黄粉虫致病菌的分离鉴定、发酵条件优化及其对植物病原菌的抑制作用

Isolation, identification and optimization of fermentation conditions of pathogenic bacteria from Tenebrio molitor and their inhibitory effects on plant pathogens

  • 摘要: 【目的】研究黄粉虫病死幼虫体内的致病菌、致病菌抑制植物病原菌的效果及其最佳发酵条件,为有效防控黄粉虫病害及利用致病菌进行农业病虫害生物防治提供科学依据。【方法】以罹病死亡的黄粉虫幼虫为材料,利用平板涂布法及划线法分离致病菌;依据柯赫氏法则测定致病菌的致病性;通过形态学观察、生理生化测定和16S rDNA序列分析确定致病菌株的分类地位;采用平板对峙法测定致病菌对12种供试植物病原真菌的抑制效果;利用单因素试验对致病菌的发酵条件进行优化。【结果】从罹病黄粉虫虫体中分离获得3株致病菌,从中筛选出致病效果最好的菌株并命名为WU-1,其对黄粉虫幼虫的致死率为58.00%; WU-1菌株对供试12种植物病原真菌具有广谱抑菌性,抑菌率为60.54%~78.33%。结合WU-1菌株菌落形态特征、生理生化测定和16S rDNA序列分析结果,将WU-1菌株鉴定为贝莱斯芽孢杆菌(Bacillus velezensis)。WU-1菌株发酵条件优化结果表明,其最适培养基为YSP培养基;最适发酵条件为250 mL三角瓶装液量30 mL、初始pH 8、接菌量50 μL、培养温度40℃、转速240 r/min、发酵时间24 h。【结论】优化的发酵方案可用于快速、大批量培养WU-1菌悬液。WU-1菌株对黄粉虫幼虫具有较好的致病效果,可为鞘翅目害虫防治提供参考。该菌株对12种植物病原真菌具有良好的抑制作用,可作为研制植物病害生物防治菌剂的备选菌株。

     

    Abstract: 【Objective】The purpose of the study was to research the pathogenic bacteria in the diseased and dead larvae of Tenebrio molitor,the inhibitory effect of pathogenic bacteria on plant pathogens and their optimal fermentation conditions,in order to provide scientific basis for effective prevention and control of T. molitor diseases and biological control of agricultural pests and diseases by using pathogenic bacteria.【Method】Using the diseased and dead T. molitor larvae as materials,the pathogenic bacteria were separated by plate coating method and streaking method. The pathogenicity of pathogenic bacteria was determined according to Koch’ s rule. The taxonomic status of pathogenic strains was determined by morphological observation,physiological and biochemical determination and 16S rDNA sequence analysis. The inhibitory effect of pathogenic bacteria on 12 kinds of plant pathogens was determined by plate confrontation method. The fermentation conditions of pathogenic bacteria were optimized by single factor experiment.【Result】Three strains of pathogenic bacteria were isolated from diseased T. molitor,and the strain with the best pathogenic effect was screened and named WU-1, which had a mortality rate of 58.00% against T. molitor larvae. WU-1 strain had broad-spectrum antibacterial activity against 12 plant pathogens tested,and the antibacterial rate was 60.54%-78.33%. Combined with the colony morphological characteristics,physiological and biochemical determination and 16S rDNA sequence analysis results,WU-1 strain was identified as Bacillus velezensis. The optimization results of fermentation conditions of WU-1 strain showed that the optimum medium was YSP medium. The optimum fermentation conditions were a filling volume of 30 mL in 250 mL triangular flask,an initial pH of 8,an inoculation amount of 50 μL,a culture temperature of 40 ℃,a rotation speed of 240 r/min,and a fermentation time of 24 h.【Conclusion】The optimized fermentation scheme can be used for rapid and large-scale culture of WU-1 bacterial suspension. WU-1 strain has a good pathogenic effect on T. molitor larvae,which can provide reference for Coleoptera pest control. This strain has a good inhibitory effect on 12 kinds of plant pathogens, and can be used as a candidate strain to develop biological control agents for plant diseases.

     

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