Project : USDA ARS

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

Location: Grain Legume Genetics Physiology Research

2023 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
This is the final report for project 2090-21000-034-000D, "Improving Genetic Resources and Disease Management for Cool Season Food Legumes Enhanced Agronomic Performance and Disease Resistance in Edible Legumes", which has been replaced by new project 2090-21000-038-000D, "Enhancing Agronomic Performance and Nutritional Qualities of Pulse Crops". For additional information, please review the new project report. ARS researchers in Pullman, Washington, made considerable progress 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 is focused on developing and releasing improved cultivars and germplasm of dry pea, lentil, and chickpea. These three crops are integral components of dryland agricultural production systems in the U.S. Pacific Northwest and Northern Plains. Development and release of three food grade winter pea cultivars was a very significant achievement of this project. These cultivars include two winter green peas, “USDA-Dint” and “USDA-MiCa” and a winter yellow pea, “USDA-Klondike”. These new cultivars provide dryland producers with an entirely new crop option, fall-sown, winter hardy peas with the same food quality traits as commercial spring-sown pea cultivars. Two new spring-sown yellow peas, “USDA-Kite” and “USDA-Peregrine” were also developed and released. USDA-Kite and USDA-Peregrine were released based on their superior disease resistance, yield, and quality. These two cultivars are primarily grown in Montana and North Dakota, where most U.S. yellow peas are produced. The small green lentil “USDA-Sage”, which is also grown primarily in Montana, was developed and released. The chickpea cultivar “USDA-Quinn” was also developed and released. USDA-Quinn was released based on its high yield and large seed size. USDA Quinn is primarily grown in Idaho and Washington, where most U.S. chickpeas are produced. Commercial licenses have been developed for winter yellow pea USDA-Klondike and chickpea USDA-Quinn. Genetic technologies to enable plant breeding were developed as a component of Objective 1. ARS researchers in Pullman, Washington, conducted research with a team of scientists from several countries to sequence the pea genome. The genome consists of approximately 4.3 billion “base pairs”, which is more than 30% larger than the human genome. More than 44,000 genes were identified, and possible functions were determined for approximately 33,000 genes. This genome sequence provides breeders with knowledge and tools to develop new pea varieties with broad portfolios of desirable traits. The researchers also identified genetic markers associated with resistance in chickpea to Pythium ultimum, a disease of emerging importance in the United States. This research provides tools to help breeders develop more disease resistant varieties. Determining how genetic and environmental factors influence nutritional traits in pulse crops was also a component of Objective 1. Pre-biotic carbohydrates are associated with "gut" health and other health benefits including reduced incidence of type II diabetes and coronary artery disease. Prebiotic carbohydrate concentrations were determined for elite USDA chickpea breeding lines and cultivars over two years at three locations in Washington and Idaho. Genetic effects were significant for several compounds including fructose, sucrose, and raffinose. Similarly, genetic effects were significant for seed protein concentrations of USDA breeding lines and cultivars. The results suggest concentrations of pre-biotic carbohydrates and total protein can be improved through breeding. A primary reason for the long-term success of pulses is they can be colonized by beneficial soil bacteria that convert atmospheric nitrogen into a form that can used as fertilizer by plants. As a component of Objective 1, ARS researchers collected nitrogen- fixing bacteria (rhizobacteria) from roots of peas and chickpeas and used DNA sequences to estimate how much genetic variation was present in the collection. The researchers found that strains collected from chickpea were much more diverse than strains from pea. This is likely due to the more recent history of chickpea production in the U.S. Pacific Northwest, which began in the 1980s, as compared to pea production beginning in the 1910s. The strains we isolated from pea likely represent strains that have closely evolved over time to successfully colonize pea roots in growing conditions in the Pacific Northwest. Objective 2 of this research focused on increasing understanding of physiological mechanisms and genes involved in host resistance to plant diseases and the ability of plant pathogens to infect and cause disease in pulse crops. For more than 30 years the fungicide metalaxyl was used to control seed rot of chickpeas caused by the soilborne pathogen Pythium ultimum. However, in 2014 metalaxyl-resistant populations of Pythium were first discovered in the U.S. Pacific Northwest and have become a major disease problem for chickpea production. ARS researchers confirmed that a new fungicide, ethaboxam, can effectively manage metalaxyl-resistant Pythium. Because of this discovery many chickpea growers are using this new fungicide to manage disease. More than 250 chickpea lines were screened for resistance to seed rot caused by metalaxyl-resistant isolates of Pythium ultimum. As well, ARS researchers identified more than 100 resistant lines, but the great majority of these were "desi" chickpeas, which are smaller and darker than "kabuli" chickpeas that are almost exclusively grown in the United States. Fortunately, they identified nine disease resistant kabuli lines. The researchers are now using these lines as parents in crosses to develop new chickpea varieties with improved disease resistance. A major focus of Objective 2 of this research is to identify genes responsible for ability of plant pathogens to cause disease. Sclerotinia sclerotiorum is a fungal plant pathogen that 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. ARS researchers “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. These results indicate the knocked-out genes played a role in disease development. All fungal pathogens produce enzymes called “polygalacturonases” (PGs) to degrade plant cell walls during infection. Conversely, as part of the “arms race” between plants and pathogens, plants produce enzymes called “polygalacturonase inhibiting proteins” (PGIPs) that inhibit fungal PGs to restrict disease development. ARS researchers discovered, for the first time in any plant pathogen, another protein called SsPINE1 that is produced by Sclerotinia and inactivates a plant PGIP. Sclerotinia mutants with a defective SsPINE1 gene caused less disease than isolates of the pathogen with the normal gene. These results help explain why Sclerotinia can cause disease across such a wide range of crops and suggests approaches breeders can use to develop disease resistant crops. This is the first example of a new class of fungal effectors that can inactivate plant defense mechanisms. This discovery has stimulated new approaches for improving disease resistance by identifying plant PGIPs that cannot be inactivated by fungal effector proteins. Collectively, these advances in understanding physiological and genetic factors associated with the development of white mold disease caused by Sclerotinia provide breeders with new strategies for improving disease resistance across many of the most globally important crops, including cereal grains, oilseed crops, and pulse crops.

Accomplishments
1. More nutritious chickpea lines identified along with genes associated with fatty acid concentrations. In general, over consumption of saturated fatty acids is associated with health risks including Type II diabetes and coronary artery disease (CAD), while consumption of unsaturated fatty acids is associated with health benefits. Chickpea is known to be a rich source of protein and dietary minerals, but little was known about the concentrations of different fatty acids in chickpea or how these traits were inherited. ARS researchers in Pullman, Washington, in cooperation with scientists at Clemson University in Clemson, South Carolina, evaluated concentrations of oleic acid, linoleic acid, alpha linolenic acid, and palmitic acid in a panel of diverse chickpea lines and commercial varieties. Lines were identified that provided up to 46% and 72% of the recommended daily allowances (RDA) for the unsaturated fatty acids linolenic acid and alpha-linoleic acid, respectively. Genetic markers were identified that were associated with concentrations of palmitic acid and several genes were identified that may be involved in regulating concentrations of fatty acids in chickpea. Palmitic acid is a saturated fatty acid and consequently, breeding efforts are focused on reducing concentrations of palmitic acid. Breeding lines with high concentrations of unsaturated fatty acids and low concentration of palmitic acid identified in this study are being used by collaborators to develop new chickpea varieties with more nutritious profiles of fatty acids that will promote improved health.

U.S. DEPARTMENT OF AGRICULTURE

Grain Legume Genetics Physiology Research: Pullman, WA