Identification and Quantification of a Toxigenic and Non-Toxigenic Aspergillus flavus Strain in Contaminated Maize Using Quantitative Real-Time PCR.

J Erik Mylroie,Seval Ozkan,Michael N Alpe,W Paul Williams,Gary L Windham,Renuka Shivaji

doi:10.3390/toxins8010015

Abstract

Aflatoxins, which are produced by Aspergillus flavus, are toxic to humans, livestock, and pets. The value of maize (Zea mays) grain is markedly reduced when contaminated with aflatoxin. Plant resistance and biological control using non-toxin producing strains are considered effective strategies for reducing aflatoxin accumulation in maize grain. Distinguishing between the toxin and non-toxin producing strains is important in determining the effectiveness of bio-control strategies and understanding inter-strain interactions. Using polymorphisms found in the fungal rRNA intergenic spacer region (IGS) between a toxigenic strain of A. flavus (NRRL 3357) and the non-toxigenic strain used in the biological control agent Afla-Guard® (NRRL 21882), we developed a set of primers that allows for the identification and quantification of the two strains using quantitative PCR. This primer set has been used to screen maize grain that was inoculated with the two strains individually and co-inoculated with both strains, and it has been shown to be effective in both the identification and quantification of both strains. Screening of co-inoculated ears from multiple resistant and susceptible genotypic crosses revealed no significant differences in fungal biomass accumulation of either strain in the field tests from 2010 and 2011 when compared across the means of all genotypes. Only one genotype/year combination showed significant differences in strain accumulation. Aflatoxin accumulation analysis showed that, as expected, genotypes inoculated with the toxigenic strain accumulated more aflatoxin than when co-inoculated with both strains or inoculated with only the non-toxigenic strain. Furthermore, accumulation of toxigenic fungal mass was significantly correlated with aflatoxin accumulation while non-toxigenic fungal accumulation was not. This primer set will allow researchers to better determine how the two fungal strains compete on the maize ear and investigate the interaction between different maize lines and these A. flavus strains.

Highlights

Aflatoxin is a toxic and carcinogenic secondary metabolite produced by Aspergillus flavus during infection of maize and other crops [1,2]
Sequencing using the primers ITS1 and ITS4 revealed no usable polymorphisms in the internal transcribed spacer (ITS) region and the Intergenic spacer (IGS) region was sequenced
Much is still not understood about the relationship between maize, toxigenic fungi and the non-toxigenic fungi used as biological control agents

Summary

Introduction

Aflatoxin is a toxic and carcinogenic secondary metabolite produced by Aspergillus flavus during infection of maize and other crops [1,2]. Efforts have been made to reduce aflatoxin accumulation in maize grain through avenues such as detoxification, biological control, and host plant resistance. Breeding programs have been successful in developing resistant maize germplasm such as Mp313E, Mp715, Mp719, and Tex6 [4,5,6,7,8]. Biological control agents, such as Afla-Guard® and AF36® , have been shown to reduce aflatoxin contamination in maize [9,10,11,12,13,14,15]. It is important to better understand the interaction between maize and both toxigenic and non-toxigenic

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Conclusion