Enhanced diversity and aflatoxigenicity in interspecific hybrids of Aspergillus flavus and Aspergillus parasiticus.

Aspergillus flavus and A. parasiticus are the two most important aflatoxin-producing fungi responsible for the contamination of agricultural commodities worldwide. Both species are heterothallic and undergo sexual reproduction in laboratory crosses. Here we examine the possibility of interspecific matings between A. flavus and A. parasiticus. These species can be distinguished morphologically and genetically, as well as by their mycotoxin profiles. Aspergillus flavus produces both B aflatoxins and cyclopiazonic acid (CPA), B aflatoxins or CPA alone, or neither mycotoxin; Aspergillus parasiticus produces B and G aflatoxins or the aflatoxin precursor O-methylsterigmatocystin, but not CPA. Only four of forty-five attempted interspecific crosses between opposite mating types of A. flavus and A. parasiticus were fertile and produced viable ascospores. Single ascospore strains from each cross were shown to be recombinant hybrids using multilocus genotyping and array comparative genome hybridization. Conidia of parents and their hybrid progeny were haploid and predominantly monokaryons and dikaryons based on flow cytometry. Multilocus phylogenetic inference showed that experimental hybrid progeny were grouped with naturally occurring A. flavus L strain and A. parasiticus. Higher total aflatoxin concentrations in some F1 progeny strains compared to midpoint parent aflatoxin levels indicate synergism in aflatoxin production; moreover, three progeny strains synthesized G aflatoxins that were not produced by the parents, and there was evidence of allopolyploidization in one strain. These results suggest that hybridization is an important diversifying force resulting in the genesis of novel toxin profiles in these agriculturally important fungi.

Sexuality generates diversity in the aflatoxin gene cluster: evidence on a global scale.

Aflatoxins are produced by Aspergillus flavus and A. parasiticus in oil-rich seed and grain crops and are a serious problem in agriculture, with aflatoxin B1 being the most carcinogenic natural compound known. Sexual reproduction in these species occurs between individuals belonging to different vegetative compatibility groups (VCGs). We examined natural genetic variation in 758 isolates of A. flavus, A. parasiticus and A. minisclerotigenes sampled from single peanut fields in the United States (Georgia), Africa (Benin), Argentina (Córdoba), Australia (Queensland) and India (Karnataka). Analysis of DNA sequence variation across multiple intergenic regions in the aflatoxin gene clusters of A. flavus, A. parasiticus and A. minisclerotigenes revealed significant linkage disequilibrium (LD) organized into distinct blocks that are conserved across different localities, suggesting that genetic recombination is nonrandom and a global occurrence. To assess the contributions of asexual and sexual reproduction to fixation and maintenance of toxin chemotype diversity in populations from each locality/species, we tested the null hypothesis of an equal number of MAT1-1 and MAT1-2 mating-type individuals, which is indicative of a sexually recombining population. All samples were clone-corrected using multi-locus sequence typing which associates closely with VCG. For both A. flavus and A. parasiticus, when the proportions of MAT1-1 and MAT1-2 were significantly different, there was more extensive LD in the aflatoxin cluster and populations were fixed for specific toxin chemotype classes, either the non-aflatoxigenic class in A. flavus or the B1-dominant and G1-dominant classes in A. parasiticus. A mating type ratio close to 1∶1 in A. flavus, A. parasiticus and A. minisclerotigenes was associated with higher recombination rates in the aflatoxin cluster and less pronounced chemotype differences in populations. This work shows that the reproductive nature of the population (more sexual versus more asexual) is predictive of aflatoxin chemotype diversity in these agriculturally important fungi.

Effect of sexual recombination on population diversity in aflatoxin production by Aspergillus flavus and evidence for cryptic heterokaryosis.

Aspergillus flavus is the major producer of carcinogenic aflatoxins (AFs) in crops worldwide. Natural populations of A. flavus show tremendous variation in AF production, some of which can be attributed to environmental conditions, differential regulation of the AF biosynthetic pathway and deletions or loss-of-function mutations in the AF gene cluster. Understanding the evolutionary processes that generate genetic diversity in A. flavus may also explain quantitative differences in aflatoxigenicity. Several population studies using multilocus genealogical approaches provide indirect evidence of recombination in the genome and specifically in the AF gene cluster. More recently, A. flavus has been shown to be functionally heterothallic and capable of sexual reproduction in laboratory crosses. In the present study, we characterize the progeny from nine A. flavus crosses using toxin phenotype assays, DNA sequence-based markers and array comparative genome hybridization. We show high AF heritability linked to genetic variation in the AF gene cluster, as well as recombination through the independent assortment of chromosomes and through crossing over within the AF cluster that coincides with inferred recombination blocks and hotspots in natural populations. Moreover, the vertical transmission of cryptic alleles indicates that while an A. flavus deletion strain is predominantly homokaryotic, it may harbour AF cluster genes at a low copy number. Results from experimental matings indicate that sexual recombination is driving genetic and functional hyperdiversity in A. flavus. The results of this study have significant implications for managing AF contamination of crops and for improving biocontrol strategies using nonaflatoxigenic strains of A. flavus.

Value-added processing of peanut meal: aflatoxin sequestration during protein extraction.

The efficacy of a bentonite clay, Astra-Ben 20A (AB20A), to sequester aflatoxin from contaminated (approximately 110 ppb) peanut meal during protein extraction was studied. Aqueous peanut meal dispersions (10% w/w) were prepared by varying the pH, temperature, enzymatic hydrolysis conditions, and concentrations of AB20A. After extraction, dispersions were centrifuged and filtered to separate both the water-soluble and the water-insoluble fractions for subsequent testing. Inclusion of AB20A at 0.2 and 2% reduced (p < 0.05) aflatoxin concentrations below 20 ppb in both fractions; however, the higher concentration of AB20A also reduced (p < 0.05) the water-soluble protein content. Inclusion of 0.2% AB20A did not affect protein solubility, total soluble solids, or degree of hydrolysis. Peanut meal adsorption isotherms measured the AB20A capacity to sequester aflatoxin. These results are discussed in the context of a process designed to sequester aflatoxin from contaminated peanut meal, which could enable derivatives of this high protein material to be utilized in enhanced feed and/or food applications.

Efficacy of a biopesticide for control of aflatoxins in corn.

A 2-year study was carried out to determine the efficacy of a biopesticide in reducing aflatoxin contamination in corn. The biopesticide, afla-guard, delivers a nontoxigenic strain of Aspergillus flavus to the field where it competes with naturally occurring toxigenic strains of the fungus. Afla-guard was applied to entire fields in two areas of Texas at either 11.2 or 22.4 kg/ha. Specific nontreated fields in close proximity to treated fields were designated as controls. Samples of corn were collected at harvest and analyzed for aflatoxins and density of toxigenic and nontoxigenic isolates of A. flavus. Aflatoxin concentrations were generally quite low in 2007, but the mean concentration in treated samples (0.5 ppb) was reduced by 85% compared with controls (3.4 ppb). In 2008, samples from treated and control fields averaged 1.5 and 12.4 ppb, respectively, an 88% reduction. There were no significant differences between the two afla-guard application rates. In conjunction with the reductions in aflatoxin contamination, treatments produced significant reductions in the incidence of toxigenic isolates of A. flavus in corn.

Biological control of aflatoxin contamination in corn using a nontoxigenic strain of Aspergillus flavus.

A 2-year study was conducted to determine the efficacy of different applications of a nontoxigenic strain of Aspergillus flavus for reducing aflatoxin contamination in corn. Treatments consisted of the nontoxigenic strain in the form of (i) conidia-coated hulled barley applied to soil when corn was about 0.8 m tall, (ii) conidia-coated hulled barley applied in plant whorls prior to tasseling, (iii) multiple applications of a spray formulation of conidia during silking, and (iv) untreated control. Treatments were replicated eight times in individual plots consisting of four rows of 18 m each. In year 1, no significant differences were associated with treatments for aflatoxin, total A. flavus colonization, or incidence of nontoxigenic isolates of A. flavus in corn, which were all relatively high, ranging from 83.8 to 93.1%. In year 2, the whorl application produced a significantly lower mean aflatoxin concentration of 49.5 ppb compared with all other treatments, while both the soil (108.3 ppb) and spray applications (173.7 ppb) were significantly reduced compared with the control (191.6 ppb). The whorl application was the only treatment that had a significantly higher incidence (86.5%) of nontoxigenic isolates of A. flavus than the control had, which was still relatively high at 69.1%. Data indicated that applications of the nontoxigenic strain influenced untreated corn, thus reducing the apparent effect of the biocontrol treatments. Larger-scale studies with greater separation between treated and untreated fields are warranted.

Clustered genes involved in cyclopiazonic acid production are next to the aflatoxin biosynthesis gene cluster in Aspergillus flavus.

Cyclopiazonic acid (CPA), an indole-tetramic acid mycotoxin, is produced by many species of Aspergillus and Penicillium. In addition to CPA Aspergillus flavus produces polyketide-derived carcinogenic aflatoxins. Aflatoxin biosynthesis genes form a gene cluster in a subtelomeric region. Isolates of A. flavus lacking aflatoxin production due to the loss of the entire aflatoxin gene cluster and portions of the subtelomeric region are often unable to produce CPA, which suggests a physical link of genes involved in CPA biosynthesis to the aflatoxin gene cluster. Examining the subtelomeric region in A. flavus isolates of different chemotypes revealed a region possibly associated with CPA production. Disruption of three of the four genes present in this region predicted to encode a monoamine oxidase, a dimethylallyl tryptophan synthase, and a hybrid polyketide non-ribosomal peptide synthase abolished CPA production in an aflatoxigenic A. flavus strain. Therefore, some of the CPA biosynthesis genes are organized in a mini-gene cluster that is next to the aflatoxin gene cluster in A. flavus.

Separate and combined applications of nontoxigenic Aspergillus flavus and A. parasiticus for biocontrol of aflatoxin in peanuts.

A 2-year study was carried out to determine the effect of applying nontoxigenic strains of Aspergillus flavus and A. parasiticus to soil separately and in combination on preharvest aflatoxin contamination of peanuts. A naturally occurring, nontoxigenic strain of A. flavus and a UV-induced mutant of A. parasiticus were applied to peanut soils during the middle of each of two growing seasons using a formulation of conidia-coated hulled barley. In addition to an untreated control, treatments included soil inoculated with nontoxigenic A. flavus only, soil inoculated with nontoxigenic A. parasiticus only, and soil inoculated with a mixture of the two nontoxigenic strains. Plants were exposed to late-season drought conditions that were optimal for aflatoxin contamination. Results from year one showed that significant displacement (70%) of toxigenic A. flavus occurred only in peanuts from plots treated with nontoxigenic A. flavus alone; however, displacement did not result in a statistically significant reduction in the mean aflatoxin concentration in peanuts. In year two, soils were re-inoculated as in year one and all treatments resulted in significant reductions in aflatoxin, averaging 91.6%. Regression analyses showed strong correlations between the presence of nontoxigenic strains in peanuts and aflatoxin reduction. It is concluded that treatment with the nontoxigenic A. flavus strain alone is more effective than the A. parasiticus strain alone and equally as effective as the mixture.

Sequence breakpoints in the aflatoxin biosynthesis gene cluster and flanking regions in nonaflatoxigenic Aspergillus flavus isolates.

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.

Isolation and toxigenicity of Aspergillus fumigatus from moldy silage.

Thirty-nine silage samples were collected from various silos on Terceira Island in the Azores. Samples were examined for the presence of total fungi, and isolates of Aspergillus fumigatus were analyzed for their ability to produce fumitremorgens B and C, fumigaclavines B and C, and gliotoxin. Thirty-four silage samples (87%) were contaminated with fungi, and A. fumigatus was isolated from 27 samples (69%). Samples that were taken from the surface of silos had significantly higher populations of both total fungi and A. fumigatus than did samples taken from the middle of silos. Analysis of 27 A. fumigatus isolates (one representing each positive sample) showed that 59.3% produced fumitremorgen B; 33.3% produced fumitremorgen C; 29.6% produced fumigaclavine B; 7.4% produced fumigaclavine C; and 11.1% produced gliotoxin. Fifty-two percent of the isolates produced multiple toxins, and 25.9% did not produce any of these toxins. Gliotoxin and fumigaclavine C were always produced in combination with other toxins. Because of the demonstrated potential of these A. fumigatus isolates to produce mycotoxins, it is important to properly construct and manage silos to prevent their contamination with A. fumigatus.

Simultaneous quantitation of Aspergillus flavus/A. parasiticus and aflatoxins in peanuts.

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).

Cleanup procedure for determination of aflatoxins in major agricultural commodities by liquid chromatography.

A simple, fast, reliable, and inexpensive chemical cleanup procedure was developed for quantitation of aflatoxins in major important agricultural commodities by liquid chromatography (LC). Aflatoxins were extracted from a ground sample with methanol-water (80 + 20, v/v), and after a single cleanup step on a minicolumn packed with basic aluminum oxide, they were quantitated by LC equipped with a C18 column, photochemical reactor, and fluorescence detector. Water-methanol-1-butanol (1,400 + 720 + 25, v/v/v) served as the mobile phase. Recoveries of aflatoxins B1, B2, G1, and G2 from peanuts spiked at 5.0, 2.5, 7.5, and 2.5 microg/kg were 87.2 +/- 2.3, 82.0 +/- 0.8, 80.0 +/- 1.8, and 80.4 +/- 2.8%, respectively. Similar recoveries, precision, and accuracy were achieved for corn, cottonseed, almonds, Brazil nuts, pistachios, and walnuts. The quantitation limit for aflatoxin B1 was 1 microg/kg. The minimal cost of the minicolumn allows for substantial savings compared with available commercial aflatoxin cleanup devices.

Recent advances in analytical methodology for cyclopiazonic acid.

Cyclopiazonic acid (CPA) is a toxic indole tetramic acid that has been isolated from numerous species of Aspergillus and Penicillium. It has been found as a natural contaminant of cheese, corn, peanuts and various feedstuffs. Historically, thin-layer chromatography has been the most widely used method for quantitative determination of CPA in fungal cultures and agricultural commodities. Several liquid chromatographic (LC) and spectrophotometric methods have also been used, but these require extensive, time-consuming cleanup procedures to achieve accurate quantitation. More recently, enzyme-linked immunosorbent assays (ELISA) have been developed for quantification of CPA, and an immunoaffinity column (IAC) has been developed for cleanup of sample extracts prior to quantification by ELISA or LC. In applying the IAC to the cleanup of peanut extracts, recovery of CPA from spiked samples ranged from 83.7% to 90.8%, and the method was successfully applied to the analysis of peanuts that were naturally contaminated with CPA.

Effect of competition and adverse culture conditions on aflatoxin production by Aspergillus flavus through successive generations.

Strains of Aspergillus flavus often degenerate with serial transfers on culture media, resulting in morphological changes and loss of aflatoxin production. However, degeneration does not readily occur in nature as indicated by the wild-type morphological characters of newly isolated strains and the high percentage of aflatoxigenic A. flavus from soil and crops in some geographic regions. In this study, three aflatoxin-producing strains of A. flavus were serially transferred using conidia for 20 generations (three independent generation lines per strain) on potato dextrose agar at 30 C. The rate of degeneration was compared to that of cultures grown in the presence of competing fungi (A. terreus, Penicillium funiculosum, and the yeast, Pichia guilliermondii) and under adverse conditions of elevated temperature, reduced water activity, low pH, and nutrient deprivation. Formation of morphological variants and the associated loss of aflatoxin production over generations varied considerably according to strain and the generation line within each strain. In the strain most sensitive to degeneration on potato dextrose agar, aflatoxin-producing ability was maintained to varying degrees under adverse culture conditions, but not when A. flavus was competing with other fungi.

Amelioration of Aflatoxicosis in Rats by Volclay NF-BC, Microfine Bentonite.

Addition of sequestering agents to feeds and foods has been proposed as a protective strategy against mycotoxins. To investigate the efficacy of Volclay, a bentonite clay, to protect against aflatoxicosis, rats were fed peanut butter (50% wt/wt)-based diets containing 1,500 ppb aflatoxin (AF), 1,500 ppb aflatoxin with 0.1% Volclay supplementation (AF-LD), or 1,500 ppb aflatoxin with 1% Volclay supplementation (AF-HD) for 8 weeks. The control group was fed a peanut butter-based diet without aflatoxin or Volclay supplementation and a fifth group was fed the control diet with 1.0% Volclay supplementation (VC). No differences in appearance, behavior, or selected hematological and serum chemical variables were observed. Decreased weight gain, decreased food consumption, and liver lesions consistent with hepatic aflatoxicosis were found in AF-fed rats. Weight gain and food consumption of the AF-HD group were comparable to the control and VC groups and were significantly increased compared to AF-fed rats, even though weekly aflatoxin ingestion of AF-HD rats equaled or exceeded that of the AF group. Body weight and food consumption of the AF-LD group were slightly increased compared to AF group and decreased compared to the control, VC, and AF-HD groups, but the differences were not statistically significant. Liver lesions were found in all AF and AF-LD rats. Lesions were also detected in eight of 10 AF-HD-fed rats, but were subtle and significantly less extensive than in AF and AF-LD rats. The data suggest that Volclay is nontoxic and may be an efficacious sequestering agent for residual aflatoxin in peanut butter.

Use of a Biocompetitive Agent to Control Preharvest Aflatoxin in Drought Stressed Peanuts.

A three-year study was conducted to evaluate the use of a nonaflatoxin-producing strain of Aspergillus parasiticus (NRRL 13539) as a biocompetitive agent for the control of preharvest aflatoxin contamination of peanuts. The agent was added to the soil of the environmental control plot facility at the National Peanut Research Laboratory and tested by subjecting peanuts to optimal conditions for the development of aflatoxin contamination. Edible peanuts from the treated soil contained aflatoxin concentrations of 11, 1, and 40 ppb for crop years 1987, 1988, and 1989, respectively, compared to untreated peanuts with 531, 96, and 241 ppb, respectively. In addition, treatment in 1989 with low and high inoculum levels of a UV-induced mutant from the NRRL 13539 strain resulted in aflatoxin concentrations of 29 and 17 ppb, respectively, in edible peanuts. Soil populations of the biocompetitive agents were not higher than populations of wild strains of A. flavus/parasiticus in untreated soil subjected to late-season drought stress. This is an important ecological consideration relative to the utilization of this biocontrol system.