蜡样芽孢杆菌NJSZ-13菌株防治松材线虫病研究

尹艳楠; 谈家金; 李梦伟; 许嘉麟; 郝德君

doi:10.12302/j.issn.1000-2006.201910013

尹艳楠, 谈家金, 李梦伟, 许嘉麟, 郝德君

南京林业大学学报（自然科学版） ›› 2021, Vol. 45 ›› Issue (3) : 152-158.

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南京林业大学学报（自然科学版） ›› 2021, Vol. 45 ›› Issue (3) : 152-158. DOI: 10.12302/j.issn.1000-2006.201910013

研究论文

蜡样芽孢杆菌NJSZ-13菌株防治松材线虫病研究

作者信息 +

A study on the biocontrol of pine wilt disease by Bacillus cereus NJSZ-13

Author information +

文章历史 +

摘要

【目的】松材线虫病是一种由松材线虫对赤松、马尾松、黑松等易感树种寄生取食继而引起树种死亡的病害,其发病速度快、易传播,传统的物理、化学防治成本高,且化学防治对环境易造成不可逆的污染,目前还没有较好的防治方法。为寻找安全有效的生物防治方法,笔者探究蜡样芽孢杆菌在温室下对马尾松盆栽苗松材线虫病的防治效果。【方法】采用人工皮接法将2 000、4 000和6 000条松材线虫AMA3虫株分别接种到2年生马尾松幼苗上,测定发生松材线虫病症状的临界值;蜡样芽孢杆菌NJSZ-13菌株是从南京湿地松上分离出的具有杀线虫作用的松树内生细菌,在接种松材线虫AMA3虫株的前7天、当天和接种后第7天用灌根法灌入蜡样芽孢杆菌NJSZ-13菌株菌粉水悬液,并设置对照试验:将发酵液、菌悬液和发酵滤液稀释10倍后,各取20 mL灌注到2年生马尾松根部,其中发酵液和菌悬液的菌体浓度为4×10⁸ cfu/mL,以无菌水和培养基处理为对照,比较蜡样芽孢杆菌不同发酵产物对松材线虫病的防治效果;以灌根法分别施入20 mL菌体浓度为4×10⁸、4×10⁷、2×10⁷ cfu/mL的蜡样芽孢杆菌NJSZ-13菌株菌粉水悬液;将蜡样芽孢杆菌以菌粉水悬液灌根法和菌粉涂抹法两种方式处理2年生马尾松幼苗。从施菌浓度、施菌方法和施菌时间等3个方面对经过低温喷雾干燥制备的蜡样芽孢杆菌菌粉进行防治松材线虫病试验,观察各处理下马尾松的发病情况。【结果】2年生马尾松接种松材线虫AMA3虫株后发病的临界值为4 000条。采用灌根法施入蜡样芽孢杆菌NJSZ-13菌株的发酵液和无菌发酵滤液后不能对松材线虫病起到预防作用,施蜡样芽孢杆菌菌悬液对松材线虫病有一定的预防效果(33%)。蜡样芽孢杆菌对马尾松松材线虫病的预防效果要大于治疗效果,健康马尾松在接种线虫之前以灌根法施入蜡样芽孢杆菌的预防效果会更好。将蜡样芽孢杆菌菌粉直接涂抹松枝,与菌粉水悬液灌根的方法相比,两者对松材线虫病的防治效果相当。同一菌体浓度下蜡样芽孢杆菌菌粉水悬液和发酵菌悬液对松材线虫病的预防效果相当;施用蜡样芽孢杆菌菌粉水悬液对松材线虫病的防治效果随菌体浓度的增加有所提高,当菌体浓度达到一定值以后,增加浓度也不会提高防治效果。【结论】在蜡样芽孢杆菌NJSZ-13菌株防治2年生马尾松幼苗松材线虫病前,先筛选2年生马尾松接种松材线虫的临界值,能有效避免因接种松材线虫浓度过大而使真实防治效果降低的缺点。蜡样芽孢杆菌NJSZ-13菌株菌悬液和经低温喷雾干燥制得的菌粉对松材线虫病均有一定的预防效果,在马尾松未感染松材线虫的健康状态施用蜡样芽孢杆菌NJSZ-13菌株效果更好,通过菌粉涂抹法处理松枝可以对松材线虫病产生预防效果。

Abstract

【Objective】The pine wilt disease caused by parasitic Bursaphelenchus xylophilus occurs in susceptible tree species such as Pinus densiflora, P. massoniana and P. thunbergii. There is no optimal control treatment available owing to the fast onset and spread of the disease, high costs of physical and chemical control methods, and environmental pollution issues. To determine safe and effective biological control methods, this study explored the prevention and control of the disease by Bacillus cereus NJSZ-13 using potted P. massoniana seedlings in a greenhouse. 【Method】Different number of Bursaphelenchus xylophilus AMA3 (2 000, 4 000 and 6 000) were inoculated on two-year-old P. massoniana seedlings to determine the critical value of symptoms of the pine wilt disease. Bacillus cereus NJSZ-13 is a nematocidal endophyte that was isolated from P. elliottii in Nanjing. To understand the effect of B. cereus NJSZ-13 on the pine wilt disease, the roots of two-year-old P. massoniana were inoculated with a 20 mL suspension of 4×10 ⁸ cfu/mL of B. cereus NJSZ-13 as the treatment and with sterile water and an agar medium as the control. Twenty milliliters of bacterial powder suspension of 4×10 ⁸,4×10⁷ and 2×10⁷ cfu/mL of B. cereus NJSZ-13 were applied by the root filling method. Two-year-old P. massoniana seedlings were treated under two conditions, namely root filling with powder suspension and application of bacterial powder. Bacillus cereus NJSZ-13 and B. xylophilus AMA3 were inoculated with the P. massoniana roots in different orders, namely together, before 7 d, and after 7 d, and the control group was set up. The bacterial concentration, inoculation method, and time were included in the evaluation of the biocontrol effect. 【Result】The critical point of B. xylophilus inoculation in P. massoniana was 4 000. The fermented culture and sterilized culture of B. cereus NJSZ-13 had no effect on the pine wilt disease development. The biocontrol effect of the B. cereus NJSZ-13 suspension on the pine wilt disease was 33%. At the same concentration, the preventive effect of B. cereus NJSZ-13 did not differ between the powder and bacterial suspensions. The biocontrol effect of B. cereus NJSZ-13 did not improve when the bacterial suspension concentration reached a certain level. The biocontrol effect of B. cereus NJSZ-13 was significantly higher than that of the control. The control effect of B. cereus NJSZ-13 powder application was the same as that of the root filling method. 【Conclusion】The critical point of B. xylophilus inoculation in two-year-old P. massoniana can effectively avoid the disadvantage of an excessive concentration of B. xylophilus inoculation, which can reduce the control effect. The B. cereus NJSZ-13 bacterial suspension and powder had control effects on the pine wilt disease. It is better to apply B. cereus NJSZ-13 when P. massoniana is still healthy and uninfected. The preventive effect of B. cereus NJSZ-13 can be achieved by smearing the bacteria on pine branches.

导出引用

尹艳楠, 谈家金, 李梦伟, 等. 蜡样芽孢杆菌NJSZ-13菌株防治松材线虫病研究[J]. 南京林业大学学报（自然科学版）. 2021, 45(3): 152-158 https://doi.org/10.12302/j.issn.1000-2006.201910013

YIN Yannan, TAN Jiajin, LI Mengwei, et al. A study on the biocontrol of pine wilt disease by Bacillus cereus NJSZ-13[J]. JOURNAL OF NANJING FORESTRY UNIVERSITY. 2021, 45(3): 152-158 https://doi.org/10.12302/j.issn.1000-2006.201910013

中图分类号： S763

参考文献

列表( 原文顺序 | 文献年度倒序 | 文中引用次数倒序 ) 可视化分析

[1]	方丽英. 松材线虫病[J]. 广西林业, 2002(1):38-38. FANG L Y. Pine wilt disease[J]. Guangxi For, 2002 (1):38. DOI: 10.3969/j.issn.1004-0390.2002.01.030. 10.3969/j.issn.1004-0390.2002.01.030 本文引用 [1]

[2]	孙永春. 南京中山陵发现松材线虫[J]. 江苏林业科技, 1982,9(4):47-27. SUN Y C. The discovery of Bursaphelenchus xylophilus in Zhongshan Mausoleum[J]. J Jiangsu For Sci Technol, 1982,9(4):47-27. 本文引用 [1]

[3]	理永霞, 张星耀. 松材线虫病致病机理研究进展[J]. 环境昆虫学报, 2018,40(2):231-241. LI Y X, ZHANG X Y. Research advance of pathogenic mechanism of pine wilt disease[J]. J Environ Entomol, 2018,40(2):231-241. DOI: 10.3969/j.issn.1674-0858.2018.02.1. 10.3969/j.issn.1674-0858.2018.02.1 本文引用 [1]

[4]	胡龙娇, 吴小芹. 松树抗松材线虫病机制研究进展[J]. 生命科学, 2018,30(6):659-666. HU L J, WU X Q. Research progress on the mechanism of pine response to the infection of Bursaphelenchus xylophilus[J]. Chin Bull Life Sci, 2018,30(6):659-666. DOI: 10.13376/j.cbls/20180601. 10.13376/j.cbls/20180601 本文引用 [1]

[5]	蒋敏, 黄斌, 余旭, 等. 松材线虫病的分布、危害及其防治对策[J]. 浙江林业科技, 2018,38(6):83-91. JIANG M, HUANG B, YU X, et al. Distribution, damage and control of pine wilt disease[J]. J Zhejiang For Sci Technol, 2018,38(6):83-91. DOI: 10.3969/j.issn.1001-3776.2018.06.015. 10.3969/j.issn.1001-3776.2018.06.015 本文引用 [1]

[6]	徐红梅, 赵青, 殷涛. 以松材线虫为靶标生物防治技术研究[J]. 湖北林业科技, 2018,47(1):51-55. XU H M, ZHAO Q, YIN T. Advances in biological control targeting on Bursaphelenchus xylophilus[J]. Hubei For Sci Technol, 2018,47(1):51-55. DOI: 10.3969/j.issn.1004-3020.2018.01.016. 10.3969/j.issn.1004-3020.2018.01.016 本文引用 [1]

[7]	GOWEN S R. Chemical control of nematodes:efficiency and side-effects[J]. Fao Plant Production & Protection Paper, 1997,144:59-65. 本文引用 [1]

[8]

牛秋红, 董冰雪, 黄思良, 等. 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(8):76-81.

NIU Q

, DONG B

, HUANG S

, et al. Screening, identification and virulence factor determination of the bacteria with nematicidal activity to Bursaphelenchus xylophilus[J]. China Biotechnol, 2010,30(8):76-81. DOI: 10.13523/j.cb.20100814.

10.13523/j.cb.20100814

本文引用 [1]

[9]

余子全, 周燚, 孙明, 等. 苏云金芽孢杆菌防治植物寄生线虫的研究进展[J]. 植物保护学报, 2004,31(4):418-424.

YU Z

, ZHOU

, SUN

, et al. Progress of researches on activity of Bacillus thuringiensis against plant-parasitic nematodes[J]. Acta Phytophylacica Sin, 2004,31(4):418-424. DOI: 10.3321/j.issn:0577-7518.2004.04.015.

10.3321/j.issn:0577-7518.2004.04.015

本文引用 [1]

[10]	史应武, 娄恺, 李春. 植物内生菌在生物防治中的应用[J]. 微生物学杂志, 2009,29(6):61-64. SHI Y W, LOU K, LI C. The application of endophytic microbes in bio-control[J]. J Microbiol, 2009,29(6):61-64. DOI: 10.3969/j.issn.1005-7021.2009.06.013. 10.3969/j.issn.1005-7021.2009.06.013 本文引用 [1]

[11]

李亮亮, 谈家金, 陈凤毛. 两株松材线虫拮抗细菌的筛选和鉴定[J]. 南京林业大学学报(自然科学版), 2017,41(4):37-41.

LI L

, TAN J

, CHEN F

. The screening and identification of two bacterial strains with nematicidal activity against Bursaphelenchus xylophilus[J]. J Nanjing For Univ(Nat Sci Ed), 2017,41(4):37-41. DOI: 10.3969/j.issn.1000-2006.201601057.

10.3969/j.issn.1000-2006.201601057

本文引用 [2]

[12]	LI L L, TAN J J, CHEN F M, et al. Colonization of Bacillus cereus NJSZ-13, a species with nematicidal activity in masson pine (Pinus massoniana Lamb.)[J]. J For Res, 2018: 1-9. DOI: 10.1007/s11676-018-0823-2. 10.1007/s11676-018-0823-2 本文引用 [2]

[13]	丁晓磊. 基于高通量测序的松材线虫致病机理与毒力差异研究[D]. 南京: 南京林业大学, 2016. DING X L. The pathogenic mechanism and virulence variations analyses of Bursaphelenchus xylophilus based on high-through putsequencing[D]. Nanjing:Nanjing Forestry University, 2016. 本文引用 [1]

[14]	毛小芳, 李辉信, 陈小云, 等. 土壤线虫三种分离方法效率比较[J]. 生态学杂志, 2004,23(3):149-151. MAO X F, LI H X, CHEN X Y, et al. Extraction efficiency of soil nematodes by different methods[J]. Chin J Ecol, 2004,23(3):149-151. DOI: 10.13292/j.1000-4890.2004.0101. 10.13292/j.1000-4890.2004.0101 本文引用 [1]

[15]

陈凤毛, 史延梅, 王姝颖, 等. 不同株系拟松材线虫对黄山松和黑松苗木的致病性[J]. 林业科学, 2010,46(12):86-90.

CHEN F

, SHI Y

, WANG S

, et al. Pathogenicity of different isolates of Bursaphelenchus mucronatus to Pinus taiwanensis and P. thunbergii seedlings[J]. Sci Silvae Sin, 2010,46(12):86-90. DOI: 10.11707/j.1001-7488.20101214.

10.11707/j.1001-7488.20101214

本文引用 [1]

[16]

翁启勇, 陈庆河, 赵健, 等. 利福平标记菌株BS1在番茄、茄子根部及土壤中的定殖动态[J]. 福建农业学报, 2003,18(2):87-88.

WENG Q

, CHEN Q

, ZHAO

, et al. Colonization of BS1 marked with Rifampicin on roots and in root zone soil of tomato and eggplant[J]. Fujian J Agric Sci, 2003,18(2):87-88. DOI: 10.19303/j.issn.1008-0384.2003.02.005.

10.19303/j.issn.1008-0384.2003.02.005

本文引用 [1]

[17]	HE H, QIU S X, CAI X Q, et al. Colonization in plant and identification of endophytic bacteria BS-1 and BS-2 from Capsicum annuum[J]. Acta Microbiologica Sin, 2004,44(1):13-18. DOI: 10.1161/01.ATV.0000116865.98067.31. 10.1161/01.ATV.0000116865.98067.31 本文引用 [2]

[18]

谈家金, 杨荣铮, 吴慧平. 不同地理种群的松材线虫对马尾松的致病力差异[J]. 植物检疫, 2000,14(6):324-325.

TAN J

, YANG R

, WU H

. Difference of virulence of several geographical populations of Bursaphelenchus xylophilus to Pinus massoniana[J]. Plant Quar, 2000,14(6):324-325. DOI: 10.3969/j.issn.1005-2755.2000.06.002.

10.3969/j.issn.1005-2755.2000.06.002

本文引用 [1]

[19]

周爱东, 徐小明, 王岚, 等. 松材线虫病发生34 a的综合防控——以江苏省镇江市为例[J]. 江苏林业科技, 2019,46(4):54-57.

ZHOU A

, XU X

, WANG

, et al. Integrated control of pine wilt disease in the past thirty years in Zhenjiang City of Jiangsu Province[J]. J Jiangsu For Sci & Tech, 2019,46(4):54-57. DOI: 10.3969/j.issn.1001-7380.2019.04.011.

10.3969/j.issn.1001-7380.2019.04.011

本文引用 [1]

[20]

朱丽梅, 吴小芹, 徐旭凌. 松材线虫拮抗细菌的筛选和鉴定[J]. 南京林业大学学报(自然科学版), 2008,32(3):91-94.

ZHU L

, WU X

, XU X

. The screening and identification of the bacterium with nematicidal activity to Bursaphelenchus xylophilus[J]. J Nanjing For Univ(Nat Sci Ed), 2008,32(3):091-94. DOI: 10.3969/j.issn.1000-2006.2008.03.022.

10.3969/j.issn.1000-2006.2008.03.022

本文引用 [1]

[21]	NIU Q H, HUANG X W, TIAN B Y, et al. Bacillus sp. B16 kills Nematodes with a serine protease identified as a pathogenic factor[J]. Appl Microbiol Biotechnol, 2006,69(6):722-730. DOI: 10.1007/s00253-005-0019-5. https://doi.org/10.1007/s00253-005-0019-5 10.1007/s00253-005-0019-5 本文引用 [1]

[22]

罗兰, 谢丙炎, 杨宇红, 等. 具杀线虫活性的苏云金杆菌筛选研究[J]. 植物病理学报, 2007,37(3):314-316.

LUO

, XIE B

, YANG Y

, et al. Screening of Bacillus thuringiensis against plant-parasitic nematodes[J]. Acta Phytopathol Sin, 2007,37(3):314-316. DOI: 10.13926/j.cnki.apps.2007.03.017.

10.13926/j.cnki.apps.2007.03.017

本文引用 [1]

[23]	ZHENG L J, LI G H, WANG X B, et al. Nematicidal endophytic bacteria obtained from plants[J]. Ann Microbiol, 2008,58(4):569-572. DOI: 10.1007/BF03175559. https://doi.org/10.1007/BF03175559 10.1007/BF03175559 本文引用 [1]

[24]

王方, 王林松, 马怡, 等. 杀松材线虫细菌的分离、鉴定及其培养条件研究[J]. 微生物学杂志, 2019,39(3):22-28.

WANG

, WANG L

, MA

, et al. Isolation, identification, and culture condition of nematicidal bacteria strains against pine wood nematode.[J]. J Microbiol, 2019,39(3):22-28. DOI: 10.3969/j.issn.1005-7021.2019.03.004.

10.3969/j.issn.1005-7021.2019.03.004

本文引用 [1]

[25]	曾腓力. 美国松材线虫携带细菌的生防潜能研究[D]. 南京: 南京林业大学, 2010. ZENG F L. Study on biocontrol potential of bacteria carried by pine wood nematode[D]. Nanjing: Nanjing Forestry University, 2010. 本文引用 [1]

[26]	曾腓力, 贲爱玲, 郑敬荣, 等. 美国松材线虫体表携带优势细菌的鉴定及致病性[J]. 浙江农林大学学报, 2012,29(5):696-702. ZENG F L, BEN A L, ZHENG J R, et al. Identification and pathogenicity of bacterial strains carried by American pine wood nematodes[J]. J Zhejiang A F Univ, 2012,29(5):696-702. 本文引用 [1]

[27]

姚伍, 郑催云, 陈红梅, 等. 福建三明市应用Smal-007菌剂防治松材线虫病的效果[J]. 林业科学, 2018,54(1):168-173.

YAO

, ZHENG C

, CHEN H

, et al. Control effect of pine wilt disease using a biological control agent of Smal-007 in Sanming, Fujian Province[J]. Sci Silvae Sin, 2018,54(1):168-173. DOI: 10.11707/j.1001-7488.20180119.

10.11707/j.1001-7488.20180119

本文引用 [1]