Identification of pathogenic bacteria and screening of control agents for wine grape crown gall disease in the Western Sichuan Plateau

Abstract:【Objective】The 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 sunlight, large temperature difference between day and night, little rainfall and large evaporation. However, the crown gall disease of grape frequently occurs in this region, directly 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 cause grape crown gall disease specifically in the Western Sichuan Plateau for better clarifying the disease-controlling agents. Recent studies on grape crown gall disease are mainly carried out in the northern China, and the causes and pathogens causing grape crown gall disease in the WesternSichuan Plateau region have barely been investigated. Moreover, the limited control measures and unclear efficacy of commercial microbicides on grape crown gall disease have impeded the prevention and control in the field. Therefore, this study also aimed to find out the suitable disease managements by evaluating the controlling effect of 11 types of commercial microbicides on pathogenic bacteria. 【Methods】Fresh grape crown galls were collected from the Western Sichuan Plateau for tissue grinding with the pathogen isolation by using MW selection medium, followed by yeast extract mannitol broth (YEB) medium. The isolated strains were then identified by morphological observation. The 16s rDNA was amplified with 27F (5'-AGAGTTTGATCCTGGCTCAG-3') /1492R (5'-AAGGAGGTGATCCAGCCGCA-3') for sequencing and phylogenetic analysis (Maximum-Likelihood) for each isolates. To better character the isolates and define their opine type, genes in T-DNA of Agrobacterium producing arginine, including octopine type and nopaline type, were amplified with primers AP-F/AP-R, OP-F/OPR and NP-F/NP-R, respectively. The growth curve of the isolates was drawn by measuring optical density at 600 nm (OD600nm). The biovar types of the isolates were applied to conduct physiological and biochemical analysis of bacterial identification. The pathogenicity detection was carried out by carrots and grapevine infection of the isolated strains. Then, the in vitro experiment was undertaken to study the inhibitory effect of different microbicides on isolated pathogenic bacteria by using in-dish bacteriostatic zone method. The bacteriostatic zone formed with different dilutions of 11 types of microbicides was measured. To further confirm the control effect of the microbicides agents on grape crown gall disease, the in vivo experiment of grape seedling inoculation was carried out for selecting the best agents.【Results】From the grape crown galls, 48 isolates were purified with characteristic of milky white, smooth and rounded colony with neat edges. In total, 9 strains were selected by PCR analysis and further confirmed with their 16s rDNA sequencing results. All of the 9 strains isolated from grape crown galls were identified as Agrobacterium vitis (A. vitis). Phylogenetic analysis showed that 9 strains were clustered in the same branch, which included A. vitis S4 (CP000633.1). Furthermore, the physiological and biochemical analysis was carried out for the 9 isolates, showing 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 by the characteristic of Agrobacterium biovar 3. The growth curve of the 9 strains was slightly different, indicating different growth characteristics. All of them could induce tumorigenic growth in carrots, and A10 strain could cause crown galls in Vitis vinifera‘Cabernet Sauvignon’and Vitis vinifera‘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 presented a better inhibition effect on pathogen growth compared with B. subtilis. In vivo experiment was carried out in Vitis vinifera‘Cabernet Sauvignon’to analyze the control effect of 5 agents on grape crown gall disease. The control effect of ethylicin was the best, with the incidence being only 6%, and the control effect was as high as 48.05%. The control effect of oxine-copper and tetramycin was acceptable as the incidences were 19% and 12%, respectively. Their control effects were 37.5% and 29.69%, respectively. The control efficacy of chlorothalonil and B. amyloliquefaciens KN-527 was very limited as the control effects were only 25.78% and 21.88%, respectively.【Conclusion】The pathogenic bacteria causing grape crown gall disease in the Western Sichuan Plateau were identified as Agrobacterium vitis. The isolated strains were slightly different in growth characteristics, but all of them showed tumor-igenic 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 has 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.