松材线虫生防细菌的筛选、鉴定及其毒性因子的初步研究

doi:Q939.96

中国生物工程杂志

2010, Vol. 30

Issue (08): 76-81 DOI: Q939.96

研究报告

松材线虫生防细菌的筛选、鉴定及其毒性因子的初步研究

牛秋红^**,董冰雪,黄思良,惠丰立,柯涛,张林

南阳师范学院生命科学与技术学院南阳 473061

Screening, Identification and Virulence Factor Determination of the Bacteria with Nematicidal Activity to Bursaphelenchus xylophilus

NIU Qiu-hong,DONG Bing-xue,HUANG Si-liang,HUI Feng-li,KE Tao,ZHANG Lin

School of Life Science and Technology, Nanyang Normal University, Nanyang 473061, China

全文: PDF(699 KB) HTML

摘要：

从河南南阳不同农田的植物根部采取土壤样本，共分离获得了198株细菌。通过毒性测试和平板生测从中筛选出松材线虫生防细菌6株，其中 NS-3菌株对松材线虫的杀灭活性最高。结合该菌株的形态学、生理学特征及16S rDNA序列分析等结果将该菌株归为芽孢杆菌属，命名为Bacillus sp. strain NS-3。将该细菌液体培养的上清液和上清蛋白粗提物分别处理松材线虫48 h后线虫的死亡率分别达到50 %和100 %; 线虫死亡后虫体消解。然而，细菌的上清蛋白粗提物经煮沸变性后，基本丧失了对松材线虫的侵染活性，结果显示细菌Bacillus sp. strain NS3的杀线虫活性物质主要要存在于细菌培养上清液中，并且为热不稳定性物质。

关键词： 松材线虫; 生防细菌; 芽孢杆菌; 毒性因子

Abstract:

One hundred and ninety eight bacteria were isolated and tested for their nematicidal activity from the soil in Nanyang, Henan Province, China. Six strains which had nematicidal activity to Bursaphelenchus xylophilus were found. A bacterium, named Bacillus sp. strain NS-3, showed the highest nematicidal activity to B xylophilus. The bacterium was classified by phenotypic and genotypic characteristics, which belonged to Bacillus genus. The culture supernatant and the crude extracellular protein extract from the strain NS-3 separately killed 50 % and 100 % of the tested nematodes (B. xylophilus) within 48 h, which suggested their high toxic activity toward the nematodes. Most of the dead nematodes were degraded and only minor fragments were left. However, the crude protein extract lost nematicidal activity after boiled, which showed the namaticidal materials produced in the cultural filtrate by the bacterium was not stable to the heat.

Key words: Bursaphelenchus xylophilus Bacillus Virulence factor

收稿日期: 2010-01-15 出版日期: 2010-08-25

基金资助:

河南省重点学科生物化学与分子生物学建设项目及河南省高校科技创新团队支持计划项目“中国南北气候过渡带农业生物资源的保护与利用” (2010JRTSTHN012)、河南省教育厅自然科学研究计划(2009B180018)资助项目

通讯作者: 牛秋红 E-mail: niuqiuhong723@yahoo.com.cn

	服务
	把本文推荐给朋友
	加入引用管理器
	E-mail Alert
	RSS
	作者相关文章
	牛秋红
	董冰雪
	黄思良
	惠丰立
	柯涛
	张林

引用本文:

牛秋红董冰雪黄思良惠丰立柯涛张林. 松材线虫生防细菌的筛选、鉴定及其毒性因子的初步研究[J]. 中国生物工程杂志, 2010, 30(08): 76-81.

NIU Qiu-Gong, DONG Bing-Xue, HUANG Sai-Liang, HUI Feng-Li, KE Chao, ZHANG Lin. Screening, Identification and Virulence Factor Determination of the Bacteria with Nematicidal Activity to Bursaphelenchus xylophilus. China Biotechnology, 2010, 30(08): 76-81.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/Q939.96 或 https://manu60.magtech.com.cn/biotech/CN/Y2010/V30/I08/76

［1］董锦艳, 李铷, 张克勤. 松材线虫生物防治研究进展. 植物保护, 2005, 31 (5) : 915. Dong J Y, Li R, Zhang K Q. Plant Protection, 2005, 31 (5): 915.
［2］ Yang J K, Li J, Liang L M, et al. Cloning and characterization of an extracellular serine protease from the nematodetrapping fungus Arthrobotrys conoides. Arch Microbiol, 2007, 188: 167174.
［3］茆振川.防治根结线虫的原核生防菌的筛选及作用.杨凌: 西北农林科技大学, 2004. Mao Z C. Screening and function of biocontrol microbes against root knot nematodes . Yangling: Northwest SciTech University of Agriculture and Forestry, 2004.
［4］牛秋红, 黄晓玮, 徐进, 等. 细菌在线虫生防中应用的研究进展.生物技术, 2006 , 16 (1) : 9095. Niu Q H, Huang X W, Xu J, et al. Biotechnology, 2006, 16 (1): 9095.
［5］ Niu Q H, Huang X W, Zhang L, et al. Functional identification of the gene bace16 from nematophagous bacterium Bacillus nematocida. Appl Microbiol Biotechnol, 2007, 75(1): 141148.
［6］ Tian B Y, Yang J K, Zhang K Q. Bacteria used in biocontrol nematode: potential, development and perspective. FEMS Microbiol Ecol, 2007, 61: 197213.
［7］ Siddiqui I A, Haas D, Heeb S. Extracellular protease of Pseudomonas fluorescens CHA0, a biocontrol factor with activity against the rootknot nematode Meloidogyne incognita. Appl Environ Microbiol, 2005, 71(9): 56465649.
［8］ Wei J Z, Hale K, Carta L, et al. Bacillus thuringiensis crystal proteins that target nematodes. Proc Natl Acad Sci USA, 2003, 100(5): 27602765.
［9］海外传真. 农药, 2007, 46 (9) : 647. Overseas fax. Pesticide, 2007, 46 (9): 647.
［10］ Gray N F. Ecology of nematophagous fungi: comparison of the soil sprinkling method with the Baerman funnel technique in the isolation of endoparasites. Soil Biol Biochem, 1984, 16: 81~83.
［11］黄秀梨. 微生物学实验指导. 北京: 高等教育出版社, 2004. Huang X L. Experimental Guidance of Microbiology. Beijing: Higher Education Press, 2004.
［12］ Xu P, Li W J, Xu L H, et al. A microwavebased method for genomic DNA extraction from Actinomycetes, Microbiology, 2003, 30 (4): 7375.
［13］ Higgins D G, Bleasby A J, Fuchs R CLUSTAL V: Improved software for multiple sequence alignment. Comput Appl Biosci, 1992, 8: 189191.
［14］ Thompson J D, Gibson T J, Plewniak F, et al. The Clustal X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Research, 1997, 24: 48764882.
［15］ Kimura M. A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequence. J Mol Evol, 1980, 16:111120.
［16］ Saitou N, Nei M. The neighborjoining method: a new method for reconstructing phylogenetic tree. Mol Biol Evol, 1987, 4: 406425.
［17］ Kumars S, Tamura K, Nei M. MEGA 3: Integrated software for molecular evolutionary genetics analysis and sequence alignment. Briefings in Bio informatics, 2004, 5: 150163.
［18］ Stirling G R, Wachtel M F. Mass production of Bacillus penetrans for the biological control of rootknot nematodes. Nematologica, 1980, 26: 308312.
［19］ KerenZur M, Antonov J, Bercovitz A, et al. Bacillus firmus formulations for the safe control of rootknot nematodes. In: Proceedings of the Brighton Crop Protection Conference on Pests and Diseases, 2000, vol. 2A, 4752.
［20］ Giannakou I O, Karpouzas D G. Demetra ProphetouAthanasiadou. A novel nonchemical nematicide for the control of rootknot nematodes. Applied Soil Ecology, 2004, 26: 6979.
［21］ Soad A A, Xie G L, Li B, et al. Comparative performance of Bacillus spp. in growth promotion and suppression of tomato bacterial wilts caused by Ralstonia solanacearum. Journal of Zhejiang University (Agric & Life Sci), 2004, 30(6): 603610.
［22］ Huang X W, Tian B Y, Niu Q H, et al. An extracellular protease from Brevibacillus laterosporus G4 without parasporal crystals can serve as a pathogenic factor in infection of nematodes. Res Microbiol, 2005, 156: 719727.
［23］ Lian L H, Tian B Y, Xiong R, et al. Proteases from Bacillus: a new insight on the mechanism of action for rhizobacterial suppression of nematode populations. Letters in Applied Mircobiology, 2007, 45: 262269.
［24］ Chen X H, Koumoutsi A, Scholz R, et al. Comparative analysis of the complete genome sequence of the plant growthpromoting bacterium Bacillus amyloliquefaciens FZB42. Nat Biotechnol, 2007, 25: 10071014.

[1]	王晓洁,孟凡强,周立邦,吕凤霞,别小妹,赵海珍,陆兆新. 利用基因组改组技术提高短杆菌素产量及其培养条件优化^*[J]. 中国生物工程杂志, 2021, 41(8): 42-51.
[2]	周惠颖,周翠霞,张婷,王雪雨,张会图,冀颐之,路福平. 强化底物利用酶系表达,提升地衣芽孢杆菌生产碱性蛋白酶性能[J]. 中国生物工程杂志, 2021, 41(2/3): 53-62.
[3]	梅雨薇,杨子云,于樊,龙旭伟. 生物表面活性剂脂肽的发酵生产及抑菌应用研究进展^*[J]. 中国生物工程杂志, 2020, 40(5): 105-116.
[4]	马翠萍,刘朵朵,潘炳菊,申会涛,宋亚囝. 来源于嗜碱芽孢杆菌N16-5甘露聚糖利用基因簇的乙酰酯酶AesA的克隆及性质分析^*[J]. 中国生物工程杂志, 2020, 40(3): 65-71.
[5]	胡艳红,龚雪梅,丁柳柳,高嵩,李婷婷. 利用短短芽孢杆菌进行酮还原酶CgKR2的高效表达与纯化 ^*[J]. 中国生物工程杂志, 2019, 39(8): 59-65.
[6]	王鑫淼,张康,陈晟,吴敬. 嗜热网球菌纤维二糖差向异构酶在枯草芽孢杆菌中的表达及发酵优化 ^*[J]. 中国生物工程杂志, 2019, 39(7): 24-31.
[7]	李法彬,刘露,杜燕,班睿. 构建重组枯草芽孢杆菌催化制备D-对羟基苯甘氨酸[J]. 中国生物工程杂志, 2019, 39(3): 75-86.
[8]	程功,焦思明,任立世,冯翠,杜昱光. 枯草芽孢杆菌壳聚糖酶水解制备低脱乙酰度壳寡糖及其组分分析 ^*[J]. 中国生物工程杂志, 2018, 38(9): 19-26.
[9]	孙帆,宿玲恰,张康,吴敬. D-阿洛酮糖 3-差向异构酶在枯草芽孢杆菌中的高效表达及固定化细胞研究 ^*[J]. 中国生物工程杂志, 2018, 38(7): 83-88.
[10]	王男,金吕华,张玲,林荣,杨海麟. *信号肽对亮氨酸脱氢酶在Bacillus subtilis中分泌表达的影响及酶学性质研究*[J]. 中国生物工程杂志, 2018, 38(4): 46-53.
[11]	张玲,王男,金吕华,林荣,杨海麟. 双启动子促进亮氨酸脱氢酶在Bacillus subtilis中表达及发酵研究 ^*[J]. 中国生物工程杂志, 2018, 38(12): 21-31.
[12]	刘宇帅,张杰,钟瑾,李晶,孟利强,张淑梅. 解淀粉芽孢杆菌TF28抗菌脂肽芬芥素的分离鉴定及抑菌作用 ^*[J]. 中国生物工程杂志, 2018, 38(10): 20-29.
[13]	李阳, 吴酬飞, 吴小芹, 叶建仁, 张立钦. Cry3a杀虫蛋白基因的合成优化及在JK-SH007中高效表达[J]. 中国生物工程杂志, 2017, 37(6): 70-77.
[14]	赵一瑾, 王腾飞, 汪俊卿, 王瑞明. 以CotC为分子载体在枯草芽孢杆菌表面展示海藻糖合酶[J]. 中国生物工程杂志, 2017, 37(1): 71-80.
[15]	刘婧莹, 刘晨浪, 张允雷, 王世奇, 夏立秋, 张友明. 牛乳铁蛋白肽(LfcinB)在苏云金芽胞杆菌中的融合表达[J]. 中国生物工程杂志, 2016, 36(8): 23-30.

Viewed

Full text

Abstract

Cited

Shared

Discussed