1a. Objectives (from AD-416):
The primary objective of the project is to investigate molecular genetics and pathogenic mechanisms of Sclerotinia spp. and related pathogens of pea, chickpea and lentil. The second objective is to investigate and develop management practices for diseases of pea, chickpea and lentil.
1b. Approach (from AD-416):
1. Pathogen strains will be collected from infected cool season grain legume plants from various geographic regions. Standard mycological techniques will be used to obtain pure cultures. Isolates will be maintained in cellulose filter paper and in 15% glycerol at -75 C. Additional isolates will be obtained from cooperators from other locations under appropriate USDA, Animal and Plant Health Inspection Service (APHIS) permits. 2. Total genomic DNA will be isolated from each isolate using standard DNA isolation procedure and quantified using spectrophotometric method to study population structure of the grain legume pathogens. Various DNA markers including microsatellite markers 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. 3. To investigate pathogenic mechanisms of the grain legume pathogens, genomic segments related to or responsible for pathogenesis will be identified through generation of non-pathogenic mutants of the 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 transcriptomics. Targeted mutagenesis (gene knockout) will be used to investigate and confirm functions of specific genes. 4. Some secondary metabolites like toxins of fungal pathogens play important roles in causing diseases. 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 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.
3. Progress Report:
Sclerotinia sclerotiorum causes white mold of cool season grain legumes and many other important crops. Despite extensive studies of S. sclerotiorum and its pathogenic mechanisms, many of the details of the pathogenic processes remain to be elucidated. Agrobacterium-mediated transformation (AMT) was used to identify potential virulence factors in Sclerotinia sclerotiorum. Screening AMT transformants identified two mutants showing significantly reduced virulence. The mutants showed similar growth rate, colony morphology, and sclerotial and oxalate production as the wild-type strain. Inverse-PCR and Southern hybridization analyses showed that the mutation was due to a single T-DNA insertion at 212-bp downstream of Cu-Zn-superoxide dismutase gene (SsSOD1, SS1G_00699). In the wild type strain, expression levels of SsSOD1 were significantly increased under oxidative stresses or during plant infection. However, SsSOD1 transcripts could not be detected in the mutant strains. A heterologous complementation test was used to confirm the function of SsSOD1. SsSOD1 functionally complemented the Cu-Zn superoxide dismutase gene in a Ssod1 Saccharomyces cerevisiae mutant. The SOD mutant of S. sclerotiorum had increased sensitivity to heavy metal toxicity induced with zinc and oxidative stress induced with paraquat in culture and reduced ability to detoxify superoxide in infected leaves as evidenced by staining with nitro blue tetrazolium chloride. The mutant also had reduced expression levels of other known pathogenicity genes endo-polygalacturanases: endo-polygalacturonase 1d (sspg1d) and endo-polygalacturonase 3(sspg3). The function of SsSOD1 was further confirmed by SsSOD1-deletion mutation. Like AMT SOD mutant, the SsSOD1-deletion mutant exhibited similar growth rate, sensitivity to metal and oxidative stress, and reduced virulence. These results suggest that this SsSOD1 gene plays critical roles in detoxification of reactive oxygen species during host-pathogen interactions and is an important virulence factor of S. sclerotiorum. The results of this project relate to Subobjective 5B (Develop diagnostic techniques for Sclerotinia spp. identification and determine population structure of Sclerotinia spp. using microsatellite and other molecular markers) of the in-house parent project.