Evaluation location: California, United States
Verticillium wilt resistance was assessed using greenhouse grown plants inoculated with V. dahliae isolate VdLs17, a highly aggressive race 2 isolate. The isolate was prepared and maintained according to Vallad et al. (2006). Seeds of each cultivar were sown in 200-well plug trays, incubated at 10 degrees C in the dark for 48 hours in a growth chamber, and then germinated at 20 degrees C with a 16-hour photoperiod. Seedlings were inoculated at 2, 3 and 4 weeks after sowing by saturating the soil in each plug tray well with a 3-ml suspension containing 2 x 106 conidia/ml in sterile, distilled water. Seedlings were incubated for another 1 to 2 weeks after the third inoculation and transplanted into 0.5 L foam-insulated cups filled with a pasteurized sand:potting soil mixture (3:1 v/v). All replicated experiments used a randomized complete block design with three blocks and five plants per block per accession. Unreplicated experiments used ten plants per accessions, and were transplanted in alpha-numerical order based on the accession number. Inoculated and non-inoculated plants of the cultivar Salinas 88 were included in each replicated and unreplicated experiment for each population as a susceptible control; inoculated plants of the cultivar La Brillante were included in replicated experiments for each population as a race-1 resistant control. Plants were maintained until flowering, and then evaluated for disease incidence (DI, proportion of symptomatic plants). Plants were uprooted, roots were cleaned of soil and cut longitudinally to evaluate for the presence of root discoloration and foliar symptoms typical of Verticillium wilt. Crown and stem sections (approximately 1 to 2 inches long) of non-symptomatic plants were sampled and plated on semi-selective NP-10 medium (Kabir et al., 2004) to determine the presence or absence of the pathogen. This was conducted only with accessions having less than approximately 20% DI within each experiment. Stems from symptomatic plants, as well as non-symptomatic uninoculated plants were collected and plated as positive and negative controls. Up to six cross sections of each plant stem sample were plated, and identification of V. dahliae from at least one section was interpreted as a positive result (infected) for that plant.
Selection for Verticillium wilt resistance was conducted in two independent populations of accessions (populations A and B). Each population was comprised of randomly selected Plant Introductions (PI) which are part of the Western Regional Plant Introduction Station (WRPIS) in Pullman, WA. The source of the seeds used in these experiments came from a working collection of the WRPIS genebank maintained at the United States Department of Agriculture (USDA), United States Agriculture Research Station in Salinas, CA.