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ARS Home » Pacific West Area » Pullman, Washington » Grain Legume Genetics Physiology Research » Research » Research Project #427454

Research Project: Molecular Interactions Between Cool Season Grain Legumes and Fungal Pathogens

Location: Grain Legume Genetics Physiology Research

Project Number: 2090-21000-034-02-S
Project Type: Non-Assistance Cooperative Agreement

Start Date: Sep 22, 2014
End Date: Sep 30, 2018

The main objective is to investigate the molecular mechanisms of interactions of grain legumes (chickpea, lentil and pea) with fungal pathogens that commonly occur in the United States. The project will focus on pathogens that frequently occur with economical significance on the grain legumes in the Pacific Northwest, such as Ascochyta, Sclerotinia, Stemphylium, Rhizoctonia and Pythium. The other objective is to study the management practices in controlling or mitigating the adverse effect of the pathogens on grain legume production and grain yields.

1. Grain legume genotypes (cultivars, breeding lines and germplasm lines) will be phenotyped for reactions to important diseases relevant to grain legume production in the US and resistant genotypes will be selected for studying molecular mechanisms of their interactions with pathogens. 2. Strains of the pathogens will be collected from infected cool season grain legume plants from various geographic regions, and isolated in pure culture using standard mycological techniques. Isolates will be maintained on cellulose filter paper and in 15% glycerol at -80 C. Additional isolates will be obtained from cooperators from other locations under appropriate USDA permits. 3. To study population structure of the grain legume pathogens, total genomic DNA will be isolated from each isolate using standard DNA isolation procedure and quantified using spectrophotometric method. Microsatellite alleles of the isolates will be determined using PCR, and haplotypes (multi locus genotype or combination of microsatellite alleles) will be determined for each isolate and used in analysis using clustering and Bayesian methods. 4. Some secondary metabolites like toxins of fungal pathogens may play important roles in causing diseases and pathogen biology. To study secondary metabolites of the fungal pathogens, pathogen cultures will be grown in appropriate culture media. Secreted metabolites from the cultures will be isolated and purified using appropriate solvents and concentrated in rotary evaporator, detected and quantified using high performance liquid chromatography. Unknown compounds will be identified using nuclear magnetic resonance spectroscopy and mass spectrometer. Biological activities of the secondary metabolites will be tested using appropriate bioassays. 5. To increase our mechanistic understanding of pathogenesis of grain legume pathogens, genomic segments related to or responsible for pathogenesis will be identified through generation of non-pathogenic mutants of the grain legume pathogens. Random mutagenesis will be used to generate tagged mutations and mutants will be screened for virulence. Mutants with altered virulence will be further characterized in terms of genetic mutations and function of the mutated genes, and other genomic methods like comparative transcriptome analyses. 6. To identify resistance sources to diseases in grain legumes, germplasm lines and cultivars of pea, chickpea and lentil will be planted in the greenhouse. At appropriate growth stages, the test plants will be inoculated with the target pathogens and incubated for disease development at environmental conditions conducive to disease development. Disease severity of the plant genotypes will be scored with appropriate rating scale and resistance will be rated and evaluated in repeated experiments.