枯草芽胞杆菌VD18R19在胡椒上的定殖动态及促生作用和对胡椒瘟病的防治效果

doi:10.16409/j.cnki.2095-039x.2017.05.011

摘要/Abstract

摘要： 生防芽胞杆菌Bacillus subtilis定殖能力是决定其应用效果的关键因素。目前，芽胞杆菌在胡椒上的定殖情况鲜有报道。本研究以生防芽胞杆菌VD18R19为对象，研究其在胡椒根系和叶片表面的定殖动态及其促生长作用和对胡椒瘟病的防治效果。首先利用荧光质粒pAD43-25标记菌株VD18R19；然后通过荧光观测和平板计数方法检测标记菌株在胡椒根系和叶片表面的定殖形态和数量动态；最后通过盆栽试验分别评估了该菌株的促生长作用和对胡椒瘟病的防治效果。结果表明，菌株VD18R19荧光标记成功，且标记具有良好的遗传稳定性（120世代后高达85.33%）；该菌株在胡椒根际偏好于根毛区定殖，定殖数量呈先升后降趋势，在处理后第9 d的定殖数量最多（9.88×10⁶ CFU/g），整个30 d的定殖密度均维持在10⁶ CFU/g水平；叶片定殖呈迅速下降趋势，处理后第9 d的定殖密度仅5.13×10⁴ CFU/g，在21 d后基本检测不到该菌株；在不接种病原菌的情况下，菌株VD18R19处理的胡椒苗的株高、叶片数和地上部分鲜重等均显著好于对照，其促生率分别为61.71%、36.18%和106.43%；胡椒瘟病病原菌Phytophthora capsici叶片接种后，菌株VD18R19处理的胡椒苗在发病率和病斑大小上均显著低于对照，对胡椒瘟病的防治效果达76.86%。以上研究结果揭示了芽胞杆菌VD18R19在胡椒上的定殖动态及其防治效果，对该菌的开发应用有重要指导意义。

关键词: 枯草芽胞杆菌, 胡椒瘟病, 定殖, 生物防治, 促生长

Abstract: Colonization is an important factor for application of beneficial Bacillus genus bacteria, however, colonization of B. subtilis in black pepper has not been reported yet. In this study, B. subtilis VD18R19 was applied to black pepper plants to investigate the colonization dynamics, growth promotion effect and the biological control effects on Phytophthora rot disease. To evaluate colonization ability, strain VD18R19 was labeled with green fluorescent protein (GFP). Green fluorescent light could be observed from the GFP-tagged strain under microscope, and the genetic stability was 85.33% after 120 generations. Distinct colonization tendencies were exhibited when treated by leaf spraying and root irrigating. At rhizosphere, increased colonization was initially observed over time to the 9th day, followed by decreased levels to the 21st day that were subsequently maintained at a stable level for the duration of the experiment. The population densities of VD18R19 were at 10⁶ CFU/g level through the experiment. At leaf surface, the colonization was decreased quickly, and few stains could be detected after 21 days. In the absence of pathogen, the VD18R19 treatments could significantly increase the plant height, leaf number and fresh weight, with the promotion rates of 61.71%, 36.18% and 106.43%, respectively. When challenging the pathogen Phytophthora capsici, plants treated by VD18R19 showed much lower disease incidence and less severe disease symptoms, with the control efficiency of 76.86%. The results are useful for development and application of B. subtilis in the future.

Key words: Bacillus subtilis, black pepper Phytophthora rot, colonization, biocontrol, growth promotion

中图分类号:

S476

高圣风, 刘爱勤, 桑利伟, 苟亚峰, 孙世伟, 王政, 孟倩倩. 枯草芽胞杆菌VD18R19在胡椒上的定殖动态及促生作用和对胡椒瘟病的防治效果[J]. 中国生物防治学报, 2017, 33(5): 650-657.

GAO Shengfeng, LIU Aiqin, SANG Liwei, GOU Yafeng, SUN Shiwei, WANG Zheng, MENG Qianqian. Colonization Dynamics and Growth Promotion Effect of Bacillus subtilis VD18R19 in Black Pepper Plant and Its Biological Control Effects on Phytophthora Rot Disease[J]. journal1, 2017, 33(5): 650-657.

参考文献

[1] 刘爱勤. 热带特色香料饮料作物主要病虫害防治图谱[M]. 北京:中国农业出版社, 2013, 4-11.
[2] Paul D, Anandaraj M, Kumar A, et al. Antagonistic mechanisms of pseudomonas fluorescent against Phytophthora capsici in black pepper (Piper nigrum L.)[J]. Journal of Spices and Aromatic Crops, 2005, 14(2):122-129.
[3] Aravind R, Kumar A, Eapen S J, et al. Endophytic bacterial flora in root and stem tissues of black pepper (Piper nigrum L.) genotype:isolation, identification and evaluation against Phytophthora capsici[J]. Letters in Applied Microbiology, 2009, 48(1):58-64.
[4] Ezziyyani M, Requena M E, Egea G C, et al. Biological control of Phytophthora root rot of pepper using Trichoderma harzianum and Streptomyces rochei in combination[J]. Journal of Phytopathology, 2007, 155(6):342-349.
[5] Lugtenberg B, Kamilova F. Plant-growth-promoting rhizobacteria[J]. Annual Review of Microbiology, 2009, 63:541-556.
[6] Kamilova F, Validov S, Azarova T, et al. Enrichment for enhanced competitive plant root tip colonizers selects for a new class of biocontrol bacteria[J]. Environmental Microbiology, 2005, 7(11):1809-1817.
[7] Benizri E, Baudoin E, Guckert A. Root colonization by inoculated plant growth-promoting rhizobacteria[J]. Biocontrol Science and Technology, 2001, 11(5):557-574.
[8] Gao S, Wu H, Wang W, et al. Efficient colonization and harpins mediated enhancement in growth and biocontrol of wilt disease in tomato by Bacillus subtilis[J]. Letters in Applied Microbiology, 2013, 57(6):526-533.
[9] Gao S, Wu H, Yu X, et al. Swarming motility plays the major role in migration during tomato root colonization by Bacillus subtilis, SWR01[J]. Biological Control, 2016, 98(1):11-17.
[10] Chen Y, Yan F, Chai Y, et al. Biocontrol of tomato wilt disease by Bacillus subtilis isolates from natural environments depends on conserved genes mediating biofilm formation[J]. Environmental Microbiology, 2013, 15(3):848.
[11] 高毓晗, 李世东, 郭荣君. sfp基因转化增强了Bacillus subtilis 168的定殖能力和对黄瓜茎内枯萎病菌的抑制作用[J]. 中国生物防治学报, 2016, 32(1):76-85.
[12] 高圣风, 刘爱勤, 桑利伟, 等. 香草兰生防细菌的筛选、分子鉴定及其抑菌机制的初步研究[J]. 热带农业科学, 2016, 36(1):41-46.
[13] 桑利伟, 刘爱勤, 谭乐和, 等. 海南省胡椒瘟病病原鉴定及发生规律[J]. 植物保护, 2011, 37(6):168-171.
[14] 高圣风. 枯草芽孢杆菌OKB105菌株在番茄根表定殖机制研究[D]. 南京:南京农业大学, 2012.
[15] Yuan J, Zhang N, Huang Q, et al. Organic acids from root exudates of banana help root colonization of PGPR strain Bacillus amyloliquefaciens NJN-6[J]. Scientific Reports, 2015, 5(13438):121-127.
[16] Li S, Zhang N, Zhang Z, et al. Antagonist Bacillus subtilis HJ5 controls Verticillium wilt of cotton by root colonization and biofilm formation[J]. Biology and Fertility of Soils, 2013, 49(3):295-303.
[17] 郝慧娟, 刘洪伟, 尹淑丽, 等. 枯草芽孢杆菌BSD-2的GFP标记及其在黄瓜上的定殖研究[J]. 华北农学报, 2016, 31(4):106-111.
[18] 刘邮洲, 梁雪杰, 乔俊卿, 等. 枯草芽胞杆菌PTS-394的GFP标记及其定殖能力[J]. 植物保护学报, 2014, 41(4):416-422.
[19] 吕雅悠, 丁方丽, 王娟, 等. 两株促植物生长根际细菌对辣椒的促生效果及其在辣椒根际定殖能力[J]. 北方园艺, 2015, 18:26-30.
[20] 乔俊卿, 陈志谊, 梁雪杰, 等. 枯草芽孢杆菌Bs916在番茄根部的定殖[J]. 江苏农业学报, 2015, 31(6):1278-1283.
[21] 田兆丰, 刘伟成, 董丹, 等. 生防枯草芽孢杆菌Kct99的GFP标记及其在甘蓝根部的定殖示踪[J]. 华北农学报, 2012, 27(6):53-57.
[22] 王颖, 杨成德, 薛莉, 等. 拮抗内生细菌Bacillus mojavensis ZA1在马铃薯根内及根际的定殖动态及其对土壤微生物的影响[J]. 中国生物防治学报, 2016, 32(3):372-378.
[23] Zhang N, Wu K, He X, et al. A new bioorganic fertilizer can effectively control banana wilt by strong colonization with Bacillus subtilis N11[J]. Plant and Soil, 2011, 344(1):87-97.
[24] Uren N C. Types, amounts, and possible functions of compounds released into the rhizosphere by soil-grown plants[M]//The Rrhizosphere. Biochemistry and Organic Substances at the Soil-plant Interface. New York:Marcel Dekker, 2007, 1-21.
[25] 陈志谊, 苗东华, 许志刚. 水稻纹枯病拮抗细菌的评价与利用[J]. 水稻科学, 2000, 14(2):58-63.
[26] 陈志谊, 刘摇荣, 刘永锋. 水稻纹枯病拮抗细菌B916的选育[J]. 中国生物防治, 2003, 19(1):15-18.
[27] 陈志谊, 刘永峰, 刘邮洲, 等. 植物病害生防芽孢杆菌研究进展[J]. 江苏农业学报, 2012, 28(5):999-1006.
[28] 梁晓琳, 孙莉, 张娟, 等. 利用Bacillus amyloliquefaciens SQR9研制复合微生物肥料[J]. 土壤, 2015, 47(3):558-563.
[29] 张楠, 吴凯, 沈怡斐, 等. 根际益生菌解淀粉芽孢杆菌SQR9在香蕉根表的定殖行为研究[J]. 南京农业大学学报, 2014, 37(6):59-65.
[30] 袁玉娟, 徐晨伟. 枯草芽孢杆菌SQR9促进黄瓜生长的效果[J]. 中国瓜菜, 2017, 30(3):25-28.
[31] 高学文, 谢珊珊, 伍辉军, 等. 一种枯草芽孢杆菌G1及其应用[P]. 2016. CN 103571780B
[32] 高学文, 谢珊珊, 伍辉军, 等. 枯草芽孢杆菌G1在促进植物生长中的应用[P]. 2014. CN103563996A
[33] Wang S, Wu H, Qiao J, et al. Molecular mechanism of plant growth promotion and induced systemic resistance to tobacco mosaic virus by Bacillus spp.[J]. Journal of Microbiology and Biotechnology, 2009, 19(10):1250-1258.
[34] Pieterse C M J, Zamioudis C, Berendsen R L, et al. Induced systemic resistance by beneficial microbes[J]. Annual Review of Phytopathology, 2014, 52:347.