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Title: Co-inoculating of aflatoxigenic and non-aflatoxigenic strains of Aspergillus flavus to study fungal invasion, colonization, and competition in maize kernels

item HRUSKA, ZUZANA - Mississippi State University
item Rajasekaran, Kanniah - Rajah
item YAO, HAIBO - Mississippi State University
item KINCAID, RUSSELL - Mississippi State University
item DARLINGTON, DAWN - Mississippi State University
item Brown, Robert
item Bhatnagar, Deepak
item Cleveland, Thomas

Submitted to: Frontiers in Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/10/2014
Publication Date: 3/10/2014
Publication URL:
Citation: Hruska, Z., Rajasekaran, K., Yao, H., Kincaid, R., Darlington, D., Brown, R.L., Bhatnagar, D., Cleveland, T.E. 2014. Co-inoculating of aflatoxigenic and non-aflatoxigenic strains of Aspergillus flavus to study fungal invasion, colonization, and competition in maize kernels. Frontiers in Microbiology. 5(122):1-7. DOI: 10.3389/fmicb.2014-00122.

Interpretive Summary: Biological control of toxin-forming Aspergillus flavus by field inoculation of non-aflatoxigenic strains has yielded promising results. Displacement of aflatoxigenic strains with non-aflatoxigenic strains is the primary reason attributed for the favorable results from these earlier studies. However, the precise interaction between the toxigenic and non-toxigenic strains is not properly understood. In the present study, corn kernels were co-inoculated with a toxin forming, green fluorescent expressing aflatoxigenic strain with a non-aflatoxigenic strain under filed conditions and the kernels were evaluated by tracking relative GFP-fluorescence, as an indication of the presence of both the strains. Aflatoxin levels were also estimated in inoculated kernels. Results of the study confirmed postulated competitive exclusion based on the robust growth and proliferation exhibited by the non-aflatoxigenic fungus thereby reducing the aflatoxin levels in infected kernels. These findings are very useful to scientists and farmers alike to understand the mode of action by non-aflatoxigenic strains of Aspergillus flavus to control aflatoxin contamination of crop species such as corn, cotton, peanut and tree nuts.

Technical Abstract: A currently utilized pre-harvest bio-control method involves field inoculations with non-aflatoxigenic Aspergillus flavus, a tactic shown to strategically displace the native aflatoxin producing strain and effectively decrease aflatoxin contamination in corn. The present in situ study focuses on tracking the invasion and colonization of an aflatoxigenic and a non-aflatoxigenic strain of A. flavus by utilizing a Green Fluorescence Protein (GFP) labeled aflatoxin producing A. flavus isolate in order to gain a deeper understanding of the mechanism involved during competition between the two strains of fungi in corn (Zea mays). Corn kernels were co-inoculated with aflatoxin producing GFP labeled AF70 + non-aflatoxigenic AF36 as well as GFP-labeled and unlabeled controls. The kernels were incubated at 30oC for 3,4,5,7 and 9 days. Cross-sections were imaged under UV and blue light (GFP) in order to visualize competition between the aflatoxigenic and non-aflatoxigenic fungi. After imaging, all kernels were chemically analyzed for aflatoxin. There appeared to be a population difference between the co-inoculated AF70 + 36 group and the GFP AF70 group both visually and with pixel count analysis. There was no GFP fluorescence evident in any of the unlabeled groups. The growth of AF70 inside the kernels was suppressed up to 82% when co-inoculated with AF36 indicating that AF36 displaced AF70. This was in agreement with images taken of whole kernels where AF36 exhibited a more robust external growth compared to AF70. The decrease in population of AF70 was reflected in a corresponding (up to 73%) decrease in aflatoxin levels. Our results indicate that the decrease in aflatoxin production is a consequence of decrease in population of the aflatoxigenic fungus caused by the non-aflatoxigenic fungal isolate supporting the theory of competitive exclusion with a possible combined strategy of robust growth, proliferation and the use of volatiles employed by the non-aflatoxigenic fungus.