异小杆线虫XJZL1409(Heterorhabditis bacteriophora)共生菌的分离及致病性研究

Isolation and pathogenicity of symbiotic bacteria associated with the nematode XJZL1409(Heterorhabditis bacteriophora)

本研究分离了来自新疆和田地区间作绿豆的4年枣0-40 cm根际土壤中的昆虫病原线虫XJZL1409Heterorhabditis bacteriophora的共生菌,并进行致病性测定。通过侵染期线虫和线虫致死的大蜡螟幼虫血腔直接分离,采用传统形态学观察结合分子生物学方法对菌株进行鉴定。同时大蜡螟Galleria mellonella 5龄幼虫接种菌株细胞发酵液测定致病性。菌株形态学特性结合16S r DNA序列系统发育分析,线虫XJZL1409的共生菌被鉴定为发光杆菌属Photorhabdus luminescens subsp. laumondii。室内菌液注射毒性测定结果显示,侵染致死的幼虫体色呈灰绿色;接种48 h后,每头幼虫接种菌液量为500 CFU时校正死亡率就可以达到100%; 48 h时致死中浓度LC50为10. 74 CFU/m L。同时菌液口服杀虫毒性结果表明,第5天时待测菌株菌液对大蜡螟幼虫的校正死亡率为21. 21%,体重抑制率为20. 02%。因此,昆虫病原线虫XJZL1409的共生菌为P. luminescens subsp. laumondii对大蜡螟幼虫具有很强的注射毒性,为研究线虫的致病机制及发掘新抗性基因奠定基础。

The study aimed to isolate and identify symbiotic bacteria associated with the entomopathogenic nematode XJZL1409 Heterorhabditis bacteriophora,which was isolated from the rhizosphere soil(0-40 cm)of four-year jujube trees intercropped with mungbean in Khotan,Xinjiang.Then we also tested the pathogenicity of the symbiotic bacterium to Galleria mellonella larvae.The symbiotic bacterium was isolated from the infective juveniles(IJ)of XJZL1409 and also from the haemolymph of infected G.mellonella larvae.The isolated strain was identified by morphology,16S rDNA sequence as well as phylogeny analysis.And the pathogenicity of the symbiotic bacterium was tested by injecting bacterial suspension into G.mellonella larvae haemolymph and feeding artificial diet with cell culture.The isolated bacterial strain was belong to the species Photorhabdus luminescens subsp.laumondii based on the results of morphology and 16S rDNA sequence.Results of injection toxicity exhibited strong pathogenicity.With the concentration of 500 CFU/larva,the corrected mortality was 100%at 48 h and the LC 50 was 10.74 CFU/mL.Furthermore,oral toxicity test showed that the corrected mortality larvae was 21.21%and the inhibiting rate of weight was 20.02%at 5 d.Therefore,the symbiotic bacterium associated with the nematode XJZL1409 was P.luminescens subsp.laumondii and the cell-free broth of the bacterium demonstrated high pathogenicity against G.mellonella larvae.This will lay the foundation for studing the pathogenic mechanism of the nematodes.