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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

2021 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. Objective 3: Establish innovative, science-based methods and standards for assessing and evaluating alfalfa quality for multiple end uses. Objective 4: Characterization and manipulation of the alfalfa/soil interactome to promote the agronomic utility of alfalfa in rotational cropping systems.

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 project terminated in 2020. See the report for the replacement project, 5062-12210-004-00D, “Genetic Improvement and Cropping Systems of Alfalfa for Livestock Utilization, Environmental Protection and Soil Health” for additional information. The terminated project successfully developed strategies to reduce yield losses and stand decline in alfalfa from biotic and abiotic stresses and to increase value of alfalfa and rotational crops by developing new products. Research was initiated to support Objectives 3 and 4 that were added in 2020. In support of Objective 1a, gene mapping was used to identify the chromosomal locations of genes for resistance to Aphanomyces root rot, a major disease of alfalfa in the U.S. Resistance to race 1 strains was identified on the top of chromosome 1, near a cluster of disease resistance genes, and resistance to race 2 was identified at the top of chromosome 2. To identify potential genes for race 2 resistance, a backcross of a highly resistant F1 plant to the susceptible parent was conducted, seeds collected, over 300 plants tested for disease resistance, and DNA isolated. Association of DNA markers with disease resistance is in progress in the replacement project. Although numerous crosses were made among plants with resistance to a single strain, no distinct populations were generated with resistance to a single race 2 strain that could be used for distinguishing new races. In support of Objective 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, Aphanomyces euteiches, 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 had similar activity to the standard treatment in assays with infested soil. Biological seed treatments were not effective against seed rot and damping off pathogens. On-farm field trials with the most effective seed treatments are in progress. 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. Evaluation of field soils with poor seedling establishment identified high concentrations of Pythium species and Aphanomyces euteiches. Seed treatments, resistant germplasm, and soil indexing for pathogens populations will provide alfalfa growers and plant breeders with new tools to reduce damage from this disease and increase forage yield and stand life. In support of Subobjective 1c, a defensin peptide was identified that displayed high activity against both bacterial and fungal crown rot pathogens in vitro. Agrobacterium-mediated transformation was used to create transgenic lines of alfalfa constitutively expressing the defensin. Disease bioassays demonstrated increased resistance in the transgenic lines expressing the defensin against fungal crown rot pathogens. In support of Subobjective 1d, 2,600 alfalfa samples from California, Utah, Oregon, Minnesota and Ohio were processed to detect 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. 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 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 diseases 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. Under Subobjective 1e, 237 transgenic alfalfa plants were tested for possible mutations in three targets within a disease susceptibility gene, DMR6. A total of 72 plants with potential mutations were identified. Plants with edits in all target sites were inoculated with Phytophthora medicaginis. Symptoms were similar to control inoculated plants. In wild type plants DMR6 RNA increases about 50% with plant infection. Taken together, these results suggest that DMR6 does not play a major role in Phytophthora root rot of alfalfa. 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 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 herbage and field separated leaves. A non-lodging variety produced more APC than conventional hay type varieties with fewer harvests. 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. Collaborations were established for extensive testing of feeds with alfalfa protein concentrate using rainbow trout. Alfalfa plants expressing phytase were identified for developing a concentrate to enhance feed digestibility and water quality. In support of Objective 3, research was initiated to increase energy production in alfalfa foliage by transgenic expression of genes for lipid biosynthesis. An increase in lipid droplets in transformed plants was observed indicating that the genes were expressed as predicted. In support of Objective 4, an experiment to measure effects of dairy manure application on productivity of alfalfa and rotational crops, forage quality, and soil health was established at three locations. A corn-alfalfa experimental rotation was initiated in four replicates and three crop phases. Within microplots treatments were applied: 1) Agronomic recommended rate of cattle manure + fertilizer; 2) High rate cattle manure + fertilizer; 3) Agronomic recommended conventional fertilizer; and 4) Control with no nutrient inputs. Soil cores were removed in fall 2019 and spring 2020 and used for DNA extraction and amplicon sequencing for bacterial and fungal communities. A corresponding laboratory incubation experiment was also conducted to evaluate the impacts of manure application on soil microbial community composition and function under more controlled conditions. This experiment incorporated soils from three Minnesota sites and also two additional Dairy Agroecosystem Working Group (DAWG) sites in Idaho and Pennsylvania. New research was established to support development of a reference genome sequence for alfalfa. Genomic DNA of a highly regenerable genotype was used for long-read DNA sequencing. Additionally, RNA was extracted from roots, leaves, stems, nodules, and seed pods and used for profiling gene expression. Biparental crosses were made to develop a genetic map of the sequenced genotype. To support DNA marker development, leaf tissue was provided from 30 genotypes and DNA of 10 genotypes used for long-read DNA sequencing. New research was established to evaluate the impacts of novel forage crop rotations on field-scale carbon dioxide and water flux. Two eddy covariance towers were set up in Rosemount, Minnesota, and extensive soil sampling was conducted. New research was also initiated to model changes in soil organic carbon under alfalfa cropping scenarios in support of a life cycle analysis (LCA) study led by University of Minnesota faculty.


Review Publications
Coburn, J., Wells, M., Phelps, N., Gaylord, T., Samac, D.A. 2021. Acceptance of a protein concentrate from alfalfa (Medicago sativa) by yellow perch (Perca flavescens) fed a formulated diet. Fishes. 6(2). Article 9.
Castell-Miller, C.V., Schlatter, D.C., Samac, D.A. 2021. Efficiency and profitability of fungicides in controlling Bipolaris diseases and enhancing grain yield in cultivated wild rice (Zizania palustris). Crop Protection. 141. Article 105455.
Coburn, J., Wells, M., Sheaffer, C.C., Ruan, R., Samac, D.A. 2021. Evaluation of alfalfa (Medicago sativa L.) foliar protein extracts for use in aquaculture feeds. Agrosystems, Geosciences & Environment. 4(2). Article e20184.