Studies On The Rapid Detection Technology Of Pathotypes Of Cotton Verticillium Dahliae

Isolates of Verticillium dahliae infecting cotton show crossvirulence and can be classified into defoliating (D) and non defoliating (ND) pathotypes based on their ability to completely defoliate the plant or to only cause wilt and partial or no defoliation. Verticillium wilt epidemics caused by the D pathotype develop earlier, more rapidly, and result in a greater reduction of cotton seed yield compared with losses caused by the ND pathotype. Therefore, the proper characterization of V. dahliae pathotypes would help in the decisiontaking process for disease management such as choice of host cultivar and avoidance of soil infested with D V. Dahliae. This paper describes the development of a PCR-based assay for the specific detection of D and ND pathotypes of V. dahliae in soil, plants, and seeds. Together with the DNA extraction protocol, the diagnostic method allows for the rapid and accurate assessment of soil, plants and seeds contamination by V. dahliae.Together with the DNA extraction protocol using Soil DNA Isolation kit, the V. dahliae detection method was also efficient with artificial and natural soils infested with spores of V. dahliae ND and D pathotypes. The sensitivity of the detection was 103 spores per gram in defoliating pathotype soil and 104 spores per gram in nondefoliating pathotype soil. After 7 days'moist incubation, Both for ND and D pathotypes, the specific nested-PCR procedure effectively detected 10 or more spores per gram of soil.Extraction of total DNA from infected plants using DNeasy plant Mini Kit (QIAGEN) and nested PCR using the above primers can identify Pathogenic Types of V.dahliae in susceptible plants and plants without symptom. The study of detecting roots, stems and leaves of cotton before symptom appearing suggested that defoliation pathotype Verticillium Wilt can be detected from roots and stems of cotton 5-10 days before symptom appearing, and nondefoliation pathotype Verticillium Wilt can be detected from cotton leaves 10-15 days before symptom appearing.The test to seeds indicated that V.dahliae can be detected from more than one seeds borne fungi using the technology above and DNeasy plant Mini Kit (QIAGEN). And the detection of different parts of seeds surggested that V.dahliae can be identified in both short fibre and seed capsule, but can not be detected in endosperm.Infected plants and infested soil of severe disease field from the cotton-planting corps in Xinjiang were tested. The results showed that there were 33 defoliation pathotype strains and 95 nondefoliation pathotype strains in 128 infected plants, which were respective 25.8% and 74.2% of supplying samples. There were 57 defoliation pathotype strains and 135 nondefoliation pathotype strains in 188 infested soil of severe disease field, which were respective 29.7% and 70.3% of involved samples. The ratio of both were nearly the same. It can be seen that defoliation pathotype strains occurred widely in cotton regions of Xinjiang and spread quickly. We should reinforce quarantine to prevent extension of disease region, especially diffusion of seeds in defoliation pathotype disease region. We also should get done with rational arragement of different resistant cultivar.