Studies On Genetic Diversity Of Verticillium Dahliae Of Cotton

Verticillium dahliae is the worldwide soil-borne fungus disease, which causes severe vascular wilt in more than 660 plants and heavy economic losses. In order to explore the differentiation and regularity of V.dahliae and to provide the theoretical testimony for establishing disease control techniques,the pathogenicity, biological cultural character- istics,ISSR molecular analysis,vegetative compatibility and fungus toxin of strains of V.dahliae collected from more than 20 provinces in China were studied and obtained the following research results.1. The pathogenicity of 60 representative strains of V.dahliae collected from more than 20 provinces in China was determined by conidia suspension of strains inoculated to the seedlings of 5 cotton differentiate hosts. The result showed that all strains can be classed into three pathogenic types. PathotypeⅠwith strong pathogenicity included 27 strains which were mainly collected from Shaanxi and Jiangsu provinces and accounted for 45.0% of the total strains tested. PathotypeⅢwith intermediate pathogenicity included 18 strains which were mainly collected from Henan, Hubei and some from Hebei provinces and accounted for 30.0% of the total. PathotypeⅡwith weak pathogenicity included 15 strains accounted for 25.0% of the total. It clearly indicated that there was the differentiation in pathogenticity among strains of V.dahliae tested and strains with strong pathogenicity occupied the dominant position.2. The result of cultural characteristics of representative strains of V. dahliae incubated on PDA medium showed that there was the great difference in colonial morphology among strains tested and it was divided into three cultural types, including microsclerotia type with strong pathogenicity, filamentous type with relatively strong pathogenicity, and intermediate type with weak pathogenicity. As for microsclerotia type, it produced a lot of microsclerotia and conidia; for filamentous type, it only had a small number of strains with flourishing white aerial mycelia and no microsclerotia formation; and for intermediate type, it had a white-gray color of colony surface with a small number of microsclerotia and conidia. Therefore, results revealed that the cultural characteristics of strains of V. dahliae had a close relation to its pathogenicity and the formation of the quantity, speed and melanin of microsclerotia of rest strains of V. dahliae had the positive relation to its pathogenicity except strains of filamentous type.3. It revealed that the temperature, pH value, lighting, carbon and nitrogen sources had the great effect on the colonial growth and sporulation of strains. The result showed that strains can grow in 10℃～30℃and the optimal temperature was at 25℃while there was no any growth when temperature exceeded 33℃. Strains can grow under 5.0～7.5 of pH value and the optimal pH was in 6.0～7.0. The lighting was suitable for growth and the dark condition was suitable for formation of microsclerotium, but it had no use for colonial growth and germination of microsclerotium. The glucose was the optimal carbon source for growth of strains and sucrose and soluble starch next, but lactose and glycerin were bad. Sodium nitrate and urea were the better nitrogen sources for colonial growth, the ammonium nitrate next and ammonium sulfate bad.4. Genetic diversity of 60 strains of V. dahliae origined from Henan, Jiangsu, Shaanxi and so on was studied by ISSR molecular analysis. Results showed that the method of DNA CTAB-SDS extraction was improved and the DNA extracted had the high purity and quality, and can meet the need of molecular biological research. The ISSR reaction system was optimized and 7 specific ISSR primers were screened from 30 ISSR primers, and there were 152 bands obtained by PCR amplification with 7 ISSR primers. Among them, there were 56 polymorphic bands which occupied 36.84% of its total. Results showed that the scope of genetic similar coefficient was 0.20～1.00 and there was more genetic difference in some strains. The 60 strains of V. dahliae were clustered into 2 genetic lineages at 0.46 genetic similarity and the typeⅠ,including 50 strains from Shaanxi, Jiangsu and Hubei provinces, others belonged to typeⅡ,including 10 strains from Xinjiang, Sichuan and Yunnan provinces. Results of clustering analysis of strains showed that there were some relations of genetic lineages of strains of V.dahliae to its geographical origin, pathogenicity and ISSR genetic variation.5. The vegetative compatibility of 60 strains of V.dahliae was studied. Based on the formation of complementary heterokaryons, 691 nitrate nonutilizing (nit) mutants which induced on KPS medium were obtained, in which 533 nit mutants belonged to nit1, accounted for 77.13% of the total;156 nit mutants belonged to nitM, accountted for 22.58% of the total; only 2 belonged to nit3 mutants, accountted for 0.29% of the total. The complementry test of nit1 and nitM was carried out and the results showed that 60 strains were assigned to three vegetative compatibilty groups (VCGs): strains from Henan and Hebei were classified as VC group three (VGC3) while Sichuan, Yunan and Xinjiang classified as VC group two (VGC2), other 42 representative strains classified as VC group one (VGC1). Results revealed that there was a relationship between the pathogenicity and vegetative compatibility of the strains which VCG1, VCG2 and VCG3 had strong, weak and intermediate pathogenicity accordingly.6. The quantity of toxin production of V.dahliae had the close relation to its cultural conditions. The temperature of 25℃, pH value of 6~7 and lighting for 24 h were the optimal conditions for toxin production of V.dahliae. Result indicated that there was the relation of pathogenicity of strains to its amount of toxin production, phosphorus extravasation rate of cotton cotyledon, the death rate of root cap cells and the wilting of cotton seedlings.