Location: Grain Legume Genetics Physiology Research
2019 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 U.S. This research focuses on variety development and control of diseases impacting these crops throughout the U.S. Considerable progress was made on both objectives, which fall under National Program 301.
Sub-objective 1A focuses on the development of improved germplasm and varieties of peas, lentils, and chickpeas. We conducted yield trials of ARS breeding lines in several states including Washington, Idaho, Montana, North Dakota, and Nebraska. Pea, lentil, and chickpea breeding lines and varieties harvested in 2018 have been evaluated for several nutritional quality traits including concentrations of protein, iron and zinc, and pre-biotic carbohydrates, which are sugars and sugar-like compounds associated with ‘gut’ health and other health benefits including reduced incidence of type II diabetes and coronary artery disease. We found that concentrations of several pre-biotic carbohydrates, including sucrose, mannitol, and raffinose, were significantly affected by genetic differences between breeding lines, which suggests we can improve these nutritional quality traits through breeding. In 2019 we produced seed of more than 400 chickpea lines that were developed from crosses made to increase protein concentration. We are beginning to characterize the protein content of these lines, which will largely determine which lines are advanced to field trials in 2020.
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 or ‘fix’ atmospheric nitrogen into a form that can 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 2A focuses on improving the ability of pulse crops to fix nitrogen through associations with beneficial rhizobacteria. This year we completed a multi-year project during which we collected nitrogen- fixing bacteria (rhizobacteria) from roots of peas and chickpeas, isolated DNA from more than 100 bacterial strains collected, determined for each strain the DNA sequence of five different genes, and used the DNA sequences to estimate how much genetic variation was present in the collection. We 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 the early production of peas in this region beginning in the 1910s. Most pea growers rely on naturally occurring soil bacteria to fix nitrogen in roots while most chickpea growers still apply a commercial inoculant of nitrogen-fixing bacteria to seed before planting. The strains we isolated from pea likely represent strains that have closely evolved over time to successfully colonize pea roots in the growing conditions prevalent in the Pacific Northwest. This year we used the knowledge obtained by examining DNA sequences to select three strains of the pea bacteria, Rhizobium leguminosarum, for use in studies where we will test the ability of different bacteria strains to colonize pea roots and fix nitrogen.
Progress on Sub-objective 2A focused on improving the understanding of fungicide resistance in pathogens that cause diseases of 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. This year we compared metalaxyl- resistant and metalaxyl-susceptible isolates for growth traits. We found that the resistant isolates could grow as fast as susceptible isolates, which suggests there is no ‘cost’ associated with being resistant to the fungicide. This indicates that metaxyl-resistant populations will persist in the soil for many years and active management is required. This year we 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.
Sub-objective 2B focused on identifying sources of disease resistance in pulse crops. This year we screened more than 250 chickpea lines for resistance to seed rot caused by metalaxyl-resistant isolates of Pythium ultimum. We 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 U.S. Fortunately, we did identify 9 kabuli lines that were disease resistant. We are using these lines now as parents in crosses to develop new chickpea varieties with improved disease resistance.
Progress on Sub-objective 2C included 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. All fungal pathogens produce enzymes called ‘polygalacturonases’ (PGs) to degrade plant cell walls during infection. Conversely, as part of the ‘arms race’ all plants studied so far have evolved to produce their own enzymes, called ‘polygalacturonase inhibiting proteins’ (PGIPs), which inhibit the fungal PGs to avoid or restrict diseases. This year we discovered, for the first time in any plant pathogen, another protein produced by
S. sclerotiorum that serves to inactivate a plant PGIP. These results help explain why S. sclerotiorum is so efficient at degrading plant cell walls and can cause disease in such a wide range of hosts and suggests approaches breeders can use to develop disease resistant crops.
Accomplishments
1. Sequencing the pea genome. Peas are integral components of dryland agriculture systems throughout the U.S. and have served as globally important sources of protein, fiber, and minerals for millennia. Peas were also the crop studied by Gregor Mendel when he developed fundamental laws of inheritance over 150 years ago, but contemporary progress in pea breeding has been impeded because the ‘genome’, or DNA sequence arrangement, of pea is both large and poorly understood. ARS researchers at Pullman, Washington, along with a team of scientists from several other countries, determined the DNA sequence of the pea genome. The genome consists of approximately 4.3 billion ‘base pairs’, which is more than 30 percent larger than the human genome. More than 44,000 genes were identified, and possible functions were determined for approximately 33,000 genes. This provides breeders with knowledge and tools to more efficiently develop new pea varieties with broad portfolios of desirable traits including improved yield, disease resistance, and nutritional quality.
Review Publications
Pinhasi Van-Oss, R., Gopher, A., Kerem, Z., Sherman, A., Zhang, H., Coyne, C.J., Vandemark, G.J., Reamy, O., Abbo, S. 2018. Independent selection for seed free tryptophan content and vernalization response in chickpea domestication. Plant Breeding. 137(3):290-300. https://doi.org/10.1111/pbr.12598.
Mugabe, D., Coyne, C.J., Piakowski, J., Zheng, P., Ma, Y., McGee, R.J., Main, D., Vandemark, G.J., Zhang, H., Abbo, S. 2018. Quantitative trait loci for cold tolerance in chickpea. Crop Science. 59(2):573–582. https://doi.org/10.2135/cropsci2018.08.0504.
Vann, R., Reberg-Horton, C., Castillo, M., Mirsky, S.B., Mcgee, R.J. 2019. Winter pea, crimson clover, and hairy vetch planted in mixture with small grains in the Southeast USA. Agronomy Journal. 111:805-815.
Ma, Y., Coyne, C.J., Main, D., Pavan, S., Sun, S., Zhu, Z., Zong, X., Leitao, J., McGee, R.J. 2017. Development and validation of breeder-friendly KASPar markers for er1, a powdery mildew resistance gene in pea (Pisum sativum L.). Molecular Breeding. 37:151. https://doi.org/10.1007/s11032-017-0740-7.
