Submitted to: Phytopathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/15/2010
Publication Date: 8/3/2010
Citation: Maruthachalam, K., Atallah, Z.K., Vallad, G.E., Klosterman, S.J., Hayes, R.J., Davis, M.R., Subbarao, K. 2010. Molecular variation among isolates of verticillium dahliae and PCR-based differentiation of races. Phytopathology. 11:1222-30. Interpretive Summary: The soilborne fungal pathogen Verticillium dahliae can survive in the soil for years and infect plants through their roots. The pathogen invades the vascular tissue and causes plant wilting. V. dahliae causes a disease on many vegetable crops and ornamental plants in California and worldwide. It is also established that there are two pathogenic types of this fungus on tomato and lettuce, and these are known as race 1 and race 2. In lettuce and tomato, no plants have been found with resistance to race 2 of the pathogen. Therefore it is important to understand the genetically variability in the pathogen as well as that of the host plants which this fungus infects. In this study, genetic variability of the soilborne fungal pathogen V. dahliae from different plant sources was characterized based on molecular analyses of the DNA. The isolates of the fungus were primarily collected from tomato and lettuce, but also from other plants, in central and coastal California. The results indicate high level of genetic variability between isolates of V. dahliae. However, the tomato isolates of the fungus were genetically distinct from marigold isolates, while cotton and olive isolates appeared more closely related to tomato isolates. In addition a DNA primer set was used to amplify a race-1 specific DNA product, based on previously published work. Virulence assays were conducted in greenhouses to validate the race-specificity of this primer pair. The results indicate that this primer set can be used to distinguish race 1 from race 2 of the pathogen and therefore provide additional tool for the breeding of plant resistance to the pathogen.
Technical Abstract: Verticillium dahliae is a soilborne fungal pathogen that causes Verticillium wilt on a variety of economically important crops worldwide. Although race 1-specific resistance has been identified in both tomato and lettuce, no such resistance is available against race 2 of the pathogen. Knowledge of the variability of the pathogen at the field level and rapid differentiation of the two races of V. dahliae pathogenic on tomato and lettuce is critical for our related aim of breeding host resistance. In this study, eighty-two isolates were collected from different hosts mainly in central and coastal California and assessed for DNA polymorphisms in the internal transcribed spacers (ITS), intergenic spacer region (IGS), and microsatellite (Simple Sequences Repeats, SSRs) sequences. High levels of heterogeneity were observed in the IGS sequences among the isolates of V. dahliae. Analyses of the 22 SSR markers illustrated that tomato populations were distinct relative to the marigold population. In contrast, cotton and olive isolates showed admixture with tomato isolates. Because all other polymorphic markers were unable to delineate races of V. dahliae, a PCR approach using published sequences was applied to amplify a race 1-specific amplicon. The use of this primer set in a PCR assay was validated on isolates from many hosts, including those isolates from tomato and lettuce with known or unknown virulence phenotypes.