Structure of Aspergillus flavus populations associated with maize in Greece, Spain, and Serbia: Implications for aflatoxin biocontrol on a regional scale.

Aspergillus flavus is the most frequently identified producer of aflatoxins. Non-aflatoxigenic members of the A. flavus L strains are used in various continents as active ingredients of bioprotectants directed at preventing aflatoxin contamination by competitive displacement of aflatoxin producers. The current research examined the genetic diversity of A. flavus L strain across southern Europe to gain insights into the population structure and evolution of this species and to evaluate the prevalence of genotypes closely related to MUCL54911, the active ingredient of AF-X1. A total of 2173L strain isolates recovered from maize collected across Greece, Spain, and Serbia in 2020 and 2021 were subjected to simple sequence repeat (SSR) genotyping. The analysis revealed high diversity within and among countries and dozens of haplotypes shared. Linkage disequilibrium analysis indicated asexual reproduction and clonal evolution of A. flavus L strain resident in Europe. Moreover, haplotypes closely related to MUCL54911 were found to belong to the same vegetative compatibility group (VCG) IT006 and were relatively common in all three countries. The results indicate that IT006 is endemic to southern Europe and may be utilized as an aflatoxin mitigation tool for maize across the region without concern for potential adverse impacts associated with the introduction of an exotic microorganism.

Telomere-to-telomere genome assembly of the aflatoxin biocontrol agent Aspergillus flavus isolate La3279 isolated from maize in Nigeria.

Here, we report the complete genome of the non-aflatoxigenic Aspergillus flavus isolate La3279, which is an active ingredient of the aflatoxin biocontrol product Aflasafe. The chromosome-scale assembly clarifies the deletion pattern in the aflatoxin biosynthesis gene cluster and corrects a misidentified assembly previously published for this isolate.

Nanopore PCR-cDNA sequencing of the biocontrol isolate Aspergillus flavus AF36 (NRRL 18543) informs gene annotation.

Toxic molds in the Aspergillus genus synthesize carcinogenic aflatoxins which contaminate crops. The widely applied biocontrol isolate Aspergillus flavus AF36 (NRRL 18543) has a high-quality public genome but lacks corresponding gene annotations. We generated high-quality gene predictions for this isolate by using long-read Nanopore PCR-cDNA sequencing.

Complete genome of the toxic mold Aspergillus pseudotamarii isolate NRRL 25517 reveals genomic instability of the aflatoxin biosynthesis cluster.

Fungi can synthesize a broad array of secondary metabolite chemicals. The genes underpinning their biosynthesis are typically arranged in tightly linked clusters in the genome. For example, ∼25 genes responsible for the biosynthesis of carcinogenic aflatoxins by Aspergillus section Flavi species are grouped in a ∼70 Kb cluster. Assembly fragmentation prevents assessment of the role of structural genomic variation in secondary metabolite evolution in this clade. More comprehensive analyses of secondary metabolite evolution will be possible by working with more complete and accurate genomes of taxonomically diverse Aspergillus species. Here, we combined short- and long-read DNA sequencing to generate a highly contiguous genome of the aflatoxigenic fungus, Aspergillus pseudotamarii (isolate NRRL 25517 = CBS 766.97; scaffold N50 = 5.5 Mb). The nuclear genome is 39.4 Mb, encompassing 12,639 putative protein-encoding genes and 74-97 candidate secondary metabolite biosynthesis gene clusters. The circular mitogenome is 29.7 Kb and contains 14 protein-encoding genes that are highly conserved across the genus. This highly contiguous A. pseudotamarii genome assembly enables comparisons of genomic rearrangements between Aspergillus section Flavi series Kitamyces and series Flavi. Although the aflatoxin biosynthesis gene cluster of A. pseudotamarii is conserved with Aspergillus flavus, the cluster has an inverted orientation relative to the telomere and occurs on a different chromosome.

Aflatoxin contamination of maize and groundnut in Burundi: Distribution of contamination, identification of causal agents and potential biocontrol genotypes of Aspergillus flavus.

Aflatoxin contamination of the staples maize and groundnut is a concern for health and economic impacts across sub-Saharan Africa. The current study (i) determined aflatoxin levels in maize and groundnut collected at harvest in Burundi, (ii) characterized populations of Aspergillus section Flavi associated with the two crops, and (iii) assessed aflatoxin-producing potentials among the recovered fungi. A total of 120 groundnut and 380 maize samples were collected at harvest from eight and 16 provinces, respectively. Most of the groundnut (93%) and maize (87%) contained aflatoxin below the European Union threshold, 4 µg/kg. Morphological characterization of the recovered Aspergillus section Flavi fungi revealed that the L-morphotype of A. flavus was the predominant species. Aflatoxin production potentials of the L-morphotype isolates were evaluated in maize fermentations. Some isolates produced over 137,000 µg/kg aflatoxin B1. Thus, despite the relatively low aflatoxin levels at harvest, the association of both crops with highly toxigenic fungi poses significant risk of post-harvest aflatoxin contamination and suggests measures to mitigate aflatoxin contamination in Burundi should be developed. Over 55% of the L-morphotype A. flavus did not produce aflatoxins. These atoxigenic L-morphotype fungi were characterized using molecular markers. Several atoxigenic genotypes were detected across the country and could be used as biocontrol agents. The results from the current study hold promise for developing aflatoxin management strategies centered on biocontrol for use in Burundi to reduce aflatoxin contamination throughout the value chain.

Characterization of the Aspergillus flavus Population from Highly Aflatoxin-Contaminated Corn in the United States.

Aflatoxin contamination of corn is a major threat to the safe food and feed. The United States Federal Grain Inspection Service (FGIS) monitors commercial grain shipments for the presence of aflatoxin. A total of 146 Aspergillus flavus were isolated from 29 highly contaminated grain samples to characterize the visual phenotypes, aflatoxin-producing potential, and genotypes to explore the etiological cause of high aflatoxin contamination of US corn. Five of the isolates had reduced sensitivity (43-49% resistant) to the fungicide azoxystrobin, with the remainder all being over 50% resistant to azoxystrobin at the discriminating dose of 2.5 µg/mL. Only six isolates of the highly aflatoxigenic S morphotype were found, and 48 isolates were non-aflatoxigenic. Analysis of the mating type locus revealed 45% MAT 1-1 and 55% MAT 1-2. The A. flavus population originating from the highly aflatoxin contaminated grain samples was compared to a randomly selected subset of isolates originating from commercial corn samples with typical levels of aflatoxin contamination (average < 50 ppb). Use of simple sequence repeat (SSR) genotyping followed by principal component analysis (PCoA) revealed a similar pattern of genotypic distribution in the two populations, but greater diversity in the FGIS-derived population. The noticeable difference between the two populations was that genotypes identical to strain NRRL 21882, the active component of the aflatoxin biocontrol product Afla-Guard™, were ten times more common in the commercial corn population of A. flavus compared to the population from the high-aflatoxin corn samples. The other similarities between the two populations suggest that high aflatoxin concentrations in corn grain are generally the result of infection with common A. flavus genotypes.

Genetic Diversity of Aspergillus flavus Associated with Chili in Nigeria and Identification of Haplotypes with Potential in Aflatoxin Mitigation.

Dried red chili (Capsicum spp.), a widely produced and consumed spice in Nigeria, is often contaminated by aflatoxins. Aflatoxins are potent mycotoxins of severe health and economic concern worldwide. Aspergillus flavus often contaminates crops with aflatoxins in warm regions; however, not all isolates are aflatoxin producers. Nonaflatoxigenic isolates have potential as biocontrol agents for aflatoxin mitigation. The current study examined the genetic diversity of A. flavus (n = 325) associated with chilies in Nigeria and identified 123 nonaflatoxigenic isolates. The Nigerian A. flavus isolates from chili were diverse at 17 microsatellite loci, with 5 to 36 alleles per locus, and included 152 haplotypes. The isolates that are active ingredients in Aflasafe, registered for aflatoxin biocontrol on maize and groundnuts in Nigeria, did not share haplotypes with the chili isolates. Of the 152 haplotypes, 65% produced aflatoxins in autoclaved maize, some of which (17%) produced >100,000 µg/kg of aflatoxins. Aflatoxins were not detected in 35% of the haplotypes. Cluster amplification pattern assay detected large deletions in the aflatoxin biosynthetic clusters of some (32%) of the nonaflatoxigenic haplotypes. Coinfection of chili with nonaflatoxigenic isolates from chilies (n = 7) and A. aflatoxiformans resulted in a significantly greater average reduction in total aflatoxins compared with that achieved by Aflasafe active ingredient isolates (P < 0.01). These nonaflatoxigenic isolates are a genetic resource for the development of biological control products for aflatoxin mitigation in chilies in Nigeria and should be evaluated under field conditions.

Degradation of Aflatoxins B1 by Atoxigenic Aspergillus flavus Biocontrol Agents.

Aflatoxins are potent Aspergillus mycotoxins that contaminate food and feed, thereby impacting health and trade. Biopesticides with atoxigenic Aspergillus flavus isolates as active ingredients are used to reduce aflatoxin contamination in crops. The mechanism of aflatoxin biocontrol is primarily attributed to competitive exclusion but, sometimes, aflatoxin is reduced by greater amounts than can be explained by displacement of aflatoxin-producing fungi on the crop. Objectives of this study were to (i) evaluate the ability of atoxigenic A. flavus genotypes to degrade aflatoxin B1 (AFB1) and (ii) characterize impacts of temperature, time, and nutrient availability on AFB1 degradation by atoxigenic A. flavus. Aflatoxin-contaminated maize was inoculated with atoxigenic isolates in three separate experiments that included different atoxigenic genotypes, temperature, and time as variables. Atoxigenic genotypes varied in aflatoxin degradation but all degraded AFB1 >44% after 7 days at 30°C. The optimum temperature for AFB1 degradation was 25 to 30°C, which is similar to the optimum range for AFB1 production. In a time-course experiment, atoxigenics degraded 40% of AFB1 within 3 days, and 80% of aflatoxin was degraded by day 21. Atoxigenic isolates were able to degrade and utilize AFB1 as a sole carbon source in a chemically defined medium but quantities of AFB1 degraded declined as glucose concentrations increased. Degradation may be an additional mechanism through which atoxigenic A. flavus biocontrol products reduce aflatoxin contamination pre- or postharvest. Thus, selection of optimal atoxigenic active ingredients can include assessment of both competitive ability in agricultural fields and their ability to degrade aflatoxins.

Conservation and Loss of a Putative Iron Utilization Gene Cluster among Genotypes of Aspergillus flavus.

Iron is an essential component for growth and development. Despite relative abundance in the environment, bioavailability of iron is limited due to oxidation by atmospheric oxygen into insoluble ferric iron. Filamentous fungi have developed diverse pathways to uptake and use iron. In the current study, a putative iron utilization gene cluster (IUC) in Aspergillus flavus was identified and characterized. Gene analyses indicate A. flavus may use reductive as well as siderophore-mediated iron uptake and utilization pathways. The ferroxidation and iron permeation process, in which iron transport depends on the coupling of these two activities, mediates the reductive pathway. The IUC identified in this work includes six genes and is located in a highly polymorphic region of the genome. Diversity among A. flavus genotypes is manifested in the structure of the IUC, which ranged from complete deletion to a region disabled by multiple indels. Molecular profiling of A. flavus populations suggests lineage-specific loss of IUC. The observed variation among A. flavus genotypes in iron utilization and the lineage-specific loss of the iron utilization genes in several A. flavus clonal lineages provide insight on evolution of iron acquisition and utilization within Aspergillus section Flavi. The potential divergence in capacity to acquire iron should be taken into account when selecting A. flavus active ingredients for biocontrol in niches where climate change may alter iron availability.

Distribution of active ingredients of a commercial aflatoxin biocontrol product in naturally occurring fungal communities across Kenya.

Human populations in Kenya are repeatedly exposed to dangerous aflatoxin levels through consumption of contaminated crops. Biocontrol with atoxigenic Aspergillus flavus is an effective method for preventing aflatoxin in crops. Although four atoxigenic A. flavus isolates (C6E, E63I, R7H and R7K) recovered from maize produced in Kenya are registered as active ingredients for a biocontrol product (Aflasafe KE01) directed at preventing contamination, natural distributions of these four genotypes prior to initiation of commercial use have not been reported. Distributions of the active ingredients of KE01 based on haplotypes at 17 SSR loci are reported. Incidences of the active ingredients and closely related haplotypes were determined in soil collected from 629 maize fields in consecutive long and short rains seasons of 2012. The four KE01 haplotypes were among the top ten most frequent. Haplotype H-1467 of active ingredient R7K was the most frequent and widespread haplotype in both seasons and was detected in the most soils (3.8%). The four KE01 haplotypes each belonged to large clonal groups containing 27-46 unique haplotypes distributed across multiple areas and in 21% of soils. Each of the KE01 haplotypes belonged to a distinct vegetative compatibility group (VCG), and all A. flavus with haplotypes matching a KE01 active ingredient belonged to the same VCG as the matching active ingredient as did all A. flavus haplotypes differing at only one SSR locus. Persistence of the KE01 active ingredients in Kenyan agroecosystems is demonstrated by detection of identical SSR haplotypes six years after initial isolation. The data provide baselines for assessing long-term influences of biocontrol applications in highly vulnerable production areas of Kenya.

Temperature Influences on Interactions Among Aflatoxigenic Species of Aspergillus Section Flavi During Maize Colonization.

Fungal species within Aspergillus section Flavi contaminate food and feed with aflatoxins. These toxic fungal metabolites compromise human and animal health and disrupt trade. Genotypically and phenotypically diverse species co-infect crops, but temporal and spatial variation in frequencies of different lineages suggests that environmental factors such as temperature may influence structure of aflatoxin-producing fungal communities. Furthermore, though most species within Aspergillus section Flavi produce sclerotia, divergent sclerotial morphologies (small or S-type sclerotia vs. large or L-type sclerotia) and differences in types and quantities of aflatoxins produced suggest lineages are adapted to different life strategies. Temperature is a key parameter influencing pre- and post-harvest aflatoxin contamination of crops. We tested the hypothesis that species of aflatoxin-producing fungi that differ in sclerotial morphology will vary in competitive ability and that outcomes of competition and aflatoxin production will be modulated by temperature. Paired competition experiments between highly aflatoxigenic S-type species (A. aflatoxiformans and Lethal Aflatoxicosis Fungus) and L-type species (A. flavus L morphotype and A. parasiticus) were conducted on maize kernels at 25 and 30°C. Proportions of each isolate growing within and sporulating on kernels were measured using quantitative pyrosequencing. At 30°C, S-type fungi were more effective at host colonization compared to L-type isolates. Total aflatoxins and the proportion of B vs. G aflatoxins were greater at 30°C compared to 25°C. Sporulation by L-type isolates was reduced during competition with S-type fungi at 30°C, while relative quantities of conidia produced by S-type species either increased or did not change during competition. Results indicate that both species interactions and temperature can shape population structure of Aspergillus section Flavi, with warmer temperatures favoring growth and dispersal of highly toxigenic species with S-type sclerotia.

Phenotypic Differentiation of Two Morphologically Similar Aflatoxin-Producing Fungi from West Africa.

Aflatoxins (AF) are hepatocarcinogenic metabolites produced by several Aspergillus species. Crop infection by these species results in aflatoxin contamination of cereals, nuts, and spices. Etiology of aflatoxin contamination is complicated by mixed infections of multiple species with similar morphology and aflatoxin profiles. The current study investigates variation in aflatoxin production between two morphologically similar species that co-exist in West Africa, A. aflatoxiformans and A. minisclerotigenes. Consistent distinctions in aflatoxin production during liquid fermentation were discovered between these species. The two species produced similar concentrations of AFB1 in defined media with either urea or ammonium as the sole nitrogen source. However, production of both AFB1 and AFG1 were inhibited (p < 0.001) for A. aflatoxiformans in a yeast extract medium with sucrose. Although production of AFG1 by both species was similar in urea, A. minisclerotigenes produced greater concentrations of AFG1 in ammonium (p = 0.039). Based on these differences, a reliable and convenient assay for differentiating the two species was designed. This assay will be useful for identifying specific etiologic agents of aflatoxin contamination episodes in West Africa and other regions where the two species are sympatric, especially when phylogenetic analyses based on multiple gene segments are not practical.

Molecular Analysis of S-morphology Aflatoxin Producers From the United States Reveals Previously Unknown Diversity and Two New Taxa.

Aflatoxins are highly toxic carcinogens that detrimentally influence profitability of agriculture and the health of humans and domestic animals. Several phylogenetically distinct fungi within Aspergillus section Flavi have S-morphology (average sclerotial size < 400 µm), and consistently produce high concentrations of aflatoxins in crops. S-morphology fungi have been implicated as important etiologic agents of aflatoxin contamination in the United States (US), but little is known about the diversity of these fungi. The current study characterized S-morphology fungi (n = 494) collected between 2002 and 2017, from soil and maize samples, in US regions where aflatoxin contamination is a perennial problem. Phylogenetic analyses based on sequences of the calmodulin (1.9 kb) and nitrate reductase (2.1 kb) genes resolved S-morphology isolates from the US into four distinct clades: (1) Aspergillus flavus S-morphotype (89.7%); (2) Aspergillus agricola sp. nov. (2.4%); (3) Aspergillus texensis (2.2%); and (4) Aspergillus toxicus sp. nov. (5.7%). All four S-morphology species produced high concentrations of aflatoxins in maize at 25, 30, and 35°C, but only the A. flavus S-morphotype produced unacceptable aflatoxin concentrations at 40°C. Genetic typing of A. flavus S isolates using 17 simple sequence repeat markers revealed high genetic diversity, with 202 haplotypes from 443 isolates. Knowledge of the occurrence of distinct species and haplotypes of S-morphology fungi that are highly aflatoxigenic under a range of environmental conditions may provide insights into the etiology, epidemiology, and management of aflatoxin contamination in North America.

Founder events influence structures of Aspergillus flavus populations.

In warm regions, agricultural fields are occupied by complex Aspergillus flavus communities composed of isolates in many vegetative compatibility groups (VCGs) with varying abilities to produce highly toxic, carcinogenic aflatoxins. Aflatoxin contamination is reduced with biocontrol products that enable atoxigenic isolates from atoxigenic VCGs to dominate the population. Shifts in VCG frequencies similar to those caused by the introduction of biocontrol isolates were detected in Sonora, Mexico, where biocontrol is not currently practiced. The shifts were attributed to founder events. Although VCGs reproduce clonally, significant diversity exists within VCGs. Simple sequence repeat (SSR) fingerprinting revealed that increased frequencies of VCG YV150 involved a single haplotype. This is consistent with a founder event. Additionally, great diversity was detected among 82 YV150 isolates collected over 20 years across Mexico and the United States. Thirty-six YV150 haplotypes were separated into two populations by Structure and SplitsTree analyses. Sixty-five percent of isolates had MAT1-1 and belonged to one population. The remaining had MAT1-2 and belonged to the second population. SSR alleles varied within populations, but recombination between populations was not detected despite co-occurrence at some locations. Results suggest that YV150 isolates with opposite mating-type have either strongly restrained or lost sexual reproduction among themselves.

Monitoring Aspergillus flavus Genotypes in a Multi-Genotype Aflatoxin Biocontrol Product With Quantitative Pyrosequencing.

Aflatoxins pose significant food security and public health risks, decrease productivity and profitability of animal industries, and hamper trade. To minimize aflatoxin contamination in several crops, a biocontrol technology based on atoxigenic strains of Aspergillus flavus is commercially used in the United States and some African countries. Significant efforts are underway to popularize the use of biocontrol in Africa by various means including incentives. The purpose of this study was to develop quantitative pyrosequencing assays for rapid, simultaneous quantification of proportions of four A. flavus biocontrol genotypes within complex populations of A. flavus associated with maize crops in Nigeria to facilitate payment of farmer incentives for Aflasafe (a biocontrol product) use. Protocols were developed to confirm use of Aflasafe by small scale farmers in Nigeria. Nested PCR amplifications followed by sequence by synthesis pyrosequencing assays were required to quantify frequencies of the active ingredients and, in so doing, confirm successful use of biocontrol by participating farmers. The entire verification process could be completed in 3-4 days proving a savings over other monitoring methods in both time and costs and providing data in a time frame that could work with the commercial agriculture scheme. Quantitative pyrosequencing assays represent a reliable tool for rapid detection, quantification, and monitoring of multiple A. flavus genotypes within complex fungal communities, satisfying the requirements of the regulatory community and crop end-users that wish to determine which purchased crops were treated with the biocontrol product. Techniques developed in the current study can be modified for monitoring other crop-associated fungi.

Potential of Atoxigenic Aspergillus flavus Vegetative Compatibility Groups Associated With Maize and Groundnut in Ghana as Biocontrol Agents for Aflatoxin Management.

Increasing knowledge of the deleterious health and economic impacts of aflatoxin in crop commodities has stimulated global interest in aflatoxin mitigation. Current evidence of the incidence of Aspergillus flavus isolates belonging to vegetative compatibility groups (VCGs) lacking the ability to produce aflatoxins (i.e., atoxigenic) in Ghana may lead to the development of an aflatoxin biocontrol strategy to mitigate crop aflatoxin content. In this study, 12 genetically diverse atoxigenic African A. flavus VCGs (AAVs) were identified from fungal communities associated with maize and groundnut grown in Ghana. Representative isolates of the 12 AAVs were assessed for their ability to inhibit aflatoxin contamination by an aflatoxin-producing isolate in laboratory assays. Then, the 12 isolates were evaluated for their potential as biocontrol agents for aflatoxin mitigation when included in three experimental products (each containing four atoxigenic isolates). The three experimental products were evaluated in 50 maize and 50 groundnut farmers' fields across three agroecological zones (AEZs) in Ghana during the 2014 cropping season. In laboratory assays, the atoxigenic isolates reduced aflatoxin biosynthesis by 87-98% compared to grains inoculated with the aflatoxin-producing isolate alone. In field trials, the applied isolates moved to the crops and had higher (P < 0.05) frequencies than other A. flavus genotypes. In addition, although at lower frequencies, most atoxigenic genotypes were repeatedly found in untreated crops. Aflatoxin levels in treated crops were lower by 70-100% in groundnut and by 50-100% in maize (P < 0.05) than in untreated crops. Results from the current study indicate that combined use of appropriate, well-adapted isolates of atoxigenic AAVs as active ingredients of biocontrol products effectively displace aflatoxin producers and in so doing limit aflatoxin contamination. A member each of eight atoxigenic AAVs with superior competitive potential and wide adaptation across AEZs were selected for further field efficacy trials in Ghana. A major criterion for selection was the atoxigenic isolate's ability to colonize soils and grains after release in crop field soils. Use of isolates belonging to atoxigenic AAVs in biocontrol management strategies has the potential to improve food safety, productivity, and income opportunities for smallholder farmers in Ghana.

Aspergillus flavus resident in Kenya: High genetic diversity in an ancient population primarily shaped by clonal reproduction and mutation-driven evolution.

Aspergillus flavus has long been considered to be an asexual species. Although a sexual stage was recently reported for this species from in vitro studies, the amount of recombination ongoing in natural populations and the genetic distance across which meiosis occurs is largely unknown. In the current study, genetic diversity, reproduction and evolution of natural A. flavus populations endemic to Kenya were examined. A total of 2744 isolates recovered from 629 maize-field soils across southern Kenya in two consecutive seasons were characterized at 17 SSR loci, revealing high genetic diversity (9-72 alleles/locus and 2140 haplotypes). Clonal reproduction and persistence of clonal lineages predominated, with many identical haplotypes occurring in multiple soil samples and both seasons. Genetic analyses predicted three distinct lineages with linkage disequilibrium and evolutionary relationships among haplotypes within each lineage suggesting mutation-driven evolution followed by clonal reproduction. Low genetic differentiation among adjacent communities reflected frequent short distance dispersal.

Distribution and incidence of atoxigenic Aspergillus flavus VCG in tree crop orchards in California: A strategy for identifying potential antagonists, the example of almonds.

To identify predominant isolates for potential use as biocontrol agents, Aspergillus flavus isolates collected from soils of almond, pistachio and fig orchard in the Central Valley of California were tested for their membership to 16 atoxigenic vegetative compatibility groups (VCGs), including YV36, the VCG to which AF36, an atoxigenic isolate commercialized in the United States as biopesticide, belongs. A surprisingly large proportion of isolates belonged to YV36 (13.3%, 7.2% and 6.6% of the total almond, pistachio and fig populations, respectively), while the percentage of isolates belonging to the other 15 VCGs ranged from 0% to 2.3%. In order to gain a better insight into the structure and diversity of atoxigenic A. flavus populations and to further identify predominant isolates, seventeen SSR markers were then used to genetically characterize AF36, the 15 type-isolates of the VCGs and 342 atoxigenic isolates of the almond population. There was considerable genetic diversity among isolates with a lack of differentiation among micro-geographical regions or years. Since isolates sharing identical SSR profiles from distinct orchards were rare, we separated them into groups of at least 3 closely-related isolates from distinct orchards that shared identical alleles for at least 15 out of the 17 loci. This led to the identification of 15 groups comprising up to 24 closely-related isolates. The group which contained the largest number of isolates were members of YV36 while five groups were also found to be members of our studied atoxigenic VCGs. These results suggest that these 15 groups, and AF36 in particular, are well adapted to various environmental conditions in California and to tree crops and, as such, are good candidates for use as biocontrol agents.

Structure of an Aspergillus flavus population from maize kernels in northern Italy.

In order to gain insight into the causal agents of aflatoxin contamination of maize in Italy, populations of Aspergillus flavus on maize produced in the most affected area were characterized. Forty-six percent of A. flavus, isolated from maize kernels collected in 5 districts of northern Italy between 2003 and 2010, were unable to produce detectable levels of aflatoxins. The genetic diversity of the population was assessed by analysis of vegetative compatibility groups (VCGs) and presence or absence of several aflatoxin biosynthesis genes. Forty-eight VCGs were identified through complementation between nitrate non-utilizing mutants. Twenty-five VCGs contained only atoxigenic isolates, and the remaining 23 only aflatoxin producers. Members of the largest atoxigenic VCG (IT6) were found in 4 of the 5 districts sampled. Six deletion patterns of genes in the aflatoxin biosynthesis gene cluster were detected. No deletions in the cluster were detected for twelve atoxigenic isolates and 10 had the entire cluster deleted. One isolate had a deletion pattern only seen once before in Nigeria. The basis for initial selection of endemic atoxigenic strains of A. flavus for biological control of aflatoxin contamination of maize in Italy is provided.

Aspergillus flavus diversity on crops and in the environment can be exploited to reduce aflatoxin exposure and improve health.

Humans and animals are exposed to aflatoxins, toxic carcinogenic fungal metabolites, through consumption of contaminated food and feed. Aspergillus flavus, the primary causal agent of crop aflatoxin contamination, is composed of phenotypically and genotypically diverse vegetative compatibility groups (VCGs). Molecular data suggest that VCGs largely behave as clones with certain VCGs exhibiting niche preference. VCGs vary in aflatoxin-producing ability, ranging from highly aflatoxigenic to atoxigenic. The prevalence of individual VCGs is dictated by competition during growth and reproduction under variable biotic and abiotic conditions. Agronomic practices influence structures and average aflatoxin-producing potentials of A. flavus populations and, as a result, incidences and severities of crop contamination. Application of atoxigenic strains has successfully reduced crop aflatoxin contamination across large areas in the United States. This strategy uses components of the endemic diversity to alter structures of A. flavus populations and improve safety of food, feed, and the overall environment.