Comparison of DNA-based methods for quantifying frequencies of a non-aflatoxigenic biocontrol strain of Aspergillus flavus

Pre-harvest crop contamination with aflatoxins, potent carcinogenic mycotoxins produced by certain species in Aspergillus section Flavi, can be reduced through competitive displacement of aflatoxin-producing fungi by non-aflatoxigenic biocontrol strains of Aspergillus flavus. Efficacy of aflatoxin biocontrol is assessed by quantifying frequencies of A. flavus biocontrol strains associated with soils and crops in treated fields. Traditional methods of strain tracking include culture-based methods, which can be laborious and time consuming. Culture-independent methods such as real-time quantitative PCR (qPCR), pyrosequencing, and Droplet Digital PCR (ddPCR) have the potential to reduce time and labor costs, but the utility of these methods for quantifying A. flavus strains has not yet been compared. In this study, the precision and accuracy of qPCR, pyrosequencing, and ddPCR for quantification of different ratios of a biocontrol (Aspergillus flavus AF36) and aflatoxin-producing strain (AF13) of A. flavus were compared. Accuracy and precision were comparable between ddPCR and qPCR, but were significantly reduced when using pyrosequencing. Absolute quantification of DNA copy number was more accurately measured by qPCR compared to ddPCR. The findings of this study suggest that qPCR and ddPCR are both effective methods of tracking biocontrol frequencies, but that qPCR may be more reliable for absolute quantification.