松材线虫生防放线菌的筛选、鉴定及其毒性因子初步研究

目的从土壤中筛选、鉴定出对松材线虫有较高杀灭活性的放线菌，并确定其毒力因子。方法以藤黄八叠球菌、枯草杆菌、大肠杆菌3个敏感菌株为抑菌检测对象，从南阳师范学院校园内、白河边、宝天曼、卧龙岗等地采集的土壤样品中筛选有抑菌活性的放线菌，采用形态学观察以及基于16S rRNA序列分析等方法对抑菌活性最高的菌株进行分类鉴定，将该菌株进行杀松材线虫活性测试，并分离纯化出杀线活性物质。结果筛选出4株有抑菌活性的放线菌菌株:C611、C612、C614、C619，其中C611菌株对藤黄八叠球菌、枯草杆菌和大肠杆菌均有较高抑制活性。结合该菌株形态学、生理学特征以及16S rRNA序列分析等结果，将其初步确定为链霉菌属。将C611发酵液处理松材线虫7 d后，线虫死亡率高达85%，空白对照组死亡率为0，对C611发酵液进行分离纯化后，初步确定活性成分为呋喃它酮。使用含量为0.1%呋喃它酮纯溶液测试杀线虫活性，作用于线虫5 d后其死亡率高达96%。结论本研究筛选到的生防放线菌C611，鉴定出其活性物质为呋喃它酮，丰富了松材线虫生防菌的种类，本研究首次发现呋喃它酮对线虫有较强毒杀作用，为松材线虫生物防治奠定了一定的理论基础。 

Screening and identification of actinomycetes for biological control of Bursaphelenchus xylophilus and preliminary study on their toxicity factors

ObjectiveThis paper aims to screen and identify the actinomycetes with high killing activity against pine wood nematodes Bursaphelenchus xylophilus from soil, and determine their virulent factors.MethodUsing Sarcina lutea, Bacillus subtilis and Escherichia coli as testing strains to determine the antibacterial activities, the actimomycetes with biocontrol activities to B. xylophilus were screened from the soil samples of Nanyang Normal University campus, Baihe Edge, Baotianman and other fields. Morphological observation and molecular identifications based on 16S rRNA sequence analysis were used to classify and identify the strain with the highest activities. The activity of the strain against pine wood nematode was tested, and the virulent substance was isolated and purified.ResultFour actinomycete strains with antibacterial activity were screened: C611, C612, C614 and C619. The strain C611 showed the highest inhibitory activity against S. lutea, B. subtilis and E. coli among the four strains. The strain was identified as Streptomyces sp. according to the morphological and phylogenetic analysis. After 7 days of treatment of pine wood nematode with C611 fermentation broth, the mortality rate of nematode reached 85%, and that of blank control group was 0. The active ingredient was determined to be furan ketone (furaltadone) after separation and purification of C611 fermentation broth.The nematode mortality reached 96% after 5 days treated with 0.1% pure furatanone solution.ConclusionThe actinomycete C611 was isolated and identified furaltadone as a key virulence factor in this study, which enriched the species of biological control microbe against pine wood nematodes. It was first reported that furantone had strong toxicity to nematodes and laid a theoretical foundation for biological control of pine wood nematode.