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ARS Home » Pacific West Area » Pullman, Washington » Grain Legume Genetics Physiology Research » Research » Research Project #423174

Research Project: Genetic Improvement of Cool Season Food Legumes

Location: Grain Legume Genetics Physiology Research

2017 Annual Report


Objectives
In the United States, pea, lentils and chickpeas are grown primarily in the Pacific Northwest and Northern Plains. Over the past five crop seasons (2007-2012), these crops have been grown on an average of 1,260,000 acres in the US with an average harvest value of over $320 million. These crops also contribute to the success of the US wheat and barley industry by serving as useful rotation crops in small grain production systems. This research project has three objectives that focus on developing new and improved varieties of cool season food legumes (peas, lentils, and chickpeas) and effective integrated disease control strategies for these crops. 1) Develop and release new varieties and germplasm of peas, lentils, and chickpea that have higher seed mineral concentrations; improved host-plant resistance to Aphanomyces root rot, Sclerotinia wilt and Ascochyta blight; and higher yields than existing commercial varieties. 2) Identify genetic markers closely associated with superior yield, optimal plant height for harvest, seed mineral concentration, resistance to Aphanomyces root rot, and improved cold tolerance for autumn-sown peas, and validate their utility for marker-assisted plant breeding. Sub-objective 2A: Identify molecular markers in adapted pea populations that are associated with important traits including concentrations of minerals in seed, resistance to Aphanomyces root rot, and winter hardiness. Sub-objective 2B: Identify molecular markers in adapted chickpea populations that are associated with seed size and early maturity. 3) 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. Sub-objective 3A: Develop efficient techniques to screen peas, lentils and chickpeas for resistance to Ascochyta blight and Sclerotinia white mold. Sub-objective 3B: Determine genetic factors responsible for pathogenicity of Sclerotinia sclerotiorum using a variety of genetic and genomic tools. Sub-objective 3C: Determine the role of solanapyrone phytotoxins produced by A. rabiei during the development of Ascochyta blight disease in chickpea. This research will result in several products, including new varieties of peas, lentils, and chickpeas along with improved methods for controlling diseases of these crops.


Approach
New varieties and germplasm will be developed from pure lines selected from among segregating populations of peas, lentils, and chickpeas. Cyclical hybridization will be conducted to combine favorable alleles for traits of interest. Parental lines will include adapted germplasm, commercial cultivars and accessions from the various international breeding programs. Promising breeding lines will be released as either germplasm or varieties based on a rigorous comparison of their performance relative to that of commercial check varieties. Linkage analysis and the detection of associations between markers and different traits will be done using simple sequence repeats (SSRs), expressed sequence tagged- SSRs, and single nucleotide polymorphisms (SNPs). Pea recombinant inbred line (RIL) populations will be developed from a cross between Aragorn and Kiflica to identify markers associated with seed mineral concentrations. RILs from a cross between the pea cultivars Medora and Melrose will be used to identify markers associated with cold tolerance. Molecular markers associated with seed size and early maturity in chickpea will be detected using a RIL population developed from an interspecific cross between C. arientinum line Flip 90-27 and PI599072 (C. reticulatum). Associations between markers and quantitative trait loci (QTL) conditioning traits of interest will be detected by composite interval mapping. Improved methods will be developed to screen chickpea for reaction to Ascochyta blight. Toxins will be purified from liquid cultures of A. rabiei. Toxins will be adjusted to various concentrations and applied to detached chickpea leaflets. Leaflets treated with water will be used as controls. The speed of lesion development and final lesion size will be used to compare the reactions of different chickpea genotypes. The relationship between field disease scores of the chickpea genotypes and their sensitivity to the toxin will be determined. Studies to develop more efficient methods to screen peas and lentils for reaction to Sclerotinia white mold will initially examine resistant and susceptible materials reported in prior studies. Plants will be grown in the greenhouse and inoculated with agar plugs containing mycelia of S. sclerotiorum. Disease reaction will be scored by measuring the length of the lesion produced by the fungus over different time points. Two approaches will be taken to investigate the genetic factors responsible for pathogenicity and virulence of S. sclerotiorum. One approach will be to use Agrobacterium mediated transformation (AMT) to generate random mutations that will be screened to detect mutants with reduced virulence. The other approach will be to identify genes of S. sclerotiorum that are differentially expressed during the processes of infection and disease development.


Progress Report
A primary objective of the research project is to develop improved germplasm and cultivars of peas, lentils, and chickpeas. In 2017, two ARS breeding lines were recommended for release as new cultivars by the Variety Release Committee of the USA Dry Pea and Lentil Council. The small green lentil line LC01602273E was recommended for release based its performance compared to the cultivar ‘Eston’. Across 33 location-years of evaluations in Washington and Idaho, yields of LC01602273E were on average 25% greater than yields of Eston. The large kabuli chickpea breeding line CA0790B0043C was recommended for release based its performance compared to the cultivar ‘Sierra’. Across 31 location-years of evaluations in Washington and Idaho, yields of CA0790B0043C were on average 17% greater than yields of Sierra. We are currently identifying prospective license holders for these two breeding lines before requesting approval from the ARS for releasing the plant materials. This progress in cultivar development is related to Objective 1 of this project “Develop and release new varieties and germplasm of peas, lentils, and chickpea that have higher seed mineral concentrations; improved host-plant resistance to Aphanomyces root rot, Sclerotinia wilt and Ascochyta blight; and higher yields than existing commercial varieties”. Diseases of chickpea caused by Pythium ultimum have been primarily controlled by the use of the fungicide metalaxyl in seed treatments. However, seed rot of chickpea caused by metalaxyl-resistant (MR) strains of P. ultimum has recently emerged as a disease of increasing importance. We are trying to develop new chickpea cultivars with disease resistance and a prerequisite for reaching this goal is to identify sources of resistance. During this year we examined several different methods for evaluating resistance in chickpeas to seed rot caused by MR P. ultimum. We have developed a growth chamber-based disease screening method that is reproducible and can be completed in two weeks. We have used this method to demonstrate that the desi chickpea cultivar ‘Myles’ is resistant while several popular kabuli chickpea cultivars are susceptible to this disease. Myles has been crossed to several kabuli cultivars this year to introduce resistance into kabuli chickpeas. This progress in identifying sources of resistance to emerging diseases is related to Objective 1 of this project “Develop and release new varieties and germplasm of peas, lentils, and chickpea that have higher seed mineral concentrations; improved host-plant resistance to Aphanomyces root rot, Sclerotinia wilt and Ascochyta blight; and higher yields than existing commercial varieties”. The development of new cultivars can be accelerated by the use of genetic maps that show where molecular markers, which can be easily observed in the laboratory, are associated with important traits including yield, disease resistance, and nutritional qualities. This year we developed a genetic map for chickpea that has eight linkage groups, each of which corresponds to one of the eight chromosomes of chickpea, and 312 different molecular markers. The chickpea population used to make the genetic map is currently being examined using a ‘Genotype by Sequencing’ approach that should result in a new genetic map that is densely saturated with molecular markers. The population is also being evaluated in the field at Pullman, Washington, for several traits, including disease resistance and yield, in order to be able to associate individual molecular markers with specific traits. This progress in developing a chickpea genetic map is related to Objective 2 of this project “Identify genetic markers closely associated with superior yield, optimal plant height for harvest, seed mineral concentration, resistance to Aphanomyces root rot, and improved cold tolerance for autumn-sown peas, and validate their utility for marker-assisted plant breeding”. DNA marker-assisted breeding is now routinely used in major crops to facilitate more efficient development of new crop cultivars. Although pea is one of the oldest consumed foods in the world, it remains behind many other crops in the availability of genomic and genetic resources. This year we completed research that led to the development of a high-density pea genetic map and the identification of several genes controlling mineral concentrations in pea seed (boron, calcium, iron, potassium, magnesium, manganese, molybdenum, phosphorous, sulfur, and zinc). The DNA markers and the genetic linkage map developed in this study permitted the identification of specific regions responsible for high mineral concentrations in pea seed. Breeders will now be able to use this technology to improve the speed and efficiency of identifying superior pea lines with high concentrations of important mineral nutrients. This progress in developing a pea genetic map and identifying markers associated with seed mineral concentrations is related to Objective 2 of this project “Identify genetic markers closely associated with superior yield, optimal plant height for harvest, seed mineral concentration, resistance to Aphanomyces root rot, and improved cold tolerance for autumn-sown peas, and validate their utility for marker-assisted plant breeding”. White mold caused by the fungus Sclerotinia sclerotiorum is a disease of over 300 different crop species, including peas, lentils, and chickpeas. Its tremendous success as a global plant pathogen suggests a better understanding is required of how the fungus infects plants and causes disease. A recent theory proposes that low pH is critical for infection of plants by the fungus. We examined what genes in the fungus were most active at different pH conditions and during the infection process. We identified 17 different genes that were especially active during the infection process and also under low pH conditions. The 17 genes identified can now be examined in greater detail to determine their roles in the ability of the fungus to infect hundreds of different crops. This progress in improving understanding of how white mold disease occurs relates to Objective 3 of this 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”.


Accomplishments
1. Identification of a new fungicide for controlling chickpea seed rot caused by metalaxyl-resistant Pythium. Pythium seed rot of chickpea has been effectively controlled for more than three decades by applying the fungicide metalaxyl onto seeds before planting. However, over the past three years seed rot caused by strains of Pythium that are resistant to metalaxyl has emerged as a threat to chickpea production in the U.S. ARS researchers at Pullman, Washington, conducted both laboratory experiments and field evaluations that clearly demonstrated that the fungicide ethaboxam is effective as a seed treatment in protecting chickpea seeds from seed rot caused by metalaxyl-resistant Pythium. The use of ethoboxam as a seed treatment has been rapidly adopted as a common practice for chickpea production in the Pacific Northwest. This has resolved this emerging disease problem for the present time.

2. Genetic characterization of beneficial nitrogen-fixing rhizobacteria. A crucial factor contributing to the historical success of peas, lentils, and chickpeas is their ability to form symbiotic relationships with beneficial rhizobacteria that convert atmospheric Nitrogen to ammonia (NH3), which can be used as fertilizer by grain legumes and subsequent rotational cereal crops. Plant breeding has been proposed as a method to increase biological nitrogen fixation (BNF), but little is known about how BNF is affected by different plant lines and strains of nitrogen fixing rhizobacteria. ARS researchers at Pullman, Washington, completed a genetic characterization of 156 isolates of rhizobacteria collected from field grown peas and chickpeas. The entire collection of characterized isolates was deposited into the National Rhizobium Germplasm Resource Collection. This work has produced a characterized reference set of rhizobia strains for improving capacity for BNF in peas and chickpeas.


Review Publications
Holdsworth, W.L., Gazave, E., Cheng, P., Myers, J.R., Gore, M.A., Coyne, C.J., McGee, R.J., Mazourek, M. 2017. A community resource for exploring and utilizing genetic diversity in the USDA Pea Single Plant Plus Collection. Horticulture Research. doi: 10.1038/hortres.2017.17.
Ma, Y., Grusak, M.A., Cheng, P., Mazourek, M., Coyne, C.J., Main, D., McGee, R.J. 2017. Genome-wide SNP identification, linkage map construction and QTL mapping for mineral nutrient concentrations and contents in pea (Pisum sativum L.). Biomed Central (BMC) Plant Biology. doi: 10.1186/s12870-016-0956-4.
Schrodinger, K., Chen, W. 2016. Evaluation of Intego Solo (ethaboxam) for management of metalaxyl-resistant Pythium spp. in chickpea. Plant Disease Management Reports. 10:FC134.
Xu, L., Chen, W. 2016. Direct repeat-mediated DNA deletion of the mating type locus MAT1-2 genes results in unidirectional mating type switching in Sclerotinia trifoliorum. Scientific Reports. 6:27083.
Wei, W., Zhu, W., Cheng, J., Xie, J., Jiang, D., Li, G., Chen, W., Fu, Y. 2016. Nox Complex signal and MAPK cascade pathway are cross-linked and essential for pathogenicity and conidiation of mycoparasite Coniothyrium minitans. Scientific Reports. 6:24325.
Chen, W., Vandemark, G.J., McGee, R.J. 2016. Field evaluation of fungicides for control of Ascochyta blight of chickpea, 2015. Plant Disease Management Reports. 10:FC196.
Chen, W., Guy, S., McGee, R.J., Paulitz, T.C., Porter, L., Vandemark, G.J., Schroeder, K.L. 2016. Field evaluation of seed treatment fungicides for control of damping-off of chickpea caused by metalaxyl-resistant Pythium spp, 2015. Plant Disease Management Reports. 10:ST017.
Tekeoglu, M., Ozkilinc, H., Tunali, B., Chen, W. 2016. Molecular identification of Fusarium spp. causing wilt of chickpea and the first report of Fusarium redolens in Turkey. Mediterranean Agricultural Sciences. 30:27-33.
Shakeel, Q., Lyu, A., Zhang, J., Wu, M., Chen, S., Chen, W., Li, G., Yang, L. 2016. Optimization of medium composition and cultural conditions for production of antifungal substances by Streptomyces platensis 3-10 and evaluation of its efficacy in suppression of clubroot disease of oilseed rape. Biological Control. 101:59-68.
You, J., Zhang, J., Wu, M., Yang, L., Chen, W., Li, G. 2016. Multiple criteria-based screening of Trichoderma isolates for biological control of Botrytis cinerea on tomato. Biological Control. 101:31-38.
Wu, M., Deng, Y., Zhou, Z., He, G., Chen, W., Li, G. 2016. Characterization of three mycoviruses co-infecting the plant pathogenic fungus Sclerotinia nivalis. Virus Research. 223:28-38.
Bouhadida, M., Jendoubi, W., Gargouri, S., Beji, M., Kharrat, M., Chen, W. 2017. First report of Fusarium redolens causing Fusarium yellowing and wilt of chickpea in Tunisia. Canadian Journal of Plant Pathology. 101:1038.
Lyu, A., Liu, H., Che, H., Yang, L., Zhang, J., Wu, M., Chen, W., Li, G. 2017. Reveromycins A and B from Streptomyces sp. 3–10: Antifungal activity against plant pathogenic fungi in vitro and in a strawberry food model system. Frontiers in Microbiology. 8:550.
Yang, D., Zhang, J., Wu, M., Chen, W., Li, G., Yang, L. 2016. Characterization of the mycelial compatibility groups and mating type alleles in populations of Sclerotinia minor in central China. Plant Disease. 100:2313-2318.
Chen, Y., Mcgee, R.J., Vandemark, G.J., Brick, M., Thompson, H. 2016. Dietary fiber analysis of common pulses using AOAC 2011.25: Implications for human health. Nutrients. doi: 10.3390/nu8120829.
Sankaran, S., Wang, M., Vandemark, G.J. 2016. Image-Based rapid phenotyping method of chickpeas seed size characterization. Engineering in Agriculture, Environment and Food. 9:50-55.