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Co-Inoculation of Aflatoxigenic and Non-Aflatoxigenic Strains of Aspergillus flavus to Study Fungal Invasion, Colonization, and Competition in Maize Kernels

Zuzana Hruska, Kanniah Rajasekaran, Haibo Yao, Russell Kincaid, Dawn Darlington, Robert L. Brown, Deepak Bhatnagar, Thomas E. Cleveland
Frontiers in microbiology 2014 v.5 no.122 pp. 1-7
Aspergillus flavus, Zea mays, aflatoxins, blue light, competitive exclusion, corn, fungi, green fluorescent protein, image analysis, microbial colonization, seeds, toxigenic strains
A currently utilized pre-harvest biocontrol method involves field inoculations with non-aflatoxigenic Aspergillus flavus strains, a tactic shown to strategically suppress native aflatoxin-producing strains and effectively decrease aflatoxin contamination in corn. The present in situ study focuses on tracking the invasion and colonization of an aflatoxigenic A. flavus strain(AF70), labeled with green fluorescent protein (GFP), in the presence of a non-aflatoxigenic A. flavus biocontrol strain (AF36), to better understand the competitive interaction between these two strains in seed tissue of corn(Zea mays). Corn kernels that had been co-inoculated with GFP-labeled AF70 and wild-type AF36 were cross-sectioned and observed under UV and blue light to determine the outcome of competition between these strains. After imaging, all kernels were analyzed for aflatoxin levels. The reappeared to be a population difference between the co-inoculated AF70-GFP + AF36 and the individual AF70-GFP tests, both visually and with pixel count analysis. The GFP allowed us to observe that AF70-GFP inside the kernels was suppressed up to 82% when co-inoculated with AF36 indicating that AF36 inhibited progression of AF70-GFP. This was in agreement with images taken of whole kernels where AF36 exhibited a more robust external growth compared to AF70-GFP. The suppressed growth of AF70-GFP was reflected in a corresponding (upto73%) suppression in aflatoxin levels. Our results indicate that the decrease in aflatoxin production correlated with population depression of the aflatoxigenic fungus by the biocontrol train supporting the theory of competitive exclusion through robust propagation and fast colonization by the non-aflatoxigenic fungus.