ISSR Analysis Of Verticillium Wilt Pathogen Preserved In Ineral Oil At Room Temperature

Verticillium wilt is an important soilborne disease on cotton. Since 1950's, our lab had collected almost 2000 strains of Verticillium dahliae, Fusarium oxysporium f.sp. vasinfectum, and Verticillium nigrescens, etc. from different areas of China, and preserved these strains in mineral oil at room temperature. Previous results indicated that survival percentage of V. dahliae and V. nigrescens in mineral oil at room temperature was 55.9%. Two strains of V. dahliae are still alive after being preserved for 52 years. In this thesis, the biologcial characteristics, pathogenicity and ISSR molercular marker were tested, and compared between strains preserved in mineral oil at room temperature and new isolates. The main results were shown as following.1. Single-spore isolates of 45 V. dahliae and V. nigrescens were abtained by using the gradient dilution and plate paint isolation methods. Total DNA of each single-spore isolates were extracted by improved SDS method in large scale.2. Biological parameters, including colony diameter, sporulation, pathogenicity, were compared between strains preserved in mineral oil at room temperature and new isolates. It showed that there was no difference in colony diameter and sporulation between them, while the pathogenicity of strains preserved was weaker than that of the new isolates. And pathogenicity does not relate to colony diameter and sporulation. So, it was not enough to evaluate the viability of pathogen with colony diameter and sporulation.3. Forty-five strains were analized using ISSR-PCR technique. The result showed that these strains represented rich molecular marker polymorphism. The phylogenetic tree of SAHN was constructed through UPGMA method based on ISSR-PCR data. These strains could be divided into three groups which corresponded to V.dahliae with weak pathogenicity, V.dahliae with strong pathogenicity and V. nigrescens.There is some kind of correlation between the polymorphism of ISSR and pathogenicity, but no relationship between ISSR polymorphism and preserved years of the strains.