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 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.     

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.     

Conidial movement of nontoxigenic Aspergillus flavus and A. parasiticus in peanut fields following application to soil

The use of nontoxigenic strains of Aspergillus flavus and A. parasiticus in biological control effectively reduces aflatoxin in peanuts when conidium-producing inoculum is applied to the soil surface. In this study, the movement of conidia in soil was examined following natural rainfall and controlled precipitation from a sprinkler irrigation system. Conidia of nontoxigenic A. flavus and A. parasiticus remained near the soil surface despite repeated rainfall and varying amounts of applied water from irrigation. In addition, rainfall washed the conidia along the peanut furrows for up to 100 meters downstream from the experimental plot boundary. The dispersal gradient was otherwise very steep upstream along the furrows and in directions perpendicular to the peanut rows. The retention of biocontrol conidia in the upper soil layers is likely important in reducing aflatoxin contamination of peanuts and aerial crops such as corn and cottonseed. This revised version was published online in August 2006 with corrections to the Cover Date.     

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.     

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     

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.     

Deciphering the origin of Aspergillus flavus NRRL21882, the active biocontrol agent of Afla-Guard®

Single nucleotide polymorphisms (SNPs) of genome sequences of eight Aspergillus flavus and seven Aspergillus oryzae strains were extracted with Mauve, a multiple-genome alignment programme. A phylogenetic analysis with sequences comprised of concatenated total SNPs by the unweighted pair group method with arithmetic mean (UPGMA) of MAFFT adequately separated them into three groups, A. flavus S-morphotype, A. flavus L-morphotype and A. oryzae. Divergence time inferred for A. flavus NRRL21882, the active agent of the biocontrol product Afla-Guard^®, and S-morphotype was about 5·1 mya. Another biocontrol strain, A. flavus AF36, diverged from aflatoxigenic L-morphotype about 2·6–3·0 mya. Despite the close relatedness of A. oryzae to A. flavus, A. oryzae strains likely evolved from aflatoxigenic Aspergillus aflatoxiformans (=A. parvisclerotigenus). A survey of A. flavus populations implies that prior Afla-Guard^® applications are associated with prevalence of NRRL21882-type isolates in Mississippi fields. In addition, a few NRRL21882 relatives were identified. A. flavus Og0222, a biocontrol ingredient of Aflasafe™, was verified as a NRRL21882-type strain, having identical sequence breakpoints that led to deletion of aflatoxin and cyclopiazonic acid gene clusters. A similar UPGMA analysis suggests that the occurrence of NRRL21882-type strains is a more recent event.     

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 was determined both before and after surface disinfections. When seeds were surface disinfected, mold fungi were detected in 73% of the seeds whereas 81% of the seed that was not disinfected produced mold fungi. Aspergillus spp. was most frequently present while Penicillium spp. occurred in seed of 4 varieties and in less than 4% of the seed. Twelve isolates oiA. 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. parasiticus isolates produced at least 2 aflatoxins whereas 4 of the A. parasiticus isolates were non-toxigenic. 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, however, the level of aflatoxin was higher in winged bean than in peanut tissue and 6 of the 12 winged bean varieties contained higher levels of aflatoxins than rice.     

Aflatoxin is degraded by mycelia from toxigenic and nontoxigenic strains of aspergilli grown on different substrates

The ability of 9-day-old mycelia of Aspergillus parasiticus NRRL 2999 to degrade aflatoxin varied depending on the substrate used to grow the mold. Substrates which allowed substantial mycelial growth yielded mycelia which actively degraded aflatoxin. Substrates which allowed minimal growth of mycelia yielded mycelia with little ability to degrade aflatoxin. Biodegradation of aflatoxin was also strain-dependent. A. parasiticus NRRL 2999 and NRRL 3000 actively degraded aflatoxin, A. flavus NRRL 3353 was less active, and A. flavus NRRL 482 and A. parasiticus NRRL 3315 degraded minimal amounts of aflatoxins. Those aspergilli producing greatest amounts of aflatoxin also degraded aflatoxins most rapidly, whereas those strains which produced minimal amounts of aflatoxin generally degraded aflatoxins less effectively. Substrates which allowed maximum aflatoxin production also yielded mycelia which actively degraded aflatoxins, whereas media which allowed limited production of aflatoxin generally yielded mycelia with minimal ability to degrade the toxin. Although exceptions exist, generally as aflatoxin production increased so did the ability of mycelia to degrade the toxin.     

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.     

Comparative studies on the changes of total soluble proteins and protease activity in Georgian commercial peanuts contaminated by Aspergillus flavus

The presence of Aspergillus species is an indicator of storage conditions, which also suggests the possibility of several biochemical changes in grains. A comparative change in total soluble proteins and protease activity was determined in commercial peanut seeds collected from Georgia State. Protein contents of healthy peanuts, naturally contaminated peanuts and then artificially inoculated peanut seeds with A. flavus were estimated by Bradford method, and protease activity was also determined by using the Protease Detection Kits. Protein contents and the protease activity of the peanuts varied from sample to sample. The soluble protein content of seeds was significantly higher in healthy peanuts than in artificially inoculated or naturally infected peanuts with A. flavus. Protease activity was found to be higher in artificially inoculated seeds than in either naturally infected or healthy peanuts. Level of soluble proteins in buffer extracts of contaminated seeds decreased with incubation time, and protease activity increased with incubation time. These changes may be attributed to host response due to infection, contribution by A. flavus or due to biochemical alterations that occur naturally during the transition from endosperm to seedling during incubation period.     

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.     

Use of microsatellite markers to assess the competitive ability of nontoxigenic Aspergillus flavus strains in studies on biocontrol of aflatoxins in maize in Thailand

Many nontoxigenic strains of Aspergillus flavus have been used in studies on biocontrol by competitive exclusion, but assessing their competitive ability is difficult. This paper reports on the use of a microsatellite marker technique for assessing competitiveness. The chosen microsatellite markers were able to differentiate, at an individual level, between the four biocontrol strains used in a study on the biocontrol of aflatoxins in maize in Thailand. The microsatellite markers were then used to determine which of the four biocontrol strains used were identical with 86 nontoxigenic strains of A. flavus taken from dried maize samples produced in that study. Fifty-one of the 86 strains (59%) were identified as one of the four biocontrol strains, with another four likely to be so. Analysis of microsatellites in A. flavus strains taken from dried samples at the conclusion of a field trial was shown to be of value in understanding the competitive ability of the specific strains used for biocontrol. This method provides an objective assessment of the competitiveness of biocontrol strains.     

Morphological changes in strains of Aspergillus flavus link ex fries and Aspergillus parasiticus speare related with aflatoxin production

Two strains ofAspergillus flavus Linkex Fr. and two strains ofA. parasiticus Speare were cultured on crushed moist wheat (Triticum durum var. Pané no. 247) for aflatoxin production studies in correlation with morphological changes. The toxicogenic strains were adapted to the substratum by means of successive transfers at regular intervals (72 h.)The amount aflatoxins synthesized by the toxicogenic strains decreased gradually after succesive subculturing. The decrease was accompanied by marked morphological changes. One of the strains studied,A. flavus NRRL 3251, lost completly the capacity of aflatoxin synthesis after several subcultures, presenting at the same time strong morphological variations.A. flavus CBS 120.62 also lost its toxicogenicity after six subcultures.     

Aflatoxins in sunflower seeds: Influence of Alternaria alternata on aflatoxin production by Aspergillus parasiticus

The aim of the present work was to determine the influence of Alternaria alternata upon aflatoxin production by Aspergillus parasiticus.A mixture of spores of both strains was inoculated in sunflower seeds at 0,90 a_w, and incubated for 42 days at 28 °C ±1.The cultures were observed and analyzed every 7 days to determine the infection level of the seeds and the production of aflatoxins. Results showed that when the seeds were inoculated only with Aspergillus parasiticus, 100% were infected from the 7th day.When Aspergillus parasiticus and Alternaria alternata were simultaneously inoculated the infection level of the seeds was 100% for Aspergillus parasiticus following 7 days of inoculation and 0% for Alternaria alternata. After the 14th day of inoculation there was no significant difference in the infection percentage of both strains (approximately 80% of each one). As far as toxin production is concerned a remarkable decrease was observed when seeds were inoculated with both strains simultaneously.In accordance to the results, Alternaria alternata would not compete with Aspergillus parasiticus in colonization of seeds but would either degrade the aflatoxins by Aspergillus parasiticus or compete for aflatoxin biosynthesis precursors. Alternaria alternata could also secrete some substance that specifically inhibits aflatoxin synthesis.     

A Survey on Distribution of Aspergillus Section Flavi in Corn Field Soils in Iran: Population Patterns Based on Aflatoxins, Cyclopiazonic Acid and Sclerotia Production

Soil isolates of Aspergillus section Flavi from Mazandaran and Semnan provinces with totally different climatic conditions in Iran were examined for aflatoxins (AFs; B and G types), cyclopiazonic acid (CPA) and sclerotia production. A total of 66 Aspergillus flavus group strains were identified from three species viz. Aspergillus flavus, Aspergillus parasiticus and Aspergillus nomius in both locations. A. flavus (87.9%) was found to be the prominent species followed by A. nomius (9.1%) and A. parasiticus (3.0%). Only 27.5% of A. flavus isolates were aflatoxigenic (B₁ or B₁ and B₂), out of which approximately 75% were capable to producing CPA. All the A. parasiticus and A. nomius isolates produced AFs of both B (B₁ and B₂) and G (G₁ and G₂) types, but did not produce CPA. Sclerotia production was observed in only 4 isolates of A. flavus among all 66 isolates from three identified species. A. flavus isolates were classified into various chemotypes based on the ability to produce aflatoxins and CPA. In this study, a new naturally occurring toxigenic A. flavus chemotype comprising of two strains capable of producing more AFB₂ than AFB₁ has been identified. A relatively larger proportion of aflatoxigenic A. flavus strains were isolated from corn field soils of Mazandaran province which indicate a possible relationship between high levels of relative humidity and the incidence of aflatoxin-producing fungi. The importance of incidence of Aspergillus section Flavi in corn field soils regard to their mycotoxin production profiles and crop contamination with special reference to climatic conditions is discussed.     

Examination of fungal growth and aflatoxin production on marihuana

Under favorable growth conditions,Aspergillus flavus andA. parasiticus produced aflatoxins on marihuana. Cultures ofA. flavus ATCC 15548 produced both aflat oxin B₁(AFB₁) and G₁(AFG₁). The production of AFG₁ was substantially greater than that of AFB₁. Cultures ofA. flavus NRRL 3251 andA. parasiticus NRRL 2999 produced only AFB₁. All natural flora cultures tested negative for aflatoxins. NoAspergilli sporulations were observed in these cultures. In the cultures inoculated with known toxigenic fungi, the highest mean level for total aflatoxins was 8.7 g/g of medium. Marihuana appears not to yield large quantities of these mycotoxins but sufficient levels are present to be a potential health hazard for both the user and the forensic analyst who is in daily contact with such plant material. Careful processing, storage, and sanitation procedures should be maintained with marihuana. If these conditions are disregarded due to the illicit status of marihuana, the potential for mycotoxin contamination must be considered.     

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.