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:
This research relates to objective 3 of the inhouse project “Develop efficient techniques to screen peas, lentils, and chickpeas for host-plant resistance to Ascochyta blight and Sclerotinia wilt, and characterize genetic and physiological factors responsible for the virulence of these pathogens”. Sclerotinia sclerotiorum is a ubiquitous necrotrophic pathogen capable of infecting over 400 plant species including many economically important crops. Oxalic acid production has been shown in numerous studies to be a pathogenicity factor for S. sclerotiorum through several mechanisms. During our random mutagenesis study of S. sclerotiorum using Agrobacterium-mediated transformation, we identified three mutants that had lost oxalate production. Southern hybridization blots showed the mutation was due to a single T-DNA insertion, and plasmid rescue and DNA sequencing confirmed that the T-DNA insertion site was located in the open reading frame (ORF) of oxaloacetate acetylhydrolase (Ssoah, SS1G_08218) of S. sclerotiorum. The mutants did not change the color of a pH-indicating medium (potato dextrose agar (PDA) amended with 50 mg/L bromophenol blue). The pH values of 6-day potato dextrose broth (PDB) culture filtrates were 1.8-2.0 for the wild type and 2.8-3.1 for the mutants. No oxalic acid was detected using high performance liquid chromatography (HPLC) in culture filtrates or in the mycelium of the mutants, but another acid compound was accumulated in culture filtrates of the mutants and detected by HPLC, and the compound was identified as fumaric acid using liquid chromatography-mass sprectrometry (LC-MS). The mutants showed reduced vegetative growth on PDA and produced sclerotia that are beige in color and soft in texture. Artificial acidic conditions (pH 3.4 and 4.2) enhanced vegetative growth and promoted normal (black and hard) sclerotial formation of the mutants. Furthermore, the oxalate-minus mutants retained pathogenicity on pea, green bean and faba bean in detached leaf assays and on intact plants of Arabidopsis thaliana, and their virulence levels were similar to that of the wild type strain on certain host plants, but varied depending on the plant species tested. The mutant had increased expression levels of cell wall-degrading enzymes such as polygalacturonases compared to the wild type strain during the process of infecting pea leaves. The results showed that a low pH condition is very important for growth and virulence of S. sclerotiorum on its wide range of hosts.