松材线虫拮抗细菌的杀线活性及其发酵培养特性
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摘要
旨为筛选具有高效杀松材线虫活性的拮抗细菌。采用浸测法对3株芽孢杆菌进行杀线活性测定,并对高效杀线菌株发酵滤液处理线虫后的虫体进行形态观察及该菌株培养条件和杀线物质稳定性测定。结果表明3株细菌发酵滤液处理松材线虫(Bursaphelenchus xylophilus)48 h后,线虫校正死亡率均达100%,且松材线虫在蜡样芽孢杆菌(Bacillus cereus)JK-XZ3发酵滤液4倍和8倍稀释液中处理48 h后,其校正死亡率分别达99.55%和88.32%,明显高于短小芽孢杆菌(B. pumilus)HR10和瓦雷兹芽孢杆菌(B. velezensis)YH-20;该JK-XZ3菌株发酵滤液处理过的线虫虫体断裂,内含物外溢,体壁消解;该菌株在30℃培养4 d的发酵滤液杀线活性最高,且松材线虫在发酵滤液4倍和8倍稀释液中处理48 h后,其校正死亡率均达100%;松材线虫在40℃、60℃、80℃和100℃处理后的发酵滤液8倍稀释液中处理48 h后,其校正死亡率均在84%以上;在pH为8-10的发酵滤液8倍稀释液处理48 h后,其校正死亡率均在88%以上;说明该菌株发酵滤液具有耐高温和耐碱特性。蜡样芽孢杆菌JK-XZ3是一株具有开发和应用潜力的高毒力杀线菌株。
The aim of this study is to screen bacteria with efficient nematicidal activity against Bursaphelenchus xylophilus. Immersion test was to assess the nematicidal activities of 3 Bacillus strains,then the morphological depiction on the polypides of nematodes killed by fermentation filtrate of the selected efficient strains were observed,as well as the culture condition and the stability of substances with nematicidal activity were also determined. The results revealed that the corrected mortality of nematodes was 100% after B. xylophilus were treated for 48 h with fermentation filtrate by 3 bacteria strains. The corrected mortality of nematodes was still up to 99.55% and 88.32%,respectively when they were treated for 48 h with B. cereus JK-XZ3 in fermentation filtrate diluted for 4 times and 8 times. It was significantly higher than the corrected mortality of nematodes treated with B. pumilus HR10 and B. velezensis YH-20 fermented filtrate. Moreover,the polypides of nematodes treated with the fermentation filtrate of JK-XZ3 were broken,and contents overflowed,and body wall was dissolved. The filtrate of JK-XZ3 fermented in 30℃ for 4 d presented the highest nematicidal activity,and the corrected mortality of nematodes,when treated for 48 h with the fermentation filtrate diluted for 4 and 8 times,were both up to 100%,respectively;the corrected mortality of nematodes was more than 84% when the nematodes were treated for 48 h in the 8 times-diluted fermentation filtrate processed at 40、60、80 and 100℃;at pH of8-10,the corrected mortality of nematodes was > 88% when they were treated for 48 h in 8 times-diluted fermentation filtrate. It indicated that the fermentation filtrate of the strain demonstrated the characteristics of high temperature and alkali resistances. B. cereus JK-XZ3 is a highly virulent nematicidal strain with potentials of research and application.
The aim of this study is to screen bacteria with efficient nematicidal activity against Bursaphelenchus xylophilus. Immersion test was to assess the nematicidal activities of 3 Bacillus strains,then the morphological depiction on the polypides of nematodes killed by fermentation filtrate of the selected efficient strains were observed,as well as the culture condition and the stability of substances with nematicidal activity were also determined. The results revealed that the corrected mortality of nematodes was 100% after B. xylophilus were treated for 48 h with fermentation filtrate by 3 bacteria strains. The corrected mortality of nematodes was still up to 99.55% and 88.32%,respectively when they were treated for 48 h with B. cereus JK-XZ3 in fermentation filtrate diluted for 4 times and 8 times. It was significantly higher than the corrected mortality of nematodes treated with B. pumilus HR10 and B. velezensis YH-20 fermented filtrate. Moreover,the polypides of nematodes treated with the fermentation filtrate of JK-XZ3 were broken,and contents overflowed,and body wall was dissolved. The filtrate of JK-XZ3 fermented in 30℃ for 4 d presented the highest nematicidal activity,and the corrected mortality of nematodes,when treated for 48 h with the fermentation filtrate diluted for 4 and 8 times,were both up to 100%,respectively;the corrected mortality of nematodes was more than 84% when the nematodes were treated for 48 h in the 8 times-diluted fermentation filtrate processed at 40、60、80 and 100℃;at pH of8-10,the corrected mortality of nematodes was > 88% when they were treated for 48 h in 8 times-diluted fermentation filtrate. It indicated that the fermentation filtrate of the strain demonstrated the characteristics of high temperature and alkali resistances. B. cereus JK-XZ3 is a highly virulent nematicidal strain with potentials of research and application.
引文
[1]Zhang L, Fu Q, Li W, et al. Identification and characterization of a novelβ-glucosidase via metagenomic analysis of Bursaphelenchus xylophilus and its microbial flora[J]. Sci Rep, 2017, 7(1):14850.
[2]Ryss AY, Kulinich OA, Sutherland J R. Pine wilt disease:a short review of world wide research[J]. Forestry Studies in China,2011, 13(2):132-138.
[3]Wang CY, Fang ZM, Wang Z, et al. Biological control of the pinewood nematode Bursaphelenchus xylophilus by application of the endoparasitic fungus Esteya vermicola[J]. Biocontrol, 2011, 56(1):91-100.
[4]Yang Z, Yu Z, Lei L, et al. Nematicidal effect of volatiles produced by Trichoderma sp[J]. Journal of Asia-Pacific Entomology, 2012,15(4):647-650.
[5]Song HC, Shen WY, Dong JY, et al. Nematicidal metabolites from Gliocladium roseum YMF1. 00133[J]. Applied Biochemistry And Microbiology, 2016, 52(3):324-330.
[6]陈聪聪.杀松材线虫海洋放线菌HT-8的分离鉴定、培养条件及其活性物质分析[D].青岛:青岛大学, 2016.
[7]Jeong MH, Yang SY, Lee YS, et al. Selection and characterization of Bacillus licheniformis MH48 for the biocontrol of pine wood nematode(Bursaphelenchus xylophilus)[J]. Journal of Korean Forestry Society, 2015, 104(3):512-518.
[8]Zeng L, Jin H, Lu D, et al. Isolation and identification of chemical constituents from the bacterium Bacillus sp. and their nematicidal activities[J]. Journal of Basic Microbiology, 2015, 55(10):1239-1244.
[9]Yu J, Du GC, Li RG, et al. Nematicidal activities of bacterial volatiles and components from two marine bacteria, Pseudoalteromonas marina strain H-42 and Vibrio atlanticus strain S-16, against the pine wood nematode, Bursaphelenchus xylophilus[J]. Nematology,2015, 17:1011-1025.
[10]朱丽梅,吴小芹,陈江天,等.一株毒杀松材线虫的解淀粉芽孢杆菌活性物质的基本性质[J].中国生物防治学报, 2009,25(4):359-363.
[11]曾丽琼,何学友,蔡守平,等.具杀松材线虫活性细菌的筛选和鉴定[J].江苏林业科技, 2018, 45(2):6-9.
[12]Huang T, Lin Q, Qian X, et al. Nematicidal activity of Cry1Ea11from Bacillus thuringiensis BRC-XQ12 against the pine wood nematode(Bursaphelenchus xylophilus)[J]. Phytopathology,2018, 108(1):44-51.
[13]谈家金,李亮亮,陈凤毛.一株蜡样芽孢杆菌NJSZ-13及其应用:中国, CN106520635A[P]. 2017-03-22.
[14]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.
[15]李恩杰,李娜,王青华,等.具杀松材线虫活性的苏云金芽孢杆菌筛选及其毒力测定[J].中国生物防治学报, 2018, 34(4)539-545.
[16]Wu XQ, Yuan WM, Tian XJ, et al. Specific and functional diversity of endophytic bacteria from pine wood nematode Bursaphelenchus xylophilus with different virulence[J]. International Journal of Biological Sciences, 2013, 9(1):34-44.
[17]Guo Q, Du G, Qi H, et al. A nematicidal tannin from Punica granatum L. rind and its physiological effect on pine wood nematode(Bursaphelenchusxylophilus)[J].Pesticide Biochemistry&Physiology, 2016, 135:64-68.
[18]Rajasekharan SK, Lee JH, Ravichandran V, et al. Assessments of iodoindoles and abamectin as inducers of methuosis in pine wood nematode, Bursaphelenchus xylophilus[J]. Sci Rep, 2017, 7(1):1-13.
[19]Geng C, Nie X, Tang Z, et al. A novel serine protease, Sep1, from Bacillus firmus DS-1 has nematicidal activity and degrades multiple intestinal-associated nematode proteins[J]. Sci Rep, 2016, 6:25012.
[20]Liu MJ, Hwang BS, Jin CZ, et al. Screening, isolation and evaluation of a nematicidal compound from actinomycetes against the pine wood nematode, Bursaphelenchus xylophilus[J]. Pest management science, 2018.
[21]朱丽梅,吴小芹,蒋萍.细菌XS-JS3对松材线虫杀线活性的测定[J].南京林业大学学报:自然科学版, 2008, 32(1):79-82.
[22]Li LL, Tan JJ, Chen FM. Bacillus pumilus strain LYMC-3 shows nematicidal activity against Bursaphelenchus xylophilus via the production of a guanidine compound[J]. Biocontrol Science and Technology, 2018, 28(12):1128-1139.
[2]Ryss AY, Kulinich OA, Sutherland J R. Pine wilt disease:a short review of world wide research[J]. Forestry Studies in China,2011, 13(2):132-138.
[3]Wang CY, Fang ZM, Wang Z, et al. Biological control of the pinewood nematode Bursaphelenchus xylophilus by application of the endoparasitic fungus Esteya vermicola[J]. Biocontrol, 2011, 56(1):91-100.
[4]Yang Z, Yu Z, Lei L, et al. Nematicidal effect of volatiles produced by Trichoderma sp[J]. Journal of Asia-Pacific Entomology, 2012,15(4):647-650.
[5]Song HC, Shen WY, Dong JY, et al. Nematicidal metabolites from Gliocladium roseum YMF1. 00133[J]. Applied Biochemistry And Microbiology, 2016, 52(3):324-330.
[6]陈聪聪.杀松材线虫海洋放线菌HT-8的分离鉴定、培养条件及其活性物质分析[D].青岛:青岛大学, 2016.
[7]Jeong MH, Yang SY, Lee YS, et al. Selection and characterization of Bacillus licheniformis MH48 for the biocontrol of pine wood nematode(Bursaphelenchus xylophilus)[J]. Journal of Korean Forestry Society, 2015, 104(3):512-518.
[8]Zeng L, Jin H, Lu D, et al. Isolation and identification of chemical constituents from the bacterium Bacillus sp. and their nematicidal activities[J]. Journal of Basic Microbiology, 2015, 55(10):1239-1244.
[9]Yu J, Du GC, Li RG, et al. Nematicidal activities of bacterial volatiles and components from two marine bacteria, Pseudoalteromonas marina strain H-42 and Vibrio atlanticus strain S-16, against the pine wood nematode, Bursaphelenchus xylophilus[J]. Nematology,2015, 17:1011-1025.
[10]朱丽梅,吴小芹,陈江天,等.一株毒杀松材线虫的解淀粉芽孢杆菌活性物质的基本性质[J].中国生物防治学报, 2009,25(4):359-363.
[11]曾丽琼,何学友,蔡守平,等.具杀松材线虫活性细菌的筛选和鉴定[J].江苏林业科技, 2018, 45(2):6-9.
[12]Huang T, Lin Q, Qian X, et al. Nematicidal activity of Cry1Ea11from Bacillus thuringiensis BRC-XQ12 against the pine wood nematode(Bursaphelenchus xylophilus)[J]. Phytopathology,2018, 108(1):44-51.
[13]谈家金,李亮亮,陈凤毛.一株蜡样芽孢杆菌NJSZ-13及其应用:中国, CN106520635A[P]. 2017-03-22.
[14]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.
[15]李恩杰,李娜,王青华,等.具杀松材线虫活性的苏云金芽孢杆菌筛选及其毒力测定[J].中国生物防治学报, 2018, 34(4)539-545.
[16]Wu XQ, Yuan WM, Tian XJ, et al. Specific and functional diversity of endophytic bacteria from pine wood nematode Bursaphelenchus xylophilus with different virulence[J]. International Journal of Biological Sciences, 2013, 9(1):34-44.
[17]Guo Q, Du G, Qi H, et al. A nematicidal tannin from Punica granatum L. rind and its physiological effect on pine wood nematode(Bursaphelenchusxylophilus)[J].Pesticide Biochemistry&Physiology, 2016, 135:64-68.
[18]Rajasekharan SK, Lee JH, Ravichandran V, et al. Assessments of iodoindoles and abamectin as inducers of methuosis in pine wood nematode, Bursaphelenchus xylophilus[J]. Sci Rep, 2017, 7(1):1-13.
[19]Geng C, Nie X, Tang Z, et al. A novel serine protease, Sep1, from Bacillus firmus DS-1 has nematicidal activity and degrades multiple intestinal-associated nematode proteins[J]. Sci Rep, 2016, 6:25012.
[20]Liu MJ, Hwang BS, Jin CZ, et al. Screening, isolation and evaluation of a nematicidal compound from actinomycetes against the pine wood nematode, Bursaphelenchus xylophilus[J]. Pest management science, 2018.
[21]朱丽梅,吴小芹,蒋萍.细菌XS-JS3对松材线虫杀线活性的测定[J].南京林业大学学报:自然科学版, 2008, 32(1):79-82.
[22]Li LL, Tan JJ, Chen FM. Bacillus pumilus strain LYMC-3 shows nematicidal activity against Bursaphelenchus xylophilus via the production of a guanidine compound[J]. Biocontrol Science and Technology, 2018, 28(12):1128-1139.