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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 rD NA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 rD NA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 caused by Bursaphelenchus xylophilus, which causes serious damage to coniferous forest resources in many countries and regions around the world. In an effort to find marine actinomycetes with high-activity pine wood nematode activity, nematocidal activity assays were performed on actinomycete strains isolated from the marine environment. The actinomycetes isolated from samples collected from the coastal waters of Qingdao were selected by dilution coating plate method. The activity of the killed pine wood nematode strain was determined by impregnation method. The morphology, culture characteristics, 16S rD NA sequence. The single factor test was used to determine the optimal culture conditions for producing nematicidal actives. The results showed that a total of 28 actinomycetes were isolated from the collected samples and one actinomycete HT-8 with high nematicide activity was screened by nematicidal activity. The strain isolated from the sea sand, the culture supernatant of pine wood nematode 30 h, the corrected mortality of nematodes as high as 88.30%. The strain HT-8 was identified as Streptomyces termitum according to morphological, culturing characteristics and 16S rDNA sequence analysis and its phylogenetic analysis. The results of single factor test showed that the optimal culture conditions for the production of nematicidal active substance were 48 hours of inoculation, 6% inoculum size, 100% seawater concentration, initial pH 7.5, culture temperature 25 ℃, culture time 7 d. This study provides a theoretical basis for the exploitation of marine microbial resources and the utilization of their natural active substances to kill pine wood nematode.