Scientia Agricultura Sinica ›› 2017, Vol. 50 ›› Issue (20): 3918-3929.doi: 10.3864/j.issn.0578-1752.2017.20.008

• PLANT PROTECTION • Previous Articles     Next Articles

Screening and identification of peach endophytic bacteria with antagonism against Agrobacterium tumefaciens

LI YuJia1, LI Qian1,2, ZHANG ZhiXiang1, LI ShiFang1   

  1. 1State Key Laboratory for biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193; 2College of Horticulture, China Agricultural University, Beijing 100193
  • Received:2017-03-20 Online:2017-10-16 Published:2017-10-16

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.

Key words: peach crown gall disease, endophytic bacteria, antagonist, biological control, colonization

[1]    王金生. 植物病原细菌学. 北京: 中国农业出版社, 1998.
Wang J S. Plant pathogenic bacteriology. Beijing: China Agriculture Press, 1998. (in Chinese)
[2]    Kerr A. Biological control of crown gall through production of agrocin 84. Plant Disease, 1980, 64(1): 25-30.
[3]    Kerr A. Biological control of crown gall: seed inoculation. Journal of Applied Bacteriology, 1972, 35(3): 493-497.
[4]    Kerr A, Htay K. Biological control of crown gall through bacteriocin production. Physiological Plant Pathology, 1974, 4(1): 37-44.
[5]    Copping L G. The manual of biocontrol agents, 3rd ed of the biopesticide manual. UK: British Crop Protection Council, 2001: 4-6.
[6]    何玲敏, 叶建仁. 植物内生细菌及其生防作用研究进展. 南京林业大学学报 (自然科学版), 2014, 38(6): 153-159.
He L M, Ye J R. Endophytic bacteria: research advances and biocontrol applications. Journal of Nanjing Forestry University (Natural Sciences Edition), 2014, 38(6): 153-159. (in Chinese)
[7]    Sturz A V, Christie B R, Nowak J. Bacterial endophytes: potential role in developing sustainable systems of crop production. Critical Reviews in Plant Sciences, 2000, 19(1): 1-30.
[8]    林玲, 乔勇升, 顾本康, 周益军, 董汉松. 植物内生细菌及其生物防治植物病害的研究进展. 江苏农业学报, 2008, 24(6): 969-974.
Lin L, Qiao Y S, Gu B K, ZHOU Y J, DONG H S. Advances in the study of plant endophytic bacteria and their effects on biological control of plant diseases. Jiangsu Journal of Agricultural Sciences, 2008, 24(6): 969-974. (in Chinese)
[9]    孔庆科, 丁爱云. 内生细菌作为生防因子的研究进展. 山东农业大学学报 (自然科学版), 2001, 32(2): 256-260.
Kong Q K, Ding A Y. Advances of study on endophytic bacteria as biological control agents. Journal of Shandong Agricultural University (Natural Science), 2001, 32(2): 256-260. (in Chinese)
[10]   Hardoim P R, Van Overbeek L S, Berg G, Pirttila A M, Compant S, Campisano A, Doring M, Sessitsch A. The hidden world within plants: ecological and evolutionary considerations for defining functioning of microbial endophytes. Microbiology and Molecular Biology Reviews, 2015, 79(3): 293-320.
[11]   Mendes R, Pizzirani-Kleiner A A, Araujo W L, Raaijmakers J M. Diversity of cultivated endophytic bacteria from sugarcane: genetic and biochemical characterization of burkholderia cepacia complex isolates. Applied and Environmental Microbiology, 2007, 73(22): 7259-7267.
[12]   Wang E T, Tan Z Y, Guo X W, Rodríguez-Duran R, Boll G, Martínez-Romero E. Diverse endophytic bacteria isolated from a leguminous tree Conzattia multiflora grown in Mexico. Archives of Microbiology, 2006, 186(4): 251-259.
[13]   Cottyn B, Debode J, Regalado E, Mew T W, Swings J. Phenotypic and genetic diversity of rice seed-associated bacteria and their role in pathogenicity and biological control. Journal of Applied Microbiology, 2009, 107(3): 885-897.
[14]   闫孟红, 蔡正求, 韩继刚, 孙磊, 宋未. 植物内生细菌在防治植物病害中的应用研究. 生物技术通报, 2004(3): 8-12, 22.
Yan M H, Cai Z Q, Han J G, Sun L, Song W. The applied research of endophytic bacteria in biological control of plant disease. Biotechnology Bulletin, 2004(3): 8-12, 22. (in Chinese)
[15]   陈延熙, 陈璧, 潘贞德, 王淑芝. 增产菌的应用与研究. 生物防治通报, 1985, 1(2): 22-24.
CHEN Y X, CHEN B, PAN Z D, WANG S Z. Application and studies on plant-growth promoting rhizobacteria. Chinese Journal of Biological Control, 1985, 1(2): 22-24. (in Chinese)
[16]   Pleban S, Ingel F, Chet I. Control of Rhizoctonia solani and Sclerotium rolfsii in the greenhouse using endophytic Bacillus spp. European Journal of Plant Pathology, 1995, 101(6): 665-672.
[17]   Hao F G, Wang L R, Cao K, Wang X W, Fang W C, Zhu G R, Chen C W. Systemic acquired resistance induced by Agrobacterium tumefaciens in peach and differential expression of PR1 genes. HortScience, 2015, 50(5): 666-672.
[18]   Bliss F A, Almehdi A A, Danderkar A M, Schuerman P L, Bellaloui N. Crown gall resistance in accessions of 20 Prunus species. Hortscience, 1999, 34(2): 326-330.
[19]   王志勇, 刘秀娟. 植物内生菌分离方法的研究现状. 贵州农业科学, 2014, 42(1): 152-155.
Wang Z Y, Liu X J. Research status on separation methods of plant endophytes. Guizhou Agricultural Sciences, 2014, 42(1): 152-155. (in Chinese)
[20]   Nadkarni A, Martin F E, Jacques N A, HUNTER N. Determination of bacterial load by real-time PCR using a broad-range (universal) probe and primers set. Microbiology, 2002, 148: 257-266.
[21]   Garrity G M, Winters M, Searles D B. Bergry’s manual of systematic bacteriology. German: Springer-Verlag, 2001.
[22]   高之蕾, 李茜, 郭荣君, 李世东, 李世访, 王红清. 2株桃树根际细菌Alcaligenes faecalis对根癌病的抑制作用. 果树学报, 2015, 32(2): 267-273.
Gao Z L, Li Q, Guo R J, Li S D, Li S F, Wang H Q. Suppression effect of two peach rhizobacteria Alcaligenes faecalis on crown gall disease caused by Agrobacterium tumefaciens. Journal of Fruit Science, 2015, 32(2): 267-273. (in Chinese)
[23]   王红艳. 樱桃和樱花根癌病病原及生物防治研究[D]. 北京: 中国农业大学, 1998.
Wang H Y.Study on pathogen and biological control of cherry and flowering cherry crown gall[D]. Beijing: China Agricultural University, 1998. (in Chinese)
[24]   陶铁男, 明发源. 主要农作物灾害评估. 北京: 中国农业科学技术出版社, 2010.
Tao T N, Ming F Y. The assessment of disease on main crops. Beijing: China Agricultural Science and Technology Press, 2010. (in Chinese)
[25]   东秀珠, 蔡妙英. 常见细菌系统鉴定手册. 北京: 科学出版社, 2001.
Dong X Z, Cai M Y. Identification manual for general bacterium system. Beijing: Science Press, 2001. (in Chinese)
[26]   张云峰, 邵磊. 根癌农杆菌电击转化条件的研究. 淮阴师范学院学报 (自然科学版), 2009, 8(3): 243-245.
Zhang Y F, Shao L. Studies on electroporation transformation of Agrobacterium tumefaciens. Journal of Huaiyin Teachers College (Natural Science Edition), 2009, 8(3): 243-245. (in Chinese)
[27]   刘邮洲, 梁雪杰, 乔俊卿, 张荣胜, 陈志谊. 枯草芽胞杆菌PTS-394GFP标记及其定殖能力. 植物保护学报, 2014, 41(4): 416-422.
Liu Y Z, Liang X J, Qiao J Q, Zhang R S, Chen Z Y. Bacillus subtilis PTS-394 labeled by green fluorescent protein and its colonization. Acta Phytophylacica Sinica, 2014, 41(4): 416-422. (in Chinese)
[28]   Brady C L, Venter S N, Cleenwerck I, Engelbeen K, Vancanneyt M, Swings J, Couyinho T A. Pantoea vagans sp. nov., Pantoea eucalypti sp. nov., Pantoea deleyi sp. nov. and Pantoea anthophila sp. nov. International Journal of Systematic and Evolutionary Microbiology, 2009, 59: 2339-2345.
[29]   Popp A, Cleenwerck I, Iversen C, Vos P D, Stepphan R. Pantoea gaviniae sp. nov. and Pantoea calida sp. nov., isolated from infant formula and an infant formula production environment. International Journal of Systematic and Evolutionary Microbiology, 2010, 60: 2786-2792.
[30]   Tanaka Y K, Horie N, Mochida K, Yoshida Y, Okugawa E, Nanjo F. Pantoea theicola sp. nov., isolated from black tea. International Journal of Systematic and Evolutionary Microbiology, 2015, 65: 3313-3319.
[31]   Inoue K, Sugiyama K, Kosako Y, Sakazaki R, Yamai S. Enterobacter cowanii sp. nov., a new species of the family Enterobacteriaceae. Current Microbiology, 2000, 41: 417-420.
[32]   Madhaiyan M, Poonguzhali S, Lee J S, Saravanan V S, Lee K C, Santhanakrishnan P. Enterobacter arachidis sp. nov., a plant-growth-promoting diazotrophic bacterium isolated from rhizosphere soil of groundnut. International Journal of Systematic and Evolutionary Microbiology, 2010, 60: 1559-1564.
[33]   孙占斌, 袁行方, 王音娴, 张辉, 张文会, 冯永君. 黄瓜初花期与结瓜期叶片可培养内生细菌多样性研究. 微生物学通报, 2012, 39(6): 764-772.
Sun Z B, Yuan X F, Wang Y X, Zhang H, Zhang W H, Feng Y J. Diversity of culturable entophytic bacteria in cucumber leaves at blossoming and fruiting stages. Microbiology China, 2012, 39(6): 764-772. (in Chinese)
[34]   Torres R, Teixido N, Usall J, Abadias M, Mir N, Larrigaudiere C, Vinas I. Anti-oxidant activity of oranges after infection with the pathogen Penicillium digitatum or treatment with the biocontrol agent Pantoea agglomerans CPA-2. Biological Control, 2011, 57(2): 103-109.
[35]   Kim I Y, Pusey P L, Zhao Y, Korban S S, Choi H, Kim K K. Controlled release of Pantoea agglomerans E325 for biocontrol of fire blight disease of apple. Journal of Controlled Release, 2012, 161(1): 109-115.
[36] Al-mughrabi K I. Biological control of Fusarium dry rot and other potato tuber diseases using Pseudomonas fluorescens and Enterobacter cloacae. Biological Control, 2010, 53(3): 280-284.
[37]   Nguyen M T, Ranamukhaarachchi S L. Soil-borne antagonists for biological control of bacterial wilt disease caused by Ralstonia solanacearum in tomato and pepper. Journal of Plant Pathology, 2010, 92(2): 395-405.
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