Development of DNA markers specific for strains within a population is a multiyear program that will require extensive DNA analysis and the development of new statistical tools. We propose to work toward this goal by conducting research in three broad areas: (i) the evolution of pathogenicity; (ii) the evolutionary processes that influence the genetic diversity; and (iii) the identification of diagnostic targets for fungal pathogens. <P>The specific objectives of this proposal are: 1. Determine the role of repetitive DNA and factors regulating their expression on pathogenicity in Magnaporthe oryzae; 2) Characterize recombination patterns and rates in the aflatoxin gene cluster in natural and experimental populations of Aspergillus flavus and A. parasiticus; 3) Characterize pathogenicity in Aspergillus flavus and the regulation of aflatoxin biosynthesis; and 4) Examine the potential utility of additional genetic loci for identification of filamentous fungi. <P>Completion of the outlined research is necessary to locate DNA regions that can be used to identify individuals within a population and to distinguish between endemic and introduced fungal pathogens.
Non-Technical Summary: Fungi represent the largest group of plant pathogens and throughout history have significantly limited the production of a safe and sustainable food supply. For example, late blight of potato, caused by Phytophthora infestans, resulted in the death of thousands of people and the emigration of millions from Ireland. The majority of serious plant epidemics in the US have been caused by non-indigenous pathogens. This threat of introduced pathogens is increasing due to a global trade, a highly mobile world population, and potential acts of bioterrorism. Rapid response to new pathogens requires accurate and reliable diagnostic procedures. This research is directed toward identifying regions of DNA diagnostic for pathogenicity, survival, and toxin production in fungi. Completion of the outlined research is necessary to locate specific DNA regions that can be used to identify individuals within a population and to distinguish between endemic and introduced fungal pathogens. <P> Approach: We plan to use the genome of M. oryzae to reconstruct the evolution of pathogenesis at the whole genome level. Examination of isolates from different hosts has revealed genetically distinct host-specific populations. We propose to examine defined loci and genomic regions of representative isolates from several lineages/VCGs as sequence data becomes available. As appropriate, we will include data generated from the genome sequences of M. poae and G. graminis. In addition, we propose to examine small RNA populations from developmental stages in different strains and correlate findings with gene expression. In other studies bottom-up approaches will be used to examine the evolutionary processes that influence the evolution of the aflatoxin gene cluster in experimental and natural populations. To do this, we will identify nucleotide sequence variation within coding and noncoding portions of the aflatoxin gene cluster in population samples of A. flavus or A. parasiticus from a single peanut field in Georgia. To better understand pathogenicity factors in A. flavus we will focus on the role of two genes identified by transcriptional profiling as upregulated during pathogenesis. The expression of these genes and the localization of their proteins will be visualized using reporter genes and protein fusion constructs. Either the green fluorescent protein (GFP) or mCherry will be used for visualization. Should the reporter genes not be effective due to autofluorescence of the seed, we will use in situ hybridization. An integral part of the research proposed with is the design of probes for detection of the most commonly encountered lineages of fungi associated with plant disease. Our experimental approach will involve the design and development of taxon-specific probes for identification of plant pathogenic fungi and will provide new tools for examining their ecology and epidemiology across time and space. We propose to develop an expanded DNA-sequence database framework for rapid detection of dominant fungal groups and phylogenetic-based probes will be designed and tested for their ability to detect these fungi. Based on a comprehensive search of genome sequence data from filamentous fungi for 18 phylogenetically informative loci defined by the Assembling the Fungal Tree of Life 2 project, we have identified at least six loci that are of potential utility to accomplish our proposed objectives.