Contamination of certain crops with aflatoxins is a serious concern for agriculture and animal and human health. The predominant species associated with this crop contamination is Aspergillus flavus. The ability of A. flavus to produce other toxins could also be an additional concern. Phylogenetic e...
Marín, Sonia; Ramos, Antonio J; Sanchis, V
Aflatoxins (AFs) are the main contaminants in pistachio nuts. AFs production in pistachio has been attributed to Aspergillus flavus. The aim of this study was to apply existing models to predict growth and AFs production by an A. flavus isolated from pistachios as a function of moisture content and storage temperature of pistachios in order to test their usefulness and complementarities. A full factorial design was used: the moisture content levels assayed were 10, 15, 20, 25 and 30% and incubation temperatures were 10, 15, 20, 25, 30, 37 and 42 °C. Both kinetic and probability models were built to predict growth of the strain under the assayed conditions. Among the assayed models, cardinal ones gave a good quality fit for radial growth rate data. Moreover, the progressive approach, which was developed based on a reduced number of experimental points led to an improved prediction in the validation step. This is quite significant as may allow for improved experimental designs, less costly than full factorial ones. Probability model proved to be concordant in 91% of the calibration set observations. Even though the validation set included conditions around the growth/no-growth interface, there was a 100% agreement in the predictions from the data set (n = 16, cut off = 0.5) after 60 days. Similarly, the probability for AF presence was rightly predicted in 89% of the cases. According to our results EC maximum aflatoxin levels would be surpassed in a period as short as 1 month if pistachio nuts reach 20 °C, unless %mc is ≤10%.
Hadrich, Inès; Neji, Sourour; Makni, Fattouma; Ayadi, Ali; Elloumi, Moez; Ranque, Stéphane
There are limited data on in vitro susceptibility testing of echinocandins against Aspergillus species. The objective of this study was to describe the phenotypes of Aspergillus flavus observed on exposure to caspofungin in vitro and to test whether these phenotypes were associated with A. flavus genotypes. The caspofungin MICs of 37 A. flavus clinical isolates collected from 14 patients with invasive aspergillosis were determined using Etest assays. Caspofungin MICs ranged from 0.012 to 0.064 mg l(-1); the modal MIC was 0.023 mg l(-1) and the MIC₅₀ and MIC₉₀ were 0.032 and 0.064 mg l(-1), respectively. A clear end point was noted in 24 (65 %) isolates, whereas seven (19 %) displayed a trailing effect and six (16 %) showed paradoxical growth when exposed to caspofungin. In these A. flavus isolates, the absence of a significant population structure or genetic differentiation indicated that trailing or paradoxical growth phenotypes were independent of microsatellite genotype.
... tolerance exemption for Aspergillus flavus AF36, a non-aflatoxin-producing strain of Aspergillus flavus, in... reduction of aflatoxin. No further toxicological data are required to support this exemption from the..., including drinking water from ground water or surface water and exposure through pesticide use in...
Wang, Bin; Han, Xiaoyun; Bai, Youhuang; Lin, Zhenguo; Qiu, Mengguang; Nie, Xinyi; Wang, Sen; Zhang, Feng; Zhuang, Zhenhong; Yuan, Jun; Wang, Shihua
Aflatoxins (AFs), produced mainly by Aspergillus flavus and Aspergillus parasiticus, are strongly toxic and carcinogenic. Here, we showed that glutamine is the optimal nitrogen source for AF-production in A. flavus grown in Czapek Dox medium. Additionally, 4mM glutamine was the threshold for high production of aflatoxin B1. However, no significant impact of glutamine synthetase inhibitor was detected for on AF biosynthesis. In contrast, rapamycin could significantly suppress the glutamine inducing effect on AFs production, simultaneously inhibiting the fungal growth and conidiation. To identify the genes and regulatory networks involved in AFs biosynthesis, especially concerning the nitrogen source metabolism pathway and the target of rapamycin (TOR) signaling pathway, we obtained transcriptomes for A. flavus under treatment of three nitrogen sources by RNA-sequencing. We identified 1429 differentially expressed genes. Through GO and KEGG pathway analyses, the relationship between nitrogen metabolism and AFs biosynthesis was revealed, and the effects of TOR inhibitor were confirmed. Additionally, the quantitative real-time PCR results verified the credibility and reliability of the RNA-seq data, and were consistent with the other experimental results. Our research laid the foundation for a primary study on the involvement of the nitrogen regulatory network and TOR signaling pathway in AF biosynthesis.
The availability of a representative gene ontology (GO) database is a prerequisite for a successful functional genomics study. Using online Blast2GO resources we constructed a GO database of Aspergillus flavus. Of the predicted total 13,485 A. flavus genes 8,987 were annotated with GO terms. The mea...
Yin, Hsin-Bai; Chen, Chi-Hung; Kollanoor-Johny, Anup; Darre, Michael J; Venkitanarayanan, Kumar
Aflatoxins (AF) are toxic metabolites primarily produced by molds, Aspergillus flavus and Aspergillus parasiticus. Contamination of poultry feed with AF is a major concern to the poultry industry due to severe economic losses stemming from poor performance, reduced egg production, and diminished egg hatchability. This study investigated the inhibitory effect of 2 generally regarded as safe (GRAS), natural plant compounds, namely carvacrol (CR) and trans-cinnamaldehyde (TC), on A. flavus and A. parasiticus growth and AF production in potato dextrose broth (PDB) and in poultry feed. In broth culture, PDB supplemented with CR (0%, 0.02%, 0.04% and 0.08%) or TC (0%, 0.005%, 0.01% and 0.02%) was inoculated with A. flavus or A. parasiticus (6 log CFU/mL), and mold counts and AF production were determined on days 0, 1, 3, and 5. Similarly, 200 g portions of poultry feed supplemented with CR or TC (0%, 0.4%, 0.8%, and 1.0%) were inoculated with each mold, and their counts and AF concentrations in the feed were determined at 0, 1, 2, 3, 4, 8, and 12 weeks of storage. Moreover, the effect of CR and TC on the expression of AF synthesis genes in A. flavus and A. parasiticus (aflC, nor1, norA, and ver1) was determined using real-time quantitative PCR (RT-qPCR). All experiments had duplicate samples and were replicated 3 times. Results indicated that CR and TC reduced A. flavus and A. parasiticus growth and AF production in broth culture and chicken feed (P<0.05). All tested concentrations of CR and TC decreased AF production in broth culture and chicken feed by at least 60% when compared to controls (P<0.05). In addition, CR and TC down-regulated the expression of major genes associated with AF synthesis in the molds (P<0.05). Results suggest the potential use of CR and TC as feed additives to control AF contamination in poultry feed. © 2015 Poultry Science Association Inc.
López-Malo, Aurelio; Maris Alzamora, Stella; Palou, Enrique
The combined effects of water activity ([a(w)] 0.99 or 0.95), pH (4.5 or 3.5) and antimicrobial agent (potassium sorbate, sodium benzoate, sodium bisulfite, carvacrol, citral, eugenol, thymol, or vanillin) concentration (0, 100, 200 up to 1800 ppm) on the growth of Aspergillus flavus were evaluated in potato dextrose agar (PDA). Mold spore germination time and radial growth rates (RGR) were significantly (p<0.05) affected by the variables. For equal antimicrobial concentration, reduction in pH or a(w) had important effects, lowering RGR and delaying germination time. Depending on a(w) and pH, increase in antimicrobial concentration slightly reduced RGR until a critical concentration where RGR was drastically reduced or mold growth was inhibited. Germination time increased as antimicrobial agent concentration increased and when a(w) and pH decreased. Important antimicrobial differences were observed, being, in general, the natural antimicrobials less pH-dependent than chemical preservatives. A. flavus exhibited higher sensitivity to thymol, eugenol, carvacrol, potassium sorbate, sodium bisulfite, and sodium benzoate (at pH 3.5) than to vanillin or citral.
Dias Ferreira, Flávio; Mossini, Simone Aparecida Galerani; Dias Ferreira, Francine Maery; Arrotéia, Carla Cristina; da Costa, Christiane Luciana; Nakamura, Celso Vataru; Machinski, Miguel
The essential oil from Curcuma longa L. was analysed by GC/MS. The major components of the oil were ar-turmerone (33.2%), α -turmerone (23.5%) and β -turmerone (22.7%). The antifungal activities of the oil were studied with regard to Aspergillus flavus growth inhibition and altered morphology, as preliminary studies indicated that the essential oil from C. longa inhibited Aspergillus flavus Link aflatoxin production. The concentration of essential oil in the culture media ranged from 0.01% to 5.0% v/v, and the concentration of curcumin was 0.01-0.5% v/v. The effects on sporulation, spore viability, and fungal morphology were determined. The essential oil exhibited stronger antifungal activity than curcumin on A. flavus. The essential oil reduced the fungal growth in a concentration-dependent manner. A. flavus growth rate was reduced by C. longa essential oil at 0.10%, and this inhibition effect was more efficient in concentrations above 0.50%. Germination and sporulation were 100% inhibited in 0.5% oil. Scanning electron microscopy (SEM) of A. flavus exposed to oil showed damage to hyphae membranes and conidiophores. Because the fungus is a plant pathogen and aflatoxin producer, C. longa essential oil may be used in the management of host plants.
Mossini, Simone Aparecida Galerani; Ferreira, Francine Maery Dias; Arrotéia, Carla Cristina; da Costa, Christiane Luciana; Nakamura, Celso Vataru; Machinski Junior, Miguel
The essential oil from Curcuma longa L. was analysed by GC/MS. The major components of the oil were ar-turmerone (33.2%), α-turmerone (23.5%) and β-turmerone (22.7%). The antifungal activities of the oil were studied with regard to Aspergillus flavus growth inhibition and altered morphology, as preliminary studies indicated that the essential oil from C. longa inhibited Aspergillus flavus Link aflatoxin production. The concentration of essential oil in the culture media ranged from 0.01% to 5.0% v/v, and the concentration of curcumin was 0.01–0.5% v/v. The effects on sporulation, spore viability, and fungal morphology were determined. The essential oil exhibited stronger antifungal activity than curcumin on A. flavus. The essential oil reduced the fungal growth in a concentration-dependent manner. A. flavus growth rate was reduced by C. longa essential oil at 0.10%, and this inhibition effect was more efficient in concentrations above 0.50%. Germination and sporulation were 100% inhibited in 0.5% oil. Scanning electron microscopy (SEM) of A. flavus exposed to oil showed damage to hyphae membranes and conidiophores. Because the fungus is a plant pathogen and aflatoxin producer, C. longa essential oil may be used in the management of host plants. PMID:24367241
Aspergillus flavus is the major producer of carcinogenic aflatoxins in crops worldwide and is also an important opportunistic human pathogen in aspergillosis. The sexual state of this heterothallic fungus is described from crosses between strains of the opposite mating type. Sexual reproduction oc...
Uraih, N; Chipley, J R
The effects of sodium chloride, sodium acetate, benzoic acid, sodium benzoate, malonic acid, and sodium malonate on growth and aflatoxin production by Aspergillus flavus were investigated in synthetic media. Sodium chloride at concentrations equivalent to or greater than 12 g/100 ml inhibited growth and aflatoxin production, while at 8 g or less/100 ml, growth and aflatoxin production were stimulated. At 2 g or less/100 ml, sodium acetate also stimulated growth and aflatoxin production, but reduction occurred with 4 g or more/100 ml. Malonic acid at 10, 20, 40, and 50 mM reduced growth and aflatoxin production (over 50%) while sodium malonate at similar concentrations but different pH values had the opposite effect. Benzoic acid (pH 3.9) and sodium benzoate (pH 5.0) at 0.4 g/100 ml completely inhibited growth and aflatoxin production. Examination of the effect of initial pH indicated that the extent of inhibitory action of malonic acid and sodium acetate was a function of initial pH. The inhibitory action of benzoic acid and sodium benzoate appeared to be a function of undissociated benzoic acid molecules. Aflatoxin reduction was usually accompanied by an unidentified orange pigment, while aflatoxin stimulation was accompanied by unidentified blue and green fluorescent spots but with lower Rf values that aflatoxins B1, G1, B2, and G2 standards.
Abellana, M; Sanchis, V; Ramos, A J
This study compared the effect of temperature and water activity and their interactions on the rate of mycelial growth of Penicillium aurantiogriseum, P. chrysogenum, P. corylophilum and Aspergillus flavus on a sponge cake analogue. As expected, growth rates showed dependence on a(w), and temperature. However, no significant differences were observed in the growth rates of different isolates. The minimum a(w) values for growth of the Penicillium spp. was 0.85-0.90. A. flavus was able to grow at 0.90 a(w) when the temperature was above 15 degrees C. This study has shown that fungal growth by these species on a sponge cake analogue, with a composition similar to usual bakery products, is prevented if the a(w) is kept at < 0.85.
Marshall, K. C.; Alexander, M.
Marshall, K. C. (Cornell University, Ithaca, N. Y.) and M. Alexander. Nitrification by Aspergillus flavus. J. Bacteriol. 83:572–578. 1962.—Aspergillus flavus has been shown to produce bound hydroxylamine, nitrite, and nitrate when grown in peptone, amino acid, or buffered ammonium media. Free hydroxylamine was not detected in these cultures, but it was found in an unbuffered ammonium medium in which neither nitrite nor nitrate was formed. Evidence was obtained for the presence of β-nitropropionic acid in the filtrate of an actively nitrifying culture. Alumina treatment of an ammonium medium prevented the formation by growing cultures of nitrite and nitrate but not bound hydroxylamine. The effect of alumina treatment was reversed by the addition of 10−3m CeCl3 to the medium. Extracts of the fungus contained peroxidase and an enzyme capable of catalyzing the production of nitrite from β-nitropropionic acid. The nitrite-forming enzyme is apparently specific for β-nitropropionate; no activity was found with nitromethane, nitroethane, and nitropropane as substrates. Nitrate was not reduced to nitrite nor was nitrite oxidized to nitrate by the hyphal extracts. The significance of these observations in nitrification by A. flavus is discussed. PMID:14470254
Chitarrini, G; Nobili, C; Pinzari, F; Antonini, A; De Rossi, P; Del Fiore, A; Procacci, S; Tolaini, V; Scala, V; Scarpari, M; Reverberi, M
Buckwheat (Fagopyrum spp.) is a "pseudo-cereal" of great interest in the production of healthy foods since its flour, derived from achenes, is enriched with bioactive compounds and, due to the absence of gluten, may be used in composition of celiac diets. Amongst buckwheat species, F. tataricum achenes possess a larger amount of the antioxidant flavenol rutin than the common buckwheat F. esculentum. Ongoing climate change may favor plant susceptibility to the attack by pathogenic, often mycotoxigenic, fungi with consequent increase of mycotoxins in previously unexploited feeds and foodstuffs. In particular, Aspergillus flavus, under suitable environmental conditions such as those currently occurring in Italy, may produce aflatoxin B1 (AFB1), the most carcinogenic compound of fungal origin which is classified by IARC as Category 1. In this study, the viable achenes of two buckwheat species, F. tataricum (var. Golden) and F. esculentum (var. Aelita) were inoculated with an AFB1-producing A. flavus NRRL 3357 to analyze their relative performances against fungal invasion and toxin contamination. Notably, we sought the existence of a correlation between the amount of tocols/flavonols in the achenes of buckwheat, infected and non-infected with A. flavus, and to analyze the ability of the pathogen to grow and produce toxin during achene infection. Results suggest that achenes of F. tataricum, the best producer of antioxidant compounds in this study, are less susceptible to A. flavus infection and consequently, but not proportionally, to mycotoxin contamination compared with F. esculentum. Moreover, rutin-derived quercetin appears to be more efficient in inhibiting aflatoxin biosynthesis than the parent compound. Copyright © 2014 Elsevier B.V. All rights reserved.
Aspergillus flavus infects several food and feed crops such as corn, cotton, peanuts and tree nut crops and contaminates the seed with carcinogenic aflatoxins. These susceptible crops contain rich reserves of lipids and fatty acids. The nature of relationship between lipids and the ability of the f...
The aspergilli show immense ecological and metabolic diversity. To date, the sequences of fifteen different Aspergillus genomes have been determined providing scientists with an exciting resource to improve the understanding of Aspergillus molecular genomics. Aspergillus flavus, one of the most wide...
Helal, G A; Sarhan, M M; Abu Shahla, A N K; Abou El-Khair, E K
The mycelial growth of Aspergillus flavus Link was completely inhibited using 1.5 (microl/ml or 2.0 (microl/ml of Cymbopogon citratus essential oil applied by fumigation or contact method in Czapek's liquid medium, respectively. This oil was found also to be fungicidal at the same concentrations. The sublethal doses 1.0 and 1.5 (microl/ml inhibited about 65% of fungal growth after five days of incubation and delayed conidiation as compared with the control. Microscopic observations using Light Microscope (LM), Scanning Electron Microscope (SEM) and Transmission Electron Microscope (TEM) were carried out to determine the ultra structural modifications of A. flavus hyphae after treatment with C. citratus essential oil. The hyphal diameter decreased and hyphal wall appeared as precipitates and disappeared in some regions. This oil also caused plasma membrane disruption and mitochondrial structure disorganization. Moreover, Ca(+2), K(+) and Mg(+2) leakages increased from the fumigated mycelium and its total lipid content decreased, while the saturated and unsaturated fatty acids increased. One of the most important results obtained during this study was the ability of C. citratus essential oil at its sublethal dose to completely inhibit aflatoxin B(1) production from A. flavus. These findings increase the possibility of exploiting C. citratus essential oil as an effective inhibitor of biodegradation and storage contaminating fungi and also in fruit juice preservation.
Gandomi, Hassan; Misaghi, Ali; Basti, Afshin Akhondzadeh; Bokaei, Saeed; Khosravi, Alireza; Abbasifar, Arash; Javan, Ashkan Jebelli
The effect of Zataria multiflora Boiss. essential oil (EO) against growth, spore production and aflatoxin formation by Aspergillus flavus ATCC 15546 was investigated in synthetic media as well as Iranian ultra-filtered white cheese in brine. EO effectively inhibited radial growth and spore production on potato dextrose agar (PDA) in a dose-dependent manner. At 200 ppm, the radial growth and sporulation reduced by 79.4% and 92.5%, respectively. The growth was completely prevented at EO400 ppm on PDA, and minimum fungicidal concentration (MFC) of the oil was estimated at 1000 ppm. The oil also significantly suppressed mycelial growth and aflatoxin synthesis in broth medium at all concentrations tested (P<0.05). At 150 ppm of EO, the mycelial growth and aflatoxin accumulation reduced by 90% and 99.4%, respectively. The EO at all concentrations tested, had an inhibitory effect against radial fungal growth and aflatoxin production by A. flavus in cheese. However, no concentration of EO examined was able to completely inhibit the growth and aflatoxin production in cheese. The results suggested the potential substitution of the antifungal chemicals by this EO as a natural inhibitor to control the growth of molds in foods such as cheese.
Aspergillus flavus is a pathogen of many agronomically important crops worldwide and can also cause human and animal diseases. A. flavus is the major producer of aflatoxins (AFs), which are carcinogenic secondary metabolites. In the United States, mycotoxins have been estimated to cause agricultur...
Gómez-Sánchez, Aída; Palou, Enrique; López-Malo, Aurelio
The antifungal activity of Mexican oregano (Lippia berlandieri Schauer) essential oil by gaseous contact on the growth of Aspergillus flavus at selected essential oil concentrations (14.7, 29.4, 58.8, or 117.6 μl of essential oil per liter of air) and temperatures (25, 30, or 35°C) was evaluated in potato dextrose agar formulated at water activity of 0.98 and pH 4.0. Mold growth curves were adequately fitted (0.984 < R(2) < 0.999) by the modified Gompertz model. The effect of the independent variables (concentration of essential oil and temperature) on the estimated model parameters (reciprocal of growth rate [1/ν(m)] and lag time [λ]) were evaluated through polynomial equations. Both ν(m) and λ were significantly (P < 0.05) affected by the independent variables; ν(m) decreased and λ increased as essential oil concentration increased and temperature decreased, which suggests that Mexican oregano essential oil retards or inhibits mold germination stage. Further, minimum fungistatic and fungicide essential oil concentrations at 30 and 35°C were determined. Mexican oregano essential oil applied in gas phase exerts important antifungal activity on the growth of A. flavus, suggesting its potential to inhibit other food spoilage molds.
Ahlberg, Sara; Joutsjoki, Vesa; Laurikkala, Sini; Varmanen, Pekka; Korhonen, Hannu
Certain strains of lactic acid bacteria have been reported to inhibit fungal growth and may so be potential as biocontrol agents. In this study, 171 LAB strains were isolated from traditional fermented Kenyan milk and maize products and tested against aflatoxin-producing A. flavus fungi. The three LAB strains showing highest antifungal activity were identified as Lactobacillus plantarum. None of the strains were able to completely inhibit fungal growth under conditions favorable for fungi and suboptimal for LAB. These conditions probably reduced the growth and metabolic activity of some LAB isolates, as several growth-related aspects like production of antifungal biomolecules and other metabolites contribute to the inhibiting activity. The results suggest that certain LAB strains could be employed in food to control the growth of aflatoxigenic fungi. Further studies to establish the efficacy of the potential LAB strains in fermented products are in progress.
Sun, Qi; Shang, Bo; Wang, Ling; Lu, Zhisong; Liu, Yang
Cinnamaldehyde (CIN) is a promising natural preservative and generally recognized as safe for commodities as well as consumers. In this work, the antifungal effects of CIN on Aspergillus flavus were evaluated both in solid and in liquid culture conditions. Our results indicated that CIN effectively inhibited radial growth, spore production, mycelium formation, and aflatoxin B1 biosynthesis by A. flavus in a dose-dependent manner. At the concentration of 104 mg L(-1), CIN exposure was able to completely inhibit fungal growth as well as aflatoxin B1 production. Furthermore, the inhibitory activities of CIN were closely connected with the treatment period and the tested fungal species. Compared with the control strains, CIN dose dependently changed the morphology and ultrastructure of mycelium in different degree. Especially, the reduction of hydrogen peroxide was considered to follow the destruction of mitochondrial. Meanwhile, CIN significantly cut the levels of lipid peroxidation and reduced glutathione. The activity of total superoxide dismutase was significantly inhibited after CIN treatment at the end of incubation, whereas the activities of catalase and glutathione peroxidase were opposite. These results indicated that the inhibitory effect of CIN could attribute to oxidative stress alleviation possibly induced by modifications of cellular structure as well as redox status.
Sindhu, S; Chempakam, B; Leela, N K; Suseela Bhai, R
Turmeric is well known for a wide range of medicinal properties. Essential oil of turmeric leaves (Curcuma longa L.) were evaluated at varying concentrations of 0.01, 0.05, 0.1, 0.5, 0.75, 1.0 and 1.5% (v/v) in Yeast Extract Sucrose (YES) broth inoculated with spore suspension of Aspergillus flavus of 10(6)conidia/ml. These were evaluated for their potential in the control of aflatoxigenic fungus A. flavus and aflatoxin production. Turmeric leaf oil exhibited 95.3% and 100% inhibition of toxin production respectively at 1.0% and 1.5%. The extent of inhibition of fungal growth and aflatoxin production was dependent on the concentration of essential oil used. The oil exhibited significant inhibition of fungal growth as well as aflatoxins B(1) and G(1) production. The LD(50) and LD(90) were also determined. GC-MS analysis of the oil showed α-phellandrene, p-cymene and terpinolene as the major components in turmeric leaf oil. The possibility of using these phytochemical components as bio-preservatives for storage of spices is discussed.
Yogendrarajah, Pratheeba; Vermeulen, An; Jacxsens, Liesbeth; Mavromichali, Evangelia; De Saeger, Sarah; De Meulenaer, Bruno; Devlieghere, Frank
The growth and mycotoxin production of three Aspergillus flavus isolates and an Aspergillus parasiticus isolate were studied in whole black peppercorns (Piper nigrum L.) using a full factorial design with seven water activity (aw) (0.826-0.984) levels and three temperatures (22, 30 and 37°C). Growth rates and lag phases were estimated using linear regression. Diverse secondary models were assessed for their ability to describe the radial growth rate as a function of individual and combined effect of aw and temperature. Optimum radial growth rate ranged from 0.75±0.04 to 2.65±0.02mm/day for A. flavus and 1.77±0.10 to 2.50±0.10mm/day for A. parasiticus based on the Rosso cardinal estimations. Despite the growth failure of some isolates at marginal conditions, all the studied models showed good performance to predict the growth rates. Validation of the models was performed on independently derived data. The bias factors (0.73-1.03), accuracy factors (0.97-1.36) and root mean square error (0.050-0.278) show that the examined models are conservative predictors of the colony growth rate of both fungal species in black peppers. The Rosso cardinal model can be recommended to describe the individual aw effect while the extended Gibson model was the best model for describing the combined effect of aw and temperature on the growth rate of both fungal species in peppercorns. Temperature optimum ranged from 30 to 33°C, while aw optimum was 0.87-0.92 as estimated by multi-factorial cardinal model for both species. The estimated minimum temperature and aw for A. flavus and A. parasiticus for growth were 11-16°C and 0.73-0.76, respectively, hence, achieving these conditions should be considered during storage to prevent the growth of these mycotoxigenic fungal species in black peppercorns. Following the growth study, production of mycotoxins (aflatoxins B1, B2, G1, G2, sterigmatocystin and O-methyl sterigmatocystin (OMST)) was quantified using LC-MS/MS. Very small
Singh, Priyanka; Shukla, Ravindra; Kumar, Ashok; Prakash, Bhanu; Singh, Shubhra; Dubey, Nawal Kishore
Essential oils extracted from Citrus reticulata and Cymbopogon citratus were tested in vitro against the toxigenic strain of Aspergillus flavus, isolated from the tuberous roots of Asparagus racemosus, used in preparation of herbal drugs. The essential oils completely inhibited the growth of A. flavus at 750 ppm and also exhibited a broad fungitoxic spectrum against nine additional fungi isolated from the roots. Citrus reticulata and Cymbopogon citratus essential oils completely inhibited aflatoxin B(1) production at 750 and 500 ppm, respectively. During in vivo investigation, the incidence of fungi and aflatoxin B(1) production decreased considerably in essential oil-treated root samples. The findings thus indicate possible exploitation of the essential oils as effective inhibitor of aflatoxin B(1) production and as post-harvest fungitoxicant of traditionally used plant origin for the control of storage fungi. These essential oils may be recommended as plant-based antifungals as well as aflatoxin B(1) suppressors in post-harvest processing of herbal samples.
Farzaneh, Mohsen; Shi, Zhi-Qi; Ahmadzadeh, Masoud; Hu, Liang-Bin; Ghassempour, Alireza
In this study, the treatment of pistachio nuts by Bacillus subtilis UTBSP1, a promising isolate to degrade aflatoxin B1 (AFB1), caused to reduce the growth of Aspergillus flavus R5 and AFB1 content on pistachio nuts. Fluorescence probes revealed that the cell free supernatant fluid from UTBSP1 affects spore viability considerably. Using high-performance liquid chromatographic (HPLC) method, 10 fractions were separated and collected from methanol extract of cell free supernatant fluid. Two fractions showed inhibition zones against A. flavus. Mass spectrometric analysis of the both antifungal fractions revealed a high similarity between these anti-A. flavus compounds and cyclic-lipopeptides of surfactin, and fengycin families. Coproduction of surfactin and fengycin acted in a synergistic manner and consequently caused a strong antifungal activity against A. flavus R5. There was a positive significant correlation between the reduction of A. flavus growth and the reduction of AFB1 contamination on pistachio nut by UTBSP1. The results indicated that fengycin and surfactin-producing B. subtilis UTBSP1 can potentially reduce A. flavus growth and AFB1 content in pistachio nut. PMID:27298596
Santos, L; Kasper, R; Sardiñas, N; Marín, S; Sanchis, V; Ramos, A J
The aim of this study was to determine the effect of a carotenoid mixture (Capsantal FS-30-NT), containing capsanthin and capsorubin, on growth and aflatoxins (AF) production of AF-producing Aspergillus flavus isolates. Each isolate, previously isolated from paprika and chilli, was inoculated on Czapek Yeast extract Agar (CYA) medium supplemented with different amounts of capsantal (0-1%) and incubated at 10, 15 and 25 °C during 21 days. Growth rates and lag phases were obtained, and AF production was determined at 7, 14 and 21 days. None of the isolates grew at 10 °C and one isolate (UdLTA 3.193) hardly grew at 15 °C. Capsantal addition had no effect over lag phases and growth rates at 15 °C. At 25 °C capsantal reduced growth rates and increased lag phases. However, the effect of capsantal on AF production was inconclusive, because it depended on temperature or time, and most of the times it was not significant. Low temperature has been a crucial factor in AF production, regardless of the capsantal concentration tested. Industrial storage temperature for paprika and chilli use to be approximately 10 °C, so if this temperature is maintained mould growth and AF production should be prevented.
Cyclopiazonic acid (CPA) is an indole-tetramic acid mycotoxin produced by some strains of Aspergillus flavus. Characterization of the CPA biosynthesis gene cluster confirmed that formation of CPA is via a three-enzyme pathway. This review examines the structure and organization of the CPA genes, elu...
Aspergillus flavus is an opportunistic pathogen of crops. It is important because it produces aflatoxin as a secondary metabolite in the seeds of a number of crops both before and after harvest. Aflatoxin is a potent carcinogen that is highly regulated in most countries. In the field, aflatoxin i...
Santos, L; Marin, S; Sanchis, V; Ramos, A J
The aim of this study was to determine the effect of some pre-harvest fungicides on growth and aflatoxin (AF) production of three Aspergillus flavus strains found in Capsicum powder. Each isolate, previously isolated from paprika, chilli and smoked paprika, was inoculated on yeast extract sucrose agar and on a 3% paprika extract agar medium supplemented with different fungicides and incubated at 20 and 30°C during 7 days. Growth measurements were obtained on days 3, 5 and 7, and the AF production was determined on day 7. The significance of the effects of the factors (strain, medium, temperature, time and fungicides) and their interaction over colony diameter and AF production was determined. Temperature constrained the effectiveness of fungicides in reducing growth, the fungicides being most effective at 20°C. The efficacy of the fungicides over AF production depended on the medium used and temperature. The most effective fungicides in inhibiting growth and AF production, regardless of the strain tested or applied conditions, were tebuconazole 25% and mancozeb 80% applied at a concentration of 0.75 and 3.5 g l(-1), respectively. Care should thus be taken in the choice of a suitable fungicide because their effectiveness may depend on intra-specific variation and temperature. Moreover, it is necessary to take into account that the most efficient fungicide in reducing growth is not always the best choice for pre-harvest treatments because it may promote AF production. Thus, the best fungicide is the one that can simultaneous prevent growth and AF production.
Cyclopiazonic acid (CPA) is an indole-tetramic acid neurotoxin produced by some of the same strains of A. flavus that produce aflatoxins and by some Aspergillus oryzae strains. Despite its discovery 40 years ago, few reviews of its toxicity and biosynthesis have been reported. This review examines w...
Lahouar, Amani; Marin, Sonia; Crespo-Sempere, Ana; Saïd, Salem; Sanchis, Vicente
Sorghum, which is consumed in Tunisia as human food, suffers from severe colonization by several toxigenic fungi and contamination by mycotoxins. The Tunisian climate is characterized by high temperature and humidity that stimulates mold proliferation and mycotoxin accumulation in foodstuffs. This study investigated the effects of temperature (15, 25 and 37°C), water activity (aw, between 0.85 and 0.99) and incubation time (7, 14, 21 and 28 d) on fungal growth and aflatoxin B1 (AFB1) production by three Aspergillus flavus isolates (8, 10 and 14) inoculated on sorghum grains. The Baranyi model was applied to identify the limits of growth and mycotoxin production. Maximum diameter growth rates were observed at 0.99 a(w) at 37°C for two of the isolates. The minimum aw needed for mycelial growth was 0.91 at 25 and 37°C. At 15°C, only isolate 8 grew at 0.99 a(w). Aflatoxin B1 accumulation could be avoided by storing sorghum at low water activity levels (≤0.91 a(w)). Aflatoxin production was not observed at 15°C. This is the first work on the effects of water activity and temperature on A. flavus growth and AFB1 production by A. flavus isolates on sorghum grains.
Aspergillus flavus is a fungal plant pathogen of many diverse crops including cotton, peanuts, maize, almond, and pistachio. During infection by A. flavus, crops are frequently contaminated with highly carcinogenic aflatoxins. A. flavus populations are composed of numerous vegetative compatibility g...
Aspergillus flavus is a major producer of aflatoxin and an opportunistic pathogen for a wide range of hosts. Understanding genotypic and phenotypic variations within strains of A. flavus is important for controlling disease and reducing aflatoxin contamination. A. flavus is multinucleate and predomi...
Aspergillus flavus is one of the most widely known species of Aspergillus. It was described as a species in 1809 and first reported as a plant pathogen in 1920. More recently, A. flavus has emerged as an important opportunistic pathogen and is now rec¬ognized as the second leading cause of aspergill...
Aspergillus flavus is a saprophyte and an opportunistic plant pathogen. It is capable of producing carcinogenic aflatoxins. We treated A. flavus CA42 with the volatile decanal and analyzed changes in the transcriptomic profiles at different stages of growth and development. Paired-end RNA-Seq reads ...
Aflatoxin contamination of food and feed occurs due to growth of Aspergillus flavus. This poses a serious health risk because of aflatoxin’s toxic and carcinogenic properties which negatively impact human and livestock health. Colonization and subsequent aflatoxin production by A. flavus is typicall...
Aldars-García, Laila; Ramos, Antonio J; Sanchis, Vicente; Marín, Sonia
Human exposure to aflatoxins in foods is of great concern. The aim of this work was to use predictive mycology as a strategy to mitigate the aflatoxin burden in pistachio nuts postharvest. The probability of growth and aflatoxin B1 (AFB1) production of aflatoxigenic Aspergillus flavus, isolated from pistachio nuts, under static and non-isothermal conditions was studied. Four theoretical temperature scenarios, including temperature levels observed in pistachio nuts during shipping and storage, were used. Two types of inoculum were included: a cocktail of 25 A. flavus isolates and a single isolate inoculum. Initial water activity was adjusted to 0.87. Logistic models, with temperature and time as explanatory variables, were fitted to the probability of growth and AFB1 production under a constant temperature. Subsequently, they were used to predict probabilities under non-isothermal scenarios, with levels of concordance from 90 to 100% in most of the cases. Furthermore, the presence of AFB1 in pistachio nuts could be correctly predicted in 70-81 % of the cases from a growth model developed in pistachio nuts, and in 67-81% of the cases from an AFB1 model developed in pistachio agar. The information obtained in the present work could be used by producers and processors to predict the time for AFB1 production by A. flavus on pistachio nuts during transport and storage.
Mehl, Hillary L.
The population dynamics of Aspergillus flavus, shaped in part by intraspecific competition, influence the likelihood and severity of crop aflatoxin contamination. Competition for nutrients may be one factor modulating intraspecific interactions, but the influences of specific types and concentrations of nutrients on competition between genotypes of A. flavus have not been investigated. Competition between paired A. flavus isolates on agar media was affected by varying concentrations of carbon (sucrose or asparagine) and nitrogen (nitrate or asparagine). Cocultivated isolate percentages from conidia and agar-embedded mycelia were quantified by measurements of isolate-specific single-nucleotide polymorphisms with quantitative pyrosequencing. Compositions and concentrations of nutrients influenced conidiation resulting from cocultivation, but the percentages of total conidia from each competing isolate were not predicted by sporulation of isolates grown individually. Success during sporulation did not reflect the outcomes of competition during mycelial growth, and the extents to which isolate percentages from conidia and mycelia differed varied among both isolate pairs and media. Whether varying concentrations of sucrose, nitrate, or asparagine increased, decreased, or had no influence on competitive ability was isolate dependent. Different responses of A. flavus isolates to nutrient variability suggest genotypes are adapted to different nutrient environments that have the potential to influence A. flavus population structure and the epidemiology of aflatoxin contamination. PMID:23263958
Chang, Perng-Kuang; Hua, Sui Sheng T; Sarreal, Siov Bouy L; Li, Robert W
The saprophytic soil fungus Aspergillus flavus infects crops and produces aflatoxin. Pichia anomala, which is a biocontrol yeast and produces the major volatile 2-phenylethanol (2-PE), is able to reduce growth of A. flavus and aflatoxin production when applied onto pistachio trees. High levels of 2-PE are lethal to A. flavus and other fungi. However, at low levels, the underlying mechanism of 2-PE to inhibit aflatoxin production remains unclear. In this study, we characterized the temporal transcriptome response of A. flavus to 2-PE at a subinhibitory level (1 μL/mL) using RNA-Seq technology and bioinformatics tools. The treatment during the entire 72 h experimental period resulted in 131 of the total A. flavus 13,485 genes to be significantly impacted, of which 82 genes exhibited decreased expression. They included those encoding conidiation proteins and involved in cyclopiazonic acid biosynthesis. All genes in the aflatoxin gene cluster were also significantly decreased during the first 48 h treatment. Gene Ontology (GO) analyses showed that biological processes with GO terms related to catabolism of propionate and branched-chain amino acids (valine, leucine and isoleucine) were significantly enriched in the down-regulated gene group, while those associated with ribosome biogenesis, translation, and biosynthesis of α-amino acids OPEN ACCESS Toxins 2015, 7 3888 were over-represented among the up-regulated genes. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that metabolic pathways negatively impacted among the down-regulated genes parallel to those active at 30 °C, a condition conducive to aflatoxin biosynthesis. In contrast, metabolic pathways positively related to the up-regulated gene group resembled those at 37 °C, which favors rapid fungal growth and is inhibitory to aflatoxin biosynthesis. The results showed that 2-PE at a low level stimulated active growth of A. flavus but concomitantly rendered decreased activities in
Mousa, Wael; Ghazali, Farinazleen Mohd; Jinap, Selamat; Ghazali, Hasanah Mohd; Radu, Son
The aim of this study was to model the radial growth rate and to assess aflatoxin production by Aspergillus flavus as a function of water activity (a(w) 0.82 to 0.92) and temperature (12 to 42 °C) on polished and brown rice. The growth of the fungi, expressed as colony diameter (mm) was measured daily, and the aflatoxins were analyzed using HPLC with a fluorescence detector. The growth rates were estimated using the primary model of Baranyi, which describes the change in colony radius as a function of time. Total of 2 secondary models were used to describe the combined effects of a(w) and temperature on the growth rates. The models were validated using independent experimental data. Linear Arrhenius-Davey model proved to be the best predictor of A. flavus growth rates on polished and brown rice followed by polynomial model. The estimated optimal growth temperature was around 30 °C. A. flavus growth and aflatoxins were not detected at 0.82 a(w) on polished rice while growth and aflatoxins were detected at this a(w) between 25 and 35 °C on brown rice. The highest amounts of toxins were formed at the highest a(w) values (0.90 to 0.92) at a temperature of 20 °C after 21 d of incubation on both types of rice. Nevertheless, the consistencies of toxin production within a wider range of a(w) values occurred between 25 to 30 °C. Brown rice seems to support A. flavus growth and aflatoxin production more than the polished rice. The developed models can be used to estimate to what extent the change in grain ecosystem conditions affect the storage stability and safety of grains without the need for running long-standing storage study. By monitoring the intergranular relative humidity and temperature at different locations in the storage facility and inputting these data into the models, it is directly possible to assess either the conditions are conductive for the growth of A. flavus or aflatoxin production. © 2012 Institute of Food Technologists®
Hua, Sui Sheng T; Beck, John J; Sarreal, Siov Bouy L; Gee, Wai
Aspergillus flavus is a ubiquitous saprophyte that is able to produce the most potent natural carcinogenic compound known as aflatoxin B1 (AFB1). This toxin frequently contaminates crops including corn, cotton, peanuts, and tree nuts causing substantial economic loss worldwide. Consequently, more than 100 countries have strict regulations limiting AFB1 in foodstuffs and feedstuffs. Plants and microbes are able to produce volatile compounds that act as a defense mechanism against other organisms. Pichia anomala strain WRL-076 is a biocontrol yeast currently being tested to reduce AF contamination of tree nuts in California. We used the SPME-GC/MS analysis and identified the major volatile compound produced by this strain to be 2-phenylethanol (2-PE). It inhibited spore germination and AF production of A. flavus. Inhibition of AF formation by 2-PE was correlated with significant down regulation of clustering AF biosynthesis genes as evidenced by several to greater than 10,000-fold decrease in gene expression. In a time-course analysis we found that 2-PE also altered the expression patterns of chromatin modifying genes, MYST1, MYST2, MYST3, gcn5, hdaA and rpdA. The biocontrol capacity of P. anomala can be attributed to the production of 2-PE, which affects spore germination, growth, toxin production, and gene expression in A. flavus.
Mateo, Eva M; Gómez, José Vicente; Gimeno-Adelantado, José Vicente; Romera, David; Mateo-Castro, Rufino; Jiménez, Misericordia
Aspergillus flavus is a highly aflatoxin (AF)-producing species infecting maize and other crops. It is dominant in tropical regions, but it is also considered an emerging problem associated with climate change in Europe. The aim of this study was to assess the efficacy of azole fungicides (prochloraz, tebuconazole and a 2:1 (w/w) mixture of prochloraz plus tebuconazole) to control the growth of A. flavus and AF production in yeast-extract-sucrose (YES) agar and in maize kernels under different water activities (aw) and temperatures. Aflatoxins B1 and B2 were determined by LC with fluorescence detection and post-column derivatisation of AFB1. In YES medium and maize grains inoculated with conidia of A. flavus, the growth rate (GR) of the fungus and AFB1 and AFB2 production were significantly influenced by temperature and treatment. In YES medium and maize kernels, optimal temperatures for GR and AF production were 37 and 25°C, respectively. In maize kernels, spore germination was not detected at the combination 37ºC/0.95 aw; however, under these conditions germination was found in YES medium. All fungicides were more effective at 0.99 than 0.95 aw, and at 37 than 25ºC. Fungicides effectiveness was prochloraz > prochloraz plus tebuconazole (2:1) > tebuconazole. AFs were not detected in cultures containing the highest fungicide doses, and only very low AF levels were found in cultures containing 0.1 mg l(-)(1) prochloraz or 5.0 mg l(-)(1) tebuconazole. Azoles proved to be highly efficient in reducing A. flavus growth and AF production, although stimulation of AF production was found under particular conditions and low-dosage treatments. Maize kernels were a more favourable substrate for AF biosynthesis than YES medium. This paper is the first comparative study on the effects of different azole formulations against A. flavus and AF production in a semi-synthetic medium and in maize grain under different environmental conditions.
Sidhu, O P; Chandra, Harish; Behl, H M
Occurrence of aflatoxin in Madhuca indica Gmel. seeds was determined by competitive ELISA. Eighty percent of mahua seed samples were found to be contaminated with aflatoxin. Total aflatoxin content ranged from 115.35 to 400.54ppb whereas the concentration of AFB(1) was in the range of 86.43 to 382.45ppb. Mahua oil was extracted by cold press expeller and analysed for contamination of aflatoxin in both the oil and cake samples. Total aflatoxin and aflatoxin B(1) were 220.66 and 201.57ppb in oil as compared to that in cake samples where it was 87.55 and 74.35ppb, respectively. Various individual and combined plant extracts were evaluated for their efficacy against growth of Aspergillus flavus and aflatoxin production in vitro. Combination of botanicals were found to be more effective in controlling fungal growth and aflatoxin production than individual extracts. Results of the present study suggests that synergistic effect of plant extracts can be used for control of fungal growth and aflatoxin production. These natural plant products may successfully replace synthetic chemicals and provide an alternative method to protect mahua as well as other agricultural commodities of nutritional significance from toxigenic fungi such as A. flavus and aflatoxin production.
Garcia, Daiana; Ramos, Antonio J; Sanchis, Vicente; Marín, Sonia
Cereals are a very important part of the human and animal diets. However, agricultural products can be contaminated by moulds and their mycotoxins. Plant extracts, particularly those of Equisetum arvense and Stevia rebaudiana have been reported previously to contain antioxidant compounds which may have antifungal properties. In this study, E. arvense and S. rebaudiana extracts were tested for their control of mycotoxigenic fungi in maize. The extracts were tested separately and as a mixture for their effect on growth of Aspergillus flavus and Fusarium verticillioides. Extracts were added to unsterilised inoculated maize at different water activity (a(w)) levels (0.85-0.95). Moulds were inoculated and incubated for 30 days. Results confirmed that the extract of E. arvense and a mixture 1:1 of Equisetum-Stevia may be effective for the inhibition of both growth of A. flavus and aflatoxin production at high water activity levels (pre-harvest conditions). In general, growth of the F. verticillioides was reduced by the use of plant extracts, especially at 0.95 a(w). However, fumonisin presence was not significantly affected. E. arvense and S. rebaudiana extracts could be developed as an alternative treatment to control aflatoxigenic mycobiota in moist maize.
Aspergillus flavus is a fungal pathogen of many agronomically important crops worldwide. We sampled A. flavus strains from a cornfield in Rocky Mount, North Carolina, over a period of two years. The field was planted in 2010 and plots were inoculated at tasselling with either AF36 or NRRL 21882 (=Af...
Aspergillus flavus is the major producer of carcinogenic aflatoxins worldwide in crops. Populations of A. flavus are characterized by high genetic variation and the source of this variation is likely sexual reproduction. The fungus is heterothallic and laboratory crosses produce ascospore-bearing ...
Aspergillus flavus is a fungal pathogen of many agronomically important crops worldwide. We sampled A. flavus strains from a cornfield in Rocky Mount, NC. This field was planted in 2010 and plots were inoculated at tasselling with either AF36 or NRRL 21882 (=Afla-Guard) biocontrol strains, both of...
Aspergillus flavus is a fungal pathogen of many agronomically important crops worldwide. We sampled A. flavus strains from a cornfield in Rocky Mount, NC. This field was planted in 2010 and plots were inoculated at tasselling with either AF36 or NRRL 21882 (=Afla-Guard) biocontrol strains, both of...
Aspergillus flavus is a common saprophyte and opportunistic pathogen that survives in the natural environment by extracting nutrition from plant debris, insect carcasses and a variety of other carbon sources. A. flavus produces numerous secondary metabolites and hydrolytic enzymes. The primary obj...
Verheecke, C; Liboz, T; Anson, P; Diaz, R; Mathieu, F
The aim of this study is to investigate aflatoxin gene expression during Streptomyces-Aspergillus interaction. Aflatoxins are carcinogenic compounds produced mainly by Aspergillus flavus and Aspergillus parasiticus. A previous study has shown that Streptomyces-A. flavus interaction can reduce aflatoxin content in vitro. Here, we first validated this same effect in the interaction with A. parasiticus. Moreover, we showed that growth reduction and aflatoxin content were correlated in A. parasiticus but not in A. flavus. Secondly, we investigated the mechanisms of action by reverse-transcriptase quantitative PCR. As microbial interaction can lead to variations in expression of household genes, the most stable [act1, βtub (and cox5 for A. parasiticus)] were chosen using geNorm software. To shed light on the mechanisms involved, we studied during the interaction the expression of five genes (aflD, aflM, aflP, aflR and aflS). Overall, the results of aflatoxin gene expression showed that Streptomyces repressed gene expression to a greater level in A. parasiticus than in A. flavus. Expression of aflR and aflS was generally repressed in both Aspergillus species. Expression of aflM was repressed and was correlated with aflatoxin B1 content. The results suggest that aflM expression could be a potential aflatoxin indicator in Streptomyces species interactions. Therefore, we demonstrate that Streptomyces can reduce aflatoxin production by both Aspergillus species and that this effect can be correlated with the repression of aflM expression.
Aspergillus flavus is a globally distributed fungus and an important food contaminant because it produces the most potent natural carcinogenic compound known as aflatoxin (AF) B1. The major volatile from a yeast strain, Pichia anomala WRL-076 was identified by SPEM-GC/MS analysis to be 2-phenylethan...
The genomic study of filamentous fungi has made significant advances in recent years, and the genomes of several species in the genus Aspergillus have been sequenced, including Aspergillus flavus. This ubiquitous mold is present as a saprobe in a wide range of agricultural and natural habits, and c...
Lillard, H. S.; Hanlin, R. T.; Lillard, D. A.
Of 120 isolates of the Aspergillus flavus group from pecans used in bakery products, 85 were shown to produce aflatoxin on yeast extract sucrose medium. Extracts from moldy sections of raw pecans obtained commercially at the retail level showed aflatoxin-like spots on thin-layer chromatography. Cooked (autoclaved) pecans inoculated with toxigenic isolates of A. flavus were also good substrates for aflatoxin production. PMID:5415207
Jantapan, Kittika; Poapolathep, Amnart; Imsilp, Kanjana; Poapolathep, Saranya; Tanhan, Phanwimol; Kumagai, Susumu; Jermnak, Usuma
The antiaflatoxigenic and antifungal activities of essential oils (EOs) of finger root (Boesenbergia rotunda (L.) Mansf.), pine (Pinus pinaster), rosewood (Aniba rosaedora), Siam benzoin (Styrax tonkinensis), Thai moringa (Moringa oleifera), and ylang ylang (Cananga odorata) were tested for Aspergillus parasiticus and Aspergillus flavus in potato dextrose broth. Aflatoxin B1 (AFB1) was extracted from culture using a QuEChERS-based extraction procedure and analyzed with high performance liquid chromatography (HPLC) coupled to a fluorescence detector. EO of pine showed the greatest inhibition of growth and AFB1 production of A. parasiticus, followed by EOs of rosewood, finger root, Siam benzoin, and ylang ylang. EO of finger root gave the best inhibitory effects on A. flavus, followed by EOs of rosewood, pine, ylang ylang, and Siam benzoin. EO of Thai moringa did not show any significant inhibition of aflatoxigenic fungi. The antiaflatoxigenic activities of EOs correlated with their antifungal activities in the dosedependent manner. Comparison of the application of the five selected EOs in peanut pods by direct and vapor exposure indicated that the AFB1 production inhibitory effects of the five EOs by direct exposure were faster and more effective than by vapor exposure. EO of finger root showed the best inhibition of AFB1 production of A. flavus in peanut pods by direct exposure, followed by EOs of pine, rosewood, ylang ylang, and Siam benzoin.
Description of Aspergillus flavus growth under the influence of different factors (water activity, incubation temperature, protein and fat concentration, pH, and cinnamon essential oil concentration) by kinetic, probability of growth, and time-to-detection models.
Kosegarten, Carlos E; Ramírez-Corona, Nelly; Mani-López, Emma; Palou, Enrique; López-Malo, Aurelio
A Box-Behnken design was used to determine the effect of protein concentration (0, 5, or 10g of casein/100g), fat (0, 3, or 6g of corn oil/100g), aw (0.900, 0.945, or 0.990), pH (3.5, 5.0, or 6.5), concentration of cinnamon essential oil (CEO, 0, 200, or 400μL/kg) and incubation temperature (15, 25, or 35°C) on the growth of Aspergillus flavus during 50days of incubation. Mold response under the evaluated conditions was modeled by the modified Gompertz equation, logistic regression, and time-to-detection model. The obtained polynomial regression models allow the significant coefficients (p<0.05) for linear, quadratic and interaction effects for the Gompertz equation's parameters to be identified, which adequately described (R(2)>0.967) the studied mold responses. After 50days of incubation, every tested model system was classified according to the observed response as 1 (growth) or 0 (no growth), then a binary logistic regression was utilized to model A. flavus growth interface, allowing to predict the probability of mold growth under selected combinations of tested factors. The time-to-detection model was utilized to estimate the time at which A. flavus visible growth begins. Water activity, temperature, and CEO concentration were the most important factors affecting fungal growth. It was observed that there is a range of possible combinations that may induce growth, such that incubation conditions and the amount of essential oil necessary for fungal growth inhibition strongly depend on protein and fat concentrations as well as on the pH of studied model systems. The probabilistic model and the time-to-detection models constitute another option to determine appropriate storage/processing conditions and accurately predict the probability and/or the time at which A. flavus growth occurs. Copyright Â© 2016 Elsevier B.V. All rights reserved.
Thippeswamy, B; Shivakumar, C K; Krishnappa, M
In the present study Aspergillus niger and Aspergillus flavus isolated from paper mill effluent showed tolerance and accumulation of toxic metals Ni, Zn, Cd, Pb, Cr and Cu from synthetic medium and paper mill effluent. Physico-chemical and heavy metals characterization of industrially treated paper mill effluent showed insignificant reduction in BOD, hardness, TDS and heavy metals as compared to permissible limits of BIS and WHO. A. niger and A. flavus were treated with synthetic medium containing 100-1000 mg l(-1) of six heavy metals. A. niger was able to tolerate and grow in 1000 mg l(-1) Pb, 500 mg l(-1) Cu, 250 mg l(-1) Zn and 100 mg l(-1) Cr, Ni respectively. No growth of A. niger was observed in 100 mg l-(-1) of Cd. A. flavus was capable to tolerate and grow in 1000 mg l(-1) Pb, Zn and Ni, 100mg l(-1) Cu. A. flavus growth was completely inhibited in 100 mg l(-1) of Cd and Cr. The Cd, Zn, Cu and Pb reduction were found significant (p < 0.05) in the paper effluent inoculated with A. niger and A. flavus biomass compared to industrial treated effluent. A. niger and A. flavus accumulated maximum of Pb (75.82%) followed by Zn (49.40%) > Cu (45.34%) > Ni (25.20%), while only 41% Cr was accumulated by A. nigerfrom 100 mg l(-1) of Cr solution.
Aspergillus flavus and A. parasiticus are two of the most important aflatoxin-producing species that contaminate agricultural commodities worldwide. Both species are heterothallic and undergo sexual reproduction in laboratory crosses. Here, we examine the possibility of interspecific matings betwe...
The navel orangeworm (Amyelois transitella), a pest of California tree nuts, is associated with the fungus Aspergillus flavus, and mounting evidence suggests that these two species are facultative mutualists. Navel orangeworm larvae exhibit improved growth and survival on diets containing this fungu...
Zablotowicz, R M; Abbas, H K; Locke, M A
Understanding the source of Aspergillus flavus is required to manage aflatoxin contamination of maize (Zea mays L.). Studies assessed A. flavus propagules, Fusarium spp., and total fungi associated with Mississippi Delta soils. Soils from 12 and 15 sites were collected in 2000 and 2001, respectively. The propagule density of A. flavus ranged from log(10) 2.0 to 4.3 colony-forming units (cfu) g(-1) soil, while total fusaria ranged from log(10) 3.0 to 5.4 cfu g(-1) soil. The highest populations of A. flavus were associated with soils containing higher organic matter, especially in sites under a no-tillage management. The frequency of aflatoxin production in isolates ranged from 13 to 81% depending on soil. In 2001, there was a highly significant correlation between A. flavus and the history of maize cultivation. Soil fertility factors such as organic matter content, nitrate and extractable phosphorus correlated with the density of Aspergillus, Fusarium spp., and total fungi. The relationship between soil parameters and Aspergillus populations may be useful in predicting the contribution of soil microflora to aflatoxin contamination.
Wang, Houmiao; Lei, Yong; Yan, Liying; Wan, Liyun; Ren, Xiaoping; Chen, Silong; Dai, Xiaofeng; Guo, Wei; Jiang, Huifang; Liao, Boshou
In the Aspergillus flavus (A. flavus)–peanut pathosystem, development and metabolism of the fungus directly influence aflatoxin contamination. To comprehensively understand the molecular mechanism of A. flavus interaction with peanut, RNA-seq was used for global transcriptome profiling of A. flavus during interaction with resistant and susceptible peanut genotypes. In total, 67.46 Gb of high-quality bases were generated for A. flavus-resistant (af_R) and -susceptible peanut (af_S) at one (T1), three (T2) and seven (T3) days post-inoculation. The uniquely mapped reads to A. flavus reference genome in the libraries of af_R and af_S at T2 and T3 were subjected to further analysis, with more than 72% of all obtained genes expressed in the eight libraries. Comparison of expression levels both af_R vs. af_S and T2 vs. T3 uncovered 1926 differentially expressed genes (DEGs). DEGs associated with mycelial growth, conidial development and aflatoxin biosynthesis were up-regulated in af_S compared with af_R, implying that A. flavus mycelia more easily penetrate and produce much more aflatoxin in susceptible than in resistant peanut. Our results serve as a foundation for understanding the molecular mechanisms of aflatoxin production differences between A. flavus-R and -S peanut, and offer new clues to manage aflatoxin contamination in crops. PMID:26891328
Pichia anomalais, which produces the antimicrobial volatile 2-phenylethanol (2-PE), is effective in reducing A. flavus growth and aflatoxin production. We treated A. flavus NRRL3357 with 2-PE and analyzed changes in the transcriptomic profiles at different stages of fungal growth. RNA-Seq reads from...
Aspergillus flavus and A. parasiticus are potent producers of carcinogenic and hepatotoxic aflatoxins, polyketide-derived secondary metabolites that contaminate a wide variety of agricultural crops. Strains with opposite mating-type genes MAT1-1 and MAT1-2 within each species were crossed in an att...
Aspergillus flavus infects both plants and animals, and is of toxicological importance due to its production of aflatoxins (AFs) and other mycotoxins. Mycotoxins can cause agricultural losses totaling upwards of $1.4 billion annually. Recent efforts to reduce AF concentrations have focused on the us...
Thirty eight Aspergillus flavus isolates collected from a pistachio orchard in California were analyzed for production of aflatoxin (AF), cyclopiazonic acid (CPA), vegetative compatibility groups (VCGs) and mating types. All toxigenic isolates produced both AFB1 and CPA. Twenty-one percent of the i...
Introduction: Aflatoxigenic Aspergillus flavus is a frequent contaminant of agricultural commodities such as corn, peanut, tree nuts and cottonseed. Ingestion of foods, especially corn, contaminated with aflatoxins has been implicated in acute toxicoses while chronic, low-level exposure can lead to...
Aspergillus flavus is a well-known pathogen of many important agricultural commodities and is a major producer of aflatoxins, which are carcinogenic polyketides that pose a serious health risk to humans and animals. Aflatoxin contamination in peanut exports worldwide accounts for as much as $450 mi...
The colonization of crops by Aspergillus flavus results in the production of aflatoxins. Aflatoxin production is also exacerbated by abiotic stresses in the field. Here, we investigated the role of reactive oxygen species (ROS), which accumulate in plant tissues in response to drought and heat stres...
Nogueira, Juliana H C; Gonçalez, Edlayne; Galleti, Silvia R; Facanali, Roseane; Marques, Márcia O M; Felício, Joana D
Aflatoxin B(1) (AFB(1)) is a highly toxic and carcinogenic metabolite produced by Aspergillus species on food and agricultural commodities. Inhibitory effects of essential oil of Ageratum conyzoides, on the mycelial growth and aflatoxin B(1) production by Aspergillus flavus were studied. Cultures were incubated in yeast extract-sucrose (YES) broth for days at 25 degrees C at the following different concentrations of the essential oil (from 0.0 to 30mug/mL). The essential oil inhibited fungal growth to different extents depending on the concentration, and completely inhibited aflatoxin production at concentrations above 0.10microg/mL. The analysis of the oil by GC/MS showed that its main components are precocene II (46.35%), precocene I (42.78%), cumarine (5.01%) and Trans-caryophyllene (3.02%). Comparison by transmission electron microscopy of the fungal cells, control and those incubated with different concentrations of essential oil, showed ultra-structural changes which were concentration dependent of the essential oil of A. conyzoides. Such ultra-structural changes were more evident in the endomembrane system, affecting mainly the mitochondria. Degradation was also observed in both surrounding fibrils. The ability to inhibit aflatoxin production as a new biological activity of A.conyzoides L. indicates that it may be considered as a useful tool for a better understanding of the complex pathway of aflatoxin biosynthesis.
Wang, Xiaoyun; Wang, Youzhi; Zhou, Yuguang; Wei, Xinli
Farnesol (FOH) is known to induce apoptosis in some fungi and mammalian cells. We treated Aspergillus flavus, one of the leading causes of human invasive aspergillosis and a key producer of the most potent naturally occurring hepatocarcinogenic compounds, with FOH to assess its effect on the viability of the fungus. FOH strongly inhibited germination and growth of A. flavus and induced markers for apoptosis including nuclear condensation, phosphatidylserine (PS) externalization, DNA fragmentation and intracellular reactive oxygen species (ROS) generation, metacaspase activation and abnormal cellular ultrastructure. Moreover, FOH-induced apoptosis in A. flavus was inhibited by the broad-spectrum caspase inhibitor Z-VAD-fmk and partially inhibited by the ROS scavenger l-proline, which suggests that FOH induces apoptosis in A. flavus via a mechanism involving metacaspase activation and ROS production.
DiCrispino, Kevin; Yao, Haibo; Hruska, Zuzana; Brabham, Kori; Lewis, David; Beach, Jim; Brown, Robert L.; Cleveland, Thomas E.
Aflatoxin contaminated corn is dangerous for domestic animals when used as feed and cause liver cancer when consumed by human beings. Therefore, the ability to detect A. flavus and its toxic metabolite, aflatoxin, is important. The objective of this study is to measure A. flavus growth using hyperspectral technology and develop spectral signatures for A. flavus. Based on the research group's previous experiments using hyperspectral imaging techniques, it has been confirmed that the spectral signature of A. flavus is unique and readily identifiable against any background or surrounding surface and among other fungal strains. This study focused on observing changes in the A. flavus spectral signature over an eight-day growth period. The study used a visible-near-infrared hyperspectral image system for data acquisition. This image system uses focal plane pushbroom scanning for high spatial and high spectral resolution imaging. Procedures previously developed by the research group were used for image calibration and image processing. The results showed that while A. flavus gradually progressed along the experiment timeline, the day-to-day surface reflectance of A. flavus displayed significant difference in discreet regions of the wavelength spectrum. External disturbance due to environmental changes also altered the growth and subsequently changed the reflectance patterns of A. flavus.
Scarpari, Marzia; Punelli, Marta; Scala, Valeria; Zaccaria, Marco; Nobili, Chiara; Ludovici, Matteo; Camera, Emanuela; Fabbri, Anna A.; Reverberi, Massimo; Fanelli, Corrado
In some filamentous fungi, the pathways related to the oxidative stress and oxylipins production are involved both in the process of host-recognition and in the pathogenic phase. In fact, recent studies have shown that the production of oxylipins in filamentous fungi, yeasts and chromists is also related to the development of the organism itself and to mechanisms of communication with the host at the cellular level. The oxylipins, also produced by the host during defense reactions, are able to induce sporulation and to regulate the biosynthesis of mycotoxins in several pathogenic fungi. In A. flavus, the oxylipins play a crucial role as signals for regulating the biosynthesis of aflatoxins, the conidiogenesis and the formation of sclerotia. To investigate the involvement of an oxylipins based cross-talk into Z. mays and A. flavus interaction, we analyzed the oxylipins profile of the wild type strain and of three mutants of A. flavus that are deleted at the Aflox1 gene level also during maize kernel invasion. A lipidomic approach has been addressed through the use of LC-ToF-MS, followed by a statistical analysis of the principal components (PCA). The results showed the existence of a difference between the oxylipins profile generated by the WT and the mutants onto challenged maize. In relation to this, aflatoxin synthesis which is largely hampered in vitro, is intriguingly restored. These results highlight the important role of maize oxylipin in driving secondary metabolism in A. flavus. PMID:24578700
Scully, Lisa R; Bidochka, Michael J
In order to study fungal pathogen evolution, we used a model system whereby the opportunistic fungus Aspergillus flavus was serially propagated through the insect (Galleria mellonella) larvae, yielding a cysteine/methionine auxotroph of A. flavus with properties of an obligate insect pathogen. The auxotroph exhibited insect host restriction but did not show any difference in virulence when compared with the wild-type (Scully LR, Bidochka MJ, 2006. Microbiology 152, 223-232). Here, we report that on 1% insect cuticle medium and synthetic Galleria medium, the auxotroph displayed increased extracellular protease production, a virulence factor necessary for insect pathogenesis. In the wild-type strain, protease production was deregulated during carbon (glucose), nitrogen (nitrate), or sulphate deprivation. If all three were present, protease production was vastly reduced. However, in the cysteine/methionine auxotroph, protease production was deregulated in complete medium. We suggest that the deficiency in sulphate assimilation in the auxotroph resulted in deregulation of protease production. The auxotroph exhibited delayed germination and slower hyphal growth when compared to the wild-type but there were no differences in virulence or cuticle penetration, suggesting a shift in pathogenic strategy that compensated decreased growth with increased virulence factor (extracellular protease) production. We concluded that the biosynthetic deficiency that mediated insect host restriction also increased protease production in the slow-growing auxotroph, resulting in an alternate, more host-specific pathogenic strategy. However, we argue that transmission is not necessarily correlated with virulence as competition bioassays in insect larvae showed that the wild-type generally out-competed the auxotroph by producing the majority of the conidia on the sporulating cadavers. This is one of the few examples that highlight the effect of genome decay on nutrition acquisition
Aspergillus flavus (Link) accumulates aflatoxins in peanuts, mainly affecting immature kernels during drought. Peanut invasion by A. flavus induces synthesis of phytoalexins, mostly stilbenoids, as a plant defense mechanism. Fungal laccases are often related to pathogenicity, and among other subst...
Aspergillus flavus produces aflatoxins and is the second leading cause of aspergillosis in immunocompromised individuals. Aspergillus oryzae, on the other hand, has been used for centuries in Japan for the fermentation of food. The recently available whole genome sequences of Aspergillus flavus an...
Zhang, Feng; Zhong, Hong; Han, Xiaoyun; Guo, Zhenni; Yang, Weiqiang; Liu, Yongfeng; Yang, Kunlong; Zhuang, Zhenhong; Wang, Shihua
Aspergillus flavus, a common contaminant of crops and stored grains, can produce aflatoxins that are harmful to humans and other animals. Water activity (aw) is one of the key factors influencing both fungal growth and mycotoxin production. In this study, we used the isobaric tagging for relative and absolute quantitation (iTRAQ) technique to investigate the effect of aw on the proteomic profile of A. flavus. A total of 3566 proteins were identified, of which 837 were differentially expressed in response to variations in aw. Among these 837 proteins, 403 were over-expressed at 0.99 aw, whereas 434 proteins were over-expressed at 0.93 aw. According to Gene Ontology (GO) analysis, the secretion of extracellular hydrolases increased as aw was raised, suggesting that extracellular hydrolases may play a critical role in induction of aflatoxin biosynthesis. On the basis of Clusters of Orthologous Groups (COG) and Kyoto Encyclopedia of Genes and Genomes (KEGG) categorizations, we identified an exportin protein, KapK, that may down-regulate aflatoxin biosynthesis by changing the location of NirA. Finally, we considered the role of two osmotic stress-related proteins (Sln1 and Glo1) in the Hog1 pathway and investigated the expression patterns of proteins related to aflatoxin biosynthesis. The data uncovered in this study are critical for understanding the effect of water stress on toxin production and for the development of strategies to control toxin contamination of agricultural products. Copyright © 2014 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.
Han, Xiaoyun; Qiu, Mengguang; Wang, Bin; Yin, Wen-Bing; Nie, Xinyi; Qin, Qiuping; Ren, Silin; Yang, Kunlong; Zhang, Feng; Zhuang, Zhenhong; Wang, Shihua
In Aspergillus nidulans, the nitrogen metabolite repression (NMR) regulator NmrA plays a major role in regulating the activity of the GATA transcription factor AreA during nitrogen metabolism. However, the function of nmrA in A. flavus has not been previously studied. Here, we report the identification and functional analysis of nmrA in A. flavus. Our work showed that the amino acid sequences of NmrA are highly conserved among Aspergillus species and that A. flavus NmrA protein contains a canonical Rossmann fold motif. Deletion of nmrA slowed the growth of A. flavus but significantly increased conidiation and sclerotia production. Moreover, seed infection experiments indicated that nmrA is required for the invasive virulence of A. flavus. In addition, the ΔnmrA mutant showed increased sensitivity to rapamycin and methyl methanesulfonate, suggesting that nmrA could be responsive to target of rapamycin signaling and DNA damage. Furthermore, quantitative real-time reverse transcription polymerase chain reaction analysis suggested that nmrA might interact with other nitrogen regulatory and catabolic genes. Our study provides a better understanding of NMR and the nitrogen metabolism network in fungi.
Han, Xiaoyun; Qiu, Mengguang; Wang, Bin; Yin, Wen-Bing; Nie, Xinyi; Qin, Qiuping; Ren, Silin; Yang, Kunlong; Zhang, Feng; Zhuang, Zhenhong; Wang, Shihua
In Aspergillus nidulans, the nitrogen metabolite repression (NMR) regulator NmrA plays a major role in regulating the activity of the GATA transcription factor AreA during nitrogen metabolism. However, the function of nmrA in A. flavus has not been previously studied. Here, we report the identification and functional analysis of nmrA in A. flavus. Our work showed that the amino acid sequences of NmrA are highly conserved among Aspergillus species and that A. flavus NmrA protein contains a canonical Rossmann fold motif. Deletion of nmrA slowed the growth of A. flavus but significantly increased conidiation and sclerotia production. Moreover, seed infection experiments indicated that nmrA is required for the invasive virulence of A. flavus. In addition, the ΔnmrA mutant showed increased sensitivity to rapamycin and methyl methanesulfonate, suggesting that nmrA could be responsive to target of rapamycin signaling and DNA damage. Furthermore, quantitative real-time reverse transcription polymerase chain reaction analysis suggested that nmrA might interact with other nitrogen regulatory and catabolic genes. Our study provides a better understanding of NMR and the nitrogen metabolism network in fungi. PMID:27933036
Jafarinasab, Mohammad-Reza; Feizi, Sepehr; Yazdizadeh, Forouzan; Rezaei Kanavi, Mozhgan; Moein, Hamid-Reza
Purpose To report the clinical, microbiologic, confocal scan and histopathologic features of Aspergillus flavus keratitis which developed immediately after deep anterior lamellar keratoplasty (DALK). Case Report A 28-year-old woman underwent DALK using the big-bubble technique for keratoconus. The operation was uneventful, yielding a bare Descemet’s membrane (DM) followed by transplantation of a corneal graft devoid of DM and endothelium. Four days after keratoplasty, mild infiltrates were noticed in the inferonasal margin of the graft, which rapidly progressed to involve the adjacent recipient cornea. Confocal scan findings suggested filamentous fungal keratitis, leading to initiation of topical and systemic antifungal medications followed by immediate replacement of the graft. Histopathologic examination disclosed keratitis caused by a filamentous fungus, which was determined by microbiologic cultures to be Aspergillus flavus. Early diagnosis and appropriate management resulted in complete recovery from this potentially devastating infection. Conclusion Aspergillus Flavus can cause graft ulcers immediately after DALK. Confocal scan proved to be a valuable tool for early diagnosis and prompt intervention to control this otherwise devastating infection. PMID:23275826
Abdel-Hadi, Ahmed; Schmidt-Heydt, Markus; Parra, Roberto; Geisen, Rolf; Magan, Naresh
A microarray analysis was used to examine the effect of combinations of water activity (aw, 0.995–0.90) and temperature (20–42°C) on the activation of aflatoxin biosynthetic genes (30 genes) in Aspergillus flavus grown on a conducive YES (20 g yeast extract, 150 g sucrose, 1 g MgSO4·7H2O) medium. The relative expression of 10 key genes (aflF, aflD, aflE, aflM, aflO, aflP, aflQ, aflX, aflR and aflS) in the biosynthetic pathway was examined in relation to different environmental factors and phenotypic aflatoxin B1 (AFB1) production. These data, plus data on relative growth rates and AFB1 production under different aw × temperature conditions were used to develop a mixed-growth-associated product formation model. The gene expression data were normalized and then used as a linear combination of the data for all 10 genes and combined with the physical model. This was used to relate gene expression to aw and temperature conditions to predict AFB1 production. The relationship between the observed AFB1 production provided a good linear regression fit to the predicted production based in the model. The model was then validated by examining datasets outside the model fitting conditions used (37°C, 40°C and different aw levels). The relationship between structural genes (aflD, aflM) in the biosynthetic pathway and the regulatory genes (aflS, aflJ) was examined in relation to aw and temperature by developing ternary diagrams of relative expression. These findings are important in developing a more integrated systems approach by combining gene expression, ecophysiological influences and growth data to predict mycotoxin production. This could help in developing a more targeted approach to develop prevention strategies to control such carcinogenic natural metabolites that are prevalent in many staple food products. The model could also be used to predict the impact of climate change on toxin production. PMID:21880616
Khurana, N; Saxena, R K; Gupta, R; Rajam, M V
Since polyamines (PAs) play a potential role in the regulation of growth and developmental processes in a wide variety of organisms, we have examined the influence of the PAs putrescine (Put) and spermidine (Spd) and the PA biosynthetic inhibitors alpha-difluoromethylornithine (DFMO), alpha-difluoromethylarginine (DFMA), methylglyoxal bis-(guanylhydrazone) (MGBG) and cyclohexylamine (CHA), singly and in combinations on microcycle conidiation (MC) in Aspergillus flavus. The exogenous application of the diamine Put (concentrations ranging from 0.1 to 5 mM) caused a sharp decline of MC in a dose-dependent fashion, but induced vegetative growth. However, the triamine Spd (0.1-5 mM) had a minimal effect on MC and induced a shift from MC to normal condition. PA inhibitors, especially DFMO, MGBG and CHA, produced greater inhibition of MC and complete inhibition of MC was observed at 5 mM of these inhibitors. DFMA even at 5 mM had only a weak inhibitory effect on MC. DFMO also inhibited conidial germination and germ tube growth. MGBG and CHA, while having an inhibitory effect on MC, induced vegetative growth. The inhibitory effect of PA inhibitors was partially reversed by exogenous Put or Spd, with Spd being more effective than Put. The analysis of free PA levels during various phases of MC revealed that undifferentiated spores contained a high Put/Spd ratio and there was a dramatic decrease in Put/Spd ratio before and during microcycle conidiophore maturity. The change in spermine titres could not be detected. These observations imply that Put is essential for vegetative growth, while Spd is involved in MC, and that a low Put/Spd ratio seems to be important for spore differentiation to MC.
Effective biological control of aflatoxinproducing Aspergillus flavus with atoxigenic members of that species requires suitable A. flavus well adapted to and resident in target agroecosystems. Eighteen atoxigenic isolates of A. flavus endemic in Italy were compared for ability to reduce aflatoxin c...
The fungus Aspergillus flavus is responsible for producing carcinogenic mycotoxins, the aflatoxins, on corn (maize) and other crops. An additional harmful toxin, cyclopiazonic acid, is produced by some isolates of A. flavus. Several A. flavus strains that do not produce one or both of these mycoto...
Lee, Chia-Yi; Ho, Yi-Ju; Sun, Chi-Chin; Lin, Hsin-Chiung; Hsiao, Ching-Hsi; Ma, David Hui-Kang; Lai, Chi-Chun; Chen, Hung-Chi
Aspergillus species produces a wide spectrum of fungal diseases like endophthalmitis and fungal keratitis ophthalmologically, but there has been no report about blepharitis caused by Aspergilus flavus to date. Herein, we report a 61-year-old ethnic Han Taiwanese male who had suffered from pain with burning and foreign body sensation after an insect bite on his left eye. Specimens from bilateral eyelids suggested infection of A. flavus, whereas corneal scraping showed the presence of Gram-negative bacteria. He was admitted for treatment of infectious keratitis with topical antibiotic and antifungal eye drops. Two weeks after discharge, recurrent blepharitis and keratitis of A. flavus was diagnosed microbiologically. Another treatment course of antifungal agent was resumed in the following 6 months, without further significant symptoms in the following 2 years. Collectively, it is possible for A. flavus to induce concurrent keratitis and blepharitis, and combined treatment of keratitis as well as blepharitis is advocated for as long as 6 months to ensure no recurrence. © The American Society of Tropical Medicine and Hygiene.
Ribeiro, J.; Cavaglieri, L.; Vital, H.; Cristofolini, A.; Merkis, C.; Astoreca, A.; Orlando, J.; Carú, M.; Dalcero, A.; Rosa, C. A. R.
The aim of this work was to study the effect of gamma radiation (2 kGy) on Aspergillus flavus and Aspergillus ochraceus ultrastructure. Moreover, the influence on aflatoxin B 1 and ochratoxin A production was also observed. Irradiated A. flavus strain showed a dull orangish colony while control strain showed the typical green color. Minor differences were observed on stipes, metulae and conidia size between control and irradiated A. flavus and A. ochraceus strains. Irradiated fungi showed ultrastructural changes on cell wall, plasmalema and cytoplasm levels. The levels of mycotoxins produced by irradiated strains were two times greater than those produced by control strains. Successive transferences of irradiated strains on malt extract agar allowed the fungus to recuperate morphological characteristics. Although minor changes in the fungal morphology were observed, ultrastructural changes at cell wall level and the increase of mycotoxin production ability were observed. Inappropriate storage of irradiated food and feed would allow the development of potentially more toxicogenic fungal propagules.
Mycotoxins, and especially the aflatoxins, are an enormous problem in agriculture, with aflatoxin B1 being the most carcinogenic known natural compound. The worldwide costs associated with aflatoxin monitoring and crop losses are in the hundreds of millions of dollars. Aspergillus flavus and A. par...
Jayanthi, Kamala P D; Ayyasamy, Arthikirubha; Kempraj, Vivek; Aurade, Ravindra M; Govindan, Selvakumar; Verghese, Abraham
Insects depend upon cuticular, humoral and cellular defenses to resist mycosis. However, entomopathogenic fungi through co-evolution have developed mechanisms to counter such defenses. Although a plethora of mechanisms of mycosis by entomopathogenic fungi are well-established, studies on the impairment of insects' antioxidative enzymes during mycosis remain elusive. Here, we used the interaction of Sternochetus mangiferae and its associated entomopathogenic fungus, Aspergillus flavus, as a model to validate our hypothesis. Uninfected insects were exposed to fungal spores for infection to occur. We observed symptoms of mycosis within 48 h of incubation period. Biochemical studies on antioxidative enzymes namely catalase, peroxidase and phenoloxidase, in infected and uninfected insects revealed decreased activity of these enzymes. It appears that A. flavus disables the host's antioxidative enzyme system that plays a crucial role in elimination of oxidative toxins produced during mycosis. Copyright © 2014 Elsevier Inc. All rights reserved.
In order to gain insight into the causal agents of aflatoxin contamination of maize in Italy, populations of Aspergillus flavus on maize produced in the most affected area were characterized. Forty-six percent of A. flavus, isolated from maize kernels collected in 5 districts of northern Italy betwe...
Reverberi, Massimo; Punelli, Marta; Smith, Carrie A.; Zjalic, Slaven; Scarpari, Marzia; Scala, Valeria; Cardinali, Giorgia; Aspite, Nicaela; Pinzari, Flavia; Payne, Gary A.; Fabbri, Anna A.; Fanelli, Corrado
In filamentous fungi, peroxisomes are crucial for the primary metabolism and play a pivotal role in the formation of some secondary metabolites. Further, peroxisomes are important site for fatty acids β-oxidation, the formation of reactive oxygen species and for their scavenging through a complex of antioxidant activities. Oxidative stress is involved in different metabolic events in all organisms and it occurs during oxidative processes within the cell, including peroxisomal β-oxidation of fatty acids. In Aspergillus flavus, an unbalance towards an hyper-oxidant status into the cell is a prerequisite for the onset of aflatoxin biosynthesis. In our preliminary results, the use of bezafibrate, inducer of both peroxisomal β-oxidation and peroxisome proliferation in mammals, significantly enhanced the expression of pex11 and foxA and stimulated aflatoxin synthesis in A. flavus. This suggests the existence of a correlation among peroxisome proliferation, fatty acids β-oxidation and aflatoxin biosynthesis. To investigate this correlation, A. flavus was transformed with a vector containing P33, a gene from Cymbidium ringspot virus able to induce peroxisome proliferation, under the control of the promoter of the Cu,Zn-sod gene of A. flavus. This transcriptional control closely relates the onset of the antioxidant response to ROS increase, with the proliferation of peroxisomes in A. flavus. The AfP33 transformant strain show an up-regulation of lipid metabolism and an higher content of both intracellular ROS and some oxylipins. The combined presence of a higher amount of substrates (fatty acids-derived), an hyper-oxidant cell environment and of hormone-like signals (oxylipins) enhances the synthesis of aflatoxins in the AfP33 strain. The results obtained demonstrated a close link between peroxisome metabolism and aflatoxin synthesis. PMID:23094106
Sebők, Flóra; Dobolyi, Csaba; Zágoni, Dóra; Risa, Anita; Krifaton, Csilla; Hartman, Mátyás; Cserháti, Mátyás; Szoboszlay, Sándor; Kriszt, Balázs
Due to the climate change, aflatoxigenic Aspergillus species and strains have appeared in several European countries, contaminating different agricultural commodities with aflatoxin. Our aim was to screen the presence of aflatoxigenic fungi in maize fields throughout the seven geographic regions of Hungary. Fungi belonging to Aspergillus section Flavi were isolated in the ratio of 26.9% and 42.3% from soil and maize samples in 2013, and these ratios decreased to 16.1% and 34.7% in 2014. Based on morphological characteristics and the sequence analysis of the partial calmodulin gene, all isolates proved to be Aspergillus flavus, except four strains, which were identified as Aspergillus parasiticus. About half of the A. flavus strains and all the A. parasiticus strains were able to synthesize aflatoxins. Aflatoxigenic Aspergillus strains were isolated from all the seven regions of Hungary. A. parasiticus strains were found in the soil of the regions Southern Great Plain and Southern Transdanubia and in a maize sample of the region Western Transdanubia. In spite of the fact that aflatoxins have rarely been detected in feeds and foods in Hungary, aflatoxigenic A. flavus and A. parasiticus strains are present in the maize culture throughout Hungary posing a potential threat to food safety.
Wilson, David M.; Huang, L. H.; Jay, Edward
Freshly harvested high-moisture corn with 29.4% moisture and corn remoistened to 19.6% moisture were inoculated with Aspergillus flavus Link ex Fr. and stored for 4 weeks at about 27 C in air (0.03% CO2, 21% O2, and 78% N2) and three modified atmospheres: (i) 99.7% N2 and 0.3% O2; (ii) 61.7% CO2, 8.7% O2, and 29.6% N2; and (iii) 13.5% CO2, 0.5% O2, and 84.8% N2. Kernel infections by A. flavus, Fusarium moniliforme (Sheld.) Snyd. et Hans., and other fungi were monitored weekly. The modified-atmosphere treatments delayed deterioration by A. flavus and F. moniliforme, but their growth was not completely stopped. A. flavus survived better in the remoistened than in the freshly harvested corn. F. moniliforme survived in both. A. flavus and F. moniliforme were the dominant fungi in corn removed from the modified atmospheres and exposed to normal air for 1 week. PMID:811165
Tian, Jun; Zeng, Xiaobin; Zeng, Hong; Feng, Zhaozhong; Miao, Xiangmin; Peng, Xue
The antifungal efficacy of nerol (NEL) has been proved against Aspergillus flavus by using in vitro and in vivo tests. The mycelial growth of A. flavus was completely inhibited at concentrations of 0.8 μL/mL and 0.1 μL/mL NEL in the air at contact and vapor conditions, respectively. The NEL also had an evident inhibitory effect on spore germination in A. flavus along with NEL concentration as well as time-dependent kinetic inhibition. The NEL presented noticeable inhibition on dry mycelium weight and synthesis of aflatoxin B1 (AFB1) by A. flavus, totally restraining AFB1 production at 0.6 μL/mL. In real food system, the efficacy of the NEL on resistance to decay development in cherry tomatoes was investigated in vivo by exposing inoculated and control fruit groups to NEL vapor at different concentration. NEL vapors at 0.1 μL/mL air concentration significantly reduced artificially contaminated A. flavus and a broad spectrum of fungal microbiota. Results obtained from presented study showed that the NEL had a great antifungal activity and could be considered as a benefit and safe tool to control food spoilage. PMID:24453813
Chang, Perng-Kuang; Scharfenstein, Leslie L.; Luo, Meng; Mahoney, Noreen; Molyneux, Russell J.; Yu, Jiujiang; Brown, Robert L.; Campbell, Bruce C.
Production of the harmful carcinogenic aflatoxins by Aspergillus parasiticus and Aspergillus flavus has been postulated to be a mechanism to relieve oxidative stress. The msnA gene of A. parasiticus and A. flavus is the ortholog of Saccharomyces cerevisiae MSN2 that is associated with multi-stress response. Compared to wild type strains, the msnA deletion (∆msnA) strains of A. parasiticus and A. flavus exhibited retarded colony growth with increased conidiation. The ∆msnA strains also produced slightly higher amounts of aflatoxins and elevated amounts of kojic acid on mixed cereal medium. Microarray assays showed that expression of genes encoding oxidative stress defense enzymes, i.e., superoxide dismutase, catalase, and cytochrome c peroxidase in A. parasiticus ∆msnA, and the catalase A gene in A. flavus ∆msnA, was up-regulated. Both A. parasiticus and A. flavus ∆msnA strains produced higher levels of reactive oxygen species (ROS), and ROS production of A. flavus msnA addback strains was decreased to levels comparable to that of the wild type A. flavus. The msnA gene appears to be required for the maintenance of the normal oxidative state. The impairment of msnA resulted in the aforementioned changes, which might be used to combat the increased oxidative stress in the cells. PMID:22069691
Gomaa, Ola M.; Momtaz, Osama A.
Aspergillus flavus was isolated from soil and exhibited laccase activity under both constitutive and copper induced conditions. Spiking the medium with 1 mM copper sulfate resulted in an increase in the activity which reached 51.84 U/mL, a distinctive protein band was detected at 60 kDa. The extracellular enzyme was purified 81 fold using gel filtration chromatography and resulted in two different laccase fractions L1 and L2, the latter had a higher enzymatic activity which reached 79.57 U/mL and specific activity of 64.17 U/μg protein. The analysis of the spectrum of the L2 fraction showed a shoulder at 330 nm which is characteristic for T2/T3 copper centers; both copper and zinc were detected suggesting that this is an unconventional white laccase. Primers of laccase gene were designed and synthesized to recover specific gene from A. flavus . Sequence analysis indicated putative laccase (Genbank ID: JF683612) at the amino acid level suggesting a close identity to laccases from other genera containing the copper binding site. Decolorization of textile waste water under different conditions showed possible application in bioremediation within a short period of time. The effect of copper on A. flavus was concentration dependent. PMID:26221119
Gomaa, Ola M; Momtaz, Osama A
Aspergillus flavus was isolated from soil and exhibited laccase activity under both constitutive and copper induced conditions. Spiking the medium with 1 mM copper sulfate resulted in an increase in the activity which reached 51.84 U/mL, a distinctive protein band was detected at 60 kDa. The extracellular enzyme was purified 81 fold using gel filtration chromatography and resulted in two different laccase fractions L1 and L2, the latter had a higher enzymatic activity which reached 79.57 U/mL and specific activity of 64.17 U/μg protein. The analysis of the spectrum of the L2 fraction showed a shoulder at 330 nm which is characteristic for T2/T3 copper centers; both copper and zinc were detected suggesting that this is an unconventional white laccase. Primers of laccase gene were designed and synthesized to recover specific gene from A. flavus . Sequence analysis indicated putative laccase (Genbank ID: JF683612) at the amino acid level suggesting a close identity to laccases from other genera containing the copper binding site. Decolorization of textile waste water under different conditions showed possible application in bioremediation within a short period of time. The effect of copper on A. flavus was concentration dependent.
Aspergillus flavus produces many secondary metabolites including aflatoxins. Besides conidia, the fungus uses sclerotia as another type of propagule. We obtained transcriptomes from four growth conditions of the aswA mutant, a strain impaired in sclerotial development and production of sclerotium-sp...
Liu, Jie; Sun, Lvhui; Zhang, Niya; Zhang, Jiacai; Guo, Jiao; Li, Chong; Rajput, Shahid Ali; Qi, Desheng
The current study was to better understand the potential factors affecting aflatoxin B1 (AFB1) accumulation varies between different grains. The nutrient composition and contents of defatted substrates were determined; additionally, according to the nutrient content of the substrates, the effects of starch, soluble sugars, amino acids, and trace elements on AFB1 production and mycelial growth in Czapek-Dox medium were examined. These results verified that removal of lipids from ground substrates significantly reduced the substrate's potential for AFB1 production by Aspergillus flavus. Maltose, glucose, sucrose, arginine, glutamic acid, aspartic acid, and zinc significantly induced AFB1 production up to 1.7- to 26.6-fold. And stachyose more significantly promoted A. flavus growth than the other nutrients. Thus, this study demonstrated that, combined with the nutrients content of grains, in addition to lipids, sucrose, stachyose, glutamic acid, and zinc might play key roles in various grains that are differentially infected by A. flavus. Particularly, two new nutrients (arginine and stachyose) of the grains we found significantly stimulate AFB1 production and A. flavus growth, respectively. The results provide new concepts for antifungal methods to protect food and animal feed from AFB1 contamination. PMID:27294129
Liu, Jie; Sun, Lvhui; Zhang, Niya; Zhang, Jiacai; Guo, Jiao; Li, Chong; Rajput, Shahid Ali; Qi, Desheng
The current study was to better understand the potential factors affecting aflatoxin B1 (AFB1) accumulation varies between different grains. The nutrient composition and contents of defatted substrates were determined; additionally, according to the nutrient content of the substrates, the effects of starch, soluble sugars, amino acids, and trace elements on AFB1 production and mycelial growth in Czapek-Dox medium were examined. These results verified that removal of lipids from ground substrates significantly reduced the substrate's potential for AFB1 production by Aspergillus flavus. Maltose, glucose, sucrose, arginine, glutamic acid, aspartic acid, and zinc significantly induced AFB1 production up to 1.7- to 26.6-fold. And stachyose more significantly promoted A. flavus growth than the other nutrients. Thus, this study demonstrated that, combined with the nutrients content of grains, in addition to lipids, sucrose, stachyose, glutamic acid, and zinc might play key roles in various grains that are differentially infected by A. flavus. Particularly, two new nutrients (arginine and stachyose) of the grains we found significantly stimulate AFB1 production and A. flavus growth, respectively. The results provide new concepts for antifungal methods to protect food and animal feed from AFB1 contamination.
Drummond, J; Pinnock, D E
Fourteen isolates of Aspergillus parasiticus and 2 isolates of Aspergillus flavus isolated from the mealybug Saccharicoccus sacchari were analyzed for production of aflatoxins B1, B2, G1, and G2 in liquid culture over a 20-day period. Twelve Aspergillus isolates including 11 A. parasiticus and 1 A. flavus produced aflatoxins which were extracted from both the mycelium and culture filtrate. Aflatoxin production was detected at day 3 and was detected continually for up to day 20. Aflatoxin B1 production was greatest between 7 and 10 days and significantly higher quantities were produced by A. flavus compared to A. parasiticus. Aflatoxin production was not a stable trait in 1 A. parasiticus isolate passaged 50 times on agar. In addition to loss of aflatoxin production, an associated loss in sporulation ability was also observed in this passaged isolate, although it did maintain pathogenicity against S. sacchari. An aflatoxin B1 concentration of 0.16 micrograms/mealybug (14.2 micrograms/g wet wt) was detected within the tissues of infected mealybugs 7 days after inoculation. In conclusion, the ability of Aspergillus isolates to produce aflatoxins was not essential to the entomopathogenic activity of this fungus against its host S. sacchari.
López-Malo, Aurelio; Alzamora, Stella M; Palou, Enrique
The effects of selected concentrations of antimicrobials from natural (vanillin, thymol, eugenol, carvacrol or citral) or synthetic (potassium sorbate or sodium benzoate) origin on Aspergillus flavus lag time inoculated in laboratory media formulated at water activity (a(w)) 0.99 and pH 4.5 or 3.5, were evaluated. Time to detect a colony with a diameter > 0.5 mm was determined. Mold response was modeled using the Fermi function. Antimicrobial minimal inhibitory concentration (MIC) was defined as the minimal required inhibiting mold growth for 2 months. Fermi function successfully captured A. flavus dose-response curves to the tested antimicrobials with a highly satisfactory fit. Fermi equation coefficients, Pc and k, were used to compare antimicrobials and assess the effect of pH. Important differences in Pc and k were observed among antimicrobials, being natural antimicrobials less pH dependent than synthetic antimicrobials. A large Pc value represents a small antimicrobial effect on A. flavus lag time; thus, high concentrations are needed to delay growth. A. flavus exhibited higher sensitivity to thymol, eugenol, carvacrol, potassium sorbate (at pH 3.5), and sodium benzoate (at pH 3.5) than to vanillin or citral. MICs varied from 200 ppm of sodium bcnzoate at pH 3.5 to 1800 ppm of citral at both evaluated pHs.
Oksanen, E.; Blakeley, M. P.; Bonneté, F.; Dauvergne, M. T.; Dauvergne, F.; Budayova-Spano, M.
Urate oxidase (Uox) catalyses the oxidation of urate to allantoin and is used to reduce toxic urate accumulation during chemotherapy. X-ray structures of Uox with various inhibitors have been determined and yet the detailed catalytic mechanism remains unclear. Neutron crystallography can provide complementary information to that from X-ray studies and allows direct determination of the protonation states of the active-site residues and substrate analogues, provided that large, well-ordered deuterated crystals can be grown. Here, we describe a method and apparatus used to grow large crystals of Uox (Aspergillus flavus) with its substrate analogues 8-azaxanthine and 9-methyl urate, and with the natural substrate urate, in the presence and absence of cyanide. High-resolution X-ray (1.05–1.20 Å) and neutron diffraction data (1.9–2.5 Å) have been collected for the Uox complexes at the European Synchrotron Radiation Facility and the Institut Laue-Langevin, respectively. In addition, room temperature X-ray data were also collected in preparation for joint X-ray and neutron refinement. Preliminary results indicate no major structural differences between crystals grown in H2O and D2O even though the crystallization process is affected. Moreover, initial nuclear scattering density maps reveal the proton positions clearly, eventually providing important information towards unravelling the mechanism of catalysis. PMID:19586953
Hemdan, R. Elmitwalli; Fatma, Helmi M.; Rizk, Mohammed A.; Hagrassy, Abeer F.
Biodeterioration of mural paintings by Aspergillus niger and Aspergillus flavus Fungi has been proved in different mural paintings in Egypt nowadays. Several researches have studied the effect of fungi on mural paintings, the mechanism of interaction and methods of control. But none of these researches gives us the solution without causing a side effect. In this paper, for the first time, a recent treatment by antibiotic "6 penthyl α pyrone phenol" was applied as a successful technique for elimination of Aspergillus niger and Aspergillus flavus. On the other hand, it is favorable for cleaning Surfaces of Murals executed by tembera technique from the fungi metabolism which caused a black pigments on surfaces.
The abilities of non-aflatoxin producing strains of Aspergillus flavus NRRL 32354; 18543; 21882; 21368 as well as domesticated koji strains Aspergillus oryzae (syn. A. flavus var. oryzae) NRRL 451; 1911; 5592; 6271; 30038 to interfere with aflatoxin formation by A. flavus NRRL 3357; 32355 were exami...
Zhang, Feng; Guo, Zhenni; Zhong, Hong; Wang, Sen; Yang, Weiqiang; Liu, Yongfeng; Wang, Shihua
Aspergillus flavus is one of the most important producers of carcinogenic aflatoxins in crops, and the effect of water activity (aw) on growth and aflatoxin production of A. flavus has been previously studied. Here we found the strains under 0.93 aw exhibited decreased conidiation and aflatoxin biosynthesis compared to that under 0.99 aw. When RNA-Seq was used to delineate gene expression profile under different water activities, 23,320 non-redundant unigenes, with an average length of 1297 bp, were yielded. By database comparisons, 19,838 unigenes were matched well (e-value < 10−5) with known gene sequences, and another 6767 novel unigenes were obtained by comparison to the current genome annotation of A. flavus. Based on the RPKM equation, 5362 differentially expressed unigenes (with |log2Ratio| ≥ 1) were identified between 0.99 aw and 0.93 aw treatments, including 3156 up-regulated and 2206 down-regulated unigenes, suggesting that A. flavus underwent an extensive transcriptome response during water activity variation. Furthermore, we found that the expression of 16 aflatoxin producing-related genes decreased obviously when water activity decreased, and the expression of 11 development-related genes increased after 0.99 aw treatment. Our data corroborate a model where water activity affects aflatoxin biosynthesis through increasing the expression of aflatoxin producing-related genes and regulating development-related genes. PMID:25421810
Lan, Huahui; Sun, Ruilin; Fan, Kun; Yang, Kunlong; Zhang, Feng; Nie, Xin Y.; Wang, Xiunai; Zhuang, Zhenhong; Wang, Shihua
Histone acetyltransferases (HATs) help regulate fungal development and the production of secondary metabolites. In this study, we determined that the HAT AflGcnE influenced morphogenesis and aflatoxin biosynthesis in Aspergillus flavus. We observed that AflGcnE localized to the nucleus and cytoplasm during the conidial production and germination stages, while it was located mainly in the nucleus during the hyphal development stage. Deletion of AflgcnE inhibited the growth of A. flavus and decreased the hydrophobicity of the cell surface. The ΔAflgcnE mutant exhibited a lack of asexual sporulation and was unable to generate sclerotia. Additionally, AflgcnE was required to maintain cell wall integrity and genotoxic stress responses. Importantly, the ΔAflgcnE mutant did not produce aflatoxins, which was consistent with a significant down-regulation of aflatoxin gene expression levels. Furthermore, our data revealed that AflgcnE is a pathogenicity factor required for colonizing maize seeds. In summary, we revealed that A. flavus AflGcnE is crucial for morphological development, aflatoxin biosynthesis, stress responses, and pathogenicity. Our findings help clarify the functional divergence of GcnE orthologs, and may provide a possible target for controlling A. flavus infections of agriculturally important crops. PMID:27625637
Tintu, Ignatius; Abhilash, Joseph; Dileep, Kalarickal V; Augustine, Anu; Haridas, Madathilkovilakath; Sadasivan, Chittalakkottu
Aspergillus flavus is a commonly found fungal pathogen which produces structurally related and highly toxic secondary metabolites, aflatoxins. It has been proposed that α-amylase inhibitors may limit the ability of the fungus to produce aflatoxins. Hence, this enzyme is a potent target for the development of antifungal agents. In this study, it was found that Spatholobus parviflorus seed lectin (SPL) can inhibit the growth of A. flavus with a MIC value of 1.5 mg/mL. The enzyme kinetics, molecular modeling and isothermal titration calorimetric studies suggest that SPL can inhibit α-amylase with Ki value of 0.0042 mm. Hence, it is suggested that the antifungal activity of SPL might be partly due to its ability to inhibit the enzyme α-amylase. © 2014 John Wiley & Sons A/S.
Esper, Renata H; Gonçalez, Edlayne; Marques, Marcia O M; Felicio, Roberto C; Felicio, Joana D
Aflatoxin B1 (AFB1) is a highly toxic and carcinogenic metabolite produced by Aspergillus species on food and agricultural commodities. Inhibitory effects of essential oils of Ageratum conyzoides (mentrasto) and Origanum vulgare (oregano) on the mycelial growth and aflatoxin B1 production by Aspergillus flavus have been studied previously in culture medium. The aim of this study was to evaluate aflatoxin B1 production by Aspergillus flavus in real food systems (corn and soybean) treated with Ageratum conyzoides (mentrasto) and Origanum vulgare (oregano) essential oils. Samples with 60 g of the grains were treated with different volumes of essential oils, 200, 100, 50, and 10 μL for oregano and 50, 30, 15, and 10 μL for mentrasto. Fungal growth was evaluated by disk diffusion method. Aflatoxin B1 production was evaluated inoculating suspensions of A. flavus containing 1.3 × 10(5) spores/mL in 60 g of grains (corn and soybeans) after adjusting the water activity at 0.94. Aflatoxin was quantified by photodensitometry. Fungal growth and aflatoxin production were inhibited by essential oils, but the mentrasto oil was more effective in soybeans than that of oregano. On the other hand, in corn samples, the oregano essential oil was more effective than that of mentrasto. Chemical compositions of the essential oils were also investigated. The GC/MS oils analysis showed that the main component of mentrasto essential oil is precocene I and of the main component of oregano essential oil is 4-terpineol. The results indicate that both essential oils can become an alternative for the control of aflatoxins in corn and soybeans.
Esper, Renata H.; Gonçalez, Edlayne; Marques, Marcia O. M.; Felicio, Roberto C.; Felicio, Joana D.
Aflatoxin B1 (AFB1) is a highly toxic and carcinogenic metabolite produced by Aspergillus species on food and agricultural commodities. Inhibitory effects of essential oils of Ageratum conyzoides (mentrasto) and Origanum vulgare (oregano) on the mycelial growth and aflatoxin B1 production by Aspergillus flavus have been studied previously in culture medium. The aim of this study was to evaluate aflatoxin B1 production by Aspergillus flavus in real food systems (corn and soybean) treated with Ageratum conyzoides (mentrasto) and Origanum vulgare (oregano) essential oils. Samples with 60 g of the grains were treated with different volumes of essential oils, 200, 100, 50, and 10 μL for oregano and 50, 30, 15, and 10 μL for mentrasto. Fungal growth was evaluated by disk diffusion method. Aflatoxin B1 production was evaluated inoculating suspensions of A. flavus containing 1.3 × 105 spores/mL in 60 g of grains (corn and soybeans) after adjusting the water activity at 0.94. Aflatoxin was quantified by photodensitometry. Fungal growth and aflatoxin production were inhibited by essential oils, but the mentrasto oil was more effective in soybeans than that of oregano. On the other hand, in corn samples, the oregano essential oil was more effective than that of mentrasto. Chemical compositions of the essential oils were also investigated. The GC/MS oils analysis showed that the main component of mentrasto essential oil is precocene I and of the main component of oregano essential oil is 4-terpineol. The results indicate that both essential oils can become an alternative for the control of aflatoxins in corn and soybeans. PMID:24926289
Donkersloot, J. A.; Mateles, R. I.
A method based on tritium suicide was developed to enrich auxotrophic mutants of Aspergillus flavus. N-methyl-N′-nitro-N-nitrosoguanidine (NG) was chosen as a mutagen, since a wide variety of mutations were induced by the action of 0.1% NG on A. flavus conidia suspended in phosphate buffer (pH 7.0). The decimal reduction time under these conditions was about 30 min, and the surviving population contained 4 to 6% auxotrophs after 1 hr of mutagenesis. This proportion was then increased by tritium suicide of wild-type cells. At a concentration of 1.3 μm, 3H-leucine was incorporated better than 3H-proline or 3H-thymidine into the germinating conidia. With about 20 hr of incubation and a short treatment in a high-speed mixer to disentangle mycelia and conidia, a 5- to 20-fold decrease in the number of survivors resulted from the incorporated 3H-leucine (5 c/mmole) after 1 week of storage at 5 C. At a 10-fold lower concentration, the uptake of radioactivity and the subsequent suicide rate were much lower. With 3H-leucine, the proportion of auxotrophs in the surviving population rose from 5 to about 20% during 2 weeks of storage at 5 C. Mutants requiring various intermediates for protein or nucleic acid synthesis or requiring vitamins were isolated. Finally, it was noted that A. flavus shows a much higher resistance to tritium suicide than does Escherichia coli. PMID:5726297
Aljuboori, Ahmad H Rajab; Idris, Azni; Al-joubory, Hamid Hussain Rijab; Uemura, Yoshimitsu; Ibn Abubakar, B S U
In this study, the flocculation behavior and mechanism of a cation-independent bioflocculant IH-7 produced by Aspergillus flavus were investigated. Results showed 91.6% was the lowest flocculating rate recorded by IH-7 (0.5 mg L(-1)) at pH range 4-8. Moreover, IH-7 showed better flocculation performance than polyaluminum chloride (PAC) at a wide range of flocculant concentration (0.06-25 mg L(-1)), temperature (5-45 °C) and salinity (10-60% w/w). The current study found that cation addition did not significantly enhance the flocculating rate and IH-7 is a positively charged bioflocculant. These findings suggest that charge neutralization is the main flocculation mechanism of IH-7 bioflocculant. IH-7 was significantly used to flocculate different types of suspended solids such as activated carbons, kaolin clays, soil solids and yeast cells.
... of Aspergillus flavus NRRL 21882 on peanut; peanut, hay; peanut, meal; and peanut, refined oil. (b... NRRL 21882 on corn, field, forage; corn, field, grain; corn, field, stover; corn, field, aspirated...
... of Aspergillus flavus NRRL 21882 on peanut; peanut, hay; peanut, meal; and peanut, refined oil. (b... NRRL 21882 on corn, field, forage; corn, field, grain; corn, field, stover; corn, field, aspirated...
... of Aspergillus flavus NRRL 21882 on peanut; peanut, hay; peanut, meal; and peanut, refined oil. (b... NRRL 21882 on corn, field, forage; corn, field, grain; corn, field, stover; corn, field, aspirated...
Aflatoxin contamination resulting from maize infection by Aspergillus flavus is both an economic concern and public health concern. Therefore, strategies for controlling maize contamination are being investigated. Abilities of 11 naturally occurring atoxigenic strains in Nigeria to reduce aflatox...
Aspergillus flavus is the primary causal agent of food and feed contamination with the toxic fungal metabolites aflatoxins. Aflatoxin-producing potential of A. flavus is known to vary among isolates. The genes involved in aflatoxin biosynthesis are clustered together and the order of genes within th...
Aspergillus flavus is a major producer of carcinogenic aflatoxins worldwide in corn, peanuts, tree nuts, cottonseed, spices and other crops. Many countries have strict limits on the amount of aflatoxins permitted in human commodities and animal feed. Sclerotia produced by A. flavus serve several f...
Aspergillus flavus Link:Fr, an opportunistic fungus that produces aflatoxin, is pathogenic to maize and other oilseed crops. Aflatoxin is a potent carcinogen, and its presence markedly reduces the value of grain. Understanding and enhancing host resistance to A. flavus infection and/or subsequent af...
Aflatoxin contamination by Aspergillus flavus is exacerbated by drought stress in the field. Given that reactive oxygen species (ROS) both accumulate in plant tissues during drought and can stimulate aflatoxin production in vitro, we examined the responses of toxigenic isolates of A. flavus to oxida...
Navel orangeworm (NOW) damage to almonds is correlated with increased incidence of aflatoxin contamination caused by Aspergillus flavus. However, no reports demonstrate a causative relationship between NOW feeding and A. flavus infection. To demonstrate the potential of NOW to act as a vector of A. ...
Aspergillus flavus colonizes agricultural commodities worldwide and contaminates them with carcinogenic aflatoxins. The high genetic diversity of A. flavus populations is largely due to sexual reproduction characterized by the formation of ascospore-bearing ascocarps embedded within sclerotia. A. ...
Bueno, Dante J; Silva, Julio O; Oliver, Guillermo; González, Silvia N
The effect of two species of lactobacilli, Lactobacillus casei CRL 431 and Lactobacillus rhamnosus CRL 1224, on growth of different Aspergillus flavus strains was determined. A. flavus strains (Ap, TR2, or CF80) were grown in LAPTg broth at 37 degrees C for 7 days as a single culture and in association with L. casei CRL 431 or L. rhamnosus CRL 1224 at initial inoculum ratios of 1:1, 1:10, and 1:100. In most cases, the mixed cultures had a lower fungal growth and a lower pH than the control cultures. Mycelial dry weight was reduced to 73 and 85% using L. casei CRL 431 and L. rhamnosus CRL 1224, respectively. The pH decrease in mixed cultures when the fungal mycelial dry weight is reduced may play an important role in inhibition. The number of viable bacteria was variably affected by fungal growth. These results indicate that L. casei CRL 431 and L. rhamnosus CRL 1224 may be useful as potential biocontrol agent against A. flavus.
Yang, Kunlong; Qin, Qiuping; Liu, Yinghang; Zhang, Limei; Liang, Linlin; Lan, Huahui; Chen, Chihao; You, Yunchao; Zhang, Feng; Wang, Shihua
Aspergillus flavus is one of the most important opportunistic pathogens of crops and animals. The carcinogenic mycotoxin, aflatoxins produced by this pathogen cause a health problem to human and animals. Since cyclic AMP signaling controls a range of physiological processes, like fungal development and infection when responding to extracellular stimuli in fungal pathogens, in this study, we investigated the function of adenylate cyclase, a core component of cAMP signaling, in aflatoxins biosynthesis and virulence on plant seeds in A. flavus. A gene replacement strategy was used to generate the deletion mutant of acyA that encodes the adenylate cyclase. Severe defects in fungal growth, sporulation and sclerotia formation were observed in the acyA deletion mutant. The defect in radical growth could be partially rescued by exogenous cAMP analog. The acyA mutant was also significantly reduced in aflatoxins production and virulence. Similar to the former studies in other fungi, The acyA mutant showed enhancing tolerance to oxidative stress, but more sensitive to heat stress. Overall, the pleiotropic defects of the acyA deletion mutant indicates that the cAMP-PKA pathway is involved in fungal development, aflatoxins biosynthesis and plant seed invasion in A. flavus.
Yang, Kunlong; Qin, Qiuping; Liu, Yinghang; Zhang, Limei; Liang, Linlin; Lan, Huahui; Chen, Chihao; You, Yunchao; Zhang, Feng; Wang, Shihua
Aspergillus flavus is one of the most important opportunistic pathogens of crops and animals. The carcinogenic mycotoxin, aflatoxins produced by this pathogen cause a health problem to human and animals. Since cyclic AMP signaling controls a range of physiological processes, like fungal development and infection when responding to extracellular stimuli in fungal pathogens, in this study, we investigated the function of adenylate cyclase, a core component of cAMP signaling, in aflatoxins biosynthesis and virulence on plant seeds in A. flavus. A gene replacement strategy was used to generate the deletion mutant of acyA that encodes the adenylate cyclase. Severe defects in fungal growth, sporulation and sclerotia formation were observed in the acyA deletion mutant. The defect in radical growth could be partially rescued by exogenous cAMP analog. The acyA mutant was also significantly reduced in aflatoxins production and virulence. Similar to the former studies in other fungi, The acyA mutant showed enhancing tolerance to oxidative stress, but more sensitive to heat stress. Overall, the pleiotropic defects of the acyA deletion mutant indicates that the cAMP-PKA pathway is involved in fungal development, aflatoxins biosynthesis and plant seed invasion in A. flavus. PMID:28066725
Aflatoxins are toxic polyketides produced by several Aspergillus species that contaminate food crops worldwide. Aspergillus flavus and A. parasiticus are the most common agents of aflatoxin contamination of oil-rich crops. The genes involved in aflatoxin biosynthesis are clustered and convert acetat...
Aspergillus flavus is found colonizing numerous oil seed crops such as corn, peanuts, sorghum, treenuts and cotton worldwide, contaminating them with aflatoxin and other harmful potent toxins. In the phylogenetically related model fungus Aspergillus nidulans, the methyltransferase, RmtA, has been de...
Aflatoxins are among the most powerful carcinogens in nature. They are produced by the fungal pathogen Aspergillus flavus Link and other Aspergillus species. Aflatoxins accumulate in many crops, including rice, wheat, oats, pecans, pistachios, soybean, cassava, almonds, peanuts, beans, corn and cot...
Species in Aspergillus section Flavi commonly infect agricultural staples such as corn, peanuts, cottonseed, and tree nuts and produce an array of mycotoxins, the most potent of which is aflatoxin. Aspergillus flavus is the dominant aflatoxin-producing species in the majority of crops. Populations...
Aspergillus flavus strains vary widely in their production of aflatoxins and cyclopiazonic acid (CPA). A total of 500 Aspergillus strains isolated from a variety of sources showed 16.4% were negative for both aflatoxin and CPA, 41.3% were positive for both mycotoxins, 13.0% were positive only fo...
The transcription factors NsdC and NsdD have been shown to be necessary for sexual development in Aspergillus nidulans. Herein we examine the role of these proteins in development and aflatoxin production of the agriculturally important, aflatoxin-producing fungus, Aspergillus flavus. We found tha...
Aflatoxin contamination in peanut products has been an important and long-standing problem around the world. Produced mainly by Aspergillus flavus and Aspergillus parasiticus, aflatoxins are the most toxic and carcinogenic compounds among toxins. This study investigated the application of fluorescen...
Yu, Jiujiang; Payne, Gary A; Nierman, William C; Machida, Masayuki; Bennett, Joan W; Campbell, Bruce C; Robens, Jane F; Bhatnagar, Deepak; Dean, Ralph A; Cleveland, Thomas E
Aspergillus flavus is a weak pathogen that infects plants, animals and humans. When it infects agricultural crops, however, it produces one of the most potent carcinogens known (aflatoxins). To devise strategies to control aflatoxin contamination of pre-harvest agricultural crops and post-harvest grains during storage, we launched the A. flavus genomics program. The major objective of this program is the identification of genes involved in aflatoxin biosynthesis and regulation, as well as in pathogenicity, to gain a better understanding of the mechanism of aflatoxin formation. The sequencing of A. flavus whole genome has been completed. Initial annotation of the sequence revealed that there are about 13,071 genes in the A. flavus genome. Genes which potentially encode for enzymes involved in secondary metabolite production in the A. flavus genome have been identified. Preliminary comparative genome analysis of A. flavus with A. oryzae is summarized here.
Kohiyama, Cássia Yumie; Yamamoto Ribeiro, Milene Mayumi; Mossini, Simone Aparecida Galerani; Bando, Erika; Bomfim, Natália da Silva; Nerilo, Samuel Botião; Rocha, Gustavo Henrique Oliveira; Grespan, Renata; Mikcha, Jane Martha Graton; Machinski, Miguel
The antifungal and antiaflatoxigenic properties of Thymus vulgaris essential oil (TEO) were evaluated upon Aspergillus flavus "in vitro". Suspension containing 10(6) of A. flavus were cultivated with TEO in concentrations ranging from 50 to 500 μg/mL. TEO reached minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) at 250 μg/mL. Inhibition of ergosterol biosynthesis was detected at a concentration of 100 μg/mL of TEO. Morphological evaluation performed by both light microscopy and scanning electron microscopy showed that antifungal activity of TEO could be detected starting at a concentration of 50 μg/mL and the fungicide effect at a concentration of 250 μg/mL. TEO completely inhibited production of both B1 and B2 aflatoxins (AFB1 and AFB2) at a concentration of 150 μg/mL. This way, fungal biomass development and aflatoxin production were dependent on TEO concentration. Therefore, TEO was capable of controlling the growth of A. flavus and its production of aflatoxins. Copyright © 2014 Elsevier Ltd. All rights reserved.
Mellon, Jay E; Dowd, Michael K; Cotty, Peter J
Utilization of the three major corn reserve materials, starch, triglycerides (refined corn oil), and zein (storage protein), by Aspergillus flavus was monitored in vitro over a 7-day fermentation. Medium composition in which proportions of reserve materials initially approximated proportions in mature corn kernels changed little over the first 18 h. Subsequently, hydrolysis of both starch and triglycerides occurred simultaneously, with peak concentrations of glucose and free fatty acids on day 2 of the fermentation period. Fatty acid concentrations dropped relatively rapidly after day 2 but increased again after day 6. Aflatoxin B(1) production increased after 36 h, with a peak at day 4. Aflatoxin B(1) production paralleled fungal biomass production during the exponential growth phase. A. flavus did not appear to preferentially utilize any of the released fatty acids. A number of fungus-specific metabolites were detected, including arabitol, erythritol, mannitol, trehalose, and kojic acid. Mannitol exceeded the other metabolites in concentration, and the timing of mannitol production closely paralleled that of aflatoxin B(1). Kojic acid concentrations peaked at day 6. In contrast to previously described selective use of simple carbohydrates by A. flavus, less discrimination was displayed when faced with utilization of complex substrates such as starch or triglycerides.
Bonneté, F.; Vivarès, D.; Robert, Ch.; Colloc'h, N.
Interparticle interactions of urate oxidase from Aspergillus flavus have been studied by small-angle X-ray scattering to determine crystallization conditions. This enzyme is a homotetramer with a total molecular weight of 128 kDa. It is a slightly basic protein (pI between 7.5 and 8). The interaction potentials have been studied as a function of the main thermodynamic and chemical parameters: temperature, protein concentration, pH, salt nature and concentration, addition of polyols. In 10 mM sodium carbonate at pH 10.5, the interactions are slightly repulsive and become less repulsive with a pH closer to pI. With the addition of carbonate, the protein loses its tetrameric structure for a dimeric one; with formate, the tetrameric structure remains stable. We also studied the effect of polyethylene glycols as it had been done with high molecular weight proteins. With the addition of PEG 8 K, the interactions became less repulsive and even turned attractive with the addition of both PEG 8 K and salt. Protein crystals of urate oxidase were observed in slightly repulsive conditions (second virial coefficient A2 about +10 -5 mol ml g -2 instead of -2 to -8×10 -4 mol ml g -2 for low molecular weight proteins).
Aljuboori, Ahmad H Rajab; Idris, Azni; Abdullah, Norhafizah; Mohamad, Rosfarizan
The production and characterization of a bioflocculant, IH-7, by Aspergillus flavus was investigated. About 0.4 g of purified bioflocculant with an average molecular weight of 2.574 × 10(4)Da could be obtained from 1L of fermentation medium. The bioflocculant mainly consisted of protein (28.5%) and sugar (69.7%), including 40% of neutral sugar, 2.48% of uronic acid and 1.8% amino sugar. The neutral sugar components are sucrose, lactose, glucose, xylose, galactose, mannose and fructose at a molar ratio of 2.4:4.4:4.1:5.8:9.9:0.8:3.1. Fourier-transform infrared spectroscopy analysis revealed that purified IH-7 contained hydroxyl, amide, carboxyl and methoxyl groups. The elemental analysis of purified IH-7 showed that the weight fractions of the elements C, H, O, N and S were 29.9%, 4.8%, 34.7%, 3.3%, and 2.0%, respectively. IH-7 had good flocculating rate in kaolin suspension without cation addition and stable over wide range of pH and temperature.
Narayanan, Karthik; Parameswaran, Binod; Pandey, Ashok
Chitosan is a biopolymer obtained by deacetylation of chitin and has been proven to have various applications in industry and biomedicine. Deacetylation of chitin using the enzyme chitin deacetylase (CDA) is favorable in comparison to the hazardous chemical method involving strong alkali and high temperature. A fungal strain producing CDA was isolated from environmental samples collected from coastal regions of South Kerala, India. It was identified as Aspergillus flavus by morphological characteristics and ITS DNA analysis. Nutritional requirement for maximum production of CDA under submerged condition was optimized using statistical methods including Plackett-Burman and response surface methodology central composite design. A 5.98-fold enhancement in CDA production was attained in shake flasks when the fermentation process parameters were used at their optimum levels. The highest CDA activity was 57.69 ± 1.68 U under optimized bioprocess conditions that included 30 g L(-1) glucose, 40 g L(-1) yeast extract, 15 g L(-1) peptone, and 7 g L(-1) MgCl2 at initial media pH of 7 and incubation temperature of 32°C after 48 hr of incubation, while the unoptimized basal medium yielded 9.64 ± 2.04 U.
Dasan, Beyhan Gunaydin; Mutlu, Mehmet; Boyaci, Ismail Hakki
In this study, an atmospheric pressure fluidized bed plasma (APFBP) system was designed and its decontamination effect on aflatoxigenic fungi (Aspergillus flavus and Aspergillus parasiticus) on the surface of hazelnuts was investigated. Hazelnuts were artificially contaminated with A. flavus and A. parasiticus and then were treated with dry air plasma for up to 5min in the APFBP system at various plasma parameters. Significant reductions of 4.50 log (cfu/g) in A. flavus and 4.19 log (cfu/g) in A. parasiticus were achieved after 5min treatments at 100% V - 25kHz (655W) by using dry air as the plasma forming gas. The decontamination effect of APFBP on A. flavus and A. parasiticus spores inoculated on hazelnuts was increased with the applied reference voltage and the frequency. No change or slight reductions were observed in A. flavus and A. parasiticus load during the storage of plasma treated hazelnuts whereas on the control samples fungi continued to grow under storage conditions (30days at 25°C). Temperature change on hazelnut surfaces in the range between 35 and 90°C was monitored with a thermal camera, and it was demonstrated that the temperature increase taking place during plasma treatment did not have a lethal effect on A. flavus and A. parasiticus spores. The damage caused by APFBP treatment on Aspergillus spp. spores was also observed by scanning electron microscopy.
Aflatoxins are carcinogenic mycotoxins produced by the fungus Aspergillus flavus. Aflatoxin contamination in pre-harvest corn profusely happens when heat and drought field conditions favor A. flavus colonization. Commercial corn hybrids are generally susceptible to A. flavus infection. An ideal cont...
Cuero, R.G.; Smith, J.E.; Lacey, J.
Aspergillus flavus was grown on maize and rice extract agars and on irradiated viable cracked maize and rice grains, either in pure culture or in dual culture with wild strains of either Hyphopichia burtonii or Bacillus amyloliquefaciens. Aflatoxin production by A. flavus and its growth and interactions with the other microorganisms were studied at three water activities (a/sub w/) (0.98, 0.95, and 0.90) and two temperatures (25 and 16/sup 0/C). Both H. burtonii and B. amyloliquefaciens markedly stimulated growth and aflotoxin production by A. flavus on cracked maize, especially at 25/sup 0/C and 0.95 and 0.98 a/sub w/. No aflatoxin was detected in pure cultures of A. flavus on cracked rice after 12 days of incubation at 25/sup 0/C, but some was produced by mixed cultures at 16/sup 0/C and 0.98 a/sub w/. The morphological interactions among A. flavus, H. burtonii, and B. amyloliquefaciens were also examined on maize and rice extract agars under similar controlled conditions.
..., a non-aflatoxin-producing strain of Aspergillus flavus, was described extensively in the Federal... drinking water from ground water or surface water and exposure through pesticide use in gardens, lawns, or... placed as a nutrient source, and displaces aflatoxin- producing strains of Aspergillus flavus (Ref....
The zinc finger transcription factor nsdC is required for both sexual development and aflatoxin production in the saprophytic fungus Aspergillus flavus. While previous work with an nsdC knockout mutant was conducted in Aspergillus nidulans and A. flavus strain 3357, here we demonstrate perturbations...
Baquião, Arianne Costa; Rodriges, Aline Guedes; Lopes, Evandro Luiz; Tralamazza, Sabina Moser; Zorzete, Patricia; Correa, Benedito
The aims of the present study were to monitor the production of aflatoxin B1 (AFB1) and mycelial growth, and to evaluate the expression of genes directly and indirectly involved in the biosynthesis of aflatoxins by Aspergillus flavus isolated from Brazil nuts. Six previously identified A. flavus strains were grown on coconut agar at 25°C for up to 10 days. Mycotoxins were separated by high-performance liquid chromatography and fungal growth was measured daily using the diametric mycelial growth rate. Transcriptional analysis was performed by real-time polymerase chain reaction (PCR) after 2 and 7 d of incubation using specific primers (aflR, aflD, aflP, lipase, metalloprotease, and LaeA). Three (50%) of the six A. flavus isolates produced AFB1 (ICB-1, ICB-12, and ICB-54) and three (50%) were not aflatoxigenic (ICB-141, ICB-161, and ICB-198). Aflatoxin production was observed from d 2 of incubation (1.5 ng/g for ICB-54) and increased gradually with time of incubation until d 10 (15,803.6 ng/g for ICB-54). Almost all A. flavus isolates exhibited a similar gene expression pattern after 2 d of incubation (p > 0.10). After 7 d of incubation, the LaeA (p < 0.05) and metalloprotease (p < 0.05) genes were the most expressed by nonaflatoxigenic strains, whereas aflatoxigenic isolates exhibited higher expression of the aflR (p < 0.05) and aflD genes (p < 0.05). Our results suggest that the expression of aflR and aflD is correlated with aflatoxin production in A. flavus and that overexpression of aflR could affect the transcriptional and aflatoxigenic pattern (ICB-54). Elucidation of the molecular mechanisms that regulate the secondary metabolism of toxigenic fungi may permit the rational silencing of the genes involved and consequently the programmed inhibition of aflatoxin production. Knowledge of the conditions, under which aflatoxin genes are expressed, should contribute to the development of innovative and more cost-effective strategies to
Suhem, Kitiya; Matan, Narumol; Nisoa, Mudtorlep; Matan, Nirundorn
This study aimed to optimize the operating parameters of cold atmospheric plasma treatment to inhibit the growth of Aspergillus flavus on agar media and brown rice cereal bars. The effects of argon plasma jet treatment on the growth of A. flavus on malt extract agar (MEA) at powers of 20 W and 40 W with exposure times at 5, 15 and 25 min were studied using response surface methodology (RSM) with a central composite face-centered (CCF) design. Multiple regression analysis indicated that plasma treatment at 40 W for 25 min is most effective for inhibiting growth of A. flavus on the agar medium. On brown rice cereal bars, plasma powered at 40 W for 20 min was capable of giving protection against A. flavus growth for up to 20 days under storage conditions of 25°C and 100% RH. These results demonstrated the potential of cold atmospheric plasma jet treatment to control mold growth on various food products. Copyright © 2012 Elsevier B.V. All rights reserved.
Gilbert, Matthew K; Mack, Brian M; Wei, Qijian; Bland, John M; Bhatnagar, Deepak; Cary, Jeffrey W
The filamentous fungus, Aspergillus flavus (A. flavus) is an opportunistic pathogen capable of invading a number of crops and contaminating them with toxic secondary metabolites such as aflatoxins. Characterizing the molecular mechanisms governing growth and development of this organism is vital for developing safe and effective strategies for reducing crop contamination. The transcription factor nsdC has been identified as being required for normal asexual development and aflatoxin production in A. flavus. Building on a previous study using a large (L)-sclerotial morphotype A. flavus nsdC mutant we observed alterations in conidiophore development and loss of sclerotial and aflatoxin production using a nsdC mutant of a small (S)-sclerotial morphotype, that normally produces aflatoxin and sclerotia in quantities much higher than the L-morphotype. RNA sequencing analysis of the nsdC knockout mutant and isogenic control strain identified a number of differentially expressed genes related to development and production of secondary metabolites, including aflatoxin, penicillin and aflatrem. Further, RNA-seq data indicating down regulation of aflatrem biosynthetic gene expression in the nsdC mutant correlated with HPLC analyses showing a decrease in aflatrem levels. The current study expands the role of nsdC as a globally acting transcription factor that is a critical regulator of both asexual reproduction and secondary metabolism in A. flavus.
Alizadeh, Alireza; Sharaifi, Rohollah; Javan-Nikkhah, Mohammad; Sedaghat, Narges
Essential oil components as result of non host disease resistance of plants have high capability to introduce as alternative of chemical pesticides. Thymus migricus essential oil was selected to investigation of its antifungal activity on survival and growth of Aspergillus flavus. For obtain essential oil first Leaves and flowers of Th. migricus collected then dried. The Essential oil was extracted by means of hydro-distillation and afterwards GC-MS analysis was performed to identify their components. The main constituents that resulted were Thymol (44.9%), Geraniol (10.8%), gamma-Terpinene (10.3%), Citronellol (8.5%) and p-Cymene (7.2%). EC50 and MIC (Minimum Inhibitory Concentration) of Th. migricus oil against A. flavus was 324.42 microl/l and 451.62 microl/l, respectively. Whereas EC50 and MIC for chemical thiabendazol was 650 microl/l and 1635 microl/l, respectively. The EC50 and MIC concentrations of Th. migricus oil in antifungal activity examination were used in aflatoxin inhibition test. Result of HPTLC measurement showed that both of concentrations inhibit aflatoxin production completely compares to control with 7.63 ppm aflatoxin production. In other word, Th. migricus oil can suppress aflatoxin production in concentrations lower than EC50 for mycelium growth.
Aldars-García, Laila; Sanchis, Vicente; Ramos, Antonio J; Marín, Sonia
The aim of this work was to assess the temporal relationship among quantified germination, mycelial growth and aflatoxin B1 (AFB1) production from colonies coming from single spores, in order to find the best way to predict as accurately as possible the presence of AFB1 at the early stages of contamination. Germination, mycelial growth, probability of growth and probability of AFB1 production of an isolate of Aspergillus flavus were determined at 25 °C and two water activities (0.85 and 0.87) on 3% Pistachio Extract Agar (PEA). The percentage of germinated spores versus time was fitted to the modified Gompertz equation for the estimation of the germination parameters (geometrical germination time and germination rate). The radial growth curve for each colony was fitted to a linear model for the estimation of the apparent lag time for growth and the growth rate, and besides the time to visible growth was estimated. Binary data obtained from growth and AFB1 studies were modeled using logistic regression analysis. Both water activities led to a similar fungal growth and AFB1 production. In this study, given the suboptimal set conditions, it has been observed that germination is a stage far from the AFB1 production process. Once the probability of growth started to increase it took 6 days to produce AFB1, and when probability of growth was 100%, only a 40-57% probability of detection of AFB1 production was predicted. Moreover, colony sizes with a radius of 1-2 mm could be a helpful indicator of the possible AFB1 contamination in the commodity. Despite growth models may overestimate the presence of AFB1, their use would be a helpful tool for producers and manufacturers; from our data 5% probability of AFB1 production (initiation of production) would occur when a minimum of 60% probability of growth is observed. Legal restrictions are quite severe for these toxins, thus their control from the early stages of contamination throughout the food chain is of paramount
Montes-Belmont, R; Carvajal, M
The effects of 11 plant essential oils for maize kernel protection against Aspergillus flavus were studied. Tests were conducted to determine optimal levels of dosages for maize protection, effects of combinations of essential oils, and residual effects and toxicity of essential oils to maize plants. Principal constituents of eight essential oils were tested for ability to protect maize kernels. Essential oils of Cinnamomum zeylanicum (cinnamon), Mentha piperita (peppermint), Ocimum basilicum (basil), Origanum vulgare (origanum), Teloxys ambrosioides (the flavoring herb epazote), Syzygium aromaticum (clove), and Thymus vulgaris (thyme) caused a total inhibition of fungal development on maize kernels. Thymol and o-methoxycinnamaldehyde significantly reduced maize grain contamination. The optimal dosage for protection of maize varied from 3 to 8%. Combinations of C. zeylanicum with the remaining oils gave efficient control. A residual effect of C. zeylanicum was detected after 4 weeks of kernel treatment. No phytotoxic effect on germination and corn growth was detected with any of these oils.
Panahirad, Sima; Zaare-Nahandi, Fariborz; Mohammadi, Nilufar; Alizadeh-Salteh, Saeedeh; Safaie, Naser
One of the most important saprophytic infections in fresh pistachio fruits after harvesting is Aspergillus flavus colonization, which significantly reduces fruit quality. Salicylic acid plays a crucial role in plant tissues and has a suppression effect on some fungi. The inhibitory effect of salicylic acid on the growth of A. flavus was assessed in vitro and in vivo. For this purpose, seven concentrations (0, 1, 3, 5, 7, 9 and 11 mmol L(-1)) of salicylic acid were used in both experiments. Also, aflatoxin B1 contents of the samples were analysed using immunoaffinity chromatography. The results obtained from in vitro experiments showed that salicylic acid significantly reduced Aspergillus growth at all concentrations, and at 9 mmol L(-1) growth was completely suppressed. In vivo evaluation showed relatively high levels of inhibition, though the intact treated fruits as compared with the injured treated fruits demonstrated higher inhibitory effects. Regarding the inhibitory effects of salicylic acid on the control of A. flavus contamination, its application on pistachio fruits after harvesting could be a promising approach to control the fungus infection and reduce aflatoxin production in treated fruits. © 2013 Society of Chemical Industry.
Gallo, Antonia; Epifani, Filomena; Bonsegna, Stefania; Pascale, Michelangelo; Santino, Angelo; Perrone, Giancarlo
Aflatoxins contamination by Aspergillus flavus is a matter of great concern for oil rich crops among which hazelnuts represent economically important agricultural commodities of Mediterranean countries, mainly used as mixed nuts or as ingredients in the bakery and confectionery industries. Since the biosynthetic pathway of aflatoxin biosynthesis has been elucidated in detail, expression analysis of the genes along the pathway can provide a thorough insight into the molecular mechanisms of toxin production and regulation. In the present work, we carried out a transcriptional analysis of the main genes belonging to aflatoxin biosynthetic cluster of A. flavus, namely the two regulatory genes aflR and aflS and the five structural genes aflD, aflM, aflO, aflP, and aflQ. The analysis was carried out at different stages of fungal growth on two different media: hazelnut agar medium and YES medium. The transcripts of all the genes paralleled the synthesis of aflatoxin and both were detected starting around 36h in YES medium, and 72h in hazelnut agar medium. Significantly, the amount of aflatoxin produced was about one order lower in hazelnut agar compared to YES medium. The expression of two genes encoding a lipase and a metalloprotease, potentially involved in lipid and protein catabolism, was also monitored during fungal growth. Noteworthy, the expression of the metalloprotease gene appeared to be specific for the hazelnut medium, whereas the lipase gene was expressed in both media. Finally, we verified the expression profiles of three genes encoding fatty acid dioxygenases/diol synthases involved in the biosynthesis of fungal oxylipins, namely ppoA, ppoB, ppoC. Recent findings have pointed out the importance of fungal oxylipins in fungal growth/mycotoxin production and our results indicated that all the three ppo genes are expressed during A. flavus growth on hazelnut medium. In particular, ppoB appeared to be specifically expressed in this medium. This study reports for
Jain, Rachna; Garg, Veena; Yadav, Deepak
Fungal degradation is emerging as a new powerful tool for the removal of potent neurotoxin pesticide, monocrotophos. Therefore, the present study is aimed at comparative characterization of monocrotophos degrading ability of three different fungal strains. Fungal strains were isolated from local agricultural soil by enrichment culture method, screened by gradient culture and identified as Aspergillus flavus, Fusarium pallidoroseum and Macrophomina sp. Growth kinetics revealed a direct positive influence of monocrotophos on the viability of fungal isolates. Fungal degradation was studied in phosphorus free liquid culture medium supplemented with 150 mg L(-1) concentration of monocrotophos for a period of 15 days under optimized culture conditions. Degradation of MCP followed first order kinetics with kdeg of 0.007, 0.002 and 0.005 day(-1) and half life (t1/2) of 4.21, 12.64 and 6.32 days for A. flavus, F. pallidoroseum and Macrophomina sp. respectively. To the best of our knowledge, it is the first report signifying the potential of monocrotophos degradation by Fusarium and Macrophomina sp. The results were further confirmed by HPTLC and FTIR which indicates disappearance of monocrotophos by hydrolytic cleavage of vinyl phosphate bond. Degradation of monocrotophos by fungal isolates was accompanied by the release of extracellular alkaline phosphatases, inorganic phosphates and ammonia. The overall comparative analysis followed the order of A. flavus > Macrophomina sp. > F. pallidoroseum. Therefore, it could be concluded from the study that these three different fungal strains could be effectively used as a potential candidate for the removal of monocrotophos from contaminated sites.
Grubisha, L C; Cotty, P J
Aspergillus flavus, a fungal pathogen of animals and both wild and economically important plants, is most recognized for producing aflatoxin, a cancer-causing secondary metabolite that contaminates food and animal feed globally. Aspergillus flavus has two self/nonself recognition systems, a sexual compatibility system and a vegetative incompatibility system, and both play a role in directing gene flow in populations. Aspergillus flavus reproduces clonally in wild and agricultural settings, but whether a cryptic sexual stage exists in nature is currently unknown. We investigated the distribution of genetic variation in 243 samples collected over 4 years from three common vegetative compatibility groups (VCGs) in Arizona and Texas from cotton using 24 microsatellite loci and the mating type locus (MAT) to assess population structure and potential gene flow among A. flavus VCGs in sympatric populations. All isolates within a VCG had the same mating type with OD02 having MAT1-2 and both CG136 and MR17 having MAT1-1. Our results support the hypothesis that these three A. flavus VCGs are genetically isolated. We found high levels of genetic differentiation and no evidence of gene flow between VCGs, including VCGs of opposite mating-type. Our results suggest that these VCGs diverged before domestication of agricultural hosts (>10,000 yr bp).
Delgado, Josué; Owens, Rebecca A; Doyle, Sean; Asensio, Miguel A; Núñez, Félix
Antifungal proteins produced by molds are generally small, highly basic, and cysteine-rich. The best known effects of these proteins include morphological changes, metabolic inactivation, and membrane perturbation on sensitive fungi. Reactive oxygen species (ROS) generation leads to apoptosis, with G -protein playing a key role in transduction of cell death signals. The antifungal protein PgAFP from Penicillium chrysogenum inhibits growth of some toxigenic molds. Here we analyzed the effect of the antifungal protein PgAFP on the growth of Aspergillus flavus. For this, comparative proteomic analysis was used to identify the whole protein profile and protein change in abundance after PgAFP treatment. PgAFP provoked metabolic changes related to reduced energy metabolism, cell wall integrity alteration, and increased stress response due to higher levels of ROS. The observed changes in protein abundance, favoring a higher glutathione concentration as well as the increased abundance in heat shock proteins, do not seem to be enough to avoid necrosis. The decreased chitin deposition observed in PgAFP-treated A. flavus is attributed to a lower relative quantity of Rho1. The reduced relative abundance of a β subunit of G -protein seems to be the underlying reason for modulation of apoptosis in PgAFP-treated A. flavus hyphae. We propose Rho1 and G -protein subunit β CpcB to be the main factors in the mode of action of PgAFP in A. flavus. Additionally, enzymes essential for the biosynthesis of aflatoxin were no longer detectable in A. flavus hyphae at 24 h, following treatment with PgAFP. This presents a promising effect of PgAFP, which may prevent A. flavus from producing mycotoxins. However, the impact of PgAFP on actual aflatoxin production requires further study.
Aflatoxin produced by Aspergillus flavus in corn poses significant health risks for both humans and livestock. Corn growers suffer huge economic losses due to increased aflatoxin accumulation in grain especially under drought and higher temperature stress conditions. Exploitation of host plant resi...
Aflatoxin, a toxin produced by the fungus Aspergillus flavus Link:Fries, occurs naturally in maize (Zea mays L.). Aflatoxin is a potent human carcinogen and is toxic to livestock, pets, and wildlife. When contaminated with aflatoxin, the value of maize grain is markedly reduced. Eight germplasm l...
Resistance to Aspergillus flavus by maize (Zea mays L.) is mediated by several defense proteins; however the mechanism regulating the expression of these defenses is poorly understood. This study examined the potential roles of six maize WRKY transcription factors, ZmWRKY19, ZmWRKY21, ZmWRKY53, ZmW...
Aspergillus flavus is one of the major fungal mold that colonize peanut in the field and during storage. The impacts to human and animal health and to economy in agriculture and commerce are significant since this mould produces the most potent natural toxins, aflatoxins, which are carcinogenic, mut...
Aspergillus flavus contaminates many important crops worldwide and is the major producer of aflatoxins, which are cancer-causing secondary metabolites. Biological control is the most effective means of reducing inoculum levels of detrimental aflatoxin-producing fungal pathogens in agricultural syst...
The colonization of maize (Zea mays L.) and peanut (Arachis hypogaea L.) by the fungal pathogen Aspergillus flavus and A. parasiticus results in the contamination with carcinogenic mycotoxins known as aflatoxins leading to economic losses as well as a potential health threat to human. The interactio...
The carcinogen, aflatoxin B1 (AFB1) produced by Aspergillus flavus, is a major food safety concern in crops. However, information on AFB1 occurrence in soil and crop residue is scarce. A series of experiments investigated the occurrence of AFB1 in soil and corn residues, and ascertained the ecology ...
Aspergillus flavus is able to synthesize a variety of polyketide-derived secondary metabolites including the hepatocarcinogen, aflatoxin B1. The fungus reproduces and disseminates predominantly by production of conidia. It also produces hardened mycelial aggregates called sclerotia that are used to ...
Field experiments were conducted in 2011 and 2012 to evaluate the efficacy of water dispersible granule (WDG) formulations of biocontrol strains of Aspergillus flavus in controlling aflatoxin contamination of corn. In 2011, when aflatoxin was present at very high levels, no WDG treatment provided s...
Plants use a variety of physical and chemical defenses in response to herbivory and pathogen attack. Infection of maize by the fungal pathogen Aspergillus flavus results in the accumulation of aflatoxins, which are among the most detrimental biogenic substances known to man. The majority of maize de...
Two new stilbene derivatives have been isolated from peanut seeds challenged by an Aspergillus flavus strain, along with chiricanine B that has not been reported from peanuts, as well as a stilbenoid that has been known as a synthetic product. The structures of these new putative phytoalexins were d...
..., refined oil; cotton, undelinted seed. (b) An exemption from the requirement of a tolerance is established... tolerance is established for residues of Aspergillus flavus AF36 in or on corn, field, forage; corn, field, grain; corn, field, stover; corn, field, aspirated grain fractions; corn, sweet, kernel plus cob with...
..., refined oil; cotton, undelinted seed. (b) Aspergillus flavus AF36 is temporarily exempt from the... flavis AF 36 is temporarily exempt from the requirement of a tolerance on corn, field, forage; corn, field, grain; corn, field, stover; corn, pop, grain; corn, pop, stover; corn, sweet, forage; corn, sweet...
The atoxigenic strain Aspergillus flavus AF36, which has been extensively used as a biocontrol agent in commercial corn and cotton fields to reduce aflatoxin contamination, was applied in research pistachio orchards from 2002 to 2005 and in commercial pistachio orchards from 2008 to 2011. AF36 was a...
Aspergillus flavus is a diverse assemblage of strains that include aflatoxin-producing and non-toxigenic strains with cosmopolitan distribution. The most promising strategy currently being used to reduce preharvest contamination of crops with aflatoxin is to introduce non-aflatoxin (biocontrol) A. f...
Aspergillus flavus produces potent mutagenic and carcinogenic polyketide-derived secondary metabolites known as aflatoxins. Development of host-plant resistance in peanut and other crops would be a cost-effective and practical approach to eliminate the serious problem of aflatoxin contamination due...
The European corn borer (ECB), Ostrinia nubilalis, is a major pest of corn in Europe and in several agricultural areas of the USA. In addition to direct yield losses, the ECB is expected to act as a vector for carrying spores of the aflatoxin-producing fungus Aspergillus flavus. Therefore the object...
Marilyn L. Warburton; Juliet D. Tang; Gary L. Windham; Leigh K. Hawkins; Seth C. Murray; Wenwei Xu; Debbie Boykin; Andy Perkins; W. Paul Williams
Contamination of maize (Zea mays L.) with aflatoxin, produced by the fungus Aspergillus flavus Link, has severe health and economic consequences. Efforts to reduce aflatoxin accumulation in maize have focused on identifying and selecting germplasm with natural host resistance factors, and several maize lines with significantly...
Aspergillus flavus is an opportunistic pathogenic fungus that infects several crops of agricultural importance, among them, corn, cotton, and peanuts. Once established as a pathogen the fungus may secrete secondary metabolites commonly known as mycotoxins, that if consumed by humans or animals may r...
In this study, we evaluated the feasibility of bioplastic-based formulations for delivering a non-aflatoxigenic strain of Aspergillus flavus and for monitoring Aspergilli with the final objective of controlling aflatoxin contamination in corn. Field application of inoculated bioplastic granules show...
The C2H2-type transcription factor NsdC (Never in Sexual Development C) has been shown to play a role in asexual development and secondary metabolite production in Aspergillus flavus, an agriculturally relevant, aflatoxin-producing species. The nsdC knoackout mutant demonstrates perturbed morphologi...
Acute aflatoxin poisonings (aflatoxicosis) in Kenya have led to the deaths of several hundred people between 2004 and 2006. Etiology of contamination in the outbreak districts (Eastern Province) identified an unusual fungal community structure dominated by the highly toxigenic Aspergillus flavus S s...
Aflatoxins are toxic secondary metabolites predominantly produced by the fungi Aspergillus flavus and A. parasiticus. Aflatoxin contaminated corn is toxic to domestic animals when ingested in feed and is a known carcinogen associated with liver and lung cancer in humans. Consequently, aflatoxin leve...
... tolerance is established for residues of Aspergillus flavus AF36 in or on corn, field, forage; corn, field, grain; corn, field, stover; corn, field, aspirated grain fractions; corn, sweet, kernel plus cob with husk removed; corn, sweet, forage; corn, sweet, stover; corn, pop, grain; and corn, pop, stover, when...
... tolerance is established for residues of Aspergillus flavus AF36 in or on corn, field, forage; corn, field, grain; corn, field, stover; corn, field, aspirated grain fractions; corn, sweet, kernel plus cob with husk removed; corn, sweet, forage; corn, sweet, stover; corn, pop, grain; and corn, pop, stover, when...
Aflatoxin contamination of peanut by Aspergillus flavus is exacerbated by drought stress. Drought also stimulates the production of reactive oxygen species (ROS) in plant tissues implying a correlation between ROS and aflatoxin production. Here, we performed gene expression analysis by RNAseq of tox...
The development of an efficient gene-targeting system is a prerequisite for success in the functional genomics study of Aspergillus flavus, an aflatoxin-producing fungus of great economic importance. To this end, the ku70 gene, a gene of the nonhomologous end-joining pathway, was deleted to increase...
Almonds, pistachios, and walnuts grown in California have an aggregate value of over $3.3 billion, with a large proportion of the crop being exported. However, these tree nuts can be subject to contamination by aflatoxins, metabolites produced primarily by Aspergillus flavus and parasiticus, and im...
Aspergillus flavus contains more than 55 gene clusters which are predicted to encode proteins involved in secondary metabolite production. One of these, cluster 27, contains a polyketide synthase (pks27) gene which encodes a protein that is highly homologous to the aflatoxin cluster PKS. Comparative...
The objectives of this study were to examine the effects of Aspergillus flavus colonization of maize kernels under different water activity (aw; 0.99 and 0.91) and temperature (30 and 37°C) conditions on (a) aflatoxin B1 (AFB1) production and (b) impacts on the transcriptome using RNAseq. This study...
Aspergillus flavus and aflatoxin contamination in the field are known to be influenced by numerous stress factors, particularly drought and heat stress. However, the purpose of aflatoxin production is unknown. Here, we report transcriptome analyses comprised of 282.6 Gb of sequencing data describing...
Contamination of crops with aflatoxin is a serious threat to global food safety. Aflatoxin production by Aspergillus flavus has been shown to be exacerbated by drought stress in the field and by oxidative stress in vitro. We examined the transcriptomes of three toxigenic and three atoxigenic isolate...
Drought stress predisposes oilseed crops such as maize and peanut to infection by Aspergillus flavus resulting in their contamination with aflatoxins. Drought stress in plants results in the accumulation of reactive oxygen species (ROS) in their tissues, and these ROS have been shown to stimulate af...
Aflatoxin contamination caused by Aspergillus flavus infection in crops is known to be exacerbated primarily by abiotic stresses such as drought stress, and biotic stresses such as arthropod infestation. These stresses result in the production and accumulation of reactive oxygen species (ROS) in the...
Contamination of maize with aflatoxin, produced by the fungus Aspergillus flavus, has severe health and economic consequences. Efforts to reduce aflatoxin accumulation in maize have focused on identifying and selecting germplasm with natural host resistance factors, and several maize lines with sign...
Schroeder, H. W.; Boller, R. A.
Peanuts, cottonseed, rice, and sorghum from Texas were sampled over a 3-year period. To insure adequate isolation of alfatoxin-producing species of fungi, low-quality lots were sampled at a rate greater than their respective proportional representation. Aflatoxins were found each year in peanut and cottonseed and were found in 2 of 3 years in rice and sorghum. Aflatoxins were detected in all four crops. The Aspergillus flavus group was much more prevalent in peanut and rice than in cottonseed and sorghum. Of the isolates of the A. flavus group, 96% from peanuts, 79% from cottonseed, 49% from sorghum, and 35% from rice produced aflatoxins. The average toxin production of isolates from rice was much less than that from peanuts, cottonseed, or sorghum. More than 90% of all isolates of the A. flavus group were identified as the species A. flavus. A. parasiticus was isolated from all four crops. Only A. parasiticus produced aflatoxin G. PMID:4197766
Davari, E; Mohsenzadeh, M; Mohammadi, Gh; Rezaeian-Doloei, R
Aflatoxins are secondary toxic metabolites produced by some Aspergillus spp. particularly, Aspergillus flavus and A. parasiticus that contaminate food and feed. The objective of this study was to evaluate the contamination of feedstuffs with Aspergillus spp. and detect genes involved in the aflatoxin biosynthesis pathway of A. flavus and A. parasiticus isolates. A total of 110 cow feed samples (comprised of silage, concentrate, hay and total mixed ration) from 30 industrial and semi-industrial dairy farms of Khorasan Razavi province, northeastern Iran, were examined using cultural and PCR methods. 68 (61.82%) Aspergillus spp. were isolated from 110 samples of feedstuff. The predominant Aspergillus isolates were A. fumigates (21.81%), followed by A. flavus (17.27%), A. niger (10%), A. parasiticus (8.18%), and A. oryzae (4.54%). Fungal contamination levels of industrial and semi-industrial dairy farm samples were not significantly different (P>0.05). Using four sets of primers, a quadruplex PCR was developed to detect genes (nor1, ver1, omtA and aflR) at different loci coding enzymes in the aflatoxin biosynthesis pathway of A. flavus and A. parasiticus strains. Out of 28 strains of A. flavus and A. parasiticus, 10 isolates (35.71%) showed a quadruplet pattern indicating the important genes involved in the aflatoxin biosynthesis pathway, encoded for functional products. These isolates were confirmed to be aflatoxigenic by Thin Layer Chromatography. 18 isolates (64.29%) had three, two and single molecular patterns. The results obtained by this study show that rapid and specific detection of aflatoxigenic molds is important to ensure the microbiological safety of feedstuffs. PMID:27175167
Wang, Shi; Park, Yong-Soon; Yang, Yang; Borrego, Eli J.; Isakeit, Tom; Gao, Xiquan; Kolomiets, Michael V.
Ethylene (ET) emitted by plant tissues has been broadly reported to play important roles in plant development, response to environmental stresses and defense against certain pathogens. Recent evidence obtained from using in vitro fungal cultures exposed to ET suggested that exogenous ET may regulate the production of aflatoxin by Aspergilli. However, the function of endogenous, seed-derived ET has not been explored. In this study, we found that the maize lipoxygenase lox3 mutant, previously reported to be susceptible to Aspergillus spp., emitted greater levels of ET upon A. flavus infection, suggesting the potential involvement of endogenous ET in the susceptibility of maize to A. flavus. Supporting this idea, both colonization and conidiation of A. flavus were reduced in wild-type (WT) kernels treated with AgNO3, an ET synthesis inhibitor. There was no ET emission from non-viable kernels colonized by A. flavus, suggesting that living seed but not the fungus itself was the primary source of ET released upon infection with A. flavus. The kernels of acs2 and acs6, two ET biosynthetic mutants carrying Mutator transposons in the ACC synthase genes, ACS2 and ACS6, respectively, displayed enhanced seed colonization and conidiation, but not the levels of aflatoxin, upon infection with A. flavus. Surprisingly, both acs2 and acs6 mutant kernels emitted greater levels of ET in response to infection by A. flavus as compared with WT seed. The increased ET in single mutants was found to be due to overexpression of functional ACS genes in response to A. flavus infection. Collectively, these findings suggested that ET emitted by infected seed facilitates colonization by A. flavus but not aflatoxin production. PMID:28400781
Hua, Sui-Sheng T.; Baker, James L.; Flores-Espiritu, Melanie
The nor mutant of Aspergillus flavus has a defective norsolorinic acid reductase, and thus the aflatoxin biosynthetic pathway is blocked, resulting in the accumulation of norsolorinic acid, a bright red-orange pigment. We developed a visual agar plate assay to monitor yeast strains for their ability to inhibit aflatoxin production by visually scoring the accumulation of this pigment of the nor mutant. We identified yeast strains that reduced the red-orange pigment accumulation in the nor mutant. These yeasts also reduced aflatoxin accumulation by a toxigenic strain of A. flavus. These yeasts may be useful for reducing aflatoxin contamination of food commodities. PMID:10347069
Aspergillus flavus and Fusariumverticillioides are fungal pathogens that colonize maize seeds and contaminate them with mycotoxins. To investigate the plant microbe interactions, we conducted histological and molecular studies to characterize the internal colonization of maize seed by the two fungal...
Rosada, L J; Sant'anna, J R; Franco, C C S; Esquissato, G N M; Santos, P A S R; Yajima, J P R S; Ferreira, F D; Machinski, M; Corrêa, B; Castro-Prado, M A A
Aspergillus flavus, a haploid organism found worldwide in a variety of crops, including maize, cottonseed, almond, pistachio, and peanut, causes substantial and recurrent worldwide economic liabilities. This filamentous fungus produces aflatoxins (AFLs) B1 and B2, which are among the most carcinogenic compounds from nature, acutely hepatotoxic and immunosuppressive. Recent efforts to reduce AFL contamination in crops have focused on the use of nonaflatoxigenic A. flavus strains as biological control agents. Such agents are applied to soil to competitively exclude native AFL strains from crops and thereby reduce AFL contamination. Because the possibility of genetic recombination in A. flavus could influence the stability of biocontrol strains with the production of novel AFL phenotypes, this article assesses the diversity of vegetative compatibility reactions in isolates of A. flavus to identify heterokaryon self-incompatible (HSI) strains among nonaflatoxigenic isolates, which would be used as biological controls of AFL contamination in crops. Nitrate nonutilizing (nit) mutants were recovered from 25 A. flavus isolates, and based on vegetative complementation between nit mutants and on the microscopic examination of the number of hyphal fusions, five nonaflatoxigenic (6, 7, 9 to 11) and two nontoxigenic (8 and 12) isolates of A. flavus were phenotypically characterized as HSI. Because the number of hyphal fusions is reduced in HSI strains, impairing both heterokaryon formation and the genetic exchanges with aflatoxigenic strains, the HSI isolates characterized here, especially isolates 8 and 12, are potential agents for reducing AFL contamination in crops.
Divakara, S T; Aiyaz, M; Moore, G G; Venkataramana, M; Hariprasad, P; Nayaka, S Chandra; Niranjana, S R
Thirty-four Aspergillus flavus isolates were recovered from sorghum seeds sampled across five states in India. Our study included (1) species confirmation through PCR assay, (2) quantification of total aflatoxin concentrations by the indirect competitive-ELISA (ic-ELISA) method, and (3) analysis of molecular diversity among the A. flavus isolates using β-tubulin, ITS, and ISSR markers. Among the isolates studied, 28 were found to be positive for the production of aflatoxins. ITS and β-tubulin phylogenetic analysis segregated the A. flavus sample population into two major groups or clades with little to no subdivision based on geography. In contrast, ISSR analysis also separated the A. flavus isolates into two main clusters, showing a distance of 0.0-0.5, with one cluster exhibiting a high level of diversity though no geographic or chemotype subdivision could be observed. The majority of sampled A. flavus isolates were highly toxigenic, and also highly diversified in terms of toxin-producing potential in-vitro. Genetic diversity among the sorghum isolates of A. flavus further warrants the development of appropriate farming management practices as well as improved aflatoxin detection measures in India. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
Leema, George; Chou, Duen-Suey; Jesudasan, Christadoss A Nelson; Geraldine, Pitchairaj; Thomas, Philip A
To document transcriptional activation (expression) of key aflatoxin biosynthetic pathway genes in corneal isolates of Aspergillus flavus. The expression of certain regulatory (aflatoxin regulatory [aflR] and aflatoxin J [aflJ]) and structural (polyketide synthase acetate [pksA] and norsolonic acid-1 [nor-1]) genes in four corneal A. flavus isolates was evaluated by reverse transcription PCR. The aflatoxin-producing potential of each strain was determined by thin-layer chromatography and quantified by spectrophotometry. Four environmental isolates were used for comparison. The mean expression levels of these genes were compared in the corneal and environmental A. flavus isolates. In addition, the mean expression levels were also correlated with the aflatoxin production levels. All isolates expressed aflJ, nor-1, and pksA, while all but one expressed aflR. Overall, significantly higher mean expression levels occurred in aflatoxigenic than in non-aflatoxigenic corneal isolates. A significant positive correlation was noted between the mean expression level of aflR and the quantum of aflatoxin production by the corneal isolates. Essentially similar patterns of expression of these genes were noted in four environmental A. flavus isolates used for comparison. For the first time, isolates of A. flavus from human keratitis patients have been shown to express regulatory and structural aflatoxin biosynthetic pathway genes. Further studies are needed to clarify the precise influence of the corneal microenvironment on expression of these genes and aflatoxin production by A. flavus infecting the cornea.
Sweany, Rebecca Ruth; Damann, Kenneth Eugene; Kaller, Michael Douglas
Aspergillus flavus is considered a generalist-opportunistic pathogen, but studies are beginning to show that A. flavus populations have strains specific to various hosts. The research objective was to determine whether A. flavus soil populations consist of solely saprophytic strains and strains which can be facultatively parasitic on corn. A. flavus was isolated from both corn kernels and soil within 11 Louisiana fields. Sixteen vegetative compatibility groups (VCGs) were identified among 255 soil isolates. Only 6 of the 16 VCGs were identified in the 612 corn isolates and 88% of corn isolates were in two VCGs, whereas only 5% of soil isolates belonged to the same two VCGs. Isolates were characterized for aflatoxin B1 production and sclerotial size. A random subset of the isolates (99 from corn and 91 from soil) were further characterized for simple-sequence repeat (SSR) haplotype and mating type. SSR polymorphisms revealed 26 haplotypes in the corn isolates and 78 in the soil isolates, and only 1 haplotype was shared between soil and corn isolates. Corn and soil populations were highly significantly different for all variables. Differences between corn and soil populations indicate that some soil isolates are not found in corn and some isolates have become specialized to infect corn. Further understanding of A. flavus virulence is important for development of resistant hybrids and for better biological control against toxigenic A. flavus.
Aspergillus flavus is a ubiquitous saprophyte and is capable of producing many secondary metabolites including the carcinogenic aflatoxins. The A. flavus population that produces small sclerotia (S strain) has been implicated as the culprit for persistent aflatoxin contamination of crops in fields. ...
A series of field studies in corn (maize) evaluated the ability of non-aflatoxigenic biocontrol strains of Aspergillus flavus to reduce, through competitive exclusion, production in kernels of aflatoxins and cyclopiazonic acid (CPA) by A. flavus and fumonisins by Fusarium verticillioides. The abili...
Six Aspergillus flavus isolates out of 17 fungal isolates were sampled from diverse food and organic matter in southwest Nigeria. All the A. flavus samples produced aflatoxin and cyclopiazonic acid. These six isolates constitute a ready mycobank of toxigenic species for analytical research involving...
Aflatoxin contamination of food and livestock feed results in significant annual crop losses internationally. Aspergillus flavus is the major fungus responsible for this loss. Additionally, A. flavus is the second leading cause of aspergillosis in immune compromised human patients. Here we report th...
Cyclopiazonic acid (a-cyclopiazonic acid, a-CPA) is an indole-hydrindane-tetramic acid neurotoxin produced by various fungal species, including the notorious food and feed contaminant Aspergillus flavus. Despite its discovery in A. flavus cultures, approximately 40 years ago, its contribution to the...
Evaluation of resistance or susceptibility of corn inbreds to infection by Aspergillus flavus was evaluated by a kernel screening assay. A GFP-expressing strain of A. flavus was used to accomplish this study to measure fungal spread and aflatoxin levels in real time. Among the four inbreds tested, ...
Non-toxigenic strains of Aspergillus flavus offer the potential to control aflatoxin contamination by competitive displacement of indigenous populations of A. flavus colonizing corn grain. Two sets of experiments were conducted to assess the competitiveness of strain K49 when challenged against two...
Aspergillus flavus produces a variety of toxic secondary metabolites, among them the aflatoxins (AFs) are the most well-known. These compounds are highly mutagenic and carcinogenic, particularly AFB1. A. flavus is capable of colonizing economically important crops contaminating them with AFs. Molecu...
Nierman, William C; Yu, Jiujiang; Fedorova-Abrams, Natalie D; Losada, Liliana; Cleveland, Thomas E; Bhatnagar, Deepak; Bennett, Joan W; Dean, Ralph; Payne, Gary A
Aflatoxin contamination of food and livestock feed results in significant annual crop losses internationally. Aspergillus flavus is the major fungus responsible for this loss. Additionally, A. flavus is the second leading cause of aspergillosis in immunocompromised human patients. Here, we report the genome sequence of strain NRRL 3357.
Aspergillus flavus, an opportunistic pathogen, contaminates maize and other key crops with carcinogenic aflatoxins (AF). Besides AF, A. flavus makes many more secondary metabolites (SMs), whose toxicity in insects or vertebrates has been studied. However, the role of SMs in the invasion of plant hos...
Mahmoud, Mohamed A
Aspergillus flavus is the main species from section Flavi responsible for aflatoxin accumulation in stored peanuts. Rapid methods to detect A. flavus could help to prevent aflatoxins from entering the food chain. A real-time polymerase chain reaction (RTi-PCR) assay was standardized for rapid, specific, and sensitive detection of A. flavus in stored peanuts. A. flavus was detected in 53.6% and 50% of peanut samples by RTi-PCR and A. flavus and Aspergillus parasiticus agar culture, respectively, with 95% agreement between them. Twenty-two A. flavus isolates were screened using high-performance liquid chromatography for their capacity to produce aflatoxin AFB1 (B1). B1 was produced by >72% of the isolates. Sixteen isolates produced B1 at concentrations ranging from 1.64 to 109.18 μg/mL. Four aflatoxin biosynthetic pathway genes (aflD, aflM, aflP, and aflQ) were evaluated using PCR and reverse-transcription PCR in 22 A. flavus isolates from peanut kernels with the aim of rapidly and accurately differentiating toxigenic and atoxigenic isolates. The PCR amplification of genes did not correlate with aflatoxin production capability. The expression of aflD and aflQ was a good marker for differentiating toxigenic from atoxigenic isolates.
Contamination of corn, cotton, peanuts and tree nuts by aflatoxins is a severe economic burden for growers. A current biocontrol strategy is to use non-aflatoxigenic Aspergillus flavus strains to competitively exclude field toxigenic Aspergillus species. Aspergillus flavus K49 does not produce aflat...
Centeno, S; Calvo, M A; Adelantado, C; Figueroa, S
The antifungal activity of ethanolic extracts of Rosmarinus officinalis and Thymus vulgaris were tested against strains of Aspergillus flavus and A. ochraceus, since these two species are common contaminants of cereals and grains and are able to produce and accumulate mycotoxins. The methodology used is based on measuring the inhibition halos produced by discs impregnated with the extracts and establishing their Minimum Inhibitory Concentration (MIC) as well as the Minimum Fungicide Concentration (MFC). The results obtained suggest that the assayed extracts affect the proper development of A. flavus and A. ochraceus; leading to a lower MIC (1200 ppm) and MFC (2400 ppm) for T. vulgaris extract against A. ochraceus than against A. flavus. The results show, that the extracts of Rosmarinus officinalis and Thymus vulgaris used at low concentrations could have significant potential for the biological control of fungi in foodstuffs.
Ismaiel, Ahmed A; Ghaly, Mohamed F; El-Naggar, Ayman K
The association of kefir microbiota was observed by electron microscopic examination. Scanning electron microscopic (SEM) observations revealed that kefir grain surface is very rough and the inner portions had scattered irregular holes on its surface. The interior of the grain comprised fibrillar materials which were interpreted as protein, lipid and a soluble polysaccharide, the kefiran complex that surrounds yeast and bacteria in the grain. Yeast was observed more clearly than bacteria on the outer portion of the grain. Transmission electron microscopic (TEM) observations of kefir revealed that the grain comprised a mixed culture of yeast and bacteria growing in close association with each other. Microbiota is dominated by budded and long-flattened yeast cells growing together with lactobacilli and lactococci bacteria. Bacterial cells with rounded ends were also observed in this mixed culture. Kefir grains, kefir suspensions, and kefiran were tested for antimicrobial activities against several bacterial and fungal species. The highest activity was obtained against Streptococcus faecalis KR6 and Fusarium graminearum CZ1. Growth of Aspergillus flavus AH3 producing for aflatoxin B1 for 10 days in broth medium supplemented with varying concentrations of kefir filtrate (%, v/v) showed that sporulation was completely inhibited at the higher concentrations of kefir filtrate (7-10%, v/v). The average values of both mycelial dry weights and aflatoxin B1 were completely inhibited at 10% (v/v). This is the first in vitro study about the antifungal characteristics of kefir against filamentous fungi which was manifested by applying its inhibitory effect on the productivity of aflatoxin B1 by A. flavus AH3.
Mahmoud, M A; Ali, H M; El-Aziz, A R M; Al-Othman, M R; Al-Wadai, A S
Twelve species from six fungal genera were found to be associated with corn (Zea mays L.) grain samples collected from three main regions of Saudi Arabia. The average frequencies of the most common genera were Aspergillus (11.4%), Fusarium (9.5%), Penicillium (5.1%), and Alternaria (5.8%). Fifteen isolates of Aspergillus flavus were screened by HPLC for their ability to produce aflatoxins (AF). The percentage of aflatoxigenic A. flavus isolates was 53%. Eight isolates produced AF, at concentrations ranging 0.7-2.9 ppb. Random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) molecular markers were used to genetically characterize isolates of A. flavus and to discriminate between the aflatoxigenic and non-aflatoxigenic isolates. RAPD and ISSR analysis revealed a high level of genetic diversity in the A. flavus population, which was useful for genetic characterization. The clustering in the RAPD and ISSR dendrograms obtained was unrelated to geographic origin. The RAPD and ISSR markers could not discriminate between aflatoxigenic and non-aflatoxigenic isolates, but the ISSR primers were somewhat better.
Hadrich, Inès; Neji, Sourour; Drira, Inès; Trabelsi, Houwaida; Mahfoud, Nedia; Ranque, Stéphane; Makni, Fattouma; Ayadi, Ali
Aspergillus flavus is the second most important Aspergillus species associated with aspergillosis and the incidence of infections caused by it are increasing in the immunocompromised population. This species is of major epidemiological importance in regions with a dry and hot climate. Despite the growing clinical significance of A. flavus, data on its molecular epidemiology are scarce. This study was aimed at examining whether isolates from distinct genotypes were involved in distinct clinical forms of aspergillosis. Sixty-three clinical isolates of A. flavus recovered from 35 patients with various clinical presentations of aspergillosis were characterized by microsatellite typing. The highest discriminatory power for a single locus was obtained with the AFLA1 marker, which had 14 distinct alleles and a 0.903 D value. The combination of all six markers yielded 48 different genotypes with a 0.994 D value. There was a considerable genetic diversity in the isolates and patients with invasive aspergillosis were usually colonized by multiples genotypes. There was no evidence that a given genotype was associated with a particular clinical presentation of A. flavus aspergillosis. The occurrence of more than one genotype in clinical samples indicates that a patient may be infected by multiple genotypes and that any particular isolate from a clinical specimen may not necessarily be the one causing aspergillosis.
Tripathi, Smita; Mishra, H.N.
Quantitative losses in various biochemical constituents like capsaicin, carotenes, ascorbic acid, polyphenols, mineral matter, sugars (soluble and insoluble), protein and fat were estimated after the successful growth of Aspergillus flavus for 30 days on powdered red pepper. The fungal biomass was measured by ergosterol content and Aflatoxin B1 by HPLC. Amongst the various nutritional constituents evaluated for nutritional losses and changes the highest nutritional loss was reported in total carotenoids (88.55%) followed by total sugars (85.5%). The protein content of the infected sample increased from 18.01% to 23%. The nutritional profile of chilli powder (Capsicum annum var. sannam L.) shows highest share of total soluble sugars (32.89%) and fiber content (21.05%), followed by protein (18.01%) and fat (13.32%) making it an ideal solid- substrate for mould growth. At the end of incubation the fungal biomass was 192. 25 mg / 100 gram powder, total plate count 17.5 X 10 4 CFU/g and Aflatoxin B1 content was 30.06 μg / kg. PMID:24031333
Luo, Man; Jiang, Li-Ke; Huang, Yao-Xiong; Xiao, Ming; Li, Bo; Zou, Guo-Lin
Citral refined from Litsea cubeba oil has been found to have a strong influence on fungi, especially Aspergillus flavus. Multiplex microanalysis and quasi-elastic light scattering techniques were applied to study the effects of citral on Aspergillus flavus spores from the levels of membrane, organelle and intracellular macromolecule. It was found that citral injured the wall and the membrane of A. flavus spore, resulting in decrease of its elasticity. After entering the cell, citral not only influenced the genetic expression of mitochondrion reduplication and its morphology, but also changed the aggregation of protein-like macromolecules. As a result, cells, organelles and macromolecules lost their normal structures and functions, eventually leading to the loss of germination ability of A. flavus spores. Since Litsea cubeba oil as food additive and antifungal agent is safe and less poisonous, it is important to elucidate the inhibitory mechanisms of Litsea cubeba oil on the germination ability of A. flavus spore.
Dorner, Joe W
A method was developed for simultaneous quantitation of Aspergillus flavus/A. parasiticus and aflatoxins in peanuts. Peanut samples were ground with an equal weight of water in a vertical cutter mixer to produce a slurry. Separate subsamples were taken for dilution-plating to determine total colony forming units (CFU)/g of A. flavus/A. parasiticus and for liquid chromatographic analysis to determine aflatoxin concentrations. Dry-grinding peanuts for homogenization of aflatoxins produced high temperatures that killed most of the A. flavus/A. parasiticus propagules. Addition of water to produce a slurry kept the temperature from rising above levels that killed the fungi. A 7 min grind time provided optimal homogenization for both the fungi and aflatoxins, so long as the temperature of the slurry did not exceed 45 degrees C. In the analysis of 60 shelled peanut samples, total aflatoxin concentrations ranged from 0 to 10,000 ng/g and total A. flavus/A. parasiticus ranged from 1.4 x 10(3) to 3.2 x 10(6) CFU/g. Regression analysis showed a significant positive correlation (p < 0.0001) between the quantities of A. flavus/A. parasiticus and aflatoxin (R2 = 0.82).
Solorzano, Cesar D.; Abbas, Hamed K.; Zablotowicz, Robert M.; Chang, Perng-Kuang; Jones, Walker A.
A nontoxigenic Aspergillus flavus strain, K49, is currently being tested as a biological control agent in corn fields in the Mississippi Delta. However, little is known about the overall genetic diversity of A. flavus from year to year in corn fields and specifically in Mississippi. Our objective was to assess the genetic variability of A. flavus isolates from different seasons, inoculum sources, and years, from a no-till corn field. Of the 175 A. flavus isolates examined, 74 and 97 had the typical norB-cypA type I (1.5 kb) and type II (1.0 kb) deletion patterns, respectively. Variability in the sequence of the omtA gene of the majority of the field isolates (n = 118) was compared to strain K49. High levels of haplotypic diversity (24 omtA haplotypes; Hd = 0.61 ± 0.04) were found. Among the 24 haplotypes, two were predominant, H1 (n = 71), which consists of mostly toxigenic isolates, and H49 (n = 18), which consists of mostly atoxigenic isolates including K49. Toxigenic isolates were prevalent (60%) in this natural population. Nonetheless, about 15% of the population likely shared the same ancestral origin with K49. This study provides valuable information on the diversity of A. flavus. This knowledge can be further used to develop additional biological control strains. PMID:25478591
Yu, Jiujiang; Cleveland, Thomas E; Nierman, William C; Bennett, Joan W
Aspergillus flavus is an imperfect filamentous fungus that is an opportunistic pathogen causing invasive and non-invasive aspergillosis in humans, animals, and insects. It also causes allergic reactions in humans. A. flavus infects agricultural crops and stored grains and produces the most toxic and potent carcinogic metabolites such as aflatoxins and other mycotoxins. Breakthroughs in A. flavus genomics may lead to improvement in human health, food safety, and agricultural economy. The availability of A. flavus genomic data marks a new era in research for fungal biology, medical mycology, agricultural ecology, pathogenicity, mycotoxin biosynthesis, and evolution. The availability of whole genome microarrays has equipped scientists with a new powerful tool for studying gene expression under specific conditions. They can be used to identify genes responsible for mycotoxin biosynthesis and for fungal infection in humans, animals and plants. A. flavus genomics is expected to advance the development of therapeutic drugs and to provide information for devising strategies in controlling diseases of humans and other animals. Further, it will provide vital clues for engineering commercial crops resistant to fungal infection by incorporating antifungal genes that may prevent aflatoxin contamination of agricultural harvest.
Tian, Jun; Ban, Xiaoquan; Zeng, Hong; He, Jingsheng; Chen, Yuxin; Wang, Youwei
The essential oil extracted from the seeds of dill (Anethum graveolens L.) was demonstrated in this study as a potential source of an eco-friendly antifungal agent. To elucidate the mechanism of the antifungal action further, the effect of the essential oil on the plasma membrane and mitochondria of Aspergillus flavus was investigated. The lesion in the plasma membrane was detected through flow cytometry and further verified through the inhibition of ergosterol synthesis. The essential oil caused morphological changes in the cells of A. flavus and a reduction in the ergosterol quantity. Moreover, mitochondrial membrane potential (MMP), acidification of external medium, and mitochondrial ATPase and dehydrogenase activities were detected. The reactive oxygen species (ROS) accumulation was also examined through fluorometric assay. Exposure to dill oil resulted in an elevation of MMP, and in the suppression of the glucose-induced decrease in external pH at 4 µl/ml. Decreased ATPase and dehydrogenase activities in A. flavus cells were also observed in a dose-dependent manner. The above dysfunctions of the mitochondria caused ROS accumulation in A. flavus. A reduction in cell viability was prevented through the addition of L-cysteine, which indicates that ROS is an important mediator of the antifungal action of dill oil. In summary, the antifungal activity of dill oil results from its ability to disrupt the permeability barrier of the plasma membrane and from the mitochondrial dysfunction-induced ROS accumulation in A. flavus. PMID:22272289
Tian, Jun; Ban, Xiaoquan; Zeng, Hong; He, Jingsheng; Chen, Yuxin; Wang, Youwei
The essential oil extracted from the seeds of dill (Anethum graveolens L.) was demonstrated in this study as a potential source of an eco-friendly antifungal agent. To elucidate the mechanism of the antifungal action further, the effect of the essential oil on the plasma membrane and mitochondria of Aspergillus flavus was investigated. The lesion in the plasma membrane was detected through flow cytometry and further verified through the inhibition of ergosterol synthesis. The essential oil caused morphological changes in the cells of A. flavus and a reduction in the ergosterol quantity. Moreover, mitochondrial membrane potential (MMP), acidification of external medium, and mitochondrial ATPase and dehydrogenase activities were detected. The reactive oxygen species (ROS) accumulation was also examined through fluorometric assay. Exposure to dill oil resulted in an elevation of MMP, and in the suppression of the glucose-induced decrease in external pH at 4 µl/ml. Decreased ATPase and dehydrogenase activities in A. flavus cells were also observed in a dose-dependent manner. The above dysfunctions of the mitochondria caused ROS accumulation in A. flavus. A reduction in cell viability was prevented through the addition of L-cysteine, which indicates that ROS is an important mediator of the antifungal action of dill oil. In summary, the antifungal activity of dill oil results from its ability to disrupt the permeability barrier of the plasma membrane and from the mitochondrial dysfunction-induced ROS accumulation in A. flavus.
Houshyarfard, Mahmoud; Rouhani, Hamid; Falahati-Rastegar, Mahrokh; Malekzadeh-Shafaroudi, Saeid; Mahdikhani-Moghaddam, Esmat
Out of fifty-two Iranian nonaflatoxigenic strains of Aspergillus flavus,collected from various substrates (soil and kernel) and sources (peanut, corn and pistachio), fifteen representatives were selected according to their different geographical origins (six provinces: Guilan and Golestan, Ardebil, Fars, Kerman and Semnan) and vegetative compatibility groups (VCGs, IR1 to IR15) for microsatellite-primed PCR analysis. Two inter-simple sequence repeat (ISSR) primers AFMPP and AFM13 were used to determine polymorphism and the relationship among strain isolates. A. flavus isolates were identified by their morphologies and their identities were confirmed by PCR amplification using the specific primer pair ITS1 and ITS4. The results revealed variations in the percentages of polymorphisms. In the ISSR analysis, primers AFMPP and AFM13 generated a total of 18 and 23 amplicons among the fungal strains, out of which 12 (66.7%) and 22 (95.7%) were polymorphic, respectively. Cluster analysis of the ISSR data was carried out using 1 D DNA gel image analysis. The two dendrograms obtained through these markers showed six different clusterings of testing nonaflatoxigenic A. flavus L strains, but we noticed that some clusters were different in some cases. The microsatellite-primed PCR data revealed that the Iranian nonaflatoxigenic isolates of A. flavus were not clustered according to their origins and sources. This study is the first to characterize Iranian nonaflatoxigenic isolates of A. flavus using ISSR markers. PMID:27843995
Solorzano, Cesar D; Abbas, Hamed K; Zablotowicz, Robert M; Chang, Perng-Kuang; Jones, Walker A
A nontoxigenic Aspergillus flavus strain, K49, is currently being tested as a biological control agent in corn fields in the Mississippi Delta. However, little is known about the overall genetic diversity of A. flavus from year to year in corn fields and specifically in Mississippi. Our objective was to assess the genetic variability of A. flavus isolates from different seasons, inoculum sources, and years, from a no-till corn field. Of the 175 A. flavus isolates examined, 74 and 97 had the typical norB-cypA type I (1.5 kb) and type II (1.0 kb) deletion patterns, respectively. Variability in the sequence of the omtA gene of the majority of the field isolates (n = 118) was compared to strain K49. High levels of haplotypic diversity (24 omtA haplotypes; Hd = 0.61 ± 0.04) were found. Among the 24 haplotypes, two were predominant, H1 (n = 71), which consists of mostly toxigenic isolates, and H49 (n = 18), which consists of mostly atoxigenic isolates including K49. Toxigenic isolates were prevalent (60%) in this natural population. Nonetheless, about 15% of the population likely shared the same ancestral origin with K49. This study provides valuable information on the diversity of A. flavus. This knowledge can be further used to develop additional biological control strains.
Aspergillus flavus and A. parasiticus fungi, carcinogen-mycotoxins producers, infect peanut seeds, causing considerable impact on both human health and the economy. Here we report 9 genome sequences of Aspergillus spp. isolated from peanut seeds. The information obtained will allow conducting biodiv...
Cyclopiazonic acid (CPA), an indole-tetramic acid toxin, is produced by many species of Aspergillus and Penicillium. In addition to CPA Aspergillus flavus produces polyketide-derived carcinogenic aflatoxins (AFs). AF biosynthesis genes form a gene cluster in a subtelomeric region. Isolates of A. fla...
Almousally, Ibrahem; Shaban, Mouhnad; Blee, Elizabeth
Caleosins are a small family of calcium-binding proteins endowed with peroxygenase activity in plants. Caleosin-like genes are present in fungi; however, their functions have not been reported yet. In this work, we identify a plant caleosin-like protein in Aspergillus flavus that is highly expressed during the early stages of spore germination. A recombinant purified 32-kDa caleosin-like protein supported peroxygenase activities, including co-oxidation reactions and reduction of polyunsaturated fatty acid hydroperoxides. Deletion of the caleosin gene prevented fungal development. Alternatively, silencing of the gene led to the increased accumulation of endogenous polyunsaturated fatty acid hydroperoxides and antioxidant activities but to a reduction of fungal growth and conidium formation. Two key genes of the aflatoxin biosynthesis pathway, aflR and aflD, were downregulated in the strains in which A. flavus PXG (AfPXG) was silenced, leading to reduced aflatoxin B1 production in vitro. Application of caleosin/peroxygenase-derived oxylipins restored the wild-type phenotype in the strains in which AfPXG was silenced. PXG-deficient A. flavus strains were severely compromised in their capacity to infect maize seeds and to produce aflatoxin. Our results uncover a new branch of the fungal oxylipin pathway and may lead to the development of novel targets for controlling fungal disease. PMID:26116672
Accinelli, Cesare; Saccà, M Ludovica; Abbas, Hamed K; Zablotowicz, Robert M; Wilkinson, Jeffery R
Previous research demonstrated that aflatoxin contamination in corn is reduced by field application of wheat grains pre-inoculated with the non-aflatoxigenic Aspergillus flavus strain NRRL 30797. To facilitate field applications of this biocontrol isolate, a series of laboratory studies were conducted on the reliability and efficiency of replacing wheat grains with the novel bioplastic formulation Mater-Bi to serve as a carrier matrix to formulate this fungus. Mater-Bi granules were inoculated with a conidial suspension of NRRL 30797 to achieve a final cell density of approximately log 7 conidia/granule. Incubation of 20-g soil samples receiving a single Mater-Bi granule for 60-days resulted in log 4.2-5.3 propagules of A. flavus/g soil in microbiologically active and sterilized soil, respectively. Increasing the number of granules had no effect on the degree of soil colonization by the biocontrol fungus. In addition to the maintenance of rapid vegetative growth and colonization of soil samples, the bioplastic formulation was highly stable, indicating that Mater-Bi is a suitable substitute for biocontrol applications of A. flavus NRRL 30797.
Ascomycetous fungi of the genus Aspergillus comprise a wide variety of species of biotechnological importance as well as pathogens and toxin producers. Recent studies report A. fumigatus to be heterothallic and possibly undergoing sexual reproduction. We therefore investigated whether compatible mat...
Gandomi, Hassan; Misaghi, Ali; Basti, Afshin Akhondzadeh; Hamedi, Hassan; Shirvani, Zahra Ramezani
The mode of inhibitory action of Zataria multiflora Boiss. essential oil (EO) on the fungus, Aspergillus flavus, was studied by colony morphology examination, light microscopy, scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The EO at concentrations used in this study suppressed the size of the colony as well as sporulation. SEM of mycelia treated with given concentrations of EO showed morphological alterations ranging from loss of turgidity and uniformity of mycelia at low concentrations of EO to evident destruction of the hyphae at higher concentration of EO. Semi-thin sections of mycelia exposed to different concentrations of EO were analysed by light microscopy and revealed that the major change at level as low as 50 ppm of EO was limited to vacuolisation of cytoplasm resulting in cell swelling, while at higher concentrations, detachment of the cell membrane from the cell wall, deformation of mycelia and shedding the cytoplasm from the cell were the main alterations. These damages were well documented by TEM, which showed that the main sites of action of EO were the plasma membrane and cell wall. In conclusion, morphological and structural changes observed in this study may be one of the mechanisms involved in growth inhibition of the fungi and reducing aflatoxin production.
Yan, Shijuan; Liang, Yating; Zhang, Jindan; Chen, Zhuang; Liu, Chun-Ming
Aflatoxins produced by Aspergillus species are among the most toxic and carcinogenic compounds in nature. Although it has been known for a long time that seeds with high oil content are more susceptible to aflatoxin contamination, the role of fatty acids in aflatoxin biosynthesis remains controversial. Here we demonstrate in A. flavus that both the saturated stearic acid (C18:0) and the polyunsaturated linolenic acid (C18:3) promoted aflatoxin production, while C18:3, but not C18:0, inhibited aflatoxin biosynthesis after exposure to air for several hours. Further experiments showed that autoxidated C18:3 promoted mycelial growth, sporulation, and kojic acid production, but inhibited the expression of genes in the AF biosynthetic gene cluster. Mass spectrometry analyses of autoxidated C18:3 fractions that were able to inhibit aflatoxin biosynthesis led to the identification of multiple oxylipin species. These results may help to clarify the role of fatty acids in aflatoxin biosynthesis, and may explain why controversial results have been obtained for fatty acids in the past.
Xing, Fuguo; Yao, Haibo; Hruska, Zuzana; Kincaid, Russell; Zhu, Fengle; Brown, Robert L.; Bhatnagar, Deepak; Liu, Yang
Aflatoxin contamination in peanut products has been an important and long-standing problem around the world. Produced mainly by Aspergillus flavus and Aspergillus parasiticus, aflatoxins are the most toxic and carcinogenic compounds among toxins. This study investigated the application of fluorescence visible near-infrared (VNIR) hyperspectral images to assess the spectral difference between peanut kernels inoculated with toxigenic and atoxigenic inocula of A. flavus and healthy kernels. Peanut kernels were inoculated with NRRL3357, a toxigenic strain of A. flavus, and AF36, an atoxigenic strain of A. flavus, respectively. Fluorescence hyperspectral images under ultraviolet (UV) excitation were recorded on peanut kernels with and without skin. Contaminated kernels exhibited different fluorescence features compared with healthy kernels. For the kernels without skin, the inoculated kernels had a fluorescence peaks shifted to longer wavelengths with lower intensity than healthy kernels. In addition, the fluorescence intensity of peanuts without skin was higher than that of peanuts with skin (10 times). The fluorescence spectra of kernels with skin are significantly different from that of the control group (p<0.001). Furthermore, the fluorescence intensity of the toxigenic, AF3357 peanuts with skin was lower than that of the atoxigenic AF36 group. Discriminate analysis showed that the inoculation group can be separated from the controls with 100% accuracy. However, the two inoculation groups (AF3357 vis AF36) can be separated with only ～80% accuracy. This study demonstrated the potential of fluorescence hyperspectral imaging techniques for screening of peanut kernels contaminated with A. flavus, which could potentially lead to the production of rapid and non-destructive scanning-based detection technology for the peanut industry.
Jaime-Garcia, Ramon; Cotty, Peter J
ABSTRACT Aspergillus flavus, the causal agent of aflatoxin contamination of cottonseed, is a natural inhabitant of soils. A. flavus can be divided into the S and L strains, of which the S-strain isolates, on average, produce greater quantities of aflatoxins than the L-strain isolates. Aflatoxin contamination can be severe in several crops in South Texas. The structure of A. flavus communities residing in soils of South Texas was determined from 326 soil samples collected from 152 fields located from the Rio Grande Valley in the south to Fort Bend County in the north from 2001 through 2003. Analysis of variance indicated significant differences in the incidence of A. flavus isolates belonging to the S strain (percent S) among regions. The Coastal Bend (30.7%) and Upper Coast (25.5%) regions had significantly higher percent S incidence than the Rio Grande Valley (4.8%). No significant differences in percent S among years were detected. The CFU per gram of soil were not significantly different among regions. Strain S incidence was positively correlated with clay content and negatively correlated with sand content. Fields cropped to cotton the previous year had a higher S-strain incidence, whereas fields cropped to corn had greater total quantities of A. flavus propagules. Maps of S-strain patterns show that the S strain constitutes >30% of the overall A. flavus community in the area extending from the central Coastal Bend region to the central Upper Coast region. The west Rio Grande Valley had the lowest S-strain incidence (<10%). Geographic variation in S-strain incidence may influence the distribution of aflatoxin contamination in South Texas.
Ehrlich, Kenneth C
Aspergillus flavus is a diverse assemblage of strains that include aflatoxin-producing and non-toxigenic strains with cosmopolitan distribution. The most promising strategy currently being used to reduce preharvest contamination of crops with aflatoxin is to introduce non-aflatoxin (biocontrol) A. flavus into the crop environment. Whether or not introduction of biocontrol strains into agricultural fields is enough to reduce aflatoxin contamination to levels required for acceptance of the contaminated food as fit for consumption is still unknown. There is no question that biocontrol strains are able to reduce the size of the populations of aflatoxin-producing strains but the available data suggests that at most only a four- to five-fold reduction in aflatoxin contamination is achieved. There are many challenges facing this strategy that are both short term and long term. First, the population biology of A. flavus is not well understood due in part to A. flavus's diversity, its ability to form heterokaryotic reproductive forms, and its unknown ability to survive for prolonged periods after application. Second, biocontrol strains must be selected that are suitable for the environment, the type of crop, and the soil into which they will be introduced. Third, there is a need to guard against inadvertent introduction of A. flavus strains that could impose an additional burden on food safety and food quality, and fourth, with global warming and resultant changes in the soil nutrients and concomitant microbiome populations, the biocontrol strategy must be sufficiently flexible to adapt to such changes. Understanding genetic variation within strains of A. flavus is important for developing a robust biocontrol strategy and it is unlikely that a "one size fits all" strategy will work for preharvest aflatoxin reduction.
Belewa, V; Baijnath, H; Frost, C; Somai, B M
This study investigates the effect that aqueous extracts of Tulbaghia violacea Harv. harbouring plant saponins, phenolics and tannins have on Aspergillus flavus β-(1,3) glucan and chitin synthesis. Aspergillus flavus was treated with various subinhibitory concentrations of an aqueous T. violacea plant extract and the β-(1,3) glucan and chitin content was determined together with glucan synthase and chitin synthase production respectively. The aqueous extract caused a significant decline (P < 0·05) in β-glucan production in A. flavus in a dose-dependent manner when compared to the untreated sample. Further investigations showed a decrease in β-glucan synthase production as the concentration of the plant extract was increased. A significant reduction in total chitin content corresponding to a decrease in chitin synthase production in the presence of the plant extract was also found. The broad spectrum activity and the efficacy of aqueous T. violacea plant extract on both β-glucan and chitin synthesis may limit the potential of the fungus developing resistance towards it and therefore the extract is an ideal candidate for use as a potential antifungal agent. © 2017 The Society for Applied Microbiology.
Villamizar, Raquel A; Maroto, Alicia; Rius, F Xavier
In the present study, we have used carbon nanotube field effect transistors (FET) that have been functionalized with protein G and IgG to detect Aspergillus flavus in contaminated milled rice. The adsorbed protein G on the carbon nanotubes walls enables the IgG anti-Aspergillus antibodies to be well oriented and therefore to display full antigen binding capacity for fungal antigens. A solution of Tween 20 and gelatine was used as an effective blocking agent to prevent the non-specific binding of the antibodies and other moulds and also to protect the transducer against the interferences present in the rice samples. Our FET devices were able to detect at least 10 μg/g of A. flavus in only 30 min. To evaluate the selectivity of our biosensors, Fusarium oxysporum and Penicillium chrysogenum were tested as potential competing moulds for A. flavus. We have proved that our devices are highly selective tools for detecting mycotoxigenic moulds at low concentrations in real samples.
Al-Wadai, A S; Al-Othman, M R; Mahmoud, M A; Abd El-Aziz, A R M
Twelve species belonging to six fungal genera were found to be associated with wheat (Triticum aestivum L.) grain samples collected from three main regions in Saudi Arabia. The most common genera (average frequency) were Aspergillus (14.3%), Fusarium (29.1%), Penicillium (9.3%), and Alternaria (8.2%). Nineteen isolates of Aspergillus flavus were screened for their ability to produce aflatoxins using HPLC. Thirteen isolates produced aflatoxins ranging from 0.5 to 2.6 µg/kg. Inter-simple sequence repeats (ISSR), and random amplified polymorphic DNA (RAPD) molecular markers were used, with the aim of genetically characterizing strains of A. flavus to discriminate between aflatoxigenic and non-aflatoxigenic isolates. RAPD and ISSR analysis revealed a high level of genetic diversity in the A. flavus population, useful for genetic characterization. Clustering based on RAPD and ISSR dendograms was unrelated to geographic origin. RAPD and ISSR markers were not suitable to discriminate aflatoxigenic and non-aflatoxigenic isolates, but ISSR primers were better compared to RAPD.
Ahmed, Sarah A; Abbas, Manal A; Jouvion, Gregory; Al-Hatmi, Abdullah M S; de Hoog, G Sybren; Kolecka, Anna; Mahgoub, El Sheikh
Chronic subcutaneous infections caused by Aspergillus species are considered to be extremely rare. Because these fungi are among the most common laboratory contaminants, their role as eumycetoma causative agents is difficult to ascertain. Here, we report the first case of A. flavus eumycetoma confirmed by isolation, molecular identification and immunohistochemical analysis. Patient was a 55-year-old male from Sudan suffering from eumycetoma on his left foot for a period of 17 years. He developed swelling, sinuses and white grain discharge was observed. He has been operated nine times and was treated with several regimens of ketoconazole and itraconazole without improvement. Initial diagnosis based on histology and radiology was Scedosporium eumycetoma. However, examination of the biopsy revealed A. flavus, which was identified by molecular analysis and MALDI-TOF MS. Immunohistochemistry using antibody directed against Aspergillus species was positive. Because of the earlier treatment failures with ketoconazole and itraconazole, therapy with voriconazole was initiated. However, in vitro susceptibility testing yielded a lower Minimum Inhibitory Concentration (MIC) value for itraconazole (0.25 μg ml(-1) ) than for voriconazole (1 μg ml(-1) ). Based on the presented results, A. flavus can be considered as one of the agents of white-grain eumycetoma. © 2015 Blackwell Verlag GmbH.
Singh, Diwakar; Radhakrishnan, T.; Kumar, Vinod; Bagwan, N.B.; Basu, M.S.; Dobaria, J.R.; Mishra, Gyan P.; Chanda, S.V.
Aflatoxin contamination of peanut, due to infection by Aspergillus flavus, is a major problem of rain-fed agriculture in India. In the present study, molecular characterisation of 187 Aspergillus flavus isolates, which were sampled from the peanut fields of Gujarat state in India, was performed using AFLP markers. On a pooled cluster analysis, the markers could successfully discriminate among the ‘A’, ‘B’ and ‘G’ group A. flavus isolates. PCoA analysis also showed equivalent results to the cluster analysis. Most of the isolates from one district could be clustered together, which indicated genetic similarity among the isolates. Further, a lot of genetic variability was observed within a district and within a group. The results of AMOVA test revealed that the variance within a population (84%) was more than that between two populations (16%). The isolates, when tested by indirect competitive ELISA, showed about 68.5% of them to be atoxigenic. Composite analysis between the aflatoxin production and AFLP data was found to be ineffective in separating the isolate types by aflatoxigenicity. Certain unique fragments, with respect to individual isolates, were also identified that may be used for development of SCAR marker to aid in rapid and precise identification of isolates. PMID:26413047
Yang, Kunlong; Liang, Linlin; Ran, Fanlei; Liu, Yinghang; Li, Zhenguo; Lan, Huahui; Gao, Peili; Zhuang, Zhenhong; Zhang, Feng; Nie, Xinyi; Kalayu Yirga, Shimuye; Wang, Shihua
DNA methylation is essential for epigenetic regulation of gene transcription and development in many animals, plants and fungi. We investigated whether DNA methylation plays a role in the development and secondary metabolism of Aspergillus flavus, identified the DmtA methyltransferase from A. flavus, and produced a dmtA knock-out mutant by replacing the dmtA coding sequence with the pyrG selectable marker. The A. flavus dmtA null mutant lines produced white fluffy mycelium in liquid medium, and displayed a slightly flavescent conidial pigmentation compared with the normal yellow of the wild-type strain when grown on agar. The ΔdmtA lines exhibited decreased conidiation and aflatoxin (AF) biosynthesis, compared with the wild-type line, suggesting that the DmtA knock-out affected the transcriptional level of genes in the AF cluster. In particular, sclerotia development and host colonization were altered in the dmtA null mutants. Green fluorescent protein tagging at the C-terminus of DmtA showed that DmtA localized to the nucleus and cytoplasm. DNA methylation content measurements in the dmtA mutants revealed no widespread DNA methylation in the mutants or wild-type lines. Thus, our findings suggest that DmtA, apart from being a C-5 cytosine methyltransferase in A. flavus, contributes to asexual development, aflatoxin biosynthesis, sclerotial production and virulence. PMID:26979781
Al-Gabr, Hamid Mohammad; Zheng, Tianling; Yu, Xin
The disinfection process for inactivating microorganisms at drinking water treatment plants is aimed for safety of drinking water for humans from a microorganism, such as bacteria, viruses, algae, fungi by using chlorination, ozonation, UV irradiation, etc. In the present study, a combination of two disinfectants, UV irradiation followed by chlorination, was evaluated for inactivating Aspergillus flavus under low contact time and low dosage of UV irradiation. The results indicated an inverse correlation between the inactivation of A. flavus by using UV irradiation only or chlorination alone. By using UV radiation, the 2 log10 control of A. flavus was achieved after 30 s of irradiation, while chlorination was observed to be more effective than UV, where the 2 log was achieved at chlorine concentration of 0.5, 1, 2 and 3 mg/l, in contact time of 60, 5, 1 and 1 min, respectively. However, combined use (UV irradiation followed by chlorination) was more effective than using either UV or chlorination alone; 5 s UV irradiation followed by chlorination produced 4 log10 reduction of A. flavus at chlorine concentrations of 2 and 3 mg/l under a contact time of 15 min. The results indicated that efficiency of UV irradiation improves when followed by chlorination at low concentrations. Copyright © 2013 Elsevier B.V. All rights reserved.
Chang, Perng-Kuang; Horn, Bruce W; Dorner, Joe W
Aspergillus flavus populations are genetically diverse. Isolates that produce either, neither, or both aflatoxins and cyclopiazonic acid (CPA) are present in the field. We investigated defects in the aflatoxin gene cluster in 38 nonaflatoxigenic A. flavus isolates collected from southern United States. PCR assays using aflatoxin-gene-specific primers grouped these isolates into eight (A-H) deletion patterns. Patterns C, E, G, and H, which contain 40 kb deletions, were examined for their sequence breakpoints. Pattern C has one breakpoint in the cypA 3' untranslated region (UTR) and another in the verA coding region. Pattern E has a breakpoint in the amdA coding region and another in the ver1 5'UTR. Pattern G contains a deletion identical to the one found in pattern C and has another deletion that extends from the cypA coding region to one end of the chromosome as suggested by the presence of telomeric sequence repeats, CCCTAATGTTGA. Pattern H has a deletion of the entire aflatoxin gene cluster from the hexA coding region in the sugar utilization gene cluster to the telomeric region. Thus, deletions in the aflatoxin gene cluster among A. flavus isolates are not rare, and the patterns appear to be diverse. Genetic drift may be a driving force that is responsible for the loss of the entire aflatoxin gene cluster in nonaflatoxigenic A. flavus isolates when aflatoxins have lost their adaptive value in nature.
Sugihara, Satoshi; Doi, Hiroyuki; Kato, Masahiko; Mitoh, Yoshihiro; Tsuda, Toshihide; Ikeda, Satoru
Aflatoxin (AFT) contamination is frequent in foods grown in tropical regions, including rice. Although AFTs are generally not found in temperate-region foods, global warming has affected typical temperate-region climates, potentially permitting the contamination of foods with AFT-producing Aspergillus flavus (A. flavus). Here we investigated the AFT production in rice during storage under natural climate conditions in Japan. We examined AFTs in brown rice and rough rice artificially contaminated with A. flavus for 1 year in Japan, and we subjected AFTs in white rice to the same treatment in airtight containers and examined the samples in warm and cold seasons, simulating the storage of white rice in general households. In the brown rice, AFTs increased after 2 months (March) and peaked after 9 months (October). The AFT contamination in the rough rice was minimal. After the polishing and cooking of the brown rice, AFTs were undetectable. In the white rice stored in airtight containers, AFTs increased after 1 month (August) and peaked after 2 months (September). Minimal AFTs were detected in the cold season. Thus, AFT contamination in rice may occur in temperate regions following A. flavus contamination. The storage of rice as rough rice could provide be useful for avoiding AFT contamination.
Choi, Seonyeong; Jun, Hyejung; Bang, Jihyun; Chung, Soo-Hyun; Kim, Yoonsook; Kim, Byeong-Sam; Kim, Hoikyung; Beuchat, Larry R; Ryu, Jee-Hoon
We investigated the survival and growth patterns of Aspergillus flavus and Fusarium graminearum, as well as mycotoxin production, on Korean rice as affected by the degree of milling (rough, brown, and white rice) and storage conditions (21 °C/85% relative humidity [RH], 21 °C/97% RH, and 30 °C/85% RH). When rice was stored at 21 °C/85% RH, the population of A. flavus remained constant and aflatoxin was not produced, regardless of the degree of milling. At 21 °C/97% RH and 30 °C/85% RH, the populations of A. flavus increased significantly (P ≤ 0.05) and aflatoxins were produced. The highest population of A. flavus and highest amount of aflatoxin B1 were observed on brown rice stored at 21 °C/97% RH. For F. graminearum, when stored at 85% RH, the populations were reduced to less than a detectable level (5 CFU/g of rice) within 120 days and no deoxynivalenol (DON) was produced, regardless of the degree of milling and storage temperature. However, at 21 °C/97% RH, the population of F. graminearum increased significantly (P ≤ 0.05) and DON was produced on all types of rice. Findings from this study provide insights concerning storage conditions necessary to prevent growth and mycotoxin production by A. flavus and F. graminearum on Korean rice with different degrees of milling. Copyright © 2014 Elsevier Ltd. All rights reserved.
Restaino, L.; Myron, J.J.J.; Lenovich, L.M.; Bills, S.; Tscherneff, K.
With an initial microbial level of ca. 10/sup 7/ microorganisms per g of Ivory Coast cacao beans, 5 kGy of gamma radiation from a Co/sup 60/ source under an atmosphere of air reduced the microflora per g by 2.49 and 3.03 logs at temperatures of 35 and 50/sup 0/C, respectively. Bahia cacao beans were artificially contaminated with dried spores of Aspergillus flavus and Penicillium citrinum, giving initial fungal levels of 1.9 x 10/sup 4/ and 1.4 x 10/sup 3/ spores per g of whole Bahia cacao beans, respectively. The average D/sub 10/ values for A. flavus and P. citrinum spores on Bahia cacao beans were 0.66 and 0.88 kGy, respectively. 12 references.
Dolezal, Andrea L.; Shu, Xiaomei; OBrian, Gregory R.; Nielsen, Dahlia M.; Woloshuk, Charles P.; Boston, Rebecca S.; Payne, Gary A.
Maize kernels are susceptible to infection by the opportunistic pathogen Aspergillus flavus. Infection results in reduction of grain quality and contamination of kernels with the highly carcinogenic mycotoxin, aflatoxin. To understanding host response to infection by the fungus, transcription of approximately 9000 maize genes were monitored during the host-pathogen interaction with a custom designed Affymetrix GeneChip® DNA array. More than 4000 maize genes were found differentially expressed at a FDR of 0.05. This included the up regulation of defense related genes and signaling pathways. Transcriptional changes also were observed in primary metabolism genes. Starch biosynthetic genes were down regulated during infection, while genes encoding maize hydrolytic enzymes, presumably involved in the degradation of host reserves, were up regulated. These data indicate that infection of the maize kernel by A. flavus induced metabolic changes in the kernel, including the production of a defense response, as well as a disruption in kernel development. PMID:25132833
... From the Federal Register Online via the Government Publishing Office ENVIRONMENTAL PROTECTION AGENCY Notice of Filing of a Pesticide Petition for Residues of a Aspergillus flavus AF36 on Corn Food... residues of the antifungal ] agent, Aspergillus flavus AF36, in or on corn food and feed commodities. The...
Degola, F; Berni, E; Dall'Asta, C; Spotti, E; Marchelli, R; Ferrero, I; Restivo, F M
To develop a multiplex reverse transciption-polymerase chain reaction (RT-PCR) protocol to discriminate aflatoxin-producing from aflatoxin-nonproducing strains of Aspergillus flavus. The protocol was first optimized on a set of strains obtained from laboratory collections and then validated on A. flavus strains isolated from corn grains collected in the fields of the Po Valley (Italy). Five genes of the aflatoxin gene cluster of A. flavus, two regulatory (aflR and aflS) and three structural (aflD, aflO and aflQ), were targeted with specific primers to highlight their expression in mycelia cultivated under inducing conditions for aflatoxins production. 48-h-old cultures expressed the complete set of the genes analysed here whereas 24-h-old ones did not. Genomic PCR (quadruplex PCR) was also performed in parallel using chromosomal DNA extracted from the same set of strains to correlate the integrity of the genes with their expression. We show that a good correlation exists between gene expression of the aflatoxin genes, here analysed by multipex RT-PCR, and aflatoxin production, except for one strain that apparently transcribed all the relevant genes but did not produce aflatoxin in the medium. This is the first example of the application of a combination of multiplex PCR and RT-PCR approaches to screen a population of A. flavus for the presence of aflatoxigenic and nonaflatoxigenic strains. The proposed protocol will be helpful in evaluating the risk posed by A. flavus in natural environments and might also be a useful tool to monitor its presence during the processing steps of food and feed commodities.
Ehrlich, Kenneth C.
Aspergillus flavus is a diverse assemblage of strains that include aflatoxin-producing and non-toxigenic strains with cosmopolitan distribution. The most promising strategy currently being used to reduce preharvest contamination of crops with aflatoxin is to introduce non-aflatoxin (biocontrol) A. flavus into the crop environment. Whether or not introduction of biocontrol strains into agricultural fields is enough to reduce aflatoxin contamination to levels required for acceptance of the contaminated food as fit for consumption is still unknown. There is no question that biocontrol strains are able to reduce the size of the populations of aflatoxin-producing strains but the available data suggests that at most only a four- to five-fold reduction in aflatoxin contamination is achieved. There are many challenges facing this strategy that are both short term and long term. First, the population biology of A. flavus is not well understood due in part to A. flavus’s diversity, its ability to form heterokaryotic reproductive forms, and its unknown ability to survive for prolonged periods after application. Second, biocontrol strains must be selected that are suitable for the environment, the type of crop, and the soil into which they will be introduced. Third, there is a need to guard against inadvertent introduction of A. flavus strains that could impose an additional burden on food safety and food quality, and fourth, with global warming and resultant changes in the soil nutrients and concomitant microbiome populations, the biocontrol strategy must be sufficiently flexible to adapt to such changes. Understanding genetic variation within strains of A. flavus is important for developing a robust biocontrol strategy and it is unlikely that a “one size fits all” strategy will work for preharvest aflatoxin reduction. PMID:24575088
Olonisakin, Oluwafunmilayo Oluwakemi; Jeff-Agboola, Yemisi Adefunke; Ogidi, Clement Olusola; Akinyele, Bamidele Juliet
The antifungal activity of isolated lactic acid bacteria (LAB) from a locally fermented cereal, “Kunu”, was tested against toxigenic Aspergillus flavus. The liquid refreshment, “Kunu”, was prepared under hygienic condition using millet, sorghum, and the combination of the two grains. The antifungal potential of isolated LAB against toxigenic A. flavus was carried out using both in vitro and in vivo antifungal assays. The LAB count from prepared “Kunu” ranged from 2.80 ×104 CFU/mL to 4.10×104 CFU/mL and Lactobacillus plantarum, Lactobacillus delbrueckii, Lactobacillus fermentum, Pediococcus acidilactici, and Leuconostoc mesenteroides were the isolated bacteria. Inhibitory zones exhibited by LAB against toxigenic A. flavus ranged from 5.0 mm to 20.0 mm. The albino mice infected with toxigenic A. flavus showed sluggishness, decrease in body weight, distortion of hair, and presence of blood in their stool, while those treated with LAB after infection were recovered and active like those in control groups. Except for the white blood cell that was increased in the infected mice as 6.73 mm3, the packed cell volume, hemoglobin, and red blood cell in infected animals were significantly reduced (P<0.05) to 29.28%, 10.06%, and 4.28%, respectively, when compared to the treated mice with LAB and control groups. The antifungal activity of LAB against toxigenic A. flavus can be attributed to the antimicrobial metabolites. These metabolites can be extracted and used as biopreservatives in food products to substitute the use of chemical preservatives that is not appealing to consumers due to several side effects. PMID:28702431
Olonisakin, Oluwafunmilayo Oluwakemi; Jeff-Agboola, Yemisi Adefunke; Ogidi, Clement Olusola; Akinyele, Bamidele Juliet
The antifungal activity of isolated lactic acid bacteria (LAB) from a locally fermented cereal, "Kunu", was tested against toxigenic Aspergillus flavus. The liquid refreshment, "Kunu", was prepared under hygienic condition using millet, sorghum, and the combination of the two grains. The antifungal potential of isolated LAB against toxigenic A. flavus was carried out using both in vitro and in vivo antifungal assays. The LAB count from prepared "Kunu" ranged from 2.80 ×10(4) CFU/mL to 4.10×10(4) CFU/mL and Lactobacillus plantarum, Lactobacillus delbrueckii, Lactobacillus fermentum, Pediococcus acidilactici, and Leuconostoc mesenteroides were the isolated bacteria. Inhibitory zones exhibited by LAB against toxigenic A. flavus ranged from 5.0 mm to 20.0 mm. The albino mice infected with toxigenic A. flavus showed sluggishness, decrease in body weight, distortion of hair, and presence of blood in their stool, while those treated with LAB after infection were recovered and active like those in control groups. Except for the white blood cell that was increased in the infected mice as 6.73 mm(3), the packed cell volume, hemoglobin, and red blood cell in infected animals were significantly reduced (P<0.05) to 29.28%, 10.06%, and 4.28%, respectively, when compared to the treated mice with LAB and control groups. The antifungal activity of LAB against toxigenic A. flavus can be attributed to the antimicrobial metabolites. These metabolites can be extracted and used as biopreservatives in food products to substitute the use of chemical preservatives that is not appealing to consumers due to several side effects.
Devipriya, Duraipandi; Roopan, Selvaraj Mohana
Recently, non-toxic source mediated synthesis of metal and a metal oxide nanoparticle attains more attention due to key applicational responsibilities. This present report stated that the eco-friendly synthesis of copper oxide nanoparticles (CuO NPs) using Cissus quadrangularis (C. quadrangularis) plant extract. Further the eco-friendly synthesized CuO NPs were characterized using a number of analytical techniques. The observed results stated that the synthesized CuO NPs were spherical in shape with 30±2nm. Then the eco-friendly synthesized CuO NPs were subjected for anti-fungal against two strains namely Aspergillus niger (A. niger) resulted in 83% at 500ppm, 86% of inhibition at 1000ppm and Aspergillus flavus (A. flavus) resulted in 81% at 500ppm, 85% of inhibition at 1000ppm respectively. Despite the fact that compared to standard Carbendazim, eco-friendly synthesized CuO NPs exhibits better results were discussed in this manuscript. Copyright © 2017 Elsevier B.V. All rights reserved.
Ramírez-Camejo, Luis A; Torres-Ocampo, Ana P; Agosto-Rivera, José L; Bayman, Paul
Aspergilloses are fungal diseases in humans and animals that is caused by members of the genus Aspergillus. Aspergillus flavus is an important opportunistic pathogen, second only to A. fumigatus as a cause of human aspergillosis. Differences in virulence among A. flavus isolates from clinical and other substrates and mating types are not well known. The fruit fly Drosophila melanogaster has become a model organism for investigating virulence of human pathogens due to similarities between its immune system and that of mammals. In this study we used D. melanogaster as a model host to compare virulence among A. flavus strains obtained from clinical sources as compared with other substrates, between isolates of different mating types, and between isolates of A. flavus and A. fumigatus. Anesthetized flies were infected with A. flavus; mortality ranged from 15% to >90%. All strains were virulent, but some were significantly more so than others, which in turn led to the wide mortality range. Clinical strains were significantly less virulent than environmental strains, probably because the clinical strains were from culture collections and the environmental strains were recent isolates. Mean virulence did not differ between MAT1-1 and MAT1-2 mating types and the phylogeny of A. flavus isolates did not predict virulence. A. flavus was on average significantly more virulent than A. fumigatus on two lines of wild-type flies, Canton-S and Oregon-R. D. melanogaster is an attractive model to test pathogenicity and could be useful for identifying genes involved in virulence.
Sobolev, Victor S; Krausert, Nicole M; Gloer, James B
Two new stilbene derivatives have been isolated from peanut seeds challenged by an Aspergillus flavus strain, along with chiricanine B, which has not been previously reported from peanuts, as well as a stilbenoid reported previously only as a synthetic product. The structures of these new putative phytoalexins were determined by analysis of (1)H and (13)C NMR, HRESIMS, MS(n), and UV data. The new stilbenoids were named arahypin-13 (21), arahypin-14 (22), and arahypin-15 (23). Together with other known bioactive peanut stilbenoids that were also produced in the challenged seeds, these new compounds may play a defensive role against invasive fungi.
Park, Eun-Sil; Bae, In Kyung; Kim, Ho Jin; Lee, Sung-Eun
The present study investigated its inhibitory role in aflatoxin (AF) biosynthesis. Treating only AFB1- and B2-producing Aspergillus flavus with piperonal completely inhibited AFB1 production with high sclerotial formation, resulting in 20-fold higher AFG2 production. On the other hand, benzodioxole and eugenol suppressed AFB1 production without AFG formation, while methyleugenol showed potent inhibition of AFB1 production with slight production of AFG1. These results indicate that natural products may change aflatoxin biosynthesis, and highlight a novel regulation of AFG2 production by piperonal. It is the first report for chemical regulation on AFG2 production in non-AFG producing-aspergilli.
Uka, Valdet; Moore, Geromy G.; Arroyo-Manzanares, Natalia; Nebija, Dashnor; De Saeger, Sarah; Diana Di Mavungu, José
Cyclopiazonic acid (α-cyclopiazonic acid, α-CPA) is an indole-hydrindane-tetramic acid neurotoxin produced by various fungal species, including the notorious food and feed contaminant Aspergillus flavus. Despite its discovery in A. flavus cultures approximately 40 years ago, its contribution to the A. flavus mycotoxin burden is consistently minimized by our focus on the more potent carcinogenic aflatoxins also produced by this fungus. Here, we report the screening and identification of several CPA-type alkaloids not previously found in A. flavus cultures. Our identifications of these CPA-type alkaloids are based on a dereplication strategy involving accurate mass high resolution mass spectrometry data and a careful study of the α-CPA fragmentation pattern. In total, 22 CPA-type alkaloids were identified in extracts from the A. flavus strains examined. Of these metabolites, 13 have been previously reported in other fungi, though this is the first report of their existence in A. flavus. Two of our metabolite discoveries, 11,12-dehydro α-CPA and 3-hydroxy-2-oxo CPA, have never been reported for any organism. The conspicuous presence of CPA and its numerous derivatives in A. flavus cultures raises concerns about the long-term and cumulative toxicological effects of these fungal secondary metabolites and their contributions to the entire A. flavus mycotoxin problem. PMID:28098779
Whitehead, M P; Shieh, M T; Cleveland, T E; Cary, J W; Dean, R A
Two genes, pecA and pecB, encoding endopolyglacturonases were cloned from a highly aggressive strain of Aspergillus flavus. The pecA gene consisted of 1,228 bp encoding a protein of 363 amino acids with a predicted molecular mass of 37.6 kDa, interrupted by two introns of 58 and 81 bp in length. Accumulation of pecA mRNA in both pectin- or glucose-grown mycelia in the highly aggressive strain matched the activity profile of a pectinase previously identified as P2c. Transformants of a weakly aggressive strain containing a functional copy of the pecA gene produced P2c in vitro, confirming that pecA encodes P2c. The coding region of pecB was determined to be 1,217 bp in length interrupted by two introns of 65 and 54 bp in length. The predicted protein of 366 amino acids had an estimated molecular mass of 38 kDa. Transcripts of this gene accumulated in mycelia grown in medium containing pectin alone, never in mycelia grown in glucose-containing medium, for both highly and weakly aggressive strains. Thus, pecB encodes the activity previously identified as P1 or P3. pecA and pecB share a high degree of sequence identity with polygalacturonase genes from Aspergillus parasiticus and Aspergillus oryzae, further establishing the close relationships between members of the A. flavus group. Conservation of intron positions in these genes also indicates that they share a common ancestor with genes encoding endopolyglacturonases of Aspergillus niger.
Fountain, Jake C.; Bajaj, Prasad; Pandey, Manish; Nayak, Spurthi N.; Yang, Liming; Kumar, Vinay; Jayale, Ashwin S.; Chitikineni, Anu; Zhuang, Weijian; Scully, Brian T.; Lee, R. Dewey; Kemerait, Robert C.; Varshney, Rajeev K.; Guo, Baozhu
Contamination of crops with aflatoxin is a serious global threat to food safety. Aflatoxin production by Aspergillus flavus is exacerbated by drought stress in the field and by oxidative stress in vitro. We examined transcriptomes of three toxigenic and three atoxigenic isolates of A. flavus in aflatoxin conducive and non-conducive media with varying levels of H2O2 to investigate the relationship of secondary metabolite production, carbon source, and oxidative stress. We found that toxigenic and atoxigenic isolates employ distinct mechanisms to remediate oxidative damage, and that carbon source affected the isolates’ expression profiles. Iron metabolism, monooxygenases, and secondary metabolism appeared to participate in isolate oxidative responses. The results suggest that aflatoxin and aflatrem biosynthesis may remediate oxidative stress by consuming excess oxygen and that kojic acid production may limit iron-mediated, non-enzymatic generation of reactive oxygen species. Together, secondary metabolite production may enhance A. flavus stress tolerance, and may be reduced by enhancing host plant tissue antioxidant capacity though genetic improvement by breeding selection. PMID:27941917
Leal, André Ferraz Goiana; Leite, Melyna Chaves; Medeiros, Caroline Sanuzi Quirino; Cavalcanti, Isabella Macário Ferro; Wanderley, Almir Gonçalves; Magalhães, Nereide Stela Santos; Neves, Rejane Pereira
The aim of this study was to assess the efficacy of topical application of a liposomal formulation of itraconazole for the treatment of experimental keratitis with endophthalmitis caused by Aspergillus flavus. The liposomes were obtained by the lipid film hydration method followed by sonication. Adult female Wistar rats (weighing 200-220 g) were immunosuppressed by intraperitoneal injection of 150 mg/kg of cyclophosphamide 3 days before infection by exposure to the fungus A. flavus (10(7) spores/ml). Forty-eight hours later, the animals were treated with the liposomal formulation. For comparison, one group of animals (n = 6) was treated with the same drug not encapsulated. At the end of the experiment, the animals were evaluated for clinical signs and number of colony forming units (CFU/g), along with direct microscopic examination. The results indicated that the liposomal formulation of itraconazole has better antifungal activity than the unencapsulated drug in the treatment of fungal keratitis with endophthalmitis caused experimentally by A. flavus in Wistar rats.
Orum, T V; Bigelow, D M; Cotty, P J; Nelson, M R
ABSTRACT Aspergillus flavus is a soil-inhabiting fungus that frequently produces aflatoxins, potent carcinogens, in cottonseed and other seed crops. A. flavus S strain isolates, characterized on the basis of sclerotial morphology, are highly toxigenic. Spatial and temporal characteristics of the percentage of the A. flavus isolates that are S strain (S strain incidence) were used to predict patterns across areas of more than 30 km(2). Spatial autocorrelation in S strain incidence in Yuma County, AZ, was shown to extend beyond field boundaries to adjacent fields. Variograms revealed both short-range (2 to 6 km) and long-range (20 to 30 km) spatial structure in S strain incidence. S strain incidence at 36 locations sampled in July 1997 was predicted with a high correlation between expected and observed values (R = 0.85, P = 0.0001) by kriging data from July 1995 and July 1996. S strain incidence at locations sampled in October 1997 and March 1998 was markedly less than predicted by kriging data from the same months in prior years. Temporal analysis of four locations repeatedly sampled from April 1995 through July 1998 also indicated a major reduction in S strain incidence in the Texas Hill area after July 1997. Surface maps generated by kriging point data indicated a similarity in the spatial pattern of S strain incidence among all sampling dates despite temporal changes in the overall S strain incidence. Geostatistics provided useful descriptions of variability in S strain incidence over space and time.
Fountain, Jake C.; Scully, Brian T.; Ni, Xinzhi; Kemerait, Robert C.; Lee, Robert D.; Chen, Zhi-Yuan; Guo, Baozhu
Since the early 1960s, the fungal pathogen Aspergillus flavus (Link ex Fr.) has been the focus of intensive research due to the production of carcinogenic and highly toxic secondary metabolites collectively known as aflatoxins following pre-harvest colonization of crops. Given this recurrent problem and the occurrence of a severe aflatoxin outbreak in maize (Zea mays L.), particularly in the Southeast U.S. in the 1977 growing season, a significant research effort has been put forth to determine the nature of the interaction occurring between aflatoxin production, A. flavus, environment and its various hosts before harvest. Many studies have investigated this interaction at the genetic, transcript, and protein levels, and in terms of fungal biology at either pre- or post-harvest time points. Later experiments have indicated that the interaction and overall resistance phenotype of the host is a quantitative trait with a relatively low heritability. In addition, a high degree of environmental interaction has been noted, particularly with sources of abiotic stress for either the host or the fungus such as drought or heat stresses. Here, we review the history of research into this complex interaction and propose future directions for elucidating the relationship between resistance and susceptibility to A. flavus colonization, abiotic stress, and its relationship to oxidative stress in which aflatoxin production may function as a form of antioxidant protection to the producing fungus. PMID:24550905
Abou-Zeid, A M
Filamentous fungi isolated from cereals were screened for their ability to produce alpha-amylase (1,4-alpha-glucan glucanohydrolase, EC 188.8.131.52). A selected strain identified as Aspergillus flavus showed high enzymatic activity. A single extracellular alpha-amylase was purified to homogeneity by a starch adsorption method. The molecular weight (M(r)) of the A. flavus alpha-amylase was approximately 75,000 +/- 3,000 by polyacrylamide gel electrophoresis (PAGE) and that of the subunit was approximately 75,000 +/- 3000 SDS-PAGE. The optimal activity of the purified enzyme was achieved at pH 7.0 and 30 degrees C. K+ ions increased the alpha-amylase activity, but Mg2+ did not greatly affect enzyme activity. Mn2+, Zn2+, Cu2+ and Fe3+ ions strongly inhibited the enzyme activity. The products of hydrolysis of native starch by the A. flavus enzyme were mainly glucose as well as unidentified oligosaccharides.
Negri, Clara E; Johnson, Adam; McEntee, Laura; Box, Helen; Whalley, Sarah; Schwartz, Julie A; Ramos-Martín, V; Livermore, Joanne; Kolamunnage-Dona, Ruwanthi; Colombo, Arnaldo L; Hope, William W
Aspergillus flavus is one of the most common agents of invasive aspergillosis and is associated with high mortality. The orotomides are a new class of antifungal agents with a novel mechanism of action. An understanding of the pharmacodynamics of the lead compound F901318 is required to plan safe and effective regimens for clinical use. The pharmacokinetics (PK) and pharmacodynamics (PD) of F901318 were evaluated by developing new in vitro and in vivo models of invasive fungal sinusitis. Galactomannan was used as a pharmacodynamic endpoint in all models. Mathematical PK-PD models were used to describe dose-exposure-response relationships. F901318 MICs ranged from 0.015 to 0.06 mg/L. F901318 induced a concentration-dependent decline in galactomannan. In the in vitro model a Cmin:MIC of 10 resulted in suppression of galactomannan; whereas, values of approximately 10 and 9-19 when assessed by survival of mice or the decline in galactomannan, respectively were equivalent or exceeded the effect induced by posaconazole. There was histological clearance of lung tissue that was consistent with the effects of F901318 on galactomannan. F901318 is a potential new agent for the treatment of invasive infections caused by Aspergillus flavus with pharmacodynamics that are comparable to other first-line triazole agents.
Karim, Kazi Muhammad Rezaul; Hossain, Md. Anowar; Sing, Ngieng Ngui; Mohd Sinang, Fazia; Hussain, Mohd Hasnain Md.; Roslan, Hairul Azman
A novel thermostable glucoamylase cDNA without starch binding domain (SBD) of Aspergillus flavus NSH9 was successfully identified, isolated, and overexpressed in Pichia pastoris GS115. The complete open reading frame of glucoamylase from Aspergillus flavus NSH9 was identified by employing PCR that encodes 493 amino acids lacking in the SBD. The first 17 amino acids were presumed to be a signal peptide. The cDNA was cloned into Pichia pastoris and the highest expression of recombinant glucoamylase (rGA) was observed after 8 days of incubation period with 1% methanol. The molecular weight of the purified rGA was about 78 kDa and exhibited optimum catalytic activity at pH 5.0 and temperature of 70°C. The enzyme was stable at higher temperature with 50% of residual activity observed after 20 min at 90°C and 100°C. Low concentration of metal (Mg++, Fe++, Zn++, Cu++, and Pb++) had positive effect on rGA activity. This rGA has the potential for use and application in the saccharification steps, due to its thermostability, in the starch processing industries. PMID:27504454
Wang, Zizhang; Yan, Shijuan; Liu, Chunming; Chen, Fang; Wang, Tai
An immune response is triggered in host cells when host receptors recognize conserved molecular motifs, pathogen-associated molecular patterns (PAMPs), such as β-glucans, and chitin at the cell surface of a pathogen. Effector-triggered immunity occurs when pathogens deliver effectors into the host cell to suppress the first immune signaling. Using a differential proteomic approach, we identified an array of proteins responding to aflatoxins in cotyledons of peanut (Arachis hypogaea) infected with aflatoxin-producing (toxigenic) but not nonaflatoxin-producing (atoxigenic) strains of Aspergillus flavus. These proteins are involved in immune signaling and PAMP perception, DNA and RNA stabilization, induction of defense, innate immunity, hypersensitive response, biosynthesis of phytoalexins, cell wall responses, peptidoglycan assembly, penetration resistance, condensed tannin synthesis, detoxification, and metabolic regulation. Gene expression analysis confirmed the differential abundance of proteins in peanut cotyledons supplemented with aflatoxins, with or without infection with the atoxigenic strain. Similarly, peanut germination and A. flavus growth were altered in response to aflatoxin B1. These findings show an additional immunity initiated by aflatoxins. With the PAMP- and effector-triggered immune responses, this immunity constitutes the third immune response of the immune system in peanut cotyledon cells. The system is also a three-grade coevolution of plant-pathogen interaction.
Kong, Qing; Chi, Chen; Yu, Jiujiang; Shan, Shihua; Li, Qiyu; Li, Qianting; Guan, Bin; Nierman, William C; Bennett, Joan W
Aspergillus flavus is one of the major moulds that colonize peanut in the field and during storage. The impact to human and animal health, and to the economy in agriculture and commerce, is significant since this mold produces the most potent known natural toxins, aflatoxins, which are carcinogenic, mutagenic, immunosuppressive, and teratogenic. A strain of marine Bacillus megaterium isolated from the Yellow Sea of East China was evaluated for its effect in inhibiting aflatoxin formation in A. flavus through down-regulating aflatoxin pathway gene expression as demonstrated by gene chip analysis. Aflatoxin accumulation in potato dextrose broth liquid medium and liquid minimal medium was almost totally (more than 98 %) inhibited by co-cultivation with B. megaterium. Growth was also reduced. Using expression studies, we identified the fungal genes down-regulated by co-cultivation with B. megaterium across the entire fungal genome and specifically within the aflatoxin pathway gene cluster (aflF, aflT, aflS, aflJ, aflL, aflX). Modulating the expression of these genes could be used for controlling aflatoxin contamination in crops such as corn, cotton, and peanut. Importantly, the expression of the regulatory gene aflS was significantly down-regulated during co-cultivation. We present a model showing a hypothesis of the regulatory mechanism of aflatoxin production suppression by AflS and AflR through B. megaterium co-cultivation.
Naseer, R; Sultana, Bushra; Khan, M Z; Naseer, D; Nigam, Poonam
Antifungal activity in lemon and pomegranate peels was considerable against Aspergillus flavus, higher in pomegranate (DIZ 37mm; MIC 135μg/mL). Powdered peels (5, 10, 20% w/w) were mixed in inoculated rice. The inhibitory effect on fungal-growth and production of aflatoxins by A. flavus was investigated at storage conditions - temperature (25, 30°C) and moisture (18%, 21%) for 9months. The maximum total aflatoxins accumulated at 30°C, 21% moisture and at 25°C, 18% moisture were 265.09 and 163.45ng/g, respectively in control. Addition of pomegranate-peels inhibited aflatoxins production to 100% during four month-storage of rice at 25°C and 18% moisture, while lemon-peels showed similar inhibitory effect for 3months at same conditions. However a linear correlation was observed in aflatoxins level with temperature and moisture. Studies showed that both fruit-wastes are potent preventer of aflatoxin production in rice, useful for a safer and longer storage of rice. Copyright © 2014 Elsevier Ltd. All rights reserved.
Iimura, Kurin; Furukawa, Tomohiro; Yamamoto, Toshiyoshi; Negishi, Lumi; Suzuki, Michio; Sakuda, Shohei
Cyclo(l-Ala-l-Pro) inhibits aflatoxin production in aflatoxigenic fungi without affecting fungal growth. The mode of action of cyclo(l-Ala-l-Pro) in inhibiting aflatoxin production of Aspergillus flavus was investigated. A glutathione S-transferase (GST) of the fungus, designated AfGST, was identified as a binding protein of cyclo(l-Ala-l-Pro) in an experiment performed using cyclo(l-Ala-l-Pro)-immobilized Sepharose beads. Cyclo(l-Ala-l-Pro) specifically bound to recombinant AfGST and inhibited its GST activity. Ethacrynic acid, a known GST inhibitor, inhibited the GST activity of recombinant AfGST and aflatoxin production of the fungus. Ethacrynic acid reduced the expression level of AflR, a key regulatory protein for aflatoxin production, similar to cyclo(l-Ala-l-Pro). These results suggest that cyclo(l-Ala-l-Pro) inhibits aflatoxin production by affecting GST function in A. flavus, and that AfGST inhibitors are possible candidates as selective aflatoxin production inhibitors.
Anand, R; Shankar, J; Tiwary, B N; Singh, A P
Aspergillus flavus is one of the leading Aspergillus spp. resulting in invasive aspergillosis of central nervous system (CNS) in human beings. Immunological status in aspergillosis of central nervous system remains elusive in case of both immunocompetent and immunocompromised patients. Since cytokines are the major mediators of host response, evaluation of disease pathology along with cytokine profile in brain may provide snapshots of neuro-immunological response. An intravenous model of A. flavus infection was utilized to determine the pathogenicity of infection and cytokine profile in the brain of male BALB/c mice. Enumeration of colony forming units and histopathological analyses were performed on the brain tissue at distinct time periods. The kinetics of cytokines (TNF-α, IFN-γ, IL-12/IL-23p40, IL-6, IL-23, IL-17A and IL-4) was evaluated at 6, 12, 24, 48, 72 and 96h post infection (hPI) in brain homogenates using murine cytokine specific enzyme linked immunosorbent assay. Histological analysis exhibited the hyphae with leukocyte infiltrations leading to formation of granulomata along with ischemia and pyknosis of neurons in the brain of infected mice. Diseased mice displayed increased secretion of IFN-γ, IL-12p40 and IL-6 with a concomitant reduction in the secretion of Th2 cytokine IL-4, and Th17 promoting cytokine, IL-23 during the late phase of infection. A.flavus induced inflammatory granulomatous cerebral aspergillosis in mice, characterized by a marked increase in the Th1 cytokines and neurons undergoing necrosis. A marked increase in necrosis of neurons with concurrent inflammatory responses might have led to the host mortality during late phase of infection.
Satterlee, Timothy; Cary, Jeffrey W.; Calvo, Ana M.
Aspergillus flavus colonizes numerous oil seed crops such as corn, peanuts, treenuts and cotton worldwide, contaminating them with aflatoxin and other harmful potent toxins. In the phylogenetically related model fungus Aspergillus nidulans, the methyltransferase, RmtA, has been described to be involved in epigenetics regulation through histone modification. Epigenetics regulation affects a variety of cellular processes, including morphogenesis and secondary metabolism. Our study shows that deletion of rmtA in A. flavus results in hyperconidiating colonies, indicating that rmtA is a repressor of asexual development in this fungus. The increase in conidiation in the absence of rmtA coincides with greater expression of brlA, abaA, and wetA compared to that in the wild type. Additionally, the rmtA deletion mutant presents a drastic reduction or loss of sclerotial production, while forced expression of this gene increased the ability of this fungus to generate these resistant structures, revealing rmtA as a positive regulator of sclerotial formation. Importantly, rmtA is also required for the production of aflatoxin B1 in A. flavus, affecting the expression of aflJ. Furthermore, biosynthesis of additional metabolites is also controlled by rmtA, indicating a broad regulatory output in the control of secondary metabolism. This study also revealed that rmtA positively regulates the expression of the global regulatory gene veA, which could contribute to mediate the effects of rmtA on development and secondary metabolism in this relevant opportunistic plant pathogen. PMID:27213959
Nicholson, Matthew J; Koulman, Albert; Monahan, Brendon J; Pritchard, Beth L; Payne, Gary A; Scott, Barry
Aflatrem is a potent tremorgenic toxin produced by the soil fungus Aspergillus flavus, and a member of a structurally diverse group of fungal secondary metabolites known as indole-diterpenes. Gene clusters for indole-diterpene biosynthesis have recently been described in several species of filamentous fungi. A search of Aspergillus complete genome sequence data identified putative aflatrem gene clusters in the genomes of A. flavus and Aspergillus oryzae. In both species the genes for aflatrem biosynthesis cluster at two discrete loci; the first, ATM1, is telomere proximal on chromosome 5 and contains a cluster of three genes, atmG, atmC, and atmM, and the second, ATM2, is telomere distal on chromosome 7 and contains five genes, atmD, atmQ, atmB, atmA, and atmP. Reverse transcriptase PCR in A. flavus demonstrated that aflatrem biosynthesis transcript levels increased with the onset of aflatrem production. Transfer of atmP and atmQ into Penicillium paxilli paxP and paxQ deletion mutants, known to accumulate paxilline intermediates paspaline and 13-desoxypaxilline, respectively, showed that AtmP is a functional homolog of PaxP and that AtmQ utilizes 13-desoxypaxilline as a substrate to synthesize aflatrem pathway-specific intermediates, paspalicine and paspalinine. We propose a scheme for aflatrem biosynthesis in A. flavus based on these reconstitution experiments in P. paxilli and identification of putative intermediates in wild-type cultures of A. flavus.
Assunção, Ednei; Reis, Tatiana Alves; Baquião, Arianne Costa; Corrêa, Benedito
The aim of this study was to evaluate the effects of gamma radiation (GR) and electron beam (EB) on Brazil nut samples contaminated with Aspergillus flavus. Fifty samples were spread with an A. flavus suspension and incubated at 30°C and a relative humidity of 93%. After 15 days of incubation, mycobiota and aflatoxin analysis were performed. The samples were divided into three groups (control, group 1, and group 2) that received radiation doses of 0 kGy (control) and 5 and 10 kGy each of GR and EB (groups 1 and 2). Noninoculated samples were irradiated with the same doses for sensory evaluation. The results showed that after 15 days of incubation, the average water activity of the samples was 0.80. The irradiation with GR and EB at doses of 5 and 10 kGy was able to eliminate A. flavus in Brazil nut samples. Aflatoxin analysis showed that EB doses of 5 and 10 kGy reduced aflatoxin B1 levels by 53.32 and 65.66%, respectively, whereas the same doses of GR reduced the levels of this toxin by 70.61 and 84.15% compared with the level in the control groups. Sensory evaluation demonstrated that the texture and odor of irradiated Brazil nut samples were acceptable. The taste evaluation indicated that 5 kGy of GR was judged acceptable. The results highlight that both irradiation processes (5- and 10-kGy doses) showed efficiency in A. flavus and aflatoxin elimination. GR and EB treatments resulted in some alterations in the sensory attributes of samples with the doses used in this study; however, Brazil nut samples irradiated with 5-kGy GR doses were considered acceptable.
de Souza, Evandro L.; Sales, Camila V.; de Oliveira, Carlos E. V.; Lopes, Laênia A. A.; da Conceição, Maria L.; Berger, Lúcia R. R.; Stamford, Thayza C. M.
Cherry tomato (Lycopersicon esculentum Mill) fruits are susceptible to contamination by Aspergillus flavus, which may cause the development of fruit rot and significant postharvest losses. Currently there are significant drawbacks for the use of synthetic fungicides to control pathogenic fungi in tomato fruits, and it has increased the interest in exploring new alternatives to control the occurrence of fungal infections in these fruits. This study evaluated the efficacy of chitosan (CHI) from Mucor circinelloides in combination with carvacrol (CAR) in inhibiting A. flavus in laboratory media and as a coating on cherry tomato fruits (25°C, 12 days and 12°C, 24 days). During a period of storage, the effect of coatings composed of CHI and CAR on autochthonous microflora, as well as on some quality characteristics of the fruits such as weight loss, color, firmness, soluble solids, and titratable acidity was evaluated. CHI and CAR displayed MIC valuesof 7.5 mg/mL and 10 μL/mL, respectively, against A. flavus. The combined application of CHI (7.5 or 3.75 mg/mL) and CAR (5 or 2.5 μL/mL) strongly inhibited the mycelial growth and spore germination of A. flavus. The coating composed of CHI (3.75 mg/mL) and CAR (2.5 or 1.25 μL/mL) inhibited the growth of A. flavus in artificially contaminated fruits, as well as the native fungal microflora of the fruits stored at room or low temperature. The application of the tested coatings preserved the quality of cherry tomato fruits as measured by some physicochemical attributes. From this, composite coatings containing CHI and CAR offer a promising alternative to control postharvest infection caused by A. flavus or native fungal microflora in fresh cherry tomato fruits without negatively affecting their quality over storage. PMID:26257717
de Souza, Evandro L; Sales, Camila V; de Oliveira, Carlos E V; Lopes, Laênia A A; da Conceição, Maria L; Berger, Lúcia R R; Stamford, Thayza C M
Cherry tomato (Lycopersicon esculentum Mill) fruits are susceptible to contamination by Aspergillus flavus, which may cause the development of fruit rot and significant postharvest losses. Currently there are significant drawbacks for the use of synthetic fungicides to control pathogenic fungi in tomato fruits, and it has increased the interest in exploring new alternatives to control the occurrence of fungal infections in these fruits. This study evaluated the efficacy of chitosan (CHI) from Mucor circinelloides in combination with carvacrol (CAR) in inhibiting A. flavus in laboratory media and as a coating on cherry tomato fruits (25°C, 12 days and 12°C, 24 days). During a period of storage, the effect of coatings composed of CHI and CAR on autochthonous microflora, as well as on some quality characteristics of the fruits such as weight loss, color, firmness, soluble solids, and titratable acidity was evaluated. CHI and CAR displayed MIC valuesof 7.5 mg/mL and 10 μL/mL, respectively, against A. flavus. The combined application of CHI (7.5 or 3.75 mg/mL) and CAR (5 or 2.5 μL/mL) strongly inhibited the mycelial growth and spore germination of A. flavus. The coating composed of CHI (3.75 mg/mL) and CAR (2.5 or 1.25 μL/mL) inhibited the growth of A. flavus in artificially contaminated fruits, as well as the native fungal microflora of the fruits stored at room or low temperature. The application of the tested coatings preserved the quality of cherry tomato fruits as measured by some physicochemical attributes. From this, composite coatings containing CHI and CAR offer a promising alternative to control postharvest infection caused by A. flavus or native fungal microflora in fresh cherry tomato fruits without negatively affecting their quality over storage.
Background Aspergillus flavus is intensively studied for its role in infecting crop plants and contaminating produce with aflatoxin, but its role as a human pathogen is less well understood. In parts of the Middle East and India, A. flavus surpasses A. fumigatus as a cause of invasive aspergillosis and is a significant cause of cutaneous, sinus, nasal and nail infections. Methods A collection of 45 clinical and 10 environmental A. flavus isolates from Iran were analysed using Variable-Number Tandem-Repeat (VNTR) markers with MICROSAT and goeBURST to determine their genetic diversity and their relatedness to clinical and environmental A. flavus isolates from Australia. Phylogeny was assessed using partial β-tubulin and calmodulin gene sequencing, and mating type was determined by PCR. Antifungal susceptibility testing was performed on selected isolates using a reference microbroth dilution method. Results There was considerable diversity in the A. flavus collection, with no segregation on goeBURST networks according to source or geographic location. Three Iranian isolates, two from sinus infections and one from a paranasal infection grouped with Aspergillus minisclerotigenes, and all produced B and G aflatoxin. Phylogenic analysis using partial β-tubulin and calmodulin sequencing confirmed two of these as A. minisclerotigenes, while the third could not be differentiated from A. flavus and related species within Aspergillus section flavi. Based on epidemiological cut-off values, the A. minisclerotigens and A. flavus isolates tested were susceptible to commonly used antifungal drugs. Conclusions This is the first report of human infection due to A. minisclerotigenes, and it raises the possiblity that other species within Aspergillus section flavi may also cause clinical disease. Clinical isolates of A. flavus from Iran are not distinct from Australian isolates, indicating local environmental, climatic or host features, rather than fungal features, govern the high
Aflatoxins are carcinogenic toxic compounds produced by Aspergillus flavus during infection of crops including maize (Zea mays L.). Contamination of maize with aflatoxin is exacerbated by late season drought stress. Previous studies have implicated numerous resistance-associated proteins (RAPs) that...
The purpose of the production of secondary metabolites in fungi are various and include stress responses, competitive antimicrobial activity, and the elimination of toxic compounds. However, the purpose of the production of aflatoxin, a carcinogenic mycotoxin, by Aspergillus flavus, is unknown. Prev...
The laeA gene encodes a nuclear protein that governs production of multiple fungal secondary metabolites. We examined the effects of laeA deletion in an Aspergillus flavus strain. Compared to wild type, expression of genes involved in secondary metabolism, conidiation and hydrophobicity was drastica...
More than 55 secondary metabolite biosynthesis gene clusters are predicted to be present in the Aspergillus flavus genome. In spite of this the biosynthesis of only a few metabolites, such as the aflatoxin, cyclopiazonic acid and aflatrem, has been correlated with a particular gene cluster. Using RN...
Aspergillus flavus infection and aflatoxin contamination of maize pose negative impacts in agriculture and health. Commercial maize hybrids are generally susceptible to this fungus. Significant levels of host plant resistance have been observed in certain maize inbred lines. This study was conducted...
Biocontrol of Aspergillus flavus using inoculated bioplastic granules has been proven to be effective under laboratory and field conditions. In the present study, the use of low-density pellets from recycled bioplastic as a biocontrol strain carrier was evaluated. Applying recycled bioplastic pell...
Several nut crops including almonds, pistachios, and walnuts can become contaminated with mycotoxins. Of greatest economic significance are aflatoxins, which are mainly produced by members of Aspergillus section Flavi. The distribution of the two sclerotial-size morphotypes of A. flavus (i.e. S and ...
Aspergillus flavus and A. parasiticus infect peanut seeds and produce aflatoxins, which are associated with various diseases in domestic animals and humans throughout the world. The most cost-effective strategy to minimize aflatoxin contamination involves the development of peanut cultivars that are...
Previous research demonstrated that aflatoxin contamination in corn grown in Mississippi is reduced by field application of wheat grains pre-inoculated with the non-aflatoxigenic Aspergillus flavus strain NRRL 30797. To facilitate field applications of the biocontrol isolate, a series of laboratory ...
The contamination of corn (maize) by fungi and the accumulation of mycotoxins are a serious agricultural problem for human and animal health. One particular devastating group of mycotoxins, called aflatoxins, has been intensely studied since the 1960s. Studies of Aspergillus flavus, the agricultura...
Maize (Zea mays L.) susceptibility to ear rot and aflatoxin accumulation by Aspergillus flavus (Link:Fr) causes significant economic and human health damage worldwide. Although host plant resistance is an ideal solution to the problem, no commercial varieties display sufficient levels of resistance ...
Aspergillus flavus is an opportunistic plant pathogen that colonizes and produces the toxic and carcinogenic secondary metabolites, aflatoxins, in oil-rich crops such as maize (Zea mays ssp. mays L.). Pathogenesis-related proteins serve as a first line of defense against invading pathogens by confer...
Aflatoxin in corn grain is a problem in many areas of the world. Any combination of environmentally stressful or agronomically unfavorable conditions can increase the likelihood of Aspergillus flavus infection and production of aflatoxin in the corn grain. In the absence of a consistent natural A....
Aspergillus flavus aswA (AFLA_085170) is a gene encoding a Zn(II)2Cys6 DNA-binding domain. Partial deletion of aswA yielded strains that made a truncated gene transcript and generated a fungus that produced a greatly increased number of sclerotia. These sclerotia were odd-shaped and non-pigmented (w...
Zaccaria, Marco; Ludovici, Matteo; Sanzani, Simona Marianna; Ippolito, Antonio; Aiese Cigliano, Riccardo; Sanseverino, Walter; Scarpari, Marzia; Scala, Valeria; Fanelli, Corrado; Reverberi, Massimo
Aspergillus flavus is an efficient producer of mycotoxins, particularly aflatoxin B1, probably the most hepatocarcinogenic naturally-occurring compound. Although the inducing agents of toxin synthesis are not unanimously identified, there is evidence that oxidative stress is one of the main actors in play. In our study, we use menadione, a quinone extensively implemented in studies on ROS response in animal cells, for causing stress to A. flavus. For uncovering the molecular determinants that drive A. flavus in challenging oxidative stress conditions, we have evaluated a wide spectrum of several different parameters, ranging from metabolic (ROS and oxylipin profile) to transcriptional analysis (RNA-seq). There emerges a scenario in which A. flavus activates several metabolic processes under oxidative stress conditions for limiting the ROS-associated detrimental effects, as well as for triggering adaptive and escape strategies. PMID:26512693
Zhang, Feng; Xu, Gaopo; Geng, Longpo; Lu, Xiaoyan; Yang, Kunlong; Yuan, Jun; Nie, Xinyi; Zhuang, Zhenhong; Wang, Shihua
This study focused on AflSkn7, which is a stress response regulator in the aflatoxin-producing Aspergillus flavus. The ΔAflSkn7 mutants exhibited partially defective conidial formation and a complete inability to generate sclerotia, indicating AflSkn7 affects A. flavus asexual and sexual development. The mutants tolerated osmotic stress but were partially susceptible to the effects of cell wall stress. Additionally, the ΔAflSkn7 mutants were especially sensitive to oxidative stress. These observations confirmed that AflSkn7 influences oxidative stress responses rather than osmotic stress responses. Additionally, AflSkn7 was observed to increase aflatoxin biosynthesis and seed infection rates. These results indicate AflSkn7 affects A. flavus morphological development, stress response, aflatoxin production, and pathogenicity. The results of this study may facilitate the development of new methods to manage A. flavus infections. PMID:27399770
Ampt, Eline A; Bush, Daniel S; Siegel, Joel P; Berenbaum, May R
The navel orangeworm, Amyelois transitella (Walker), is a polyphagous pest of California nut crops and is responsible for extensive losses in the United States. It directly damages crops by feeding and contaminating nuts with frass and webbing and vectors saprophytic fungi that infect crops. The navel orangeworm is commonly associated with Aspergillus species, including the toxigenic Aspergillus flavus, which causes crop loss by producing carcinogens, including aflatoxin B1. This lepidopteran-fungus association is the most economically serious pest complex in Central Valley orchards, and evidence indicates that this relationship is mutualistic. We assessed preference and performance of navel orangeworm larvae associated with A. flavus in behavioral bioassays in which neonates were allowed to orient within arenas to media with or without fungal tissue, and performance bioassays in which larvae were reared with and without A. flavus on potato dextrose agar (PDA) and a semidefined almond PDA diet to evaluate effects on development and pupal weight. Navel orangeworm larvae were attracted to A. flavus and developed faster in its presence, indicating a nutritional benefit to the caterpillars. Larvae reached pupation ∼33% faster on diet containing A. flavus, and pupal weights were ∼18% higher for males and ∼13% higher for females on this diet. Our findings indicate that A. flavus plays an important role in larval orientation and development on infected hosts. The preference-performance relationship between navel orangeworms and Aspergillus flavus is consistent with a facultative mutualism that has broad implications for pest management efforts and basic understanding of Lepidoptera-plant interactions.
Fountain, Jake C.; Scully, Brian T.; Chen, Zhi-Yuan; Gold, Scott E.; Glenn, Anthony E.; Abbas, Hamed K.; Lee, R. Dewey; Kemerait, Robert C.; Guo, Baozhu
Drought stress in the field has been shown to exacerbate aflatoxin contamination of maize and peanut. Drought and heat stress also produce reactive oxygen species (ROS) in plant tissues. Given the potential correlation between ROS and exacerbated aflatoxin production under drought and heat stress, the objectives of this study were to examine the effects of hydrogen peroxide (H2O2)-induced oxidative stress on the growth of different toxigenic (+) and atoxigenic (−) isolates of Aspergillus flavus and to test whether aflatoxin production affects the H2O2 concentrations that the isolates could survive. Ten isolates were tested: NRRL3357 (+), A9 (+), AF13 (+), Tox4 (+), A1 (−), K49 (−), K54A (−), AF36 (−), and Aflaguard (−); and one A. parasiticus isolate, NRRL2999 (+). These isolates were cultured under a H2O2 gradient ranging from 0 to 50 mM in two different media, aflatoxin-conducive yeast extract-sucrose (YES) and non-conducive yeast extract-peptone (YEP). Fungal growth was inhibited at a high H2O2 concentration, but specific isolates grew well at different H2O2 concentrations. Generally the toxigenic isolates tolerated higher concentrations than did atoxigenic isolates. Increasing H2O2 concentrations in the media resulted in elevated aflatoxin production in toxigenic isolates. In YEP media, the higher concentration of peptone (15%) partially inactivated the H2O2 in the media. In the 1% peptone media, YEP did not affect the H2O2 concentrations that the isolates could survive in comparison with YES media, without aflatoxin production. It is interesting to note that the commercial biocontrol isolates, AF36 (−), and Aflaguard (−), survived at higher levels of stress than other atoxigenic isolates, suggesting that this testing method could potentially be of use in the selection of biocontrol isolates. Further studies will be needed to investigate the mechanisms behind the variability among isolates with regard to their degree of oxidative stress
Nepote, M C; Piontelli, E; Saubois, A
It has been demonstrated in several agricultural regions all around the world that Aspergillus flavus can infect corn grains and produce aflatoxins even before the harvest. It is also known that the incidence and levels of contamination of cereals factors. In the present work, the incidence of aflatoxins in corn grain from the central and northern areas of Santa Fe province in Argentina was studied. The relationship between the extent of kernel infection by the fungus and the presence of aflatoxins in the samples was examined. The isolation and identification of A.flavus were carried out by plating dilutions of the ground kernels on dichloran-rose bengal-chloramphenicol agar (DRBC). Simultaneously, kernels were superficially sterilized with 10% commercial CIONa and plated on potato-dextrose-chloramphenicol agar (PDA + C). The analysis of aflatoxins B1, B2, G1 and G2 was performed by thin layer chromatography (TLC) according with Norma IRAM 14803 (Argentina). A.flavus Link:Fr. was identified in 63.3% of the corn samples. Colonized kernels ranged from 2.5 to 25% and counts on DRBC were in the order of 10(3) CFU/g. Two samples colonized by A.flavus contained aflatoxins B1 and B2 (50 micrograms/kg of aflatoxin B1 and 30 micrograms/kg of aflatoxin B2, and 30 micrograms/kg of aflatoxin B1, and traces of aflatoxin B2, respectively). One sample contained only aflatoxin B1 (22 mu/kg). According to these results, it may be concluded that the incidence of A.flavus observed constitutes a call in attention with respect to the conditions required for storage and transportation of the grains, to minimize the proliferation of the fungus and the production of aflatoxins in these stages. Although the incidence of aflatoxins in the samples of grains was rather low, the levels of aflatoxin B1 recorded in the positive samples were higher than those recommended--or given as advisory levels for human foods, by most countries in the world.
Background Aspergillus flavus Link:Fr, an opportunistic fungus that produces aflatoxin, is pathogenic to maize and other oilseed crops. Aflatoxin is a potent carcinogen, and its presence markedly reduces the value of grain. Understanding and enhancing host resistance to A. flavus infection and/or subsequent aflatoxin accumulation is generally considered an efficient means of reducing grain losses to aflatoxin. Different proteomic, genomic and genetic studies of maize (Zea mays L.) have generated large data sets with the goal of identifying genes responsible for conferring resistance to A. flavus, or aflatoxin. Results In order to maximize the usage of different data sets in new studies, including association mapping, we have constructed a relational database with web interface integrating the results of gene expression, proteomic (both gel-based and shotgun), Quantitative Trait Loci (QTL) genetic mapping studies, and sequence data from the literature to facilitate selection of candidate genes for continued investigation. The Corn Fungal Resistance Associated Sequences Database (CFRAS-DB) (http://agbase.msstate.edu/) was created with the main goal of identifying genes important to aflatoxin resistance. CFRAS-DB is implemented using MySQL as the relational database management system running on a Linux server, using an Apache web server, and Perl CGI scripts as the web interface. The database and the associated web-based interface allow researchers to examine many lines of evidence (e.g. microarray, proteomics, QTL studies, SNP data) to assess the potential role of a gene or group of genes in the response of different maize lines to A. flavus infection and subsequent production of aflatoxin by the fungus. Conclusions CFRAS-DB provides the first opportunity to integrate data pertaining to the problem of A. flavus and aflatoxin resistance in maize in one resource and to support queries across different datasets. The web-based interface gives researchers different query
Asters, Matthew C.; Williams, W. Paul; Perkins, Andy D.; Mylroie, J. Erik; Windham, Gary L.; Shan, Xueyan
Aspergillus flavus is a pathogenic fungus infecting maize and producing aflatoxins that are health hazards to humans and animals. Characterizing host defense mechanism and prioritizing candidate resistance genes are important to the development of resistant maize germplasm. We investigated methods amenable for the analysis of the significance and relations among maize candidate genes based on the empirical gene expression data obtained by RT-qPCR technique from maize inbred lines. We optimized a pipeline of analysis tools chosen from various programs to provide rigorous statistical analysis and state of the art data visualization. A network-based method was also explored to construct the empirical gene expression relational structures. Maize genes at the centers in the network were considered as important candidate genes for maize DNA marker studies. The methods in this research can be used to analyze large RT-qPCR datasets and establish complex empirical gene relational structures across multiple experimental conditions. PMID:24770700
Lillehoj, E B; Calvert, O H; Kwolek, W F; Zuber, M S
Aflatoxin levels and physical properties of corn kernels inoculated with Aspergillus flavus during development and noninoculated kernels were compared in samples with various proportions of the 2 kernel types. The relationship between mean toxin levels and associated standard deviations of 5 samples demonstrated a linear association from the lowest toxin in noninoculated corn through a mixture of 60% inoculated/40% noninoculated. However, at the highest toxin level in the 100% inoculated material, a reduction in sample variation was observed. Examination of individual kernal weights showed that inoculated kernels were distinctly lighter than noninoculated seed. A uniform grinding procedure of the samples yielded heterogeneous particle sizes based on the starting corn. The large particle fraction (greater than 500 micrometers) decreased from 100% noninoculated kernels through the mixtures to the 100% inoculated seed; particles below 150 micrometers were most abundant in the ground samples from inoculated kernels. In addition, the density of particles within a size category varied; lower densities were observed in samples obtained from A. flavus-inoculated kernels.
Matsumura, M; Mori, T
We isolated two strains of Aspergillus flavus from a lung lesion and a skin lesion at autopsy from a patient with acute myelogenous leukemia complicated with fungal infection. An attempt was made to detect aflatoxins in culture filtrates of those isolates and the tissue extract of the lung lesion through the techniques of thin-layer chromatography (TLC), densitometry and high-performance liquid chromatography (HPLC). Aflatoxins B1, B2 and M1 were demonstrated in all of these materials qualitatively and quantitatively. The concentrations of aflatoxins in the cultures of the isolates and in the lung lesion extract determined by HPLC were aflatoxin B1: 11.715 microg/ml (lung isolate), 21.383 micro g/ml (skin isolate), 0.635 microg/g (lung extract), aflatoxin B2: 0.341 microg/ml (lung isolate), 0.577 micro g/ml (skin isolate), 0.0273 microg/g (lung extract) and aflatoxin M1: 0.277 microg/ml (lung isolate), 0.491 micro g/ml (skin isolate), 0.0525 microg/g (lung extract), respectively. B1, known as the most toxic among the aflatoxin group, showed the highest concentration through these experiments. This case may be considered as the first to detect aflatoxins in autopsied materials associated with A. flavus infection.
Mencarelli, Mariangela; Accinelli, Cesare; Vicari, Alberto
A novel biocontrol strategy consisting of field application of bioplastic-based granules inoculated with a non-toxigenic Aspergillus flavus L. strain has recently been shown to be effective for reducing aflatoxin contamination in corn. This study focused on other factors that may affect the feasibility of this biocontrol technique, and more specifically the role of the European corn borer (ECB), Ostrinia nubilalis H., in the dispersal and infestation of A. flavus in corn and its impact on crop yield. In spite of the high percentage of corn ears showing larval feeding damage, ECB-bored kernels accounted for only 3 and 4% in 2009 and 2010 respectively. Most of the damaged kernels were localised in the ear tip or immediately below. More precisely, the average incidence of ECB-bored kernels in the upper end of the ear was 32%. However, less than 5% of kernels from the central body of the ear, which includes the majority of kernels, were injured by ECB. Although ECB larvae showed a high tolerance to aflatoxin B1 and thus had the potential to serve as vectors of the mould, fungal infection of kernels was poorly associated with insect damage. ECB infestation resulted in grain yield losses not exceeding 2.5%. © 2012 Society of Chemical Industry.
Sohbatzadeh, F.; Mirzanejhad, S.; Shokri, H.; Nikpour, M.
The main objective of this study is to investigate the inactivation efficacy of cold streamers in a sealed package on pathogenic fungi Aspergillus flavus ( A. flavus) spores that artificially contaminated pistachio surface. To produce penetrating cold streamers, electric power supply was adapted to deposit adequate power into the package. The plasma streamers were generated by an alternating high voltage with carrier frequency of 12.5 kHz which was suppressed by a modulated pulsed signal at frequency of 110 Hz. The plasma exposition time was varied from 8 to 18 min to show the effect of the plasma treatment on fungal clearance while the electrode and sample remained at room temperature. This proved a positive effect of the cold streamers treatment on fungal clearance. Benefits of deactivation of fungal spores by streamers inside the package include no heating, short treatment time and adaptability to existing processes. Given its ability to ensure the safety and longevity of food products, this technology has great potential for utilization in food packaging and processing industry. In this study, moisture and pH changes of pistachio samples after plasma streamers treatment were also investigated.
Shafique, Sobiya; Bajwa, Rukhsana; Shafique, Shazia
Aspergillus flavus FCBP-231, a filamentous fungus, was genetically modified for its ability to reveal extra cellular alpha-amylase activity. For strain improvement, the selected strains were subjected to UV irradiation (5-40 min exposure) and EMS treatment (50-300 microg mL(-1)) for hyper activity of an alpha-amylase enzyme. The mutants were quantitatively compared with the parental strain. UV and chemical mutagenesis brought about a dramatic enhancement in enzymatic activity. The mutant strains Af-UV-5.3 and Af-Ch-5.7 exhibited 79 and 110% more enzyme activity than the native strain A. flavus FCBP-231. This improvement in enzyme activity of the mutants suggests that they are suitable strains to be used in biotechnology. RAPD-PCR analysis revealed different patterns of amplicons of native as well as mutant derivatives, which suggested that the mutation imparted changes in the genetic make up of the mutants probably involved enzyme production control.
Gordon, S H; Schudy, R B; Wheeler, B C; Wicklow, D T; Greene, R V
Aspergillus flavus and other pathogenic fungi display typical infrared spectra which differ significantly from spectra of substrate materials such as corn. On this basis, specific spectral features have been identified which permit detection of fungal infection on the surface of corn kernels by photoacoustic infrared spectroscopy. In a blind study, ten corn kernels showing bright greenish yellow fluorescence (BGYF) in the germ or endosperm and ten BGYF-negative kernels were correctly classified as infected or not infected by Fourier transform infrared photoacoustic spectroscopy. Earlier studies have shown that BGYF-positive kernels contain the bulk of the aflatoxin contaminating grain at harvest. Ten major spectral features, identified by visual inspection of the photoacoustic spectra of A. flavus mycelium grown in culture versus uninfected corn, were interpreted and assigned by theoretical comparisons of the relative chemical compositions of fungi and corn. The spectral features can be built into either empirical or knowledge-based computer models (expert systems) for automatic infrared detection and segregation of grains or kernels containing aflatoxin from the food and feed supply.
Paul, R. A.; Meis, J. F.
This study aimed to explore any mutation in the CYP51 gene conferring azole resistance in Aspergillus flavus. Two voriconazole-resistant and 45 voriconazole-susceptible isolates were included in the study. Sequence analysis demonstrated a T1025C nucleotide change in CYP51C, resulting in the Y319H amino acid substitution in one resistant isolate. However, the earlier described T788G mutation in CYP51C conferring voriconazole resistance in A. flavus isolates was present in all isolates, irrespective of their susceptibility status. PMID:26248359
Aflatoxins are toxic metabolites and potent carcinogen produced from asexual fungi Aspergillus flavus and A. parasiticus. Aflatoxins can contaminate cottonseed under conducive preharvest and postharvest conditions. U.S. federal regulations restrict the use of aflatoxin contaminated cottonseed at >20...
Ren, Silin; Yang, Mingkun; Li, Yu; Zhang, Feng; Chen, Zhuo; Zhang, Jia; Yang, Guang; Yue, Yuewei; Li, Siting; Ge, Feng; Wang, Shihua
Aspergillus flavus is a pathogenic fungus that produces toxic and carcinogenic aflatoxins and is the causative agent of aflatoxicosis. A growing body of evidence indicates that reversible phosphorylation plays important roles in regulating diverse functions in this pathogen. However, only a few phosphoproteins of this fungus have been identified, which hampers our understanding of the roles of phosphorylation in A. flavus. So we performed a global and site-specific phosphoproteomic analysis of A. flavus. A total of 598 high-confidence phosphorylation sites were identified in 283 phosphoproteins. The identified phosphoproteins were involved in various biological processes, including signal transduction and aflatoxins biosynthesis. Five identified phosphoproteins associated with MAPK signal transduction and aflatoxins biosynthesis were validated by immunoblotting using phospho-specific antibodies. Further functional studies revealed that phosphorylation of the MAP kinase kinase kinase Ste11 affected aflatoxins biosynthesis in A. flavus. Our data represent the results of the first global survey of protein phosphorylation in A. flavus and reveal previously unappreciated roles for phosphorylation in the regulation of aflatoxins production. The generated dataset can serve as an important resource for the functional analysis of protein phosphorylation in A. flavus and facilitate the elucidation of phosphorylated signaling networks in this pathogen. PMID:27667718
The filamentous fungus, Aspergillus flavus, produces the toxic and carcinogenic, polyketide synthase (PKS)-derived family of secondary metabolites termed aflatoxins. While analysis of the A. flavus genome has identified many other PKSs capable of producing secondary metabolites, to date, only a few ...
Aspergillus flavus fluG deletion strains showed decreased conidiation but had elevated sclerotial production. These developmental changes were not remediated by co-culturing with fluG-positive strains. The fluG mutant still retained its aflatoxin-producing ability. The A. flavus fluG gene functions ...
Aspergillus flavus is a saprophytic fungus that can invade and contaminate agronomically important crops. The fungus produces a number of toxic secondary metabolites, such as aflatoxin, which are synthesized from genes located in close proximity with each other on the chromosome. A. flavus has appro...
The fungus Aspergillus flavus is known for its ability to produce the toxic and carcinogenic aflatoxins in food and feed. While aflatoxins are of most concern, A. flavus is predicted to be capable of producing many more metabolites based on a study of its complete genome sequence. Some of these meta...
The contamination of crops with aflatoxins during Aspergillus flavus infection is exacerbated by drought stress. Reactive oxygen species have been shown to be produced in plant tissues in response to drought and to stimulate the production of aflatoxin by A. flavus in vitro. To better understand the...
Maize infected by aflatoxin-producing Aspergillus flavus may become contaminated with aflatoxins and as a result, threaten human health, food security, and farmers’ income in developing countries where maize is a staple. Environmental distribution and genetic diversity of A. flavus can influence the...
The global regulatory veA gene governs development and secondary metabolism in numerous fungal species, including Aspergillus flavus. This is especially relevant since A. flavus infects crops of agricultural importance worldwide, contaminating them with potent mycotoxins. The most well-known are afl...
The filamentous fungus Aspergillus flavus is an agriculturally important opportunistic plant pathogen that produces potent carcinogenic compounds called aflatoxins. We identified the A. flavus rtfA gene, the ortholog of rtf1 in S. cerevisiae and rtfA in A. nidulans. Interestingly, rtfA has multiple ...
Huang, Changwei; Jha, Archana; Sweany, Rebecca; DeRobertis, Catherine; Damann,, Kenneth E.
Biological control of preharvest aflatoxin contamination by atoxigenic stains of Aspergillus flavus has been demonstrated in several crops. The assumption is that some form of competition suppresses the fungus's ability to infect or produce aflatoxin when challenged. Intraspecific aflatoxin inhibition was demonstrated by others. This work investigates the mechanistic basis of that phenomenon. A toxigenic and atoxigenic isolate of A. flavus which exhibited intraspecific aflatoxin inhibition when grown together in suspended disc culture were not inhibited when grown in a filter insert-plate well system separated by a .4 or 3 µm membrane. Toxigenic and atoxigenic conidial mixtures (50∶50) placed on both sides of these filters restored inhibition. There was ∼50% inhibition when a 12 µm pore size filter was used. Conidial and mycelial diameters were in the 3.5–7.0 µm range and could pass through the 12 µm filter. Larger pore sizes in the initially separated system restored aflatoxin inhibition. This suggests isolates must come into physical contact with one another. This negates a role for nutrient competition or for soluble diffusible signals or antibiotics in aflatoxin inhibition. The toxigenic isolate was maximally sensitive to inhibition during the first 24 hrs of growth while the atoxigenic isolate was always inhibition competent. The atoxigenic isolate when grown with a green fluorescent protein (GFP) toxigenic isolate failed to inhibit aflatoxin indicating that there is specificity in the touch inhibiton. Several atoxigenic isolates were found which inhibited the GFP isolate. These results suggest that an unknown signaling pathway is initiated in the toxigenic isolate by physical interaction with an appropriate atoxigenic isolate in the first 24 hrs which prevents or down-regulates normal expression of aflatoxin after 3–5 days growth. We suspect thigmo-downregulation of aflatoxin synthesis is the mechanistic basis of intraspecific aflatoxin inhibition
Baquião, Arianne Costa; Lopes, Evandro Luiz; Corrêa, Benedito
The objective of this study was to carry out a transcription analysis of eight genes belonging to the aflatoxin (AF) and cyclopiazonic acid (CPA) biosynthesis pathway, and to detect aflatoxin B1 (AFB1) and CPA production in Aspergillus flavus strains isolated from Brazil nuts. Additionally, these genes were correlated with the different mycotoxigenic profiles of the same strains. Four previously identified A. flavus strains (ICB-01, ICB-151, ICB-161, and ICB-165) were grown on Brazil nut agar at 25°C for 10days. Mycotoxins were separated by high-performance liquid chromatography. Transcriptional analysis was performed by real-time RT-PCR using specific primers designed based on the conserved regions of two regulatory genes (aflR and aflS), three structural genes of the AFB1 biosynthesis pathway (aflH, aflJ and aflP), and three structural genes of the CPA biosynthesis pathway (maoA, dmaT and pks-nrps). The expression of most genes in the A. flavus isolates varied according to the mycotoxin profile of each strain. The most expressed genes in the aflatoxigenic strain ICB-151 were aflJ (77.11%) and aflH (32.75%), while the CPA-producing strain ICB-161 mainly expressed dmaT (100%), maoA (63.72%), aflS (43.52%), and aflR (42.63%). The ICB-01 isolate was a producer of AFB1 and CPA and the most expressed genes were aflS (47.79%), dmaT (42.77%), aflP (39.5%), and aflR (38.02%). ICB-198 did not produce any mycotoxin and exhibited lower expression of almost all genes analyzed. Furthermore, the ratio of aflS/aflR expression was correlated with the biosynthesis of AF and CPA in A. flavus strains producing exclusively AF or CPA or producing both AF and CPA. The ratio of aflS/aflR expression therefore seems to be related to the production of mycotoxins in Brazil nuts. Our results provide important data for the development of innovative and more cost-effective strategies to reduce and prevent AFB and CPA contamination in Brazil nuts. Copyright © 2016. Published by Elsevier Ltd.
Vermani, Maansi; Vijayan, Vannan Kandi; Agarwal, Mahendra Kumar
Aspergillus species (A. flavus and A. niger) are important sources of inhalant allergens. Current diagnostic modalities employ crude Aspergillus extracts which only indicate the source to which the patient has been sensitized, without identifying the number and type of allergens in crude extracts. We report a study on the identification of major and minor allergens of the two common airborne Aspergillus species and heterogeneity of patients' IgE response to them. Skin prick tests were performed on 300 patients of bronchial asthma and/or allergic rhinitis and 20 healthy volunteers. Allergen specific IgE in patients' sera was estimated by enzyme allergosorbent test (EAST). Immunoblots were performed to identify major/minor allergens of Aspergillus extracts and to study heterogeneity of patients'IgE response to them. Positive cutaneous responses were observed in 17% and 14.7% of patients with A. flavus and A. niger extracts, respectively. Corresponding EAST positivity was 69.2% and 68.7%. In immunoblots, 5 allergenic proteins were identified in A. niger extract, major allergens being 49, 55.4 and 81.5 kDa. Twelve proteins bound patients' IgE in A. flavus extract, three being major allergens (13.3, 34 and 37 kDa). The position and slopes of EAST binding and inhibition curves obtained with individual sera varied from patient to patient. The number and molecular weight of IgE-binding proteins in both the Aspergillus extracts varied among patients. These results gave evidence of heterogeneity of patients' IgE response to major/minor Aspergillus allergens. This approach will be helpful to identify disease eliciting molecules in the individual patients (component resolved diagnosis) and may improve allergen-specific immunotherapy.
Kandhavelu, Jeyalakshmi; Demonte, Naveen Luke; Namperumalsamy, Venkatesh Prajna; Prajna, Lalitha; Thangavel, Chitra; Jayapal, Jeya Maheshwari; Kuppamuthu, Dharmalingam
Fungal keratitis is one of the leading causes of blindness in the tropical countries affecting individuals in their most productive age. The host immune response during this infection is poorly understood. We carried out comparative tear proteome analysis of Aspergillus flavus keratitis patients and uninfected controls. Proteome was separated into glycosylated and non-glycosylated fractions using lectin column chromatography before mass spectrometry. The data revealed the major processes activated in the human host in response to fungal infection and reflected in the tear. Extended analysis of this dataset presented here complements the research article entitled "Aspergillus flavus induced alterations in tear protein profile reveal pathogen-induced host response to fungal infection " (Jeyalakhsmi Kandhavelu, Naveen Luke Demonte, Venkatesh Prajna Namperumalsamy, Lalitha Prajna, Chitra Thangavel, Jeya Maheshwari Jayapal, Dharmalingam Kuppamuthu, 2016). The mass spectrometry proteomics data have been deposited in the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PRIDE:PXD003825.
Krishnamurthy, Y L; Shashikala, J
The inhibitory effect of cowdung fumes, Captan, leaf powder of Withania somnifera, Hyptis suaveolens, Eucalyptus citriodora, peel powder of Citrus sinensis, Citrus medica and Punica granatum, neem cake and pongamia cake and spore suspension of Trichoderma harzianum and Aspergillus niger on aflatoxin B(1) production by toxigenic strain of Aspergillus flavus isolated from soybean seeds was investigated. Soybean seed was treated with different natural products and fungicide captan and was inoculated with toxigenic strain of A. flavus and incubated for different periods. The results showed that all the treatments were effective in controlling aflatoxin B(1) production. Captan, neem cake, spore suspension of T. harzianum, A. niger and combination of both reduced the level of aflatoxin B(1) to a great extent. Leaf powder of W. somnifera, H. suaveolens, peel powder of C. sinensis, C. medica and pongamia cake also controlled the aflatoxin B(1) production. All the natural product treatments applied were significantly effective in inhibiting aflatoxin B(1) production on soybean seeds by A. flavus. These natural plant products may successfully replace chemical fungicides and provide an alternative method to protect soybean and other agricultural commodities from aflatoxin B(1) production by A. flavus.
Kedia, Akash; Dwivedy, Abhishek Kumar; Jha, Dhruva Kumar; Dubey, Nawal Kishore
The present study reports in vivo antifungal and antiaflatoxigenic efficacy of Mentha spicata essential oil (EO) against toxigenic Aspergillus flavus strain LHP(C)-D6 in chickpea food system up to 12 months of storage. In addition, the mode of antifungal action of EO was also determined to understand the mechanism of fungal growth inhibition. The in vivo study with different concentrations of M. spicata EO showed dose-dependent decrease in fungal colony count as well as aflatoxin B1 concentration. The EO caused >50% protection in inoculated sets and >70% protection in uninoculated sets of chickpea food system against A. flavus at 1.0 μL mL(-1) air concentration. However, at the same concentration, EO caused 100% inhibition to aflatoxin B1 production in both sets when analyzed through high-performance liquid chromatography (HPLC). The antifungal target of EO in fumigated cells of A. flavus was found to be the plasma membrane when analyzed through electron microscopic observations and ions leakage test. The EO fumigated chickpea seeds showed 100% seed germination and seedling growth after 12 months of storage. Based on these observations, M. spicata EO can be recommended as plant-based preservative for safe protection of food commodities during storage conditions against fungal and most importantly mycotoxin contaminations.
Paranagama, P A; Abeysekera, K H T; Abeywickrama, K; Nugaliyadde, L
To develop a natural fungicide against aflatoxigenic fungi, to protect stored rice, using the essential oil of lemongrass. Aspergillus flavus Link. was isolated from stored rice and identified as an aflatoxigenic strain. Lemongrass oil was tested against A. flavus and the test oil was fungistatic and fungicidal against the test pathogen at 0.6 and 1.0 mg ml(-1), respectively. Aflatoxin production was completely inhibited at 0.1 mg ml(-1). The results obtained from the thin layer chromatographic bioassay and gas chromatography indicated citral a and b as the fungicidal constituents in lemongrass oil. During the fumigant toxicity assay of lemongrass oil, the sporulation and the mycelial growth of the test pathogen were inhibited at the concentrations of 2.80 and 3.46 mg ml(-1), respectively. Lemongrass oil could be used to manage aflatoxin formation and fungal growth of A. flavus in stored rice. Currently, fungicides are not used to control fungal pests or mycotoxin production on stored rice. Rice treated with the essential oil of lemongrass could be used to manage fungal pests as well as the insect pests in stored rice. The essential oil is chemically safe and acceptable to consumers, as synthetic chemical fungicides can cause adverse health effects to consumers.
Diaz-Guerra, Teresa M.; Mellado, Emilia; Cuenca-Estrella, Manuel; Gaztelurrutia, Lourdes; Navarro, Jose Ignacio Villate; Tudela, Juan L. Rodríguez
We report the simultaneous isolation of one Aspergillus flavus strain from the aortic prosthesis of a heart surgery patient and another two isolates recovered from a dual-reservoir cooler-heater used in the operating room where this patient was operated on. Genetic typing of these three isolates by randomly amplified polymorphic DNA (RAPD) revealed identical genotypes. Eight unrelated control strains of A. flavus had eight different genotypes. These results clearly indicated the nosocomial origin of the A. flavus strain isolated from the patient. We suggest that the RAPD technique is a rapid and reliable tool to ascertain the epidemiology of infections caused by A. flavus. PMID:10835021
Varghese, Lalee; Chacko, Rabin; Varghese, George M; Job, Anand
Septic arthritis of the temporomandibular joint (TMJ) is a very rare complication of otitis externa that can lead to ankylosis and destruction of the joint. We report the case of a 74-year-old man who developed aspergillosis of the TMJ following otitis externa. To the best of our knowledge, this is the first reported case of TMJ septic arthritis secondary to otitis externa caused by Aspergillus flavus. The patient was successfully managed with condylectomy, debridement, and drug treatment with voriconazole.
Shantha, T; Murthy, V S
Resting cells of Aspergillus flavus synthesized aflatoxin from acetate as the sole carbon source after 36 h of incubation. Addition of pyruvate (5.5 mg/m) as cosubstrate to [1-14C]acetate and unlabeled acetate considerably reduced toxin production but increased the radioactivity on the tricarboxylic acid intermediates. This suggests that high tricarboxylic acid activity drastically affected toxin synthesis. PMID:6797348
Liu, Peiqing; Li, Benjin; Yin, Rongmei; Weng, Qiyong; Chen, Qinghe
Aspergillus flavus is a common filamentous fungus that produces aflatoxins and presents a major threat to agriculture and human health. Previous studies focused mainly on the detection of A. flavus or aflatoxin separately. Here, we developed internal transcribed spacer (ITS)- and aflP-based rapid detection of A. flavus in food samples using the loop-mediated isothermal amplification (LAMP) method. The ITS1-5.8S-ITS2 rDNA region of A. flavus and the aflatoxin-encoding gene aflP were used as target regions. The detection limits of A. flavus and aflP were 10 fg and 1 pg pure DNA, respectively, which allows aflatoxin-contaminated samples to be differentiated from infected samples and reduces false-negative or false-positive results. For specificity testing, DNA extracted from 7 A. flavus, 5 different Aspergillus spp., and 21 other fungi were used, and our results showed that A. flavus strains are detected by ITS-based detection and aflatoxigenic A. flavus strains are detected by aflP-based detection. Furthermore, the ITS- and aflP-based LAMP assays were used for detection analysis of DNA from food samples artificially and naturally contaminated with A. flavus. Our results showed that the detection rate of A. flavus based on the multi-ITS-based LAMP detection is 100% and that the aflatoxigenic strains in all A. flavus are detected by the aflP-based LAMP assay. The LAMP protocol described in our study represents a rapid and highly specific and sensitive diagnostic method for A. flavus detection, which can be used as a diagnostic tool that simplifies A. flavus monitoring and guarantees the quality and safety of foods.
Zhuang, Zhenhong; Lohmar, Jessica M; Satterlee, Timothy; Cary, Jeffrey W; Calvo, Ana M
Aspergillus flavus produces a variety of toxic secondary metabolites; among them, the aflatoxins (AFs) are the most well known. These compounds are highly mutagenic and carcinogenic, particularly AFB₁. A. flavus is capable of colonizing a number of economically-important crops, such as corn, cotton, peanut and tree nuts, and contaminating them with AFs. Molecular genetic studies in A. flavus could identify novel gene targets for use in strategies to reduce AF contamination and its adverse impact on food and feed supplies worldwide. In the current study, we investigated the role of the master transcription factor gene mtfA in A. flavus. Our results revealed that forced overexpression of mtfA results in a drastic decrease or elimination of several secondary metabolites, among them AFB₁. The reduction in AFB₁ was accompanied by a decrease in aflR expression. Furthermore, mtfA also regulates development; conidiation was influenced differently by this gene depending on the type of colonized substrate. In addition to its effect on conidiation, mtfA is necessary for the normal maturation of sclerotia. Importantly, mtfA positively affects the pathogenicity of A. flavus when colonizing peanut seeds. AF production in colonized seeds was decreased in the deletion mtfA strain and particularly in the overexpression strain, where only trace amounts were detected. Interestingly, a more rapid colonization of the seed tissue occurred when mtfA was overexpressed, coinciding with an increase in lipase activity and faster maceration of the oily part of the seed.
Shen, Qingshan; Zhou, Wei; Li, Hongbo; Hu, Liangbin; Mo, Haizhen
Aspergillus flavus is a well-known pathogenic fungus for both crops and human beings. The acquisition of resistance to azoles by A. flavus is leading to more failures occurring in the prevention of infection by A. flavus. In this study, we found that thymol, one of the major chemical constituents of the essential oil of Monarda punctate, had efficient fungicidal activity against A. flavus and led to sporular lysis. Further studies indicated that thymol treatment induced the generation of both ROS and NO in spores, whereas NO accumulation was far later than ROS accumulation in response to thymol. By blocking ROS production with the inhibitors of NADPH oxidase, NO generation was also significantly inhibited in the presence of thymol, which indicated that ROS induced NO generation in A. flavus in response to thymol treatment. Moreover, the removal of either ROS or NO attenuated lysis and death of spores exposed to thymol. The addition of SNP (exogenous NO donor) eliminated the protective effects of the inhibitors of NADPH oxidase on thymol-induced lysis and death of spores. Taken together, it could be concluded that ROS is involved in spore death induced by thymol via the induction of NO. PMID:27196096
Accinelli, Cesare; Abbas, Hamed K; Vicari, Alberto; Shier, W Thomas
Applying non-aflatoxin-producing Aspergillus flavus isolates to the soil has been shown to be effective in reducing aflatoxin levels in harvested crops, including peanuts, cotton and corn. The aim of this study was to evaluate the possibility of controlling aflatoxin contamination using a novel sprayable formulation consisting of a partially gelatinized starch-based bioplastic dispersion embedded with spores of biocontrol A. flavus strains, which is applied to the leaf surfaces of corn plants. The formulation was shown to be adherent, resulting in colonization of leaf surfaces with the biocontrol strain of A. flavus, and to reduce aflatoxin contamination of harvested kernels by up to 80% in Northern Italy and by up to 89% in the Mississippi Delta. The percentage of aflatoxin-producing isolates in the soil reservoir under leaf-treated corn was not significantly changed, even when the soil was amended with additional A. flavus as a model of changes to the soil reservoir that occur in no-till agriculture. This study indicated that it is not necessary to treat the soil reservoir in order to achieve effective biocontrol of aflatoxin contamination in kernel corn. Spraying this novel bioplastic-based formulation to leaves can be an effective alternative in the biocontrol of A. flavus in corn. © 2015 Society of Chemical Industry. © 2015 Society of Chemical Industry.
Akhund, Shaista; Akram, Abida; Hanif, Nafeesa Qudsia; Qureshi, Rahmatullah; Naz, Farah; Nayyar, Brian Gagosh
Various cultivars of red chilli were collected from a small town named Kunri, located in the province Sindh, Pakistan. This town is a hub of red chilli production in Asia. A total of 69 samples belonging to 6 cultivars were obtained and analysed for the occurrence of aflatoxins and Aspergillus flavus, to explore the potential of resistant and susceptible germplasm. Aflatoxins were detected by thin layer chromatography (TLC) and high performance liquid chromatography (HPLC), while A. flavus was isolated and identified using agar plate, blotter paper, deep freezing and dilution techniques. Molecular characterization using internal transcribed spacer (ITS) 1/4 and A. flavus specific FL1-F/R primers confirmed the identity of A. flavus. The data revealed that 67 and 75% samples contaminated with aflatoxin B1 (AFB1) and with A. flavus, respectively. A highly susceptible chilli cultivar was 'Nagina', showing 78.8% frequency of total aflatoxins (1.2-600 μg/kg) and a mean of 87.7 μg/kg for AFB1 and 121.9 μg/kg for total aflatoxins. A. flavus was detected with 93% frequency and 2.14 × 10(4) colony forming units. In contrast, cultivars 'Kunri' and 'Drooping Type' were found to be resistant, with low levels of aflatoxins and fungal counts. The study was conducted for the first time to explore two potential cultivars that were less susceptible towards A. flavus and aflatoxin contamination. These cultivars could be preferably cultivated and thereby boost Pakistan's chilli production.
Shu, Xiaomei; Livingston, David P; Franks, Robert G; Boston, Rebecca S; Woloshuk, Charles P; Payne, Gary A
Aspergillus flavus and Fusarium verticillioides are fungal pathogens that colonize maize kernels and produce the harmful mycotoxins aflatoxin and fumonisin, respectively. Management practice based on potential host resistance to reduce contamination by these mycotoxins has proven difficult, resulting in the need for a better understanding of the infection process by these fungi and the response of maize seeds to infection. In this study, we followed the colonization of seeds by histological methods and the transcriptional changes of two maize defence-related genes in specific seed tissues by RNA in situ hybridization. Maize kernels were inoculated with either A. flavus or F. verticillioides 21-22 days after pollination, and harvested at 4, 12, 24, 48, 72, 96 and 120 h post-inoculation. The fungi colonized all tissues of maize seed, but differed in their interactions with aleurone and germ tissues. RNA in situ hybridization showed the induction of the maize pathogenesis-related protein, maize seed (PRms) gene in the aleurone and scutellum on infection by either fungus. Transcripts of the maize sucrose synthase-encoding gene, shrunken-1 (Sh1), were observed in the embryo of non-infected kernels, but were induced on infection by each fungus in the aleurone and scutellum. By comparing histological and RNA in situ hybridization results from adjacent serial sections, we found that the transcripts of these two genes accumulated in tissue prior to the arrival of the advancing pathogens in the seeds. A knowledge of the patterns of colonization and tissue-specific gene expression in response to these fungi will be helpful in the development of resistance. © 2014 BSPP AND JOHN WILEY & SONS LTD.
Medina, Angel; Gilbert, Matthew K; Mack, Brian M; OBrian, Gregory R; Rodríguez, Alicia; Bhatnagar, Deepak; Payne, Gary; Magan, Naresh
Effects of Aspergillus flavus colonization of maize kernels under different water activities (aw; 0.99 and 0.91) and temperatures (30, 37°C) on (a) aflatoxin B1 (AFB1) production and (b) the transcriptome using RNAseq were examined. There was no significant difference (p=0.05) in AFB1 production at 30 and 37°C and 0.99 aw. However, there was a significant (p=0.05) increase in AFB1 at 0.91 aw at 37°C when compared with 30°C/0.99 aw. Environmental stress effects using gene ontology enrichment analysis of the RNA-seq results for increasing temperature at 0.99 and 0.91 aw showed differential expression of 2224 and 481 genes, respectively. With decreasing water availability, 4307 were affected at 30°C and 702 genes at 37°C. Increasing temperature from 30 to 37°C at both aw levels resulted in 12 biological processes being upregulated and 9 significantly downregulated. Decreasing aw at both temperatures resulted in 22 biological processes significantly upregulated and 25 downregulated. The interacting environmental factors influenced functioning of the secondary metabolite gene clusters for aflatoxins and cyclopiazonic acid (CPA). An elevated number of genes were co-regulated by both aw and temperature. An interaction effect for 4 of the 25 AFB1 genes, including regulatory and transcription activators occurred. For CPA, all 5 biosynthetic genes were affected by aw stress, regardless of temperature. The molecular regulation of A. flavus in maize is discussed. Copyright © 2017 Elsevier B.V. All rights reserved.
El Khoury, Rhoda; Caceres, Isaura; Puel, Olivier; Bailly, Sylviane; Atoui, Ali; Oswald, Isabelle P.; El Khoury, André; Bailly, Jean-Denis
Of all the food-contaminating mycotoxins, aflatoxins, and most notably aflatoxin B1 (AFB1), are found to be the most toxic and economically costly. Green farming is striving to replace fungicides and develop natural preventive strategies to minimize crop contamination by these toxic fungal metabolites. In this study, we demonstrated that an aqueous extract of the medicinal plant Micromeria graeca—known as hyssop—completely inhibits aflatoxin production by Aspergillus flavus without reducing fungal growth. The molecular inhibitory mechanism was explored by analyzing the expression of 61 genes, including 27 aflatoxin biosynthesis cluster genes and 34 secondary metabolism regulatory genes. This analysis revealed a three-fold down-regulation of aflR and aflS encoding the two internal cluster co-activators, resulting in a drastic repression of all aflatoxin biosynthesis genes. Hyssop also targeted fifteen regulatory genes, including veA and mtfA, two major global-regulating transcription factors. The effect of this extract is also linked to a transcriptomic variation of several genes required for the response to oxidative stress such as msnA, srrA, catA, cat2, sod1, mnsod, and stuA. In conclusion, hyssop inhibits AFB1 synthesis at the transcriptomic level. This aqueous extract is a promising natural-based solution to control AFB1 contamination. PMID:28257049
Hu, Yichen; Zhang, Jinming; Kong, Weijun; Zhao, Gang; Yang, Meihua
The antifungal activity and potential mechanisms in vitro as well as anti-aflatoxigenic efficiency in vivo of natural essential oil (EO) derived from turmeric (Curcuma longa L.) against Aspergillus flavus was intensively investigated. Based on the previous chemical characterization of turmeric EO by gas chromatography-mass spectrometry, the substantially antifungal activities of turmeric EO on the mycelial growth, spore germination and aflatoxin production were observed in a dose-dependent manner. Furthermore, these antifungal effects were related to the disruption of fungal cell endomembrane system including the plasma membrane and mitochondria, specifically i.e. the inhibition of ergosterol synthesis, mitochondrial ATPase, malate dehydrogenase, and succinate dehydrogenase activities. Moreover, the down-regulation profiles of turmeric EO on the relative expression of mycotoxin genes in aflatoxin biosynthetic pathway revealed its anti-aflatoxigenic mechanism. Finally, the suppression effect of fungal contamination in maize indicated that turmeric EO has potential as an eco-friendly antifungal agent. Copyright © 2016 Elsevier Ltd. All rights reserved.
Misidentification of Aspergillus nomius and Aspergillus tamarii as Aspergillus flavus: characterization by internal transcribed spacer, β-Tubulin, and calmodulin gene sequencing, metabolic fingerprinting, and matrix-assisted laser desorption ionization-time of flight mass spectrometry.
Tam, Emily W T; Chen, Jonathan H K; Lau, Eunice C L; Ngan, Antonio H Y; Fung, Kitty S C; Lee, Kim-Chung; Lam, Ching-Wan; Yuen, Kwok-Yung; Lau, Susanna K P; Woo, Patrick C Y
Aspergillus nomius and Aspergillus tamarii are Aspergillus species that phenotypically resemble Aspergillus flavus. In the last decade, a number of case reports have identified A. nomius and A. tamarii as causes of human infections. In this study, using an internal transcribed spacer, β-tubulin, and calmodulin gene sequencing, only 8 of 11 clinical isolates reported as A. flavus in our clinical microbiology laboratory by phenotypic methods were identified as A. flavus. The other three isolates were A. nomius (n = 2) or A. tamarii (n = 1). The results corresponded with those of metabolic fingerprinting, in which the A. flavus, A. nomius, and A. tamarii strains were separated into three clusters based on ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC MS) analysis. The first two patients with A. nomius infections had invasive aspergillosis and chronic cavitary and fibrosing pulmonary and pleural aspergillosis, respectively, whereas the third patient had A. tamarii colonization of the airway. Identification of the 11 clinical isolates and three reference strains by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) showed that only six of the nine strains of A. flavus were identified correctly. None of the strains of A. nomius and A. tamarii was correctly identified. β-Tubulin or the calmodulin gene should be the gene target of choice for identifying A. flavus, A. nomius, and A. tamarii. To improve the usefulness of MALDI-TOF MS, the number of strains for each species in MALDI-TOF MS databases should be expanded to cover intraspecies variability.
Atehnkeng, J; Ojiambo, P S; Ikotun, T; Sikora, R A; Cotty, P J; Bandyopadhyay, R
Aflatoxin contamination resulting from maize infection by Aspergillus flavus is both an economic and a public health concern. Therefore, strategies for controlling aflatoxin contamination in maize are being investigated. The abilities of eleven naturally occurring atoxigenic isolates in Nigeria to reduce aflatoxin contamination in maize were evaluated in grain competition experiments and in field studies during the 2005 and 2006 growing seasons. Treatments consisted of inoculation of either grains in vials or ears at mid-silking stage in field plots, with the toxigenic isolate (La3228) or atoxigenic isolate alone and co-inoculation of each atoxigenic isolate and La3328. Aflatoxin B(1) + B(2) concentrations were significantly (p < 0.05) lower in the co-inoculation treatments compared with the treatment in which the aflatoxin-producing isolate La3228 was inoculated alone. Relative levels of aflatoxin B(1) + B(2) reduction ranged from 70.1% to 99.9%. Among the atoxigenics, two isolates from Lafia, La3279 and La3303, were most effective at reducing aflatoxin B(1) + B(2) concentrations in both laboratory and field trials. These two isolates have potential value as agents for the biocontrol of aflatoxin contamination in maize. Because these isolates are endemic to West Africa, they are both more likely than introduced isolates to be well adapted to West African environments and to meet regulatory concerns over their use throughout that region.
Hawkins, Leigh K.; Mylroie, J. Erik; Oliveira, Dafne A.; Smith, J. Spencer; Ozkan, Seval; Windham, Gary L.; Williams, W. Paul; Warburton, Marilyn L.
Maize (Zea mays L.) is a crop of global importance, but prone to contamination by aflatoxins produced by fungi in the genus Aspergillus. The development of resistant germplasm and the identification of genes contributing to resistance would aid in the reduction of the problem with a minimal need for intervention by farmers. Chitinolytic enzymes respond to attack by potential pathogens and have been demonstrated to increase insect and fungal resistance in plants. Here, all chitinase genes in the maize genome were characterized via sequence diversity and expression patterns. Recent evolution within this gene family was noted. Markers from within each gene were developed and used to map the phenotypic effect on resistance of each gene in up to four QTL mapping populations and one association panel. Seven chitinase genes were identified that had alleles associated with increased resistance to aflatoxin accumulation and A. flavus infection in field grown maize. The chitinase in bin 1.05 identified a new and highly significant QTL, while chitinase genes in bins 2.04 and 5.03 fell directly beneath the peaks of previously published QTL. The expression patterns of these genes corroborate possible grain resistance mechanisms. Markers from within the gene sequences or very closely linked to them are presented to aid in the use of marker assisted selection to improve this trait. PMID:26090679
Song, Hui; Wang, Pengfei; Li, Changsheng; Han, Suoyi; Zhao, Chuanzhi; Xia, Han; Bi, Yuping; Guo, Baozhu; Zhang, Xinyou
Studies have demonstrated that nucleotide-binding site–leucine-rich repeat (NBS–LRR) genes respond to pathogen attack in plants. Characterization of NBS–LRR genes in peanut is not well documented. The newly released whole genome sequences of Arachis duranensis and Arachis ipaënsis have allowed a global analysis of this important gene family in peanut to be conducted. In this study, we identified 393 (AdNBS) and 437 (AiNBS) NBS–LRR genes from A. duranensis and A. ipaënsis, respectively, using bioinformatics approaches. Full-length sequences of 278 AdNBS and 303 AiNBS were identified. Fifty-one orthologous, four AdNBS paralogous, and six AiNBS paralogous gene pairs were predicted. All paralogous gene pairs were located in the same chromosomes, indicating that tandem duplication was the most likely mechanism forming these paralogs. The paralogs mainly underwent purifying selection, but most LRR 8 domains underwent positive selection. More gene clusters were found in A. ipaënsis than in A. duranensis, possibly owing to tandem duplication events occurring more frequently in A. ipaënsis. The expression profile of NBS–LRR genes was different between A. duranensis and A. hypogaea after Aspergillus flavus infection. The up-regulated expression of NBS–LRR in A. duranensis was continuous, while these genes responded to the pathogen temporally in A. hypogaea. PMID:28158222
Horn, Bruce W; Gell, Richard M; Singh, Rakhi; Sorensen, Ronald B; Carbone, Ignazio
Aspergillus flavus colonizes agricultural commodities worldwide and contaminates them with carcinogenic aflatoxins. The high genetic diversity of A. flavus populations is largely due to sexual reproduction characterized by the formation of ascospore-bearing ascocarps embedded within sclerotia. A. flavus is heterothallic and laboratory crosses between strains of the opposite mating type produce progeny showing genetic recombination. Sclerotia formed in crops are dispersed onto the soil surface at harvest and are predominantly produced by single strains of one mating type. Less commonly, sclerotia may be fertilized during co-infection of crops with sexually compatible strains. In this study, laboratory and field experiments were performed to examine sexual reproduction in single-strain and fertilized sclerotia following exposure of sclerotia to natural fungal populations in soil. Female and male roles and mitochondrial inheritance in A. flavus were also examined through reciprocal crosses between sclerotia and conidia. Single-strain sclerotia produced ascospores on soil and progeny showed biparental inheritance that included novel alleles originating from fertilization by native soil strains. Sclerotia fertilized in the laboratory and applied to soil before ascocarp formation also produced ascospores with evidence of recombination in progeny, but only known parental alleles were detected. In reciprocal crosses, sclerotia and conidia from both strains functioned as female and male, respectively, indicating A. flavus is hermaphroditic, although the degree of fertility depended upon the parental sources of sclerotia and conidia. All progeny showed maternal inheritance of mitochondria from the sclerotia. Compared to A. flavus populations in crops, soil populations would provide a higher likelihood of exposure of sclerotia to sexually compatible strains and a more diverse source of genetic material for outcrossing.
Horn, Bruce W.; Gell, Richard M.; Singh, Rakhi; Sorensen, Ronald B.; Carbone, Ignazio
Aspergillus flavus colonizes agricultural commodities worldwide and contaminates them with carcinogenic aflatoxins. The high genetic diversity of A. flavus populations is largely due to sexual reproduction characterized by the formation of ascospore-bearing ascocarps embedded within sclerotia. A. flavus is heterothallic and laboratory crosses between strains of the opposite mating type produce progeny showing genetic recombination. Sclerotia formed in crops are dispersed onto the soil surface at harvest and are predominantly produced by single strains of one mating type. Less commonly, sclerotia may be fertilized during co-infection of crops with sexually compatible strains. In this study, laboratory and field experiments were performed to examine sexual reproduction in single-strain and fertilized sclerotia following exposure of sclerotia to natural fungal populations in soil. Female and male roles and mitochondrial inheritance in A. flavus were also examined through reciprocal crosses between sclerotia and conidia. Single-strain sclerotia produced ascospores on soil and progeny showed biparental inheritance that included novel alleles originating from fertilization by native soil strains. Sclerotia fertilized in the laboratory and applied to soil before ascocarp formation also produced ascospores with evidence of recombination in progeny, but only known parental alleles were detected. In reciprocal crosses, sclerotia and conidia from both strains functioned as female and male, respectively, indicating A. flavus is hermaphroditic, although the degree of fertility depended upon the parental sources of sclerotia and conidia. All progeny showed maternal inheritance of mitochondria from the sclerotia. Compared to A. flavus populations in crops, soil populations would provide a higher likelihood of exposure of sclerotia to sexually compatible strains and a more diverse source of genetic material for outcrossing. PMID:26731416
Al-Gabr, Hamid Mohammad; Zheng, Tianling; Yu, Xin
Aquatic fungi are common in various aqueous environments and play potentially crucial roles in nutrient and carbon cycling as well as interacting with other organisms. Species of Aspergillus are the most common fungi that occur in water. The present study was undertaken to elucidate the efficacy of two coagulants, aluminum sulfate and ferric chloride, used at different concentrations to treat drinking water, in removing Aspergillus flavus, as well as testing three different filtration media: sand, activated carbon, and ceramic granules, for their removal of fungi from water. The results revealed that both coagulants were effective in removing fungi and decreasing the turbidity of drinking water, and turbidity decreased with increasing coagulant concentration. Also, at the highest concentration of the coagulants, A. flavus was decreased by 99.6% in the treated water. Among ceramic granules, activated carbon, and sand used as media for water filtration, the sand and activated carbon filters were more effective in removing A. flavus than ceramic granules while simultaneously decreasing the turbidity levels in the test water samples. Post-treatment total organic carbon (TOC) and total nitrogen (TN) concentrations in the experimental water did not decrease; on the contrary, TN concentrations increased with the increasing dosage of coagulants. The filtration process had no effect in reducing TOC and TN in tested water.
Verheecke, C; Liboz, T; Darriet, M; Sabaou, N; Mathieu, F
This work aimed to study the interaction between Actinomycetal isolates and Aspergillus flavus to promote mutual antagonism in contact. Thirty-seven soilborn Streptomyces spp. isolates were chosen as potential candidates. After a 10-day in vitro co-incubation period, 27 isolates respond to the criteria, that is, mutual antagonism in contact. Further aflatoxins B1 and B2 analysis revealed that those 27 isolates reduced aflatoxin B1 residual concentration from 38·6 to 4·4%, depending on the isolate. We selected 12 isolates and tested their capacity to reduce AFB1 in pure culture to start identifying the mechanisms involved in its reduction. AFB1 was reduced by eight isolates. The remaining AFB1 concentration varied between 82·2 and 15·6%. These findings led us to suggest that these eight isolates could be used as biocontrol agents against AFB1 and B2 with low risk of impacting the natural microbial equilibrium. Interaction between Aspergillus flavus and Actinomycetes isolates was conducted in vitro. Actinomycetes isolates having a mutual antagonism in contact with A. flavus were chosen for further aflatoxins production study. This is a new approach based to develop biocontrol against aflatoxins accumulation in maize while respecting natural microbial equilibrium. © 2014 The Society for Applied Microbiology.
Khodavaisy, S; Badali, H; Hashemi, S J; Aala, F; Nazeri, M; Nouripour-Sisakht, S; Sorkherizi, M S; Amirizad, K; Aslani, N; Rezaie, S
Aspergillus flavus is the second leading cause of invasive and non-invasive aspergillosis, as well as the most common cause of fungal sinusitis, cutaneous infections, and endophthalmitis in tropical countries. Since resistance to antifungal agents has been observed in patients, susceptibility testing is helpful in defining the activity spectrum of antifungals and determining the appropriate drug for treatment. A collection of 199 clinical and environmental strains of Aspergillus flavus consisted of clinical (n=171) and environmental (n=28) were verified by DNA sequencing of the partial b-tubulin gene. MICs of amphotericin B, itraconazole, voriconazole, posaconazole, and MEC of caspofungin were determined in accordance with the Clinical and Laboratory Standards Institute M38-A2 document. Caspofungin, followed by posaconazole, exhibited the lowest minimum inhibitory concentrations (MIC). All isolates had caspofungin MEC90 (0.063μg/ml) lower than the epidemiologic cutoff values, and 3.5% of the isolates had amphotericin B MIC higher than the epidemiologic cutoff values. However, their clinical effectiveness in the treatment of A. flavus infection remains to be determined.
Chen, Yen-Ting; Lin, Mei-Ju; Yang, Ching-Hui; Ko, Wen-Hsiung
A fungus capable of using vegetable tissues for multiplication in soil was isolated and identified as Aspergillus flavus based on morphological characteristics and sequence similarity of ITS and 28S. When grown in liquid medium prepared from the same vegetable tissues used in soil amendment, the isolate of A. flavus produced a substance capable of preventing disease development of black leaf spot of mustard cabbage caused by Alternaria brassicicola and inhibiting the germination of A. brassicicola conidia. The inhibitory substance was fungistatic, and was very stable under high temperature and high or low pH value. It was soluble in ethanol or methanol, moderately soluble in water, and insoluble in acetone, ethyl acetate or ether. The inhibitor is not a protein and has no charges on its molecule. This is the first discovery of the production of a fungistatic substance by this deleterious fungus. Results from this study suggest the possession of a strong competitive saprophytic ability by A. flavus, which in turn may explain the widespread occurrence of this fungus in soils. Production of a fungistatic substance when A. flavus was grown in medium prepared from vegetable tissues suggests the importance of antibiotic production in its competitive saprophytic colonization of organic matters in soils.
Hill, R A; Blankenship, P D; Cole, R J; Sanders, T H
Four soil temperature and moisture treatment regimens were imposed on Florunner peanuts 94 days after planting in experimental plots in 1980. At harvest (145 days after planting), the incidence of the Aspergillus flavus group and the aflatoxin concentration were greatest in damaged kernels. Extensive colonization of sound mature kernels (SMK) by the A. flavus group occurred with the drought stress treatment (56% kernels colonized); colonization was less in the irrigated plot (7%) and the drought stress plot with cooled soil (11%) and was intermediate in the irrigated plot with heated soil (26%). Aflatoxin was virtually absent from SMK with the last three treatments, but it was found at an average concentration of 244 ppb (ng/g) in drought-stressed SMK. Colonization of SMK by the A. flavus group and aflatoxin production were greater with hot dry conditions. Neither elevated temperature alone nor drought stress alone caused aflatoxin contamination in SMK. When the ratio of SMK colonized by A. flavus compared with A. niger was greater than 19:1, there was aflatoxin contamination, but there was none if this ratio was less than 9:1. Irrigation caused a higher incidence of A. niger than drought did. This may have prevented the aflatoxin contamination of undamaged peanuts. PMID:6402980
Gallo, Antonia; Solfrizzo, Michele; Epifani, Filomena; Panzarini, Giuseppe; Perrone, Giancarlo
Almonds are among the commodities at risk of aflatoxin contamination by Aspergillus flavus. Temperature and water activity are the two key determinants in pre and post-harvest environments influencing both the rate of fungal spoilage and aflatoxin production. Varying the combination of these parameters can completely inhibit or fully activate the biosynthesis of aflatoxin, so it is fundamental to know which combinations can control or be conducive to aflatoxin contamination. Little information is available about the influence of these parameters on aflatoxin production on almonds. The objective of this study was to determine the influence of different combinations of temperature (20 °C, 28 °C, and 37 °C) and water activity (0.90, 0.93, 0.96, 0.99 aw) on growth, aflatoxin B1 (AFB1) production and expression of the two regulatory genes, aflR and aflS, and two structural genes, aflD and aflO, of the aflatoxin biosynthetic cluster in A. flavus grown on an almond medium solidified with agar. Maximum accumulation of fungal biomass and AFB1 production was obtained at 28 °C and 0.96 aw; no fungal growth and AFB1 production were observed at 20 °C at the driest tested conditions (0.90 and 0.93 aw). At 20° and 37 °C AFB1 production was 70-90% lower or completely suppressed, depending on aw. Reverse transcriptase quantitative PCR showed that the two regulatory genes (aflR and aflS) were highly expressed at maximum (28 °C) and minimum (20 °C and 37 °C) AFB1 production. Conversely the two structural genes (aflD and aflO) were highly expressed only at maximum AFB1 production (28 °C and 0.96-0.99 aw). It seems that temperature acts as a key factor influencing aflatoxin production which is strictly correlated to the induction of expression of structural biosynthesis genes (aflD and aflO), but not to that of aflatoxin regulatory genes (aflR and aflS), whose functional products are most likely subordinated to other regulatory processes acting at post-translational level
Ehrlich, Kenneth C.; Chang, Perng-Kuang; Scharfenstein, Lester L.; Cary, Jeffrey W.; Crawford, Jason M.; Townsend, Craig A.
Biosynthesis of the highly toxic and carcinogenic aflatoxins in select Aspergillus species from the common intermediate O-methylsterigmatocystin (OMST) has been postulated to require only the cytochrome P450 monooxygenase, OrdA (AflQ). We now provide evidence that the aryl alcohol dehydrogenase NorA (AflE) encoded by the aflatoxin biosynthetic gene cluster in A. flavus affects the accumulation of aflatoxins in the final steps of aflatoxin biosynthesis. Mutants with inactive norA produced reduced quantities of aflatoxin B1 (AFB1), but elevated quantities of a new metabolite, deoxyAFB1. To explain this result, we suggest that, in the absence of NorA, the AFB1 reduction product, aflatoxicol, is produced and is readily dehydrated to deoxyAFB1 in the acidic medium, enabling us to observe this otherwise minor toxin produced in wild-type A. flavus. PMID:20158523
Kelley, Rowena Y; Williams, W Paul; Mylroie, J Erik; Boykin, Deborah L; Harper, Jonathan W; Windham, Gary L; Ankala, Arunkanth; Shan, Xueyan
Aspergillus flavus infection and aflatoxin contamination of maize pose negative impacts in agriculture and health. Commercial maize hybrids are generally susceptible to this fungus. Significant levels of host plant resistance have been observed in certain maize inbred lines. This study was conducted to identify maize genes associated with host plant resistance or susceptibility to A. flavus infection and aflatoxin accumulation. Genome wide gene expression levels with or without A. flavus inoculation were compared in two resistant maize inbred lines (Mp313E and Mp04:86) in contrast to two susceptible maize inbred lines (Va35 and B73) by microarray analysis. Principal component analysis (PCA) was used to find genes contributing to the larger variances associated with the resistant or susceptible maize inbred lines. The significance levels of gene expression were determined by using SAS and LIMMA programs. Fifty candidate genes were selected and further investigated by quantitative RT-PCR (qRT-PCR) in a time-course study on Mp313E and Va35. Sixteen of the candidate genes were found to be highly expressed in Mp313E and fifteen in Va35. Out of the 31 highly expressed genes, eight were mapped to seven previously identified quantitative trait locus (QTL) regions. A gene encoding glycine-rich RNA binding protein 2 was found to be associated with the host hypersensitivity and susceptibility in Va35. A nuclear pore complex protein YUP85-like gene was found to be involved in the host resistance in Mp313E. Maize genes associated with host plant resistance or susceptibility were identified by a combination of microarray analysis, qRT-PCR analysis, and QTL mapping methods. Our findings suggest that multiple mechanisms are involved in maize host plant defense systems in response to Aspergillus flavus infection and aflatoxin accumulation. These findings will be important in identification of DNA markers for breeding maize lines resistant to aflatoxin accumulation.
Bamba, Rozy; Sumbali, Geeta
During hot and humid seasons, extensive rot of sour lime was observed to be caused by Aspergillus flavus. In view of this, investigations were undertaken to obtain data on the production of various toxins by A. flavus during post harvest pathogenesis of sour lime. Sixty percent of the pathogenic A. flavus isolates were detected to be aflatoxin B(1) producers in sour lime tissue. It was also noted that thirty three percent of aflatoxigenic A. flavus isolates had the potential to coproduce cyclopiazonic acid (CPA). Such aflatoxigenic isolates produced quantitatively more CPA (ranging from 250.0 to 2501.3 microg/kg) than aflatoxin B(1) (ranging from 141.3 to 811.7 microg/kg) in the affected sour lime. This study demonstrates for the first time that sour lime are a favourable substrate for aflatoxin B(1) and cyclopiazonic acid production by A. flavus isolates. This is of great concern to the health of consumers.
Zhuang, Zhenhong; Lohmar, Jessica M.; Satterlee, Timothy; Cary, Jeffrey W.; Calvo, Ana M.
Aspergillus flavus produces a variety of toxic secondary metabolites; among them, the aflatoxins (AFs) are the most well known. These compounds are highly mutagenic and carcinogenic, particularly AFB1. A. flavus is capable of colonizing a number of economically-important crops, such as corn, cotton, peanut and tree nuts, and contaminating them with AFs. Molecular genetic studies in A. flavus could identify novel gene targets for use in strategies to reduce AF contamination and its adverse impact on food and feed supplies worldwide. In the current study, we investigated the role of the master transcription factor gene mtfA in A. flavus. Our results revealed that forced overexpression of mtfA results in a drastic decrease or elimination of several secondary metabolites, among them AFB1. The reduction in AFB1 was accompanied by a decrease in aflR expression. Furthermore, mtfA also regulates development; conidiation was influenced differently by this gene depending on the type of colonized substrate. In addition to its effect on conidiation, mtfA is necessary for the normal maturation of sclerotia. Importantly, mtfA positively affects the pathogenicity of A. flavus when colonizing peanut seeds. AF production in colonized seeds was decreased in the deletion mtfA strain and particularly in the overexpression strain, where only trace amounts were detected. Interestingly, a more rapid colonization of the seed tissue occurred when mtfA was overexpressed, coinciding with an increase in lipase activity and faster maceration of the oily part of the seed. PMID:26805883
Das, Arijit; Bhattacharya, Sourav; Palaniswamy, Muthusamy; Angayarkanni, Jayaraman
Aflatoxin B1 (AFB1) produced by Aspergillus flavus is known to have carcinogenic and teratogenic effects on animal health. Accidental feeding of AFB1-contaminated rice straw may be detrimental to dairy cattle. White-rot basidiomycetous fungus Pleurotus ostreatus can grow on different agronomic wastes by synthesizing different ligninolytic enzymes. These extracellular enzymes are capable of degrading many environmentally hazardous compounds including AFB1. The present study examines the ability of different strains of P. ostreatus to degrade AFB1 in contaminated rice straw. Different strains of A. flavus were inoculated on rice straw for AFB1 production. The moldy straw was then subjected to co-cultivation by different strains of P. ostreatus. The extent of AFB1 degradation was determined by high performance liquid chromatography. Results indicated the presence of AFB1 in the moldy straw samples at levels of 27.95 ± 0.23 and 21.26 ± 0.55 µg/g of dry substrate for A. flavus MTCC 2798 and A. flavus GHBF09, respectively. Co-cultivation of P. ostreatus strains on AFB1-contaminated rice straw revealed their ability to rapidly colonize the substrate by profuse hyphal ramification. Highest degradation of AFB1 (89.41 %) was recorded in the straw containing co-cultures of A. flavus MTCC 2798 and P. ostreatus GHBBF10. Natural isolate P. ostreatus GHBBF10 demonstrated higher AFB1-degradation potential than P.ostreatus MTCC 142. This basidiomycete strain can be further exploited to effectively degrade moderate concentrations of AFB1 in contaminated moldy rice straw.
Impoolsup, Attawut; Bhumiratana, Amaret; Flegel, Timothy W.
Two different extracellular proteases, protease I (P-I), an alkaline protease, and protease II (P-II) a neutral protease, from Aspergillus flavus var. columnaris were partially purified by using (NH4)2SO4 precipitation, diethylaminoethyl-Sephadex A-50 chromatography, carboxymethylcellulose CM-52 chromatography, and Sephadex G-100 gel filtration. The degree of purity was followed using polyacrylamide gel electrophoresis. The activity of P-I was completely inhibited by 0.1 mM phenylmethylsulfonyl fluoride, and that of P-II was completely inhibited by 1 mM ethylenediaminetetraacetate. By using these inhibitors with extracts of wheat bran koji, the proportions of total activity that could be assigned to P-I and P-II were 80 and 20%, respectively. This compared favorably with activities estimated by using polyacrylamide gel electrophoresis slices (82 and 18%, respectively). Extracts from factory-run soybean koji gave comparable results. Both enzymes demonstrated maximum activity at 50 to 55°C and only small changes in activity between pH 6 and 11. For P-I, activity was somewhat higher from pH 8.0 to 11.0, whereas for P-II it was somewhat higher from pH 6 to 9. In the presence of 18% NaCl, the activities of both P-I and P-II dropped by approximately 90 and 85%, respectively. P-I was inferred to possess aminopeptidase activity since it could hydrolyze l-leucyl-p-nitroanilide hydrochloride. P-II was devoid of such activity. The ramifications of the results for factory-produced soy sauce koji are discussed. Images PMID:16345858
Hua, Sui Sheng T; McAlpin, Cesaria E; Chang, Perng-Kuang; Sarreal, Siov Bouy L
Pistachio is a popular snack food. Aflatoxin contamination of pistachio nuts is a serious problem for many producing countries. The development of biological control methods based on ecological parameters is an environmentally friendly approach. Thirty-eight Aspergillus flavus isolates collected from a pistachio orchard in California (CA) were analyzed for production of aflatoxin (AF), cyclopiazonic acid (CPA), vegetative compatibility groups (VCGs), and mating types. All aflatoxigenic isolates produced both AFB1 and CPA. The most toxigenic one was CA28 which produced 164 μg AFB1 per 5 ml PDA fungal culture and small sclerotia (S strain, sclertoium size less than 400 μm). The other aflatoxigenic strains produce AFB1 ranging from 1.2 μg to 80 μg per 5 ml fungal culture. Twenty-one percent of the CA isolates produced AFB1, 84% produced CPA and half formed sclerotia on at least one of three tested media. The 38 CA isolates formed 26 VCGs, 6 of which had two or more isolates and 20 contained single isolates. The S strain isolates belong to 4 different VCGs. Genomic profiling by a retrotransposon DNA probe revealed fingerprint patterns that were highly polymorphic. The predicted VCGs (Pred-VCGs) based on a similarity coefficient >80% matched the VCGs of multiple isolates determined by complementation. All isolates within a VCG had the same mating-type gene of either MAT1-1 or MAT1-2. Uncorrected and VCG-corrected MAT1-1 and MAT1-2 among the isolates were equally distributed.
Aloui, Hajer; Khwaldia, Khaoula; Licciardello, Fabio; Mazzaglia, Agata; Muratore, Giuseppe; Hamdi, Moktar; Restuccia, Cristina
This study reports the efficacy of the combined application of chitosan (CH) and Locust Bean Gum (LBG) in combination with different citrus essential oils (EOs) to inhibit Aspergillus flavus in vitro and on artificially infected dates for a storage period of 12 days. The effect of these treatments on the fruits' sensory characteristics was evaluated to verify the complete absence of off-odours and off-flavours. Bergamot EO was the most effective in reducing mycelial growth, followed by bitter orange EO. Both bergamot and bitter orange oils significantly reduced conidial germination and a complete inhibition was obtained at concentrations higher than 2%. The mixtures based on CH-2% (v/v) bergamot EO or CH-2% (v/v) bitter orange EO proved to be the most effective coatings to reduce conidial germination resulting in an 87-90% inhibition compared with the control. In fruit decay assays coatings based on CH incorporating citrus oils were able to reduce fungal decay in the range of 52-62% at day 12. The study results and the complete absence of off-flavours and off-odours demonstrate the potential of CH coatings carrying citrus EOs at sub-inhibitory concentrations to control postharvest growth of A. flavus in dates.
Hruska, Zuzana; Rajasekaran, Kanniah; Yao, Haibo; Kincaid, Russell; Darlington, Dawn; Brown, Robert L; Bhatnagar, Deepak; Cleveland, Thomas E
A currently utilized pre-harvest biocontrol method involves field inoculations with non-aflatoxigenic Aspergillus flavus strains, a tactic shown to strategically suppress native aflatoxin-producing strains and effectively decrease aflatoxin contamination in corn. The present in situ study focuses on tracking the invasion and colonization of an aflatoxigenic A. flavus strain (AF70), labeled with green fluorescent protein (GFP), in the presence of a non-aflatoxigenic A. flavus biocontrol strain (AF36), to better understand the competitive interaction between these two strains in seed tissue of corn (Zea mays). Corn kernels that had been co-inoculated with GFP-labeled AF70 and wild-type AF36 were cross-sectioned and observed under UV and blue light to determine the outcome of competition between these strains. After imaging, all kernels were analyzed for aflatoxin levels. There appeared to be a population difference between the co-inoculated AF70-GFP+AF36 and the individual AF70-GFP tests, both visually and with pixel count analysis. The GFP allowed us to observe that AF70-GFP inside the kernels was suppressed up to 82% when co-inoculated with AF36 indicating that AF36 inhibited progression of AF70-GFP. This was in agreement with images taken of whole kernels where AF36 exhibited a more robust external growth compared to AF70-GFP. The suppressed growth of AF70-GFP was reflected in a corresponding (upto 73%) suppression in aflatoxin levels. Our results indicate that the decrease in aflatoxin production correlated with population depression of the aflatoxigenic fungus by the biocontrol strain supporting the theory of competitive exclusion through robust propagation and fast colonization by the non-aflatoxigenic fungus.
Pildain, M Belén; Vaamonde, Graciela; Cabral, Daniel
Isolates of Aspergillus flavus obtained from a new growing peanut region in Argentina (Formosa province) were examined for aflatoxin types B and G and cyclopiazonic acid (CPA) production. Sclerotia diameters and the number of sclerotia produced per square centimetre were also determined for each isolate. They were tested by vegetative compatibility group analysis to investigate their genetic relatedness and correlate the results with vegetative compatibility groups previously described from the major peanut-growing area (Córdoba province) in our country. Two isolates were considered atypical because they simultaneously produce aflatoxins B and G and CPA. A. flavus population from Formosa province was very diverse genetically. Vegetative compatibility groups (VCGs) formed by typical isolations of A. flavus were different among agroecological sites. Formosa isolates could not be grouped to any of the Córdoba VCGs, while that one of the VCGs that contain atypical isolates included strains from the two geographical regions. Each VCG included isolates of the same mycotoxin and sclerotia production pattern. The two regions analysed have different climatic conditions, soil type, crop sequence history and also are in different latitude. These parameters may reflect different geographic adaptation between isolates from both sites.
Savi, Geovana D.; Cardoso, Willian A.; Furtado, Bianca G.; Bortolotto, Tiago; Da Agostin, Luciana O. V.; Nones, Janaína; Torres Zanoni, Elton; Montedo, Oscar R. K.; Angioletto, Elidio
Zeolites are microporous crystalline hydrated aluminosilicates with absorbent and catalytic properties. This material can be used in many applications in stored-pest management such as: pesticide and fertilizer carriers, animal feed additives, mycotoxin binders and food packaging materials. Herein, four 4A zeolite forms were prepared by ion-exchange and their antifungal effect against Aspergillus flavus was highlighted. Additionally, the antimycotoxin activity and the aflatoxin B1 (AFB1) adsorption capacity of these zeolites as well as their toxic effects on Artemia sp. were investigated. The ion-exchanged zeolites with Li+ and Cu2+ showed the best antifungal activity against A. flavus, including effects on conidia germination and hyphae morphological alterations. Regarding to antimycotoxin activity, all zeolite samples efficiently inhibited the AFB1 production by A. flavus. However, the ion-exchanged zeolites exhibited better results than the 4A zeolite. On the other hand, the AFB1 adsorption capacity was only observed by the 4A zeolite and zeolite-Li+. Lastly, our data showed that all zeolites samples used at effective concentrations for antifungal and antimycotoxin assays (2 mg ml-1) showed no toxic effects towards Artemia sp. Results suggest that some these ion-exchanged zeolites have great potential as an effective fungicide and antimycotoxin agent for agricultural and food safety applications.
Georgianna, D. Ryan; Fedorova, Natalie D.; Burroughs, James L.; Dolezal, Andrea L.; Bok, J.; Horowitz-Brown, S.; Woloshuk, Charles P.; Yu, Jiujiang; Keller, Nancy P.; Payne, Gary A.
SUMMARY Species of Aspergillus produce a diverse array of secondary metabolites, and recent genomic analysis predicts that these species have the capacity to synthesize many more compounds. It has been possible to infer the presence of 55 gene clusters associated with secondary metabolism in A. flavus, however, only three metabolic pathways - aflatoxin, cyclopiazonic acid (CPA), and aflatrem - have been assigned to these clusters. To gain insight into the regulation of, and infer ecological significance for the 55 secondary metabolite gene clusters predicted in A. flavus, we examined their expression over 28 diverse conditions. Variables included culture media and temperature, fungal development, colonization of developing maize seeds, and misexpression of laeA, a global regulator of secondary metabolism. Hierarchical clustering analysis of expression profiles allowed us to categorize the gene clusters into four distinct clades. Gene clusters for the production of aflatoxins, CPA, and seven other unknown compound(s) were identified as belonging to one clade. To further explore the relationships found by gene expression analysis, aflatoxin and CPA production were quantified under five different cell culture environments known to be conducive or non-conducive for aflatoxin biosynthesis and during colonization of developing maize seeds. Results from these studies showed that secondary metabolism gene clusters have distinctive gene expression profiles. Aflatoxin and CPA were found to have unique regulation but are similar enough that they would be expected to co-occur in substrates colonized with A. flavus. PMID:20447271
A keratinolytic enzyme secreted by Aspergillus flavus K-03 cultured in feather meal basal medium (FMBM) containing 2% (w/v) chicken feather was purified and characterized. Keratinolytic enzyme secretion was the maximal at day 16 of the incubation period at pH 8 and 28℃. No relationship was detected between enzyme yield and increase of fungal biomass. The fraction obtained at 80% ammonium sulfate saturation showed 2.39-fold purification and was further purified by gel filtration in Sephadex G-100 followed by ion exchange chromatography on DEAE-Sephadex A-50, yielding an active protein peak showing 11.53-fold purification. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and zymograms indicated that the purified keratinase is a monomeric enzyme with 31 kDa molecular weight. The extracellular keratinase of A. flavus was active in a board range of pH (7~10) and temperature (30℃~70℃) profiles with the optimal for keratinase activity at pH 8 and 45℃. The keratinase activity was totally inhibited by protease inhibitors such as phenylmethylsulfonyl fluoride (PMSF), iodoacetic acid, and ethylenediaminetetraacetate (EDTA) while no reduction of activity by the addition of dithiothreitol (DTT) was observed. N-terminal amino acid sequences were up to 80% homologous with the fungal subtilisins produced by Fusarium culmorum. Therefore, on the basis of these characteristics, the keratinase of A. flavus K-03 is determined to be subtilisins-like. PMID:24015101
Shcherbakova, Larisa; Statsyuk, Natalia; Mikityuk, Oleg; Nazarova, Tatyana; Dzhavakhiya, Vitaly
Background: Aflatoxin B1 (AFB1), produced by Aspergillus flavus, is one of the most life threatening food contaminants causing significant economic losses worldwide. Biological AFB1 degradation by microorganisms, or preferably microbial enzymes, is considered as one of the most promising approaches. Objectives: The current work aimed to study the AFB1-degrading metabolites, produced by Phoma glomerata PG41, sharing a natural substrate with aflatoxigenic A. flavus, and the preliminary determination of the nature of these metabolites. Materials and Methods: The AFB1-degrading potential of PG41 metabolites was determined by a quantitative high performance liquid chromatography (HPLC) of residual AFB1 after 72 hours incubation at 27ºC. The effects of pH, heat, and protease treatment on the AFB1-destroying activity of extracellular metabolites were examined. Results: The AFB1-degrading activity of protein-enriched fractions, isolated from culture liquid filtrate and cell-free extract, is associated with high-molecular-weight components, is time- and pH-dependent, thermolabile, and is significantly reduced by proteinase K treatment. The AFB1 degradation efficiency of these fractions reaches 78% and 66%, respectively. Conclusions: Phoma glomerata PG41 strain sharing natural substrate with toxigenic A. flavus secretes metabolites possessing a significant aflatoxin-degrading activity. The activity is associated mainly with a protein-enriched high-molecular-weight fraction of extracellular metabolites and appears to be of enzymatic origin. PMID:25789135
Dzhavakhiya, Vitaly G.; Voinova, Tatiana M.; Popletaeva, Sofya B.; Statsyuk, Natalia V.; Limantseva, Lyudmila A.; Shcherbakova, Larisa A.
Aflatoxins and melanins are the products of a polyketide biosynthesis. In this study, the search of potential inhibitors of the aflatoxin B1 (AFB1) biosynthesis was performed among compounds blocking the pigmentation in fungi. Four compounds—three natural (thymol, 3-hydroxybenzaldehyde, compactin) and one synthetic (fluconazole)—were examined for their ability to block the pigmentation and AFB1 production in Aspergillus flavus. All compounds inhibited the mycelium pigmentation of a fungus growing on solid medium. At the same time, thymol, fluconazole, and 3-hydroxybenzaldehyde stimulated AFB1 accumulation in culture broth of A. flavus under submerged fermentation, whereas the addition of 2.5 μg/mL of compactin resulted in a 50× reduction in AFB1 production. Moreover, compactin also suppressed the sporulation of A. flavus on solid medium. In vivo treatment of corn and wheat grain with compactin (50 μg/g of grain) reduced the level of AFB1 accumulation 14 and 15 times, respectively. Further prospects of the compactin study as potential AFB1 inhibitor are discussed. PMID:27801823
Gniadek, Agnieszka; Krzyściak, Paweł; Twarużek, Magdalena; Macura, Anna B
The basic care requirement for patients with weakened immune systems is to create the environment where the risk of mycosis is reduced to a minimum. Between 2007 and 2013 air samples were collected from various wards of a number of hospitals in Kraków, Poland, by means of the collision method using MAS-100 Iso MH Microbial Air Sampler (Merck Millipore, Germany). The air mycobiota contained several species of fungi, and almost 1/3 of it was made up of the species of the Aspergillus genus. Sixty-one strains of species other than A. fumigatus were selected for the research purposes, namely: 28 strains of A. ochraceus, 22 strains of A. niger and 11 strains of A. flavus species. Selected fungi underwent a cytotoxicity evaluation with the application of the MTT colorimetric assay (3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide). The assay assesses cell viability by means of reducing the yellow tetrazolium salt to insoluble formazan. A semi-quantitative scale for cytotoxicity grading was adopted: low cytotoxic effect (+) with half maximal inhibitory concentration (IC50) for values ranging from 31.251 cm2/ml to 7.813 cm2/ml, medium cytotoxic effect (++) for values ranging from 3.906 cm2/ml to 0.977 cm2/ml and the high one (+++) for values ranging from 0.488 cm2/ml to 0.061 cm2/ml. The absence of cytotoxicity was determined when the IC50 values was at ≥ 50. For 48 samples the analyzed fungi displayed the cytotoxic effect with A. ochraceus in 26 out of 28 cases, with 11 strains displaying the high cytotoxic effect. The lowest cytotoxicity was displayed by fungi of A. niger in 13 out of 22 cases, and the major fungi of A. flavus species were toxic (9 out of 11 cases). A half of the fungi displayed the low cytotoxic effect. On the basis of the comparison of average cytotoxicity levels it was determined that there were
Sudhakar, P; Latha, P; Sreenivasulu, Y; Reddy, B V Bhaskar; Hemalatha, T M; Balakrishna, M; Reddy, K Raja
Methyleugenol is naturally occurring substance in oils and fruits and in various foods as flavoring agent. Effect of this methyleugenol in inhibiting A. flavus colonization and aflatoxin production on peanut pods and kernels has been studied. Spray of methyleugenol (0.5%) on peanut pods and kernels checked the colonization of A. flavus and aflatoxin synthesis. This chemical can be used as both prophylactic or post infection spray on peanut pods before storage. It is the first report on the inhibition of A. flavus by methyleugenol on peanut.
Petrović, Jovana; Glamočlija, Jasmina; Stojković, Dejan S; Ćirić, Ana; Nikolić, Miloš; Bukvički, Danka; Guerzoni, Maria Elisabetta; Soković, Marina D
The volatile compounds of fruiting bodies of wild Laetiporus sulphureus (Bull.) Murrill, growing on willow trees from Serbia, were isolated and extracted using methanol, acetone and dichloromethane and investigated by GC/MS-SPME. A total of 56 components were identified in the extracts. Hydrocarbons predominated (76.90%, 77.20%, and 43.10%) in dichloromethane, acetone and methanol extracts, respectively. Fatty acids, esters and sesquiterpenes were present in amounts equal or lower than 2.00%. Ketones were represented with moderate amount with the exception of methanol extract where it reached as much as 28.90% of the total investigated compounds. Extracts were also tested for antimicrobial activity with and without the addition of food additive - potassium disulfite in vitro against eight bacterial and eight fungal species, and in situ in tomato paste against Aspergillus flavus. All the tested extracts showed good antimicrobial activity, but methanol extract with addition of E224 showed the best antimicrobial activity in vitro. In situ results indicate complete inhibition of A. flavus growth in tomato paste after 15 days of the treatment. This study is the first report on volatile composition of L. sulphureus growing wild in Serbia. We describe for the first time the application of its extract as antifungal food preservative.
Padhiar, Jigita; Das, Arijit; Bhattacharya, Sourav
The present study was aimed at optimization, production and partial purification of lipases from Pseudomonas aeruginosa, Candida albicans and Aspergillus flavus. Various nutritional and physical parameters affecting lipase production such as carbon and nitrogen supplements, pH, temperature, agitation speed and incubation time were studied. Refined sunflower oil (1% v/v) and tryptone at a pH of 6.2 favored maximum lipase production in Pseudomonas at 30 degrees C and 150 rpm, when incubated for 5 days. In C. albicans refined sunflower oil (3% v/v) and peptone resulted in maximum lipase production at pH 5.2, 30 degrees C and 150 rpm, when incubated for 5 days. In A. flavus coconut oil (3% v/v) and peptone yielded maximum lipase at pH 6.2, 37 degrees C, 200 rpm after an incubation period of 5 days. The lipases were partially purified by ammonium sulphate precipitation and dialysis. In P. aeruginosa enzyme activity of the dialyzed fraction was found to be 400 U mL-' and for C. albicans 410 U mL(-1). The dialysed lipase fraction from A. flavus demonstrated an activity of 460 U mL(-1). The apparent molecular weights of the dialyzed lipases were determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The dialyzed lipase fraction obtained from P. aeruginosa revealed molecular weights of 47, 49 and 51 kDa, whereas, lipases from C. albicans and A. flavus demonstrated 3 bands (16.5, 27 and 51 kDa) and one band (47 kDa), respectively. These extracellular lipases may find wide industrial applications.
Llewellyn, G C; Eadie, T; Dashek, W V
The susceptibility of blackberries, cherries, and strawberries to Aspergillus growth and aflatoxin production has been examined. Three aflatoxigenic isolates of Aspergillus, A. flavus ATCC 15548 and NRRL 3251 as well as A. parasiticus NRRL 2999, were cultured on homogenates of the fruits for 14 days at 28 +/- 2 degrees C. Percent mycelial growth and spore infestation were determined each day with a calibrated grid. At day 14 each culture was frozen at -5 degrees C until aflatoxins were extracted with methylene chloride and water. Aflatoxins were separated by thin layer chromatography (TLC) with benzene-methanol-acetic acid (90 + 5 + 5). This extraction and solvent system provided satisfactory separations of the aflatoxins and was free of background interference on the TLC plates. Although all fruits served as substrates for both Aspergillus growth and aflatoxin production, cherries appeared to be a more favorable substrate than did blackberries, and the latter was more favorable than strawberries. Whereas A. flavus produced both B1 and G1 on all substrates, it yielded B2 and G2 only on cherries. Although A. parasiticus NRRL 2999 synthesized B1, B2, G1, and G2 on both blackberries and cherries, no aflatoxins were detected on strawberries. In contrast, A. flavus NRRL 3251 failed to produce detectable levels of aflatoxin on any substrate. All substrates supported both mycelial growth and subsequent sporulation with cherries greater than blackberries greater than strawberries.
Li, Jinhan; Li, Jialin; Lu, Zhisong; Liu, Yang; Li, Chang Ming
A polydopamine-Fe3O4 nanocomposite-based H2O2 electrochemical sensor is fabricated to real-time monitor the transmembrane release of reactive oxygen species from citral-treated Aspergillus flavus, revealing a mechanism involving transient transmembrane secretion of H2O2 for the citral-caused inhibition of aflatoxin production from a fungus for the first time.
Aflatoxins are toxic and carcinogenic secondary metabolites. These compounds, produced by Aspergillus flavus and A. parasiticus, contaminate pre-harvest agricultural crops in the field and post-harvest grains during storage. In order to reduce and eliminate aflatoxin contamination of food and feed...
The genome of the filamentous fungus, Aspergillus flavus, has been shown to harbor as many as 55 putative secondary metabolic gene clusters including the one responsible for production of the toxic and carcinogenic, polyketide synthase (PKS)-derived family of secondary metabolites termed aflatoxins....
A public candidate gene testing pipeline for resistance to aflatoxin accumulation or Aspergillus flavus infection in maize is presented here. The pipeline consists of steps for identifying, testing, and verifying the association of any maize gene sequence with resistance under field conditions. Reso...
Over a three year period, we compared aflatoxin accumulation and kernel infection in maize hybrids inoculated with six inoculum concentrations of Aspergillus flavus isolate NRRL 3357 or A. parasiticus isolate NRRL 6111 which is a norsolorinic acid producer. Aflatoxin resistant and susceptible mai...
A currently utilized pre-harvest bio-control method involves field inoculations with non-aflatoxigenic Aspergillus flavus, a tactic shown to strategically displace the native aflatoxin producing strain and effectively decrease aflatoxin contamination in corn. The present in situ study focuses on tra...
Oilseed crops such as maize and peanut are staple food crops which are vital for global food security. The contamination of these crops with carcinogenic aflatoxins during infection by Aspergillus flavus under drought stress conditions is a serious threat to the safety of these commodities. In order...
Aflatoxins, which are produced by the fungus Aspergillus flavus, are toxic to humans, livestock, and pets. The value of maize (Zea mays) grain is markedly reduced when contaminated with aflatoxin. Plant resistance and biological control using non-toxin producing strains are considered effective st...
The infection of maize and peanut with Aspergillus flavus and subsequent contamination with aflatoxin pose a threat to global food safety and human health, and is exacerbated by drought stress. Drought stress-responding compounds such as reactive oxygen species (ROS) are associated with fungal stres...
Maize (Zea mays L.) is one of the major crops susceptible to Aspergillus flavus Link ex. Fries infection and subsequent aflatoxin contamination. Previous studies found the expression of an antifungal 14 kDa trypsin inhibitor (TI) was associated with maize aflatoxin resistance. To further investigate...
Competitive exclusion of aflatoxin producers by endemic atoxigenic strains of Aspergillus flavus is a proven tool for aflatoxin management being adapted for use in Africa. Field efficacy of an experimental formulation consisting of four native atoxigenic strains (La3303, La3304, La3279 and Ka16127) ...
Aflatoxin contamination of peanut and other crops is a major concern for producers globally, and has been shown to be exacerbated by drought stress. Previous transcriptomic and proteomic examination of the responses of isolates of Aspergillus flavus to drought-related oxidative stress in vitro have ...
Biological control of toxigenic Aspergillus flavus in maize through competitive displacement by non-aflatoxigenic strains was evaluated in a series of field studies. Four sets of experiments were conducted between 2007 to 2009 to assess the competitiveness of non-aflatoxigenic strains when challen...
Aflatoxin contamination of peanut and maize is exacerbated by drought stress. Reactive oxygen species (ROS) are produced in host plants during drought/heat stress, and are hypothesized to stimulate aflatoxin production. In order to better understand why Aspergillus flavus produces aflatoxin and the ...
Liang, Dandan; Xing, Fuguo; Selvaraj, Jonathan Nimal; Liu, Xiao; Wang, Limin; Hua, Huijuan; Zhou, Lu; Zhao, Yueju; Wang, Yan; Liu, Yang
In order to reveal the inhibitory effects of cinnamaldehyde, citral, and eugenol on aflatoxin biosynthesis, the expression levels of 5 key aflatoxin biosynthetic genes were evaluated by real-time PCR. Aspergillus flavus growth and AFB1 production were completely inhibited by 0.80 mmol/L of cinnamaldehyde and 2.80 mmol/L of citral. However, at lower concentration, cinnamaldehyde (0.40 mmol/L), eugenol (0.80 mmol/L), and citral (0.56 mmol/L) significantly reduced AFB1 production with inhibition rate of 68.9%, 95.4%, and 41.8%, respectively, while no effect on fungal growth. Real-time PCR showed that the expressions of aflR, aflT, aflD, aflM, and aflP were down-regulated by cinnamaldehyde (0.40 mmol/L), eugenol (0.80 mmol/L), and citral (0.56 mmol/L). In the presence of cinnamaldehyde, AflM was highly down-regulated (average of 5963 folds), followed by aflP, aflR, aflD, and aflT with the average folds of 55, 18, 6.5, and 5.8, respectively. With 0.80 mmol/L of eugenol, aflP was highly down-regulated (average of 2061-folds), followed by aflM, aflR, aflD, and aflT with average of 138-, 15-, 5.2-, and 4.8-folds reduction, respectively. With 0.56 mmol/L of citral, aflT was completely inhibited, followed by aflM, aflP, aflR, and aflD with average of 257-, 29-, 3.5-, and 2.5-folds reduction, respectively. These results suggest that the reduction in AFB1 production by cinnamaldehyde, eugenol, and citral at low concentration may be due to the down-regulations of the transcription level of aflatoxin biosynthetic genes. Cinnamaldehyde and eugenol may be employed successfully as a good candidate in controlling of toxigenic fungi and subsequently contamination with aflatoxins in practice. © 2015 Institute of Food Technologists®
Wang, Houmiao; Lei, Yong; Wan, Liyun; Yan, Liying; Lv, Jianwei; Dai, Xiaofeng; Ren, Xiaoping; Guo, Wei; Jiang, Huifang; Liao, Boshou
Aflatoxin contamination caused by Aspergillus flavus in peanut (Arachis hypogaea) including in pre- and post-harvest stages seriously affects industry development and human health. Even though resistance to aflatoxin production in post-harvest peanut has been identified, its molecular mechanism has been poorly understood. To understand the mechanism of peanut response to aflatoxin production by A. flavus, RNA-seq was used for global transcriptome profiling of post-harvest seed of resistant (Zhonghua 6) and susceptible (Zhonghua 12) peanut genotypes under the fungus infection and aflatoxin production stress. A total of 128.72 Gb of high-quality bases were generated and assembled into 128, 725 unigenes (average length 765 bp). About 62, 352 unigenes (48.43%) were annotated in the NCBI non-redundant protein sequences, NCBI non-redundant nucleotide sequences, Swiss-Prot, KEGG Ortholog, Protein family, Gene Ontology, or eukaryotic Ortholog Groups database and more than 93% of the unigenes were expressed in the samples. Among obtained 30, 143 differentially expressed unigenes (DEGs), 842 potential defense-related genes, including nucleotide binding site-leucine-rich repeat proteins, polygalacturonase inhibitor proteins, leucine-rich repeat receptor-like kinases, mitogen-activated protein kinase, transcription factors, ADP-ribosylation factors, pathogenesis-related proteins and crucial factors of other defense-related pathways, might contribute to peanut response to aflatoxin production. Notably, DEGs involved in phenylpropanoid-derived compounds biosynthetic pathway were induced to higher levels in the resistant genotype than in the susceptible one. Flavonoid, stilbenoid and phenylpropanoid biosynthesis pathways were enriched only in the resistant genotype. This study provided the first comprehensive analysis of transcriptome of post-harvest peanut seeds in response to aflatoxin production, and would contribute to better understanding of molecular interaction between
Lvova, L S; Sosedov, N I; Shatilova, T I; Shulgina, A P
The influence of temperature ranging from 15 to 35 degrees C and relative humidity of 75 to 90% on the accumulation of aflatoxins on wheat grain inoculated by Aspergillus flavus NRRL 2999 was investigated. The lowest accumulation of aflatoxins took place at 20 degrees C and relative humidity of 80%. The highest accumulation of aflatoxins appeared in the grain after 3 day storage. Some products of three-graded 78% grinding showed a decrease in the aflatoxin content (flour of the first and second grade). During baking of leavened bread 70-80% of aflatoxins were decomposed. The feasible use of the rapid conditioning and Remix-method to reduce the content of aflatoxins in flour and bread is discussed.
Vincent, P G; Kulik, M M
Four fungi of the Aspergillus flavus group were differentiated to the species level and strain level by pyrolysis-gas-liquid chromotography. Comparisons of pyrochromatograms revealed more similarities than dissimilarities among both species and strains in the pyrolytic elution patterns. Quantitative analysis was made by comparing the number of peaks in which two strains or reference species agreed or disagreed, the degree of superimposability between the pyrolytic elution patterns of strains and reference species, and the presence or absence of peaks for strain pairs within each species. The accuracy and precision of these techniques suggest that pyrolysis-gas-liquid chromatography may have wide application in the detection, enumeration, and identification of fungi by nonmycologically trained personnel.
Alaniz Zanon, María Silvina; Barros, Germán Gustavo; Chulze, Sofía Noemí
Biological control is one of the most promising strategies for preventing aflatoxin contamination in peanuts at field stage. A population of 46 native Aspergillus flavus nonaflatoxin producers were analysed based on phenotypic, physiological and genetic characteristics. Thirty-three isolates were characterized as L strain morphotype, 3 isolates as S strain morphotype, and 10 isolates did not produce sclerotia. Only 11 of 46 non-aflatoxigenic isolates did not produce cyclopiazonic acid. The vegetative compatibility group (VCG) diversity index for the population was 0.37. For field trials we selected the non-aflatoxigenic A. flavus AR27, AR100G and AFCHG2 strains. The efficacy of single and mixed inocula as potential biocontrol agents in Northern Argentina was evaluated through a 2-year study (2014-2015). During the 2014 peanut growing season, most of the treatments reduced the incidence of aflatoxigenic strains in both soil and peanut kernel samples, and no aflatoxin was detected in kernels. During the 2015 growing season, there was a reduction of aflatoxigenic strains in kernel samples from the plots treated with the potential biocontrol agents. Reductions of aflatoxin contamination between 78.36% and 89.55% were observed in treated plots in comparison with the un-inoculated control plots. This study provides the first data on aflatoxin biocontrol based on competitive exclusion in the peanut growing region of Northern Argentina, and proposes bioproducts with potential use as biocontrol agents. Copyright © 2016. Published by Elsevier B.V.
Ortega-Beltran, A; Grubisha, L C; Callicott, K A; Cotty, P J
To assess frequencies of the Aspergillus flavus atoxigenic vegetative compatibility group (VCG) YV36, to which the biocontrol agent AF36 belongs, in maize-growing regions of Mexico. Over 3500 A. flavus isolates recovered from maize agroecosystems in four states of Mexico during 2005 through 2008 were subjected to vegetative compatibility analyses based on nitrate nonutilizing mutants. Results revealed that 59 (1·6%) isolates belong to VCG YV36. All 59 isolates had the MAT1-2 idiomorph at the mating-type locus and the single nucleotide polymorphism in the polyketide synthase gene that confers atoxigenicity. Additional degradation of the aflatoxin gene cluster was detected in three isolates. Microsatellite loci analyses revealed low levels of genetic diversity and no linkage disequilibrium within VCG YV36. The VCG to which the biocontrol agent AF36 belongs, YV36, is also native to Mexico. The North American Free Trade Agreement should facilitate adoption of AF36 for use by Mexico in aflatoxin prevention programs. An USEPA registered biocontrol agent effective at preventing aflatoxin contamination of crops in the US, is also native to Mexico. This should facilitate the path to registration of AF36 as the first biopesticide for aflatoxin mitigation of maize in Mexico. Economic and health benefits to the population of Mexico should result once aflatoxin mitigation programs based on AF36 applications are implemented. Published 2016. This article is a U.S. Government work and is in the public domain in the USA.
Aflatoxin contamination caused by the opportunistic pathogen A. flavus is a major concern in maize production prior to harvest and during storage, and also a concern in many other crops, such as peanuts, cottonseed, tree nuts, and rice. Although a number of resistant maize lines with low aflatoxin c...
A total of 34 A. flavus isolates were recovered from sorghum seeds sampled across five states in India. Our study included (1) species confirmation through PCR assay, (2) an aflatoxin cluster genotype assay using developed multiplex PCR, (3) quantification of total aflatoxin concentrations by the iC...
De Lucca, Anthony J; Boue, Stephen M; Carter-Wientjes, Carol; Bhatnagar, Deepak
Aspergillus flavus is a saprophytic fungus which can grow on corn and produce aflatoxins which render it unsafe for consumption as food and feed. In this study, aflatoxin and non-aflatoxin producing isolates of A. flavus were grown separately on wet (20% water added), sterile or non-sterile cracked corn. Wet and dry cracked corn controls were included as needed. Secondary metabolic volatiles were identified and aflatoxin concentrations determined over a 12-day period. Volatiles unique to the toxigenic A. flavus isolates were determined by comparison with volatiles produced by the respective corn controls and the non-toxigenic A. flavus isolate. The number and identity of the volatiles produced by these A. flavus isolates varied by isolate, whether sterile or non-sterile corn was the substrate, and the sampling day. Overall, most of the volatiles were produced before day 8 after inoculation. Aflatoxin production was 10-fold lower on the sterile corn, compared to the non-sterile corn. Volatiles unique to the aflatoxin producing isolates were identified on both substrates after comparison with those produced by the non-aflatoxin producing isolate, as well as the corn control samples. Results indicate that several factors (substrate, fungal isolate, culture age) affect volatile and aflatoxin production by A. flavus.
Musungu, Bryan M.; Bhatnagar, Deepak; Brown, Robert L.; Payne, Gary A.; OBrian, Greg; Fakhoury, Ahmad M.; Geisler, Matt
A gene co-expression network (GEN) was generated using a dual RNA-seq study with the fungal pathogen Aspergillus flavus and its plant host Zea mays during the initial 3 days of infection. The analysis deciphered novel pathways and mapped genes of interest in both organisms during the infection. This network revealed a high degree of connectivity in many of the previously recognized pathways in Z. mays such as jasmonic acid, ethylene, and reactive oxygen species (ROS). For the pathogen A. flavus, a link between aflatoxin production and vesicular transport was identified within the network. There was significant interspecies correlation of expression between Z. mays and A. flavus for a subset of 104 Z. mays, and 1942 A. flavus genes. This resulted in an interspecies subnetwork enriched in multiple Z. mays genes involved in the production of ROS. In addition to the ROS from Z. mays, there was enrichment in the vesicular transport pathways and the aflatoxin pathway for A. flavus. Included in these genes, a key aflatoxin cluster regulator, AflS, was found to be co-regulated with multiple Z. mays ROS producing genes within the network, suggesting AflS may be monitoring host ROS levels. The entire GEN for both host and pathogen, and the subset of interspecies correlations, is presented as a tool for hypothesis generation and discovery for events in the early stages of fungal infection of Z. mays by A. flavus. PMID:27917194
Musungu, Bryan M; Bhatnagar, Deepak; Brown, Robert L; Payne, Gary A; OBrian, Greg; Fakhoury, Ahmad M; Geisler, Matt
A gene co-expression network (GEN) was generated using a dual RNA-seq study with the fungal pathogen Aspergillus flavus and its plant host Zea mays during the initial 3 days of infection. The analysis deciphered novel pathways and mapped genes of interest in both organisms during the infection. This network revealed a high degree of connectivity in many of the previously recognized pathways in Z. mays such as jasmonic acid, ethylene, and reactive oxygen species (ROS). For the pathogen A. flavus, a link between aflatoxin production and vesicular transport was identified within the network. There was significant interspecies correlation of expression between Z. mays and A. flavus for a subset of 104 Z. mays, and 1942 A. flavus genes. This resulted in an interspecies subnetwork enriched in multiple Z. mays genes involved in the production of ROS. In addition to the ROS from Z. mays, there was enrichment in the vesicular transport pathways and the aflatoxin pathway for A. flavus. Included in these genes, a key aflatoxin cluster regulator, AflS, was found to be co-regulated with multiple Z. mays ROS producing genes within the network, suggesting AflS may be monitoring host ROS levels. The entire GEN for both host and pathogen, and the subset of interspecies correlations, is presented as a tool for hypothesis generation and discovery for events in the early stages of fungal infection of Z. mays by A. flavus.
Lee, Seung-Hun; Park, Shin Young; Byun, Kye-Hwan; Chun, Hyang Sook; Ha, Sang-Do
Aspergillus flavus and Aspergillus parasiticus are primary pathogen moulds on brown rice and barley. This study investigated the effects of microwave irradiation (MWI) (2450 MHz, 700 W, 10-50 s) on inactivation of A. flavus and A. parasiticus on brown rice and barley and the quality of these samples. The counts of both strains were significantly (p < 0.05) reduced by the stepwise increase in MWI treatment time. The log reductions of A. flavus on brown rice and barley were 0.05 and 0.04 after 10 s; 1.06 and 1.05 after 20 s; 1.59 and 1.52 after 30 s; and 3.04 and 2.78 after 40 s. The log reductions of A. parasiticus on brown rice and barley were 0.06 and 0.10 after 10 s; 1.20 and 1.00 after 20 s; 2.04 and 1.61 after 30 s; and 2.89 and 2.90 after 40 s. Moreover, neither strain survived after 50 s of MWI. The Hunter colour 'L' gradually increased with increasing MWI treatment time. However, there were no significant differences in the 'L' of brown rice after 10-40 s of MWI treatment and of barley after 10-30 s of MWI treatment. The Hunter colour 'a' and 'b' gradually increased with increasing microwave time. No significant change was observed in the moisture content of either cereal treated with 10-20 s of MWI. The differences in the sensory quality (colour, appearance, flavour, texture and overall acceptability) after 0-30 s of MWI were not significant. However, values for colour, appearance, texture and overall acceptability were significantly reduced when treated with 40-50 s of MWI. Therefore, with 20 s of MWI at 2450 MHz, 700 W could be effective for > 90% reduction of mould without causing deleterious changes to the colour, moisture content and sensory qualities of these cereals.
Gqaleni, N; Smith, J E; Lacey, J; Gettinby, G
The combined effects of water activity (a(w)) and temperature on mycotoxin production by Penicilium commune (cyclopiazonic acid - CPA) and Aspergillus flavus (CPA and aflatoxins - AF) were studied on maize over a 14-day period using a statistical experimental design. Analysis of variance showed a highly significant interaction (P ≤ 0.001) between these factors and mycotoxin production. The minimum a(w)/temperature for CPA production (2264 ng g(-1) P. commune, 709 ng g(-1) A. flavus) was 0.90 a(w)/30 °C while greatest production (7678 ng g(-1) P. commune, 1876 ng g(-1) A. flavus) was produced at 0.98 a(w)/20 °C. Least AF (411 ng g(-1)) was produced at 0.90 a(w)/20 °C and most (3096 ng g(-1)) at 0.98 a(w)/30 °C.
Aspergillus, a mycotoxicogenic fungal genus, produces carcinogenic aflatoxins in crops like peanuts and maize. Development of fungal resistant maize cultivars is one strategy used to decrease contamination. Successful development and identification of resistant maize genotypes requires evaluation o...
Zarrin, Majid; Erfaninejad, Maryam
Aspergillus flavus is the second most common disease-causing species of Aspergillus in humans. The fungus is frequently associated with life-threatening infections in immunocompromised hosts. The primary aim of the present study was to analyze the genetic variability among different isolates of A. flavus using polymerase chain reaction (PCR)-based restriction fragment length polymorphism (RFLP). A total of 62 A. flavus isolates were tested in the study. Molecular variability was searched for by analysis of the PCR amplification of the internal transcribed spacer (ITS) regions of ribosomal DNA using restriction enzymes. PCR using primers for ITS1 and ITS4 resulted in a product of ~600 bp. Amplicons were subjected to digestion with restriction endonucleases EcoRI, HaeIII and TaqI. Digestion of the PCR products using these restriction enzymes produced different patterns of fragments among the isolates, with different sizes and numbers of fragments, revealing genetic variability. In conclusion, ITS-RFLP is a useful molecular tool in screening for nucleotide polymorphisms among A. flavus isolates. PMID:27588085
Umemura, Myco; Nagano, Nozomi; Koike, Hideaki; Kawano, Jin; Ishii, Tomoko; Miyamura, Yuki; Kikuchi, Moto; Tamano, Koichi; Yu, Jiujiang; Shin-ya, Kazuo; Machida, Masayuki
Ustiloxin B is a secondary metabolite known to be produced by Ustilaginoidea virens. In our previous paper, we observed the production of this compound by Aspergillus flavus, and identified two A. flavus genes responsible for ustiloxin B biosynthesis (Umemura et al., 2013). The compound is a cyclic tetrapeptide of Tyr-Ala-Ile-Gly, whose tyrosine is modified with a non-protein coding amino acid, norvaline. Although its chemical structure strongly suggested that ustiloxin B is biosynthesized by a non-ribosomal peptide synthetase, in the present study, we observed its synthesis through a ribosomal peptide synthetic (RiPS) pathway by precise sequence analyses after experimental validation of the cluster. The cluster possessed a gene (AFLA_094980), termed ustA, whose translated product, UstA, contains a 16-fold repeated peptide embedding a tetrapeptide, Tyr-Ala-Ile-Gly, that is converted into the cyclic moiety of ustiloxin B. This result strongly suggests that ustiloxin B is biosynthesized through a RiPS pathway and that UstA provides the precursor peptide of the compound. The present work is the first characterization of RiPS in Ascomycetes and the entire RiPS gene cluster in fungi. Based on the sequence analyses, we also proposed a biosynthetic mechanism involving the entire gene cluster. Our finding indicates the possibility that a number of unidentified RiPSs exist in Ascomycetes as the biosynthetic genes of secondary metabolites, and that the feature of a highly repeated peptide sequence in UstA will greatly contribute to the discovery of additional RiPS. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.
Tian, Jun; Wang, Yanzhen; Lu, Zhaoqun; Sun, Chunhui; Zhang, Man; Zhu, Aihua; Peng, Xue
In the present study, we provide detailed insights into perillaldehyde (PAE)'s mechanisms of action on Aspergillus flavus and offer evidence in favor of the induction of an apoptosis-like phenotype. Specifically, PAE's antifungal mode of action was investigated through the detection of mitochondrial membrane potential (MtΔψ) and phosphatidylserine (PS) exposure, as well as intracellular Ca(2+) level, reactive oxygen species accumulation, and metacaspase activation. This was done by way of fluorometry, measuring DNA fragmentation, and condensation by fluorescent microscopy. Furthermore, we searched for phenotypic changes characteristic of apoptosis by transmission electron microscopy and flow cytometry, determining the amount of cytochrome c released using Western blotting. Results indicated that cultivation of A. flavus in the presence of PAE caused depolarization of MtΔψ, rapid DNA condensation, large-scale DNA fragmentation, and an elevation of intracellular Ca(2+) level. The percentage of early apoptotic cells with exposure of PS were 27.4% and 48.7%, respectively, after 9 h incubations with 0.25 and 0.5 μL/mL of PAE. The percentage of stained cells with activated intracellular metacaspases exposed to PAE at concentrations of 0.25 and 0.5 μL/mL compared with control subjects were increased by 28.4 ± 3.25% and 37.9 ± 4.24%, respectively. The above results has revealed that PAE induces fungal apoptosis through a caspase-dependent mitochondrial pathway. In all, our findings provide a novel mechanism for exploring a possible antifungal agent used in food preservation.
Xu, Maowen; Jia, Min; Mao, Cuiping; Liu, Sangui; Bao, Shujuan; Jiang, Jian; Liu, Yang; Lu, Zhisong
A novel approach was developed to prepare porous carbon materials with an extremely high surface area of 2459.6 m2g‑1 by using Aspergillus flavus conidia as precursors. The porous carbon serves as a superior cathode material to anchor sulfur due to its uniform and tortuous morphology, enabling high capacity and good cycle lifetime in lithium sulfur-batteries. Under a current rate of 0.2 C, the carbon-sulfur composites with 56.7 wt% sulfur loading deliver an initial capacity of 1625 mAh g‑1, which is almost equal to the theoretical capacity of sulfur. The good performance may be ascribed to excellent electronic networks constructed by the high-surface-area carbon species. Moreover, the semi-closed architecture of derived carbons can effectively retard the polysulfides dissolution during charge/discharge, resulting in a capacity of 940 mAh g‑1 after 120 charge/discharge cycles.
Lai, Xianwen; Zhang, He; Liu, Ruicen; Liu, Chenglan
In this study, we investigated the potential for aflatoxin B1 (AFB1) and B2 (AFB2) production in rice grain by 127 strains of Aspergillus flavus isolated from rice grains collected from China. These strains were inoculated onto rice grains and incubated at 28 °C for 21 days. AFB1 and AFB2 were extracted and quantified by high-performance liquid chromatography coupled with fluorescence detection. Among the tested strains, 37% produced AFB1 and AFB2 with levels ranging from 175 to 124 101 μg kg−1 for AFB1 and from not detected to 10 329 μg kg−1 for AFB2. The mean yields of these isolates were 5884 μg kg−1 for AFB1 and 1968 μg kg−1 for AFB2. Overall, most of the aflatoxigenic strains produced higher levels of AFB1 than AFB2 in rice. The obtained information is useful for assessing the risk of aflatoxin contamination in rice samples. PMID:25737649
Prieto, R; Woloshuk, C P
Among the enzymatic steps in the aflatoxin biosynthetic pathway, the conversion of O-methylsterigmatocystin to aflatoxin has been proposed to be catalyzed by an oxidoreductase. Transformants of Aspergillus flavus 649WAF2 containing a 3.3-kb genomic DNA fragment and the aflatoxin biosynthesis regulatory gene aflR converted exogenously supplied O-methylsterigmatocystin to aflatoxin B1. A gene, ord1, corresponding to a transcript of about 2 kb was identified within the 3.3-kb DNA fragment. The promoter region presented a putative AFLR binding site and a TATA sequence. The nucleotide sequence of the gene revealed an open reading frame encoding a protein of 528 amino acids with a deduced molecular mass of 60.2 kDa. The gene contained six introns and seven exons. Heterologous expression of the ord1 open reading frame under the transcriptional control of the Saccharomyces cerevisiae galactose-inducible gal1 promoter results in the ability to convert O-methylsterigmatocystin to aflatoxin B1. The data indicate that ord1 is sufficient to accomplish the last step of the aflatoxin biosynthetic pathway. A search of various databases for similarity indicated that ord1 encodes a cytochrome P-450-type monooxygenase, and the gene has been assigned to a new P-450 gene family named CYP64. PMID:9143099
Xu, Maowen; Jia, Min; Mao, Cuiping; Liu, Sangui; Bao, Shujuan; Jiang, Jian; Liu, Yang; Lu, Zhisong
A novel approach was developed to prepare porous carbon materials with an extremely high surface area of 2459.6 m2g−1 by using Aspergillus flavus conidia as precursors. The porous carbon serves as a superior cathode material to anchor sulfur due to its uniform and tortuous morphology, enabling high capacity and good cycle lifetime in lithium sulfur-batteries. Under a current rate of 0.2 C, the carbon-sulfur composites with 56.7 wt% sulfur loading deliver an initial capacity of 1625 mAh g−1, which is almost equal to the theoretical capacity of sulfur. The good performance may be ascribed to excellent electronic networks constructed by the high-surface-area carbon species. Moreover, the semi-closed architecture of derived carbons can effectively retard the polysulfides dissolution during charge/discharge, resulting in a capacity of 940 mAh g−1 after 120 charge/discharge cycles. PMID:26732547
Wong, Yoke Phooi; Saw, Horng Yuan; Janaun, Jidon; Krishnaiah, Kamatam; Prabhakar, Auti
Solid-state fermentation (SSF) was employed to enhance the nutritive values of palm kernel cake (PKC) for poultry feeding. Aspergillus flavus was isolated from local PKC and utilized to increase the mannose content of PKC via the degradation of β-mannan in PKC; evaluation was done for batch SSF in Erlenmeyer flasks and in a novel laterally aerated moving bed (LAMB) bioreactor. The optimum condition for batch SSF in flasks was 110% initial moisture content, initial pH 6.0, 30 °C, 855 μm particle size, and 120 h of fermentation, yielding 90.91 mg mannose g⁻¹ dry PKC (5.9-fold increase). Batch SSF in the LAMB at the optimum condition yielded 79.61 mg mannose g⁻¹ dry PKC (5.5-fold increase) within just 96 h due to better heat and mass transfer when humidified air flowed radially across the PKC bed. In spite of a compromise of 12% reduction in mannose content when compared with the flasks, the LAMB facilitated good heat and mass transfer, and improved the mannose content of PKC in a shorter fermentation period. These attributes are useful for batch production of fermented PKC feed in an industrial scale.
Song, Hui; Wang, Pengfei; Li, Changsheng; Han, Suoyi; Lopez-Baltazar, Javier; Zhang, Xinyou; Wang, Xingjun
Lipoxygenase (LOX) genes are widely distributed in plants and play crucial roles in resistance to biotic and abiotic stress. Although they have been characterized in various plants, little is known about the evolution of legume LOX genes. In this study, we identified 122 full-length LOX genes in Arachis duranensis, Arachis ipaënsis, Cajanus cajan, Cicer arietinum, Glycine max, Lotus japonicus and Medicago truncatula. In total, 64 orthologous and 36 paralogous genes were identified. The full-length, polycystin-1, lipoxygenase, alpha-toxin (PLAT) and lipoxygenase domain sequences from orthologous and paralogous genes exhibited a signature of purifying selection. However, purifying selection influenced orthologues more than paralogues, indicating greater functional conservation of orthologues than paralogues. Neutrality and effective number of codons plot results showed that natural selection primarily shapes codon usage, except for C. arietinum, L. japonicas and M. truncatula LOX genes. GCG, ACG, UCG, CGG and CCG codons exhibited low relative synonymous codon usage (RSCU) values, while CCA, GGA, GCU, CUU and GUU had high RSCU values, indicating that the latter codons are strongly preferred. LOX expression patterns differed significantly between wild-type peanut and cultivated peanut infected with Aspergillus flavus, which could explain the divergent disease resistance of wild progenitor and cultivars. PMID:27731413
James, Michael J.; Lasker, Brent A.; McNeil, Michael M.; Shelton, Mark; Warnock, David W.; Reiss, Errol
Aspergillus flavus is second to A. fumigatus as a cause of invasive aspergillosis, but no standard method exists for molecular typing of strains from human sources. A repetitive DNA sequence cloned from A. flavus and subcloned into a pUC19 vector, pAF28, was used to type 18 isolates from diverse clinical, environmental, and geographic sources. The restriction fragment length polymorphisms generated with EcoRI- or PstI-digested genomic DNA and probed with digoxigenin-labeled pAF28 revealed complete concordance between patterns. Eighteen distinct fingerprints were observed. The probe was used to investigate two cases of cutaneous A. flavus infection in low-birth-weight infants in a neonatal intensive care unit (NICU). Both infants were transported by the same ambulance and crew to the NICU on the same day. A. flavus strains of the same genotype were isolated from both infants, from a roll of tape used to fasten their umbilical catheters, from a canvas bag used to store the tape in the ambulance, and from the tape tray in the ambulance isolette. These cases highlight the need to consider exposures in critically ill neonates that might occur during their transport to the NICU and for stringent infection control practices. The hybridization profiles of strains from a second cluster of invasive A. flavus infections in two pediatric hematology-oncology patients revealed a genotype common to strains from a definite case patient and a health care worker. A probable case patient was infected with a strain with a genotype different from that of the strain from the definite case patient but highly related to that of an environmental isolate. The high degree of discrimination and reproducibility obtained with the pAF28 probe underscores its utility for typing clinical and environmental isolates of A. flavus. PMID:11015372
Bhatnagar-Mathur, Pooja; Sunkara, Sowmini; Bhatnagar-Panwar, Madhurima; Waliyar, Farid; Sharma, Kiran Kumar
Aflatoxins are toxic, carcinogenic, mutagenic, teratogenic and immunosuppressive byproducts of Aspergillus spp. that contaminate a wide range of crops such as maize, peanut, and cotton. Aflatoxin not only affects crop production but renders the produce unfit for consumption and harmful to human and livestock health, with stringent threshold limits of acceptability. In many crops, breeding for resistance is not a reliable option because of the limited availability of genotypes with durable resistance to Aspergillus. Understanding the fungal/crop/environment interactions involved in aflatoxin contamination is therefore essential in designing measures for its prevention and control. For a sustainable solution to aflatoxin contamination, research must be focused on identifying and improving knowledge of host-plant resistance factors to aflatoxin accumulation. Current advances in genetic transformation, proteomics, RNAi technology, and marker-assisted selection offer great potential in minimizing pre-harvest aflatoxin contamination in cultivated crop species. Moreover, developing effective phenotyping strategies for transgenic as well as precision breeding of resistance genes into commercial varieties is critical. While appropriate storage practices can generally minimize post-harvest aflatoxin contamination in crops, the use of biotechnology to interrupt the probability of pre-harvest infection and contamination has the potential to provide sustainable solution.
Bedre, Renesh; Rajasekaran, Kanniah; Mangu, Venkata Ramanarao; Sanchez Timm, Luis Eduardo; Bhatnagar, Deepak; Baisakh, Niranjan
Aflatoxins are toxic and potent carcinogenic metabolites produced from the fungi Aspergillus flavus and A. parasiticus. Aflatoxins can contaminate cottonseed under conducive preharvest and postharvest conditions. United States federal regulations restrict the use of aflatoxin contaminated cottonseed at >20 ppb for animal feed. Several strategies have been proposed for controlling aflatoxin contamination, and much success has been achieved by the application of an atoxigenic strain of A. flavus in cotton, peanut and maize fields. Development of cultivars resistant to aflatoxin through overexpression of resistance associated genes and/or knocking down aflatoxin biosynthesis of A. flavus will be an effective strategy for controlling aflatoxin contamination in cotton. In this study, genome-wide transcriptome profiling was performed to identify differentially expressed genes in response to infection with both toxigenic and atoxigenic strains of A. flavus on cotton (Gossypium hirsutum L.) pericarp and seed. The genes involved in antifungal response, oxidative burst, transcription factors, defense signaling pathways and stress response were highly differentially expressed in pericarp and seed tissues in response to A. flavus infection. The cell-wall modifying genes and genes involved in the production of antimicrobial substances were more active in pericarp as compared to seed. The genes involved in auxin and cytokinin signaling were also induced. Most of the genes involved in defense response in cotton were highly induced in pericarp than in seed. The global gene expression analysis in response to fungal invasion in cotton will serve as a source for identifying biomarkers for breeding, potential candidate genes for transgenic manipulation, and will help in understanding complex plant-fungal interaction for future downstream research.
Kumar, Vikas; Loomba, Poonam Sood; Singh, Daljit; Saran, Ravindra Kumar
Cerebrospinal fluid (CSF) shunt failure is commonly associated with infection or mechanical obstruction of the shunt system. A 4-year-old male child who had undergone multiple shunt revisions at another hospital for congenital hydrocephalus and later for shunt obstruction, presented with exposed shunt at the supraclavicular region. Shunt revision was performed. The CSF culture showed no growth; however, the histopathological examination of shunt tube showed Aspergillus growth inside the lumen of silicone tube well away from the tip of ventriculoperitoneal shunt. The skin biopsy from the exposed site revealed foreign body giant cell granulomatous reaction. The patient was discharged on postoperative day 6 without any complications. At 3 months follow-up, the patient is doing well. A growth of Aspergillus within the shunt tube prompted us to think of how the hardware can get infected and may remain a source of constant infection. PMID:28553391
Background Proteases play an important role in virulence of many human, plant and insect pathogens. The proteinaceous protease inhibitors of plant origin have been reported widely from many plant species. The inhibitors may potentially be used for multiple therapeutic applications in viral, bacterial, fungal diseases and physiological disorders. In traditional Indian medicine system, Cassia tora (Senna tora) is reportedly effective in treatment of skin and gastrointestinal disorders. The present study explores the protease inhibitory activity of the above plant seeds against trypsin, Aspergillus flavus and Bacillus sp. proteases. Methods The crushed seeds of Cassia tora were washed thoroughly with acetone and hexane for depigmentation and defatting. The proteins were fractionated by ammonium sulphate (0-30, 30-60, 60-90%) followed by dialysis and size exclusion chromatography (SEC). The inhibitory potential of crude seed extract and most active dialyzed fraction against trypsin and proteases was established by spot test using unprocessed x-ray film and casein digestion methods, respectively. Electrophoretic analysis of most active fraction (30-60%) and SEC elutes were carried employing Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and Gelatin SDS-PAGE. Inhibition of fungal spore germination was studied in the presence of dialyzed active inhibitor fraction. Standard deviation (SD) and ANOVA were employed as statistical tools. Results The crude seeds' extract displayed strong antitryptic, bacterial and fungal protease inhibitory activity on x-ray film. The seed protein fraction 30-60% was found most active for trypsin inhibition in caseinolytic assay (P < 0.001). The inhibition of caseinolytic activity of the proteases increased with increasing ratio of seed extract. The residual activity of trypsin, Aspergillus flavus and Bacillus sp. proteases remained only 4, 7 and 3.1%, respectively when proteases were incubated with 3 mg ml-1 seed protein
Bluma, R; Amaiden, M R; Daghero, J; Etcheverry, M
The antifungal effect of Pimpinella anisum (anise), Pëumus boldus (boldus), Mentha piperita (peppermint), Origanum vulgare (oregano) and Minthosthachys verticillata (peperina) essential oils against Aspergillus section Flavi (two isolates of Aspergillus parasiticus and two isolates of Aspergillus flavus) was evaluated in maize meal extract agar at 0.982 and 0.955 water activities, at 25 degrees C. The percentage of germination, germ-tube elongation rate, growth rate and aflatoxin B(1) (AFB(1)) accumulation at different essential oils concentrations were evaluated. Anise and boldus essential oils were the most inhibitory at 500 mg kg(-1) to all growth parameters of the fungus. These essential oils inhibited the percentage of germination, germ-tube elongation rate and fungal growth. AFB(1) accumulation was completely inhibited by anise, boldus and oregano essential oils. Peperina and peppermint essential oils inhibited AFB(1) production by 85-90% in all concentrations assayed. Anise and boldus essential oils could be considered as effective fungitoxicans for Aspergillus section flavi. Our results suggest that these phytochemical compounds could be used alone or in conjunction with other substances to control the presence of aflatoxigenic fungi in stored maize.
Suhem, Kitiya; Matan, Narumol; Matan, Nirundorn; Danworaphong, Sorasak; Aewsiri, Tanong
The aim of this study was to improve the antifungal activity of the volatile Litsea cubeba essential oil and its main components (citral and limonene) on brown rice snack bars by applying He-Ne laser treatment. Different volumes (50-200 μL) of L. cubeba, citral or limonene were absorbed into a filter paper and placed inside an oven (18 L). Ten brown rice snack bars (2 cm wide × 4 cm long × 0.5 cm deep) were put in an oven and heated at 180 °C for 20 min. The shelf-life of the treated snack bars at 30 °C was assessed and sensory testing was carried out to investigate their consumer acceptability. A count of total phenolic content (TPC) and Fourier transform infrared spectroscopy (FTIR) on the properties of essential oil, citral, and limonene before and after the laser treatment was studied for possible modes of action. It was found that the laser treatment improved the antifungal activity of the examined volatile L. cubeba and citral with Aspergillus flavus inhibition by 80% in comparison with those of the control not treated with the laser. L. cubeba vapor at 100 μL with the laser treatment was found to completely inhibit the growth of natural molds on the snack bars for at least 25 days; however, without essential oil vapor and laser treatment, naturally contaminating mold was observed in 3 days. Results from the sensory tests showed that the panelists were unable to detect flavor and aroma differences between essential oil treatment and the control. Laser treatment caused an increase in TPC of citral oil whereas the TPC in limonene showed a decrease after the laser treatment. These situations could result from the changing peak of the aliphatic hydrocarbons that was revealed by the FTIR spectra.
Scully, Lisa R; Bidochka, Michael J
The evolution of host specialization in pathogens is a topic of considerable interest, particularly since it can represent a decisive step in the emergence of infectious diseases. Aspergillus flavus is an opportunistic fungus capable of infecting a wide variety of hosts, including plants, insects and mammals, although with low virulence. Here the derivation of an A. flavus strain that exhibits severe host restriction is reported. This strain exhibited a severe diminution or a complete lack of conidial production on a variety of standard agar media and on various plant species. However, it retained its ability to infect insects from various orders and to re-emerge from and adequately conidiate on the insect cadavers as a culmination of the pathogenic life cycle. This strain, demonstrating insect-dependent conidiation, was discovered to be a cysteine/methionine auxotroph due to an inability to reduce sulfate to sulfite. However, other A. flavus auxotrophs tested for plant and insect host range failed to show insect-dependent conidiation. An association between this specific auxotroph and a decreased host range is shown, emphasizing the role of nutrition in the host-pathogen relationship with respect to host restriction and evolution towards obligate pathogenesis.
Avdiyuk, K V; Varbanets, L D
The effect of cations and anions on the activity of Aspergillus flavus var. oryzae and Bacillus subtilis α-amylases showed that the tested enzymes are sensitive to most of cations and resistant to anions. The most significant inhibitory effects on the activity of A. flavus var. oryzae α-amylase have been demonstrated by Al3+ and Fe3+ ions, while on the activity of B. subtilis α-amylase - Hg2+, Cu2+ and Fe3+ ions. Inactivation of A. flavus var. oryzae and B. subtilis α-amylases in the presence of EGTA is indicated on the presence within their structure of metal ions. An important role in the enzymatic catalysis of both enzymes play carboxyl groups as evidenced by their inhibition of 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide methiodide. Inhibition of B. subtilis α-amylase by p-chloromercuribenzoate, N-ethylmaleimide and sodium sulfite is indicated on the probable involvement of the sulfhydryl groups in the functioning of the enzyme. Unlike most studied glycosidases the tested enzymes do not contain histidine imidazole group in the active center.
Abbas, Hamed K; Accinelli, Cesare; Zablotowicz, Robert M; Abel, Craig A; Bruns, H Arnold; Dong, Yanhong; Shier, W Thomas
Mycotoxin and Aspergillus flavus levels in soil-surface corn debris left by no-till agriculture methods (stover, cobs, and cobs with grain) were determined during the December-March fallow period for near-isogenic Bt and non-Bt hybrid corn. By December, average mycotoxin levels in non-Bt corn were many times higher in cobs with grain than in grain harvested in September (total aflatoxins, 774 vs 211 ng/g; total fumonisins, 216 vs 3.5 microg/g; cyclopiazonic acid, 4102 vs 72.2 microg/g; zearalenone, 0.2 vs < 0.1 microg/g). No trichothecenes were detected. Levels of mycotoxins and A. flavus propagules were approximately 10- to 50-fold lower in cobs without grain and stover, respectively, for all mycotoxins except zearalenone. Mycotoxin levels in corn debris fractions decreased during winter but began to rise in March. Levels of all mycotoxins and A. flavus propagules were lower in harvested grain and debris from Bt than non-Bt corn, but differences were significant (p < 0.05) only for aflatoxins.
Cary, J. W.; Han, Z.; Yin, Y.; Lohmar, J. M.; Shantappa, S.; Harris-Coward, P. Y.; Mack, B.; Ehrlich, K. C.; Wei, Q.; Arroyo-Manzanares, N.; Uka, V.; Vanhaecke, L.; Bhatnagar, D.; Yu, J.; Nierman, W. C.; Johns, M. A.; Sorensen, D.; Shen, H.; De Saeger, S.; Diana Di Mavungu, J.
The global regulatory veA gene governs development and secondary metabolism in numerous fungal species, including Aspergillus flavus. This is especially relevant since A. flavus infects crops of agricultural importance worldwide, contaminating them with potent mycotoxins. The most well-known are aflatoxins, which are cytotoxic and carcinogenic polyketide compounds. The production of aflatoxins and the expression of genes implicated in the production of these mycotoxins are veA dependent. The genes responsible for the synthesis of aflatoxins are clustered, a signature common for genes involved in fungal secondary metabolism. Studies of the A. flavus genome revealed many gene clusters possibly connected to the synthesis of secondary metabolites. Many of these metabolites are still unknown, or the association between a known metabolite and a particular gene cluster has not yet been established. In the present transcriptome study, we show that veA is necessary for the expression of a large number of genes. Twenty-eight out of the predicted 56 secondary metabolite gene clusters include at least one gene that is differentially expressed depending on presence or absence of veA. One of the clusters under the influence of veA is cluster 39. The absence of veA results in a downregulation of the five genes found within this cluster. Interestingly, our results indicate that the cluster is expressed mainly in sclerotia. Chemical analysis of sclerotial extracts revealed that cluster 39 is responsible for the production of aflavarin. PMID:26209694
Cary, J W; Han, Z; Yin, Y; Lohmar, J M; Shantappa, S; Harris-Coward, P Y; Mack, B; Ehrlich, K C; Wei, Q; Arroyo-Manzanares, N; Uka, V; Vanhaecke, L; Bhatnagar, D; Yu, J; Nierman, W C; Johns, M A; Sorensen, D; Shen, H; De Saeger, S; Diana Di Mavungu, J; Calvo, A M
The global regulatory veA gene governs development and secondary metabolism in numerous fungal species, including Aspergillus flavus. This is especially relevant since A. flavus infects crops of agricultural importance worldwide, contaminating them with potent mycotoxins. The most well-known are aflatoxins, which are cytotoxic and carcinogenic polyketide compounds. The production of aflatoxins and the expression of genes implicated in the production of these mycotoxins are veA dependent. The genes responsible for the synthesis of aflatoxins are clustered, a signature common for genes involved in fungal secondary metabolism. Studies of the A. flavus genome revealed many gene clusters possibly connected to the synthesis of secondary metabolites. Many of these metabolites are still unknown, or the association between a known metabolite and a particular gene cluster has not yet been established. In the present transcriptome study, we show that veA is necessary for the expression of a large number of genes. Twenty-eight out of the predicted 56 secondary metabolite gene clusters include at least one gene that is differentially expressed depending on presence or absence of veA. One of the clusters under the influence of veA is cluster 39. The absence of veA results in a downregulation of the five genes found within this cluster. Interestingly, our results indicate that the cluster is expressed mainly in sclerotia. Chemical analysis of sclerotial extracts revealed that cluster 39 is responsible for the production of aflavarin. Copyright © 2015, American Society for Microbiology. All Rights Reserved.
Abbas, Hamed K; Accinelli, Cesare; Shier, W Thomas
Aflatoxin contamination has a major economic impact on crop production in the southern United States. Reduction of aflatoxin contamination in harvested crops has been achieved by applying nonaflatoxigenic biocontrol Aspergillus flavus strains that can out-compete wild aflatoxigenic A. flavus, reducing their numbers at the site of application. Currently, the standard method for applying biocontrol A. flavus strains to soil is using a nutrient-supplying carrier (e.g., pearled barley for Afla-Guard). Granules of Bioplastic (partially acetylated corn starch) have been investigated as an alternative nutritive carrier for biocontrol agents. Bioplastic granules have also been used to prepare a sprayable biocontrol formulation that gives effective reduction of aflatoxin contamination in harvested corn kernels with application of much smaller amounts to leaves later in the growing season. The ultimate goal of biocontrol research is to produce biocontrol systems that can be applied to crops only when long-range weather forecasting indicates they will be needed.
Pichia anomala WRL-076 is a biocontrol yeast which has been shown to inhibit growth and aflatoxin production of A. flavus. Using the SPME-GC/MS analysis we identified that the volatile, 2-phenylethanol (2-PE) produced by this yeast and demonstrated that the compound inhibited aflatoxin production. W...
Cary, Jeffrey W; Harris-Coward, Pamela Y; Ehrlich, Kenneth C; Di Mavungu, José Diana; Malysheva, Svetlana V; De Saeger, Sarah; Dowd, Patrick F; Shantappa, Sourabha; Martens, Stacey L; Calvo, Ana M
The filamentous fungus, Aspergillus flavus, produces the toxic and carcinogenic, polyketide synthase (PKS)-derived family of secondary metabolites termed aflatoxins. While analysis of the A. flavus genome has identified many other PKSs capable of producing secondary metabolites, to date, only a few other metabolites have been identified. In the process of studying how the developmental regulator, VeA, affects A. flavus secondary metabolism we discovered that mutation of veA caused a dramatic down-regulation of transcription of a polyketide synthase gene belonging to cluster 27 and the loss of the ability of the fungi to produce sclerotia. Inactivation of the cluster 27 pks (pks27) resulted in formation of greyish-yellow sclerotia rather than the dark brown sclerotia normally produced by A. flavus while conidial pigmentation was unaffected. One metabolite produced by Pks27 was identified by thin layer chromatography and mass spectral analysis as the known anthraquinone, asparasone A. Sclerotia produced by pks27 mutants were significantly less resistant to insect predation than were the sclerotia produced by the wild-type and more susceptible to the deleterious effects of ultraviolet light and heat. Normal sclerotia were previously thought to be resistant to damage because of a process of melanization similar to that known for pigmentation of conidia. Our results show that the dark brown pigments in sclerotia derive from anthraquinones produced by Pks27 rather than from the typical tetrahydronapthalene melanin production pathway. To our knowledge this is the first report on the genes involved in the biosynthesis of pigments important for sclerotial survival.
Kandhavelu, Jeyalakshmi; Demonte, Naveen Luke; Namperumalsamy, Venkatesh Prajna; Prajna, Lalitha; Thangavel, Chitra; Jayapal, Jeya Maheshwari; Kuppamuthu, Dharmalingam
Aspergillus flavus and Fusarium sp. are primary causative agents of keratitis that results in corneal tissue damage leading to vision loss particularly in individuals from the tropical parts of the world. Proteins in the tear film collected from control and keratitis patients was profiled and compared. A total of 1873 proteins from control and 1400 proteins from patient tear were identified by mass spectrometry. While 847 proteins were found to be glycosylated in the patient tear, only 726 were glycosylated in control tear. And, some of the tear proteins showed alterations in their glycosylation pattern after infection. Complement system proteins, proteins specific for neutrophil extracellular traps and proteins involved in would healing were found only in the patient tear. The presence of these innate immune system proteins in the tear film of patients supports the previous data indicating the involvement of neutrophil and complement pathways in antifungal defense. High levels of wound healing proteins in keratitis patient tear implied activation of tissue repair during infection. The early appearance of the host defense proteins and wound healing response indicates that tear proteins could be used as an early marker system for monitoring the progression of pathogenesis. Identification of negative regulators of the above defense pathways in keratitis tear indicates an intricate balance of pro and anti-defense mechanisms operating in fungal infection of the eye. Tear proteins from control and mycotic keratitis patients were separated into glycoproteins and non-glycosylated proteins and then identified by mass spectrometry. Tear proteins from keratitis patients showed alteration in the glycosylation pattern indicating the alteration of glycosylation machinery due to infection. Neutrophil extracellular traps specific proteins, complement pathway proteins, as well as wound healing proteins, were found only in patient tear showing the activation of antifungal defense
de Alencar Guimaraes, Nelciele Cavalieri; Sorgatto, Michele; Peixoto-Nogueira, Simone de Carvalho; Betini, Jorge Henrique Almeida; Zanoelo, Fabiana Fonseca; Marques, Maria Rita; de Moraes Polizeli, Maria de Lourdes Teixeira; Giannesi, Giovana C
This study compares two xylanases produced by filamentous fungi such as A. niger and A. flavus using agroindustrial residues as substract and evaluated the effect of these enzymes on cellulose pulp biobleaching process. Wheat bran was the best carbon source for xylanase production by A. niger and A. flavus. The production of xylanase was 18 and 21% higher on wheat bran when we compare the xylanase production with xylan. At 50°C, the xylanase of A. niger retained over 85% activity with 2 h of incubation, and A. flavus had a half-life of more than 75 minutes. At 55°C, the xylanase produced by A. niger showed more stable than from A. flavus showing a half-life of more than 45 minutes. The xylanase activity of A. niger and A. flavus were somehow protected in the presence of glycerol 5% when compared to the control (without additives). On the biobleaching assay it was observed that the xylanase from A. flavus was more effective in comparison to A. niger. The kappa efficiency corresponded to 36.32 and 25.93, respectively. That is important to emphasize that the cellulase activity was either analyzed and significant levels were not detected, which explain why the viscosity was not significantly modified.
Fountain, Jake C.; Bajaj, Prasad; Nayak, Spurthi N.; Yang, Liming; Pandey, Manish K.; Kumar, Vinay; Jayale, Ashwin S.; Chitikineni, Anu; Lee, Robert D.; Kemerait, Robert C.; Varshney, Rajeev K.; Guo, Baozhu
The infection of maize and peanut with Aspergillus flavus and subsequent contamination with aflatoxin pose a threat to global food safety and human health, and is exacerbated by drought stress. Drought stress-responding compounds such as reactive oxygen species (ROS) are associated with fungal stress responsive signaling and secondary metabolite production, and can stimulate the production of aflatoxin by A. flavus in vitro. These secondary metabolites have been shown to possess diverse functions in soil-borne fungi including antibiosis, competitive inhibition of other microbes, and abiotic stress alleviation. Previously, we observed that isolates of A. flavus showed differences in oxidative stress tolerance which correlated with their aflatoxin production capabilities. In order to better understand these isolate-specific oxidative stress responses, we examined the transcriptional responses of field isolates of A. flavus with varying levels of aflatoxin production (NRRL3357, AF13, and Tox4) to H2O2-induced oxidative stress using an RNA sequencing approach. These isolates were cultured in an aflatoxin-production conducive medium amended with various levels of H2O2. Whole transcriptomes were sequenced using an Illumina HiSeq platform with an average of 40.43 million filtered paired-end reads generated for each sample. The obtained transcriptomes were then used for differential expression, gene ontology, pathway, and co-expression analyses. Isolates which produced higher levels of aflatoxin tended to exhibit fewer differentially expressed genes than isolates with lower levels of production. Genes found to be differentially expressed in response to increasing oxidative stress included antioxidant enzymes, primary metabolism components, antibiosis-related genes, and secondary metabolite biosynthetic components specifically for aflatoxin, aflatrem, and kojic acid. The expression of fungal development-related genes including aminobenzoate degradation genes and conidiation
Carmo, Egberto Santos; de Oliveira Lima, Edeltrudes; de Souza, Evandro Leite
Origanum vulgare L. (Lamiaceae) has been currently known for their interesting antimicrobial activity being regarded as alternative antimicrobial for use is food conservation systems. This study aimed to evaluate the effectiveness of O. vulgare essential oil in inhibiting the growth of some food-related Aspergillus species (A. flavus, A. parasiticus, A. terreus, A. ochraceus, A. fumigatus and A. niger). The essential oil revealed a strong anti-Aspergillus property providing an inhibition of all assayed mould strains. MIC values were between 80 and 20 μL/mL being found a MIC50 of 40 μL/mL. The essential oil at concentration of 80 and 40 μL/mL provided a fungicidal effect on A. flavus, A. fumigatus and A. niger noted by a total inhibition of the radial mycelial growth along 14 days of interaction. In addition, the essential oil was able to inhibit the mould spores germination when assayed at concentrations of 80 and 40 μL/mL. Our results showed the interesting anti-Aspergillus activity of O. vulgare essential oil supporting their possible use as anti-mould compound in food conservation. PMID:24031231
Mylroie, J. Erik; Ozkan, Seval; Shivaji, Renuka; Windham, Gary L.; Alpe, Michael N.; Williams, W. Paul
Aflatoxins, which are produced by Aspergillus flavus, are toxic to humans, livestock, and pets. The value of maize (Zea mays) grain is markedly reduced when contaminated with aflatoxin. Plant resistance and biological control using non-toxin producing strains are considered effective strategies for reducing aflatoxin accumulation in maize grain. Distinguishing between the toxin and non-toxin producing strains is important in determining the effectiveness of bio-control strategies and understanding inter-strain interactions. Using polymorphisms found in the fungal rRNA intergenic spacer region (IGS) between a toxigenic strain of A. flavus (NRRL 3357) and the non-toxigenic strain used in the biological control agent Afla-Guard® (NRRL 21882), we developed a set of primers that allows for the identification and quantification of the two strains using quantitative PCR. This primer set has been used to screen maize grain that was inoculated with the two strains individually and co-inoculated with both strains, and it has been shown to be effective in both the identification and quantification of both strains. Screening of co-inoculated ears from multiple resistant and susceptible genotypic crosses revealed no significant differences in fungal biomass accumulation of either strain in the field tests from 2010 and 2011 when compared across the means of all genotypes. Only one genotype/year combination showed significant differences in strain accumulation. Aflatoxin accumulation analysis showed that, as expected, genotypes inoculated with the toxigenic strain accumulated more aflatoxin than when co-inoculated with both strains or inoculated with only the non-toxigenic strain. Furthermore, accumulation of toxigenic fungal mass was significantly correlated with aflatoxin accumulation while non-toxigenic fungal accumulation was not. This primer set will allow researchers to better determine how the two fungal strains compete on the maize ear and investigate the interaction
Warburton, Marilyn L.; Williams, William Paul; Hawkins, Leigh; Bridges, Susan; Gresham, Cathy; Harper, Jonathan; Ozkan, Seval; Mylroie, J. Erik; Shan, Xueyan
A public candidate gene testing pipeline for resistance to aflatoxin accumulation or Aspergillus flavus infection in maize is presented here. The pipeline consists of steps for identifying, testing, and verifying the association of selected maize gene sequences with resistance under field conditions. Resources include a database of genetic and protein sequences associated with the reduction in aflatoxin contamination from previous studies; eight diverse inbred maize lines for polymorphism identification within any maize gene sequence; four Quantitative Trait Loci (QTL) mapping populations and one association mapping panel, all phenotyped for aflatoxin accumulation resistance and associated phenotypes; and capacity for Insertion/Deletion (InDel) and SNP genotyping in the population(s) for mapping. To date, ten genes have been identified as possible candidate genes and put through the candidate gene testing pipeline, and results are presented here to demonstrate the utility of the pipeline. PMID:22069738
King, Eileen D; Bobby Bassi, Albeit B; Ross, David C; Druebbisch, Bernd
Several nonaflatoxigenic strains of Aspergillus flavus have been registered in the United States to reduce aflatoxin accumulation in maize and other crops, but there may be unintended negative consequences if these strains produce cyclopiazonic acid (CPA). AF36, a nonaflatoxigenic, CPA-producing strain has been shown to produce CPA in treated maize and peanuts. Alternative strains, including Afla-Guard® brand biocontrol agent and K49, do not produce CPA and can reduce both aflatoxin and CPA in treated crops. Chronic toxicity of CPA has not been studied, and recent animal studies show significant harmful effects from short-term exposure to CPA at low doses. Grower and industry confidence in this approach must be preserved through transparency.
Akar, Tamer; Tunali, Sibel
The Pb(II) and Cu(II) biosorption characteristics of Aspergillus flavus fungal biomass were examined as a function of initial pH, contact time and initial metal ion concentration. Heat inactivated (killed) biomass was used in the determination of optimum conditions before investigating the performance of pretreated biosorbent. The maximum biosorption values were found to be 13.46 +/- 0.99 mg/g for Pb(II) and 10.82 +/- 1.46 mg/g for Cu(II) at pH 5.0 +/- 0.1 with an equilibrium time of 2 h. Detergent, sodium hydroxide and dimethyl sulfoxide pretreatments enhanced the biosorption capacity of biomass in comparison with the heat inactivated biomass. The biosorption data obtained under the optimum conditions were well described by the Freundlich isotherm model. Competitive biosorption of Pb(II) and Cu(II) ions was also investigated to determine the selectivity of the biomass. The results indicated that A. flavus is a suitable biosorbent for the removal of Pb(II) and Cu(II) ions from aqueous solution.
Falade, Titilayo D O; Syed Mohdhamdan, Sharifah H; Sultanbawa, Yasmina; Fletcher, Mary T; Harvey, Jagger J W; Chaliha, Mridusmita; Fox, Glen P
In vitro experimental environments are used to study interactions between microorganisms, and to predict dynamics in natural ecosystems. This study highlights that experimental in vitro environments should be selected to match closely the natural environment of interest during in vitro studies to strengthen extrapolations about aflatoxin production by Aspergillus and competing organisms. Fungal competition and aflatoxin accumulation were studied in soil, cotton wool or tube (water-only) environments, for Aspergillus flavus competition with Penicillium purpurogenum, Fusarium oxysporum or Sarocladium zeae within maize grains. Inoculated grains were incubated in each environment at two temperature regimes (25 and 30°C). Competition experiments showed interaction between the main effects of aflatoxin accumulation and the environment at 25°C, but not so at 30°C. However, competition experiments showed fungal populations were always interacting with their environments. Fungal survival differed after the 72-h incubation in different experimental environments. Whereas all fungi incubated within the soil environment survived, in the cotton wool environment none of the competitors of A. flavus survived at 30°C. With aflatoxin accumulation, F. oxysporum was the only fungus able to interdict aflatoxin production at both temperatures. This occurred only in the soil environment and fumonisins accumulated instead. Smallholder farmers in developing countries face serious mycotoxin contamination of their grains, and soil is a natural reservoir for the associated fungal propagules, and a drying and storage surface for grains on these farms. Studying fungal dynamics in the soil environment and other environments in vitro can provide insights into aflatoxin accumulation post-harvest.
Dhaliwal, Manpreet; Chakrabarti, Arunaloke; Mouton, Johan W.
ABSTRACT Invasive aspergillosis (IA) due to Aspergillus flavus is associated with high mortality. Although voriconazole (VRC) is widely recommended as the first-line treatment for IA, emergence of azole resistance in Aspergillus spp. is translating to treatment failure. We evaluated the efficacy of voriconazole in a nonneutropenic murine model of disseminated A. flavus infection using two voriconazole-resistant isolates (one harboring the Y319H substitution in the cyp51C gene) and two wild-type isolates without mutations. All isolates exhibited a dose-response relationship, and voriconazole treatment improved mouse survival in a dose-dependent manner. At 40 mg/kg of body weight, 100% efficacy was observed for 1 susceptible isolate and 1 resistant isolate (with mutation), whereas for another susceptible isolate and resistant isolate (without mutation), survival rates were 81% and 72%, respectively. The Hill equation with a variable slope fitted the relationship between the area under the concentration-time curve (AUC)/MIC ratio and 14-day survival well for each strain. An F test showed the 50% effective doses to be significantly different from each other (P = 0.0023). However, contrary to expectation, there was a significant difference in exposure-response relationships between strains, and it appeared that the susceptible strains required a relatively higher exposure than the resistant ones to result in the same treatment effect, the 50% effective pharmacokinetic/pharmacodynamic (PK/PD) index (EI50) required being negatively and log-linearly related to the MIC (P = 0.04). We conclude that the efficacy of voriconazole depended on drug exposure and the voriconazole MIC of the isolates, but lower exposures are required for strains with higher MICs. These findings may have profound significance in clinical practice with respect to dosing and drug choice. PMID:27821453
Rudramurthy, Shivaprakash M; Seyedmousavi, Seyedmojtaba; Dhaliwal, Manpreet; Chakrabarti, Arunaloke; Meis, Jacques F; Mouton, Johan W
Invasive aspergillosis (IA) due to Aspergillus flavus is associated with high mortality. Although voriconazole (VRC) is widely recommended as the first-line treatment for IA, emergence of azole resistance in Aspergillus spp. is translating to treatment failure. We evaluated the efficacy of voriconazole in a nonneutropenic murine model of disseminated A. flavus infection using two voriconazole-resistant isolates (one harboring the Y319H substitution in the cyp51C gene) and two wild-type isolates without mutations. All isolates exhibited a dose-response relationship, and voriconazole treatment improved mouse survival in a dose-dependent manner. At 40 mg/kg of body weight, 100% efficacy was observed for 1 susceptible isolate and 1 resistant isolate (with mutation), whereas for another susceptible isolate and resistant isolate (without mutation), survival rates were 81% and 72%, respectively. The Hill equation with a variable slope fitted the relationship between the area under the concentration-time curve (AUC)/MIC ratio and 14-day survival well for each strain. An F test showed the 50% effective doses to be significantly different from each other (P = 0.0023). However, contrary to expectation, there was a significant difference in exposure-response relationships between strains, and it appeared that the susceptible strains required a relatively higher exposure than the resistant ones to result in the same treatment effect, the 50% effective pharmacokinetic/pharmacodynamic (PK/PD) index (EI50) required being negatively and log-linearly related to the MIC (P = 0.04). We conclude that the efficacy of voriconazole depended on drug exposure and the voriconazole MIC of the isolates, but lower exposures are required for strains with higher MICs. These findings may have profound significance in clinical practice with respect to dosing and drug choice. Copyright © 2016 American Society for Microbiology.
Cotty, Peter J.
Some filamentous fungi in Aspergillus section Flavi produce carcinogenic secondary compounds called aflatoxins. Aflatoxin contamination is routinely managed in commercial agriculture with strains of Aspergillus flavus that do not produce aflatoxins. These non-aflatoxin-producing strains competitively exclude aflatoxin producers and reshape fungal communities so that strains with the aflatoxin-producing phenotype are less frequent. This study evaluated the genetic variation within naturally occurring atoxigenic A. flavus strains from the endemic vegetative compatibility group (VCG) YV36. AF36 is a strain of VCG YV36 and was the first fungus used in agriculture for aflatoxin management. Genetic analyses based on mating-type loci, 21 microsatellite loci, and a single nucleotide polymorphism (SNP) in the aflC gene were applied to a set of 237 YV36 isolates collected from 1990 through 2005 from desert legumes and untreated fields and from fields previously treated with AF36 across the southern United States. One haplotype dominated across time and space. No recombination with strains belonging to VCGs other than YV36 was detected. All YV36 isolates carried the SNP in aflC that prevents aflatoxin biosynthesis and the mat1-2 idiomorph at the mating-type locus. These results suggest that VCG YV36 has a clonal population structure maintained across both time and space. These results demonstrate the genetic stability of atoxigenic strains belonging to a broadly distributed endemic VCG in both untreated populations and populations where the short-term frequency of VCG YV36 has increased due to applications of a strain used to competitively exclude aflatoxin producers. This work supports the hypothesis that strains of this VCG are not involved in routine genetic exchange with aflatoxin-producing strains. PMID:26092465
Grubisha, Lisa C; Cotty, Peter J
Some filamentous fungi in Aspergillus section Flavi produce carcinogenic secondary compounds called aflatoxins. Aflatoxin contamination is routinely managed in commercial agriculture with strains of Aspergillus flavus that do not produce aflatoxins. These non-aflatoxin-producing strains competitively exclude aflatoxin producers and reshape fungal communities so that strains with the aflatoxin-producing phenotype are less frequent. This study evaluated the genetic variation within naturally occurring atoxigenic A. flavus strains from the endemic vegetative compatibility group (VCG) YV36. AF36 is a strain of VCG YV36 and was the first fungus used in agriculture for aflatoxin management. Genetic analyses based on mating-type loci, 21 microsatellite loci, and a single nucleotide polymorphism (SNP) in the aflC gene were applied to a set of 237 YV36 isolates collected from 1990 through 2005 from desert legumes and untreated fields and from fields previously treated with AF36 across the southern United States. One haplotype dominated across time and space. No recombination with strains belonging to VCGs other than YV36 was detected. All YV36 isolates carried the SNP in aflC that prevents aflatoxin biosynthesis and the mat1-2 idiomorph at the mating-type locus. These results suggest that VCG YV36 has a clonal population structure maintained across both time and space. These results demonstrate the genetic stability of atoxigenic strains belonging to a broadly distributed endemic VCG in both untreated populations and populations where the short-term frequency of VCG YV36 has increased due to applications of a strain used to competitively exclude aflatoxin producers. This work supports the hypothesis that strains of this VCG are not involved in routine genetic exchange with aflatoxin-producing strains.
The objectives of this study were to probe the effect of the yeast, P. anomala against A flavus by using real time RT-PCR technique and vitality fluorescent stains. Yeast and fungi were inoculated into a 250 ml-flask containing 50 ml potato dextrose broth (PDB) at yeast to fungus (Y : F) ratios of ...
Khosravi, A.R.; Minooeianhaghighi, M.H.; Shokri, H.; Emami, S.A.; S.M., Alavi; Asili, J.
The goals of this study were to evaluate the effectiveness of Cuminum cyminum, Ziziphora clinopodioides and Nigella sativa essential oils to inhibit the growth of Aspergillus fumigatus and A. flavus and to evoke ultrastructural changes. The fungi were cultured into RPMI 1640 media in the presence of oils at concentrations of 8, 6, 5, 4, 3, 2, 1.5, 1.25, 1, 0.75 and 0.5 mg/ml in broth microdilution and 2, 1.5, 1 and 0.5 mg/ml in broth macrodilution methods with shaking for 48 h at 28oC. Conidial and mycelial samples exposed to 0.25, 0.5, 1, 1.5 and 2 mg essential oils/ml for 5 days in 2% yeast extract granulated plus 15% Saccharose media were processed for transmission electron microscopy (TEM). Based on broth dilution methods, C. cyminum and to a lesser extent Z. clinopodioides oils exhibited the strongest activity against A. fumigatus and A. flavus with MIC90 ranging from 0.25 to 1.5 mg/ml, while the oil from N. sativa exhibited relatively moderate activity against two above fungi with MIC90 ranging from 1.5 to 2 mg/ml. The main changes observed by TEM were in the cell wall, plasma membrane and membranous organelles; in particular, in the nuclei and mitochondria. These modifications in fungal structure were associated with the interference of the essential oils with the enzymes responsible for cell wall synthesis, which disturbed normal growth. Moreover, the essential oils caused high vacuolation of the cytoplasm, detachment of fibrillar layer of cell wall, plasma membrane disruption and disorganization of the nuclear and mitochondrial structures. Aspergillus fumigatus and A. flavus growth inhibition induced by these oils were found to be well-correlated with subsequent morphological changes of the fungi exposed to different fungistatic concentrations of the oils. Our results show the anti-Aspergillus activities of C. cyminum, Z. clinopodioides and N. sativa essential oils, which strengthens the potential use of these substances as anti-mould in the future. PMID
Khosravi, A R; Minooeianhaghighi, M H; Shokri, H; Emami, S A; S M, Alavi; Asili, J
The goals of this study were to evaluate the effectiveness of Cuminum cyminum, Ziziphora clinopodioides and Nigella sativa essential oils to inhibit the growth of Aspergillus fumigatus and A. flavus and to evoke ultrastructural changes. The fungi were cultured into RPMI 1640 media in the presence of oils at concentrations of 8, 6, 5, 4, 3, 2, 1.5, 1.25, 1, 0.75 and 0.5 mg/ml in broth microdilution and 2, 1.5, 1 and 0.5 mg/ml in broth macrodilution methods with shaking for 48 h at 28(o)C. Conidial and mycelial samples exposed to 0.25, 0.5, 1, 1.5 and 2 mg essential oils/ml for 5 days in 2% yeast extract granulated plus 15% Saccharose media were processed for transmission electron microscopy (TEM). Based on broth dilution methods, C. cyminum and to a lesser extent Z. clinopodioides oils exhibited the strongest activity against A. fumigatus and A. flavus with MIC90 ranging from 0.25 to 1.5 mg/ml, while the oil from N. sativa exhibited relatively moderate activity against two above fungi with MIC90 ranging from 1.5 to 2 mg/ml. The main changes observed by TEM were in the cell wall, plasma membrane and membranous organelles; in particular, in the nuclei and mitochondria. These modifications in fungal structure were associated with the interference of the essential oils with the enzymes responsible for cell wall synthesis, which disturbed normal growth. Moreover, the essential oils caused high vacuolation of the cytoplasm, detachment of fibrillar layer of cell wall, plasma membrane disruption and disorganization of the nuclear and mitochondrial structures. Aspergillus fumigatus and A. flavus growth inhibition induced by these oils were found to be well-correlated with subsequent morphological changes of the fungi exposed to different fungistatic concentrations of the oils. Our results show the anti-Aspergillus activities of C. cyminum, Z. clinopodioides and N. sativa essential oils, which strengthens the potential use of these substances as anti-mould in the future.
Application of loop-mediated isothermal amplification assays for direct identification of pure cultures of Aspergillus flavus, A. nomius, and A. caelatus and for their rapid detection in shelled Brazil nuts.
Luo, Jie; Taniwaki, Marta H; Iamanaka, Beatriz T; Vogel, Rudi F; Niessen, Ludwig
Brazil nuts have a high nutritional content and are a very important trade commodity for some Latin American countries. Aflatoxins are carcinogenic fungal secondary metabolites. In Brazil nuts they are produced predominantly by Aspergillus (A.) nomius and A. flavus. In the present study we applied and evaluated two sets of primers previously published for the specific detection of the two species using loop-mediated isothermal amplification (LAMP) technology. Moreover, a primer set specific for A. caelatus as a frequently occurring non-aflatoxigenic member of Aspergillus section Flavi in Brazil nuts was newly developed. LAMP assays were combined with a simplified DNA release method and used for rapid identification of pure cultures and rapid detection of A. nomius and A. flavus from samples of shelled Brazil nuts. An analysis of pure cultures of 68 isolates representing the major Aspergillus species occurring on Brazil nuts showed that the three LAMP assays had individual accuracies of 61.5%, 84.4%, and 93.3% for A. flavus, A. nomius, and A. caelatus, respectively when morphological identification was used as a reference. The detection limits for conidia added directly to the individual LAMP reactions were found to be 10⁵ conidia per reaction with the primer set ID9 for A. nomius and 10⁴ conidia per reaction with the primer set ID58 for A. flavus. Sensitivity was increased to 10¹ and 10² conidia per reaction for A. nomius and A. flavus, respectively, when sample preparation included a spore disruption step. The results of LAMP assays obtained during the analysis of 32 Brazil nut samples from different regions of Brazil and from different steps in the production process of the commodity were compared with results obtained from mycological analysis and aflatoxin analysis of corresponding samples. Compared with mycological analysis of the samples, the Negative Predictive Values of LAMP assays were 42.1% and 12.5% while the Positive Predictive Values were 61
Gemeda, Negero; Woldeamanuel, Yimtubezinash; Asrat, Daniel; Debella, Asfaw
Objective To investigate effect of essential oils on Aspergillus spore germination, growth and mycotoxin production. Method In vitro antifungal and antiaflatoxigenic activity of essential oils was carried out using poisoned food techniques, spore germination assay, agar dilution assay, and aflatoxin arresting assay on toxigenic strains of Aspergillus species. Results Cymbopogon martinii, Foeniculum vulgare and Trachyspermum ammi (T. ammi) essential oils were tested against toxicogenic isolates of Aspergillus species. T. ammi oil showed highest antifungal activity. Absolute mycelial inhibition was recorded at 1 µl/mL by essential oils of T. ammi. The oil also showed, complete inhibition of spore germination at a concentration of 2 µl/mL. In addition, T. ammi oil showed significant antiaflatoxigenic potency by totally inhibiting aflatoxin production from Aspergillus niger and Aspergillus flavus at 0.5 and 0.75 µl/mL, respectively. Cymbopogon martinii, Foeniculum vulgare and T. ammi oils as antifungal were found superior over synthetic preservative. Moreover, a concentration of 5 336.297 µl/kg body weight was recorded for LC50 on mice indicating the low mammalian toxicity and strengthening its traditional reputations. Conclusions In conclusion, the essential oils from T. ammi can be a potential source of safe natural food preservative for food commodities contamination by storage fungi. PMID:25183114
Gemeda, Negero; Woldeamanuel, Yimtubezinash; Asrat, Daniel; Debella, Asfaw
To investigate effect of essential oils on Aspergillus spore germination, growth and mycotoxin production. In vitro antifungal and antiaflatoxigenic activity of essential oils was carried out using poisoned food techniques, spore germination assay, agar dilution assay, and aflatoxin arresting assay on toxigenic strains of Aspergillus species. Cymbopogon martinii, Foeniculum vulgare and Trachyspermum ammi (T. ammi) essential oils were tested against toxicogenic isolates of Aspergillus species. T. ammi oil showed highest antifungal activity. Absolute mycelial inhibition was recorded at 1 µl/mL by essential oils of T. ammi. The oil also showed, complete inhibition of spore germination at a concentration of 2 µl/mL. In addition, T. ammi oil showed significant antiaflatoxigenic potency by totally inhibiting aflatoxin production from Aspergillus niger and Aspergillus flavus at 0.5 and 0.75 µl/mL, respectively. Cymbopogon martinii, Foeniculum vulgare and T. ammi oils as antifungal were found superior over synthetic preservative. Moreover, a concentration of 5 336.297 µl/kg body weight was recorded for LC50 on mice indicating the low mammalian toxicity and strengthening its traditional reputations. In conclusion, the essential oils from T. ammi can be a potential source of safe natural food preservative for food commodities contamination by storage fungi.
Camiletti, Boris X; Asensio, Claudia M; Pecci, María de la Paz Giménez; Lucini, Enrique I
The objective in this study was to evaluate the antifungal activity of essential oils from native and commercial aromatic plants grown in Argentina against corn postharvest fungi and to link the essential oil bioactivity with lipid oxidation and morphological changes in fungus cell membrane. Essential oil (EO) of oregano variety Mendocino (OMen), Cordobes (OCor), and Compacto (OCom), mint variety Inglesa (Mi), and Pehaujo (Mp), Suico (Sui); rosemary (Ro), and Aguaribay (Ag) were tested in vitro against 4 corn fungi: A. flavus (CCC116-83 and BXC01), P. oxalicum (083296), and P. minioluteum (BXC03). The minimum fungicidal concentration (MFC) and the minimum inhibitory concentration (MIC) were determined. The chemical profiles of the EOs were analyzed by GC-MS. Lipid oxidation in cell membrane of fungi was determined by hydroperoxides and related with essential oil antifungal activity. The major compounds were Thymol in OCor (18.66%), Omen (12.18%), and OCom (9.44%); menthol in Mi and Mp; verbenone in Sui; dehydroxy-isocalamendiol in Ag; and eucaliptol in Ro. OCor, Omen, and OCom showed the best antifungal activity. No antifungal activity was observed in Ag and Ro EO. The hydroperoxide value depended on the fungi (P < 0.001) and the antimicrobial agent (P < 0.001).Membrane lipids were oxidized by Sui EO in A. flavus BXC01 and A. flavus CCC116-83 (0.021 and 0.027 meqO2 /kg, respectively). The results suggest that the EOs of OCor, OMen, OCom, Mi, Mp, and Sui grown in Argentina can be used as natural alternatives to control fungi that produce mycotoxin in maize. © 2014 Institute of Food Technologists®
Atehnkeng, Joseph; Donner, Matthias; Ojiambo, Peter S; Ikotun, Babatunde; Augusto, Joao; Cotty, Peter J; Bandyopadhyay, Ranajit
Maize infected by aflatoxin-producing Aspergillus flavus may become contaminated with aflatoxins, and as a result, threaten human health, food security and farmers' income in developing countries where maize is a staple. Environmental distribution and genetic diversity of A. flavus can influence the effectiveness of atoxigenic isolates in mitigating aflatoxin contamination. However, such information has not been used to facilitate selection and deployment of atoxigenic isolates. A total of 35 isolates of A. flavus isolated from maize samples collected from three agro-ecological zones of Nigeria were used in this study. Ecophysiological characteristics, distribution and genetic diversity of the isolates were determined to identify vegetative compatibility groups (VCGs). The generated data were used to inform selection and deployment of native atoxigenic isolates to mitigate aflatoxin contamination in maize. In co-inoculation with toxigenic isolates, atoxigenic isolates reduced aflatoxin contamination in grain by > 96%. A total of 25 VCGs were inferred from the collected isolates based on complementation tests involving nitrate non-utilizing (nit(-)) mutants. To determine genetic diversity and distribution of VCGs across agro-ecological zones, 832 nit(-) mutants from 52 locations in 11 administrative districts were paired with one self-complementary nitrate auxotroph tester-pair for each VCG. Atoxigenic VCGs accounted for 81.1% of the 153 positive complementations recorded. Genetic diversity of VCGs was highest in the derived savannah agro-ecological zone (H = 2.61) compared with the southern Guinea savannah (H = 1.90) and northern Guinea savannah (H = 0.94) zones. Genetic richness (H = 2.60) and evenness (E5 = 0.96) of VCGs were high across all agro-ecological zones. Ten VCGs (40%) had members restricted to the original location of isolation, whereas 15 VCGs (60%) had members located between the original source of isolation and a distance
Conversion of O-methylsterigmatocystin (OMST) to aflatoxin B1 (AFB1), a highly toxic and carcinogenic fungal metabolite of some Aspergillus species, begins with its oxidation catalyzed by the cytochrome P450 monooxygenase, OrdA (AflQ). The complexity of the subsequent oxidation, hydration, ring-ope...
Species of Aspergillus produce a diverse array of secondary metabolites, and recent genomic analysis has predicted that these species have the capacity to synthesize many more compounds. It has been possible to infer the presence of 55 gene clusters associated with secondary metabolism in Aspergill...
Aflatoxins are naturally occurring and carcinogenic mycotoxins produced by several members of Aspergillus section Flavi. These potent toxins frequently contaminate maize in warm production areas. Maize provides over half the caloric intake for the majority of the population of Mexico. However, most ...
Members of the Aspergillus section Nigri, known as black-spored aspergilli, can contaminate several substrates including maize. Although some species within the group can produce plant disease symptoms such as black mold in onions and maize ear rot, the main concern with A. niger aggregate contamina...
Efficacy of Some Essential Oils Against Aspergillus flavus with Special Reference to Lippia alba Oil an Inhibitor of Fungal Proliferation and Aflatoxin B1 Production in Green Gram Seeds during Storage.
Pandey, Abhay K; Sonker, Nivedita; Singh, Pooja
During mycofloral analysis of green gram (Vigna radiata (L.) R. Wilczek) seed samples taken from different grocery stores by agar and standard blotter paper methods, 5 fungal species were identified, of which Aspergillus flavus exhibited higher relative frequency (75.20% to 80.60%) and was found to produce aflatoxin B1 . On screening of 11 plant essential oils against this mycotoxigenic fungi, Lippia alba essential oil was found to be most effective and showed absolute inhibition of mycelia growth at 0.28 μL/mL. The oil of L. alba was fungistatic and fungicidal at 0.14 and 0.28 μL/mL, respectively. Oil had broad range of fungitoxicity at its MIC value and was absolutely inhibited the AFB1 production level at 2.0 μL/mL. Chemical analysis of this oil revealed geranial (36.9%) and neral (29.3%) as major components followed by myrcene (18.6%). Application of a dose of 80 μL/0.25 L air of Lippia oil in the storage system significantly inhibited the fungal proliferation and aflatoxin production without affecting the seed germination rate. By the virtue of fungicidal, antiaflatoxigenic nature and potent efficacy in storage food system, L. alba oil can be commercialized as botanical fungicide for the protection of green gram seeds during storage.
Lanubile, Alessandra; Maschietto, Valentina; De Leonardis, Silvana; Battilani, Paola; Paciolla, Costantino; Marocco, Adriano
Developing kernels of resistant and susceptible maize genotypes were inoculated with Fusarium proliferatum, F. subglutinans, and Aspergillus flavus. Selected defense systems were investigated using real-time reverse transcription-polymerase chain reaction to monitor the expression of pathogenesis-related (PR) genes (PR1, PR5, PRm3, PRm6) and genes protective from oxidative stress (peroxidase, catalase, superoxide dismutase and ascorbate peroxidase) at 72 h postinoculation. The study was also extended to the analysis of the ascorbate-glutathione cycle and catalase, superoxide dismutase, and cytosolic and wall peroxidases enzymes. Furthermore, the hydrogen peroxide and malondialdehyde contents were studied to evaluate the oxidation level. Higher gene expression and enzymatic activities were observed in uninoculated kernels of resistant line, conferring a major readiness to the pathogen attack. Moreover expression values of PR genes remained higher in the resistant line after inoculation, demonstrating a potentiated response to the pathogen invasions. In contrast, reactive oxygen species-scavenging genes were strongly induced in the susceptible line only after pathogen inoculation, although their enzymatic activity was higher in the resistant line. Our data provide an important basis for further investigation of defense gene functions in developing kernels in order to improve resistance to fungal pathogens. Maize genotypes with overexpressed resistance traits could be profitably utilized in breeding programs focused on resistance to pathogens and grain safety.
Padhi, Somanath; Uppin, Shantveet G; Uppin, Megha S; Umabala, P; Challa, Sundaram; Laxmi, V; Prasad, V B N
Mycetoma is a chronic suppurative and/or granulomatous inflammatory lesion of skin, subcutaneous tissue, fascia, and tendons caused by the traumatic inoculation of either fungal (eumycotic) or bacterial (actinomycotic) organisms present in the soil. The disease is characterized by triad of tumefaction, discharging sinuses, and grains. Thirteen new cases of biopsy proven mycetomas were analyzed, retrospectively, from January 2000 to October 2009. Clinical parameters, bone involvement, microbiological properties, and histopathological features were evaluated. Categorization into eumycotic or actinomycotic was based upon features on hematoxylin and eosin stained sections with special stains. Therapeutic outcome was presented wherever available. There were eight actinomycetomas and five eumycetoma cases including 11 men and two women. Foot and lower extremities were the most common site of involvement (9 of 13, 69%). Culture results were available in 8 of 13 cases (61.5%). Madurella mycetomatis, Neoscytalidium dimidiatum, and Aspergillus flavus were the isolates among eumycetomas whereas Acinomadura madurae, Actinomadura pelletieri, and Nocardia species were the isolates among actinomycetomas. Two cases had underlying bone involvement. On follow-up, four of five eumycetoma cases showed partial improvement following surgery and antifungal therapy, one had amputation of the lower leg. Of the actinomycetomas, six of eight had dramatic improvement following sulfamethoxazole-trimethoprim based therapy, one had complete cure, and one was lost to follow-up. Strong clinical suspicion, exact categorization of lesion into eumycotic or actinomycotic along with culture correlation, is essential for prognosis and effective therapy. © 2010 The International Society of Dermatology.
Astoreca, Andrea; Vaamonde, Graciela; Dalcero, Ana; Marin, Sonia; Ramos, Antonio
The objectives of this study were i) to determine the effects of the interactions of water activity, temperature and incubation time on the co-production of AFB1 and CPA by isolates of Aspergillus flavus with different profile of mycotoxin production and ii) to identify the aW and temperature limiting conditions for the production of both mycotoxins. Fungi used in this study were selected because they belonged to different chemotypes: chemotype I (AFB1+/CPA+), III (AFB1+/CPA-) and IV (AFB1-/CPA+), respectively. Two culture media were used; Czapek yeast agar (CYA) and corn extract agar (CEM), at different incubated temperatures (10-40 °C) and aW levels (0.80-0.98). AFB1 and CPA production were analyzed after 7, 14, 21 and 28 days of incubation. Significant differences were observed with respect to mycotoxin production depending on the media evaluated. The AFB1 production occurred more favorably on CYA while the highest CPA concentrations were recorded on CEM. Within the range of aW evaluated in this study, 0.83 was the limiting level for both toxins production. The optimum conditions for AFB1 production occurred at 0.96 aW and 30 °C after 21 days of incubation, regardless of the media and isolate. Although different amounts of toxins were produced in each medium, the limiting and optimum conditions for their production were similar in both. No differences in the response of the three isolates to the abiotic factors discussed were observed despite belonging to different chemotypes. The determination of the thresholds of mycotoxins co-production, especially in the case of data obtained with the corn extract medium can be useful to avoid the conditions conducive to co-occurrence of these mycotoxins in corn.
Carmo, Egberto Santos; de Oliveira Lima, Edeltrudes; de Souza, Evandro Leite; de Sousa, Frederico Barbosa
Cinnamomum zeylanicum Blume is known for a wide range of medicinal properties. This study aimed to assess the interference of C. zeylanicum essential oil on the growth and morphogenesis of some potentially pathogenic Aspergillus species. The essential oil presented strong antifungal effect causing the growth inhibition of the assayed strains and development of large growth inhibition zones. MIC50 and MIC90 values were 40 and 80 μL/mL, respectively. 80, 40 and 20 μL/mL of the oil strongly inhibited the radial mycelial growth of A. niger, A. flavus and A. fumigatus along 14 days. 80 and 40 μL/mL of the oil caused a 100% inhibition of the fungal spore germination. Main morphological changes observed under light microscopy provided by the essential oil in the fungal strains were decreased conidiation, leakage of cytoplasm, loss of pigmentation and disrupted cell structure indicating fungal wall degeneration. It is concluded that C. zeylanicum essential oil could be known as potential antifungal compound, particularly, to protect against the growth of Aspergillus species. PMID:24031186
Carmo, Egberto Santos; de Oliveira Lima, Edeltrudes; de Souza, Evandro Leite; de Sousa, Frederico Barbosa
Cinnamomum zeylanicum Blume is known for a wide range of medicinal properties. This study aimed to assess the interference of C. zeylanicum essential oil on the growth and morphogenesis of some potentially pathogenic Aspergillus species. The essential oil presented strong antifungal effect causing the growth inhibition of the assayed strains and development of large growth inhibition zones. MIC50 and MIC90 values were 40 and 80 μL/mL, respectively. 80, 40 and 20 μL/mL of the oil strongly inhibited the radial mycelial growth of A. niger, A. flavus and A. fumigatus along 14 days. 80 and 40 μL/mL of the oil caused a 100% inhibition of the fungal spore germination. Main morphological changes observed under light microscopy provided by the essential oil in the fungal strains were decreased conidiation, leakage of cytoplasm, loss of pigmentation and disrupted cell structure indicating fungal wall degeneration. It is concluded that C. zeylanicum essential oil could be known as potential antifungal compound, particularly, to protect against the growth of Aspergillus species.
The objectives of this study were (1) to evaluate differential gene expression levels for resistance to A. flavus kernel infection in susceptible (Va35) and resistant (Mp313E) maize lines using Oligonucleotide and cDNA microarray analysis, (2) to evaluate differences in A. flavus accumulation betwee...
Survival of fungal species depends on the ability of these organisms to respond to environmental stresses. Osmotic stress or high levels of reactive oxygen species (ROS) can cause stress in fungi resulting in growth inhibition. Both eukaryotic and prokaryotic cells have developed numerous mechanisms...
Aflatoxins are the most toxic and carcinogenic secondary metabolites produced primarily by the filamentous fungi Aspergillus flavus and Aspergillus parasiticus. The toxins cause devastating economic losses because of strict regulations on distribution of contaminated products. Aspergillus sojae are...
Korani, Walid Ahmed; Chu, Ye; Holbrook, Corley; Clevenger, Josh; Ozias-Akins, Peggy
Aflatoxin contamination is a major economic and food safety concern for the peanut industry that largely could be mitigated by genetic resistance. To screen peanut for aflatoxin resistance, ten genotypes were infected with a green fluorescent protein (GFP)-expressing Aspergillus flavus strain. Percentages of fungal infected area and fungal GFP signal intensity were documented by visual ratings every 8 h for 72 h after inoculation. Significant genotypic differences in fungal growth rates were documented by repeated measures and area under the disease progress curve (AUDPC) analyses. SICIA (Seed Infection Coverage and Intensity Analyzer), an image processing software, was developed to digitize fungal GFP signals. Data from SICIA image analysis confirmed visual rating results validating its utility for quantifying fungal growth. Among the tested peanut genotypes, NC 3033 and GT-C20 supported the lowest and highest fungal growth on the surface of peanut seeds, respectively. Although differential fungal growth was observed on the surface of peanut seeds, total fungal growth in the seeds was not significantly different across genotypes based on a fluorometric GFP assay. Significant differences in aflatoxin B levels were detected across peanut genotypes. ICG 1471 had the lowest aflatoxin level whereas Florida-07 had the highest. Two-year aflatoxin tests under simulated late-season drought also showed that ICG 1471 had reduced aflatoxin production under pre-harvest field conditions. These results suggest that all peanut genotypes support A. flavus fungal growth yet differentially influence aflatoxin production.
Detection of Aspergillus flavus and A. fumigatus in Bronchoalveolar Lavage Specimens of Hematopoietic Stem Cell Transplants and Hematological Malignancies Patients by Real-Time Polymerase Chain Reaction, Nested PCR and Mycological Assays.
Zarrinfar, Hossein; Mirhendi, Hossein; Fata, Abdolmajid; Khodadadi, Hossein; Kordbacheh, Parivash
Pulmonary aspergillosis (PA) is one of the most serious complications in immunocompromised patients, in particular among hematopoietic stem cell transplants (HSCT) and patients with hematological malignancies. The current study aimed to evaluate the incidence of PA and utility of molecular methods in HSCT and patients with hematological malignancies, four methods including direct examination, culture, nested polymerase chain reaction (PCR) and real-time PCR were performed on bronchoalveolar lavage (BAL) specimens in Tehran, Iran. During 16 months, 46 BAL specimens were obtained from individuals with allogeneic HSCT (n = 18) and patients with hematological malignancies (n = 28). Direct wet mounts with 20% potassium hydroxide (KOH) and culture on mycological media were performed. The molecular detection of Aspergillus fumigatus and A. flavus was done by amplifying the conserved sequences of internal transcribed spacer 1 (ITS1) ribosomal DNA by nested-PCR and the β-tubulin gene by TaqMan real-time PCR. Seven (15.2%) out of 46 specimens were positive in direct examination and showed branched septate hyphae; 11 (23.9%) had positive culture including eight (72.7%) A. flavus and three (27.3%) A. fumigatus; 22 (47.8%) had positive nested-PCR and eight (17.4%) had positive real-time PCR. The incidence of invasive pulmonary aspergillosis (IPA) in these patients included proven IPA in 1 (2.2%), probable IPA in 10 (21.7%), possible IPA in 19 (41.3%) and not IPA in 16 cases (34.8%). The incidence of IPA in allogeneic HSCT and patients with hematological malignancies was relatively high and A. flavus was the most common cause of PA. As molecular methods had higher sensitivity, it may be useful as screening methods in HSCT and patients with hematological malignancies, or to determine when empirical antifungal therapy can be withheld.
A gene co-expression network was generated using a dual RNA-seq study with the fungal pathogen A. flavus and its plant host Z. mays during the initial 3 days of infection. The analysis deciphered novel pathways and mapped genes of interest in both organisms during the infection. This network reveal...
Woldeamanuel, Yimtubezinash; Asrat, Daniel; Debella, Asfaw
This study was performed to investigate effect of essential oils on Aspergillus spore germination, growth, and mycotoxin production. In vitro antifungal and antiaflatoxigenic activities of Cymbopogon martinii, Foeniculum vulgare, and Trachyspermum ammi essential oils were carried out on toxigenic strains of Aspergillus species. Plant materials were hydrodistilled for 4-5 h in Clevenger apparatus. 0.25 μL/mL, 0.5 μL/mL, 1 μL/mL, 2 μL/mL, and 4 μL/mL concentrations of each essential oil were prepared in 0.1% Tween 80 (V/V). T. ammi oil showed highest antifungal activity. Absolute mycelial inhibition was recorded at 1 μL/mL by essential oils of T. ammi. The oil also showed complete inhibition of spore germination at a concentration of 2 μL/mL. In addition, T. ammi oil showed significant antiaflatoxigenic potency by totally inhibiting toxin production from A. niger and A. flavus at 0.5 and 0.75 μL/mL, respectively. C. martinii, F. vulgare, and T. ammi oils as antifungals were found superior over synthetic preservative. Moreover, a concentration of 5336.297 μL/kg body weight was recorded for LC50 on mice indicating the low mammalian toxicity. In conclusion, the essential oils from T. ammi can be a potential source of safe natural food preservative for food commodities contamination by Aspergillus species. PMID:26904653
Gómez-Ramírez, C; Sosa-Morales, M E; Palou, E; López-Malo, A
Individual and combined effects of aw and incorporation of selected concentrations of Mexican oregano essential oil on the time to growth (TTG) of Aspergillus niger intentionally inoculated into dried tomatoes were studied during storage at 25°C for 100 days. For aw 0.96, 1,000 ppm of Mexican oregano essential oil inhibited A. niger growth during 100 days, whereas 500 ppm were sufficient at aw 0.91 and 250 ppm for tomatoes with aw 0.78. A. niger growth was evident at different incubation times depending on tested tomato aw and concentration of essential oil; these data were utilized to model TTG. Regression analysis revealed good agreement between experimental and predicted data with a correlation coefficient higher than 0.98. Analysis of mold growth data through TTG models makes possible to include observations detected as no growth and can be utilized to predict mold time to growth for specific preservation factor combinations or to select preservation factor levels for an expected shelf-life based on A. niger growth.
Alborch, L; Bragulat, M R; Abarca, M L; Cabañes, F J
As there is no knowledge of the influence of abiotic factors on the two new ochratoxin A (OTA)-producing species Aspergillus sclerotioniger and Aspergillus lacticoffeatus, the aim of this study was to evaluate the effect of temperature and incubation time on growth and OTA production by these species on culture media. The study was carried out on yeast extract sucrose agar (YES) and Czapek yeast extract agar (CYA) incubated at ten different temperatures from 5 to 50°C (at 5°C intervals). Growth assessment and OTA production were determined after 5, 10, 15, 20 and 30 days of incubation at each temperature. Aspergillus sclerotioniger grew from 10 to 35°C; OTA was detected from 10 to 35°C and the highest concentration was achieved at 15°C in CYA. Aspergillus lacticoffeatus grew from 10 to 45°C; OTA was detected from 15 to 45°C, and the maximum concentration was produced after 5 days at 25°C in YES. The studied species can produce OTA over a wide range of temperatures and significant amounts can be produced in only 5 days. This is the first report on the influence of ecophysiological factors on these two ochratoxigenic species. The pattern of effects of temperature on growth and OTA production by A. sclerotioniger and A. lacticoffeatus was similar to those reported for the closely related species Aspergillus carbonarius and Aspergillus niger, respectively. The two new OTA-producing species have both been isolated from coffee beans, and the closely related ochratoxigenic species of section Nigri, A. carbonarius and A. niger are important sources of OTA in this substrate. © 2011 The Authors. Letters in Applied Microbiology © 2011 The Society for Applied Microbiology.
The objectives of this study were to determine the antiaflatoxin B1 activity in vitro of the essential oil (EO) extracted from the seeds of Carum copticum and to evaluate its antifungal activity in vivo as a potential food preservative. The C. copticum EO exhibited noticeable inhibition on dry mycelium and synthesis of aflatoxin B1 (AFB1) by Aspergillus flavus, completely inhibiting AFB1 production at 4 μL/mL. C. copticum EOs showed the lowest percentages of decayed cherry tomatoes for all fungi compared with the control at 100 μL/mL with values of 5.01 ± 67% for A. flavus and 5.98 ± 54% for Aspergillus niger. The results indicated that the percentage of infected fruits is significantly (p < 0.01) reduced by the EO at 16°C for 30 days. In this case, the oil at 100 μL/mL concentration showed the highest inhibition of fungal infection with a value of 80.45% compared with the control. Thus, the EO of dill could be used to control food spoilage and as a potential source of food preservative.
Asexual and sexual differentiation in Aspergillus nidulans involve complex control by a number of factors and is light-dependent. The velvet protein, VeA, in A. nidulans is a negative regulator of conidiation and a positive regulator of sexual development. It forms a complex with VelB and LaeA to co...
Akbar, A; Medina, A; Magan, N
The objective of this study was to evaluate the effect of different caffeine concentrations (0-4%) on (i) lag phase prior to growth, (ii) growth rates and (iii) ochratoxin A (OTA) production by strains from the Aspergillus section Circumdati and Aspergillus section Nigri groups, isolated from coffee, when grown on a conducive medium at 0·98 water activity and 30°C. The lag phases prior to growth increased with caffeine concentration. A strain of Aspergillus niger and Aspergillus carbonarius were the most sensitive to caffeine with growth being inhibited by <1% caffeine. For strains of Aspergillus westerdijkiae, Aspergillus ochraceus and Aspergillus steynii, although growth was inhibited significantly, some growth (10-15% of controls) occurred in 4% caffeine. OTA production was significantly inhibited by only 0·5% caffeine for strains of A. westerdijkiae, A. niger and A. carbonarius. For A. steynii at least 1·5% caffeine was required to inhibit OTA production. In contrast, for the strain of A. ochraceus there was a stimulation of OTA at 3% with a reduction at 4% caffeine. These results are discussed in the context of the different concentrations of caffeine found in Arabica and Robusta coffee and the development of minimization strategies. Arabic (0·6%) and Robusta coffee (4%) have significantly different amounts of endogenous caffeine. The growth of six ochratoxigenic fungi which contaminate coffee with ochratoxin A (OTA) had differential tolerance/sensitivity to concentrations of caffeine in vitro in this range. However, low concentrations of caffeine (<0·5%) was inhibitory to OTA production. These results are discussed in the context of the potential for using such information for the design of minimization strategies to control mycotoxin production in such products. © 2016 The Society for Applied Microbiology.
Císarová, Miroslava; Tančinová, Dana; Medo, Juraj; Kačániová, Miroslava
The aim of the present study was to assess the antifungal and anti-toxinogenic activity of 15 essential oils (EOs) against three fungi of the genus Aspergillus (A. parasiticus KMi-227-LR, A. parasiticus KMi-220-LR and A. flavus KMi-202-LR). The minimum inhibitory doses (MIDs) of the tested essential oils and their antifungal activity were determined using the micro-atmosphere method. The original commercial essential oil samples of Jasminum officinale L., Thymus vulgaris L., Syzygium aromaticum (L.) Merrill & Perry, Rosmarinus officinalis L., Ocimum basilicum L., Eucalyptus globulus Labill., Salvia officinalis L., Citrus limon (L.) Burm, Origanum vulgare L., Lavandula angustifolia Mill., Carum carvi L., Citrus sinensis (L.) Osbeck., Zingiber officinalis Rosc., Mentha piperita L. and Cinnamomum zeylanicum Nees. (C. verum J.S.Presl.) were produced in Slovakia (Calendula a.s., Nová Ľubovňa, Slovakia). All essential oils exhibited activity against all tested strains of fungi. After 14 days of incubation, A. flavus (KMi-202-LR) showed the highest susceptibility with a growth inhibition percentage (GIP) of 18.70% to C. limon and 5.92% to C. sinensis, while A. parasiticus (KMi-220-LR) exhibited a GIP of 20.56% to J. officinale. The minimum inhibitory doses (MIDs) of EOs with the most significant activity were recorded. The best antifungal activity, using the micro-atmosphere method was found in S. aromaticum with an MID of 62.5 μL L(-1) air, T. vulgaris (MID of 62.5 μL L(-1) air) and O. vulgare (MID of 31.5 μL L(-1) air) against all tested strains. Mycotoxin production of the tested strains was evaluated by the thin layer chromatography (TLC) method. Mycotoxin production of AFB1 and AFG1 was inhibited following all treatments with C. carvi, R. officinale and S. officinale, Eucalyptus globulus L. and O. basilicum L. Essential oils exhibited a potential inhibition activity against toxic fungi, although, these affected only the production of AFB1.
Miyamoto, Kana; Murakami, Tomoko; Kakumyan, Pattana; Keller, Nancy P; Matsui, Kenji
Eight-carbon (C8) volatiles, such as 1-octen-3-ol, are ubiquitous among fungi. They are the volatiles critical for aroma and flavor of fungi, and assumed to be signals controlling germination of several fungi. In this study, we found that intact Aspergillus flavus conidia scarcely synthesized C8 volatiles but repeated freeze-thaw treatment that made the cell membrane permeable promoted (R)-1-octen-3-ol formation. Loss or down regulation of any one of five fatty acid oxygenases (PpoA, PpoB, PpoC, PpoD or lipoxygenase) hypothesized contribute to 1-octen-3-ol formation had little impact on production of this volatile. This suggested that none of the oxygenases were directly involved in the formation of 1-octen-3-ol or that compensatory pathways exist in the fungus. Germination of the conidia was markedly inhibited at high density (1.0 × 10(9)spores mL(-1)). It has been postulated that 1-octen-3-ol is an autoinhibitor suppressing conidia germination at high density. 1-Octen-3-ol at concentration of no less than 10 mM was needed to suppress the germination while the concentration of 1-octen-3-ol in the suspension at 1.0 × 10(9) mL(-1) was under the detection limit (<1 µM). Thus, 1-octen-3-ol was not the principal component responsible for inhibition of germination. Instead, it was evident that the other heat-labile factor(s) suppressed conidial germination.
Miyamoto, Kana; Murakami, Tomoko; Kakumyan, Pattana; Keller, Nancy P.
Eight-carbon (C8) volatiles, such as 1-octen-3-ol, are ubiquitous among fungi. They are the volatiles critical for aroma and flavor of fungi, and assumed to be signals controlling germination of several fungi. In this study, we found that intact Aspergillus flavus conidia scarcely synthesized C8 volatiles but repeated freeze-thaw treatment that made the cell membrane permeable promoted (R)-1-octen-3-ol formation. Loss or down regulation of any one of five fatty acid oxygenases (PpoA, PpoB, PpoC, PpoD or lipoxygenase) hypothesized contribute to 1-octen-3-ol formation had little impact on production of this volatile. This suggested that none of the oxygenases were directly involved in the formation of 1-octen-3-ol or that compensatory pathways exist in the fungus. Germination of the conidia was markedly inhibited at high density (1.0 × 109spores mL−1). It has been postulated that 1-octen-3-ol is an autoinhibitor suppressing conidia germination at high density. 1-Octen-3-ol at concentration of no less than 10 mM was needed to suppress the germination while the concentration of 1-octen-3-ol in the suspension at 1.0 × 109 mL−1 was under the detection limit (<1 µM). Thus, 1-octen-3-ol was not the principal component responsible for inhibition of germination. Instead, it was evident that the other heat-labile factor(s) suppressed conidial germination. PMID:24883255
Cubaiu, Loredana; Abbas, Hamid; Dobson, Alan D. W.; Budroni, Marilena; Migheli, Quirico
The aim of this study was to select wine yeast strains as biocontrol agents against fungal contaminants responsible for the accumulation of ochratoxin A (OTA) in grape and wine and to dissect the mechanism of OTA detoxification by a Saccharomyces cerevisiae strain (DISAABA1182), which had previously been reported to reduce OTA in a synthetic must. All of the yeast strains tested displayed an ability to inhibit the growth of Aspergillus carbonarius both in vivo and in vitro and addition of culture filtrates from the tested isolates led to complete inhibition of OTA production. S. cerevisiae DISAABA1182 was selected and further tested for its capacity to inhibit OTA production and pks (polyketide synthase) transcription in A. carbonarius and Aspergillus ochraceus in vitro. In order to dissect the mechanism of OTA detoxification, each of these two fungi was co-cultured with living yeast cells exposed to yeast crude or to autoclaved supernatant: S. cerevisiae DISAABA1182 was found to inhibit mycelial growth and OTA production in both Aspergilli when co-cultured in the OTA-inducing YES medium. Moreover, a decrease in pks transcription was observed in the presence of living cells of S. cerevisiae DISAABA1182 or its supernatant, while no effects were observed on transcription of either of the constitutively expressed calmodulin and β-tubulin genes. This suggests that transcriptional regulation of OTA biosynthetic genes takes place during the interaction between DISAABA1182 and OTA-producing Aspergilli. PMID:23223175
Dogi, Cecilia A; Pellegrino, Matías; Poloni, Valeria; Poloni, Luis; Pereyra, Carina M; Sanabria, Analía; Pianzzola, María Julia; Dalcero, Ana; Cavaglieri, Lilia
Laboratory-scale silos were prepared to evaluate the efficacy of two different lactic acid bacteria (LAB) on the fermentation quality and mycobiota of corn silage. Their influence on Aspergillus species' variability by using the q-PCR technique was studied. Silage inoculated with Lactobacillus rhamnosus RC007 or L. plantarum RC009 were compared with uninoculated silage. Silos were opened after 1, 7, 45, 90 and 120 days after ensiling. At the end of the ensiling period, silos were left open for 7 days to evaluate aerobic stability. Rapid lactic acid production and decline in pH values were seen in the early stages of fermentation in silage inoculated with L. rhamnosus RC007. After aerobic exposure, a significant decline in lactic acid content was observed in untreated and L. plantarum RC009-inoculated silages. Counts for yeasted and toxigenic fungus remained lower, after aerobic exposure, in L. rhamnosus RC007-inoculated silage, in comparison with L. plantarum RC009 and uninoculated silages. Comparing the influence exerted by both BAL, it was observed that L. rhamnosus RC007 was more efficient at inhibiting the three fungal species tested whose DNA concentrations, determined by q-PCR, oscillated near the initial value (pre-ensiling maize). The ability of L. rhamnosus RC007 to produce lactic acid rapidly and the decline in pH values in the early stages of the fermentation along with the reduction of yeast and mycotoxicogenic fungus after aerobic exposure shows its potential as a bio-control inoculant agent in animal feed.
Kong, Qing; Wang, Long; Liu, Zengran; Kwon, Nak-Jung; Kim, Sun Chang; Yu, Jae-Hyuk
Growth, development, virulence and secondary metabolism in fungi are governed by heterotrimeric G proteins (G proteins). A Gβ-like protein called Gib2 has been shown to function as an atypical Gβ in Gpa1-cAMP signaling in Cryptococcus neoformans. We found that the previously reported CpcB (cross pathway control B) protein is the ortholog of Gib2 in Aspergillus nidulans and Aspergillus fumigatus. In this report, we further characterize the roles of CpcB in governing growth, development and toxigenesis in the two aspergilli. The deletion of cpcB results in severely impaired cellular growth, delayed spore germination, and defective asexual sporulation (conidiation) in both aspergilli. Moreover, CpcB is necessary for proper expression of the key developmental activator brlA during initiation and progression of conidiation in A. nidulans and A. fumigatus. Somewhat in accordance with the previous study, the absence of cpcB results in the formation of fewer, but not micro-, cleistothecia in A. nidulans in the presence of wild type veA, an essential activator of sexual development. However, the cpcB deletion mutant cleistothecia contain no ascospores, validating that CpcB is required for progression and completion of sexual fruiting including ascosporogenesis. Furthermore, unlike the canonical GβSfaD, CpcB is not needed for the biosynthesis of the mycotoxin sterigmatocystin (ST) as the cpcB null mutant produced reduced amount of ST with unaltered STC gene expression. However, in A. fumigatus, the deletion of cpcB results in the blockage of gliotoxin (GT) production. Further genetic analyses in A. nidulans indicate that CpcB may play a central role in vegetative growth, which might be independent of FadA- and GanB-mediated signaling. A speculative model summarizing the roles of CpcB in conjunction with SfaD in A. nidulans is presented. PMID:23936193
Kong, Qing; Wang, Long; Liu, Zengran; Kwon, Nak-Jung; Kim, Sun Chang; Yu, Jae-Hyuk
Growth, development, virulence and secondary metabolism in fungi are governed by heterotrimeric G proteins (G proteins). A Gβ-like protein called Gib2 has been shown to function as an atypical Gβ in Gpa1-cAMP signaling in Cryptococcus neoformans. We found that the previously reported CpcB (cross pathway control B) protein is the ortholog of Gib2 in Aspergillus nidulans and Aspergillus fumigatus. In this report, we further characterize the roles of CpcB in governing growth, development and toxigenesis in the two aspergilli. The deletion of cpcB results in severely impaired cellular growth, delayed spore germination, and defective asexual sporulation (conidiation) in both aspergilli. Moreover, CpcB is necessary for proper expression of the key developmental activator brlA during initiation and progression of conidiation in A. nidulans and A. fumigatus. Somewhat in accordance with the previous study, the absence of cpcB results in the formation of fewer, but not micro-, cleistothecia in A. nidulans in the presence of wild type veA, an essential activator of sexual development. However, the cpcB deletion mutant cleistothecia contain no ascospores, validating that CpcB is required for progression and completion of sexual fruiting including ascosporogenesis. Furthermore, unlike the canonical GβSfaD, CpcB is not needed for the biosynthesis of the mycotoxin sterigmatocystin (ST) as the cpcB null mutant produced reduced amount of ST with unaltered STC gene expression. However, in A. fumigatus, the deletion of cpcB results in the blockage of gliotoxin (GT) production. Further genetic analyses in A. nidulans indicate that CpcB may play a central role in vegetative growth, which might be independent of FadA- and GanB-mediated signaling. A speculative model summarizing the roles of CpcB in conjunction with SfaD in A. nidulans is presented.
Niknejad, F; Zaini, F; Faramarzi, MA; Amini, M; Kordbacheh, P; Mahmoudi, M; Safara, M
Background: Aflatoxin contamination of food and feed stuff is a serious health problem and significant economic concerns. In the present study, the inhibitory effect of Candida parapsilosis IP1698 on mycelial growth and aflatoxin production in aflatoxigenic strains of Aspergillus species was investigated. Methods: Mycelial growth inhibitions of nine strains of aflatoxigenic and non-aflatoxigenic Aspergillus species in the presence of C. parapsilosis investigated by pour plate technique at different pH, temperature and time of incubation. Reduction of aflatoxin was evaluated in co-cultured fungi in yeast extract sucrose broth after seven days of incubation using HPLC method. The data were analyzed by SPSS 11.5. Results: The presence of the C. parapsilosis at different pH did not affect significantly the growth rate of Aspergillus isolates. On the other hand, temperature and time of incubation showed to be significantly effective when compared to controls without C. parapsilosis (P≤0.05). In aflatoxigenic strains, minimum percentage of reductions in total aflatoxin and B1, B2, G1, G2 fractions were 92.98, 92.54, 77.48, 54.54 and 72.22 and maximum percentage of reductions were 99.59, not detectable, 94.42, and not detectable in both G1 and G2, respectively. Conclusion: C. parapsilosis might employ as a good biocontrol agent against growth and aflatoxin production by aflatoxigenic Aspergillus species PMID:23308351
Panda, T.; Bisaria, V. S.; Ghose, T. K.
A simple differential method based on measurement of an intracellular pigment of Aspergillus wentii was developed for estimation of the individual growths of two fungi, Trichoderma reesei and A. wentii, in mixed-culture fermentation of an insoluble cellulosic substrate. PMID:16347888
Tantaoui-Elaraki, A; Beraoud, L
We studied the effect of 13 chemically different essential oils (EO) on the mycelial growth of and aflatoxin synthesis by Aspergillus parasiticus. Cinnamon, thyme, oregano, and cumin EO were able to stop mycelial growth at only 0.1% in the medium, while curcumin, ginger, lemon, and orange EO were unable to inhibit totally the growth even at 1% concentration. Coriander, black pepper, mugwort, bay, and rosemary EO caused the growth to stop at concentrations between 0.2 and 1%. The EO most active upon mycelial growth were also the most active against aflatoxinogenesis. However, aflatoxin synthesis was inhibited by all the EO at higher extent than the mycelial growth.
Aflatoxin contamination of food products causes liver cancer and weakened immunity in humans, and stunted growth and reduced productivity in animals (CAST, 2003). Effective control of pre-harvest aflatoxin contamination of peanut and corn due to AflaGuard and Aflasafe in the United States and Africa...
Nieminen, Susanna M.; Kärki, Riikka; Auriola, Seppo; Toivola, Mika; Laatsch, Hartmut; Laatikainen, Reino; Hyvärinen, Anne; von Wright, Atte
Genotoxic and cytotoxic compounds were isolated and purified from the culture medium of an indoor air mold, Aspergillus fumigatus. One of these compounds was identified as gliotoxin, a known fungal secondary metabolite. Growth of A. fumigatus and gliotoxin production on some building materials were also studied. Strong growth of the mold and the presence of gliotoxin were detected on spruce wood, gypsum board, and chipboard under saturation conditions. PMID:12324333
Hitokoto, H; Morozumi, S; Wauke, T; Sakai, S; Kurata, H
The inhibitory effects of 29 commercial powdered spices on the growth and toxin production of three species of toxigenic Aspergillus were observed by introducing these materials into culture media for mycotoxin production. Of the 29 samples tested, cloves, star anise seeds, and allspice completely inhibited the fungal growth, whereas most of the others inhibited only the toxin production. Eugenol extracted from cloves and thymol from thyme caused complete inhibition of the growth of both Aspergillus flavus and Aspergillus versicolor at 0.4 mg/ml or less. At a concentration of 2 mg/ml, anethol extracted from star anise seeds inhibited the growth of all the strains.
Degola, F; Berni, E; Spotti, E; Ferrero, I; Restivo, F M
The aim of our research project was to consolidate a multiplex RT-PCR protocol to detect aflatoxigenic strains of Aspergillus flavus. Several independent A. flavus strains were isolated from corn and flour samples from the North of Italy and from three European countries. Aflatoxin producing/not producing phenotype was assessed by qualitative and quantitative assays at day five of growth in aflatoxin inducing conditions. Expression of 16 genes belonging to the aflatoxin cluster was assayed by multiplex or monomeric RT-PCR. There is a good correlation between gene expression and aflatoxin production. Strains that apparently transcribed all the relevant genes but did not release aflatoxin in the medium ("false positives") were re-assessed for mycotoxin production after extended growth in inducing condition. All the "false positive" strains in actual fact were positive when aflatoxin determination was performed after 10 days of growth. These strains should then be re-classified as "slow aflatoxin accumulators". To optimise the diagnostic procedure, a quintuplex RT-PCR procedure was designed consisting of a primer set directed against four informative aflatoxin cluster genes and the beta-tubulin gene as an internal amplification control. In conclusion we have provided evidence for the robustness and reliability of our RT-PCR protocol in discriminating mycotoxin producer from non-producer strains of A. flavus. and the molecular procedure we devised is a promising tool with which to screen and control the endemic population of A. flavus colonising different areas of the World.
Cvek, Domagoj; Markov, Ksenija; Frece, Jadranka; Landeka Dragicević, Tibela; Majica, Matea; Delas, Frane
Fungi produce a large variety of extracellular proteins, organic acids, and other metabolites and can adapt to several environmental conditions. Mycotoxin-producing moulds of the genera Aspergillus and Penicillium are common food contaminants. One of the natural ways to protect food from mould contamination is to use essential oils. In this study, we evaluated the effect of essential oils of cinnamon, lavender, rosemary, and sage at 1 % (v/v) concentration in yeast media inoculated with spores (final concentration 106 mL-1 media) of Aspergillus ochraceus ZMPBF 318 and Penicillium expansum ZMPBF 565, alone or in combination, on fungal biomass. Cinnamon showed the best inhibitory effect (100 %). Lavender oil best inhibited the growth of Aspergillus ochraceus (nearly 100 %), and was less successful with Penicillium expansum (having dropped to 57 % on day 28). With cultivation time the inhibitory effect of sage and rosemary oil grew for Aspergillus ochraceus and dropped for Penicillium expansum.These results suggest that fungi can be controlled with essential oils, especially with cinnamon oil.
Aflatoxins are extremely potent natural carcinogens and a major food safety concern because of potential contamination of food commodities. Threshold levels set by the U. S. Food and Drug Administration for aflatoxins in foods for domestic consumption are less than 20 parts/ billion (ppb). However, ...
[Influence of the interaction of temperature and water activity on the production of ochratoxin A and the growth of Aspergillus niger, Aspergillus carbonarius and Aspergillus ochraceus on coffee-based culture medium].
Kouadio, Ahou Irène; Lebrihi, Ahmed; Agbo, Georges N' Zi; Mathieu, Florence; Pfohl-Leszkowiz, Annie; Dosso, Mireille Bretin
In the present study, the effect of temperature and water activity on fungal growth and ochratoxin production on coffee-based medium was assessed. Optimal growth of three Aspergillus strains was observed in the same ecological conditions, namely 30 degrees C and 0.99 water activity. Maximal daily growth is 11.2, 6.92, and 7.22 mm/day for Aspergillus niger, Aspergillus carbonarius, and Aspergillus ochraceus, respectively. However, ecological conditions for optimal ochratoxin production vary according to the toxinogenic strain, with water activity as a limiting factor. Such an ochratoxin A production is inhibited at 42 degrees C and 0.75 water activity. Correspondence between laboratory tested water activity and that measured on a sun-dried ripe cherry batch shows that the first 5 days of drying are critical for fungal growth and ochratoxin A production. Accordingly, artificial drying of cherries at temperatures above 42 degrees C will impede not only fungal growth but also contamination with ochratoxin A.
Binks, P R; Robson, G D; Goosey, M W; Trinci, A P
The phosphatidylcholine (PC) content of Aspergillus nidulans choC was varied by growing the auxotroph in medium containing various concentrations of choline chloride. Direct linear correlations were observed between PC content and in vivo chitin synthase activity, between in vivo chitin synthase activity and mean hyphal extension rate, and between mean hyphal extension rate and hyphal growth unit length; hyphal growth unit length is a measure of hyphal branching. Further, there was a correlation between PC content and colony radial growth rate. Thus, membrane composition is an important determinant of both hyphal (and colony) extension rate and mycelial morphology.
Asurmendi, Paula; García, María J; Ruíz, Francisco; Dalcero, Ana; Pascual, Liliana; Barberis, Lucila
The aim of the present study was to investigate the inhibitory activity of lactic acid bacteria (LAB) isolated from brewer's grains on Aspergillus section Flavi growth and aflatoxin B1 production. The Aspergillus strains tested were inhibited by all the LAB strains assayed. The isolates Lactobacillus brevis B20, P. pentosaceus B86, Lactococcus lactis subsp. lactis B87, L. brevis B131, and Lactobacillus sp. B144 completely suppressed the fungal growth and reduced aflatoxin B1 production. In conclusion, LAB isolated from brewer's grains show a high inhibitory activity on fungal growth and aflatoxin biosynthesis by Aspergillus flavus and Aspergillus parasiticus. Further studies must be conducted to evaluate the success of in vitro assays under food environment conditions and to elucidate the antifungal mechanism of these strains.
Nugaeva, Natalia; Gfeller, Karin Y; Backmann, Natalia; Düggelin, Marcel; Lang, Hans Peter; Güntherodt, Hans-Joachim; Hegner, Martin
We demonstrate a new sensitive biosensor for detection of vital fungal spores of Aspergillus niger. The biosensor is based on silicon microfabricated cantilever arrays operated in dynamic mode. The change in resonance frequency of the sensor is a function of mass binding to the cantilever surface. For specific A. niger spore immobilization on the cantilever, each cantilever was individually coated with anti-Aspergillus niger polyclonal antibodies. We demonstrate the detection of single A. niger spores and their subsequent growth on the functionalized cantilever surface by online measurements of resonance frequency shifts. The new biosensor operating in humid air allows quantitative and qualitative detection of A. niger spores as well as detection of vital, functional spores in situ within approximately 4 h. The detection limit of the sensor is 103 CFU mL-1. Mass sensitivity of the cantilever sensor is approximately 53 pg Hz-1.
Carranza, Cecilia S; Barberis, Carla L; Chiacchiera, Stella M; Magnoli, Carina E
Agriculture is one of the bases of the Argentine economy. Glyphosate is undoubtedly one of the most important herbicides used. The increasing consumption and the efficiency of glyphosate-based herbicides have encouraged several studies on their persistence in soils, their effects on soil microbiota and their degradation processes. Fungi have been reported as being the main herbicide-degrading microorganisms as well as the most tolerant to environmental stress conditions. This study evaluated the growth performance of Aspergillus section Flavi and Aspergillus niger aggregate strains on Czapek Dox media supplied with a commercial glyphosate formulation as sole source of carbon (CZC), phosphorus (CZP) or nitrogen (CZN). Six Aspergillus spp. strains were evaluated. Each medium was stab-inoculated with fungal spores from 7-day old cultures. Two measures of colony radii were taken daily. All of the Aspergillus section Flavi strains showed a significant increase (from 24 to 44%) in growth rate on the CZN medium, as compared to controls. The A. niger aggregate strains exhibited the same behavioral pattern under all the conditions tested, except on the CZN medium. Velutinous or slightly floccose colonies with abundant sporulation were observed on CZP. Moreover, the colonies produced sparse sporulation on CZC or CZN media, being their appearances completely different from those on the CZP medium. This study establishes that A. section Flavi and A. niger aggregate strains can grow in vitro in the presence of glyphosate, especially when it is used as a sole source of phosphorus or nitrogen. Copyright © 2017 Asociación Argentina de Microbiología. Publicado por Elsevier España, S.L.U. All rights reserved.
Mohammadpour, Hossein; Moghimipour, Eskandar; Rasooli, Iraj; Fakoor, Mohammad Hadi; Alipoor Astaneh, Shakiba; Shehni Moosaie, Sara; Jalili, Zeynab
Aflatoxin B1 (AFB1) is a highly toxic and hepatocarcinogenic metabolite produced by Aspergillus species. Some natural products are known to kill fungi and destroy toxins and toxin-producing agents. The purpose of this study is to provide experimental data on the antifungal activity of cumin oils and their components that could be considered suitable for application in foods and drugs. The essential oil (EO) of Cuminum cyminum L. collected from Alborz Mountain, Iran, was obtained by hydro-distillation. The oil was analyzed by gas chromatography (GC) and chromatography/mass spectrophotometry (GC/MS). The antifungal activity of the oil was studied with regard to the inhibition of the growth of Aspergillus flavus PICC-AF39 , Aspergillus flavus PICC-AF24, Aspergillus parasiticus NRRL-2999 and Aspergillus niger. The minimal inhibitory (MIC) and minimal fungicidal (MFC) concentrations of the oil were determined. α-Pinene (29.2%), limonene (21.7%), 1,8-cineole (18.1%), linalool (10.5%), linalyl acetate (4.8%), and α-terpineole (3.17%) were the major components of the essential oil from C. cyminum L., and the oil showed a strong inhibitory effect on fungal growth. Essential oils could be safely used as preservatives in pharmaceuticals as well as health and food products to protect them against toxigenic fungal infections.
Mohammadpour, Hossein; Moghimipour, Eskandar; Rasooli, Iraj; Fakoor, Mohammad Hadi; Alipoor Astaneh, Shakiba; Shehni Moosaie, Sara; Jalili, Zeynab
Background Aflatoxin B1 (AFB1) is a highly toxic and hepatocarcinogenic metabolite produced by Aspergillus species. Some natural products are known to kill fungi and destroy toxins and toxin-producing agents. Objectives The purpose of this study is to provide experimental data on the antifungal activity of cumin oils and their components that could be considered suitable for application in foods and drugs. Materials and Methods The essential oil (EO) of Cuminum cyminum L. collected from Alborz Mountain, Iran, was obtained by hydro-distillation. The oil was analyzed by gas chromatography (GC) and chromatography/mass spectrophotometry (GC/MS). The antifungal activity of the oil was studied with regard to the inhibition of the growth of Aspergillus flavus PICC-AF39 , Aspergillus flavus PICC-AF24, Aspergillus parasiticus NRRL-2999 and Aspergillus niger. The minimal inhibitory (MIC) and minimal fungicidal (MFC) concentrations of the oil were determined. Results α–Pinene (29.2%), limonene (21.7%), 1,8-cineole (18.1%), linalool (10.5%), linalyl acetate (4.8%), and α-terpineole (3.17%) were the major components of the essential oil from C. cyminum L., and the oil showed a strong inhibitory effect on fungal growth. Conclusions Essential oils could be safely used as preservatives in pharmaceuticals as well as health and food products to protect them against toxigenic fungal infections. PMID:24624154
The inhibitory effects of 16 spice hydrosols [anise, basil, cumin, dill, Aegean sage, fennel (sweet), laurel, mint, oregano, pickling herb, rosemary, sage, savory, sea fennel, sumac, and thyme (black)] on the mycelial growth of Aspergillus parasiticus strain NRRL 2999 were investigated in vitro. The hydrosols of anise, cumin, fennel, mint, pickling herb, oregano, savory, and thyme showed a stronger inhibitory effect on mycelial growth, while sumac, sea fennel, rosemary, sage, Aegean sage, laurel, basil, and rosemary hydrosols were unable to inhibit totally the growth. Of these, sumac had the least effect on the mycelial growth of A. parasiticus. The effectiveness of the inhibitors followed the sequence anise = cumin = fennel = mint = pickling herb = oregano = savory = thyme > laurel > dill > sage > rosemary > basil > sea fennel > rosemary > sumac.
García-Cela, Esther; Marin, Sonia; Sanchis, Vicente; Crespo-Sempere, Ana; Ramos, Antonio J
The effects of two exposure times per day (6 and 16 h) of UV-A or UV-B radiation, combined with dark and dark plus light incubation periods during 7-21 d on fungal growth and mycotoxins production of Aspergillus species were studied. Aspergillus carbonarius and Aspergillus parasiticus were inoculated on grape and pistachio media under diurnal and nocturnal temperatures choosing light photoperiod according to harvest conditions of these crops in Spain. Ultraviolet irradiation had a significant effect on A. carbonarius and A. parasiticus colony size (diameter, biomass dry weight, and colony density) and mycotoxin accumulation, although intraspecies differences were observed. Inhibition of A. carbonarius fungal growth decreased when exposure time was reduced from 16 h to 6 h, but this was not always true for ochratoxin A (OTA) production. OTA reduction was higher under UV-A than UV-B radiation and the reduction increased along time conversely to the aflatoxins (AFs). Aflatoxin B1 (AFB1) was the main toxin produced by A. parasiticus except in the UV-B light irradiated colonies which showed a higher percentage of AFG than AFB. Morphological changes were observed in colonies grown under UV-B light.
Tsang, Chi-Ching; Hui, Teresa W S; Lee, Kim-Chung; Chen, Jonathan H K; Ngan, Antonio H Y; Tam, Emily W T; Chan, Jasper F W; Wu, Andrea L; Cheung, Mei; Tse, Brian P H; Wu, Alan K L; Lai, Christopher K C; Tsang, Dominic N C; Que, Tak-Lun; Lam, Ching-Wan; Yuen, Kwok-Yung; Lau, Susanna K P; Woo, Patrick C Y
Thirteen Aspergillus isolates recovered from nails of 13 patients (fingernails, n=2; toenails, n=11) with onychomycosis were characterized. Twelve strains were identified by multilocus sequencing as Aspergillus spp. (Aspergillus sydowii [n=4], Aspergillus welwitschiae [n=3], Aspergillus terreus [n=2], Aspergillus flavus [n=1], Aspergillus tubingensis [n=1], and Aspergillus unguis [n=1]). Isolates of A. terreus, A. flavus, and A. unguis were also identifiable by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The 13th isolate (HKU49(T)) possessed unique morphological characteristics different from other Aspergillus spp. Molecular characterization also unambiguously showed that HKU49(T) was distinct from other Aspergillus spp. We propose the novel species Aspergillus hongkongensis to describe this previously unknown fungus. Antifungal susceptibility testing showed most Aspergillus isolates had low MICs against itraconazole and voriconazole, but all Aspergillus isolates had high MICs against fluconazole. A diverse spectrum of Aspergillus species is associated with onychomycosis. Itraconazole and voriconazole are probably better drug options for Aspergillus onychomycosis.
Select colony-associated fungi (bee isolates). Absidia sp., Ascosphaera apis, Aspergillus flavus, Fusarium sp., Penicillium glabrum, Mucor sp., showed a 40% reduction in radial growth rate with formic acid, a 28% reduction with oxalic acid, and a 15% reduction with fructose and high fructose corn sy...
Wilson, David M; Mubatanhema, Wellington; Jurjevic, Zeljko
The fungal genus Aspergillus was established in 1729, and includes species that are adapted to a wide range of environmental conditions. Many aspergilli produce mycotoxins in foods that may be toxic, mutagenic or carcinogenic in animals. Most of the Aspergillus species are soil fungi or saprophytes but some are capable of causing decay in storage, disease in plants or invasive disease in humans and animals. Major agricultural commodities affected before or after harvest by fungal growth and mycotoxins include corn, peanuts, cottonseed, rice, tree nuts, cereal grains, and fruits. Animal products (meat, milk and eggs) can become contaminated because of diet. Aspergillus flavus, A. parasiticus, A. ochraceus, A. niger, A. fumigatus and other aspergilli produce mycotoxins of concern. These include the aflatoxins and ochratoxins, as well as cyclopiazonic acid, patulin, sterigmatocystin, gliotoxin, citrinin and other potentially toxic metabolites.
Muhialdin, Belal J; Hassan, Zaiton; Abu Bakar, Fatimah; Algboory, Hussein L; Saari, Nazamid
The ability of Leuconostoc mesenteroides DU15 to produce antifungal peptides that inhibit growth of Aspergillus niger was evaluated under optimum growth conditions of 30 °C for 48 h. The cell-free supernatant showed inhibitory activity against A. niger. Five novel peptides were isolated with the sequences GPFPL, YVPLF, LLHGVPLP, GPFPLEMTLGPT, and TVYPFPGPL as identified by de novo sequencing using PEAKS 6 software. Peptide LLHGVPLP was the only positively charged (cationic peptides) and peptide GPFPLEMTLGPT negatively charged (anionic), whereas the rest are neutral. The identified peptides had high hydrophobicity ratio and low molecular weights with amino acids sequences ranging from 5 to 12 residues. The mode of action of these peptides is observed under the scanning electron microscope and is due to cell lysis of fungi. This work reveals the potential of peptides from L. mesenteroides DU15 as natural antifungal preservatives in inhibiting the growth of A. niger that is implicated to the spoilage during storage.
Meijer, M; Houbraken, J A M P; Dalhuijsen, S; Samson, R A; de Vries, R P
Wild type Aspergillus niger isolates from different biotopes from all over the world were compared to each other and to the type strains of other black Aspergillus species with respect to growth and extracellular enzyme profiles. The origin of the A. niger isolate did not result in differences in growth profile with respect to monomeric or polymeric carbon sources. Differences were observed in the growth rate of the A. niger isolates, but these were observed on all carbon sources and not specific for a particular carbon source. In contrast, carbon source specific differences were observed between the different species. Aspergillus brasiliensis is the only species able to grow on D-galactose, and A. aculeatus had significantly better growth on Locus Bean gum than the other species. Only small differences were found in the extracellular enzyme profile of the A. niger isolates during growth on wheat bran, while large differences were observed in the profiles of the different black aspergilli. In addition, differences were observed in temperature profiles between the black Aspergillus species, but not between the A. niger isolates, demonstrating no isolate-specific adaptations to the environment.These data indicate that the local environment does not result in stable adaptations of A. niger with respect to growth profile or enzyme production, but that the potential is maintained irrespective of the environmental parameters. It also demonstrates that growth, extracellular protein and temperature profiles can be used for species identification within the group of black aspergilli.
McCormick, Allison; Heesemann, Leonie; Wagener, Johannes; Marcos, Veronica; Hartl, Dominik; Loeffler, Jürgen; Heesemann, Jürgen; Ebel, Frank
Neutrophil extracellular traps (NETs) represent a distinct mechanism to control and eliminate microbial infections. Our results show that conidia and germ tubes of the human pathogenic mold Aspergillus fumigatus are able to trigger the formation of NETs. Viable fungal cells are not essentially required for this host-pathogen interaction. Neutrophils engulf conidia and thereby inhibit their germination, a process that is independent of NETosis. In the experimental set-up used in this study neutrophils do not kill germ tubes, but reduce their polar growth and this inhibition depends on NETs as it can be overcome by the addition of DNase-1. The Zn(2+) chelator calprotectin is associated with the Aspergillus-induced NETs and addition of Zn(2+) abrogates the NET-mediated growth inhibition. In summary, our data provide evidence that NETs are not sufficient to kill A. fumigatus, but might be a valuable tool to confine infection. Copyright © 2010 Institut Pasteur. Published by Elsevier SAS. All rights reserved.
Bayman, Paul; Baker, James L; Mahoney, Noreen E
California exports tree nuts to countries where they face stringent standards for aflatoxin contamination. Trade concerns have stimulated efforts to eliminate aflatoxins and Aspergillus flavus from almonds, pistachios and walnuts. Incidence of fungi on tree nuts and associations among fungi on tree nuts were studied. Eleven hundred pistachios, almonds, walnuts and brazil nuts without visible insect damage were plated on salt agar and observed for growth of fungi. Samples came both from California nut orchards and from supermarkets. To distinguish internal fungal colonization of nuts from superficial colonization, half the nuts were surface-sterilized before plating. The most common genera found were Aspergillus, Rhizopus and Penicillium. Each species of nut had a distinct mycoflora. Populations of most fungi were reduced by surface sterilization in all except brazil nuts, suggesting that they were present as superficial inoculum on (rather than in) the nuts. In general, strongly positive associations were observed among species of Aspergillus; nuts infected by one species were likely to be colonized by other species as well. Presence of Penicillium was negatively associated with A. niger and Rhizopus in some cases. Results suggest that harvest or postharvest handling has a major influence on nut mycoflora, and that nuts with fungi are usually colonized by several fungi rather than by single species.
Kamilla, L; Mansor, S M; Ramanathan, S; Sasidharan, S
Clitoria ternatea is known for its antimicrobial activity but the antifungal effects of leaf extract on growth and morphogenesis of Aspergillus niger have not been observed. The extract showed a favorable antifungal activity against A. niger with a minimum inhibition concentration 0.8 mg/mL and minimum fungicidal concentration 1.6 mg/mL, respectively. The leaf extract exhibited considerable antifungal activity against filamentous fungi in a dose-dependent manner with 0.4 mg/mL IC50 value on hyphal growth of A. niger. The main changes observed under scanning electron microscopy after C. ternatea extract treatment were loss of cytoplasm in fungal hyphae and the hyphal wall and its diameter became markedly thinner, distorted, and resulted in cell wall disruption. In addition, conidiophore alterations were also observed when A. niger was treated with C. ternatea leaf extract.
Barberis, C L; Astoreca, A L; Dalcero, A M; Magnoli, C E
Each year, a significant portion of the peanuts produced cannot be marketed because of fungal disease at the postharvest stage and mycotoxin contamination. Antioxidants could be used as an alternative to fungicides to control ochratoxigenic fungi in peanuts during storage. This study was carried out to determine the effect of the antioxidant butylated hydroxyanisole (BHA) and the antimicrobial propyl paraben (PP) on the lag phase before growth, growth rate, and ochratoxin A (OTA) production by Aspergillus section Nigri strains in peanut kernels under different conditions of water activity (aw) and temperature. At 20 mM/g BHA, 18 degrees C, and 0.93 aw, complete inhibition of growth occurred. For PP, there was no growth at 20 mM/g, 18 degrees C, and 0.93, 0.95, and 0.98 aw. BHA at 20 mM/g inhibited OTA production in peanuts by Aspergillus carbonarius and Aspergillus niger aggregate strains at 0.93 aw and 18 degrees C. PP at 20 mM/g completely inhibited OTA production at 18 degrees C. The results of this work suggest that PP is more appropriate than BHA for controlling growth and OTA production by Aspergillus section Nigri species in peanut kernels.
Renshaw, Hilary; Vargas-Muñiz, José M.; Richards, Amber D.; Asfaw, Yohannes G.; Juvvadi, Praveen R.
Myosins are a family of actin-based motor proteins found in many organisms and are categorized into classes based on their structures. Class II and V myosins are known to be important for critical cellular processes, including cytokinesis, endocytosis, exocytosis, and organelle trafficking, in the model fungi Saccharomyces cerevisiae and Aspergillus nidulans. However, the roles of myosins in the growth and virulence of the pathogen Aspergillus fumigatus are unknown. We constructed single- and double-deletion strains of the class II and class V myosins in A. fumigatus and found that while the class II myosin (myoB) is dispensable for growth, the class V myosin (myoE) is required for proper hyphal extension; deletion of myoE resulted in hyperbranching and loss of hyphal polarity. Both myoB and myoE are necessary for proper septation, conidiation, and conidial germination, but only myoB is required for conidial viability. Infection with the ΔmyoE strain in the invertebrate Galleria mellonella model and also in a persistently immunosuppressed murine model of invasive aspergillosis resulted in hypovirulence, while analysis of bronchoalveolar lavage fluid revealed that tumor necrosis factor alpha (TNF-α) release and cellular infiltration were similar compared to those of the wild-type strain. The ΔmyoE strain showed fungal growth in the murine lung, while the ΔmyoB strain exhibited little fungal burden, most likely due to the reduced conidial viability. These results show, for the first time, the important role these cytoskeletal components play in the growth of and disease caused by a known pathogen, prompting future studies to understand their regulation and potential targeting for novel antifungal therapies. PMID:26953327
Species in Aspergillus section Flavi commonly infect agricultural staples such as corn, peanuts, cottonseed, and tree nuts and produce an array of mycotoxins, the most potent of which are aflatoxins. Aspergillus flavus is the dominant aflatoxin-producing species in the majority of crops. Populatio...
Parjo, U. K.; Sunar, N. M.; Leman, A. M.; Gani, P.; Embong, Z.; Tajudin, S. A. A.
The aim of this study was to evaluate the Aspergillus niger (A. niger) growth on substrates after incorporates with different compounds of antifungals which is normally used in food industry. The antifungals named as potassium sorbate (PS), calcium benzoate (CB) and zinc salicylate (ZS) were applied on concrete substrate covered with different wall finishing such as acrylic paint (AP), glycerol based paint (GBP), thin wallpaper (THIN) and thick wallpaper (THICK). The concrete substrate were inoculated with spore suspension, incubated at selected temperature (30oC) and relative humidity (90%)in plant growth chamber. The observations were done from the Day 3 until Day 27. The results showed that the growth of the A. niger for concrete treated by PS for AP, GBP, THIN, and THICK were 64%, 32%, 11% and 100%, respectively. Meanwhile for CB, the growth of A. niger on AP, GBP, THIN, and THICK were 100%, 12%, 41%, and 13%, respectively. Similarly, treated concrete by ZS revealed that the growth of A. niger on the same substrate cover were 33%, 47%, 40%, and 39%, respectively. The results obtained in this study provide a valuable knowledge on the abilities of antifungals to remediate A. niger that inoculated on the concrete substrate. Consequently, this study proved that the PS covering with THIN more efficiency compares CB and ZS to prevent A. niger growth.
Aspergillus oryzae and Aspergillus flavus are closely related fungal species. The A. flavus population that produces numerous small sclerotia (S strain) and aflatoxin has a unique 1.5 kb deletion in the norB-cypA region of the aflatoxin gene cluster (the S genotype). Phylogenetic studies have indica...
Aramayo, R.; Adams, T. H.; Timberlake, W. E.
We investigated the functions of the highly expressed, sporulation-specific SpoC1 genes of Aspergillus nidulans by deleting the entire 38-kb SpoC1 gene cluster. The resultant mutant strain did not differ from the wild type in (1) growth rate, (2) morphology of specialized reproductive structures formed during completion of the asexual or sexual life cycles, (3) sporulation efficiency, (4) spore viability or (5) spore resistance to environmental stress. Thus, deletion of the SpoC1 gene cluster, representing 0.15% of the A. nidulans genome, had no readily detectable phenotypic effects. Implications of this result are discussed in the context of major alterations in gene expression that occur during A. nidulans development. PMID:2471671
Briard, Benoit; Heddergott, Christoph
ABSTRACT Chronic lung infections with opportunistic bacterial and fungal pathogens are a major cause of morbidity and mortality especially in patients with cystic fibrosis. Pseudomonas aeruginosa is the most frequently colonizing bacterium in these patients, and it is often found in association with the filamentous fungus Aspergillus fumigatus. P. aeruginosa is known to inhibit the growth of A. fumigatus in situations of direct contact, suggesting the existence of interspecies communication that may influence disease outcome. Our study shows that the lung pathogens P. aeruginosa and A. fumigatus can interact at a distance via volatile-mediated communication and expands our understanding of interspecific signaling in microbial communities. PMID:26980832
Saleh, Ahmed A.; Amber, Khairy; El-Magd, Mohammed A.; Atta, Mostafa S.; Mohammed, Ahmed A.; Ragab, Mohamed M.; Abd El-Kader, Hanaa
This study was conducted to show the effect of Aspergillus awamori (AA), fructooligosaccharide (FOS), and combined Aspergillus awamori and fructooligosaccharide (AA + FOS) on growth, digestibility, blood parameters, and expression of some growth-related genes. A total of 60 broiler chicks at the age of 15 d were divided into a control group (n = 15) and 3 treatment groups. The control group was fed a basal diet, and the treatment groups were fed basal diets supplemented with 0.05% AA, 0.05% FOS, and combined of 0.05% AA and 0.05% FOS. Results from measurement of growth performance and digestibility revealed a significant increase in the body weight gain with improved feed conversion rate in the experimental groups. Interestingly, dry matter digestibility (DMD) and crude protein utilization (CPU) were improved. In addition, plasma total cholesterol and low density lipoprotein-cholesterol (LDL-C) were decreased, while plasma high density lipoprotein-cholesterol (HDL-C) was increased by feeding AA, FOS, and AA + FOS. Expressions of growth hormone secretagogue receptor (GHSR), insulin-like growth factor 1 (IGF-1), and insulin-like growth factor 1 receptor (IGF1R) were increased in experimental groups. In conclusion, the supplementation of either Aspergillus awamori or fructooligosaccharide or both improves digestibility and growth performance probably by promoting skeletal muscle protein metabolism. PMID:24895630
Passamani, Fabiana Reinis Franca; Hernandes, Thais; Lopes, Noelly Alves; Bastos, Sabrina Carvalho; Santiago, Wilder Douglas; Cardoso, Maria das Graças; Batista, Luís Roberto
The growth of ochratoxigenic fungus and the presence of ochratoxin A (OTA) in grapes and their derivatives can be caused by a wide range of physical, chemical, and biological factors. The determination of interactions between these factors and fungal species from different climatic regions is important in designing models for minimizing the risk of OTA in wine and grape juice. This study evaluated the influence of temperature, water activity (aw), and pH on the development and production of OTA in a semisynthetic grape culture medium by Aspergillus carbonarius and Aspergillus niger strains. To analyze the growth conditions and production of OTA, an experimental design was conducted using response surface methodology as a tool to assess the effects of these abiotic variables on fungal behavior. A. carbonarius showed the highest growth at temperatures from 20 to 33°C, aw between 0.95 and 0.98, and pH levels between 5 and 6.5. Similarly, for A. niger, temperatures between 24 and 37°C, aw greater than 0.95, and pH levels between 4 and 6.5 were optimal. The greatest toxin concentrations for A. carbonarius and A. niger (10 μg/g and 7.0 μg/g, respectively) were found at 15°C, aw 0.99, and pH 5.35. The lowest pH was found to contribute to greater OTA production. These results show that the evaluated fungi are able to grow and produce OTA in a wide range of temperature, aw, and pH. However, the optimal conditions for toxin production are generally different from those optimal for fungal growth. The knowledge of optimal conditions for fungal growth and production of OTA, and of the stages of cultivation in which these conditions are optimal, allows a more precise assessment of the potential risk to health from consumption of products derived from grapes.
Doupnik, Ben; Bell, D. K.
Isolates of Aspergillus chevalieri, A. flavus, A. ochraceus, A. repens, and Penicillium funiculosum and complexes of P. citrinum-P. implicatum isolated from moldy pecan meats were toxic to chicks. PMID:5564681