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United States Department of Agriculture

Agricultural Research Service

Research Project: IDENTIFICATION AND ENHANCEMENT OF SEED-BASED BIOCHEMICAL RESISTANCE IN CROPS TO AFLATOXIN PRODUCING PATHOGENS Title: Development of a GFP-Expressing Aspergillus flavus Strain to Study Fungal Invasion, Colonization, and Resistance in Cottonseed

Authors
item Rajasekaran, Kanniah
item Cary, Jeffrey
item Cotty, Peter
item Cleveland, Thomas

Submitted to: Mycopathologia
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: April 12, 2007
Publication Date: February 12, 2008
Citation: Rajasekaran, K., Cary, J.W., Cotty, P.J., Cleveland, T.E. 2008. Development of a GFP-Expressing Aspergillus flavus Strain to Study Fungal Invasion, Colonization, and Resistance in Cottonseed. Mycopathologia. 165:89-97.

Interpretive Summary: The fungus Aspergillus flavus produces the carcinogenic aflatoxins in lipid-rich seeds of cotton, corn, peanuts and tree nuts. Aflatoxin contamination of cottonseed occurs in two phases - the first one prior to seed maturity in the field and the second one during boll maturation, harvesting and storage. The contamination process is exacerbated by moist, warm conditions. In spite of several studies, it has been very difficult to track the fungal growth during the infection process. A strain of toxin-producing A. flavus genetically engineered to express the green fluorescent protein (GFP) from jellyfish was used to study invasion and aflatoxin production in cotton bolls and seeds. The GFP strain glows under UV or blue light and could be tracked in real time without sacrificing the samples. Analyses of inoculated cotton bolls indicated that the seed coat is colonized rapidly within two days and the mode of entry is invariably through the porous chalazal cap in the seed coat. Once the fungus has penetrated, colonization of cotyledons occurs within 72 hours. GFP in cotyledons could be detected and measured and the amount of fluorescence is followed closely by aflatoxin production. The A. flavus strain expressing GFP should be very useful for tracking the fungal infection, colonization and for rapidly identifying cotton lines with enhanced resistance to A. flavus colonization developed through genetic engineering or traditional plant breeding.

Technical Abstract: Cotton bolls were inoculated with a green fluorescent protein (GFP)-expressing Aspergillus flavus (strain 70) to monitor fungal growth, mode of entry, colonization of cottonseeds and production of aflatoxins. The GFP strain and the wild type did not differ significantly in pathogen aggressiveness as indicated by similar reductions in inoculated locule weight. GFP fluorescence was at least 10 times higher than the blue green yellow fluorescence (BGYF) produced in response to infection by A. flavus. The GFP produced by the strain made it possible to identify and monitor specific plant tissues colonized by the fungus. For example, the inner seed coat and cotyledon were colonized by the fungus within 72 hours of inoculation and the mode of entry was invariably through the porous chalazal cap in intact seeds. The amount of GFP fluorescence was shown to be an indicator of fungal growth, colonization and, to some extent, aflatoxin production. The A. flavus strain expressing GFP should be very useful for rapidly identifying cotton lines with enhanced resistance to A. flavus colonization developed through genetic engineering or traditional plant breeding. The ease with which tissues colonized by GFP expressing A. flavus strains can be identified should also make GFP an attractive tool for studying the ecology, etiology and epidemiology of cotton boll rot caused by A. flavus that results in aflatoxin production.

Last Modified: 12/21/2014