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. The U.S. Government’s right to retain a non-exclusive, royalty-free license in and to any copyright is acknowledged.     

Evaluation of biological control formulations to reduce aflatoxin contamination in peanuts

A two-year study was conducted to evaluate the efficacy of three formulations of nontoxigenic strains of Aspergillus flavus and Aspergillus parasiticus to reduce preharvest aflatoxin contamination of peanuts. Formulations included: (1) solid-state fermented rice; (2) fungal conidia encapsulated in an extrusion product termed Pesta; (3) conidia encapsulated in pregelatinized corn flour granules. Formulations were applied to peanut plots in 1996 and reapplied to the same plots in 1997 in a randomized design with four replications, including untreated controls. Analysis of soils for A. flavus and A. parasiticus showed that a large soil population of the nontoxigenic strains resulted from all formulations. In the first year, the percentage of kernels infected by wild-type A. flavus and A. parasiticus was significantly reduced in plots treated with rice and corn flour granules, but it was reduced only in the rice-treated plots in year two. There were no significant differences in total infection of kernels by all strains of A. flavus and A. parasiticus in either year. Aflatoxin concentrations in peanuts were significantly reduced in year two by all formulation treatments with an average reduction of 92%. Reductions were also noted for all formulation treatments in year one (average 86%), but they were not statistically significant because of wide variation in the aflatoxin concentrations in the untreated controls. Each of the formulations tested, therefore, was effective in delivering competitive levels of nontoxigenic strains of A. flavus and A. parasiticus to soil and in reducing subsequent aflatoxin contamination of peanuts.     

Effect of nontoxigenic Aspergillus flavus and A. parasiticus on aflatoxin contamination of wounded peanut seeds inoculated with agricultural soil containing natural fungal populations

Peanuts and other seed and grain crops are commonly contaminated with carcinogenic aflatoxins, secondary metabolites produced by Aspergillus flavus and A. parasiticus. Aflatoxin contamination of peanuts in the field can be reduced by 77–98% with biological control through the application of nontoxigenic strains of these species, which competitively exclude native aflatoxin-producing strains from developing peanuts. In this study, viable peanut seeds were artificially wounded and inoculated with field soil containing natural fungal populations that were supplemented with conidia of nontoxigenic A. flavus NRRL 21882 (niaD nitrate-nonutilizing mutant) and A. parasiticus NRRL 21369 (conidial color mutant). Increasing soil densities of applied nontoxigenic strains generally resulted in an increase in the incidence of seed colonization by applied nontoxigenic strains, a decrease in seed colonization by native A. flavus and A. parasiticus, and a decrease in aflatoxin concentration in seeds. Reduction of aflatoxins in peanut seeds depended on both the density and the aflatoxin-producing potential of native populations and on the fungal strain used for biological control. Wild-type strain A. flavus NRRL 21882 and its niaD mutant were equally effective in reducing aflatoxins in peanuts, indicating that nitrate-nonutilizing mutants, which are easily monitored in the field, can be used for evaluating the efficacy of biocontrol strains.     

Effect ofAspergillus parasiticus soil inoculum on invasion of peanut seeds

Environmental control plots adjusted to late season drought and elevated soil temperatures where inoculated at peanut planting with low and high levels of conidia, sclerotia, and mycelium from a brown conidial mutant ofAspergillus parasiticus. Percentage infection of peanut seeds from undamaged pods was greatest for the subplot containing the high sclerotial inoculum (15/cm² soil surface). Sclerotia did not germinate sporogenically and may have invaded seeds through mycelium. In contrast, the mycelial inoculum (colonized peanut seed particles) released large numbers of conidia into soil. Soil conidial populations of brownA. parasiticus from treatments with conidia and mycelium were positively correlated with the incidence of seed infection in undamaged pods. The ratio ofA. flavus to wild-typeA. parasiticus in soil shifted from 7:3 to 1:1 in the uninoculated subplot after instigation of drought, whereas in all subplots treated with brownA. parasiticus, the ratio of the two species became approximately 8:2. Despite high levels of brownA. parasiticus populations in soil, nativeA. flavus often dominated peanut seeds, suggesting that it is a more aggressive species. Sclerotia of wild-typeA. parasiticus formed infrequently on preharvest peanut seeds from insect-damaged pods.     

Evaluation of different genotypes of nontoxigenic Aspergillus flavus for their ability to reduce aflatoxin contamination in peanuts

Aflatoxins produced by the fungus Aspergillus flavus are potent carcinogens and account for large monetary losses worldwide in peanuts, maize, and cottonseed. Biological control in which a nontoxigenic strain of A. flavus is applied to crops at high concentrations effectively reduces aflatoxins through competition with native aflatoxigenic populations. In this study, eight nontoxigenic strains of A. flavus belonging to different vegetative compatibility groups and differing in deletion patterns within the aflatoxin gene cluster were evaluated for their ability to reduce aflatoxin B₁ when paired with eight aflatoxigenic strains on individual peanut seeds. Inoculation of wounded viable peanut seeds with conidia demonstrated that nontoxigenic strains differed in their ability to reduce aflatoxin B₁. Reductions in aflatoxin B₁ often exceeded expected reductions based on a 50:50 mixture of the two A. flavus strains, although one nontoxigenic strain significantly increased aflatoxin B₁ when paired with an aflatoxigenic strain. Therefore, nontoxigenicity alone is insufficient for selecting a biocontrol agent and it is also necessary to test the effectiveness of a nontoxigenic strain against a variety of aflatoxigenic strains.     

Mycotoxins in Australia: biocontrol of aflatoxin in peanuts

The major mycotoxin problem in Australia is the formation of aflatoxins in peanuts by Aspergillus flavus and A. parasiticus. This is controlled by good farm management practice, segregation into grades on aflatoxin content at intake to shelling facilities, colour sorting and aflatoxin assays. A second problem is the potential presence of ochratoxin A in grapes and grape products, resulting from infection by Aspergillus carbonarius. Good quality control before and during wine making ensures ochratoxin A is kept to very low levels, but in dried vine fruit, ochratoxin A levels may be higher. Biocontrol by competitive exclusion has been developed as the most promising means of controlling aflatoxins in peanuts. Some details of the process are given, including some basic laboratory experiments.     

Distribution and characteristics of aflatoxin-producingAspergillus flavus in the soil in Kanagawa Prefecture,Central Japan

In Kanagawa Prefecture, located in central Japan, aflatoxin-producingAspergillus flavus was isolated in 4 (2.5%) of 160 field soil samples. In the 4 fields, whose soil contained aflatoxin-producingA. flavus, the annual average temperature of the sampling sites of the soil ranged from 13.8 to 15.1°C. Of all the isolated strains of aflatoxin-producingA. flavus, 4 strains, isolated from a single soil sample, produced large amounts of aflatoxin B₁ and B₂ when incubated in coconut agar, peanut agar, peanuts or trilaurin-added rice, although they did not produce aflatoxin when incubated in rice, yeast extract-sucrose broth or sucrose-low salts broth.     

Effect of Inoculum Rate of Biological Control Agents on Preharvest Aflatoxin Contamination of Peanuts

Studies were conducted during 1994 and 1995 in the environmental control plot facility at the National Peanut Research Laboratory to determine the effect of different inoculum rates of biological control agents on preharvest aflatoxin contamination of Florunner peanuts. Biocontrol agents were nontoxigenic color mutants ofAspergillus flavusandAspergillus parasiticusthat were grown on rice for use as soil inoculum. Three replicate plots (4.0 × 5.5 m) were treated with 0, 2, 10, and 50 g/m of row (0, 20, 100, and 500 lb/acre, respectively) of an equal mixture of the color mutant-infested rice in 1994, and the same plots were retreated in 1995. Aflatoxin concentrations were determined by high performance liquid chromatographic analysis of all peanuts. Treatment means for total kernels in 1994 were 337.6, 73.7, 34.8, and 33.3 ppb for the 0, 2, 10, and 50 g/m treatments, respectively. Regression analysis indicated a trend toward lower aflatoxin concentrations with increasing rates of inoculum (R²= 0.40;P< 0.05). For the same repeated treatments in 1995 aflatoxin concentrations in total kernels averaged 718.3, 184.4, 35.9, and 0.4 ppb. Regression analysis revealed a stronger relationship between inoculum rate and aflatoxin concentrations (R²= 0.66;P< 0.05) in the second year of treatment. Compared with untreated controls, the 2, 10, and 50 g/m treatments produced respective reductions in aflatoxin of 74.3, 95.0, and 99.9% in the second year. The data indicated not only a treatment-related effect, but also that a higher degree of control might be achieved when plots or fields are retreated with biocontrol agents in subsequent years.     

Comparison of four media for the isolation ofAspergillus flavus group fungi

Four agar media used to isolate aflatoxin producing fungi were compared for utility in isolating fungi in theAspergillus flavus group from agricultural soils collected in 15 fields and four states in the southern United States. The four media wereAspergillus flavus andparasiticus Agar (AFPA, 14), the rose bengal agar described by Bell and Crawford (BCRB; 3), a modified rose bengal agar (M-RB), and Czapek's-Dox Agar supplemented with the antibiotics in BC-RB (CZ-RB). M-RB was the most useful for studying the population biology of this group because it permitted both identification of the greatest number ofA. flavus group strains and growth of the fewest competing fungi. M-RB supported an average of 12% moreA. flavus group colonies than the original rose bengal medium while reducing the number of mucorales colonies and the number of total fungi by 99% and 70%, respectively. M-RB was successfully employed to isolate all three aflatoxin producing species,A. flavus, A. parasiticus andA. nomius, and both the S and L strains ofA. flavus. M-RB is a defined medium without complex nitrogen and carbon sources (e.g. peptone and yeast extract) present in BC-RB. M-RB should be useful for studies on the population biology of theA. flavus group.Abbreviations M-RB Modified Rose Bengal Agar - CZ-RB Czapeks Rose Bengal Agar - BC-RB Bell and Crawford's Rose Bengal Agar - AFPA Aspergillus flavus andparasiticus agar     

Survey of Vietnamese Peanuts,Corn and Soil for the Presence of <Emphasis Type="Italic">Aspergillus flavus</Emphasis> and <Emphasis Type="Italic">Aspergillus parasiticus</Emphasis>

Aspergillus flavus and Aspergillus parasiticus cause perennial infection of agriculturally important crops in tropical and subtropical areas. Invasion of crops by these fungi may result in contamination of food and feed by potent carcinogenic aflatoxins. Consumption of aflatoxin contaminated foods is a recognised risk factor for human hepatocellular carcinoma (HCC) and may contribute to the high incidence of HCC in Southeast Asia. This study conducted a survey of Vietnamese crops (peanuts and corn) and soil for the presence of aflatoxigenic fungi and used microsatellite markers to investigate the genetic diversity of Vietnamese Aspergillus strains. From a total of 85 samples comprising peanut (25), corn (45) and soil (15), 106 strains were isolated. Identification of strains by colony morphology and aflatoxin production found all Vietnamese strains to be A. flavus with no A. parasiticus isolated. A. flavus was present in 36.0% of peanut samples, 31.1% of corn samples, 27.3% of farmed soil samples and was not found in virgin soil samples. Twenty-five per cent of the strains produced aflatoxins. Microsatellite analysis revealed a high level of genetic diversity in the Vietnamese A. flavus population. Clustering, based on microsatellite genotype, was unrelated to aflatoxin production, geographic origin or substrate origin.     

Elucidation of <Emphasis Type="Italic">veA</Emphasis>-dependent genes associated with aflatoxin and sclerotial production in <Emphasis Type="Italic">Aspergillus flavus</Emphasis> by functional genomics

The aflatoxin-producing fungi, Aspergillus flavus and A. parasiticus, form structures called sclerotia that allow for survival under adverse conditions. Deletion of the veA gene in A. flavus and A. parasiticus blocks production of aflatoxin as well as sclerotial formation. We used microarray technology to identify genes differentially expressed in wild-type veA and veA mutant strains that could be involved in aflatoxin production and sclerotial development in A. flavus. The DNA microarray analysis revealed 684 genes whose expression changed significantly over time; 136 of these were differentially expressed between the two strains including 27 genes that demonstrated a significant difference in expression both between strains and over time. A group of 115 genes showed greater expression in the wild-type than in the veA mutant strain. We identified a subgroup of veA-dependent genes that exhibited time-dependent expression profiles similar to those of known aflatoxin biosynthetic genes or that were candidates for involvement in sclerotial production in the wild type.     

A rapid identification method for aflatoxin-producing strains ofAspergillus flavus andA. parasiticus by ammonia vapor

The colony reverse of aflatoxin (AF)-producing strains ofAspergillus flavus andA. parasiticus turned pink when their cultures were exposed to ammonia vapor. The color change was visible for colonies grown on media suitable for AF production such as potato dextrose, coconut, and yeast extract sucrose agars after 2 d incubation at 25°C. Of the 120 strains ofA. flavus, A. parasiticus, and two related species inA. flavus group:A. oryzae andA. sojae tested in this study, only the AF-producing strains ofA. flavus andA. parasiticus showed the pink pigmentation. The color change occurred immediately after the colony was contacted with ammonia vapor. This method was useful for rapid screening the AF-producing strains ofA. flavus andA. parasiticus.     

Inhibition of Aspergillus flavus growth and detoxification of aflatoxin B1 by the medicinal plant zimmu (Allium sativum L. × Allium cepa L.)

Aflatoxins are carcinogenic, teratogenic and immunosuppressive secondary metabolites produced by Aspergillus flavus and Aspergillus parasiticus. Aflatoxin contamination of peanut is one of the most important constraints to peanut production worldwide. In order to develop an eco-friendly method of prevention of A. flavus infection and aflatoxin contamination in peanut, aqueous extracts obtained from leaves of 30 medicinal plants belonging to different families were evaluated for their ability to inhibit the growth of A. flavus in vitro. Among them the leaf extract of zimmu (Allium sativum L. × Allium cepa L.) was the only one that showed antifungal activity against A. flavus and recorded 73% inhibition of A. flavus growth. The antifungal activity of the zimmu extract was significantly decreased upon dialysis with a dialysis membrane having molecular cut off 12 kDa or autoclaving at 121°C for 20 min or boiling at 100°C for 10 min and recorded inhibition of 52, 16 and 21%, respectively. When A. flavus was grown in medium containing zimmu extract the production of aflatoxin B₁ (AFB₁) was completely inhibited even at a concentration of 0.5%. When AFB₁ was incubated with zimmu extract a complete degradation of AFB₁ was observed 5 days after incubation. When the roots of zimmu were incubated in water containing 70 ng of AFB₁/ml, a reduction (by 58.5%) in AFB₁ concentration was observed 5 days after incubation. A significant reduction in the population of A. flavus in the soil, kernel infection by A. flavus and aflatoxin contamination in kernels was observed when peanut was intercropped with zimmu. The population of the fungal antagonist, Trichoderma viride in the zimmu-intercropped field increased approximately twofold.     

Purified moniliformin does not affect the force or rate of contraction of isolated guinea pig atria

Aspergillus flavus Link ex Fries and A. parasiticus Speare can invade peanut kernels and under certain environmental conditions produce unacceptable levels of the mycotoxin aflatoxin. A concerted effort is underway to reduce aflatoxin contamination in peanut and peanut products. A potentially effective method of control in peanut is the discovery and use of genes for resistance to either fungal invasion or aflatoxin formation. The objective of the present experimental study was to develop an effective and efficient procedure for screening individual plants or pods of single plants for resistance to invasion by the aflatoxigenic fungi and subsequent aflatoxin production. Methods of obtaining adequate drought-stress and fungal infection were developed through this series of experiments. By completely isolating the pods from the root zone and imposing drought-stress only on pegs and pods, high levels of fungal infection were observed. High amounts of preharvest aflatoxin accumulation were also produced by completely isolating the pods from the root zone. Mid-bloom inoculation with A. parasiticus-contaminated cracked corn and drought-stress periods of 40 to 60 days were the most effective procedures. This technique was used to assess peanut genotypes previously identified as being partially resistant to A. parasiticus infection or aflatoxin contamination, and segregating populations from four crosses. Variability in aflatoxin contamination was found among the 11 genotypes evaluated, however, none were significantly lower than the standard cultivars. Broad-sense heritability of four crosses was estimated through evaluation of seed from individual plants in the F₂ generation. The heritability estimates of crosses GFA-2 × NC-V11 and Tifton-8 × NC-V11 were 0.46 and 0.29, respectively, but mean aflatoxin contamination levels were high (73,295 and 27,305 ppb). This greenhouse screening method could be an effective tool when genes for superior aflatoxin resistance are identified.Cooperative investigation of the USDA-ARS and the University of Georgia, College of Agriculture.     

Association between vegetative compatibility and aflatoxin production by <Emphasis Type="Italic">Aspergillus</Emphasis> species during intraspecific competition

Intraspecific competition is the basis for biological control of aflatoxins, but there is little understanding of the mechanism(s) by which competing strains inhibit toxin production. Evidence is presented that demonstrates a relationship between strength of the vegetative compatibility reaction and aflatoxin production in Aspergillus flavus and A. parasiticus using the suspended disk culture method. Combining wild-type aflatoxin-producing isolates belonging to different vegetative compatibility groups (VCGs) resulted in a substantial reduction in aflatoxin yield. Pairs of aflatoxin-producing isolates within the same VCG, but showing weak compatibility reactions using complementary nitrate-nonutilizing mutants, also were associated with reduced levels of aflatoxin B₁. In contrast, pairings of isolates displaying a strong compatibility reaction typically produced high levels of aflatoxins. These results suggest that interactions between vegetatively compatible wild-type isolates of A. flavus and A. parasiticus are cooperative and result in more aflatoxin B₁ than pairings between isolates that are incompatible. Successful hyphal fusions among spore germlings produce a common mycelial network with a larger resource base to support aflatoxin biosynthesis. By comparison, vegetative incompatibility reactions might result in the death of those heterokaryotic cells composed of incompatible nuclei and thereby disrupt the formation of mycelial networks at the expense of aflatoxin biosynthesis. The content of this paper was presented at the 50th Anniversary Meeting of the Mycological Society of Japan, June 3–4, 2006, Chiba, Japan     

Association of aflatoxin biosynthesis and sclerotialdevelopment in Aspergillus parasiticus

Secondary metabolism in fungi is frequently associated with asexual and sexual development. Aspergillus parasiticus produces aflatoxins known to contaminate a variety of agricultural commodities. This strictly mitotic fungus, besides producing conidia asexually, produces sclerotia, structures resistant to harsh conditions and for propagation. Sclerotia are considered to be derived from the sexual structure, cleistothecia, and may represent a vestige of ascospore production. Introduction of the aflatoxin pathway-specific regulatory gene, aflR, and aflJ, which encoded a putative co-activator, into an O-methylsterigmatocystin (OMST)-accumulating strain,A. parasiticus SRRC 2043, resulted in elevated levels of accumulation of major aflatoxin precursors, including norsolorinic acid (NOR), averantin (AVN), versicolorin A (VERA) and OMST. The total amount of these aflatoxin precursors, NOR, VERA, AVN and OMST, produced by the aflR plus aflJ transformants was two to three-fold that produced by the aflR transformants. This increase indicated a synergisticeffect of aflR and aflJ on the synthesis of aflatoxin precursors. Increased production of the aflatoxin precursors was associated with progressive decrease in sclerotial size, alteration in sclerotial shape and weakening in the sclerotial structure of the transformants. The results showed that sclerotial development and aflatoxin biosynthesis are closely related. We proposed that competition for a common substrate, such as acetate, by the aflatoxin biosynthetic pathway could adversely affect sclerotial development in A. parasiticus. This revised version was published online in June 2006 with corrections to the Cover Date.     

Understanding the genetics of regulation of aflatoxin production and Aspergillus flavus development

Aflatoxins are polyketide-derived, toxic, and carcinogenic secondary metabolites produced primarily by two fungal species, Aspergillus flavus and A. parasiticus, on crops such as corn, peanuts, cottonseed, and treenuts. Regulatory guidelines issued by the U.S. Food and Drug Administration (FDA) prevent sale of commodities if contamination by these toxins exceeds certain levels. The biosynthesis of these toxins has been extensively studied. About 15 stable precursors have been identified. The genes involved in encoding the proteins required for the oxidative and regulatory steps in the biosynthesis are clustered in a 70 kb portion of chromosome 3 in the A. flavus genome. With the characterization of the gene cluster, new insights into the cellular processes that govern the genes involved in aflatoxin biosynthesis have been revealed, but the signaling processes that turn on aflatoxin biosynthesis during fungal contamination of crops are still not well understood. New molecular technologies, such as gene microarray analyses, quantitative polymerase chain reaction (PCR), and chromatin immunoprecipitation are being used to understand how physiological stress, environmental and soil conditions, receptivity of the plant, and fungal virulence lead to episodic outbreaks of aflatoxin contamination in certain commercially important crops. With this fundamental understanding, we will be better able to design improved non-aflatoxigenic biocompetitive Aspergillus strains and develop inhibitors of aflatoxin production (native to affected crops or otherwise) amenable to agricultural application for enhancing host-resistance against fungal invasion or toxin production. Comparisons of aflatoxin-producing species with other fungal species that retain some of the genes required for aflatoxin formation is expected to provide insight into the evolution of the aflatoxin gene cluster, and its role in fungal physiology. Therefore, information on how and why the fungus makes the toxin will be valuable for developing an effective and lasting strategy for control of aflatoxin contamination.     

Effect of peanut tannin extracts on growth of Aspergillus parasiticus and aflatoxin production

Twenty-three peanut (Arachis hypogaea L.) genotypes were evaluated for kernel resistance to Aspergillus parasiticus Spear. colonization and aflatoxin contamination when incubated under high relative humidity. Also, tannin-containing extracts from kernel coats (testae) and cotyledons of these genotypes were prepared and tested for their effect on A. parasiticus growth and aflatoxin production in vitro. The lowest degree of colonization, less than 30% was noted in kernels from the genotypes, Toalson x UF 73-4022 (selections TX-798731 and TX-798736), A72118, SN 55-437, PI337409, and Florunner. Genotypes with low levels of colonization also had the lowest aflatoxin contamination. The coefficient of correlation between infection frequency and aflatoxin contamination was 0.66. Higher levels of tannins were detected in the testae (23.9–97.2 mg g tissue) compared to the cotyledons (0.17–0.82 mg g tissue). Some of the methanol-extracted and water-soluble tannin extracts from testae and cotyledons, when incorporated in yeast extract sucrose liquid medium (100 mg l), significantly inhibited A. parasiticus growth and reduced the levels of aflatoxin produced. There was no overall correlation between the peanut genotypes and the influence of tannin extracts on A. parasiticus growth and aflatoxin production. However, correlations were higher for specific genotypes. For example, the coefficient of correlation between the ability of tannin extracts from testae of genotypes PI337409 and TX-798736 to inhibit aflatoxin production was 0.93 and 0.85 respectively.     

Winged bean (Psophocarpus tetragonolobus (L.) DC) as a substrate for growth and aflatoxin production by aflatoxigenic strains of Aspergillus spp.

The mold flora of seeds of twelve varieties of winged beans were determined both before and after surface disinfections. When seeds were surface disinfected, molds were detected in 73% of the seeds whereas 81% of the seed that was not disinfected produced molds. Aspergillus spp. were most frequently present while Penicillium spp. occurred in seed of 4 varieties and in less than 4% of the seed. Twelve isolates of A. flavus and A. parasiticus were examined for their ability to produce aflatoxins. Whether aflatoxins were produced and the amount of each varied according to the origin of the isolate and the species of Aspergillus. For example all A. flavus isolates produced at least 2 aflatoxins whereas 4 of the A. parasiticus isolates were nontoxigenic. When ground seeds of winged beans were inoculated with an aflatoxigenic strain of A. parasiticus the level of aflatoxins that occurred varied with the variety. All of the varieties supported greater aflatoxin production than peanuts and 6 of the 12 winged bean varieties gave higher levels of aflatoxins than rice.     

Studies on the biocontrol of aflatoxin in maize in Thailand

Aflatoxins in maize and peanuts remain a major cause of liver cancer and other human and animal health issues. The principal causal fungi are Aspergillus flavus and A. parasiticus. Relatively little attention has been paid to reducing aflatoxin formation before harvest. The most promising approach is biocontrol by competitive exclusion. This project aimed to demonstrate the efficacy of locally isolated strains of A. flavus for biocontrol of aflatoxin in maize in Thailand. After a rigorous process utilising molecular methods was used to select non-toxigenic A. flavus strains, field inoculum was produced by using hulled rice coated with A. flavus spores in molasses. Field experiments were conducted over two years in two districts, one of light sandy soil (Chokchai), the other a heavy, close textured, soil (Pakchong). Postharvest treatments representative of local practice were also undertaken. Crops 1 and 2 were not significantly contaminated with aflatoxin at the time of harvest, so any impact of biocontrol could not be assessed. However, wet shelling plus storage before drying resulted in increased aflatoxin contamination; biocontrol had no impact on this increase. In crops 3 and 4, biocontrol had a beneficial impact in some freshly harvested maize. Biocontrol treatments also significantly reduced aflatoxin contamination in samples from some treatments stored for two or four days after shelling, but had minimal effect in others. These experiments demonstrated that biocontrol can be highly effective in reducing aflatoxin contamination in maize in Thailand, both at harvest and during poor postharvest crop handling. However, results were inconsistent.