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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

2020 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
In support of Objective 1a, a backcross of a highly resistant F1 plant to the susceptible parent was completed and seeds collected. New biparental crosses and seed collected for an experiment to determine if differential populations with resistance to a single Aphanomyces isolate could be developed. The original biparental population was tested for resistance to two races of anthracnose for mapping resistance genes. In support of Objective 1b, five field experiments were established in sites with high soilborne seedling disease pressure for testing five fungicide seed treatments. In support of Objective 1c, transgenic plants with the highest defensin expression were tested for Fusarium and bacterial wilt resistance. In support of Objective 1d, a second experiment testing bactericides for reducing disease was carried out. Additional pathogen isolates were collected from 2019 samples and genes amplified for phylogenetic analysis. In support of Objective 1e, plants with edits in all four target sites were inoculated with Phytophthora medicaginis and root collected for transcript analysis. In support of Objective 2, 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 RNA-Seq and iso-Seq. 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. In surveys for alfalfa diseases, two newly identified root rots, Mycoleptodiscus root rot and Paraphoma root rot were identified in commercial production fields. 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 in April 2020 for this effort. 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.

1. Alfalfa leaf protein concentrate is a sustainable alternative to fish meal for aquaculture diets. Aquaculture, the production of fish and shellfish in controlled conditions, is the fastest growing food sector worldwide but fishmeal used in diets of most aquaculture species is a limited resource with volatile pricing. ARS scientists at Saint Paul, Minnesota, and collaborators from the University of Minnesota pressed fresh alfalfa foliage to produce a juice and concentrated proteins by various methods. A heat treatment resulted in the highest protein concentration with the most favorable amino acid profile for aquaculture feeds that lacks the antinutritional factors found in seed meals. Approximately 800 pounds of protein concentrate can be produced from an acre of alfalfa and the press residue can be used as a feed ingredient for cattle or as a bioenergy feed stock. Feeding studies with yellow perch and rainbow trout found that alfalfa protein concentrate in feeds was accepted by both species. Although further evaluation is needed to optimize alfalfa protein concentrate in aquaculture feeds, this research indicates that alfalfa protein concentrate is a viable alternative to fishmeal in aquaculture diets and could be adopted by alfalfa processors and the aquaculture industry as a sustainable alternative to fish meal.

2. Carbon export measured in cropping systems supporting dairy production. The U.S. dairy industry is increasingly reliant on corn silage as a primary forage source. Across the Upper Midwestern United States, this has contributed to a loss of nearly two million acres of alfalfa since 1980. This change may impact carbon balances and soil organic carbon / organic matter levels on forage-cropped acres. ARS researchers at Saint Paul, Minnesota, evaluated carbon dioxide flux, harvest removals of carbon, and manure additions of carbon from two 160-acre forage production fields and one grain production field for nine years. Long-term carbon losses were over threefold greater from silage corn-based forage cropping systems than from the predominant grain cropping system in the region, the soybean-corn rotation, even when alfalfa was included in rotation and liquid dairy manure was field-applied. These results show substantial carbon export from fields in silage corn and imply a substantial reduction in soil carbon / soil organic matter on forage-cropped acres in the Upper Midwest. This information is useful to producers and regulators for developing strategies to enhance soil organic carbon retention in cropping systems.

Review Publications
Campbell, B.W., Hoyle, J.W., Bucciarelli, B., Stec, A.O., Samac, D.A., Parrott, W.A., Stupar, R.M. 2019. Functional analysis and development of a CRISPR/Cas9 allelic series for a CPR5 ortholog necessary for proper growth of soybean trichomes. Scientific Reports. 9:14757.
Lipps, S.M., Lenz, P., Samac, D.A. 2019. First report of bacterial stem blight of alfalfa caused by Pseudomonas viridiflava in California and Utah. Plant Disease. 103(12):3274.
Jungers, J.M., Kaiser, D.E., Lamb, J.F., Lamb, J.A., Noland, R., Samac, D.A., Wells, M.S., Sheaffer, C.C. 2019. Potassium fertilization affects alfalfa forage yield, nutritive value, root traits, and persistence. Agronomy Journal. 111(6):2843-2852.
Dundore-Arias, J.P., Eloe-Fadrosh, E., Schriml, L.M., Beattie, G.A., Brennan, F.P., Busby, P.E., Calderon, R.B., Castle, S.C., Emerson, J.B., Everhart, S.E., Eversole, K., Frost, K.E., Herr, J.R., Huerta, A.I., Iyer-Pascuzzi, A.S., Kalil, A.K., Leach, J.E., Leonard, J., Maul, J.E., Prithiviraj, B., Potrykus, M., Redekar, N.R., Rojas, J.A., Silverstein, K.T., Tomso, D.J., Tringe, S.G., Vinatzer, B.A., Kinkel, L.L. 2020. Community-driven metadata standards for agricultural microbiome research. Phytobiomes Journal. 4:115-121.
Sathoff, A.E., Lewenza, S., Samac, D.A. 2020. Plant defensin antibacterial mode of action against Pseudomonas species. BMC Microbiology. 20:173.
Sathoff, A.E., Dornbusch, M.R., Miller, S.S., Samac, D.A. 2020. Functional analysis of Medicago-derived pathogen-induced gene promoters for usage in transgenic alfalfa. Molecular Breeding. 40(66):1-12.