陈聪聪 郭群群 王超等

摘要[目的] 从海洋中分离并筛选具有杀松材线虫活性的细菌。[方法] 采用稀释涂布平板法对采自青岛近海海域的样品进行细菌分离，采用浸渍法测定菌株的杀线虫活性，并对筛选菌株的形态学、生理生化特性和16S rDNA序列进行测定，通过单因素试验筛选产生杀线虫活性物质的最适培养条件。[结果] 共分离到36株海洋细菌，根据杀线虫活性筛选出1株具有高杀线虫活性的细菌HT11。该菌株分离自牡蛎，其培养滤液处理松材线虫24 h的校正死亡率高达92.41%。根据形态学、生理生化特性和16S rDNA序列测定及其系统发育分析结果，将菌株HT11鑒定为考克氏菌属（Kocuria）。单因素试验结果表明，该菌株在营养肉汤（NB）培养基中产生杀线虫活性物质的最适培养条件为：海水浓度100%，初始pH 7.0，培养温度25 ℃，培养时间3 d。[结论] 该研究可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。

关键词海洋细菌；杀线虫活性；松材线虫；鉴定；培养条件

中图分类号S767.3+2文献标识码A文章编号0517-6611（2016）04-024-05

Identification and Culture Conditions of Marine Bacterium HT11 with Nematicidal Activity against Bursaphelenchus xylophilus

CHEN Congcong，GUO Qunqun，WANG Chao，GUO Daosen* et al （College of Life Sciences，Qingdao University，Qingdao，Shandong 266071）

Abstract [Objective] To isolate and screen the marine bacteria with nematicidal activity against Bursaphelenchus xylophilus from the sea.[Method] The marine bacterial strains were isolated from the samples collected from the subtidal zones near Qingdao coast using the dilution and streak plate method； nematicidal activity of strains were detected by immersion method.The morphology，physiological and biochemical characters，and 16S rDNA sequence of the screened strains were also detected.The optimal cultivation conditions to produce nematicidal activity substances were detected by single factor test.[Result] A total of 36 marine bacterial strains were isolated from the samples.One of these strains HT11 had high nematicidal activity，which was isolated from oyster.Its corrected mortality reached as high as 92.41% after treated with the culture filtrate for 24 h .According to the results of morphology，physiological and biochemical characters，16S rDNA sequence and phylogenetic analysis，strain HT11 was classified into the genus of Kocuria.Results of single factor experiment indicated that the optimal cultivation conditions of HT11 for the production of nematicidal substances were 100% seawater concentration，initial pH 7， 25 ℃ cultivation temperature，and 3 d cultivation time.[Conclusion] This research provides theoretical foundation for the development of marine microorganism resources and the utilization of natural nematicidal substances.

Key wordsMarine bacteria； Nematicidal activity； Bursaphelenchus xylophilus； Identification； Culture conditions

松树萎蔫病（Pine wilt disease，PWD）是松材线虫（Bursaphelenchus xylophilus）入侵松属植物后引起寄主萎蔫并快速枯死的一种毁灭性疾病[1]，传播媒介为松墨天牛（Monochamus alternatus）。该病在全球范围内造成了巨大的经济损失和生态环境的破坏，在东亚尤其是日本、中国、韩国[2]以及西欧的葡萄牙[3]和西班牙[4]损失较为严重。我国于1982年首次在江苏省南京市发现该病，截至2011年已扩散至全国14个省（市），且呈扩大趋势，随着国际贸易的往来，该病传播扩散的速度也在不断加快，因此松树萎蔫病已经引起许多国家的高度重视[5]。……