Abstract: 【Objective】 The objective of this study is to construct a rapid screening system of pathogenicity-related mutants in Verticillium dahliae, which will provide a technical support to identification of T-DNA mutant library.【Method】The cotton roots were inoculated with V. dahliae conidial suspension by root-dipping method for 30 min, including five concentrationsof 5.0×10⁴, 5.0×10⁵, 5.0×10⁶, 5.0×10⁷and 5.0×10⁸ conidia/mL, to determine the suitable concentration range of causing the obvious Verticillium wilt. The culture flasks were used for the V. dahliae culture, and 15, 25, 35 and 45 mL of sterilized water were added into the flasks to elute the conidial. The concentration of conidial was calculated by the hemocytometer to detect the effect of sterilized water volume on the conidial concentration. Based on the unit of mutants in 96-well plates, the single spore of mutants was isolated on the petri dish and expanding cultured in the flasks, then the sterilized water at suitable volume was directly added into the flasks to elute the conidial and the cotton seedling roots were dipped into the conidial suspension in the flasks for 30 min, subsequently the infected cotton seedlings were cultured for 14 d and the disease index was investigated. A rapid screening system of pathogenicity-related mutantsin V. dahliae was constructed after optimization of cotton planting, mutants single spore isolation, expanding culture, inoculation, and results investigation. The quantitative inoculation method was used for validation of the reliability results of pathogenicity-related mutants, 30 cotton seedlings with three replicates for each mutant, the concentration of suspension was 5.0×10⁶ conidia/mL, 5 mL suspension for each seedling, and inoculated for 30 min, finally the disease indexes of pathogenicity-related mutants were investigated on 5, 8, 11 and 14 d after inoculation.【Result】The conidial concentration up to 5.0×10⁵ conidia/mL could obviously infect the cotton and cause the Verticillium wilt symptom. A standard culture method of V. dahliae was constructed which includes single spore isolation for 5 days and expanding culture in standard culture flasks for 9 days. The appropriate volume of sterilized water used for eluting the conidial suspension was 25 mL. The test results showed that the concentration for eluting the conidial suspension of 20 mutants were（2.55±0.58)×10⁶-(1.72±0.25)×10⁷ conidia/mL. Subsequently, the inoculation steps were optimized for construction of the rapid process of V. dahliae pathogenicity identification, which includes single spore isolation, expanding culture, conidial suspension preparation, inoculation, and results investigation. Moreover, a rapid screening system of pathogenicity-related mutants in V. dahliae was constructed after coordinative design. The test results showed that one people could finish 1 334 mutants within 54 days by running one cycle which includes seven processes, and the work load is only 21 man-days. The validation of target mutants by quantitative inoculation method showed that the pathogenicity of these mutants was significantly reduced, consistent with the results of the rapid screening system, which indicated that the rapid screening system is appropriate for the screening of pathogenicity-related mutants in V. dahliae.【Conclusion】A rapid screening system ofpathogenicity-related mutants in V. dahliae was constructed after optimization and standardization of cotton planting, mutants single spore isolation, expanding culture, preparation of the conidial suspension, inoculation, and results investigation, which will provide a technical support for the pathogenicity-related gene research in future.

Key words: Verticillium dahliae, pathogenicity-related mutant, pathogenic identification, rapid screening system