Abstract:【Objective】Western Sichuan plateau in China is considered as a high-quality wine grape producing area in China, due to its climatic characteristics such as sufficient light, large temperature difference between day and night, little rainfall and large evaporation. However, the crown gall disease of grape was frequently occurred in this region, threatening plant health and grape production and causing huge economic losses. Therefore, the premier objective of this study is to identify the pathogen species that causing grape crown gall disease specifically in western Sichuan plateau for better clarify the disease causing agents. Recent studies of grape crown gall disease were focus in the north of China, the causes and pathogens of grape crown gall disease in west Sichuan Plateau region were barely investigated. Moreover, the limit control measures and unclear effect of commercial microbicides on grape crown gall disease impeded to the prevention and control in field. Therefore, this study also aim to find the suitable disease managements by evaluating the control effect of 11 commercial microbicides on pathogenic bacteria and crown gall disease.【Method】Fresh grape crown galls were collected from western Sichuan plateau for tissue grinding with the pathogen isolation by using MW selection medium and followed by yeast extract mannitol broth (YEB) medium. The isolated strains were then identified by morphological observation. The the16s rDNA was amplified with 27F (5’-AGAGTTTGATCCTGGCTCAG-3’) / 1492R (5’-AAGGAGGTGATCCAGCCGCA-3’) for sequencing and phylogenetic analysis (Maximum-Likelihood) of each isolates. To better character the isolates and define their opine type, genes in T-DNA of Agrobacterium that producing arginine, octopine type and nopaline type were amplified with primers JAS-F/JAS-R, ZYJ-F/ZYJ-R and YZJ-F/YZJ-R, respectively. The growth curve of the isolates were carried by measure optical density at 600 nm (OD600nm). The biovar types of the isolates were carried physiological and biochemical analysis of bacterial identification. The pathogenicity detection carried by carrots and grapevine infection of the isolated strains. Then, the in vitro experiment was carried to study the inhibitory effect of different microbicides on isolated pathogenic bacteria by using in-dish bacteriostatic zone method. The bacteriostatic zone formed under different dilutions of 11 microbicides was measured. Furtherly, to further confirmed the control effect of the microbicides agents on grape crown gall disease, the in vivo experiment of grape seedling inoculation was carried for the best effect agents selected by in vitro experiment. 【Results】From the grape crown galls, 48 isolates was purified with characteristic of milky white, smooth, rounded colony with neat edges. In total 9 strains were selected by PCR analysis and further confirmed with their 16s rDNA sequencing results. All the 9 strains isolated from grape crown galls were identified as Agrobacterium vitis (A. vitis). Phylogenetic analysis shown that 9 strains were clustered in the same branch, which including A. vitis S4 (CP000633.1). Furthermore, physiological and biochemical analysis were carried out for the 9 isolates, resulting as tested positive for the growth in 2% NaCl and alkaline reaction of litmus milk test, and tested negative for the 3-ketolactose production test, reaction to ferric ammonium citrate, citrate utilization test and reaction to acid from ethanol, followed the characteristic of Agrobacterium biovar 3. Although the growth curve of the 9 strains were slightly different indicating different growth characteristics. All of them could induce tumorigenic growth in carrots, with A10 strain could cause crown galls in Vitis vinifera cv. Cabernet Sauvignon and Vitis vinifera cv. Marselan. In vitro study on the control effect of 11 commercial microbicides on the isolated strains indicated that zhongshengmycin, thiram, carbendazim, kasugamycin and copper hydroxide had weak or no effect on the isolated strains. The tetramycin, chlorothalonil, oxine-copper and ethylicin could inhibit pathogen growth. As for the biocontrol agents Bacillus amyloliquefaciens（B. amyloliquefaciens） KN-527 present a better inhibition effect on pathogen growth compared with B. subtilis. In vivo experiment was carried in Vitis vinifera cv. Cabernet Sauvignon to analysis the control effect of 5 agents to grape crown gall disease. The control effect of ethylicin was the best, with the incidence was only 6%, and the control effect was as high as 48.05%. The control effect of oxine-copper and tetramycin was acceptable as the incidence were 19% and 12%, respectively. Their control effect were 37.5% and 29.69%, respectively. The control effect of chlorothalonil and B. amyloliquefaciens KN-527 were very limited as the control effect were only 25.78% and 21.88%, respectively.【Conclusion】The pathogenic bacteria causing grape crown gall disease in western Sichuan Plateau was identified as Agrobacterium vitis. The isolated strains were slightly difference in growth characteristics, but all tumorigenic and pathogenic. Phylogenetic analysis indicated that 9 strains were closely related to A. vitis S4 (CP000633.1). Moreover, our results indicated the agents with the best control effect were ethylicin, followed by oxine-copper, tetramycin, chlorothalonil and B. amyloliquefaciens KN-527. Among them, ethylicin have a great prospect to be used in field application. The commercial biocontrol agents B. amyloliquefaciens KN-527 has an inhibitory effect on the early stage of grape crown gall disease development.
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