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ARS Home » Midwest Area » St. Paul, Minnesota » Plant Science Research » Research » Research Project #434150

Research Project: Enhanced Alfalfa Germplasm and Genomic Resources for Yield, Quality, and Environmental Protection

Location: Plant Science Research

2019 Annual Report

Objective 1: Reduce yield losses and stand decline in alfalfa from biotic and abiotic stresses. Objective 2: Increase value of alfalfa and rotational crops by developing new products.

Subobjective 1a: Determine the location of QTL for resistance to Aphanomyces root rot. A combination of genotyping by sequencing and interval mapping will be used to identify the chromosomal locations of resistance genes. Crosses will be made with plants segregating for single resistance genes to develop differential lines to identify specific pathogen races. Subobjective 1b: Evaluate sensitivity of seed rot, damping-off, and root rot pathogens to fungicides and biological agents. Test efficacy of fungicides and biologicals when used as seed treatments. Measure disease resistance in experimental germplasm that has undergone selection for resistance to Pythium species causing seed rot and damping off. Subobjective 1c: Evaluate resistance of alfalfa plants expressing defensin peptides to crown rot pathogens. Plants expressing defensins will be identified by quantitative RT-PCR and Western blotting. Disease resistance will be measured using detached leaf assays and whole plant inoculations. Populations for field-testing will be developed by crossing the most resistant plants to adapted germplasm. Subobjective 1d: Measure resistance in alfalfa germplasm to diverse strains of Pseudomonas syringae, the pathogen causing bacterial stem blight (BSB) of alfalfa. Tag bacterial strains with GFP to facilitate tracking plant invasion and measuring bacterial growth. Investigate the effect of glyphosate treatment on gene expression, disease resistance, and cold tolerance. Subobjective 1e: Test mutated plants developed using genome editing for deletions in a susceptibility gene. Evaluate resistance to biotrophic and necrotrophic pathogens using detached leaf assays and whole plant inoculations. Cross mutants to track inheritance of gene mutations. Subobjective 2a: Determine yield and composition of alfalfa leaf protein extracts purified using different methods from biomass and conventional alfalfa. Proteins will be extracted with heat, cold, and pH treatments from juice of leaf and total herbage. Total protein, amino acids, lipids, fiber and carbohydrate content will be measured. Subobjective 2b: Evaluate the economic and health benefits of alfalfa leaf protein concentrate in aquaculture feeds. Feeding trials will be carried out with yellow perch and rainbow trout in which alfalfa leaf protein replaces fishmeal in the diets.

Progress Report
This is a bridging project that was initiated March 2018. Significant progress was made under both objectives. In support of Subobjective 1a, seed was produced to develop new lines of alfalfa with resistance to a single strain of the pathogen causing Aphanomyces root rot. A manuscript describing the research is in progress. In support of Subobjective 1b, nine commercial fungicides were tested for activity against multiple strains of pathogens causing seed rot and damping off of alfalfa. One fungicide was active against all alfalfa seed rot and damping off pathogens tested: four species of Pythium, four strains of Aphanomyces euteiches, three strains of Phytophthora medicaginis, and three species of Fusarium. Another was active against all pathogens except Fusarium species. When used as a seed treatment, these two fungicides were similar in activity to the standard treatment in assays with infested soil. Biological seed treatments were not effective against seed rot and damping off pathogens. A single cycle of selection for resistance to one strain of Pythium irregulare resulted in a significant increase in resistance to multiple strains and species of Pythium. A second cycle of selection using three strains generally did not improve the percentage of resistant plants. The results of this project provide alfalfa growers and plant breeders with new tools to reduce damage from this disease and increase forage yield and stand life. A manuscript and germplasm release are in preparation. In support of Subobjective 1c, a bi-domain defensin from Medicago truncatula displayed high activity against both bacterial and fungal crown rot pathogens in vitro. This defensin had IC50 values against Pseudomonas syringae pv. syringae of 0.198 uM and 1.50 uM against Phoma medicaginis. Agrobacterium-mediated transformation was used to create transgenic lines of alfalfa constitutively expressing the defensin. Transgene expression was confirmed by qRT-PCR and by Western blots. Disease bioassays demonstrated increased resistance in the transgenic lines expressing the defensin against fungal crown rot pathogens, especially against P. medicaginis. Transgenic lines with greater levels of expression corresponded to increased fungal resistance. A manuscript reporting this research has been prepared and crossing to adapted germplasm is in progress. In support of Subobjective 1d, we evaluated 1500 samples from California, Utah, Oregon, Minnesota and Ohio for bacterial stem blight. The pathogen was found in all states causing disease symptoms. Bactericides were found to reduce symptoms when applied on a 10-day schedule. A second pathogen, Pseudomonas viridiflava, was discovered to be associated with diseased samples at a high frequency and shown to cause bacterial stem blight symptoms. A PCR assay was developed to quickly distinguish between P. syringae pv. syringae and P. viridiflava. A manuscript describing the first report of P. viridiflava causing bacterial stem blight on alfalfa was submitted. Phylogenies constructed using rep-PCR and multilocus sequence analysis found that the P. syringae pv. syringae strains do not cluster by location and are genetically similar, suggesting that the population is widespread and has been established on alfalfa plants in the region for a long period of time. All isolates were highly aggressive on alfalfa indicating that this is a well-adapted pathogen that is genetically separated from other types of P. syringae pv. syringae causing disease on other crops. Methods were developed to quantify P. syringae pv. syringae cells to compare pathogen density with disease symptoms. Sensitivity of detection by quantitative PCR was 2 cells and 5 ng of pathogen genomic DNA. New inoculation methods were tested and results showed resistant alfalfa plants selected previously with one strain were resistant to multiple recent strains from California. A manuscript reporting this information is in preparation. Under Subobjective 1e, 237 transgenic alfalfa plants were tested for possible mutations in three targets within a disease susceptibility gene. A total of 72 plants with potential mutations were identified. In support of Subobjective 2a, experiments were completed to measure yield and composition of alfalfa protein concentrate (APC). Acid based precipitation methods resulted in the largest recovery of APC, while heating the foliage extract produced the highest concentration of protein and limiting amino acids in the product. Extraction of APC from reduced-lignin varieties did not result in higher protein extraction compared to conventional varieties. Yields of APC on a dry matter basis were similar for total forage and stripped leaves only. The implication is that APC does not need to be produced from lower yielding immature foliage but can be produced from standard varieties harvested at later maturities to maximize yields. In support of Subobjective 2b, experiments were completed to measure the growth and feed efficiency response of yellow perch fed a diet with APC replacing 100% of the fishmeal, and to evaluate the use of APC as an additive to support efficient growth of rainbow trout with APC replacing 3% and 6% of the fishmeal. Results showed that yellow perch reared for 14-16 weeks accepted the APC diet but gained weight at a slightly lower specific growth rate and had an elevated feed conversion ratio than fish on the control diet. The trout diets supplemented with APC were also accepted well and no growth or efficiency differences were detected. This research indicates that APC is a viable feed protein or additive for both yellow perch and rainbow trout; however, further evaluation is needed to find the appropriate levels to optimize benefits. Manuscripts reporting these results have been prepared for submission.

1. Alternative fungicide for protection of alfalfa seeds identified. Seed rot and damping off of alfalfa is a soilborne disease caused by multiple pathogens that results in thin initial stands of alfalfa plants, decreases forage yields, and reduces survival of plants over the winter. Almost all alfalfa seed is treated with mefanoxam but this fungicide is not active against the pathogen causing Aphanomyces root rot, a lethal widespread disease of alfalfa, nor against fungal seed rot pathogens. ARS researchers at Saint Paul, Minnesota, tested eight alternative fungicides for activity against alfalfa pathogens. A commercial fungicide containing a mixture of metalaxyl, prothioconazole, and penflufen reduced growth of all alfalfa seed rot and damping off pathogens at a low concentration, including the pathogens not controlled by mefanoxam. Seeds treated with mefanoxam or the fungicide mixture that were planted in soil infested with damping off pathogens were protected from disease. This research provides alfalfa growers with a new tool to reduce damage from Aphanomyces root rot and damping off pathogens of alfalfa.

2. Emerging bacterial disease of alfalfa increases frost damage and reduces forage yields. Bacterial stem blight of alfalfa, once considered to be a minor disease, has been identified as a major cause of forage yield loss in the spring in the Intermountain West. In 2019, ARS researchers at Saint Paul, Minnesota, and collaborators at the University of California and Utah State University identified the disease in California, Utah, Oregon, Minnesota, and Ohio. Two bacteria, Pseudomonas syringae and P. viridiflava, were associated with disease symptoms in all locations. Frost damage to plants was associated with high populations of P. syringae, which facilitates frost formation on plants. Bactericide treatments were effective in reducing bacterial populations but did not always reduce plant damage. Disease resistant plant populations were developed and may provide greater protection from the disease than chemical controls. This research should improve yields in frost-prone areas, which represent a significant percentage of the alfalfa acreage nationwide.

Review Publications
Sathoff, A.E., Velivelli, S., Shah, D.M., Samac, D.A. 2019. Plant defensin peptides have antifungal and antibacterial activity against human and plant pathogens. Phytopathology. 109:402-408.
Schmitt, A., Sathoff, A., Holl, C., Bauer, B., Samac, D.A., Carter, C.J. 2018. The major nectar protein of Brassica rapa is a non-specific lipid transfer protein with strong antifungal activity. Journal of Experimental Botany. 69(22):5587-5597.
Bonhomme, M., Navier, H., Hajri, A., Badis, Y., Miteul, H., Fariello, M., Samac, D.A., Dumas, B., Baranger, A., Jacquet, C., Pilet-Nayel, M. 2019. GWAS powered by a local score approach unravels the genomic bases of Medicago truncatula quantitative disease resistance to multiple Aphanomyces euteiches strains. Heredity. 2019(5):1-15.
Castell-Miller, C.V., Samac, D.A. 2019. Sensitivity of Bipolaris oryzae isolates pathogenic on cultivated wild rice to the quinone outside inhibitor azoxystrobin. Plant Disease. 103:1910-1917.
Sathoff, A.E., Samac, D.A. 2019. Antibacterial activity of plant defensins. Molecular Plant-Microbe Interactions. 32(5):507-514.
Castle, S.C., Samac, D.A., Sadowsky, M.J., Rosen, C.J., Guknecht, J.L., Kinkel, L.L. 2019. Impacts of sampling design on estimates of microbial community diversity and composition in agricultural soils. Microbial Ecology. 78:1-11.