杀松材线虫细菌的分离、鉴定及其培养条件研究
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摘要
为了对松萎蔫病进行安全、无污染的生物防治,从青岛大学校园内采集的样品中分离出120株菌。采用浸渍法从中筛选出1株具有较高杀线虫活性的细菌G8-1。杀线虫活性测定结果表明,该菌株发酵上清液48 h后的杀线虫率为90.81%。通过16S rRNA的序列分析,将其鉴定为Flectobacillus rhizosphaerae。通过单因素分析法和正交实验对该菌株的培养条件进行研究,确定最佳培养条件:最适初始pH为8.0,最适培养温度为35℃,最佳接种菌龄为9 h,最佳培养时间为3 d,菌株G8-1发酵上清液的杀线虫率可达到96.31%。对菌株产生的活性物质的稳定性进行初步分析,结果表明该菌株的活性物质都具有较好的热稳定性,并且在弱碱性条件下具有较高的杀线虫活性稳定性。本研究为松材线虫的生物防治提供参考。
In order to implement safety and pollution-free biological control on pine wilt, a total of 120 strains of bacteria were isolated samples collected from the campus of Qingdao University. One strain of bacteria G8-1 with high nematicidal activity was screened by immersion method. The results of nematicidal activity showed that the rate of nematodes was at 90.81% by fermented supernatant 48 h after. It was identified as Fecmumillus rhizosphaerae by sequence analysis of 16 S rRNA. Using single factor analysis method and orthogonal experiment the culture conditions of the strain and the optimum culture conditions were studied and determined, the optimal initial was at pH 8.0, the optimal temperature at 35 ℃, the best inoculation strain age was 9 h, the best cultivation time was 3 d, the nematicidal rate of the fermentation supernatant of strain G8-1 was as high as 96.31%. The stability of the active substances produced by the strain was initially analyzed, and the results showed that the active substances produced by the strain had better thermal stability and higher activity stability against nematodes under weakly alkaline conditions. This paper provided reference for bio-controlling pine nematode.
In order to implement safety and pollution-free biological control on pine wilt, a total of 120 strains of bacteria were isolated samples collected from the campus of Qingdao University. One strain of bacteria G8-1 with high nematicidal activity was screened by immersion method. The results of nematicidal activity showed that the rate of nematodes was at 90.81% by fermented supernatant 48 h after. It was identified as Fecmumillus rhizosphaerae by sequence analysis of 16 S rRNA. Using single factor analysis method and orthogonal experiment the culture conditions of the strain and the optimum culture conditions were studied and determined, the optimal initial was at pH 8.0, the optimal temperature at 35 ℃, the best inoculation strain age was 9 h, the best cultivation time was 3 d, the nematicidal rate of the fermentation supernatant of strain G8-1 was as high as 96.31%. The stability of the active substances produced by the strain was initially analyzed, and the results showed that the active substances produced by the strain had better thermal stability and higher activity stability against nematodes under weakly alkaline conditions. This paper provided reference for bio-controlling pine nematode.
引文
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[15] Kim OS,Cho YJ,Lee K,et al.Introducing EzTaxon-e:a prokaryotic 16S rRNA gene sequence database with phylotypes that represent uncultured species[J].Int J Syst Evol Microbiol,2012,62(Pt 3):716.
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[17] 韩其飞,林玲玲,刁辉胜,等.树干注射和根施农药控制松材线虫病试验[J].林业与环境科学,2010,26(5):72-74.
[18] Guo Q,Du G,Qi H,et al.A nematicidal tannin from Punica granatum L.rind and its physiological effect on pine wood nematode (Bursaphelenchus xylophilus) [J].Pestic Biochem.Physiol,2016,135:64.
[19] Guo Q,Du G,He H,et al.Two nematicidal furocoumarins from Ficus carica L.leaves and their physiological effects on pine wood nematode (Bursaphelenchus xylophilus) [J].Nat Prod Res,2015,30(17):1.
[20] Barbosa P,Lima AS,Vieira P,et al.Nematicidal activity of essential oils and volatiles derived from Portuguese aromatic flora against the pinewood nematode,Bursaphelenchus xylophilus[J].J Nematol,2010,42(1):8-16.
[21] Wang CY,Yin C,Fang ZM,et al.Using the nematophagous fungus Esteya vermicola to control the disastrous pine wilt disease[J].Biocontrol Science & Technology,2018,28(1):1-10.
[22] Cao Y,Tian B,Ji X,et al.Associated bacteria of different life stages of Meloidogyne incognita using pyrosequencing-based analysis[J].J Basic Microbiol,2015,55(8):950-960.
[23] 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.
[24] Huang T,Lin Q,Qian X,et al.Nematicidal activity of Cry1Ea11 from Bacillus thuringiensis BRC-XQ12 against the pine wood nematode (Bursaphelenchus xylophilus)[J].Phytopathology,2018,108(1)44-51.
[2] Mota M.Pine Wilt Disease,and the pinewood nematode:a worldwide issue,a nematological challenge[J].J Nematol,2014,46(2):208-208.
[3] Kwon TS,Shin JH,Lim JH,et al.Management of pine wilt disease in Korea through preventative silvicultural control[J].For Ecol Manage,2011,261(3):562-569.
[4] Mota MM,Braasch H,Bravo MA.First report of Bursaphelenchus xylophilus in Portugal and in Europe[J].Nematology,1999,1(1):727-734.
[5] 丹阳,黄忠良,魏孝义,等.松材线虫的天然毒素研究进展[J].热带亚热带植物学报,2004,12(4):381-387.
[6] Ichihara Y,Fukuda K,Suzuki K,et al.Early symptom development and histological changes associated with migration of Bursaphelenchus xylophilus in seedling tissues of Pinus thunbergii[J].Plant Dis,2000,84:675-680.
[7] Noling JW,Becker JO.The challenge of research and extension to define and implement alternatives to methyl bromide[J].J Nematol,1994,26(4 Suppl):573-586.
[8] Gowen SR.Chemical control of nematodes:efficiency and side-effects[J].Fao Plant Production & Protection Paper,1997,144:59-65.
[9] Yu J,Du GC,Li RG,et al.Nematicidal activities of bacterial volatiles and components from two marine bacteria,Pseudoalteromonas marinastrain H-42 and Vibrio atlanticus strain S-16,against the pine wood nematode,Bursaphelenchus xylophilus[J].Nematology,2015,17(9):1011-1025.
[10] 李子,周长景,杜桂彩,等.杀松材线虫海洋真菌ML-5的鉴定及培养条件研究[J].青岛大学学报(工程技术版),2015,30(2):102-107.
[11] 陈聪聪,王超,郭群群,等.杀松材线虫海洋放线菌HT-8的鉴定及培养条件的研究[J].微生物学杂志,2016,36(6):54-60.
[12] Zhao BG,Li RG.The Roles of Bacteria Associated with the Pine Wood Nematode in Pathogenecity and Toxin-Production Related to Pine Wilt.In Pine Wilt Disease[M].Tokyo,Japan:Springer,2008:250-259.
[13] Dong JY,Li XP,Li L,et al.Preliminary results on nematicidal activity from culture filtrates of Basidiomycetes against the pine wood nematode,Bursaphelenchus xylophilus (Aphelenchoididae) [J].Ann Microbiol,2006,56(2):163.
[14] Wang J,Pan X,Yi H,et al.Rosmarinic Acid from Eelgrass Shows Nematicidal and Antibacterial Activities against Pine Wood Nematode and Its Carrying Bacteria[J].Mar Drugs,2012,10(12):2729-2740.
[15] Kim OS,Cho YJ,Lee K,et al.Introducing EzTaxon-e:a prokaryotic 16S rRNA gene sequence database with phylotypes that represent uncultured species[J].Int J Syst Evol Microbiol,2012,62(Pt 3):716.
[16] Kumar S,Stecher G,Tamura K.MEGA7:Molecular Evolutionary Genetics Analysis Version 7.0 for Bigger Datasets[J].Mol Biol Evol,2016,33(7):1870.
[17] 韩其飞,林玲玲,刁辉胜,等.树干注射和根施农药控制松材线虫病试验[J].林业与环境科学,2010,26(5):72-74.
[18] Guo Q,Du G,Qi H,et al.A nematicidal tannin from Punica granatum L.rind and its physiological effect on pine wood nematode (Bursaphelenchus xylophilus) [J].Pestic Biochem.Physiol,2016,135:64.
[19] Guo Q,Du G,He H,et al.Two nematicidal furocoumarins from Ficus carica L.leaves and their physiological effects on pine wood nematode (Bursaphelenchus xylophilus) [J].Nat Prod Res,2015,30(17):1.
[20] Barbosa P,Lima AS,Vieira P,et al.Nematicidal activity of essential oils and volatiles derived from Portuguese aromatic flora against the pinewood nematode,Bursaphelenchus xylophilus[J].J Nematol,2010,42(1):8-16.
[21] Wang CY,Yin C,Fang ZM,et al.Using the nematophagous fungus Esteya vermicola to control the disastrous pine wilt disease[J].Biocontrol Science & Technology,2018,28(1):1-10.
[22] Cao Y,Tian B,Ji X,et al.Associated bacteria of different life stages of Meloidogyne incognita using pyrosequencing-based analysis[J].J Basic Microbiol,2015,55(8):950-960.
[23] 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.
[24] Huang T,Lin Q,Qian X,et al.Nematicidal activity of Cry1Ea11 from Bacillus thuringiensis BRC-XQ12 against the pine wood nematode (Bursaphelenchus xylophilus)[J].Phytopathology,2018,108(1)44-51.