Relationships between pathogenic bacterium and the endophytic bacteria isolated from Bursaphelenchus xylophilus.
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摘要: 采用16S rRNA基因文库和 illumina Miseq技术测序对松材线虫内生细菌群落的宏基因组进行初步分析,文库显示优势菌群为黄单胞菌科(22.41%)、鞘脂杆菌科(17.66%)、丛毛单胞菌科(14.71%)、根瘤菌科(10.17%)。采用稀释平板法,从松材线虫体内分离出5株内生细菌。利用形态学、生理学及16S rRNA 序列特征等对这些内生细菌进行了分类鉴定。研究发现这些内生细菌在体外均显示较强的杀线虫活性,其中克雷伯氏菌属sct5的杀虫活性最大,作用4 d松材线虫的死亡率即可达到100％。该细菌产生毒性蛋白酶并在体外快速形成生物膜。进一步研究发现,该内生细菌与杀线虫芽孢杆菌B16之间具有强亲和性,混合2个菌株sct5+B16作用1 d对松材线虫的防效为75％,明显好于单菌株sct5 (60％)和B16(40％)。而另一内生细菌肠杆菌属sct29与B16存在拮抗关系,两者不亲和,混合菌株sct29+B16作用1 d对松材线虫的防效仅为13％。本研究首次系统地报道了松材线虫可培养内生细菌的杀虫活性及其与杀线虫芽孢杆菌B16的亲和关系,对研制开发组合生防菌剂、提高生防菌株的稳定性等奠定理论基础,本研究从新的视角阐释了松材线虫内生细菌与病原细菌之间的互作关系。
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关键词:
- 宏基因组 /
- 松材线虫 /
- 内生细菌 /
- 杀线虫芽孢杆菌B16 /
- 互作关系
Abstract: The metagenome of ecosystem of endophytic bacteria associated with the pine wood nematode Bursaphelenchus xylophilus was analyzed by 16S rRNA gene library and illumina Miseq sequencing. The results showed that the dominant bacteria were Xanthomonadaceae (22.41%), Sphingobacteriaceae (17.66%), Comamonadaceae (14.71%) and Rhizobiaceae (10.17%). Five endophytic bacteria were isolated from B. xylophilus using dilution plate method. These bacteria were classified by phenotypic and genotypic characteristics. The five endophytic bacteria all showed nematocidal activities in vitro. The bacterium Klebsiella sp. sct5 showed highest nematocidal activity, and 100% of the tested B. xylophilus were killed within 4 days. Furthermore, the strain sct5 could produce proteases and form biofilm quickly in vitro. Moreover, the bacterial strain sct5 had compatible relationship with Bacillus nematocida B16. The compatible combination of sct5 with B16 resulted in enhanced nematocidal activities within 1 d (75％) against Bursaphelenchus xylophilus as compared to the single strain sct5 (60％) or B16 (40％). In contrast, the incompatible combination of sct29 with B16 led to reduced disease nematocidal activities (only 13％ within 1 d). This study firstly reported nematocidal activities of endophytic bacteria isolated from Bursaphelenchus xylophilus and their relationships with pathogenic bacteria Bacillus nematocida B16, which lays theoretical foundations for developing co-inoculated bacterial strains in biocontrol and improving the stability of bio-control strains. Also, our research explains the relationships between endophytic bacteria in Bursaphelenchus xylophilus and pathogenic bacteria from a new perspective. -
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