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

Location: Grain Legume Genetics Physiology Research

2021 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. Sub-objective 1C: Work with the Pulse quality lab at Fargo, North Dakota, and other pulse breeders to identify, evaluate, and screen the intrinsic end-use quality of peas, chickpea, and lentil and evaluate nutritional and industrial properties to enable development of improved cultivars needed by the pulse industry to expand the uses of peas, chickpeas, and lentils as food ingredients and other by-products and meat and milk substitutes. 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). Objective 1 focuses on the development of improved germplasm and varieties of peas, lentils, and chickpeas that have desirable agronomic traits and improved nutritional qualities. In support of Sub-objective 1A, yield trials of USDA pea, lentil, and chickpea breeding lines were conducted in 2021 at several locations in Washington, with the majority of these trials conducted in fields owned by grower-cooperators. Preliminary replicated yield trials of promising breeding pea, lentil, and chickpea breeding lines were also conducted in Pullman, Washington. During 2021, two new spring-sown pea varieties were released, “USDA-Kite” and “USDA-Peregrine”. These are both yellow peas and will be primarily grown in the U.S. Northern Plains, especially in Montana and North Dakota. In addition, a first preliminary yield trial was conducted with “stay-green” chickpea breeding lines. There are developing markets for chickpeas that are harvested green for use as snacks and spreads. Producers historically have had to harvest immature seeds to obtain green seeds, a process that reduces yield and is complicated by immature seeds being crushed during harvest. These new breeding lines can be harvested in a dry state while retaining their green color, which should improve efficiencies for these producers. In additional support of Sub-objective 1A, seed protein concentrations were determined for 23 advanced chickpea breeding lines and commercial varieties grown in replicated field trials at four locations in Washington in 2020 (Pullman, Walla Walla, Colton, and Fairfield). These results are being analyzed to determine the relative contributions of genetic and environmental effects on chickpea protein concentrations. This important information will help determine if a particular growing area can produce chickpeas with high protein concentrations and if a specific variety or breeding line produces high protein seeds across diverse locations. Similarly, concentrations of total protein in seeds were determined for advanced pea breeding lines and varieties. Seed protein concentrations ranged from 17-26%. A concerning result is that similar to other pulse crops, pea lines that produced high amounts of seed protein tended to have lower yields than lines that produced lower protein seeds. In additional support of Sub-objective 1A, advanced replicated yield trials of USDA autumn-sown (or winter) pea and lentil breeding lines were conducted in Washington, Idaho, Nebraska, South Dakota, and Kansas. Although winter peas have been sporadically grown in the United States for more than 20 years, none of these peas have been considered “food grade”, or acceptable for human diets, and were only used as animal feed. During this year the research unit released three new food grade winter pea varieties, “USDA-MiCa”, “USDA-Dint”, and “USDA-Klondike”. USDA Dint and USDA MiCa are green peas and USDA Klondike is a yellow pea. These are the first food grade winter pea varieties released by the USDA. The initial commercial plantings of all three varieties will be in 2022. These new winter pea varieties provide growers with an alternative fall-sown crop to winter wheat or winter barley. This will confer benefits associated with using legumes in crop production systems such as the natural production of nitrogen fertilizer that results from the mutually beneficial interactions between pea roots and soil bacteria, especially Rhizobium leguminosarum. In support of Sub-objective 1B, winter peas were planted at several locations in Washington in Fall 2020. Extremely high temperatures and drought stress during the growing season severely reduced colonization of pea roots by Rhizobium leguminosarum. Seed protein concentrations will be determined for winter pea experiments harvested in 2021 and these results will reflect of the total amount of nitrogen made available to developing seeds during the growing season, along with identifying any breeding lines that combine high yield and high seed protein concentration. 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 2A focuses on examining fungicide resistance in soilborne fungi that cause diseases of pulse crops. The recent emergence of metalaxyl resistance in Pythium populations has threatened chickpea production in the U..S. Pacific Northwest. In support of Sub-objective 2A, the relative “fitness”, or ability to reproduce and thrive in culture, was compared between fungicide resistant and fungicide susceptible isolates of Pythium ultimum. It was determined that metalaxyl resistant isolates have the same growth rate and reproduction capacity as metalaxyl sensitive isolates, which suggests that fungicide resistance does not come at the cost of reduced fitness and resistant isolates can survive and proliferate similar to susceptible isolates. Research in support of Sub-objective 2B focused on identifying sources of disease resistance in lentil and chickpea and the transfer of resistance into adapted plant materials. More than 300 accessions of lentil were screened for resistance to root rot caused by Aphanomyces euteiches. Sources of resistance were identified and used as parents for crosses with commercial lentil varieties. F3 lines were produced from F2 plants that were resistant to Aphanomyces root rot. As mentioned above, seed rot of chickpea caused by metalaxyl resistant isolates of Pythium has become a problem in Washington and Idaho. In 2021, chickpea accessions were identified that are resistant to Pythium seed rot at disease concentrations five times as high as previously tested. This allowed for more rigorous identification of highly resistant materials to use in breeding programs. In addition, four chickpea populations were screened for resistance to Ascochyta blight and several highly resistant lines were identified. These lines are being grown in a greenhouse to produce seed for subsequent evaluation of other seed traits including size, shape, and color. Sub-objective 2C is focused on improving the understanding of how the fungus Sclerotinia sclerotiorum causes white mold disease. More than 300 crop species suffer from white mold disease, making Sclerotinia sclerotiorum one of the most globally destructive plant pathogens. The fungus possesses numerous factors that aid in its ability to infect plants and cause white mold disease. In support of Sub-objective 3A, this year, a protein was identified in the fungus that interferes with the ability of plants to induce a defense response. Currently, we are examining how this fungal protein functions to inhibit defense responses in bean plants. Understanding how this protein reduces the ability of plants to inhibit disease will assist in developing more resistant varieties for the hundreds of crop species that suffer from white mold disease.

Accomplishments
1. Three new food-grade winter pea varieties released. Fall-sown, or “winter”, peas have been sporadically grown in the United States for more than 20 years but only for animal feed, as no winter pea varieties were considered “food grade”, or acceptable for human diets. An ARS researcher in Pullman, Washington, developed and released three winter pea varieties: “USDA Dint”, “USDA MiCa”, and “USDA Klondike”. USDA Dint and USDA MiCa are green peas and USDA Klondike is a yellow pea. These are the first food grade winter pea varieties released by the USDA. These new winter pea varieties provide growers with an alternative fall-sown crop to winter wheat or winter barley and will confer benefits associated with using legumes in crop production systems such as the natural production of nitrogen fertilizer and control of grassy weeds.

2. Two new spring yellow pea varieties released. Yellow peas have a wide range of uses including human food, animal feed, and recently, as a source of plant-based protein. An ARS researcher at Pullman, Washington, developed and released two new spring-sown yellow peas, “USDA-Kite” and “USDA-Peregrine”. These are both yellow peas and produce yields that are typically at least 10% greater than popular commercial yellow pea varieties. "USDA Kite" and "USDA Peregrine" will be primarily grown in the United States Northern Plains, especially in Montana and North Dakota, where they will provide greater yield potential to growers.

Review Publications
Wang, M., Van Vleet, S., McGee, R.J., Paulitz, T.C., Porter, L.D., Schroeder, K., Vandemark, G.J., Chen, W. 2021. Chickpea seed rot and damping-off caused by metalaxyl-resistant Pythium ultimum and its management with ethaboxam. Plant Disease. 105(6):1728-1737. https://doi.org/10.1094/PDIS-08-20-1659-RE.
McGinley, J., Fitzgerald, V., Neil, E., Omerigic, H., Heuberger, A., Weir, T., McGee, R.J., Vandemark, G.J. 2020. Pulse crop effects on gut microbial populations, intestinal function, and adiposity in a mouse model of dietary induced obesity. Nutrients. 12(3). Article 593. https://doi.org/10.3390/nu12030593.
Rajendran, K., Coyne, C.J., Zheng, P., Saha, G., Main, D., Amin, N., Ma, Y., Kisha, T.J., Bett, K., Kumar Agrawal, S., McGee, R.J. 2021. Genetic diversity and GWAS of agronomic traits using an ICARDA lentil (Lens culinaris Medik.) Reference Plus collection. Plant Genetic Resources. 1-10. https://doi.org/10.1017/S147926212100006X.
Vann, R., Reberg-Horton, S., Castillo, M., Murphy, J., Mirsky, S.B., Saha, U., McGee, R.J. 2021. Differences among eighteen winter pea genotypes for forage and cover crop use in the southeastern United States. Crop Science. 61(2):947-965. https://doi.org/10.1002/csc2.20355.
Zhang, C., Craine, W., Quiros, J., McGee, R.J., Vandemark, G.J., Davis, J., Brown, J., Hulbert, S., Sankaran, S. 2020. Imaged-based phenotyping of flowering intensity in cool-season crops. Sensors. 20(5). Article 1450. https://doi.org/10.3390/s20051450.
Wright, D., Neupane, S., Heidecker, T., Haile, T., Coyne, C.J., McGee, R.J., Udupa, S., Henkrar, F., Barilli, E., Rubiales, D., Gioia, T., Mehra, R., Sarker, A., Dhakal, R., Anwar, B., Sarker, D., Vandenberg, A., Bett, K.E. 2020. Understanding photothermal interactions will help expand production range and increase genetic diversity of lentil (Lens culinaris Medik.). Plants, People, Planet. 3(2):171-181. https://doi.org/10.1002/ppp3.10158.
Gali, K.K., Sackville, A., Tafesse, E.G., Lachagari, R.V., McPhee, K., Hybl, M., Mikic, A., Smykal, P., McGee, R.J., Bustin, J., Domoney, C., Ellis, T.N., Warkentin, T.D. 2019. Association mapping for agronomic and seed quality traits of field pea (Pisum sativum, L). Frontiers in Plant Science. 10. Article 1538. https://doi.org/10.3389/fpls.2019.01538.
Kim, W., Chen, W. 2019. Phytotoxic metabolites produced by legume-associated Ascochyta and its related genera in the Dothideomycetes. Toxins. 11(11). Article 627. https://doi.org/10.3390/toxins11110627.