Pathogenicity Differentiation Of Verticillium Dahliae And Its Wilting Toxin Analysis From Cotton

Verticillium wilt, caused by Verticillium dahliae kleb, is one of the most damaging diseases of cotton in most cotton-growing countries worldwide. This disease has been a major problem on the improvement of the yield and the quality of cotton in China. The resistance of cotton varieties to V. dahliae is easy to loss due to the variation of V. dahliae and results in the epidemic of Verticillium wilt in the fields. Therefore, clarification the difference in virulence among the isolates of V. dahliae and the distribution of virulent isolates in the field is valuable for the control of Verticillium wilt. 490 isolates of V. dahliae were collected from the major cotton production areas in China during 2007-2009, and partial isolates were characterized by morphology, pathogenicity test, molecular approach and toxins analysis.Three morphology characters were included in the 490 isolates of V.dahliae and the black colony (sclerotium type) covered the majority of isolates, including 438 isolates and covered 89.39% to the total collection, the isolates belong to this group could produce a great of micro-sclerotia on PDA medium and distributed at all sampling sites. The white colony (mycelial type) was a secondary group, which including 36 isolates and covered 7.35% to the total collection, the isolates belong to this group could not produce micro-sclerotia and mainly collected from Hebei, Henan and Jiangsu Province. The black-white middle type only including 16 isolates and covered 3.27 to the total collection, the isolates belong to this group could produce a small amount of micro-sclerotia and mainly distributed at Wuyi county in Hebei Province.The pathogenicity of 91 V. dahliae isolates were tested in greenhouse, results indicated that the pathogenicity were different among isolates when tested against the same variety and three different pathogenicity group from weak to strong were observed. 73 isolates including 3 nondefoliating pathotypes belong to the strong pathogenicity group and covered 80.22% to the tested isolates, distributed at all sampling sites. 10 isolates belong to the moderate pathogenicity group and covered 10.99% to the tested isolates, mainly distributed in Hebei, Henan and Jiangsu Province. 8 isolates including two defoliating pathotypes belong to the weak pathogenicity group and covered 8.79% to the tested isolates, mainly distributed in Hebei, Liaoning, Sichuan, Hubei and Jiangsu Province. The pathogenicity also indicated that a majority of isolates had a stronger pathogenicity and the pathogenicity was different among isolates. Moreover, the pathogenicity for the three different morphology groups were different and the sclerotium type isolates were highly virulent, the black-white middle type isolates were mildly virulent and the white mycelial type isolates were weakly virulent.The defoliating and nondefoliating pathotypes of V. dahliae isolates were identification by specific PCR amplification. Results indicated that 466 isolates were defoliating pathotypes and covered 95.01% to the total collection, distributed in Hebei (381 isolates), Jiangsu (37 isolates), Henan (31 isolates), Tianjin (5 isolates), Shanxi (4 isolates), Sichuan (4 isolates), Hubei (2 isolates), Shandong Province (2 isolates) and Xinjiang Autonomous regions (1 isolate), which were covered 99.48%, 92.5%, 100%, 100%, 100%, 66.67%, 100%, 100% and 22.22% to the collection. 24 isolates were nondefoliating pathotypes and covered 4.90% to the total collection, distributed in Sichuan (6 isolates), Gansu (3 isolates), Jiangsu (3 isolates), Hebei (2 isolates), Liaoning (1 isolate), Hubei Province (1 isolate) and Xinjiang Autonomous regions (8 isolates), which were covered 88.89%, 66.67%, 100%, 7.5%, 0.52%, 100%,and 33.33% to the collection. Pathogenicity assay indicated 70 defoliating pathotypes isolates and 3 nondefoliating pathotypes isolates were highly virulent, 10 defoliating pathotypes were mildly virulent, the 83 isolates had the ability to completely defoliate the cotton in greenhouse and in the field. 2 defoliating pathotypes isolates and 6 nondefoliating pathotypes were weakly virulent and couldn`t defoliate the cotton in greenhouse.The toxins were extracted from the culture medium of V. dahliae which including 7 defoliating and 4 nondefoliating pathotype and were tested on the germination of cotton seeds and the wilting of seedlings. Results showed the toxins from defoliating pathotype isolates had the ability of restraining the germination of cotton seeds and bring obvious wilt of seedling, but the toxins from nondefoliating pathotype isolates had a weak capability of restraining the germination of cotton seeds and the wilt of seedling. The pathogenicity of toxins from defoliating pathotypes isolates were higher than the toxins from nondefoliating pathotypes isolates, although the quantity of toxins were lower in defoliating pathotype isolates when compared with the nondefoliating pathotype isolates.