Phenylpyrrole-resistance and aflatoxin production in Aspergillus parasiticus Speare

Mutants of Aspergillus parasiticus highly resistant to phenylpyrroles were isolated at a high mutation frequency, after UV-mutagenesis and selection on media containing fludioxonil. Studies on the effect of mutation(s) on the aflatoxin production resulted in the identification of two fludioxonil-resistant phenotypes: aflatoxigenic (FLD(afl)(+)) and non-aflatoxigenic (FLD(afl)(-)) mutant strains. Most of the FLD(afl)(+) mutant strains produced the aflatoxin B(1) at similar or even higher (up to 2.5-fold) concentrations than the wild-type parent strain on yeast extract sucrose medium. Interestingly, in most of these mutant strains the aflatoxigenic ability significantly increased (up to 4-fold) when the mutants were grown on fungicide-amended medium. However, a significant reduction in the aflatoxin production was observed in wheat grains by all FLD(afl)(+) mutant strains. Tests on the response of mutant strains to high osmotic pressure showed that most fludioxonil-resistant mutants were more sensitive to high osmolarity than the wild-type parent strain. Study of other fitness determining parameters showed that the mutation(s) for resistance to phenylpyrroles may or may not affect the mycelial growth rate, sporulation and conidial germination. However, in a number of aflatoxigenic-mutant strains these fitness parameters were unaffected or only slightly affected. Cross resistance studies with fungicides from different chemical groups showed that the mutation(s) for resistance to fludioxonil also highly reduced the sensitivity of mutant strains to the aromatic hydrocarbon and dicarboximide fungicides. No effect of phenylpyrroles resistance mutation(s) on fungitoxicity of triazoles, benzimidazoles, anilinopyrimidines, phenylpyridinamines, strobilurin-type fungicides and to the non site-specific inhibitors chlorothalonil and maneb was observed. The above mentioned data indicate, for the first time, the potential risk of increased aflatoxin contamination of agricultural products by the appearance and predominance of highly aflatoxigenic mutant strains of A. parasiticus resistant to aromatic hydrocarbon, dicarboximide and phenylpyrrole fungicides.     

Ethylene inhibits aflatoxin biosynthesis in Aspergillus parasiticus grown on peanuts

The filamentous fungi Aspergillus parasiticus and Aspergillus flavus synthesize aflatoxins when they grow on a variety of susceptible food and feed crops. These mycotoxins are among the most carcinogenic naturally occurring compounds known and they pose significant health risks to humans and animals. We previously demonstrated that ethylene and CO2 act alone and together to reduce aflatoxin synthesis by A. parasiticus grown on laboratory media. To demonstrate the potential efficacy of treatment of stored seeds and grains with these gases, we tested ethylene and CO2 for ability to inhibit aflatoxin accumulation on Georgia Green peanuts stored for up to 5 days. We demonstrated an inverse relationship between A. parasiticus spore inoculum size and the level of toxin accumulation. We showed that ethylene inhibits aflatoxin synthesis in a dose-dependent manner on peanuts; CO2 also inhibits aflatoxin synthesis over a narrow dose range. Treatments had no discernable effect on mold growth. These observations support further exploration of this technology to reduce aflatoxin contamination of susceptible crops in the field and during storage.     

Inhibition of growth and aflatoxin production of Aspergillus parasiticus by citrus oils

Summary Aspergillus parasiticus was inoculated into grapefruit juice and a glucose-yeast extract medium; both contained 500–7000 ppm of citrus oils that were incorporated into the media by sonication. Orange and lemon oil were more inhibitory to mold growth and aflatoxin production than was d-limonene, the main constituent of the two peel oils. After 7 days at 28° C, 2000 ppm of lemon and 3000 ppm of orange oil in grapefruit juice afforded maximum suppression of mold growth and toxin formation. When the glucose-yeast extract medium was used, 3000 ppm of either oil were needed to achieve the same result. After 4 days at 28° C, orange oil at 3500 ppm in either medium markedly inhibited mold growth (as evidenced by dry weight of mold mycelium) and aflatoxin production (only 14 and 1% of the amount normally produced in the juice and artificial medium, respectively). Higher concentrations of orange oil further reduced mold growth and aflatoxin production and also delayed the onset of sporulation, if it occurred. Although aflatoxin was detected in all samples, only 0.2 to 0.5% of the amount found in controls (without the citrus oil) was present when the medium contained 7000 ppm orange oil. The mold consistently grew, albeit very poorly, on the glass at the liquid-atmosphere interface even when the substrate contained a large amount of citrus oil.

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Hemmung des Wachstums und der Aflatoxinproduktion von Aspergillus parasiticus durch Orangenöl, Citronenöl und d-Limonen

Zusammenfassung Pampelmusensaft und ein Glucose-Hefeextrakt-Medium wurden beide mit 500–7000 ppm Orangenöl, Citronenöl oder d-Limonen angereichert und dann mitAspergillus parasiticus beimpft. Wachstum und Aflatoxinproduktion des Pilzes wurden stärker durch die Öle als durch d-Limonen gehemmt, obwohl dieser der Hauptbestandteil der beiden Öle ist. 2000 ppm Citronenöl bzw. 3000 ppm Orangenöl in Pampelmusensaft genügten zur starken Hemmung der Wachstums- and der Aflatoxinproduktion vonA. parasiticus während 7 Tage bei 28° C. Wenn Glucose-Hefeextrakt als Nährboden diente, dann wiesen bei 3000 ppm beide Öle gleiche Hemmung auf. Wenn beide Nahrboden nur 4 Tage bei 28° C gehalten wurden, dann waren 3500 ppm Orangendl notwendig, um Wachstum and Aflatoxinproduktion zu hemmen. Pampelmusensaft mit einem Orangenöl-Gehalt von 3500 ppm enthielt nur 14% der Aflatoxin-Menge des beimpften Saftes ohne Öl. Das Medium mit Glucose, Hefeextrakt und Orangendl hatte nur 1 % des Aflatoxin-Gehaltes der Kontrolle. Höhere Konzentrationen von Orangenöl hemmten noch stärker und verzögerten den Beginn der Konidienbildung. Wenn das Medium 7000 ppm Orangenöl enthielt, dann konnte nur geringes Pilzwachstum und Aflatoxinproduktion (0,2–0,5% der Kontrolle) beobachtet wurden; das minimale Wachstum des Pilzes geschah an der Grenzfläche Nährboden und Atmosphare.

     

Potential use of phenolic antioxidants on peanut to control growth and aflatoxin B1 accumulation by Aspergillus flavus and Aspergillus parasiticus

Food‐grade antioxidants: butylated hydroxyanisole (BHA), propyl paraben (PP) and butylated hydroxytoluene (BHT) (10 and 20 mmol g^?1) and all the mixtures of these chemicals were tested for inhibitory activity on the growth of and aflatoxin B₁ (AFB₁) accumulation by Aspergillus parasiticus and A. flavus on irradiated (7 kGy) peanut grains. Also, the influence of these treatments was evaluated in different water conditions (0.982, 0.955, 0.937a_w) at 11 and 35 days of incubation at 28 °C. Water activity (a_w) affected the fungal growth, no fungal development was observed at the highest stress water condition (0.937a_w). Butylated hydroxyanisole at 10 mmol g^?1 level and all the mixtures with PP and/or BHT were significantly effective (P = 0.05) in increasing lag phase and reducing growth rate and colony forming units per gram of peanut of both Aspergillus section Flavi strains and AFB₁ accumulation. The application of BHA at concentrations of 20 mmol g^?1 alone or with PP and/or BHT totally inhibited fungal growth at 11 and 35 days of incubation. The results suggest that the addition of these chemical mixtures on peanut grains at low levels has potential to impact synergically on the control of Aspergillus section Flavi. Copyright © 2007 Society of Chemical Industry     

Growth and biosynthesis of aflatoxin by Aspergillus parasiticus in cultures containing nisin

Nisin, 200 or 5000 Reading units/ml, was added to Aspergillus parasiticus cultures. The cultures were incubated at 28 degrees C for 3, 7 or 10 days and analyzed for mycelial dry weight, pH and accumulation of aflatoxin B1 and G1. During the first 3 days of incubation, dry weight, pH decrease and aflatoxin accumulation were suppressed by nisin, when compared with similar values for the nisin-free control. After longer incubation, differences in dry weight nd pH values decreased, whereas accumulation of aflatoxin in the nisin-containing cultures surpassed that of the control.     

Growth and biosynthesis of aflatoxin by Aspergillus parasiticus in cultures containing nisin

Summary Nisin, 200 or 5000 Reading units/ml, was added toAspergillus parasiticus cultures. The cultures were incubated at 28 °C for 3, 7 or 10 days and analyzed for mycelial dry weight, pH and accumulation of aflatoxin B₁ and G₁. During the first 3 days of incubation, dry weight, pH decrease and aflatoxin accumulation were suppressed by nisin, when compared with similar values for the nisin-free control. After longer incubation, differences in dry weight and pH values decreased, whereas accumulation of aflatoxin in the nisin-containing cultures surpassed that of the control.

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Wachstum und Aflatoxin-Biosynthese von Aspergillus parasiticus in Nisin-enthaltenden Kulturen

Zusammenfassung Nisin (200 oder 5000 Reading-Einheiten/ml) wurdeAspergillus parasiticus Kulturen zugegeben. Die Kulturen wurden bei 28 °C für 3, 7 oder 10 Tage bebrütet und auf Mycel-Trockengewicht, pH und Aflatoxin-Inhalt geprüft. Während der ersten drei Tage wurden das Wachstum und die Aflatoxin-Biosynthese durch Nisin etwas gehemmt, nach längere Incubation jedoch gefördert.

     

Novel techniques for controlling the growth of and aflatoxin production by Aspergillus parasiticus in packaged peanuts

The effects of modified atmosphere packaging involving oxygen absorbents, storage temperature and packaging film barrier characteristics on the growth of and aflatoxin production by Aspergillus parasiticus in packaged peanuts was investigated. Mold growth was barely visible in air-packaged peanuts using a high gas barrier film (ASI) while extensive mold growth was observed in peanuts packaged under similar gaseous conditions using a low barrier film (ASIII). Incorporation of an oxygen absorbent (Ageless type S) inhibited mold growth in peanuts packaged in film ASI, while mold growth occurred in peanuts packaged with an absorbent/carbon dioxide generator (Ageless type G) in film ASI and in all absorbent-packaged peanuts in film ASIII. Aflatoxin B₁ production was detected at levels greater than the regulatory limit of 20 ng g^-1 in air-packaged peanuts using film ASI at 20°C and 25°C with the maximum level of aflatoxin (52·95 ng g^-1) being detected in air-packaged peanuts using film ASIII. However, aflatoxin production in all absorbent-packaged peanut samples was less than the regulatory level of 20 ng g^-1 irrespective of the barrier characteristics of the packaging film. Discoloration was more intensive in air-packaged peanuts in film ASIII especially at 25°C and 30°C than those packaged under similar or modified gaseous conditions using film ASI. This study has shown that oxygen absorbent technology is a simple and effective means of controlling the growth of and aflatoxin production by A. parasiticus. However, the effectiveness of these absorbents is dependent on the gas barrier properties of the packaging film surrounding the product.     

Potential of pulsed electric field to control Aspergillus parasiticus,aflatoxin and mutagenicity levels: Sesame seed quality

Seed processing technologies are essential for seed safety and functionality through protection of physicochemical quality, pathogen inactivation, aflatoxin detoxification and alleviation of mutagenicity. Design of a pilot-scale unit of pulsed electric fields (PEF) to treat sesame seeds with respect to quality parameters, Aspergillus parasiticus inactivation and aflatoxin reduction as well as alleviation of aflatoxin mutagenicity were prompted in this study. PEF energy ranged from 0.97 to 17.28 J achieved maximum reductions of peroxide value and acidity number of 67.4 and 85.7%, respectively, and did not change color L*, a*, b* and hue values. A 60% reduction of A. parasiticus counts occurred at the maximum PEF energy. Aflatoxins G1, G2, B1, and B2 contents decreased by 94.7, 92.7, 86.9, and 98.7%, respectively. Except for the samples treated by 2.16 J with 100 μg/plate and by 6.80 J with 10 μg/plate, PEF treatment provided elimination of aflatoxin mutagenity. It is concluded that PEF treatment can be used to treat sesame seeds with preservation of physicochemical properties, inactivation of A. parasiticus and decomposition of aflatoxins with reduced mutagenicity.     

Effect of anilinopyrimidine resistance on aflatoxin production and fitness parameters in Aspergillus parasiticus Speare

Mutants of Aspergillus parasiticus resistant to the anilinopyrimidine fungicides were isolated at a high mutation frequency after UV-mutagenesis and selection on media containing cyprodinil. In vitro fungitoxicity tests resulted in the identification of two predominant resistant phenotypes that were highly (R₁-phenotype) and moderately (R₂-phenotype) resistant to the anilinopyrimidines cyprodinil, pyrimethanil and mepanipyrim. Cross-resistance studies with fungicides from other chemical groups showed that the highly resistance mutation(s) did not affect the sensitivity of R₁-mutant strains to fungicides affecting other cellular pathways. Contrary to that, a reduction in the sensitivity to the triazoles epoxiconazole and flusilazole, the benzimidazole carbendazim, the phenylpyrrole fludioxonil, the dicarboximide iprodione and to the strobilurin-type fungicide pyraclostrobin was observed in R₂-mutant strains. Study of fitness parameters of anilinopyrimidine-resistant strains of both phenotypic classes showed that all R₁ mutant strains had mycelial growth rate, sporulation and conidial germination similar to or even higher than the wild-type parent strain, while these fitness parameters were negatively affected in R₂ mutant strains. Analysis of the aflatoxin production showed that most R₁ mutant strains produced aflatoxins at concentrations markedly higher than the wild-type parent strain. A considerable reduction in the aflatoxin production was observed on cultured medium and on wheat grains by all R₂ mutant strains, indicating a possible correlation between fitness penalties and aflatoxigenic ability of A. parasiticus. The potential risk of increased aflatoxin contamination of agricultural products and their byproducts by the appearance and predominance of highly aflatoxigenic mutant strains of A. parasiticus resistant to the anilinopyrimidines is discussed.     

Changes in enzyme activities and aflatoxin elaboration in sorghum genotypes following Aspergillus parasiticus infestation

Sorghum is a relatively poor substrate for aflatoxin production compared with high‐risk agricultural commodities like maize and groundnut, even though it is susceptible to fungal attack. Fungal infestation of sorghum results in a varied biochemical composition of the deteriorated grain. In this study, six sorghum genotypes (red—AON 486, IS 620; yellow—LPJ, IS 17 779; white—SPV 86, SPV 462) were inoculated with a toxigenic strain of Aspergillus parasiticus (NRRL 2999) in order to evaluate the changes in the activities of various hydrolytic enzymes (α‐ and β‐amylases, protease and lipase) in comparison with those in uninfected grains. Enzyme activities were measured at different times after fungal infestation, and the enzymatic activities were correlated with the aflatoxin production. Alpha‐amylase activity was observed to be greater than β‐amylase activity in all six genotypes under both healthy and infected conditions. The increase in α‐amylase activity during the period of infection was higher in white genotypes than in red sorghum genotypes. Alpha‐amylase activity in all the genotypes increased up to day 6 after fungal infection, but was significantly lower in infected grains than in healthy grains. The variability in the basal enzyme activities among the six sorghum genotypes was quite high compared with the amount of induction of each specific enzyme due to infection and germination. Higher protease activity was observed in the infected grains than in healthy grains. The enzyme activities in high tannin red genotypes were less than those in yellow and white genotypes. The α‐ and β‐amylase activities were positively correlated (r = 0.406 and 0.436; P < 0.05) to aflatoxin production. Inherent lipase activity was highest (on day 0) in AON 486, SPV 462 and SPV 86, as compared with the activity in infected grains. The total aflatoxins produced (quantified by TLC‐fluorodensitometry) were lower in red genotypes than in yellow and white genotypes, suggesting that red genotypes were least susceptible to aflatoxin elaboration among the various genotypes tested. All four aflatoxins, (B₁, B₂, G₁ and G₂) were present in five genotypes (IS 620, LPJ, IS 17 779, SPV 86 and SPV 462) at all the stages of infection, but, aflatoxin could not be detected in the red genotype AON 486 on day 3 after infection. White genotypes SPV 86 and SPV 462) showed maximal aflatoxin (total) production on day 6 after infection. © 2000 Society of Chemical Industry     

Production of aflatoxin B1 and G1 by Aspergillus parasiticus on silica gels.

Silica gels were prepared by acidifying alkaline silicate solutions with phosphoric or tartaric acid. Various combinations of glucose, sucrose, yeast extract, and salts were included in the gels an nutrients. Maximum production of aflatoxins B1 and G1 occurred when silica gel (0.4 to 0.5 cm deep in a petri dish) containing 20% sucrose and 2% yeast extract, and gelled with tartaric acid, was inoculated with approximately 120 to 12000 spores of Aspergillus parasiticus per plate; and plates were incubated at 28 degrees C for 10 days.     

Inhibition of Aspergillus parasiticus by Thymol

Thymol concentrations ≥ 500 μg/ml completely inhibited the growth of Aspergillus parasiticus, while lower concentrations of the flavor compound produced either partial or transitory growth inhibition. Aflatoxin production was inhibited to a degree ≤ that of growth.     

Inhibitory effects of Satureja hortensis L. essential oil on growth and aflatoxin production by Aspergillus parasiticus

In an effort to screen the essential oils of some Iranian medicinal plants for novel aflatoxin (AF) inhibitors, Satureja hortensis L. was found as a potent inhibitor of aflatoxins B1 (AFB1) and G1(AFG1) production by Aspergillus parasiticus NRRL 2999. Fungal growth was also inhibited in a dose-dependent manner. Separation of the plant inhibitory substance(s) was achieved using initial fractionation of its effective part (leaf essential oil; LEO) by silica gel column chromatography and further separation by reverse phase-high performance liquid chromatography (RP-HPLC). These substances were finally identified as carvacrol and thymol, based on the interpretation of 1H and 13C NMR spectra. Microbioassay (MBA) on cell culture microplates contained potato-dextrose broth (PDB) medium (4 days at 28 degrees C) and subsequent analysis of cultures with HPLC technique revealed that both carvacrol and thymol were able to effectively inhibit fungal growth, AFB1 and AFG1 production in a dose-dependent manner at all two-fold concentrations from 0.041 to 1.32 mM. The IC50 values for growth inhibition were calculated as 0.79 and 0.86 mM for carvacrol and thymol, while for AFB1 and AFG1, it was reported as 0.50 and 0.06 mM for carvacrol and 0.69 and 0.55 mM for thymol. The results obtained in this study clearly show a new biological activity for S. hortensis L. as strong inhibition of aflatoxin production by A. parasiticus. Carvacrol and thymol, the effective constituents of S. hortensis L., may be useful to control aflatoxin contamination of susceptible crops in the field.