Progress 09/25/03 to 06/30/08
Outputs
Progress Report Objectives (from AD-416) Search for and identify resistance sources of grain legumes to white mold caused by Sclerotinia sclerotiorum by screening the commercial cultivars and core germplasm collections of peas, chickpeas, and lentils. Develop efficient field and greenhouse screening technique suitable for each of the grain legume crops. Determine the genetic nature of the identified resistance and potentially related genes by using traditional and modern genetic techniques including hybridization and progeny analysis, gene cloning and disruption. Investigate genetic variability of the pathogen. Formulate an approach to incorporate the resistance into future cultivars of peas, chickpea, and lentils for efficient management of white mold in grain legume production. Publish findings in international journals. Approach (from AD-416) Compare various techniques for inoculum production and inoculation onto peas, chickpea, and lentils. Select the best technique to screen the commercial cultivars and core germplasm collections of the grain legumes. Evaluate plant growth habit in relation to white mold resistance. Use traditional genetic techniques such as hybridization to determine the inheritance pattern of the identified resistance. Employ modern genetic techniques to locate resistance genes in grain legumes, and to isolate and disrupt the pathogenicity gene in the pathogen. Utilize vegetative compatibility grouping technique and DNA fingerprinting to determine genetic variability of S. clerotiorum. Document the SCA with the Department of Plant Pathology at Washington State University, Pullman. Formerly 5348-21000-007-02S (August 2003). Significant Activities that Support Special Target Populations Research efforts of this research agreement were on developing techniques to unequivocally determine the species identities of Sclerotinia isolates obtained from chickpea, and demonstrate their pathogenicity on chickpea plants. The identities of Sclerotinia isolates obtained from chickpea (Cicer arietinum L.) plants showing stem and crown rot were determined using cultural characteristics, ascospore morphology, variations in group I introns and ITS sequences. Isolates could be separated into two groups based on growth rates, fast growing vs. slow growing. Growth on a pH-indicating medium showed that all fast growing isolates produced abundant acid and all slow growing isolate did not. The slow growing isolates contained at least one group I intron in the nuclear 18s subunit rDNA, whereas all fast growing isolates lacked group I introns in the same DNA region. ITS sequences of the slow growing isolates were identical to previously deposited sequences of Sclerotinia trifoliorum. ITS sequences of the fast growing isolates were identical to deposited sequences of S. sclerotiorum. A technique was developed to induce carpogenic germination of sclerotia of Sclertoinia isolates, which allowed direct observation of ascospore morphology. The slow growing isolates showed ascospore dimorphism, a definitive character of S. trifoliorum, whereas the fast growing isolates showed no ascospore dimorphism. Isolates of both the slow growing and the fast growing are pathogenic on chickpea and caused symptoms similar to those observed in the field. This research showed for the first time in North America that S. trifoliorum causes stem and crown rot of chickpea. This work addresses Component 3 of the NP 301 Action Plan (Genetic Improvement of Crops) and Problem 3a (Genetic Theory and Methods of Crop Improvement). Research progress has been monitored via weekly informal discussions, monthly progress and planning meetings, and regular visits to the collaborator's greenhouse facility where disease screening is conducted, which located at WSU-IAREC, Prosser, WA
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Progress 10/01/06 to 09/30/07
Outputs
Progress Report Objectives (from AD-416) Search for and identify resistance sources of grain legumes to white mold caused by Sclerotinia sclerotiorum by screening the commercial cultivars and core germplasm collections of peas, chickpeas, and lentils. Develop efficient field and greenhouse screening technique suitable for each of the grain legume crops. Determine the genetic nature of the identified resistance and potentially related genes by using traditional and modern genetic techniques including hybridization and progeny analysis, gene cloning and disruption. Investigate genetic variability of the pathogen. Formulate an approach to incorporate the resistance into future cultivars of peas, chickpea, and lentils for efficient management of white mold in grain legume production. Publish findings in international journals. Approach (from AD-416) Compare various techniques for inoculum production and inoculation onto peas, chickpea, and lentils. Select the best technique to screen the commercial cultivars and core germplasm collections of the grain legumes. Evaluate plant growth habit in relation to white mold resistance. Use traditional genetic techniques such as hybridization to determine the inheritance pattern of the identified resistance. Employ modern genetic techniques to locate resistance genes in grain legumes, and to isolate and disrupt the pathogenicity gene in the pathogen. Utilize vegetative compatibility grouping technique and DNA fingerprinting to determine genetic variability of S. clerotiorum. Document the SCA with the Department of Plant Pathology at Washington State University, Pullman. Formerly 5348-21000-007-02S (August 2003). Significant Activities that Support Special Target Populations This is a specific cooperative agreement between ARS and Washington State University, Department of Plant Pathology. Additional details of research can be found in the report for the parent CRIS 5348-21000-014-00D, Germplasm Enhancement, Genetics and Disease Management of Cool Season Food Legumes. Research efforts of this research agreement were on developing techniques to unequivocally determine the species identities of Sclerotinia isolates obtained from chickpea, and demonstrate their pathogenicity on chickpea plants. The identities of Sclerotinia isolates obtained from chickpea (Cicer arietinum L.) plants showing stem and crown rot were determined using cultural characteristics, ascospore morphology, variations in group I introns and ITS sequences. Isolates could be separated into two groups based on growth rates, fast growing vs. slow growing. Growth on a pH-indicating medium showed that all fast growing isolates produced abundant acid and all slow growing isolate did not. The slow growing isolates contained at least one group I intron in the nuclear 18s subunit rDNA, whereas all fast growing isolates lacked group I introns in the same DNA region. ITS sequences of the slow growing isolates were identical to previously deposited sequences of Sclerotinia trifoliorum. ITS sequences of the fast growing isolates were identical to deposited sequences of S. sclerotiorum. A technique was developed to induce carpogenic germination of sclerotia of Sclertoinia isolates, which allowed direct observation of ascospore morphology. The slow growing isolates showed ascospore dimorphism, a definitive character of S. trifoliorum, whereas the fast growing isolates showed no ascospore dimorphism. Isolates of both the slow growing and the fast growing are pathogenic on chickpea and caused symptoms similar to those observed in the field. This research showed for the first time in North America that S. trifoliorum causes stem and crown rot of chickpea. Research progress is monitored via weekly informal discussions, monthly progress and planning meetings, and regular visits to the collaborator's greenhouse facility where disease screening is conducted, which located at WSU-IAREC, Prosser, WA
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Progress 10/01/05 to 09/30/06
Outputs
Progress Report 4d Progress report. This is a specific cooperative agreement between ARS and Washington State University, Department of Plant Pathology. Additional details of research can be found in the report for the parent CRIS 5348-21000-014-00D, Germplasm Enhancement, Genetics and Disease Management of Cool Season Food Legumes. Research efforts of this research agreement were on developing mapping populations of lentils and on developing tools to study pathogenic mechanisms of the white mould pathogen Sclerotinia sclerotiorum. Populations of lentil from nine crosses have been advanced to the F6 to generate recombinant inbred lines (RILs) for possible use in mapping the genes for resistance to white mold. Populations from three additional crosses between parents with known differential responses to infection by S. sclerotiorum have been advanced to the F4 for developing RIL populations. One of the populations is being used to develop a co- dominant molecular
marker map of the lentil genome. The genetic map of that population will be used in a quantitative trait loci analysis of resistance to white mold. Parallel research efforts were directed at determining the pathogen species causing white mold of chickpea in central California. Isolates obtained from chickpea in central California could be separated into two groups based on growth rate: fast- growing (40 mm in 24 hours) and slow-growing (20 mm in 24 hours). Molecular differences between these two groups were also detected. The slow-growing isolates possess two introns in the nuclear small subunit rDNA whereas the fast growing isolates do not have introns in the same DNA region. These differences in growth rate and the introns in rDNA helped us to identify the fast growing isolates as S. sclerotiorum and the slow growing isolates as S. trifoliorum. Isolates of the two groups were also compared to previously identified isolates of the species to assure the species identification.
This is the first report of Sclerotinia trifoliorum causing collar rot of chickpea in the United States. Future research effort is directed at understanding virulence of the isolates on different chickpea genotypes to identify sources of resistance/tolerance.
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Progress 10/01/04 to 09/30/05
Outputs
4d Progress report. This report serves to document research under a specific cooperative agreement between ARS and Washington State University, Department of Plant Pathology. Additional details of research can be found in the report for the parent CRIS 5348-21000-014-00D, Germplasm Enhancement, Genetics and Disease Management of Cool Season Food Legumes. Research efforts of this research agreement were on developing mapping populations of lentils and on developing tools to study pathogenic mechanisms of the white mould pathogen Sclerotinia sclerotiorum. Populations of lentil from nine crosses have been advanced to the F6 in the process to generate recombinant inbred lines (RILs) for use in mapping the genes for resistance to white mold. Populations from three additional crosses between parents with known differential responses to infection by S. sclerotiorum have been advanced to the F3 for developing RIL populations. These populations will be the materials for
mapping quantitative trait loci for resistance to white mold. Parallel research efforts were directed at understanding the pathogenic mechanisms by studying mutants with altered virulence or pathogenicity. An efficient transformation system was developed to transform S. sclerotiorum using Agrobacterium- mediated transformation. The transformation vector was constructed and the conditions for efficient transformation were optimized. The system allows us to generate tagged mutations that will facilitate isolating the mutated genes. More than forty transformants were generated and characterized and the virulence of the transformants on lentil plants was compared with the wild type strain. Two transformants with significantly reduced virulence were identified. Research efforts are underway to identify and isolate the mutated genes and to determine and verify their role in pathogenicity. The goal is to understand the pathogenic mechanisms in order to develop more effective
resistance and control strategies.
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Progress 10/01/03 to 09/30/04
Outputs
4. What were the most significant accomplishments this past year? D. This report serves to document research conducted under a Specific Cooperative Agreement between ARS and Washington State University, Department of Plant Pathology. Additional details of research can be found in the report for the parent CRIS 5348-21000-014-00D, Germplasm Enhancement, Genetics and Disease Management of Cool Season Food Legumes. Major research efforts of this research agreement in the past year have been focused on developing a screening technique for resistance in lentil to white mold caused by Sclerotinia sclerotiorum, and to test variation among isolates of the pathogen in mycelial compatibility and fungicide sensitivity. An efficient screening technique was developed for screening lentils for resistance to white mold. Several germplasm lines have been identified as resistance sources for studying the genetics of resistance in lentil to white mold. Key crosses between
resistant and susceptible lentil lines are being made. Isolates obtained from lentil fields of the Palouse region have been studied for their mycelial compatibility as a means to study their genetic variability. Considerable genetic variability in terms of mycelial compatibility has been found among the isolates from the Pacific Northwest. A technique is also developed for detecting double-stranded RNA (viruses) inhibiting in the isolates, and the isolates were found to vary in harboring the viruses. Selected isolates were also tested for their sensitivity to four fungicides Benlate, Endura, Ronilan and Topsin, and variations were also found among the isolates in their sensitivity to the fungicides. This is important information for lentil growers in managing white mold using fungicides.
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