群体感应信号物质对吡咯伯克霍尔德氏菌JK-SH007在杨树内定殖的影响

doi:10.16409/j.cnki.2095-039x.2019.06.023

摘要/Abstract

摘要： 为了明确杨树溃疡病生防菌吡咯伯克霍尔德氏菌JK-SH007的群体感应系统是否与其内生定殖相关，本文通过群体感应指示菌紫色杆菌CV026和液相色谱串联四级杆飞行时间质谱技术（HPLC-Q-TOF-MS）确定其群体感应信号物质种类，并利用结晶紫染色、菌体回收、GFP标记等技术，研究其群体感应信号物质对该菌株生物膜及在杨树苗内的定殖能力的影响。结果表明，菌株JK-SH007产生的群体感应信号物质为含8个碳原子酰基侧链的辛酰基-L-高丝氨酸内酯（C₈-HSL）。群体感应信号物质C₈-HSL的添加对该菌株生物膜及在杨树苗内的定殖能力有影响，并且呈现低浓度促进，较高浓度抑制的规律。C₈-HSL的添加与菌株JK-SH007生物膜的OD值和菌体量具有显著相关性，相关系数分别为0.923和0.756。当添加1.0% C₈-HSL终浓度达到5×10^{-8 g/L}时，菌株JK-SH007生物膜的形成量达到峰值。荧光显微镜镜检发现，当添加100 μL C₈-HSL时，杨树组培苗根和茎中GFP荧光标记的菌株JK-SH007的定殖数量明显较CK多。菌株JK-SH007的群体感应与其在杨树内的内生定殖能力密切相关，群体感应信号物质C₈-HSL具有增强菌株JK-SH007内生定殖的能力，同时也表明该物质有利于该菌株生防效果的发挥。

关键词: 吡咯伯克霍尔德氏菌, 杨树溃疡病, 群体感应, N-酰基高丝氨酸内酯, 内生定殖

Abstract: In order to clarify whether the quorum sensing of Burkholderia pyrrocinia JK-SH007 with high biocontrol activity against poplar cancer is related to its endogenous colonization, the type of quorum sensing signal substance of JK-SH007 strain was determined by the quorum sensing indicator Chromobacterium violaceum CV026 and liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry (HPLC-Q-TOF-MS). The effects of quorum sensing signal substance on the biofilm and endogenous colonization of JK-SH007 strain in poplar seedlings were studied by crystal violet staining, colonization recovery and GFP labeling. The results showed that the quorum sensing signal substance produced by JK-SH007 strain was N-octanoyl-DL-homoserine lactone (C₈-HSL) containing acyl side chain of 8 carbon atoms. The addition of quorum sensing signal substance C₈-HSL had a certain effect on the biofilm of the strain and the colonization ability of the strain in poplar seedlings, showing promotion at low concentrations and inhibition at higher concentrations. The addition of C₈-HSL was significantly correlated with the OD value and the amount of bacteria in the strain JK-SH007 biofilm, which were 0.923 and 0.756, respectively. When 1.0% (volume fraction) of C₈-HSL was added (final concentration reached 5×10^-8 g/L), the amount of JK-SH007 biofilm formation peaked. Fluorescence microscopy revealed that when 100 μL of C₈-HSL were added, the number of colonized GFP fluorescently labeled strain JK-SH007 in roots and stems of poplar tissue culture was significantly higher than that of CK. The above results indicate that the quorum sensing of JK-SH007 strain is closely related to its endogenous colonization ability in poplar seedlings. The quorum sensing signal substance C₈-HSL can enhance the endogenous colonization effect of strain JK-SH007 in poplar seedlings, and also indicates that the substance is beneficial to the biocontrol effect of the strain.

Key words: Burkholderia pyrrocinia, poplar canker disease, quorum sensing, N-acyl-homoserine lactones, endogenous colonization

中图分类号:

S476

杨欣, 张鹏飞, 白变霞, 叶建仁, 任嘉红. 群体感应信号物质对吡咯伯克霍尔德氏菌JK-SH007在杨树内定殖的影响[J]. 中国生物防治学报, 2019, 35(6): 949-957.

YANG Xin, ZHANG Pengfei, BAI Bianxia, YE Jianren, REN Jiahong. Effect of Quorum Sensing Signal Substance on Endogenous Colonization of Burkholderia pyrrocinia JK-SH007 in Poplar Seedlings[J]. Chinese Journal Of Biological Control, 2019, 35(6): 949-957.

参考文献

[1] Schenk S T, Schikora A. AHL-priming functions via oxylipin and salicylic acid[J]. Frontiers in Plant Science, 2015, 5:1-7.
[2] Zhu J, Mekalanos J J. Quorum sensing-dependent biofilms enhance colonization in Vibrio cholerae[J]. Developmental Cell, 2003, 5(4):647-656.
[3] Pierson L S, Keppenne V D, Wood D W. Phenazine antibiotic biosynthesis in Pseudomonas aureofaciens 30-84 is regulated by PhzR in response to cell density[J]. Journal of Bacteriology, 1994, 176(13):3966-3974.
[4] Swift S, Downie J A, Whitehead N A, et al. Quorum sensing as a population-density-dependent determinant of bacterial physiology[J]. Advances in Microbial Physiology, 2001, 45:199-270.
[5] Zhang Z, Pierson L S. A second quorum-sensing system regulates cell surface properties but not phenazine antibiotic production in Pseudomonas aureofaciens[J]. Applied and Environmental Microbiology, 2001, 67(9):4305-4315.
[6] Wei H L, Zhang L Q. Quorum-sensing system influences root colonization and biological control ability in Pseudomonas fluorescens 2P24[J]. Antonie van Leeuwenhoek, 2006, 89(2):267-280.
[7] Ravn L, Christensen A B, Molin S, et al. Methods for detecting acylated homoserine lactones produced by gram-negative bacteria and their application in studies of AHL-production kinetics[J]. Journal of Microbiological Methods, 2001, 44(3):239-251.
[8] Huber B, Feldmann F, Kothe M, et al. Identification of a novel virulence factor in Burkholderia cenocepacia H111 required for efficient slow killing of Caenorhabditis elegans[J]. Infection and Immunity, 2004, 72(12):7220-7230.
[9] 谢关林, 金扬秀, 徐传雨, 等. 我国水稻纹枯病拮抗细菌种类研究[J]. 中国生物防治学报, 2003, 19(4):166-170.
[10] Lutter E, Lewenza S, Dennis J J, et al. Distribution of quorum-sensing genes in the Burkholderia cepacia complex[J]. Infection and Immunity, 2001, 69(7):4661-4666.
[11] 刘长建, 权春善, 范圣第. Burkholderia属luxIR同源群体感应系统的研究进展[J]. 安徽农业科学, 2008, 36(14):5754-5756.
[12] 任嘉红, 班虎栋, 叶建仁. 吡咯伯克霍尔德氏菌JK-SH007的发酵条件及其对杨树溃疡病的防治效果[J]. 中国生物防治, 2010, 26(3):300-306.
[13] Ren J H, Ye J R, Liu H, et al. Isolation and characterization of a new Burkholderia pyrrociniastrain JK-SH007 as a potential biocontrol agent[J]. World Journal of Microbiology and Biotechnology, 2011, 27(9):2203-2215.
[14] Tomlin K L, Clark S R D, Ceri H. Green and red fluorescent protein vectors for use in biofilm studies of the intrinsically resistant Burkholderia cepacia complex[J]. Journal of Microbiol Methods, 2004, 57(1):95-106.
[15] 綦国红, 董名盛, 王岁楼. N-酰基高丝氨酸内酯类群体感应信号分子检测方法的建立[J]. 农业生物技术学报, 2007, 15(4):694-699.
[16] Chen R, Zhou Z, Cao Y, et al. High yield expression of an AHL-lactonase from Bacillus sp. B546 in Pichia pastoris and its application to reduce Aeromonas hydrophila mortality in aquaculture[J]. Microbial Cell Factories, 2010, 9(1):39.
[17] Hamon M, Lazazzera B. The sporulation transcription factor Spo0A is required for biofilm development in Bacillus subtilis[J]. Molecular Microbiology, 2001, 42(5):1199-1209.
[18] 李京宝, 韩峰, 于文功. 细菌生物膜研究技术[J]. 微生物学报, 2007, 47(3):558-561.
[19] 任嘉红, 刘辉, 姜楠, 等. GFP标记溶磷草木樨中华根瘤菌CHW10B及其定殖[J]. 林业科学, 2015, 51(1):74-79.
[20] 赵芊, 贾振华, 宋水山. 细菌信号分子N-酰基高丝氨酸内酯调控植物抗性的研究进展[J]. 植物生理学报, 2014(2):143-149.
[21] 丁丽娜, 杨瑞英, 杨国兴. N-乙酰基高丝氨酸内酯调节细菌与植物间互作的研究进展[J]. 湖北农业科学, 2016, 55(14):3537-3543.
[22] Schuhegger R, Ihring A, Gantner S, et al. Induction of systemic resistance in tomato by N-acyl-L-homoserine lactone-producing rhizosphere bacteria[J]. Plant Cell Environment, 2006, 29(5):909-918.
[23] Rad U V, Klein I, Dobrev P I, et al. Response of Arabidopsis thaliana to N-hexanoyl-DL-homoserine-lactone, a bacterial quorum sensing molecule produced in the rhizosphere[J]. Planta, 2008, 229(1):73-85.
[24] Mcclean K H, Winson M K, Fish L, et al. Quorum sensing and Chromobacterium violaceum:exploitation of violacein production and inhibition for the detection of N-acylhomoserine lactones[J]. Microbiology, 1997, 143(12):3703-3711.
[25] Hartmann A, Schikora A. Quorum sensing of bacteria and trans-kingdom interactions of N-acyl homoserine lactones with eukaryotes[J]. Journal of Chemical Ecology, 2012, 38(6):704-713.
[26] 李佳梅. 群感信号AHLs对杨树幼苗细根形态建成的影响与调控[D]. 泰安:山东农业大学, 2018.
[27] Brackman G, Celen S, Baruah K, et al. AI-2 quorum-sensing inhibitors affect the starvation response and reduce virulence in several Vibrio species, most likely by interfering with LuxPQ[J]. Microbiology, 2009, 155(12):4114-4122.
[28] Lynch M J, Swift S, Kirke D F, et al. The regulation of biofilm development by quorum sensing in Aeromonas hydrophila[J]. Environmental Microbiology, 2002, 4(1):18-28.
[29] 叶晓敏, 陆春. 霍乱弧菌群体感应系统影响生物膜形成的研究进展[J]. 微生物学杂志, 2010, 30(1):80-83.
[30] 凡肖, 束长龙, 关雄, 等. 细菌生物被膜在生物防治中的作用[J]. 中国生物防治学报, 2018, 34(5):791-801.
[31] 邬慧颖, 韩雪, 张丽娟, 等. 群体感应系统及其在乳酸菌生物膜形成中的作用[J]. 食品工业科技, 2018, 39(1):318-322.
[32] Chen Y, Cao S, Chai Y, et al. A Bacillus subtilis sensor kinase involved in triggering biofilm formation on the roots of tomato plants[J]. Molecular Microbiology, 2012, 85(3):418-430.
[33] 周礼杰, 王鑫, 韩倩, 等. C8-oxo-HSL对P. aeruginosa在污水处理中生长特性影响[J]. 中国环境科学, 2017, 37(5):1930-1936.
[34] Flickinger S T, Copeland M F, Downes E M, et al. Quorum sensing between Pseudomonas aeruginosa biofilms accelerates cell growth[J]. Journal of the American Chemical Society, 2011, 133(15):5966-5975.
[35] 葛静慧, 崔玉娜, 王晶, 等. 即食海参优势腐败菌群体感应信号分子识别[J]. 中国食品学报, 2015, 15(11):186-191.
[36] 任嘉红, 吴小芹, 刘辉, 等. 吡咯伯克霍尔德氏菌JK-SH007抗菌蛋白的分离纯化[J]. 微生物学通报, 2010, 37(6):872-880.
[37] Boon C, Deng Y, Wang L H, et al. A novel DSF-like signal from Burkholderia cenocepacia interferes with Candida albicans morphological transition[J]. Isme Journal, 2008, 2(1):27-36.
[38] Deng Y, Boon C, Eberl L, et al. Differential modulation of Burkholderia cenocepacia virulence and energy metabolism by the quorum-sensing signal BDSF and its synthase[J]. Journal of Bacteriology, 2009, 191(23):7270-7278.
[39] Deng Y, Wu J, Eberl L, et al. Structural and functional characterization of diffusible signal factor family quorum-sensing signals produced by members of the Burkholderia cepacia complex[J]. Applied and Environmental Microbiology, 2010, 76(14):4675-4683.