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ARS Home » Midwest Area » St. Paul, Minnesota » Plant Science Research » Research » Research Project #432293

Research Project: Developing a Rapid Standard Test for Verticillium Wilt Resistance in Alfalfa

Location: Plant Science Research

Project Number: 5062-12210-004-018-N
Project Type: Non-Funded Cooperative Agreement

Start Date: May 1, 2017
End Date: Apr 30, 2022

The objective of this project is to develop a faster and more effective test for identifying alfalfa plants with resistance to Verticillium wilt (VW) compared to the standard test currently used to select plants for breeding and to characterize alfalfa cultivars. The goals of this project are to: (1) develop a more rapid and accurate standard test for Verticillium wilt using qPCR and/or wheat germ agglutin-FITC (WGA-FITC) and (2) compare the effectiveness of the conventional and new standard tests for developing disease resistant alfalfa populations.

Seeds of a susceptible check and a highly resistant check will be planted in the greenhouse and vegetative cuttings made from each plant. The vegetative cuttings will be inoculated by root soak and/or stem inoculation methods as per the standard test. At 2, 3, and 4 weeks after inoculation the stems of two clones from each source plant will be harvested and frozen at -20°C for DNA extraction and two clones will be harvested and stored in 1 N KOH for WGA-FITC evaluation. At 4 or 6 weeks after inoculation, at least two clones from each source plant will be scored for VW symptoms on the 1-5 scale. DNA will be extracted from total stem samples and used for the qPCR assay for V. alfalfa. The stem samples in 1 N KOH will be processed for the WGA-FITC assay and the amount of chitin/sample determined. Symptoms of each plant will be compared to qPCR and WGA-FITC results. The experiment will be repeated with additional cultivars using the time point and assays determined to be optimal in the first experiment. Resistant plants identified by the standard test and new test will be retained. If the two tests identify different resistant plants, the plants from each test will be intermated to create new resistant populations. The progeny from the intermating will be tested by the conventional standard test and the new test to determine the percentage of resistant plants in the populations.