Isolation, identification and fermentation condition optimization of symbiotic bacteria from entomopathogenic nematodes resistant to rice sheath blight

【Objective】 The strains of symbiotic bacteria from entomopathogenic nematodes were isolated from Chilo suppressalis individuals，the strain with high antibacterial activity against rice sheath blight was screened to further enrich the resources of entomopathogenic nematode symbiotic bacteria，which could provide theoretical basis for the application of entomopathogenic nematode symbiotic bacteria in the control of Chilo suppressalis and rice sheath blight. 【Method】 Strains of symbiotic bacteria from entomopathogenic nematodes were isolated by hemolymph separation method from the dead Chilo suppressalis individuals infected with Steinernema. NBTA culture morphology combined with 16S rRNA amplification sequencing was used to identify the symbiotic species of entomopathogenic nematodes. Plate culture method was used to determine the inhibitory rate of symbiotic bacteria from entomopathogenic nematodes against rice sheath blight strain，and strain with high antibacterial activity against rice sheath blight was screened. The whole genome of strain with high antibacterial activity was sequenced，and the growth curve of strain with high antibacterial activity was obtained by liquid culture. The carbon source，nitrogen source and inorganic salt of the strain liquid fermentation medium of strain with high antibacterial activity were optimized by single factor test and orthogonal test，and the optimal conditions for liquid fermentation，inoculation amount，fermentation temperature and rotation speed were screened. 【Result】 Five strains of symbiotic bacteria from entomopathogenic nematodes were isolated and identified as Xenorhabdus nematophila strains. NBTA culture characteristics showed that 1 strain of the 5 entomopathogenic nematode symbiotic bacteria was secondary bacteria，and the other 4 strains were primary bacteria. The primary strain HN04 had the highest inhibitory rate against rice sheath blight（73.62%），and the secondary strain HN01 had the lowest inhibitory rate（55.29%），strain HN01 was significantly different from that of the 4 primary strains（P<0.05）. The strain HN04 with the highest inhibitory rate was selected for follow-up study. The results of whole genome sequencing showed that strain HN04 consisted of a ring chromosome with a size of 7998302 bp and an average GC content of 44.03%. The logarithmic growth period of strain HN04 was 4-28 h，the stable period was 28-52 h，and it entered the decline period after 52 h. Single factor and orthogonal experiment optimized the liquid fermentation conditions of strain HN04，and the medium combination was 2.0% maltose，2.0% peptone，1.0% NaCl. The main order of influence of each factor on the concentration of bacteria in the fermentation solution was nitrogen source factor > inorganic salt factor > carbon source factor. The optimal liquid fermentation conditions were 30%-40% of liquid in triangular bottle，4%-12% of inoculum，fermentation temperature 28-32 ° C and rotation speed 180 r/min. 【Conclusion】 Xenorhabdus nematophila strains are isolated from Chilo suppressalis. The primary Xenorhabdus nematophila bacteria HN04 has the highest antibacterial activity against rice sheath blight. The optimized fermentation scheme can be used to rapidly culture HN04 strain suspensions in large quantities. HN04 has potential application value in controlling Chilo suppressalis and rice sheath blight.