Comparison of growth and aflatoxin B1 production profiles of Aspergillus flavus strains on conventional and isogenic GM-maize-based nutritional matrices

Maize grown in both North and South America are now predominantly genetically modified (GM) cultivars with some resistance to herbicide, pesticide, or both. There is little information on the relative colonisation and aflatoxin B₁ (AFB₁) production with maize meal-based nutritional matrices based on kernels of non-GM maize and isogenic GM-ones by strains of Aspergillus flavus. The objectives were to examine the effect of interacting conditions of temperature (25–35 °C) and water availability (0.99–0.90 water activity, a_w) on (a) mycelial growth, (b) AFB₁ production and (c) develop contour maps of optimum and marginal conditions of these parameters for four strains of A. flavus on three different non-GM and isogenic GM-maize based nutritional media. The growth of the four strains of A. flavus (three aflatoxigenic; one non-aflatoxigenic) was relatively similar in relation to the temperature × a_w conditions examined on both non-GM and GM-based matrices. Optimum growth overall was at 30–35 °C and 0.99 a_w for all four strains. Under water stress (0.90 a_w) growth was optimum at 35 °C. Statistically: non-GM, GM cultivars, temperature and a_w all significantly affected growth rates. For AFB₁ production, all single and interacting factors were statistically significant except for non-GM × GM cultivar. In conclusion, colonisation of GM- and non-GM nutritional sources was similar for the different A. flavus strains examined. The contour maps will be very useful for understanding the ecological niches for both toxigenic and non-toxigenic strains in the context of the competitive exclusion of those producing aflatoxins.     

Biocontrol of aflatoxin in corn by inoculation with non-aflatoxigenic Aspergillus flavus isolates

The ability of two non-aflatoxigenic Aspergillus flavus Link isolates (CT3 and K49) to reduce aflatoxin contamination of corn was assessed in a 4-year field study (2001–2004). Soil was treated with six wheat inoculant treatments: aflatoxigenic isolate F3W4; two non-aflatoxigenic isolates (CT3 and K49); two mixtures of CT3 or K49 with F3W4; and an autoclaved wheat control, applied at 20 kg ha^?1. In 2001, inoculation with the aflatoxigenic isolate increased corn grain aflatoxin levels by 188% compared to the non-inoculated control, while CT3 and K49 inoculation reduced aflatoxin levels in corn grain by 86 and 60%, respectively. In 2002, the non-toxigenic CT3 and K49 reduced aflatoxin levels by 61 and 76% compared to non-inoculated controls, respectively. In 2001, mixtures of aflatoxigenic and non-aflatoxigenic isolates had little effect on aflatoxin levels, but in 2002, inoculation with mixtures of K49 and CT3 reduced aflatoxin levels 68 and 37% compared to non-inoculated controls, respectively. In 2003 and 2004, a low level of natural aflatoxin contamination was observed (8 ng g^?1). However, inoculation with mixtures of K49?+?F3W4 and CT3?+?F3W4, reduced levels of aflatoxin 65–94% compared to the aflatoxigenic strain alone. Compared to the non-sclerotia producing CT3, strain K49 produces large sclerotia, has more rapid in vitro radial growth, and a greater ability to colonize corn when artificially inoculated, perhaps indicating greater ecological competence. Results indicate that non-aflatoxigenic, indigenous A. flavus isolates, such as strain K49, have potential use for biocontrol of aflatoxin contamination in southern US corn.     

Comparison of Aflatoxigenic and Nonaflatoxigenic Isolates of Aspergillus flavus using DNA Amplification Fingerprinting Techniques

Aspergillus flavus is a filamentous fungus that produces mycotoxins in many food and feed crops, such as maize (Zea mays L.). Isolates were analyzed for toxin production by nucleic acid profiles in an attempt to differentiate aflatoxigenic from nonaflatoxigenic isolates. A total of 41 aflatoxigenic and 34 nonalfatoxigenic isolates were included in the study. The isolates were evaluated initially using DNA amplification fingerprinting (DAF) without clear resolution of the groups. A weak association of aflatoxigenic isolates was observed, as evidenced by their clustering in 18 of 81 trees recovered from maximum parsimony analysis of binary characters derived from arbitrary signatures from amplification profiles (ASAP) data; nonaflatoxigenic isolates exhibited a pattern of paraphyletic laddering. Up to five markers unambiguously supported the aflatoxigenic isolate grouping, but the presence of alternative conflicting topologies in equally parsimonious trees precluded the observation of meaningful statistical support. With additional markers for genome of A. flavus, this method could be used to resolve toxigenic from nontoxigenic strains. This additional work could resolve aflatoxigenic isolates of A. flavus present on maize plants using ASAP, which would reduce labor intense costs and potentially lead to faster determination of resistant cultivars in breeding efforts.     

The proportion of non-aflatoxigenic strains of the Aspergillus flavus/oryzae complex from meju by analyses of the aflatoxin biosynthetic genes

Strains of the Aspergillus flavus/oryzae complex are frequently isolated from meju, a fermented soybean product, that is used as the starting material for ganjang (soy sauce) and doenjang (soybean paste) production. In this study, we examined the aflatoxin producing capacity of A. flavus/oryzae strains isolated from meju. 192 strains of A. flavus/oryzae were isolated from more than 100 meju samples collected from diverse regions of Korea from 2008 to 2011, and the norB-cypA, omtA, and aflR genes in the aflatoxin biosynthesis gene cluster were analyzed. We found that 178 strains (92.7%) belonged to non-aflatoxigenic group (Type I of norB-cypA, IB-L-B-, IC-AO, or IA-L-B- of omtA, and AO type of aflR), and 14 strains (7.3%) belonged to aflatoxin-producible group (Type II of norB-cypA, IC-L-B+/B- or IC-L-B+ of omtA, and AF type of aflR). Only 7 strains (3.6%) in the aflatoxin-producible group produced aflatoxins on Czapek yeast-extract medium. The aflatoxin-producing capability of A. flavus/oryzae strains from other sources in Korea were also investigated, and 92.9% (52/56) strains from air, 93.9% (31/33) strains from rice straw, 91.7% (11/12) strains from soybean, 81.3% (13/16) strains from corn, 82% (41/50) strains from peanut, and 73.2% (41/56) strains from arable soil were included in the non-aflatoxigenic group. The proportion of non-aflatoxigenicity of meju strains was similar to that of strains from soybean, air and rice straw, all of which have an effect on the fermentation of meju. The data suggest that meju does not have a preference for non-aflatoxigenic or aflatoxin-producible strains of A. flavus/oryzae from the environment of meju. The non-aflatoxigenic meju strains are proposed to be named A. oryzae, while the meju strains that can produce aflatoxins should be referred to A. flavus in this study.     

Production of Aflatoxins B1 and G1 by Aspergillus flavus and Aspergillus parasiticus Isolated from Market Pecans

One hundred and forty-eight isolates of Aspergillus flavus and A. parasiticus were isolated from 5,608 pecans obtained from Chicago and Georgia markets. The percentage of internal contamination by these species was 7.3% in the Chicago market pecans and 1.7% in those from markets in Georgia. Of the 148 isolates, 93% of the A. parasiticus, but only 54% of the A. flavus, were capable of producing aflatoxin. Overall, 57% of the isolates were potentially aflatoxigenic. A. parasiticus isolates generally produced a greater amount of aflatoxins than A. flavus.     

An insight into the distribution,genetic diversity,and mycotoxin production of <Emphasis Type="Italic">Aspergillus</Emphasis> section <Emphasis Type="Italic">Flavi</Emphasis> in soils of pistachio orchards

In the present study, 193 Aspergillus strains were isolated from a total of 100 soil samples of pistachio orchards, which all of them were identified as Aspergillus flavus as the most abundant species of Aspergillus section Flavi existing in the environment. Approximately 59%, 81%, and 61% of the isolates were capable of producing aflatoxins (AFs), cyclopiazonic acid (CPA), and sclerotia, respectively. The isolates were classified into four chemotypes (I to IV) based on the ability to produce AFs and CPA. The resulting dendrogram of random amplified polymorphic DNA (RAPD) analysis of 24 selected A. flavus isolates demonstrated the formation of two separate clusters. Cluster 1 contained both aflatoxigenic and non-aflatoxigenic isolates (17 isolates), whereas cluster 2 comprised only aflatoxigenic isolates (7 isolates). All the isolates of cluster 2 produced significantly higher levels of AFs than those of cluster 1 and the isolates that produced both AFB₁ and AFB₂ were found only in cluster 2. RAPD genotyping allowed the differentiation of A. flavus from Aspergillus parasiticus as a closely related species within section Flavi. The present study has provided for the first time the relevant information on distribution and genetic diversity of different A. flavus populations from nontoxigenic to highly toxigenic enable to produce hazardous amounts of AFB₁ and CPA in soils of pistachio orchards. These fungi, either toxigenic or not-toxigenic, should be considered as potential threats for agriculture and public health.     

Effect of solute and matric potential on in vitro growth and sporulation of strains from a new population of Aspergillus flavus isolated in Italy

The effect of temperature and different solute (Ψ_s) and matric potentials (Ψ_m) on growth and sporulation of three aflatoxigenic strains of Aspergillus flavus isolated from contaminated maize in northern Italy was determined. The Ψ_s of maize-based media were modified ionically (NaCl) and non-ionically (glycerol) and the Ψ_m with PEG 8000 in the range −1.4 to −21.0 MPa at 25 and 30 °C. Both temperature and Ψ_s/Ψ_m stress had statistically significant effects on growth rates of the three strains. Faster growth occurred at 30 °C and −1.4 and −2.8 MPa. A. flavus strains were more sensitive to Ψ_m than Ψ_s stress with limits of −9.8 MPa and −14 to−18 MPa, respectively. Sporulation was significantly influenced by Ψ_s potential, solute type and temperature. This suggests that these aflatoxigenic strains of A. flavus isolated from aflatoxin-contaminated maize are probably able to colonise crop debris rapidly at prevailing temperatures and water stress conditions. This type of information on the ecology of aflatoxin producing A. flavus strains isolated in Italy will contribute to the development of a systems model to predict their activity in crop residue and colonisation of maize grain.     

Recombination and lineage‐specific gene loss in the aflatoxin gene cluster of Aspergillus flavus

Aflatoxins produced by Aspergillus flavus are potent carcinogens that contaminate agricultural crops. Recent efforts to reduce aflatoxin concentrations in crops have focused on biological control using nonaflatoxigenic A. flavus strains AF36 (=NRRL 18543) and NRRL 21882 (the active component of afla‐guard^®). However, the evolutionary potential of these strains to remain nonaflatoxigenic in nature is unknown. To elucidate the underlying population processes that influence aflatoxigenicity, we examined patterns of linkage disequilibrium (LD) spanning 21 regions in the aflatoxin gene cluster of A. flavus. We show that recombination events are unevenly distributed across the cluster in A. flavus. Six distinct LD blocks separate late pathway genes aflE, aflM, aflN, aflG, aflL, aflI and aflO, and there is no discernable evidence of recombination among early pathway genes aflA, aflB, aflC, aflD, aflR and aflS. The discordance in phylogenies inferred for the aflW/aflX intergenic region and two noncluster regions, tryptophan synthase and acetamidase, is indicative of trans‐species evolution in the cluster. Additionally, polymorphisms in aflW/aflX divide A. flavus strains into two distinct clades, each harbouring only one of the two approved biocontrol strains. The clade with AF36 includes both aflatoxigenic and nonaflatoxigenic strains, whereas the clade with NRRL 21882 comprises only nonaflatoxigenic strains and includes all strains of A. flavus missing the entire gene cluster or with partial gene clusters. Our detection of LD blocks in partial clusters indicates that recombination may have played an important role in cluster disassembly, and multilocus coalescent analyses of cluster and noncluster regions indicate lineage‐specific gene loss in A. flavus. These results have important implications in assessing the stability of biocontrol strains in nature.     

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.     

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.     

Hybridization of genes involved in aflatoxin biosynthesis to DNA of aflatoxigenic and non-aflatoxigenic aspergilli

Southern blots of DNA from a number of aspergilli belonging to Aspergillus section Flavi, including aflatoxin-producing and non-aflatoxigenic isolates of A. flavus and A. parasiticus, were probed with the aflatoxin pathway genes aflR and omt-1. DNA of all A. flavus, A. parasiticus and A. sojae isolates examined hybridized with both genes. None of the A. oryzae isolates examined hybridized to the aflR probe and one of the three did not hybridize to the omt-1 probe. None of the A. tamarii isolates examined hybridized to either gene. Our results suggest that some isolates in this section do not produce aflatoxin because they lack at least one of the genes necessary for biosynthesis, and that non-producing A. flavus, A. parasiticus and A. sojae strains either lack a gene we did not examine or have genes that are not being expressed.     

Serologischer Nachweis des Gurkenmosaik- Virus unter Verwendung vereinfachter Präzipitationsteste (Kurze Mitteilung)

A?atoxins are carcinogenic metabolites produced by Aspergillus flavus. Rice seeds may be contaminated by it at the time of harvesting or during storage. Detection of aflatoxigenic strains by TLC and analysis of genetic variability among 50 A. flavus cultures from seeds of various rice genotypes by PCR using 18 RAPD primers is reported here. About 58% isolates were aflatoxigenic whereas 42% were non-toxic. There were 246 bands and 48 haplotypes. Approximately 99% bands were polymorphic, and thus high degree of genetic variability was observed. All the primers were informative with PIC values of range 0.75–0.95. Seventeen RAPD markers were not found to be useful for the detection of aflatoxigenic A. flavus. However, one RAPD primer 3B could differentiate aflatoxigenic and non-toxigenic isolates to some extent. One allele of Primer 3B at approximately 480?bp was present in 85% aflatoxin non-producers and in 6% aflatoxigenic isolates. This information may be utilised for designing markers for differentiating toxic/non-toxic isolates of A. flavus.     

Production of aflatoxin and cyclopiazonic acid by various aspergilli: An ELISA analysis

An enzyme-linked Immunosorbent assay (ELISA) was used to monitor a total of 153 fungi in theAspergillus flavus group, Including 130A. flavus, 15A. parasiticus and 8A. tamarii, for their ability to produce aflatoxins (AFs) and cyclopiazonic acid (CPA) in a mycologlcal broth-sucrose-yeast extract medium. Of 15A. parasiticus isolates, ten produced AFs In a range of 12.4 to 89.3 μg/vial (average 56.9 μg/vial); two isolates produced only trace amounts of AFs and three isolates produced none at all. Production of CPA was not demonstrated in anyA. parasiticus isolate. On the other hand, all A. tamarii isolates produced only CPA with a range of 310 to 1100 gmg/vial. Fifteen percent (14.6%) of theA. flavus isolates (19/130) produced more than 500 μg CPA/vial, but yielded no or little AF (less than 0.1 μg/vial). About 22.3% ofA. flavus (29/130) that produced less than 500 μg of CPA also yielded little or no aflatoxin. MostA. flavus isolates (44.6%) produced both CPA (50 to 300 μg/vial) and AFs (10 to 40 μg/vial). About 9.2% of theA. flavus are low CPA producers (less than 100 μg/vial) but yielded higher amounts of AFs. A small percentage (12/130 or 9.2%) of A. flavus isolates produced neither CPA nor aflatoxin. Excluding the isolates that produced neither AFs nor CPA, there is a negative correlation between the production of CPA and AFs by most A.flavus isolates. Data obtained from ELISA for the production of CPA were consistent with TLC results. Thus, the ELISA method for CPA and AFB could be applied to the screening of toxigenic fungi. Data on the simultaneous production of both toxins by a large percentage of the toxigenicA. flavus isolates suggest that there is a potential health hazard for co-existence of both toxins in foods and feeds.     

Development of a droplet digital PCR assay for population analysis of aflatoxigenic and atoxigenic <Emphasis Type="Italic">Aspergillus flavus</Emphasis> mixtures in soil

Aflatoxin B₁ is a potent hepatotoxin and carcinogen that poses a serious safety hazard to both humans and animals. Aspergillus flavus is the most common aflatoxin-producing species on corn, cotton, peanuts, and tree nuts. Application of atoxigenic strains to compete against aflatoxigenic strains of A. flavus has emerged as one of the most practical strategies for ameliorating aflatoxin contamination in food. Genes directly involved in aflatoxin biosynthesis are clustered on an 82-kb region of the genome. Three atoxigenic strains (CA12, M34, and AF123) were each paired with each of four aflatoxigenic strains (CA28, CA42, CA90, and M52), inoculated into soil and incubated at 28 °C for 2 weeks and 1 month. TaqMan probes, omtA-FAM, and norA-HEX were designed for developing a droplet digital PCR (ddPCR) assay to analyze the soil population of mixtures of A. flavus strains. DNA was extracted from each soil sample and used for ddPCR assays. The data indicated that competition between atoxigenic and aflatoxigenic was strain dependent. Variation in competitive ability among different strains of A. flavus influenced the population reduction of the aflatoxigenic strain by the atoxigenic strain. Higher ratios of atoxigenic to aflatoxigenic strains increased soil population of atoxigenic strains. This is the first study to demonstrate the utility of ddPCR to quantify mixtures of both atoxigenic and aflatoxigenic A. flavus strains in soil and allows for rapid and accurate determination of population sizes of atoxigenic and aflatoxigenic strains. This method eliminates the need for isolation and identification of individual fungal isolates from experimental soil samples.     

Nutrient Environments Influence Competition among Aspergillus flavus Genotypes

The population dynamics of Aspergillus flavus, shaped in part by intraspecific competition, influence the likelihood and severity of crop aflatoxin contamination. Competition for nutrients may be one factor modulating intraspecific interactions, but the influences of specific types and concentrations of nutrients on competition between genotypes of A. flavus have not been investigated. Competition between paired A. flavus isolates on agar media was affected by varying concentrations of carbon (sucrose or asparagine) and nitrogen (nitrate or asparagine). Cocultivated isolate percentages from conidia and agar-embedded mycelia were quantified by measurements of isolate-specific single-nucleotide polymorphisms with quantitative pyrosequencing. Compositions and concentrations of nutrients influenced conidiation resulting from cocultivation, but the percentages of total conidia from each competing isolate were not predicted by sporulation of isolates grown individually. Success during sporulation did not reflect the outcomes of competition during mycelial growth, and the extents to which isolate percentages from conidia and mycelia differed varied among both isolate pairs and media. Whether varying concentrations of sucrose, nitrate, or asparagine increased, decreased, or had no influence on competitive ability was isolate dependent. Different responses of A. flavus isolates to nutrient variability suggest genotypes are adapted to different nutrient environments that have the potential to influence A. flavus population structure and the epidemiology of aflatoxin contamination.     

Contamination of Groundnut in South‐Western Nigeria by Aflatoxigenic Fungi and Aflatoxins in Relation to Processing

Groundnut is commonly consumed in its roasted form by many Nigerians. This study was therefore conducted to determine the levels of aflatoxin in roasted groundnut retailed in south‐western Nigeria with a view to assessing the fitness of the processed nut for human consumption. The effects of roasting and de‐coating as alternative methods for reducing the ‘aflatoxin scare’ in the nut were further assessed on aflatoxigenic fungal load and aflatoxin content of the nuts. Forty‐eight samples of retailed raw and roasted groundnut were collected and assessed by mycological and thin‐layer chromatographic analysis for changes in aflatoxigenic fungal population and aflatoxin concentration, respectively. Consequently, 480 isolates of the Aspergillus section Flavi group, A. flavus L strain (n = 410), A. tamarii (n = 56), A. parasiticus (n = 7) and A. parvisclerotigenus (n = 7), were recovered from all samples. Aflatoxigenic isolates of A. flavus L strain (58.8%) had a significantly (P < 0.05) higher incidence than the non‐aflatoxigenic isolates (41.2%). Aflatoxins were detected in 43 (89.6%) of the samples. Approximately 25% of all samples exceeded the 20 ng/g limit for aflatoxin B₁ (AFB₁) adopted by the National Agency for Food and Drug Administration and Control while 83 and 79% of all samples contained AFB₁ and total aflatoxins above the European Union limits of 2 and 4 ng/g, respectively. Aflatoxin concentrations in the raw and coated samples were as much as five times higher than those in the roasted and de‐coated nuts, respectively. However, no significant difference was recorded between aflatoxin levels in the coated and de‐coated samples. This study has shown that roasting of groundnut and testa removal (de‐coating) are effective processing interventions that can significantly lower aflatoxin quantities in the kernels, thus making it fit for human consumption.     

Survival of Aspergillus flavus and Fusarium moniliforme in High-Moisture Corn Stored Under Modified Atmospheres

Freshly harvested high-moisture corn with 29.4% moisture and corn remoistened to 19.6% moisture were inoculated with Aspergillus flavus Link ex Fr. and stored for 4 weeks at about 27 C in air (0.03% CO₂, 21% O₂, and 78% N₂) and three modified atmospheres: (i) 99.7% N₂ and 0.3% O₂; (ii) 61.7% CO₂, 8.7% O₂, and 29.6% N₂; and (iii) 13.5% CO₂, 0.5% O₂, and 84.8% N₂. Kernel infections by A. flavus, Fusarium moniliforme (Sheld.) Snyd. et Hans., and other fungi were monitored weekly. The modified-atmosphere treatments delayed deterioration by A. flavus and F. moniliforme, but their growth was not completely stopped. A. flavus survived better in the remoistened than in the freshly harvested corn. F. moniliforme survived in both. A. flavus and F. moniliforme were the dominant fungi in corn removed from the modified atmospheres and exposed to normal air for 1 week.     

A mycological and bacteriological survey on feed ingredients and mixed poultry feeds in Reunion island

A survey was carried out in Reunion island to obtain data on the occurrence of fungi, aflatoxigenic strains of Aspergillus flavus, aflatoxins, total aerobic bacteria and salmonellae of 150 samples of mixed poultry feeds and raw materials. These were collected at five farms over a 3-month period during the warm rainy season.White corn and Brazilian soybean meal seemed to present a better microbiological quality than yellow corn and US soybean meal.Mixed poultry feeds presented a high total mold count reflecting the mold flora of raw materials. The most frequent and abundant fungi were Aspergillus flavus, A. glaucus group, Fusarium spp., Penicillium spp., A. candidus, Mucor spp., A. restrictus, Scopulariopsis spp., Cladosporium spp. and A. versicolor. Of the 118 A. flavus strains screened, 42 (35.6%) were aflatoxigenic. Yellow corn samples were the most frequently contamined with aflatoxigenic strains (54.5%), followed by mixed feeds (44%).Of the 66 samples tested, 24 (36%) contained aflatoxins (traces to 22 ng/g). A good correlation seemed to exist between presence of at least one aflatoxigenic strain per sample and presence of aflatoxins.     

Characterization of aflatoxigenic and non-aflatoxigenic <Emphasis Type="Italic">Aspergillus flavus</Emphasis> isolates from pistachio

Pistachio is a popular snack food. Aflatoxin contamination of pistachio nuts is a serious problem for many producing countries. The development of biological control methods based on ecological parameters is an environmentally friendly approach. Thirty-eight Aspergillus flavus isolates collected from a pistachio orchard in California (CA) were analyzed for production of aflatoxin (AF), cyclopiazonic acid (CPA), vegetative compatibility groups (VCGs), and mating types. All aflatoxigenic isolates produced both AFB₁ and CPA. The most toxigenic one was CA28 which produced 164 μg AFB₁ per 5 ml PDA fungal culture and small sclerotia (S strain, sclertoium size less than 400 μm). The other aflatoxigenic strains produce AFB₁ ranging from 1.2 μg to 80 μg per 5 ml fungal culture. Twenty-one percent of the CA isolates produced AFB₁, 84% produced CPA and half formed sclerotia on at least one of three tested media. The 38 CA isolates formed 26 VCGs, 6 of which had two or more isolates and 20 contained single isolates. The S strain isolates belong to 4 different VCGs. Genomic profiling by a retrotransposon DNA probe revealed fingerprint patterns that were highly polymorphic. The predicted VCGs (Pred-VCGs) based on a similarity coefficient >80% matched the VCGs of multiple isolates determined by complementation. All isolates within a VCG had the same mating-type gene of either MAT1-1 or MAT1-2. Uncorrected and VCG-corrected MAT1-1 and MAT1-2 among the isolates were equally distributed.     

Isolation of Bacterial Antagonists of Aspergillus flavus from Almonds

Bacteria were isolated from California almond orchard samples to evaluate their potential antifungal activity against aflatoxin-producing Aspergillus flavus. Fungal populations from the same samples were examined to determine the incidence of aflatoxigenic Aspergillus species. Antagonistic activities of the isolated bacterial strains were screened against a nonaflatoxigenic nor mutant of A. flavus, which accumulates the pigmented aflatoxin precursor norsolorinic acid (NOR) under conditions conducive to aflatoxin production. Using solid and liquid media in coculture assays, 171 bacteria isolated from almond flowers, immature nut fruits, and mature nut fruits showed inhibition of A. flavus growth and/or inhibition of NOR accumulation. Bacterial isolates were further characterized for production of extracellular enzymes capable of hydrolyzing chitin or yeast cell walls. Molecular and physiological identification of the bacterial strains indicated that the predominant genera isolated were Bacillus, Pseudomonas, Ralstonia, and Burkholderia, as well as several plant-associated enteric and nonenteric bacteria. A set of 20 isolates was selected for further study based on their species identification, antifungal phenotypes, and extracellular enzyme production. Quantitative assays using these isolates in liquid coculture with a wild-type, aflatoxin-producing A. flavus strain showed that a number of strains completely inhibited fungal growth in three different media. These results indicate the potential for development of bacterial antagonists as biological control agents against aflatoxigenic aspergilli on almonds.