01-01

    Ralstonia solanacearum, a species complex of bacterial plant pathogens divided into four monophyletic phylotypes, causes plant diseases in tropical climates around the world. Some strains exhibit a broad host range on solanaceous hosts, while others are highly host-specific as for example some banana-pathogenic strains. Previous studies showed that transcription activator-like (TAL) effectors from Ralstonia, termed RipTALs, are capable of activating reporter genes in planta, if these are preceded by a matching effector binding element (EBE). RipTALs target DNA via their central repeat domain (CRD), where one repeat pairs with one DNA-base of the given EBE. The repeat variable diresidue dictates base repeat specificity in a predictable fashion, known as the TALE code. In this work, we analyze RipTALs across all phylotypes of the Ralstonia solanacearum species complex. We find that RipTALs are prevalent in phylotypes I and IV but absent from most phylotype III and II strains (10/12, 8/14, 1/24, and 1/5 strains contained a RipTAL, respectively). RipTALs originating from strains of the same phylotype show high levels of sequence similarity (>98%) in the N-terminal and C-terminal regions, while RipTALs isolated from different phylotypes show 47-91% sequence similarity in those regions, giving rise to four RipTAL classes. We show that, despite sequence divergence, the base preference for guanine, mediated by the N-terminal region, is conserved across RipTALs of all classes. Using the number and order of repeats found in the CRD, we functionally sub-classify RipTALs, introduce a new simple nomenclature, and predict matching EBEs for all seven distinct RipTALs identified. We experimentally study RipTAL EBEs and uncover that some RipTALs are able to target the EBEs of other RipTALs, referred to as cross-reactivity. In particular, RipTALs from strains with a broad host range on solanaceous hosts cross-react on each other's EBEs. Investigation of sequence divergence between

  9. TALE-Like Effectors Are an Ancestral Feature of the Ralstonia solanacearum Species Complex and Converge in DNA Targeting Specificity.

    PubMed

    Schandry, Niklas; de Lange, Orlando; Prior, Philippe; Lahaye, Thomas

    2016-01-01

    Ralstonia solanacearum, a species complex of bacterial plant pathogens divided into four monophyletic phylotypes, causes plant diseases in tropical climates around the world. Some strains exhibit a broad host range on solanaceous hosts, while others are highly host-specific as for example some banana-pathogenic strains. Previous studies showed that transcription activator-like (TAL) effectors from Ralstonia, termed RipTALs, are capable of activating reporter genes in planta, if these are preceded by a matching effector binding element (EBE). RipTALs target DNA via their central repeat domain (CRD), where one repeat pairs with one DNA-base of the given EBE. The repeat variable diresidue dictates base repeat specificity in a predictable fashion, known as the TALE code. In this work, we analyze RipTALs across all phylotypes of the Ralstonia solanacearum species complex. We find that RipTALs are prevalent in phylotypes I and IV but absent from most phylotype III and II strains (10/12, 8/14, 1/24, and 1/5 strains contained a RipTAL, respectively). RipTALs originating from strains of the same phylotype show high levels of sequence similarity (>98%) in the N-terminal and C-terminal regions, while RipTALs isolated from different phylotypes show 47-91% sequence similarity in those regions, giving rise to four RipTAL classes. We show that, despite sequence divergence, the base preference for guanine, mediated by the N-terminal region, is conserved across RipTALs of all classes. Using the number and order of repeats found in the CRD, we functionally sub-classify RipTALs, introduce a new simple nomenclature, and predict matching EBEs for all seven distinct RipTALs identified. We experimentally study RipTAL EBEs and uncover that some RipTALs are able to target the EBEs of other RipTALs, referred to as cross-reactivity. In particular, RipTALs from strains with a broad host range on solanaceous hosts cross-react on each other's EBEs. Investigation of sequence divergence between

  10. TALE-Like Effectors Are an Ancestral Feature of the Ralstonia solanacearum Species Complex and Converge in DNA Targeting Specificity

    PubMed Central

    Schandry, Niklas; de Lange, Orlando; Prior, Philippe; Lahaye, Thomas

    2016-01-01

    Ralstonia solanacearum, a species complex of bacterial plant pathogens divided into four monophyletic phylotypes, causes plant diseases in tropical climates around the world. Some strains exhibit a broad host range on solanaceous hosts, while others are highly host-specific as for example some banana-pathogenic strains. Previous studies showed that transcription activator-like (TAL) effectors from Ralstonia, termed RipTALs, are capable of activating reporter genes in planta, if these are preceded by a matching effector binding element (EBE). RipTALs target DNA via their central repeat domain (CRD), where one repeat pairs with one DNA-base of the given EBE. The repeat variable diresidue dictates base repeat specificity in a predictable fashion, known as the TALE code. In this work, we analyze RipTALs across all phylotypes of the Ralstonia solanacearum species complex. We find that RipTALs are prevalent in phylotypes I and IV but absent from most phylotype III and II strains (10/12, 8/14, 1/24, and 1/5 strains contained a RipTAL, respectively). RipTALs originating from strains of the same phylotype show high levels of sequence similarity (>98%) in the N-terminal and C-terminal regions, while RipTALs isolated from different phylotypes show 47–91% sequence similarity in those regions, giving rise to four RipTAL classes. We show that, despite sequence divergence, the base preference for guanine, mediated by the N-terminal region, is conserved across RipTALs of all classes. Using the number and order of repeats found in the CRD, we functionally sub-classify RipTALs, introduce a new simple nomenclature, and predict matching EBEs for all seven distinct RipTALs identified. We experimentally study RipTAL EBEs and uncover that some RipTALs are able to target the EBEs of other RipTALs, referred to as cross-reactivity. In particular, RipTALs from strains with a broad host range on solanaceous hosts cross-react on each other’s EBEs. Investigation of sequence divergence

  11. Constraints on Genome Dynamics Revealed from Gene Distribution among the Ralstonia solanacearum Species

    PubMed Central

    Lefeuvre, Pierre; Cellier, Gilles; Remenant, Benoît; Chiroleu, Frédéric; Prior, Philippe

    2013-01-01

    Because it is suspected that gene content may partly explain host adaptation and ecology of pathogenic bacteria, it is important to study factors affecting genome composition and its evolution. While recent genomic advances have revealed extremely large pan-genomes for some bacterial species, it remains difficult to predict to what extent gene pool is accessible within or transferable between populations. As genomes bear imprints of the history of the organisms, gene distribution pattern analyses should provide insights into the forces and factors at play in the shaping and maintaining of bacterial genomes. In this study, we revisited the data obtained from a previous CGH microarrays analysis in order to assess the genomic plasticity of the R. solanacearum species complex. Gene distribution analyses demonstrated the remarkably dispersed genome of R. solanacearum with more than half of the genes being accessory. From the reconstruction of the ancestral genomes compositions, we were able to infer the number of gene gain and loss events along the phylogeny. Analyses of gene movement patterns reveal that factors associated with gene function, genomic localization and ecology delineate gene flow patterns. While the chromosome displayed lower rates of movement, the megaplasmid was clearly associated with hot-spots of gene gain and loss. Gene function was also confirmed to be an essential factor in gene gain and loss dynamics with significant differences in movement patterns between different COG categories. Finally, analyses of gene distribution highlighted possible highways of horizontal gene transfer. Due to sampling and design bias, we can only speculate on factors at play in this gene movement dynamic. Further studies examining precise conditions that favor gene transfer would provide invaluable insights in the fate of bacteria, species delineation and the emergence of successful pathogens. PMID:23723974

  12. Genome sequence of the tobacco bacterial wilt pathogen Ralstonia solanacearum.

    PubMed

    Li, Zefeng; Wu, Sanling; Bai, Xuefei; Liu, Yun; Lu, Jianfei; Liu, Yong; Xiao, Bingguang; Lu, Xiuping; Fan, Longjiang

    2011-11-01

    Ralstonia solanacearum is a causal agent of plant bacterial wilt with thousands of distinct strains in a heterogeneous species complex. Here we report the genome sequence of a phylotype IB strain, Y45, isolated from tobacco (Nicotiana tabacum) in China. Compared with the published genomes of eight strains which were isolated from other hosts and habitats, 794 specific genes and many rearrangements/inversion events were identified in the tobacco strain, demonstrating that this strain represents an important node within the R. solanacearum complex. PMID:21994922

  13. Genome sequence of the tobacco bacterial wilt pathogen Ralstonia solanacearum.

    PubMed

    Li, Zefeng; Wu, Sanling; Bai, Xuefei; Liu, Yun; Lu, Jianfei; Liu, Yong; Xiao, Bingguang; Lu, Xiuping; Fan, Longjiang

    2011-11-01

    Ralstonia solanacearum is a causal agent of plant bacterial wilt with thousands of distinct strains in a heterogeneous species complex. Here we report the genome sequence of a phylotype IB strain, Y45, isolated from tobacco (Nicotiana tabacum) in China. Compared with the published genomes of eight strains which were isolated from other hosts and habitats, 794 specific genes and many rearrangements/inversion events were identified in the tobacco strain, demonstrating that this strain represents an important node within the R. solanacearum complex.

  14. Breeding for resistances to Ralstonia solanacearum

    PubMed Central

    Huet, Gaëlle

    2014-01-01

    Ralstonia solanacearum is one of the most devastating bacterial plant pathogens due to its large host range, worldwide geographic distribution and persistence in fields. This soilborne pathogen is the causal agent of bacterial wilt and it can infect major agricultural crops thereby reducing significantly their yield. To favor infection, the bacterium delivers, through the type three secretion system, effectors that manipulate plant immunity. In this review, the relative efficiency of control strategies and existing resistances to R. solanacearum will be presented. Then, the genetic and molecular insights gained from the study of bacterial wilt in model plants will be described. Finally, I will explore how the knowledge gathered from unraveling avirulence and virulence mechanisms of R. solanacearum effectors could help to develop more durable resistances in crop plants toward this destructive pathogen. PMID:25566289

  15. Molecular Diversity of Ralstonia solanacearum Isolated from Ginger in Hawaii.

    PubMed

    Yu, Q; Alvarez, A M; Moore, P H; Zee, F; Kim, M S; de Silva, A; Hepperly, P R; Ming, R

    2003-09-01

    ABSTRACT The genetic diversity of Ralstonia solanacearum strains isolated from ginger (Zingiber officinale) growing on the island of Hawaii was determined by analysis of amplified fragment length polymorphisms (AFLPs). Initially 28 strains of R. solanacearum collected from five host plant species worldwide were analyzed by AFLP. A second analysis was conducted on 55 R. solanacearum strains collected from three ginger farms along the Hamakua Coast of Hawaii, the principle area of ginger cultivation in the state. From the initial analysis, R. solanacearum strains from ginger in Hawaii showed a high degree of similarity at 0.853. In contrast, the average genetic similarity between R. solanacearum strains from heliconia and ginger was only 0.165, and strains from ginger showed little similarity with strains from all other hosts. The second analysis of 55 strains from ginger on different Hawaiian farms confirmed that they were distinct from race 1 strains from tomato. Strains from ginger also showed greater diversity among themselves in the second analysis, and the greatest diversity occurred among strains from a farm where ginger is frequently imported and maintained. Our results provide evidence that R. solanacearum strains from ginger in Hawaii are genetically distinct from local strains from tomato (race 1) and heliconia (race 2).

  16. [Phytotoxic properties of Ralstonia solanacearum lipopolysaccharides].

    PubMed

    Hrytsaĭ, R V; Iakovleva, L M; Varbanets', L D

    2014-01-01

    The study is dedicated to research of phytotoxic properties of Ralstonia solanacearum lipopolysaccharides. This causative agent is one of the most dangerous among potato bacterial diseases. It is revealed that the inhibitory effect of LPS solution on seedlings germination is more noticeable on crops susceptible to brown rot. Maximal total phytotoxic properties have been shown by LPS from strains 35, 52b, TX1 and TS3, which were characterized by relatively low rhamnose content. Relative to the control plants LPS may diminish and some ones--increase the root length, height and weight of seedlings, subject to particular strain. But the stimulation revealed is minor.

  17. Polyphenol Oxidase Activity Expression in Ralstonia solanacearum

    PubMed Central

    Hernández-Romero, Diana; Solano, Francisco; Sanchez-Amat, Antonio

    2005-01-01

    Sequencing of the genome of Ralstonia solanacearum revealed several genes that putatively code for polyphenol oxidases (PPOs). To study the actual expression of these genes, we looked for and detected all kinds of PPO activities, including laccase, cresolase, and catechol oxidase activities, in cellular extracts of this microorganism. The conditions for the PPO assays were optimized for the phenolic substrate, pH, and sodium dodecyl sulfate concentration used. It was demonstrated that three different PPOs are expressed. The genes coding for the enzymes were unambiguously correlated with the enzymatic activities detected by generation of null mutations in the genes by using insertional mutagenesis with a suicide plasmid and estimating the changes in the levels of enzymatic activities compared to the levels in the wild-type strain. The protein encoded by the RSp1530 locus is a multicopper protein with laccase activity. Two other genes, RSc0337 and RSc1501, code for nonblue copper proteins exhibiting homology to tyrosinases. The product of RSc0337 has strong tyrosine hydroxylase activity, and it has been shown that this enzyme is involved in melanin synthesis by R. solanacearum. The product of the RSc1501 gene is an enzyme that shows a clear preference for oxidation of o-diphenols. Preliminary characterization of the mutants obtained indicated that PPOs expressed by R. solanacearum may participate in resistance to phenolic compounds since the mutants exhibited higher sensitivity to l-tyrosine than the wild-type strain. These results suggest a possible role in the pathogenic process to avoid plant resistance mechanisms involving the participation of phenolic compounds. PMID:16269713

  18. Deciphering phenotypic diversity of Ralstonia solanacearum strains pathogenic to potato.

    PubMed

    Cellier, G; Prior, P

    2010-11-01

    Based on the phylotype classification, we questioned how genetically and phenotypically diverse strains of Ralstonia solanacearum pathogenic to potato may be. We studied 129 European and Mediterranean strains along with 57 reference strains known to cover genetic diversity in this species. Phylogeny analysis was done on endoglucanase gene sequences. Pathogenicity to potato, tomato, and eggplant was established at 24 to 30°C and 15 to 24°C, whereas tests on banana were conducted at 24 to 30°C. The ability to cause wilt on species of Solanaceae was shared by strains in all four phylotypes. Brown rot phylotypes IIB-1 and IIB-2 and phylotype IIB-27 established latent infections in banana, and Moko disease-causing phylotypes IIA-6, IIB-3, and IIB-4 were virulent to susceptible potato and tomato, addressing the question of host adaptation mechanisms, which may have undergone a similar bottleneck evolution. Cold-tolerance ability is only shared on species of Solanaceae among brown rot phylotype IIB-1, which gathered the majority of European and Mediterranean strains. We surveyed strain LNPV24.25 as the first report of an emerging phylotype IIB-4NPB strain in France. These findings showed that pathogenicity traits of genetically identified strains still need to be understood, especially in the perspective of post-genomics comparative analysis, to understand bacterial speciation in the R. solanacearum species complex.

  19. Susceptibility of Geranium Cultivars (Pelargonium spp.) to Ralstonia solanacearum

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Sixty-one cultivars of geraniums including zonal, regal, ivy, and scented were tested for susceptibility to three strains of Ralstonia solanacearum: a Race 1 Biovar 1 (R1B1) strain P597 isolated from tomato in Florida, a R1B1 strain P673 obtained from pothos originating in Costa Rica, and a Race 3 B...

  20. Comparison between Solanum torvum Sw. and S. melongena L. after Ralstonia solanacearum inoculation.

    PubMed

    Aribaud, M; Noirot, M; Fock-Bastide, I; Vaniet, S; Kodja, H

    2014-09-01

    Bacterial wilt, caused by Ralstonia solanacearum, is one of the most devastating plant diseases, affecting some economically important Solanaceae crops. In contrast, Solanum torvum, also known as wild eggplant, does not wilt when infested with R. solanacearum. In order to describe the mechanism underlying the response of S. torvum, it was compared with the cultivated eggplant, S. melongena, when both were infected with the same R. solanacearum strain. No wilting occurred in S. torvum, although the bacteria colonised roots and stems in both species within the first 24 h. There were marked differences beyond 24 h, consisting of high bacterial mortality in S. torvum. Using the calli model, our investigations revealed an increase in cell wall monoamine oxidase activity in S. torvum after R. solanacearum inoculation, which did not occur in S. melongena.

  1. Ralfuranone thioether production by the plant pathogen Ralstonia solanacearum.

    PubMed

    Pauly, Julia; Spiteller, Dieter; Linz, Jeanine; Jacobs, Jonathan; Allen, Caitilyn; Nett, Markus; Hoffmeister, Dirk

    2013-11-01

    Ralfuranones are aryl-substituted furanone secondary metabolites of the Gram-negative plant pathogen Ralstonia solanacearum. New sulfur-containing ralfuranone derivatives were identified, including the methyl thioether-containing ralfuranone D. Isotopic labeling in vivo, as well as headspace analyses of volatiles from R. solanacearum liquid cultures, established a mechanism for the transfer of an intact methylthio group from L-methionine or α-keto-γ-methylthiobutyric acid. The methylthio acceptor molecule ralfuranone I, a previously postulated biosynthetic intermediate in ralfuranone biosynthesis, was isolated and characterized by NMR. The highly reactive Michael acceptor system of this intermediate readily reacts with various thiols, including glutathione.

  2. Studies on the biosynthesis of ralfuranones in Ralstonia solanacearum.

    PubMed

    Kai, Kenji; Ohnishi, Hideyuki; Kiba, Akinori; Ohnishi, Kouhei; Hikichi, Yasufumi

    2016-01-01

    Ralfuranones, aryl-furanone secondary metabolites, are involved in the virulence of Ralstonia solanacearum in solanaceous plants. Ralfuranone I (6) has been suggested as a biosynthetic precursor for other ralfuranones; however, this conversion has not been confirmed. We herein investigate the biosynthesis of ralfuranones using feeding experiments with ralfuranone I (6) and its putative metabolite, ralfuranone B (2). The results obtained demonstrated that the biosynthesis of ralfuranones proceeded in enzymatic and non-enzymatic manners. PMID:26645956

  3. Studies on the biosynthesis of ralfuranones in Ralstonia solanacearum.

    PubMed

    Kai, Kenji; Ohnishi, Hideyuki; Kiba, Akinori; Ohnishi, Kouhei; Hikichi, Yasufumi

    2016-01-01

    Ralfuranones, aryl-furanone secondary metabolites, are involved in the virulence of Ralstonia solanacearum in solanaceous plants. Ralfuranone I (6) has been suggested as a biosynthetic precursor for other ralfuranones; however, this conversion has not been confirmed. We herein investigate the biosynthesis of ralfuranones using feeding experiments with ralfuranone I (6) and its putative metabolite, ralfuranone B (2). The results obtained demonstrated that the biosynthesis of ralfuranones proceeded in enzymatic and non-enzymatic manners.

  4. A novel multilocus variable number tandem repeat analysis typing scheme for African phylotype III strains of the Ralstonia solanacearum species complex

    PubMed Central

    Ravelomanantsoa, Santatra; Robène, Isabelle; Chiroleu, Frédéric; Guérin, Fabien; Poussier, Stéphane; Pruvost, Olivier

    2016-01-01

    Background. Reliable genotyping that provides an accurate description of diversity in the context of pathogen emergence is required for the establishment of strategies to improve disease management. MultiLocus variable number tandem repeat analysis (MLVA) is a valuable genotyping method. It can be performed at small evolutionary scales where high discriminatory power is needed. Strains of the Ralstonia solanacearum species complex (RSSC) are highly genetically diverse. These destructive pathogens are the causative agent of bacterial wilt on an unusually broad range of host plants worldwide. In this study, we developed an MLVA scheme for genotyping the African RSSC phylotype III. Methods. We selected different publicly available tandem repeat (TR) loci and additional TR loci from the genome of strain CMR15 as markers. Based on these loci, a new phylotype III-MLVA scheme is presented. MLVA and multiLocus sequence typing (MLST) were compared at the global, regional, and local scales. Different populations of epidemiologically related and unrelated RSSC phylotype III strains were used. Results and Discussion. Sixteen polymorphic TR loci, which included seven microsatellites and nine minisatellites, were selected. These TR loci were distributed throughout the genome (chromosome and megaplasmid) and located in both coding and intergenic regions. The newly developed RS3-MLVA16 scheme was more discriminative than MLST. RS3-MLVA16 showed good ability in differentiating strains at global, regional, and local scales, and it especially highlighted epidemiological links between closely related strains at the local scale. RS3-MLVA16 also underlines genetic variability within the same MLST-type and clonal complex, and gives a first overview of population structure. Overall, RS3-MLVA16 is a promising genotyping method for outbreak investigation at a fine scale, and it could be used for outbreak investigation as a first-line, low-cost assay for the routine screening of RSSC

  5. A novel multilocus variable number tandem repeat analysis typing scheme for African phylotype III strains of the Ralstonia solanacearum species complex.

    PubMed

    Ravelomanantsoa, Santatra; Robène, Isabelle; Chiroleu, Frédéric; Guérin, Fabien; Poussier, Stéphane; Pruvost, Olivier; Prior, Philippe

    2016-01-01

    Background. Reliable genotyping that provides an accurate description of diversity in the context of pathogen emergence is required for the establishment of strategies to improve disease management. MultiLocus variable number tandem repeat analysis (MLVA) is a valuable genotyping method. It can be performed at small evolutionary scales where high discriminatory power is needed. Strains of the Ralstonia solanacearum species complex (RSSC) are highly genetically diverse. These destructive pathogens are the causative agent of bacterial wilt on an unusually broad range of host plants worldwide. In this study, we developed an MLVA scheme for genotyping the African RSSC phylotype III. Methods. We selected different publicly available tandem repeat (TR) loci and additional TR loci from the genome of strain CMR15 as markers. Based on these loci, a new phylotype III-MLVA scheme is presented. MLVA and multiLocus sequence typing (MLST) were compared at the global, regional, and local scales. Different populations of epidemiologically related and unrelated RSSC phylotype III strains were used. Results and Discussion. Sixteen polymorphic TR loci, which included seven microsatellites and nine minisatellites, were selected. These TR loci were distributed throughout the genome (chromosome and megaplasmid) and located in both coding and intergenic regions. The newly developed RS3-MLVA16 scheme was more discriminative than MLST. RS3-MLVA16 showed good ability in differentiating strains at global, regional, and local scales, and it especially highlighted epidemiological links between closely related strains at the local scale. RS3-MLVA16 also underlines genetic variability within the same MLST-type and clonal complex, and gives a first overview of population structure. Overall, RS3-MLVA16 is a promising genotyping method for outbreak investigation at a fine scale, and it could be used for outbreak investigation as a first-line, low-cost assay for the routine screening of RSSC

  6. A novel multilocus variable number tandem repeat analysis typing scheme for African phylotype III strains of the Ralstonia solanacearum species complex.

    PubMed

    Ravelomanantsoa, Santatra; Robène, Isabelle; Chiroleu, Frédéric; Guérin, Fabien; Poussier, Stéphane; Pruvost, Olivier; Prior, Philippe

    2016-01-01

    Background. Reliable genotyping that provides an accurate description of diversity in the context of pathogen emergence is required for the establishment of strategies to improve disease management. MultiLocus variable number tandem repeat analysis (MLVA) is a valuable genotyping method. It can be performed at small evolutionary scales where high discriminatory power is needed. Strains of the Ralstonia solanacearum species complex (RSSC) are highly genetically diverse. These destructive pathogens are the causative agent of bacterial wilt on an unusually broad range of host plants worldwide. In this study, we developed an MLVA scheme for genotyping the African RSSC phylotype III. Methods. We selected different publicly available tandem repeat (TR) loci and additional TR loci from the genome of strain CMR15 as markers. Based on these loci, a new phylotype III-MLVA scheme is presented. MLVA and multiLocus sequence typing (MLST) were compared at the global, regional, and local scales. Different populations of epidemiologically related and unrelated RSSC phylotype III strains were used. Results and Discussion. Sixteen polymorphic TR loci, which included seven microsatellites and nine minisatellites, were selected. These TR loci were distributed throughout the genome (chromosome and megaplasmid) and located in both coding and intergenic regions. The newly developed RS3-MLVA16 scheme was more discriminative than MLST. RS3-MLVA16 showed good ability in differentiating strains at global, regional, and local scales, and it especially highlighted epidemiological links between closely related strains at the local scale. RS3-MLVA16 also underlines genetic variability within the same MLST-type and clonal complex, and gives a first overview of population structure. Overall, RS3-MLVA16 is a promising genotyping method for outbreak investigation at a fine scale, and it could be used for outbreak investigation as a first-line, low-cost assay for the routine screening of RSSC

  7. Ralstonia solanacearum lipopeptide induces chlamydospore development in fungi and facilitates bacterial entry into fungal tissues.

    PubMed

    Spraker, Joseph E; Sanchez, Laura M; Lowe, Tiffany M; Dorrestein, Pieter C; Keller, Nancy P

    2016-09-01

    Ralstonia solanacearum is a globally distributed soil-borne plant pathogenic bacterium, which shares a broad ecological range with many plant- and soil-associated fungi. We sought to determine if R. solanacearum chemical communication directs symbiotic development of polymicrobial consortia. R. solanacearum produced a diffusible metabolite that induced conserved morphological differentiation in 34 species of fungi across three diverse taxa (Ascomycetes, Basidiomycetes and Zygomycetes). Fungi exposed to this metabolite formed chlamydospores, survival structures with thickened cell walls. Some chlamydospores internally harbored R. solanacearum, indicating a newly described endofungal lifestyle for this important plant pathogen. Using imaging mass spectrometry and peptidogenomics, we identified an undescribed lipopeptide, ralsolamycin, produced by an R. solanacearum non-ribosomal peptide synthetase-polyketide synthase hybrid. Inactivation of the hybrid non-ribosomal peptide synthetase-polyketide synthase gene, rmyA, abolished ralsolamycin synthesis. R. solanacearum mutants lacking ralsolamycin no longer induced chlamydospore development in fungal coculture and invaded fungal hyphae less well than wild-type. We propose that ralsolamycin contributes to the invasion of fungal hyphae and that the formation of chlamydospores may provide not only a specific niche for bacterial colonization but also enhanced survival for the partnering fungus. PMID:26943626

  8. Ralstonia solanacearum lipopeptide induces chlamydospore development in fungi and facilitates bacterial entry into fungal tissues.

    PubMed

    Spraker, Joseph E; Sanchez, Laura M; Lowe, Tiffany M; Dorrestein, Pieter C; Keller, Nancy P

    2016-09-01

    Ralstonia solanacearum is a globally distributed soil-borne plant pathogenic bacterium, which shares a broad ecological range with many plant- and soil-associated fungi. We sought to determine if R. solanacearum chemical communication directs symbiotic development of polymicrobial consortia. R. solanacearum produced a diffusible metabolite that induced conserved morphological differentiation in 34 species of fungi across three diverse taxa (Ascomycetes, Basidiomycetes and Zygomycetes). Fungi exposed to this metabolite formed chlamydospores, survival structures with thickened cell walls. Some chlamydospores internally harbored R. solanacearum, indicating a newly described endofungal lifestyle for this important plant pathogen. Using imaging mass spectrometry and peptidogenomics, we identified an undescribed lipopeptide, ralsolamycin, produced by an R. solanacearum non-ribosomal peptide synthetase-polyketide synthase hybrid. Inactivation of the hybrid non-ribosomal peptide synthetase-polyketide synthase gene, rmyA, abolished ralsolamycin synthesis. R. solanacearum mutants lacking ralsolamycin no longer induced chlamydospore development in fungal coculture and invaded fungal hyphae less well than wild-type. We propose that ralsolamycin contributes to the invasion of fungal hyphae and that the formation of chlamydospores may provide not only a specific niche for bacterial colonization but also enhanced survival for the partnering fungus.

  9. Ralstonia solanacearum lipopeptide induces chlamydospore development in fungi and facilitates bacterial entry into fungal tissues

    PubMed Central

    Spraker, Joseph E; Sanchez, Laura M; Lowe, Tiffany M; Dorrestein, Pieter C; Keller, Nancy P

    2016-01-01

    Ralstonia solanacearum is a globally distributed soil-borne plant pathogenic bacterium, which shares a broad ecological range with many plant- and soil-associated fungi. We sought to determine if R. solanacearum chemical communication directs symbiotic development of polymicrobial consortia. R. solanacearum produced a diffusible metabolite that induced conserved morphological differentiation in 34 species of fungi across three diverse taxa (Ascomycetes, Basidiomycetes and Zygomycetes). Fungi exposed to this metabolite formed chlamydospores, survival structures with thickened cell walls. Some chlamydospores internally harbored R. solanacearum, indicating a newly described endofungal lifestyle for this important plant pathogen. Using imaging mass spectrometry and peptidogenomics, we identified an undescribed lipopeptide, ralsolamycin, produced by an R. solanacearum non-ribosomal peptide synthetase-polyketide synthase hybrid. Inactivation of the hybrid non-ribosomal peptide synthetase-polyketide synthase gene, rmyA, abolished ralsolamycin synthesis. R. solanacearum mutants lacking ralsolamycin no longer induced chlamydospore development in fungal coculture and invaded fungal hyphae less well than wild-type. We propose that ralsolamycin contributes to the invasion of fungal hyphae and that the formation of chlamydospores may provide not only a specific niche for bacterial colonization but also enhanced survival for the partnering fungus. PMID:26943626

  10. A computer program for fast and easy typing of partial endoglucanase gene sequence into phylotypes and sequevars 1&2 (select agents) of Ralstonia solanacearum

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The phytopathogen Ralstonia solanacearum is a species complex that contains a subset of strains that are quarantined or select agent pathogens. An unidentified R. solanacearum strain is considered a select agent in the US until proven otherwise, which can be done by phylogenetic analysis of a partia...

  11. Complete genome sequence of the potato pathogen Ralstonia solanacearum UY031.

    PubMed

    Guarischi-Sousa, Rodrigo; Puigvert, Marina; Coll, Núria S; Siri, María Inés; Pianzzola, María Julia; Valls, Marc; Setubal, João C

    2016-01-01

    Ralstonia solanacearum is the causative agent of bacterial wilt of potato. Ralstonia solanacearum strain UY031 belongs to the American phylotype IIB, sequevar 1, also classified as race 3 biovar 2. Here we report the completely sequenced genome of this strain, the first complete genome for phylotype IIB, sequevar 1, and the fourth for the R. solanacearum species complex. In addition to standard genome annotation, we have carried out a curated annotation of type III effector genes, an important pathogenicity-related class of genes for this organism. We identified 60 effector genes, and observed that this effector repertoire is distinct when compared to those from other phylotype IIB strains. Eleven of the effectors appear to be nonfunctional due to disruptive mutations. We also report a methylome analysis of this genome, the first for a R. solanacearum strain. This analysis helped us note the presence of a toxin gene within a region of probable phage origin, raising the hypothesis that this gene may play a role in this strain's virulence. PMID:26779304

  12. Complete genome sequence of the potato pathogen Ralstonia solanacearum UY031.

    PubMed

    Guarischi-Sousa, Rodrigo; Puigvert, Marina; Coll, Núria S; Siri, María Inés; Pianzzola, María Julia; Valls, Marc; Setubal, João C

    2016-01-01

    Ralstonia solanacearum is the causative agent of bacterial wilt of potato. Ralstonia solanacearum strain UY031 belongs to the American phylotype IIB, sequevar 1, also classified as race 3 biovar 2. Here we report the completely sequenced genome of this strain, the first complete genome for phylotype IIB, sequevar 1, and the fourth for the R. solanacearum species complex. In addition to standard genome annotation, we have carried out a curated annotation of type III effector genes, an important pathogenicity-related class of genes for this organism. We identified 60 effector genes, and observed that this effector repertoire is distinct when compared to those from other phylotype IIB strains. Eleven of the effectors appear to be nonfunctional due to disruptive mutations. We also report a methylome analysis of this genome, the first for a R. solanacearum strain. This analysis helped us note the presence of a toxin gene within a region of probable phage origin, raising the hypothesis that this gene may play a role in this strain's virulence.

  13. Roles of different forms of lipopolysaccharides in Ralstonia solanacearum pathogenesis.

    PubMed

    Li, Chien-Hui; Wang, Kuan-Chung; Hong, Yu-Hau; Chu, Tai-Hsiang; Chu, Yu-Ju; Chou, I-Chun; Lu, Der-Kang; Chen, Chiao-Yen; Yang, Wen-Chieh; Lin, Yu-Mei; Cheng, Chiu-Ping

    2014-05-01

    Lipopolysaccharides (LPS) are critical components for the fitness of most gram-negative bacteria. Ralstonia solanacearum causes a deadly wilting disease in many crops; however, the pathogenic roles of different forms of LPS and their pathways of biogenesis remain unknown. By screening for phage-resistant mutants of R. solanacearum Pss4, whose genome sequence is unavailable, mutants with various types of structural defects in LPS were isolated. Pathogenesis assays of the mutants revealed that production of rough LPS (R-LPS), which does not contain O-polysaccharides, was sufficient to cause necrosis on Nicotiana benthamiana and induce the hypersensitive response on N. tabacum. However, biosynthesis of smooth LPS (S-LPS), which contains O-polysaccharides, was required for bacterial proliferation at infection sites on N. benthamiana leaves and for proliferation and causing wilt on tomato. Complementation tests confirmed the involvement of the previously unidentified cluster RSc2201 to RSc2204 in the formation of R. solanacearum S-LPS. With these data and the availability of the annotated genomic sequence of strain GMI1000, certain loci involved in key steps of R. solanacearum LPS biosynthesis were identified. The strategy of this work could be useful for similar studies in other bacteria without available genome sequences.

  14. Ralstonia solanacearum: secrets of a major pathogen unveiled by analysis of its genome.

    PubMed

    Genin, Stéphane; Boucher, Christian

    2002-05-01

    Summary Ralstonia solanacearum Taxonomy: Bacteria; Proteobacteria; beta subdivision; Ralstonia group; genus Ralstonia Microbiological properties: Gram-negative, aerobic, motile rod. Disease symptoms: Agent of bacterial wilt of solanaceous plants, which appears as a sudden wilt. Typically, stem cross-sections ooze a whitish bacterial exudate. R. solanacearum is also the agent of the Moko disease of banana and brown rot of potato. Disease control: Pathogen-free seed and transplants. Few resistant and tolerant plant lines. Sanitation and cultural rotations. Useful web sites: http://ibws.nexenservices.com/;http://sequence.toulouse.inra.fr/R.solanacearum.html.

  15. A real-time PCR assay for the quantitative detection of Ralstonia solanacearum in the horticultural soil and plant tissues.

    PubMed

    Chen, Yun; Zhang, Wen-Zhi; Liu, Xin; Ma, Zhong-Hua; Li, Bo; Allen, Caitilyn; Guo, Jian-Hua

    2010-01-01

    A specific and rapid real-time PCR assay for detecting Ralstonia solanacearum in the horticultural soil and plant tissues was developed in this study. The specific primers RSF/RSR were designed based on the upstream region of UDP-3-O-acyl-GlcNAc deacetylase gene from R. solanacearum, and a PCR product of 159 bp was amplified specifically from 28 strains of R. solanacearum, which represent all genetically diverse AluI types and all 6 biovars, but not from any other nontarget species. The detection limit of 102 CFU/g tomato stem and horticultural soil was achieved in this real-time PCR assay. The high sensitivity and specificity observed with filed samples as well as with artificially infected samples suggested that this method might be a useful tool for detection and quantification of R. solanacearum in precise forecast and diagnosis.

  16. Complete Genome Sequence of the Plant Pathogen Ralstonia solanacearum Strain Po82 ▿

    PubMed Central

    Xu, Jin; Zheng, Hua-jun; Liu, Lei; Pan, Zhe-chao; Prior, Philippe; Tang, Biao; Xu, Jing-sheng; Zhang, Hao; Tian, Qian; Zhang, Li-qing; Feng, Jie

    2011-01-01

    Ralstonia solanacearum strain Po82, a phylotype IIB/sequevar 4 strain, was found to be pathogenic to both solanaceous plants and banana. Here, we report the complete genome sequence of Po82 and its comparison with seven published R. solanacearum genomes. PMID:21685279

  17. A multiplex PCR assay to detect and differentiate select agent strains of Ralstonia solanacearum

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Ralstonia solanacearum causes bacterial wilt in a variety of cash crops. R. solanacearum race 3 biovar 2 strains are considered select agents by the U.S. Government because they are not endemic to the U.S. and have the potential to cause brown rot disease in our potato production fields. Simple and...

  18. Genetic diversity and host range variation of Ralstonia solanacearum strains entering the United States

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Each year, large volumes of plant propagative stock are imported into the United States; occasionally, Ralstonia solanacearum is found systemically infecting this plant material. In this study, 106 R. solanacearum strains were collected over a 10-year period from imported ornamental propagative sto...

  19. Involvement of ralfuranone production in the virulence of Ralstonia solanacearum OE1-1.

    PubMed

    Kai, Kenji; Ohnishi, Hideyuki; Mori, Yuka; Kiba, Akinori; Ohnishi, Kouhei; Hikichi, Yasufumi

    2014-11-24

    Ralstonia solanacearum causes a destructive disease called "bacterial wilt" in numerous plant species. Its virulence is controlled by the transcriptional regulator PhcA, the activity of which is, in turn, regulated in a cell-density dependent manner, termed quorum sensing. We herein described the identification and characterization of ralfuranones J-L, new PhcA-regulated secondary metabolites, and the known derivatives, ralfuranones A and B, from R. solanacearum strain OE1-1. Their structures were determined by spectroscopic and chemical methods. These ralfuranones were also detected in vascular exudates from host plants infected with OE1-1. Deletion of ralA, which encodes an enzyme for ralfuranone biosynthesis, reduced the virulence of OE1-1 in tomato plants. Virulence was restored by complementation of the ralA gene. The results suggest that ralfuranones play important roles in the virulence of OE1-1.

  20. Protein O-linked glycosylation in the plant pathogen Ralstonia solanacearum.

    PubMed

    Elhenawy, Wael; Scott, Nichollas E; Tondo, M Laura; Orellano, Elena G; Foster, Leonard J; Feldman, Mario F

    2016-03-01

    Ralstonia solanacearum is one of the most lethal phytopathogens in the world. Due to its broad host range, it can cause wilting disease in many plant species of economic interest. In this work, we identified the O-oligosaccharyltransferase (O-OTase) responsible for protein O-glycosylation in R. solanacearum. An analysis of the glycoproteome revealed that 20 proteins, including type IV pilins are substrates of this general glycosylation system. Although multiple glycan forms were identified, the majority of the glycopeptides were modified with a pentasaccharide composed of HexNAc-(Pen)-dHex(3), similar to the O antigen subunit present in the lipopolysaccharide of multiple R. solanacearum strains. Disruption of the O-OTase led to the total loss of protein glycosylation, together with a defect in biofilm formation and reduced pathogenicity towards tomato plants. Comparative proteomic analysis revealed that the loss of glycosylation is not associated with widespread proteome changes. Only the levels of a single glycoprotein, the type IV pilin, were diminished in the absence of glycosylation. In parallel, disruption of glycosylation triggered an increase in the levels of a surface lectin homologous to Pseudomonas PA-IIL. These results reveal the important role of glycosylation in the pathogenesis of R. solanacearum. PMID:26531228

  1. Protein O-linked glycosylation in the plant pathogen Ralstonia solanacearum.

    PubMed

    Elhenawy, Wael; Scott, Nichollas E; Tondo, M Laura; Orellano, Elena G; Foster, Leonard J; Feldman, Mario F

    2016-03-01

    Ralstonia solanacearum is one of the most lethal phytopathogens in the world. Due to its broad host range, it can cause wilting disease in many plant species of economic interest. In this work, we identified the O-oligosaccharyltransferase (O-OTase) responsible for protein O-glycosylation in R. solanacearum. An analysis of the glycoproteome revealed that 20 proteins, including type IV pilins are substrates of this general glycosylation system. Although multiple glycan forms were identified, the majority of the glycopeptides were modified with a pentasaccharide composed of HexNAc-(Pen)-dHex(3), similar to the O antigen subunit present in the lipopolysaccharide of multiple R. solanacearum strains. Disruption of the O-OTase led to the total loss of protein glycosylation, together with a defect in biofilm formation and reduced pathogenicity towards tomato plants. Comparative proteomic analysis revealed that the loss of glycosylation is not associated with widespread proteome changes. Only the levels of a single glycoprotein, the type IV pilin, were diminished in the absence of glycosylation. In parallel, disruption of glycosylation triggered an increase in the levels of a surface lectin homologous to Pseudomonas PA-IIL. These results reveal the important role of glycosylation in the pathogenesis of R. solanacearum.

  2. Ralstonia solanacearum Strains from Martinique (French West Indies) Exhibiting a New Pathogenic Potential▿ †

    PubMed Central

    Wicker, Emmanuel; Grassart, Laurence; Coranson-Beaudu, Régine; Mian, Danièle; Guilbaud, Caroline; Fegan, Mark; Prior, Philippe

    2007-01-01

    We investigated a destructive pathogenic variant of the plant pathogen Ralstonia solanacearum that was consistently isolated in Martinique (French West Indies). Since the 1960s, bacterial wilt of solanaceous crops in Martinique has been caused primarily by strains of R. solanacearum that belong to either phylotype I or phylotype II. Since 1999, anthurium shade houses have been dramatically affected by uncharacterized phylotype II strains that also affected a wide range of species, such as Heliconia caribea, cucurbitaceous crops, and weeds. From 1989 to 2003, a total of 224 R. solanacearum isolates were collected and compared to 6 strains isolated in Martinique in the 1980s. The genetic diversity and phylogenetic position of selected strains from Martinique were assessed (multiplex PCRs, mutS and egl DNA sequence analysis) and compared to the genetic diversity and phylogenetic position of 32 reference strains covering the known diversity within the R. solanacearum species complex. Twenty-four representative isolates were tested for pathogenicity to Musa species (banana) and tomato, eggplant, and sweet pepper. Based upon both PCR and sequence analysis, 119 Martinique isolates from anthurium, members of the family Cucurbitaceae, Heliconia, and tomato, were determined to belong to a group termed phylotype II/sequevar 4 (II/4). While these strains cluster with the Moko disease-causing strains, they were not pathogenic to banana (NPB). The strains belonging to phylotype II/4NPB were highly pathogenic to tomato, eggplant, and pepper, were able to wilt the resistant tomato variety Hawaii7996, and may latently infect cooking banana. Phylotype II/4NPB constitutes a new pathogenic variant of R. solanacearum that has recently appeared in Martinique and may be latently prevalent throughout Caribbean and Central/South America. PMID:17720825

  3. Ralstonia solanacearum strains from Martinique (French West Indies) exhibiting a new pathogenic potential.

    PubMed

    Wicker, Emmanuel; Grassart, Laurence; Coranson-Beaudu, Régine; Mian, Danièle; Guilbaud, Caroline; Fegan, Mark; Prior, Philippe

    2007-11-01

    We investigated a destructive pathogenic variant of the plant pathogen Ralstonia solanacearum that was consistently isolated in Martinique (French West Indies). Since the 1960s, bacterial wilt of solanaceous crops in Martinique has been caused primarily by strains of R. solanacearum that belong to either phylotype I or phylotype II. Since 1999, anthurium shade houses have been dramatically affected by uncharacterized phylotype II strains that also affected a wide range of species, such as Heliconia caribea, cucurbitaceous crops, and weeds. From 1989 to 2003, a total of 224 R. solanacearum isolates were collected and compared to 6 strains isolated in Martinique in the 1980s. The genetic diversity and phylogenetic position of selected strains from Martinique were assessed (multiplex PCRs, mutS and egl DNA sequence analysis) and compared to the genetic diversity and phylogenetic position of 32 reference strains covering the known diversity within the R. solanacearum species complex. Twenty-four representative isolates were tested for pathogenicity to Musa species (banana) and tomato, eggplant, and sweet pepper. Based upon both PCR and sequence analysis, 119 Martinique isolates from anthurium, members of the family Cucurbitaceae, Heliconia, and tomato, were determined to belong to a group termed phylotype II/sequevar 4 (II/4). While these strains cluster with the Moko disease-causing strains, they were not pathogenic to banana (NPB). The strains belonging to phylotype II/4NPB were highly pathogenic to tomato, eggplant, and pepper, were able to wilt the resistant tomato variety Hawaii7996, and may latently infect cooking banana. Phylotype II/4NPB constitutes a new pathogenic variant of R. solanacearum that has recently appeared in Martinique and may be latently prevalent throughout Caribbean and Central/South America.

  4. Ralstonia solanacearum strains from Martinique (French West Indies) exhibiting a new pathogenic potential.

    PubMed

    Wicker, Emmanuel; Grassart, Laurence; Coranson-Beaudu, Régine; Mian, Danièle; Guilbaud, Caroline; Fegan, Mark; Prior, Philippe

    2007-11-01

    We investigated a destructive pathogenic variant of the plant pathogen Ralstonia solanacearum that was consistently isolated in Martinique (French West Indies). Since the 1960s, bacterial wilt of solanaceous crops in Martinique has been caused primarily by strains of R. solanacearum that belong to either phylotype I or phylotype II. Since 1999, anthurium shade houses have been dramatically affected by uncharacterized phylotype II strains that also affected a wide range of species, such as Heliconia caribea, cucurbitaceous crops, and weeds. From 1989 to 2003, a total of 224 R. solanacearum isolates were collected and compared to 6 strains isolated in Martinique in the 1980s. The genetic diversity and phylogenetic position of selected strains from Martinique were assessed (multiplex PCRs, mutS and egl DNA sequence analysis) and compared to the genetic diversity and phylogenetic position of 32 reference strains covering the known diversity within the R. solanacearum species complex. Twenty-four representative isolates were tested for pathogenicity to Musa species (banana) and tomato, eggplant, and sweet pepper. Based upon both PCR and sequence analysis, 119 Martinique isolates from anthurium, members of the family Cucurbitaceae, Heliconia, and tomato, were determined to belong to a group termed phylotype II/sequevar 4 (II/4). While these strains cluster with the Moko disease-causing strains, they were not pathogenic to banana (NPB). The strains belonging to phylotype II/4NPB were highly pathogenic to tomato, eggplant, and pepper, were able to wilt the resistant tomato variety Hawaii7996, and may latently infect cooking banana. Phylotype II/4NPB constitutes a new pathogenic variant of R. solanacearum that has recently appeared in Martinique and may be latently prevalent throughout Caribbean and Central/South America. PMID:17720825

  5. Ralstonia solanacearum RSp0194 Encodes a Novel 3-Keto-Acyl Carrier Protein Synthase III.

    PubMed

    Mao, Ya-Hui; Ma, Jin-Cheng; Li, Feng; Hu, Zhe; Wang, Hai-Hong

    2015-01-01

    Fatty acid synthesis (FAS), a primary metabolic pathway, is essential for survival of bacteria. Ralstonia solanacearum, a β-proteobacteria member, causes a bacterial wilt affecting more than 200 plant species, including many economically important plants. However, thus far, the fatty acid biosynthesis pathway of R. solanacearum has not been well studied. In this study, we characterized two forms of 3-keto-ACP synthase III, RsFabH and RsFabW, in R. solanacearum. RsFabH, the homologue of Escherichia coli FabH, encoded by the chromosomal RSc1050 gene, catalyzes the condensation of acetyl-CoA with malonyl-ACP in the initiation steps of fatty acid biosynthesis in vitro. The RsfabH mutant lost de novo fatty acid synthetic ability, and grows in medium containing free fatty acids. RsFabW, a homologue of Pseudomonas aeruginosa PA3286, encoded by a megaplasmid gene, RSp0194, condenses acyl-CoA (C2-CoA to C10-CoA) with malonyl-ACP to produce 3-keto-acyl-ACP in vitro. Although the RsfabW mutant was viable, RsfabW was responsible for RsfabH mutant growth on medium containing free fatty acids. Our results also showed that RsFabW could condense acyl-ACP (C4-ACP to C8-ACP) with malonyl-ACP, to produce 3-keto-acyl-ACP in vitro, which implies that RsFabW plays a special role in fatty acid synthesis of R. solanacearum. All of these data confirm that R. solanacearum not only utilizes acetyl-CoA, but also, utilizes medium-chain acyl-CoAs or acyl-ACPs as primers to initiate fatty acid synthesis.

  6. Contrasting recombination patterns and demographic histories of the plant pathogen Ralstonia solanacearum inferred from MLSA

    PubMed Central

    Wicker, Emmanuel; Lefeuvre, Pierre; de Cambiaire, Jean-Charles; Lemaire, Christophe; Poussier, Stéphane; Prior, Philippe

    2012-01-01

    We used multilocus sequence analysis (MLSA) on a worldwide collection of the plant pathogenic Ralstonia solanacearum (Betaproteobacteria) to retrace its complex evolutionary history. Using genetic imprints left during R. solanacearum evolution, we were able to delineate distinct evolutionary complex displaying contrasting dynamics. Among the phylotypes already described (I, IIA, IIB, III, IV), eight groups of strains with distinct evolutionary patterns, named clades, were identified. From our recombination analysis, we identified 21 recombination events that occurred within and across these lineages. Although appearing the most divergent and ancestral phylotype, phylotype IV was inferred as a gene donor for the majority of the recombination events that we detected. Whereas this phylotype apparently fuelled the species diversity, ongoing diversification was mainly detected within phylotype I, IIA and III. These three groups presented a recent expanding population structure, a high level of homologous recombination and evidences of long-distance migrations. Factors such as adaptation to a specific host or intense trading of infected crops may have promoted this diversification. Whether R. solanacearum lineages will eventually evolve in distinct species remains an open question. The intensification of cropping and increase of geographical dispersion may favour situations of phylotype sympatry and promote higher exchange of key factors for host adaptation from their common genetic pool. PMID:22094345

  7. Draft Genome Sequence of Ralstonia solanacearum Strain Rs-T02, Which Represents the Most Prevalent Phylotype in Guangxi, China

    PubMed Central

    Zou, Chengwu; Wang, Kaihao; Meng, Jiaorong; Yuan, Gaoqing; Lin, Wei; Peng, Haowen

    2016-01-01

    Ralstonia solanacearum strain Rs-T02 was originally isolated from a bacterial wilt of tomato plant in Nanning City of Guangxi Province, China. It represents the most prevalent phylotype in Guangxi. Here, we present the draft genome sequence of this strain, which comprises 5,225 genes and 5,976,011 nucleotides with an average G+C content of 66.79%. There are 968 different genes between this isolate and the previously reported genome sequence of Ralstonia solanacearum GMl l000 (race l, biovar 3, phylotype I), and the genome sequence information of this isolate may be useful for comparative genomic studies to determine the genetic diversity in this species. PMID:27081126

  8. Ralstonia solanacearum Extracellular Polysaccharide Is a Specific Elicitor of Defense Responses in Wilt-Resistant Tomato Plants

    PubMed Central

    Milling, Annett; Babujee, Lavanya; Allen, Caitilyn

    2011-01-01

    Ralstonia solanacearum, which causes bacterial wilt of diverse plants, produces copious extracellular polysaccharide (EPS), a major virulence factor. The function of EPS in wilt disease is uncertain. Leading hypotheses are that EPS physically obstructs plant water transport, or that EPS cloaks the bacterium from host plant recognition and subsequent defense. Tomato plants infected with R. solanacearum race 3 biovar 2 strain UW551 and tropical strain GMI1000 upregulated genes in both the ethylene (ET) and salicylic acid (SA) defense signal transduction pathways. The horizontally wilt-resistant tomato line Hawaii7996 activated expression of these defense genes faster and to a greater degree in response to R. solanacearum infection than did susceptible cultivar Bonny Best. However, EPS played different roles in resistant and susceptible host responses to R. solanacearum. In susceptible plants the wild-type and eps− mutant strains induced generally similar defense responses. But in resistant Hawaii7996 tomato plants, the wild-type pathogens induced significantly greater defense responses than the eps− mutants, suggesting that the resistant host recognizes R. solanacearum EPS. Consistent with this idea, purified EPS triggered significant SA pathway defense gene expression in resistant, but not in susceptible, tomato plants. In addition, the eps− mutant triggered noticeably less production of defense-associated reactive oxygen species in resistant tomato stems and leaves, despite attaining similar cell densities in planta. Collectively, these data suggest that bacterial wilt-resistant plants can specifically recognize EPS from R. solanacearum. PMID:21253019

  9. Genetic diversity of the bacterial wilt pathogen Ralstonia solanacearum using a RAPD marker.

    PubMed

    Nishat, Sayeda; Hamim, Islam; Khalil, M Ibrahim; Ali, Md Ayub; Hossain, Muhammed Ali; Meah, M Bahadur; Islam, Md Rashidul

    2015-11-01

    Bacterial wilt caused by Ralstonia solanacearum is a destructive disease of many economically important crop species. A significant variation in wilt incidence and severity in eggplant and potato was observed among the growing areas surveyed. R. solanacearum isolates obtained both from eggplant and potato belong to biovar III, while isolates from eggplant belong to race 1 and isolates obtained from potato belong to race 3. Random amplified polymorphic DNA (RAPD) technique was used as a tool for assessing genetic variation and relationship among seven isolate groups of R. solanacearum viz., RsB-1, RsB-2, RsB-3, RsP-1, RsP-2, RsP-3 and RsP-4, consisting in a total of 28 isolates. Out of the RAPD markers used, amplification with four decamer primers produced 70 bands with sizes ranging from 100 to 1400 bp. Out of 70 bands, 68 bands (97.06%) were polymorphic and two bands (2.94%) were monomorphic amongst the seven R. solanacearum isolates group. The Unweighted Pair Group Method of Arithmetic Means (UPGMA) dendrogram constructed from Nei's genetic distance produced two main clusters of the seven isolates of R. solanacearum. The isolates RsB-1, RsB-2, RsB-3 and R-4 grouped in cluster І, while RsP-2, RsP-3 and RsP-4 grouped in cluster ІІ. The highest intra-variety similarity index (Si) was found in RsB-1 isolate (86.35%) and the lowest one in RsP-2 (56.59%). The results indicated that relatively higher and lower levels of genetic variation were found in RsP-3 and RsB-3, respectively. The coefficient of gene differentiation (G(st)) was 0.5487, reflecting the existence of a high level of genetic variations among seven isolates of R. solanacearum. Comparatively higher genetic distance (0.4293) and lower genetic identity (0.6510) were observed between RsB-2 and RsP-4 combinations. The lowest genetic distance (0.0357) and highest genetic identity (0.9650) were found in RsB-1 vs. RsB-2 pair. Thus, RAPD offers a potentially simple, rapid and reliable method to evaluate

  10. Genetic diversity of the bacterial wilt pathogen Ralstonia solanacearum using a RAPD marker.

    PubMed

    Nishat, Sayeda; Hamim, Islam; Khalil, M Ibrahim; Ali, Md Ayub; Hossain, Muhammed Ali; Meah, M Bahadur; Islam, Md Rashidul

    2015-11-01

    Bacterial wilt caused by Ralstonia solanacearum is a destructive disease of many economically important crop species. A significant variation in wilt incidence and severity in eggplant and potato was observed among the growing areas surveyed. R. solanacearum isolates obtained both from eggplant and potato belong to biovar III, while isolates from eggplant belong to race 1 and isolates obtained from potato belong to race 3. Random amplified polymorphic DNA (RAPD) technique was used as a tool for assessing genetic variation and relationship among seven isolate groups of R. solanacearum viz., RsB-1, RsB-2, RsB-3, RsP-1, RsP-2, RsP-3 and RsP-4, consisting in a total of 28 isolates. Out of the RAPD markers used, amplification with four decamer primers produced 70 bands with sizes ranging from 100 to 1400 bp. Out of 70 bands, 68 bands (97.06%) were polymorphic and two bands (2.94%) were monomorphic amongst the seven R. solanacearum isolates group. The Unweighted Pair Group Method of Arithmetic Means (UPGMA) dendrogram constructed from Nei's genetic distance produced two main clusters of the seven isolates of R. solanacearum. The isolates RsB-1, RsB-2, RsB-3 and R-4 grouped in cluster І, while RsP-2, RsP-3 and RsP-4 grouped in cluster ІІ. The highest intra-variety similarity index (Si) was found in RsB-1 isolate (86.35%) and the lowest one in RsP-2 (56.59%). The results indicated that relatively higher and lower levels of genetic variation were found in RsP-3 and RsB-3, respectively. The coefficient of gene differentiation (G(st)) was 0.5487, reflecting the existence of a high level of genetic variations among seven isolates of R. solanacearum. Comparatively higher genetic distance (0.4293) and lower genetic identity (0.6510) were observed between RsB-2 and RsP-4 combinations. The lowest genetic distance (0.0357) and highest genetic identity (0.9650) were found in RsB-1 vs. RsB-2 pair. Thus, RAPD offers a potentially simple, rapid and reliable method to evaluate

  11. Genomic characterization of Ralstonia solanacearum phage ϕRS138 of the family Siphoviridae.

    PubMed

    Van Truong Thi, Bich; Pham Khanh, Nguyen Huan; Namikawa, Ryuta; Miki, Kaito; Kondo, Akihiro; Dang Thi, Phuong Thao; Kamei, Kaeko

    2016-02-01

    ϕRS138, a bacteriophage of the family Siphoviridae that lyses Ralstonia solanacearum, was isolated. The genomic DNA of ϕRS138 was 41,941 bp long with a GC content of 65.1 % and contained 56 putative open reading frames. The ϕRS138 genome could be divided into three regions based on similarities to other genomes: (1) a region containing genes encoding a putative transcriptional regulator and an integrase, similar to the prophage genes in Ralstonia solanacearum K60-1; (2) a region encoding proteins related to structural modules and virion morphogenesis, similar to genes in the Pseudomonas phages of the family Siphoviridae; and (3) a region highly similar to the genomes of other Ralstonia solanacearum strains. PMID:26526151

  12. Genomic characterization of Ralstonia solanacearum phage ϕRS138 of the family Siphoviridae.

    PubMed

    Van Truong Thi, Bich; Pham Khanh, Nguyen Huan; Namikawa, Ryuta; Miki, Kaito; Kondo, Akihiro; Dang Thi, Phuong Thao; Kamei, Kaeko

    2016-02-01

    ϕRS138, a bacteriophage of the family Siphoviridae that lyses Ralstonia solanacearum, was isolated. The genomic DNA of ϕRS138 was 41,941 bp long with a GC content of 65.1 % and contained 56 putative open reading frames. The ϕRS138 genome could be divided into three regions based on similarities to other genomes: (1) a region containing genes encoding a putative transcriptional regulator and an integrase, similar to the prophage genes in Ralstonia solanacearum K60-1; (2) a region encoding proteins related to structural modules and virion morphogenesis, similar to genes in the Pseudomonas phages of the family Siphoviridae; and (3) a region highly similar to the genomes of other Ralstonia solanacearum strains.

  13. Genome sequence of the plant pathogen Ralstonia solanacearum.

    PubMed

    Salanoubat, M; Genin, S; Artiguenave, F; Gouzy, J; Mangenot, S; Arlat, M; Billault, A; Brottier, P; Camus, J C; Cattolico, L; Chandler, M; Choisne, N; Claudel-Renard, C; Cunnac, S; Demange, N; Gaspin, C; Lavie, M; Moisan, A; Robert, C; Saurin, W; Schiex, T; Siguier, P; Thébault, P; Whalen, M; Wincker, P; Levy, M; Weissenbach, J; Boucher, C A

    2002-01-31

    Ralstonia solanacearum is a devastating, soil-borne plant pathogen with a global distribution and an unusually wide host range. It is a model system for the dissection of molecular determinants governing pathogenicity. We present here the complete genome sequence and its analysis of strain GMI1000. The 5.8-megabase (Mb) genome is organized into two replicons: a 3.7-Mb chromosome and a 2.1-Mb megaplasmid. Both replicons have a mosaic structure providing evidence for the acquisition of genes through horizontal gene transfer. Regions containing genetically mobile elements associated with the percentage of G+C bias may have an important function in genome evolution. The genome encodes many proteins potentially associated with a role in pathogenicity. In particular, many putative attachment factors were identified. The complete repertoire of type III secreted effector proteins can be studied. Over 40 candidates were identified. Comparison with other genomes suggests that bacterial plant pathogens and animal pathogens harbour distinct arrays of specialized type III-dependent effectors. PMID:11823852

  14. Genetic Diversity of Japanese Strains of Ralstonia solanacearum.

    PubMed

    Horita, M; Tsuchiya, K

    2001-04-01

    ABSTRACT The genetic diversity of 74 Japanese strains of Ralstonia solanacearum was assessed by pathogenicity tests and the repetitive sequencebased polymerase chain reaction (rep-PCR) fingerprint method. Based on their genomic fingerprints, biovar N2 strains were divided into two distinct groups, one consisting of potato isolates belonging to race 3, and the other consisting of tomato, eggplant, pepper, and tobacco isolates belonging to race 1. Biovar 3 strains had low average similarity and were divided into five groups that differed in original host or pathogenicity. Biovar 4 strains consisted of only one group at the 80% similarity level. Comparative analysis of the rep-PCR fingerprints of 78 strains, including six biovars from Japan and various countries, revealed two main clusters. Cluster 1 comprised all biovar 3, 4, and 5 strains, biovar 1 strains from Reunion, and some biovar N2 strains from Japan. Cluster 2 included most of the biovar 1, 2, and N2 strains. The fingerprints showed low average similarity with biovar N2 strains from Japan and Brazil. PMID:18943853

  15. Genetic Determinants for Pyomelanin Production and Its Protective Effect against Oxidative Stress in Ralstonia solanacearum.

    PubMed

    Ahmad, Shabir; Lee, Seung Yeup; Kong, Hyun Gi; Jo, Eun Jeong; Choi, Hye Kyung; Khan, Raees; Lee, Seon-Woo

    2016-01-01

    Ralstonia solanacearum is a soil-borne plant pathogen that infects more than 200 plant species. Its broad host range and long-term survival under different environmental stress conditions suggest that it uses a variety of mechanisms to protect itself against various types of biotic and abiotic stress. R. solanacearum produces a melanin-like brown pigment in the stationary phase when grown in minimal medium containing tyrosine. To gain deeper insight into the genetic determinants involved in melanin production, transposon-inserted mutants of R. solanacearum strain SL341 were screened for strains with defective melanin-producing capability. In addition to one mutant already known to be involved in pyomelanin production (viz., strain SL341D, with disruption of the hydroxphenylpyruvate dioxygenase gene), we identified three other mutants with disruption in the regulatory genes rpoS, hrpG, and oxyR, respectively. Wild-type SL341 produced pyomelanin in minimal medium containing tyrosine whereas the mutant strains did not. Likewise, homogentisate, a major precursor of pyomelanin, was detected in the culture filtrate of the wild-type strain but not in those of the mutant strains. A gene encoding hydroxyphenylpyruvate dioxygenase exhibited a significant high expression in wild type SL341 compared to other mutant strains, suggesting that pyomelanin production is regulated by three different regulatory proteins. However, analysis of the gene encoding homogentisate dioxygenase revealed no significant difference in its relative expression over time in the wild-type SL341 and mutant strains, except for SL341D, at 72 h incubation. The pigmented SL341 strain also exhibited a high tolerance to hydrogen peroxide stress compared with the non-pigmented SL341D strain. Our study suggests that pyomelanin production is controlled by several regulatory factors in R. solanacearum to confer protection under oxidative stress. PMID:27513990

  16. Genetic Determinants for Pyomelanin Production and Its Protective Effect against Oxidative Stress in Ralstonia solanacearum

    PubMed Central

    Kong, Hyun Gi; Jo, Eun Jeong; Choi, Hye Kyung; Khan, Raees; Lee, Seon-Woo

    2016-01-01

    Ralstonia solanacearum is a soil-borne plant pathogen that infects more than 200 plant species. Its broad host range and long-term survival under different environmental stress conditions suggest that it uses a variety of mechanisms to protect itself against various types of biotic and abiotic stress. R. solanacearum produces a melanin-like brown pigment in the stationary phase when grown in minimal medium containing tyrosine. To gain deeper insight into the genetic determinants involved in melanin production, transposon-inserted mutants of R. solanacearum strain SL341 were screened for strains with defective melanin-producing capability. In addition to one mutant already known to be involved in pyomelanin production (viz., strain SL341D, with disruption of the hydroxphenylpyruvate dioxygenase gene), we identified three other mutants with disruption in the regulatory genes rpoS, hrpG, and oxyR, respectively. Wild-type SL341 produced pyomelanin in minimal medium containing tyrosine whereas the mutant strains did not. Likewise, homogentisate, a major precursor of pyomelanin, was detected in the culture filtrate of the wild-type strain but not in those of the mutant strains. A gene encoding hydroxyphenylpyruvate dioxygenase exhibited a significant high expression in wild type SL341 compared to other mutant strains, suggesting that pyomelanin production is regulated by three different regulatory proteins. However, analysis of the gene encoding homogentisate dioxygenase revealed no significant difference in its relative expression over time in the wild-type SL341 and mutant strains, except for SL341D, at 72 h incubation. The pigmented SL341 strain also exhibited a high tolerance to hydrogen peroxide stress compared with the non-pigmented SL341D strain. Our study suggests that pyomelanin production is controlled by several regulatory factors in R. solanacearum to confer protection under oxidative stress. PMID:27513990

  17. Genetic Determinants for Pyomelanin Production and Its Protective Effect against Oxidative Stress in Ralstonia solanacearum.

    PubMed

    Ahmad, Shabir; Lee, Seung Yeup; Kong, Hyun Gi; Jo, Eun Jeong; Choi, Hye Kyung; Khan, Raees; Lee, Seon-Woo

    2016-01-01

    Ralstonia solanacearum is a soil-borne plant pathogen that infects more than 200 plant species. Its broad host range and long-term survival under different environmental stress conditions suggest that it uses a variety of mechanisms to protect itself against various types of biotic and abiotic stress. R. solanacearum produces a melanin-like brown pigment in the stationary phase when grown in minimal medium containing tyrosine. To gain deeper insight into the genetic determinants involved in melanin production, transposon-inserted mutants of R. solanacearum strain SL341 were screened for strains with defective melanin-producing capability. In addition to one mutant already known to be involved in pyomelanin production (viz., strain SL341D, with disruption of the hydroxphenylpyruvate dioxygenase gene), we identified three other mutants with disruption in the regulatory genes rpoS, hrpG, and oxyR, respectively. Wild-type SL341 produced pyomelanin in minimal medium containing tyrosine whereas the mutant strains did not. Likewise, homogentisate, a major precursor of pyomelanin, was detected in the culture filtrate of the wild-type strain but not in those of the mutant strains. A gene encoding hydroxyphenylpyruvate dioxygenase exhibited a significant high expression in wild type SL341 compared to other mutant strains, suggesting that pyomelanin production is regulated by three different regulatory proteins. However, analysis of the gene encoding homogentisate dioxygenase revealed no significant difference in its relative expression over time in the wild-type SL341 and mutant strains, except for SL341D, at 72 h incubation. The pigmented SL341 strain also exhibited a high tolerance to hydrogen peroxide stress compared with the non-pigmented SL341D strain. Our study suggests that pyomelanin production is controlled by several regulatory factors in R. solanacearum to confer protection under oxidative stress.

  18. [Advances in studies of the type III secretion system in Ralstonia solanacearum--A review].

    PubMed

    Zhang, Yong; Li, Muyuan; Luo, Feng

    2015-06-01

    Bacterial wilt caused by Ralstonia solanacearum is one of the most devastating plant diseases worldwide. The syringe-like type III secretion system (T3SS) plays a crucial role in its pathogenicity. R. solanacearum uses the T3SS to inject effector proteins (Type III effectors) into the cytoplasm of host cells, causing diseases in susceptible plants or triggering the hypersensitive response in resistant plants. In this article we review recent advances in studies of R. solanacearum T3SS and highlight their unique features. PMID:26562991

  19. [Advances in studies of the type III secretion system in Ralstonia solanacearum--A review].

    PubMed

    Zhang, Yong; Li, Muyuan; Luo, Feng

    2015-06-01

    Bacterial wilt caused by Ralstonia solanacearum is one of the most devastating plant diseases worldwide. The syringe-like type III secretion system (T3SS) plays a crucial role in its pathogenicity. R. solanacearum uses the T3SS to inject effector proteins (Type III effectors) into the cytoplasm of host cells, causing diseases in susceptible plants or triggering the hypersensitive response in resistant plants. In this article we review recent advances in studies of R. solanacearum T3SS and highlight their unique features.

  20. Draft Genome Sequences of Nine Strains of Ralstonia solanacearum Differing in Virulence to Eggplant (Solanum melongena).

    PubMed

    Guinard, Jérémy; Vinatzer, Boris A; Poussier, Stéphane; Lefeuvre, Pierre; Wicker, Emmanuel

    2016-01-01

    Ralstonia solanacearum displays variability in its virulence to solanaceous crops. We report here the draft genome sequences of eight phylotype I strains and one phylotype III strain differing in virulence to the resistant eggplant genotype AG91-25. These data will allow the identification of virulence- and avirulence-related genes. PMID:26823572

  1. Draft Genome Sequences of Nine Strains of Ralstonia solanacearum Differing in Virulence to Eggplant (Solanum melongena)

    PubMed Central

    Guinard, Jérémy; Vinatzer, Boris A.; Poussier, Stéphane; Lefeuvre, Pierre

    2016-01-01

    Ralstonia solanacearum displays variability in its virulence to solanaceous crops. We report here the draft genome sequences of eight phylotype I strains and one phylotype III strain differing in virulence to the resistant eggplant genotype AG91-25. These data will allow the identification of virulence- and avirulence-related genes. PMID:26823572

  2. Draft Genome Sequences of Nine Strains of Ralstonia solanacearum Differing in Virulence to Eggplant (Solanum melongena).

    PubMed

    Guinard, Jérémy; Vinatzer, Boris A; Poussier, Stéphane; Lefeuvre, Pierre; Wicker, Emmanuel

    2016-01-28

    Ralstonia solanacearum displays variability in its virulence to solanaceous crops. We report here the draft genome sequences of eight phylotype I strains and one phylotype III strain differing in virulence to the resistant eggplant genotype AG91-25. These data will allow the identification of virulence- and avirulence-related genes.

  3. Antagonistic activity and mechanisms of Bacillus subtilis SB1 against Ralstonia solanacearum

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A potential biocontrol agent of bacterial wilt, Bacillus subtilis SB1, showed a broad-spectrum of antimicrobial activity in vitro experiments. In addition to Ralstonia solanacearum, strain SB1 inhibited the growth of many other plant pathogens, including Fusarium oxysporum, Botrytis cinerea, Phytoph...

  4. Effect of plant essential oils on Ralstonia solanacearum race 4 causing bacterial wilt of edible ginger

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Palmarosa (Cymbopogon martini), lemongrass (C. citratus) and eucalyptus (Eucalyptus globulus) oils were investigated for their effects on Ralstonia solanacearum race 4, and their potential use as bio-fumigants for treating pathogen- infested edible ginger (Zingiber officinale R.) fields. Three conce...

  5. Ralstonia solanacearum type III secretion system effector Rip36 induces a hypersensitive response in the nonhost wild eggplant Solanum torvum.

    PubMed

    Nahar, Kamrun; Matsumoto, Iyo; Taguchi, Fumiko; Inagaki, Yoshishige; Yamamoto, Mikihiro; Toyoda, Kazuhiro; Shiraishi, Tomonori; Ichinose, Yuki; Mukaihara, Takafumi

    2014-04-01

    Ralstonia solanacearum is a Gram-negative soil-borne bacterium that causes bacterial wilt disease in more than 200 plant species, including economically important Solanaceae species. In R. solanacearum, the hypersensitive response and pathogenicity (Hrp) type III secretion system is required for both the ability to induce the hypersensitive response (HR) in nonhost plants and pathogenicity in host plants. Recently, 72 effector genes, called rip (Ralstonia protein injected into plant cells), have been identified in R. solanacearum RS1000. RS1002, a spontaneous nalixidic acid-resistant derivative of RS1000, induced strong HR in the nonhost wild eggplant Solanum torvum in an Hrp-dependent manner. An Agrobacterium-mediated transient expression system revealed that Rip36, a putative Zn-dependent protease effector of R. solanacearum, induced HR in S. torvum. A mutation in the putative Zn-binding motif (E149A) completely abolished the ability to induce HR. In agreement with this result, the RS1002-derived Δrip36 and rip36E149A mutants lost the ability to induce HR in S. torvum. An E149A mutation had no effect on the translocation of Rip36 into plant cells. These results indicate that Rip36 is an avirulent factor that induces HR in S. torvum and that a putative Zn-dependent protease motif is essential for this activity.

  6. Evaluation of Resistance to Ralstonia solanacearum in Tomato Genetic Resources at Seedling Stage.

    PubMed

    Kim, Sang Gyu; Hur, On-Sook; Ro, Na-Young; Ko, Ho-Cheol; Rhee, Ju-Hee; Sung, Jung Sook; Ryu, Kyoung-Yul; Lee, Sok-Young; Baek, Hyung Jin

    2016-02-01

    Bacterial wilt of tomatoes caused by Ralstonia solanacearum is a devastating disease that limits the production of tomato in Korea. The best way to control this disease is using genetically resistant tomato plant. The resistance degree to R. solanacearum was evaluated for 285 tomato accessions conserved in the National Agrobiodiversity Center of Rural Development Administration. These accessions of tomato were originated from 23 countries. Disease severity of tomato accessions was investigated from 7 days to 14 days at an interval of 7 days after inoculation of R. solanacearum under greenhouse conditions. A total of 279 accessions of tomato germplasm were susceptible to R. solanacearum, resulting in wilt and death in 70 to 90% of these plants. Two tomato accessions were moderately resistant to R. solanacearum. Only four accessions showed high resistance against R. solanacearum. No distinct symptom of bacterial wilt appeared on the resistant tomato germplasms for up to 14 days after inoculation of R. solanacearum. Microscopy of resistant tomato stems infected with R. solanacearum revealed limited bacterial spread with thickening of pit membrane and gum production. Therefore, these four resistant tomato germplasms could be used in tomato breeding program against bacterial wilt. PMID:26889116

  7. Evaluation of Resistance to Ralstonia solanacearum in Tomato Genetic Resources at Seedling Stage

    PubMed Central

    Kim, Sang Gyu; Hur, On-Sook; Ro, Na-Young; Ko, Ho-Cheol; Rhee, Ju-Hee; Sung, Jung Sook; Ryu, Kyoung-Yul; Lee, Sok-Young; Baek, Hyung Jin

    2016-01-01

    Bacterial wilt of tomatoes caused by Ralstonia solanacearum is a devastating disease that limits the production of tomato in Korea. The best way to control this disease is using genetically resistant tomato plant. The resistance degree to R. solanacearum was evaluated for 285 tomato accessions conserved in the National Agrobiodiversity Center of Rural Development Administration. These accessions of tomato were originated from 23 countries. Disease severity of tomato accessions was investigated from 7 days to 14 days at an interval of 7 days after inoculation of R. solanacearum under greenhouse conditions. A total of 279 accessions of tomato germplasm were susceptible to R. solanacearum, resulting in wilt and death in 70 to 90% of these plants. Two tomato accessions were moderately resistant to R. solanacearum. Only four accessions showed high resistance against R. solanacearum. No distinct symptom of bacterial wilt appeared on the resistant tomato germplasms for up to 14 days after inoculation of R. solanacearum. Microscopy of resistant tomato stems infected with R. solanacearum revealed limited bacterial spread with thickening of pit membrane and gum production. Therefore, these four resistant tomato germplasms could be used in tomato breeding program against bacterial wilt. PMID:26889116

  8. Evaluation of Resistance to Ralstonia solanacearum in Tomato Genetic Resources at Seedling Stage.

    PubMed

    Kim, Sang Gyu; Hur, On-Sook; Ro, Na-Young; Ko, Ho-Cheol; Rhee, Ju-Hee; Sung, Jung Sook; Ryu, Kyoung-Yul; Lee, Sok-Young; Baek, Hyung Jin

    2016-02-01

    Bacterial wilt of tomatoes caused by Ralstonia solanacearum is a devastating disease that limits the production of tomato in Korea. The best way to control this disease is using genetically resistant tomato plant. The resistance degree to R. solanacearum was evaluated for 285 tomato accessions conserved in the National Agrobiodiversity Center of Rural Development Administration. These accessions of tomato were originated from 23 countries. Disease severity of tomato accessions was investigated from 7 days to 14 days at an interval of 7 days after inoculation of R. solanacearum under greenhouse conditions. A total of 279 accessions of tomato germplasm were susceptible to R. solanacearum, resulting in wilt and death in 70 to 90% of these plants. Two tomato accessions were moderately resistant to R. solanacearum. Only four accessions showed high resistance against R. solanacearum. No distinct symptom of bacterial wilt appeared on the resistant tomato germplasms for up to 14 days after inoculation of R. solanacearum. Microscopy of resistant tomato stems infected with R. solanacearum revealed limited bacterial spread with thickening of pit membrane and gum production. Therefore, these four resistant tomato germplasms could be used in tomato breeding program against bacterial wilt.

  9. Tropical strains of Ralstonia solanacearum Outcompete race 3 biovar 2 strains at lowland tropical temperatures.

    PubMed

    Huerta, Alejandra I; Milling, Annett; Allen, Caitilyn

    2015-05-15

    Bacterial wilt, caused by members of the heterogenous Ralstonia solanacearum species complex, is an economically important vascular disease affecting many crops. Human activity has widely disseminated R. solanacearum strains, increasing their global agricultural impact. However, tropical highland race 3 biovar 2 (R3bv2) strains do not cause disease in tropical lowlands, even though they are virulent at warm temperatures. We tested the hypothesis that differences in temperature adaptation and competitive fitness explain the uneven geographic distribution of R. solanacearum strains. Using three phylogenetically and ecologically distinct strains, we measured competitive fitness at two temperatures following paired-strain inoculations of their shared host, tomato. Lowland tropical strain GMI1000 was only weakly virulent on tomato under temperate conditions (24°C for day and 19°C for night [24/19°C]), but highland tropical R3bv2 strain UW551 and U.S. warm temperate strain K60 were highly virulent at both 24/19°C and 28°C. Strain K60 was significantly more competitive than both GMI1000 and UW551 in tomato rhizospheres and stems at 28°C, and GMI1000 also outcompeted UW551 at 28°C. The results were reversed at cooler temperatures, at which highland strain UW551 generally outcompeted GMI1000 and K60 in planta. The superior competitive index of UW551 at 24/19°C suggests that adaptation to cool temperatures could explain why only R3bv2 strains threaten highland agriculture. Strains K60 and GMI1000 each produced different bacteriocins that inhibited growth of UW551 in culture. Such interstrain inhibition could explain why R3bv2 strains do not cause disease in tropical lowlands.

  10. Tropical Strains of Ralstonia solanacearum Outcompete Race 3 Biovar 2 Strains at Lowland Tropical Temperatures

    PubMed Central

    Huerta, Alejandra I.; Milling, Annett

    2015-01-01

    Bacterial wilt, caused by members of the heterogenous Ralstonia solanacearum species complex, is an economically important vascular disease affecting many crops. Human activity has widely disseminated R. solanacearum strains, increasing their global agricultural impact. However, tropical highland race 3 biovar 2 (R3bv2) strains do not cause disease in tropical lowlands, even though they are virulent at warm temperatures. We tested the hypothesis that differences in temperature adaptation and competitive fitness explain the uneven geographic distribution of R. solanacearum strains. Using three phylogenetically and ecologically distinct strains, we measured competitive fitness at two temperatures following paired-strain inoculations of their shared host, tomato. Lowland tropical strain GMI1000 was only weakly virulent on tomato under temperate conditions (24°C for day and 19°C for night [24/19°C]), but highland tropical R3bv2 strain UW551 and U.S. warm temperate strain K60 were highly virulent at both 24/19°C and 28°C. Strain K60 was significantly more competitive than both GMI1000 and UW551 in tomato rhizospheres and stems at 28°C, and GMI1000 also outcompeted UW551 at 28°C. The results were reversed at cooler temperatures, at which highland strain UW551 generally outcompeted GMI1000 and K60 in planta. The superior competitive index of UW551 at 24/19°C suggests that adaptation to cool temperatures could explain why only R3bv2 strains threaten highland agriculture. Strains K60 and GMI1000 each produced different bacteriocins that inhibited growth of UW551 in culture. Such interstrain inhibition could explain why R3bv2 strains do not cause disease in tropical lowlands. PMID:25769835

  11. Functional assignment to positively selected sites in the core type III effector RipG7 from Ralstonia solanacearum.

    PubMed

    Wang, Keke; Remigi, Philippe; Anisimova, Maria; Lonjon, Fabien; Kars, Ilona; Kajava, Andrey; Li, Chien-Hui; Cheng, Chiu-Ping; Vailleau, Fabienne; Genin, Stéphane; Peeters, Nemo

    2016-05-01

    The soil-borne pathogen Ralstonia solanacearum causes bacterial wilt in a broad range of plants. The main virulence determinants of R. solanacearum are the type III secretion system (T3SS) and its associated type III effectors (T3Es), translocated into the host cells. Of the conserved T3Es among R. solanacearum strains, the Fbox protein RipG7 is required for R. solanacearum pathogenesis on Medicago truncatula. In this work, we describe the natural ripG7 variability existing in the R. solanacearum species complex. We show that eight representative ripG7 orthologues have different contributions to pathogenicity on M. truncatula: only ripG7 from Asian or African strains can complement the absence of ripG7 in GMI1000 (Asian reference strain). Nonetheless, RipG7 proteins from American and Indonesian strains can still interact with M. truncatula SKP1-like/MSKa protein, essential for the function of RipG7 in virulence. This indicates that the absence of complementation is most likely a result of the variability in the leucine-rich repeat (LRR) domain of RipG7. We identified 11 sites under positive selection in the LRR domains of RipG7. By studying the functional impact of these 11 sites, we show the contribution of five positively selected sites for the function of RipG7CMR15 in M. truncatula colonization. This work reveals the genetic and functional variation of the essential core T3E RipG7 from R. solanacearum. This analysis is the first of its kind on an essential disease-controlling T3E, and sheds light on the co-evolutionary arms race between the bacterium and its hosts.

  12. Draft Genome Sequence of Ralstonia solanacearum Strain Rs-T02, Which Represents the Most Prevalent Phylotype in Guangxi, China.

    PubMed

    Zou, Chengwu; Wang, Kaihao; Meng, Jiaorong; Yuan, Gaoqing; Lin, Wei; Peng, Haowen; Li, Qiqin

    2016-01-01

    Ralstonia solanacearumstrain Rs-T02 was originally isolated from a bacterial wilt of tomato plant in Nanning City of Guangxi Province, China. It represents the most prevalent phylotype in Guangxi. Here, we present the draft genome sequence of this strain, which comprises 5,225 genes and 5,976,011 nucleotides with an average G+C content of 66.79%. There are 968 different genes between this isolate and the previously reported genome sequence ofRalstonia solanacearumGMl l000 (race l, biovar 3, phylotype I), and the genome sequence information of this isolate may be useful for comparative genomic studies to determine the genetic diversity in this species. PMID:27081126

  13. 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.

  14. Transcriptome Analysis of Quantitative Resistance-Specific Response upon Ralstonia solanacearum Infection in Tomato

    PubMed Central

    Ishihara, Takeaki; Mitsuhara, Ichiro; Takahashi, Hideki; Nakaho, Kazuhiro

    2012-01-01

    Bacterial wilt, caused by the soil-borne bacterium Ralstonia solanacearum, is a lethal disease of tomato, but the molecular mechanisms of the host resistance responses to R. solanacearum remain unclear. In this study, we report the first work describing the transcriptome of cultivar resistance and susceptible tomato cultivar after inoculation with R. solanacearum. To elucidate the characteristics of resistance early in the interaction, we analyzed microarrays for resistant cultivar LS-89 and susceptible cultivar Ponderosa 1 day after stem inoculation. No change in gene expression was detected for Ponderosa, but expression levels of over 140 genes, including pathogenesis-related, hormone signaling and lignin biosynthesis genes, increased in LS-89. Expression of β-1,3-glucanase genes increased substantially. In an immunohistochemical study, glucanase in LS-89 accumulated in the xylem and pith tissues surrounding xylem vessels filled with R. solanacearum. The expression of these genes also increased in four other resistant cultivars, but changed little in four susceptible cultivars in response to R. solanacearum, suggesting that similar reactions occur in other cultivars. These gene expression profiles will serve as fundamental information to elucidate the molecular mechanisms in the resistance response to R. solanacearum in tomato. PMID:23071630

  15. Chemotaxis is required for virulence and competitive fitness of the bacterial wilt pathogen Ralstonia solanacearum.

    PubMed

    Yao, Jian; Allen, Caitilyn

    2006-05-01

    Ralstonia solanacearum, a soilborne plant pathogen of considerable economic importance, invades host plant roots from the soil. Qualitative and quantitative chemotaxis assays revealed that this bacterium is specifically attracted to diverse amino acids and organic acids, and especially to root exudates from the host plant tomato. Exudates from rice, a nonhost plant, were less attractive. Eight different strains from this heterogeneous species complex varied significantly in their attraction to a panel of carbohydrate stimuli, raising the possibility that chemotactic responses may be differentially selected traits that confer adaptation to various hosts or ecological conditions. Previous studies found that an aflagellate mutant lacking swimming motility is significantly reduced in virulence, but the role of directed motility mediated by the chemotaxis system was not known. Two site-directed R. solanacearum mutants lacking either CheA or CheW, which are core chemotaxis signal transduction proteins, were completely nonchemotactic but retained normal swimming motility. In biologically realistic soil soak virulence assays on tomato plants, both nonchemotactic mutants had significantly reduced virulence indistinguishable from that of a nonmotile mutant, demonstrating that directed motility, not simply random motion, is required for full virulence. In contrast, nontactic strains were as virulent as the wild-type strain was when bacteria were introduced directly into the plant stem through a cut petiole, indicating that taxis makes its contribution to virulence in the early stages of host invasion and colonization. When inoculated individually by soaking the soil, both nontactic mutants reached the same population sizes as the wild type did in the stems of tomato plants just beginning to wilt. However, when tomato plants were coinoculated with a 1:1 mixture of a nontactic mutant and its wild-type parent, the wild-type strain outcompeted both nontactic mutants by 100-fold

  16. Necessity of OxyR for the hydrogen peroxide stress response and full virulence in Ralstonia solanacearum.

    PubMed

    Flores-Cruz, Zomary; Allen, Caitilyn

    2011-09-01

    The plant pathogen Ralstonia solanacearum, which causes bacterial wilt disease, is exposed to reactive oxygen species (ROS) during tomato infection and expresses diverse oxidative stress response (OSR) genes during midstage disease on tomato. The R. solanacearum genome predicts that the bacterium produces multiple and redundant ROS-scavenging enzymes but only one known oxidative stress response regulator, OxyR. An R. solanacearum oxyR mutant had no detectable catalase activity, did not grow in the presence of 250 μM hydrogen peroxide, and grew poorly in the oxidative environment of solid rich media. This phenotype was rescued by the addition of exogenous catalase, suggesting that oxyR is essential for the hydrogen peroxide stress response. Unexpectedly, the oxyR mutant strain grew better than the wild type in the presence of the superoxide generator paraquat. Gene expression studies indicated that katE, kaG, ahpC1, grxC, and oxyR itself were each differentially expressed in the oxyR mutant background and in response to hydrogen peroxide, suggesting that oxyR is necessary for hydrogen peroxide-inducible gene expression. Additional OSR genes were differentially regulated in response to hydrogen peroxide alone. The virulence of the oxyR mutant strain was significantly reduced in both tomato and tobacco host plants, demonstrating that R. solanacearum is exposed to inhibitory concentrations of ROS in planta and that OxyR-mediated responses to ROS during plant pathogenesis are important for R. solanacearum host adaptation and virulence.

  17. Degradation of the Plant Defense Signal Salicylic Acid Protects Ralstonia solanacearum from Toxicity and Enhances Virulence on Tobacco

    PubMed Central

    Lowe-Power, Tiffany M.; Jacobs, Jonathan M.; Ailloud, Florent; Fochs, Brianna; Prior, Philippe

    2016-01-01

    ABSTRACT Plants use the signaling molecule salicylic acid (SA) to trigger defenses against diverse pathogens, including the bacterial wilt pathogen Ralstonia solanacearum. SA can also inhibit microbial growth. Most sequenced strains of the heterogeneous R. solanacearum species complex can degrade SA via gentisic acid to pyruvate and fumarate. R. solanacearum strain GMI1000 expresses this SA degradation pathway during tomato pathogenesis. Transcriptional analysis revealed that subinhibitory SA levels induced expression of the SA degradation pathway, toxin efflux pumps, and some general stress responses. Interestingly, SA treatment repressed expression of virulence factors, including the type III secretion system, suggesting that this pathogen may suppress virulence functions when stressed. A GMI1000 mutant lacking SA degradation activity was much more susceptible to SA toxicity but retained the wild-type colonization ability and virulence on tomato. This may be because SA is less important than gentisic acid in tomato defense signaling. However, another host, tobacco, responds strongly to SA. To test the hypothesis that SA degradation contributes to virulence on tobacco, we measured the effect of adding this pathway to the tobacco-pathogenic R. solanacearum strain K60, which lacks SA degradation genes. Ectopic addition of the GMI1000 SA degradation locus, including adjacent genes encoding two porins and a LysR-type transcriptional regulator, significantly increased the virulence of strain K60 on tobacco. Together, these results suggest that R. solanacearum degrades plant SA to protect itself from inhibitory levels of this compound and also to enhance its virulence on plant hosts like tobacco that use SA as a defense signal molecule. PMID:27329752

  18. New Insights into the Antibacterial Activity of Hydroxycoumarins against Ralstonia solanacearum.

    PubMed

    Yang, Liang; Ding, Wei; Xu, Yuquan; Wu, Dousheng; Li, Shili; Chen, Juanni; Guo, Bing

    2016-01-01

    Coumarins are important plant-derived natural products with wide-ranging bioactivities and extensive applications. In this study, we evaluated for the first time the antibacterial activity and mechanisms of action of coumarins against the phytopathogen Ralstonia solanacearum, and investigated the effect of functional group substitution. We first tested the antibacterial activity of 18 plant-derived coumarins with different substitution patterns, and found that daphnetin, esculetin, xanthotol, and umbelliferone significantly inhibited the growth of R. solanacearum. Daphnetin showed the strongest antibacterial activity, followed by esculetin and umbelliferone, with MICs of 64, 192, and 256 mg/L, respectively, better than the archetypal coumarin with 384 mg/L. We further demonstrated that the hydroxylation of coumarins at the C-6, C-7 or C-8 position significantly enhanced the antibacterial activity against R. solanacearum. Transmission electron microscope (TEM) and fluorescence microscopy images showed that hydroxycoumarins may interact with the pathogen by mechanically destroying the cell membrane and inhibiting biofilm formation. The antibiofilm effect of hydroxycoumarins may relate to the repression of flagellar genes fliA and flhC. These physiological changes in R. solanacearum caused by hydroxycoumarins can provide information for integral pathogen control. The present findings demonstrated that hydroxycoumarins have superior antibacterial activity against the phytopathogen R. solanacearum, and thus have the potential to be applied for controlling plant bacterial wilt. PMID:27070570

  19. New Insights into the Antibacterial Activity of Hydroxycoumarins against Ralstonia solanacearum.

    PubMed

    Yang, Liang; Ding, Wei; Xu, Yuquan; Wu, Dousheng; Li, Shili; Chen, Juanni; Guo, Bing

    2016-01-01

    Coumarins are important plant-derived natural products with wide-ranging bioactivities and extensive applications. In this study, we evaluated for the first time the antibacterial activity and mechanisms of action of coumarins against the phytopathogen Ralstonia solanacearum, and investigated the effect of functional group substitution. We first tested the antibacterial activity of 18 plant-derived coumarins with different substitution patterns, and found that daphnetin, esculetin, xanthotol, and umbelliferone significantly inhibited the growth of R. solanacearum. Daphnetin showed the strongest antibacterial activity, followed by esculetin and umbelliferone, with MICs of 64, 192, and 256 mg/L, respectively, better than the archetypal coumarin with 384 mg/L. We further demonstrated that the hydroxylation of coumarins at the C-6, C-7 or C-8 position significantly enhanced the antibacterial activity against R. solanacearum. Transmission electron microscope (TEM) and fluorescence microscopy images showed that hydroxycoumarins may interact with the pathogen by mechanically destroying the cell membrane and inhibiting biofilm formation. The antibiofilm effect of hydroxycoumarins may relate to the repression of flagellar genes fliA and flhC. These physiological changes in R. solanacearum caused by hydroxycoumarins can provide information for integral pathogen control. The present findings demonstrated that hydroxycoumarins have superior antibacterial activity against the phytopathogen R. solanacearum, and thus have the potential to be applied for controlling plant bacterial wilt.

  20. Potential Interactions between Salmonella enterica and Ralstonia solanacearum in tomato plants.

    PubMed

    Pollard, Stephanie; Barak, Jeri; Boyer, Renee; Reiter, Mark; Gu, Ganyu; Rideout, Steven

    2014-02-01

    Over the past decade, the Eastern Shore of Virginia (ESV) has been implicated in at least four outbreaks of salmonellosis associated with tomato, all originating from the same serovar, Salmonella enterica serovar Newport. In addition to Salmonella Newport contamination, the devastating plant disease bacterial wilt, caused by the phytopathogen Ralstonia solanacearum, threatens the sustainability of ESV tomato production. Bacterial wilt is present in most ESV tomato fields and causes devastating yield losses each year. Although the connection between bacterial wilt and tomato-related salmonellosis outbreaks in ESV is of interest, the relationship between the two pathogens has never been investigated. In this study, tomato plants were root dip inoculated with one of four treatments: (i) 8 log CFU of Salmonella Newport per ml, (ii) 5 log CFU of R. solanacearum per ml, (iii) a coinoculation of 8 log CFU of Salmonella Newport per ml plus 5 log CFU of R. solanacearum per ml, and (iv) sterile water as control. Leaf, stem, and fruit samples were collected at the early-green-fruit stage, and S. enterica contamination in the internal tissues was detected. S. enterica was recovered in 1.4 and 2.9% of leaf samples from plants inoculated with Salmonella Newport only and from plants coinoculated with Salmonella Newport plus R. solanacearum, respectively. S. enterica was recovered from 1.7 and 3.5% of fruit samples from plants inoculated with Salmonella Newport only and from plants coinoculated with Salmonella Newport plus R. solanacearum, respectively. There were significantly more stem samples from plants coinoculated with Salmonella Newport plus R. solanacearum that were positive for S. enterica (18.6%) than stem samples collected from plants inoculated with Salmonella Newport only (5.7%). Results suggested that R. solanacearum could influence S. enterica survival and transportation throughout the internal tissues of tomato plants.

  1. Ralstonia solanacearum Uses Inorganic Nitrogen Metabolism for Virulence, ATP Production, and Detoxification in the Oxygen-Limited Host Xylem Environment

    PubMed Central

    Dalsing, Beth L.; Truchon, Alicia N.; Gonzalez-Orta, Enid T.; Milling, Annett S.

    2015-01-01

    ABSTRACT Genomic data predict that, in addition to oxygen, the bacterial plant pathogen Ralstonia solanacearum can use nitrate (NO3−), nitrite (NO2−), nitric oxide (NO), and nitrous oxide (N2O) as terminal electron acceptors (TEAs). Genes encoding inorganic nitrogen reduction were highly expressed during tomato bacterial wilt disease, when the pathogen grows in xylem vessels. Direct measurements found that tomato xylem fluid was low in oxygen, especially in plants infected by R. solanacearum. Xylem fluid contained ~25 mM NO3−, corresponding to R. solanacearum’s optimal NO3− concentration for anaerobic growth in vitro. We tested the hypothesis that R. solanacearum uses inorganic nitrogen species to respire and grow during pathogenesis by making deletion mutants that each lacked a step in nitrate respiration (ΔnarG), denitrification (ΔaniA, ΔnorB, and ΔnosZ), or NO detoxification (ΔhmpX). The ΔnarG, ΔaniA, and ΔnorB mutants grew poorly on NO3− compared to the wild type, and they had reduced adenylate energy charge levels under anaerobiosis. While NarG-dependent NO3− respiration directly enhanced growth, AniA-dependent NO2− reduction did not. NO2− and NO inhibited growth in culture, and their removal depended on denitrification and NO detoxification. Thus, NO3− acts as a TEA, but the resulting NO2− and NO likely do not. None of the mutants grew as well as the wild type in planta, and strains lacking AniA (NO2− reductase) or HmpX (NO detoxification) had reduced virulence on tomato. Thus, R. solanacearum exploits host NO3− to respire, grow, and cause disease. Degradation of NO2− and NO is also important for successful infection and depends on denitrification and NO detoxification systems. PMID:25784703

  2. The Ectopic Expression of CaRop1 Modulates the Response of Tobacco Plants to Ralstonia solanacearum and Aphids.

    PubMed

    Qiu, Ailian; Liu, Zhiqin; Li, Jiazhi; Chen, Yanshen; Guan, Deyi; He, Shuilin

    2016-01-01

    In plants, Rho-related GTPases (Rops) are versatile molecular switches that regulate various biological processes, although their exact roles are not fully understood. Herein, we provide evidence that the ectopic expression of a Rop derived from Capsicum annuum, designated CaRop1, in tobacco plants modulates the response of these plants to Ralstonia solanacearum or aphid attack. The deduced amino acid sequence of CaRop1 harbors a conserved Rho domain and is highly homologous to Rops of other plant species. Transient expression of a CaRop1-GFP fusion protein in Nicotiana benthamiana leaf epidermal cells revealed localization of the GFP signal to the plasma membrane, cytoplasm, and nucleus. Overexpression (OE) of the wild-type CaRop1 or its dominant-negative mutant (DN-CaRop1) conferred substantial resistance to R. solanacearum infection and aphid attack, and this effect was accompanied by enhanced transcriptional expression of the hypersensitive-reaction marker gene HSR201; the jasmonic acid (JA)-responsive PR1b and LOX1; the insect resistance-associated NtPI-I, NtPI-II, and NtTPI; the ethylene (ET) production-associated NtACS1; and NPK1, a mitogen-activated protein kinase kinase kinase (MAPKKK) that interferes with N-, Bs2-, and Rx-mediated disease resistance. In contrast, OE of the constitutively active mutant of CaRop1(CA-CaRop1) enhanced susceptibility of the transgenic tobacco plants to R. solanacearum infection and aphid attack and downregulated or sustained the expression of HSR201, PR1b, NPK1, NtACS1, NtPI-I, NtPI-II, and NtTPI. These results collectively suggest that CaRop1 acts as a signaling switch in the crosstalk between Solanaceaes's response to R. solanacearum infection and aphid attack possibly via JA/ET-mediated signaling machinery. PMID:27551287

  3. The Ectopic Expression of CaRop1 Modulates the Response of Tobacco Plants to Ralstonia solanacearum and Aphids.

    PubMed

    Qiu, Ailian; Liu, Zhiqin; Li, Jiazhi; Chen, Yanshen; Guan, Deyi; He, Shuilin

    2016-01-01

    In plants, Rho-related GTPases (Rops) are versatile molecular switches that regulate various biological processes, although their exact roles are not fully understood. Herein, we provide evidence that the ectopic expression of a Rop derived from Capsicum annuum, designated CaRop1, in tobacco plants modulates the response of these plants to Ralstonia solanacearum or aphid attack. The deduced amino acid sequence of CaRop1 harbors a conserved Rho domain and is highly homologous to Rops of other plant species. Transient expression of a CaRop1-GFP fusion protein in Nicotiana benthamiana leaf epidermal cells revealed localization of the GFP signal to the plasma membrane, cytoplasm, and nucleus. Overexpression (OE) of the wild-type CaRop1 or its dominant-negative mutant (DN-CaRop1) conferred substantial resistance to R. solanacearum infection and aphid attack, and this effect was accompanied by enhanced transcriptional expression of the hypersensitive-reaction marker gene HSR201; the jasmonic acid (JA)-responsive PR1b and LOX1; the insect resistance-associated NtPI-I, NtPI-II, and NtTPI; the ethylene (ET) production-associated NtACS1; and NPK1, a mitogen-activated protein kinase kinase kinase (MAPKKK) that interferes with N-, Bs2-, and Rx-mediated disease resistance. In contrast, OE of the constitutively active mutant of CaRop1(CA-CaRop1) enhanced susceptibility of the transgenic tobacco plants to R. solanacearum infection and aphid attack and downregulated or sustained the expression of HSR201, PR1b, NPK1, NtACS1, NtPI-I, NtPI-II, and NtTPI. These results collectively suggest that CaRop1 acts as a signaling switch in the crosstalk between Solanaceaes's response to R. solanacearum infection and aphid attack possibly via JA/ET-mediated signaling machinery.

  4. The Ectopic Expression of CaRop1 Modulates the Response of Tobacco Plants to Ralstonia solanacearum and Aphids

    PubMed Central

    Qiu, Ailian; Liu, Zhiqin; Li, Jiazhi; Chen, Yanshen; Guan, Deyi; He, Shuilin

    2016-01-01

    In plants, Rho-related GTPases (Rops) are versatile molecular switches that regulate various biological processes, although their exact roles are not fully understood. Herein, we provide evidence that the ectopic expression of a Rop derived from Capsicum annuum, designated CaRop1, in tobacco plants modulates the response of these plants to Ralstonia solanacearum or aphid attack. The deduced amino acid sequence of CaRop1 harbors a conserved Rho domain and is highly homologous to Rops of other plant species. Transient expression of a CaRop1-GFP fusion protein in Nicotiana benthamiana leaf epidermal cells revealed localization of the GFP signal to the plasma membrane, cytoplasm, and nucleus. Overexpression (OE) of the wild-type CaRop1 or its dominant-negative mutant (DN-CaRop1) conferred substantial resistance to R. solanacearum infection and aphid attack, and this effect was accompanied by enhanced transcriptional expression of the hypersensitive-reaction marker gene HSR201; the jasmonic acid (JA)-responsive PR1b and LOX1; the insect resistance-associated NtPI-I, NtPI-II, and NtTPI; the ethylene (ET) production-associated NtACS1; and NPK1, a mitogen-activated protein kinase kinase kinase (MAPKKK) that interferes with N-, Bs2-, and Rx-mediated disease resistance. In contrast, OE of the constitutively active mutant of CaRop1(CA-CaRop1) enhanced susceptibility of the transgenic tobacco plants to R. solanacearum infection and aphid attack and downregulated or sustained the expression of HSR201, PR1b, NPK1, NtACS1, NtPI-I, NtPI-II, and NtTPI. These results collectively suggest that CaRop1 acts as a signaling switch in the crosstalk between Solanaceaes’s response to R. solanacearum infection and aphid attack possibly via JA/ET-mediated signaling machinery. PMID:27551287

  5. Oleanolic Acid Induces the Type III Secretion System of Ralstonia solanacearum.

    PubMed

    Wu, Dousheng; Ding, Wei; Zhang, Yong; Liu, Xuejiao; Yang, Liang

    2015-01-01

    Ralstonia solanacearum, the causal agent of bacterial wilt, can naturally infect a wide range of host plants. The type III secretion system (T3SS) is a major virulence determinant in this bacterium. Studies have shown that plant-derived compounds are able to inhibit or induce the T3SS in some plant pathogenic bacteria, though no specific T3SS inhibitor or inducer has yet been identified in R. solanacearum. In this study, a total of 50 different compounds were screened and almost half of them (22 of 50) significantly inhibited or induced the T3SS expression of R. solanacearum. Based on the strong induction activity on T3SS, the T3SS inducer oleanolic acid (OA) was chosen for further study. We found that OA induced the expression of T3SS through the HrpG-HrpB pathway. Some type III effector genes were induced in T3SS inducing medium supplemented with OA. In addition, OA targeted only the T3SS and did not affect other virulence determinants. Finally, we observed that induction of T3SS by OA accelerated disease progress on tobacco. Overall our results suggest that plant-derived compounds are an abundant source of R. solanacearum T3SS regulators, which could prove useful as tools to interrogate the regulation of this key virulence pathway. PMID:26732647

  6. Oleanolic Acid Induces the Type III Secretion System of Ralstonia solanacearum

    PubMed Central

    Wu, Dousheng; Ding, Wei; Zhang, Yong; Liu, Xuejiao; Yang, Liang

    2015-01-01

    Ralstonia solanacearum, the causal agent of bacterial wilt, can naturally infect a wide range of host plants. The type III secretion system (T3SS) is a major virulence determinant in this bacterium. Studies have shown that plant-derived compounds are able to inhibit or induce the T3SS in some plant pathogenic bacteria, though no specific T3SS inhibitor or inducer has yet been identified in R. solanacearum. In this study, a total of 50 different compounds were screened and almost half of them (22 of 50) significantly inhibited or induced the T3SS expression of R. solanacearum. Based on the strong induction activity on T3SS, the T3SS inducer oleanolic acid (OA) was chosen for further study. We found that OA induced the expression of T3SS through the HrpG-HrpB pathway. Some type III effector genes were induced in T3SS inducing medium supplemented with OA. In addition, OA targeted only the T3SS and did not affect other virulence determinants. Finally, we observed that induction of T3SS by OA accelerated disease progress on tobacco. Overall our results suggest that plant-derived compounds are an abundant source of R. solanacearum T3SS regulators, which could prove useful as tools to interrogate the regulation of this key virulence pathway. PMID:26732647

  7. Quantitative Immunofluorescence of Regulated eps Gene Expression in Single Cells of Ralstonia solanacearum

    PubMed Central

    Kang, Yaowei; Saile, Elke; Schell, Mark A.; Denny, Timothy P.

    1999-01-01

    Ralstonia solanacearum, a phytopathogenic bacterium, uses an environmentally sensitive and complex regulatory network to control expression of multiple virulence genes. Part of this network is an unusual autoregulatory system that produces and senses 3-hydroxypalmitic acid methyl ester. In culture, this autoregulatory system ensures that expression of virulence genes, such as those of the eps operon encoding biosynthesis of the acidic extracellular polysaccharide, occurs only at high cell density (>107 cells/ml). To determine if regulation follows a similar pattern within tomato plants, we first developed a quantitative immunofluorescence (QIF) method that measures the relative amount of a target protein within individual bacterial cells. For R. solanacearum, QIF was used to determine the amount of β-galactosidase protein within wild-type cells containing a stable eps-lacZ reporter allele. When cultured cells were examined to test the method, QIF accurately detected both low and high levels of eps gene expression. QIF analysis of R. solanacearum cells recovered from stems of infected tomato plants showed that expression of eps during pathogenesis was similar to that in culture. These results suggest that there are no special signals or conditions within plants that override or short-circuit the regulatory processes observed in R. solanacearum in culture. Because QIF is a robust, relatively simple procedure that uses generally accessible equipment, it should be useful in many situations where gene expression in single bacterial cells must be determined. PMID:10347013

  8. Opening the Ralstonia solanacearum type III effector tool box: insights into host cell subversion mechanisms.

    PubMed

    Deslandes, Laurent; Genin, Stephane

    2014-08-01

    Effectors delivered to host cells by the Type III secretion system are essential to Ralstonia solanacearum pathogenicity, as in several other plant pathogenic bacteria. The establishment of exhaustive effector repertoires in multiple R. solanacearum strains drew a first picture of the evolutionary dynamics of the pathogen effector suites. Effector repertoires are diversified, with a core of 20-30 effectors present in most of the strains and the obtention of mutants lacking one or more effector genes revealed the functional overlap among this effector network. Recent functional studies have provided insights into the ability of single effectors to manipulate the host proteasome, elicit cell death, trigger the expression of plant genes, and/or display biochemical activities on plant protein targets.

  9. Development and Comparison of TaqMan-Based Real-Time PCR Assays for Detection and Differentiation of Ralstonia solanacearum strains.

    PubMed

    Stulberg, Michael J; Rascoe, John; Li, Wenbin; Yan, Zonghe; Nakhla, Mark K; Huang, Qi

    2016-10-01

    Bacterial wilt caused by Ralstonia solanacearum is destructive to many plant species worldwide. The race 3 biovar 2 (r3b2) strains of R. solanacearum infect potatoes in temperate climates and are listed as select agents by the U.S. government. TaqMan-based real-time quantitative PCR (qPCR) is commonly used in federal and state diagnostic laboratories over conventional PCR due to its speed and sensitivity. We developed the Rs16S primers and probe set and compared it with a widely used set (RS) for detecting R. solanacearum species complex strains. We also developed the RsSA3 primers and probe set and compared it with the previously published B2 and RsSA2 sets for specific detection of r3b2 strains. Both comparisons were done under standardized qPCR master mix and cycling conditions. The Rs16S and RS assays detected all 90 R. solanacearum species complex strains and none of the five outgroups, but the former was more sensitive than the latter. For r3b2 strain detection, the RsSA2 and RsSA3 sets specifically detected the 34 r3b2 strains and none of the 56 R. solanacearum non-r3b2 strains or out-group strains. The B2 set, however, detected five non-r3b2 R. solanacearum strains and was less sensitive than the other two sets under the same testing conditions. We conclude that the Rs16S, RsSA2, and RsSA3 sets are best suited under the standardized conditions for the detection of R. solanacearum species complex and r3b2 strains by TaqMan-based qPCR assays. PMID:27402488

  10. Development and Comparison of TaqMan-Based Real-Time PCR Assays for Detection and Differentiation of Ralstonia solanacearum strains.

    PubMed

    Stulberg, Michael J; Rascoe, John; Li, Wenbin; Yan, Zonghe; Nakhla, Mark K; Huang, Qi

    2016-10-01

    Bacterial wilt caused by Ralstonia solanacearum is destructive to many plant species worldwide. The race 3 biovar 2 (r3b2) strains of R. solanacearum infect potatoes in temperate climates and are listed as select agents by the U.S. government. TaqMan-based real-time quantitative PCR (qPCR) is commonly used in federal and state diagnostic laboratories over conventional PCR due to its speed and sensitivity. We developed the Rs16S primers and probe set and compared it with a widely used set (RS) for detecting R. solanacearum species complex strains. We also developed the RsSA3 primers and probe set and compared it with the previously published B2 and RsSA2 sets for specific detection of r3b2 strains. Both comparisons were done under standardized qPCR master mix and cycling conditions. The Rs16S and RS assays detected all 90 R. solanacearum species complex strains and none of the five outgroups, but the former was more sensitive than the latter. For r3b2 strain detection, the RsSA2 and RsSA3 sets specifically detected the 34 r3b2 strains and none of the 56 R. solanacearum non-r3b2 strains or out-group strains. The B2 set, however, detected five non-r3b2 R. solanacearum strains and was less sensitive than the other two sets under the same testing conditions. We conclude that the Rs16S, RsSA2, and RsSA3 sets are best suited under the standardized conditions for the detection of R. solanacearum species complex and r3b2 strains by TaqMan-based qPCR assays.

  11. Antibacterial activity of Lansiumamide B to tobacco bacterial wilt (Ralstonia solanacearum).

    PubMed

    Li, Lichun; Feng, Xiujie; Tang, Ming; Hao, Wenbo; Han, Yun; Zhang, Guobin; Wan, Shuqing

    2014-01-01

    Tobacco bacterial wilt caused by Ralstonia solanacearum is one of the most serious diseases of tobacco in the area of tobacco cultivation. As there is no effective control method for tobacco bacterial wilt diseases, developing new antibacterial agents in tobacco will make great practical sense. The antibacterial activity against R. solanacearum of Lansiumamide B which is isolated from the seeds of Clausena lansium is reported in this paper for the first time. The bioassay results indicate that Lansiumamide B could completely inhibit the growth of R. solanacearum at the concentration of 125 mg/L in vitro, the EC50 and EC90 are 48.82 mg/L and 86.26 mg/L, respectively. The result of pot experiments indicates that the control efficiency of the Lansiumamide B on tobacco bacterial wilt are 95.84%, 91.67% and 86.38% at 7 days, 14 days and 21 days after treatment at the concentration of 100mg/kg, respectively, nearly 40 times higher than Streptomycin, a special fungicide to the disease, at 21 days after treatment with root irrigation method. These results suggest that Lansiumamide B has the potential of developing as a new type of plant-type fungicide on controlling the diseases of tobacco bacterial wilt.

  12. Elicitor-Induced Defense Responses in Solanum lycopersicum against Ralstonia solanacearum

    PubMed Central

    Kar, Itishree; Mukherjee, Arup K.; Acharya, Priyambada

    2013-01-01

    We investigated on important parameters of induced resistance in hydroponic tomato (Solanum lycopersicum) against Ralstonia solanacearum using the elicitors chitosan (CHT), salicylic acid (SA), and jasmonic acid (JA). The increase in total phenolic content of roots by the elicitors was significantly higher than control. Most pronounced increase in lignin synthesis was triggered by SA followed by CHT. At 24 h post-elicitation (hpe), the activity of phenylalanine ammonia lyase was 4.5 times higher than control elicited by CHT. The peroxidase activity was about 86 nkat/mg protein at 24 hpe in case of SA and 78 nkat/mg protein in case of CHT. The activity of polyphenol oxidase increased several folds by the elicitors. Cinnamyl alcohol dehydrogenase activity increased to the maximum at 48 hpe under the influence of CHT. The results indicate that the elicitors SA and CHT induced effective defense responses in tomato plants against R. solanacearum. This was evident from reduced vascular browning and wilting symptoms of tomato plants treated with SA and CHT and challenged subsequently with R. solanacearum. This reduced disease incidence in tomato by SA and CHT may be a result of cell wall strengthening through deposition of lignin and the coincident induction of defense enzymes. PMID:24187521

  13. Methyl 3-Hydroxymyristate, a Diffusible Signal Mediating phc Quorum Sensing in Ralstonia solanacearum.

    PubMed

    Kai, Kenji; Ohnishi, Hideyuki; Shimatani, Mika; Ishikawa, Shiho; Mori, Yuka; Kiba, Akinori; Ohnishi, Kouhei; Tabuchi, Mitsuaki; Hikichi, Yasufumi

    2015-11-01

    Ralstonia solanacearum, a plant pathogenic bacterium causing "bacterial wilt" on crops, uses a quorum sensing (QS) system consisting of phc regulatory elements to control its virulence. Methyl 3-hydroxypalmitate (3-OH PAME) was previously identified as the QS signal in strain AW1. However, 3-OH PAME has not been reportedly detected from any other strains, and this suggests that they produce another unknown QS signal. Here we identify (R)-methyl 3-hydroxymyristate [(R)-3-OH MAME] as a new QS signal that regulates the production of virulence factors and secondary metabolites. (R)-3-OH MAME was synthesized by the methyltransferase PhcB and sensed by the histidine kinase PhcS. The phylogenetic trees of these proteins from R. solanacearum strains were divided into two groups, according to their QS signal types--(R)-3-OH MAME or (R)-3-OH PAME. These results demonstrate that (R)-3-OH MAME is another crucial QS signal and highlight the unique evolution of QS systems in R. solanacearum.

  14. Phylogeny and population structure of brown rot- and Moko disease-causing strains of Ralstonia solanacearum phylotype II.

    PubMed

    Cellier, G; Remenant, B; Chiroleu, F; Lefeuvre, P; Prior, P

    2012-04-01

    The ancient soilborne plant vascular pathogen Ralstonia solanacearum has evolved and adapted to cause severe damage in an unusually wide range of plants. In order to better describe and understand these adaptations, strains with very similar lifestyles and host specializations are grouped into ecotypes. We used comparative genomic hybridization (CGH) to investigate three particular ecotypes in the American phylotype II group: (i) brown rot strains from phylotypes IIB-1 and IIB-2, historically known as race 3 biovar 2 and clonal; (ii) new pathogenic variants from phylotype IIB-4NPB that lack pathogenicity for banana but can infect many other plant species; and (iii) Moko disease-causing strains from phylotypes IIB-3, IIB-4, and IIA-6, historically known as race 2, that cause wilt on banana, plantain, and Heliconia spp. We compared the genomes of 72 R. solanacearum strains, mainly from the three major ecotypes of phylotype II, using a newly developed pangenomic microarray to decipher their population structure and gain clues about the epidemiology of these ecotypes. Strain phylogeny and population structure were reconstructed. The results revealed a phylogeographic structure within brown rot strains, allowing us to distinguish European outbreak strains of Andean and African origins. The pangenomic CGH data also demonstrated that Moko ecotype IIB-4 is phylogenetically distinct from the emerging IIB-4NPB strains. These findings improved our understanding of the epidemiology of important ecotypes in phylotype II and will be useful for evolutionary analyses and the development of new DNA-based diagnostic tools. PMID:22286995

  15. Phylogeny and Population Structure of Brown Rot- and Moko Disease-Causing Strains of Ralstonia solanacearum Phylotype II

    PubMed Central

    Remenant, B.; Chiroleu, F.; Lefeuvre, P.; Prior, P.

    2012-01-01

    The ancient soilborne plant vascular pathogen Ralstonia solanacearum has evolved and adapted to cause severe damage in an unusually wide range of plants. In order to better describe and understand these adaptations, strains with very similar lifestyles and host specializations are grouped into ecotypes. We used comparative genomic hybridization (CGH) to investigate three particular ecotypes in the American phylotype II group: (i) brown rot strains from phylotypes IIB-1 and IIB-2, historically known as race 3 biovar 2 and clonal; (ii) new pathogenic variants from phylotype IIB-4NPB that lack pathogenicity for banana but can infect many other plant species; and (iii) Moko disease-causing strains from phylotypes IIB-3, IIB-4, and IIA-6, historically known as race 2, that cause wilt on banana, plantain, and Heliconia spp. We compared the genomes of 72 R. solanacearum strains, mainly from the three major ecotypes of phylotype II, using a newly developed pangenomic microarray to decipher their population structure and gain clues about the epidemiology of these ecotypes. Strain phylogeny and population structure were reconstructed. The results revealed a phylogeographic structure within brown rot strains, allowing us to distinguish European outbreak strains of Andean and African origins. The pangenomic CGH data also demonstrated that Moko ecotype IIB-4 is phylogenetically distinct from the emerging IIB-4NPB strains. These findings improved our understanding of the epidemiology of important ecotypes in phylotype II and will be useful for evolutionary analyses and the development of new DNA-based diagnostic tools. PMID:22286995

  16. Moko Disease-Causing Strains of Ralstonia solanacearum from Brazil Extend Known Diversity in Paraphyletic Phylotype II.

    PubMed

    Albuquerque, Greecy M R; Santos, Liliana A; Felix, Kátia C S; Rollemberg, Christtianno L; Silva, Adriano M F; Souza, Elineide B; Cellier, Gilles; Prior, Philippe; Mariano, Rosa L R

    2014-11-01

    The epidemic situation of Moko disease-causing strains in Latin America and Brazil is unclear. Thirty-seven Ralstonia solanacearum strains from Brazil that cause the Moko disease on banana and heliconia plants were sampled and phylogenetically typed using the endoglucanase (egl) and DNA repair (mutS) genes according to the phylotype and sequevar classification. All of the strains belonged to phylotype II and a portion of the strains was typed as the Moko disease-related sequevars IIA-6 and IIA-24. Nevertheless, two unsuspected sequevars also harbored the Moko disease-causing strains IIA-41 and IIB-25, and a new sequevar was described and named IIA-53. All of the strains were pathogenic to banana and some of the strains of sequevars IIA-6, IIA-24, and IIA-41 were also pathogenic to tomato. The Moko disease-causing strains from sequevar IIB-25 were pathogenic to potato but not to tomato. These results highlight the high diversity of strains of Moko in Brazil, reinforce the efficiency of the egl gene to reveal relationships among these strains, and contribute to a better understanding of the diversity of paraphyletic Moko disease-causing strains of the R. solanacearum species complex, where the following seven distinct genetic clusters have been described: IIA-6, IIA-24, IIA-41, IIA-53, IIB-3, IIB-4, and IIB-25.

  17. Phylogeny and population structure of brown rot- and Moko disease-causing strains of Ralstonia solanacearum phylotype II.

    PubMed

    Cellier, G; Remenant, B; Chiroleu, F; Lefeuvre, P; Prior, P

    2012-04-01

    The ancient soilborne plant vascular pathogen Ralstonia solanacearum has evolved and adapted to cause severe damage in an unusually wide range of plants. In order to better describe and understand these adaptations, strains with very similar lifestyles and host specializations are grouped into ecotypes. We used comparative genomic hybridization (CGH) to investigate three particular ecotypes in the American phylotype II group: (i) brown rot strains from phylotypes IIB-1 and IIB-2, historically known as race 3 biovar 2 and clonal; (ii) new pathogenic variants from phylotype IIB-4NPB that lack pathogenicity for banana but can infect many other plant species; and (iii) Moko disease-causing strains from phylotypes IIB-3, IIB-4, and IIA-6, historically known as race 2, that cause wilt on banana, plantain, and Heliconia spp. We compared the genomes of 72 R. solanacearum strains, mainly from the three major ecotypes of phylotype II, using a newly developed pangenomic microarray to decipher their population structure and gain clues about the epidemiology of these ecotypes. Strain phylogeny and population structure were reconstructed. The results revealed a phylogeographic structure within brown rot strains, allowing us to distinguish European outbreak strains of Andean and African origins. The pangenomic CGH data also demonstrated that Moko ecotype IIB-4 is phylogenetically distinct from the emerging IIB-4NPB strains. These findings improved our understanding of the epidemiology of important ecotypes in phylotype II and will be useful for evolutionary analyses and the development of new DNA-based diagnostic tools.

  18. PopW of Ralstonia solanacearum, a new two-domain harpin targeting the plant cell wall.

    PubMed

    Li, Jian-Gang; Liu, Hong-Xia; Cao, Jing; Chen, Li-Feng; Gu, Chun; Allen, Caitilyn; Guo, Jian-Hua

    2010-05-01

    Harpins are extracellular glycine-rich proteins eliciting a hypersensitive response (HR). In this study, we identified a new harpin, PopW, from Ralstonia solanacearum strain ZJ3721. This 380-amino-acid protein is acidic, rich in glycine and serine, and lacks cysteine. When infiltrated into the leaves of tobacco (non-host), PopW induced a rapid tissue collapse via a heat-stable but protease-sensitive HR-eliciting activity. PopW has an N-terminal harpin domain (residues 1-159) and a C-terminal pectate lyase (PL) domain (residues 160-366); its HR-eliciting activity depends on its N-terminal domain. Analyses of subcellular localization and plasmolysis demonstrated that PopW targeted the onion cell wall. This was further confirmed by its ability to specifically bind to calcium pectate, a major component of the plant cell wall. However, PopW had no detectable PL activity. Western blotting revealed that PopW was secreted by the type III secretion system in an hrpB-dependent manner. Gene sequencing indicated that popW is conserved among 20 diverse strains of R. solanacearum. A popW-deficient mutant retained the ability of wild-type strain ZJ3721 to elicit HR in tobacco and to cause wilt disease in tomato (a host). We conclude that PopW is a new cell wall-associated, hrpB-dependent, two-domain harpin that is conserved across the R. solanacearum species complex. PMID:20447285

  19. Detection of Quorum Sensing Molecules and Biofilm Formation in Ralstonia solanacearum.

    PubMed

    Kumar, J Shiva; Umesha, S; Prasad, K Shiva; Niranjana, P

    2016-03-01

    Many bacteria use small diffusible signaling molecules to communicate each other termed as quorum sensing (QS). Most Gram-negative bacteria use acyl homoserine lactone (AHL) as QS signal molecules. Using these signaling molecules, bacteria are able to express specific genes in response to population density. This work aimed to detect the production of QS signal molecules and biofilm formation in Ralstonia solanacearum isolated from various diseased tomato plants with symptoms of bacterial wilt. A total of 30 R. solanacearum strains were investigated for the production of QS signal molecules using Chromobacterium violaceum CV026 and Agrobacterium tumefaciens NT1 (pZLR4) biosensor systems. All 30 bacterial isolates from various bacterial wilt-affected tomato plants produced AHL molecules that induced the biosensor. The microtiter plate assay demonstrated that of the 30 bacterial isolates, 60 % formed biofilm, among which four isolates exhibited a higher degree of biofilm formation. The biofilm-inducing factor was purified from these four culture supernatants. The structure of the responsible molecule was solved using nuclear magnetic resonance and mass spectroscopy and was determined to be 2-hydroxy-4-((methylamino)(phenyl)methyl) cyclopentanone (HMCP), which was confirmed by chemical synthesis and NMR. The Confocal laser scanning microscopic analysis showed well-developed biofilm architecture of bacteria when treated with HMCP. The knowledge we obtained from this study will be useful for further researcher on the role of HMCP molecule in biofilm formation. PMID:26620535

  20. Recent trends in control methods for bacterial wilt diseases caused by Ralstonia solanacearum.

    PubMed

    Yuliar; Nion, Yanetri Asi; Toyota, Koki

    2015-01-01

    Previous studies have described the development of control methods against bacterial wilt diseases caused by Ralstonia solanacearum. This review focused on recent advances in control measures, such as biological, physical, chemical, cultural, and integral measures, as well as biocontrol efficacy and suppression mechanisms. Biological control agents (BCAs) have been dominated by bacteria (90%) and fungi (10%). Avirulent strains of R. solanacearum, Pseudomonas spp., Bacillus spp., and Streptomyces spp. are well-known BCAs. New or uncommon BCAs have also been identified such as Acinetobacter sp., Burkholderia sp., and Paenibacillus sp. Inoculation methods for BCAs affect biocontrol efficacy, such as pouring or drenching soil, dipping of roots, and seed coatings. The amendment of different organic matter, such as plant residue, animal waste, and simple organic compounds, have frequently been reported to suppress bacterial wilt diseases. The combined application of BCAs and their substrates was shown to more effectively suppress bacterial wilt in the tomato. Suppression mechanisms are typically attributed to the antibacterial metabolites produced by BCAs or those present in natural products; however, the number of studies related to host resistance to the pathogen is increasing. Enhanced/modified soil microbial communities are also indirectly involved in disease suppression. New promising types of control measures include biological soil disinfection using substrates that release volatile compounds. This review described recent advances in different control measures. We focused on the importance of integrated pest management (IPM) for bacterial wilt diseases. PMID:25762345

  1. Towards the Identification of Type III Effectors Associated with Ralstonia solanacearum Virulence on Tomato and Eggplant.

    PubMed

    Pensec, Flora; Lebeau, Aurore; Daunay, M C; Chiroleu, Frédéric; Guidot, Alice; Wicker, Emmanuel

    2015-12-01

    For the development of pathogen-informed breeding strategies, identifying the microbial genes involved in interactions with the plant is a critical step. To identify type III effector (T3E) repertoires associated with virulence of the bacterial wilt pathogen Ralstonia solanacearum on Solanaceous crops, we used an original association genetics approach combining DNA microarray data and pathogenicity data on resistant eggplant, pepper, and tomato accessions. From this first screen, 25 T3Es were further full-length polymerase chain reaction-amplified within a 35-strain field collection, to assess their distribution and allelic diversity. Six T3E repertoire groups were identified, within which 11 representative strains were chosen to challenge the bacterial wilt-resistant egg plants 'Dingras multiple Purple' and 'AG91-25', and tomato Hawaii 7996. The virulence or avirulence phenotypes could not be explained by specific T3E repertoires, but rather by individual T3E genes. We identified seven highly avirulence-associated genes, among which ripP2, primarily referenced as conferring avirulence to Arabidopsis thaliana. Interestingly, no T3E was associated with avirulence to both egg-plants. Highly virulence-associated genes were also identified: ripA5_2, ripU, and ripV2. This study should be regarded as a first step toward investigating both avirulence and virulence function of the highlighted genes, but also their evolutionary dynamics in natural R. solanacearum populations. PMID:26368514

  2. Towards the Identification of Type III Effectors Associated with Ralstonia solanacearum Virulence on Tomato and Eggplant.

    PubMed

    Pensec, Flora; Lebeau, Aurore; Daunay, M C; Chiroleu, Frédéric; Guidot, Alice; Wicker, Emmanuel

    2015-12-01

    For the development of pathogen-informed breeding strategies, identifying the microbial genes involved in interactions with the plant is a critical step. To identify type III effector (T3E) repertoires associated with virulence of the bacterial wilt pathogen Ralstonia solanacearum on Solanaceous crops, we used an original association genetics approach combining DNA microarray data and pathogenicity data on resistant eggplant, pepper, and tomato accessions. From this first screen, 25 T3Es were further full-length polymerase chain reaction-amplified within a 35-strain field collection, to assess their distribution and allelic diversity. Six T3E repertoire groups were identified, within which 11 representative strains were chosen to challenge the bacterial wilt-resistant egg plants 'Dingras multiple Purple' and 'AG91-25', and tomato Hawaii 7996. The virulence or avirulence phenotypes could not be explained by specific T3E repertoires, but rather by individual T3E genes. We identified seven highly avirulence-associated genes, among which ripP2, primarily referenced as conferring avirulence to Arabidopsis thaliana. Interestingly, no T3E was associated with avirulence to both egg-plants. Highly virulence-associated genes were also identified: ripA5_2, ripU, and ripV2. This study should be regarded as a first step toward investigating both avirulence and virulence function of the highlighted genes, but also their evolutionary dynamics in natural R. solanacearum populations.

  3. Diversity of Ralstonia solanacearum in French Guiana expands knowledge of the "emerging ecotype".

    PubMed

    Deberdt, P; Guyot, J; Coranson-Beaudu, R; Launay, J; Noreskal, M; Rivière, P; Vigné, F; Laplace, D; Lebreton, L; Wicker, E

    2014-06-01

    Although bacterial wilt remains a major plant disease throughout South America and the Caribbean, the diversity of prevalent Ralstonia solanacearum populations is largely unknown. The genetic and phenotypic diversity of R. solanacearum strains in French Guiana was assessed using diagnostic polymerase chain reactions and sequence-based (egl and mutS) genotyping on a 239-strain collection sampled on the families Solanaceae and Cucurbitaceae, revealing an unexpectedly high diversity. Strains were distributed within phylotypes I (46.9%), IIA (26.8%), and IIB (26.3%), with one new endoglucanase sequence type (egl ST) found within each group. Phylotype IIB strains consisted mostly (97%) of strains with the emerging ecotype (IIB/sequevar 4NPB). Host range of IIB/4NPB strains from French Guiana matched the original emerging reference strain from Martinique. They were virulent on cucumber; virulent and highly aggressive on tomato, including the resistant reference Hawaii 7996; and only controlled by eggplant SM6 and Surya accessions. The emerging ecotype IIB/4NPB is fully established in French Guiana in both cultivated fields and uncultivated forest, rendering the hypothesis of introduction via ornamental or banana cuttings unlikely. Thus, this ecotype may have originated from the Amazonian region and spread throughout the Caribbean region.

  4. Recent trends in control methods for bacterial wilt diseases caused by Ralstonia solanacearum.

    PubMed

    Yuliar; Nion, Yanetri Asi; Toyota, Koki

    2015-01-01

    Previous studies have described the development of control methods against bacterial wilt diseases caused by Ralstonia solanacearum. This review focused on recent advances in control measures, such as biological, physical, chemical, cultural, and integral measures, as well as biocontrol efficacy and suppression mechanisms. Biological control agents (BCAs) have been dominated by bacteria (90%) and fungi (10%). Avirulent strains of R. solanacearum, Pseudomonas spp., Bacillus spp., and Streptomyces spp. are well-known BCAs. New or uncommon BCAs have also been identified such as Acinetobacter sp., Burkholderia sp., and Paenibacillus sp. Inoculation methods for BCAs affect biocontrol efficacy, such as pouring or drenching soil, dipping of roots, and seed coatings. The amendment of different organic matter, such as plant residue, animal waste, and simple organic compounds, have frequently been reported to suppress bacterial wilt diseases. The combined application of BCAs and their substrates was shown to more effectively suppress bacterial wilt in the tomato. Suppression mechanisms are typically attributed to the antibacterial metabolites produced by BCAs or those present in natural products; however, the number of studies related to host resistance to the pathogen is increasing. Enhanced/modified soil microbial communities are also indirectly involved in disease suppression. New promising types of control measures include biological soil disinfection using substrates that release volatile compounds. This review described recent advances in different control measures. We focused on the importance of integrated pest management (IPM) for bacterial wilt diseases.

  5. Recent Trends in Control Methods for Bacterial Wilt Diseases Caused by Ralstonia solanacearum

    PubMed Central

    Yuliar; Nion, Yanetri Asi; Toyota, Koki

    2015-01-01

    Previous studies have described the development of control methods against bacterial wilt diseases caused by Ralstonia solanacearum. This review focused on recent advances in control measures, such as biological, physical, chemical, cultural, and integral measures, as well as biocontrol efficacy and suppression mechanisms. Biological control agents (BCAs) have been dominated by bacteria (90%) and fungi (10%). Avirulent strains of R. solanacearum, Pseudomonas spp., Bacillus spp., and Streptomyces spp. are well-known BCAs. New or uncommon BCAs have also been identified such as Acinetobacter sp., Burkholderia sp., and Paenibacillus sp. Inoculation methods for BCAs affect biocontrol efficacy, such as pouring or drenching soil, dipping of roots, and seed coatings. The amendment of different organic matter, such as plant residue, animal waste, and simple organic compounds, have frequently been reported to suppress bacterial wilt diseases. The combined application of BCAs and their substrates was shown to more effectively suppress bacterial wilt in the tomato. Suppression mechanisms are typically attributed to the antibacterial metabolites produced by BCAs or those present in natural products; however, the number of studies related to host resistance to the pathogen is increasing. Enhanced/modified soil microbial communities are also indirectly involved in disease suppression. New promising types of control measures include biological soil disinfection using substrates that release volatile compounds. This review described recent advances in different control measures. We focused on the importance of integrated pest management (IPM) for bacterial wilt diseases. PMID:25762345

  6. In planta comparative transcriptomics of host-adapted strains of Ralstonia solanacearum

    PubMed Central

    Ailloud, Florent; Lowe, Tiffany M.; Robène, Isabelle; Cruveiller, Stéphane; Allen, Caitilyn

    2016-01-01

    Background. Ralstonia solanacearum is an economically important plant pathogen with an unusually large host range. The Moko (banana) and NPB (not pathogenic to banana) strain groups are closely related but are adapted to distinct hosts. Previous comparative genomics studies uncovered very few differences that could account for the host range difference between these pathotypes. To better understand the basis of this host specificity, we used RNAseq to profile the transcriptomes of an R. solanacearum Moko strain and an NPB strain under in vitro and in planta conditions. Results. RNAs were sequenced from bacteria grown in rich and minimal media, and from bacteria extracted from mid-stage infected tomato, banana and melon plants. We computed differential expression between each pair of conditions to identify constitutive and host-specific gene expression differences between Moko and NPB. We found that type III secreted effectors were globally up-regulated upon plant cell contact in the NPB strain compared with the Moko strain. Genes encoding siderophore biosynthesis and nitrogen assimilation genes were highly up-regulated in the NPB strain during melon pathogenesis, while denitrification genes were up-regulated in the Moko strain during banana pathogenesis. The relatively lower expression of oxidases and the denitrification pathway during banana pathogenesis suggests that R. solanacearum experiences higher oxygen levels in banana pseudostems than in tomato or melon xylem. Conclusions. This study provides the first report of differential gene expression associated with host range variation. Despite minimal genomic divergence, the pathogenesis of Moko and NPB strains is characterized by striking differences in expression of virulence- and metabolism-related genes. PMID:26788428

  7. S-glycoprotein-like protein regulates defense responses in Nicotiana plants against Ralstonia solanacearum.

    PubMed

    Maimbo, Milimo; Ohnishi, Kouhei; Hikichi, Yasufumi; Yoshioka, Hirofumi; Kiba, Akinori

    2010-04-01

    RsRGA4 (for Ralstonia solanacearum-responsive gene A4) encodes a polypeptide similar to S-locus glycoprotein (SGP) from Brassica rapa and SGP-like proteins from Ipomoea trifida and Medicago truncatula. Therefore, we designated RsRGA4 as NtSGLP (for Nicotiana tabacum SGP-like protein) and NbSGLP (its Nicotiana benthamiana ortholog). NbSGLP is expressed in root, leaf, petal, gynoecium, and stamen. Expression of NbSGLP was strongly induced by inoculation with an avirulent strain of R. solanacearum (Rs8107) and slightly enhanced by inoculation with virulent R. solanacearum (RsOE1-1). Expression of NbSGLP was induced by inoculation with an hrpY-deficient mutant of RsOE1-1 and Rs8107. Expression was also induced by aminocyclopropane carboxylic acid and salicylic acid. Virus-induced gene silencing of NbSGLP enhanced the growth of Rs8107. Growth of RsOE1-1 and appearance of wilt symptoms were also accelerated in silenced plants. Expression of PR-1a and EREBP was reduced, and markers for basal defense, such as callose deposition and reduced vascular flow, were compromised in NbSGLP-silenced plants. Moreover, growth of Pseudomonas cichorii, Pseudomonas syringae pv tabaci, and P. syringae pv mellea was also enhanced in the silenced plants. On the other hand, silencing of NbSGLP did not interfere with the appearance of the hypersensitive response. NbSGLP was secreted in a signal peptide-dependent manner. Agrobacterium tumefaciens-mediated expression of NbSGLP induced PR-1a and EREBP expression, callose deposition, and reduced vascular flow. NbSGLP-induced callose deposition and reduced vascular flow were not observed in salicylic acid-deficient N. benthamiana NahG plants. Taken together, SGLP might have a role in the induction of basal defense in Nicotiana plants.

  8. In planta comparative transcriptomics of host-adapted strains of Ralstonia solanacearum.

    PubMed

    Ailloud, Florent; Lowe, Tiffany M; Robène, Isabelle; Cruveiller, Stéphane; Allen, Caitilyn; Prior, Philippe

    2016-01-01

    Background. Ralstonia solanacearum is an economically important plant pathogen with an unusually large host range. The Moko (banana) and NPB (not pathogenic to banana) strain groups are closely related but are adapted to distinct hosts. Previous comparative genomics studies uncovered very few differences that could account for the host range difference between these pathotypes. To better understand the basis of this host specificity, we used RNAseq to profile the transcriptomes of an R. solanacearum Moko strain and an NPB strain under in vitro and in planta conditions. Results. RNAs were sequenced from bacteria grown in rich and minimal media, and from bacteria extracted from mid-stage infected tomato, banana and melon plants. We computed differential expression between each pair of conditions to identify constitutive and host-specific gene expression differences between Moko and NPB. We found that type III secreted effectors were globally up-regulated upon plant cell contact in the NPB strain compared with the Moko strain. Genes encoding siderophore biosynthesis and nitrogen assimilation genes were highly up-regulated in the NPB strain during melon pathogenesis, while denitrification genes were up-regulated in the Moko strain during banana pathogenesis. The relatively lower expression of oxidases and the denitrification pathway during banana pathogenesis suggests that R. solanacearum experiences higher oxygen levels in banana pseudostems than in tomato or melon xylem. Conclusions. This study provides the first report of differential gene expression associated with host range variation. Despite minimal genomic divergence, the pathogenesis of Moko and NPB strains is characterized by striking differences in expression of virulence- and metabolism-related genes. PMID:26788428

  9. S-glycoprotein-like protein regulates defense responses in Nicotiana plants against Ralstonia solanacearum.

    PubMed

    Maimbo, Milimo; Ohnishi, Kouhei; Hikichi, Yasufumi; Yoshioka, Hirofumi; Kiba, Akinori

    2010-04-01

    RsRGA4 (for Ralstonia solanacearum-responsive gene A4) encodes a polypeptide similar to S-locus glycoprotein (SGP) from Brassica rapa and SGP-like proteins from Ipomoea trifida and Medicago truncatula. Therefore, we designated RsRGA4 as NtSGLP (for Nicotiana tabacum SGP-like protein) and NbSGLP (its Nicotiana benthamiana ortholog). NbSGLP is expressed in root, leaf, petal, gynoecium, and stamen. Expression of NbSGLP was strongly induced by inoculation with an avirulent strain of R. solanacearum (Rs8107) and slightly enhanced by inoculation with virulent R. solanacearum (RsOE1-1). Expression of NbSGLP was induced by inoculation with an hrpY-deficient mutant of RsOE1-1 and Rs8107. Expression was also induced by aminocyclopropane carboxylic acid and salicylic acid. Virus-induced gene silencing of NbSGLP enhanced the growth of Rs8107. Growth of RsOE1-1 and appearance of wilt symptoms were also accelerated in silenced plants. Expression of PR-1a and EREBP was reduced, and markers for basal defense, such as callose deposition and reduced vascular flow, were compromised in NbSGLP-silenced plants. Moreover, growth of Pseudomonas cichorii, Pseudomonas syringae pv tabaci, and P. syringae pv mellea was also enhanced in the silenced plants. On the other hand, silencing of NbSGLP did not interfere with the appearance of the hypersensitive response. NbSGLP was secreted in a signal peptide-dependent manner. Agrobacterium tumefaciens-mediated expression of NbSGLP induced PR-1a and EREBP expression, callose deposition, and reduced vascular flow. NbSGLP-induced callose deposition and reduced vascular flow were not observed in salicylic acid-deficient N. benthamiana NahG plants. Taken together, SGLP might have a role in the induction of basal defense in Nicotiana plants. PMID:20118275

  10. Nitrate Assimilation Contributes to Ralstonia solanacearum Root Attachment, Stem Colonization, and Virulence

    PubMed Central

    Dalsing, Beth L.

    2014-01-01

    Ralstonia solanacearum, an economically important plant pathogen, must attach, grow, and produce virulence factors to colonize plant xylem vessels and cause disease. Little is known about the bacterial metabolism that drives these processes. Nitrate is present in both tomato xylem fluid and agricultural soils, and the bacterium's gene expression profile suggests that it assimilates nitrate during pathogenesis. A nasA mutant, which lacks the gene encoding the catalytic subunit of R. solanacearum's sole assimilatory nitrate reductase, did not grow on nitrate as a sole nitrogen source. This nasA mutant exhibited reduced virulence and delayed stem colonization after soil soak inoculation of tomato plants. The nasA virulence defect was more severe following a period of soil survival between hosts. Unexpectedly, once bacteria reached xylem tissue, nitrate assimilation was dispensable for growth, virulence, and competitive fitness. However, nasA-dependent nitrate assimilation was required for normal production of extracellular polysaccharide (EPS), a major virulence factor. Quantitative analyses revealed that EPS production was significantly influenced by nitrate assimilation when nitrate was not required for growth. The plant colonization delay of the nasA mutant was externally complemented by coinoculation with wild-type bacteria but not by coinoculation with an EPS-deficient epsB mutant. The nasA mutant and epsB mutant did not attach to tomato roots as well as wild-type strain UW551. However, adding either wild-type cells or cell-free EPS improved the root attachment of these mutants. These data collectively suggest that nitrate assimilation promotes R. solanacearum virulence by enhancing root attachment, the initial stage of infection, possibly by modulating EPS production. PMID:24363343

  11. Multihost experimental evolution of the pathogen Ralstonia solanacearum unveils genes involved in adaptation to plants.

    PubMed

    Guidot, Alice; Jiang, Wei; Ferdy, Jean-Baptiste; Thébaud, Christophe; Barberis, Patrick; Gouzy, Jérôme; Genin, Stéphane

    2014-11-01

    Ralstonia solanacearum, the causal agent of a lethal bacterial wilt plant disease, infects an unusually wide range of hosts. These hosts can further be split into plants where R. solanacearum is known to cause disease (original hosts) and those where this bacterium can grow asymptomatically (distant hosts). Moreover, this pathogen is able to adapt to many plants as supported by field observations reporting emergence of strains with enlarged pathogenic properties. To investigate the genetic bases of host adaptation, we conducted evolution experiments by serial passages of a single clone of the pathogen on three original and two distant hosts over 300 bacterial generations and then analyzed the whole-genome of nine evolved clones. Phenotypic analysis of the evolved clones showed that the pathogen can increase its fitness on both original and distant hosts although the magnitude of fitness increase was greater on distant hosts. Only few genomic modifications were detected in evolved clones compared with the ancestor but parallel evolutionary changes in two genes were observed in independent evolved populations. Independent mutations in the regulatory gene efpR were selected for in three populations evolved on beans, a distant host. Reverse genetic approaches confirmed that these mutations were associated with fitness gain on bean plants. This work provides a first step toward understanding the within-host evolutionary dynamics of R. solanacearum during infection and identifying bacterial genes subjected to in planta selection. The discovery of EfpR as a determinant conditioning host adaptation of the pathogen illustrates how experimental evolution coupled with whole-genome sequencing is a potent tool to identify novel molecular players involved in central life-history traits.

  12. In planta comparative transcriptomics of host-adapted strains of Ralstonia solanacearum.

    PubMed

    Ailloud, Florent; Lowe, Tiffany M; Robène, Isabelle; Cruveiller, Stéphane; Allen, Caitilyn; Prior, Philippe

    2016-01-01

    Background. Ralstonia solanacearum is an economically important plant pathogen with an unusually large host range. The Moko (banana) and NPB (not pathogenic to banana) strain groups are closely related but are adapted to distinct hosts. Previous comparative genomics studies uncovered very few differences that could account for the host range difference between these pathotypes. To better understand the basis of this host specificity, we used RNAseq to profile the transcriptomes of an R. solanacearum Moko strain and an NPB strain under in vitro and in planta conditions. Results. RNAs were sequenced from bacteria grown in rich and minimal media, and from bacteria extracted from mid-stage infected tomato, banana and melon plants. We computed differential expression between each pair of conditions to identify constitutive and host-specific gene expression differences between Moko and NPB. We found that type III secreted effectors were globally up-regulated upon plant cell contact in the NPB strain compared with the Moko strain. Genes encoding siderophore biosynthesis and nitrogen assimilation genes were highly up-regulated in the NPB strain during melon pathogenesis, while denitrification genes were up-regulated in the Moko strain during banana pathogenesis. The relatively lower expression of oxidases and the denitrification pathway during banana pathogenesis suggests that R. solanacearum experiences higher oxygen levels in banana pseudostems than in tomato or melon xylem. Conclusions. This study provides the first report of differential gene expression associated with host range variation. Despite minimal genomic divergence, the pathogenesis of Moko and NPB strains is characterized by striking differences in expression of virulence- and metabolism-related genes.

  13. Nitrate assimilation contributes to Ralstonia solanacearum root attachment, stem colonization, and virulence.

    PubMed

    Dalsing, Beth L; Allen, Caitilyn

    2014-03-01

    Ralstonia solanacearum, an economically important plant pathogen, must attach, grow, and produce virulence factors to colonize plant xylem vessels and cause disease. Little is known about the bacterial metabolism that drives these processes. Nitrate is present in both tomato xylem fluid and agricultural soils, and the bacterium's gene expression profile suggests that it assimilates nitrate during pathogenesis. A nasA mutant, which lacks the gene encoding the catalytic subunit of R. solanacearum's sole assimilatory nitrate reductase, did not grow on nitrate as a sole nitrogen source. This nasA mutant exhibited reduced virulence and delayed stem colonization after soil soak inoculation of tomato plants. The nasA virulence defect was more severe following a period of soil survival between hosts. Unexpectedly, once bacteria reached xylem tissue, nitrate assimilation was dispensable for growth, virulence, and competitive fitness. However, nasA-dependent nitrate assimilation was required for normal production of extracellular polysaccharide (EPS), a major virulence factor. Quantitative analyses revealed that EPS production was significantly influenced by nitrate assimilation when nitrate was not required for growth. The plant colonization delay of the nasA mutant was externally complemented by coinoculation with wild-type bacteria but not by coinoculation with an EPS-deficient epsB mutant. The nasA mutant and epsB mutant did not attach to tomato roots as well as wild-type strain UW551. However, adding either wild-type cells or cell-free EPS improved the root attachment of these mutants. These data collectively suggest that nitrate assimilation promotes R. solanacearum virulence by enhancing root attachment, the initial stage of infection, possibly by modulating EPS production.

  14. 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.

  15. Volatile organic compounds produced by Pseudomonas fluorescens WR-1 restrict the growth and virulence traits of Ralstonia solanacearum.

    PubMed

    Raza, Waseem; Ling, Ning; Liu, Dongyang; Wei, Zhong; Huang, Qiwei; Shen, Qirong

    2016-11-01

    The volatile organic compounds (VOCs) produced by soil microbes have a significant role in the control of plant diseases and plant growth promotion. In this study, we examined the effect of VOCs produced by Pseudomonas fluorescens strain WR-1 on the growth and virulence traits of tomato wilt pathogen Ralstonia solanacearum. The VOCs produced by P. fluorescens WR-1 exhibited concentration dependent bacteriostatic effect on the growth of R. solanacearum on agar medium and in infested soil. The VOCs of P. fluorescens WR-1 also significantly inhibited the virulence traits of R. solanacearum. The proteomics analysis showed that the VOCs of P. fluorescens WR-1 downregulated cellular proteins of R. solanacearum related to the antioxidant activity, virulence, inclusion body proteins, carbohydrate and amino acid synthesis and metabolism, protein folding and translation, methylation and energy transfer, while the proteins involved in the ABC transporter system, detoxification of aldehydes and ketones, protein folding and translation were upregulated. This study revealed the significance of VOCs of P. fluorescens WR-1 to control the tomato wilt pathogen R. solanacearum. Investigation of the modes of action of biocontrol agents is important to better comprehend the interactions mediated by VOCs in nature to design better control strategies for plant pathogens. PMID:27664728

  16. 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

  17. 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).

  18. Volatile organic compounds produced by Pseudomonas fluorescens WR-1 restrict the growth and virulence traits of Ralstonia solanacearum.

    PubMed

    Raza, Waseem; Ling, Ning; Liu, Dongyang; Wei, Zhong; Huang, Qiwei; Shen, Qirong

    2016-11-01

    The volatile organic compounds (VOCs) produced by soil microbes have a significant role in the control of plant diseases and plant growth promotion. In this study, we examined the effect of VOCs produced by Pseudomonas fluorescens strain WR-1 on the growth and virulence traits of tomato wilt pathogen Ralstonia solanacearum. The VOCs produced by P. fluorescens WR-1 exhibited concentration dependent bacteriostatic effect on the growth of R. solanacearum on agar medium and in infested soil. The VOCs of P. fluorescens WR-1 also significantly inhibited the virulence traits of R. solanacearum. The proteomics analysis showed that the VOCs of P. fluorescens WR-1 downregulated cellular proteins of R. solanacearum related to the antioxidant activity, virulence, inclusion body proteins, carbohydrate and amino acid synthesis and metabolism, protein folding and translation, methylation and energy transfer, while the proteins involved in the ABC transporter system, detoxification of aldehydes and ketones, protein folding and translation were upregulated. This study revealed the significance of VOCs of P. fluorescens WR-1 to control the tomato wilt pathogen R. solanacearum. Investigation of the modes of action of biocontrol agents is important to better comprehend the interactions mediated by VOCs in nature to design better control strategies for plant pathogens.

  19. Comparative Transcriptome Analysis Reveals Cool Virulence Factors of Ralstonia solanacearum Race 3 Biovar 2.

    PubMed

    Meng, Fanhong; Babujee, Lavanya; Jacobs, Jonathan M; Allen, Caitilyn

    2015-01-01

    While most strains of the plant pathogenic bacterium Ralstonia solanacearum are tropical, the race 3 biovar 2 (R3bv2) subgroup attacks plants in cooler climates. To identify mechanisms underlying this trait, we compared the transcriptional profiles of R. solanacearum R3bv2 strain UW551 and tropical strain GMI1000 at 20°C and 28°C, both in culture and during tomato pathogenesis. 4.2% of the ORFs in the UW551 genome and 7.9% of the GMI1000 ORFs were differentially expressed by temperature in planta. The two strains had distinct transcriptional responses to temperature change. GMI1000 up-regulated several stress response genes at 20°C, apparently struggling to cope with plant defenses. At the cooler temperature, R3bv2 strain UW551 up-regulated a cluster encoding a mannose-fucose binding lectin, LecM; a quorum sensing-dependent protein, AidA; and a related hypothetical protein, AidC. The last two genes are absent from the GMI1000 genome. In UW551, all three genes were positively regulated by the adjacent SolI/R quorum sensing system. These temperature-responsive genes were required for full virulence in R3bv2. Mutants lacking lecM, aidA, or aidC were each significantly more reduced in virulence on tomato at 20°C than at 28°C in both a naturalistic soil soak inoculation assay and when they were inoculated directly into tomato stems. The lecM and aidC mutants also survived poorly in potato tubers at the seed tuber storage temperature of 4°C, and the lecM mutant was defective in biofilm formation in vitro. Together, these results suggest novel mechanisms, including a lectin, are involved in the unique temperate epidemiology of R3bv2. PMID:26445498

  20. Comparative Transcriptome Analysis Reveals Cool Virulence Factors of Ralstonia solanacearum Race 3 Biovar 2

    PubMed Central

    Meng, Fanhong; Babujee, Lavanya; Jacobs, Jonathan M.; Allen, Caitilyn

    2015-01-01

    While most strains of the plant pathogenic bacterium Ralstonia solanacearum are tropical, the race 3 biovar 2 (R3bv2) subgroup attacks plants in cooler climates. To identify mechanisms underlying this trait, we compared the transcriptional profiles of R. solanacearum R3bv2 strain UW551 and tropical strain GMI1000 at 20°C and 28°C, both in culture and during tomato pathogenesis. 4.2% of the ORFs in the UW551 genome and 7.9% of the GMI1000 ORFs were differentially expressed by temperature in planta. The two strains had distinct transcriptional responses to temperature change. GMI1000 up-regulated several stress response genes at 20°C, apparently struggling to cope with plant defenses. At the cooler temperature, R3bv2 strain UW551 up-regulated a cluster encoding a mannose-fucose binding lectin, LecM; a quorum sensing-dependent protein, AidA; and a related hypothetical protein, AidC. The last two genes are absent from the GMI1000 genome. In UW551, all three genes were positively regulated by the adjacent SolI/R quorum sensing system. These temperature-responsive genes were required for full virulence in R3bv2. Mutants lacking lecM, aidA, or aidC were each significantly more reduced in virulence on tomato at 20°C than at 28°C in both a naturalistic soil soak inoculation assay and when they were inoculated directly into tomato stems. The lecM and aidC mutants also survived poorly in potato tubers at the seed tuber storage temperature of 4°C, and the lecM mutant was defective in biofilm formation in vitro. Together, these results suggest novel mechanisms, including a lectin, are involved in the unique temperate epidemiology of R3bv2. PMID:26445498

  1. Comparative Transcriptome Analysis Reveals Cool Virulence Factors of Ralstonia solanacearum Race 3 Biovar 2.

    PubMed

    Meng, Fanhong; Babujee, Lavanya; Jacobs, Jonathan M; Allen, Caitilyn

    2015-01-01

    While most strains of the plant pathogenic bacterium Ralstonia solanacearum are tropical, the race 3 biovar 2 (R3bv2) subgroup attacks plants in cooler climates. To identify mechanisms underlying this trait, we compared the transcriptional profiles of R. solanacearum R3bv2 strain UW551 and tropical strain GMI1000 at 20°C and 28°C, both in culture and during tomato pathogenesis. 4.2% of the ORFs in the UW551 genome and 7.9% of the GMI1000 ORFs were differentially expressed by temperature in planta. The two strains had distinct transcriptional responses to temperature change. GMI1000 up-regulated several stress response genes at 20°C, apparently struggling to cope with plant defenses. At the cooler temperature, R3bv2 strain UW551 up-regulated a cluster encoding a mannose-fucose binding lectin, LecM; a quorum sensing-dependent protein, AidA; and a related hypothetical protein, AidC. The last two genes are absent from the GMI1000 genome. In UW551, all three genes were positively regulated by the adjacent SolI/R quorum sensing system. These temperature-responsive genes were required for full virulence in R3bv2. Mutants lacking lecM, aidA, or aidC were each significantly more reduced in virulence on tomato at 20°C than at 28°C in both a naturalistic soil soak inoculation assay and when they were inoculated directly into tomato stems. The lecM and aidC mutants also survived poorly in potato tubers at the seed tuber storage temperature of 4°C, and the lecM mutant was defective in biofilm formation in vitro. Together, these results suggest novel mechanisms, including a lectin, are involved in the unique temperate epidemiology of R3bv2.

  2. Characterization of biofumigated Ralstonia solanacearum cells using micro-Raman spectroscopy and electron microscopy.

    PubMed

    Paret, Mathews L; Sharma, Shiv K; Alvarez, Anne M

    2012-01-01

    Essential oils of palmarosa, lemongrass, and eucalyptus have shown promise as biofumigants for control of the bacterial wilt disease of edible ginger (Zingiber officinale) caused by Ralstonia solanacearum race 4 in previous potting medium studies. Biochemical changes in R. solanacearum cells were evaluated with micro-Raman spectroscopy following treatment with essential oils at different concentrations (0.04, 0.07, and 0.14% [vol/vol] of culture medium) and changes in cell structure were observed using electron microscopy. All treatments except palmarosa oil at 0.04% caused significant reductions in levels of amino acids, purine and pyrimidine bases of nucleic acids, carbohydrates, and lipids, as indicated by significant reduction in Raman peak heights at 621, 1,003, and 1,031 inverse centimeters (cm(-1)) (phenylalanine); 643, 827, 852, 1,158, and 1,172 cm(-1) (tyrosine); 758 cm(-1) (tryptophan); 725, 782, 1,337, and 1,578 cm(-1) (adenine, cytosine plus uracil, adenine, and adenine plus guanine, respectively); 1,097 cm(-1) (carbohydrates); and 1,127, 1,450, and 2,932 cm(-1) (lipids) compared with untreated controls. Lemongrass oil treatments were the most effective in degrading cellular components. Scanning electron microscopy of palmarosa and lemongrass-oil-treated cells showed rupture of cell walls and cell debris but no degradation was noted for eucalyptus-oil-treated cells. Palmarosa- and lemongrass-oil-treated cells were positively stained with uranyl acetate when viewed by transmission electron microscopy whereas controls and eucalyptus-oil-treated cells were negatively stained, indicating that the cell membranes were intact. The viability of eucalyptus-oil-treated cells was confirmed by cell culture following treatment. Micro-Raman spectroscopy is a powerful tool which can be further employed to better understand effects of fumigants and other bactericides on bacterial cells.

  3. Biocontrol of Ralstonia solanacearum by Treatment with Lytic Bacteriophages ▿ †

    PubMed Central

    Fujiwara, Akiko; Fujisawa, Mariko; Hamasaki, Ryosuke; Kawasaki, Takeru; Fujie, Makoto; Yamada, Takashi

    2011-01-01

    Ralstonia solanacearum is a Gram-negative bacterium and the causative agent of bacterial wilt in many important crops. We treated R. solanacearum with three lytic phages: φRSA1, φRSB1, and φRSL1. Infection with φRSA1 and φRSB1, either alone or in combination with the other phages, resulted in a rapid decrease in the host bacterial cell density. Cells that were resistant to infection by these phages became evident approximately 30 h after phage addition to the culture. On the other hand, cells infected solely with φRSL1 in a batch culture were maintained at a lower cell density (1/3 of control) over a long period. Pretreatment of tomato seedlings with φRSL1 drastically limited penetration, growth, and movement of root-inoculated bacterial cells. All φRSL1-treated tomato plants showed no symptoms of wilting during the experimental period, whereas all untreated plants had wilted by 18 days postinfection. φRSL1 was shown to be relatively stable in soil, especially at higher temperatures (37 to 50°C). Active φRSL1 particles were recovered from the roots of treated plants and from soil 4 months postinfection. Based on these observations, we propose an alternative biocontrol method using a unique phage, such as φRSL1, instead of a phage cocktail with highly virulent phages. Using this method, φRSL1 killed some but not all bacterial cells. The coexistence of bacterial cells and the phage resulted in effective prevention of wilting. PMID:21498752

  4. Proteome Analysis of Disease Resistance against Ralstonia solanacearum in Potato Cultivar CT206-10.

    PubMed

    Park, Sangryeol; Gupta, Ravi; Krishna, R; Kim, Sun Tae; Lee, Dong Yeol; Hwang, Duk-Ju; Bae, Shin-Chul; Ahn, Il-Pyung

    2016-02-01

    Potato is one of the most important crops worldwide. Its commercial cultivars are highly susceptible to many fungal and bacterial diseases. Among these, bacterial wilt caused by Ralstonia solanacearum causes significant yield loss. In the present study, integrated proteomics and genomics approaches were used in order to identify bacterial wilt resistant genes from Rs resistance potato cultivar CT-206-10. 2-DE and MALDI-TOF/TOF-MS analysis identified eight differentially abundant proteins including glycine-rich RNA binding protein (GRP), tomato stress induced-1 (TSI-1) protein, pathogenesis-related (STH-2) protein and pentatricopeptide repeat containing (PPR) protein in response to Rs infection. Further, semi-quantitative RT-PCR identified up-regulation in transcript levels of all these genes upon Rs infection. Taken together, our results showed the involvement of the identified proteins in the Rs stress tolerance in potato. In the future, it would be interesting to raise the transgenic plants to further validate their involvement in resistance against Rs in potato. PMID:26889112

  5. 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

  6. Proteome Analysis of Disease Resistance against Ralstonia solanacearum in Potato Cultivar CT206-10.

    PubMed

    Park, Sangryeol; Gupta, Ravi; Krishna, R; Kim, Sun Tae; Lee, Dong Yeol; Hwang, Duk-Ju; Bae, Shin-Chul; Ahn, Il-Pyung

    2016-02-01

    Potato is one of the most important crops worldwide. Its commercial cultivars are highly susceptible to many fungal and bacterial diseases. Among these, bacterial wilt caused by Ralstonia solanacearum causes significant yield loss. In the present study, integrated proteomics and genomics approaches were used in order to identify bacterial wilt resistant genes from Rs resistance potato cultivar CT-206-10. 2-DE and MALDI-TOF/TOF-MS analysis identified eight differentially abundant proteins including glycine-rich RNA binding protein (GRP), tomato stress induced-1 (TSI-1) protein, pathogenesis-related (STH-2) protein and pentatricopeptide repeat containing (PPR) protein in response to Rs infection. Further, semi-quantitative RT-PCR identified up-regulation in transcript levels of all these genes upon Rs infection. Taken together, our results showed the involvement of the identified proteins in the Rs stress tolerance in potato. In the future, it would be interesting to raise the transgenic plants to further validate their involvement in resistance against Rs in potato.

  7. 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.

  8. Proteome Analysis of Disease Resistance against Ralstonia solanacearum in Potato Cultivar CT206-10

    PubMed Central

    Park, Sangryeol; Gupta, Ravi; Krishna, R.; Kim, Sun Tae; Lee, Dong Yeol; Hwang, Duk-ju; Bae, Shin-Chul; Ahn, Il-Pyung

    2016-01-01

    Potato is one of the most important crops worldwide. Its commercial cultivars are highly susceptible to many fungal and bacterial diseases. Among these, bacterial wilt caused by Ralstonia solanacearum causes significant yield loss. In the present study, integrated proteomics and genomics approaches were used in order to identify bacterial wilt resistant genes from Rs resistance potato cultivar CT-206-10. 2-DE and MALDI-TOF/TOF-MS analysis identified eight differentially abundant proteins including glycine-rich RNA binding protein (GRP), tomato stress induced-1 (TSI-1) protein, pathogenesis-related (STH-2) protein and pentatricopeptide repeat containing (PPR) protein in response to Rs infection. Further, semi-quantitative RT-PCR identified up-regulation in transcript levels of all these genes upon Rs infection. Taken together, our results showed the involvement of the identified proteins in the Rs stress tolerance in potato. In the future, it would be interesting to raise the transgenic plants to further validate their involvement in resistance against Rs in potato. PMID:26889112

  9. A TaqMan-Based Multiplex qPCR Assay and DNA Extraction Method for Phylotype IIB Sequevars 1&2 (Select Agent) Strains of Ralstonia solanacearum

    PubMed Central

    Stulberg, Michael J.; Huang, Qi

    2015-01-01

    Ralstonia solanacearum race 3 biovar 2 strains belonging to phylotype IIB, sequevars 1 and 2 (IIB-1&2) cause brown rot of potato in temperate climates, and are quarantined pathogens in Canada and Europe. Since these strains are not established in the U.S. and because of their potential risk to the potato industry, the U.S. government has listed them as select agents. Cultivated geraniums are also a host and have the potential to spread the pathogen through trade, and its extracts strongly inhibits DNA-based detection methods. We designed four primer and probe sets for an improved qPCR method that targets stable regions of DNA. RsSA1 and RsSA2 recognize IIB-1&2 strains, RsII recognizes the current phylotype II (the newly proposed R. solanacearum species) strains (and a non-plant associated R. mannitolilytica), and Cox1 recognizes eight plant species including major hosts of R. solanacearum such as potato, tomato and cultivated geranium as an internal plant control. We multiplexed the RsSA2 with the RsII and Cox1 sets to provide two layers of detection of a positive IIB-1&2 sample, and to validate plant extracts and qPCR reactions. The TaqMan-based uniplex and multiplex qPCR assays correctly identified 34 IIB-1&2 and 52 phylotype II strains out of 90 R. solanacearum species complex strains. Additionally, the multiplex qPCR assay was validated successfully using 169 artificially inoculated symptomatic and asymptomatic plant samples from multiple plant hosts including geranium. Furthermore, we developed an extraction buffer that allowed for a quick and easy DNA extraction from infected plants including geranium for detection of R. solanacearum by qPCR. Our multiplex qPCR assay, especially when coupled with the quick extraction buffer method, allows for quick, easy and reliable detection and differentiation of the IIB-1&2 strains of R. solanacearum. PMID:26426354

  10. A TaqMan-based multiplex qPCR assay and DNA extraction method for phylotype IIB sequevars 1&2 (select agent) strains of Ralstonia solanacearum

    DOE PAGES

    Stulberg, Michael J.; Huang, Qi

    2015-10-01

    Ralstonia solanacearum race 3 biovar 2 strains belonging to phylotype IIB, sequevars 1 and 2 (IIB-1&2) cause brown rot of potato in temperate climates, and are quarantined pathogens in Canada and Europe. Since these strains are not established in the U.S. and because of their potential risk to the potato industry, the U.S. government has listed them as select agents. Cultivated geraniums are also a host and have the potential to spread the pathogen through trade, and its extracts strongly inhibits DNA-based detection methods. We designed four primer and probe sets for an improved qPCR method that targets stable regionsmore » of DNA. RsSA1 and RsSA2 recognize IIB-1&2 strains, RsII recognizes the current phylotype II (the newly proposed R. solanacearum species) strains (and a non-plant associated R. mannitolilytica), and Cox1 recognizes eight plant species including major hosts of R. solanacearum such as potato, tomato and cultivated geranium as an internal plant control. We multiplexed the RsSA2 with the RsII and Cox1 sets to provide two layers of detection of a positive IIB-1&2 sample, and to validate plant extracts and qPCR reactions. The TaqMan-based uniplex and multiplex qPCR assays correctly identified 34 IIB-1&2 and 52 phylotype II strains out of 90 R. solanacearum species complex strains. Additionally, the multiplex qPCR assay was validated successfully using 169 artificially inoculated symptomatic and asymptomatic plant samples from multiple plant hosts including geranium. Moreover, we developed an extraction buffer that allowed for a quick and easy DNA extraction from infected plants including geranium for detection of R. solanacearum by qPCR. Our multiplex qPCR assay, especially when coupled with the quick extraction buffer method, allows for quick, easy and reliable detection and differentiation of the IIB-1&2 strains of R. solanacearum.« less

  11. A TaqMan-Based Multiplex qPCR Assay and DNA Extraction Method for Phylotype IIB Sequevars 1&2 (Select Agent) Strains of Ralstonia solanacearum.

    PubMed

    Stulberg, Michael J; Huang, Qi

    2015-01-01

    Ralstonia solanacearum race 3 biovar 2 strains belonging to phylotype IIB, sequevars 1 and 2 (IIB-1&2) cause brown rot of potato in temperate climates, and are quarantined pathogens in Canada and Europe. Since these strains are not established in the U.S. and because of their potential risk to the potato industry, the U.S. government has listed them as select agents. Cultivated geraniums are also a host and have the potential to spread the pathogen through trade, and its extracts strongly inhibits DNA-based detection methods. We designed four primer and probe sets for an improved qPCR method that targets stable regions of DNA. RsSA1 and RsSA2 recognize IIB-1&2 strains, RsII recognizes the current phylotype II (the newly proposed R. solanacearum species) strains (and a non-plant associated R. mannitolilytica), and Cox1 recognizes eight plant species including major hosts of R. solanacearum such as potato, tomato and cultivated geranium as an internal plant control. We multiplexed the RsSA2 with the RsII and Cox1 sets to provide two layers of detection of a positive IIB-1&2 sample, and to validate plant extracts and qPCR reactions. The TaqMan-based uniplex and multiplex qPCR assays correctly identified 34 IIB-1&2 and 52 phylotype II strains out of 90 R. solanacearum species complex strains. Additionally, the multiplex qPCR assay was validated successfully using 169 artificially inoculated symptomatic and asymptomatic plant samples from multiple plant hosts including geranium. Furthermore, we developed an extraction buffer that allowed for a quick and easy DNA extraction from infected plants including geranium for detection of R. solanacearum by qPCR. Our multiplex qPCR assay, especially when coupled with the quick extraction buffer method, allows for quick, easy and reliable detection and differentiation of the IIB-1&2 strains of R. solanacearum.

  12. Overexpression of CaWRKY27, a subgroup IIe WRKY transcription factor of Capsicum annuum, positively regulates tobacco resistance to Ralstonia solanacearum infection.

    PubMed

    Dang, Fengfeng; Wang, Yuna; She, Jianju; Lei, Yufen; Liu, Zhiqin; Eulgem, Thomas; Lai, Yan; Lin, Jing; Yu, Lu; Lei, Dan; Guan, Deyi; Li, Xia; Yuan, Qian; He, Shuilin

    2014-03-01

    WRKY proteins are encoded by a large gene family and are linked to many biological processes across a range of plant species. The functions and underlying mechanisms of WRKY proteins have been investigated primarily in model plants such as Arabidopsis and rice. The roles of these transcription factors in non-model plants, including pepper and other Solanaceae, are poorly understood. Here, we characterize the expression and function of a subgroup IIe WRKY protein from pepper (Capsicum annuum), denoted as CaWRKY27. The protein localized to nuclei and activated the transcription of a reporter GUS gene construct driven by the 35S promoter that contained two copies of the W-box in its proximal upstream region. Inoculation of pepper cultivars with Ralstonia solanacearum induced the expression of CaWRKY27 transcript in 76a, a bacterial wilt-resistant pepper cultivar, whereas it downregulated the expression of CaWRKY27 transcript in Gui-1-3, a bacterial wilt-susceptible pepper cultivar. CaWRKY27 transcript levels were also increased by treatments with salicylic acid (SA), methyl jasmonate (MeJA) and ethephon (ETH). Transgenic tobacco plants overexpressing CaWRKY27 exhibited resistance to R. solanacearum infection compared to that of wild-type plants. This resistance was coupled with increased transcript levels in a number of marker genes, including hypersensitive response genes, and SA-, JA- and ET-associated genes. By contrast, virus-induced gene silencing (VIGS) of CaWRKY27 increased the susceptibility of pepper plants to R. solanacearum infection. These results suggest that CaWRKY27 acts as a positive regulator in tobacco resistance responses to R. solanacearum infection through modulation of SA-, JA- and ET-mediated signaling pathways.

  13. Defining the Metabolic Functions and Roles in Virulence of the rpoN1 and rpoN2 Genes in Ralstonia solanacearum GMI1000

    PubMed Central

    Lundgren, Benjamin R.; Connolly, Morgan P.; Choudhary, Pratibha; Brookins-Little, Tiffany S.; Chatterjee, Snigdha; Raina, Ramesh; Nomura, Christopher T.

    2015-01-01

    The alternative sigma factor RpoN is a unique regulator found among bacteria. It controls numerous processes that range from basic metabolism to more complex functions such as motility and nitrogen fixation. Our current understanding of RpoN function is largely derived from studies on prototypical bacteria such as Escherichia coli. Bacillus subtilis and Pseudomonas putida. Although the extent and necessity of RpoN-dependent functions differ radically between these model organisms, each bacterium depends on a single chromosomal rpoN gene to meet the cellular demands of RpoN regulation. The bacterium Ralstonia solanacearum is often recognized for being the causative agent of wilt disease in crops, including banana, peanut and potato. However, this plant pathogen is also one of the few bacterial species whose genome possesses dual rpoN genes. To determine if the rpoN genes in this bacterium are genetically redundant and interchangeable, we constructed and characterized ΔrpoN1, ΔrpoN2 and ΔrpoN1 ΔrpoN2 mutants of R. solanacearum GMI1000. It was found that growth on a small range of metabolites, including dicarboxylates, ethanol, nitrate, ornithine, proline and xanthine, were dependent on only the rpoN1 gene. Furthermore, the rpoN1 gene was required for wilt disease on tomato whereas rpoN2 had no observable role in virulence or metabolism in R. solanacearum GMI1000. Interestingly, plasmid-based expression of rpoN2 did not fully rescue the metabolic deficiencies of the ΔrpoN1 mutants; full recovery was specific to rpoN1. In comparison, only rpoN2 was able to genetically complement a ΔrpoN E. coli mutant. These results demonstrate that the RpoN1 and RpoN2 proteins are not functionally equivalent or interchangeable in R. solanacearum GMI1000. PMID:26659655

  14. Defining the Metabolic Functions and Roles in Virulence of the rpoN1 and rpoN2 Genes in Ralstonia solanacearum GMI1000.

    PubMed

    Lundgren, Benjamin R; Connolly, Morgan P; Choudhary, Pratibha; Brookins-Little, Tiffany S; Chatterjee, Snigdha; Raina, Ramesh; Nomura, Christopher T

    2015-01-01

    The alternative sigma factor RpoN is a unique regulator found among bacteria. It controls numerous processes that range from basic metabolism to more complex functions such as motility and nitrogen fixation. Our current understanding of RpoN function is largely derived from studies on prototypical bacteria such as Escherichia coli. Bacillus subtilis and Pseudomonas putida. Although the extent and necessity of RpoN-dependent functions differ radically between these model organisms, each bacterium depends on a single chromosomal rpoN gene to meet the cellular demands of RpoN regulation. The bacterium Ralstonia solanacearum is often recognized for being the causative agent of wilt disease in crops, including banana, peanut and potato. However, this plant pathogen is also one of the few bacterial species whose genome possesses dual rpoN genes. To determine if the rpoN genes in this bacterium are genetically redundant and interchangeable, we constructed and characterized ΔrpoN1, ΔrpoN2 and ΔrpoN1 ΔrpoN2 mutants of R. solanacearum GMI1000. It was found that growth on a small range of metabolites, including dicarboxylates, ethanol, nitrate, ornithine, proline and xanthine, were dependent on only the rpoN1 gene. Furthermore, the rpoN1 gene was required for wilt disease on tomato whereas rpoN2 had no observable role in virulence or metabolism in R. solanacearum GMI1000. Interestingly, plasmid-based expression of rpoN2 did not fully rescue the metabolic deficiencies of the ΔrpoN1 mutants; full recovery was specific to rpoN1. In comparison, only rpoN2 was able to genetically complement a ΔrpoN E. coli mutant. These results demonstrate that the RpoN1 and RpoN2 proteins are not functionally equivalent or interchangeable in R. solanacearum GMI1000.

  15. Defining the Metabolic Functions and Roles in Virulence of the rpoN1 and rpoN2 Genes in Ralstonia solanacearum GMI1000.

    PubMed

    Lundgren, Benjamin R; Connolly, Morgan P; Choudhary, Pratibha; Brookins-Little, Tiffany S; Chatterjee, Snigdha; Raina, Ramesh; Nomura, Christopher T

    2015-01-01

    The alternative sigma factor RpoN is a unique regulator found among bacteria. It controls numerous processes that range from basic metabolism to more complex functions such as motility and nitrogen fixation. Our current understanding of RpoN function is largely derived from studies on prototypical bacteria such as Escherichia coli. Bacillus subtilis and Pseudomonas putida. Although the extent and necessity of RpoN-dependent functions differ radically between these model organisms, each bacterium depends on a single chromosomal rpoN gene to meet the cellular demands of RpoN regulation. The bacterium Ralstonia solanacearum is often recognized for being the causative agent of wilt disease in crops, including banana, peanut and potato. However, this plant pathogen is also one of the few bacterial species whose genome possesses dual rpoN genes. To determine if the rpoN genes in this bacterium are genetically redundant and interchangeable, we constructed and characterized ΔrpoN1, ΔrpoN2 and ΔrpoN1 ΔrpoN2 mutants of R. solanacearum GMI1000. It was found that growth on a small range of metabolites, including dicarboxylates, ethanol, nitrate, ornithine, proline and xanthine, were dependent on only the rpoN1 gene. Furthermore, the rpoN1 gene was required for wilt disease on tomato whereas rpoN2 had no observable role in virulence or metabolism in R. solanacearum GMI1000. Interestingly, plasmid-based expression of rpoN2 did not fully rescue the metabolic deficiencies of the ΔrpoN1 mutants; full recovery was specific to rpoN1. In comparison, only rpoN2 was able to genetically complement a ΔrpoN E. coli mutant. These results demonstrate that the RpoN1 and RpoN2 proteins are not functionally equivalent or interchangeable in R. solanacearum GMI1000. PMID:26659655

  16. PIRIN2 stabilizes cysteine protease XCP2 and increases susceptibility to the vascular pathogen Ralstonia solanacearum in Arabidopsis

    PubMed Central

    Zhang, Bo; Tremousaygue, Dominique; Denancé, Nicolas; van Esse, H Peter; Hörger, Anja C; Dabos, Patrick; Goffner, Deborah; Thomma, Bart P H J; van der Hoorn, Renier A L; Tuominen, Hannele

    2014-01-01

    PIRIN (PRN) is a member of the functionally diverse cupin protein superfamily. There are four members of the Arabidopsis thaliana PRN family, but the roles of these proteins are largely unknown. Here we describe a function of the Arabidopsis PIRIN2 (PRN2) that is related to susceptibility to the bacterial plant pathogen Ralstonia solanacearum. Two prn2 mutant alleles displayed decreased disease development and bacterial growth in response to R.  solanacearum infection. We elucidated the underlying molecular mechanism by analyzing PRN2 interactions with the papain-like cysteine proteases (PLCPs) XCP2, RD21A, and RD21B, all of which bound to PRN2 in yeast two-hybrid assays and in Arabidopsis protoplast co-immunoprecipitation assays. We show that XCP2 is stabilized by PRN2 through inhibition of its autolysis on the basis of PLCP activity profiling assays and enzymatic assays with recombinant protein. The stabilization of XCP2 by PRN2 was also confirmed in planta. Like prn2 mutants, an xcp2 single knockout mutant and xcp2 prn2 double knockout mutant displayed decreased susceptibility to R. solanacearum, suggesting that stabilization of XCP2 by PRN2 underlies susceptibility to R. solanacearum in Arabidopsis. PMID:24947605

  17. TssB is essential for virulence and required for type VI secretion system in Ralstonia solanacearum.

    PubMed

    Zhang, Liqing; Xu, Jingsheng; Xu, Jin; Zhang, Hao; He, Liyuan; Feng, Jie

    2014-09-01

    The type VI secretion system (T6SS) is recently discovered machinery in Gram-negative bacteria for translocation of proteins and also is required for full virulence. TssB is a highly conserved protein among the T6SSs, and indispensable for composition and function of T6S. The plant pathogenic bacterium Ralstonia solanacearum also harbours T6SS gene clusters, and a homologue of TssB, hereafter designated as TssBRS, but up to date its characterization and function remain unclear. In this study, we showed that TssBRS of R. solanacearum was required for secretion of Hcp, the haemolysin coregulated protein and a hallmark of T6S pathway. Deletion of tssBRS in R. solanacearum GMI1000 strain resulted in defect of biofilm formation, and the expression of the flagella operon is decreased, leading to decreased motility. More importantly, tssBRS mutant strain had significantly attenuated its virulence on tomato plants. TssB is essential for virulence and required for type VI secretion system in R. solanacearum.

  18. PIRIN2 stabilizes cysteine protease XCP2 and increases susceptibility to the vascular pathogen Ralstonia solanacearum in Arabidopsis.

    PubMed

    Zhang, Bo; Tremousaygue, Dominique; Denancé, Nicolas; van Esse, H Peter; Hörger, Anja C; Dabos, Patrick; Goffner, Deborah; Thomma, Bart P H J; van der Hoorn, Renier A L; Tuominen, Hannele

    2014-09-01

    PIRIN (PRN) is a member of the functionally diverse cupin protein superfamily. There are four members of the Arabidopsis thaliana PRN family, but the roles of these proteins are largely unknown. Here we describe a function of the Arabidopsis PIRIN2 (PRN2) that is related to susceptibility to the bacterial plant pathogen Ralstonia solanacearum. Two prn2 mutant alleles displayed decreased disease development and bacterial growth in response to R.  solanacearum infection. We elucidated the underlying molecular mechanism by analyzing PRN2 interactions with the papain-like cysteine proteases (PLCPs) XCP2, RD21A, and RD21B, all of which bound to PRN2 in yeast two-hybrid assays and in Arabidopsis protoplast co-immunoprecipitation assays. We show that XCP2 is stabilized by PRN2 through inhibition of its autolysis on the basis of PLCP activity profiling assays and enzymatic assays with recombinant protein. The stabilization of XCP2 by PRN2 was also confirmed in planta. Like prn2 mutants, an xcp2 single knockout mutant and xcp2 prn2 double knockout mutant displayed decreased susceptibility to R. solanacearum, suggesting that stabilization of XCP2 by PRN2 underlies susceptibility to R. solanacearum in Arabidopsis.

  19. A novel, sensitive method to evaluate potato germplasm for bacterial wilt resistance using a luminescent Ralstonia solanacearum reporter strain.

    PubMed

    Cruz, Andrea Paola Zuluaga; Ferreira, Virginia; Pianzzola, María Julia; Siri, María Inés; Coll, Núria S; Valls, Marc

    2014-03-01

    Several breeding programs are under way to introduce resistance to bacterial wilt caused by Ralstonia solanacearum in solanaceous crops. The lack of screening methods allowing easy measurement of pathogen colonization and the inability to detect latent (i.e., symptomless) infections are major limitations when evaluating resistance to this disease in plant germplasm. We describe a new method to study the interaction between R. solanacearum and potato germplasm that overcomes these restrictions. The R. solanacearum UY031 was genetically modified to constitutively generate light from a synthetic luxCDABE operon stably inserted in its chromosome. Colonization of this reporter strain on different potato accessions was followed using life imaging. Bacterial detection in planta by this nondisruptive system correlated with the development of wilting symptoms. In addition, we demonstrated that quantitative detection of the recombinant strain using a luminometer can identify latent infections on symptomless potato plants. We have developed a novel, unsophisticated, and accurate method for high-throughput evaluation of pathogen colonization in plant populations. We applied this method to compare the behavior of potato accessions with contrasting resistance to R. solanacearum. This new system will be especially useful to detect latency in symptomless parental lines before their inclusion in long-term breeding programs for disease resistance.

  20. First Report of Bacterial Wilt Caused by Ralstonia solanacearum Biovar 2 Race 1 on Tomato in Egypt

    PubMed Central

    Seleim, Mohamed A. A.; Abo-Elyousr, Kamal A. M.; Abd-El-Moneem, Kenawy M.; Saead, Farag A.

    2014-01-01

    This study aims to isolate and identify the causal pathogen of tomato bacterial wilt in Egypt. In 2008, tomato plants showing typical symptoms of bacterial wilt disease with no foliar yellowing were observed in Minia, Assiut and Sohag governorates, Egypt. When cut stems of symptomatic plants were submerged in water, whitish ooze was evident and longitudinal sections showed a brown discoloration in the vascular tissues. Bacteria were isolated on triphenyl tetrazolium chloride medium and fifteen isolates shown typical morphological and cultural characteristics were confirmed as Ralstonia solanacearum biovar 2 race 1. Pathogenicity tests showed that all isolates proved to be pathogenic to tomato plants, varied from 52 to 97% wilting. This is the first report of R. solanacearum biovar 2 race 1 causing bacterial wilt in tomato crop in Egypt. PMID:25289016

  1. Root-associated bacterial endophytes from Ralstonia solanacearum resistant and susceptible tomato cultivars and their pathogen antagonistic effects.

    PubMed

    Upreti, Reshmi; Thomas, Pious

    2015-01-01

    This study was undertaken to assess if the root-associated native bacterial endophytes in tomato have any bearing in governing the host resistance to the wilt pathogen Ralstonia solanacearum. Internal colonization of roots by bacterial endophytes was confirmed through confocal imaging after SYTO-9 staining. Endophytes were isolated from surface-sterilized roots of 4-weeks-old seedlings of known wilt resistant (R) tomato cultivar Arka Abha and susceptible (S) cv. Arka Vikas on nutrient agar after plating the tissue homogenate. Arka Abha displayed more diversity with nine distinct organisms while Arka Vikas showed five species with two common organisms (Pseudomonas oleovorans and Agrobacterium tumefaciens). Screening for general indicators of biocontrol potential showed more isolates from Arka Abha positive for siderophore, HCN and antibiotic biosynthesis than from Arka Vikas. Direct challenge against the pathogen indicated strong antagonism by three Arka Abha isolates (P. oleovorans, Pantoea ananatis, and Enterobacter cloacae) and moderate activity by three others, while just one isolate from Arka Vikas (P. oleovorans) showed strong antagonism. Validation for the presence of bacterial endophytes on three R cultivars (Arka Alok, Arka Ananya, Arka Samrat) showed 8-9 antagonistic bacteria in them in comparison with four species in the three S cultivars (Arka Ashish, Arka Meghali, Arka Saurabhav). Altogether 34 isolates belonging to five classes, 16 genera and 27 species with 23 of them exhibiting pathogen antagonism were isolated from the four R cultivars against 17 isolates under three classes, seven genera and 13 species from the four S cultivars with eight isolates displaying antagonistic effects. The prevalence of higher endophytic bacterial diversity and more antagonistic organisms associated with the seedling roots of resistant cultivars over susceptible genotypes suggest a possible role by the root-associated endophytes in natural defense against the pathogen

  2. Root-associated bacterial endophytes from Ralstonia solanacearum resistant and susceptible tomato cultivars and their pathogen antagonistic effects.

    PubMed

    Upreti, Reshmi; Thomas, Pious

    2015-01-01

    This study was undertaken to assess if the root-associated native bacterial endophytes in tomato have any bearing in governing the host resistance to the wilt pathogen Ralstonia solanacearum. Internal colonization of roots by bacterial endophytes was confirmed through confocal imaging after SYTO-9 staining. Endophytes were isolated from surface-sterilized roots of 4-weeks-old seedlings of known wilt resistant (R) tomato cultivar Arka Abha and susceptible (S) cv. Arka Vikas on nutrient agar after plating the tissue homogenate. Arka Abha displayed more diversity with nine distinct organisms while Arka Vikas showed five species with two common organisms (Pseudomonas oleovorans and Agrobacterium tumefaciens). Screening for general indicators of biocontrol potential showed more isolates from Arka Abha positive for siderophore, HCN and antibiotic biosynthesis than from Arka Vikas. Direct challenge against the pathogen indicated strong antagonism by three Arka Abha isolates (P. oleovorans, Pantoea ananatis, and Enterobacter cloacae) and moderate activity by three others, while just one isolate from Arka Vikas (P. oleovorans) showed strong antagonism. Validation for the presence of bacterial endophytes on three R cultivars (Arka Alok, Arka Ananya, Arka Samrat) showed 8-9 antagonistic bacteria in them in comparison with four species in the three S cultivars (Arka Ashish, Arka Meghali, Arka Saurabhav). Altogether 34 isolates belonging to five classes, 16 genera and 27 species with 23 of them exhibiting pathogen antagonism were isolated from the four R cultivars against 17 isolates under three classes, seven genera and 13 species from the four S cultivars with eight isolates displaying antagonistic effects. The prevalence of higher endophytic bacterial diversity and more antagonistic organisms associated with the seedling roots of resistant cultivars over susceptible genotypes suggest a possible role by the root-associated endophytes in natural defense against the pathogen.

  3. Root-associated bacterial endophytes from Ralstonia solanacearum resistant and susceptible tomato cultivars and their pathogen antagonistic effects

    PubMed Central

    Upreti, Reshmi; Thomas, Pious

    2015-01-01

    This study was undertaken to assess if the root-associated native bacterial endophytes in tomato have any bearing in governing the host resistance to the wilt pathogen Ralstonia solanacearum. Internal colonization of roots by bacterial endophytes was confirmed through confocal imaging after SYTO-9 staining. Endophytes were isolated from surface-sterilized roots of 4-weeks-old seedlings of known wilt resistant (R) tomato cultivar Arka Abha and susceptible (S) cv. Arka Vikas on nutrient agar after plating the tissue homogenate. Arka Abha displayed more diversity with nine distinct organisms while Arka Vikas showed five species with two common organisms (Pseudomonas oleovorans and Agrobacterium tumefaciens). Screening for general indicators of biocontrol potential showed more isolates from Arka Abha positive for siderophore, HCN and antibiotic biosynthesis than from Arka Vikas. Direct challenge against the pathogen indicated strong antagonism by three Arka Abha isolates (P. oleovorans, Pantoea ananatis, and Enterobacter cloacae) and moderate activity by three others, while just one isolate from Arka Vikas (P. oleovorans) showed strong antagonism. Validation for the presence of bacterial endophytes on three R cultivars (Arka Alok, Arka Ananya, Arka Samrat) showed 8–9 antagonistic bacteria in them in comparison with four species in the three S cultivars (Arka Ashish, Arka Meghali, Arka Saurabhav). Altogether 34 isolates belonging to five classes, 16 genera and 27 species with 23 of them exhibiting pathogen antagonism were isolated from the four R cultivars against 17 isolates under three classes, seven genera and 13 species from the four S cultivars with eight isolates displaying antagonistic effects. The prevalence of higher endophytic bacterial diversity and more antagonistic organisms associated with the seedling roots of resistant cultivars over susceptible genotypes suggest a possible role by the root-associated endophytes in natural defense against the pathogen

  4. In Vivo Detection of the Cyclic Osmoregulated Periplasmic Glucan of Ralstonia solanacearum by High-Resolution Magic Angle Spinning NMR

    NASA Astrophysics Data System (ADS)

    Wieruszeski, J.-M.; Bohin, A.; Bohin, J.-P.; Lippens, G.

    2001-07-01

    We investigate the mobility of the osmoregulated periplasmic glucans of Ralstonia solanacearum in the bacterial periplasm through the use of high-resolution (HR) NMR spectroscopy under static and magic angle spinning (MAS) conditions. Because the nature of periplasm is far from an isotropic aqueous solution, the molecules could be freely diffusing or rather associated to a periplasmic protein, a membrane protein, a lipid, or the peptidoglycan. HR MAS NMR spectroscopy leads to more reproducible results and allows the in vivo detection and characterization of the complex molecule.

  5. Molecular chaperons and co-chaperons, Hsp90, RAR1, and SGT1 negatively regulate bacterial wilt disease caused by Ralstonia solanacearum in Nicotiana benthamiana.

    PubMed

    Ito, Makoto; Ohnishi, Kouhei; Hikichi, Yasufumi; Kiba, Akinori

    2015-01-01

    Ralstonia solanacearum is the causal agent of bacterial wilt disease. To better understand the molecular mechanisms involved in interaction between Nicotiana benthamiana and R. solanacearum, we focused on Hsp90, RAR1 and SGT1. Appearances of wilt symptom were significantly suppressed in Hsp90, RAR1 and SGT1-silenced plants compared with control plants. In RAR1-silenced plants, population of R. solanacearum increased in a similar manner to control plants. In contrast, multiplication of R. solanacearum was significantly suppressed in Hsp90 and SGT1-silenced plants. In addition, expression of PR genes were increased in Hsp90 and SGT1-silenced plants challenged with R. solanacearum. Therefore, RAR1 might be required for disease development or suppression of disease tolerance. These results also suggested that Hsp90 and/or SGT1 might play an important role in suppression of plant defenses leading to disease susceptibility and disease development.

  6. An evaluation of the wilt-causing bacterium Ralstonia solanacearum as a potential biological control agent for the alien Kahili ginger (Hedychium gardnerianum) in Hawaiian forests

    USGS Publications Warehouse

    1999-01-01

    Kahili ginger (Hedychium gardnerianum) is an invasive weed in tropical forests in Hawaii and elsewhere. Bacterial wilt caused by the ginger strain of Ralstonia(=Pseudomonas) solanacearum systemically infects edible ginger (Zingiber officinale) and ornamental gingers (Hedychium spp.), causing wilt in infected plants. The suitability of R. solanacearum as a biological control agent for kahili ginger was investigated by inoculating seedlings and rooted cuttings of native forest plants, ornamental ginger, and solanaceous species to confirm host specificity. Inoculation via stem injection or root wounding with a bacterial–water suspension was followed by observation for 8 weeks. Inoculations on H. gardnerianum were then carried out in ohia-lehua (Metrosideros polymorpha) wet forests of Hawaii Volcanoes National Park to determine the bacterium's efficacy in the field. No native forest or solanaceous species developed wilt or other symptoms during the study. The bacterium caused limited infection near the inoculation site on H. coronarium, Z. zerumbet, Heliconia latispatha, and Musa sapientum. However, infection did not become systemic in any of these species, and normal growth resumed following appearance of initial symptoms. All inoculated H. gardnerianum plants developed irreversible chlorosis and severe wilting 3–4 weeks following inoculation. Systemic infection also caused death and decay of rhizomes. Most plants were completely dead 16–20 weeks following inoculation. The destructiveness of the ginger strain of R. solanacearum to edible ginger has raised questions regarding its use for biological control. However, because locations of kahili ginger infestations are often remote, the risk of contaminating edible ginger plantings is unlikely. The ability of this bacterium to cause severe disease in H. gardnerianum in the field, together with its lack of virulence in other ginger species, contributes to its potential as a biological control agent.

  7. Effect of Seed Treatment by Cold Plasma on the Resistance of Tomato to Ralstonia solanacearum (Bacterial Wilt)

    PubMed Central

    Jiang, Jiafeng; Lu, Yufang; Li, Jiangang; Li, Ling; He, Xin; Shao, Hanliang; Dong, Yuanhua

    2014-01-01

    This study investigated the effect of cold plasma seed treatment on tomato bacterial wilt, caused by Ralstonia solanacearum (R. solanacearum), and the regulation of resistance mechanisms. The effect of cold plasma of 80W on seed germination, plant growth, nutrient uptake, disease severity, hydrogen peroxide (H2O2) concentration and activities of peroxidase (POD; EC 1.11.1.7), polyphenol oxidase (PPO; EC 1.10.3.2) and phenylalanine ammonia lyase (PAL; EC 4.3.1.5) were examined in tomato plants. Plasma treatment increased tomato resistance to R. solanacearum with an efficacy of 25.0%. Plasma treatment significantly increased both germination and plant growth in comparison with the control treatment, and plasma-treated plants absorbed more calcium and boron than the controls. In addition, H2O2 levels in treated plants rose faster and reached a higher peak, at 2.579 µM gFW−1, 140% greater than that of the control. Activities of POD (421.3 U gFW−1), PPO (508.8 U gFW−1) and PAL (707.3 U gFW−1) were also greater in the treated plants than in the controls (103.0 U gFW−1, 166.0 U gFW−1 and 309.4 U gFW−1, respectively). These results suggest that plasma treatment affects the regulation of plant growth, H2O2 concentration, and POD, PPO and PAL activity in tomato, resulting in an improved resistance to R. solanacearum. Consequently, cold plasma seed treatment has the potential to control tomato bacterial wilt caused by R. solanacearum. PMID:24840508

  8. Effect of seed treatment by cold plasma on the resistance of tomato to Ralstonia solanacearum (Bacterial Wilt).

    PubMed

    Jiang, Jiafeng; Lu, Yufang; Li, Jiangang; Li, Ling; He, Xin; Shao, Hanliang; Dong, Yuanhua

    2014-01-01

    This study investigated the effect of cold plasma seed treatment on tomato bacterial wilt, caused by Ralstonia solanacearum (R. solanacearum), and the regulation of resistance mechanisms. The effect of cold plasma of 80W on seed germination, plant growth, nutrient uptake, disease severity, hydrogen peroxide (H2O2) concentration and activities of peroxidase (POD; EC 1.11.1.7), polyphenol oxidase (PPO; EC 1.10.3.2) and phenylalanine ammonia lyase (PAL; EC 4.3.1.5) were examined in tomato plants. Plasma treatment increased tomato resistance to R. solanacearum with an efficacy of 25.0%. Plasma treatment significantly increased both germination and plant growth in comparison with the control treatment, and plasma-treated plants absorbed more calcium and boron than the controls. In addition, H2O2 levels in treated plants rose faster and reached a higher peak, at 2.579 µM gFW-1, 140% greater than that of the control. Activities of POD (421.3 U gFW-1), PPO (508.8 U gFW-1) and PAL (707.3 U gFW-1) were also greater in the treated plants than in the controls (103.0 U gFW-1, 166.0 U gFW-1 and 309.4 U gFW-1, respectively). These results suggest that plasma treatment affects the regulation of plant growth, H2O2 concentration, and POD, PPO and PAL activity in tomato, resulting in an improved resistance to R. solanacearum. Consequently, cold plasma seed treatment has the potential to control tomato bacterial wilt caused by R. solanacearum. PMID:24840508

  9. Effect of seed treatment by cold plasma on the resistance of tomato to Ralstonia solanacearum (Bacterial Wilt).

    PubMed

    Jiang, Jiafeng; Lu, Yufang; Li, Jiangang; Li, Ling; He, Xin; Shao, Hanliang; Dong, Yuanhua

    2014-01-01

    This study investigated the effect of cold plasma seed treatment on tomato bacterial wilt, caused by Ralstonia solanacearum (R. solanacearum), and the regulation of resistance mechanisms. The effect of cold plasma of 80W on seed germination, plant growth, nutrient uptake, disease severity, hydrogen peroxide (H2O2) concentration and activities of peroxidase (POD; EC 1.11.1.7), polyphenol oxidase (PPO; EC 1.10.3.2) and phenylalanine ammonia lyase (PAL; EC 4.3.1.5) were examined in tomato plants. Plasma treatment increased tomato resistance to R. solanacearum with an efficacy of 25.0%. Plasma treatment significantly increased both germination and plant growth in comparison with the control treatment, and plasma-treated plants absorbed more calcium and boron than the controls. In addition, H2O2 levels in treated plants rose faster and reached a higher peak, at 2.579 µM gFW-1, 140% greater than that of the control. Activities of POD (421.3 U gFW-1), PPO (508.8 U gFW-1) and PAL (707.3 U gFW-1) were also greater in the treated plants than in the controls (103.0 U gFW-1, 166.0 U gFW-1 and 309.4 U gFW-1, respectively). These results suggest that plasma treatment affects the regulation of plant growth, H2O2 concentration, and POD, PPO and PAL activity in tomato, resulting in an improved resistance to R. solanacearum. Consequently, cold plasma seed treatment has the potential to control tomato bacterial wilt caused by R. solanacearum.

  10. A duplex PCR assay for the detection of Ralstonia solanacearum phylotype II strains in Musa spp.

    PubMed

    Cellier, Gilles; Moreau, Aurélie; Chabirand, Aude; Hostachy, Bruno; Ailloud, Florent; Prior, Philippe

    2015-01-01

    Banana wilt outbreaks that are attributable to Moko disease-causing strains of the pathogen Ralstonia solanacearum (Rs) remain a social and economic burden for both multinational corporations and subsistence farmers. All known Moko strains belong to the phylotype II lineage, which has been previously recognized for its broad genetic basis. Moko strains are paraphyletic and are distributed among seven related but distinct phylogenetic clusters (sequevars) that are potentially major threats to Musaceae, Solanaceae, and ornamental crops in many countries. Although clustered within the Moko IIB-4 sequevar, strains of the epidemiologically variant IIB-4NPB do not cause wilt on Cavendish or plantain bananas; instead, they establish a latent infection in the vascular tissues of plantains and demonstrate an expanded host range and high aggressiveness toward Solanaceae and Cucurbitaceae. Although most molecular diagnostic methods focus on strains that wilt Solanaceae (particularly potato), no relevant protocol has been described that universally detects strains of the Musaceae-infecting Rs phylotype II. Thus, a duplex PCR assay targeting Moko and IIB-4NPB variant strains was developed, and its performance was assessed using an extensive collection of 111 strains representing the known diversity of Rs Moko-related strains and IIB-4NPB variant strains along with certain related strains and families. The proposed diagnostic protocol demonstrated both high accuracy (inclusivity and exclusivity) and high repeatability, detected targets on either pure culture or spiked plant extracts. Although they did not belong to the Moko clusters described at the time of the study, recently discovered banana-infecting strains from Brazil were also detected. According to our comprehensive evaluation, this duplex PCR assay appears suitable for both research and diagnostic laboratories and provides reliable detection of phylotype II Rs strains that infect Musaceae. PMID:25811378

  11. A duplex PCR assay for the detection of Ralstonia solanacearum phylotype II strains in Musa spp.

    PubMed

    Cellier, Gilles; Moreau, Aurélie; Chabirand, Aude; Hostachy, Bruno; Ailloud, Florent; Prior, Philippe

    2015-01-01

    Banana wilt outbreaks that are attributable to Moko disease-causing strains of the pathogen Ralstonia solanacearum (Rs) remain a social and economic burden for both multinational corporations and subsistence farmers. All known Moko strains belong to the phylotype II lineage, which has been previously recognized for its broad genetic basis. Moko strains are paraphyletic and are distributed among seven related but distinct phylogenetic clusters (sequevars) that are potentially major threats to Musaceae, Solanaceae, and ornamental crops in many countries. Although clustered within the Moko IIB-4 sequevar, strains of the epidemiologically variant IIB-4NPB do not cause wilt on Cavendish or plantain bananas; instead, they establish a latent infection in the vascular tissues of plantains and demonstrate an expanded host range and high aggressiveness toward Solanaceae and Cucurbitaceae. Although most molecular diagnostic methods focus on strains that wilt Solanaceae (particularly potato), no relevant protocol has been described that universally detects strains of the Musaceae-infecting Rs phylotype II. Thus, a duplex PCR assay targeting Moko and IIB-4NPB variant strains was developed, and its performance was assessed using an extensive collection of 111 strains representing the known diversity of Rs Moko-related strains and IIB-4NPB variant strains along with certain related strains and families. The proposed diagnostic protocol demonstrated both high accuracy (inclusivity and exclusivity) and high repeatability, detected targets on either pure culture or spiked plant extracts. Although they did not belong to the Moko clusters described at the time of the study, recently discovered banana-infecting strains from Brazil were also detected. According to our comprehensive evaluation, this duplex PCR assay appears suitable for both research and diagnostic laboratories and provides reliable detection of phylotype II Rs strains that infect Musaceae.

  12. The Ralstonia solanacearum pathogenicity regulator HrpB induces 3-hydroxy-oxindole synthesis.

    PubMed

    Delaspre, Fabien; Nieto Peñalver, Carlos G; Saurel, Olivier; Kiefer, Patrick; Gras, Emmanuel; Milon, Alain; Boucher, Christian; Genin, Stéphane; Vorholt, Julia A

    2007-10-01

    The transcriptional activator HrpB of the bacterial wilt causing betaproteobacterium Ralstonia solanacearum represents a key regulator for pathogenicity. In particular, it drives expression of hrp genes encoding a type III secretion system (T3SS) as well as effector molecules for delivery into the host cytosol to promote disease. However, the HrpB regulon extends beyond this T3SS. We describe here an HrpB-activated operon of six genes that is responsible for the synthesis of a fluorescent isatin derivative of 149 Amu that we named HDF for HrpB-dependent factor and that we purified from culture supernatants. The structure of the labile molecule was solved by using NMR and CD spectroscopy to be (3S)-3-hydroxy-indolin-2-one and confirmed by its chemical synthesis and MS spectrometry. HDF was found to be present at 20 nM in wild-type cultures grown on minimal medium, and its synthesis increased 15-fold upon overproduction of HrpB, confirming that HrpB activates HDF synthesis. The addition of tryptophan significantly stimulated HDF biosynthesis and was shown to represent the precursor molecule for HDF synthesis. A search for the biological function of the molecule revealed that HDF induces acyl-homoserine lactone receptor-mediated reporter activity of the well studied LuxR transcriptional regulator of Vibrio fischeri. Thus, our results provide evidence that the specificity of acyl-homoserine lactone (acyl-HSL) receptors is clearly broader than previously considered. The failure to detect induction by HDF of the described endogenous quorum-sensing circuits of the pathogen points to a role in interfering with cell-cell signaling of rivalling bacteria. PMID:17890323

  13. A Duplex PCR Assay for the Detection of Ralstonia solanacearum Phylotype II Strains in Musa spp.

    PubMed Central

    Cellier, Gilles; Moreau, Aurélie; Chabirand, Aude; Hostachy, Bruno; Ailloud, Florent; Prior, Philippe

    2015-01-01

    Banana wilt outbreaks that are attributable to Moko disease-causing strains of the pathogen Ralstonia solanacearum (Rs) remain a social and economic burden for both multinational corporations and subsistence farmers. All known Moko strains belong to the phylotype II lineage, which has been previously recognized for its broad genetic basis. Moko strains are paraphyletic and are distributed among seven related but distinct phylogenetic clusters (sequevars) that are potentially major threats to Musaceae, Solanaceae, and ornamental crops in many countries. Although clustered within the Moko IIB-4 sequevar, strains of the epidemiologically variant IIB-4NPB do not cause wilt on Cavendish or plantain bananas; instead, they establish a latent infection in the vascular tissues of plantains and demonstrate an expanded host range and high aggressiveness toward Solanaceae and Cucurbitaceae. Although most molecular diagnostic methods focus on strains that wilt Solanaceae (particularly potato), no relevant protocol has been described that universally detects strains of the Musaceae-infecting Rs phylotype II. Thus, a duplex PCR assay targeting Moko and IIB-4NPB variant strains was developed, and its performance was assessed using an extensive collection of 111 strains representing the known diversity of Rs Moko-related strains and IIB-4NPB variant strains along with certain related strains and families. The proposed diagnostic protocol demonstrated both high accuracy (inclusivity and exclusivity) and high repeatability, detected targets on either pure culture or spiked plant extracts. Although they did not belong to the Moko clusters described at the time of the study, recently discovered banana-infecting strains from Brazil were also detected. According to our comprehensive evaluation, this duplex PCR assay appears suitable for both research and diagnostic laboratories and provides reliable detection of phylotype II Rs strains that infect Musaceae. PMID:25811378

  14. Genome Sequencing of Ralstonia solanacearum Biovar 3, Phylotype I, Strains Rs-09-161 and Rs-10-244, Isolated from Eggplant and Chili in India.

    PubMed

    Ramesh, Raman; Gaitonde, Sapna; Achari, Gauri; Asolkar, Trupti; Singh, Narendra Pratap; Carrere, Sebastien; Genin, Stephane; Peeters, Nemo

    2014-01-01

    Ralstonia solanacearum Indian strains Rs-09-161 and Rs-10-244 were isolated from the coastal region of Goa and from the Andaman Islands. We report the draft genome sequences of these representative isolates infecting solanaceous vegetables in India. PMID:24874667

  15. A TaqMan-based multiplex qPCR assay and DNA extraction method for phylotype IIB sequevars 1&2 (select agent) strains of Ralstonia solanacearum

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Ralstonia solanacearum race 3 biovar 2 strains have the ability to cause brown rot of potato in temperate climates. Since these strains are not established in the U.S. and because of the potential risk they pose to the potato industry, the U.S. government has listed them as select agents. Cultivated...

  16. Cold Tolerance of some Ralstonia solanacearum strains, including Race3 Biovar2, is conferred in part by variation in cold shock gene cspD3.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Ralstonia solanacearum race 3 biovar 2 (R3B2) strains are one of only 10 USDA Select Agents, a category of quarantined pathogens reserved for the most serious threats to U.S. plant industry. The threat of R3B2 strains was not considered to be likely due to race (these are poorly defined) or biovar ...

  17. Rapid differentiation of Ralstonia solanacearum avirulent and virulent strains by cell fractioning of an isolate using high performance liquid chromatography.

    PubMed

    Zheng, Xuefang; Zhu, Yujing; Liu, Bo; Yu, Qian; Lin, Naiquan

    2016-01-01

    Ralstonia solanacearum is one of the most destructive plant bacterial pathogens worldwide. The population dynamics and genetic stability are important issues, especially when an avirulent strain is used for biocontrol. In this study, we developed a rapid method to differentiate the virulent and avirulent strains of R. solanacearum and to predict the biocontrol efficiency of an avirulent strain using high performance liquid chromatography (HPLC). Three chromatographic peaks P1, P2 and P3 were observed on the HPLC spectra among 68 avirulent and 28 virulent R. solanacearum strains. Based on the HPLC peaks, 96 strains total were assigned to three categories. For avirulent strains, the intense peak is P1, while for virulent strains, P3 is the majority. Based on the HLPC spectra of R. solanacearum strains, a chromatography titer index (CTI) was established as CTIi = Si/(S1+S2+S3) × 100% (i represents an individual HPLC peak; S1, S2 and S3 represent peak areas of P1, P2 and P3, respectively). The avirulent strains had high values of CTI1 ranging from 63.6 to 100.0%, while the virulent strains displayed high values of CTI3 ranging from 90.2 to 100.0%. Biological inoculation studies of 68 avirulent strains revealed that the biocontrol efficacy was the best when CTI1 = 100%. The purity and genetic stability of R. solanacearum strains were confirmed in the P1 fraction of avirulent strain FJAT-1957 and P3 fraction of virulent strain FJAT-1925 after 30 generations of consecutive subculture. These results confirmed that fractioning by HPLC and their deduced CTI can be used for rapid and efficient evaluation and prediction of an isolate of R. solanacearum. To the best of our knowledge, this is the first report that HPLC fractioning can be used for rapid differentiation of virulent and avirulent strains of R. solanacearum. PMID:26606869

  18. Rapid differentiation of Ralstonia solanacearum avirulent and virulent strains by cell fractioning of an isolate using high performance liquid chromatography.

    PubMed

    Zheng, Xuefang; Zhu, Yujing; Liu, Bo; Yu, Qian; Lin, Naiquan

    2016-01-01

    Ralstonia solanacearum is one of the most destructive plant bacterial pathogens worldwide. The population dynamics and genetic stability are important issues, especially when an avirulent strain is used for biocontrol. In this study, we developed a rapid method to differentiate the virulent and avirulent strains of R. solanacearum and to predict the biocontrol efficiency of an avirulent strain using high performance liquid chromatography (HPLC). Three chromatographic peaks P1, P2 and P3 were observed on the HPLC spectra among 68 avirulent and 28 virulent R. solanacearum strains. Based on the HPLC peaks, 96 strains total were assigned to three categories. For avirulent strains, the intense peak is P1, while for virulent strains, P3 is the majority. Based on the HLPC spectra of R. solanacearum strains, a chromatography titer index (CTI) was established as CTIi = Si/(S1+S2+S3) × 100% (i represents an individual HPLC peak; S1, S2 and S3 represent peak areas of P1, P2 and P3, respectively). The avirulent strains had high values of CTI1 ranging from 63.6 to 100.0%, while the virulent strains displayed high values of CTI3 ranging from 90.2 to 100.0%. Biological inoculation studies of 68 avirulent strains revealed that the biocontrol efficacy was the best when CTI1 = 100%. The purity and genetic stability of R. solanacearum strains were confirmed in the P1 fraction of avirulent strain FJAT-1957 and P3 fraction of virulent strain FJAT-1925 after 30 generations of consecutive subculture. These results confirmed that fractioning by HPLC and their deduced CTI can be used for rapid and efficient evaluation and prediction of an isolate of R. solanacearum. To the best of our knowledge, this is the first report that HPLC fractioning can be used for rapid differentiation of virulent and avirulent strains of R. solanacearum.

  19. Development of the sensitive lateral flow immunoassay with silver enhancement for the detection of Ralstonia solanacearum in potato tubers.

    PubMed

    Panferov, Vasily G; Safenkova, Irina V; Varitsev, Yury A; Drenova, Natalia V; Kornev, Konstantin P; Zherdev, Anatoly V; Dzantiev, Boris B

    2016-05-15

    Ralstonia solanacearum is a dangerous and economically important pathogen of potatoes and other agricultural crops. Therefore, rapid and sensitive methods for its routine diagnostics are necessary. The aim of this study was to develop a rapid control method for R. solanacearum with a low limit of detection (LOD) based on a lateral flow immunoassay (LFIA) with silver enhancement. To minimize the LOD, the membrane type, antibody amount for conjugation with gold nanoparticles, conjugate concentration and antibody concentration in the analytical zone were optimized. Silver enhancement was used to decrease the LOD of the LFIA. For silver enhancement, release fiberglass membranes with pre-absorbed silver lactate and hydroquinone were placed on the analytical zone, and a drop of silver lactate was added. The LFIA with silver enhancement was found to be 10-fold more sensitive (LOD 2×10(2) CFU/mL; 20 min) in comparison with the common analysis (LOD 2×10(3) CFU/mL; 10 min). The specificity of the developed LFIA was studied using different strains of R. solanacearum (54 samples) and other widespread bacterial pathogens (18 samples). The LFIA detected all tested strains, whereas non-specific reactions were not observed. The developed tests were used for the control of bacteria in extracts of infected and non-infected potato tubers, and the quantitative analysis results (based on the densitometry of line colouration) were confirmed by ELISA with a correlation coefficient equal to 0.965. PMID:26992550

  20. l-Histidine Induces Resistance in Plants to the Bacterial Pathogen Ralstonia solanacearum Partially Through the Activation of Ethylene Signaling.

    PubMed

    Seo, Shigemi; Nakaho, Kazuhiro; Hong, Si Won; Takahashi, Hideki; Shigemori, Hideyuki; Mitsuhara, Ichiro

    2016-09-01

    Wilt disease in plants, which is caused by the soil-borne bacterial pathogen Ralstonia solanacearum, is one of the most devastating plant diseases. We previously detected bacterial wilt disease-inhibiting activity in an extract from yeast cells. In the present study, we purified this activity and identified one of the substances responsible for the activity as the amino acid histidine. The exogenous application of l-histidine, but not d-histidine, inhibited wilt disease in tomato and Arabidopsis plants without exhibiting any antibacterial activity. l-Histidine induced the expression of genes related to ethylene (ET) biosynthesis and signaling as well as the production of ET in tomato and Arabidopsis plants. l-Histidine-induced resistance to R. solanacearum was partially abolished in ein3-1, an ET-insensitive Arabidopsis mutant line. Resistance to the fungal pathogen Botrytis cinerea, which is known to require ET biosynthesis or signaling, was also induced by exogenously applied l-histidine. These results suggest that l-histidine induces resistance to R. solanacearum and B. cinerea partially through the activation of ET signaling in plants. PMID:27335353

  1. l-Histidine Induces Resistance in Plants to the Bacterial Pathogen Ralstonia solanacearum Partially Through the Activation of Ethylene Signaling.

    PubMed

    Seo, Shigemi; Nakaho, Kazuhiro; Hong, Si Won; Takahashi, Hideki; Shigemori, Hideyuki; Mitsuhara, Ichiro

    2016-09-01

    Wilt disease in plants, which is caused by the soil-borne bacterial pathogen Ralstonia solanacearum, is one of the most devastating plant diseases. We previously detected bacterial wilt disease-inhibiting activity in an extract from yeast cells. In the present study, we purified this activity and identified one of the substances responsible for the activity as the amino acid histidine. The exogenous application of l-histidine, but not d-histidine, inhibited wilt disease in tomato and Arabidopsis plants without exhibiting any antibacterial activity. l-Histidine induced the expression of genes related to ethylene (ET) biosynthesis and signaling as well as the production of ET in tomato and Arabidopsis plants. l-Histidine-induced resistance to R. solanacearum was partially abolished in ein3-1, an ET-insensitive Arabidopsis mutant line. Resistance to the fungal pathogen Botrytis cinerea, which is known to require ET biosynthesis or signaling, was also induced by exogenously applied l-histidine. These results suggest that l-histidine induces resistance to R. solanacearum and B. cinerea partially through the activation of ET signaling in plants.

  2. The Viable But Nonculturable State of Ralstonia solanacearum May Be Involved in Long-Term Survival and Plant Infection

    PubMed Central

    Grey, Brian E.; Steck, Todd R.

    2001-01-01

    The role of the dormant-like viable but nonculturable (VBNC) condition in the etiology of bacterial infection was examined using a plant system. The plant-pathogenic bacterium Ralstonia solanacearum was first shown to enter into the VBNC state both in response to cupric sulfate when in a saline solution and when placed in autoclaved soil. To determine if the VBNC condition is related to pathogenesis, the physiological status of bacteria recovered from different regions of inoculated tomato plants was determined at different stages of infection. The fraction of in planta bacteria that were VBNC increased during infection and became greater than 99% by the late stage of disease. The possibility that soil-dwelling VBNC bacteria may resuscitate and infect plants was also examined. When tomato seeds were germinated in sterile soil that contained VBNC but no detectable culturable forms of R. solanacearum cells, resuscitation was observed to occur in soil adjacent to plant roots; these resuscitated bacteria were able to infect plants. This is the first report of R. solanacearum entering the VBNC state and of resuscitation of any VBNC plant-pathogenic bacteria and provides evidence that the VBNC state may be involved in explaining the persistent nature of some infections. PMID:11525979

  3. Control Efficacy of an Endophytic Bacillus amyloliquefaciens Strain BZ6-1 against Peanut Bacterial Wilt, Ralstonia solanacearum

    PubMed Central

    Liang, Guobin

    2014-01-01

    We aimed to isolate and identify endophytic bacteria that might have efficacy against peanut bacterial wilt (BW) caused by Ralstonia solanacearum. Thirty-seven endophytic strains were isolated from healthy peanut plants in R. solanacearum-infested fields and eight showed antagonistic effects against R. solanacearum. Strain BZ6-1 with the highest antimicrobial activity was identified as Bacillus amyloliquefaciens based on morphology, biochemistry, and 16S rRNA analysis. Culture conditions of BZ6-1 were optimized using orthogonal test method and inhibitory zone diameter in dual culture plate assay reached 34.2 mm. Furthermore, main antimicrobial substances of surfactin and fengycin A homologues produced by BZ6-1 were analyzed by high performance liquid chromatography electrospray ionization tandem mass spectrometry. Finally, pot experiments were adopted to test the control efficiency of BZ6-1 against peanut BW. Disease incidence decreased significantly from 84.5% in the control to 12.1% with addition of 15 mL (108 cfu mL−1) culture broth for each seedling, suggesting the feasibility of strain BZ6-1 in the biological control of peanut plants BW. PMID:24527448

  4. Development of the sensitive lateral flow immunoassay with silver enhancement for the detection of Ralstonia solanacearum in potato tubers.

    PubMed

    Panferov, Vasily G; Safenkova, Irina V; Varitsev, Yury A; Drenova, Natalia V; Kornev, Konstantin P; Zherdev, Anatoly V; Dzantiev, Boris B

    2016-05-15

    Ralstonia solanacearum is a dangerous and economically important pathogen of potatoes and other agricultural crops. Therefore, rapid and sensitive methods for its routine diagnostics are necessary. The aim of this study was to develop a rapid control method for R. solanacearum with a low limit of detection (LOD) based on a lateral flow immunoassay (LFIA) with silver enhancement. To minimize the LOD, the membrane type, antibody amount for conjugation with gold nanoparticles, conjugate concentration and antibody concentration in the analytical zone were optimized. Silver enhancement was used to decrease the LOD of the LFIA. For silver enhancement, release fiberglass membranes with pre-absorbed silver lactate and hydroquinone were placed on the analytical zone, and a drop of silver lactate was added. The LFIA with silver enhancement was found to be 10-fold more sensitive (LOD 2×10(2) CFU/mL; 20 min) in comparison with the common analysis (LOD 2×10(3) CFU/mL; 10 min). The specificity of the developed LFIA was studied using different strains of R. solanacearum (54 samples) and other widespread bacterial pathogens (18 samples). The LFIA detected all tested strains, whereas non-specific reactions were not observed. The developed tests were used for the control of bacteria in extracts of infected and non-infected potato tubers, and the quantitative analysis results (based on the densitometry of line colouration) were confirmed by ELISA with a correlation coefficient equal to 0.965.

  5. Escaping Underground Nets: Extracellular DNases Degrade Plant Extracellular Traps and Contribute to Virulence of the Plant Pathogenic Bacterium Ralstonia solanacearum.

    PubMed

    Tran, Tuan Minh; MacIntyre, April; Hawes, Martha; Allen, Caitilyn

    2016-06-01

    Plant root border cells have been recently recognized as an important physical defense against soil-borne pathogens. Root border cells produce an extracellular matrix of protein, polysaccharide and DNA that functions like animal neutrophil extracellular traps to immobilize pathogens. Exposing pea root border cells to the root-infecting bacterial wilt pathogen Ralstonia solanacearum triggered release of DNA-containing extracellular traps in a flagellin-dependent manner. These traps rapidly immobilized the pathogen and killed some cells, but most of the entangled bacteria eventually escaped. The R. solanacearum genome encodes two putative extracellular DNases (exDNases) that are expressed during pathogenesis, suggesting that these exDNases contribute to bacterial virulence by enabling the bacterium to degrade and escape root border cell traps. We tested this hypothesis with R. solanacearum deletion mutants lacking one or both of these nucleases, named NucA and NucB. Functional studies with purified proteins revealed that NucA and NucB are non-specific endonucleases and that NucA is membrane-associated and cation-dependent. Single ΔnucA and ΔnucB mutants and the ΔnucA/B double mutant all had reduced virulence on wilt-susceptible tomato plants in a naturalistic soil-soak inoculation assay. The ΔnucA/B mutant was out-competed by the wild-type strain in planta and was less able to stunt root growth or colonize plant stems. Further, the double nuclease mutant could not escape from root border cells in vitro and was defective in attachment to pea roots. Taken together, these results demonstrate that extracellular DNases are novel virulence factors that help R. solanacearum successfully overcome plant defenses to infect plant roots and cause bacterial wilt disease. PMID:27336156

  6. Escaping Underground Nets: Extracellular DNases Degrade Plant Extracellular Traps and Contribute to Virulence of the Plant Pathogenic Bacterium Ralstonia solanacearum.

    PubMed

    Tran, Tuan Minh; MacIntyre, April; Hawes, Martha; Allen, Caitilyn

    2016-06-01

    Plant root border cells have been recently recognized as an important physical defense against soil-borne pathogens. Root border cells produce an extracellular matrix of protein, polysaccharide and DNA that functions like animal neutrophil extracellular traps to immobilize pathogens. Exposing pea root border cells to the root-infecting bacterial wilt pathogen Ralstonia solanacearum triggered release of DNA-containing extracellular traps in a flagellin-dependent manner. These traps rapidly immobilized the pathogen and killed some cells, but most of the entangled bacteria eventually escaped. The R. solanacearum genome encodes two putative extracellular DNases (exDNases) that are expressed during pathogenesis, suggesting that these exDNases contribute to bacterial virulence by enabling the bacterium to degrade and escape root border cell traps. We tested this hypothesis with R. solanacearum deletion mutants lacking one or both of these nucleases, named NucA and NucB. Functional studies with purified proteins revealed that NucA and NucB are non-specific endonucleases and that NucA is membrane-associated and cation-dependent. Single ΔnucA and ΔnucB mutants and the ΔnucA/B double mutant all had reduced virulence on wilt-susceptible tomato plants in a naturalistic soil-soak inoculation assay. The ΔnucA/B mutant was out-competed by the wild-type strain in planta and was less able to stunt root growth or colonize plant stems. Further, the double nuclease mutant could not escape from root border cells in vitro and was defective in attachment to pea roots. Taken together, these results demonstrate that extracellular DNases are novel virulence factors that help R. solanacearum successfully overcome plant defenses to infect plant roots and cause bacterial wilt disease.

  7. Escaping Underground Nets: Extracellular DNases Degrade Plant Extracellular Traps and Contribute to Virulence of the Plant Pathogenic Bacterium Ralstonia solanacearum

    PubMed Central

    Tran, Tuan Minh; MacIntyre, April; Hawes, Martha; Allen, Caitilyn

    2016-01-01

    Plant root border cells have been recently recognized as an important physical defense against soil-borne pathogens. Root border cells produce an extracellular matrix of protein, polysaccharide and DNA that functions like animal neutrophil extracellular traps to immobilize pathogens. Exposing pea root border cells to the root-infecting bacterial wilt pathogen Ralstonia solanacearum triggered release of DNA-containing extracellular traps in a flagellin-dependent manner. These traps rapidly immobilized the pathogen and killed some cells, but most of the entangled bacteria eventually escaped. The R. solanacearum genome encodes two putative extracellular DNases (exDNases) that are expressed during pathogenesis, suggesting that these exDNases contribute to bacterial virulence by enabling the bacterium to degrade and escape root border cell traps. We tested this hypothesis with R. solanacearum deletion mutants lacking one or both of these nucleases, named NucA and NucB. Functional studies with purified proteins revealed that NucA and NucB are non-specific endonucleases and that NucA is membrane-associated and cation-dependent. Single ΔnucA and ΔnucB mutants and the ΔnucA/B double mutant all had reduced virulence on wilt-susceptible tomato plants in a naturalistic soil-soak inoculation assay. The ΔnucA/B mutant was out-competed by the wild-type strain in planta and was less able to stunt root growth or colonize plant stems. Further, the double nuclease mutant could not escape from root border cells in vitro and was defective in attachment to pea roots. Taken together, these results demonstrate that extracellular DNases are novel virulence factors that help R. solanacearum successfully overcome plant defenses to infect plant roots and cause bacterial wilt disease. PMID:27336156

  8. [Genetic variability of the bacterium Ralstonia solanacearum (Burkholderiales: Burholderiaceae) in the banana-growing region of Uraba (Colombia)].

    PubMed

    Cardozo, Carolina; Rodríguez, Paola; Cotes, José Miguel; Marín, Mauricio

    2010-03-01

    The banana moko disease, caused by the bacterium Ralstonia solanacearum, is one of the most important phytopathological problems of the banana agribusiness in tropical countries. In Uraba and Magdalena (Colombia), the main exporting regions of banana in Colombia, this disease causes a destruction estimated in 16.5 ha/year. The bacterium presents an extremely high level of genetic variation that affects control measures. This is the first study of its variation in Colombia and was done with AFLP molecular markers on a population of 100 isolates from banana plants, soils and "weeds". The high level of genetic diversity, with Nei and Shannon indexes of h=0.32 and I=0.48, respectively, and the AMOVA, showed that this population is subestructured (Fst=0.66): the host is the main factor of differentiation. Even so, previous tests show that all varieties have pathogenicity on Musa.

  9. Loop-Mediated Isothermal Amplification of Specific Endoglucanase Gene Sequence for Detection of the Bacterial Wilt Pathogen Ralstonia solanacearum

    PubMed Central

    Pirc, Manca; Llop, Pablo; Ravnikar, Maja; Dreo, Tanja

    2014-01-01

    The increased globalization of crops production and processing industries also promotes the side-effects of more rapid and efficient spread of plant pathogens. To prevent the associated economic losses, and particularly those related to bacterial diseases where their management relies on removal of the infected material from production, simple, easy-to-perform, rapid and cost-effective tests are needed. Loop-mediated isothermal amplification (LAMP) assays that target 16S rRNA, fliC and egl genes were compared and evaluated as on-site applications. The assay with the best performance was that targeted to the egl gene, which shows high analytical specificity for diverse strains of the betaproteobacterium Ralstonia solanacearum, including its non-European and non-race 3 biovar 2 strains. The additional melting curve analysis provides confirmation of the test results. According to our extensive assessment, the egl LAMP assay requires minimum sample preparation (a few minutes of boiling) for the identification of pure cultures and ooze from symptomatic material, and it can also be used in a high-throughput format in the laboratory. This provides sensitive and reliable detection of R. solanacearum strains of different phylotypes. PMID:24763488

  10. The effector AWR5 from the plant pathogen Ralstonia solanacearum is an inhibitor of the TOR signalling pathway.

    PubMed

    Popa, Crina; Li, Liang; Gil, Sergio; Tatjer, Laura; Hashii, Keisuke; Tabuchi, Mitsuaki; Coll, Núria S; Ariño, Joaquín; Valls, Marc

    2016-01-01

    Bacterial pathogens possess complex type III effector (T3E) repertoires that are translocated inside the host cells to cause disease. However, only a minor proportion of these effectors have been assigned a function. Here, we show that the T3E AWR5 from the phytopathogen Ralstonia solanacearum is an inhibitor of TOR, a central regulator in eukaryotes that controls the switch between cell growth and stress responses in response to nutrient availability. Heterologous expression of AWR5 in yeast caused growth inhibition and autophagy induction coupled to massive transcriptomic changes, unmistakably reminiscent of TOR inhibition by rapamycin or nitrogen starvation. Detailed genetic analysis of these phenotypes in yeast, including suppression of AWR5-induced toxicity by mutation of CDC55 and TPD3, encoding regulatory subunits of the PP2A phosphatase, indicated that AWR5 might exert its function by directly or indirectly inhibiting the TOR pathway upstream PP2A. We present evidence in planta that this T3E caused a decrease in TOR-regulated plant nitrate reductase activity and also that normal levels of TOR and the Cdc55 homologues in plants are required for R. solanacearum virulence. Our results suggest that the TOR pathway is a bona fide T3E target and further prove that yeast is a useful platform for T3E function characterisation. PMID:27257085

  11. The involvement of the PilQ secretin of type IV pili in phage infection in Ralstonia solanacearum.

    PubMed

    Narulita, Erlia; Addy, Hardian Susilo; Kawasaki, Takeru; Fujie, Makoto; Yamada, Takashi

    2016-01-22

    PilQ is a member of the secretin family of outer membrane proteins and specifically involved in type IV secretion. Here we report the effects of pilQ mutation in Ralstonia solanacearum on the host physiology including susceptibility to several phage types (Inoviridae, Podoviridae and Myoviridae). With three lines of cells, namely wild type, ΔpilQ and pilQ-complemented cells, the cell surface proteins, twitching motility and sensitivity to phages were compared. SDS-PAGE analysis revealed that the major TFP pilin (PilA) was specifically lost in pilQ mutants and was recovered in the complemented cells. Drastically inactivated twitching motility in pilQ mutants was recovered to the wild type level in the complemented cells. Several phages of different types including those of Inoviridae, Podoviridae, and Myoviridae that infect wild type cells could not form plaques on pilQ mutants but showed infectivity to pilQ-complemented cells. These results indicate that PilQ function is generally required for phage infection in R. solanacearum. PMID:26718404

  12. The effector AWR5 from the plant pathogen Ralstonia solanacearum is an inhibitor of the TOR signalling pathway

    PubMed Central

    Popa, Crina; Li, Liang; Gil, Sergio; Tatjer, Laura; Hashii, Keisuke; Tabuchi, Mitsuaki; Coll, Núria S.; Ariño, Joaquín; Valls, Marc

    2016-01-01

    Bacterial pathogens possess complex type III effector (T3E) repertoires that are translocated inside the host cells to cause disease. However, only a minor proportion of these effectors have been assigned a function. Here, we show that the T3E AWR5 from the phytopathogen Ralstonia solanacearum is an inhibitor of TOR, a central regulator in eukaryotes that controls the switch between cell growth and stress responses in response to nutrient availability. Heterologous expression of AWR5 in yeast caused growth inhibition and autophagy induction coupled to massive transcriptomic changes, unmistakably reminiscent of TOR inhibition by rapamycin or nitrogen starvation. Detailed genetic analysis of these phenotypes in yeast, including suppression of AWR5-induced toxicity by mutation of CDC55 and TPD3, encoding regulatory subunits of the PP2A phosphatase, indicated that AWR5 might exert its function by directly or indirectly inhibiting the TOR pathway upstream PP2A. We present evidence in planta that this T3E caused a decrease in TOR-regulated plant nitrate reductase activity and also that normal levels of TOR and the Cdc55 homologues in plants are required for R. solanacearum virulence. Our results suggest that the TOR pathway is a bona fide T3E target and further prove that yeast is a useful platform for T3E function characterisation. PMID:27257085

  13. Loop-mediated isothermal amplification of specific endoglucanase gene sequence for detection of the bacterial wilt pathogen Ralstonia solanacearum.

    PubMed

    Lenarčič, Rok; Morisset, Dany; Pirc, Manca; Llop, Pablo; Ravnikar, Maja; Dreo, Tanja

    2014-01-01

    The increased globalization of crops production and processing industries also promotes the side-effects of more rapid and efficient spread of plant pathogens. To prevent the associated economic losses, and particularly those related to bacterial diseases where their management relies on removal of the infected material from production, simple, easy-to-perform, rapid and cost-effective tests are needed. Loop-mediated isothermal amplification (LAMP) assays that target 16S rRNA, fliC and egl genes were compared and evaluated as on-site applications. The assay with the best performance was that targeted to the egl gene, which shows high analytical specificity for diverse strains of the betaproteobacterium Ralstonia solanacearum, including its non-European and non-race 3 biovar 2 strains. The additional melting curve analysis provides confirmation of the test results. According to our extensive assessment, the egl LAMP assay requires minimum sample preparation (a few minutes of boiling) for the identification of pure cultures and ooze from symptomatic material, and it can also be used in a high-throughput format in the laboratory. This provides sensitive and reliable detection of R. solanacearum strains of different phylotypes. PMID:24763488

  14. The effector AWR5 from the plant pathogen Ralstonia solanacearum is an inhibitor of the TOR signalling pathway.

    PubMed

    Popa, Crina; Li, Liang; Gil, Sergio; Tatjer, Laura; Hashii, Keisuke; Tabuchi, Mitsuaki; Coll, Núria S; Ariño, Joaquín; Valls, Marc

    2016-06-03

    Bacterial pathogens possess complex type III effector (T3E) repertoires that are translocated inside the host cells to cause disease. However, only a minor proportion of these effectors have been assigned a function. Here, we show that the T3E AWR5 from the phytopathogen Ralstonia solanacearum is an inhibitor of TOR, a central regulator in eukaryotes that controls the switch between cell growth and stress responses in response to nutrient availability. Heterologous expression of AWR5 in yeast caused growth inhibition and autophagy induction coupled to massive transcriptomic changes, unmistakably reminiscent of TOR inhibition by rapamycin or nitrogen starvation. Detailed genetic analysis of these phenotypes in yeast, including suppression of AWR5-induced toxicity by mutation of CDC55 and TPD3, encoding regulatory subunits of the PP2A phosphatase, indicated that AWR5 might exert its function by directly or indirectly inhibiting the TOR pathway upstream PP2A. We present evidence in planta that this T3E caused a decrease in TOR-regulated plant nitrate reductase activity and also that normal levels of TOR and the Cdc55 homologues in plants are required for R. solanacearum virulence. Our results suggest that the TOR pathway is a bona fide T3E target and further prove that yeast is a useful platform for T3E function characterisation.

  15. The involvement of the PilQ secretin of type IV pili in phage infection in Ralstonia solanacearum.

    PubMed

    Narulita, Erlia; Addy, Hardian Susilo; Kawasaki, Takeru; Fujie, Makoto; Yamada, Takashi

    2016-01-22

    PilQ is a member of the secretin family of outer membrane proteins and specifically involved in type IV secretion. Here we report the effects of pilQ mutation in Ralstonia solanacearum on the host physiology including susceptibility to several phage types (Inoviridae, Podoviridae and Myoviridae). With three lines of cells, namely wild type, ΔpilQ and pilQ-complemented cells, the cell surface proteins, twitching motility and sensitivity to phages were compared. SDS-PAGE analysis revealed that the major TFP pilin (PilA) was specifically lost in pilQ mutants and was recovered in the complemented cells. Drastically inactivated twitching motility in pilQ mutants was recovered to the wild type level in the complemented cells. Several phages of different types including those of Inoviridae, Podoviridae, and Myoviridae that infect wild type cells could not form plaques on pilQ mutants but showed infectivity to pilQ-complemented cells. These results indicate that PilQ function is generally required for phage infection in R. solanacearum.

  16. Mutations in Ralstonia solanacearum loci involved in lipopolysaccharide biogenesis, phospholipid trafficking and peptidoglycan recycling render bacteriophage infection.

    PubMed

    Hong, Yu-Hau; Huang, Chi; Wang, Kuan-Chung; Chu, Tai-Hsiang; Li, Chien-Hui; Chu, Yu-Ju; Cheng, Chiu-Ping

    2014-09-01

    Ralstonia solanacearum causes deadly wilting on many crops worldwide. However, the information on its components important for cell integrity and interactions with phages is limited. By systematically characterizing mutants resistant to a T7-like phage, we showed that the biosynthesis of rough lipopolysaccharides (R-LPS) was crucial for maintaining the membrane integrity, while the production of smooth LPS (S-LPS) was required for the resistance to polymyxin B and phage adsorption. Furthermore, RSc0154/ampG disruption did not affect LPS production and phage adsorption but may have caused aberrant release of peptidoglycan fragments, thus hindering phage DNA injection into or virion release from the cell. Mutations in the RSc2958-RSc2962/mla cluster, although not affecting LPS production, may have caused elevated phospholipid level in the outer leaflet of the outer membrane, consequently sheltering the mutants from phage adsorption on the O-antigen. These results specify important roles of the biogenesis and homeogenesis of envelope components for R. solanacearum-phage interaction.

  17. The C-terminal extension of PrhG impairs its activation of hrp expression and virulence in Ralstonia solanacearum.

    PubMed

    Zhang, Yong; Luo, Feng; Hikichi, Yasufumi; Kiba, Akinori; Yasuo, Igarashi; Ohnishi, Kouhei

    2015-04-01

    Ralstonia solanacearum is the second most destructive bacterial plant pathogens worldwide and HrpG is the master regulator of its pathogenicity. PrhG is a close paralogue of HrpG and both belong to OmpR/PhoB family of two-component response regulators. Despite a high similarity (72% global identity and 96% similarity in helix-loop-helix domain), they display distinct roles in pathogenicity. HrpG is necessary for the bacterial growth in planta and pathogenicity, while PrhG is dispensable for bacterial growth in planta and contributes little to pathogenicity. The main difference between HrpG and PrhG is the 50-amino-acid-long C-terminal extension in PrhG (amino-acid residues 230-283), which is absent in HrpG. When this extension is deleted, truncated PrhGs (under the control of its native promoter) allowed complete recovery of bacterial growth in planta and wild-type virulence of hrpG mutant. This novel finding demonstrates that the extension region in PrhG is responsible for the functional difference between HrpG and PrhG, which may block the binding of PrhG to target promoters and result in impaired activation of hrp expression by PrhG and reduced virulence of R. solanacearum.

  18. Phylogenomic analysis of the genus Ralstonia based on 686 single-copy genes.

    PubMed

    Zhang, Yucheng; Qiu, Sai

    2016-01-01

    The genus Ralstonia contains species that are devastating plant pathogens, opportunistic human pathogens, and/or important degraders of xenobiotic and recalcitrant compounds. However, significant nomenclature problems exist, especially for the Ralstonia solanacearum species complex which consists of four phylotypes. Phylogenomics of the Ralstonia genus was investigated via a comprehensive analysis of 39 Ralstonia genomes as well as four genomes of Cupriavidus necator (more commonly known by its previous name Ralstonia eutropha). These data revealed 686 single-copy orthologs that could be extracted from the Ralstonia core-genome and used to reconstruct the phylogeny of the genus Ralstonia. The generated tree has strong bootstrap support for almost all branches. We also estimated the in silico DNA-DNA hybridization (isDDH) and the average nucleotide identity (ANI) values between each genome. Our data confirmed that whole genome sequence data provides a powerful tool to resolve the complex taxonomic questions of the genus Ralstonia, e.g. strains of Ralstonia solanacearum phylotype IIA and IIB may represent two subspecies of R. solanacearum, and strains of R. solanacearum phylotype I and III may be classified into two subspecies of Ralstonia pseudosolanacearum. Recently, strains of R. solanacearum phylotype IV were proposed to be reclassified into different subspecies of Ralstonia syzygii; our study, however, showed that phylotype IV strains had high isDDH values (83.8-96.1 %), indicating it may be not appropriate to classify these closely related strains into different subspecies. We also evaluated the performance of six chromosomal housekeeping genes (gdhA, mutS, adk, leuS, rplB and gyrB) used in Ralstonia phylogenetic inference. The multilocus sequence analysis of these six marker genes was able to reliably infer the phylogenetic relationships of the genus Ralstonia. PMID:26494208

  19. Effects of volatile organic compounds produced by Bacillus amyloliquefaciens on the growth and virulence traits of tomato bacterial wilt pathogen Ralstonia solanacearum.

    PubMed

    Raza, Waseem; Wang, Jichen; Wu, Yuncheng; Ling, Ning; Wei, Zhong; Huang, Qiwei; Shen, Qirong

    2016-09-01

    The production of volatile organic compounds (VOCs) by microbes is an important characteristic for their selection as biocontrol agents against plant pathogens. In this study, we identified the VOCs produced by the biocontrol strain Bacillus amyloliquefaciens T-5 and evaluated their impact on the growth and virulence traits of tomato bacterial wilt pathogen Ralstonia solanacearum. The results showed that the VOCs of strain T-5 significantly inhibited the growth of R. solanacearum in agar medium and in soil. In addition, VOCs significantly inhibited the motility traits, root colonization, biofilm formation, and production of antioxidant enzymes and exopolysaccharides by R. solanacearum. However, no effect of VOCs on the production of hydrolytic enzymes by R. solanacearum was observed. The strain T-5 produced VOCs, including benzenes, ketones, aldehydes, alkanes, acids, and one furan and naphthalene compound; among those, 13 VOCs showed 1-10 % antibacterial activity against R. solanacearum in their produced amounts by T-5; however, the consortium of all VOCs produced on agar medium, in sterilized soil, and in natural soil showed 75, 62, and 85 % growth inhibition of R. solanacearum, respectively. The real-time PCR analysis further confirmed the results when the expression of different virulence- and metabolism-related genes in R. solanacearum cells was decreased after exposure to the VOCs of strain T-5. The results of this study clearly revealed the significance of VOCs in the control of plant pathogens. This information would help to better comprehend the microbial interactions mediated by VOCs in nature and to develop safer strategies to control plant disease. PMID:27183998

  20. Effects of volatile organic compounds produced by Bacillus amyloliquefaciens on the growth and virulence traits of tomato bacterial wilt pathogen Ralstonia solanacearum.

    PubMed

    Raza, Waseem; Wang, Jichen; Wu, Yuncheng; Ling, Ning; Wei, Zhong; Huang, Qiwei; Shen, Qirong

    2016-09-01

    The production of volatile organic compounds (VOCs) by microbes is an important characteristic for their selection as biocontrol agents against plant pathogens. In this study, we identified the VOCs produced by the biocontrol strain Bacillus amyloliquefaciens T-5 and evaluated their impact on the growth and virulence traits of tomato bacterial wilt pathogen Ralstonia solanacearum. The results showed that the VOCs of strain T-5 significantly inhibited the growth of R. solanacearum in agar medium and in soil. In addition, VOCs significantly inhibited the motility traits, root colonization, biofilm formation, and production of antioxidant enzymes and exopolysaccharides by R. solanacearum. However, no effect of VOCs on the production of hydrolytic enzymes by R. solanacearum was observed. The strain T-5 produced VOCs, including benzenes, ketones, aldehydes, alkanes, acids, and one furan and naphthalene compound; among those, 13 VOCs showed 1-10 % antibacterial activity against R. solanacearum in their produced amounts by T-5; however, the consortium of all VOCs produced on agar medium, in sterilized soil, and in natural soil showed 75, 62, and 85 % growth inhibition of R. solanacearum, respectively. The real-time PCR analysis further confirmed the results when the expression of different virulence- and metabolism-related genes in R. solanacearum cells was decreased after exposure to the VOCs of strain T-5. The results of this study clearly revealed the significance of VOCs in the control of plant pathogens. This information would help to better comprehend the microbial interactions mediated by VOCs in nature and to develop safer strategies to control plant disease.

  1. Detection of Ralstonia solanacearum from asymptomatic tomato plants, irrigation water, and soil through non-selective enrichment medium with hrp gene-based bio-PCR.

    PubMed

    Singh, Dinesh; Sinha, Shweta; Yadav, D K; Chaudhary, Garima

    2014-08-01

    Bacterial wilt of tomato caused by Ralstonia solanacearum (Smith) Yabuuchi et al. (Microbiol Immunol 39:897-904, 1995) is a serious disease, which causes losses up to 60 % depending on environmental conditions, soil property, and cultivars. In present investigation, nucleotide sequences of virulence, hypersensitive response and pathogenicity (hrp) gene were used to design a pair of primer (Hrp_rs 2F: 5'-AGAGGTCGACGCGATACAGT-3' and Hrp_rs 2R: 5'-CATGAGCAAGGACGAAGTCA-3') for amplification of bacterial genome. The genomic DNA of 27 isolates of R. solanacearum race 1 biovar 3 & 4 was amplified at 323 bp. The specificity of primer was tested on 13 strains of R. solanacearum with other group of bacteria such as Xanthomonas oryzae pv. oryzae, X. campestris pv. campestris, and X. citri subsp. citri. Primer amplified DNA fragment of R. solanacearum at 323 bp. The sensitivity of the primer was 200 cfu/ml and improved further detection level by using non-specific enrichment medium casamino acids-pepton-glucose broth followed by PCR (BIO-PCR). Out of 130 samples of asymptomatic tomato plants, irrigation water, and soil collected from bacterial wilt infested field in different agro-climatic regions of India, R. solanacearum was detected from 86.9, 88.5, and 90.9 per cents samples using BIO-PCR, respectively. The primer was found specific for detecting viable and virulent strains of R. solanacearum and useful for the diagnosis of R. solanacearum in tomato seedlings and monitoring of pathogen in irrigation water and soil.

  2. Cloning and characterization of PR5 gene from Curcuma amada and Zingiber officinale in response to Ralstonia solanacearum infection.

    PubMed

    Prasath, D; El-Sharkawy, I; Sherif, S; Tiwary, K S; Jayasankar, S

    2011-10-01

    Ginger (Zingiber officinale Roscoe), is an important spice crop that is badly affected by Ralstonia solanacearum wilt. Ginger does not set seed and sexual recombination has never been reported. In spite of extensive search in its habitats, no resistance source to Ralstonia induced bacterial wilt, could be located in ginger. Curcuma amada Roxb. is a potential donor for bacterial wilt resistance to Z. officinale, if the exact mechanism of resistance is understood. Pathogenesis-related (PR)-5 proteins are a family of proteins that are induced by different phytopathogens in many plants and share significant sequence similarity with thaumatin. Two putative PR5 genes, CaPR5 and ZoPR5, were amplified from C. amada and ginger, which encode precursor proteins of 227 and 224 amino acid residues, respectively, and share high homology with a number of other PR5 genes. The secondary and three-dimensional structure comparison did not reveal any striking differences between these two proteins. The expression of Ca and ZoPR5s under R. solanacearum inoculation was analyzed at different time points using quantitative real-time PCR (qRT-PCR). Our results reveal that CaPR5 is readily induced by the bacterium in C. amada, while ZoPR5 induction was very weak and slow in ginger. These results suggest that the CaPR5 could play a role in the molecular defense response of C. amada to pathogen attack. This is the first report of the isolation of PR5 gene from the C. amada and Z. officinale. Promoter analysis indicates the presence of a silencing element binding factor in ZoPR5-promoter, but not in CaPR5. Prospective promoter elements, such as GT-1 box and TGTCA, implicated as being positive regulatory elements for expression of PR proteins, occur in the 5'-flanking sequences of the CaPR5. Transient GUS expression study confirms its action with a weaker GUS expression in ginger, indicating that the PR5 expression may be controlled in the promoter. PMID:21594675

  3. Genome-Enabled Phylogeographic Investigation of the Quarantine Pathogen Ralstonia solanacearum Race 3 Biovar 2 and Screening for Sources of Resistance Against Its Core Effectors.

    PubMed

    Clarke, Christopher R; Studholme, David J; Hayes, Byron; Runde, Brendan; Weisberg, Alexandra; Cai, Rongman; Wroblewski, Tadeusz; Daunay, Marie-Christine; Wicker, Emmanuel; Castillo, Jose A; Vinatzer, Boris A

    2015-05-01

    Phylogeographic studies inform about routes of pathogen dissemination and are instrumental for improving import/export controls. Genomes of 17 isolates of the bacterial wilt and potato brown rot pathogen Ralstonia solanacearum race 3 biovar 2 (R3bv2), a Select Agent in the United States, were thus analyzed to get insight into the phylogeography of this pathogen. Thirteen of fourteen isolates from Europe, Africa, and Asia were found to belong to a single clonal lineage while isolates from South America were genetically diverse and tended to carry ancestral alleles at the analyzed genomic loci consistent with a South American origin of R3bv2. The R3bv2 isolates share a core repertoire of 31 type III-secreted effector genes representing excellent candidates to be targeted with resistance genes in breeding programs to develop durable disease resistance. Toward this goal, 27 R3bv2 effectors were tested in eggplant, tomato, pepper, tobacco, and lettuce for induction of a hypersensitive-like response indicative of recognition by cognate resistance receptors. Fifteen effectors, eight of them core effectors, triggered a response in one or more plant species. These genotypes may harbor resistance genes that could be identified and mapped, cloned, and expressed in tomato or potato, for which sources of genetic resistance to R3bv2 are extremely limited.

  4. Evaluation of the Antibacterial Effects and Mechanism of Action of Protocatechualdehyde against Ralstonia solanacearum.

    PubMed

    Li, Shili; Yu, Yanmei; Chen, Juanni; Guo, Bing; Yang, Liang; Ding, Wei

    2016-01-01

    Protocatechualdehyde (PCA) is an important plant-derived natural product that has been associated with a wide variety of biological activities and has been widely used in medicine as an antioxidant, anti-aging and an anti-inflammatory agent. However, fewer reports concerning its antibacterial effects on plant-pathogenic bacteria exist. Therefore, in this study, protocatechualdehyde was evaluated for its antibacterial activity against plant pathogens along with the mechanism of its antibacterial action. PCA at 40 μg/mL was highly active against R. solanacearum and significantly inhibited its growth. The minimum bactericidal concentration and minimum inhibitory concentration values for PCA were 40 μg/mL and 20 μg/mL, respectively. Further investigation of the mechanism of action of PCA via transmission electron microscopy and biological assays indicated that the destruction of the cell structure, the shapes and the inhibition of biofilm formation were important. In addition, the application of PCA effectively reduced the incidence of bacterial wilt on tobacco under greenhouse conditions, and the control efficiency was as high as 92.01% at nine days after inoculation. Taken together, these findings suggest that PCA exhibits strong antibacterial activity against R. solanacearum and has the potential to be applied as an effective antibacterial agent for controlling bacterial wilt caused by R. solanacearum. PMID:27294898

  5. Evaluation of the Antibacterial Effects and Mechanism of Action of Protocatechualdehyde against Ralstonia solanacearum.

    PubMed

    Li, Shili; Yu, Yanmei; Chen, Juanni; Guo, Bing; Yang, Liang; Ding, Wei

    2016-01-01

    Protocatechualdehyde (PCA) is an important plant-derived natural product that has been associated with a wide variety of biological activities and has been widely used in medicine as an antioxidant, anti-aging and an anti-inflammatory agent. However, fewer reports concerning its antibacterial effects on plant-pathogenic bacteria exist. Therefore, in this study, protocatechualdehyde was evaluated for its antibacterial activity against plant pathogens along with the mechanism of its antibacterial action. PCA at 40 μg/mL was highly active against R. solanacearum and significantly inhibited its growth. The minimum bactericidal concentration and minimum inhibitory concentration values for PCA were 40 μg/mL and 20 μg/mL, respectively. Further investigation of the mechanism of action of PCA via transmission electron microscopy and biological assays indicated that the destruction of the cell structure, the shapes and the inhibition of biofilm formation were important. In addition, the application of PCA effectively reduced the incidence of bacterial wilt on tobacco under greenhouse conditions, and the control efficiency was as high as 92.01% at nine days after inoculation. Taken together, these findings suggest that PCA exhibits strong antibacterial activity against R. solanacearum and has the potential to be applied as an effective antibacterial agent for controlling bacterial wilt caused by R. solanacearum.

  6. Non-Instrumented Nucleic Acid Amplification (NINA) for Rapid Detection of Ralstonia solanacearum Race 3 Biovar 2

    PubMed Central

    Kubota, Ryo; LaBarre, Paul; Singleton, Jered; Beddoe, Andy; Weigl, Bernhard H.; Alvarez, Anne M.; Jenkins, Daniel M.

    2014-01-01

    We report on the use of a non-instrumented device for the implementation of a loop-mediated amplification (LAMP) based assay for the select-agent bacterial-wilt pathogen Ralstonia solanacearum race 3 biovar 2. Heat energy is generated within the device by the exothermic hydration of calcium oxide, and the reaction temperature is regulated by storing latent energy at the melting temperature of a renewable lipid-based engineered phase-change material. Endpoint detection of the LAMP reaction is achieved without opening the reaction tube by observing the fluorescence of an innovative FRET-based hybridization probe with a simple custom fluorometer. Non-instrumented devices could maintain reactions near the design temperature of 63°C for at least an hour. Using this approach DNA extracted from the pathogen could be detected at fewer than ten copies within a 25 μL reaction mix, illustrating the potential of these technologies for simple, powerful agricultural diagnostics in the field. Furthermore, the assay was just as reliable when implemented in a tropical environment at 31°C as it was when implemented in an air-conditioned lab maintained at 22°C, illustrating the potential value of the technology for field conditions in the tropics and subtropics. PMID:25485176

  7. A TaqMan-based multiplex qPCR assay and DNA extraction method for phylotype IIB sequevars 1&2 (select agent) strains of Ralstonia solanacearum

    SciTech Connect

    Stulberg, Michael J.; Huang, Qi

    2015-10-01

    Ralstonia solanacearum race 3 biovar 2 strains belonging to phylotype IIB, sequevars 1 and 2 (IIB-1&2) cause brown rot of potato in temperate climates, and are quarantined pathogens in Canada and Europe. Since these strains are not established in the U.S. and because of their potential risk to the potato industry, the U.S. government has listed them as select agents. Cultivated geraniums are also a host and have the potential to spread the pathogen through trade, and its extracts strongly inhibits DNA-based detection methods. We designed four primer and probe sets for an improved qPCR method that targets stable regions of DNA. RsSA1 and RsSA2 recognize IIB-1&2 strains, RsII recognizes the current phylotype II (the newly proposed R. solanacearum species) strains (and a non-plant associated R. mannitolilytica), and Cox1 recognizes eight plant species including major hosts of R. solanacearum such as potato, tomato and cultivated geranium as an internal plant control. We multiplexed the RsSA2 with the RsII and Cox1 sets to provide two layers of detection of a positive IIB-1&2 sample, and to validate plant extracts and qPCR reactions. The TaqMan-based uniplex and multiplex qPCR assays correctly identified 34 IIB-1&2 and 52 phylotype II strains out of 90 R. solanacearum species complex strains. Additionally, the multiplex qPCR assay was validated successfully using 169 artificially inoculated symptomatic and asymptomatic plant samples from multiple plant hosts including geranium. Moreover, we developed an extraction buffer that allowed for a quick and easy DNA extraction from infected plants including geranium for detection of R. solanacearum by qPCR. Our multiplex qPCR assay, especially when coupled with the quick extraction buffer method, allows for quick, easy and reliable detection and differentiation of the IIB-1&2 strains of R. solanacearum.

  8. Bacillus thuringiensis Suppresses Bacterial wilt Disease Caused by Ralstonia solanacearum with Systemic Induction of Defense-Related Gene Expression in Tomato

    PubMed Central

    Hyakumachi, Mitsuro; Nishimura, Mitsuyoshi; Arakawa, Tatsuyuki; Asano, Shinichiro; Yoshida, Shigenobu; Tsushima, Seiya; Takahashi, Hideki

    2013-01-01

    Bacillus thuringiensis is a naturally abundant Gram-positive bacterium and a well-known, effective bio-insecticide. Recently, B. thuringiensis has attracted considerable attention as a potential biological control agent for the suppression of plant diseases. In this study, the bacterial wilt disease-suppressing activity of B. thuringiensis was examined in tomato plants. Treatment of tomato roots with B. thuringiensis culture followed by challenge inoculation with Ralstonia solanacearum suppressed the development of wilt symptoms to less than one third of the control. This disease suppression in tomato plants was reproduced by pretreating their roots with a cell-free filtrate (CF) that had been fractionated from B. thuringiensis culture by centrifugation and filtration. In tomato plants challenge-inoculated with R. solanacearum after pretreatment with CF, the growth of R. solanacearum in stem tissues clearly decreased, and expression of defense-related genes such as PR-1, acidic chitinase, and β-1,3-glucanase was induced in stem and leaf tissues. Furthermore, the stem tissues of tomato plants with their roots were pretreated with CF exhibited resistance against direct inoculation with R. solanacearum. Taken together, these results suggest that treatment of tomato roots with the CF of B. thuringiensis systemically suppresses bacterial wilt through systemic activation of the plant defense system. PMID:23257909

  9. Hydroxycinnamic acid degradation, a broadly conserved trait, protects Ralstonia solanacearum from chemical plant defenses and contributes to root colonization and virulence

    PubMed Central

    Lowe, Tiffany M.; Ailloud, Florent; Allen, Caitilyn

    2014-01-01

    Plants produce hydroxycinnamic acid defense compounds (HCAs) to combat pathogens, such as the bacterium Ralstonia solanacearum. We showed that an HCA degradation pathway is genetically and functionally conserved across diverse R. solanacearum strains. Further, a Δfcs (feruloyl-CoA synthetase) mutant that cannot degrade HCAs was less virulent on tomato plants. To understand the role of HCA degradation in bacterial wilt disease, we tested the following hypotheses: HCA degradation helps the pathogen (1) grow, as a carbon source; (2) spread, by reducing physical barriers HCA-derived; and (3) survive plant antimicrobial compounds. Although HCA degradation enabled R. solanacearum growth on HCAs in vitro, HCA degradation was dispensable for growth in xylem sap and root exudate, suggesting that HCAs are not significant carbon sources in planta. Acetyl-bromide quantification of lignin demonstrated that R. solanacearum infections did not affect the gross quantity or distribution of stem lignin. However, the Δfcs mutant was significantly more susceptible to inhibition by two HCAs: caffeate and p-coumarate. Finally, plant colonization assays suggested that HCA degradation facilitates early stages of infection and root colonization. Together, these results indicated that ability to degrade HCAs contributes to bacterial wilt virulence by facilitating root entry and by protecting the pathogen from HCA toxicity. PMID:25423265

  10. Arabidopsis wat1 (walls are thin1)-mediated resistance to the bacterial vascular pathogen, Ralstonia solanacearum, is accompanied by cross-regulation of salicylic acid and tryptophan metabolism.

    PubMed

    Denancé, Nicolas; Ranocha, Philippe; Oria, Nicolas; Barlet, Xavier; Rivière, Marie-Pierre; Yadeta, Koste A; Hoffmann, Laurent; Perreau, François; Clément, Gilles; Maia-Grondard, Alessandra; van den Berg, Grardy C M; Savelli, Bruno; Fournier, Sylvie; Aubert, Yann; Pelletier, Sandra; Thomma, Bart P H J; Molina, Antonio; Jouanin, Lise; Marco, Yves; Goffner, Deborah

    2013-01-01

    Inactivation of Arabidopsis WAT1 (Walls Are Thin1), a gene required for secondary cell-wall deposition, conferred broad-spectrum resistance to vascular pathogens, including the bacteria Ralstonia solanacearum and Xanthomonas campestris pv. campestris, and the fungi Verticillium dahliae and Verticillium albo-atrum. Introduction of NahG, the bacterial salicylic acid (SA)-degrading salicylate hydroxylase gene, into the wat1 mutant restored full susceptibility to both R. solanacearum and X. campestris pv. campestris. Moreover, SA content was constitutively higher in wat1 roots, further supporting a role for SA in wat1-mediated resistance to vascular pathogens. By combining transcriptomic and metabolomic data, we demonstrated a general repression of indole metabolism in wat1-1 roots as shown by constitutive down-regulation of several genes encoding proteins of the indole glucosinolate biosynthetic pathway and reduced amounts of tryptophan (Trp), indole-3-acetic acid and neoglucobrassicin, the major form of indole glucosinolate in roots. Furthermore, the susceptibility of the wat1 mutant to R. solanacearum was partially restored when crossed with either the trp5 mutant, an over-accumulator of Trp, or Pro35S:AFB1-myc, in which indole-3-acetic acid signaling is constitutively activated. Our original hypothesis placed cell-wall modifications at the heart of the wat1 resistance phenotype. However, the results presented here suggest a mechanism involving root-localized metabolic channeling away from indole metabolites to SA as a central feature of wat1 resistance to R. solanacearum.

  11. Hydroxycinnamic Acid Degradation, a Broadly Conserved Trait, Protects Ralstonia solanacearum from Chemical Plant Defenses and Contributes to Root Colonization and Virulence.

    PubMed

    Lowe, Tiffany M; Ailloud, Florent; Allen, Caitilyn

    2015-03-01

    Plants produce hydroxycinnamic acid (HCA) defense compounds to combat pathogens, such as the bacterium Ralstonia solanacearum. We showed that an HCA degradation pathway is genetically and functionally conserved across diverse R. solanacearum strains. Further, a feruloyl-CoA synthetase (Δfcs) mutant that cannot degrade HCA was less virulent on tomato plants. To understand the role of HCA degradation in bacterial wilt disease, we tested the following hypotheses: HCA degradation helps the pathogen i) grow, as a carbon source; ii) spread, by reducing HCA-derived physical barriers; and iii) survive plant antimicrobial compounds. Although HCA degradation enabled R. solanacearum growth on HCA in vitro, HCA degradation was dispensable for growth in xylem sap and root exudate, suggesting that HCA are not significant carbon sources in planta. Acetyl-bromide quantification of lignin demonstrated that R. solanacearum infections did not affect the gross quantity or distribution of stem lignin. However, the Δfcs mutant was significantly more susceptible to inhibition by two HCA, namely, caffeate and p-coumarate. Finally, plant colonization assays suggested that HCA degradation facilitates early stages of infection and root colonization. Together, these results indicated that ability to degrade HCA contributes to bacterial wilt virulence by facilitating root entry and by protecting the pathogen from HCA toxicity.

  12. Comparative Secretome Analysis of Ralstonia solanacearum Type 3 Secretion-Associated Mutants Reveals a Fine Control of Effector Delivery, Essential for Bacterial Pathogenicity.

    PubMed

    Lonjon, Fabien; Turner, Marie; Henry, Céline; Rengel, David; Lohou, David; van de Kerkhove, Quitterie; Cazalé, Anne-Claire; Peeters, Nemo; Genin, Stéphane; Vailleau, Fabienne

    2016-02-01

    Ralstonia solanacearum, the causal agent of bacterial wilt, exerts its pathogenicity through more than a hundred secreted proteins, many of them depending directly on the functionality of a type 3 secretion system. To date, only few type 3 effectors have been identified as required for bacterial pathogenicity, notably because of redundancy among the large R. solanacearum effector repertoire. In order to identify groups of effectors collectively promoting disease on susceptible hosts, we investigated the role of putative post-translational regulators in the control of type 3 secretion. A shotgun secretome analysis with label-free quantification using tandem mass spectrometry was performed on the R. solanacearum GMI1000 strain. There were 228 proteins identified, among which a large proportion of type 3 effectors, called Rip (Ralstonia injected proteins). Thanks to this proteomic approach, RipBJ was identified as a new effector specifically secreted through type 3 secretion system and translocated into plant cells. A focused Rip secretome analysis using hpa (hypersensitive response and pathogenicity associated) mutants revealed a fine secretion regulation and specific subsets of Rips with different secretion patterns. We showed that a set of Rips (RipF1, RipW, RipX, RipAB, and RipAM) are secreted in an Hpa-independent manner. We hypothesize that these Rips could be preferentially involved in the first stages of type 3 secretion. In addition, the secretion of about thirty other Rips is controlled by HpaB and HpaG. HpaB, a candidate chaperone was shown to positively control secretion of numerous Rips, whereas HpaG was shown to act as a negative regulator of secretion. To evaluate the impact of altered type 3 effectors secretion on plant pathogenesis, the hpa mutants were assayed on several host plants. HpaB was required for bacterial pathogenicity on multiple hosts whereas HpaG was found to be specifically required for full R. solanacearum pathogenicity on the legume

  13. CaWRKY6 transcriptionally activates CaWRKY40, regulates Ralstonia solanacearum resistance, and confers high-temperature and high-humidity tolerance in pepper.

    PubMed

    Cai, Hanyang; Yang, Sheng; Yan, Yan; Xiao, Zhuoli; Cheng, Junbin; Wu, Ji; Qiu, Ailian; Lai, Yan; Mou, Shaoliang; Guan, Deyi; Huang, Ronghua; He, Shuilin

    2015-06-01

    High temperature (HT), high humidity (HH), and pathogen infection often co-occur and negatively affect plant growth. However, these stress factors and plant responses are generally studied in isolation. The mechanisms of synergistic responses to combined stresses are poorly understood. We isolated the subgroup IIb WRKY family member CaWRKY6 from Capsicum annuum and performed quantitative real-time PCR analysis. CaWRKY6 expression was upregulated by individual or simultaneous treatment with HT, HH, combined HT and HH (HTHH), and Ralstonia solanacearum inoculation, and responded to exogenous application of jasmonic acid (JA), ethephon, and abscisic acid (ABA). Virus-induced gene silencing of CaWRKY6 enhanced pepper plant susceptibility to R. solanacearum and HTHH, and downregulated the hypersensitive response (HR), JA-, ethylene (ET)-, and ABA-induced marker gene expression, and thermotolerance-associated expression of CaHSP24, ER-small CaSHP, and Chl-small CaHSP. CaWRKY6 overexpression in pepper attenuated the HTHH-induced suppression of resistance to R. solanacearum infection. CaWRKY6 bound to and activated the CaWRKY40 promoter in planta, which is a pepper WRKY that regulates heat-stress tolerance and R. solanacearum resistance. CaWRKY40 silencing significantly blocked HR-induced cell death and reduced transcriptional expression of CaWRKY40. These data suggest that CaWRKY6 is a positive regulator of R. solanacearum resistance and heat-stress tolerance, which occurs in part by activating CaWRKY40.

  14. Induction of the viable but nonculturable state of Ralstonia solanacearum by low temperature in the soil microcosm and its resuscitation by catalase.

    PubMed

    Kong, Hyun Gi; Bae, Ju Young; Lee, Hyoung Ju; Joo, Hae Jin; Jung, Eun Joo; Chung, Eunsook; Lee, Seon-Woo

    2014-01-01

    Ralstonia solanacearum is the causal agent of bacterial wilt on a wide variety of plants, and enters a viable but nonculturable (VBNC) state under stress conditions in soil and water. Here, we adopted an artificial soil microcosm (ASM) to investigate the VBNC state of R. solanacearum induced by low temperature. The culturability of R. solanacearum strains SL341 and GMI1000 rapidly decreased at 4°C in modified ASM (mASM), while it was stably maintained at 25°C in mASM. We hypothesized that bacterial cells at 4°C in mASM are viable but nonculturable. Total protein profiles of SL341 cells at 4°C in mASM did not differ from those of SL341 culturable cells at 25°C in mASM. Moreover, the VBNC cells maintained in the mASM retained respiration activity. Catalase treatment effectively restored the culturability of nonculturable cells in mASM, while temperature increase or other treatments used for resuscitation of other bacteria were not effective. The resuscitated R. solanacearum from VBNC state displayed normal level of bacterial virulence on tomato plants compared with its original culturable bacteria. Expression of omp, oxyR, rpoS, dps, and the 16S rRNA gene quantified by RT-qPCR did not differ significantly between the culturable and VBNC states of R. solanacearum. Our results suggested that the VBNC bacterial cells in mASM induced by low temperature exist in a physiologically unique state.

  15. Expression of a wheat MYB gene in transgenic tobacco enhances resistance to Ralstonia solanacearum, and to drought and salt stresses.

    PubMed

    Liu, Hongxia; Zhou, Xianyao; Dong, Na; Liu, Xin; Zhang, Huaiyu; Zhang, Zengyan

    2011-09-01

    MYB transcription factors play diverse roles in plant growth, developmental processes and stress responses. A full-length cDNA sequence of a MYB gene, namely TaPIMP1, was isolated from wheat (Triticum aestivum L.). The TaPIMP1 transcript level was significantly up-regulated by inoculation with a fungal pathogen Bipolaris sorokiniana and by drought treatment. TaPIMP1 encodes the MYB protein TaPIMP1 consisting of 323 amino acids. TaPIMP1 contains two MYB DNA binding domains (R2, R3), two putative nuclear localization sites and two putative transcription activation domains. TaPIMP1 is a new member of the R2R3-MYB transcription factor subfamily. Transient expression in onion epidermal cells of GFP fused with TaPIMP1 proved that subcellular localization of TaPIMP1 occurred in the nucleus. The TaPIMP1 gene was transferred into tobacco (Nicotiana tabacum L.) cultivar W38 by Agrobacterium-mediated transformation. After screening through PCR and RT-PCR analyses, transgenic tobacco lines expressing TaPIMP1 were identified and evaluated for pathogen resistance, and drought and salt tolerance. Compared to untransformed tobacco host plants, TaPIMP1 expressing plants displayed significantly enhanced resistance to Ralstonia solanacearum and exhibited improved tolerances to drought and salt stresses. In these transgenic lines, the activities of phenylalanine ammonia-lyase (PAL) and superoxide dismutase (SOD) were significantly increased relative to wild-type tobacco plants. The results suggested that the wheat R2R3-MYB transcription factor plays an important role in modulating responses to biotic and abiotic stresses.

  16. Expression of a wheat MYB gene in transgenic tobacco enhances resistance to Ralstonia solanacearum, and to drought and salt stresses.

    PubMed

    Liu, Hongxia; Zhou, Xianyao; Dong, Na; Liu, Xin; Zhang, Huaiyu; Zhang, Zengyan

    2011-09-01

    MYB transcription factors play diverse roles in plant growth, developmental processes and stress responses. A full-length cDNA sequence of a MYB gene, namely TaPIMP1, was isolated from wheat (Triticum aestivum L.). The TaPIMP1 transcript level was significantly up-regulated by inoculation with a fungal pathogen Bipolaris sorokiniana and by drought treatment. TaPIMP1 encodes the MYB protein TaPIMP1 consisting of 323 amino acids. TaPIMP1 contains two MYB DNA binding domains (R2, R3), two putative nuclear localization sites and two putative transcription activation domains. TaPIMP1 is a new member of the R2R3-MYB transcription factor subfamily. Transient expression in onion epidermal cells of GFP fused with TaPIMP1 proved that subcellular localization of TaPIMP1 occurred in the nucleus. The TaPIMP1 gene was transferred into tobacco (Nicotiana tabacum L.) cultivar W38 by Agrobacterium-mediated transformation. After screening through PCR and RT-PCR analyses, transgenic tobacco lines expressing TaPIMP1 were identified and evaluated for pathogen resistance, and drought and salt tolerance. Compared to untransformed tobacco host plants, TaPIMP1 expressing plants displayed significantly enhanced resistance to Ralstonia solanacearum and exhibited improved tolerances to drought and salt stresses. In these transgenic lines, the activities of phenylalanine ammonia-lyase (PAL) and superoxide dismutase (SOD) were significantly increased relative to wild-type tobacco plants. The results suggested that the wheat R2R3-MYB transcription factor plays an important role in modulating responses to biotic and abiotic stresses. PMID:21597961

  17. Ralstonia solanacearum Type III Effector RipAY Is a Glutathione-Degrading Enzyme That Is Activated by Plant Cytosolic Thioredoxins and Suppresses Plant Immunity

    PubMed Central

    Hatanaka, Tadashi; Nakano, Masahito; Oda, Kenji

    2016-01-01

    ABSTRACT The plant pathogen Ralstonia solanacearum uses a large repertoire of type III effector proteins to succeed in infection. To clarify the function of effector proteins in host eukaryote cells, we expressed effectors in yeast cells and identified seven effector proteins that interfere with yeast growth. One of the effector proteins, RipAY, was found to share homology with the ChaC family proteins that function as γ-glutamyl cyclotransferases, which degrade glutathione (GSH), a tripeptide that plays important roles in the plant immune system. RipAY significantly inhibited yeast growth and simultaneously induced rapid GSH depletion when expressed in yeast cells. The in vitro GSH degradation activity of RipAY is specifically activated by eukaryotic factors in the yeast and plant extracts. Biochemical purification of the yeast protein identified that RipAY is activated by thioredoxin TRX2. On the other hand, RipAY was not activated by bacterial thioredoxins. Interestingly, RipAY was activated by plant h-type thioredoxins that exist in large amounts in the plant cytosol, but not by chloroplastic m-, f-, x-, y- and z-type thioredoxins, in a thiol-independent manner. The transient expression of RipAY decreased the GSH level in plant cells and affected the flg22-triggered production of reactive oxygen species (ROS) and expression of pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) marker genes in Nicotiana benthamiana leaves. These results indicate that RipAY is activated by host cytosolic thioredoxins and degrades GSH specifically in plant cells to suppress plant immunity. PMID:27073091

  18. Development of variable number of tandem repeats typing schemes for Ralstonia solanacearum, the agent of bacterial wilt, banana Moko disease and potato brown rot.

    PubMed

    N'guessan, Carine Aya; Brisse, Sylvain; Le Roux-Nio, Anne-Claire; Poussier, Stéphane; Koné, Daouda; Wicker, Emmanuel

    2013-03-01

    Ralstonia solanacearum is an important soil borne bacterial plant pathogen causing bacterial wilt on many important crops. To better monitor epidemics, efficient tools that can identify and discriminate populations are needed. In this study, we assessed variable number of tandem repeats (VNTR) genotyping as a new tool for epidemiological surveillance of R. solanacearum phylotypes, and more specifically for the monitoring of the monomorphic ecotypes "Moko" (banana-pathogenic) and "brown rot" (potato-pathogenic under cool conditions). Screening of six R. solanacearum genome sequences lead to select 36 VNTR loci that were preliminarily amplified on 24 strains. From this step, 26 single-locus primer pairs were multiplexed, and applied to a worldwide collection of 337 strains encompassing the whole phylogenetic diversity, with revelation on a capillary-electrophoresis genotype. Four loci were monomorphic within all phylotypes and were not retained; the other loci were highly polymorphic but displayed a clear phylotype-specificity. Phylotype-specific MLVA schemes were thus defined, based on 13 loci for phylotype I, 12 loci for phylotype II, 11 loci for phylotype III and 6 for phylotype IV. MLVA typing was significantly more discriminative than egl-based sequevar typing, particularly on monomorphic "brown rot" ecotype (phylotype IIB/sequevar 1) and "Moko disease" clade 4 (Phylotype IIB/sequevar 4). Our results raise promising prospects for studies of population genetic structures and epidemiological monitoring.

  19. Meta-analysis Reveals That the Genus Pseudomonas Can Be a Better Choice of Biological Control Agent against Bacterial Wilt Disease Caused by Ralstonia solanacearum.

    PubMed

    Chandrasekaran, Murugesan; Subramanian, Dharaneedharan; Yoon, Ee; Kwon, Taehoon; Chun, Se-Chul

    2016-06-01

    Biological control agents (BCAs) from different microbial taxa are increasingly used to control bacterial wilt caused by Ralstonia solanacearum. However, a quantitative research synthesis has not been conducted on the role of BCAs in disease suppression. Therefore, the present study aimed to meta-analyze the impacts of BCAs on both Ralstonia wilt disease suppression and plant (host) growth promotion. The analysis showed that the extent of disease suppression by BCAs varied widely among studies, with effect size (log response ratio) ranging from -2.84 to 2.13. The disease incidence and severity were significantly decreased on average by 53.7% and 49.3%, respectively. BCAs inoculation also significantly increased fresh and dry weight by 34.4% and 36.1%, respectively on average. Also, BCAs inoculation significantly increased plant yield by 66%. Mean effect sizes for genus Pseudomonas sp. as BCAs were higher than for genus Bacillus spp. Among antagonists tested, P. fluorescens, P. putida, B. cereus, B. subtilis and B. amyloliquefaciens were found to be more effective in general for disease reduction. Across studies, highest disease control was found for P. fluorescens, annual plants, co-inoculation with more than one BCA, soil drench and greenhouse condition were found to be essential in understanding plant responses to R. solanacearum. Our results suggest that more efforts should be devoted to harnessing the potential beneficial effects of these antagonists, not just for plant growth promoting traits but also in mode of applications, BCAs formulations and their field studies should be considered in the future for R. solanacearum wilt disease suppression. PMID:27298597

  20. Meta-analysis Reveals That the Genus Pseudomonas Can Be a Better Choice of Biological Control Agent against Bacterial Wilt Disease Caused by Ralstonia solanacearum

    PubMed Central

    Chandrasekaran, Murugesan; Subramanian, Dharaneedharan; Yoon, Ee; Kwon, Taehoon; Chun, Se-Chul

    2016-01-01

    Biological control agents (BCAs) from different microbial taxa are increasingly used to control bacterial wilt caused by Ralstonia solanacearum. However, a quantitative research synthesis has not been conducted on the role of BCAs in disease suppression. Therefore, the present study aimed to meta-analyze the impacts of BCAs on both Ralstonia wilt disease suppression and plant (host) growth promotion. The analysis showed that the extent of disease suppression by BCAs varied widely among studies, with effect size (log response ratio) ranging from −2.84 to 2.13. The disease incidence and severity were significantly decreased on average by 53.7% and 49.3%, respectively. BCAs inoculation also significantly increased fresh and dry weight by 34.4% and 36.1%, respectively on average. Also, BCAs inoculation significantly increased plant yield by 66%. Mean effect sizes for genus Pseudomonas sp. as BCAs were higher than for genus Bacillus spp. Among antagonists tested, P. fluorescens, P. putida, B. cereus, B. subtilis and B. amyloliquefaciens were found to be more effective in general for disease reduction. Across studies, highest disease control was found for P. fluorescens, annual plants, co-inoculation with more than one BCA, soil drench and greenhouse condition were found to be essential in understanding plant responses to R. solanacearum. Our results suggest that more efforts should be devoted to harnessing the potential beneficial effects of these antagonists, not just for plant growth promoting traits but also in mode of applications, BCAs formulations and their field studies should be considered in the future for R. solanacearum wilt disease suppression. PMID:27298597

  1. Meta-analysis Reveals That the Genus Pseudomonas Can Be a Better Choice of Biological Control Agent against Bacterial Wilt Disease Caused by Ralstonia solanacearum.

    PubMed

    Chandrasekaran, Murugesan; Subramanian, Dharaneedharan; Yoon, Ee; Kwon, Taehoon; Chun, Se-Chul

    2016-06-01

    Biological control agents (BCAs) from different microbial taxa are increasingly used to control bacterial wilt caused by Ralstonia solanacearum. However, a quantitative research synthesis has not been conducted on the role of BCAs in disease suppression. Therefore, the present study aimed to meta-analyze the impacts of BCAs on both Ralstonia wilt disease suppression and plant (host) growth promotion. The analysis showed that the extent of disease suppression by BCAs varied widely among studies, with effect size (log response ratio) ranging from -2.84 to 2.13. The disease incidence and severity were significantly decreased on average by 53.7% and 49.3%, respectively. BCAs inoculation also significantly increased fresh and dry weight by 34.4% and 36.1%, respectively on average. Also, BCAs inoculation significantly increased plant yield by 66%. Mean effect sizes for genus Pseudomonas sp. as BCAs were higher than for genus Bacillus spp. Among antagonists tested, P. fluorescens, P. putida, B. cereus, B. subtilis and B. amyloliquefaciens were found to be more effective in general for disease reduction. Across studies, highest disease control was found for P. fluorescens, annual plants, co-inoculation with more than one BCA, soil drench and greenhouse condition were found to be essential in understanding plant responses to R. solanacearum. Our results suggest that more efforts should be devoted to harnessing the potential beneficial effects of these antagonists, not just for plant growth promoting traits but also in mode of applications, BCAs formulations and their field studies should be considered in the future for R. solanacearum wilt disease suppression.

  2. Application of Variable-Number Tandem-Repeat Typing To Discriminate Ralstonia solanacearum Strains Associated with English Watercourses and Disease Outbreaks

    PubMed Central

    Bryant, Ruth; Bew, Janice; Conyers, Christine; Stones, Robert; Alcock, Michael; Elphinstone, John

    2013-01-01

    Variable-number tandem-repeat (VNTR) analysis was used for high-resolution discrimination among Ralstonia solanacearum phylotype IIB sequevar 1 (PIIB-1) isolates and further evaluated for use in source tracing. Five tandem-repeat-containing loci (comprising six tandem repeats) discriminated 17 different VNTR profiles among 75 isolates from potato, geranium, bittersweet (Solanum dulcamara), tomato, and the environment. R. solanacearum isolates from crops at three unrelated outbreak sites where river water had been used for irrigation had distinct VNTR profiles that were shared with PIIB-1 isolates from infected bittersweet growing upriver of each site. The VNTR profiling results supported the implication that the source of R. solanacearum at each outbreak was contaminated river water. Analysis of 51 isolates from bittersweet growing in river water at different locations provided a means to evaluate the technique for studying the epidemiology of the pathogen in the environment. Ten different VNTR profiles were identified among bittersweet PIIB-1 isolates from the River Thames. Repeated findings of contiguous river stretches that produced isolates that shared single VNTR profiles supported the hypothesis that the pathogen had disseminated from infected bittersweet plants located upriver. VNTR profiles shared between bittersweet isolates from two widely separated Thames tributaries (River Ray and River Colne) suggested they were independently contaminated with the same clonal type. Some bittersweet isolates had VNTR profiles that were shared with potato isolates collected outside the United Kingdom. It was concluded that VNTR profiling could contribute to further understanding of R. solanacearum epidemiology and assist in control of future disease outbreaks. PMID:23892739

  3. Deciphering the route of Ralstonia solanacearum colonization in Arabidopsis thaliana roots during a compatible interaction: focus at the plant cell wall.

    PubMed

    Digonnet, Catherine; Martinez, Yves; Denancé, Nicolas; Chasseray, Marine; Dabos, Patrick; Ranocha, Philippe; Marco, Yves; Jauneau, Alain; Goffner, Deborah

    2012-11-01

    The compatible interaction between the model plant, Arabidopsis thaliana, and the GMI1000 strain of the phytopathogenic bacterium, Ralstonia solanacearum, was investigated in an in vitro pathosystem. We describe the progression of the bacteria in the root from penetration at the root surface to the xylem vessels and the cell type-specific, cell wall-associated modifications that accompanies bacterial colonization. Within 6 days post inoculation, R. solanacearum provoked a rapid plasmolysis of the epidermal, cortical, and endodermal cells, including those not directly in contact with the bacteria. Plasmolysis was accompanied by a global degradation of pectic homogalacturonanes as shown by the loss of JIM7 and JIM5 antibody signal in the cell wall of these cell types. As indicated by immunolabeling with Rsol-I antibodies that specifically recognize R. solanacearum, the bacteria progresses through the root in a highly directed, centripetal manner to the xylem poles, without extensive multiplication in the intercellular spaces along its path. Entry into the vascular cylinder was facilitated by cell collapse of the two pericycle cells located at the xylem poles. Once the bacteria reached the xylem vessels, they multiplied abundantly and moved from vessel to vessel by digesting the pit membrane between adjacent vessels. The degradation of the secondary walls of xylem vessels was not a prerequisite for vessel colonization as LM10 antibodies strongly labeled xylem cell walls, even at very late stages in disease development. Finally, the capacity of R. solanacearum to specifically degrade certain cell wall components and not others could be correlated with the arsenal of cell wall hydrolytic enzymes identified in the bacterial genome.

  4. Deciphering the route of Ralstonia solanacearum colonization in Arabidopsis thaliana roots during a compatible interaction: focus at the plant cell wall.

    PubMed

    Digonnet, Catherine; Martinez, Yves; Denancé, Nicolas; Chasseray, Marine; Dabos, Patrick; Ranocha, Philippe; Marco, Yves; Jauneau, Alain; Goffner, Deborah

    2012-11-01

    The compatible interaction between the model plant, Arabidopsis thaliana, and the GMI1000 strain of the phytopathogenic bacterium, Ralstonia solanacearum, was investigated in an in vitro pathosystem. We describe the progression of the bacteria in the root from penetration at the root surface to the xylem vessels and the cell type-specific, cell wall-associated modifications that accompanies bacterial colonization. Within 6 days post inoculation, R. solanacearum provoked a rapid plasmolysis of the epidermal, cortical, and endodermal cells, including those not directly in contact with the bacteria. Plasmolysis was accompanied by a global degradation of pectic homogalacturonanes as shown by the loss of JIM7 and JIM5 antibody signal in the cell wall of these cell types. As indicated by immunolabeling with Rsol-I antibodies that specifically recognize R. solanacearum, the bacteria progresses through the root in a highly directed, centripetal manner to the xylem poles, without extensive multiplication in the intercellular spaces along its path. Entry into the vascular cylinder was facilitated by cell collapse of the two pericycle cells located at the xylem poles. Once the bacteria reached the xylem vessels, they multiplied abundantly and moved from vessel to vessel by digesting the pit membrane between adjacent vessels. The degradation of the secondary walls of xylem vessels was not a prerequisite for vessel colonization as LM10 antibodies strongly labeled xylem cell walls, even at very late stages in disease development. Finally, the capacity of R. solanacearum to specifically degrade certain cell wall components and not others could be correlated with the arsenal of cell wall hydrolytic enzymes identified in the bacterial genome. PMID:22729825

  5. PrhN, a putative marR family transcriptional regulator, is involved in positive regulation of type III secretion system and full virulence of Ralstonia solanacearum

    PubMed Central

    Zhang, Yong; Luo, Feng; Wu, Dousheng; Hikichi, Yasufumi; Kiba, Akinori; Igarashi, Yasuo; Ding, Wei; Ohnishi, Kouhei

    2015-01-01

    The MarR-family of transcriptional regulators are involved in various cellular processes, including resistance to multiple antibiotics and other toxic chemicals, adaptation to different environments and pathogenesis in many plant and animal pathogens. Here, we reported a new MarR regulator PrhN, which was involved in the pathogenesis of Ralstonia solanacearum. prhN mutant exhibited significantly reduced virulence and stem colonization compared to that of wild type in tomato plants. prhN mutant caused identical hypersensitive response (HR) on resistant plants to the wild type. Deletion of prhN gene substantially reduced the expression of type III secretion system (T3SS) in vitro and in planta (mainly in tomato plants), which is essential for pathogenicity of R. solanacearum, and the complemented PrhN could restore its virulence and T3SS expression to that of wild type. T3SS is directly controlled by AraC-type transcriptional regulator HrpB, and the transcription of hrpB is activated by HrpG and PrhG. HrpG and PrhG are homologs but are regulated by the PhcA positively and negatively, respectively. Deletion of prhN gene also abolished the expression of hrpB and prhG, but didn't change the expression of hrpG and phcA. Together, these results indicated that PrhN positively regulates T3SS expression through PrhG and HrpB. PrhN and PhcA should regulate prhG expression in a parallel way. This is the first report on the pathogenesis of MarR regulator in R. solanacearum, and this new finding will improve our understanding on the various biological functions of MarR regulator and the complex regulatory network on hrp regulon in R. solanacearum. PMID:25972849

  6. Two Host-Induced Ralstonia solanacearum Genes, acrA and dinF, Encode Multidrug Efflux Pumps and Contribute to Bacterial Wilt Virulence▿ †

    PubMed Central

    Brown, Darby G.; Swanson, Jill K.; Allen, Caitilyn

    2007-01-01

    Multidrug efflux pumps (MDRs) are hypothesized to protect pathogenic bacteria from toxic host defense compounds. We created mutations in the Ralstonia solanacearum acrA and dinF genes, which encode putative MDRs in the broad-host-range plant pathogen. Both mutations reduced the ability of R. solanacearum to grow in the presence of various toxic compounds, including antibiotics, phytoalexins, and detergents. Both acrAB and dinF mutants were significantly less virulent on the tomato plant than the wild-type strain. Complementation restored near-wild-type levels of virulence to both mutants. Addition of either dinF or acrAB to Escherichia coli MDR mutants KAM3 and KAM32 restored the resistance of these strains to several toxins, demonstrating that the R. solanacearum genes can function heterologously to complement known MDR mutations. Toxic and DNA-damaging compounds induced expression of acrA and dinF, as did growth in both susceptible and resistant tomato plants. Carbon limitation also increased expression of acrA and dinF, while the stress-related sigma factor RpoS was required at a high cell density (>107 CFU/ml) to obtain wild-type levels of acrA expression both in minimal medium and in planta. The type III secretion system regulator HrpB negatively regulated dinF expression in culture at high cell densities. Together, these results show that acrAB and dinF encode MDRs in R. solanacearum and that they contribute to the overall aggressiveness of this phytopathogen, probably by protecting the bacterium from the toxic effects of host antimicrobial compounds. PMID:17337552

  7. Application of variable-number tandem-repeat typing to discriminate Ralstonia solanacearum strains associated with English watercourses and disease outbreaks.

    PubMed

    Parkinson, Neil; Bryant, Ruth; Bew, Janice; Conyers, Christine; Stones, Robert; Alcock, Michael; Elphinstone, John

    2013-10-01

    Variable-number tandem-repeat (VNTR) analysis was used for high-resolution discrimination among Ralstonia solanacearum phylotype IIB sequevar 1 (PIIB-1) isolates and further evaluated for use in source tracing. Five tandem-repeat-containing loci (comprising six tandem repeats) discriminated 17 different VNTR profiles among 75 isolates from potato, geranium, bittersweet (Solanum dulcamara), tomato, and the environment. R. solanacearum isolates from crops at three unrelated outbreak sites where river water had been used for irrigation had distinct VNTR profiles that were shared with PIIB-1 isolates from infected bittersweet growing upriver of each site. The VNTR profiling results supported the implication that the source of R. solanacearum at each outbreak was contaminated river water. Analysis of 51 isolates from bittersweet growing in river water at different locations provided a means to evaluate the technique for studying the epidemiology of the pathogen in the environment. Ten different VNTR profiles were identified among bittersweet PIIB-1 isolates from the River Thames. Repeated findings of contiguous river stretches that produced isolates that shared single VNTR profiles supported the hypothesis that the pathogen had disseminated from infected bittersweet plants located upriver. VNTR profiles shared between bittersweet isolates from two widely separated Thames tributaries (River Ray and River Colne) suggested they were independently contaminated with the same clonal type. Some bittersweet isolates had VNTR profiles that were shared with potato isolates collected outside the United Kingdom. It was concluded that VNTR profiling could contribute to further understanding of R. solanacearum epidemiology and assist in control of future disease outbreaks.

  8. Quick adaptation of Ralstonia Solanacearum to copper stress to recover culturability and growth in water and soil

    PubMed Central

    Ascarrunz, Sergio Daniel Moreira; Natsuaki, Tomohide; Honjo, Hitoshi; Fukui, Ryo

    2011-01-01

    Cells of Ralstonia solanacearum were exposed to Cu in distilled water, and the resulting Cu-stressed non-culturable cells were inoculated to natural (non-pasteurized) and pasteurized soils in order to examine their culturability and recovery. Exposing the cells to 20 μM CuSO4 produced transitory non-culturable cells, which exhibited a remarkable recovery in culturability after incubation in the solution for 36 h, reaching a density near the initial level by 108 h. To determine whether such non-culturable cells actually “resuscitated” or multiplied after adapting to Cu toxicity, growth curves were constructed in order to contrast the rates of increase in culturable cell numbers between Cu-stressed or non-stressed inocula. Additionally, fresh non-stressed cells were exposed to CuSO4 in the presence of nalidixic acid by adding the antibiotic at different times after the onset of Cu stress to verify any cell multiplication during the population increase. The results revealed that the non-culturable cells surviving Cu toxicity adapted very quickly to Cu and began multiplying within 12 h, because only the Cu-stressed cells that were increasing in the exponential growth phase, but not those in the stationary phase, were killed by the antibiotic. Such cells exhibited an apparent tolerance to this metal when inoculated to a freshly prepared solution of CuSO4, and also detoxified the ion in the solution in which they grew. The presence of nutrients greatly counteracted the effect of Cu in water microcosms, since culturable cells were detected and increased in number even when exposed to 40 μM CuSO4. In contrast, when inoculated to non-pasteurized soil, Cu-stressed cells showed no such recoveries. However, when the soil was pasteurized before inoculation or added with nutrients, culturable cells were recovered and increased in number. This indicates that increased nutrient availability in soil allows Cu-stressed cells to quickly overcome the stress and increase in

  9. Comparative Secretome Analysis of Ralstonia solanacearum Type 3 Secretion-Associated Mutants Reveals a Fine Control of Effector Delivery, Essential for Bacterial Pathogenicity.

    PubMed

    Lonjon, Fabien; Turner, Marie; Henry, Céline; Rengel, David; Lohou, David; van de Kerkhove, Quitterie; Cazalé, Anne-Claire; Peeters, Nemo; Genin, Stéphane; Vailleau, Fabienne

    2016-02-01

    Ralstonia solanacearum, the causal agent of bacterial wilt, exerts its pathogenicity through more than a hundred secreted proteins, many of them depending directly on the functionality of a type 3 secretion system. To date, only few type 3 effectors have been identified as required for bacterial pathogenicity, notably because of redundancy among the large R. solanacearum effector repertoire. In order to identify groups of effectors collectively promoting disease on susceptible hosts, we investigated the role of putative post-translational regulators in the control of type 3 secretion. A shotgun secretome analysis with label-free quantification using tandem mass spectrometry was performed on the R. solanacearum GMI1000 strain. There were 228 proteins identified, among which a large proportion of type 3 effectors, called Rip (Ralstonia injected proteins). Thanks to this proteomic approach, RipBJ was identified as a new effector specifically secreted through type 3 secretion system and translocated into plant cells. A focused Rip secretome analysis using hpa (hypersensitive response and pathogenicity associated) mutants revealed a fine secretion regulation and specific subsets of Rips with different secretion patterns. We showed that a set of Rips (RipF1, RipW, RipX, RipAB, and RipAM) are secreted in an Hpa-independent manner. We hypothesize that these Rips could be preferentially involved in the first stages of type 3 secretion. In addition, the secretion of about thirty other Rips is controlled by HpaB and HpaG. HpaB, a candidate chaperone was shown to positively control secretion of numerous Rips, whereas HpaG was shown to act as a negative regulator of secretion. To evaluate the impact of altered type 3 effectors secretion on plant pathogenesis, the hpa mutants were assayed on several host plants. HpaB was required for bacterial pathogenicity on multiple hosts whereas HpaG was found to be specifically required for full R. solanacearum pathogenicity on the legume

  10. Hierarchical autoinduction in Ralstonia solanacearum: control of acyl-homoserine lactone production by a novel autoregulatory system responsive to 3-hydroxypalmitic acid methyl ester.

    PubMed Central

    Flavier, A B; Ganova-Raeva, L M; Schell, M A; Denny, T P

    1997-01-01

    Bacteria employ autoinduction systems to sense the onset of appropriate cell density for expression of developmental genes. In many gram-negative bacteria, autoinduction involves the production of and response to diffusible acylated-homoserine lactones (acyl-HSLs) and is mediated by members of the LuxR and LuxI families. Ralstonia (Pseudomonas) solanacearum, a phytopathogenic bacterium that appears to autoregulate its virulence genes, produces compounds that promote expression of several heterologous acyl-HSL-responsive reporter gene constructs. High-pressure liquid chromatography of highly concentrated ethyl acetate extracts revealed that culture supernatants of strain AW1 contained two compounds with retention times similar to N-hexanoyl- and N-octanoyl-HSL. To investigate the role of these acyl-HSLs in R. solanacearum virulence gene expression, transposon mutants that were deficient for inducing an acyl-HSL-responsive reporter in Agrobacterium tumefaciens were generated. Three loci involved in normal acyl-HSL production were identified, one of which was shown to contain the divergently transcribed solR and solI genes, the luxR and luxI homologs, respectively. A 4.1-kb fragment containing solR and solI enabled all of the mutants (regardless of the locus inactivated) and a naturally acyl-HSL-defective strain of R. solanacearum to produce acyl-HSLs. Inactivation of solI abolished production of all detectable acyl-HSLs but affected neither the expression of virulence genes in culture nor the ability to wilt tomato plants. AW1 has a functional autoinduction system, because (i) expression of solI required SolR and acyl-HSL and (ii) expression of a gene linked to solR and solI, designated aidA, was acyl-HSL dependent. Because AidA has no homologs in the protein databases, its discovery provided no clues as to the role of acyl-HSLs in R. solanacearum gene regulation. However, expression of solR and solI required the global LysR-type virulence regulator PhcA, and both

  11. Arabidopsis CLAVATA1 and CLAVATA2 receptors contribute to Ralstonia solanacearum pathogenicity through a miR169-dependent pathway.

    PubMed

    Hanemian, Mathieu; Barlet, Xavier; Sorin, Céline; Yadeta, Koste A; Keller, Harald; Favery, Bruno; Simon, Rüdiger; Thomma, Bart P H J; Hartmann, Caroline; Crespi, Martin; Marco, Yves; Tremousaygue, Dominique; Deslandes, Laurent

    2016-07-01

    Bacterial wilt caused by Ralstonia solanacearum is one of the most destructive bacterial plant diseases. Although many molecular determinants involved in R. solanacearum adaptation to hosts and pathogenesis have been described, host components required for disease establishment remain poorly characterized. Phenotypical analysis of Arabidopsis mutants for leucine-rich repeat (LRR)-receptor-like proteins revealed that mutations in the CLAVATA1 (CLV1) and CLAVATA2 (CLV2) genes confer enhanced disease resistance to bacterial wilt. We further investigated the underlying mechanisms using genetic, transcriptomic and molecular approaches. The enhanced resistance of both clv1 and clv2 mutants to the bacteria did not require the well characterized CLV signalling modules involved in shoot meristem homeostasis, and was conditioned by neither salicylic acid nor ethylene defence-related hormones. Gene expression microarray analysis performed on clv1 and clv2 revealed deregulation of genes encoding nuclear transcription factor Y subunit alpha (NF-YA) transcription factors whose post-transcriptional regulation is known to involve microRNAs from the miR169 family. Both clv mutants showed a defect in miR169 accumulation. Conversely, overexpression of miR169 abrogated the resistance phenotype of clv mutants. We propose that CLV1 and CLV2, two receptors involved in CLV3 perception during plant development, contribute to bacterial wilt through a signalling pathway involving the miR169/NF-YA module. PMID:26990325

  12. The symbiotic transcription factor MtEFD and cytokinins are positively acting in the Medicago truncatula and Ralstonia solanacearum pathogenic interaction.

    PubMed

    Moreau, Sandra; Fromentin, Justine; Vailleau, Fabienne; Vernié, Tatiana; Huguet, Stéphanie; Balzergue, Sandrine; Frugier, Florian; Gamas, Pascal; Jardinaud, Marie-Françoise

    2014-03-01

    • A plant-microbe dual biological system was set up involving the model legume Medicago truncatula and two bacteria, the soil-borne root pathogen Ralstonia solanacearum and the beneficial symbiont Sinorhizobium meliloti. • Comparison of transcriptomes under symbiotic and pathogenic conditions highlighted the transcription factor MtEFD (Ethylene response Factor required for nodule Differentiation) as being upregulated in both interactions, together with a set of cytokinin-related transcripts involved in metabolism, signaling and response. MtRR4 (Response Regulator), a cytokinin primary response gene negatively regulating cytokinin signaling and known as a target of MtEFD in nodulation processes, was retrieved in this set of transcripts. • Refined studies of MtEFD and MtRR4 expression during M. truncatula and R. solanacearum interaction indicated differential kinetics of induction and requirement of central regulators of bacterial pathogenicity, HrpG and HrpB. Similar to MtRR4, MtEFD upregulation during the pathogenic interaction was dependent on cytokinin perception mediated by the MtCRE1 (Cytokinin REsponse 1) receptor. • The use of M. truncatula efd-1 and cre1-1 mutants evidenced MtEFD and cytokinin perception as positive factors for bacterial wilt development. These factors therefore play an important role in both root nodulation and root disease development.

  13. Arabidopsis CLAVATA1 and CLAVATA2 receptors contribute to Ralstonia solanacearum pathogenicity through a miR169-dependent pathway.

    PubMed

    Hanemian, Mathieu; Barlet, Xavier; Sorin, Céline; Yadeta, Koste A; Keller, Harald; Favery, Bruno; Simon, Rüdiger; Thomma, Bart P H J; Hartmann, Caroline; Crespi, Martin; Marco, Yves; Tremousaygue, Dominique; Deslandes, Laurent

    2016-07-01

    Bacterial wilt caused by Ralstonia solanacearum is one of the most destructive bacterial plant diseases. Although many molecular determinants involved in R. solanacearum adaptation to hosts and pathogenesis have been described, host components required for disease establishment remain poorly characterized. Phenotypical analysis of Arabidopsis mutants for leucine-rich repeat (LRR)-receptor-like proteins revealed that mutations in the CLAVATA1 (CLV1) and CLAVATA2 (CLV2) genes confer enhanced disease resistance to bacterial wilt. We further investigated the underlying mechanisms using genetic, transcriptomic and molecular approaches. The enhanced resistance of both clv1 and clv2 mutants to the bacteria did not require the well characterized CLV signalling modules involved in shoot meristem homeostasis, and was conditioned by neither salicylic acid nor ethylene defence-related hormones. Gene expression microarray analysis performed on clv1 and clv2 revealed deregulation of genes encoding nuclear transcription factor Y subunit alpha (NF-YA) transcription factors whose post-transcriptional regulation is known to involve microRNAs from the miR169 family. Both clv mutants showed a defect in miR169 accumulation. Conversely, overexpression of miR169 abrogated the resistance phenotype of clv mutants. We propose that CLV1 and CLV2, two receptors involved in CLV3 perception during plant development, contribute to bacterial wilt through a signalling pathway involving the miR169/NF-YA module.

  14. CaWRKY58, encoding a group I WRKY transcription factor of Capsicum annuum, negatively regulates resistance to Ralstonia solanacearum infection.

    PubMed

    Wang, Yuna; Dang, Fengfeng; Liu, Zhiqin; Wang, Xu; Eulgem, Thomas; Lai, Yan; Yu, Lu; She, Jianju; Shi, Youliang; Lin, Jinhui; Chen, Chengcong; Guan, Deyi; Qiu, Ailian; He, Shuilin

    2013-02-01

    WRKY transcription factors are encoded by large gene families across the plant kingdom. So far, their biological and molecular functions in nonmodel plants, including pepper (Capsicum annuum) and other Solanaceae, remain poorly understood. Here, we report on the functional characterization of a new group I WRKY protein from pepper, termed CaWRKY58. Our data indicate that CaWRKY58 can be localized to the nucleus and can activate the transcription of the reporter β-glucuronidase (GUS) gene driven by the 35S core promoter with two copies of the W-box in its proximal upstream region. In pepper plants infected with the bacterial pathogen Ralstonia solanacearum, CaWRKY58 transcript levels showed a biphasic response, manifested in an early/transient down-regulation and late up-regulation. CaWRKY58 transcripts were suppressed by treatment with methyl jasmonate and abscisic acid. Tobacco plants overexpressing CaWRKY58 did not show any obvious morphological phenotypes, but exhibited disease symptoms of greater severity than did wild-type plants. The enhanced susceptibility of CaWRKY58-overexpressing tobacco plants correlated with the decreased expression of hypersensitive response marker genes, as well as various defence-associated genes. Consistently, CaWRKY58 pepper plants silenced by virus-induced gene silencing (VIGS) displayed enhanced resistance to the highly virulent R. solanacearum strain FJC100301, and this was correlated with enhanced transcripts of defence-related pepper genes. Our results suggest that CaWRKY58 acts as a transcriptional activator of negative regulators in the resistance of pepper to R. solanacearum infection.

  15. The vascular plant-pathogenic bacterium Ralstonia solanacearum produces biofilms required for its virulence on the surfaces of tomato cells adjacent to intercellular spaces.

    PubMed

    Mori, Yuka; Inoue, Kanako; Ikeda, Kenichi; Nakayashiki, Hitoshi; Higashimoto, Chikaki; Ohnishi, Kouhei; Kiba, Akinori; Hikichi, Yasufumi

    2016-08-01

    The mechanism of colonization of intercellular spaces by the soil-borne and vascular plant-pathogenic bacterium Ralstonia solanacearum strain OE1-1 after invasion into host plants remains unclear. To analyse the behaviour of OE1-1 cells in intercellular spaces, tomato leaves with the lower epidermis layers excised after infiltration with OE1-1 were observed under a scanning electron microscope. OE1-1 cells formed microcolonies on the surfaces of tomato cells adjacent to intercellular spaces, and then aggregated surrounded by an extracellular matrix, forming mature biofilm structures. Furthermore, OE1-1 cells produced mushroom-type biofilms when incubated in fluids of apoplasts including intercellular spaces, but not xylem fluids from tomato plants. This is the first report of biofilm formation by R. solanacearum on host plant cells after invasion into intercellular spaces and mushroom-type biofilms produced by R. solanacearum in vitro. Sugar application led to enhanced biofilm formation by OE1-1. Mutation of lecM encoding a lectin, RS-IIL, which reportedly exhibits affinity for these sugars, led to a significant decrease in biofilm formation. Colonization in intercellular spaces was significantly decreased in the lecM mutant, leading to a loss of virulence on tomato plants. Complementation of the lecM mutant with native lecM resulted in the recovery of mushroom-type biofilms and virulence on tomato plants. Together, our findings indicate that OE1-1 produces mature biofilms on the surfaces of tomato cells after invasion into intercellular spaces. RS-IIL may contribute to biofilm formation by OE1-1, which is required for OE1-1 virulence. PMID:26609568

  16. Induction of a small heat shock protein and its functional roles in Nicotiana plants in the defense response against Ralstonia solanacearum.

    PubMed

    Maimbo, Milimo; Ohnishi, Kouhei; Hikichi, Yasufumi; Yoshioka, Hirofumi; Kiba, Akinori

    2007-12-01

    In tobacco (Nicotiana tabacum), Ralstonia solanacearum OE1-1 (RsOE1-1) is pathogenic, whereas R. solanacearum 8107 (Rs8107) is nonpathogenic and induces the hypersensitive response (HR). To elucidate the molecular mechanisms of plant-R. solanacearum interactions, we used differential display to isolate a cDNA fragment, A6, regulated in tobacco by inoculation with RsOE1-1. The deduced amino acid sequence predicted from full-length A6-cDNA showed similarity to small heat shock proteins from Arabidopsis (Arabidopsis thaliana; hypothetical protein), Medicago truncatula, and Cucumis melo; we therefore designated A6 to correspond to Ntshsp17 (for tobacco small heat shock protein 17). Recombinant Ntshsp17 overproduced in Escherichia coli exhibited molecular chaperone function. Expression of Ntshsp17 was increased in tobacco leaves inoculated with both RsOE1-1 and Rs8107. Expression was induced by heat treatment and by treatment with aminocyclopropane carboxylic acid, hydrogen peroxide, methyl jasmonate, and salicylic acid. Ntshsp17 expression was induced by inoculation with a HR and pathogenicity gene mutant of Rs8107 that does not induce the HR, but not by Agrobacterium-mediated transient expression of INF1, an HR elicitor. In Nbshsp17-silenced plants (an Ntshsp17 ortholog in Nicotiana benthamiana), expression of ETHYLENE-RESPONSE ELEMENT-BINDING PROTEIN, PATHOGENESIS-RELATED1a (PR1a), and PR4 genes was compromised, but expression of ELONGATION FACTOR1alpha was scarcely affected. Appearance of the HR was not affected in the silenced plants. In the silenced plants, growth of Rs8107 was accelerated. Bacterial growth and wilt symptoms elicited by RsOE1-1 were also accelerated in the silenced plants. These results indicate that this small heat shock protein might have a role in HR-independent defenses in Nicotiana plants. PMID:17965181

  17. The vascular plant-pathogenic bacterium Ralstonia solanacearum produces biofilms required for its virulence on the surfaces of tomato cells adjacent to intercellular spaces.

    PubMed

    Mori, Yuka; Inoue, Kanako; Ikeda, Kenichi; Nakayashiki, Hitoshi; Higashimoto, Chikaki; Ohnishi, Kouhei; Kiba, Akinori; Hikichi, Yasufumi

    2016-08-01

    The mechanism of colonization of intercellular spaces by the soil-borne and vascular plant-pathogenic bacterium Ralstonia solanacearum strain OE1-1 after invasion into host plants remains unclear. To analyse the behaviour of OE1-1 cells in intercellular spaces, tomato leaves with the lower epidermis layers excised after infiltration with OE1-1 were observed under a scanning electron microscope. OE1-1 cells formed microcolonies on the surfaces of tomato cells adjacent to intercellular spaces, and then aggregated surrounded by an extracellular matrix, forming mature biofilm structures. Furthermore, OE1-1 cells produced mushroom-type biofilms when incubated in fluids of apoplasts including intercellular spaces, but not xylem fluids from tomato plants. This is the first report of biofilm formation by R. solanacearum on host plant cells after invasion into intercellular spaces and mushroom-type biofilms produced by R. solanacearum in vitro. Sugar application led to enhanced biofilm formation by OE1-1. Mutation of lecM encoding a lectin, RS-IIL, which reportedly exhibits affinity for these sugars, led to a significant decrease in biofilm formation. Colonization in intercellular spaces was significantly decreased in the lecM mutant, leading to a loss of virulence on tomato plants. Complementation of the lecM mutant with native lecM resulted in the recovery of mushroom-type biofilms and virulence on tomato plants. Together, our findings indicate that OE1-1 produces mature biofilms on the surfaces of tomato cells after invasion into intercellular spaces. RS-IIL may contribute to biofilm formation by OE1-1, which is required for OE1-1 virulence.

  18. HpaP modulates type III effector secretion in Ralstonia solanacearum and harbours a substrate specificity switch domain essential for virulence.

    PubMed

    Lohou, David; Turner, Marie; Lonjon, Fabien; Cazalé, Anne-Claire; Peeters, Nemo; Genin, Stéphane; Vailleau, Fabienne

    2014-08-01

    Many pathogenic bacteria have evolved a type III secretion system (T3SS) to successfully invade their host. This extracellular apparatus allows the translocation of proteins, called type III effectors (T3Es), directly into the host cells. T3Es are virulence factors that have been shown to interfere with the host's immunity or to provide nutrients from the host to the bacteria. The Gram-negative bacterium Ralstonia solanacearum is a worldwide major crop pest whose virulence strongly relies on the T3SS. In R. solanacearum, transcriptional regulation has been extensively studied. However, very few data are available concerning the role played by type III-associated regulators, such as type III chaperones and T3SS control proteins. Here, we characterized HpaP, a putative type III secretion substrate specificity switch (T3S4) protein of R. solanacearum which is not secreted by the bacterium or translocated in the plant cells. HpaP self-interacts and interacts with the PopP1 T3E. HpaP modulates the secretion of early (HrpY pilin) and late (AvrA and PopP1 T3Es) type III substrates. HpaP is dispensable for the translocation of T3Es into the host cells. Finally, we identified two regions of five amino acids in the T3S4 domain that are essential for efficient PopP1 secretion and for HpaP's role in virulence on tomato and Arabidopsis thaliana, but not required for HpaP-HpaP and HpaP-PopP1 interactions. Taken together, our results indicate that HpaP is a putative R. solanacearum T3S4 protein important for full pathogenicity on several hosts, acting as a helper for PopP1 secretion, and repressing AvrA and HrpY secretion.

  19. Sensitive quantitative detection of Ralstonia solanacearum in soil by the most probable number-polymerase chain reaction (MPN-PCR) method.

    PubMed

    Inoue, Yasuhiro; Nakaho, Kazuhiro

    2014-05-01

    We developed a sensitive quantitative assay for detecting Ralstonia solanacearum in soil by most probable number (MPN) analysis based on bio-PCR results. For development of the detection method, we optimized an elution buffer containing 5 g/L skim milk for extracting bacteria from soil and reducing contamination of polymerase inhibitors in soil extracts. Because R. solanacearum can grow in water without any added nutrients, we used a cultivation buffer in the culture step of the bio-PCR that contained only the buffer and antibiotics to suppress the growth of other soil microorganisms. To quantify the bacterial population in soil, the elution buffer was added to 10 g soil on a dry weight basis so that the combined weight of buffer, soil, and soil-water was 50 g; 5 mL of soil extract was assumed to originate from 1 g of soil. The soil extract was divided into triplicate aliquots each of 5 mL and 500, 50, and 5 μL. Each aliquot was diluted with the cultivation buffer and incubated at 35 °C for about 24 h. After incubation, 5 μL of culture was directly used for nested PCR. The number of aliquots showing positive results was collectively checked against the MPN table. The method could quantify bacterial populations in soil down to 3 cfu/10 g dried soil and was successfully applied to several types of soil. We applied the method for the quantitative detection of R. solanacearum in horticultural soils, which could quantitatively detect small populations (9.3 cfu/g), but the semiselective media were not able to detect the bacteria.

  20. Genomic characterization of Ralstonia solanacearum phage phiRSA1 and its related prophage (phiRSX) in strain GMI1000.

    PubMed

    Fujiwara, Akiko; Kawasaki, Takeru; Usami, Shoji; Fujie, Makoto; Yamada, Takashi

    2008-01-01

    PhiRSA1 is a wide-host-range bacteriophage isolated from Ralstonia solanacearum. In this study, the complete nucleotide sequence of the phiRSA1 genomic DNA was determined. The genome was 38,760 bp of double-stranded DNA (65.3% G+C) with 19-bp 5'-extruding cohesive ends (cos) and contained 51 open reading frames (ORFs). Two-thirds of the phiRSA1 genomic region encodes the phage structural modules, and they are very similar to those reported for coliphage P2 and P2-like phages. A phiRSA1 minireplicon with an 8.2-kbp early-expressing region was constructed. A late-expression promoter sequence motif was predicted for these phiRSA1 genes as 5' TGTTGT-(X)13-ACAACA. The genomic sequence similarity between phiRSA1 and related phages phi52237 and phiCTX was interrupted by three AT islands, one of which contained an insertion sequence element, suggesting that they were recombinational hot spots. phiRSA1 was found to be integrated into at least three different strains of R. solanacearum, and the chromosomal integration site (attB) was identified as the 3' portion of the arginine tRNA(CCG) gene. In the light of the phiRSA1 gene arrangement, one possible prophage sequence previously detected on the chromosome of R. solanacearum strain GMI1000 was characterized as a phiRSA1-related prophage (designated phiRSX). phiRSX was found to be integrated at the serine tRNA (GGA) gene as an att site, and its size was determined to be 40,713 bp. phiRSX ORFs shared very high amino acid identity with their phiRSA1 counterparts. The relationships and evolution of these P2-like phages are discussed.

  1. Effect of calcium cyanamide, ammonium bicarbonate and lime mixture, and ammonia water on survival of Ralstonia solanacearum and microbial community.

    PubMed

    Liu, Lijuan; Sun, Chengliang; Liu, Xingxing; He, Xiaolin; Liu, Miao; Wu, Hao; Tang, Caixian; Jin, Chongwei; Zhang, Yongsong

    2016-01-01

    The inorganic nitrogenous amendments calcium cyanamide (CC), ammonia water (AW), and a mixture of ammonium bicarbonate with lime (A+L) are popularly used as fumigants to control soil-borne disease in China. However, it is unclear which of these fumigants is more effective in controlling R. solanacearum. This present study compared the efficiencies of the three nitrogenous amendments listed above at four nitrogen levels in suppressing the survival of R. solanacearum in soil. The CC showed the best ability to suppress R. solanacearum due to its highest capacity to increase soil and NO2(-) contents and pH. However, AW was more suitable to controlling bacterial wilt caused by R. solanacearum because it had a lower cost and its application rate of 0.25 g N kg(-1) soil could effectively suppress the survival of R. solanacearum. Additionally, soil microbial activity and community populations were restored to their initial state four weeks after the application of each fumigant, indicating that the three fumigants had few detrimental impacts on soil microbial activity and community structure with an exception of the suppression of R. solanacearum. The present study provides guidance for the selection of a suitable alkaline nitrogenous amendment and its application rate in controlling bacterial wilt. PMID:26738601

  2. Effect of calcium cyanamide, ammonium bicarbonate and lime mixture, and ammonia water on survival of Ralstonia solanacearum and microbial community

    PubMed Central

    Liu, Lijuan; Sun, Chengliang; Liu, Xingxing; He, Xiaolin; Liu, Miao; Wu, Hao; Tang, Caixian; Jin, Chongwei; Zhang, Yongsong

    2016-01-01

    The inorganic nitrogenous amendments calcium cyanamide (CC), ammonia water (AW), and a mixture of ammonium bicarbonate with lime (A+L) are popularly used as fumigants to control soil-borne disease in China. However, it is unclear which of these fumigants is more effective in controlling R. solanacearum. This present study compared the efficiencies of the three nitrogenous amendments listed above at four nitrogen levels in suppressing the survival of R. solanacearum in soil. The CC showed the best ability to suppress R. solanacearum due to its highest capacity to increase soil and NO2− contents and pH. However, AW was more suitable to controlling bacterial wilt caused by R. solanacearum because it had a lower cost and its application rate of 0.25 g N kg−1 soil could effectively suppress the survival of R. solanacearum. Additionally, soil microbial activity and community populations were restored to their initial state four weeks after the application of each fumigant, indicating that the three fumigants had few detrimental impacts on soil microbial activity and community structure with an exception of the suppression of R. solanacearum. The present study provides guidance for the selection of a suitable alkaline nitrogenous amendment and its application rate in controlling bacterial wilt. PMID:26738601

  3. Effect of calcium cyanamide, ammonium bicarbonate and lime mixture, and ammonia water on survival of Ralstonia solanacearum and microbial community.

    PubMed

    Liu, Lijuan; Sun, Chengliang; Liu, Xingxing; He, Xiaolin; Liu, Miao; Wu, Hao; Tang, Caixian; Jin, Chongwei; Zhang, Yongsong

    2016-01-01

    The inorganic nitrogenous amendments calcium cyanamide (CC), ammonia water (AW), and a mixture of ammonium bicarbonate with lime (A+L) are popularly used as fumigants to control soil-borne disease in China. However, it is unclear which of these fumigants is more effective in controlling R. solanacearum. This present study compared the efficiencies of the three nitrogenous amendments listed above at four nitrogen levels in suppressing the survival of R. solanacearum in soil. The CC showed the best ability to suppress R. solanacearum due to its highest capacity to increase soil and NO2(-) contents and pH. However, AW was more suitable to controlling bacterial wilt caused by R. solanacearum because it had a lower cost and its application rate of 0.25 g N kg(-1) soil could effectively suppress the survival of R. solanacearum. Additionally, soil microbial activity and community populations were restored to their initial state four weeks after the application of each fumigant, indicating that the three fumigants had few detrimental impacts on soil microbial activity and community structure with an exception of the suppression of R. solanacearum. The present study provides guidance for the selection of a suitable alkaline nitrogenous amendment and its application rate in controlling bacterial wilt.

  4. Moderate temperature fluctuations rapidly reduce the viability of Ralstonia solanacearum race 3, biovar 2, in infected geranium, tomato, and potato plants.

    PubMed

    Scherf, Jacob M; Milling, Annett; Allen, Caitilyn

    2010-11-01

    Most Ralstonia solanacearum strains are tropical plant pathogens, but race 3, biovar 2 (R3bv2), strains can cause bacterial wilt in temperate zones or tropical highlands where other strains cannot. R3bv2 is a quarantine pathogen in North America and Europe because of its potential to damage the potato industry in cooler climates. However, R3bv2 will not become established if it cannot survive temperate winters. Previous experiments showed that in water at 4°C, R3bv2 does not survive as long as native U.S. strains, but R3bv2 remains viable longer than U.S. strains in potato tubers at 4°C. To further investigate the effects of temperature on this high-concern pathogen, we assessed the ability of R3bv2 and a native U.S. strain to survive typical temperate winter temperature cycles of 2 days at 5°C followed by 2 days at -10°C. We measured pathogen survival in infected tomato and geranium plants, in infected potato tubers, and in sterile water. The population sizes of both strains declined rapidly under these conditions in all three plant hosts and in sterile water, and no culturable R. solanacearum cells were detected after five to seven temperature cycles in plant tissue. The fluctuations played a critical role in loss of bacterial viability, since at a constant temperature of -20°C, both strains could survive in infected geranium tissue for at least 6 months. These results suggest that even when sheltered in infected plant tissue, R3bv2 is unlikely to survive the temperature fluctuations typical of a northern temperate winter.

  5. So near and yet so far: the specific case of Ralstonia Solanacearum populations from Côte d'Ivoire in Africa.

    PubMed

    N'guessan, C A; Abo, K; Fondio, L; Chiroleu, F; Lebeau, A; Poussier, S; Wicker, E; Koné, D

    2012-08-01

    The genetic and phenotypic diversity of Côte d'Ivoire Ralstonia solanacearum strains was assessed on a 168-strain collection sampled on Solanaceae both in the southern lowlands and western highlands. Phylotypes I, II, and III were prevalent, though at unexpected frequencies. Phylotype I strains (87.5%) were genetically diverse and overrepresented in all agroecological areas, including highlands (AEZ III). Phylotype II strains (10.7%) only belonged to one tropical lowland-adapted broad host range lineage (IIA-35), whereas no highland-adapted potato brown rot (IIB-1) or Moko strains were detected. African phylotype III strains were rare (1.8%). They originated from a single Burkina Faso lineage (III-23) and were only found in lowlands. Three phylotype I strains were found harboring pRSC35, a plasmid identified in phylotype III strains in Cameroon. From pathogenicity tests performed on commercial varieties and tomato/eggplant/pepper references, the virulence diversity observed was high, with five pathoprofiles described. Eggplant accessions MM152 and EG203 and tomato HW7996 displayed the largest resistance spectrum and highest level. Two highly virulent phylotype I strains were able to bypass resistance of HW7996 and the eggplant reference AG91-25. Collectively, these points lead to the conclusion that the situation in Côte d'Ivoire is specific towards other African countries, and specifically from the Cameroon reference, and that within phylotype I can exist a high virulence diversity. This calls for similar studies in neighboring West African countries, linking R. solanacearum pathogen genetic diversity to strain virulence at the regional level, for the rationalization of regional resistance deployment strategies and future resistance durability studies. PMID:22533876

  6. Response of tomato wilt pathogen Ralstonia solanacearum to the volatile organic compounds produced by a biocontrol strain Bacillus amyloliquefaciens SQR-9

    PubMed Central

    Raza, Waseem; Ling, Ning; Yang, Liudong; Huang, Qiwei; Shen, Qirong

    2016-01-01

    It is important to study the response of plant pathogens to the antibiosis traits of biocontrol microbes to design the efficient biocontrol strategies. In this study, we evaluated the role of volatile organic compounds (VOCs) produced by a biocontrol strain Bacillus amyloliquefaciens SQR-9 on the growth and virulence traits of tomato wilt pathogen Ralstonia solanacearum (RS). The VOCs of SQR-9 significantly inhibited the growth of RS on agar medium and in soil. In addition, the VOCs significantly inhibited the motility traits, production of antioxidant enzymes and exopolysaccharides, biofilm formation and tomato root colonization by RS. The strain SQR-9 produced 22 VOCs, but only nine VOCs showed 1–11% antibacterial activity against RS in their corresponding amounts; however, the consortium of all VOCs showed 70% growth inhibition of RS. The proteomics analysis showed that the VOCs of SQR-9 downregulated RS proteins related to the antioxidant activity, virulence, carbohydrate and amino acid metabolism, protein folding and translation, while the proteins involved in the ABC transporter system, amino acid synthesis, detoxification of aldehydes and ketones, methylation, protein translation and folding, and energy transfer were upregulated. This study describes the significance and effectiveness of VOCs produced by a biocontrol strain against tomato wilt pathogen. PMID:27103342

  7. Response of tomato wilt pathogen Ralstonia solanacearum to the volatile organic compounds produced by a biocontrol strain Bacillus amyloliquefaciens SQR-9.

    PubMed

    Raza, Waseem; Ling, Ning; Yang, Liudong; Huang, Qiwei; Shen, Qirong

    2016-01-01

    It is important to study the response of plant pathogens to the antibiosis traits of biocontrol microbes to design the efficient biocontrol strategies. In this study, we evaluated the role of volatile organic compounds (VOCs) produced by a biocontrol strain Bacillus amyloliquefaciens SQR-9 on the growth and virulence traits of tomato wilt pathogen Ralstonia solanacearum (RS). The VOCs of SQR-9 significantly inhibited the growth of RS on agar medium and in soil. In addition, the VOCs significantly inhibited the motility traits, production of antioxidant enzymes and exopolysaccharides, biofilm formation and tomato root colonization by RS. The strain SQR-9 produced 22 VOCs, but only nine VOCs showed 1-11% antibacterial activity against RS in their corresponding amounts; however, the consortium of all VOCs showed 70% growth inhibition of RS. The proteomics analysis showed that the VOCs of SQR-9 downregulated RS proteins related to the antioxidant activity, virulence, carbohydrate and amino acid metabolism, protein folding and translation, while the proteins involved in the ABC transporter system, amino acid synthesis, detoxification of aldehydes and ketones, methylation, protein translation and folding, and energy transfer were upregulated. This study describes the significance and effectiveness of VOCs produced by a biocontrol strain against tomato wilt pathogen. PMID:27103342

  8. Two asian jumbo phages, ϕRSL2 and ϕRSF1, infect Ralstonia solanacearum and show common features of ϕKZ-related phages.

    PubMed

    Bhunchoth, Anjana; Blanc-Mathieu, Romain; Mihara, Tomoko; Nishimura, Yosuke; Askora, Ahmed; Phironrit, Namthip; Leksomboon, Chalida; Chatchawankanphanich, Orawan; Kawasaki, Takeru; Nakano, Miyako; Fujie, Makoto; Ogata, Hiroyuki; Yamada, Takashi

    2016-07-01

    Jumbo phages infecting Ralstonia solanacearum were isolated in Thailand (ϕRSL2) and Japan (ϕRSF1). They were similar regarding virion morphology, genomic arrangement, and host range. Phylogenetic and proteomic tree analyses demonstrate that the ϕRSL2 and ϕRSF1 belong to a group of evolutionary related phages, including Pseudomonas phages ϕKZ, 201ϕ2-1 and all previously described ϕKZ-related phages. Despite conserved genomic co-linearity between the ϕRSL2 and ϕRSF1, they differ in protein separation patterns. A major difference was seen in the detection of virion-associated-RNA polymerase subunits. All β- and β'-subunits were detected in ϕRSF1, but one β'-subunit was undetected in ϕRSL2. Furthermore, ϕRSF1 infected host cells faster (latent period: 60 and 150min for ϕRSF1 and ϕRSL2, respectively) and more efficiently than ϕRSL2. Therefore, the difference in virion-associated-RNA polymerase may affect infection efficiency. Finally, we show that ϕRSF1 is able to inhibit bacterial wilt progression in tomato plants. PMID:27081857

  9. Two asian jumbo phages, ϕRSL2 and ϕRSF1, infect Ralstonia solanacearum and show common features of ϕKZ-related phages.

    PubMed

    Bhunchoth, Anjana; Blanc-Mathieu, Romain; Mihara, Tomoko; Nishimura, Yosuke; Askora, Ahmed; Phironrit, Namthip; Leksomboon, Chalida; Chatchawankanphanich, Orawan; Kawasaki, Takeru; Nakano, Miyako; Fujie, Makoto; Ogata, Hiroyuki; Yamada, Takashi

    2016-07-01

    Jumbo phages infecting Ralstonia solanacearum were isolated in Thailand (ϕRSL2) and Japan (ϕRSF1). They were similar regarding virion morphology, genomic arrangement, and host range. Phylogenetic and proteomic tree analyses demonstrate that the ϕRSL2 and ϕRSF1 belong to a group of evolutionary related phages, including Pseudomonas phages ϕKZ, 201ϕ2-1 and all previously described ϕKZ-related phages. Despite conserved genomic co-linearity between the ϕRSL2 and ϕRSF1, they differ in protein separation patterns. A major difference was seen in the detection of virion-associated-RNA polymerase subunits. All β- and β'-subunits were detected in ϕRSF1, but one β'-subunit was undetected in ϕRSL2. Furthermore, ϕRSF1 infected host cells faster (latent period: 60 and 150min for ϕRSF1 and ϕRSL2, respectively) and more efficiently than ϕRSL2. Therefore, the difference in virion-associated-RNA polymerase may affect infection efficiency. Finally, we show that ϕRSF1 is able to inhibit bacterial wilt progression in tomato plants.

  10. A Resource Allocation Trade-Off between Virulence and Proliferation Drives Metabolic Versatility in the Plant Pathogen Ralstonia solanacearum

    PubMed Central

    Marmiesse, Lucas; Gouzy, Jérôme

    2016-01-01

    Bacterial pathogenicity relies on a proficient metabolism and there is increasing evidence that metabolic adaptation to exploit host resources is a key property of infectious organisms. In many cases, colonization by the pathogen also implies an intensive multiplication and the necessity to produce a large array of virulence factors, which may represent a significant cost for the pathogen. We describe here the existence of a resource allocation trade-off mechanism in the plant pathogen R. solanacearum. We generated a genome-scale reconstruction of the metabolic network of R. solanacearum, together with a macromolecule network module accounting for the production and secretion of hundreds of virulence determinants. By using a combination of constraint-based modeling and metabolic flux analyses, we quantified the metabolic cost for production of exopolysaccharides, which are critical for disease symptom production, and other virulence factors. We demonstrated that this trade-off between virulence factor production and bacterial proliferation is controlled by the quorum-sensing-dependent regulatory protein PhcA. A phcA mutant is avirulent but has a better growth rate than the wild-type strain. Moreover, a phcA mutant has an expanded metabolic versatility, being able to metabolize 17 substrates more than the wild-type. Model predictions indicate that metabolic pathways are optimally oriented towards proliferation in a phcA mutant and we show that this enhanced metabolic versatility in phcA mutants is to a large extent a consequence of not paying the cost for virulence. This analysis allowed identifying candidate metabolic substrates having a substantial impact on bacterial growth during infection. Interestingly, the substrates supporting well both production of virulence factors and growth are those found in higher amount within the plant host. These findings also provide an explanatory basis to the well-known emergence of avirulent variants in R. solanacearum

  11. CaCDPK15 positively regulates pepper responses to Ralstonia solanacearum inoculation and forms a positive-feedback loop with CaWRKY40 to amplify defense signaling.

    PubMed

    Shen, Lei; Yang, Sheng; Yang, Tong; Liang, Jiaqi; Cheng, Wei; Wen, Jiayu; Liu, Yanyan; Li, Jiazhi; Shi, Lanping; Tang, Qian; Shi, Wei; Hu, Jiong; Liu, Cailing; Zhang, Yangwen; Mou, Shaoliang; Liu, Zhiqin; Cai, Hanyang; He, Li; Guan, Deyi; Wu, Yang; He, Shuilin

    2016-01-01

    CaWRKY40 is a positive regulator of pepper (Capsicum annum) response to Ralstonia solanacearum inoculation (RSI), but the underlying mechanism remains largely unknown. Here, we functionally characterize CaCDPK15 in the defense signaling mediated by CaWRKY40. Pathogen-responsive TGA, W, and ERE boxes were identified in the CaCDPK15 promoter (pCaCDPK15), and pCaCDPK15-driven GUS expression was significantly enhanced in response to RSI and exogenously applied salicylic acid, methyl jasmonate, abscisic acid, and ethephon. Virus-induced gene silencing (VIGS) of CaCDPK15 significantly increased the susceptibility of pepper to RSI and downregulated the immunity-associated markers CaNPR1, CaPR1, and CaDEF1. By contrast, transient CaCDPK15 overexpression significantly activated hypersensitive response associated cell death, upregulated the immunity-associated marker genes, upregulated CaWRKY40 expression, and enriched CaWRKY40 at the promoters of its targets genes. Although CaCDPK15 failed to interact with CaWRKY40, the direct binding of CaWRKY40 to pCaCDPK15 was detected by chromatin immunoprecipitation, which was significantly potentiated by RSI in pepper plants. These combined results suggest that RSI in pepper induces CaCDPK15 and indirectly activates downstream CaWRKY40, which in turn potentiates CaCDPK15 expression. This positive-feedback loop would amplify defense signaling against RSI and efficiently activate strong plant immunity. PMID:26928570

  12. Ralstonia solanacearum ΔPGI-1 strain KZR-5 is affected in growth, response to cold stress and invasion of tomato.

    PubMed

    Stevens, Patricia; van Overbeek, Leonard Simon; van Elsas, Jan Dirk

    2011-01-01

    The survival and persistence of Ralstonia solanacearum biovar 2 in temperate climates is still poorly understood. To assess whether genomic variants of the organism show adaptation to local conditions, we compared the behaviour of environmental strain KZR-5, which underwent a deletion of the 17.6 kb genomic island PGI-1, with that of environmental strain KZR-1 and potato-derived strains 1609 and 715. PGI-1 harbours two genes of potential ecological relevance, i.e. one encoding a hypothetical protein with a RelA/SpoT domain and one a putative cellobiohydrolase. We thus assessed bacterial fate under conditions of amino acid starvation, during growth, upon incubation at low temperature and invasion of tomato plants. In contrast to the other strains, environmental strain KZR-5 did not grow on media that induce amino acid starvation. In addition, its maximum growth rate at 28°C in rich medium was significantly reduced. On the other hand, long-term survival at 4°C was significantly enhanced as compared to that of strains 1609, 715 and KZR-1. Although strain KZR-5 showed growth rates (at 28°C) in two different media, which were similar to those of strains 1609 and 715, its ability to compete with these strains under these conditions was reduced. In singly inoculated tomato plants, no significant differences in invasiveness were observed among strains KZR-5, KZR-1, 1609 and 715. However, reduced competitiveness of strain KZR-5 was found in experiments on tomato plant colonisation and wilting when using 1:1 or 5:1 mixtures of strains. The potential role of PGI-1 in plant invasion, response to stress and growth in competition at high and moderate temperatures is discussed.

  13. CaCDPK15 positively regulates pepper responses to Ralstonia solanacearum inoculation and forms a positive-feedback loop with CaWRKY40 to amplify defense signaling

    PubMed Central

    Shen, Lei; Yang, Sheng; Yang, Tong; Liang, Jiaqi; Cheng, Wei; Wen, Jiayu; Liu, Yanyan; Li, Jiazhi; Shi, Lanping; Tang, Qian; Shi, Wei; Hu, Jiong; Liu, Cailing; Zhang, Yangwen; Mou, Shaoliang; Liu, Zhiqin; Cai, Hanyang; He, Li; Guan, Deyi; Wu, Yang; He, Shuilin

    2016-01-01

    CaWRKY40 is a positive regulator of pepper (Capsicum annum) response to Ralstonia solanacearum inoculation (RSI), but the underlying mechanism remains largely unknown. Here, we functionally characterize CaCDPK15 in the defense signaling mediated by CaWRKY40. Pathogen-responsive TGA, W, and ERE boxes were identified in the CaCDPK15 promoter (pCaCDPK15), and pCaCDPK15-driven GUS expression was significantly enhanced in response to RSI and exogenously applied salicylic acid, methyl jasmonate, abscisic acid, and ethephon. Virus-induced gene silencing (VIGS) of CaCDPK15 significantly increased the susceptibility of pepper to RSI and downregulated the immunity-associated markers CaNPR1, CaPR1, and CaDEF1. By contrast, transient CaCDPK15 overexpression significantly activated hypersensitive response associated cell death, upregulated the immunity-associated marker genes, upregulated CaWRKY40 expression, and enriched CaWRKY40 at the promoters of its targets genes. Although CaCDPK15 failed to interact with CaWRKY40, the direct binding of CaWRKY40 to pCaCDPK15 was detected by chromatin immunoprecipitation, which was significantly potentiated by RSI in pepper plants. These combined results suggest that RSI in pepper induces CaCDPK15 and indirectly activates downstream CaWRKY40, which in turn potentiates CaCDPK15 expression. This positive-feedback loop would amplify defense signaling against RSI and efficiently activate strong plant immunity. PMID:26928570

  14. Pepper CabZIP63 acts as a positive regulator during Ralstonia solanacearum or high temperature–high humidity challenge in a positive feedback loop with CaWRKY40

    PubMed Central

    Shen, Lei; Liu, Zhiqin; Yang, Sheng; Yang, Tong; Liang, Jiaqi; Wen, Jiayu; Liu, Yanyan; Li, Jiazhi; Shi, Lanping; Tang, Qian; Shi, Wei; Hu, Jiong; Liu, Cailing; Zhang, Yangwen; Lin, Wei; Wang, Rongzhang; Yu, Huanxin; Mou, Shaoliang; Hussain, Ansar; Cheng, Wei; Cai, Hanyang; He, Li; Guan, Deyi; Wu, Yang; He, Shuilin

    2016-01-01

    CaWRKY40 is known to act as a positive regulator in the response of pepper (Capsicum annuum) to Ralstonia solanacearum inoculation (RSI) or high temperature–high humidity (HTHH), but the underlying mechanism remains elusive. Herein, we report that CabZIP63, a pepper bZIP family member, participates in this process by regulating the expression of CaWRKY40. CabZIP63 was found to localize in the nuclei, be up-regulated by RSI or HTHH, bind to promoters of both CabZIP63 (pCabZIP63) and CaWRKY40 (pCaWRKY40), and activate pCabZIP63- and pCaWRKY40-driven β-glucuronidase expression in a C- or G-box-dependent manner. Silencing of CabZIP63 by virus-induced gene silencing (VIGS) in pepper plants significantly attenuated their resistance to RSI and tolerance to HTHH, accompanied by down-regulation of immunity- or thermotolerance-associated CaPR1, CaNPR1, CaDEF1, and CaHSP24. Hypersensitive response-mediated cell death and expression of the tested immunity- and thermotolerance-associated marker genes were induced by transient overexpression (TOE) of CabZIP63, but decreased by that of CabZIP63-SRDX. Additionally, binding of CabZIP63 to pCaWRKY40 was up-regulated by RSI or HTHH, and the transcript level of CaWRKY40 and binding of CaWRKY40 to the promoters of CaPR1, CaNPR1, CaDEF1 and CaHSP24 were up-regulated by TOE of CabZIP63. On the other hand, CabZIP63 was also up-regulated transcriptionally by TOE of CaWRKY40. The data suggest collectively that CabZIP63 directly or indirectly regulates the expression of CaWRKY40 at both the transcriptional and post-transcriptional level, forming a positive feedback loop with CaWRKY40 during pepper’s response to RSI or HTHH. Altogether, our data will help to elucidate the underlying mechanism of crosstalk between pepper’s response to RSI and HTHH. PMID:26936828

  15. Pepper CabZIP63 acts as a positive regulator during Ralstonia solanacearum or high temperature-high humidity challenge in a positive feedback loop with CaWRKY40.

    PubMed

    Shen, Lei; Liu, Zhiqin; Yang, Sheng; Yang, Tong; Liang, Jiaqi; Wen, Jiayu; Liu, Yanyan; Li, Jiazhi; Shi, Lanping; Tang, Qian; Shi, Wei; Hu, Jiong; Liu, Cailing; Zhang, Yangwen; Lin, Wei; Wang, Rongzhang; Yu, Huanxin; Mou, Shaoliang; Hussain, Ansar; Cheng, Wei; Cai, Hanyang; He, Li; Guan, Deyi; Wu, Yang; He, Shuilin

    2016-04-01

    CaWRKY40 is known to act as a positive regulator in the response of pepper (Capsicum annuum) to Ralstonia solanacearum inoculation (RSI) or high temperature-high humidity (HTHH), but the underlying mechanism remains elusive. Herein, we report that CabZIP63, a pepper bZIP family member, participates in this process by regulating the expression of CaWRKY40. CabZIP63 was found to localize in the nuclei, be up-regulated by RSI or HTHH, bind to promoters of both CabZIP63(pCabZIP63) and CaWRKY40(pCaWRKY40), and activate pCabZIP63- and pCaWRKY40-driven β-glucuronidase expression in a C- or G-box-dependent manner. Silencing of CabZIP63 by virus-induced gene silencing (VIGS) in pepper plants significantly attenuated their resistance to RSI and tolerance to HTHH, accompanied by down-regulation of immunity- or thermotolerance-associated CaPR1, CaNPR1, CaDEF1, and CaHSP24. Hypersensitive response-mediated cell death and expression of the tested immunity- and thermotolerance-associated marker genes were induced by transient overexpression (TOE) of CabZIP63, but decreased by that of CabZIP63-SRDX. Additionally, binding of CabZIP63 to pCaWRKY40 was up-regulated by RSI or HTHH, and the transcript level of CaWRKY40 and binding of CaWRKY40 to the promoters of CaPR1, CaNPR1, CaDEF1 and CaHSP24 were up-regulated by TOE of CabZIP63. On the other hand, CabZIP63 was also up-regulated transcriptionally by TOE of CaWRKY40. The data suggest collectively that CabZIP63 directly or indirectly regulates the expression of CaWRKY40 at both the transcriptional and post-transcriptional level, forming a positive feedback loop with CaWRKY40 during pepper's response to RSI or HTHH. Altogether, our data will help to elucidate the underlying mechanism of crosstalk between pepper's response to RSI and HTHH. PMID:26936828

  16. Pepper CabZIP63 acts as a positive regulator during Ralstonia solanacearum or high temperature-high humidity challenge in a positive feedback loop with CaWRKY40.

    PubMed

    Shen, Lei; Liu, Zhiqin; Yang, Sheng; Yang, Tong; Liang, Jiaqi; Wen, Jiayu; Liu, Yanyan; Li, Jiazhi; Shi, Lanping; Tang, Qian; Shi, Wei; Hu, Jiong; Liu, Cailing; Zhang, Yangwen; Lin, Wei; Wang, Rongzhang; Yu, Huanxin; Mou, Shaoliang; Hussain, Ansar; Cheng, Wei; Cai, Hanyang; He, Li; Guan, Deyi; Wu, Yang; He, Shuilin

    2016-04-01

    CaWRKY40 is known to act as a positive regulator in the response of pepper (Capsicum annuum) to Ralstonia solanacearum inoculation (RSI) or high temperature-high humidity (HTHH), but the underlying mechanism remains elusive. Herein, we report that CabZIP63, a pepper bZIP family member, participates in this process by regulating the expression of CaWRKY40. CabZIP63 was found to localize in the nuclei, be up-regulated by RSI or HTHH, bind to promoters of both CabZIP63(pCabZIP63) and CaWRKY40(pCaWRKY40), and activate pCabZIP63- and pCaWRKY40-driven β-glucuronidase expression in a C- or G-box-dependent manner. Silencing of CabZIP63 by virus-induced gene silencing (VIGS) in pepper plants significantly attenuated their resistance to RSI and tolerance to HTHH, accompanied by down-regulation of immunity- or thermotolerance-associated CaPR1, CaNPR1, CaDEF1, and CaHSP24. Hypersensitive response-mediated cell death and expression of the tested immunity- and thermotolerance-associated marker genes were induced by transient overexpression (TOE) of CabZIP63, but decreased by that of CabZIP63-SRDX. Additionally, binding of CabZIP63 to pCaWRKY40 was up-regulated by RSI or HTHH, and the transcript level of CaWRKY40 and binding of CaWRKY40 to the promoters of CaPR1, CaNPR1, CaDEF1 and CaHSP24 were up-regulated by TOE of CabZIP63. On the other hand, CabZIP63 was also up-regulated transcriptionally by TOE of CaWRKY40. The data suggest collectively that CabZIP63 directly or indirectly regulates the expression of CaWRKY40 at both the transcriptional and post-transcriptional level, forming a positive feedback loop with CaWRKY40 during pepper's response to RSI or HTHH. Altogether, our data will help to elucidate the underlying mechanism of crosstalk between pepper's response to RSI and HTHH.

  17. Isolation of a Pseudomonas solanacearum-specific DNA probe by subtraction hybridization and construction of species-specific oligonucleotide primers for sensitive detection by the polymerase chain reaction.

    PubMed Central

    Seal, S E; Jackson, L A; Daniels, M J

    1992-01-01

    A subtraction hybridization technique was employed to make a library enriched for Pseudomonas solanacearum-specific sequences. One cloned fragment, PS2096, hybridized under stringent conditions to DNA of 82 P. solanacearum strains representing all subgroups of the species. Other plant-associated bacteria, including closely related species such as Pseudomonas capacia, Pseudomonas picketti, or Pseudomonas syzygii, did not hybridize to PS2096. A minimum number of between 4 x 10(5) and 4 x 10(6) P. solanacearum cells could routinely be detected with PS2096 labelled either with [32P]dCTP or with digoxigenin-11-dUTP. To improve the sensitivity of detection, PS2096 was sequenced to allow the construction of specific oligonucleotide primers to be used for polymerase chain reaction (PCR) amplification. After 50 cycles of amplification, 5 to 116 cells, depending on the strain, could reproducibly be detected by visualization of a 148-bp PCR product on an agarose gel. A preliminary field trial in Burundi with the probe and PCR primers has confirmed that they are sensitive tools for specifically detecting low-level infections of P. solanacearum in potato tubers. Images PMID:1482193

  18. Identification of the mcpA and mcpM genes, encoding methyl-accepting proteins involved in amino acid and l-malate chemotaxis, and involvement of McpM-mediated chemotaxis in plant infection by Ralstonia pseudosolanacearum (formerly Ralstonia solanacearum phylotypes I and III).

    PubMed

    Hida, Akiko; Oku, Shota; Kawasaki, Takeru; Nakashimada, Yutaka; Tajima, Takahisa; Kato, Junichi

    2015-11-01

    Sequence analysis has revealed the presence of 22 putative methyl-accepting chemotaxis protein (mcp) genes in the Ralstonia pseudosolanacearum GMI1000 genome. PCR analysis and DNA sequencing showed that the highly motile R. pseudosolanacearum strain Ps29 possesses homologs of all 22 R. pseudosolanacearum GMI1000 mcp genes. We constructed a complete collection of single mcp gene deletion mutants of R. pseudosolanacearum Ps29 by unmarked gene deletion. Screening of the mutant collection revealed that R. pseudosolanacearum Ps29 mutants of RSp0507 and RSc0606 homologs were defective in chemotaxis to l-malate and amino acids, respectively. RSp0507 and RSc0606 homologs were designated mcpM and mcpA. While wild-type R. pseudosolanacearum strain Ps29 displayed attraction to 16 amino acids, the mcpA mutant showed no response to 12 of these amino acids and decreased responses to 4 amino acids. We constructed mcpA and mcpM deletion mutants of highly virulent R. pseudosolanacearum strain MAFF106611 to investigate the contribution of chemotaxis to l-malate and amino acids to tomato plant infection. Neither single mutant exhibited altered virulence for tomato plants when tested by root dip inoculation assays. In contrast, the mcpM mutant (but not the mcpA mutant) was significantly less infectious than the wild type when tested by a sand soak inoculation assay, which requires bacteria to locate and invade host roots from sand. Thus, McpM-mediated chemotaxis, possibly reflecting chemotaxis to l-malate, facilitates R. pseudosolanacearum motility to tomato roots in sand.

  19. Comparative large-scale analysis of interactions between several crop species and the effector repertoires from multiple pathovars of Pseudomonas and Ralstonia.

    PubMed

    Wroblewski, Tadeusz; Caldwell, Katherine S; Piskurewicz, Urszula; Cavanaugh, Keri A; Xu, Huaqin; Kozik, Alexander; Ochoa, Oswaldo; McHale, Leah K; Lahre, Kirsten; Jelenska, Joanna; Castillo, Jose A; Blumenthal, Daniel; Vinatzer, Boris A; Greenberg, Jean T; Michelmore, Richard W

    2009-08-01

    Bacterial plant pathogens manipulate their hosts by injection of numerous effector proteins into host cells via type III secretion systems. Recognition of these effectors by the host plant leads to the induction of a defense reaction that often culminates in a hypersensitive response manifested as cell death. Genes encoding effector proteins can be exchanged between different strains of bacteria via horizontal transfer, and often individual strains are capable of infecting multiple hosts. Host plant species express diverse repertoires of resistance proteins that mediate direct or indirect recognition of bacterial effectors. As a result, plants and their bacterial pathogens should be considered as two extensive coevolving groups rather than as individual host species coevolving with single pathovars. To dissect the complexity of this coevolution, we cloned 171 effector-encoding genes from several pathovars of Pseudomonas and Ralstonia. We used Agrobacterium tumefaciens-mediated transient assays to test the ability of each effector to induce a necrotic phenotype on 59 plant genotypes belonging to four plant families, including numerous diverse accessions of lettuce (Lactuca sativa) and tomato (Solanum lycopersicum). Known defense-inducing effectors (avirulence factors) and their homologs commonly induced extensive necrosis in many different plant species. Nonhost species reacted to multiple effector proteins from an individual pathovar more frequently and more intensely than host species. Both homologous and sequence-unrelated effectors could elicit necrosis in a similar spectrum of plants, suggesting common effector targets or targeting of the same pathways in the plant cell. PMID:19571308

  20. Comparative Large-Scale Analysis of Interactions between Several Crop Species and the Effector Repertoires from Multiple Pathovars of Pseudomonas and Ralstonia1[W][OA

    PubMed Central

    Wroblewski, Tadeusz; Caldwell, Katherine S.; Piskurewicz, Urszula; Cavanaugh, Keri A.; Xu, Huaqin; Kozik, Alexander; Ochoa, Oswaldo; McHale, Leah K.; Lahre, Kirsten; Jelenska, Joanna; Castillo, Jose A.; Blumenthal, Daniel; Vinatzer, Boris A.; Greenberg, Jean T.; Michelmore, Richard W.

    2009-01-01

    Bacterial plant pathogens manipulate their hosts by injection of numerous effector proteins into host cells via type III secretion systems. Recognition of these effectors by the host plant leads to the induction of a defense reaction that often culminates in a hypersensitive response manifested as cell death. Genes encoding effector proteins can be exchanged between different strains of bacteria via horizontal transfer, and often individual strains are capable of infecting multiple hosts. Host plant species express diverse repertoires of resistance proteins that mediate direct or indirect recognition of bacterial effectors. As a result, plants and their bacterial pathogens should be considered as two extensive coevolving groups rather than as individual host species coevolving with single pathovars. To dissect the complexity of this coevolution, we cloned 171 effector-encoding genes from several pathovars of Pseudomonas and Ralstonia. We used Agrobacterium tumefaciens-mediated transient assays to test the ability of each effector to induce a necrotic phenotype on 59 plant genotypes belonging to four plant families, including numerous diverse accessions of lettuce (Lactuca sativa) and tomato (Solanum lycopersicum). Known defense-inducing effectors (avirulence factors) and their homologs commonly induced extensive necrosis in many different plant species. Nonhost species reacted to multiple effector proteins from an individual pathovar more frequently and more intensely than host species. Both homologous and sequence-unrelated effectors could elicit necrosis in a similar spectrum of plants, suggesting common effector targets or targeting of the same pathways in the plant cell. PMID:19571308

  1. Biosynthesis of a Complex Yersiniabactin-Like Natural Product via the mic Locus in Phytopathogen Ralstonia solanacearum▿†

    PubMed Central

    Kreutzer, Martin F.; Kage, Hirokazu; Gebhardt, Peter; Wackler, Barbara; Saluz, Hans P.; Hoffmeister, Dirk; Nett, Markus

    2011-01-01

    A genome mining study in the plant pathogenic bacterium Ralstonia solanacearum GMI1000 unveiled a polyketide synthase/nonribosomal peptide synthetase gene cluster putatively involved in siderophore biosynthesis. Insertional mutagenesis confirmed the respective locus to be operational under iron-deficient conditions and spurred the isolation of the associated natural product. Bioinformatic analyses of the gene cluster facilitated the structural characterization of this compound, which was subsequently identified as the antimycoplasma agent micacocidin. The metal-chelating properties of micacocidin were evaluated in competition experiments, and the cellular uptake of gallium-micacocidin complexes was demonstrated in R. solanacearum GMI1000, indicating a possible siderophore role. Comparative genomics revealed a conservation of the micacocidin gene cluster in defined, but globally dispersed phylotypes of R. solanacearum. PMID:21724891

  2. Native Valve Endocarditis due to Ralstonia pickettii: A Case Report and Literature Review.

    PubMed

    Orme, Joseph; Rivera-Bonilla, Tomas; Loli, Akil; Blattman, Negin N

    2015-01-01

    Ralstonia pickettii is a rare pathogen and even more rare in healthy individuals. Here we report a case of R. pickettii bacteremia leading to aortic valve abscess and complete heart block. To our knowledge this is the first case report of Ralstonia species causing infective endocarditis with perivalvular abscess. PMID:25648998

  3. Native Valve Endocarditis due to Ralstonia pickettii: A Case Report and Literature Review.

    PubMed

    Orme, Joseph; Rivera-Bonilla, Tomas; Loli, Akil; Blattman, Negin N

    2015-01-01

    Ralstonia pickettii is a rare pathogen and even more rare in healthy individuals. Here we report a case of R. pickettii bacteremia leading to aortic valve abscess and complete heart block. To our knowledge this is the first case report of Ralstonia species causing infective endocarditis with perivalvular abscess.

  4. Native Valve Endocarditis due to Ralstonia pickettii: A Case Report and Literature Review

    PubMed Central

    Orme, Joseph; Rivera-Bonilla, Tomas; Loli, Akil; Blattman, Negin N.

    2015-01-01

    Ralstonia pickettii is a rare pathogen and even more rare in healthy individuals. Here we report a case of R. pickettii bacteremia leading to aortic valve abscess and complete heart block. To our knowledge this is the first case report of Ralstonia species causing infective endocarditis with perivalvular abscess. PMID:25648998

  5. 'Candidatus Liberibacter solanacearum', associated with plants in the family Solanaceae.

    PubMed

    Liefting, Lia W; Weir, Bevan S; Pennycook, Shaun R; Clover, Gerard R G

    2009-09-01

    A liberibacter (isolate NZ082226) was detected in a symptomatic tomato plant and subsequently in five other members of the family Solanaceae: capsicum, potato, tamarillo, cape gooseberry and chilli. Phylogenetic analyses of the 16S rRNA gene sequence, the deduced amino acid sequence of the rplJ gene and a partial nucleotide sequence of the beta operon indicated that isolate NZ082226 represents a novel candidate species of 'Candidatus Liberibacter', for which the name 'Candidatus Liberibacter solanacearum' is proposed.

  6. New report of additional enterobacterial species causing wilt in West Bengal, India.

    PubMed

    Sarkar, Shamayeeta; Chaudhuri, Sujata

    2015-07-01

    Ralstonia solanacearum is known to be the most prominent causal agent of bacterial wilt worldwide. It has a wide host range comprising solanaceous and nonsolanaceous plants. Typical symptoms of the disease are leaf wilt, browning of vascular tissues, and collapsing of the plant. With the objective of studying the diversity of pathogens causing bacterial wilt in West Bengal, we collected samples of diseased symptomatic crops and adjacent symptomatic and asymptomatic weeds from widespread locations in West Bengal. By means of a routine molecular identification test specific to "R. solanacearum species complex", the majority of these strains (68 out of 71) were found to not be R. solanacearum. Presumptive identification of these isolates with conventional biochemicals, extensive testing of pathogenicity of a subset involving greenhouse trials fulfilling Koch's postulate test, and scanning electron microscopic analysis for the presence of pathogen in diseased plants were done. 16S rDNA sequencing of a subset of these strains (GenBank accession Nos. JX880249-JX880251) and analysis of sequences with the nBLAST programme showed a high similarity (97%-99%) to sequences of the Enterobacteriaceae group available in GenBank. Molecular phylogeny further established the taxonomic position of the strains. The 3 bacterial strain cultures have been submitted to MTCC, Institute of Microbial Technology, Chandigarh, India, and were identified as Klebsiella oxytoca, Enterobacter cowanii, and Klebsiella oxytoca, respectively. Although Enterobacter sp. has previously been reported to cause wilt in many plants, susceptibility of most of the dedicated hosts of R. solanacearum to wilt caused by Enterobacter and other bacteria from Enterobacteriaceae is being reported for the first time in this work.

  7. Effects of environmental parameters on the dual-species biofilms formed by Escherichia coli O157:H7 and Ralstonia insidiosa, a strong biofilm producer isolated from a fresh-cut produce processing plant.

    PubMed

    Liu, Nancy T; Nou, Xiangwu; Bauchan, Gary R; Murphy, Charles; Lefcourt, Alan M; Shelton, Daniel R; Lo, Y Martin

    2015-01-01

    Biofilm-forming bacteria resident to food processing facilities are a food safety concern due to the potential of biofilms to harbor foodborne bacterial pathogens. When cultured together, Ralstonia insidiosa, a strong biofilm former frequently isolated from produce processing environments, has been shown to promote the incorporation of Escherichia coli O157:H7 into dual-species biofilms. In this study, interactions between E. coli O157:H7 and R. insidiosa were examined under different incubating conditions. Under static culture conditions, the incorporation of E. coli O157:H7 into biofilms with R. insidiosa was not significantly affected by either low incubating temperature (10°C) or by limited nutrient availability. Greater enhancement of E. coli O157:H7 incorporation in dual-species biofilms was observed by using a continuous culture system with limited nutrient availability. Under the continuous culture conditions used in this study, E coli O157:H7 cells showed a strong tendency of colocalizing with R. insidiosa on a glass surface at the early stage of biofilm formation. As the biofilms matured, E coli O157:H7 cells were mostly found at the bottom layer of the dual-species biofilms, suggesting an effective protection by R. insidiosa in the mature biofilms.

  8. Effects of environmental parameters on the dual-species biofilms formed by Escherichia coli O157:H7 and Ralstonia insidiosa, a strong biofilm producer isolated from a fresh-cut processing plant

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Biofilm forming bacteria resident to food processing facilities are a food safety concern due to the potential of biofilms to harbor foodborne bacterial pathogens. When cultured together, Ralstonia insidiosa, a strong biofilm former frequently isolated from produce processing environments, has been ...

  9. Enzymatic modification of chitosan by cinnamic acids: Antibacterial activity against Ralstonia solanacearum.

    PubMed

    Yang, Caifeng; Zhou, Yu; Zheng, Yu; Li, Changlong; Sheng, Sheng; Wang, Jun; Wu, Fuan

    2016-06-01

    This study aimed to identify chitosan polymers that have antibacterial activity against the bacterial wilt pathogen. The chitosan polymers were enzymatically synthesized using chitosan and five cinnamic acids (CADs): caffeic acid (CA), ferulic acid (FA), cinnamic acid (CIA), p-coumaric acid (COA) and chlorogenic acid (CHA), using laccase from Pleurotus ostreatus as a catalyst. The reaction was performed in a phosphate buffered solution under heterogenous reaction conditions. The chitosan derivatives (CTS-g-CADs) were characterized by FT-IR, XRD, TGA and SEM. FT-IR demonstrated that the reaction products bound covalently to the free amino groups or hydroxyl groups of chitosan via band of amide I or ester band. XRD showed a reduced packing density for grafted chitosan comparing to original chitosan. TGA demonstrated that CTS-g-CADs have a higher thermostability than chitosan. Additionally, chitosan and its derivatives showed similar antibacterial activity. However, the IC50 value of the chitosan-caffeic acid derivative (CTS-g-CA) against the mulberry bacterial wilt pathogen RS-5 was 0.23mg/mL, which was two-fifths of the IC50 value of chitosan. Therefore, the enzymatically synthesized chitosan polymers can be used to control plant diseases in biotechnological domains. PMID:26993531

  10. Enzymatic modification of chitosan by cinnamic acids: Antibacterial activity against Ralstonia solanacearum.

    PubMed

    Yang, Caifeng; Zhou, Yu; Zheng, Yu; Li, Changlong; Sheng, Sheng; Wang, Jun; Wu, Fuan

    2016-06-01

    This study aimed to identify chitosan polymers that have antibacterial activity against the bacterial wilt pathogen. The chitosan polymers were enzymatically synthesized using chitosan and five cinnamic acids (CADs): caffeic acid (CA), ferulic acid (FA), cinnamic acid (CIA), p-coumaric acid (COA) and chlorogenic acid (CHA), using laccase from Pleurotus ostreatus as a catalyst. The reaction was performed in a phosphate buffered solution under heterogenous reaction conditions. The chitosan derivatives (CTS-g-CADs) were characterized by FT-IR, XRD, TGA and SEM. FT-IR demonstrated that the reaction products bound covalently to the free amino groups or hydroxyl groups of chitosan via band of amide I or ester band. XRD showed a reduced packing density for grafted chitosan comparing to original chitosan. TGA demonstrated that CTS-g-CADs have a higher thermostability than chitosan. Additionally, chitosan and its derivatives showed similar antibacterial activity. However, the IC50 value of the chitosan-caffeic acid derivative (CTS-g-CA) against the mulberry bacterial wilt pathogen RS-5 was 0.23mg/mL, which was two-fifths of the IC50 value of chitosan. Therefore, the enzymatically synthesized chitosan polymers can be used to control plant diseases in biotechnological domains.

  11. First report of bacterial wilt caused by Ralstonia solanacearum on Mesona chinensis in China

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Jellywort (Mesona chinensis Benth) is a herbaceous plant in the Lamiaceae Family. The plant is referred to as ‘Xiancao’ (Weed from Angels) in Chinese and is primarily used to make grass jelly, a popular refreshing drink. Currently, Xiancao cultivation is a fast growing industry with a high profit ma...

  12. Metabolism of Thioamides by Ralstonia pickettii TA▿

    PubMed Central

    Dodge, Anthony G.; Richman, Jack E.; Johnson, Gilbert; Wackett, Lawrence P.

    2006-01-01

    Information on bacterial thioamide metabolism has focused on transformation of the antituberculosis drug ethionamide and related compounds by Mycobacterium tuberculosis. To study this metabolism more generally, a bacterium that grew using thioacetamide as the sole nitrogen source was isolated via enrichment culture. The bacterium was identified as Ralstonia pickettii and designated strain TA. Cells grown on thioacetamide also transformed other thioamide compounds. Transformation of the thioamides tested was dependent on oxygen. During thioamide degradation, sulfur was detected in the medium at the oxidation level of sulfite, further suggesting an oxygenase mechanism. R. pickettii TA did not grow on thiobenzamide as a nitrogen source, but resting cells converted thiobenzamide to benzamide, with thiobenzamide S-oxide and benzonitrile detected as intermediates. Thioacetamide S-oxide was detected as an intermediate during thioacetamide degradation, but the only accumulating metabolite of thioacetamide was identified as 3,5-dimethyl-1,2,4-thiadiazole, a compound shown to derive from spontaneous reaction of thioacetamide and oxygenated thioacetamide species. This dead-end metabolite accounted for only ca. 12% of the metabolized thioacetamide. Neither acetonitrile nor acetamide was detected during thioacetamide degradation, but R. pickettii grew on both compounds as nitrogen and carbon sources. It is proposed that R. pickettii TA degrades thioamides via a mechanism involving consecutive oxygenations of the thioamide sulfur atom. PMID:16997975

  13. Genomic Plasticity in Ralstonia eutropha and Ralstonia pickettii: Evidence for Rapid Genomic Change and Adaptation

    SciTech Connect

    Terence L. MArsh

    2007-06-27

    a sequence identical to the intracellular replicative form. Using PCR targeting phage-specific genes we showed that seven out of the eleven isolates carried the phage. The seven isolates positive for phage were formed one of the genomovar groups, hence the presence of the phage may have generated the divergent lineage. One representative of each of these genomovars was sequence by JGI. While the genomes have not been closed completely, the results so far are provocative. Strain 12J, which carries the phage, revealed four integrated copies of the phage genome, each copy at a different level of divergence from the active replicative form. A comparative analysis of the common genes found in these integrated phage copies revealed that the gene complements were incongruent with one another within a phage copy, suggesting that the copies had become sites of recombination or that the cell had recruited genes for different functions. One of the integrated copies had the exact sequence as the replicative form and we assume this to be the most recent integration event. Evidence for a recent integration was revealed in a repeated sequence element found on each terminus of the phage genome. Finally, these isolates are of interest in bioremediation and reclamation of metals from waste streams. We have determined that each Ralstonia cell is capable of binding 6 x 107 Cu (II) ions and that 90% of the binding occurs within 12 hours of exposure to Cu(II). Our studies have revealed a species with a uniquely fluid genome. This Ralstonia population has been under extreme selective pressure over the past hundred years as it responded to the accumulation of copper in the lake sediment as a result of unregulated mining practices. In addition to the selective pressure of copper, the population was repeatedly infected with a filamentous phage that may have contributed to the divergence of the genomovars. This dynamic population could help reveal the selective forces and their consequences

  14. Intraspecific Variability within Globodera tabacum solanacearum Using Random Amplified Polymorphic DNA

    PubMed Central

    Syracuse, A. J.; Johnson, C. S.; Eisenback, J. D.; Nessler, C. L.; Smith, E. P.

    2004-01-01

    Random amplified polymorphic DNA (RAPDs) were used to investigate the intraspecific variability among 19 geographic isolates of Globodera tabacum solanacearum from eight counties in Virginia and one county in North Carolina. Globodera tabacum tabacum, G. t. virginiae, and the Mexican cyst nematode (MCN) were included as outgroups. Six primers were used and 119 amplification products were observed. Each primer yielded reproducible differences in fragment patterns that differentiated the isolates and species. Hierarchical cluster analysis was performed to illustrate the relatedness among isolates and species. The average Jaccard's similarity index among isolates of G. t. solanacearum was 74%, possibly representing greater variation than that reported in the literature across different pathotypes of the potato cyst nematode, Globodera pallida, in studies where RAPD were also employed. The RAPD markers described here may be useful for the development of specific primers or probes that could improve the identification of TCN populations. Such improvements in the characterization of TCN genotypes would facilitate the effective deployment of existing and future resistant cultivars to control these economically important pests. PMID:19262823

  15. Reproduction of Globodera tabacum solanacearum in Seven Flue-Cured Tobacco-Producing Soils

    PubMed Central

    Rideout, S. L.; JOHNSON, C. S.; Eisenback, J. D.; Reed, T. D.

    2000-01-01

    The tobacco cyst nematode (Globodera tabacum solanacearum) continues to pose a serious threat to flue-cured tobacco production in Virginia and nearby states. Soils were sampled from five uninfested and two infested flue-cured tobacco-producing locations. Twenty-three edaphic factors were characterized to determine if any were correlated with G. t. solanacearum reproduction. Comparisons were also made between pasteurized and natural soils to determine if biological suppression of G. t. solanacearum reproduction might be occurring in currently uninfested areas. Differences in G. t. solanacearum reproduction were noted among the soils, but results were inconsistent across the three trials conducted in this study. Only soil pH correlated significantly with nematode reproduction, and then only in one of three trials. Globodera tabacum solanacearum reproduced with similar efficiency in natural and pasteurized soils. PMID:19270999

  16. Expression of L-ornithine Ndelta-oxygenase (PvdA) in fluorescent Pseudomonas species: an immunochemical and in silico study.

    PubMed

    Putignani, Lorenza; Ambrosi, Cecilia; Ascenzi, Paolo; Visca, Paolo

    2004-01-01

    Omega-amino acid monooxygenases (EC 1.14.13.-), catalysing the formation of hydroxamate precursors of microbial siderophores (e.g., pyoverdine), have so far eluded structural and biochemical characterisation. Here, the expression of recombinant L-ornithine-Ndelta-oxygenase (PvdA) from Pseudomonas aeruginosa PAO1 is reported. A library of eight monoclonal antibodies (MAbs) directed against PvdA has been generated. Two MAb families recognising the N- and C-terminal regions of PvdA were identified. The MAbs made it possible to demonstrate that 45-48 kDa PvdA homologues are expressed in response to iron limitation by different species and strains of fluorescent pseudomonads. Despite the different degrees in sequence similarity between P. aeruginosa PvdA and putative homologues from Pseudomonas fluorescens, Pseudomonas putida, Pseudomonas syringae, Burkholderia cepacia, and Ralstonia solanacearum, in silico domain scanning predicts an impressive conservation of putative cofactor and substrate binding domains. The MAb library was also used to monitor PvdA expression during the transition of P. aeruginosa from iron-sufficient to iron-deficient growth. PMID:14684153

  17. Sequence analysis of Candidatus Liberibacter solanacearum (Lso-C) isolated from carrot psyllids collected in Scandinavia

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The fastidious prokaryote Candidatus Liberibacter solanacearum (Lso), transmitted by the tomato potato psyllid (Bactericera cockerelli), is associated with the Zebra Chip disease of potato. Plants infected with Liberibacter may experience significant yield losses and these plants also serve as pote...

  18. A putative monofunctional glycosyltransferase is expressed in Ralstonia eutropha.

    PubMed Central

    Paik, J; Jendrossek, D; Hakenbeck, R

    1997-01-01

    A gene, mgt, encoding a protein homologous to the N-terminal module of class A high-molecular-mass penicillin-binding proteins was identified in Ralstonia eutropha. By using specific antibodies, the corresponding Mgt protein was detected in association with the membrane, confirming that the N-terminal hydrophobic segment functioned as a membrane anchor. A derivative in which the hydrophobic sequence was deleted was overexpressed as a maltose-binding fusion protein in Escherichia coli. Cleavage of the product resulted in substantial amounts of soluble Mgt derivative, indicating that folding occurs independently on other proteins or on homologous domains of penicillin-binding proteins. PMID:9190828

  19. Draft Genome Sequences of Ralstonia pickettii Strains SSH4 and CW2, Isolated from Space Equipment

    PubMed Central

    Monsieurs, Pieter; Mijnendonckx, Kristel; Provoost, Ann; Venkateswaran, Kasthuri; Ott, C. Mark; Leys, Natalie

    2014-01-01

    Ralstonia pickettii SSH4 and CW2 were isolated from space equipment. Here, we report their draft genome sequences with the aim of gaining insight into their potential to adapt to these environments. PMID:25189592

  20. Phenotypic evaluation of the Chinese mini-mini core collection of peanut (Arachis hypogaea L.) and assessment for resistance to bacterial wilt disease caused by Ralstonia solanacearum

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In order to utilize the germplasm more efficiently for peanut (Arachis hypogaea L.) genetic improvement, a core collection of 576 accessions and a primary mini core collection of 298 accessions was developed previously from a collection of 6,839 cultivated peanut lines stored at the Oil Crops Resear...

  1. Arsenite oxidase from Ralstonia sp. 22: characterization of the enzyme and its interaction with soluble cytochromes.

    PubMed

    Lieutaud, Aurélie; van Lis, Robert; Duval, Simon; Capowiez, Line; Muller, Daniel; Lebrun, Régine; Lignon, Sabrina; Fardeau, Marie-Laure; Lett, Marie-Claire; Nitschke, Wolfgang; Schoepp-Cothenet, Barbara

    2010-07-01

    We characterized the aro arsenite oxidation system in the novel strain Ralstonia sp. 22, a beta-proteobacterium isolated from soil samples of the Salsigne mine in southern France. The inducible aro system consists of a heterodimeric membrane-associated enzyme reacting with a dedicated soluble cytochrome c(554). Our biochemical results suggest that the weak association of the enzyme to the membrane probably arises from a still unknown interaction partner. Analysis of the phylogeny of the aro gene cluster revealed that it results from a lateral gene transfer from a species closely related to Achromobacter sp. SY8. This constitutes the first clear cut case of such a transfer in the Aro phylogeny. The biochemical study of the enzyme demonstrates that it can accommodate in vitro various cytochromes, two of which, c(552) and c(554,) are from the parent species. Cytochrome c(552) belongs to the sox and not the aro system. Kinetic studies furthermore established that sulfite and sulfide, substrates of the sox system, are both inhibitors of Aro activity. These results reinforce the idea that sulfur and arsenic metabolism are linked.

  2. Mobilization of Poly(3-Hydroxybutyrate) in Ralstonia eutropha

    PubMed Central

    Handrick, René; Reinhardt, Simone; Jendrossek, Dieter

    2000-01-01

    Ralstonia eutropha H16 degraded (mobilized) previously accumulated poly(3-hydroxybutyrate) (PHB) in the absence of an exogenous carbon source and used the degradation products for growth and survival. Isolated native PHB granules of mobilized R. eutropha cells released 3-hydroxybutyrate (3HB) at a threefold higher rate than did control granules of nonmobilized bacteria. No 3HB was released by native PHB granules of recombinant Escherichia coli expressing the PHB biosynthetic genes. Native PHB granules isolated from chromosomal knockout mutants of an intracellular PHB (i-PHB) depolymerase gene of R. eutropha H16 and HF210 showed a reduced but not completely eliminated activity of 3HB release and indicated the presence of i-PHB depolymerase isoenzymes. PMID:11004196

  3. Mobilization of Selenite by Ralstonia metallidurans CH34

    PubMed Central

    Roux, Murielle; Sarret, Géraldine; Pignot-Paintrand, Isabelle; Fontecave, Marc; Coves, Jacques

    2001-01-01

    Ralstonia metallidurans CH34 (formerly Alcaligenes eutrophus CH34) is a soil bacterium characteristic of metal-contaminated biotopes, as it is able to grow in the presence of a variety of heavy metals. R. metallidurans CH34 is reported now to resist up to 6 mM selenite and to reduce selenite to elemental red selenium as shown by extended X-ray absorption fine-structure analysis. Growth kinetics analysis suggests an adaptation of the cells to the selenite stress during the lag-phase period. Depending on the culture conditions, the medium can be completely depleted of selenite. Selenium accumulates essentially in the cytoplasm as judged from electron microscopy and energy-dispersive X-ray analysis. Elemental selenium, highly insoluble, represents a nontoxic storage form for the bacterium. The ability of R. metallidurans CH34 to reduce large amounts of selenite may be of interest for bioremediation processes targeting selenite-polluted sites. PMID:11157242

  4. De Novo Genome Sequence of "Candidatus Liberibacter solanacearum" from a Single Potato Psyllid in California.

    PubMed

    Wu, F; Deng, X; Liang, G; Wallis, C; Trumble, J T; Prager, S; Chen, J

    2015-01-01

    The draft genome sequence of "Candidatus Liberibacter solanacearum" strain RSTM from a potato psyllid (Bactericera cockerelli) in California is reported here. The RSTM strain has a genome size of 1,286,787 bp, a G+C content of 35.1%, 1,211 predicted open reading frames (ORFs), and 43 RNA genes. PMID:26679599

  5. Comparison of Candiatus Liberibacter solanacearum genomic sequences isolated from carrot psyllids in Scandinavia and potato psyllids from Texas

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The fastidious prokaryote Candidatus Liberibacter solanacearum (Lso), transmitted by the tomato potato psyllid (Bactericera cockerelli), is associated with the Zebra Chip disease of potato. Plants infected with Liberibacter may experience significant yield losses and these plants also serve as poten...

  6. Zebra chip disease and potato biochemistry: tuber physiological changes in response to 'Candidatus Liberibacter solanacearum' infection over time.

    PubMed

    Rashed, A; Wallis, C M; Paetzold, L; Workneh, F; Rush, C M

    2013-05-01

    Zebra chip disease, putatively caused by the bacterium 'Candidatus Liberibacter solanacearum', is of increasing concern to potato production in Mexico, the United States, and New Zealand. However, little is known about the etiology of this disease and changes that occur within host tubers that result in its symptoms. Previous studies found that increased levels of phenolics, amino acids, defense proteins, and carbohydrates in 'Ca. L. solanacearum'-infected tubers are associated with symptoms of zebra chip. This study was conducted to quantify variations in levels of these biochemical components in relation to the time of infestation, symptom severity, and 'Ca. L. solanacearum' titer. Levels of phenolics, peroxidases, polyphenol oxidases, and reducing sugars (glucose and, to some extent, fructose) changed during infection, with higher levels occurring in tubers infected at least 5 weeks before harvest than in those infected only a week before harvest and those of controls. Compared with the apical tuber ends, greater levels of phenolics, peroxidases, and sucrose occurred at the basal (stolon attachment) end of infected tubers. With the exception of phenolics, concentrations of the evaluated compounds were not associated with 'Ca. L. solanacearum' titer. However, there were significant associations between biochemical responses and symptom severity. The lack of a linear correlation between most plant biochemical responses and 'Ca. L. solanacearum' titer suggests that shifts in metabolic profiles are independent of variations in 'Ca. L. solanacearum' levels. PMID:23425237

  7. Zebra chip disease and potato biochemistry: tuber physiological changes in response to 'Candidatus Liberibacter solanacearum' infection over time.

    PubMed

    Rashed, A; Wallis, C M; Paetzold, L; Workneh, F; Rush, C M

    2013-05-01

    Zebra chip disease, putatively caused by the bacterium 'Candidatus Liberibacter solanacearum', is of increasing concern to potato production in Mexico, the United States, and New Zealand. However, little is known about the etiology of this disease and changes that occur within host tubers that result in its symptoms. Previous studies found that increased levels of phenolics, amino acids, defense proteins, and carbohydrates in 'Ca. L. solanacearum'-infected tubers are associated with symptoms of zebra chip. This study was conducted to quantify variations in levels of these biochemical components in relation to the time of infestation, symptom severity, and 'Ca. L. solanacearum' titer. Levels of phenolics, peroxidases, polyphenol oxidases, and reducing sugars (glucose and, to some extent, fructose) changed during infection, with higher levels occurring in tubers infected at least 5 weeks before harvest than in those infected only a week before harvest and those of controls. Compared with the apical tuber ends, greater levels of phenolics, peroxidases, and sucrose occurred at the basal (stolon attachment) end of infected tubers. With the exception of phenolics, concentrations of the evaluated compounds were not associated with 'Ca. L. solanacearum' titer. However, there were significant associations between biochemical responses and symptom severity. The lack of a linear correlation between most plant biochemical responses and 'Ca. L. solanacearum' titer suggests that shifts in metabolic profiles are independent of variations in 'Ca. L. solanacearum' levels.

  8. Formaldehyde degradation by Ralstonia eutropha in an immobilized cell bioreactor.

    PubMed

    Habibi, Alireza; Vahabzadeh, Farzaneh

    2013-01-01

    The formaldehyde (FA) degradation ability of the loofa-immobilized Ralstonia eutropha cells in a packed bed reactor was modeled using a statistically based design of the experiment (DOE) considering application of response surface methodology (RSM). The simultaneous effects of four operative test factors on the cells performance in terms of FA degradation rate and extent of the chemical oxygen demand (COD) removal were monitored. The combination of factors at initial FA concentration of 629.7 mg L(-1)h(-1), recycling substrate flow rate of 4.4 mL min(-1), aeration rate of 1.05 vvm, and the system's temperature of 28.8°C resulted the optimal conditions for the FA biodegradation rate and COD removal efficiency. Loofa porous structure was found to be a protective environment for the cells in exposing to the toxic substances and the scanning electron microscopy (SEM) images revealed extensive cells penetration within this support. Oxygen transfer analysis in the form of evaluating K la value was also carried out and at the optimum conditions of the DOE was equaled to 9.96 h(-1)and oxygen uptake rate was 35.6 mg L(-1)h(-1).

  9. Tests in vitro and in pots with certain chemicals for inhibition of Pseudomonas solanacearum.

    PubMed

    el-Goorani, M A; Abo-el-Dahab, M K; Wagih, E E

    1978-01-01

    Twenty one isolates of Pseudomonas solanacearum E. F. Smith (Race 3) from various parts of Egypt were inhibited in vitro by Ampicillin, Chloramphenicol, Kanamycin, Oxytetracycline. Tetracycline, Penicillin G, Streptomycin, Nabam (Dithane A-40), Maneb (Dithane M-22), Zinc-ion maneb complex (Dithane M-45), and the insecticide Chlorthion. On the basis of in vitro-sensitivity to the selected 11 chemicals the 21 isolates could not be separated into different groups. Carbendazin (Bavistin), Benomyl, Drazoxolon (Mil-Col), and Temik proved ineffective in inhibiting the in vitro growth of all isolates at all tested concentrations. Preliminary investigations indicate that drenching the soil with solutions of Dithane M-22 (0.25% w/v, Dithane M-45 (0.25%) w/v, or insecticide Chlorthion (50 microgram/ml active ingredient) decreased the incidence of potato wilt disease that developed in soil, artificially infested with P. solanacearum. PMID:696045

  10. Regulation of extracellular polygalacturonase production in Pseudomonas solanacearum. Progress report, [May 1, 1992--April 30, 1994

    SciTech Connect

    Allen, C.

    1994-06-01

    Pseudomonas solanacearum is an economically important plant pathogen that causes bacterial wilt disease of diverse crops. The bacterium produces at least three isozymes of polygalacturonase, which degrade plant cell walls and contribute substantially to bacterial wilt disease development. The central objective of this research project is to determine how expression of these enzymes is regulated. To this end, we isolated a positive trans-acting regulator of polygalacturonase production (pehR). We have focused on further characterization of the pehR mutant pheonotype, and studies of pehR expression. Preliminary results suggest pehR also regulates bacterial motility. An investigation of two unusual tyrosine phosphoproteins in P. solanacearum is also described.

  11. Highly virulent strains of Pseudomonas solanacearum that are defective in extracellular-polysaccharide production.

    PubMed Central

    Xu, P L; Iwata, M; Leong, S; Sequeira, L

    1990-01-01

    Extracellular polysaccharide (EPS) has long been regarded as one of the most important factors involved in wilting of plants by Pseudomonas solanacearum. By means of transposon Tn5 mutagenesis, we have isolated a class of mutants that have an afluidal colony morphology but retain the ability to cause severe wilting and death of tobacco plants. One such mutant, KD700, was studied in detail. By marker exchange mutagenesis, the altered colony morphology was shown to be the result of a single Tn5 insertion in a 14.3-kilobase EcoRI fragment. This defect could be corrected by introducing a homologous clone from a cosmid library of the wild-type, parental strain K60. The Tn5-containing fragment was introduced into other P. solanacearum wild-type strains by marker exchange, and these altered strains had the same afluidal phenotype as KD700. N-Acetylgalactosamine (GalNac), the major constituent of EPS of all wild-type strains of P. solanacearum, was not detected by gas chromatography-mass spectrometry analysis of vascular fluids from wilting plants infected by KD700. In contrast, GalNac was readily detected in similar fluids of plants infected by K60. Polysaccharides extracted from culture filtrates of KD700 contained approximately one-fifth of the GalNac present in polysaccharides from K60. No differences in growth rates in culture or in planta between the mutant and the parental strains were observed. Since strains that are deficient in EPS production can remain highly virulent to tobacco, we conclude that EPS, or at least its GalNac-containing component, may not be required for disease development by P. solanacearum. Images PMID:2163393

  12. Assessing the Likelihood of Transmission of Candidatus Liberibacter solanacearum to Carrot by Potato Psyllid, Bactericera cockerelli (Hemiptera: Triozidae).

    PubMed

    Munyaneza, Joseph E; Mustafa, Tariq; Fisher, Tonja W; Sengoda, Venkatesan G; Horton, David R

    2016-01-01

    'Candidatus Liberibacter solanacearum' (Lso) is a phloem-limited bacterium that severely affects important Solanaceae and Apiaceae crops, including potato, tomato, pepper, tobacco, carrot and celery. This bacterium is transmitted to solanaceous species by potato psyllid, Bactericera cockerelli, and to Apiaceae by carrot psyllids, including Trioza apicalis and Bactericera trigonica. Five haplotypes of Lso have so far been described, two are associated with solanaceous species and potato psyllids, whereas the other three are associated with carrot and celery crops and carrot psyllids. Little is known about cross-transmission of Lso to carrot by potato psyllids or to potato by carrot psyllids. Thus, the present study assessed whether potato psyllid can transmit Lso to carrot and whether Lso haplotypes infecting solanaceous species can also infect carrot and lead to disease symptom development. In addition, the stylet probing behavior of potato psyllid on carrot was assessed using electropenetrography (EPG) technology to further elucidate potential Lso transmission to Apiaceae by this potato insect pest. Results showed that, while potato psyllids survived on carrot for several weeks when confined on the plants under controlled laboratory and field conditions, the insects generally failed to infect carrot plants with Lso. Only three of the 200 carrot plants assayed became infected with Lso and developed characteristic disease symptoms. Lso infection in the symptomatic carrot plants was confirmed by polymerase chain reaction assay and Lso in the carrots was determined to be of the haplotype B, which is associated with solanaceous species. EPG results further revealed that potato psyllids readily feed on carrot xylem but rarely probe into the phloem tissue, explaining why little to no Lso infection occurred during the controlled laboratory and field cage transmission trials. Results of our laboratory and field transmission studies, combined with our EPG results, suggest

  13. Isolation, identification and characterization of a novel Ralstonia sp. FD-1, capable of degrading 4-fluoroaniline.

    PubMed

    Song, Erxi; Wang, Meizhen; Shen, Dongsheng

    2014-02-01

    A gram-negative strain, designated as FD-1, isolated from aerobic activated sludge was capable of metabolizing 4-fluoroaniline (4-FA) as its sole carbon and nitrogen source and energy supply. According to the Biolog GNIII detection method 17 of 71 carbon substrates were easily utilized, while 12 of 23 substrates did not inhibit strain FD-1. The 16S rDNA sequence from strain FD-1 was 99 % similar to Ralstonia sp., suggesting that it belonged to the genus Ralstonia. The optimal conditions for growth and 4-FA degradation were pH 7 and 30 °C. The tolerance to 4-FA were 1,250 mg/L, while the tolerance to salinity was 15 g/L. Catechol 2,3-dioxygenase activity was detected and degradation intermediates were analyzed by liquid chromatography mass spectrometry leading to a proposed degradation pathway and suggesting that extradiol cleavage was involved in 4-FA degradation. This is the first report on the degradation of 4-FA by a bacterium from the Ralstonia genus.

  14. Genome sequences of Ralstonia insidiosa type strain ATCC 49129 and strain FC1138, a strong biofilm producer isolated from a fresh-cut produce-processing plant

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Ralstonia insidiosa FC1138 is a strong biofilm producer, isolated from a local fresh-cut produce processing plant. Here, we present the complete genome sequence of Ralstonia insidiosa FC1138 which includes two circular chromosomes and a plasmid. To our knowledge, this is the first reported complete ...

  15. Cloning and Expression of a Ralstonia eutropha HF39 Gene Mediating Indigo Formation in Escherichia coli

    PubMed Central

    Drewlo, Sascha; Brämer, Christian O.; Madkour, Mohamed; Mayer, Frank; Steinbüchel, Alexander

    2001-01-01

    On complex medium Escherichia coli strains carrying hybrid plasmid pBEC/EE:11.0, pSKBEC/BE:9.0, pSKBEC/PP:3.3, or pSKBEC/PP:2.4 harboring genomic DNA of Ralstonia eutropha HF39 produced a blue pigment characterized as indigo by several chemical and spectroscopic methods. A 1,251-bp open reading frame (bec) was cloned and sequenced. The deduced amino acid sequence of bec showed only weak similarities to short-chain acyl-coenzyme A dehydrogenases, and the gene product catalyzed formation of indoxyl, a reactive preliminary stage for production of indigo. PMID:11282658

  16. Comparative Proteome Analysis Reveals Four Novel Polyhydroxybutyrate (PHB) Granule-Associated Proteins in Ralstonia eutropha H16

    PubMed Central

    Sznajder, Anna; Pfeiffer, Daniel

    2014-01-01

    Identification of proteins that were present in a polyhydroxybutyrate (PHB) granule fraction isolated from Ralstonia eutropha but absent in the soluble, membrane, and membrane-associated fractions revealed the presence of only 12 polypeptides with PHB-specific locations plus 4 previously known PHB-associated proteins with multiple locations. None of the previously postulated PHB depolymerase isoenzymes (PhaZa2 to PhaZa5, PhaZd1, and PhaZd2) and none of the two known 3-hydroxybutyrate oligomer hydrolases (PhaZb and PhaZc) were significantly present in isolated PHB granules. Four polypeptides were found that had not yet been identified in PHB granules. Three of the novel proteins are putative α/β-hydrolases, and two of those (A0671 and B1632) have a PHB synthase/depolymerase signature. The third novel protein (A0225) is a patatin-like phospholipase, a type of enzyme that has not been described for PHB granules of any PHB-accumulating species. No function has been ascribed to the fourth protein (A2001), but its encoding gene forms an operon with phaB2 (acetoacetyl-coenzyme A [CoA] reductase) and phaC2 (PHB synthase), and this is in line with a putative function in PHB metabolism. The localization of the four new proteins at the PHB granule surface was confirmed in vivo by fluorescence microscopy of constructed fusion proteins with enhanced yellow fluorescent protein (eYFP). Deletion of A0671 and B1632 had a minor but detectable effect on the PHB mobilization ability in the stationary growth phase of nutrient broth (NB)-gluconate cells, confirming the functional involvement of both proteins in PHB metabolism. PMID:25548058

  17. Draft Genome Sequence of Ralstonia sp. MD27, a Poly(3-Hydroxybutyrate)-Degrading Bacterium, Isolated from Compost

    PubMed Central

    Zhu, Morgan; McCully, Lucy M.; Silby, Mark W.; Charles-Ogan, Tamunonengiyeofori I.

    2015-01-01

    Ralstonia sp. strain MD27, a novel biopolymer-degrading betaproteobacterium, was isolated from compost samples. This organism has been shown to utilize the biopolymer poly(3-hydroxybutyrate) [P(3HB)] as a carbon source for growth. We report the draft genome sequence of MD27 with an estimated total sequence length of 5.9 Mb. PMID:26450738

  18. Evaluation of Chloropicrin as a Soil Fumigant against Ralstonia solanacarum in Ginger (Zingiber officinale Rosc.) Production in China

    PubMed Central

    Ma, Taotao; Liu, Pengfei; Shen, Jin; Li, Yuan; Ouyang, Canbin; Guo, Meixia; Cao, Aocheng

    2014-01-01

    Background Chloropicrin (Pic) offers a potential alternative to methyl bromide (MB) against Ralstonia solanacarum in ginger (Zingiber officinale Rosc.) production. MB is scheduled to be withdrawn from routine use by 2015 in developing countries. Methods Pic treatments were evaluated in a laboratory study and in three commercial ginger fields. Results Laboratory studies showed that the EC50 value and EC80 value of Pic were 2.7 and 3.7 mg a.i. kg−1 soil, respectively. Field trials in highly infested soil revealed that treatments of Pic at the dose of 50 g m−2 covered with totally impermeable film (TIF) or polyethylene film (PE) sharply reduced Ralstonia solanacarum and maintained high ginger yields. Both of the Pic treatments provided results similar to, or in some cases slightly lower than, MB with respect to Ralstonia solanacarum control, plant survival, plant growth and yield. All of the fumigant treatments were significantly better than the non-treated control. Conclusions The present study confirms that the Pic is a promising alternative with good efficacy against Ralstonia solanacarum for ginger production and could be used in integrated pest management programs in China. PMID:24618853

  19. Ralstonia insidiosa serves as bridges in biofilm formation by foodborne pathogens Listeria monocytogenes, Salmonella enterica, and enterohemorrhagic Escherichia coli

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Biofilm formation on abiotic surfaces in fresh produce processing facilities might play a role in foodborne outbreaks by providing protective microniches for pathogenic bacteria. Our previous study showed that a strain of Ralstonia insidiosa isolated from a fresh produce processing plant could enhan...

  20. Ralstonia mannitolilytica-Induced Septicemia and Homology Analysis in Infected Patients: 3 Case Reports

    PubMed Central

    Liu, Cai-Xia; Yan, Chun; Zhang, Pan; Li, Fang-Qu; Yang, Jing-Hong; Li, Xiang-Yang

    2016-01-01

    Background Ralstonia mannitolilytica is an emerging opportunistic pathogen. Hospital outbreaks of Ralstonia spp. are mainly associated with contaminated treatment water or auxiliary instruments. Objectives In this report, we summarize the clinical infection characteristics of R. mannitolilytica, the drug-susceptibility testing of the bacterial strains, and the results of related infection investigations. Patients and Methods We retrospectively analyzed the clinical information of 3 patients with R. mannitolilytica. Results The patients’ primary-onset symptoms were chills and fever. The disease progressed rapidly and septic shock symptoms developed. Laboratory tests indicated progressively decreased white blood cells and platelets, as well as significant increases in certain inflammation indicators. The effect of treatment with Tazocin was good. The growth period of R. mannitolilytica in sterile distilled water was > 6 months. The pulsed-field gel electrophoresis (PFGE) results revealed that the infectious strains from these 3 patients were not the same clonal strain. This bacterium was not detected in the nosocomial infection samples. Conclusions Our results suggest that R. mannitolilytica-induced septicemia had an acute disease onset and rapid progression. The preferred empirical antibiotic was Tazocin. In these 3 cases, the R. mannitolilytica-induced septicemia was not due to clonal transmission. PMID:27679705

  1. Ralstonia mannitolilytica-Induced Septicemia and Homology Analysis in Infected Patients: 3 Case Reports

    PubMed Central

    Liu, Cai-Xia; Yan, Chun; Zhang, Pan; Li, Fang-Qu; Yang, Jing-Hong; Li, Xiang-Yang

    2016-01-01

    Background Ralstonia mannitolilytica is an emerging opportunistic pathogen. Hospital outbreaks of Ralstonia spp. are mainly associated with contaminated treatment water or auxiliary instruments. Objectives In this report, we summarize the clinical infection characteristics of R. mannitolilytica, the drug-susceptibility testing of the bacterial strains, and the results of related infection investigations. Patients and Methods We retrospectively analyzed the clinical information of 3 patients with R. mannitolilytica. Results The patients’ primary-onset symptoms were chills and fever. The disease progressed rapidly and septic shock symptoms developed. Laboratory tests indicated progressively decreased white blood cells and platelets, as well as significant increases in certain inflammation indicators. The effect of treatment with Tazocin was good. The growth period of R. mannitolilytica in sterile distilled water was > 6 months. The pulsed-field gel electrophoresis (PFGE) results revealed that the infectious strains from these 3 patients were not the same clonal strain. This bacterium was not detected in the nosocomial infection samples. Conclusions Our results suggest that R. mannitolilytica-induced septicemia had an acute disease onset and rapid progression. The preferred empirical antibiotic was Tazocin. In these 3 cases, the R. mannitolilytica-induced septicemia was not due to clonal transmission.

  2. Association of 'Candidatus Liberibacter solanacearum' with a vegetative disorder of celery in Spain and development of a real-time PCR method for its detection.

    PubMed

    Teresani, Gabriela R; Bertolini, Edson; Alfaro-Fernández, Ana; Martínez, Carmen; Tanaka, Francisco André Ossamu; Kitajima, Elliot W; Roselló, Montserrat; Sanjuán, Susana; Ferrándiz, Juan Carlos; López, María M; Cambra, Mariano; Font, María Isabel

    2014-08-01

    A new symptomatology was observed in celery (Apium graveolens) in Villena, Spain in 2008. Symptomatology included an abnormal amount of shoots per plant and curled stems. These vegetative disorders were associated with 'Candidatus Liberibacter solanacearum' and not with phytoplasmas. Samples from plant sap were immobilized on membranes based on the spot procedure and tested using a newly developed real-time polymerase chain reaction assay to detect 'Ca. L. solanacearum'. Then, a test kit was developed and validated by intralaboratory assays with an accuracy of 100%. Bacterial-like cells with typical morphology of 'Ca. Liberibacter' were observed using electron microscopy in celery plant tissues. A fifth haplotype of 'Ca. L. solanacearum', named E, was identified in celery and in carrot after analyzing partial sequences of 16S and 50S ribosomal RNA genes. From our results, celery (family Apiaceae) can be listed as a new natural host of this emerging bacterium.

  3. Localization of 'Candidatus Liberibacter solanacearum' and Evidence for Surface Appendages in the Potato Psyllid Vector.

    PubMed

    Cicero, J M; Fisher, T W; Brown, J K

    2016-02-01

    The potato psyllid Bactericera cockerelli is implicated as the vector of the causal agent of zebra chip of potato and vein-greening of tomato diseases. Until now, visual identification of bacteria in the genus 'Candidatus Liberibacter' has relied on direct imaging by light and electron microscopy without labeling, or with whole-organ fluorescence labeling only. In this study, aldehyde fixative followed by a coagulant fixative, was used to process adult psyllids for transmission electron microscopy (TEM) colloidal gold in situ hybridization experiments. Results indicated that 'Ca. Liberibacter solanacearum' (CLso)-specific DNA probes annealed to a bacterium that formed extensive, monocultural biofilms on gut, salivary gland, and oral region tissues, confirming that it is one morphotype of potentially others, that is rod-shaped, approximately 2.5 µm in diameter and of variable length, and has a rough, granular cytosol. In addition, CLso, prepared from shredded midguts, and negatively stained for TEM, possessed pili- and flagella-like surface appendages. Genes implicating coding capacity for both types of surface structures are encoded in the CLso genome sequence. Neither type was seen for CLso associated with biofilms within or on digestive organs, suggesting that their production is stimulated only in certain environments, putatively, in the gut during adhesion leading to multiplication, and in hemolymph to afford systemic invasion. PMID:26551449

  4. Genome sequence of the bioplastic-producing "Knallgas" bacterium Ralstonia eutropha H16.

    PubMed

    Pohlmann, Anne; Fricke, Wolfgang Florian; Reinecke, Frank; Kusian, Bernhard; Liesegang, Heiko; Cramm, Rainer; Eitinger, Thomas; Ewering, Christian; Pötter, Markus; Schwartz, Edward; Strittmatter, Axel; Voss, Ingo; Gottschalk, Gerhard; Steinbüchel, Alexander; Friedrich, Bärbel; Bowien, Botho

    2006-10-01

    The H(2)-oxidizing lithoautotrophic bacterium Ralstonia eutropha H16 is a metabolically versatile organism capable of subsisting, in the absence of organic growth substrates, on H(2) and CO(2) as its sole sources of energy and carbon. R. eutropha H16 first attracted biotechnological interest nearly 50 years ago with the realization that the organism's ability to produce and store large amounts of poly[R-(-)-3-hydroxybutyrate] and other polyesters could be harnessed to make biodegradable plastics. Here we report the complete genome sequence of the two chromosomes of R. eutropha H16. Together, chromosome 1 (4,052,032 base pairs (bp)) and chromosome 2 (2,912,490 bp) encode 6,116 putative genes. Analysis of the genome sequence offers the genetic basis for exploiting the biotechnological potential of this organism and provides insights into its remarkable metabolic versatility.

  5. Antibacterial enzymes from the functional screening of metagenomic libraries hosted in Ralstonia metallidurans

    PubMed Central

    Iqbal, Hala A.; Craig, Jeffrey W.; Brady, Sean F.

    2014-01-01

    Phenotype-based screening of bacterial metagenomic libraries provides an avenue for the discovery of novel genes, enzymes and metabolites that have a variety of potential clinical and industrial uses. Here we report the identification of a functionally diverse collection of antibacterially active enzymes from the phenotypic screening of 700,000 cosmid clones prepared from Arizona soil DNA and hosted in Ralstonia metallidurans. Environmental DNA clones surrounded by zones of growth inhibition in a bacterial overlay assay were found, through bioinformatics and functional analyses, to encode enzymes with predicted peptidase, lipase and glycolytic activities conferring antibiosis. The antibacterial activities observed in our R. metallidurans-based assay could not be replicated with the same clones in screens using Escherichia coli as a heterologous host, suggesting that the large-scale screening of metagenomic libraries for antibiosis using phylogenetically diverse hosts should be a productive strategy for identifying enzymes with functionally diverse antibacterial activities. PMID:24661178

  6. Large scale extraction of poly(3-hydroxybutyrate) from Ralstonia eutropha H16 using sodium hypochlorite

    PubMed Central

    2012-01-01

    Isolation of polyhydroxyalkanoates (PHAs) from bacterial cell matter is a critical step in order to achieve a profitable production of the polymer. Therefore, an extraction method must lead to a high recovery of a pure product at low costs. This study presents a simplified method for large scale poly(3-hydroxybutyrate), poly(3HB), extraction using sodium hypochlorite. Poly(3HB) was extracted from cells of Ralstonia eutropha H16 at almost 96% purity. At different extraction volumes, a maximum recovery rate of 91.32% was obtained. At the largest extraction volume of 50 L, poly(3HB) with an average purity of 93.32% ± 4.62% was extracted with a maximum recovery of 87.03% of the initial poly(3HB) content. This process is easy to handle and requires less efforts than previously described processes. PMID:23164136

  7. NAD(P)-Dependent Aldehyde Dehydrogenases Induced during Growth of Ralstonia eutropha Strain Bo on Tetrahydrofurfuryl Alcohol

    PubMed Central

    Schräder, Thomas; Zarnt, Grit; Andreesen, Jan R.

    2001-01-01

    Different aldehyde dehydrogenases (AlDHs) were formed during growth of Ralstonia eutropha Bo on tetrahydrofurfuryl alcohol (THFA). One of these enzymes, AlDH 4, was purified and characterized as a homodimer containing no prosthetic groups, showing a strong substrate inhibition, and having an N-terminal sequence similar to those of various NAD(P)-dependent AlDHs. The conversion rate of THFA by the quinohemoprotein THFA dehydrogenase was increased by AlDH 4. PMID:11717302

  8. Whole-Genome Microarray and Gene Deletion Studies Reveal Regulation of the Polyhydroxyalkanoate Production Cycle by the Stringent Response in Ralstonia eutropha H16

    SciTech Connect

    Brigham, CJ; Speth, DR; Rha, C; Sinskey, AJ

    2012-10-22

    Poly(3-hydroxybutyrate) (PHB) production and mobilization in Ralstonia eutropha are well studied, but in only a few instances has PHB production been explored in relation to other cellular processes. We examined the global gene expression of wild-type R. eutropha throughout the PHB cycle: growth on fructose, PHB production using fructose following ammonium depletion, and PHB utilization in the absence of exogenous carbon after ammonium was resupplied. Our results confirm or lend support to previously reported results regarding the expression of PHB-related genes and enzymes. Additionally, genes for many different cellular processes, such as DNA replication, cell division, and translation, are selectively repressed during PHB production. In contrast, the expression levels of genes under the control of the alternative sigma factor sigma(54) increase sharply during PHB production and are repressed again during PHB utilization. Global gene regulation during PHB production is strongly reminiscent of the gene expression pattern observed during the stringent response in other species. Furthermore, a ppGpp synthase deletion mutant did not show an accumulation of PHB, and the chemical induction of the stringent response with DL-norvaline caused an increased accumulation of PHB in the presence of ammonium. These results indicate that the stringent response is required for PHB accumulation in R. eutropha, helping to elucidate a thus-far-unknown physiological basis for this process.

  9. Cell-associated glucans of Burkholderia solanacearum and Xanthomonas campestris pv. citri: a new family of periplasmic glucans.

    PubMed Central

    Talaga, P; Stahl, B; Wieruszeski, J M; Hillenkamp, F; Tsuyumu, S; Lippens, G; Bohin, J P

    1996-01-01

    The cell-associated glucans produced by Burkholderia solanacearum and Xanthomonas campestris pv. citri were isolated by trichloroacetic acid treatment and gel permeation chromatography. The compounds obtained were characterized by compositional analysis, matrix-assisted laser desorption ionization mass spectrometry, and high-performance anion-exchange chromatography. B. solanacearum synthesizes only a neutral cyclic glucan containing 13 glucose residues, and X. campestris pv. citri synthesizes a neutral cyclic glucan containing 16 glucose residues. The two glucans were further purified by high-performance anion-exchange chromatography. Methylation analysis revealed that these glucans are linked by 1,2-glycosidic bonds and one 1,6-glycosidic bond. Our 600-MHz homonuclear and 1H-13C heteronuclear nuclear magnetic resonance experiments revealed the presence of a single alpha-1,6-glycosidic linkage, whereas all other glucose residues are beta-1,2 linked. The presence of this single alpha-1,6 linkage, however, induces such structural constraints in these cyclic glucans that all individual glucose residues could be distinguished. The different anomeric proton signals allowed complete sequence-specific assignment of both glucans. The structural characteristics of these glucans contrast with those of the previously described osmoregulated periplasmic glucans. PMID:8636027

  10. Development of a broad-host synthetic biology toolbox for ralstonia eutropha and its application to engineering hydrocarbon biofuel production

    PubMed Central

    2013-01-01

    Background The chemoautotrophic bacterium Ralstonia eutropha can utilize H2/CO2 for growth under aerobic conditions. While this microbial host has great potential to be engineered to produce desired compounds (beyond polyhydroxybutyrate) directly from CO2, little work has been done to develop genetic part libraries to enable such endeavors. Results We report the development of a toolbox for the metabolic engineering of Ralstonia eutropha H16. We have constructed a set of broad-host-range plasmids bearing a variety of origins of replication, promoters, 5’ mRNA stem-loop structures, and ribosomal binding sites. Specifically, we analyzed the origins of replication pCM62 (IncP), pBBR1, pKT (IncQ), and their variants. We tested the promoters PBAD, T7, Pxyls/PM, PlacUV5, and variants thereof for inducible expression. We also evaluated a T7 mRNA stem-loop structure sequence and compared a set of ribosomal binding site (RBS) sequences derived from Escherichia coli, R. eutropha, and a computational RBS design tool. Finally, we employed the toolbox to optimize hydrocarbon production in R. eutropha and demonstrated a 6-fold titer improvement using the appropriate combination of parts. Conclusion We constructed and evaluated a versatile synthetic biology toolbox for Ralstonia eutropha metabolic engineering that could apply to other microbial hosts as well. PMID:24219429

  11. Cleaning-resistant Cupriavidus and Ralstonia bacteria contaminating spacecrafts and the ultra clean rooms they are assembled in.

    NASA Astrophysics Data System (ADS)

    Leys, N.; Dams, A.; Bossus, A.; Provoost, A.; Venkateswaran, K.; Mergeay, M.

    Background Planetary Protection is preventing microbial contamination of both the target planet and the Earth when sending spacecrafts on interplanetary space mission It is important to preserve the natural conditions of other planets and to not bring with robots earthly microbes forward contamination when looking for spores of extra terrestrial life Spacecrafts and the ultra clean rooms they are assembled in are routinely monitored for microbial contamination It was shown that the floor air and surfaces of such spacecraft assembly rooms often contain Cupriavidu s and Ralstonia bacteria These bacteria not only contaminated the clean rooms but have also been found prior-to-flight on surfaces of space robots such as the Mars Odyssey Orbiter La Duc et al 2003 and even in-flight in ISS cooling water and Shuttle drinking water unpublished Aim In this study several Cupriavidus and Ralstonia strains isolated from space craft assembling rooms and spacecrafts were characterized and analysed in detail Results The analysis showed that all the Cupriavidus and Ralstonia clean-room isolates are able to use a wide variety of substrates as carbon sources including ethanol and acetone In addition they all have accumulated moderate resistances to an extraordinary collection of physical and chemical antimicrobial agents Some of the test strains were able to form biofilms on plastic and metal materials used for space robots a nutritional and

  12. Transcriptome analyses of Bactericera cockerelli adults in response to "Candidatus Liberibacter solanacearum" infection.

    PubMed

    Nachappa, Punya; Levy, Julien; Tamborindeguy, Cecilia

    2012-10-01

    The potato/tomato psyllid, Bactericera cockerelli (Šulc) is an economically important crop pest that not only causes damage through its feeding but also transmits the bacterium, "Candidatus Liberibacter solanacearum" (CLs), which causes zebra chip disease in potato. There is some information about the phenotypic effects of phytopathogenic bacteria on their insect vectors; however, there are no published reports of the molecular mechanisms underlying phytopathogenic bacteria-insect vector interaction. In order to investigate the effects of CLs infection on B. cockerelli, transcriptomic analyses of CLs-infected and uninfected adult psyllids that were reared on potato were performed. De novo assembly of cDNA sequences generated 136,518 and 109,983 contigs for infected and uninfected insect libraries with an average contig length of 514 bp. BlastX analysis against the NCBI-nr database revealed that 33.33 % had significant matches. Gene ontology data illustrated that the majority of the expressed psyllid genes are involved in metabolic process, biological regulation, binding and catalytic activity. The psyllid transcriptome had an abundance of genes such as vitellogenin, heat shock protein, ejaculatory bulb-specific protein, ferritin, and cytochrome oxidase. Notably absent in the psyllid transcriptome were innate immunity genes induced in response to Gram-negative bacteria (IMD pathway). Several functionally diverse contigs related to symbiotic bacteria including the primary endosymbiont Carsonella ruddii, Wolbachia, and CLs in the psyllid transcriptome were identified. A total of 247 contigs showed differential expression in response to CLs infection including immune and stress-related genes and vitellogenins. Expression analyses of selected psyllid genes were performed on psyllids that were exclusively reared on potato (host of the insects used for RNAseq) and psyllids exclusively reared on tomato (alternative host of psyllids). These genes showed similar expression

  13. Production of Poly (3-Hydroxybutyric Acid) by Ralstonia eutropha in a Biocalorimeter and its Thermokinetic Studies.

    PubMed

    Anusha, Subramanian Mohanakrishnan; Leelaram, Santharam; Surianarayanan, Mahadevan

    2016-07-01

    Bioplastic production from microbial sources is an emerging area which provides opportunities even to convert the wastes into bioplastics. Poly (3-hydroxybutyric acid), commonly called as PHB, is a bioplastic, which is stored as intracellular cytoplasmic inclusions in microorganisms. The objectives of this study are to calorimetrically monitor the PHB production and evaluate the thermokinetic data in a bioreaction calorimeter (BioRC1e). Thus, a well-known PHB-producing bacteria Ralstonia eutropha was selected for batch process in a bioreaction calorimeter. The metabolic heat generated was found to be correlated with the biomass, substrate consumption, oxygen uptake rate (OUR), carbon dioxide evolution rate (CER) and PHB production. The OUR pattern explained the oxidative metabolism of the strain R. eutropha. The heat yields due to biomass and glucose consumption during PHB production were found to be 12.56 and 13.56 kJ/g, respectively. The oxycalorific value obtained for the PHB production was 443.80 kJ/mol of O2. The concentration of PHB obtained in BioRC1e was 4.33 g/L with a production rate of 0.09 g/L/h. The chemical structure of the extracted PHB by R. eutropha was confirmed using fourier transform infrared spectroscopy (FT-IR) and (1)H and (13)C nuclear magnetic resonance (NMR) analysis.

  14. Characterization of the survival ability of Cupriavidus metallidurans and Ralstonia pickettii from space-related environments.

    PubMed

    Mijnendonckx, K; Provoost, A; Ott, C M; Venkateswaran, K; Mahillon, J; Leys, N; Van Houdt, R

    2013-02-01

    Four Cupriavidus metallidurans and eight Ralstonia pickettii isolates from the space industry and the International Space Station (ISS) were characterized in detail. Nine of the 12 isolates were able to form a biofilm on plastics and all were resistant to several antibiotics. R. pickettii isolates from the surface of the Mars Orbiter prior to flight were 2.5 times more resistant to UV-C(254nm) radiation compared to the R. pickettii type strain. All isolates showed moderate to high tolerance against at least seven different metal ions. They were tolerant to medium to high silver concentrations (0.5-4 μM), which are higher than the ionic silver disinfectant concentrations measured regularly in the drinking water aboard the ISS. Furthermore, all isolates survived a 23-month exposure to 2 μM AgNO(3) in drinking water. These resistance properties are putatively encoded by their endogenous megaplasmids. This study demonstrated that extreme resistance is not required to withstand the disinfection and sterilization procedures implemented in the ISS and space industry. All isolates acquired moderate to high tolerance against several stressors and can grow in oligotrophic conditions, enabling them to persist in these environments.

  15. Multiple beta-ketothiolases mediate poly(beta-hydroxyalkanoate) copolymer synthesis in Ralstonia eutropha.

    PubMed

    Slater, S; Houmiel, K L; Tran, M; Mitsky, T A; Taylor, N B; Padgette, S R; Gruys, K J

    1998-04-01

    Polyhydroxyalkanoates (PHAs) are a class of carbon and energy storage polymers produced by numerous bacteria in response to environmental limitation. The type of polymer produced depends on the carbon sources available, the flexibility of the organism's intermediary metabolism, and the substrate specificity of the PHA biosynthetic enzymes. Ralstonia eutropha produces both the homopolymer poly-beta-hydroxybutyrate (PHB) and, when provided with the appropriate substrate, the copolymer poly(beta-hydroxybutyrate-co-beta-hydroxyvalerate) (PHBV). A required step in production of the hydroxyvalerate moiety of PHBV is the condensation of acetyl coenzyme A (acetyl-CoA) and propionyl-CoA to form beta-ketovaleryl-CoA. This activity has generally been attributed to the beta-ketothiolase encoded by R. eutropha phbA. However, we have determined that PhbA does not significantly contribute to catalyzing this condensation reaction. Here we report the cloning and genetic analysis of bktB, which encodes a beta-ketothiolase from R. eutropha that is capable of forming beta-ketovaleryl-CoA. Genetic analyses determined that BktB is the primary condensation enzyme leading to production of beta-hydroxyvalerate derived from propionyl-CoA. We also report an additional beta-ketothiolase, designated BktC, that probably serves as a secondary route toward beta-hydroxyvalerate production.

  16. Catalytic and Molecular Properties of the Quinohemoprotein Tetrahydrofurfuryl Alcohol Dehydrogenase from Ralstonia eutropha Strain Bo

    PubMed Central

    Zarnt, Grit; Schräder, Thomas; Andreesen, Jan R.

    2001-01-01

    The quinohemoprotein tetrahydrofurfuryl alcohol dehydrogenase (THFA-DH) from Ralstonia eutropha strain Bo was investigated for its catalytic properties. The apparent kcat/Km and Ki values for several substrates were determined using ferricyanide as an artificial electron acceptor. The highest catalytic efficiency was obtained with n-pentanol exhibiting a kcat/Km value of 788 × 104 M−1 s−1. The enzyme showed substrate inhibition kinetics for most of the alcohols and aldehydes investigated. A stereoselective oxidation of chiral alcohols with a varying enantiomeric preference was observed. Initial rate studies using ethanol and acetaldehyde as substrates revealed that a ping-pong mechanism can be assumed for in vitro catalysis of THFA-DH. The gene encoding THFA-DH from R. eutropha strain Bo (tfaA) has been cloned and sequenced. The derived amino acid sequence showed an identity of up to 67% to the sequence of various quinoprotein and quinohemoprotein dehydrogenases. A comparison of the deduced sequence with the N-terminal amino acid sequence previously determined by Edman degradation analysis suggested the presence of a signal sequence of 27 residues. The primary structure of TfaA indicated that the protein has a tertiary structure quite similar to those of other quinoprotein dehydrogenases. PMID:11222593

  17. Polyhydroxyalkanoates production with Ralstonia eutropha from low quality waste animal fats.

    PubMed

    Riedel, Sebastian L; Jahns, Stefan; Koenig, Steven; Bock, Martina C E; Brigham, Christopher J; Bader, Johannes; Stahl, Ulf

    2015-11-20

    Polyhydroxyalkanoates (PHAs) are biodegradable and biocompatible polyesters considered as alternatives to petroleum-based plastics. Ralstonia eutropha is a model organism for PHA production. Utilizing industrially rendered waste animal fats as inexpensive carbon feedstocks for PHA production is demonstrated here. An emulsification strategy, without any mechanical or chemical pre-treatment, was developed to increase the bioavailability of solid, poorly-consumable fats. Wild type R. eutropha strain H16 produced 79-82% (w/w) polyhydroxybutyrate (PHB) per cell dry weight (CDW) when cultivated on various fats. A productivity of 0.3g PHB/(L × h) with a total PHB production of 24 g/L was achieved using tallow as carbon source. Using a recombinant strain of R. eutropha that produces poly(hydroxybutyrate-co-hydroxyhexanoate) [P(HB-co-HHx)], 49-72% (w/w) of PHA per CDW with a HHx content of 16-27 mol% were produced in shaking flask experiments. The recombinant strain was grown on waste animal fat of the lowest quality available at lab fermenter scale, resulting in 45 g/L CDW with 60% (w/w) PHA per CDW and a productivity of 0.4 g PHA/(L × h). The final HHx content of the polymer was 19 mol%. The use of low quality waste animal fats as an inexpensive carbon feedstock exhibits a high potential to accelerate the commercialization of PHAs.

  18. Cloning and functional analysis of the pbr lead resistance determinant of Ralstonia metallidurans CH34.

    PubMed

    Borremans, B; Hobman, J L; Provoost, A; Brown, N L; van Der Lelie, D

    2001-10-01

    The lead resistance operon, pbr, of Ralstonia metallidurans (formerly Alcaligenes eutrophus) strain CH34 is unique, as it combines functions involved in uptake, efflux, and accumulation of Pb(II). The pbr lead resistance locus contains the following structural resistance genes: (i) pbrT, which encodes a Pb(II) uptake protein; (ii) pbrA, which encodes a P-type Pb(II) efflux ATPase; (iii) pbrB, which encodes a predicted integral membrane protein of unknown function; and (iv) pbrC, which encodes a predicted prolipoprotein signal peptidase. Downstream of pbrC, the pbrD gene, encoding a Pb(II)-binding protein, was identified in a region of DNA, which was essential for functional lead sequestration. Pb(II)-dependent inducible transcription of pbrABCD from the PpbrA promoter is regulated by PbrR, which belongs to the MerR family of metal ion-sensing regulatory proteins. This is the first report of a mechanism for specific lead resistance in any bacterial genus.

  19. Ethanedisulfonate is degraded via sulfoacetaldehyde in Ralstonia sp. strain EDS1.

    PubMed

    Denger, K; Cook, A M

    2001-07-01

    Aerobic enrichment cultures (11) yielded three cultures able to utilise ethane-1,2-disulfonate as sole source of carbon and energy in salts medium. Two pure cultures were obtained and we worked with strain EDS1, which was assigned to the genus Ralstonia on the basis of its 16S rDNA sequence and simple taxonomic tests. Strain EDS1 utilised at least seven alkane(di)sulfonates, ethane-1,2-disulfonate, taurine, isethionate, sulfoacetate, sulfoacetaldehyde and propane-1,3-disulfonate, as well as methanesulfonate and formate. Growth with ethanedisulfonate was concomitant with substrate disappearance and the formation of 2 mol sulfate per mol substrate. The growth yield, 7 g protein (mol C)(-1), indicated quantitative utilisation of the substrate. Ethanedisulfonate-dependent oxygen uptake of whole cells during growth rose to a maximum before the end of growth and then sank rapidly; this was interpreted as evidence for an inducible desulfonative oxygenase that was not active in cell extracts. Inducible sulfoacetaldehyde sulfo-lyase was detected at high activity. Inducible degradation of taurine or isethionate or sulfoacetate via sulfoacetaldehyde sulfo-lyase is interpreted from the data.

  20. Multiple β-Ketothiolases Mediate Poly(β-Hydroxyalkanoate) Copolymer Synthesis in Ralstonia eutropha

    PubMed Central

    Slater, Steven; Houmiel, Kathryn L.; Tran, Minhtien; Mitsky, Timothy A.; Taylor, Nancy B.; Padgette, Stephen R.; Gruys, Kenneth J.

    1998-01-01

    Polyhydroxyalkanoates (PHAs) are a class of carbon and energy storage polymers produced by numerous bacteria in response to environmental limitation. The type of polymer produced depends on the carbon sources available, the flexibility of the organism’s intermediary metabolism, and the substrate specificity of the PHA biosynthetic enzymes. Ralstonia eutropha produces both the homopolymer poly-β-hydroxybutyrate (PHB) and, when provided with the appropriate substrate, the copolymer poly(β-hydroxybutyrate-co-β-hydroxyvalerate) (PHBV). A required step in production of the hydroxyvalerate moiety of PHBV is the condensation of acetyl coenzyme A (acetyl-CoA) and propionyl-CoA to form β-ketovaleryl-CoA. This activity has generally been attributed to the β-ketothiolase encoded by R. eutropha phbA. However, we have determined that PhbA does not significantly contribute to catalyzing this condensation reaction. Here we report the cloning and genetic analysis of bktB, which encodes a β-ketothiolase from R. eutropha that is capable of forming β-ketovaleryl-CoA. Genetic analyses determined that BktB is the primary condensation enzyme leading to production of β-hydroxyvalerate derived from propionyl-CoA. We also report an additional β-ketothiolase, designated BktC, that probably serves as a secondary route toward β-hydroxyvalerate production. PMID:9555876

  1. Engineering of Ralstonia eutropha H16 for Autotrophic and Heterotrophic Production of Methyl Ketones

    PubMed Central

    Müller, Jana; MacEachran, Daniel; Burd, Helcio; Sathitsuksanoh, Noppadon; Bi, Changhao; Yeh, Yi-Chun; Lee, Taek Soon; Hillson, Nathan J.; Chhabra, Swapnil R.; Singer, Steven W.

    2013-01-01

    Ralstonia eutropha is a facultatively chemolithoautotrophic bacterium able to grow with organic substrates or H2 and CO2 under aerobic conditions. Under conditions of nutrient imbalance, R. eutropha produces copious amounts of poly[(R)-3-hydroxybutyrate] (PHB). Its ability to utilize CO2 as a sole carbon source renders it an interesting new candidate host for the production of renewable liquid transportation fuels. We engineered R. eutropha for the production of fatty acid-derived, diesel-range methyl ketones. Modifications engineered in R. eutropha included overexpression of a cytoplasmic version of the TesA thioesterase, which led to a substantial (>150-fold) increase in fatty acid titer under certain conditions. In addition, deletion of two putative β-oxidation operons and heterologous expression of three genes (the acyl coenzyme A oxidase gene from Micrococcus luteus and fadB and fadM from Escherichia coli) led to the production of 50 to 65 mg/liter of diesel-range methyl ketones under heterotrophic growth conditions and 50 to 180 mg/liter under chemolithoautotrophic growth conditions (with CO2 and H2 as the sole carbon source and electron donor, respectively). Induction of the methyl ketone pathway diverted substantial carbon flux away from PHB biosynthesis and appeared to enhance carbon flux through the pathway for biosynthesis of fatty acids, which are the precursors of methyl ketones. PMID:23686271

  2. Metabolic engineering of Ralstonia eutropha for the biosynthesis of 2-hydroxyacid-containing polyhydroxyalkanoates.

    PubMed

    Park, Si Jae; Jang, Young-Ah; Lee, Hyuk; Park, A-Reum; Yang, Jung Eun; Shin, Jihoon; Oh, Young Hoon; Song, Bong Keun; Jegal, Jonggeon; Lee, Seung Hwan; Lee, Sang Yup

    2013-11-01

    Polyhydroxyalkanoates (PHAs) are bio-based and biodegradable polyesters synthesized by numerous microorganisms. PHAs containing 2-hydroxyacids as monomer units have attracted much attention, but their production has not been efficient. Here, we metabolically engineered Ralstonia eutropha strains for the in vivo synthesis of PHAs containing 2-hydroxyacids as monomers. This was accomplished by replacing the R. eutropha phaC gene in the chromosome with either the R. eutropha phaC S506G A510K gene, which contains two point mutations, or the Pseudomonas sp. MBEL 6-19 phaC1437 gene. In addition, the R. eutropha phaAB genes in the chromosome were replaced with the Clostridium propionicum pct540 gene. All of the engineered R. eutropha strains produced PHAs containing 2-hydroxyacid monomers, including lactate and 2-hydroxybutyrate (2HB), along with 3-hydroxybutyrate (3HB) and/or 3-hydroxyvalerate (3HV), when they were cultured in nitrogen-free medium containing 5 g/L lactate or 4 g/L 2HB and 20 g/L glucose as carbon sources. Expression of the Escherichia coli ldhA gene in engineered R. eutropha strains allowed production of poly(3-hydroxybutyrate-co-lactate) [P(3HB-co-LA)] from glucose as the sole carbon source. This is the first report on the production of 2-hydroxyacid-containing PHAs by metabolically engineered R. eutropha.

  3. Crystal structure of (R)-3-hydroxybutyryl-CoA dehydrogenase PhaB from Ralstonia eutropha.

    PubMed

    Kim, Jieun; Chang, Jeong Ho; Kim, Eun-Jung; Kim, Kyung-Jin

    2014-01-17

    (R)-3-hydroxybutyryl-CoA dehydrogenase PhaB from Ralstonia eutropha H16 (RePhaB) is an enzyme that catalyzes the NADPH-dependent reduction of acetoacetyl-CoA, an intermediate of polyhydroxyalkanoates (PHA) synthetic pathways. Polymeric PHA is used to make bioplastics, implant biomaterials, and biofuels. Here, we report the crystal structures of RePhaB apoenzyme and in complex with either NADP(+) or acetoacetyl-CoA, which provide the catalytic mechanism of the protein. RePhaB contains a Rossmann fold and a Clamp domain for binding of NADP(+) and acetoacetyl-CoA, respectively. The NADP(+)-bound form of RePhaB structure reveals that the protein has a unique cofactor binding mode. Interestingly, in the RePhaB structure in complex with acetoacetyl-CoA, the conformation of the Clamp domain, especially the Clamp-lid, undergoes a large structural change about 4.6 Å leading to formation of the substrate pocket. These structural observations, along with the biochemical experiments, suggest that movement of the Clamp-lid enables the substrate binding and ensures the acetoacetyl moiety is located near to the nicotinamide ring of NADP(+). PMID:24211201

  4. Production of Poly (3-Hydroxybutyric Acid) by Ralstonia eutropha in a Biocalorimeter and its Thermokinetic Studies.

    PubMed

    Anusha, Subramanian Mohanakrishnan; Leelaram, Santharam; Surianarayanan, Mahadevan

    2016-07-01

    Bioplastic production from microbial sources is an emerging area which provides opportunities even to convert the wastes into bioplastics. Poly (3-hydroxybutyric acid), commonly called as PHB, is a bioplastic, which is stored as intracellular cytoplasmic inclusions in microorganisms. The objectives of this study are to calorimetrically monitor the PHB production and evaluate the thermokinetic data in a bioreaction calorimeter (BioRC1e). Thus, a well-known PHB-producing bacteria Ralstonia eutropha was selected for batch process in a bioreaction calorimeter. The metabolic heat generated was found to be correlated with the biomass, substrate consumption, oxygen uptake rate (OUR), carbon dioxide evolution rate (CER) and PHB production. The OUR pattern explained the oxidative metabolism of the strain R. eutropha. The heat yields due to biomass and glucose consumption during PHB production were found to be 12.56 and 13.56 kJ/g, respectively. The oxycalorific value obtained for the PHB production was 443.80 kJ/mol of O2. The concentration of PHB obtained in BioRC1e was 4.33 g/L with a production rate of 0.09 g/L/h. The chemical structure of the extracted PHB by R. eutropha was confirmed using fourier transform infrared spectroscopy (FT-IR) and (1)H and (13)C nuclear magnetic resonance (NMR) analysis. PMID:27003281

  5. Growth and polyhydroxybutyrate production by Ralstonia eutropha in emulsified plant oil medium.

    PubMed

    Budde, Charles F; Riedel, Sebastian L; Hübner, Florian; Risch, Stefan; Popović, Milan K; Rha, ChoKyun; Sinskey, Anthony J

    2011-03-01

    Polyhydroxyalkanoates (PHAs) are natural polyesters synthesized by bacteria for carbon and energy storage that also have commercial potential as bioplastics. One promising class of carbon feedstocks for industrial PHA production is plant oils, due to the high carbon content of these compounds. The bacterium Ralstonia eutropha accumulates high levels of PHA and can effectively utilize plant oil. Growth experiments that include plant oil, however, are difficult to conduct in a quantitative and reproducible manner due to the heterogeneity of the two-phase medium. In order to overcome this obstacle, a new culture method was developed in which palm oil was emulsified in growth medium using the glycoprotein gum arabic as the emulsifying agent. Gum arabic did not influence R. eutropha growth and could not be used as a nutrient source by the bacteria. R. eutropha was grown in the emulsified oil medium and PHA production was measured over time. Additionally, an extraction method was developed to monitor oil consumption. The new method described in this study allows quantitative, reproducible R. eutropha experiments to be performed with plant oils. The method may also prove useful for studying growth of different bacteria on plant oils and other hydrophobic carbon sources. PMID:21279345

  6. Functional Analysis by Site-Directed Mutagenesis of the NAD+-Reducing Hydrogenase from Ralstonia eutropha

    PubMed Central

    Burgdorf, Tanja; De Lacey, Antonio L.; Friedrich, Bärbel

    2002-01-01

    The tetrameric cytoplasmic [NiFe] hydrogenase (SH) of Ralstonia eutropha couples the oxidation of hydrogen to the reduction of NAD+ under aerobic conditions. In the catalytic subunit HoxH, all six conserved motifs surrounding the [NiFe] site are present. Five of these motifs were altered by site-directed mutagenesis in order to dissect the molecular mechanism of hydrogen activation. Based on phenotypic characterizations, 27 mutants were grouped into four different classes. Mutants of the major class, class I, failed to grow on hydrogen and were devoid of H2-oxidizing activity. In one of these isolates (HoxH I64A), H2 binding was impaired. Class II mutants revealed a high D2/H+ exchange rate relative to a low H2-oxidizing activity. A representative (HoxH H16L) displayed D2/H+ exchange but had lost electron acceptor-reducing activity. Both activities were equally affected in class III mutants. Mutants forming class IV showed a particularly interesting phenotype. They displayed O2-sensitive growth on hydrogen due to an O2-sensitive SH protein. PMID:12399498

  7. Polyhydroxyalkanoates production with Ralstonia eutropha from low quality waste animal fats.

    PubMed

    Riedel, Sebastian L; Jahns, Stefan; Koenig, Steven; Bock, Martina C E; Brigham, Christopher J; Bader, Johannes; Stahl, Ulf

    2015-11-20

    Polyhydroxyalkanoates (PHAs) are biodegradable and biocompatible polyesters considered as alternatives to petroleum-based plastics. Ralstonia eutropha is a model organism for PHA production. Utilizing industrially rendered waste animal fats as inexpensive carbon feedstocks for PHA production is demonstrated here. An emulsification strategy, without any mechanical or chemical pre-treatment, was developed to increase the bioavailability of solid, poorly-consumable fats. Wild type R. eutropha strain H16 produced 79-82% (w/w) polyhydroxybutyrate (PHB) per cell dry weight (CDW) when cultivated on various fats. A productivity of 0.3g PHB/(L × h) with a total PHB production of 24 g/L was achieved using tallow as carbon source. Using a recombinant strain of R. eutropha that produces poly(hydroxybutyrate-co-hydroxyhexanoate) [P(HB-co-HHx)], 49-72% (w/w) of PHA per CDW with a HHx content of 16-27 mol% were produced in shaking flask experiments. The recombinant strain was grown on waste animal fat of the lowest quality available at lab fermenter scale, resulting in 45 g/L CDW with 60% (w/w) PHA per CDW and a productivity of 0.4 g PHA/(L × h). The final HHx content of the polymer was 19 mol%. The use of low quality waste animal fats as an inexpensive carbon feedstock exhibits a high potential to accelerate the commercialization of PHAs. PMID:26428087

  8. Phasin proteins activate Aeromonas caviae polyhydroxyalkanoate (PHA) synthase but not Ralstonia eutropha PHA synthase.

    PubMed

    Ushimaru, Kazunori; Motoda, Yoko; Numata, Keiji; Tsuge, Takeharu

    2014-05-01

    In this study, we performed in vitro and in vivo activity assays of polyhydroxyalkanoate (PHA) synthases (PhaCs) in the presence of phasin proteins (PhaPs), which revealed that PhaPs are activators of PhaC derived from Aeromonas caviae (PhaCAc). In in vitro assays, among the three PhaCs tested, PhaCAc was significantly activated when PhaPs were added at the beginning of polymerization (prepolymerization PhaCAc), whereas the prepolymerization PhaCRe (derived from Ralstonia eutropha) and PhaCDa (Delftia acidovorans) showed reduced activity with PhaPs. The PhaP-activated PhaCAc showed a slight shift of substrate preference toward 3-hydroxyhexanoyl-CoA (C6). PhaPAc also activated PhaCAc when it was added during polymerization (polymer-elongating PhaCAc), while this effect was not observed for PhaCRe. In an in vivo assay using Escherichia coli TOP10 as the host strain, the effect of PhaPAc expression on PHA synthesis by PhaCAc or PhaCRe was examined. As PhaPAc expression increased, PHA production was increased by up to 2.3-fold in the PhaCAc-expressing strain, whereas it was slightly increased in the PhaCRe-expressing strain. Taken together, this study provides evidence that PhaPs function as activators for PhaCAc both in vitro and in vivo but do not activate PhaCRe. This activating effect may be attributed to the new role of PhaPs in the polymerization reaction by PhaCAc.

  9. Metabolic engineering of Ralstonia eutropha for the production of polyhydroxyalkanoates from sucrose.

    PubMed

    Park, Si Jae; Jang, Young-Ah; Noh, Won; Oh, Young Hoon; Lee, Hyuk; David, Yokimiko; Baylon, Mary Grace; Shin, Jihoon; Yang, Jung Eun; Choi, So Young; Lee, Seung Hwan; Lee, Sang Yup

    2015-03-01

    A sucrose utilization pathway was established in Ralstonia eutropha NCIMB11599 and R. eutropha 437-540 by introducing the Mannheimia succiniciproducens MBEL55E sacC gene that encodes β-fructofuranosidase. These engineered strains were examined for the production of poly(3-hydroxybutyrate) [P(3HB)] and poly(3-hydroxybutyrate-co-lactate) [P(3HB-co-LA)], respectively, from sucrose as a carbon source. It was found that β-fructofuranosidase excreted into the culture medium could hydrolyze sucrose to glucose and fructose, which were efficiently used as carbon sources by recombinant R. eutropha strains. When R. eutropha NCIMB11599 expressing the sacC gene was cultured in nitrogen-free chemically defined medium containing 20 g/L of sucrose, a high P(3HB) content of 73.2 wt% could be obtained. In addition, R. eutropha 437-540 expressing the Pseudomonas sp. MBEL 6-19 phaC1437 gene and the Clostridium propionicum pct540 gene accumulated P(3HB-co-21.5 mol% LA) to a polymer content of 19.5 wt% from sucrose by the expression of the sacC gene and the Escherichia coli ldhA gene. The molecular weights of P(3HB) and P(3HB-co-21.5 mol%LA) synthesized in R. eutropha using sucrose as a carbon source were 3.52 × 10(5) (Mn ) and 2.19 × 10(4) (Mn ), respectively. The engineered R. eutropha strains reported here will be useful for the production of polyhydroxyalkanoates (PHAs) from sucrose, one of the most abundant and relatively inexpensive carbon sources.

  10. Enhancement of glycerol utilization ability of Ralstonia eutropha H16 for production of polyhydroxyalkanoates.

    PubMed

    Fukui, Toshiaki; Mukoyama, Masaharu; Orita, Izumi; Nakamura, Satoshi

    2014-09-01

    Ralstonia eutropha H16 is a well-studied bacterium with respect to biosynthesis of polyhydroxyalkanoates (PHAs), which has attracted attentions as biodegradable bio-based plastics. However, this strain shows quite poor growth on glycerol of which bulk supply has been increasing as a major by-product of biodiesel industries. This study examined enhancement of glycerol assimilation ability of R. eutropha H16 by introduction of the genes of aquaglyceroporin (glpF) and glycerol kinase (glpK) from Escherichia coli. Although introduction of glpFK Ec into the strain H16 using a multi-copy vector was not successful, a recombinant strain possessing glpFK Ec within the chromosome showed much faster growth on glycerol than H16. Further analyses clarified that weak expression of glpK Ec alone allowed to establish efficient glycerol assimilation pathway, indicating that the poor growth of H16 on glycerol was caused by insufficient kination activity to glycerol, as well as this strain had a potential ability for uptake of extracellular glycerol. The engineered strains expressing glpFK Ec or glpK Ec produced large amounts of poly[(R)-3-hydroxybutyrate] [P(3HB)] from glycerol with much higher productivity than H16. Unlike other glycerol-utilizable wild strains of R. eutropha, the H16-derived engineered strains accumulated P(3HB) with no significant decrease in molecular weights on glycerol, and the polydispersity index of the glycerol-based P(3HB) synthesized by the strains expressing glpFK Ec was lower than those by the parent strains. The present study demonstrated possibility of R. eutropha H16-based platform for production of useful compounds from inexpensive glycerol.

  11. Molecular characterization influencing metal resistance in the Cupriavidus/Ralstonia genomes.

    PubMed

    Chakraborti, Pratim; Banerjee, Rachana; Roy, Ayan; Mandal, Sunanda; Mukhopadhyay, Subhasish

    2015-01-01

    Our environment is stressed with a load of heavy and toxic metals. Microbes, abundant in our environment, are found to adapt well to this metal-stressed condition. A comparative study among five Cupriavidus/Ralstonia genomes can offer a better perception of their evolutionary mechanisms to adapt to these conditions. We have studied codon usage among 1051 genes common to all these organisms and identified 15 optimal codons frequently used in highly expressed genes present within 1051 genes. We found the core genes of Cupriavidus metallidurans CH34 have a different optimal codon choice for arginine, glycine and alanine in comparison with the other four bacteria. We also found that the synonymous codon usage bias within these 1051 core genes is highly correlated with their gene expression. This supports that translational selection drives synonymous codon usage in the core genes of these genomes. Synonymous codon usage is highly conserved in the core genes of these five genomes. The only exception among them is C. metallidurans CH34. This genomewide shift in synonymous codon choice in C. metallidurans CH34 may have taken place due to the insertion of new genes in its genomes facilitating them to survive in heavy metal containing environment and the co-evolution of the other genes in its genome to achieve a balance in gene expression. Structural studies indicated the presence of a longer N-terminal region containing a copper-binding domain in the cupC proteins of C. metallidurans CH3 that helps it to attain higher binding efficacy with copper in comparison with its orthologs.

  12. Substrate and Cofactor Range Differences of Two Cysteine Dioxygenases from Ralstonia eutropha H16

    PubMed Central

    Wenning, Leonie; Stöveken, Nadine; Wübbeler, Jan Hendrik

    2015-01-01

    Cysteine dioxygenases (Cdos), which catalyze the sulfoxidation of cysteine to cysteine sulfinic acid (CSA), have been extensively studied in eukaryotes because of their roles in several diseases. In contrast, only a few prokaryotic enzymes of this type have been investigated. In Ralstonia eutropha H16, two Cdo homologues (CdoA and CdoB) have been identified previously. In vivo studies showed that Escherichia coli cells expressing CdoA could convert 3-mercaptopropionate (3MP) to 3-sulfinopropionate (3SP), whereas no 3SP could be detected in cells expressing CdoB. The objective of this study was to confirm these findings and to study both enzymes in detail by performing an in vitro characterization. The proteins were heterologously expressed and purified to apparent homogeneity by immobilized metal chelate affinity chromatography (IMAC). Subsequent analysis of the enzyme activities revealed striking differences with regard to their substrate ranges and their specificities for the transition metal cofactor, e.g., CdoA catalyzed the sulfoxidation of 3MP to a 3-fold-greater extent than the sulfoxidation of cysteine, whereas CdoB converted only cysteine. Moreover, the dependency of the activities of the Cdos from R. eutropha H16 on the metal cofactor in the active center could be demonstrated. The importance of CdoA for the metabolism of the sulfur compounds 3,3′-thiodipropionic acid (TDP) and 3,3′-dithiodipropionic acid (DTDP) by further converting their degradation product, 3MP, was confirmed. Since 3MP can also function as a precursor for polythioester (PTE) synthesis in R. eutropha H16, deletion of cdoA might enable increased synthesis of PTEs. PMID:26590284

  13. Production of branched-chain alcohols by recombinant Ralstonia eutropha in fed-batch cultivation

    SciTech Connect

    Fei, Q; Brigham, CJ; Lu, JN; Fu, RZ; Sinskey, AJ

    2013-09-01

    Branched-chain alcohols are considered promising green energy sources due to their compatibility with existing infrastructure and their high energy density. We utilized a strain of Ralstonia eutropha capable of producing branched-chain alcohols and examined its production in flask cultures. In order to increase isobutanol and 3-methyl-1-butanol (isoamyl alcohol) productivity in the engineered strain, batch, fed-batch, and two-stage fed-batch cultures were carried out in this work. The effects of nitrogen source concentration on branched-chain alcohol production were investigated under four different initial concentrations in fermenters. A maximum 380 g m(-3) of branched-chain alcohol production was observed with 2 kg m(-3) initial NH4Cl concentration in batch cultures. A pH-stat control strategy was utilized to investigate the optimum carbon source amount fed during fed-batch cultures for higher cell density. In cultures of R. eutropha strains that did not produce polyhydroxyalkanoate or branched-chain alcohols, a maximum cell dry weight of 36 kg m(-3) was observed using a fed-batch strategy, when 10 kg m(-3) carbon source was fed into culture medium. Finally, a total branched-chain alcohol titer of 790 g m(-3), the highest branched-chain alcohol yield of 0.03 g g(-1), and the maximum branched-chain alcohol productivity of 8.23 g m(-3) h(-1) were obtained from the engineered strain Re2410/pJL26 in a two-stage fed-batch culture system with pH-stat control. Isobutanol made up over 95% (mass fraction) of the total branched-chain alcohols titer produced in this study. (C) 2013 Published by Elsevier Ltd.

  14. Versatile and stable vectors for efficient gene expression in Ralstonia eutropha H16.

    PubMed

    Gruber, Steffen; Hagen, Jeremias; Schwab, Helmut; Koefinger, Petra

    2014-09-30

    The Gram-negative β-proteobacterium Ralstonia eutropha H16 is primarily known for polyhydroxybutyrate (PHB) production and its ability to grow chemolithoautotrophically by using CO2 and H2 as sole carbon and energy sources. The majority of metabolic engineering and heterologous expression studies conducted so far rely on a small number of suitable expression systems. Particularly the plasmid based expression systems already developed for the use in R. eutropha H16 suffer from high segregational instability and plasmid loss after a short time of fermentation. In order to develop efficient and highly stable plasmid expression vectors for the use in R. eutropha H16, a new plasmid design was created including the RP4 partitioning system, as well as various promoters and origins of replication. The application of minireplicons derived from broad-host-range plasmids RSF1010, pBBR1, RP4 and pSa for the construction of expression vectors and the use of numerous, versatile promoters extend the range of feasible expression levels considerably. In particular, the use of promoters derived from the bacteriophage T5 was described for the first time in this work, characterizing the j5 promoter as the strongest promoter yet to be applied in R. eutropha H16. Moreover, the implementation of the RP4 partition sequence in plasmid design increased plasmid stability significantly and enables fermentations with marginal plasmid loss of recombinant R. eutropha H16 for at least 96 h. The utility of the new vector family in R. eutropha H16 is demonstrated by providing expression data with different model proteins and consequently further raises the value of this organism as cell factory for biotechnological applications including protein and metabolite production.

  15. Reprint of "versatile and stable vectors for efficient gene expression in Ralstonia eutropha H16".

    PubMed

    Gruber, Steffen; Hagen, Jeremias; Schwab, Helmut; Koefinger, Petra

    2014-12-20

    The Gram-negative β-proteobacterium Ralstonia eutropha H16 is primarily known for polyhydroxybutyrate (PHB) production and its ability to grow chemolithoautotrophically by using CO2 and H2 as sole carbon and energy sources. The majority of metabolic engineering and heterologous expression studies conducted so far rely on a small number of suitable expression systems. Particularly the plasmid based expression systems already developed for the use in R. eutropha H16 suffer from high segregational instability and plasmid loss after a short time of fermentation. In order to develop efficient and highly stable plasmid expression vectors for the use in R. eutropha H16, a new plasmid design was created including the RP4 partitioning system, as well as various promoters and origins of replication. The application of minireplicons derived from broad-host-range plasmids RSF1010, pBBR1, RP4 and pSa for the construction of expression vectors and the use of numerous, versatile promoters extend the range of feasible expression levels considerably. In particular, the use of promoters derived from the bacteriophage T5 was described for the first time in this work, characterizing the j5 promoter as the strongest promoter yet to be applied in R. eutropha H16. Moreover, the implementation of the RP4 partition sequence in plasmid design increased plasmid stability significantly and enables fermentations with marginal plasmid loss of recombinant R. eutropha H16 for at least 96h. The utility of the new vector family in R. eutropha H16 is demonstrated by providing expression data with different model proteins and consequently further raises the value of this organism as cell factory for biotechnological applications including protein and metabolite production.

  16. Studies on the production of branched-chain alcohols in engineered Ralstonia eutropha

    SciTech Connect

    Lu, JN; Brigham, CJ; Gai, CS; Sinskey, AJ

    2012-08-04

    Wild-type Ralstonia eutropha H16 produces polyhydroxybutyrate (PHB) as an intracellular carbon storage material during nutrient stress in the presence of excess carbon. In this study, the excess carbon was redirected in engineered strains from PHB storage to the production of isobutanol and 3-methyl-1-butanol (branched-chain higher alcohols). These branched-chain higher alcohols can directly substitute for fossil-based fuels and be employed within the current infrastructure. Various mutant strains of R. eutropha with isobutyraldehyde dehydrogenase activity, in combination with the overexpression of plasmid-borne, native branched-chain amino acid biosynthesis pathway genes and the overexpression of heterologous ketoisovalerate decarboxylase gene, were employed for the biosynthesis of isobutanol and 3-methyl-1-butanol. Production of these branched-chain alcohols was initiated during nitrogen or phosphorus limitation in the engineered R. eutropha. One mutant strain not only produced over 180 mg/L branched-chain alcohols in flask culture, but also was significantly more tolerant of isobutanol toxicity than wild-type R. eutropha. After the elimination of genes encoding three potential carbon sinks (ilvE, bkdAB, and aceE), the production titer improved to 270 mg/L isobutanol and 40 mg/L 3-methyl-1-butanol. Semicontinuous flask cultivation was utilized to minimize the toxicity caused by isobutanol while supplying cells with sufficient nutrients. Under this semicontinuous flask cultivation, the R. eutropha mutant grew and produced more than 14 g/L branched-chain alcohols over the duration of 50 days. These results demonstrate that R. eutropha carbon flux can be redirected from PHB to branched-chain alcohols and that engineered R. eutropha can be cultivated over prolonged periods of time for product biosynthesis.

  17. Aerobic mineralization of 2,6-dichlorophenol by Ralstonia sp. strain RK1

    SciTech Connect

    Steinle, P.; Stucki, G.; Stettler, R.; Hanselmann, K.W.

    1998-07-01

    A new aerobic bacterium was isolated from the sediment of a freshwater pond close to a contaminated site at Amponville (France). It was enriched in a fixed-bed reactor fed with 2,6-dichlorophenol (2,6-DCP) as the sole carbon and energy source at pH 7.5 and room temperature. The degradation of 2,6-DCP followed Monod kinetics at low initial concentrations. At concentrations above 300 {micro}M, 2,6-DCP increasingly inhibited its own degradation. The base sequence of the 16S ribosomal DNA allowed us to assign the bacterium to the genus Ralstonia (formerly Alcaligenes). The substrate spectrum of the bacterium includes toluene, benzene, chlorobenzene, phenol, and all four ortho- and para-substituted mono- and dichlorophenol isomers. Substituents other than chlorine prevented degradation. The capacity to degrade 2,6-DCP was examined in two fixed-bed reactors. The microbial population grew on and completely mineralized 2,6-DCP at 2,6-DCP concentrations up to 740 {micro}M in continuous reactor culture supplied with H{sub 2}O{sub 2} as an oxygen source. Lack of peroxide completely stopped further degradation of 2,6-DCP. Lowering the acid-neutralizing capacity of the medium to 1/10th the original capacity led to a decrease in the pH of the effluent from 7 to 6 and to a significant reduction in the degradation activity. A second fixed-bed reactor successfully removed low chlorophenol concentrations with hydraulic residence times of 8 to 30 min.

  18. Metabolic carbon fluxes and biosynthesis of polyhydroxyalkanoates in Ralstonia eutropha on short chain fatty acids.

    PubMed

    Yu, Jian; Si, Yingtao

    2004-01-01

    Short chain fatty acids such as acetic, propionic, and butyric acids can be synthesized into polyhydroxyalkanoates (PHAs) by Ralstonia eutropha. Metabolic carbon fluxes of the acids in living cells have significant effect on the yield, composition, and thermomechanical properties of PHA bioplastics. Based on the general knowledge of central metabolism pathways and the unusual metabolic pathways in R. eutropha, a metabolic network of 41 bioreactions is constructed to analyze the carbon fluxes on utilization of the short chain fatty acids. In fed-batch cultures with constant feeding of acid media, carbon metabolism and distribution in R. eutropha were measured involving CO2, PHA biopolymers, and residual cell mass. As the cells underwent unsteady state metabolism and PHA biosynthesis under nitrogen-limited conditions, accumulative carbon balance was applied for pseudo-steady-state analysis of the metabolic carbon fluxes. Cofactor NADP/NADPH balanced between PHA synthesis and the C3/C4 pathway provided an independent constraint for solution of the underdetermined metabolic network. A major portion of propionyl-CoA was directed to pyruvate via the 2-methylcitrate cycle and further decarboxylated to acetyl-CoA. Only a small amount of propionate carbon (<15% carbon) was directly condensed with acetyl-CoA for 3-hydroxyvalerate. The ratio of glyoxylate shunt to TCA cycle varies from 0 to 0.25, depending on the intracellular acetyl-CoA level and acetic acid in the medium. Malate is the node of the C3/C4 pathway and TCA cycle and its decarboxylation to dehydrogenation ranges from 0.33 to 1.28 in response to the demands on NADPH and oxaloacetate for short chain fatty acids utilization. PMID:15296425

  19. Inventorying the molecular potential of Cupriavidus and Ralstonia strains surviving harsh space-related environments

    NASA Astrophysics Data System (ADS)

    Mijnendonckx, Kristel; van Houdt, Rob; Provoost, Ann; Bossus, Albert; Ott, C. Mark; Venkateswaran, Kasthuri; Leys, Natalie

    The craving of modern man to explore life beyond earth presents a lot of challenges. The control of microbial contamination of the confined manned spacecraft is an important aspect that has to be taken into account in this journey. Because the human body contains a huge amount of microorganisms, the crew itself is the most important contamination source. But contamination can also originate from residing environmental microorganisms or from materials that are supplied from the Earth. These microbial contaminations can cause problems for the astronauts -well documented to have a decreased immunity -and the infrastructure of the space station. In this study, 14 different Cupriavidus metallidurans and Ralstonia pickettii strains, isolated from such space-related environments, where characterised in detail. These unique strains were isolated from drinking water that returned from ISS (3), from the cooling water system of the American ISS segment (4), from a swab sample of the Mars Odyssey Orbitor surface prior to flight (4), and from an air sample taken in the space assembly facility PHSF during Mars exploration Rover assembly (3). Their resistance to heavy metals and antibiotics was screened. The C. metallidurans isolates were more resistant to Zn2+ and Hg+ but more sensitive to Ni2+ than the R. pickettii strains. The MIC values for Cu2+ ranged from 1,5mM to 12mM, for Co2+ from 1,58mM to 12,63mM and for Cd2+ from 0,25mM to 1mM. For Ni2+ , the MIC values were between 2 and 8mM, except for the strain C. metallidurans IV (0502478) that was able to grow on Ni+2 concentrations up to 48mM. A metal of special interest was Ag+ because it is used to sanitize ISS drinking water. The strains isolated from air and surface samples showed a MIC value ranging from 0,35µM to 4µM. The isolates from the water samples had MIC values from 0,3µM to 2µM, which is lower than (or comparable with) the lowest limit of the silver concentration used in the ISS (1,9µM -4,6µM). However, all

  20. Phenotype conversion in Pseudomonas solanacearum due to spontaneous inactivation of PhcA, a putative LysR transcriptional regulator.

    PubMed Central

    Brumbley, S M; Carney, B F; Denny, T P

    1993-01-01

    Phenotype conversion (PC) in Pseudomonas solanacearum is the coordinated change in production of extracellular polysaccharide and a variety of extracellular proteins, some of which contribute to virulence. Although PC is normally spontaneous, it is mimicked by transposon inactivation of the phcA locus (S. M. Brumbley and T. P. Denny, J. Bacteriol. 172:5677-5685, 1990). The DNA sequence of a 1.8-kb region from strain AW1 that contains phcA revealed one open reading frame that should encode a polypeptide of 38.6 kDa. The PhcA protein produced in Escherichia coli by using a T7 RNA polymerase expression system was of the predicted size. The deduced amino acid sequence of PhcA is similar to that of some members of the LysR transcriptional activator gene family, especially in the amino terminus, where a putative helix-turn-helix DNA-binding motif was identified. An analogous allele (phcA1) was cloned from the spontaneous PC mutant strain AW1-PC and found to be nonfunctional in complementation studies. When phcA1 was expressed in E. coli, the PhcA1 protein was 35.5 kDa, 3 kDa smaller than PhcA. Sequence analysis of phcA1 and chimeric constructs of phcA and phcA1 confirmed that PhcA1 is truncated by a 2-bp insertion 147 nucleotides upstream of the carboxyl terminus of PhcA. Southern blot analysis of 10 additional independently isolated PC mutants of strain AW1 revealed that two strains have larger insertions (0.2 and 1.0 kb) within phcA. These results suggest that phcA encodes a DNA-binding protein that regulates the transcription of one or more of the genes involved in P. solanacearum virulence and that spontaneous PC can be attributed to one of several different insertions within this locus. Images PMID:8366033

  1. Characterization of an extracellular lipase and its chaperone from Ralstonia eutropha H16.

    PubMed

    Lu, Jingnan; Brigham, Christopher J; Rha, Chokyun; Sinskey, Anthony J

    2013-03-01

    Lipase enzymes catalyze the reversible hydrolysis of triacylglycerol to fatty acids and glycerol at the lipid-water interface. The metabolically versatile Ralstonia eutropha strain H16 is capable of utilizing various molecules containing long carbon chains such as plant oil, organic acids, or Tween as its sole carbon source for growth. Global gene expression analysis revealed an upregulation of two putative lipase genes during growth on trioleate. Through analysis of growth and activity using strains with gene deletions and complementations, the extracellular lipase (encoded by the lipA gene, locus tag H16_A1322) and lipase-specific chaperone (encoded by the lipB gene, locus tag H16_A1323) produced by R. eutropha H16 was identified. Increase in gene dosage of lipA not only resulted in an increase of the extracellular lipase activity, but also reduced the lag phase during growth on palm oil. LipA is a non-specific lipase that can completely hydrolyze triacylglycerol into its corresponding free fatty acids and glycerol. Although LipA is active over a temperature range from 10 °C to 70 °C, it exhibited optimal activity at 50 °C. While R. eutropha H16 prefers a growth pH of 6.8, its extracellular lipase LipA is most active between pH 7 and 8. Cofactors are not required for lipase activity; however, EDTA and EGTA inhibited LipA activity by 83 %. Metal ions Mg(2+), Ca(2+), and Mn(2+) were found to stimulate LipA activity and relieve chelator inhibition. Certain detergents are found to improve solubility of the lipid substrate or increase lipase-lipid aggregation, as a result SDS and Triton X-100 were able to increase lipase activity by 20 % to 500 %. R. eutropha extracellular LipA activity can be hyper-increased, making the overexpression strain a potential candidate for commercial lipase production or in fermentations using plant oils as the sole carbon source.

  2. Characterization and modification of enzymes in the 2-ketoisovalerate biosynthesis pathway of Ralstonia eutropha H16.

    PubMed

    Lu, Jingnan; Brigham, Christopher J; Plassmeier, Jens K; Sinskey, Anthony J

    2015-01-01

    2-Ketoisovalerate is an important cellular intermediate for the synthesis of branched-chain amino acids as well as other important molecules, such as pantothenate, coenzyme A, and glucosinolate. This ketoacid can also serve as a precursor molecule for the production of biofuels, pharmaceutical agents, and flavor agents in engineered organisms, such as the betaproteobacterium Ralstonia eutropha. The biosynthesis of 2-ketoisovalerate from pyruvate is carried out by three enzymes: acetohydroxyacid synthase (AHAS, encoded by ilvBH), acetohydroxyacid isomeroreductase (AHAIR, encoded by ilvC), and dihydroxyacid dehydratase (DHAD, encoded by ilvD). In this study, enzymatic activities and kinetic parameters were determined for each of the three R. eutropha enzymes as heterologously purified proteins. AHAS, which serves as a gatekeeper for the biosynthesis of all three branched-chain amino acids, demonstrated the tightest regulation through feedback inhibition by L-valine (IC50=1.2 mM), L-isoleucine (IC50=2.3 mM), and L-leucine (IC50=5.4 mM). Intermediates in the valine biosynthesis pathway also exhibit feedback inhibitory control of the AHAS enzyme. In addition, AHAS has a very weak affinity for pyruvate (KM=10.5 μM) and is highly selective towards 2-ketobutyrate (R=140) as a second substrate. AHAIR and DHAD are also inhibited by the branched-chain amino acids, although to a lesser extent when compared to AHAS. Experimental evolution and rational site-directed mutagenesis revealed mutants of the regulatory subunit of AHAS (IlvH) (N11S, T34I, A36V, T104S, N11F, G14E, and N29H), which, when reconstituted with wild-type IlvB, lead to AHAS having reduced valine, leucine, and isoleucine sensitivity. The study of the kinetics and inhibition mechanisms of R. eutropha AHAS, AHAIR, and DHAD has shed light on interactions between these enzymes and the products they produce; it, therefore, can be used to engineer R. eutropha strains with optimal production of 2-ketoisovalerate for

  3. Characterization and modification of enzymes in the 2-ketoisovalerate biosynthesis pathway of Ralstonia eutropha H16

    SciTech Connect

    Lu, JN; Brigham, CJ; Plassmeier, JK; Sinskey, AJ

    2014-08-01

    2-Ketoisovalerate is an important cellular intermediate for the synthesis of branched-chain amino acids as well as other important molecules, such as pantothenate, coenzyme A, and glucosinolate. This ketoacid can also serve as a precursor molecule for the production of biofuels, pharmaceutical agents, and flavor agents in engineered organisms, such as the betaproteobacterium Ralstonia eutropha. The biosynthesis of 2-ketoisovalerate from pyruvate is carried out by three enzymes: acetohydroxyacid synthase (AHAS, encoded by ilvBH), acetohydroxyacid isomeroreductase (AHAIR, encoded by ilvC), and dihydroxyacid dehydratase (DHAD, encoded by ilvD). In this study, enzymatic activities and kinetic parameters were determined for each of the three R. eutropha enzymes as heterologously purified proteins. AHAS, which serves as a gatekeeper for the biosynthesis of all three branched-chain amino acids, demonstrated the tightest regulation through feedback inhibition by l-valine (IC50 = 1.2 mM), l-isoleucine (IC50 = 2.3 mM), and l-leucine (IC50 = 5.4 mM). Intermediates in the valine biosynthesis pathway also exhibit feedback inhibitory control of the AHAS enzyme. In addition, AHAS has a very weak affinity for pyruvate (K-M = 10.5 mu M) and is highly selective towards 2-ketobutyrate (R = 140) as a second substrate. AHAIR and DHAD are also inhibited by the branched-chain amino acids, although to a lesser extent when compared to AHAS. Experimental evolution and rational site-directed mutagenesis revealed mutants of the regulatory subunit of AHAS (IlvH) (N11S, T34I, A36V, T104S, N11F, G14E, and N29H), which, when reconstituted with wild-type IlvB, lead to AHAS having reduced valine, leucine, and isoleucine sensitivity. The study of the kinetics and inhibition mechanisms of R. eutropha AHAS, AHAIR, and DHAD has shed light on interactions between these enzymes and the products they produce; it, therefore, can be used to engineer R. eutropha strains with optimal production of 2

  4. Regulation of the cnr Cobalt and Nickel Resistance Determinant of Ralstonia eutropha (Alcaligenes eutrophus) CH34

    PubMed Central

    Tibazarwa, C.; Wuertz, S.; Mergeay, M.; Wyns, L.; van der Lelie, D.

    2000-01-01

    The linked resistance to nickel and cobalt of Ralstonia eutropha-like strain CH34 (Alcaligenes eutrophus CH34) is encoded by the cnr operon, which is localized on the megaplasmid pMOL28. The regulatory genes cnrYXH have been cloned, overexpressed, and purified in Escherichia coli. CnrY fractionated as a 10.7-kDa protein in in vitro translation assays. CnrX, a periplasmic protein of 16.5 kDa, was overproduced and purified as a histidine-tagged fusion protein in E. coli. His-CnrX was found to posses a secondary structure content rich in alpha-helical and beta-sheet structures. CnrH, a sigma factor of the extracytoplasmic function family, was purified as an N-terminally histidine-tagged fusion. In gel shift mobility assays, His-CnrH, in the presence of E. coli core RNA polymerase enzyme, could retard at least two different promoter DNA targets, cnrYp and cnrHp, localized within the cnrYXH locus. These promoters and their transcription start sites were confirmed by primer extension. Purified His-CnrX did not inhibit the DNA-binding activity of His-CnrH and is therefore unlikely to be an anti-sigma factor, as previously hypothesized (EMBL M91650 description entry). To study the transcriptional response of the regulatory locus to metals and to probe promoter regions, transcriptional fusions were constructed between fragments of cnrYXH and the luxCDABE, luciferase reporter genes. Nickel and cobalt specifically induced the cnrYXH-luxCDABE fusion at optimal concentrations of 0.3 mM Ni2+ and 2.0 mM Co2+ in a noncomplexing medium for metals. The two promoter regions PY (upstream cnrY) and PH (upstream cnrH) were probed and characterized using this vector and were found to control the nickel-inducible regulatory response of the cnr operon. The cnrHp promoter was responsible for full transcription of the cnrCBA structural resistance genes, while the cnrYp promoter was necessary to obtain metal-inducible transcription from the cnrHp promoter. The zinc resistance phenotype (Zin

  5. Native interaction of Escherichia coli O157:H7 and Ralstonia insidiosa in forming dual-species biofilms

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Biofilm formation by native microflora in food processing environments can increase the risk of foodborne outbreaks by providing a protective microenvironment to foodborne pathogens. Hence the presence of strong biofilm producing bacteria in such an environment can be regarded as a risk factor. In t...

  6. Morphology of Females and Cysts of Globodera tabacum tabacum, G. t. virginiae, and G. t. solanacearum (Nemata: Heteroderinae)

    PubMed Central

    Mota, Manuel M.; Eisenback, Jonathan D.

    1993-01-01

    Detailed morphological comparisons with light and scanning electron microscopy were made of white females and cysts of several isolates of Globodera tabacum sspp. tabacum (GTT), virginiae (GTV), and solanacearum (GTS). Observations focused on body shape, anterior region including head shape, lip pattern, stylet morphology, and the terminal area in females; and body shape and terminal area of cysts. The most useful characters to separate the three subspecies were forms of the female body, cyst, stylet knobs, tail region, perineal tubercles, anal-fenestral ridge patterns, and the distinctiveness of the anus. GTT is characterized by having round females and cysts, sharply back sloped stylet knobs, clumped perineal tubercles in the vulval region, tight parallel ridges in the cyst anal-fenestral region, and a uniformly conoid tail region. GTV is characterized by its ovoid to ellipsoid female and cyst shape, the "Dutch shoe" shape of the dorsal stylet knob, the more dispersed perineal tubercles, a maze-like pattern of ridges in the anal-fenestral region, and an indistinct anus. GTS is characterized by its ovoid to ellipsoid female and cyst shape, moderately backward sloped stylet knobs, more widely separated ridges, a distinct anus, and a usually crescent shaped tail region. Much variability in shape and patterns is visible among all the isolates of the different subspecies. Tubercles in the neck, as well as bullae, are reported, and their taxonomic value is discussed. PMID:19279752

  7. Production and purification of a soluble hydrogenase from Ralstonia eutropha H16 for potential hydrogen fuel cell applications.

    PubMed

    Jugder, Bat-Erdene; Lebhar, Helene; Aguey-Zinsou, Kondo-Francois; Marquis, Christopher P

    2016-01-01

    The soluble hydrogenase (SH) from Ralstonia eutropha H16 is a promising candidate enzyme for H2-based biofuel application as it favours H2 oxidation and is relatively oxygen-tolerant. In this report, bioprocess development studies undertaken to produce and purify an active SH are described, based on the methods previously reported [1], [2], [3], [4]. Our modifications are: •Upstream method optimizations were undertaken on heterotrophic growth media and cell lysis involving ultrasonication.•Two anion exchangers (Q Sepharose and RESOURCE Q) and size exclusion chromatographic (Superdex 200) matrices were successfully employed for purification of a hexameric SH from R. eutropha.•The H2 oxidizing activity of the SH was demonstrated spectrophotometrically in solution and also immobilized on an EPG electrode using cyclic voltammetry. PMID:27077052

  8. Whole-cell kinetics of trichloroethylene degradation by phenol hydroxylase in a Ralstonia eutropha JMP134 derivative

    SciTech Connect

    Ayoubi, P.J.; Harker, A.R.

    1998-11-01

    The rate, progress, and limits of trichloroethylene (TCE) degradation by Ralstonia eutropha AEK301/pYK3021 whole cells were examined in the absence of aromatic induction. At TCE concentrations up to 800 {micro}M, degradation rates were sustained until TCE was no longer detectable. The K{sub s} and V{sub max} for TCE degradation by AEK301/pYK3021 whole cells were determined to be 630 {micro}M and 22.6 nmol/min/mg of total protein, respectively. The sustained linear rates of TCE degradation by AEK301/pYK3021 up to a concentration of 800 {micro}M TCE suggest that solvent effects are limited during the degradation of TCE and that this construct is little affected by the formation of toxic intermediates at the TCE levels and assay duration tested. TCE degradation by this strain is subject to carbon catabolite repression.

  9. Whole-cell kinetics of trichloroethylene degradation by phenol hydroxylase in a ralstonia eutropha JMP134 derivative

    PubMed

    Ayoubi; Harker

    1998-11-01

    The rate, progress, and limits of trichloroethylene (TCE) degradation by Ralstonia eutropha AEK301/pYK3021 whole cells were examined in the absence of aromatic induction. At TCE concentrations up to 800 &mgr;M, degradation rates were sustained until TCE was no longer detectable. The Ks and Vmax for TCE degradation by AEK301/pYK3021 whole cells were determined to be 630 &mgr;M and 22.6 nmol/min/mg of total protein, respectively. The sustained linear rates of TCE degradation by AEK301/pYK3021 up to a concentration of 800 &mgr;M TCE suggest that solvent effects are limited during the degradation of TCE and that this construct is little affected by the formation of toxic intermediates at the TCE levels and assay duration tested. TCE degradation by this strain is subject to carbon catabolite repression. PMID:9797289

  10. Whole-Cell Kinetics of Trichloroethylene Degradation by Phenol Hydroxylase in a Ralstonia eutropha JMP134 Derivative

    PubMed Central

    Ayoubi, Patricia J.; Harker, Alan R.

    1998-01-01

    The rate, progress, and limits of trichloroethylene (TCE) degradation by Ralstonia eutropha AEK301/pYK3021 whole cells were examined in the absence of aromatic induction. At TCE concentrations up to 800 μM, degradation rates were sustained until TCE was no longer detectable. The Ks and Vmax for TCE degradation by AEK301/pYK3021 whole cells were determined to be 630 μM and 22.6 nmol/min/mg of total protein, respectively. The sustained linear rates of TCE degradation by AEK301/pYK3021 up to a concentration of 800 μM TCE suggest that solvent effects are limited during the degradation of TCE and that this construct is little affected by the formation of toxic intermediates at the TCE levels and assay duration tested. TCE degradation by this strain is subject to carbon catabolite repression. PMID:9797289

  11. Liquid Fuel From Bacteria: Engineering Ralstonia eutropha for Production of Isobutanol (IBT) Motor Fuel from CO2, Hydrogen, and Oxygen

    SciTech Connect

    2010-07-15

    Electrofuels Project: MIT is using solar-derived hydrogen and common soil bacteria called Ralstonia eutropha to turn carbon dioxide (CO2) directly into biofuel. This bacteria already has the natural ability to use hydrogen and CO2 for growth. MIT is engineering the bacteria to use hydrogen to convert CO2 directly into liquid transportation fuels. Hydrogen is a flammable gas, so the MIT team is building an innovative reactor system that will safely house the bacteria and gas mixture during the fuel-creation process. The system will pump in precise mixtures of hydrogen, oxygen, and CO2, and the online fuel-recovery system will continuously capture and remove the biofuel product.

  12. Déjà vu: Ralstonia mannitolilytica infection associated with a humidifying respiratory therapy device, Israel, June to July 2011.

    PubMed

    Block, C; Ergaz-Shaltiel, Z; Valinsky, L; Temper, V; Hidalgo-Grass, C; Minster, N; Weissman, C; Benenson, S; Jaffe, J; Moses, A E; Bar-Oz, B

    2013-05-02

    Following a bloodstream infection in June 2011 with Ralstonia mannitolilytica in a premature infant treated with a humidifying respiratory therapy device, an investigation was initiated at the Hadassah Medical Centres in Jerusalem. The device delivers a warmed and humidified mixture of air and oxygen to patients by nasal cannula. The investigation revealed colonisation with R. mannitolilytica of two of 15 patients and contamination of components of five of six devices deployed in the premature units of the Hadassah hospitals. Ten isolates from the investigation were highly related and indistinguishable from isolates described in an outbreak in 2005 in the United States (US). Measures successful in containing the US outbreak were not included in user instructions provided to our hospitals by the distributor of the device.

  13. Enhanced Oxygen-Tolerance of the Full Heterotrimeric Membrane-Bound [NiFe]-Hydrogenase of Ralstonia eutropha

    PubMed Central

    2014-01-01

    Hydrogenases are oxygen-sensitive enzymes that catalyze the conversion between protons and hydrogen. Water-soluble subcomplexes of membrane-bound [NiFe]-hydrogenases (MBH) have been extensively studied for applications in hydrogen–oxygen fuel cells as they are relatively tolerant to oxygen, although even these catalysts are still inactivated in oxidative conditions. Here, the full heterotrimeric MBH of Ralstonia eutropha, including the membrane-integral cytochrome b subunit, was investigated electrochemically using electrodes modified with planar tethered bilayer lipid membranes (tBLM). Cyclic voltammetry and chronoamperometry experiments show that MBH, in equilibrium with the quinone pool in the tBLM, does not anaerobically inactivate under oxidative redox conditions. In aerobic environments, the MBH is reversibly inactivated by O2, but reactivation was found to be fast even under oxidative redox conditions. This enhanced resistance to inactivation is ascribed to the oligomeric state of MBH in the lipid membrane. PMID:24866391

  14. Effect of clove oil on plant pathogenic bacteria and bacterial wilt of tomato and geranium

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We determined the antibacterial activity of clove oil against seven different genera of plant pathogenic bacteria including Gram-negative Agrobacterium tumefaciens, Erwinia carotovora pv. carotovora, Pseudomonas syringae pv. syringae, Ralstonia solanacearum, and Xanthomonas campestris pv. pelargonii...

  15. Genome Sequences of Ralstonia insidiosa Type Strain ATCC 49129 and Strain FC1138, a Strong Biofilm Producer Isolated from a Fresh-Cut Produce-Processing Plant

    PubMed Central

    Xu, Yunfeng; Nagy, Attila; Yan, Xianghe; Haley, Bradd J.; Kim, Seon Woo; Liu, Nancy T.

    2016-01-01

    Ralstonia insidiosa is an opportunistic pathogen and a strong biofilm producer. Here, we present the complete genome sequences of R. insidiosa FC1138 and ATCC 49129. Both strains have two circular chromosomes of approximately 3.9 and 1.9 Mb and a 50-kb plasmid. ATCC 49129 also possesses a megaplasmid of approximately 318 kb. PMID:27540070

  16. Comparative studies of lipopolysaccharide and exopolysaccharide from a virulent strain of Pseudomonas solanacearum and from three avirulent mutants.

    PubMed Central

    Drigues, P; Demery-Lafforgue, D; Trigalet, A; Dupin, P; Samain, D; Asselineau, J

    1985-01-01

    The composition of the Pseudomonas solanacearum lipolysaccharide (LPS) was found to be similar to that described for the LPS of enterobacteria. The lipid A contained fatty acids and glucosamine in a molar ratio of 5:2. The LPS fraction contained 2-keto-3-deoxyoctulosonic acid, L-glycero-D-mannoheptose, hexoses (glucose, rhamnose, and glucosamine), and a pentose (xylose). The LPSs from the wild-type strain (GMI1000), from the spontaneous rough mutant (GMI2000), and from their respective acridine orange-resistant (Acrr) mutants (GMI1178 and GMI2179) contained the same component sugars in their polysaccharide moieties, but the relative amounts of each sugar varied greatly. Spontaneous mutation to the rough type was characterized by a decrease in the ratio of rhamnose to glucose, whereas a reverse effect was seen for the acridine orange resistance mutation from the parent strains (GMI1000 and GMI2000) to the respective mutant strains (GMI1178 and GMI2179). The exopolysaccharide (EPS) from GMI1000 was found to be composed of two fractions: a heteropolysaccharide (galactosamine, glucose, and rhamnose) excluded from Sephadex G-50 and an additional glucan with a lower molecular weight. Strains GMI1000 and GMI1178 produced comparable amounts of EPS, GMI2179 synthesized less EPS, and GMI2000 produced no detectable EPS. High-pressure liquid chromatography and 13C nuclear magnetic resonance analyses revealed some differences between these EPSs. The glucan fraction seemed to be the major component of the EPS from GMI2179, whereas GMI1000 and GMI1178 EPSs contained both fractions and appeared to differ in the structures of their heteropolysaccharide fractions. Viscosity measurements confirmed differences between whole EPSs produced by the three strains. PMID:3988700

  17. Crystal structure and biochemical characterization of beta-keto thiolase B from polyhydroxyalkanoate-producing bacterium Ralstonia eutropha H16

    SciTech Connect

    Kim, Eun-Jung; Son, Hyeoncheol Francis; Kim, Sangwoo; Ahn, Jae-Woo; Kim, Kyung-Jin

    2014-02-14

    Highlights: • We determined a crystal structure of β-keto thiolase from Ralstonia eutropha H16 (ReBktB). • Distinct substrate binding mode ReBktB was elucidated. • Enzymatic kinetic parameters of ReBktB were revealed. - Abstract: ReBktB is a β-keto thiolase from Ralstonia eutropha H16 that catalyzes condensation reactions between acetyl-CoA with acyl-CoA molecules that contains different numbers of carbon atoms, such as acetyl-CoA, propionyl-CoA, and butyryl-CoA, to produce valuable bioproducts, such as polyhydroxybutyrate, polyhydroxybutyrate-hydroxyvalerate, and hexanoate. We solved a crystal structure of ReBktB at 2.3 Å, and the overall structure has a similar fold to that of type II biosynthetic thiolases, such as PhbA from Zoogloea ramigera (ZrPhbA). The superposition of this structure with that of ZrPhbA complexed with CoA revealed the residues that comprise the catalytic and substrate binding sites of ReBktB. The catalytic site of ReBktB contains three conserved residues, Cys90, His350, and Cys380, which may function as a covalent nucleophile, a general base, and second nucleophile, respectively. For substrate binding, ReBktB stabilized the ADP moiety of CoA in a distinct way compared to ZrPhbA with His219, Arg221, and Asp228 residues, whereas the stabilization of β-mercaptoethyamine and pantothenic acid moieties of CoA was quite similar between these two enzymes. Kinetic study of ReBktB revealed that K{sub m}, V{sub max}, and K{sub cat} values of 11.58 μM, 1.5 μmol/min, and 102.18 s{sup −1}, respectively, and the catalytic and substrate binding sites of ReBktB were further confirmed by site-directed mutagenesis experiments.

  18. Cloning, expression, purification, crystallization and X-ray crystallographic analysis of β-ketothiolase B from Ralstonia eutropha H16

    PubMed Central

    Kim, Eun-Jung; Son, Hyeoncheol Francis; Chang, Jeong Ho; Kim, Kyung-Jin

    2014-01-01

    Polyhydroxyalkanoates are linear polyesters that are produced by bacterial fermentation and are used as biodegradable bioplastics. β-Ketothiolase B (BktB) from Ralstonia eutropha (ReBktB) is a key enzyme for the production of various types of copolymers by catalyzing the condensation reactions of acetyl-CoA with propionyl-CoA and butyryl-CoA. The ReBktB protein was crystallized using the hanging-drop vapour-diffusion method in the presence of 25% polyethylene glycol 3350, 0.1 M bis-tris pH 6.5, 0.2 M lithium sulfate at 295 K. X-ray diffraction data were collected to a maximum resolution of 2.3 Å on a synchrotron beamline. The crystal belonged to space group C2221, with unit-cell parameters a = 106.95, b = 107.24, c = 144.14 Å. With two molecules per asymmetric unit, the crystal volume per unit protein weight (V M) is 2.54 Å3 Da−1, which corresponds to a solvent content of approximately 51.5%. The structure was solved by the molecular-replacement method and refinement of the structure is in progress. PMID:24598917

  19. Heavy metal-resistant bacteria as extremophiles: molecular physiology and biotechnological use of Ralstonia sp. CH34.

    PubMed

    Nies, D H

    2000-04-01

    In contrast to thermophilic or psychrophilic organisms, heavy metal-resistant bacteria do not supply enzymes that are active under harsh conditions, but are themselves tools for the evaluation and remediation of heavy metal-contaminated environments. Ralstonia sp. CH34 is a gram-negative bacterium with a remarkable set of resistance determinants, allowing this bacterium to live in extreme environments that are heavily contaminated with toxic metal ions. These heavy metal ions are mostly detoxified by inducible ion efflux systems that reduce the intracellular concentration of a given ion by active export. Because all metal resistance determinants in this bacterium are inducible, their regulatory systems can be used to develop biosensors that measure the biologically important concentrations of heavy metals in an environment. Resistance based on metal ion efflux detoxifies only the cytoplasm of the respective cell. Therefore, this resistance mechanism cannot be used directly to develop biotechnological procedures; however, metal ion efflux can protect a cell in a metal-contaminated environment. Thus, the cell can be enabled to mediate biochemical reactions such as precipitation of heavy metals with the carbon dioxide produced during growth or degradation of xenobiotics.

  20. Cloning, expression, purification, crystallization and X-ray crystallographic analysis of β-ketothiolase B from Ralstonia eutropha H16.

    PubMed

    Kim, Eun-Jung; Son, Hyeoncheol Francis; Chang, Jeong Ho; Kim, Kyung-Jin

    2014-03-01

    Polyhydroxyalkanoates are linear polyesters that are produced by bacterial fermentation and are used as biodegradable bioplastics. β-Ketothiolase B (BktB) from Ralstonia eutropha (ReBktB) is a key enzyme for the production of various types of copolymers by catalyzing the condensation reactions of acetyl-CoA with propionyl-CoA and butyryl-CoA. The ReBktB protein was crystallized using the hanging-drop vapour-diffusion method in the presence of 25% polyethylene glycol 3350, 0.1 M bis-tris pH 6.5, 0.2 M lithium sulfate at 295 K. X-ray diffraction data were collected to a maximum resolution of 2.3 Å on a synchrotron beamline. The crystal belonged to space group C2221, with unit-cell parameters a = 106.95, b = 107.24, c = 144.14 Å. With two molecules per asymmetric unit, the crystal volume per unit protein weight (VM) is 2.54 Å(3) Da(-1), which corresponds to a solvent content of approximately 51.5%. The structure was solved by the molecular-replacement method and refinement of the structure is in progress. PMID:24598917

  1. Cloning, expression, purification, crystallization and X-ray crystallographic analysis of PhaA from Ralstonia eutropha.

    PubMed

    Kim, Eun-Jung; Kim, Kyung-Jin

    2014-11-01

    Polyhydroxybutyrate (PHB) is a biopolymer that is in the spotlight because of its broad applications in bioplastics, fine chemicals, implant biomaterials and biofuels. PhaA from Ralstonia eutropha (RePhaA) is the first enzyme in the PHB biosynthetic pathway and catalyzes the condensation reaction of two acetyl-CoA molecules to give acetoacetyl-CoA. RePhaA was crystallized using the hanging-drop vapour-diffusion method in the presence of 20% polyethylene glycol monomethyl ether 2K, 0.1 M Tris-HCl pH 8.5 and 0.2 M trimethylamine N-oxide dihydrate at 295 K. X-ray diffraction data were collected to a maximum resolution of 1.96 Å on a synchrotron beamline. The crystal belonged to space group P2₁, with unit-cell parameters a=68.38, b=105.47, c=106.91 Å, α=γ=90, β=106.18°. With four subunits per asymmetric unit, the crystal volume per unit protein weight (VM) is 2.3 Å3 Da(-1), which corresponds to a solvent content of approximately 46.2%. The structure was solved by the molecular-replacement method and refinement of the structure is in progress. PMID:25372833

  2. Cloning, expression, purification, crystallization and X-ray crystallographic analysis of PhaA from Ralstonia eutropha

    PubMed Central

    Kim, Eun-Jung; Kim, Kyung-Jin

    2014-01-01

    Polyhydroxybutyrate (PHB) is a biopolymer that is in the spotlight because of its broad applications in bioplastics, fine chemicals, implant biomaterials and biofuels. PhaA from Ralstonia eutropha (RePhaA) is the first enzyme in the PHB biosynthetic pathway and catalyzes the condensation reaction of two acetyl-CoA molecules to give acetoacetyl-CoA. RePhaA was crystallized using the hanging-drop vapour-diffusion method in the presence of 20% polyethylene glycol monomethyl ether 2K, 0.1 M Tris–HCl pH 8.5 and 0.2 M trimethylamine N-oxide dihydrate at 295 K. X-ray diffraction data were collected to a maximum resolution of 1.96 Å on a synchrotron beamline. The crystal belonged to space group P21, with unit-cell parameters a = 68.38, b = 105.47, c = 106.91 Å, α = γ = 90, β = 106.18°. With four subunits per asymmetric unit, the crystal volume per unit protein weight (V M) is 2.3 Å3 Da−1, which corresponds to a solvent content of approximately 46.2%. The structure was solved by the molecular-replacement method and refinement of the structure is in progress. PMID:25372833

  3. Spectroscopic and Kinetic Properties of the Molybdenum-containing, NAD+-dependent Formate Dehydrogenase from Ralstonia eutropha.

    PubMed

    Niks, Dimitri; Duvvuru, Jayant; Escalona, Miguel; Hille, Russ

    2016-01-15

    We have examined the rapid reaction kinetics and spectroscopic properties of the molybdenum-containing, NAD(+)-dependent FdsABG formate dehydrogenase from Ralstonia eutropha. We confirm previous steady-state studies of the enzyme and extend its characterization to a rapid kinetic study of the reductive half-reaction (the reaction of formate with oxidized enzyme). We have also characterized the electron paramagnetic resonance signal of the molybdenum center in its Mo(V) state and demonstrated the direct transfer of the substrate Cα hydrogen to the molybdenum center in the course of the reaction. Varying temperature, microwave power, and level of enzyme reduction, we are able to clearly identify the electron paramagnetic resonance signals for four of the iron/sulfur clusters of the enzyme and find suggestive evidence for two others; we observe a magnetic interaction between the molybdenum center and one of the iron/sulfur centers, permitting assignment of this signal to a specific iron/sulfur cluster in the enzyme. In light of recent advances in our understanding of the structure of the molybdenum center, we propose a reaction mechanism involving direct hydride transfer from formate to a molybdenum-sulfur group of the molybdenum center.

  4. Structure-Activity Relationships of Antimicrobial Gallic Acid Derivatives from Pomegranate and Acacia Fruit Extracts against Potato Bacterial Wilt Pathogen.

    PubMed

    Farag, Mohamed A; Al-Mahdy, Dalia A; Salah El Dine, Riham; Fahmy, Sherifa; Yassin, Aymen; Porzel, Andrea; Brandt, Wolfgang

    2015-06-01

    Bacterial wilts of potato, tomato, pepper, and or eggplant caused by Ralstonia solanacearum are among the most serious plant diseases worldwide. In this study, the issue of developing bactericidal agents from natural sources against R. solanacearum derived from plant extracts was addressed. Extracts prepared from 25 plant species with antiseptic relevance in Egyptian folk medicine were screened for their antimicrobial properties against the potato pathogen R. solancearum by using the disc-zone inhibition assay and microtitre plate dilution method. Plants exhibiting notable antimicrobial activities against the tested pathogen include extracts from Acacia arabica and Punica granatum. Bioactivity-guided fractionation of A. arabica and P. granatum resulted in the isolation of bioactive compounds 3,5-dihydroxy-4-methoxybenzoic acid and gallic acid, in addition to epicatechin. All isolates displayed significant antimicrobial activities against R. solanacearum (MIC values 0.5-9 mg/ml), with 3,5-dihydroxy-4-methoxybenzoic acid being the most effective one with a MIC value of 0.47 mg/ml. We further performed a structure-activity relationship (SAR) study for the inhibition of R. solanacearum growth by ten natural, structurally related benzoic acids.

  5. Structure-Activity Relationships of Antimicrobial Gallic Acid Derivatives from Pomegranate and Acacia Fruit Extracts against Potato Bacterial Wilt Pathogen.

    PubMed

    Farag, Mohamed A; Al-Mahdy, Dalia A; Salah El Dine, Riham; Fahmy, Sherifa; Yassin, Aymen; Porzel, Andrea; Brandt, Wolfgang

    2015-06-01

    Bacterial wilts of potato, tomato, pepper, and or eggplant caused by Ralstonia solanacearum are among the most serious plant diseases worldwide. In this study, the issue of developing bactericidal agents from natural sources against R. solanacearum derived from plant extracts was addressed. Extracts prepared from 25 plant species with antiseptic relevance in Egyptian folk medicine were screened for their antimicrobial properties against the potato pathogen R. solancearum by using the disc-zone inhibition assay and microtitre plate dilution method. Plants exhibiting notable antimicrobial activities against the tested pathogen include extracts from Acacia arabica and Punica granatum. Bioactivity-guided fractionation of A. arabica and P. granatum resulted in the isolation of bioactive compounds 3,5-dihydroxy-4-methoxybenzoic acid and gallic acid, in addition to epicatechin. All isolates displayed significant antimicrobial activities against R. solanacearum (MIC values 0.5-9 mg/ml), with 3,5-dihydroxy-4-methoxybenzoic acid being the most effective one with a MIC value of 0.47 mg/ml. We further performed a structure-activity relationship (SAR) study for the inhibition of R. solanacearum growth by ten natural, structurally related benzoic acids. PMID:26080741

  6. vsrB, a regulator of virulence genes of Pseudomonas solanacearum, is homologous to sensors of the two-component regulator family.

    PubMed Central

    Huang, J; Denny, T P; Schell, M A

    1993-01-01

    Pseudomonas solanacearum, an important wilt pathogen of many plants, produces several extracellular proteins (EXPs) and extracellular polysaccharides (EPSs) that contribute to its virulence. Using TnphoA mutagenesis, we discovered a new gene, vsrB, that when inactivated causes a major reduction in the virulence and production of an EPS. Analysis of eps::lacZ reporters showed that vsrB is required for maximal expression (transcription) of eps, whose products are required for production of EPS I, a major virulence determinant. Analysis of EXPs in culture supernatants revealed that inactivation of vsrB also causes reduced production of two major EXPs, with molecular masses of 28 and 97 kDa, and a simultaneous 15-fold increase in levels of another EXP, PglA endopolygalacturonase. The vsrB gene was cloned from a P. solanacearum genomic library by complementation of the nonmucoid phenotype of the vsrB::TnphoA mutant and then subcloned on a 2.4-kb DNA fragment. TnphoA fusion analysis and subcellular localization of the vsrB gene product in Escherichia coli maxicells suggest that it is a ca. 60-kDa transmembrane protein. The nucleotide sequence of the 2.4-kb DNA fragment was determined, and a 638-amino-acid open reading frame was found for VsrB. A search of the GenBank data base found that the central part of VsrB has homology with the histidine kinase domain of sensors in the two-component regulator family, while the C terminus has homology with the phosphate receiver domain of response regulators in the same family. Genetic analysis suggests that the receiver domain is not required for vsrB function. Images PMID:8407789

  7. Genome-Based Analysis and Gene Dosage Studies Provide New Insight into 3-Hydroxy-4-Methylvalerate Biosynthesis in Ralstonia eutropha

    PubMed Central

    Ushimaru, Kazunori; Mizuno, Shoji

    2015-01-01

    Recombinant Ralstonia eutropha strain PHB−4 expressing the broad-substrate-specificity polyhydroxyalkanoate (PHA) synthase 1 from Pseudomonas sp. strain 61-3 (PhaC1Ps) synthesizes a PHA copolymer containing the branched side-chain unit 3-hydroxy-4-methylvalerate (3H4MV), which has a carbon backbone identical to that of leucine. Mutant strain 1F2 was derived from R. eutropha strain PHB−4 by chemical mutagenesis and shows higher levels of 3H4MV production than does the parent strain. In this study, to understand the mechanisms underlying the enhanced production of 3H4MV, whole-genome sequencing of strain 1F2 was performed, and the draft genome sequence was compared to that of parent strain PHB−4. This analysis uncovered four point mutations in the 1F2 genome. One point mutation was found in the ilvH gene at amino acid position 36 (A36T) of IlvH. ilvH encodes a subunit protein that regulates acetohydroxy acid synthase III (AHAS III). AHAS catalyzes the conversion of pyruvate to 2-acetolactate, which is the first reaction in the biosynthesis of branched amino acids such as leucine and valine. Thus, the A36T IlvH mutation may show AHAS tolerance to feedback inhibition by branched amino acids, thereby increasing carbon flux toward branched amino acid and 3H4MV biosynthesis. Furthermore, a gene dosage study and an isotope tracer study were conducted to investigate the 3H4MV biosynthesis pathway. Based on the observations in these studies, we propose a 3H4MV biosynthesis pathway in R. eutropha that involves a condensation reaction between isobutyryl coenzyme A (isobutyryl-CoA) and acetyl-CoA to form the 3H4MV carbon backbone. PMID:25645560

  8. Formation of Polyphosphate by Polyphosphate Kinases and Its Relationship to Poly(3-Hydroxybutyrate) Accumulation in Ralstonia eutropha Strain H16

    PubMed Central

    Tumlirsch, Tony; Sznajder, Anna

    2015-01-01

    A protein (PhaX) that interacted with poly(3-hydroxybutyrate) (PHB) depolymerase PhaZa1 and with PHB granule-associated phasin protein PhaP2 was identified by two-hybrid analysis. Deletion of phaX resulted in an increase in the level of polyphosphate (polyP) granule formation and in impairment of PHB utilization in nutrient broth-gluconate cultures. A procedure for enrichment of polyP granules from cell extracts was developed. Twenty-seven proteins that were absent in other cell fractions were identified in the polyP granule fraction by proteome analysis. One protein (A2437) harbored motifs characteristic of type 1 polyphosphate kinases (PPK1s), and two proteins (A1212, A1271) had PPK2 motifs. In vivo colocalization with polyP granules was confirmed by expression of C- and N-terminal fusions of enhanced yellow fluorescent protein (eYFP) with the three polyphosphate kinases (PPKs). Screening of the genome DNA sequence for additional proteins with PPK motifs revealed one protein with PPK1 motifs and three proteins with PPK2 motifs. Construction and subsequent expression of C- and N-terminal fusions of the four new PPK candidates with eYFP showed that only A1979 (PPK2 motif) colocalized with polyP granules. The other three proteins formed fluorescent foci near the cell pole (apart from polyP) (A0997, B1019) or were soluble (A0226). Expression of the Ralstonia eutropha ppk (ppkReu) genes in an Escherichia coli Δppk background and construction of a set of single and multiple chromosomal deletions revealed that both A2437 (PPK1a) and A1212 (PPK2c) contributed to polyP granule formation. Mutants with deletion of both genes were unable to produce polyP granules. The formation and utilization of PHB and polyP granules were investigated in different chromosomal backgrounds. PMID:26407880

  9. Production of Poly(3-Hydroxybutyrate-co-3-Hydroxyhexanoate) from Plant Oil by Engineered Ralstonia eutropha Strains▿†

    PubMed Central

    Budde, Charles F.; Riedel, Sebastian L.; Willis, Laura B.; Rha, ChoKyun; Sinskey, Anthony J.

    2011-01-01

    The polyhydroxyalkanoate (PHA) copolymer poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) [P(HB-co-HHx)] has been shown to have potential to serve as a commercial bioplastic. Synthesis of P(HB-co-HHx) from plant oil has been demonstrated with recombinant Ralstonia eutropha strains expressing heterologous PHA synthases capable of incorporating HB and HHx into the polymer. With these strains, however, short-chain-length fatty acids had to be included in the medium to generate PHA with high HHx content. Our group has engineered two R. eutropha strains that accumulate high levels of P(HB-co-HHx) with significant HHx content directly from palm oil, one of the world's most abundant plant oils. The strains express a newly characterized PHA synthase gene from the bacterium Rhodococcus aetherivorans I24. Expression of an enoyl coenzyme A (enoyl-CoA) hydratase gene (phaJ) from Pseudomonas aeruginosa was shown to increase PHA accumulation. Furthermore, varying the activity of acetoacetyl-CoA reductase (encoded by phaB) altered the level of HHx in the polymer. The strains with the highest PHA titers utilized plasmids for recombinant gene expression, so an R. eutropha plasmid stability system was developed. In this system, the essential pyrroline-5-carboxylate reductase gene proC was deleted from strain genomes and expressed from a plasmid, making the plasmid necessary for growth in minimal media. This study resulted in two engineered strains for production of P(HB-co-HHx) from palm oil. In palm oil fermentations, one strain accumulated 71% of its cell dry weight as PHA with 17 mol% HHx, while the other strain accumulated 66% of its cell dry weight as PHA with 30 mol% HHx. PMID:21398488

  10. A stereoselective carbon-nitrogen lyase from Ralstonia sp. SLRS7 cleaves two of three isomers of iminodisuccinate.

    PubMed

    Cokesa, Zeljko; Lakner, Silvia; Knackmuss, Hans-Joachim; Rieger, Paul-Gerhard

    2004-08-01

    Following biodegradation tests according to the OECD guidelines for testing of chemicals 301F different degradation rates were observed for the three stereoisomers of iminodisuccinate (IDS). A strain was isolated from activated sludge, which used two of three isomers, R,S-IDS and S,S-IDS, as sole source of carbon, nitrogen, and energy. The isolated strain was identified by 16S-rDNA and referred to as Ralstonia sp. SLRS7. An IDS-degrading lyase was isolated from the cell-free extract. The enzyme was purified by three chromatographic steps, which included anion-exchange chromatography, hydrophobic interaction chromatography and gel filtration. The lyase catalysed the non-hydrolytic cleavage of IDS without requirement of any cofactors. Cleavage of S,S-IDS led to the formation of fumaric acid and L-aspartic acid. Interestingly R,S-IDS yielded only D-aspartic acid besides fumaric acid. R,R-IDS was not transformed. Thus, the IDS-degrading enzyme is a carbon-nitrogen lyase attacking only the asymmetric carbon atom exhibiting the S-configuration. Besides S,S-IDS and R,S-IDS cleavage, the lyase catalysed also the transformation of certain S,S-IDS metal complexes, namely Ca(2+)-, Mg(2+)- and Mn(2+)-IDS. The maximum enzyme activity was found at pH 8.0-8.5 and 35 degrees C. SDS-PAGE analysis revealed a single 57-kDa protein band. The native enzyme was estimated to be around 240 kDa indicating a homotetramer enzyme.

  11. Production of fatty acids in Ralstonia eutropha H16 by engineering β-oxidation and carbon storage

    PubMed Central

    Chen, Janice S.; Colón, Brendan; Dusel, Brendon; Ziesack, Marika; Torella, Joseph P.

    2015-01-01

    Ralstonia eutropha H16 is a facultatively autotrophic hydrogen-oxidizing bacterium capable of producing polyhydroxybutyrate (PHB)-based bioplastics. As PHB’s physical properties may be improved by incorporation of medium-chain-length fatty acids (MCFAs), and MCFAs are valuable on their own as fuel and chemical intermediates, we engineered R. eutropha for MCFA production. Expression of UcFatB2, a medium-chain-length-specific acyl-ACP thioesterase, resulted in production of 14 mg/L laurate in wild-type R. eutropha. Total fatty acid production (22 mg/L) could be increased up to 2.5-fold by knocking out PHB synthesis, a major sink for acetyl-CoA, or by knocking out the acyl-CoA ligase fadD3, an entry point for fatty acids into β-oxidation. As ΔfadD3 mutants still consumed laurate, and because the R. eutropha genome is predicted to encode over 50 acyl-CoA ligases, we employed RNA-Seq to identify acyl-CoA ligases upregulated during growth on laurate. Knockouts of the three most highly upregulated acyl-CoA ligases increased fatty acid yield significantly, with one strain (ΔA2794) producing up to 62 mg/L free fatty acid. This study demonstrates that homologous β-oxidation systems can be rationally engineered to enhance fatty acid production, a strategy that may be employed to increase yield for a range of fuels, chemicals, and PHB derivatives in R. eutropha. PMID:26664804

  12. Production of fatty acids in Ralstonia eutropha H16 by engineering β-oxidation and carbon storage.

    PubMed

    Chen, Janice S; Colón, Brendan; Dusel, Brendon; Ziesack, Marika; Way, Jeffrey C; Torella, Joseph P

    2015-01-01

    Ralstonia eutropha H16 is a facultatively autotrophic hydrogen-oxidizing bacterium capable of producing polyhydroxybutyrate (PHB)-based bioplastics. As PHB's physical properties may be improved by incorporation of medium-chain-length fatty acids (MCFAs), and MCFAs are valuable on their own as fuel and chemical intermediates, we engineered R. eutropha for MCFA production. Expression of UcFatB2, a medium-chain-length-specific acyl-ACP thioesterase, resulted in production of 14 mg/L laurate in wild-type R. eutropha. Total fatty acid production (22 mg/L) could be increased up to 2.5-fold by knocking out PHB synthesis, a major sink for acetyl-CoA, or by knocking out the acyl-CoA ligase fadD3, an entry point for fatty acids into β-oxidation. As ΔfadD3 mutants still consumed laurate, and because the R. eutropha genome is predicted to encode over 50 acyl-CoA ligases, we employed RNA-Seq to identify acyl-CoA ligases upregulated during growth on laurate. Knockouts of the three most highly upregulated acyl-CoA ligases increased fatty acid yield significantly, with one strain (ΔA2794) producing up to 62 mg/L free fatty acid. This study demonstrates that homologous β-oxidation systems can be rationally engineered to enhance fatty acid production, a strategy that may be employed to increase yield for a range of fuels, chemicals, and PHB derivatives in R. eutropha. PMID:26664804

  13. Phosphotransferase protein EIIANtr interacts with SpoT, a key enzyme of the stringent response, in Ralstonia eutropha H16.

    PubMed

    Karstens, Katja; Zschiedrich, Christopher P; Bowien, Botho; Stülke, Jörg; Görke, Boris

    2014-04-01

    EIIA(Ntr) is a member of a truncated phosphotransferase (PTS) system that serves regulatory functions and exists in many Proteobacteria in addition to the sugar transport PTS. In Escherichia coli, EIIA(Ntr) regulates K(+) homeostasis through interaction with the K(+) transporter TrkA and sensor kinase KdpD. In the β-Proteobacterium Ralstonia eutropha H16, EIIA(Ntr) influences formation of the industrially important bioplastic poly(3-hydroxybutyrate) (PHB). PHB accumulation is controlled by the stringent response and induced under conditions of nitrogen deprivation. Knockout of EIIA(Ntr) increases the PHB content. In contrast, absence of enzyme I or HPr, which deliver phosphoryl groups to EIIA(Ntr), has the opposite effect. To clarify the role of EIIA(Ntr) in PHB formation, we screened for interacting proteins that co-purify with Strep-tagged EIIA(Ntr) from R. eutropha cells. This approach identified the bifunctional ppGpp synthase/hydrolase SpoT1, a key enzyme of the stringent response. Two-hybrid and far-Western analyses confirmed the interaction and indicated that only non-phosphorylated EIIA(Ntr) interacts with SpoT1. Interestingly, this interaction does not occur between the corresponding proteins of E. coli. Vice versa, interaction of EIIA(Ntr) with KdpD appears to be absent in R. eutropha, although R. eutropha EIIA(Ntr) can perfectly substitute its homologue in E. coli in regulation of KdpD activity. Thus, interaction with KdpD might be an evolutionary 'ancient' task of EIIA(Ntr) that was subsequently replaced by interaction with SpoT1 in R. eutropha. In conclusion, EIIA(Ntr) might integrate information about nutritional status, as reflected by its phosphorylation state, into the stringent response, thereby controlling cellular PHB content in R. eutropha. PMID:24515609

  14. Degradation of formaldehyde at high concentrations by phenol-adapted Ralstonia eutropha closely related to pink-pigmented facultative methylotrophs.

    PubMed

    Habibi, Alireza; Vahabzadeh, Farzaneh

    2013-01-01

    The ability of the phenol-adapted Ralstonia eutropha to utilize formaldehyde (FD) as the sole source of carbon and energy was studied. Adaptation to FD was accomplished by substituting FD for glucose in a stepwise manner. The bacterium in the liquid test culture could tolerate concentrations of FD up to 900 mg L(-1). Degradation of FD was complete in 528 h at 30°C with shaking at 150 rpm (r = 1.67 mg L(-1) h(-1)), q = 0.035 g(FD) g(cell) (-1) h(-1). Substrate inhibition kinetics (Haldane and Luong equations) are used to describe the experimental data. At non-inhibitory concentrations of FD, the Monod equation was used. According to the Luong model, the values of the maximum specific growth rate (μ(max)), half-saturation coefficient (k(S)), the maximum allowable formaldehyde concentration (S(m)), and the shape factor (n) were 0.117 h(-1), 47.6 mg L(-1), 900 mg L(-1), and 2.2, respectively. The growth response of the test bacterium to consecutive FD feedings was examined, and the FD-adapted R. eutropha cells were able to degrade 1000 mg L(-1) FD in 150 h through 4 cycles of FD feeds. During FD degradation, formic acid metabolite was formed. Assimilation of FD, methanol, formic acid, and oxalate by the test bacterium was accompanied by the formation of a pink pigment. The carotenoid nature of the cellular pigment has been confirmed and the test bacterium appeared to be closely related to pink-pigmented facultative methylotrophs (PPFM). The extent of harm to soil exposed to biotreated wastewaters containing FD may be moderated due to the association between methylotrophic/oxalotrophic bacteria and plants.

  15. Efficient biological conversion of carbon monoxide (CO) to carbon dioxide (CO2) and for utilization in bioplastic production by Ralstonia eutropha through the display of an enzyme complex on the cell surface.

    PubMed

    Hyeon, Jeong Eun; Kim, Seung Wook; Park, Chulhwan; Han, Sung Ok

    2015-06-25

    An enzyme complex for biological conversion of CO to CO2 was anchored on the cell surface of the CO2-utilizing Ralstonia eutropha and successfully resulted in a 3.3-fold increase in conversion efficiency. These results suggest that this complexed system may be a promising strategy for CO2 utilization as a biological tool for the production of bioplastics. PMID:26017299

  16. Efficient biological conversion of carbon monoxide (CO) to carbon dioxide (CO2) and for utilization in bioplastic production by Ralstonia eutropha through the display of an enzyme complex on the cell surface.

    PubMed

    Hyeon, Jeong Eun; Kim, Seung Wook; Park, Chulhwan; Han, Sung Ok

    2015-06-25

    An enzyme complex for biological conversion of CO to CO2 was anchored on the cell surface of the CO2-utilizing Ralstonia eutropha and successfully resulted in a 3.3-fold increase in conversion efficiency. These results suggest that this complexed system may be a promising strategy for CO2 utilization as a biological tool for the production of bioplastics.

  17. Candidatus liberibacter solanacearum

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Zebra chip (ZC) is a new and economically important disease of potato in the United States, Mexico, Central America, and New Zealand. This disease has caused millions of dollars in losses to the potato industry. Whole crops might be rejected because of high levels of ZC, often leading to abandonment...

  18. Comparison of the transcriptomes of ginger (Zingiber officinale Rosc.) and mango ginger (Curcuma amada Roxb.) in response to the bacterial wilt infection.

    PubMed

    Prasath, Duraisamy; Karthika, Raveendran; Habeeba, Naduva Thadath; Suraby, Erinjery Jose; Rosana, Ottakandathil Babu; Shaji, Avaroth; Eapen, Santhosh Joseph; Deshpande, Uday; Anandaraj, Muthuswamy

    2014-01-01

    Bacterial wilt in ginger (Zingiber officinale Rosc.) caused by Ralstonia solanacearum is one of the most important production constraints in tropical, sub-tropical and warm temperature regions of the world. Lack of resistant genotype adds constraints to the crop management. However, mango ginger (Curcuma amada Roxb.), which is resistant to R. solanacearum, is a potential donor, if the exact mechanism of resistance is understood. To identify genes involved in resistance to R. solanacearum, we have sequenced the transcriptome from wilt-sensitive ginger and wilt-resistant mango ginger using Illumina sequencing technology. A total of 26387032 and 22268804 paired-end reads were obtained after quality filtering for C. amada and Z. officinale, respectively. A total of 36359 and 32312 assembled transcript sequences were obtained from both the species. The functions of the unigenes cover a diverse set of molecular functions and biological processes, among which we identified a large number of genes associated with resistance to stresses and response to biotic stimuli. Large scale expression profiling showed that many of the disease resistance related genes were expressed more in C. amada. Comparative analysis also identified genes belonging to different pathways of plant defense against biotic stresses that are differentially expressed in either ginger or mango ginger. The identification of many defense related genes differentially expressed provides many insights to the resistance mechanism to R. solanacearum and for studying potential pathways involved in responses to pathogen. Also, several candidate genes that may underline the difference in resistance to R. solanacearum between ginger and mango ginger were identified. Finally, we have developed a web resource, ginger transcriptome database, which provides public access to the data. Our study is among the first to demonstrate the use of Illumina short read sequencing for de novo transcriptome assembly and comparison in

  19. Comparison of the transcriptomes of ginger (Zingiber officinale Rosc.) and mango ginger (Curcuma amada Roxb.) in response to the bacterial wilt infection.

    PubMed

    Prasath, Duraisamy; Karthika, Raveendran; Habeeba, Naduva Thadath; Suraby, Erinjery Jose; Rosana, Ottakandathil Babu; Shaji, Avaroth; Eapen, Santhosh Joseph; Deshpande, Uday; Anandaraj, Muthuswamy

    2014-01-01

    Bacterial wilt in ginger (Zingiber officinale Rosc.) caused by Ralstonia solanacearum is one of the most important production constraints in tropical, sub-tropical and warm temperature regions of the world. Lack of resistant genotype adds constraints to the crop management. However, mango ginger (Curcuma amada Roxb.), which is resistant to R. solanacearum, is a potential donor, if the exact mechanism of resistance is understood. To identify genes involved in resistance to R. solanacearum, we have sequenced the transcriptome from wilt-sensitive ginger and wilt-resistant mango ginger using Illumina sequencing technology. A total of 26387032 and 22268804 paired-end reads were obtained after quality filtering for C. amada and Z. officinale, respectively. A total of 36359 and 32312 assembled transcript sequences were obtained from both the species. The functions of the unigenes cover a diverse set of molecular functions and biological processes, among which we identified a large number of genes associated with resistance to stresses and response to biotic stimuli. Large scale expression profiling showed that many of the disease resistance related genes were expressed more in C. amada. Comparative analysis also identified genes belonging to different pathways of plant defense against biotic stresses that are differentially expressed in either ginger or mango ginger. The identification of many defense related genes differentially expressed provides many insights to the resistance mechanism to R. solanacearum and for studying potential pathways involved in responses to pathogen. Also, several candidate genes that may underline the difference in resistance to R. solanacearum between ginger and mango ginger were identified. Finally, we have developed a web resource, ginger transcriptome database, which provides public access to the data. Our study is among the first to demonstrate the use of Illumina short read sequencing for de novo transcriptome assembly and comparison in

  20. Impact of Ralstonia eutropha's Poly(3-Hydroxybutyrate) (PHB) Depolymerases and Phasins on PHB Storage in Recombinant Escherichia coli

    PubMed Central

    Eggers, Jessica

    2014-01-01

    The model organism for polyhydroxybutyrate (PHB) biosynthesis, Ralstonia eutropha H16, possesses multiple isoenzymes of granules coating phasins as well as of PHB depolymerases, which degrade accumulated PHB under conditions of carbon limitation. In this study, recombinant Escherichia coli BL21(DE3) strains were used to study the impact of selected PHB depolymerases of R. eutropha H16 on the growth behavior and on the amount of accumulated PHB in the absence or presence of phasins. For this purpose, 20 recombinant E. coli BL21(DE3) strains were constructed, which harbored a plasmid carrying the phaCAB operon from R. eutropha H16 to ensure PHB synthesis and a second plasmid carrying different combinations of the genes encoding a phasin and a PHB depolymerase from R. eutropha H16. It is shown in this study that the growth behavior of the respective recombinant E. coli strains was barely affected by the overexpression of the phasin and PHB depolymerase genes. However, the impact on the PHB contents was significantly greater. The strains expressing the genes of the PHB depolymerases PhaZ1, PhaZ2, PhaZ3, and PhaZ7 showed 35% to 94% lower PHB contents after 30 h of cultivation than the control strain. The strain harboring phaZ7 reached by far the lowest content of accumulated PHB (only 2.0% [wt/wt] PHB of cell dry weight). Furthermore, coexpression of phasins in addition to the PHB depolymerases influenced the amount of PHB stored in cells of the respective strains. It was shown that the phasins PhaP1, PhaP2, and PhaP4 are not substitutable without an impact on the amount of stored PHB. In particular, the phasins PhaP2 and PhaP4 seemed to limit the degradation of PHB by the PHB depolymerases PhaZ2, PhaZ3, and PhaZ7, whereas almost no influence of the different phasins was observed if phaZ1 was coexpressed. This study represents an extensive analysis of the impact of PHB depolymerases and phasins on PHB accumulation and provides a deeper insight into the complex interplay

  1. Stable Carbon Isotope Discrimination by Form IC Rubisco Enzymes of the Extremely Metabolically Versatile Rhodobacter sphaeroides and Ralstonia eutropha}

    NASA Astrophysics Data System (ADS)

    Thomas, P. J.; Boller, A. J.; Zhao, Z.; Tabita, F. R.; Cavanaugh, C. M.; Scott, K. M.

    2006-12-01

    Variations in the relative amounts of 12C and 13C in microbial biomass can be used to infer the pathway(s) autotrophs use to fix and assimilate dissolved inorganic carbon. Discrimination against 13C by the enzymes catalyzing autotrophic carbon fixation is a major factor dictating biomass stable carbon isotopic compositions (δ13C = {[13C/12Csample/13C/12Cstandard] - 1} × 1000). Five different forms of RubisCO (IA, IB, IC, ID, and II) are utilized by algae and autotrophic bacteria reliant on the Calvin-Benson cycle for carbon fixation. To date, isotope discrimination has been measured for form IA, IB, and II RubisCOs, and their ɛ values (={[12k/13k] - 1} × 1000; 12k and 13k = rates of 12C and 13C fixation) range from 18 to 29‰, explaining the variation in biomass δ13C values of autotrophs utilizing these enzymes. Isotope discrimination by form IC RubisCO has not been measured, despite the presence of this enzyme in many proteobacteria of ecological interest, including marine manganese-oxidizing bacteria, some nitrifying and nitrogen-fixing bacteria, and extremely metabolically versatile organisms such as Rhodobacter sphaeroides and Ralstonia eutropha. The purpose of this work was to determine the ɛ values for form IC RubisCO enzymes from R. sphaeroides and R. eutropha. Recombinant form IC RubisCOs were purified by conventional column chromatography procedures. Assay conditions (pH, dissolved inorganic carbon concentration) were tested to determine which parameters were conducive to the high rates of carbon fixation necessary for ɛ determination. Under standard conditions (pH 8.5 and 5 mM DIC), form IC RubisCO activities were sufficient for ɛ determination. Experiments are currently being conducted to measure the ɛ values of these enzymes. Sampling the full phylogenetic breadth of RubisCO enzymes for isotopic discrimination makes it possible to constrain the range of δ13C values of organisms fixing carbon via the Calvin-Benson cycle. These results are

  2. Recovery of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) from Ralstonia eutropha cultures with non-halogenated solvents.

    PubMed

    Riedel, Sebastian L; Brigham, Christopher J; Budde, Charles F; Bader, Johannes; Rha, Chokyun; Stahl, Ulf; Sinskey, Anthony J

    2013-02-01

    Reduced downstream costs, together with high purity recovery of polyhydroxyalkanoate (PHA), will accelerate the commercialization of high quality PHA-based products. In this work, a process was designed for effective recovery of the copolymer poly(hydroxybutyrate-co-hydroxyhexanoate) (P(HB-co-HHx)) containing high levels of HHx (>15 mol%) from Ralstonia eutropha biomass using non-halogenated solvents. Several non-halogenated solvents (methyl isobutyl ketone, methyl ethyl ketone, and butyl acetate and ethyl acetate) were found to effectively dissolve the polymer. Isoamyl alcohol was found to be not suitable for extraction of polymer. All PHA extractions were performed from both dry and wet cells at volumes ranging from 2 mL to 3 L using a PHA to solvent ratio of 2% (w/v). Ethyl acetate showed both high recovery levels and high product purities (up to 99%) when using dry cells as starting material. Recovery from wet cells, however, eliminates a biomass drying step during the downstream process, potentially saving time and cost. When wet cells were used, methyl isobutyl ketone (MIBK) was shown to be the most favorable solvent for PHA recovery. Purities of up to 99% and total recovery yields of up to 84% from wet cells were reached. During polymer recovery with either MIBK or butyl acetate, fractionation of the extracted PHA occurred, based on the HHx content of the polymer. PHA with higher HHx content (17-30 mol%) remained completely in solution, while polymer with a lower HHx content (11-16 mol%) formed a gel-like phase. All PHA in solution could be precipitated by addition of threefold volumes of n-hexane or n-heptane to unfiltered PHA solutions. Effective recycling of the solvents in this system is predicted due to the large differences in the boiling points between solvent and precipitant. Our findings show that two non-halogenated solvents are good candidates to replace halogenated solvents like chloroform for recovery of high quality PHA. PMID:22903730

  3. Impact of Ralstonia eutropha's poly(3-Hydroxybutyrate) (PHB) Depolymerases and Phasins on PHB storage in recombinant Escherichia coli.

    PubMed

    Eggers, Jessica; Steinbüchel, Alexander

    2014-12-01

    The model organism for polyhydroxybutyrate (PHB) biosynthesis, Ralstonia eutropha H16, possesses multiple isoenzymes of granules coating phasins as well as of PHB depolymerases, which degrade accumulated PHB under conditions of carbon limitation. In this study, recombinant Escherichia coli BL21(DE3) strains were used to study the impact of selected PHB depolymerases of R. eutropha H16 on the growth behavior and on the amount of accumulated PHB in the absence or presence of phasins. For this purpose, 20 recombinant E. coli BL21(DE3) strains were constructed, which harbored a plasmid carrying the phaCAB operon from R. eutropha H16 to ensure PHB synthesis and a second plasmid carrying different combinations of the genes encoding a phasin and a PHB depolymerase from R. eutropha H16. It is shown in this study that the growth behavior of the respective recombinant E. coli strains was barely affected by the overexpression of the phasin and PHB depolymerase genes. However, the impact on the PHB contents was significantly greater. The strains expressing the genes of the PHB depolymerases PhaZ1, PhaZ2, PhaZ3, and PhaZ7 showed 35% to 94% lower PHB contents after 30 h of cultivation than the control strain. The strain harboring phaZ7 reached by far the lowest content of accumulated PHB (only 2.0% [wt/wt] PHB of cell dry weight). Furthermore, coexpression of phasins in addition to the PHB depolymerases influenced the amount of PHB stored in cells of the respective strains. It was shown that the phasins PhaP1, PhaP2, and PhaP4 are not substitutable without an impact on the amount of stored PHB. In particular, the phasins PhaP2 and PhaP4 seemed to limit the degradation of PHB by the PHB depolymerases PhaZ2, PhaZ3, and PhaZ7, whereas almost no influence of the different phasins was observed if phaZ1 was coexpressed. This study represents an extensive analysis of the impact of PHB depolymerases and phasins on PHB accumulation and provides a deeper insight into the complex interplay

  4. A Closer Look on the Polyhydroxybutyrate- (PHB-) Negative Phenotype of Ralstonia eutropha PHB-4

    PubMed Central

    Raberg, Matthias; Voigt, Birgit; Hecker, Michael; Steinbüchel, Alexander

    2014-01-01

    The undefined poly(3-hydroxybutyrate)- (PHB-) negative mutant R. eutropha PHB-4 was generated in 1970 by 1-nitroso-3-nitro-1-methylguanidine (NMG) treatment. Although being scientific relevant, its genotype remained unknown since its isolation except a recent first investigation. In this study, the mutation causing the PHA-negative phenotype of R. eutropha PHB-4 was confirmed independently: sequence analysis of the phaCAB operon identified a G320A mutation in phaC yielding a stop codon, leading to a massively truncated PhaC protein of 106 amino acids (AS) in R. eutropha PHB-4 instead of 589 AS in the wild type. No other mutations were observed within the phaCAB operon. As further mutations probably occurred in the genome of mutant PHB-4 potentially causing secondary effects on the cells' metabolism, the main focus of the study was to perform a 2D PAGE-based proteome analysis in order to identify differences in the proteomes of the wild type and mutant PHB-4. A total of 20 differentially expressed proteins were identified which provide valuable insights in the metabolomic changes of mutant PHB-4. Besides excretion of pyruvate, mutant PHB-4 encounters the accumulation of intermediates such as pyruvate and acetyl-CoA by enhanced expression of the observed protein species: (i) ThiJ supports biosynthesis of cofactor TPP and thereby reinforces the 2-oxoacid dehydrogenase complexes as PDHC, ADHC and OGDHC in order to convert pyruvate at a higher rate and the (ii) 3-isopropylmalate dehydrogenase LeuB3 apparently directs pyruvate to synthesis of several amino acids. Different (iii) acylCoA-transferases enable transfer reactions between organic acid intermediates, and (iv) citrate lyase CitE4 regenerates oxaloacetate from citrate for conversion with acetyl-CoA in the TCC in an anaplerotic reaction. Substantial amounts of reduction equivalents generated in the TCC are countered by (v) synthesis of more ubiquinones due to enhanced synthesis of MenG2 and MenG3, thereby

  5. Diazinon degradation by a novel strain Ralstonia sp. DI-3 and X-ray crystal structure determination of the metabolite of diazinon.

    PubMed

    Wang, Guangli; Liu, Yuan

    2016-09-01

    Diazinon is a widely used organophosphorus insecticide often detected in the environment. A highly effective diazinon-degrading Ralstonia sp. strain DI-3 was isolated from agricultural soil. Strain DI-3 can utilize dimethoate as its sole carbon source for growth and degrade an initial concentration of 100 mg L-1 diazinon to non-detectable levels within 60 h in liquid culture. A small amount of second carbon source as co-substrate could slightly enhance the biodegradation of diazinon. In addition, a less toxic metabolic intermediate formed during the degradation of diazinon mediated by strain DI-3 was purified using thin-layer chromatography (TLC) and identified based on single-crystal Xray diffraction analysis, allowing a degradation pathway for diazinon by pure culture to be proposed. Finally, this is the first providing authentic evidence to describe the metabolite. PMID:27581928

  6. Engineering Ralstonia eutropha for Production of Isobutanol (IBT) Motor Fuel from Carbon Dioxide, Hydrogen, and Oxygen Project Final Report

    SciTech Connect

    Sinskey, Anthony J.; Worden, Robert Mark; Brigham, Christopher; Lu, Jingnan; Quimby, John Westlake; Gai, Claudia; Speth, Daan; Elliott, Sean; Fei, John Qiang; Bernardi, Amanda; Li, Sophia; Grunwald, Stephan; Grousseau, Estelle; Maiti, Soumen; Liu, Chole

    2013-12-16

    This research project is a collaboration between the Sinskey laboratory at MIT and the Worden laboratory at Michigan State University. The goal of the project is to produce Isobutanol (IBT), a branched-chain alcohol that can serve as a drop-in transportation fuel, through the engineered microbial biosynthesis of Carbon Dioxide, Hydrogen, and Oxygen using a novel bioreactor. This final technical report presents the findings of both the biological engineering work at MIT that extended the native branched-chain amino acid pathway of the wild type Ralstonia eutropha H16 to perform this biosynthesis, as well as the unique design, modeling, and construction of a bioreactor for incompatible gasses at Michigan State that enabled the operational testing of the complete system. This 105 page technical report summarizing the three years of research includes 72 figures and 11 tables of findings. Ralstonia eutropha (also known as Cupriavidus necator) is a Gram-negative, facultatively chemolithoautotrophic bacteria. It has been the principle organism used for the study of polyhydroxybutyrate (PHB) polymer biosynthesis. The wild-type Ralstonia eutropha H16 produces PHB as an intracellular carbon storage material while under nutrient stress in the presence of excess carbon. Under this stress, it can accumulate approximately 80 % of its cell dry weight (CDW) as this intracellular polymer. With the restoration of the required nutrients, the cells are then able to catabolize this polymer. If extracted from the cell, this PHB polymer can be processed into biodegradable and biocompatible plastics, however for this research, it is the efficient metabolic pathway channeling the captured carbon that is of interest. R. eutropha is further unique in that it contains two carbon-fixation Calvin–Benson–Bassham cycle operons, two oxygen-tolerant hydrogenases, and several formate dehydrogenases. It has also been much studied for its ability in the presence of oxygen, to fix carbon dioxide

  7. 'Candidatus Liberibacter solanacearum' Titers in and Infection Effects on Potato Tuber Chemistry of Promising Germplasm Exhibiting Tolerance to Zebra Chip Disease.

    PubMed

    Wallis, C M; Munyaneza, J E; Chen, J; Novy, R; Bester, G; Buchman, J L; Nordgaard, J; van Hest, P

    2015-12-01

    Long-term sustainable management of zebra chip (ZC) disease of potato requires development of tolerant or resistant germplasm. To this end, 283 potato varieties and breeding clones were infected with the ZC putative causal agent 'Candidatus Liberibacter solanacearum' (Lso) by potato psyllid vector inoculations in 2010, 2011, 2012, and 2013. Potato germplasm was then examined for development of fresh and fried ZC symptoms. Over multiple years 29 breeding clones exhibited little to no symptoms in freshly cut tuber slices, and five exhibited little to no symptoms in fried slices. These five presumed tolerant breeding clones were chosen for further screening to determine whether the lack of physiological responses to Lso infection was the cause of observed tolerance. To this end, tuber amino acid, sugar, and phenolic levels were compared between noninfected and Lso-infected plants. The five putative tolerant clones had less dramatic shifts in host physiology following Lso infection than the susceptible Atlantic cultivar. This suggested lack of host responses to Lso infection that result in major changes in tuber biochemistry is a potential mechanism of ZC resistance. However, the susceptible Atlantic cultivar did have consistently greater Lso titers compared with two of the tolerant entries, so for these reductions in Lso pathogen progression also might be a factor. Regardless, lack of host responses could still remain one trait that could be used to aid in selection of ZC-resistant potato varieties, as other tolerant lines had infection levels consistent with susceptible Atlantic cultivar. These results also suggest that germplasm derived from relatives of cultivated potato plants are viable sources of ZC disease resistance.

  8. Methods for rapid and effective PCR-based detection of 'Candidatus Liberibacter solanacearum' from the insect vector Bactericera cockerelli: streamlining the DNA extraction/purification process.

    PubMed

    Lévy, Julien; Hancock, Joseph; Ravindran, Aravind; Gross, Dennis; Tamborindeguy, Cecilia; Pierson, Elizabeth

    2013-06-01

    This study provides a protocol for rapid DNA isolation from psyllid vectors (Bactericera cockerelli and Diaphorina citri) that can be used directly with DNA-based methods for the detection of 'Candidatus (Ca.) Liberibacter solanacearum,' the bacterial causal agent of potato zebra chip disease and eventually for 'Ca. Liberibacter asiaticus' the causal agent of huanglongbing disease in citrus. The fast DNA extraction protocol was designed to work with conventional polymerase chain reaction (cPCR) DNA amplification as well as Loop mediated PCR DNA amplification. Direct cPCR of the psyllid 28S rDNA gene from samples prepared using the fast DNA extraction method was as reliable as from samples prepared using standard DNA purification (> 97% from live insects) as tested in B. cockerelli. However, samples prepared using the fast DNA extraction method had to be diluted 1:100 in sterile water for reliable amplification, presumably to dilute PCR inhibitors in the crude extract. Similarly, both cPCR and loop mediated PCR DNA amplification detected 'Ca. Liberibacter' in psyllids infected with either the zebra chip or huanglongbing pathogen equally well from diluted samples prepared using the fast DNA extraction method or from samples prepared using a DNA purification step. In addition to being reliable, the time required to complete the fast DNA extraction for 10 samples was on average approximately 5 min and required no special reagents or laboratory equipment. Thus, the fast DNA extraction method shows strong promise as a rapid, reliable, and expedient method when coupled with PCR-based analyses for detection of 'Ca. Liberibacter' pathogens in psyllids. PMID:23865212

  9. 'Candidatus Liberibacter solanacearum' Titers in and Infection Effects on Potato Tuber Chemistry of Promising Germplasm Exhibiting Tolerance to Zebra Chip Disease.

    PubMed

    Wallis, C M; Munyaneza, J E; Chen, J; Novy, R; Bester, G; Buchman, J L; Nordgaard, J; van Hest, P

    2015-12-01

    Long-term sustainable management of zebra chip (ZC) disease of potato requires development of tolerant or resistant germplasm. To this end, 283 potato varieties and breeding clones were infected with the ZC putative causal agent 'Candidatus Liberibacter solanacearum' (Lso) by potato psyllid vector inoculations in 2010, 2011, 2012, and 2013. Potato germplasm was then examined for development of fresh and fried ZC symptoms. Over multiple years 29 breeding clones exhibited little to no symptoms in freshly cut tuber slices, and five exhibited little to no symptoms in fried slices. These five presumed tolerant breeding clones were chosen for further screening to determine whether the lack of physiological responses to Lso infection was the cause of observed tolerance. To this end, tuber amino acid, sugar, and phenolic levels were compared between noninfected and Lso-infected plants. The five putative tolerant clones had less dramatic shifts in host physiology following Lso infection than the susceptible Atlantic cultivar. This suggested lack of host responses to Lso infection that result in major changes in tuber biochemistry is a potential mechanism of ZC resistance. However, the susceptible Atlantic cultivar did have consistently greater Lso titers compared with two of the tolerant entries, so for these reductions in Lso pathogen progression also might be a factor. Regardless, lack of host responses could still remain one trait that could be used to aid in selection of ZC-resistant potato varieties, as other tolerant lines had infection levels consistent with susceptible Atlantic cultivar. These results also suggest that germplasm derived from relatives of cultivated potato plants are viable sources of ZC disease resistance. PMID:26312966

  10. Use of Electrical Penetration Graph Technology to Examine Transmission of 'Candidatus Liberibacter solanacearum' to Potato by Three Haplotypes of Potato Psyllid (Bactericera cockerelli; Hemiptera: Triozidae).

    PubMed

    Mustafa, Tariq; Horton, David R; Cooper, W Rodney; Swisher, Kylie D; Zack, Richard S; Pappu, Hanu R; Munyaneza, Joseph E

    2015-01-01

    The potato psyllid, Bactericera cockerelli (Šulc) (Hemiptera: Triozidae), is a vector of the phloem-limited bacterium 'Candidatus Liberibacter solanacearum' (Lso), the putative causal agent of zebra chip disease of potato. Little is known about how potato psyllid transmits Lso to potato. We used electrical penetration graph (EPG) technology to compare stylet probing behaviors and efficiency of Lso transmission of three haplotypes of potato psyllid (Central, Western, Northwestern). All haplotypes exhibited the full suite of stylet behaviors identified in previous studies with this psyllid, including intercellular penetration and secretion of the stylet pathway, xylem ingestion, and phloem activities, the latter comprising salivation and ingestion. The three haplotypes exhibited similar frequency and duration of probing behaviors, with the exception of salivation into phloem, which was of higher duration by psyllids of the Western haplotype. We manipulated how long psyllids were allowed access to potato ("inoculation access period", or IAP) to examine the relationship between phloem activities and Lso transmission. Between 25 and 30% of psyllids reached and salivated into phloem at an IAP of 1 hr, increasing to almost 80% of psyllids as IAP was increased to 24 h. Probability of Lso-transmission was lower across all IAP levels than probability of phloem salivation, indicating that a percentage of infected psyllids which salivated into the phloem failed to transmit Lso. Logistic regression showed that probability of transmission increased as a function of time spent salivating into the phloem; transmission occurred as quickly as 5 min following onset of salivation. A small percentage of infected psyllids showed extremely long salivation events but nonetheless failed to transmit Lso, for unknown reasons. Information from these studies increases our understanding of Lso transmission by potato psyllid, and demonstrates the value of EPG technology in exploring questions

  11. Water Extract from Spent Mushroom Substrate of Hericium erinaceus Suppresses Bacterial Wilt Disease of Tomato.

    PubMed

    Kwak, A Min; Min, Kyeong Jin; Lee, Sang Yeop; Kang, Hee Wan

    2015-09-01

    Culture filtrates of six different edible mushroom species were screened for antimicrobial activity against tomato wilt bacteria Ralstonia solanacearum B3. Hericium erinaceus, Lentinula edodes (Sanjo 701), Grifola frondosa, and Hypsizygus marmoreus showed antibacterial activity against the bacteria. Water, n-butanol, and ethyl acetate extracts of spent mushroom substrate (SMS) of H. erinaceus exhibited high antibacterial activity against different phytopathogenic bacteria: Pectobacterium carotovorum subsp. carotovorum, Agrobacterium tumefaciens, R. solanacearum, Xanthomonas oryzae pv. oryzae, X. campestris pv. campestris, X. axonopodis pv. vesicatoria, X. axonopodis pv. citiri, and X. axonopodis pv. glycine. Quantitative real-time PCR revealed that water extracts of SMS (WESMS) of H. erinaceus induced expressions of plant defense genes encoding β-1,3-glucanase (GluA) and pathogenesis-related protein-1a (PR-1a), associated with systemic acquired resistance. Furthermore, WESMS also suppressed tomato wilt disease caused by R. solanacearum by 85% in seedlings and promoted growth (height, leaf number, and fresh weight of the root and shoot) of tomato plants. These findings suggest the WESMS of H. erinaceus has the potential to suppress bacterial wilt disease of tomato through multiple effects including antibacterial activity, plant growth promotion, and defense gene induction. PMID:26539048

  12. Water Extract from Spent Mushroom Substrate of Hericium erinaceus Suppresses Bacterial Wilt Disease of Tomato

    PubMed Central

    Kwak, A Min; Min, Kyeong Jin; Lee, Sang Yeop

    2015-01-01

    Culture filtrates of six different edible mushroom species were screened for antimicrobial activity against tomato wilt bacteria Ralstonia solanacearum B3. Hericium erinaceus, Lentinula edodes (Sanjo 701), Grifola frondosa, and Hypsizygus marmoreus showed antibacterial activity against the bacteria. Water, n-butanol, and ethyl acetate extracts of spent mushroom substrate (SMS) of H. erinaceus exhibited high antibacterial activity against different phytopathogenic bacteria: Pectobacterium carotovorum subsp. carotovorum, Agrobacterium tumefaciens, R. solanacearum, Xanthomonas oryzae pv. oryzae, X. campestris pv. campestris, X. axonopodis pv. vesicatoria, X. axonopodis pv. citiri, and X. axonopodis pv. glycine. Quantitative real-time PCR revealed that water extracts of SMS (WESMS) of H. erinaceus induced expressions of plant defense genes encoding β-1,3-glucanase (GluA) and pathogenesis-related protein-1a (PR-1a), associated with systemic acquired resistance. Furthermore, WESMS also suppressed tomato wilt disease caused by R. solanacearum by 85% in seedlings and promoted growth (height, leaf number, and fresh weight of the root and shoot) of tomato plants. These findings suggest the WESMS of H. erinaceus has the potential to suppress bacterial wilt disease of tomato through multiple effects including antibacterial activity, plant growth promotion, and defense gene induction. PMID:26539048

  13. Assessing the Likelihood of Transmission of Candidatus Liberibacter solanacearum to Carrot by Potato Psyllid, Bactericera cockerelli (Hemiptera: Triozidae)

    PubMed Central

    Munyaneza, Joseph E.; Mustafa, Tariq; Fisher, Tonja W.; Sengoda, Venkatesan G.; Horton, David R.

    2016-01-01

    ‘Candidatus Liberibacter solanacearum’ (Lso) is a phloem-limited bacterium that severely affects important Solanaceae and Apiaceae crops, including potato, tomato, pepper, tobacco, carrot and celery. This bacterium is transmitted to solanaceous species by potato psyllid, Bactericera cockerelli, and to Apiaceae by carrot psyllids, including Trioza apicalis and Bactericera trigonica. Five haplotypes of Lso have so far been described, two are associated with solanaceous species and potato psyllids, whereas the other three are associated with carrot and celery crops and carrot psyllids. Little is known about cross-transmission of Lso to carrot by potato psyllids or to potato by carrot psyllids. Thus, the present study assessed whether potato psyllid can transmit Lso to carrot and whether Lso haplotypes infecting solanaceous species can also infect carrot and lead to disease symptom development. In addition, the stylet probing behavior of potato psyllid on carrot was assessed using electropenetrography (EPG) technology to further elucidate potential Lso transmission to Apiaceae by this potato insect pest. Results showed that, while potato psyllids survived on carrot for several weeks when confined on the plants under controlled laboratory and field conditions, the insects generally failed to infect carrot plants with Lso. Only three of the 200 carrot plants assayed became infected with Lso and developed characteristic disease symptoms. Lso infection in the symptomatic carrot plants was confirmed by polymerase chain reaction assay and Lso in the carrots was determined to be of the haplotype B, which is associated with solanaceous species. EPG results further revealed that potato psyllids readily feed on carrot xylem but rarely probe into the phloem tissue, explaining why little to no Lso infection occurred during the controlled laboratory and field cage transmission trials. Results of our laboratory and field transmission studies, combined with our EPG results

  14. Impact of the core components of the phosphoenolpyruvate-carbohydrate phosphotransferase system, HPr and EI, on differential protein expression in Ralstonia eutropha H16.

    PubMed

    Kaddor, Chlud; Voigt, Birgit; Hecker, Michael; Steinbüchel, Alexander

    2012-07-01

    In Ralstonia eutropha H16, seven genes encoding proteins being involved in the phosphoenolpyruvate-carbohydrate phosphotransferase system (PEP-PTS) were identified. In order to provide more insights into the poly(3-hydroxybutyrate) (PHB)-leaky phenotype of the HPr/EI deletion mutants H16ΔptsH, H16ΔptsI, and H16ΔptsHI when grown on the non-PTS substrate gluconate, parallel fermentations for comparison of their growth behavior were performed. Samples from the exponential, the early stationary, and late stationary growth phases were investigated by microscopy, gas chromatography and (phospho-) proteome analysis. A total of 71 differentially expressed proteins were identified using 2D-PAGE, Pro-Q Diamond and Coomassie staining, and MALDI-TOF analysis. Detected proteins were classified into five major functional groups: carbon metabolism, energy metabolism, amino acid metabolism, translation, and membrane transport/outer membrane proteins. Proteome analyses revealed enhanced expression of proteins involved in the Entner-Doudoroff pathway and in subsequent reactions in cells of strain H16 compared to the mutant H16ΔptsHI. Furthermore, proteins involved in PHB accumulation showed increased abundance in the wild-type. This expression pattern allowed us to identify proteins affecting carbon metabolism/PHB biosynthesis in strain H16 and translation/amino acid metabolism in strain H16ΔptsHI, and to gain insight into the molecular response of R. eutropha to the deletion of HPr/EI. PMID:22630130

  15. Improved artificial pathway for biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) with high C6-monomer composition from fructose in Ralstonia eutropha.

    PubMed

    Insomphun, Chayatip; Xie, Huan; Mifune, Jun; Kawashima, Yui; Orita, Izumi; Nakamura, Satoshi; Fukui, Toshiaki

    2015-01-01

    Poly((R)-3-hydroxybutyrate-co-(R)-3-hydroxyhexanoate) [P(3HB-co-3HHx)], a flexible and practical kind of polyhydroxyalkanoates, is generally produced from plant oils and fatty acids by several wild and recombinant bacteria. This study established an improved artificial pathway for the biosynthesis of P(3HB-co-3HHx) with high 3HHx composition from structurally unrelated fructose in Ralstonia eutropha. Depression of (R)-specific reduction of acetoacetyl-CoA by the deletion of phaB1 was an effective modification for formation of the C6-monomer unit from fructose driven by crotonyl-CoA carboxylase/reductase (Ccr). Co-overexpression of phaJ4a, which encodes medium-chain-length (R)-enoyl-CoA hydratase, with ccr promoted the incorporation of both 3HB and 3HHx units. Further introduction of emdMm, a synthetic gene encoding ethylmalonyl-CoA decarboxylase derived from mouse, was remarkably effective for P(3HB-co-3HHx) biosynthesis, probably by converting ethylmalonyl-CoA generated by the reductive carboxylase activity of Ccr back into butyryl-CoA. A high cellular content of P(3HB-co-3HHx) composed of 22mol% 3HHx could be produced from fructose by the engineered strain of R. eutropha with ΔphaB1 genotype expressing ccr, phaJ4a, and emd. PMID:25446974

  16. Electrochemical and Infrared Spectroscopic Studies Provide Insight into Reactions of the NiFe Regulatory Hydrogenase from Ralstonia eutropha with O2 and CO.

    PubMed

    Ash, Philip A; Liu, Juan; Coutard, Nathan; Heidary, Nina; Horch, Marius; Gudim, Ingvild; Simler, Thomas; Zebger, Ingo; Lenz, Oliver; Vincent, Kylie A

    2015-10-29

    The regulatory hydrogenase (RH) from Ralstonia eutropha acts as the H2-sensing unit of a two-component system that regulates biosynthesis of the energy conserving hydrogenases of the organism according to the availability of H2. The H2 oxidation activity, which was so far determined in vitro with artificial electron acceptors, has been considered to be insensitive to O2 and CO. It is assumed that bulky isoleucine and phenylalanine amino acid residues close to the NiFe active site "gate" gas access, preventing molecules larger than H2 interacting with the active site. We have carried out sensitive electrochemical measurements to demonstrate that O2 is in fact an inhibitor of H2 oxidation by the RH, and that both H(+) reduction and H2 oxidation are inhibited by CO. Furthermore, we have demonstrated that the inhibitory effect of O2 arises due to interaction of O2 with the active site. Using protein film infrared electrochemistry (PFIRE) under H2 oxidation conditions, in conjunction with solution infrared measurements, we have identified previously unreported oxidized inactive and catalytically active reduced states of the RH active site. These findings suggest that the RH has a rich active site chemistry similar to that of other NiFe hydrogenases. PMID:26115011

  17. Directed evolution and structural analysis of NADPH-dependent Acetoacetyl Coenzyme A (Acetoacetyl-CoA) reductase from Ralstonia eutropha reveals two mutations responsible for enhanced kinetics.

    PubMed

    Matsumoto, Ken'ichiro; Tanaka, Yoshikazu; Watanabe, Tsuyoshi; Motohashi, Ren; Ikeda, Koji; Tobitani, Kota; Yao, Min; Tanaka, Isao; Taguchi, Seiichi

    2013-10-01

    NADPH-dependent acetoacetyl-coenzyme A (acetoacetyl-CoA) reductase (PhaB) is a key enzyme in the synthesis of poly(3-hydroxybutyrate) [P(3HB)], along with β-ketothiolase (PhaA) and polyhydroxyalkanoate synthase (PhaC). In this study, PhaB from Ralstonia eutropha was engineered by means of directed evolution consisting of an error-prone PCR-mediated mutagenesis and a P(3HB) accumulation-based in vivo screening system using Escherichia coli. From approximately 20,000 mutants, we obtained two mutant candidates bearing Gln47Leu (Q47L) and Thr173Ser (T173S) substitutions. The mutants exhibited kcat values that were 2.4-fold and 3.5-fold higher than that of the wild-type enzyme, respectively. In fact, the PhaB mutants did exhibit enhanced activity and P(3HB) accumulation when expressed in recombinant Corynebacterium glutamicum. Comparative three-dimensional structural analysis of wild-type PhaB and highly active PhaB mutants revealed that the beneficial mutations affected the flexibility around the active site, which in turn played an important role in substrate recognition. Furthermore, both the kinetic analysis and crystal structure data supported the conclusion that PhaB forms a ternary complex with NADPH and acetoacetyl-CoA. These results suggest that the mutations affected the interaction with substrates, resulting in the acquirement of enhanced activity.

  18. Characterization of two novel alcohol short-chain dehydrogenases/reductases from Ralstonia eutropha H16 capable of stereoselective conversion of bulky substrates.

    PubMed

    Magomedova, Zalina; Grecu, Andreea; Sensen, Christoph W; Schwab, Helmut; Heidinger, Petra

    2016-03-10

    Biocatalysis has significant advantages over organic synthesis in the field of chiral molecule production and several types of stereoselective enzymes are already in use in industrial biotechnology. However, there is still a high demand for new enzymes capable of transforming bulky molecules with sufficient operability. In order to reveal novel high-potential biocatalysts, the complete genome of the β-proteobacterium Ralstonia eutropha H16 was screened for potential short-chain dehydrogenases/reductases (SDRs). We were able to identify two (S)-enantioselective SDRs named A5 and B3. These showed clear preference towards long-chain and aromatic secondary alcohols, aldehydes and ketones, with diaryl diketone benzil as one of the best substrates. In addition the phylogenetic analysis of all enzyme types, which are known to facilitate benzil reduction, revealed at least two separate evolutionary clusters. Our results indicate the biotechnological potential of SDRs A5 and B3 for the production of chiral compounds with potential commercial value.

  19. Expression and activity of the Calvin-Benson-Bassham cycle transcriptional regulator CbbR from Acidithiobacillus ferrooxidans in Ralstonia eutropha.

    PubMed

    Esparza, Mario; Jedlicki, Eugenia; Dopson, Mark; Holmes, David S

    2015-08-01

    Autotrophic fixation of carbon dioxide into cellular carbon occurs via several pathways but quantitatively, the Calvin-Benson-Bassham cycle is the most important. CbbR regulates the expression of the cbb genes involved in CO2 fixation via the Calvin-Benson-Bassham cycle in a number of autotrophic bacteria. A gene potentially encoding CbbR (cbbR(AF)) has been predicted in the genome of the chemolithoautotrophic, extreme acidophile Acidithiobacillus ferrooxidans. However, this microorganism is recalcitrant to genetic manipulation impeding the experimental validation of bioinformatic predictions. Two novel functional assays were devised to advance our understanding of cbbR(AF) function using the mutated facultative autotroph Ralstonia eutropha H14 ΔcbbR as a surrogate host to test gene function: (i) cbbR(AF) was expressed in R. eutropha and was able to complement ΔcbbR; and (ii) CbbR(AF) was able to regulate the in vivo activity of four A. ferrooxidans cbb operon promoters in R. eutropha. These results open up the use of R. eutropha as a surrogate host to explore cbbR(AF) activity.

  20. Use of a packed-bed airlift reactor with net draft tube to study kinetics of naphthalene degradation by Ralstonia eutropha.

    PubMed

    Jalilnejad, Elham; Vahabzadeh, Farzaneh

    2014-03-01

    Biodegradation of naphthalene by Ralstonia eutropha (also known as Cupriavidus necator) in a packed-bed airlift reactor with net draft tube (PBALR-nd) was studied; the Kissiris pieces were the packing material. The reactor hydrodynamics has been characterized under abiotic conditions and the dependencies of the superficial gas velocity (U G) on the gas holdup (εG), liquid mixing time, and mass transfer coefficient were determined. The improving role of the net draft tube in this small column reactor (height 42 cm, ID 5 cm) was confirmed. The flow regime was described using the εG α U G (n) expression, and bubbly flow was observed in PBALR-nd at U G < 2.83 cm/s. In the second step of the present work, the kinetics of biodegradation was modeled using the Haldane and Aiba equations. The fitting of the experimental results to the models were done according to the nonlinear least square regression technique. The biokinetic constants (q m, K s, and K i) were estimated and q m as the specific biodegradation rate was equaled to 0.415 and 0.24 mgnaph./mgcell h for the Haldane and Aiba equations, respectively. The goodness of fit reported as R (2) and root-mean-square error (RMSE) showed the adequate fitness of the Haldane and Aiba models in predicting naphthalene biodegradation kinetics. On the basis of the HPLC results, a hypothetical pathway for the biodegradation was presented.

  1. Antibacterial activity of cyclo(L-Pro-L-Tyr) and cyclo(D-Pro-L-Tyr) from Streptomyces sp. strain 22-4 against phytopathogenic bacteria.

    PubMed

    Wattana-Amorn, Pakorn; Charoenwongsa, Waranya; Williams, Christopher; Crump, Matthew P; Apichaisataienchote, Busaya

    2016-09-01

    Two bioactive cyclic dipeptides, cyclo(L-Pro-L-Tyr) and cyclo(D-Pro-L-Tyr), were isolated from the culture broth of Streptomyces sp. strain 22-4 and tested against three economically important plant pathogens, Xanthomonas axonopodis pv. citri, Ralstonia solanacearum and Clavibacter michiganensis. Both cyclic dipeptides were active against X. axonopodis pv. citri and R. Solanacearum with MIC of 31.25 μg/mL. No activity could be observed against C. michiganensis. PMID:26469746

  2. Antibacterial activity of cyclo(L-Pro-L-Tyr) and cyclo(D-Pro-L-Tyr) from Streptomyces sp. strain 22-4 against phytopathogenic bacteria.

    PubMed

    Wattana-Amorn, Pakorn; Charoenwongsa, Waranya; Williams, Christopher; Crump, Matthew P; Apichaisataienchote, Busaya

    2016-09-01

    Two bioactive cyclic dipeptides, cyclo(L-Pro-L-Tyr) and cyclo(D-Pro-L-Tyr), were isolated from the culture broth of Streptomyces sp. strain 22-4 and tested against three economically important plant pathogens, Xanthomonas axonopodis pv. citri, Ralstonia solanacearum and Clavibacter michiganensis. Both cyclic dipeptides were active against X. axonopodis pv. citri and R. Solanacearum with MIC of 31.25 μg/mL. No activity could be observed against C. michiganensis.

  3. Potential economic pests of solanaceous crops: a new species of Solanum-feeding psyllid from Australia and first record from New Zealand of Acizzia solanicola (Hemiptera: Psyllidae).

    PubMed

    Taylor, Gary S; Kent, Deborah S

    2013-01-01

    Acizzia credoensis sp. n. is described from a single population on the native plant, Solanum lasiophyllum, from semi-arid Western Australia. The host range of Acizzia solanicola Kent & Taylor, initially recorded as damaging eggplant, S. melongena, in commercial crops and gardens and on wild tobacco bush, S. mauritianum in eastern Australia, is expanded to include the following Solanaceae: rock nightshade, S. petrophilum, cape gooseberry, Physalis peruviana, and an undetermined species of angel's trumpet Brugmansia and Datura. New Zealand specimens of A. solanicola collected in early 2012 from S. mauritianum are the first record for this species from outside Australia, and possibly represent a very recent incursion. The potential for the solanaceous-inhabiting Psyllidae to vector Candidatus Liberibacter solanacearum, an economically important plant pathogen, on native Australian Solanaceae is discussed. The occurrence of A. credoensis and A. solanicola on native Australian Solanum supports the Australian origin for the solanaceous-inhabiting Acizzia psyllids.

  4. Cometabolic degradation of ethyl mercaptan by phenol-utilizing Ralstonia eutropha in suspended growth and gas-recycling trickle-bed reactor.

    PubMed

    Sedighi, Mahsa; Zamir, Seyed Morteza; Vahabzadeh, Farzaneh

    2016-01-01

    The degradability of ethyl mercaptan (EM), by phenol-utilizing cells of Ralstonia eutropha, in both suspended and immobilized culture systems, was investigated in the present study. Free-cells experiments conducted at EM concentrations ranging from 1.25 to 14.42 mg/l, showed almost complete removal of EM at concentrations below 10.08 mg/l, which is much higher than the maximum biodegradable EM concentration obtained in experiments that did not utilize phenol as the primary substrate, i.e. 2.5 mg/l. The first-order kinetic rate constant (kSKS) for EM biodegradation by the phenol-utilizing cells (1.7 l/g biomass/h) was about 10 times higher than by cells without phenol utilization. Immobilized-cells experiments performed in a gas recycling trickle-bed reactor packed with kissiris particles at EM concentrations ranging from 1.6 to 36.9 mg/l, showed complete removal at all tested concentrations in a much shorter time, compared with free cells. The first-order kinetic rate constant (rmaxKs) for EM utilization was 0.04 l/h for the immobilized system compared to 0.06 for the suspended-growth culture, due to external mass transfer diffusion. Diffusion limitation was decreased by increasing the recycling-liquid flow rate from 25 to 65 ml/min. The removed EM was almost completely mineralized according to TOC and sulfate measurements. Shut down and starvation experiments revealed that the reactor could effectively handle the starving conditions and was reliable for full-scale application.

  5. Polythioester synthesis in Ralstonia eutropha H16: novel insights into 3,3'-thiodipropionic acid and 3,3'-dithiodipropionic acid catabolism.

    PubMed

    Doberstein, Christina; Grote, Jessica; Wübbeler, Jan Hendrik; Steinbüchel, Alexander

    2014-08-20

    Ralstonia eutropha H16 is capable of utilizing 3,3'-thiodipropionic acid (TDP) and 3,3'-dithiodipropionic acid (DTDP) as precursor substrates for biosynthesis of a polythioester (PTE) heteropolymer consisting of 3-hydroxybutyric acid (3HB) and 3-mercaptopropionic acid (3MP). To elucidate the hitherto unknown catabolic pathways of TDP and DTDP in R. eutropha H16, 19 defined deletion mutants were generated based on extensive functional genome analyses. Deletions of two ABC-type transporter clusters (H16_A0357-0359, H16_A3658-3660) resulted in an alteration of poly(3HB-co-3MP) composition with TDP as precursor to only 10.2±1.9mol% 3MP in comparison to 15.1±5.5mol% in the wild type. A mutant strain of H16 lacking Bordetella uptake gene-like substrate binding proteins (H16_A2779, H16_A0337) incorporated only 7.4±3.8mol% 3MP into PTE heteropolymers with DTDP as precursor in comparison to 24.5±14.5mol% in the wild type. Therefore, both gene products are probably involved in transport processes of this compound into the cells. However, the most significant reduction in 3MP contents of the heteropolymers with DTDP as precursor occurred upon the deletion of a gene encoding the putative thiol-disulfide interchange protein DsbD (H16_A3455, 3.9±2.6mol% 3MP). DsbD is proposed to be involved in the reduction of DTDP into two molecules of 3MP, the common cleavage product of TDP and DTDP.

  6. Implications of various phosphoenolpyruvate-carbohydrate phosphotransferase system mutations on glycerol utilization and poly(3-hydroxybutyrate) accumulation in Ralstonia eutropha H16

    PubMed Central

    2011-01-01

    The enhanced global biodiesel production is also yielding increased quantities of glycerol as main coproduct. An effective application of glycerol, for example, as low-cost substrate for microbial growth in industrial fermentation processes to specific products will reduce the production costs for biodiesel. Our study focuses on the utilization of glycerol as a cheap carbon source during cultivation of the thermoplastic producing bacterium Ralstonia eutropha H16, and on the investigation of carbohydrate transport proteins involved herein. Seven open reading frames were identified in the genome of strain H16 to encode for putative proteins of the phosphoenolpyruvate-carbohydrate phosphotransferase system (PEP-PTS). Although the core components of PEP-PTS, enzyme I (ptsI) and histidine phosphocarrier protein (ptsH), are available in strain H16, a complete PTS-mediated carbohydrate transport is lacking. Growth experiments employing several PEP-PTS mutants indicate that the putative ptsMHI operon, comprising ptsM (a fructose-specific EIIA component of PTS), ptsH, and ptsI, is responsible for limited cell growth and reduced PHB accumulation (53%, w/w, less PHB than the wild type) of this strain in media containing glycerol as a sole carbon source. Otherwise, the deletion of gene H16_A0384 (ptsN, nitrogen regulatory EIIA component of PTS) seemed to largely compensate the effect of the deleted ptsMHI operon (49%, w/w, PHB). The involvement of the PTS homologous proteins on the utilization of the non-PTS sugar alcohol glycerol and its effect on cell growth as well as PHB and carbon metabolism of R. eutropha will be discussed. PMID:21906371

  7. Characterization of a Second tfd Gene Cluster for Chlorophenol and Chlorocatechol Metabolism on Plasmid pJP4 in Ralstonia eutropha JMP134(pJP4)

    PubMed Central

    Laemmli, Caroline M.; Leveau, Johan H. J.; Zehnder, Alexander J. B.; van der Meer, Jan Roelof

    2000-01-01

    Within the 5.9-kb DNA region between the tfdR and tfdK genes on the 2,4-dichlorophenoxyacetic acid (2,4-D) catabolic plasmid pJP4 from Ralstonia eutropha JMP134, we identified five open reading frames (ORFs) with significant homology to the genes for chlorocatechol and chlorophenol metabolism (tfdCDEF and tfdB) already present elsewhere on pJP4. The five ORFs were organized and assigned as follows: tfdDIICIIEIIFII and tfdBII (in short, the tfdII cluster), by analogy to tfdCDEF and tfdB (the tfdI cluster). Primer extension analysis of mRNA isolated from 2,4-D-grown R. eutropha JMP134 identified a single transcription start site in front of the first gene of the cluster, tfdDII, suggesting an operon-like organization for the tfdII genes. By expressing each ORF in Escherichia coli, we confirmed that tfdDII coded for a chloromuconate cycloisomerase, tfdCII coded for a chlorocatechol 1,2-dioxygenase, tfdEII coded for a dienelactone hydrolase, tfdFII coded for a maleylacetate reductase, and tfdBII coded for a chlorophenol hydroxylase. Dot blot hybridizations of mRNA isolated from R. eutropha JMP134 showed that both tfdI and tfdII genes are transcribed upon induction with 2,4-D. Thus, the functions encoded by the tfdII genes seem to be redundant with respect to those of the tfdI cluster. One reason why the tfdII genes do not disappear from plasmid pJP4 might be the necessity for keeping the regulatory genes for the 2,4-D pathway expression tfdR and tfdS. PMID:10894723

  8. Nanofilms of hyaluronan/chitosan assembled layer-by-layer: An antibacterial surface for Xylella fastidiosa.

    PubMed

    Hernández-Montelongo, Jacobo; Nascimento, Vicente F; Murillo, Duber; Taketa, Thiago B; Sahoo, Prasana; de Souza, Alessandra A; Beppu, Marisa M; Cotta, Monica A

    2016-01-20

    In this work, nanofilms of hyaluronan/chitosan (HA/CHI) assembled layer by layer were synthesized; their application as a potential antimicrobial material was demonstrated for the phytopathogen Xylella fastidiosa, a gram-negative bacterium, here used as a model. For the synthesis, the influence of pH and ionic strength of these natural polymer stem-solutions on final characteristics of the HA/CHI nanofilms was studied in detail. The antibacterial effect was evaluated using widefield fluorescence microscopy. These results were correlated with the chemical properties of the nanofilms, studied by FTIR and Raman spectroscopy, as well as with their morphology and surface properties characterized using SEM and AFM. The present findings can be extended to design and optimize HA/CHI nanofilms with enhanced antimicrobial behavior for other type of phytopathogenic gram-negative bacteria species, such as Xanthomonas citri, Xanthomas campestri and Ralstonia solanacearum. PMID:26572322

  9. Root Border Cells and Their Role in Plant Defense.

    PubMed

    Hawes, Martha; Allen, Caitilyn; Turgeon, B Gillian; Curlango-Rivera, Gilberto; Minh Tran, Tuan; Huskey, David A; Xiong, Zhongguo

    2016-08-01

    Root border cells separate from plant root tips and disperse into the soil environment. In most species, each root tip can produce thousands of metabolically active cells daily, with specialized patterns of gene expression. Their function has been an enduring mystery. Recent studies suggest that border cells operate in a manner similar to mammalian neutrophils: Both cell types export a complex of extracellular DNA (exDNA) and antimicrobial proteins that neutralize threats by trapping pathogens and thereby preventing invasion of host tissues. Extracellular DNases (exDNases) of pathogens promote virulence and systemic spread of the microbes. In plants, adding DNase I to root tips eliminates border cell extracellular traps and abolishes root tip resistance to infection. Mutation of genes encoding exDNase activity in plant-pathogenic bacteria (Ralstonia solanacearum) and fungi (Cochliobolus heterostrophus) results in reduced virulence. The study of exDNase activities in plant pathogens may yield new targets for disease control. PMID:27215971

  10. Root Border Cells and Their Role in Plant Defense.

    PubMed

    Hawes, Martha; Allen, Caitilyn; Turgeon, B Gillian; Curlango-Rivera, Gilberto; Minh Tran, Tuan; Huskey, David A; Xiong, Zhongguo

    2016-08-01

    Root border cells separate from plant root tips and disperse into the soil environment. In most species, each root tip can produce thousands of metabolically active cells daily, with specialized patterns of gene expression. Their function has been an enduring mystery. Recent studies suggest that border cells operate in a manner similar to mammalian neutrophils: Both cell types export a complex of extracellular DNA (exDNA) and antimicrobial proteins that neutralize threats by trapping pathogens and thereby preventing invasion of host tissues. Extracellular DNases (exDNases) of pathogens promote virulence and systemic spread of the microbes. In plants, adding DNase I to root tips eliminates border cell extracellular traps and abolishes root tip resistance to infection. Mutation of genes encoding exDNase activity in plant-pathogenic bacteria (Ralstonia solanacearum) and fungi (Cochliobolus heterostrophus) results in reduced virulence. The study of exDNase activities in plant pathogens may yield new targets for disease control.

  11. Trophic network architecture of root-associated bacterial communities determines pathogen invasion and plant health.

    PubMed

    Wei, Zhong; Yang, Tianjie; Friman, Ville-Petri; Xu, Yangchun; Shen, Qirong; Jousset, Alexandre

    2015-01-01

    Host-associated bacterial communities can function as an important line of defence against pathogens in animals and plants. Empirical evidence and theoretical predictions suggest that species-rich communities are more resistant to pathogen invasions. Yet, the underlying mechanisms are unclear. Here, we experimentally test how the underlying resource competition networks of resident bacterial communities affect invasion resistance to the plant pathogen Ralstonia solanacearum in microcosms and in tomato plant rhizosphere. We find that bipartite resource competition networks are better predictors of invasion resistance compared with resident community diversity. Specifically, communities with a combination of stabilizing configurations (low nestedness and high connectance), and a clear niche overlap with the pathogen, reduce pathogen invasion success, constrain pathogen growth within invaded communities and have lower levels of diseased plants in greenhouse experiments. Bacterial resource competition network characteristics can thus be important in explaining positive diversity-invasion resistance relationships in bacterial rhizosphere communities. PMID:26400552

  12. Trophic network architecture of root-associated bacterial communities determines pathogen invasion and plant health

    PubMed Central

    Wei, Zhong; Yang, Tianjie; Friman, Ville-Petri; Xu, Yangchun; Shen, Qirong; Jousset, Alexandre

    2015-01-01

    Host-associated bacterial communities can function as an important line of defence against pathogens in animals and plants. Empirical evidence and theoretical predictions suggest that species-rich communities are more resistant to pathogen invasions. Yet, the underlying mechanisms are unclear. Here, we experimentally test how the underlying resource competition networks of resident bacterial communities affect invasion resistance to the plant pathogen Ralstonia solanacearum in microcosms and in tomato plant rhizosphere. We find that bipartite resource competition networks are better predictors of invasion resistance compared with resident community diversity. Specifically, communities with a combination of stabilizing configurations (low nestedness and high connectance), and a clear niche overlap with the pathogen, reduce pathogen invasion success, constrain pathogen growth within invaded communities and have lower levels of diseased plants in greenhouse experiments. Bacterial resource competition network characteristics can thus be important in explaining positive diversity–invasion resistance relationships in bacterial rhizosphere communities. PMID:26400552

  13. Physiological conditions conducive to high cell density and high cyanophycin content in Ralstonia eutropha strain H16 possessing a KDPG aldolase gene-dependent addiction system.

    PubMed

    Lin, Kaichien; Elbahloul, Yasser; Steinbüchel, Alexander

    2012-03-01

    The recombinant strain of Ralstonia eutropha H16-PHB(-)4-∆eda (pBBR1MCS-2::cphA (6308)/eda (H16)) presenting a 2-keto-3-desoxy-phosphogluconate (KDPG) aldolase (eda) gene-dependent catabolic addiction system for plasmid maintenance when using gluconate or fructose as sole carbon source was used in this study. The effects of the initial pH, the nitrogen-to-carbon ratio, the inorganic components of medium, the oxygen supply, and the different carbon and nitrogen sources on the cell dry matter (CDM) and the cyanophycin granule polypeptide (CGP) content of the cells were studied in a mineral salts medium (MSM) without any additional amino acids or CGP precursor substrates. The experiments were designed to systematically find out the optimal conditions for growth of cells to high densities and for high CGP contents of the cells. Maximum contents of water-insoluble CGP and water-soluble CGP, contributing to 47.5% and 5.8% (w/w) of CDM, respectively, were obtained at the 30-L scale cultivation when cells were cultivated in MSM medium containing sufficient supplements of fructose, NH(3), K(2)SO(4), MgSO(4)[Symbol: see text]7H(2)O, Fe(Ш)NH(4)-citrate, CaCl(2)[Symbol: see text]2H(2)O, and trace elements (SL6). The molecular masses of water-insoluble and water-soluble CGP ranged from 25 to 31 kDa and from 15 to 21 kDa, respectively. High cell densities of up to 82.8 g CDM/L containing up to 37.8% (w/w) water-insoluble CGP at the 30-L scale cultivation were also obtained. This is by far the best combination of high cell density and high cellular CGP contents ever reported, and it showed that efficient production of CGP at the industrial scale in white biotechnology could be achieved. PMID:22080348

  14. Modification of β-oxidation pathway in Ralstonia eutropha for production of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) from soybean oil.

    PubMed

    Insomphun, Chayatip; Mifune, Jun; Orita, Izumi; Numata, Keiji; Nakamura, Satoshi; Fukui, Toshiaki

    2014-02-01

    Ralstonia eutropha H16 is a useful platform for metabolic engineering aiming at efficient production of polyhydroxyalkanaotes being attracted as practical bioplastics. This study focused on bifunctional (S)-specific 2-enoyl-CoA hydratase/(S)-3-hydroxyacyl-CoA dehydrogenase encoded by fadB to obtain information regarding β-oxidation in this bacterium and to achieve compositional regulation of poly((R)-3-hydroxybutyrate-co-(R)-3-hydroxyhexanoate) [P(3HB-co-3HHx)] synthesized from soybean oil. In addition to two FadB homologs (FadB1 and FadB') encoded within the previously identified β-oxidation gene clusters on the chromosome 1, a gene of third homolog (FadB2) was found on chromosome 2 of R. eutropha. The fadB homologs were disrupted in R. eutropha strain NSDG expressing a mutant gene of PHA synthase from Aeromonas caviae. The gene disruptions affected neither growth nor PHA production on fructose. On soybean oil, fadB' deletion led to reduction of PHA quantity attributed to decrease of 3HB unit, while fadB1 deletion slightly increased 3HHx composition without serious negative impact on both cell growth and PHA biosynthesis. Double deletion of fadB1 and fadB' significantly impaired the cell growth and PHA biosynthesis, indicating the major roles of fadB1 and fadB' in β-oxidation. When fadB1 was deleted in several engineered strains of R. eutropha possessing additional (R)-enoyl-CoA hydratase gene(s), the net amounts of 3HHx unit in the PHA fractions showed 6-21% increase probably due to slightly enhanced supply of medium-chain-length 2-enoyl-CoAs through the partially impaired β-oxidation. These results demonstrated that modification of β-oxidation by fadB1 deletion was effective for increasing 3HHx composition in the copolyesters produced from soybean oil. PMID:23999062

  15. (S)-3-hydroxyacyl-CoA dehydrogenase/enoyl-CoA hydratase (FadB’) from fatty acid degradation operon of Ralstonia eutropha H16

    PubMed Central

    2014-01-01

    In this study (S)-3-hydroxyacyl-CoA dehydrogenase/enoyl-CoA hydratase (H16_A0461/FadB’, gene ID: 4247876) from one of two active fatty acid degradation operons of Ralstonia eutropha H16 has been heterologously expressed in Escherichia coli, purified as protein possessing a His-Tag and initially characterized. FadB’ is an enzyme with two catalytic domains exhibiting a single monomeric structure and possessing a molecular weight of 86 kDa. The C-terminal part of the enzyme harbors enoyl-CoA hydratase activity and is able to convert trans-crotonyl-CoA to 3-hydroxybutyryl-CoA. The N-terminal part of FadB’ comprises an NAD+ binding site and is responsible for 3-hydroxyacyl-CoA dehydrogenase activity converting (S)-3-hydroxybutyryl-CoA to acetoacetyl-CoA. Enoyl-CoA hydratase activity was detected spectrophotometrically with trans-crotonyl-CoA. (S)-3-Hydroxyacyl-CoA dehydrogenase activity was measured in both directions with acetoacetyl-CoA and 3-hydroxybutyryl-CoA. FadB’ was found to be strictly stereospecific to (S)-3-hydroxybutyryl-CoA and to prefer NAD+. The Km value for acetoacetyl-CoA was 48 μM and Vmax 149 μmol mg−1 min−1. NADP(H) was utilized at a rate of less than 10% in comparison to activity with NAD(H). FadB’ exhibited optimal activity at pH 6–7 and the activity decreased at alkaline and acidic pH values. Acetyl-CoA, propionyl-CoA and CoA were found to have an inhibitory effect on FadB’. This study is a first report on biochemical properties of purified (S)-stereospecific 3-hydroxyacyl-CoA dehydrogenase/enoyl-CoA hydratase with the inverted domain order from R. eutropha H16. In addition to fundamental information about FadB’ and fatty acid metabolism, FadB’ might be also interesting for biotechnological applications. PMID:25401070

  16. Genome-scale reconstruction and in silico analysis of the Ralstonia eutropha H16 for polyhydroxyalkanoate synthesis, lithoautotrophic growth, and 2-methyl citric acid production

    PubMed Central

    2011-01-01

    Background Ralstonia eutropha H16, found in both soil and water, is a Gram-negative lithoautotrophic bacterium that can utillize CO2 and H2 as its sources of carbon and energy in the absence of organic substrates. R. eutropha H16 can reach high cell densities either under lithoautotrophic or heterotrophic conditions, which makes it suitable for a number of biotechnological applications. It is the best known and most promising producer of polyhydroxyalkanoates (PHAs) from various carbon substrates and is an environmentally important bacterium that can degrade aromatic compounds. In order to make R. eutropha H16 a more efficient and robust biofactory, system-wide metabolic engineering to improve its metabolic performance is essential. Thus, it is necessary to analyze its metabolic characteristics systematically and optimize the entire metabolic network at systems level. Results We present the lithoautotrophic genome-scale metabolic model of R. eutropha H16 based on the annotated genome with biochemical and physiological information. The stoichiometic model, RehMBEL1391, is composed of 1391 reactions including 229 transport reactions and 1171 metabolites. Constraints-based flux analyses were performed to refine and validate the genome-scale metabolic model under environmental and genetic perturbations. First, the lithoautotrophic growth characteristics of R. eutropha H16 were investigated under varying feeding ratios of gas mixture. Second, the genome-scale metabolic model was used to design the strategies for the production of poly[R-(-)-3hydroxybutyrate] (PHB) under different pH values and carbon/nitrogen source uptake ratios. It was also used to analyze the metabolic characteristics of R. eutropha when the phosphofructokinase gene was expressed. Finally, in silico gene knockout simulations were performed to identify targets for metabolic engineering essential for the production of 2-methylcitric acid in R. eutropha H16. Conclusion The genome-scale metabolic model

  17. Antagonistic activity of Bacillus subtilis SB1 and its biocontrol effect on tomato bacterial wilt

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A potential biocontrol agent of bacterial wilt, Bacillus subtilis SB1, isolated from tomato roots, showed a broad-spectrum of antimicrobial activity in in vitro experiments. It inhibited the growth of many plant pathogens, including Ralstonia solanacearum, Xanthomonas oryzae pv. oryzae, Fusarium ox...

  18. Identification and Chacterization of new strains of Enterobacter spp. causing Mulberry (Morus alba) wilt disease in China

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A new mulberry wilt disease (MWD) was recently identified in Hangzhou, Zhejiang province, China. Typical symptoms of the disease are dark brown discolorations in vascular tissues, leaf wilt, defoliation, and tree decline. Unlike the bacterial wilt disease caused by Ralstonia solanacearum, the leaf w...

  19. A case study of a bacterial pathogen in irrigation water

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This chapter presents a case study of how exotic strains of Ralstonia solanacearum were disseminated throughout Europe and Florida via waterways used for irrigation. Several studies have demonstrated that aquatic weeds that commonly grow in rivers and ponds are able to harbor the pathogen and allow ...

  20. Classification of Plant Associated Bacteria Using RIF, a Computationally Derived DNA Marker

    PubMed Central

    Schneider, Kevin L.; Marrero, Glorimar; Alvarez, Anne M.; Presting, Gernot G.

    2011-01-01

    A DNA marker that distinguishes plant associated bacteria at the species level and below was derived by comparing six sequenced genomes of Xanthomonas, a genus that contains many important phytopathogens. This DNA marker comprises a portion of the dnaA replication initiation factor (RIF). Unlike the rRNA genes, dnaA is a single copy gene in the vast majority of sequenced bacterial genomes, and amplification of RIF requires genus-specific primers. In silico analysis revealed that RIF has equal or greater ability to differentiate closely related species of Xanthomonas than the widely used ribosomal intergenic spacer region (ITS). Furthermore, in a set of 263 Xanthomonas, Ralstonia and Clavibacter strains, the RIF marker was directly sequenced in both directions with a success rate approximately 16% higher than that for ITS. RIF frameworks for Xanthomonas, Ralstonia and Clavibacter were constructed using 682 reference strains representing different species, subspecies, pathovars, races, hosts and geographic regions, and contain a total of 109 different RIF sequences. RIF sequences showed subspecific groupings but did not place strains of X. campestris or X. axonopodis into currently named pathovars nor R. solanacearum strains into their respective races, confirming previous conclusions that pathovar and race designations do not necessarily reflect genetic relationships. The RIF marker also was sequenced for 24 reference strains from three genera in the Enterobacteriaceae: Pectobacterium, Pantoea and Dickeya. RIF sequences of 70 previously uncharacterized strains of Ralstonia, Clavibacter, Pectobacterium and Dickeya matched, or were similar to, those of known reference strains, illustrating the utility of the frameworks to classify bacteria below the species level and rapidly match unknown isolates to reference strains. The RIF sequence frameworks are available at the online RIF database, RIFdb, and can be queried for diagnostic purposes with RIF sequences obtained

  1. Cationic Oligo(thiophene ethynylene) with Broad-Spectrum and High Antibacterial Efficiency under White Light and Specific Biocidal Activity against S. aureus in Dark.

    PubMed

    Zhao, Qi; Li, Junting; Zhang, Xiaoqian; Li, Zhengping; Tang, Yanli

    2016-01-13

    We designed and synthesized a novel oligo(thiophene ethynylene) (OTE) to investigate the antibacterial activities against Gram-positive (Staphylococcus aureus and Staphylococcus epidermidis) and Gram-negative (Ralstonia solanacearum and Escherichia coli) bacteria in vitro by photodynamic therapy (PDT). Notably, OTE presents broad-spectrum and greatly high antibacterial activities after white light irradiation at nanogram per milliliter concentrations. The half inhibitory concentrations (IC50) values obtained for S. aureus, S. epidermidis, E. coli, and R. solanacearum are 8, 13, 24, and 52 ng/mL after illumination for 30 min, respectively, which are lower than that of other PDT agents. Interestingly, OTE shows the specific and very strong dark killing capability against S. aureus at the concentration of 180 ng/mL for 30 min, which is the highest efficiency biocide against S. aureus without the need of irradiation to date. The antibacterial mechanism investigated demonstrated that reactive oxygen species or singlet-oxygen generated by OTE kills bacteria irreversibly upon white light irradiation, and OTE as a v-type oligomer exerts its toxicity directly on destroying bacterial cytoplasmic membrane in the dark. Importantly, the OTE shows no cell cytotoxicity and excellent biocompatibility. The results indicate that it is potential to provide versatile applications in the efficient control of pathogenic organisms and specific application for killing S. aureus. PMID:26671682

  2. Optimising droplet digital PCR analysis approaches for detection and quantification of bacteria: a case study of fire blight and potato brown rot.

    PubMed

    Dreo, Tanja; Pirc, Manca; Ramšak, Živa; Pavšič, Jernej; Milavec, Mojca; Zel, Jana; Gruden, Kristina

    2014-10-01

    Here we report on the first assessment of droplet digital PCR (ddPCR) for detection and absolute quantification of two quarantine plant pathogenic bacteria that infect many species of the Rosaceae and Solanaceae families: Erwinia amylovora and Ralstonia solanacearum. An open-source R script was written for the ddPCR data analysis. Analysis of a set of samples with known health status aided the assessment and selection of different threshold settings (QuantaSoft analysis, definetherain pipeline and manual threshold), which led to optimal diagnostic specificity. The interpretation of the E. amylovora ddPCR was straightforward, and the analysis approach had little influence on the final results and the concentrations determined. The sensitivity and linear range were similar to those for real-time PCR (qPCR), for the analysis of both bacterial suspensions and plant material, making ddPCR a viable choice when both detection and quantification are desired. With the R. solanacearum ddPCR, the use of a high global threshold was necessary to exclude false-positive reactions that are sometimes observed in healthy plant material. ddPCR significantly improved the analytical sensitivity over that of qPCR, and improved the detection of low concentrations of R. solanacearum in potato tuber samples. Accurate and rapid absolute quantification of both of these bacteria in pure culture was achieved by direct ddPCR. Our data confirm the suitability of these ddPCR assays for routine detection and quantification of plant pathogens and for preparation of defined in-house reference materials with known target concentrations.

  3. Genetic organization of the catabolic plasmid pJP4 from Ralstonia eutropha JMP134 (pJP4) reveals mechanisms of adaptation to chloroaromatic pollutants and evolution of specialized chloroaromatic degradation pathways.

    PubMed

    Trefault, N; De la Iglesia, R; Molina, A M; Manzano, M; Ledger, T; Pérez-Pantoja, D; Sánchez, M A; Stuardo, M; González, B

    2004-07-01

    Ralstonia eutropha JMP134 (pJP4) is a useful model for the study of bacterial degradation of substituted aromatic pollutants. Several key degrading capabilities, encoded by tfd genes, are located in the 88 kb, self-transmissible, IncP-1 beta plasmid pJP4. The complete sequence of the 87,688 nucleotides of pJP4, encoding 83 open reading frames (ORFs), is reported. Most of the coding sequence corresponds to a well-conserved IncP-1 beta backbone and the previously reported tfd genes. In addition, we found hypothetical proteins putatively involved in the transport of aromatic compounds and short-chain fatty acid oxidation. ORFs related to mobile elements, including the Tn501-encoded mercury resistance determinants, an IS1071-based composite transposon and a cryptic class II transposon, are also present in pJP4. These mobile elements are inefficient in transposition and are located in two regions of pJP4 that are rich in remnants of lateral gene transfer events. pJP4 plasmid was able to capture chromosomal genes and form hybrid plasmids with the IncP-1 alpha plasmid RP4. These observations are integrated into a model for the evolution of pJP4, which reveals mechanisms of bacterial adaptation to degrade pollutants. PMID:15186344

  4. Nitrogen species

    NASA Technical Reports Server (NTRS)

    Harries, J. E.; Brasseur, G.; Coffey, M. T.; Fischer, H.; Gille, J.; Jones, R.; Louisnard, N.; Mccormick, M. P.; Noxon, J.; Owens, A. J.

    1985-01-01

    Total odd nitrogen, NO(y), may be defined as the sum of all active nitrogen species that interchange photochemically with one another on a time scale of the order of weeks or less. As noted, NO + NO2 reactions dominate the processes controlling the ozone balance in the contemporary stratosphere. The observational data from non-satellite platforms are reviewed. The growth in available satellite data in the past four years is considered. Some of the most important scientific issues are discussed, taking into account new results from atmospheric models (mainly 2-D). The model results are compared with the observational data.

  5. The Complete Mitochondrial Genome Sequence of Bactericera cockerelli and Comparison with Three Other Psylloidea Species.

    PubMed

    Wu, Fengnian; Cen, Yijing; Wallis, Christopher M; Trumble, John T; Prager, Sean; Yokomi, Ray; Zheng, Zheng; Deng, Xiaoling; Chen, Jianchi; Liang, Guangwen

    2016-01-01

    Potato psyllid (Bactericera cockerelli) is an important pest of potato, tomato and pepper. Not only could a toxin secreted by nymphs results in serious phytotoxemia in some host plants, but also over the past few years B. cockerelli was shown to transmit "Candidatus Liberibacter solanacearum", the putative bacterial pathogen of potato zebra chip (ZC) disease, to potato and tomato. ZC has caused devastating losses to potato production in the western U.S., Mexico, and elsewhere. New knowledge of the genetic diversity of the B. cockerelli is needed to develop improved strategies to manage pest populations. Mitochondrial genome (mitogenome) sequencing provides important knowledge about insect evolution and diversity in and among populations. This report provides the first complete B. cockerelli mitogenome sequence as determined by next generation sequencing technology (Illumina MiSeq). The circular B. cockerelli mitogenome had a size of 15,220 bp with 13 protein-coding gene (PCGs), 2 ribosomal RNA genes (rRNAs), 22 transfer RNA genes (tRNAs), and a non-coding region of 975 bp. The overall gene order of the B. cockerelli mitogenome is identical to three other published Psylloidea mitogenomes: one species from the Triozidae, Paratrioza sinica; and two species from the Psyllidae, Cacopsylla coccinea and Pachypsylla venusta. This suggests all of these species share a common ancestral mitogenome. However, sequence analyses revealed differences between and among the insect families, in particular a unique region that can be folded into three stem-loop secondary structures present only within the B. cockerelli mitogenome. A phylogenetic tree based on the 13 PCGs matched an existing taxonomy scheme that was based on morphological characteristics. The available complete mitogenome sequence makes it accessible to all genes for future population diversity evaluation of B. cockerelli. PMID:27227976

  6. Oxidation of benzene to phenol, catechol, and 1,2,3-trihydroxybenzene by toluene 4-monooxygenase of Pseudomonas mendocina KR1 and toluene 3-monooxygenase of Ralstonia pickettii PKO1.

    PubMed

    Tao, Ying; Fishman, Ayelet; Bentley, William E; Wood, Thomas K

    2004-07-01

    Aromatic hydroxylations are important bacterial metabolic processes but are difficult to perform using traditional chemical synthesis, so to use a biological catalyst to convert the priority pollutant benzene into industrially relevant intermediates, benzene oxidation was investigated. It was discovered that toluene 4-monooxygenase (T4MO) of Pseudomonas mendocina KR1, toluene 3-monooxygenase (T3MO) of Ralstonia pickettii PKO1, and toluene ortho-monooxygenase (TOM) of Burkholderia cepacia G4 convert benzene to phenol, catechol, and 1,2,3-trihydroxybenzene by successive hydroxylations. At a concentration of 165 microM and under the control of a constitutive lac promoter, Escherichia coli TG1/pBS(Kan)T4MO expressing T4MO formed phenol from benzene at 19 +/- 1.6 nmol/min/mg of protein, catechol from phenol at 13.6 +/- 0.3 nmol/min/mg of protein, and 1,2,3-trihydroxybenzene from catechol at 2.5 +/- 0.5nmol/min/mg of protein. The catechol and 1,2,3-trihydroxybenzene products were identified by both high-pressure liquid chromatography and mass spectrometry. When analogous plasmid constructs were used, E. coli TG1/pBS(Kan)T3MO expressing T3MO formed phenol, catechol, and 1,2,3-trihydroxybenzene at rates of 3 +/- 1, 3.1 +/- 0.3, and 0.26 +/- 0.09 nmol/min/mg of protein, respectively, and E. coli TG1/pBS(Kan)TOM expressing TOM formed 1,2,3-trihydroxybenzene at a rate of 1.7 +/- 0.3 nmol/min/mg of protein (phenol and catechol formation rates were 0.89 +/- 0.07 and 1.5 +/- 0.3 nmol/min/mg of protein, respectively). Hence, the rates of synthesis of catechol by both T3MO and T4MO and the 1,2,3-trihydroxybenzene formation rate by TOM were found to be comparable to the rates of oxidation of the natural substrate toluene for these enzymes (10.0 +/- 0.8, 4.0 +/- 0.6, and 2.4 +/- 0.3 nmol/min/mg of protein for T4MO, T3MO, and TOM, respectively, at a toluene concentration of 165 microM). PMID:15240250

  7. In defense of species.

    PubMed

    LaPorte, Joseph

    2007-03-01

    In this paper, I address the charge that the category species should be abandoned in biological work. The widespread appeal to species in scientific discourse provides a presumption in favor of the category's usefulness, but a defeasible presumption. Widely acknowledged troubles attend species: these troubles might render the concept unusable by showing that 'species' is equivocal or meaningless or in some similar way fatally flawed. Further, there might be better alternatives to species. I argue that the presumption in favor of species is not defeated on these scores. Troubles attending species, which arise on account of contextual variation attending the use of 'species', do not indicate that the concept is unusable. And alternatives to the use of 'species', which have been proposed in connection with rank-free systematics and in connection with conservation efforts, fail to provide a proper replacement for species.

  8. Endangered species: Deciding which species to save

    NASA Astrophysics Data System (ADS)

    Thibodeau, Francis R.

    1983-03-01

    Many species face extinction because preservation organizations do not have the resources to mount all of the interventions that are needed. Decision analysis provides techniques that can help managers of these organizations to make judgments about which species they will attempt to rescue. A formal analysis of the choices available to the US Fish and Wildlife Services' endangered species program with regard to Isotria medeoloides illustrates how the difficulties of making preservation decisions can be lessened. I. medeoloides is perhaps the rarest orchid in the United States. Little is known of the species' biology and less about effective management. Yet unless a preservation effort is mounted, the species will continue to be threatened by habitat destruction and botanical collecting. The analysis employs formal probabalistic techniques to weigh the utility of possible intervention strategies, that is, their likelihood of achieving different amounts of increase in the longevity of the species, and to balance these gains against their costs. If similar decision analyses are performed on other endangered species, the technique can be used to choose among them, as well as among strategies for individual species.

  9. 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

  10. Antibacterial activity of caffeine against plant pathogenic bacteria.

    PubMed

    Sledz, Wojciech; Los, Emilia; Paczek, Agnieszka; Rischka, Jacek; Motyka, Agata; Zoledowska, Sabina; Piosik, Jacek; Lojkowska, Ewa

    2015-01-01

    The objective of the present study was to evaluate the antibacterial properties of a plant secondary metabolite - caffeine. Caffeine is present in over 100 plant species. Antibacterial activity of caffeine was examined against the following plant-pathogenic bacteria: Ralstonia solanacearum (Rsol), Clavibacter michiganesis subsp. sepedonicus (Cms), Dickeya solani (Dsol), Pectobacterium atrosepticum (Pba), Pectobacterium carotovorum subsp. carotovorum (Pcc), Pseudomonas syringae pv. tomato (Pst), and Xanthomonas campestris subsp. campestris (Xcc). MIC and MBC values ranged from 5 to 20 mM and from 43 to 100 mM, respectively. Caffeine increased the bacterial generation time of all tested species and caused changes in cell morphology. The influence of caffeine on the synthesis of DNA, RNA and proteins was investigated in cultures of plant pathogenic bacteria with labelled precursors: [(3)H]thymidine, [(3)H]uridine or (14)C leucine, respectively. RNA biosynthesis was more affected than DNA or protein biosynthesis in bacterial cells treated with caffeine. Treatment of Pba with caffeine for 336 h did not induce resistance to this compound. Caffeine application reduced disease symptoms caused by Dsol on chicory leaves, potato slices, and whole potato tubers. The data presented indicate caffeine as a potential tool for the control of diseases caused by plant-pathogenic bacteria, especially under storage conditions. PMID:26307771

  11. Evaluation of somatic hybrids of potato with Solanum stenotomum after a long-term in vitro conservation.

    PubMed

    Fock, Isabelle; Collonnier, Cécile; Lavergne, Danielle; Vaniet, Sébastien; Ambroise, Annick; Luisetti, Jacques; Kodja, Hippolyte; Sihachakr, Darasinh

    2007-01-01

    Somatic hybrids of potato with a cultivated relative, Solanum stenotomum also called Solanum tuberosum Stenotomum group, were evaluated for their physiological and agronomical characteristics as well as the stability of the introgressed resistance to bacterial wilt, caused by Ralstonia solanacearum, after a long-term in vitro conservation for more than 5 years. Analysis of photosynthesis showed that the PEPC/Rubisco ratio remained lower than 0.5 for all vitroplants of potato and the somatic hybrids, except for the relative species. This indicates that the carbon metabolism is heterotrophic (ratio>1) for S. stenotomum, and autotrophic for potato and the somatic hybrids (ratio<1). In both in vitro and greenhouse conditions, potato and the somatic hybrids produced few bigger tubers, while many small tubers were obtained from the relative. The hybrid tubers were morphologically intermediate. The starch content of hybrid tubers was much lower than that of potato, but similar to that of the relative species. Interestingly, the level of bacterial resistance, introgressed from S. stenotomum into potato, was shown to be very stable and remained as high as that of the relative after a long-term period of in vitro conservation.

  12. Antibacterial activity of caffeine against plant pathogenic bacteria.

    PubMed

    Sledz, Wojciech; Los, Emilia; Paczek, Agnieszka; Rischka, Jacek; Motyka, Agata; Zoledowska, Sabina; Piosik, Jacek; Lojkowska, Ewa

    2015-01-01

    The objective of the present study was to evaluate the antibacterial properties of a plant secondary metabolite - caffeine. Caffeine is present in over 100 plant species. Antibacterial activity of caffeine was examined against the following plant-pathogenic bacteria: Ralstonia solanacearum (Rsol), Clavibacter michiganesis subsp. sepedonicus (Cms), Dickeya solani (Dsol), Pectobacterium atrosepticum (Pba), Pectobacterium carotovorum subsp. carotovorum (Pcc), Pseudomonas syringae pv. tomato (Pst), and Xanthomonas campestris subsp. campestris (Xcc). MIC and MBC values ranged from 5 to 20 mM and from 43 to 100 mM, respectively. Caffeine increased the bacterial generation time of all tested species and caused changes in cell morphology. The influence of caffeine on the synthesis of DNA, RNA and proteins was investigated in cultures of plant pathogenic bacteria with labelled precursors: [(3)H]thymidine, [(3)H]uridine or (14)C leucine, respectively. RNA biosynthesis was more affected than DNA or protein biosynthesis in bacterial cells treated with caffeine. Treatment of Pba with caffeine for 336 h did not induce resistance to this compound. Caffeine application reduced disease symptoms caused by Dsol on chicory leaves, potato slices, and whole potato tubers. The data presented indicate caffeine as a potential tool for the control of diseases caused by plant-pathogenic bacteria, especially under storage conditions.

  13. The Earth's Vanishing Species.

    ERIC Educational Resources Information Center

    USA Today, 1981

    1981-01-01

    Elaborates on the problem of expanding human activity to the world's plant and animal species. Concludes that preserving an individual species is largely a waste of time and effort and that the best way to protect the most species of plants and animals is to save their environments over large tracts of land. (DB)

  14. Aquatic invasive species

    USGS Publications Warehouse

    Thorsteinson, Lyman

    2005-01-01

    Invasive species are plants or animals that are present in an ecosystem beyond their native range. They may have few natural controls in their new environment and proliferate. They can threaten native species and interfere with human activities. The Western Fisheries Research Center (WFRC) has been conducting research to understand how non-native species invade and affect ecosystems, thus aiding management efforts.

  15. Crossing species boundaries.

    PubMed

    Robert, Jason Scott; Baylis, Françoise

    2003-01-01

    This paper critically examines the biology of species identity and the morality of crossing species boundaries in the context of emerging research that involves combining human and nonhuman animals at the genetic or cellular level. We begin with the notion of species identity, particularly focusing on the ostensible fixity of species boundaries, and we explore the general biological and philosophical problem of defining species. Against this backdrop, we survey and criticize earlier attempts to forbid crossing species boundaries in the creation of novel beings. We do not attempt to establish the immorality of crossing species boundaries, but we conclude with some thoughts about such crossings, alluding to the notion of moral confusion regarding social and ethical obligations to novel interspecies beings.

  16. Simultaneous Detection of Brown Rot- and Soft Rot-Causing Bacterial Pathogens from Potato Tubers Through Multiplex PCR.

    PubMed

    Ranjan, R K; Singh, Dinesh; Baranwal, V K

    2016-11-01

    Ralstonia solanacearum (Smith) Yabuuchi et al. and Erwinia carotovora subsp. carotovora (Jones) Bergey et al. (Pectobacterium carotovorum subsp. carotovorum) are the two major bacterial pathogens of potato causing brown rot (wilt) and soft rot diseases, respectively, in the field and during storage. Reliable and early detection of these pathogens are keys to avoid occurrence of these diseases in potato crops and reduce yield loss. In the present study, multiplex polymerase chain reaction (PCR) protocol was developed for simultaneous detection of R. solanacearum and E. carotovora subsp. carotovora from potato tubers. A set of oligos targeting the pectatelyase (pel) gene of E. carotovora subsp. carotovora and the universal primers based on 16S r RNA gene of R. solanacearum were used. The standardized multiplex PCR protocol could detect R. solanacearum and E. carotovora subsp. carotovora up to 0.01 and 1.0 ng of genomic DNA, respectively. The protocol was further validated on 96 stored potato tuber samples, collected from different potato-growing states of India, viz. Uttarakhand, Odisha, Meghalaya and Delhi. 53.1 % tuber samples were positive for R. solanacearum, and 15.1 % of samples were positive for E. carotovora subsp. carotovora, and both the pathogens were positive in 26.0 % samples when BIO-PCR was used. This method offers sensitive, specific, reliable and fast detection of two major bacterial pathogens from potato tubers simultaneously, particularly pathogen-free seed certification in large scale. PMID:27480266

  17. Simultaneous Detection of Brown Rot- and Soft Rot-Causing Bacterial Pathogens from Potato Tubers Through Multiplex PCR.

    PubMed

    Ranjan, R K; Singh, Dinesh; Baranwal, V K

    2016-11-01

    Ralstonia solanacearum (Smith) Yabuuchi et al. and Erwinia carotovora subsp. carotovora (Jones) Bergey et al. (Pectobacterium carotovorum subsp. carotovorum) are the two major bacterial pathogens of potato causing brown rot (wilt) and soft rot diseases, respectively, in the field and during storage. Reliable and early detection of these pathogens are keys to avoid occurrence of these diseases in potato crops and reduce yield loss. In the present study, multiplex polymerase chain reaction (PCR) protocol was developed for simultaneous detection of R. solanacearum and E. carotovora subsp. carotovora from potato tubers. A set of oligos targeting the pectatelyase (pel) gene of E. carotovora subsp. carotovora and the universal primers based on 16S r RNA gene of R. solanacearum were used. The standardized multiplex PCR protocol could detect R. solanacearum and E. carotovora subsp. carotovora up to 0.01 and 1.0 ng of genomic DNA, respectively. The protocol was further validated on 96 stored potato tuber samples, collected from different potato-growing states of India, viz. Uttarakhand, Odisha, Meghalaya and Delhi. 53.1 % tuber samples were positive for R. solanacearum, and 15.1 % of samples were positive for E. carotovora subsp. carotovora, and both the pathogens were positive in 26.0 % samples when BIO-PCR was used. This method offers sensitive, specific, reliable and fast detection of two major bacterial pathogens from potato tubers simultaneously, particularly pathogen-free seed certification in large scale.

  18. Darwin's species category realism.

    PubMed

    Stamos, D N

    1999-01-01

    Ever since Charles Darwin's On the Origin of Species was published, the received view has been that Darwin literally thought of species as not extra-mentally real. In 1969 Michael Ghiselin upset the received view by interpreting Darwin to mean that species taxa are indeed real but not the species category. In 1985 John Beatty took Ghiselin's thesis a step further by providing a strategy theory to explain why Darwin would say one thing (his repeated nominalistic definition of species) and do another (hold that species taxa are real). In the present paper I attempt to take this line of interpretation to a new level. Guided by the principle of charity, I provide and analyze a considerable amount of evidence from Darwin's mature writings (both private and published) to show that (contra Ghiselin and Beatty) Darwin did not simply accept the species delimitations of his fellow naturalists but actually employed, repeatedly and consistently, a species concept in a thoroughly modern sense, albeit with an implicit definition, a concept uniquely his own and fully in accord with his theory of evolution by natural selection. This implicit concept and definition is carefully reconstructed in the present paper. A new strategy theory is then provided to account for why Darwin would define species (both taxa and category) nominalistically on the one hand but delimit species realistically on the other.

  19. Dual-species biofilm formation by Escherichia coli O157:H7 and environmental bacteria isolated from fresh-cut processing facilities.

    PubMed

    Liu, Nancy T; Nou, Xiangwu; Lefcourt, Alan M; Shelton, Daniel R; Lo, Y Martin

    2014-02-01

    Biofilm formation is a mechanism adapted by many microorganisms that enhances the survival in stressful environments. In food processing facilities, foodborne bacterial pathogens, which many are poor biofilm formers, could potentially take advantage of this protective mechanism by interacting with other strong biofilm producers. The objective of this study was to determine the influence of bacteria native to fresh produce processing environments on the incorporation of Escherichia coli O157:H7 in biofilms. Bacteria strains representing 13 Gram-negative species isolated from two fresh produce processing facilities in a previous study were tested for forming dual-species biofilms with E. coli O157:H7. Strong biofilm producing strains of Burkholderia caryophylli and Ralstonia insidiosa exhibited 180% and 63% increase in biofilm biomass, and significant thickening of the biofilms (B. caryophylli not tested), when co-cultured with E. coli O157:H7. E. coli O157:H7 populations increased by approximately 1 log in dual-species biofilms formed with B. caryophylli or R. insidiosa. While only a subset of environmental isolates with strong biofilm formation abilities increased the presence of E. coli O157:H7 in biofilms, all tested E. coli O157:H7 exhibited higher incorporation in dual-species biofilms with R. insidiosa. These observations support the notion that E. coli O157:H7 and specific strong biofilm producing bacteria interact synergistically in biofilm formation, and suggest a route for increased survival potential of E. coli O157:H7 in fresh produce processing environments.

  20. The expansion of brown rot disease throughout Bolivia: possible role of climate change.

    PubMed

    Castillo, José Antonio; Plata, Giovanna

    2016-05-01

    Bacterial wilt is a devastating plant disease caused by the bacterial pathogen Ralstonia solanacearum species complex and affects different crops. Bacterial wilt infecting potato is also known as brown rot (BR) and is responsible for significant economic losses in potato production, especially in developing countries. In Bolivia, BR affects up to 75% of the potato crop in areas with high incidence and 100% of stored potatoes. The disease has disseminated since its introduction to the country in the mid-1980s mostly through contaminated seed tubers. To avoid this, local farmers multiply seed tubers in highlands because the strain infecting potatoes cannot survive near-freezing temperatures that are typical in the high mountains. Past disease surveys have shown an increase in seed tubers with latent infection in areas at altitudes lower than 3000 m a.s.l. Since global warming is increasing in the Andes Mountains, in this work, we explored the incidence of BR in areas at altitudes above 3000 m a.s.l. Results showed BR presence in the majority of these areas, suggesting a correlation between the increase in disease incidence and the increase in temperature and the number of irregular weather events resulting from climate change. However, it cannot be excluded that the increasing availability of latently infected seed tubers has boosted the spread of BR.

  1. Rutin-Mediated Priming of Plant Resistance to Three Bacterial Pathogens Initiating the Early SA Signal Pathway

    PubMed Central

    Li, Yang; Wang, Yingzi; Li, Ming; Wang, Yong; Ding, Xinhua; Chu, Zhaohui

    2016-01-01

    Flavonoids are ubiquitous in the plant kingdom and have many diverse functions, including UV protection, auxin transport inhibition, allelopathy, flower coloring and insect resistance. Here we show that rutin, a proud member of the flavonoid family, could be functional as an activator to improve plant disease resistances. Three plant species pretreated with 2 mM rutin were found to enhance resistance to Xanthomonas oryzae pv. oryzae, Ralstonia solanacearum, and Pseudomonas syringae pv. tomato strain DC3000 in rice, tobacco and Arabidopsis thaliana respectively. While they were normally propagated on the cultural medium supplemented with 2 mM rutin for those pathogenic bacteria. The enhanced resistance was associated with primed expression of several pathogenesis-related genes. We also demonstrated that the rutin-mediated priming resistance was attenuated in npr1, eds1, eds5, pad4-1, ndr1 mutants, and NahG transgenic Arabidopsis plant, while not in either snc1-11, ein2-5 or jar1 mutants. We concluded that the rutin-priming defense signal was modulated by the salicylic acid (SA)-dependent pathway from an early stage upstream of NDR1 and EDS1. PMID:26751786

  2. Application of Universal Stress Proteins in Probing the Dynamics of Potent Degraders in Complex Terephthalate Metagenome

    PubMed Central

    Mbah, Andreas N.; Isokpehi, Raphael D.

    2013-01-01

    The culture-independent strategies to study microbial diversity and function have led to a revolution in environmental genomics, enabling fundamental questions about the distribution of microbes and their influence on bioremediation to be addressed. In this research we used the expression of universal stress proteins as a probe to determine the changes in degrading microbial population from a highly toxic terephthalate wastewater to a less toxic activated sludge bioreactor. The impact of relative toxicities was significantly elaborated at the levels of genus and species. The results indicated that 23 similar prokaryotic phyla were represented in both metagenomes irrespective of their relative abundance. Furthermore, the following bacteria taxa Micromonosporaceae, Streptomyces, Cyanothece sp. PCC 7822, Alicyclobacillus acidocaldarius, Bacillus halodurans, Leuconostoc mesenteroides, Lactococcus garvieae, Brucellaceae, Ralstonia solanacearum, Verminephrobacter eiseniae, Azoarcus, Acidithiobacillus ferrooxidans, Francisella tularensis, Methanothermus fervidus, and Methanocorpusculum labreanum were represented only in the activated sludge bioreactor. These highly dynamic microbes could serve as taxonomic biomarkers for toxic thresholds related to terephthalate and its derivatives. This paper, highlights the application of universal stress proteins in metagenomics analysis. Dynamics of microbial consortium of this nature can have future in biotechnological applications in bioremediation of toxic chemicals and radionuclides. PMID:24151583

  3. Rutin-Mediated Priming of Plant Resistance to Three Bacterial Pathogens Initiating the Early SA Signal Pathway.

    PubMed

    Yang, Wei; Xu, Xiaonan; Li, Yang; Wang, Yingzi; Li, Ming; Wang, Yong; Ding, Xinhua; Chu, Zhaohui

    2016-01-01

    Flavonoids are ubiquitous in the plant kingdom and have many diverse functions, including UV protection, auxin transport inhibition, allelopathy, flower coloring and insect resistance. Here we show that rutin, a proud member of the flavonoid family, could be functional as an activator to improve plant disease resistances. Three plant species pretreated with 2 mM rutin were found to enhance resistance to Xanthomonas oryzae pv. oryzae, Ralstonia solanacearum, and Pseudomonas syringae pv. tomato strain DC3000 in rice, tobacco and Arabidopsis thaliana respectively. While they were normally propagated on the cultural medium supplemented with 2 mM rutin for those pathogenic bacteria. The enhanced resistance was associated with primed expression of several pathogenesis-related genes. We also demonstrated that the rutin-mediated priming resistance was attenuated in npr1, eds1, eds5, pad4-1, ndr1 mutants, and NahG transgenic Arabidopsis plant, while not in either snc1-11, ein2-5 or jar1 mutants. We concluded that the rutin-priming defense signal was modulated by the salicylic acid (SA)-dependent pathway from an early stage upstream of NDR1 and EDS1. PMID:26751786

  4. The expansion of brown rot disease throughout Bolivia: possible role of climate change.

    PubMed

    Castillo, José Antonio; Plata, Giovanna

    2016-05-01

    Bacterial wilt is a devastating plant disease caused by the bacterial pathogen Ralstonia solanacearum species complex and affects different crops. Bacterial wilt infecting potato is also known as brown rot (BR) and is responsible for significant economic losses in potato production, especially in developing countries. In Bolivia, BR affects up to 75% of the potato crop in areas with high incidence and 100% of stored potatoes. The disease has disseminated since its introduction to the country in the mid-1980s mostly through contaminated seed tubers. To avoid this, local farmers multiply seed tubers in highlands because the strain infecting potatoes cannot survive near-freezing temperatures that are typical in the high mountains. Past disease surveys have shown an increase in seed tubers with latent infection in areas at altitudes lower than 3000 m a.s.l. Since global warming is increasing in the Andes Mountains, in this work, we explored the incidence of BR in areas at altitudes above 3000 m a.s.l. Results showed BR presence in the majority of these areas, suggesting a correlation between the increase in disease incidence and the increase in temperature and the number of irregular weather events resulting from climate change. However, it cannot be excluded that the increasing availability of latently infected seed tubers has boosted the spread of BR. PMID:26991236

  5. Overexpression of Cotton GhMPK11 Decreases Disease Resistance through the Gibberellin Signaling Pathway in Transgenic Nicotiana benthamiana.

    PubMed

    Wang, Fang; Wang, Chen; Yan, Yan; Jia, Haihong; Guo, Xingqi

    2016-01-01

    Many changes in development, growth, hormone activity and environmental stimuli responses are mediated by mitogen-activated protein kinase (MAPK) cascades. However, in plants, studies on MAPKs have mainly focused on MPK3, MPK4 and MPK6. Here, a novel group B MAPK gene, GhMPK11, was isolated from cotton (Gossypium hirsutum L.) and characterized. Both promoter and expression pattern analyses revealed that GhMPK11 is involved in defense responses and signaling pathways. GhMPK11 overexpression in Nicotiana benthamiana plants could increase gibberellin 3 (GA3) content through the regulation of GA-related genes. Interestingly, either GhMPK11 overexpression or exogenous GA3 treatment in N. benthamiana plants could enhance the susceptibility of these plants to the infectious pathogens Ralstonia solanacearum and Rhizoctonia solani. Moreover, reactive oxygen species (ROS) accumulation was increased after pathogen infiltration due to the increased expression of ROS-related gene respiratory burst oxidative homologs (RbohB) and the decreased expression or activity of ROS detoxification enzymes regulated by GA3, such as superoxide dismutases (SODs), peroxidases (PODs), catalase (CAT) and glutathione S-transferase (GST). Taken together, these results suggest that GhMPK11 overexpression could enhance the susceptibility of tobacco to pathogen infection through the GA3 signaling pathway via down-regulation of ROS detoxification enzymes. PMID:27242882

  6. A novel elicitor protein from Phytophthora parasitica induces plant basal immunity and systemic acquired resistance.

    PubMed

    Chang, Yi-Hsuan; Yan, Hao-Zhi; Liou, Ruey-Fen

    2015-02-01

    The interaction between Phytophthora pathogens and host plants involves the exchange of complex molecular signals from both sides. Recent studies of Phytophthora have led to the identification of various apoplastic elicitors known to trigger plant immunity. Here, we provide evidence that the protein encoded by OPEL of Phytophthora parasitica is a novel elicitor. Homologues of OPEL were identified only in oomycetes, but not in fungi and other organisms. Quantitative reverse transcription-polymerase chain reaction (RT-PCR) revealed that OPEL is expressed throughout the development of P. parasitica and is especially highly induced after plant infection. Infiltration of OPEL recombinant protein from Escherichia coli into leaves of Nicotiana tabacum (cv. Samsun NN) resulted in cell death, callose deposition, the production of reactive oxygen species and induced expression of pathogen-associated molecular pattern (PAMP)-triggered immunity markers and salicylic acid-responsive defence genes. Moreover, the infiltration conferred systemic resistance against a broad spectrum of pathogens, including Tobacco mosaic virus, the bacteria wilt pathogen Ralstonia solanacearum and P. parasitica. In addition to the signal peptide, OPEL contains three conserved domains: a thaumatin-like domain, a glycine-rich protein domain and a glycosyl hydrolase (GH) domain. Intriguingly, mutation of a putative laminarinase active site motif in the predicted GH domain abolished its elicitor activity, which suggests enzymatic activity of OPEL in triggering the defence response.

  7. Rutin-Mediated Priming of Plant Resistance to Three Bacterial Pathogens Initiating the Early SA Signal Pathway.

    PubMed

    Yang, Wei; Xu, Xiaonan; Li, Yang; Wang, Yingzi; Li, Ming; Wang, Yong; Ding, Xinhua; Chu, Zhaohui

    2016-01-01

    Flavonoids are ubiquitous in the plant kingdom and have many diverse functions, including UV protection, auxin transport inhibition, allelopathy, flower coloring and insect resistance. Here we show that rutin, a proud member of the flavonoid family, could be functional as an activator to improve plant disease resistances. Three plant species pretreated with 2 mM rutin were found to enhance resistance to Xanthomonas oryzae pv. oryzae, Ralstonia solanacearum, and Pseudomonas syringae pv. tomato strain DC3000 in rice, tobacco and Arabidopsis thaliana respectively. While they were normally propagated on the cultural medium supplemented with 2 mM rutin for those pathogenic bacteria. The enhanced resistance was associated with primed expression of several pathogenesis-related genes. We also demonstrated that the rutin-mediated priming resistance was attenuated in npr1, eds1, eds5, pad4-1, ndr1 mutants, and NahG transgenic Arabidopsis plant, while not in either snc1-11, ein2-5 or jar1 mutants. We concluded that the rutin-priming defense signal was modulated by the salicylic acid (SA)-dependent pathway from an early stage upstream of NDR1 and EDS1.

  8. Overexpression of Cotton GhMPK11 Decreases Disease Resistance through the Gibberellin Signaling Pathway in Transgenic Nicotiana benthamiana

    PubMed Central

    Wang, Fang; Wang, Chen; Yan, Yan; Jia, Haihong; Guo, Xingqi

    2016-01-01

    Many changes in development, growth, hormone activity and environmental stimuli responses are mediated by mitogen-activated protein kinase (MAPK) cascades. However, in plants, studies on MAPKs have mainly focused on MPK3, MPK4 and MPK6. Here, a novel group B MAPK gene, GhMPK11, was isolated from cotton (Gossypium hirsutum L.) and characterized. Both promoter and expression pattern analyses revealed that GhMPK11 is involved in defense responses and signaling pathways. GhMPK11 overexpression in Nicotiana benthamiana plants could increase gibberellin 3 (GA3) content through the regulation of GA-related genes. Interestingly, either GhMPK11 overexpression or exogenous GA3 treatment in N. benthamiana plants could enhance the susceptibility of these plants to the infectious pathogens Ralstonia solanacearum and Rhizoctonia solani. Moreover, reactive oxygen species (ROS) accumulation was increased after pathogen infiltration due to the increased expression of ROS-related gene respiratory burst oxidative homologs (RbohB) and the decreased expression or activity of ROS detoxification enzymes regulated by GA3, such as superoxide dismutases (SODs), peroxidases (PODs), catalase (CAT) and glutathione S-transferase (GST). Taken together, these results suggest that GhMPK11 overexpression could enhance the susceptibility of tobacco to pathogen infection through the GA3 signaling pathway via down-regulation of ROS detoxification enzymes. PMID:27242882

  9. Cotton GhMKK1 Induces the Tolerance of Salt and Drought Stress, and Mediates Defence Responses to Pathogen Infection in Transgenic Nicotiana benthamiana

    PubMed Central

    Lu, Wenjing; Chu, Xiaoqian; Li, Yuzhen; Wang, Chen; Guo, Xingqi

    2013-01-01

    Mitogen-activated protein kinase kinases (MAPKK) mediate a variety of stress responses in plants. So far little is known on the functional role of MAPKKs in cotton. In the present study, Gossypium hirsutum MKK1 (GhMKK1) function was investigated. GhMKK1 protein may activate its specific targets in both the nucleus and cytoplasm. Treatments with salt, drought, and H2O2 induced the expression of GhMKK1 and increased the activity of GhMKK1, while overexpression of GhMKK1 in Nicotiana benthamiana enhanced its tolerance to salt and drought stresses as determined by many physiological data. Additionally, GhMKK1 activity was found to up-regulate pathogen-associated biotic stress, and overexpression of GhMKK1 increased the susceptibility of the transgenic plants to the pathogen Ralstonia solanacearum by reducing the expression of PR genes. Moreover, GhMKK1-overexpressing plants also exhibited an enhanced reactive oxygen species scavenging capability and markedly elevated activities of several antioxidant enzymes. These results indicate that GhMKK1 is involved in plants defence responses and provide new data to further analyze the function of plant MAPK pathways. PMID:23844212

  10. Comparative Genome Analyses of Serratia marcescens FS14 Reveals Its High Antagonistic Potential

    PubMed Central

    Li, Pengpeng; Kwok, Amy H. Y.; Jiang, Jingwei; Ran, Tingting; Xu, Dongqing; Wang, Weiwu; Leung, Frederick C.

    2015-01-01

    S. marcescens FS14 was isolated from an Atractylodes macrocephala Koidz plant that was infected by Fusarium oxysporum and showed symptoms of root rot. With the completion of the genome sequence of FS14, the first comprehensive comparative-genomic analysis of the Serratia genus was performed. Pan-genome and COG analyses showed that the majority of the conserved core genes are involved in basic cellular functions, while genomic factors such as prophages contribute considerably to genome diversity. Additionally, a Type I restriction-modification system, a Type III secretion system and tellurium resistance genes are found in only some Serratia species. Comparative analysis further identified that S. marcescens FS14 possesses multiple mechanisms for antagonism against other microorganisms, including the production of prodigiosin, bacteriocins, and multi-antibiotic resistant determinants as well as chitinases. The presence of two evolutionarily distinct Type VI secretion systems (T6SSs) in FS14 may provide further competitive advantages for FS14 against other microbes. To our knowledge, this is the first report of comparative analysis on T6SSs in the genus, which identifies four types of T6SSs in Serratia spp.. Competition bioassays of FS14 against the vital plant pathogenic bacterium Ralstonia solanacearum and fungi Fusarium oxysporum and Sclerotinia sclerotiorum were performed to support our genomic analyses, in which FS14 demonstrated high antagonistic activities against both bacterial and fungal phytopathogens. PMID:25856195

  11. The Brassicaceae-Specific EWR1 Gene Provides Resistance to Vascular Wilt Pathogens

    PubMed Central

    Yadeta, Koste A.; Valkenburg, Dirk-Jan; Hanemian, Mathieu; Marco, Yves; Thomma, Bart P. H. J.

    2014-01-01

    Soil-borne vascular wilt diseases caused by Verticillium spp. are among the most destructive diseases worldwide in a wide range of plant species. The most effective means of controlling Verticillium wilt diseases is the use of genetic resistance. We have previously reported the identification of four activation-tagged Arabidopsis mutants which showed enhanced resistance to Verticillium wilt. Among these, one mutant also showed enhanced resistance to Ralstonia solanacearum, a bacterial vascular wilt pathogen. Cloning of the activation tag revealed an insertion upstream of gene At3g13437, which we designated as EWR1 (for Enhancer of vascular Wilt Resistance 1) that encodes a putatively secreted protein of unknown function. The search for homologs of Arabidopsis EWR1 (AtEWR1) in public databases only identified homologs within the Brassicaceae family. We subsequently cloned the EWR1 homolog from Brassica oleracea (BoEWR1) and show that over-expression in Arabidopsis results in V. dahliae resistance. Moreover, over-expression of AtEWR1 and BoEWR1 in N. benthamiana, a member of the Solanaceae family, results in V. dahliae resistance, suggesting that EWR1 homologs can be used to engineer Verticillium wilt resistance in non-Brassicaceae crops as well. PMID:24505441

  12. Resistance to bacterial wilt in somatic hybrids between Solanum tuberosum and Solanum phureja.

    PubMed

    Fock, I; Collonnier, C; Purwito, A; Luisetti, J; Souvannavong, V; Vedel, F; Servaes, A; Ambroise, A; Kodja, H; Ducreux, G; Sihachakr, D

    2000-12-01

    Somatic hybrid plants were produced after protoplast electrofusion between a dihaploid potato, cv. BF15, and a wild tuber-bearing relative, Solanum phureja, with a view to transferring bacterial wilt resistance into potato lines. A total of ten putative hybrids were selected. DNA analysis using flow cytometry revealed that six were tetraploids, two mixoploids, one amphiploid and one octoploid. In the greenhouse, the putative hybrids exhibited strong vigor and were morphologically intermediate, including leaf form, flowers and tuber characteristics. The hybrid nature of the ten selected plants was confirmed by examining isoenzyme patterns for esterases and peroxidases, and analysis of RAPD and SSR markers. Analysis of chloroplast genome revealed that eight hybrids possessed chloroplast (ct) DNA of the wild species, S. phureja, and only two contained Solanum tuberosum ct type. Six hybrid clones, including five tetraploids and one amphiploid, were evaluated for resistance to bacterial wilt by using race 1 and race 3 strains of Ralstonia solanacearum, originating from Reunion Island. Inoculations were performed by an in vitro root dipping method. The cultivated potato was susceptible to both bacterial strains tested. All somatic hybrids except two were tolerant to race 1 strain, and susceptible to race 3 strain. Interestingly, the amphiploid hybrid clone showed a good tolerance to both strains.

  13. Comparative genome analyses of Serratia marcescens FS14 reveals its high antagonistic potential.

    PubMed

    Li, Pengpeng; Kwok, Amy H Y; Jiang, Jingwei; Ran, Tingting; Xu, Dongqing; Wang, Weiwu; Leung, Frederick C

    2015-01-01

    S. marcescens FS14 was isolated from an Atractylodes macrocephala Koidz plant that was infected by Fusarium oxysporum and showed symptoms of root rot. With the completion of the genome sequence of FS14, the first comprehensive comparative-genomic analysis of the Serratia genus was performed. Pan-genome and COG analyses showed that the majority of the conserved core genes are involved in basic cellular functions, while genomic factors such as prophages contribute considerably to genome diversity. Additionally, a Type I restriction-modification system, a Type III secretion system and tellurium resistance genes are found in only some Serratia species. Comparative analysis further identified that S. marcescens FS14 possesses multiple mechanisms for antagonism against other microorganisms, including the production of prodigiosin, bacteriocins, and multi-antibiotic resistant determinants as well as chitinases. The presence of two evolutionarily distinct Type VI secretion systems (T6SSs) in FS14 may provide further competitive advantages for FS14 against other microbes. To our knowledge, this is the first report of comparative analysis on T6SSs in the genus, which identifies four types of T6SSs in Serratia spp.. Competition bioassays of FS14 against the vital plant pathogenic bacterium Ralstonia solanacearum and fungi Fusarium oxysporum and Sclerotinia sclerotiorum were performed to support our genomic analyses, in which FS14 demonstrated high antagonistic activities against both bacterial and fungal phytopathogens.

  14. Temporal and multiple quantitative trait loci analyses of resistance to bacterial wilt in tomato permit the resolution of linked loci.

    PubMed

    Mangin, B; Thoquet, P; Olivier, J; Grimsley, N H

    1999-03-01

    Ralstonia solanacearum is a soil-borne bacterium that causes the serious disease known as bacterial wilt in many plant species. In tomato, several QTL controlling resistance have been found, but in different studies, markers spanning a large region of chromosome 6 showed strong association with the resistance. By using two different approaches to analyze the data from a field test F3 population, we show that at least two separate loci approximately 30 cM apart on this chromosome are most likely involved in the resistance. First, a temporal analysis of the progression of symptoms reveals a distal locus early in the development of the disease. As the disease progresses, the maximum LOD peak observed shifts toward the proximal end of the chromosome, obscuring the distal locus. Second, although classical interval mapping could only detect the presence of one locus, a statistical "two-QTL model" test, specifically adapted for the resolution of linked QTL, strongly supported the hypothesis for the presence of two loci. These results are discussed in the context of current molecular knowledge about disease resistance genes on chromosome 6 and observations made by tomato breeders during the production of bacterial wilt-resistant varieties. PMID:10049932

  15. Temporal and multiple quantitative trait loci analyses of resistance to bacterial wilt in tomato permit the resolution of linked loci.

    PubMed Central

    Mangin, B; Thoquet, P; Olivier, J; Grimsley, N H

    1999-01-01

    Ralstonia solanacearum is a soil-borne bacterium that causes the serious disease known as bacterial wilt in many plant species. In tomato, several QTL controlling resistance have been found, but in different studies, markers spanning a large region of chromosome 6 showed strong association with the resistance. By using two different approaches to analyze the data from a field test F3 population, we show that at least two separate loci approximately 30 cM apart on this chromosome are most likely involved in the resistance. First, a temporal analysis of the progression of symptoms reveals a distal locus early in the development of the disease. As the disease progresses, the maximum LOD peak observed shifts toward the proximal end of the chromosome, obscuring the distal locus. Second, although classical interval mapping could only detect the presence of one locus, a statistical "two-QTL model" test, specifically adapted for the resolution of linked QTL, strongly supported the hypothesis for the presence of two loci. These results are discussed in the context of current molecular knowledge about disease resistance genes on chromosome 6 and observations made by tomato breeders during the production of bacterial wilt-resistant varieties. PMID:10049932

  16. The Brassicaceae-specific EWR1 gene provides resistance to vascular wilt pathogens.

    PubMed

    Yadeta, Koste A; Valkenburg, Dirk-Jan; Hanemian, Mathieu; Marco, Yves; Thomma, Bart P H J

    2014-01-01

    Soil-borne vascular wilt diseases caused by Verticillium spp. are among the most destructive diseases worldwide in a wide range of plant species. The most effective means of controlling Verticillium wilt diseases is the use of genetic resistance. We have previously reported the identification of four activation-tagged Arabidopsis mutants which showed enhanced resistance to Verticillium wilt. Among these, one mutant also showed enhanced resistance to Ralstonia solanacearum, a bacterial vascular wilt pathogen. Cloning of the activation tag revealed an insertion upstream of gene At3g13437, which we designated as EWR1 (for Enhancer of vascular Wilt Resistance 1) that encodes a putatively secreted protein of unknown function. The search for homologs of Arabidopsis EWR1 (AtEWR1) in public databases only identified homologs within the Brassicaceae family. We subsequently cloned the EWR1 homolog from Brassica oleracea (BoEWR1) and show that over-expression in Arabidopsis results in V. dahliae resistance. Moreover, over-expression of AtEWR1 and BoEWR1 in N. benthamiana, a member of the Solanaceae family, results in V. dahliae resistance, suggesting that EWR1 homologs can be used to engineer Verticillium wilt resistance in non-Brassicaceae crops as well. PMID:24505441

  17. Barbaloin in aloe species.

    PubMed

    Groom, Q J; Reynolds, T

    1987-08-01

    Barbaloin levels in the exudates from the leaves of 68 species of ALOE in the Kew collection have been determined by light absorption at 375 nm following separation by HPLC. The exudates from most species contained between 10-20% although a few concentrations of around 30% were found. The level in the leaf was usually around 1% of thedry weight in plants grown in glasshouses at Kew although some species were found to contain up to 5%. The highest concentrations of barbaloin were found in exudates from young mature leaves just below the apex and the level decreased in older leaves towards the base of the plant. Species in the natural groupings of the genus, Section ANGUIALOE and Group 4, all had appreciable concentrations of barbaloin in the leaf exudates. Species containing barbaloinwere distributed throughout the large heterogeneous Sections EUALOE and PACHYDENDRON with no apparent taxonomic significance.

  18. The species in primatology.

    PubMed

    Groves, Colin

    2014-01-01

    Biologists of the late eighteenth and early nineteenth centuries all bandied about the term "species," but very rarely actually said what they meant by it. Often, however, one can get inside their thinking by piecing together some of their remarks. One of the most nearly explicit-appropriately, for the man who wrote a book called The Origin of Species - was Charles Darwin: "Practically, when a naturalist can unite two forms together by others having intermediate characters, he treats the one as a variety of the other… He later translated this into evolutionary terms: "Hereafter, we shall be compelled to acknowledge that the only distinction between species and well-marked varieties is, that the latter are known, or believed, to be connected at the present day by intermediate gradations, whereas species were formerly thus connected"(1:484-5.) PMID:24591131

  19. How reticulated are species?

    PubMed

    Mallet, James; Besansky, Nora; Hahn, Matthew W

    2016-02-01

    Many groups of closely related species have reticulate phylogenies. Recent genomic analyses are showing this in many insects and vertebrates, as well as in microbes and plants. In microbes, lateral gene transfer is the dominant process that spoils strictly tree-like phylogenies, but in multicellular eukaryotes hybridization and introgression among related species is probably more important. Because many species, including the ancestors of ancient major lineages, seem to evolve rapidly in adaptive radiations, some sexual compatibility may exist among them. Introgression and reticulation can thereby affect all parts of the tree of life, not just the recent species at the tips. Our understanding of adaptive evolution, speciation, phylogenetics, and comparative biology must adapt to these mostly recent findings. Introgression has important practical implications as well, not least for the management of genetically modified organisms in pest and disease control.

  20. The species in primatology.

    PubMed

    Groves, Colin

    2014-01-01

    Biologists of the late eighteenth and early nineteenth centuries all bandied about the term "species," but very rarely actually said what they meant by it. Often, however, one can get inside their thinking by piecing together some of their remarks. One of the most nearly explicit-appropriately, for the man who wrote a book called The Origin of Species - was Charles Darwin: "Practically, when a naturalist can unite two forms together by others having intermediate characters, he treats the one as a variety of the other… He later translated this into evolutionary terms: "Hereafter, we shall be compelled to acknowledge that the only distinction between species and well-marked varieties is, that the latter are known, or believed, to be connected at the present day by intermediate gradations, whereas species were formerly thus connected"(1:484-5.)

  1. Beyond Single Species Interpretation.

    ERIC Educational Resources Information Center

    Richie, Deborah

    1995-01-01

    Species diversity, learning about wildlife in its natural habitats and conservation goals are integral to Watchable Wildlife programs. Examines the role of wildlife observation in spreading the message of biodiversity importance. Twenty-three references cited. (LZ)

  2. How reticulated are species?

    PubMed

    Mallet, James; Besansky, Nora; Hahn, Matthew W

    2016-02-01

    Many groups of closely related species have reticulate phylogenies. Recent genomic analyses are showing this in many insects and vertebrates, as well as in microbes and plants. In microbes, lateral gene transfer is the dominant process that spoils strictly tree-like phylogenies, but in multicellular eukaryotes hybridization and introgression among related species is probably more important. Because many species, including the ancestors of ancient major lineages, seem to evolve rapidly in adaptive radiations, some sexual compatibility may exist among them. Introgression and reticulation can thereby affect all parts of the tree of life, not just the recent species at the tips. Our understanding of adaptive evolution, speciation, phylogenetics, and comparative biology must adapt to these mostly recent findings. Introgression has important practical implications as well, not least for the management of genetically modified organisms in pest and disease control. PMID:26709836

  3. How reticulated are species?

    PubMed Central

    Besansky, Nora; Hahn, Matthew W.

    2015-01-01

    Many groups of closely related species have reticulate phylogenies. Recent genomic analyses are showing this in many insects and vertebrates, as well as in microbes and plants. In microbes, lateral gene transfer is the dominant process that spoils strictly tree‐like phylogenies, but in multicellular eukaryotes hybridization and introgression among related species is probably more important. Because many species, including the ancestors of ancient major lineages, seem to evolve rapidly in adaptive radiations, some sexual compatibility may exist among them. Introgression and reticulation can thereby affect all parts of the tree of life, not just the recent species at the tips. Our understanding of adaptive evolution, speciation, phylogenetics, and comparative biology must adapt to these mostly recent findings. Introgression has important practical implications as well, not least for the management of genetically modified organisms in pest and disease control. PMID:26709836

  4. USGS invasive species solutions

    USGS Publications Warehouse

    Simpson, Annie

    2011-01-01

    Land managers must meet the invasive species challenge every day, starting with identification of problem species, then the collection of best practices for their control, and finally the implementation of a plan to remove the problem. At each step of the process, the availability of reliable information is essential to success. The U.S. Geological Survey (USGS) has developed a suite of resources for early detection and rapid response, along with data management and sharing.

  5. Species integrity in trees.

    PubMed

    Ortiz-Barrientos, Daniel; Baack, Eric J

    2014-09-01

    From California sequoia, to Australian eucalyptus, to the outstanding diversity of Amazonian forests, trees are fundamental to many processes in ecology and evolution. Trees define the communities that they inhabit, are host to a multiplicity of other organisms and can determine the ecological dynamics of other plants and animals. Trees are also at the heart of major patterns of biodiversity such as the latitudinal gradient of species diversity and thus are important systems for studying the origin of new plant species. Although the role of trees in community assembly and ecological succession is partially understood, the origin of tree diversity remains largely opaque. For instance, the relative importance of differing habitats and phenologies as barriers to hybridization between closely related species is still largely uncharacterized in trees. Consequently, we know very little about the origin of trees species and their integrity. Similarly, studies on the interplay between speciation and tree community assembly are in their infancy and so are studies on how processes like forest maturation modifies the context in which reproductive isolation evolves. In this issue of Molecular Ecology, Lindtke et al. (2014) and Lagache et al. (2014) overcome some traditional difficulties in studying mating systems and sexual isolation in the iconic oaks and poplars, providing novel insights about the integrity of tree species and on how ecology leads to variation in selection on reproductive isolation over time and space. PMID:25155715

  6. Theoretical ecology without species

    NASA Astrophysics Data System (ADS)

    Tikhonov, Mikhail

    The sequencing-driven revolution in microbial ecology demonstrated that discrete ``species'' are an inadequate description of the vast majority of life on our planet. Developing a novel theoretical language that, unlike classical ecology, would not require postulating the existence of species, is a challenge of tremendous medical and environmental significance, and an exciting direction for theoretical physics. Here, it is proposed that community dynamics can be described in a naturally hierarchical way in terms of population fluctuation eigenmodes. The approach is applied to a simple model of division of labor in a multi-species community. In one regime, effective species with a core and accessory genome are shown to naturally appear as emergent concepts. However, the same model allows a transition into a regime where the species formalism becomes inadequate, but the eigenmode description remains well-defined. Treating a community as a black box that expresses enzymes in response to resources reveals mathematically exact parallels between a community and a single coherent organism with its own fitness function. This coherence is a generic consequence of division of labor, requires no cooperative interactions, and can be expected to be widespread in microbial ecosystems. Harvard Center of Mathematical Sciences and Applications;John A. Paulson School of Engineering and Applied Sciences.

  7. Bounding species distribution models

    USGS Publications Warehouse

    Stohlgren, T.J.; Jarnevich, C.S.; Esaias, W.E.; Morisette, J.T.

    2011-01-01

    Species distribution models are increasing in popularity for mapping suitable habitat for species of management concern. Many investigators now recognize that extrapolations of these models with geographic information systems (GIS) might be sensitive to the environmental bounds of the data used in their development, yet there is no recommended best practice for "clamping" model extrapolations. We relied on two commonly used modeling approaches: classification and regression tree (CART) and maximum entropy (Maxent) models, and we tested a simple alteration of the model extrapolations, bounding extrapolations to the maximum and minimum values of primary environmental predictors, to provide a more realistic map of suitable habitat of hybridized Africanized honey bees in the southwestern United States. Findings suggest that multiple models of bounding, and the most conservative bounding of species distribution models, like those presented here, should probably replace the unbounded or loosely bounded techniques currently used. ?? 2011 Current Zoology.

  8. Bounding Species Distribution Models

    NASA Technical Reports Server (NTRS)

    Stohlgren, Thomas J.; Jarnevich, Cahterine S.; Morisette, Jeffrey T.; Esaias, Wayne E.

    2011-01-01

    Species distribution models are increasing in popularity for mapping suitable habitat for species of management concern. Many investigators now recognize that extrapolations of these models with geographic information systems (GIS) might be sensitive to the environmental bounds of the data used in their development, yet there is no recommended best practice for "clamping" model extrapolations. We relied on two commonly used modeling approaches: classification and regression tree (CART) and maximum entropy (Maxent) models, and we tested a simple alteration of the model extrapolations, bounding extrapolations to the maximum and minimum values of primary environmental predictors, to provide a more realistic map of suitable habitat of hybridized Africanized honey bees in the southwestern United States. Findings suggest that multiple models of bounding, and the most conservative bounding of species distribution models, like those presented here, should probably replace the unbounded or loosely bounded techniques currently used [Current Zoology 57 (5): 642-647, 2011].

  9. Genomic definition of species

    SciTech Connect

    Crkvenjakov, R.; Drmanac, R.

    1991-07-01

    The subject of this paper is the definition of species based on the assumption that genome is the fundamental level for the origin and maintenance of biological diversity. For this view to be logically consistent it is necessary to assume the existence and operation of the new law which we call genome law. For this reason the genome law is included in the explanation of species phenomenon presented here even if its precise formulation and elaboration are left for the future. The intellectual underpinnings of this definition can be traced to Goldschmidt. We wish to explore some philosophical aspects of the definition of species in terms of the genome. The point of proposing the definition on these grounds is that any real advance in evolutionary theory has to be correct in both its philosophy and its science.

  10. Bioterrorism and invasive species.

    PubMed

    Chomel, B B; Sun, B

    2010-08-01

    The risk of dispersing invasive species, especially human pathogens, through acts of bioterrorism, cannot be neglected. However, that risk appears quite low in comparison with the risk of dispersing animal pathogens that could dramatically burden the agricultural economy of food animal producing countries, such as Australia and countries in Europe and North and South America. Although it is not directly related to bioterrorism, the intentional release of non-native species, particularly undesired companion animals or wildlife, may also have a major economic impact on the environment and, possibly, on animal and human health, in the case of accidental release of zoonotic agents.

  11. Dual Species NMR Oscillator

    NASA Astrophysics Data System (ADS)

    Weber, Joshua; Korver, Anna; Thrasher, Daniel; Walker, Thad

    2016-05-01

    We present progress towards a dual species nuclear magnetic oscillator using synchronous spin exchange optical pumping. By applying the bias field as a sequence of alkali 2 π pulses, we generate alkali polarization transverse to the bias field. The alkali polarization is then modulated at the noble gas resonance so that through spin exchange collisions the noble gas becomes polarized. This novel method of NMR suppresses the alkali field frequency shift by at least a factor of 2500 as compared to longitudinal NMR. We will present details of the apparatus and measurements of dual species co-magnetometry using this method. Research supported by the NSF and Northrop-Grumman Corp.

  12. Endangered Species. Teacher's Guide.

    ERIC Educational Resources Information Center

    Brown, Mark; And Others

    This unit is intended to examine the causes of the endangerment of Florida's plant and animal species with a detailed look at varied ecological systems. Individual lessons are designed to be used either by individual students progressing at their own rate or by small groups. Units may be modified for use by large groups. (Author/RE)

  13. Endangered Species: Wild & Rare.

    ERIC Educational Resources Information Center

    Braus, Judy, Ed.

    1987-01-01

    Ranger Rick's NatureScope is a creative education series dedicated to inspiring in children an understanding and appreciation of the natural world while developing the skills they will need to make responsible decisions about the environment. The topic of this issue is "Endangered Species: Wild and Rare." Contents are organized into the following…

  14. Man as a Species.

    ERIC Educational Resources Information Center

    Solem, Alan; And Others

    Written in 1964, the document represents experimental material of the Anthropology Curriculum Study Project. The objectives of the project were to discuss the evolution of man as distinguished from the evolution of other species and as related to culture, and to emphasize human diversity. Three brief essays are presented. The first, "The Species…

  15. Endangered Species. Issue Pac.

    ERIC Educational Resources Information Center

    Fish and Wildlife Service (Dept. of Interior), Washington, DC.

    The materials in this educational packet are designed for use with students in grades 4 through 7. They consist of an overview, three lesson plans and student data sheets, and a poster. The overview presents the history, causes, and present state of species endangerment and a review of legislation by Congress designed to protect threatened or…

  16. Electrophoretic study of Clostridium species.

    PubMed Central

    Cato, E P; Hash, D E; Holdeman, L V; Moore, W E

    1982-01-01

    Polyacrylamide gel electrophoretic analysis of soluble cellular proteins (without sodium dodecyl sulfate) of 70 Clostridium species indicated that the procedure was readily applicable to the differentiation of species in the genus. The protein patterns correlated well with the available DNA homology data and with most accepted differential tests. Results indicated that several earlier names for species were synonyms of those of accepted species and that two accepted species may be synonymous. Images PMID:6175658

  17. Anaerobic digestion in mesophilic and room temperature conditions: Digestion performance and soil-borne pathogen survival.

    PubMed

    Chen, Le; Jian, Shanshan; Bi, Jinhua; Li, Yunlong; Chang, Zhizhou; He, Jian; Ye, Xiaomei

    2016-05-01

    Tomato plant waste (TPW) was used as the feedstock of a batch anaerobic reactor to evaluate the effect of anaerobic digestion on Ralstonia solanacearum and Phytophthora capsici survival. Batch experiments were carried out for TS (total solid) concentrations of 2%, 4% and 6% respectively, at mesophilic (37±1°C) and room (20-25°C) temperatures. Results showed that higher digestion performance was achieved under mesophilic digestion temperature and lower TS concentration conditions. The biogas production ranged from 71 to 416L/kg VS (volatile solids). The inactivation of anaerobic digestion tended to increase as digestion performance improved. The maximum log copies reduction of R. solanacearum and P. capsici detected by quantitative PCR (polymerase chain reaction) were 3.80 and 4.08 respectively in reactors with 4% TS concentration at mesophilic temperatures. However, both in mesophilic and room temperature conditions, the lowest reduction of R. solanacearum was found in the reactors with 6% TS concentration, which possessed the highest VFA (volatile fatty acid) concentration. These findings indicated that simple accumulation of VFAs failed to restrain R. solanacearum effectively, although the VFAs were considered poisonous. P. capsici was nearly completely dead under all conditions. Based on the digestion performance and the pathogen survival rate, a model was established to evaluate the digestate biosafety. PMID:27155428

  18. Tracking and identification of antibacterial components in the essential oil of Tanacetum vulgare L. by the combination of high-performance thin-layer chromatography with direct bioautography and mass spectrometry.

    PubMed

    Móricz, Ágnes M; Häbe, Tim T; Böszörményi, Andrea; Ott, Péter G; Morlock, Gertrud E

    2015-11-27

    Two tansy (Tanacetum vulgare L.) essential oils were obtained by steam distillation of the capitula with subsequent liquid-liquid extraction (oil 1) or with use of an auxiliary phase for the trapping of the steam components (oil 2). These oils were investigated against Bacillus subtilis F1276, B. subtilis spizizenii (DSM 618), Xanthomonas euvesicatoria, Pseudomonas syringae pv. maculicola, Ralstonia solanacearum strain GMI1000 and Aliivibrio fischeri, using the coupling of high-performance thin-layer chromatography to direct bioautography (HPTLC-DB). Using this method with the potato and tomato pathogen R. solanacearum is shown for the first time. Due to the advanced extraction process, oil 2 was richer in components and provided more inhibition zones. The main bioactive components were identified by scanning HPTLC-Direct Analysis in Real Time mass spectrometry (HPTLC-DART-MS) and solid-phase microextraction gas chromatography electron impact MS (SPME-GC-EI-MS) as cis- and trans-chrysanthenol as well as trans-chrysanthenyl acetate. cis-Chrysanthenol exhibited antibacterial effects against all tested bacteria, whereas trans-chrysanthenol inhibited B. subtilis, R. solanacearum and A. fischeri. trans-Chrysanthenyl acetate was an inhibitor for X. euvesicatoria, R. solanacearum and A. fischeri. Although HPTLC-DART-MS resulted in a comparable fragmentation, the ionization characteristics and the recorded mass spectra clearly showed that DART is a softer ionization technique than EI. It is also more affected by ambient conditions and thus prone to additional oxidation products. PMID:26499972

  19. Estimating species richness: The importance of heterogeneity in species detectability

    USGS Publications Warehouse

    Boulinier, T.; Nichols, J.D.; Sauer, J.R.; Hines, J.E.; Pollock, K.H.

    1998-01-01

    Estimating species richness (i.e. the actual number of species present in a given area) is a basic objective of many field studies carried out in community ecology and is also of crucial concern when dealing with the conservation and management of biodiversity. In most studies, the total number of species recorded in an area at a given time is taken as a measure of species richness. Here we use a capture-recapture approach to species richness estimation with North American Breeding Bird Survey (BBS) data in order to estimate species detectability and thus gain insight about its importance. We carried out analyses on all survey routes of four states, Arizona, Maryland, North Dakota, and Wisconsin, in two years, 1970 and 1990. These states were chosen to provide contrasting habitats, bird species composition and survey quality. We investigated the effect of state, year and observer ability on the proportions of different models selected, and on estimates of detectability and species richness. Our results indicate that model Mh, which assumes heterogeneous detection probability among species, is frequently appropriate for estimating species richness from BBS data. Species detectability varied among states and was higher for the more skilled observers. These results emphasize the need to take into account potential heterogeneities in detectability among species in studies of factors affecting species richness.

  20. Estimating Effects of Species Interactions on Populations of Endangered Species.

    PubMed

    Roth, Tobias; Bühler, Christoph; Amrhein, Valentin

    2016-04-01

    Global change causes community composition to change considerably through time, with ever-new combinations of interacting species. To study the consequences of newly established species interactions, one available source of data could be observational surveys from biodiversity monitoring. However, approaches using observational data would need to account for niche differences between species and for imperfect detection of individuals. To estimate population sizes of interacting species, we extended N-mixture models that were developed to estimate true population sizes in single species. Simulations revealed that our model is able to disentangle direct effects of dominant on subordinate species from indirect effects of dominant species on detection probability of subordinate species. For illustration, we applied our model to data from a Swiss amphibian monitoring program and showed that sizes of expanding water frog populations were negatively related to population sizes of endangered yellow-bellied toads and common midwife toads and partly of natterjack toads. Unlike other studies that analyzed presence and absence of species, our model suggests that the spread of water frogs in Central Europe is one of the reasons for the decline of endangered toad species. Thus, studying population impacts of dominant species on population sizes of endangered species using data from biodiversity monitoring programs should help to inform conservation policy and to decide whether competing species should be subject to population management. PMID:27028074

  1. Estimating Effects of Species Interactions on Populations of Endangered Species.

    PubMed

    Roth, Tobias; Bühler, Christoph; Amrhein, Valentin

    2016-04-01

    Global change causes community composition to change considerably through time, with ever-new combinations of interacting species. To study the consequences of newly established species interactions, one available source of data could be observational surveys from biodiversity monitoring. However, approaches using observational data would need to account for niche differences between species and for imperfect detection of individuals. To estimate population sizes of interacting species, we extended N-mixture models that were developed to estimate true population sizes in single species. Simulations revealed that our model is able to disentangle direct effects of dominant on subordinate species from indirect effects of dominant species on detection probability of subordinate species. For illustration, we applied our model to data from a Swiss amphibian monitoring program and showed that sizes of expanding water frog populations were negatively related to population sizes of endangered yellow-bellied toads and common midwife toads and partly of natterjack toads. Unlike other studies that analyzed presence and absence of species, our model suggests that the spread of water frogs in Central Europe is one of the reasons for the decline of endangered toad species. Thus, studying population impacts of dominant species on population sizes of endangered species using data from biodiversity monitoring programs should help to inform conservation policy and to decide whether competing species should be subject to population management.

  2. Genomics of Bacillus Species

    NASA Astrophysics Data System (ADS)

    Økstad, Ole Andreas; Kolstø, Anne-Brit

    Members of the genus Bacillus are rod-shaped spore-forming bacteria belonging to the Firmicutes, the low G+C gram-positive bacteria. The Bacillus genus was first described and classified by Ferdinand Cohn in Cohn (1872), and Bacillus subtilis was defined as the type species (Soule, 1932). Several Bacilli may be linked to opportunistic infections. However, pathogenicity among Bacillus spp. is mainly a feature of bacteria belonging to the Bacillus cereus group, including B. cereus, Bacillus anthracis, and Bacillus thuringiensis. Here we review the genomics of B. cereus group bacteria in relation to their roles as etiological agents of two food poisoning syndromes (emetic and diarrhoeal).

  3. Flavonoids in Sophora Species

    NASA Astrophysics Data System (ADS)

    Shirataki, Yoshiaki; Motohashi, Noboru

    Sophora species of Leguminosae are abundantly present in the natural kingdom. Today, among Sophora plants, the flavonoids of the plant phenols occupy a remarkable position. For a very long time flavonoids have been used as natural pigments and dyes. Some of the colorful anthocyanins of the glucosides are used for color and flavor in foodstuffs. Therefore, these flavonoids are beneficial to daily human life. Herein we concentrate on flavonoids in Sophora plants, and the relationship between their chemical structures and nutraceutical effect. For this purpose, soy-based infant formulas, osteoporosis, antitumor activity, antimicrobial activity, anti-HIV activity, radical generation and O2 - scavenging activity, and enzyme inhibitory activity have been described.

  4. Genetically Altered Plant Species

    NASA Technical Reports Server (NTRS)

    2003-01-01

    Researchers in Robert Ferl's lab at the University of Florida in Gainesville, genetically altered this Arabdopsis Thaliana (a brassica species) plant to learn how extreme environments, such as the low atmospheric pressure on Mars, affect plant genes. They inserted green fluorescent protein (GFP) near the on/off switches for anoxia and drought genes. When those genes were turned on after exposure to reduced atmospheric pressure, GFP was turned on as well, causing cells expressing those genes to glow green under a blue light. The natural fluorescence of chlorophyll accounts for the red glow.

  5. Nutrition of aquaculture species.

    PubMed

    Lovell, R T

    1991-10-01

    Dietary requirements for amino acids and fatty acids have been reported for channel catfish (Ictalurus punctatus), salmonids (Oncorhynchus spp.), common carp (Cyprinus carpio), tilapias (Oreochromis spp.), and eel (Anguilla japonicus). Most of the vitamin and mineral requirements are available for channel catfish and salmonids, and some are available for common carp, tilapia, eel, and other finfish and crustaceans. From this available information, cost-effective feeds can be formulated for the major commercial aquaculture species. Major differences in nutrient requirements between fish and mammals or birds are as follows: fish have a lower digestible energy:protein ratio (8 to 10 kcal of DE/g of CP for fish vs 15 to 20 kcal of DE/g of CP for livestock); fish require n-3 fatty acids and land animals require n-6; fish can absorb minerals from the water, which negates the need for some minerals in the diet; and fish have limited ability to synthesize vitamin C and must depend on a dietary source. Areas for further research include 1) refinement of nutrient requirements of the major culture species considering effects of fish size, temperature, and management; 2) nutrient requirements of crustaceans; 3) effects of nutrition on fish health and product quality; and 4) feeding technology. PMID:1778835

  6. ULTRASTRUCTURE OF MYCOPLASMA SPECIES

    PubMed Central

    Domermuth, C. H.; Nielsen, M. H.; Freundt, E. A.; Birch-Andersen, A.

    1964-01-01

    Domermuth, C. H. (Statens Seruminstitut, Copenhagen, Denmark), M. H. Nielsen, E. A. Freundt, and A. Birch-Andersen. Ultrastructure of Mycoplasma species. J. Bacteriol. 88:727–744. 1964.—The ultrastructure of 19 strains (15 species) of Mycoplasmatales grown on solid medium was studied with the aid of an electron microscope. The cells possessed a triple-layered limiting membrane 75 to 100 A thick. This membrane appeared to be symmetrical in some strains and asymmetrical in others. An electron-dense material found in close contact with the cell surface was tentatively interpreted to be a capsular substance. Ribosomes and strands of nuclear material were observed in the cytoplasm of cells of all strains. Ribosomes observed in the JA strain of M. gallisepticum were frequently arranged in a regular geometric pattern of characteristic appearance. Dense inclusions sometimes limited by triple-layered membranes (possibly developing elementary bodies), as well as membrane-surrounded vesicles, were observed in the cytoplasm of cells of some strains. Images PMID:14208513

  7. Save Our Species: Protecting Endangered Species from Pesticides.

    ERIC Educational Resources Information Center

    Environmental Protection Agency, Washington, DC.

    This full-size poster profiles 11 wildlife species that are endangered. Color illustrations of animals and plants are accompanied by narrative describing their habitats and reasons for endangerment. The reverse side of the poster contains information on the Endangered Species Act, why protecting endangered and threatened species is important, how…

  8. Synthesis and antibacterial activity of pyridinium-tailored aromatic amphiphiles.

    PubMed

    Wang, Peiyi; Gao, Manni; Zhou, Lei; Wu, Zhibing; Hu, Deyu; Hu, Jun; Yang, Song

    2016-02-15

    In this Letter, the antibacterial activities of pyridinium-tailored aromatic amphiphiles were evaluated by turbidimeter tests in vitro. The bioassays revealed that most of the target compounds exhibit appreciable inhibition activities against the plant pathogenic bacteria Xanthomonas oryzae pv. oryzae, Ralstonia solanacearum, and Xanthomonas axonopodis pv. citri. The half-maximal effective concentrations (EC50) of 2-NP-10, 9-AP-10, and 9-AP-7 against these three bacteria were relatively high, which may be ascribed to the favourable hydrophobicity/hydrophilicity balance in these compounds. Our results suggest that pyridinium-tailored aromatic amphiphiles are promising bactericide candidates against plant bacterial diseases. PMID:26832217

  9. Synthesis and antibacterial activity of pyridinium-tailored aromatic amphiphiles.

    PubMed

    Wang, Peiyi; Gao, Manni; Zhou, Lei; Wu, Zhibing; Hu, Deyu; Hu, Jun; Yang, Song

    2016-02-15

    In this Letter, the antibacterial activities of pyridinium-tailored aromatic amphiphiles were evaluated by turbidimeter tests in vitro. The bioassays revealed that most of the target compounds exhibit appreciable inhibition activities against the plant pathogenic bacteria Xanthomonas oryzae pv. oryzae, Ralstonia solanacearum, and Xanthomonas axonopodis pv. citri. The half-maximal effective concentrations (EC50) of 2-NP-10, 9-AP-10, and 9-AP-7 against these three bacteria were relatively high, which may be ascribed to the favourable hydrophobicity/hydrophilicity balance in these compounds. Our results suggest that pyridinium-tailored aromatic amphiphiles are promising bactericide candidates against plant bacterial diseases.

  10. Does climate limit species richness by limiting individual species' ranges?

    PubMed

    Boucher-Lalonde, Véronique; Kerr, Jeremy T; Currie, David J

    2014-02-01

    Broad-scale geographical variation in species richness is strongly correlated with climate, yet the mechanisms underlying this correlation are still unclear. We test two broad classes of hypotheses to explain this pattern. Bottom-up hypotheses propose that the environment determines individual species' ranges. Ranges then sum up to yield species richness patterns. Top-down hypotheses propose that the environment limits the number of species that occur in a region, but not which ones. We test these two classes of hypotheses using a natural experiment: seasonal changes in environmental variables and seasonal range shifts of 625 migratory birds in the Americas. We show that richness seasonally tracks the environment. By contrast, individual species' geographical distributions do not. Rather, species occupy different sets of environmental conditions in two seasons. Our results are inconsistent with extant bottom-up hypotheses. Instead, a top-down mechanism appears to constrain the number of species that can occur in a given region.

  11. Invasive species and climate change

    USGS Publications Warehouse

    Middleton, Beth A.

    2006-01-01

    Invasive species challenge managers in their work of conserving and managing natural areas and are one of the most serious problems these managers face. Because invasive species are likely to spread in response to changes in climate, managers may need to change their approaches to invasive species management accordingly.

  12. 76 FR 2348 - Endangered Species

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-01-13

    ... National Oceanic and Atmospheric Administration RIN 0648-XA140 Endangered Species AGENCY: National Marine... Fort Fisher. The requested permit has been issued under the authority of the Endangered Species Act of... exporting of endangered and threatened species (50 CFR parts 222-226). The North Carolina Aquarium at...

  13. 76 FR 1405 - Endangered Species

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-01-10

    ... National Oceanic and Atmospheric Administration RIN 0648-XA128 Endangered Species AGENCY: National Marine... issued under the authority of the Endangered Species Act of 1973, as amended (ESA; 16 U.S.C. 1531 et seq... not operate to the disadvantage of such endangered or threatened species, and (3) is consistent...

  14. California Endangered Species Resource Guide.

    ERIC Educational Resources Information Center

    California State Dept. of Education, Los Angeles.

    This document was developed in response to California Senate Bill No. 885, "The Endangered Species Education Project," that called for a statewide program in which schools adopt a local endangered species, research past and current efforts to preserve the species' habitat, develop and implement an action plan to educate the community about the…

  15. Ubiquitous dispersal of microbial species

    NASA Astrophysics Data System (ADS)

    Finlay, Bland J.; Clarke, Ken J.

    1999-08-01

    The biosphere supports astronomical numbers of free-living microorganisms that belong to an indeterminate number of species. One view is that the abundance of microorganisms drives their dispersal, making them ubiquitous and resulting in a moderate global richness of species. But ubiquity is hard to demonstrate, not only because active species have a rapid turnover, but also because most species in a habitat at any moment in time are relatively rare or in some cryptic state. Here we use microbes that leave traces of their recent population growth in the form of siliceous scale structures to show that all species in the chrysomonad flagellate genus Paraphysomonas are probably ubiquitous.

  16. Introduced species as evolutionary traps

    USGS Publications Warehouse

    Schlaepfer, Martin A.; Sherman, P.W.; Blossey, B.; Runge, M.C.

    2005-01-01

    Invasive species can alter environments in such a way that normal behavioural decision-making rules of native species are no longer adaptive. The evolutionary trap concept provides a useful framework for predicting and managing the impact of harmful invasive species. We discuss how native species can respond to changes in their selective regime via evolution or learning. We also propose novel management strategies to promote the long-term co-existence of native and introduced species in cases where the eradication of the latter is either economically or biologically unrealistic.

  17. The Species Delimitation Uncertainty Principle

    PubMed Central

    Adams, Byron J.

    2001-01-01

    If, as Einstein said, "it is the theory which decides what we can observe," then "the species problem" could be solved by simply improving our theoretical definition of what a species is. However, because delimiting species entails predicting the historical fate of evolutionary lineages, species appear to behave according to the Heisenberg Uncertainty Principle, which states that the most philosophically satisfying definitions of species are the least operational, and as species concepts are modified to become more operational they tend to lose their philosophical integrity. Can species be delimited operationally without losing their philosophical rigor? To mitigate the contingent properties of species that tend to make them difficult for us to delimit, I advocate a set of operations that takes into account the prospective nature of delimiting species. Given the fundamental role of species in studies of evolution and biodiversity, I also suggest that species delimitation proceed within the context of explicit hypothesis testing, like other scientific endeavors. The real challenge is not so much the inherent fallibility of predicting the future but rather adequately sampling and interpreting the evidence available to us in the present. PMID:19265874

  18. Importance of species traits for species distribution in fragmented landscapes.

    PubMed

    Tremlová, Katerina; Münzbergová, Zuzana

    2007-04-01

    Knowledge of the relationship between species traits and species distribution in fragmented landscapes is important for understanding current distribution patterns and as background information for predictive models of the effect of future landscape changes. The existing studies on the topic suffer from several drawbacks. First, they usually consider only traits related to dispersal ability and not growth. Furthermore, they do not apply phylogenetic corrections, and we thus do not know how considerations of phylogenetic relationships can alter the conclusions. Finally, they usually apply only one technique to calculate habitat isolation, and we do not know how other isolation measures would change the results. We studied the issues using 30 species forming congeneric pairs occurring in fragmented dry grasslands. We measured traits related to dispersal, survival, and growth in the species and recorded distribution of the species in 215 grassland fragments. We show many strong relationships between species traits related to both dispersal and growth and species distribution in the landscape, such as the positive relationship between habitat occupancy and anemochory and negative relationships between habitat occupancy and seed dormancy. The directions of these relationships, however, often change after application of phylogenetic correction. For example, more isolated habitats host species with smaller seeds. After phylogenetic correction, however, they turn out to host species with larger seeds. The conclusions also partly change depending on how we calculate habitat isolation. Specifically, habitat isolation calculated from occupied habitats only has the highest predictive power. This indicates slow dynamics of the species. All the results support the expectation that species traits have a high potential to explain patterns of species distribution in the landscape and that they can be used to build predictive models of species distribution. The specific conclusions

  19. Eighteen new oleaginous yeast species.

    PubMed

    Garay, Luis A; Sitepu, Irnayuli R; Cajka, Tomas; Chandra, Idelia; Shi, Sandy; Lin, Ting; German, J Bruce; Fiehn, Oliver; Boundy-Mills, Kyria L

    2016-07-01

    Of 1600 known species of yeasts, about 70 are known to be oleaginous, defined as being able to accumulate over 20 % intracellular lipids. These yeasts have value for fundamental and applied research. A survey of yeasts from the Phaff Yeast Culture Collection, University of California Davis was performed to identify additional oleaginous species within the Basidiomycota phylum. Fifty-nine strains belonging to 34 species were grown in lipid inducing media, and total cell mass, lipid yield and triacylglycerol profiles were determined. Thirty-two species accumulated at least 20 % lipid and 25 species accumulated over 40 % lipid by dry weight. Eighteen of these species were not previously reported to be oleaginous. Triacylglycerol profiles were suitable for biodiesel production. These results greatly expand the number of known oleaginous yeast species, and reveal the wealth of natural diversity of triacylglycerol profiles within wild-type oleaginous Basidiomycetes.

  20. Eighteen new oleaginous yeast species.

    PubMed

    Garay, Luis A; Sitepu, Irnayuli R; Cajka, Tomas; Chandra, Idelia; Shi, Sandy; Lin, Ting; German, J Bruce; Fiehn, Oliver; Boundy-Mills, Kyria L

    2016-07-01

    Of 1600 known species of yeasts, about 70 are known to be oleaginous, defined as being able to accumulate over 20 % intracellular lipids. These yeasts have value for fundamental and applied research. A survey of yeasts from the Phaff Yeast Culture Collection, University of California Davis was performed to identify additional oleaginous species within the Basidiomycota phylum. Fifty-nine strains belonging to 34 species were grown in lipid inducing media, and total cell mass, lipid yield and triacylglycerol profiles were determined. Thirty-two species accumulated at least 20 % lipid and 25 species accumulated over 40 % lipid by dry weight. Eighteen of these species were not previously reported to be oleaginous. Triacylglycerol profiles were suitable for biodiesel production. These results greatly expand the number of known oleaginous yeast species, and reveal the wealth of natural diversity of triacylglycerol profiles within wild-type oleaginous Basidiomycetes. PMID:27072563

  1. Seed dormancy in alpine species

    PubMed Central

    Schwienbacher, Erich; Navarro-Cano, Jose Antonio; Neuner, Gilbert; Erschbamer, Brigitta

    2011-01-01

    In alpine species the classification of the various mechanisms underlying seed dormancy has been rather questionable and controversial. Thus, we investigated 28 alpine species to evaluate the prevailing types of dormancy. Embryo type and water impermeability of seed coats gave an indication of the potential seed dormancy class. To ascertain the actual dormancy class and level, we performed germination experiments comparing the behavior of seeds without storage, after cold-dry storage, after cold-wet storage, and scarification. We also tested the light requirement for germination in some species. Germination behavior was characterized using the final germination percentage and the mean germination time. Considering the effects of the pretreatments, a refined classification of the prevailing dormancy types was constructed based on the results of our pretreatments. Only two out of the 28 species that we evaluated had predominantly non-dormant seeds. Physiological dormancy was prevalent in 20 species, with deep physiological dormancy being the most abundant, followed by non-deep and intermediate physiological dormancy. Seeds of four species with underdeveloped embryos were assigned to the morphophysiologial dormancy class. An impermeable seed coat was identified in two species, with no additional physiological germination block. We defined these species as having physical dormancy. Light promoted the germination of seeds without storage in all but one species with physiological dormancy. In species with physical dormancy, light responses were of minor importance. We discuss our new classification in the context of former germination studies and draw implications for the timing of germination in the field. PMID:24415831

  2. To Be or Not To Be a Poly(3-Hydroxybutyrate) (PHB) Depolymerase: PhaZd1 (PhaZ6) and PhaZd2 (PhaZ7) of Ralstonia eutropha, Highly Active PHB Depolymerases with No Detectable Role in Mobilization of Accumulated PHB

    PubMed Central

    Sznajder, Anna

    2014-01-01

    The putative physiological functions of two related intracellular poly(3-hydroxybutyrate) (PHB) depolymerases, PhaZd1 and PhaZd2, of Ralstonia eutropha H16 were investigated. Purified PhaZd1 and PhaZd2 were active with native PHB granules in vitro. Partial removal of the proteinaceous surface layer of native PHB granules by trypsin treatment or the use of PHB granules isolated from ΔphaP1 or ΔphaP1-phaP5 mutant strains resulted in increased specific PHB depolymerase activity, especially for PhaZd2. Constitutive expression of PhaZd1 or PhaZd2 reduced or even prevented the accumulation of PHB under PHB-permissive conditions in vivo. Expression of translational fusions of enhanced yellow fluorescent protein (EYFP) with PhaZd1 and PhaZd2 in which the active-site serines (S190 and Ser193) were replaced with alanine resulted in the colocalization of only PhaZd1 fusions with PHB granules. C-terminal fusions of inactive PhaZd2(S193A) with EYFP revealed the presence of spindle-like structures, and no colocalization with PHB granules was observed. Chromosomal deletion of phaZd1, phaZd2, or both depolymerase genes had no significant effect on PHB accumulation and mobilization during growth in nutrient broth (NB) or NB-gluconate medium. Moreover, neither proteome analysis of purified native PHB granules nor lacZ fusion studies gave any indication that PhaZd1 or PhaZd2 was detectably present in the PHB granule fraction or expressed at all during growth on NB-gluconate medium. In conclusion, PhaZd1 and PhaZd2 are two PHB depolymerases with a high capacity to degrade PHB when artificially expressed but are apparently not involved in PHB mobilization in the wild type. The true in vivo functions of PhaZd1 and PhaZd2 remain obscure. PMID:24907326

  3. Confronting species distribution model predictions with species functional traits.

    PubMed

    Wittmann, Marion E; Barnes, Matthew A; Jerde, Christopher L; Jones, Lisa A; Lodge, David M

    2016-02-01

    Species distribution models are valuable tools in studies of biogeography, ecology, and climate change and have been used to inform conservation and ecosystem management. However, species distribution models typically incorporate only climatic variables and species presence data. Model development or validation rarely considers functional components of species traits or other types of biological data. We implemented a species distribution model (Maxent) to predict global climate habitat suitability for Grass Carp (Ctenopharyngodon idella). We then tested the relationship between the degree of climate habitat suitability predicted by Maxent and the individual growth rates of both wild (N = 17) and stocked (N = 51) Grass Carp populations using correlation analysis. The Grass Carp Maxent model accurately reflected the global occurrence data (AUC = 0.904). Observations of Grass Carp growth rate covered six continents and ranged from 0.19 to 20.1 g day(-1). Species distribution model predictions were correlated (r = 0.5, 95% CI (0.03, 0.79)) with observed growth rates for wild Grass Carp populations but were not correlated (r = -0.26, 95% CI (-0.5, 0.012)) with stocked populations. Further, a review of the literature indicates that the few studies for other species that have previously assessed the relationship between the degree of predicted climate habitat suitability and species functional traits have also discovered significant relationships. Thus,