<ol> <LI>Develop and validate a PCR-based screening method for the rapid differentiation of race 1 and 2 isolates of V. dahliae. <LI>Determine the distribution of races 1 and 2 of V. dahliae in fields of the coastal California production area. 3) Determine the distribution of race 1 and race 2 isolates of V. dahliae in weed samples collected from the Salinas Valley. <LI> Determine the distribution of race 1 and race 2 isolates of V. dahliae in weed samples collected from the Salinas Valley. <LI>Evaluate lettuce germplasm for resistance to race 2 isolates of V. dahliae.
Non-Technical Summary: The goal of this project is to integrate knowledge of Verticillium dahliae populations with host resistance for managing Verticillium wilt of lettuce. There are two lettuce-adapted races but their relative distribution in California is unknown. The project will develop and validate a polymerase chain reaction (PCR)-based method to distinguish race 1 and 2 isolates, determine their relative distribution and conduct screens of lettuce germplasm for resistance to race 2. <P> Approach: Development of a PCR-based screening method using commercially available Taq polymerase and PCR reagents for differentiating race 1 and 2 lettuce isolates will be based on the IGS sequences that were previously cloned and sequenced. Based on the analyses of these sequences (1.8 kb each) from over 70 isolates, three primers will be designed for detection and differential amplification of IGS sequences from race 1 and race 2 isolates. When the three primers are combined in one PCR mix with the DNA template from the isolate in question, PCR amplifications yield a 500 bp DNA product in all isolates and the appearance of an additional 800 bp amplicon in race 2 isolates. The goal of objective 1, to develop and validate a PCR method for differentiating races of V. dahliae, will benefit from and support objectives 2 and 3. That is, additional isolates collected from the Salinas Valley will provide additional samples for validation of the PCR technique. In addition to validating the technique with known race 1 and 2 isolates, implementation of objectives 2 and 3 of the proposed research will allow technique validation using the DNA from novel V. dahliae isolates from lettuce and weeds as well. In this process, we will clone and characterize additional IGS sequences. The results of the PCR analysis, for differentiating race 1 and 2 lettuce isolates, will be validated using greenhouse-based pathogenicity assays with resistant and susceptible plant cultivars.