Founder events influence structures of Aspergillus flavus populations

Open Access Article; Published online: 09 June 2020In 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

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

Open Access ArticleAspergillus 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

Potential of atoxigenic Aspergillus flavus vegetative compatibility groups associated with maize and groundnut in Ghana as biocontrol agents for aflatoxin management

Open Access Journal; Published online: 06 Sept 2019Increasing 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

Monitoring Aspergillus flavus genotypes in a multi-genotype aflatoxin biocontrol product with quantitative pyrosequencing

Open Access Journal; Published online: 15 Nov 2019Aflatoxins 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

The vegetative compatibility group to which the US biocontrol agent Aspergillus flavus AF36 belongs is also endemic to Mexico

Published online: 17 March 2016Aims: To assess frequencies of the Aspergillus flavus atoxigenic vegetative compatibility group (VCG) YV36, to which the biocontrol agent AF36 belongs, in maize-growing regions of Mexico. Methods and Results: Over 3500 A. flavus isolates recovered from maize agroecosystems in four states of Mexico during 2005 through 2008 were subjected to vegetative compatibility analyses based on nitrate nonutilizing mutants. Results revealed that 59 (1 6%) isolates belong to VCG YV36. All 59 isolates had the MAT1-2 idiomorph at the mating-type locus and the single nucleotide polymorphism in the polyketide synthase gene that confers atoxigenicity. Additional degradation of the aflatoxin gene cluster was detected in three isolates. Microsatellite loci analyses revealed low levels of genetic diversity and no linkage disequilibrium within VCG YV36. Conclusions: The VCG to which the biocontrol agent AF36 belongs, YV36, is also native to Mexico. The North American Free Trade Agreement should facilitate adoption of AF36 for use by Mexico in aflatoxin prevention programs. Significance and Impact of the Study: An USEPA registered biocontrol agent effective at preventing aflatoxin contamination of crops in the US, is also native to Mexico. This should facilitate the path to registration of AF36 as the first biopesticide for aflatoxin mitigation of maize in Mexico. Economic and health benefits to the population of Mexico should result once aflatoxin mitigation programs based on AF36 applications are implemented

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. © 2020 The Authors. Microbial Biotechnology published by Society for Applied Microbiology and John Wiley & Sons Ltd. This article has been contributed to by US Government employees and their work is in the public domain in the USA.Open access journalThis item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

The atoxigenic biocontrol product Aflasafe SN01 is a valuable tool to mitigate aflatoxin contamination of both maize and groundnut cultivated in Senegal

Open Access Article; Published online: 30 Nov 2019Aflatoxin contamination of groundnut and maize infected by Aspergillus section Flavi fungi is common throughout Senegal. The use of biocontrol products containing atoxigenic Aspergillus flavus strains to reduce crop aflatoxin content has been successful in several regions, but no such products are available in Senegal. The biocontrol product Aflasafe SN01 was developed for use in Senegal. The four active ingredients of Aflasafe SN01 are atoxigenic A. flavus genotypes native to Senegal and distinct from active ingredients used in other biocontrol products. Efficacy tests on groundnut and maize in farmers’ fields were carried out in Senegal during the course of 5 years. Active ingredients were monitored with vegetative compatibility analyses. Significant (P < 0.05) displacement of aflatoxin producers occurred in all years, districts, and crops. In addition, crops from Aflasafe SN01-treated fields contained significantly (P < 0.05) fewer aflatoxins both at harvest and after storage. Most crops from treated fields contained aflatoxin concentrations permissible in both local and international markets. Results suggest that Aflasafe SN01 is an effective tool for aflatoxin mitigation in groundnut and maize. Large-scale use of Aflasafe SN01 should provide health, trade, and economic benefits for Senegal