1a. Objectives (from AD-416):
The primary objective of the project is to investigate the ecology, population biology and pathogenic mechanisms of plant pathogens as they interaction with cool season grain legumes that include pea, chickpea and lentil. The project will focus on pathogens, such as species of Ascochyta, Erysiphe, Sclerotinia, and Stemphylium, with economical significance to US production of cool season grain legumes. The second objective of the project is to develop management practices for controlling or mitigating the adverse effect of pathogens on crop production.
1b. Approach (from AD-416):
1. Strains of the pathogens will be collected from infected cool season grain legume plants from various geographic regions, and isolated in pure culture using mycological techniques. Isolates will be maintained in cellulose filter paper and in 15% glycerol at -80 C. Additional isolates will be obtained from cooperators from other locations under appropriate USDA permits. 2. To study population structure of the grain legume pathogens, total genomic DNA will be isolated from each isolate using standard methods for DNA isolation and quantification. Microsatellite alleles of isolates will be determined using PCR, and haplotypes (multi locus genotype or combination of microsatellite alleles) will be determined for each isolate and used to examine genetic relatedness among isolates. 3. 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 will be detected and quantified using high performance liquid chromatography. Unknown compounds will be identified using nuclear magnetic resonance spectroscopy and mass spectrometry. Biological activities of the secondary metabolites will be tested using appropriate bioassays. 4. 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. 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, function of the mutated genes, and patterns of gene expression. 5. To identify resistant sources 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 scales and resistance will be rated and evaluated in repeated experiments.
3. Progress Report:
Ascochyta rabiei causes Ascochyta blight of chickpea, the most destructive Ascochyta blights of cool season grain legumes. Despite intensive studies of Ascochyta blight of chickpea, the pathogenic mechanisms of this pathogen are still not fully understood. To gain further understanding of the pathogenic mechanisms of A. rabiei, a RNA-sequencing approach was employed. Total mRNAs were isolated from isolates AR19 of pathotype I and AR628 of pathotype II of A. rabiei, and also from diseased tissues of chickpea ‘Spanish White’ inoculated with these two isolates at times from 6 to 96 hours post inoculation (hpi), and were sequenced with the 454 Titanium RNA sequencing technology. The transcripts in the interacting transcriptomes were separated into either plant RNA or pathogen RNA based on BLAST searches, and the pathogen transcripts were compared with the transcripts of the pathogen from pure culture. The pathogen transcripts in the interacting transcriptomes that were not found in the transcripts of pure culture were considered as induced transcripts (in response to infecting chickpea). A total of 21,226 and 28,061 unique transcripts (unigenes) were obtained from AR19 and AR628, respectively, and about 70% of them marched annotated genes in NCBI database. An average of 29,725 unigenes for each library was assembled from a total of 867,855 raw reads in the interacting transcriptomes. About 10% of unigenes were from the pathogen. There were 132 induced unigenes of isolate AR19 in the first 12 hpi, nine of them were highly expressed during 24 to 96 hpi, and 42 of them were consistently expressed up to 96 hpi. There were 178 induced unigenes of isolate AR628, six of them were highly expressed during 24 to 96 hpi, and 69 of them were consistently expressed up to 96 hpi. Analysis showed a strong induction for the expression of pathogen genes that belonged to different functional categories. The functions of these induced genes and their roles in pathogenesis remain to be investigated. The results of this project relate to Subobjective 5C (Elucidate pathogenic mechanisms of Ascochyta rabiei utilizing tagged non-pathogenic mutants) of the in-house parent project.