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

Authors: Shenge, Kenneth; Adhikari, Bishwo; AKANDE, ADEBOWALE - International Institute Of Tropical Agriculture (IITA); Callicott, Kenneth; ATEHNKENG, JOSEPH - International Institute Of Tropical Agriculture (IITA); ORTEGA-BELTRAN, ALEJANDRO - International Institute Of Tropical Agriculture (IITA); KUMAR, P.LAVA - International Institute Of Tropical Agriculture (IITA); BANDYOPADHYAY, RANAJIT - International Institute Of Tropical Agriculture (IITA); Cotty, Peter

Submitted to: Frontiers in Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/21/2019
Publication Date: 11/15/2019
Citation: Shenge, K.C., Adhikari, B.N., Akande, A., Callicott, K.A., Atehnkeng, J., Ortega-Beltran, A., Kumar, P., Bandyopadhyay, R., Cotty, P.J. 2019. Monitoring Aspergillus flavus genotypes in a multi-genotype aflatoxin biocontrol product with quantitative pyrosequencing. Frontiers in Microbiology. 10. https://doi.org/10.3389/fmicb.2019.02529.
DOI: https://doi.org/10.3389/fmicb.2019.02529

Interpretive Summary: Aspergillus flavus section Flavi produce aflatoxins that are known to cause cancer in humans. However, members of the group are highly variable in their ability to produce Aflatoxins; some members of the group do not produce aflatoxins, and are known are atoxigenic. Use of indigenous atoxigenic genotypes to displace toxin producers in fungal communities in agricultural soils is increasingly becoming popular. However, there is a need for improved methods for rapidly assessing the efficacy of biocontrol and their residual effects. To address this need, the current study designed twenty-four quantitative pyrosequencing assays for rapid post-harvest detection and quantification of Aspergillus flavus genotypes which serve as active ingredients in the biocontrol product AflasafeTM that is registered for use in Nigeria. The fungal genotypes are quantified from mixed fungal populations associated with crop surfaces. Sixteen of the assays are for individual genotypes and eight are for simultaneous detection and quantification of multiple active ingredients. Refinement of the assay with mixtures of DNA from target and non-target genotypes indicated both accuracy and sensitivity of the assays. Quantification of target alleles by single and multiple genotype assays was highly correlated with proportions of target DNA in the mixtures. Results from the current study showed that quantitative pyrosequencing is a robust and reliable tool for rapid detection, quantification, and monitoring of multiple Aspergillus flavus genotypes within complex fungal communities associated with crops.

Technical 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 withmaize 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 endusers 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.