Classification And Identification Of A Novel Species Piscinibacter Soli XHJ-5 Isolated From Grassland Soil And Its Forage Promoting Effect

Grassland soil contains a large number of microorganisms,some of which have great potential to accelerate the restoration of degraded grassland.However,most microorganisms in soil are not culturable.In a previous study by our group,bacterial strains isolated from three types of grassland soil in Inner Mongolia,in which strain XHJ-5 was a potential novel species.The detailed taxonomic status of strain XHJ-5and its forage growth-promoting potential are unclear.In this study,we conducted new bacterial characterization,whole genome sequencing and Alfalfa growth promotion experiment of strain XHJ-5,and obtained the following results:（1）Phylogenetic analysis based on 16S rRNA gene sequence showed that strain XHJ-5 was closely related to Piscinibacter caeni MQ-18^T（98.49%16S rRNA similarity）,Piscinibacter defluvii SH-1^T（98.27%）and Piscinibacter aquaticus NBRC102349^T（97.70%）.The ANI,d DDH and AAI values of strain XHJ-5 and the type strains of the genus Piscinibacter were less than 77%,22%and 69%,respectively,well below the threshold of novel species classified by these indicators.Cells of XHJ-5 were gram-negative,aerobic,rod-shaped and non-flagellated.Growth was observed at 25-45°C（optimum 35°C）,salinity 0-1%（optimum 0%）and pH 6-10（optimum 7）.Its major polar lipids were phosphatidylethanolamine,diphosphatidylglycerol and phosphatidylglycerol,and the major fatty acids were C_16:0,Summed Feature 3(C_16:1ω7c and/or C_16:1ω6c)and C_17:0 cyclo.Accordingly,strain XHJ-5 is considered to represent a novel species of the genus Piscinibacter,for which the name Piscinibacter soli XHJ-5 is proposed.（2）Whole genome sequencing results showed that strain XHJ-5 genome size was6648202 bp with 6204 protein-coding genes and 63 RNAs,no plasmid.GC content of strain XHJ-5 was 68.6 mol%.COG,GO and KEGG annotation results indicated that strain XHJ-5 was metabolically active.CARD annotation indicated that the strain may be resistant to tetracyclines.Enzymes related to carbohydrates were predicted from dbCAN2,which were correlated with bacterial acid production ability.Four clusters of secondary metabolite synthesis genes were predicted from antiSMASH.（3）Functional gene analysis showed that strain XHJ-5 could fix CO₂ through the Calvin cycle and could synthesize PHB through a three-step synthesis pathway starting with acetyl CoA.The strain could reduce nitrate and nitrite to ammonia.It could synthesize 16 kinds of amino acids and had a type Ⅱ secretion system.In addition,strain XHJ-5 contained VB₂,VB₃ and several organic acid synthesis pathways as well as several genes related to phosphorus solubilization,which may have the potential to promote plant growth.（4）The results of the pot experiment showed that inoculation of strain XHJ-5increased the plant height,root length,aboveground biomass,underground biomass,root nodule number,root nodule weight and nitrogen fixation activity of Alfalfa;It also significantly increased soil available nitrogen,available phosphorus and water content by 43.3%,80.1%and 287.1%,respectively.The results indicated that strain XHJ-5 had growth promoting effect on Alfalfa.In summary,a novel strain of the genus Piscinibacter was characterized in this study,which enriched the prokaryotic species resource bank.The growth promoting effect of the strain on forage was also studied,which provided basic data for the restoration of degraded grassland.