【目的】筛选对松材线虫具有较高杀线活性的拮抗细菌。【方法】以分离自南京、洛阳、上海的松树茎部的137株内生细菌为研究对象,采用培养滤液和菌悬液浸渍法对这些菌株进行杀线活性的测定,并对筛选的高效拮抗细菌进行菌种鉴定。【结果】通过培养滤液浸渍试验筛选出3株细菌对松材线虫有较高的杀线活性,使用3种菌滤液处理线虫48 h后,线虫死亡率均达到100%,其中菌株LYMC-3的培养滤液还可使线虫虫体出现消解。将3株菌的培养滤液分别稀释2倍、4倍和10倍后处理松材线虫,随稀释倍数的增加,培养滤液的杀线活性逐渐降低。处理线虫48 h后,菌株LYMC-3的10倍稀释滤液与其他两株菌滤液相比对线虫的致死率最高,为94.7%;在菌悬液浸渍试验中筛选出菌株NJSZ-13对松材线虫有较高杀线活性,105cfu/mL浓度的菌悬液处理线虫48 h后,线虫死亡率达81.5%。通过对LYMC-3和NJSZ-13菌株的形态特征、生理生化特征、Biolog鉴定和16S rDNA序列以及系统发育分析,确定菌株LYMC-3、NJSZ-13分别为短小芽孢杆菌(Bacillus pumilus)和蜡样芽孢杆菌(Bacillus cereus)。【结论】从松树体内筛选到两株对松材线虫有高效拮抗活性的菌株LYMC-3和NJSZ-13,对生物防治松材线虫病具有潜在的应用价值。
【Objective】The aim of the present study was to screen bacteria with nematicidal activity against the pine wood nematode(Bursaphelenchus xylophilus).【Method】A total of 137 bacteria strains,which were isolated from the stems of pine trees in Nanjing,Luoyang and Shanghai,China,were assessed for nematicidal activity by immersion test with cultural filtrate and buffer suspension.【Result】Cultural filtrates of three strains had stronger nematicidal activity than the other strains,and nematodes treated with these strains reached 100% mortality after 48 h and even exhibited decomposition when treated with the cultural filtrate of strain LYMC-3.The cultural filtrates of three strains,which were diluted 2,4 and 10 times still showed high nematicidal activity.Nematicidal activity decreased with increasing dilution,and when nematodes were treated with 10×diluted filtrates for 48 h,the greatest observed mortality was 94.7% with strain LYMC-3.Moreover,the buffer suspension of strain NJSZ-13 had strong nematicidal activity against B.xylophilus,and 81.5% nematode mortality was achieved after 48 h treatment with 105cfu/mL buffer suspension of strain NJSZ-13.Based on morphology,physiological and biochemical characteristics,Biolog system identification,16S rDNA sequences,and a phylogenetic tree,the strains LYMC-3 and NJSZ-13 were identified as Bacillus pumilus and B.cereus,respectively.【Conclusion】Two strains of bacteria LYMC-3 and NJSZ-13 with high nematicidal activity which were isolated from the stems of pine trees could provide potential solution for biological control of Bursaphelenchus xylophilus.