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【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 objective of this study was to screen for antagonistic bacteria with high cytotoxic activity against pine wood nematodes. 【Method】 The 137 endophytic bacteria isolated from the stems of pine trees in Nanjing, Luoyang and Shanghai were used as research objects. The killing activity of these strains was tested by culture filtrate and bacterial suspension dipping method. Bacterial species identification. 【Result】 Three strains of bacterium were screened out by culture filtrate immersion test. The mortality of nematode reached 100% after three kinds of bacteria were treated by three kinds of bacteria filtrate. The mortality of strain LYMC-3 Culture filtrate can also make nematode body digestion. The culture filtrates of three strains were diluted by 2, 4 and 10 times, respectively. After treated with the dilution multiples, the killing activity of the culture filtrate decreased gradually. After treatment of nematodes for 48 h, the 10-fold dilution filtrate of strain LYMC-3 had the highest lethality to nematode as compared with the other two strains, which was 94.7%. In the bacterial suspension immersion test, the strain NJSZ-13 Nematodes have higher activity to kill nematodes, and the nematode mortality rate reached 81.5% after treated with 105cfu / mL bacterial suspension for 48 hours. Through the morphological, physiological and biochemical characteristics of LYMC-3 and NJSZ-13 strains, Biolog identification and 16S rDNA sequences and phylogenetic analysis, the strains LYMC-3 and NJSZ-13 were identified as Bacillus pumilus and waxy Bacillus cereus. 【Conclusion】 Two strains of LYMC-3 and NJSZ-13, which have high antagonistic activity against pine wood nematode, were screened from pine trees, which have potential application in the biological control of pine wilt disease.