Screening and Identification of Endophytic Bacteria BR-1 for the Biocontrol of Tomato (Solanum lycopersicum) Gray Mold and Analysis of Its Biocontrol Performance
LI La1, SHI Hong-Li1, YU Ting-Ting1, LIU Qing1, JIAN Wei1,*, YANG Xing-Yong1,2,*
1 College of Life Sciences, Chongqing Normal University, Chongqing 401331, China; 2 School of Pharmacy, Chengdu University, Chengdu 601106, China
Abstract：Screening endophytic bacteria with excellent antagonistic effect against Botrytis cinerea plays important roles in controlling of B. cinerea, increasing tomato (Solanum lycopersicum) yield and reducing postharvest tomato loss. The present study adopted LB medium and gradient dilution method to isolate endophytic bacteria from the roots of Stemona japonica, and the antagonistic strains were screened by plate confrontation method. The antagonistic strains were identified by morphological characteristics, physiological and biochemical analysis, and 16S rDNA sequence analysis. The bacteriostatic ability of the volatile compounds of antagonistic strains was measured by two-sealed-base-plates method. The control effect of antagonistic strains on B. cinerea and their colonization ability on tomato fruits were analyzed by using fruit-pricking method. Seed germination rate, root length, and bud length were measured by seed germination experiment to evaluate the growth promotion effect of antagonistic strains on tomato seedlings. Results indicated there was an excellent strain BR-1 was screened and identified as Burkholderia gladioli, which had nitrogen fixation and phosphorus solubilization effects. The results showed that BR-1 can inhibit the expansion of B.cinerea by inhibiting mycelial growth, and the inhibition rate of BR-1 could reach 73.38%, and further study showed that the volatiles of BR-1 strain exhibited significant antibacterial activity. The broad-spectrum antifungal experiments showed that BR-1 had different inhibitory effects on Fusarium moniliforme, Coccinea arachniformis, Phytophthora capsici, Fusarium oxysporum, Corynosporum cucurbitum and Alternaria brassicae, especially the inhibitory rate of F. moniliforme reached 72.97%. Seed germination experiment showed that BR-1 strain significantly improved the germination rate of tomato seeds, promoted the growth of tomato seedlings and successfully colonized tomato fruits. This study had isolated and screened a biocontrol endophytic bacterium BR-1 with significant disease resistance and growth-promoting effects, and provides important biological resources for the biocontrol of tomato gray mold.
李腊, 史洪丽, 余婷婷, 刘庆, 简伟, 杨星勇. 番茄灰霉病生防内生细菌BR-1的筛选鉴定及其生防性能分析[J]. 农业生物技术学报, 2022, 30(8): 1594-1605.
LI La, SHI Hong-Li, YU Ting-Ting, LIU Qing, JIAN Wei, YANG Xing-Yong. Screening and Identification of Endophytic Bacteria BR-1 for the Biocontrol of Tomato (Solanum lycopersicum) Gray Mold and Analysis of Its Biocontrol Performance. 农业生物技术学报, 2022, 30(8): 1594-1605.
[1] 白变霞, 窦旭峰, 刘荣, 等. 2021. 1株潞党参根部内生细菌的鉴定及促生长指标测定[J]. 江苏农业科学, 49(12): 196-200. (Bai B X, Dou X F, Liu R, et al.2021. Identification and growth promoting index determination of an endophytic bacteria isolated from root of Codonopsis pilosula (Franch.) Nannf[J]. Journal of Jiangsu Agricultural Sciences, 49(12): 196-200.) [2] 陈利军, 郭世保, 田雪亮, 等. 2016. 产香真菌GS-1菌株鉴定及其挥发性物质对番茄灰霉病的生防效果[J]. 植物保护学报, 43(04): 608-613. (Chen L J, Guo S B, Tian X L, 等. 2016. Identification of aroma-producing fungus GS-1 strain and its biocontrol efficacy against tomato gray mold[J]. Journal of Plant Protection, 43(04): 608-613.) [3] 陈彧, 李冬琴, 周国英, 等. 2017. 降香黄檀炭疽病拮抗细菌的分离筛选及鉴定[J]. 热带林业, 5(01): 38-40. (Chen Y, Li D Q, Zhou G Y, et al.2017. Isolation, screening and identification of endophytic bacteria antagonistic to Dalbergia Odorifera anthracnose[J]. Tropical Forestry, 45(01): 38-40.) [4] 陈耀丽, 俞龙春, 钱悦, 等. 2019. 大尖囊蝴蝶兰内生真菌和细菌的分离与鉴定[J]. 热带生物学报, 10(04): 372-379. (Chen Y L, Yu L C, Qian Y, et al.2019. Isolation and identification of endophytic fungi and bacteria from Phalaenopsis deliciosa[J]. Journal of Tropical Biology, 10(04): 372-379.) [5] 程雷, 潘磊庆, 苏晶, 等. 2014. 两种酵母对“红富士”苹果采后病害的控制[J]. 食品工业科技, 35(15): 334-337. (Lei C, Lei Q P, Jing S, et al.2014. Effects of two kinds of yeast on the postharvest diseases of ‘Red Fuji' apples[J]. Science and Technology of Food Industry, 35(15): 334-337.) [6] 东秀珠, 蔡妙英. 2001. 常见细菌系统鉴定手册[M]. 北京: 科学出版社, pp. 349-388. (Dong X Z, Cai M Y.2001. Systematic Identification Mannual of Commen Bacteria[M]. Science Press, Beijing, China, pp. 349-388.) [7] 高沙尔·卡依尔哈力, 热子亚·麦麦吐逊, 祖丽皮亚·玉努斯. 2021. 地锦草内生细菌多样性、拮抗及促生特性测定[J]. 微生物学通报, 48(02): 392-406. (Gaoshaer K, Raziye M, Zulfiya Y.2021. Endophytic bacteria from Euphorbia humifusa: Diversity, antagonism and growth-promoting activities[J]. Microbiology China, 48(02): 392-406.) [8] 宫安东, 董飞燕, 吴楠楠, 等. 2019. 吡咯伯克霍尔德菌WY6-5产二甲基二硫对储藏期花生黄曲霉及毒素的抑制作用[J]. 中国农业科学, 52(17): 2972-2982. (Gong A D, Zhu Z Y, Lu Y N, et al.2019. Inhibitory effect of dimethyl disulfide from Burkholderia pyrrocinia WY6-5 on Aspergillus flavus and aflatoxins in peanuts during storage period[J]. Scientia Agricultura Sinica, 52(09): 1574-1586.) [9] 李振高, 骆永明, 滕应. 2008. 土壤与环境微生物研究法[M]. 北京: 科学出版社, pp. 52-63. (Li Z G, Luo Y M, Teng Y.2008. Soil and Microbial Research Methods[M]. Science Press, Beijing, China, pp. 52-63.) [10] 李露丹, 吴宣, 徐丹, 等. 2021. 珍稀药用石斛内生菌研究进展[J]. 生物资源, 43(03): 246-256. (Li L D, Wu X, Xu D, et al.2021. Research advances on endophytes of rare medicinal Dendrobium[J]. Biotic Resources, 43(03): 246-256.) [11] 刘彩云, 许瑞瑞, 季洪亮, 等. 2015. 一株生防内生真菌的分离筛选、鉴定及抑菌特性[J]. 植物保护学报, 42(05): 806-812. (Liu C Y, Xu R R, Ji H L, et al.2015. Isolation, screening and identification of an endophytic fungus and the detection of its antifungal effects[J]. Journal of Plant Protection, 42(05): 806-812.) [12] 刘人萱, 张小蕊, 王睿, 等. 2021. 生防菌剂对人参灰霉病菌抑制作用及田间防控效果[J]. 菌物研究, 19(03): 191-196. (Liu R X, Zhang X R, Wang R, et al.2021. Inhibitory and field control effects of biocontrol agents against Botrytis cinerea on ginseng online first[J]. Bacteria Research, 19(03): 191-196.) [13] 罗红丽, 林显钊, 张利敏, 等. 2012.百部内生放线菌的分离、分类及次级代谢潜力[J]. 微生物学报, 52(03): 389-395. (Luo H L, Lin X Z, Zhang L M, et al.2012. Isolation, classification and secondary metabolic potential of 100 endophytic actinomycetes[J]. Acta Microbiologica sinica, 52(03): 389-395.) [14] 罗琳, 王其慧, 赵海霞, 等. 2017. 葡萄灰霉病生防菌株的筛选及其拮抗机理初探[J]. 中国酿造, 36(04): 93-98. (Luo L, Wang Q H, Zhao H X, et al.2017. Screening of bio-control strain against Botrytis cinerea and preliminary research on its antagonistic mechanism[J]. China Brewing, 36(04): 93-98.) [15] 沈艳, 何鹏搏, 吴毅歆, 等. 2021. 番茄产后灰霉病的病原鉴定及生物防治[J]. 中国农学通报, 37(13): 102-107. (Shen Y, He P B, Wu Y X, et al.2021. Pathogen identification and biological control of gray mold on postharvest tomato[J]. Chinese Agricultural Science Bulletin, 37(13): 102-107.) [16] 孙正祥, 孟祥佳, 龙欣钰, 等. 2021. 伯克霍尔德氏菌YZU-S230对西瓜枯萎病的防效及其促生作用[J]. 长江大学学报(自然科学版), 18(02): 82-88. (Sun Z X, Meng X J, Long X Y, et al.2021. Effects of Burkholderia sp. YZU-S230 on the control and growth promotion of watermelon Fusarium wilt[J]. Journal of Yangtze University (Natural Science Edition), 18(02): 82-88.) [17] 王红莹, 李春燕, 宋发军, 等. 2021. 一株白及内生细菌的分离鉴定及抑菌机理初步研究[J]. 中南民族大学学报(自然科学版), 40(03): 246-251. (Wang H Y, Li C Y, Song F J, et al.2021. Isolation and identification of an endophytic bacterium from Bletilla striata and preliminary study of antibacterial mechanism[J]. Journal of South-Central University for Nationalities (Natural Science Edition), 40(03): 246-251.) [18] 吴翔, 甘炳成, 唐亚, 等. 2019. 四川烟草主栽区根际促生菌筛选及促生菌系构建[J]. 烟草科技, 52(03): 1-9. (Wu X, Gan B C, Tang Y, et al.2019. Screening growth-promoting rhizobacteria from main tobacco-growing areas in Sichuan and construction of growth-promoting rhizobacteria groups[J]. Tobacco Science & Technology, 52(03): 1-9.) [19] 辛华总. 2021. 水稻种植技术与病虫害防治要点浅析[J]. 南方农业, 15(03): 56-57. (Xin H Z.2021. Brief analysis of crop planting and pest control technology[J]. South China Agriculture, 15(03): 56-57.) [20] 徐伟慧, 王恒煦, 赵井明, 等. 2019. 西瓜枯萎病拮抗菌筛选及其拮抗性能研究[J]. 农业生物技术学报, 27(12): 2238-2247. (Xu W H, Wang H X, Zhao J M, et al.2019. Screening of antagonistic bacteria against Fusarium wilt of watermelon (Citrullus lanatus) and its antagonistic properties[J]. Journal of Agricultural Biotechnology, 27(12): 2238-2247.) [21] 赵沛, 冯自力, 师勇强, 等. 2019. 棉花内生真菌CEF-373菌株对棉花黄萎病的防效及其作用机理[J]. 植物保护学报, 46(06): 1203-1213. (Zhao P, Feng Z L, Shi Y Q, et al.2019. Biological control effect and mechanism of cotton endophytic fungus Fusarium solani CEF-373 against Verticillium wilt in Gossypium hirsutum[J]. Journal of Plant Protection, 46(06): 1203-1213.) [22] 张慧, 马连杰, 卢文才, 等. 2021. 草假单胞菌HT1在蚕豆根和茎的定殖特性及对内生细菌多样性的影响[J].微生物学通报, 48(12): 4677-4687. (Zhang H, Ma L J, Lu W C, et al.2021. Colonization characteristics of Pseudomonas grass HT1 in root and stem of Vicia faba and its effect on endophytic bacteria diversity[J]. Chinese Journal of Microbiology, 48(12): 4677-4687.) [23] 朱文涛, 王红兰, 连艳, 等. 2021.百部杀虫作用研究进展[J]. 中药材, 44(08): 2012-2017. (Zhu W T, Wang H L, Lian Y, et al.2021. Research progress on insecticidal effect of Radix stemonae[J]. Chinese Journal of Medicinal Materials, 44(08): 2012-2017.) [24] 邹璐, 唐凌, 韩涵, 等. 2021. 百部治疗呼吸系统疾病的基础研究进展[J]. 上海医药, 42(01): 10-13. (Zou L, Tang L, Han H, et al.2021. The progress of basic researches on Radix stemonae for the treatment of respiratory diseases[J]. Shanghai Medical & Pharmaceutical Journal, 42(01): 10-13.) [25] Ahmad T, Bashir A, Farooq S, et al.2021. Burkholderia gladioli E39CS3, an endophyte of Crocus sativus Linn, induces host resistance against corm-rot caused by Fusarium oxysporum[J]. Journal of applied microbiology, 132(01): 495-508. [26] Bari L M, Rakan A, Faeza N T.2019. Biological control of Fusarium wilt in tomato by endophytic rhizobactria[J]. Energy Procedia, 157(5): 171-179. [27] Cui G, Yin K, Lin N, Liang M, et al.2020. Burkholderia gladioli CGB10: Anovel strain biocontrolling the sugarcane smut disease[J]. Microorganisms, 8(12): 19-43. [28] Dilfuza E, Kakhramon D, Stephan W, et al.2017. Impact of soil salinity on the plant-growth promoting and biological control abilities of root associated bacteria[J]. SAUDI Journal of Biological Sciences, 24(7): 1601-1608. [29] Ge B B, Cheng Y, Liu Y, et al.2015. Biological control of Botrytis cinerea on tomato plants using Streptomyces ahygroscopicus strain CK-15[J]. Letters in Applied Microbiology, 61(6): 596-602. [30] Jian W, Cao H, Yuan S, et al.2019. SlMYB75, an MYB-type transcription factor, promotes anthocyanin accumulation and enhances volatile aroma production in tomato fruits[J]. Horticulture research, 6(1): 22-37. [31] Karniel U, Koch A, Zamir D, et al.2020. Development of zeaxanthin-rich tomato fruit through genetic manipulations of carotenoid biosynthesis[J]. Plant Biotechnology Journal, 18(11): 2292-2303. [32] Liu H, Carvalhais L C, Crawford M, et al.2017. Inner plant values: Diversity, colonization and benefits from endophytic bacteria[J]. Frontiers in Microbiology, 8(5): 2552-2569. [33] Łukasz P H, Michalina K, Michał K, et al.2015. Cuticular wax variation in the tomato (Solanum lycopersicum L.), related wild species and their interspecific hybrids[J]. Biochemical Systematics and Ecology, 60(5): 215-224. [34] Ravikumar M J, Ramasamy S.2018. Biopriming of micropropagated banana plants at pre-or post-BBTV inoculation stage with rhizosphere and endophytic bacteria determines their ability to induce systemic resistance against BBTV in cultivar Grand Naine[J]. Biocontrol Science and Technology, 28(11): 1074-1090. [35] Ruangwong O, Pornsuriya C, Pitija K, et al.2021. Biocontrol mechanisms of Trichoderma koningiopsis PSU3-2 against postharvest anthracnose of chili pepper[J]. Journal of Fungi, 7(4): 276-286. [36] Wang S, Ji B, Su X, et al.2020. Isolation of endophytic bacteria from Rehmannia glutinosa Libosch and their potential to promote plant growth[J]. The Journal of General and Applied Microbiology, 66(5): 279-288. [37] Zhuo S, Li M Y, Mei H, et al.2019. Biological control ginseng grey mold and plant colonization by antagonistic bacteria isolated from rhizospheric soil of Panax ginseng Meyer[J]. Biological Control, 138(C): 138-151. [38] Zulma R S, Jesús C M, Bruna G C, et al.2012. Common features of environmental and potentially beneficial plant-associated Burkholderia[J]. Microbial Ecology, 63(2): 249-266.
