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

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.

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

“…For instance, several VCGs of A. flavus isolated from agricultural soils occur across a large section of the United States (Horn and Dorner, 1998, 1999; Ehrlich et al ., 2007; Grubisha and Cotty, 2010, 2015; Ogunbayo et al ., 2013) and others occur across Africa (Ogunbayo et al ., 2013). Dispersal of A. flavus is consistent with the production of large quantities of airborne conidia (Bock et al ., 2004) and association with both insects (Stephenson and Russell, 1974) and human transported crop materials (Garber and Cotty, 2014; Shenge et al ., 2019). Detection of the KE01 active ingredients in all areas except Area‐8 (Tana River County) of the Tana River Irrigation Scheme near Bura is most likely due to either physical barriers to dispersal (i.e.…”

“…Vegetative compatibility analyses also require maintenance of living, functional tester mutants for each VCG (Bayman and Cotty, 1993; Horn et al ., 1996; Probst et al ., 2011), and these mutants must be shared among laboratories wishing to identify or monitor active ingredients. In this regard, DNA technologies can be advantageous (Shenge et al ., 2019).…”

Biological Control and Mitigation of Aflatoxin Contamination in Commodities

“…A non-aflatoxigenic strain ( A. flavus NRRL 21882) under the commercial name Afla-Guard (Syngenta, Basel, Switzerland), which is marketed in the USA, has been used successfully on maize, groundnuts, pistachios, and cottonseed for many years [ 36 , 40 ]. A mixture of four endemic non-aflatoxigenic A. flavus strains called Aflasafe (Ibadan, Nigeria) also has been used on maize and groundnuts in various African countries, with an AF contamination reduction rate of 80–99% [ 35 , 41 , 51 , 53 , 54 , 55 , 56 ]. A commercial product AF-X1 ( A. flavus MUCL54911, Pioneer Hi-Bred, Italy) is applied in Italy to prevent AF contamination [ 57 ].…”

Practical considerations will ensure the continued success of pre-harvest biocontrol using non-aflatoxigenic Aspergillus flavus strains

“…Since the first characterization of MAT genes in A. flavus (Ramirez-Prado et al 2008), the numbers of reports that openly disclose the mating type(s) of strains being considered for biocontrol formulations, or currently in use as biocontrol, are extremely limited. For example, reports involving the composite biocontrol formulations of AflaSafe and FourSure never disclose the mating types of the four non-aflatoxigenic strains that comprise each formulation (Agbetiameh et al 2020;Atehnkeng et al 2008Atehnkeng et al , 2014Bandyopadhyay et al 2016;Senghor et al 2020;Shenge, Mehl and Cotty 2017;Shenge et al 2019;USEPA 2015). They only report the genetic conditions that predispose these strains to exhibit non-aflatoxigenic phenotypes.…”