具有杀线活性马尾松内生细菌的筛选与鉴定  

Screening and Identification of Endophytic Bacteria with Nematicidal Activity Against Bursaphelenchus xylophilus in Pinus massoniana

作　　者：贺丽娜 冯源 石慧敏 叶建仁 HE Li-na;FENG Yuan;SHI Hui-min;YE Jian-ren(Co-innovation Center for Sustainable Forestry in Southern China,College of Forestry,Nanjing Forestry University,Nanjing 210037)

机构地区：[1]南方现代林业协同创新中心南京林业大学林学院,南京210037

出　　处：《生物技术通报》2022年第8期159-166,共8页Biotechnology Bulletin

基　　金：国家林草局和国家科技部揭榜挂帅项目(ZD202001-01)。

摘　　要：为探究马尾松内生细菌的多样性,寻找具有杀松材线虫活性的内生细菌。以前期从南京中山陵健康马尾松上分离的58株内生细菌为研究对象,利用浸渍法对内生细菌发酵滤液的杀线活性进行测定,采用形态学观察、生理生化特征测定、16S rDNA序列以及系统发育树等方法对筛选出的具有杀线活性的内生细菌进行种类鉴定。从中筛选出一株具有明显杀线功能的内生细菌NZM13-11。内生细菌NZM13-11发酵滤液在与松材线虫混合培养12 h后,校正死亡率达到92.2%,36 h后,校正死亡率达到100%。在12 h时,松材线虫的虫体被消解,消解率达85%,在培养48 h后,消解率达到100%,在显微镜视野下已经没有完整的虫体。对发酵滤液进行不同倍数稀释后,在8倍稀释倍数处理36 h,死亡率依然高达72.9%。通过对NZM13-11进行形态学观察、生理生化特征测定、16S rDNA序列以及系统发育树分析,将菌株NZM13-11鉴定为铜绿假单胞菌(Pseudomonas aeruginosa)。研究表明,在松树体内筛选出一株内生细菌NZM13-11能够高效地杀死松材线虫,该菌株被鉴定为铜绿假单胞菌(Pseudomonas aeruginosa)。该研究结果可以进一步深入,以探究利用马尾松内生细菌资源来防控松材线虫病的可能性。The purpose of this study is to explore the diversity of endophytic bacteria in Pinus massoniana,and to search for endophytic bacteria with nematicidal activity against Bursaphelenchus xylophilus.With 58 endophytic bacteria isolated from healthy P.massoniana in Nanjing Zhongshan Mausoleum as the research object,and the nematicidal activity against B.xylophilus of endophytic bacteria in fermentation filtrate was determined by immersion method.The species of endophytic bacteria with high nematicidal activity against B.xylophilus were identified by morphological observation,physiological and biochemical characteristics,16S rDNA sequence and phylogenetic tree methods.An endophytic bacterium NZM13-11 was selected with obvious nematicidal activity against B.xylophilus.The adjusted mortality rate of B.xylophilus reached 92.2%after mixing the fermented filtrate of NZM13-11 with B.xylophilus for 12 h,and reached 100%after 36 h.In addition,the body of B.xylophilus was decomposed at 12 h,the decomposition rate reached 85%.After 48 h,the decomposition rate reached 100%,and there was no complete body under the microscope.After the fermentation filtrate was diluted at different multiples,the mortality rate was still as high as 72.9%after 36 h of 8-fold dilution treatment.The strain NZM13-11 was identified as Pseudomonas aeruginosa by morphological observation,physiological and biochemical characterization,16S rDNA sequence and phylogenetic tree analysis.This study shows that an endophytic bacterium NZM13-11 screened from p P.massoniana trees can efficiently kill B.xylophilus,and the strain is identified as Pseudomonas aeruginosa.This research results can be further deepened to explore the possibility of using endophytic bacteria resources of P.massoniana to prevent and control pine wilt diseases.

关 键 词：内生细菌 铜绿假单胞菌 松材线虫 马尾松 

分 类 号：S763.18[农业科学—森林保护学]