Research Project: Improving Genetic Resources and Disease Management for Cool Season Food Legumes

Location: Grain Legume Genetics Physiology Research

2020 Annual Report

Objectives
Dry peas, lentils, and chickpeas are integral components of dryland agriculture systems throughout the U.S. and have served as globally important nutrition sources of protein, fiber, and minerals for millennia. These crops form symbiotic associations with rhizobacteria that results in biological nitrogen fixation that contributes to productivity and profitability of cropping systems. Peas, lentils, and chickpeas are typically sown in the spring, and the development of autumn sown legumes may provide alternatives to winter wheat. Diseases cause considerable losses in these crops every year and are primarily managed by the use of resistant varieties. However, resistance is lacking to several important diseases, including root rots caused by Aphanomyces and Fusarium, Ascochyta blight, Pythium seed rot, and Sclerotinia white mold. Improved understanding of fungicide resistance and mechanisms of pathogenicity and virulence will accelerate the development of effective and efficient practices for managing diseases of these crops. Over the next five years this research project has the following objectives. Objective 1: Develop and release improved germplasm and cultivars of peas, lentils, and chickpeas that have desirable agronomic traits coupled with enhancements in nutritional characteristics and the ability to form symbiotic effective relationships with nitrogen-fixing rhizobacteria. Subobjective 1A: Develop improved germplasm and cultivars of peas, lentils, and chickpeas that have enhanced field performance and nutritional quality. Subobjective 1B: Characterize factors that influence biological nitrogen fixation resulting from symbiosis between autumn sown pea and Rhizobium leguminosarum. Objective 2: Develop increased understanding of the population structure of selected pathogens, host resistance, and mechanisms of virulence and pathogenicity, and use the knowledge to improve integrated disease management practices and methods for identifying resistant plants. Subobjective 2A: Characterize fungicide resistant populations of Pythium ultimum and Ascochyta rabiei and develop management strategies for fungicide resistance. Subobjective 2B: Identify sources of resistance in pea, lentil, and chickpea to Fusarium root rot, Pythium seed rot, and Aphanomyces root rot, respectively. Subobjective 2C: Increase understanding of factors conditioning virulence and pathogenicity of Sclerotinia sclerotiorum. The advances resulting from these studies will provide comprehensive technology platforms for developing new and improved cultivars of cool season food legumes and effective integrated disease control strategies for these crops.

Approach
1A. Research Goal: Develop and release new cultivars of peas, lentils and chickpeas that have superior agronomic performance and nutritional qualities. Crossing blocks will be established for peas, lentils, and chickpeas. Families and lines will be selected for plant height, disease resistance, tolerance to lodging, early flowering, and seed traits. Remote sensing will be used to estimate canopy vigor of field plots. Correlations will be determined between remote sensing and ground truth data. Promising breeding lines will be released as either germplasm or cultivars. Molecular markers will be detected that are associated with disease resistance and desirable seed nutritional qualities. If desirable traits such as disease resistance are linked to undesirable commercial traits then large population sizes and backcross breeding approaches may be necessary to introduce traits into adapted backgrounds. 1B. Hypothesis: Biological nitrogen fixation in elite winter pea genotypes is conditioned by effects of plant genotype, genotype of the Rhizobium leguminosarum strain, the environment, and interaction effects between these sources of variance. Tests will be performed in growth chambers to evaluate plant and rhizobia genotype effects on biological nitrogen (N) fixation. 15N/14N ratios will be estimated from ground pea tissues to determine %Ndfa. Winter pea lines will be tested in the field for ability to be colonized by endemic rhizobacteria. Plots will be mechanically harvested and the contribution of biological nitrogen fixation to total seed N will be determined. If results based on field studies do not support growth chamber results, the growth chamber conditions will be changed to better reflect field conditions. 2A. Hypothesis: Characterizing and understanding fungicide-resistant pathogen populations will improve efficacy of management of fungicide resistance. The stability of metalaxyl resistance (MR) in MR isolates of Pythium ultimum will be determined, as will the fitness of MR isolates of the pathogen. Microsatelllite DNA markers will be used to survey genetic variation in P. ultimum. Isolates of Ascochyta rabiei will be evaluated for sensitivity to Qol fungicides. 2B. Research Goal: Improve resistance to Pythium seed rot in chickpea and Aphanomyces root rot in lentil. The chickpea single plant core collection will be tested for resistance to Pythium seed rot using a recently developed growth chamber assay. More than 300 accessions from the National Plant Germplasm System (NPGS) lentil core collection will be evaluated for resistance to Aphanomyces root rot using a greenhouse screening assay. Sources of resistance in lentil to Aphanomyces root rot may only be detected in the secondary gene pool. 2C. Research Goal: Increase our understanding of virulence mechanisms of Sclerotinia sclerotiorum by investigating and validating roles of pathogenicity effectors of the pathogen. Seventeen mRNA transcripts of the fungus will be targets of gene-knockout (KO) experiments and the virulence of the KO mutants will be determined. Yeast two-hybrid systems will be used to identify host receptors targeted by pathogen effectors.

Progress Report
Pulse crops, including peas, lentils, and chickpeas have served as globally important sources of protein, fiber, and minerals for millennia and are integral components of dryland agriculture systems throughout the United States. This research focuses on variety development and control of diseases impacting these crops throughout the United States. Considerable progress was made on Objective 1, which addresses Problem Statement 1B (New crops, new varieties, and enhanced germplasm with superior traits) of Component 1 (Crop Genetic Improvement) of the National Program 301, Plant Genetic Resources, Genomics, and Genetic Improvement Action Plan (2018-2022). Sub-objective 1A focuses on the development of improved germplasm and varieties of peas, lentils, and chickpeas. Yield trials of USDA pea, lentil, and chickpea breeding lines were conducted in several states including Idaho, North Dakota, and Washington. The majority of these trials were conducted in fields owned by grower-cooperators. Concentrations of total protein in seeds were determined for several hundred chickpea lines and the results, although only based on one year of data, suggest that seed protein concentration is a heritable trait that can be improved through plant breeding. In addition, a collection of more than 250 different chickpea lines representing all the genetic diversity available globally for chickpea was planted in the field. Harvested seed from these lines will be evaluated for several nutritional traits including seed concentrations of protein, important minerals such as iron and zinc, and pre-biotic carbohydrates, which are sugar-like compounds associated with “gut” health and other health benefits including reduced incidence of type II diabetes and coronary artery disease. A primary reason for the long-term success of pulses is that they are among the few food crops that can be colonized by beneficial soil bacteria, known as “rhizobacteria”, which convert atmospheric nitrogen into a form that can be used as fertilizer by plants. The fertilizer is used by the pulse crop and residual fertilizer left in soil and plant debris is used by the next crop in the rotation cycle, such as wheat. Sub-objective 1B focuses on improving the ability of pulse crops to fix nitrogen through associations with beneficial rhizobacteria. This year we began studies to determine if breeding lines and varieties of peas and chickpeas vary in their ability to be colonized by beneficial rhizobacteria. Plots have been established at different locations in Washington and both plants and soil were collected from each plot. DNA will be extracted from plants and soil samples and tested for the presence of soilborne bacteria and fungi. This information will assist in determining if plant genetics can influence associations with beneficial microbes that contribute to naturally fertilizing crops. Considerable progress was made on Objective 2, which addresses Problem Statement 1B (New crops, new varieties, and enhanced germplasm with superior traits) of Component 1 (Crop Genetic Improvement) of the National Program 301, Plant Genetic Resources, Genomics, and Genetic Improvement Action Plan (2018-2022). Sub-objective 2B focuses on identifying sources of disease resistance in pulse crops. This year we generated lentil and chickpea crosses to develop populations with improved disease resistance. Aphanomyces root rot, which is globally the most destructive disease of peas, has emerged over the past decade as a serious disease of lentils in the United States. Disease resistance was identified in Lens ervoides, a wild relative of cultivated lentil, and this was crossed with several popular lentil varieties including Avondale and Pardina. Plants resulting from these crosses are being grown in the greenhouse to produce populations from which disease resistant plants will be identified. Seed rot of chickpea caused by fungicide-resistant isolates of Pythium has been increasing in severity throughout the Pacific Northwest since first identified in 2014. This year we made crosses using two disease resistant chickpea lines and the popular varieties Nash and Sierra. We are currently evaluating lines from these crosses in greenhouses and growth chambers for resistance to Pythium seed rot and Ascochyta blight. These studies will contribute to the development of new chickpea varieties with improved disease resistance. Progress of Sub-objective 2C focused on identifying genes that are responsible for ability of plant pathogens to cause disease. Sclerotinia sclerotiorum causes white mold disease on more than 400 different crops, which makes it one of the most globally destructive of all plant pathogens. One of the reasons Sclerotinia can cause disease on such a wide range of crops is that it produces enzymes that degrade plant cell walls. We “knocked-out” four different genes involved in the production of cell wall degrading enzymes to study how these genes are involved in the development of white mold disease. Bean plants had less severe white mold disease when inoculated with knocked-out mutant isolates of Sclerotinia. One fungal gene (SsGAR2) was also identified as being involved in maintaining the integrity of the fungal cell walls, but also was found to make the fungus more susceptible to salt stress. This is the first example of identifying multiple roles for these genes involved in the ability of Sclerotinia to cause diseases of crops throughout the world.

Accomplishments
1. Two new spring yellow pea varieties, “USDA-Kite” and “USDA-Peregrine” released. Approximately one million acres of dry peas are grown each year in the United States Pacific Northwest and Northern Plains. More than 70% of peas produced annually in the United States are exported, and yellow peas are an especially popular export crop because they are widely used for industrial production of plant-based protein and starch. Commercially grown varieties must be improved to continually produce high yields despite evolving disease pressure and expanding areas of production. USDA scientists in Pullman, Washington, released two new spring yellow pea varieties, “USDA-Kite” and “USDA-Peregrine”. These two varieties were developed through several years of field testing at locations in Idaho, Montana, North Dakota, and Washington. USDA-Kite and USDA-Peregrine both have yield and seed size similar to the popular yellow pea variety, “Carousel” and are more resistant than Carousel to powdery mildew disease. A research cooperator is currently producing certified seed of both varieties in the field that will be commercially available for planting in 2021.

Review Publications
Ma, Y., Coyne, C.J., Sankaran, S., Main, D., Porter, L.D., Mugabe, D., Smitchger, J., Zhang, C., Amin, M., Fasheed, N., Ficklin, S., McGee, R.J. 2020. Dissecting genetic architecture of Aphanomyces root rot resistance in lentil by QTL mapping and genome-wide association. International Journal of Molecular Sciences. 21(6):2129. https://doi.org/10.3390/ijms21062129.
Zhu, Z., Chen, W. 2018. Downy Mildew. North Dakota State University Extension Service. Online: https://www.ag.ndsu.edu/publications/crops/lentil-disease-diagnostic-series#section-9.
Zhu, Z., Chen, W. 2018. Botrytis gray mold. North Dakota State University Extension Service. Online: https://www.ag.ndsu.edu/publications/crops/lentil-disease-diagnostic-series#section-6.
Burrows, M., Chen, W., Wunsch, M. 2019. White Mold. North Dakota State University Cooperative Extension Bulletin. Online. https://www.ag.ndsu.edu/publications/crops/lentil-disease-diagnostic-series#section-10.
White, D., Chen, W., Schroeder, K. 2018. Assessing the contribution of ethaboxam in seed treatment cocktails for the management of metalaxyl-resistant Pythium sp. in Pacific Northwest spring wheat production. Crop Protection. 115:7-12. https://doi.org/10.1016/j.cropro.2018.08.026.
Zhang, C., Chen, W., Sankaran, S. 2019. High-throughput field phenotyping of Ascochyta blight disease severity in chickpea. Crop Protection. 125. https://doi.org/10.1016/j.cropro.2019.104885.
Mahalingam, T., Chen, W., Rjapakse, C.S., Somachandra, K.P., Attanayake, R. 2020. Genetic diversity and recombination in the plant pathogen Sclerotinia sclerotiorum detected in Sri Lanka. Pathogens. 9(4):306. https://doi.org/10.3390/pathogens9040306.
Rubiales, D., Fondevilla, S., Chen, W., Davidson, J. 2018. Advances in Ascochyta research. Frontiers in Plant Science. 9. https://doi.org/10.3389/fpls.2018.00022.
Zhou, Y., Yang, L., Wu, M., Chen, W., Li, G., Zhang, J. 2017. A single-nuclerotide deletion in the transcription factor gene Bcsmr1 causes formation of orange-colored sclerotia in Botrytis cinerea. Frontiers in Microbiology. 8:2492. https://doi.org/10.3389/fmicb.2017.02492.
Xu, L., Li, G., Jiang, D., Chen, W. 2018. Sclerotinia sclerotiorum: An evaluation of virulence theories. Annual Review of Phytopathology. 56:311-38. https://doi.org/10.1146/annurev-phyto-080417-050052.
Yang, D., Wu, M., Zhang, J., Chen, W., Li, G., Yang, L. 2018. Sclerotinia minor endornavirus 1, a novel pathogenicity debilitation-associated mycovirus with a wide spectrum of transmissibility. mBio. 10(11):589. https://doi.org/10.3390%2Fv10110589.
Caballo, C., Madrid, E., Gil, J., Chen, W., Rubio, J., Milan, T. 2019. Saturation of genomic region implicated in resistance to Fusarium race 5 in chickpea. Molecular Breeding. 39:16. https://doi.org/10.1007/s11032-019-0932-4.
Hao, F., Ding, T., Wu, M., Zhang, J., Yang, L., Chen, W., Li, Q. 2018. Suppressing infection cushion formation by two novel hypovirulence-associated mycoviruses in the phytopathogenic fungus Botrytis cinerea. Viruses. 10:254. https://doi.org/10.3390%2Fv10050254.
You, J., Liu, X., Wu, M., Yang, L., Zhang, J., Chen, W., Li, G. 2019. Defective RNA of a novel mycovirus with high transmissibility detrimental to biocontrol properties of Trichoderma spp. Microorganisms. 7(11):507. https://doi.org/10.3390/microorganisms7110507.
Zhu, W., Xu, X., Peng, F., Zhang, S., Xu, R., Chen, W., Wei, W. 2019. The clcase-associated protein ChCAP is important for regulation of hyphal growth, appressorial development, penetration, pathogenicity, conidiation, intracellular cAMP level and stress tolerance in Colletotrichum higginsianum. Plant Science. 283:1-10. https://doi.org/10.1016/j.plantsci.2019.02.012.
Kim, W., Lichtenzveig, J., Syme, R.A., Berim, A., Peever, T., Hur, J., Chen, W. 2019. Identification of a polyketide synthase gene responsible for ascochitine biosynthesis in Ascochyta fabae and its abrogation in sister taxa. mSphere. 4:5. https://doi.org/10.1128/mSphere.00622-19.
Zhao, X., Ni, Y., Liu, X., Zhao, H., Wang, J., Chen, Y., Chen, W., Liu, H. 2020. A simplified and effective technique for production of pycnidia and pycnidiospores by Macrophomina phaseolina. Plant Disease. 104:1183-1187. https://doi.org/10.1094/PDIS-08-19-1795-RE.