川西高原酿酒葡萄根癌病病原菌的鉴定及防治药剂筛选

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

【目的】明确川西高原酿酒葡萄根癌病的致病菌种类,评价不同药剂对病原菌的抑制及对根癌病的防治效果。【方法】采集川西高原酿酒葡萄根瘤组织进行病原菌的分离纯化,通过形态观察、生理生化鉴定、PCR检测及16s rDNA测序分析确定分离菌株种类,并对分离菌株进行系统进化分析(极大似然法maximum-likelihood)、生长特性分析及致病性检测。利用皿内抑菌圈法初步研究不同药剂对病原菌的抑制作用,并通过葡萄苗接种试验明确药剂对根癌病的防治效果。【结果】成功由葡萄根瘤组织中分离得到9株菌株,经鉴定为生物Ⅲ型葡萄土壤杆菌(Agrobacterium vitis),进化分析表明,9株菌株聚类在同一分支。虽然9株菌株生长特性略有差异,但均能使胡萝卜及葡萄形成瘿瘤,有致病性。进一步研究11种商品化药剂对分离菌株的抑制效果,发现中生菌素、福美双、多菌灵、春雷霉素、枯草芽孢杆菌和氢氧化铜对分离菌株抑制效果微弱或无效果,对分离菌株抑制效果较好的依次为四霉素、百菌清、喹啉铜、解淀粉芽孢杆菌KN-527及乙蒜素。进一步验证这5种效果较好的药剂对葡萄根癌病的防治效果,其中乙蒜素对葡萄根癌病的防治效果最好,发病率仅为6%,防治效果高达48.05%;其次,喹啉铜及四霉素对葡萄根癌病有防治效果;在所试药剂中,百菌清及解淀粉芽孢杆菌KN-527的防治效果较差。【结论】川西高原酿酒葡萄根癌病的致病菌为葡萄土壤杆菌,对酿酒葡萄根癌病防治效果最好的为乙蒜素,然后依次为喹啉铜、四霉素、百菌清及解淀粉芽孢杆菌KN-527。其中乙蒜素在根癌病田间防治可能具有较广阔的应用前景,生防菌株解淀粉芽孢杆菌KN-527对早期葡萄根癌病的发生有抑制作用。

【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 Western Sichuan 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 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 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.