Abstract: 【Objective】 The objective of this study is to clarify the induced immunity effect and mechanism of the weak pathogenicity isolate of Verticillium dahliae Vd171 against Verticillium wilt in cotton, and to provide a basis for exploring new ways of biological control of Verticillium wilt in cotton. 【Method】 The weak pathogenicity isolate of V. dahlia Vd171 was used to evaluate the induced immune effects of different inoculation methods, inoculation periods, inoculation times and inoculum types in greenhouse. Different inoculation methods included root-dipping, root-irrigation, stem-injection, foliar-spraying and seed-soaking by Vd171 spore suspension with 1×10⁷ conidia/mL. Ten mL spore suspension for each pot was used in root-dipping, root-irrigation and foliar-spraying and 0.2 mL for each plant in stem-injection. The seeds were soaked for 12 h with spore suspension. Seven treatments were set to confirm the inoculation periods of Vd171 which including 2, 4, 6, 8 days before inoculated Vd080 (a high pathogenicity isolate of V. dahliae) and 1, 2, 4 days after inoculated Vd080. The inoculation times of Vd171 was one or two times before inoculated Vd080. The inoculum types included spore suspension and filtrate of Czapek of Vd171. The cotton seedlings were cultivated by hydroponics and inoculated by Vd080_GFP 4 days after Vd171. Then the roots were collected in different times and the spores of Vd080_GFP were washed in sterile water. The colonization of Vd080_GFP on cotton rootwas analyzed by counting the spores of Vd080_GFP under microscope. Furthermore, in order to analyze the extension of Vd080 in cotton stem, the small hypocotyl pieces was cultivated on PDA plates at the 7th day after inoculated by Vd080_GFP. The transcript level of 4CL, CHI, POD, PPO and PAL was analyzed by qPCR. The enzyme activity of POD, PPO, PAL and CHI was measured. The sedimentation of lignin was observed by phloroglucinol staining and the reactive oxygen species (ROS) of leaves was observed by DAB staining. 【Result】 The highest control efficacy was obtained when Vd171 was inoculated 4 days prior to inoculation by Vd080 (control efficacy of 89.4%). The control efficacy had significant differences among the five inoculation methods. Root-dipping treatment provided a control efficacy of 70.0%, followed by foliar-spraying (54.3%), seed-soaking (45.0%), root-irrigation (39.0%), and stem-injection (2.2%). There was no statistical significant difference between the control efficacy against Vd080 of pre-inoculated by Vd171 for one and two times (85.6% and 81.4%). Particularly, both cotton pre-inoculated with conidiospore and culture filtrate of Vd171 treatments could reduce disease indices of Verticillium wilt, with the control efficacy of 85.6% and 81.1%. There was no significant difference between the two treatments. Cotton plants pre-inoculated with Vd171 could reduce the colonization of Vd080 in cotton root and prevent its expansion in cotton stem. In addition, the transcript level of GhPAL, Gh4CL and GhCHI increased significantly by induction of Vd171, which was 2.2, 8.5 and 2.6 times of the control treatment, respectively. The activity of PPO, PAL, POD and CHI in hypocotyl increased by 31.9%, 131.0%, 57.1% and 102.1% than that of control treatment, respectively, and activity of PAL, CHI and POD in cotyledon also remarkably increased by 22.1%, 39.6% and 7.1% than that of control treatment, respectively. Meanwhile, sedimentation of lignin and ROS were induced by Vd171.【Conclusion】 The weak virulent isolate Vd171 can effectively induce cotton resistant to Verticillium wilt and has a good application prospect in the control of Verticillium wilt in cotton.

Key words:  cotton, Verticillium wilt, Verticillium dahliae, weak pathogenicity isolate,