Abstract
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.
Highlights
Several species of Aspergillus section Flavi produce immunosuppressive, hepatotoxic and carcinogenic aflatoxins (Liu and Wu, 2010; Liu et al, 2012) in maize and other crops cultivated in warm environments (Cotty et al, 1994; Cotty and Jaime-Garcia, 2007; Bandyopadhyay et al, 2016)
The current study aimed to develop multi-genotype quantitative pyrosequencing assays for quantification of A. flavus genotypes
The current study developed quantitative pyrosequencing assays for quantifying frequencies of active ingredients of Aflasafe in complex microbial populations
Summary
Several species of Aspergillus section Flavi produce immunosuppressive, hepatotoxic and carcinogenic aflatoxins (Liu and Wu, 2010; Liu et al, 2012) in maize and other crops cultivated in warm environments (Cotty et al, 1994; Cotty and Jaime-Garcia, 2007; Bandyopadhyay et al, 2016). Adoption of biocontrol strategies utilizing indigenous atoxigenic genotypes to displace aflatoxin producers in crop-associated fungal communities (Atehnkeng et al, 2008a,b; Mehl et al, 2012) are becoming widespread across the world, due to proven efficacy, low cost, and area-wide benefits (Cotty and Bayman, 1993; Cotty and Bhatnagar, 1994; Dorner, 2008; Atehnkeng et al, 2014; Mauro et al, 2015; Bandyopadhyay et al, 2016) This strategy alters compositions of crop-associated fungal communities through founder effects, competitive displacement and other mechanisms (Cotty and Mellon, 2006; Ortega-Beltran and Cotty, 2018), increasing frequencies of atoxigenic active ingredients and decreasing incidences of aflatoxin-producers through displacement (Abbas et al, 2011; Chang et al, 2012; Atehnkeng et al, 2014). Monitoring is required to verify use of the biocontrol products on crops where use is rewarded, as with farmers supplying crops under the Nigeria AflasafeTM Challenge Project (AgResults, 2019; Schreurs et al, 2019)
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