杀松材线虫海洋放线菌HT-8的鉴定及培养条件的研究

摘要

松树萎蔫病（Pine wilt disease， PWD）是由松材线虫（Bursaphelenchus xylophilus）引起的一种毁灭性疾病，该病对世界上许多国家和地区的针叶林资源造成严重破坏。为了寻找具有高杀松材线虫活性的海洋放线菌，对分离自海洋环境中的放线菌菌株进行杀线虫活性测定。选用稀释涂布平板法对采自青岛近海海域的样品进行放线菌分离，采用浸渍法测定菌株的杀松材线虫活性，并对所筛选出的高杀线虫活性菌株的形态学、培养特征和16S rDNA序列进行测定，运用单因素试验测定菌株产生杀线虫活性物质的最适培养条件。结果显示，从采集的样品中共分离到28株放线菌，经杀线虫活性筛选得到1株具有高杀线虫活性的放线菌HT-8。该菌株分离自海沙，其培养上清液处理松材线虫30 h时，线虫的校正死亡率高达88．30％。根据形态学、培养特征和16S rDNA序列测定及其系统发育分析结果，将菌株HT-8鉴定为Streptomyces termitum。单因素试验结果表明，该菌株产生杀线虫活性物质的最适培养条件为接种体菌龄48 h，接种量6％，培养基海水浓度100％，初始pH 7．5，培养温度25℃，培养时间7 d。该研究为海洋微生物资源的开发及其杀松材线虫天然活性物质的利用提供理论依据。%Pine wilt disease ( PWD) is a devastating disease to pine trees , which was caused by the pine wood nema-tode (PWN), Bursaphelenchus xylophilus, and causes major losses in coniferous forests in many countries and regions in the world.In order to screen actinomycete strains with high nematicidal activity against PWN , marine actinomycetes were isolated from the marine environment and their nematicidal activity were tested .The marine actinomycetes were i-solated from the samples collected from the subtidal zones near Qingdao coast using the dilution and streak plate meth -od and their culture supernatant were assayed in vitro for nematicidal activity against PWN using immersion test .The strain with high nematicidal activity was identified on the basis of morphology , cultural characteristics and 16S rDNA sequence analysis , and its optimal culture conditions for the production of nematicidal substances were investigated through the single-factor experiments .A total of 28 marine actinomycete strains were isolated from the samples .One of these strains, designated as strain HT-8 and isolated from sea sand , exhibited stronger nematicidal activity with a 88.30%corrected mortality of PWN treated with the culture supernatant for 30 h.The strain HT-8 was identified as Streptomyces termitum on the basis of morphology , cultural characteristics , 16S rDNA sequence and phylogenetic anal-ysis.The results of the single-factor experiment demonstrated that the optimal cultivation conditions of HT-8 for the production of nematicidal substances were inoculum age was 48 h, inoculum concentration was 6%, concentration of seawater was 100%, initial pH was 7.5 and incubating at 25℃for 7 days.This study provides a theoretical basis for the development of marine microorganism resources and the utilization of natural nematicidal substances .