1a.Objectives (from AD-416)
Characterize influences of agrichemicals, including glyphosate and copper, on communities of aflatoxin-producing fungi and evaluate the potential for improved aflatoxin management through altered use of agrichemicals and improved biocontrol with chemical resistant atoxigenic strains. Improve understanding of the genetic relationships among aflatoxin-producing and closely related fungi with varying sensitivity to agrichemicals.
1b.Approach (from AD-416)
Collections of Aspergillus flavus from agricultural environments in Arizona and Texas will be screened for sensitivity to agrichemicals. Relationships between chemical sensitivity and chemical exposure will be assessed. Genetic relationships among sensitive and resistant fungi will be assessed and barriers to gene flow among resistant and sensitive isolates evaluated. Competitiveness of resistant and sensitive fungi will be compared and attempts will be made to select atoxigenic strains with improved resistance to agrichemicals as elite biocontrol strains.
Research is carried out in laboratories of the School of Plant Sciences at the University of Arizona and in the Agricultural Research Service laboratory at the University of Arizona. Initial screening of Aspergillus (A.) flavus (a producer of the potent carcinogen, aflatoxin) fungal isolates from Texas and Arizona for sensitivity to a common herbicide Zeocin failed to detect significant differences in sensitivity. In our previous progress report, we noted that sensitivity to zeocin varied among A. flavus isolates with L strain isolates, including AF36, particularly sensitive. With this information, we have been developing a zeocin-based transformation system. We have used both Agrobacterium tumefaciens mediated transformation (ATMT) and electroporation (both routine techniques to transfer desirable genes into fungal cells) to attempt to generate AF36 lines that are zeocin resistant. Interestingly, we note that although AF36 spores are quite sensitive to zeocin, hyphae are much less sensitive. Because ATMT relies on spore germination for T-deoxyribonucleic acid (T-DNA) transfer, it was not feasible to select for resistant lines using that protocol. Electroporation can be performed directly on spores (or protoplasts), so we have been using spore-based methods to develop conditions to optimize transformation. We have produced a number of zeocin-resistant AF36 lines, verified by polymerase chain reaction (PCR) (a technique to multiply DNA) with the zeocin marker primers (starter units for multiplying DNA). It is anticipated that a zeocin-based transformation system will be of value in dissecting competitive differences among A. flavus isolates and in identifying characteristics of value in elite atoxigenic biocontrol agents. Research progress was also monitored through weekly meetings with the principal investigator and through periodic discussions with the student involved in the studies. Progress is monitored through periodic meetings and participation of both laboratories in trouble shooting and methods development.