中国农业科学2017，Vol.50Issue(20)：3918-3929,12.DOI:10.3864/j.issn.0578-1752.2017.20.008
拮抗根癌土壤杆菌的桃内生细菌的筛选鉴定
Screening and Identification of Peach Endophytic Bacteria with Antagonism Against Agrobacterium tumefaciens
摘要
Abstract
[Objective]The objectives of this study are to identify the population composition of cultivable endophytes in peach twigs of 'Xibei 13-1' cultivar, and to screen the new antagonists for biological control of crown gall disease.[Method]Peach twigs of 'Xibei 13-1' cultivar were inoculated with Agrobacterium tumefaciens, and mock inoculation of the control was performed in a similar manner, but sterilized distilled water was used instead of the bacterial suspension. Twigs with different treatments were collected at different time points (before inoculation, 10 and 60 days after inoculation, respectively). Surface sterilization, endophytes isolation and counts, and 16S rDNA sequencing, were conducted to analyze the quantity and diversity of endophytic bacteria among samples. Their antagonisms against the causal agent of crown gall disease, A. tumefaciens, were tested by pair co-culturing method, and the efficacy of the antagonists in suppressing crown gall disease was further evaluated in greenhouse using sunflowers as a susceptible plant. The taxonomic status was clarified by physiological and biochemical and molecular methods. In order to reveal the antagonistic mechanism, the plasmid of pBBR1MCS-2 that contains green fluorescent protein gene (GFP) was transformed into the antagonistic strains by electroporation. Antibacterial activities, dynamic analysis and stability of the GFP-labeled strain were tested. The labeled strain suspension was inoculated using root irrigation method, then the A. tumefaciens suspension was inoculated at the 2nd day. Their re-colonization on the tomato roots was examined by fluorescence microscope and plate dilution method. [Result]A total of 108 endophytic bacterial isolates were obtained from peach twigs of 'Xibei 13-1' cultivar. All isolates were identified as 17 genera of the 5 bacterial groups-phylogenetically based on 16S rDNA. The 5 groups are Gammaproteobacteria, Alpharoteobacteria, Actinobacteria, Firmicutes, and Bacteroidetes. This result showed the biodiversity of endophytic bacteria from peach twigs of 'Xibei 13-1' cultivar. Among them, genera Enterobacter, Pantoea, and Rhizobium were the most abundant and significantly increased following inoculation of A. tumefaciens. In addition, most of the antagonists belong to these three genera as well (10/14). In vivo, strains 10DM4-1 and 10DI2-2 showed good performances in disease control with the efficacy of 86.08% and 89.87%, respectively. Strains 10DM4-1 and 10DI2-2 were then identified as Pantoea deleyi and Enterobacter cowanii by their 16S rDNA sequences in combination with their biochemical and physiological characteristics, respectively. The population of transformants 10DM4-1-gfp and 10DI2-2-gfp on tomato roots decreased sharply in the first 10 days, then declined slowly after 10th day and remained constant at lower level (104 CFU/g), indicating that they could stably colonize and survive in the intercellular of tomato roots.[Conclusion]The resistance of peach 'Xibei 13-1' cultivar against crown gall disease may be related to its endogenous Enterobacter, Pantoea and Rhizobium. P. deleyi 10DM4-1 and E. cowanii 10DI2-2 can effectively suppress crown gall disease caused by A. tumefaciens, produce antagonistic substances, colonize the favorable ecological niche and has high potential application value for biological control of crown gall disease.关键词
桃根癌病/内生细菌/拮抗菌/生物防治/定殖Key words
peach crown gall disease/endophytic bacteria/antagonist/biological control/colonization引用本文复制引用
李昱佳,李茜,张志想,李世访..拮抗根癌土壤杆菌的桃内生细菌的筛选鉴定[J].中国农业科学,2017,50(20):3918-3929,12.基金项目
国家桃产业技术体系(CARS30) （CARS30）
