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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

2018 Annual Report


1a. Objectives (from AD-416):
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.


1b. Approach (from AD-416):
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.


3. Progress Report:
This is a new bridging project initiated March 2018. Significant progress has been made under Objective 1 in which commercial fungicides have been obtained from four companies and used in agar plate based assays to determine which products have activity against seed rot and seedling damping off organisms. Also, 45 new isolates of Pseudomonas syringae have been recovered from alfalfa stems showing symptoms of bacterial stem blight. Interestingly, a new pathogen was identified in diseased stems, Pseudomonas viridiflava, previously shown to be involved in alfalfa crown rot. The interaction of the two pathogens in bacterial stem blight is being investigated. Genetic diversity of the two pathogens is being measured using a multi-locus sequence typing approach as well as repetitive PCR fingerprinting. Pathogenic diversity and ability to act as an ice nucleus is also being tested. The green fluorescent protein gene was introduced into five Pseudomonas syringae strains in order to be able to visually follow pathogen invasion in plant tissue. A new vector for making mutations in a disease susceptibility gene was generated and transgenic alfalfa plants have been produced. Under Objective 2, a second yellow perch feeding study and a rainbow trout feeding study were completed. Results are being analyzed and manuscripts are in preparation.


4. Accomplishments


Review Publications
Castle, S.C., Song, Z., Gohl, D.M., Gutknecht, J., Rosen, C., Sadowsky, M.J., Samac, D.A., Kinkel, L.L. 2018. DNA template dilution greatly impacts amplicon-sequencing based estimates of soil fungal diversity. Phytobiomes Journal. 2:100-107. doi: 10.1094/pbiomes-09-17-0037-r.