Oral: Fungal Genomics
Quantitative pyrosequencing for rapid detection and quantification of Aspergillus flavus atoxigenic active ingredients in complex fungal communities
K. SHENGE (1), B. Adhikari (1), K. Callicott (1), R. Bandyopadhyay (2), P. Cotty (3) (1) USDA-ARS, U.S.A.; (2) International Institute of Tropical Agriculture (IITA), Nigeria; (3) USDA-ARS, U.S.A.
Use of atoxigenic Aspergillus flavus genotypes to displace aflatoxin producers is a highly effective method for reducing aflatoxin contamination in crops. However, there is a need for improved methods for rapid assessment of treatment efficacy and 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 fungi must be quantified in 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. Assay refinement with mixtures of DNA from target and non-target genotypes indicated both accuracy and sensitivity of the assays in detecting the active ingredients in fungal populations. Quantification of target alleles by single and multiple genotype assays was highly correlated with proportions of target DNA in the mixtures. Results from tests to validate the assays with DNA extracted from treated corn samples were not consistent among assays. The current results demonstrate that quantitative pyrosequencing is a robust and reliable tool for rapid detection, quantification, and monitoring of multiple genotypes within complex fungal communities associated with crops.