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ARS Home » Midwest Area » St. Paul, Minnesota » Plant Science Research » Research » Research Project #433427

Research Project: Bacterial Stem Blight of Alfalfa: Connection with Frost Damage, Development of Resistant Germplasm, and Mapping Resistance Genes

Location: Plant Science Research

Project Number: 5062-12210-004-024-I
Project Type: Interagency Reimbursable Agreement

Start Date: Sep 1, 2017
End Date: Aug 31, 2022

Objective 1: Measure bacterial populations on alfalfa foliage over the season in relationship with temperature, moisture, age of the stand, and cultivar. Objective 2: Develop germplasm with enhanced resistance to bacterial stem blight. Objective 3: Identify DNA markers and candidate genes associated with disease resistance loci.

To measure bacterial populations, alfalfa stems will be removed from test plots on a weekly basis. Weather data will be recorded throughout the season. Bactericides will be applied at three times during the season for control of the pathogen. Stems will be placed in sterile buffer solution and sonicated for 10 minutes to dislodge bacteria from plant material. Tubes will be immediate placed in a -4.5C cold bath for 15 minutes. Bacterial suspensions from tubes freezing at this temperature will be dilution plated onto sorbitol medium. After 60 hours, bacteria will be tested by replica freezing to identify Pseudomonas syringae and positive colonies collected and stored in 20% glycerol at -80C. Stored bacteria will be tested for pathogenicity on alfalfa and for frequency of ice nucleation active cells under standard conditions. Stored bacteria will be fingerprinted by repPCR to identify clonal strains and measure genetic diversity. To develop disease resistant germplasm, three germplasm sources adapted to a wide range of conditions will be screened for resistance using an inoculum of mixed strains of P. syringae isolated from alfalfa. Strains identified as resistant by foliar inoculation will be retested by stem inoculation. Resistant plants from each source germplasm will be intermated by hand in the greenhouse. Seed will be collected by female parent and mixed in equal proportions. Plants from the mixed seed will be tested for resistance to determine the gain in resistance from selection. A second or third round of selection will be done to achieve >50% resistant plants. To identify markers associated with resistance, crosses will be made between resistant and susceptible plants in two germplasm sources. The resulting F1 populations will be planted and tested for response to the pathogen. DNA will be collected from 150 resistant and 150 susceptible plants and submitted for genotyping by sequencing. Marker-trait association will be done using linkage disequilibrium to identify markers associated with resistance and DNA sequence information will be used to identify candidate resistance genes.