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.     

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.

     

Interaction of water activity and temperature on aflatoxin production by Aspergillus flavus and A. parasiticus in irradiated maize seeds.

The effects of aw (0.90, 0.95, 0.98) and temperature (25 degrees C, 30 degrees C, 35 degrees C) on aflatoxin production by Aspergillus flavus and Aspergillus parasiticus growing on irradiated maize seeds, were examined. Highest levels of aflatoxin were produced by A. parasitious at 25 degrees C and 0.98 aw and by A. flavus at 30 degrees C at 0.95 and 0.98 aw. At 0.90 aw toxin production was consistently low for both species at all temperatures. Temperature cycling of A. flavus between 25 degrees C and 35 degrees C each for 12 h resulted in higher aflatoxin synthesis than when incubated either at 25 degrees C or 35 degrees C.     

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.     

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     

Nannocystis exedens: a potential biocompetitive agent against Aspergillus flavus and Aspergillus parasiticus.

This study examined the potential for controlling toxigenic Aspergillus flavus and Aspergillus parasiticus by biological means using a myxobacterium commonly found in soil. The ability of Nannocystis exedens to antagonize A. flavus ATCC 16875, A. flavus ATCC 26946, and A. parasiticus NRRL 3145 was discovered. Cultures of aflatoxigenic fungi were grown on 0.3% Trypticase peptone yeast extract agar for 14 days at 28 degrees C. When N. exedens was grown in close proximity with an aflatoxigenic mold, zones of inhibition (10 to 20 mm) developed between the bacterium and mold colony. A flattening of the mold colony on the sides nearest N. exedens and general stunting of growth of the mold colony were also observed. When N. exedens was added to the center of the cross-streak of a mold colony, lysis of the colony by the bacterium was observed after 24 h. Microscopic observations revealed that N. exedens grew on spores, germinating spores, hyphae, and sclerotia of the molds. These results indicate that N. exedens may be a potential biocontrol agent against A. flavus and A. parasiticus.     

Aspergillus flavus and aflatoxin production in acid-treated maize

Isobutyric acid (IBA) and propionic-acetic acid (PA) were applied to comparable 52.8 m³ lots of freshly harvested yellow dent maize containing 27% moisture. After 6 months storage, 30% Aspergillus flavus infection and low levels of aflatoxin were detected in adjacent bins of IBA-treated and PA-treated maize. Extensive samples were taken after 7 months from moldy spots in each bin and evaluated for aflatoxin, zearalenone, ochratoxin and microorganisms. Aspergillus flavus (10⁶ propagules/g) was detected in 40% of the PA samples, but no aflatoxin was found. Also, counts of Aspergillus fumigatus, Absidia and Penicillia were high. In addition to the molds found on PA maize, Aspergillus niger was identified on IBA-treated maize. Aspergillus flavus (10⁴–10⁷ propagules/g) was present in 79% of the IBA samples; aflatoxin (from 2 to 857 ng/g) was detected in 57%. Aflatoxin contamination varied between locations within a moldy area. Among 20 individual kernels picked at random from each location, aflatoxin contamination ranged from 150 to 21.800 ng/g in positive kernels. Evidently, bulk quantities of maize must be appraised on the basis of individual kernels because toxin-free kernels often are adjacent to highly contaminated kernels.     

Microwave irradiation of hazelnuts for the control of aflatoxin producing Aspergillus parasiticus

In this study, the effects of microwave treatment on hazelnuts artificially contaminated with aflatoxigenic fungi were evaluated qualitatively and quantitatively. The physical quality attributes (color, moisture loss, and sensory attributes) of microwave treated hazelnuts were also evaluated. A significant 3-log reduction in Aspergillus parasiticus contamination was observed after 120 s treatment, no or similar changes were observed during the storage of microwave treated hazelnuts under the storage conditions. While taste and odour of microwaved in shell hazelnuts were unaffected during treatment and subsequent storage, microwave treatment duration of 120 s was found to be capable of reducing fungal count of A. parasiticus on in-shell hazelnut without any noticeable change in nutritional and organoleptic properties of nuts. Based on this and the earlier study, a hybrid process is proposed, where UV-C surface treatment and vacuum assisted microwave are combined with air drying to increase the shelf life and control the quality.Industrial relevanceA hybrid industrial process is proposed, where UV-C surface treatment and vacuum assisted microwave treatment are combined to increase the shelf life and control the quality of hazelnuts.     

Distribution of aflatoxin-producing Aspergillus flavus and Aspergillus parasiticus in sugarcane fields in the southernmost islands of Japan

The distribution of Aspergillus flavus and Aspergillus parasiticus in sugarcane field soils and on harvested sugarcane stems was studied on seven islands of Okinawa and Kagoshima Prefectures, the southernmost prefectures in Japan. With the use of a combination of dilution plate and plant debris plate techniques, the fungi were detected on all seven islands studied and in 74% of 53 soil samples. The fungi were also found on the cut surfaces of sugarcane stems from one of the islands. A. parasiticus was the predominant fungus, although many atypical A. parasiticus isolates that produced metulated conidial heads were also obtained. The proportions of isolates testing positive for aflatoxin production were ca. 89% (146 of 164) of all isolates and ca. 69% of A. flavus isolates. More than 40% of A. flavus isolates also produced G aflatoxins. Scanning electron microscopic observation of conidial wall texture was useful in distinguishing A. parasiticus from A. flavus. Cyclopiazonic acid, an indole mycotoxin, was never synthesized by any of the A. parasiticus or G aflatoxin-producing A. flavus isolates tested.     

Aqueous extracts of Tulbaghia violacea inhibit germination of Aspergillus flavus and Aspergillus parasiticus conidia

Aspergillus flavus and Aspergillus parasiticus are important plant pathogens and causal agents of pre- and postharvest rots of corn, peanuts, and tree nuts. These fungal pathogens cause significant crop losses and produce aflatoxins, which contaminate many food products and contribute to liver cancer worldwide. Aqueous preparations of Tulbaghia violacea (wild garlic) were antifungal and at 10 mg/ml resulted in sustained growth inhibition of greater than 50% for both A. flavus and A. parasiticus. Light microscopy revealed that the plant extract inhibited conidial germination in a dose-dependent manner. When exposed to T. violacea extract concentrations of 10 mg/ml and above, A. parasiticus conidia began germinating earlier and germination was completed before that of A. flavus, indicating that A. parasiticus conidia were more resistant to the antifungal effects of T. violacea than were A. flavus conidia. At a subinhibitory extract dose of 15 mg/ml, hyphae of both fungal species exhibited increased granulation and vesicle formation, possibly due to increased reactivity between hyphal cellular components and T. violacea extract. These hyphal changes were not seen when hyphae were formed in the absence of the extract. Transmission electron microscopy revealed thickening of conidial cell walls in both fungal species when grown in the presence of the plant extract. Cell walls of A. flavus also became considerably thicker than those of A. parasiticus, indicating differential response to the extract. Aqueous preparations of T. violacea can be used as antifungal treatments for the control of A. flavus and A. parasiticus. Because the extract exhibited a more pronounced effect on A. flavus than on A. parasiticus, higher doses may be needed for control of A. parasiticus infections.     

Aflatoxin-producing potential of Aspergillus flavus and Aspergillus parasiticus isolated from samples of smoked-dried meat

Over a period of three years 420 samples of various smoke-dried meat products, collected from individual households in different region of Croatia were analysed for the presence of aflatoxigenic strains of the Aspergillus flavus group. Strains of A. flavus and A. parasiticus were present in 17,8% of the samples, and aflatoxin-producing ability was tested in 75 strains. In relation to sequential method of aflatoxin detection, 5 of 8 isolates were found in the first step (fluorescence in aflatoxin-producing ability medium - APA) and all of them in the second step (extraction method from syntheses on moist shredded wheat - SW). A. flavus strains produced mainly aflatoxin B₁, and had various levels of toxigenicity (1.4–3.12 mg/kg). Some strains of A. parasiticus produced all four aflatoxins B₁ B₂ G₁ G₂, while the other ones produced AF B₁ + G₁ only, with concentrations of aflatoxins from 0.1 to 450 mg/kg.     

Culture of Aspergillus parasiticus in apple juice. I. The influence of sodium benzoate and potassium sorbate on fungal growth and aflatoxin biosynthesis

Sodium benzoate or potassium sorbate (100, 200, 300 and 400 mg/l) were added to cultures of Aspergillus parasiticus NRRL 2999 on apple juice (from syrup) and incubated quiescently at 25 degrees C for 3, 6, 9, 12 or 15 days. The cultures were analyzed for pH, mycelial dry weight and accumulation of aflatoxin B1 and G1. The initial pH of 2.5 remained constant in all instances throughout the incubation period. Sodium benzoate, at all concentrations, suppressed fungal growth and stimulated the biosynthesis of G1, whereas little influence was exerted upon the accumulation of B1. Potassium sorbate stimulated fungal growth at 100 mg/l, while at all concentrations it considerably inhibited toxin production (no detectable amounts of B1 and 3 to 5 times less G1 than in controls). The concentration of G1 surpassed that of B1 without exception.     

PCR-restriction fragment length analysis of aflR gene for differentiation and detection of Aspergillus flavus and Aspergillus parasiticus in maize

Contamination of food and feedstuffs by Aspergillus species and their toxic metabolites is a serious problem as they have adverse effects on human and animal health. Hence, food contamination monitoring is an important activity, which gives information on the level and type of contamination. A PCR-based method of detection of Aspergillus species was developed in spiked samples of sterile maize flour. Gene-specific primers were designed to target aflR gene, and restriction fragment length polymorphism (RFLP) of the PCR product was done to differentiate Aspergillus flavus and Aspergillus parasiticus. Sterile maize flour was inoculated separately with A. flavus and A. parasiticus, each at several spore concentrations. Positive results were obtained only after 12-h incubation in enriched media, with extracts of maize inoculated with A. flavus (101 spores/g) and A. parasiticus (104 spores/g). PCR products were subjected to restriction endonuclease (HincII and PvuII) analysis to look for RFLPs. PCR-RFLP patterns obtained with these two enzymes showed enough differences to distinguish A. flavus and A. parasiticus. This approach of differentiating these two species would be simpler, less costly and quicker than conventional sequencing of PCR products.