9株昆虫病原线虫共生菌菌株的分离、鉴定及其抗菌谱的筛选

Isolation,identification and screening of antifungal spectrum of nine entomopathogenic nematode symbiotic bacterial strains

[目的]本文旨在探讨9株昆虫病原线虫(entomopathogenic nematode,EPN)共生菌菌株的高效分离鉴定方法以及其对植物真菌病害的拮抗能力。[方法]采用研磨法、血淋巴涂布法和改良涂布法对7种斯氏线虫科(Steinernematidae)和2种异小杆科(Heterorhabditidae)EPN的共生菌进行分离。通过测定其16S rDNA序列进行同源性分析和分子系统发育树分析以鉴定共生菌的种属。采用平板菌丝抑制法测定这9株共生菌的抗病原真菌谱。挑选综合抑菌效果最好的2株共生菌菌株进行稀释和组分分离,测试各组分对真菌的菌丝生长和镰刀菌属分生孢子萌发的抑制效率。[结果]改良涂布法分离到的共生菌平均菌落数为124.0,明显多于传统的研磨法和血淋巴涂布法(分别为8.2和67.7),同时被污染的概率也低。9株共生菌中有7株属于致病杆菌属(Xenorhadus)的5个种,2株属于发光杆菌属(Photorhabdus)的2个种。9株共生菌对稻瘟病菌、尖孢镰刀菌西瓜专化型、尖孢镰刀菌黄瓜专化型、灰葡萄孢菌和辣椒疫霉均有抑制能力。其中,伯氏致病杆菌(Xenorhadus bovienii)NN6和NN8对5种真菌菌丝生长的抑制效率最高,均大于45%。NN6发酵液的5倍稀释液对辣椒疫霉菌丝生长的抑制效率为51.99%,显著高于NN8(45.46%)。NN8发酵液原液对尖孢镰刀菌黄瓜专化型孢子萌发的抑制效率为88.16%,显著高于NN6(81.79%)。[结论]改良的血淋巴涂布法是1种高效分离昆虫病原线虫共生菌的方法,分离到的9株共生菌对5种常见植物病原真菌的生长具有很强的抑制作用,其中伯氏致病杆菌NN6和NN8对菌丝生长的综合抑制能力最强。

[Objectives]The present study aimed to explore the efficient method for the isolation and identification of the symbiotic bacteria from their entomopathogenic nematode(EPN)partner and their inhibition ability against plant pathogenic fungi,so as to provide theory basis for the biological control of these fungal diseases in plants.[Methods]The symbiotic bacteria of 7 EPN of the family Steinernematidae and 2 of the family Heterorhabditidae were isolated by grinding,hemolymph coating and modified coating methods.The 16S rDNA sequences were determined and used to identify the taxonomy position of the isolated symbiotic bacteria in species level by homology analysis and molecular phylogenetic analysis.The antifungal spectrum of these 9 symbiotic bacterial strains was determined by plate mycelium inhibition method.Two symbiotic strains with the best comprehensive antifungal efficiency were selected for component separation,then the inhibitory efficiency of diluted bacterial suspension and sterile filtrate on mycelia growth and conidia germination of Fusarium spp.were tested.[Results]The average number of symbiotic bacterial colonies isolated by the modified coating method in this study was 124.0,which was significantly more than the traditional grinding method and hemolymph coating method,which were 8.2 and 67.7 respectively,and the probability of contamination was also low.Seven of the 9 strains belong to 5 species of the genus Xenorhabdus,and 2 belong to 2 species of the genus Photorhabdus.The 9 bacterial strains had significant inhibitory ability against Magnaporthe grisea,Fusarium oxysporum f.sp.niveum,Fusarium oxysporium f.sp.cucumerinum,Botrytis cinerea and Phytophthora capsici.Xenorhadus bovienii,strains NN6 and NN8 showed the highest comprehensive inhibition rate on the mycelium growth of the above-mentioned five plant pathogenic fungi with inhibitory rates more than 45%.The 5-fold dilution of NN6 fermentation broth inhibited the growth of Phytophthora capsici by 51.99%,which was significantly higher than NN8(45.46%).The inhibitory rate of NN8 fermentation broth on the germination of conidia of Fusarium oxysporium f.sp.cucumerinum was 88.16%,which was significantly higher than that of NN6(81.79%).[Conclusions]The modified coating method determined in this study was an efficient method for isolating symbiotic bacteria from EPN.The 9 symbiotic bacterial strains isolated had a strong inhibitory effect on the growth of five common plant pathogenic fungi,among which X.bovienii NN6 and X.bovienii NN8 had the strongest comprehensive inhibitory ability on mycelium growth.