Characteristics Of Phosphate-Solubilizing Bacteria From The Rhizosphere Of Haloxylon Ammodendron And Spruce And The Phosphate Solubilization Mechanism

Phosphorus（P）is an essential macronutrient for plant growth,development and reproduction.In soils,P exists in various forms that can generally be categorized as insoluble organic phosphorus（P_o）and insoluble inorganic phosphorus（P_i）.However,the available P for plants is very low,and orthophosphates(H₂PO₄^-or HPO₄^2–)are the main forms of phosphorus that is taken up by plants.Therefore,the contradiction between supply and demand of P nutrients is turning increasingly prominent in agricultural production.Chemical phosphate fertilizer has traditionally been employed to obtain the high yield and quality of crops,but its application has been limited by the non-renewability of raw materials and the adverse influence on the ecological health of the environment.Phosphate solubilization is one of the important pathways of plant growth-promoting rhizobacteria（PGPR）to promote plant growth.How to fully and effectively use phosphate-solubilizing bacteria（PSB）to mobilize unavailable forms of soil P for plants has become a hot spot in the fields of plant nutrition and ecology.Different host plants can recruit and form characteristic microbes in the rhizosphere.Haloxylon ammodendron,a dominant species in China’s north-west desert,has superior drought resistance and barren tolerance.Its rhizosphere lacks soluble P for a long time,making it challenging to fulfil plant growth requirements.Moreover,the relevant mechanism of efficient conversion and utilization on phosphate dissolution by PSB in extreme habitats remains unclear.In light of this,the aim of this study is to excavate and compare the culturable bacteria,mainly PSB resources,in the rhizosphere soil of Haloxylon ammodendron with P deficiency,collected at Badain Jaran Desert,Alxa region,Inner Mongolia;and the rhizosphere soil of spruce with P normal,the dominant tree species in typical forest ecosystem of Qilian Mountains.We obtained an excellent strain AWH-NS6 with the strong phosphate solubilization capability,belonging to the genus Serratia by growth promotion experiments.And the mechanism of P-solubilization of AWH-NS6 was systematically studied by genomics,transcriptomics and other omics analysis,combined with the construction of key gene mutants of strain AWH-NS6.The main findings are as follows:1.There were great differences in culturable bacteria in rhizosphere soil of two dominant plants.The soil fertility of Qilian Mountains was much better than that of Alxa region,especially the total phosphorus（TP）,available phosphorus（AP）,soil water content（SWC）and soil organic carbon（SOC）,which resulted in a greater abundance of soil microorganisms than that in Badain Jaran Desert.187 strains of culturable bacteria were isolated from the rhizosphere soil of H.ammodendron,belonging to 5phyla:Firmicutes、γ-Proteobacteria、Actinobacteria、α-Proteobacteria and Bacteroidetes,mainly distributed in Bacillus、Lysinibacillus、Streptomyces、Pseudomonas and Paracoccus etc.203 strains of culturable bacteria were isolated from Qilian Mountain,belonging to 6 phyla:Firmicutes、γ-Proteobacteria、Actinobacteria、α-Proteobacteria、β-Proteobacteria and Bacteroidetes,mainly distributed in Pseudomonas,Flavobacterium,Arthrobacter,Bacillus,and Stenotrophomonas.A novel PGPR species in the genus Paenibacillus was isolated from spruce forest at the height of 3,150 m in the Qilian Mountains based on phenotypic,phylogenetic,genotypic,chemotaxonomic data,and inoculation experiment,for which the name Paenibacillus monticola sp.nov.was proposed.The volatile organic compound（VOC）emitted from strain LC-T2^T could significantly improve the growth of Arabidopsis.It is worth noting that strain LC-T2^T could also significantly increase plant growth of legume crops like white clover.2.9 species of 18 PSB strains were obtained from the rhizosphere soil with the highest content of AP and abundant culturable bacteria in the two regions.The plate qualitative analysis showed that strains AWH-NS6,LT-4,LT-T17,LT-A20,F1-P7 and LT-0 had strong phosphate-solubilizing ability.Among them,strain AWH-NS6 was isolated from the rhizosphere of H.ammodendron,belonging to the genus Serratia,while the other strains were isolated from spruce forests,belonging to the genus Pseudomonas.Inoculation of strain AWH-NS6,LT-4,LT-T17,LT-A20 and F1-P7with strong growth promoting characteristics（phosphate solubilization,nitrogen fixation and secretion of IAA）can promote the growth of Medicago sativa in different degrees,especially strain AWH-NS6.The whole genome data of strain AWH-NS6 were obtained and analyzed by the combination of second-and three-generation sequencing technology.45 genes related to growth promotion and 38 functional genes related to abiotic stress were found in the genome.Strain AWH-NS6 was determined as Serratia sp.AWH-NS6 based on phylogenetic and comparative genomic analysis.3.Adding 2 m M K₂HPO₄ can maximize the phosphate-solubilizing ability of strain AWH-NS6.Transcriptomic analysis of AWH-NS6 on Ca₃（PO₄）₂ at different concentrations mediated by 2 m M K₂HPO₄ showed that:the liberation of H⁺from weak acids secreted by strain AWH-NS6 can convert fixed P into accessible forms for plant in 1 and 5 g·L^-1 Ca₃（PO₄）₂ treatment groups,which affected by sulfur metabolism.Some organic substances were oxidized and related genes were upregulated,resulting in a decrease in p H,which affected the release of H⁺and phosphate solubilization during dissimilatory sulfate reduction（DSR）.However,the sulfur metabolism and phosphate solubilization were inhibited in the 15 g·L^-1 Ca₃（PO₄）₂ groups.Further,cysteine metabolism pathway was enriched in the yellow module based on weighted gene co-expression network analysis（WGCNA）.And cysteine motif was a critical component of the organic acid-related gene pqqE.The expression of pqqE gene was up-regulated in the 5 g·L^-1 Ca₃（PO₄）₂ groups.4.Knockout mutant and complementary strain of pqqE gene of strain AWH-NS6were successfully constructed by molecular cloning,homologous recombination and tri-parental conjugation.The knock and complement of pqqE gene could affect the ability of strain AWH-NS6 to phosphate solubilization,the original physiological and biochemical characterizations,and the utilization efficiency of resources.Inoculation experiments demonstrated that the mutant strain had no obvious effect on the growth of Medicago sativa,and its growth promotion effect was significantly lower than that of wild type strain AWH-NS6（P<0.05）.The complementary strain can partially restore the phosphate-solubilizing ability and can significantly increase plant growth of Medicago sativa（P<0.05）.This study indicated that there are differences in the rhizosphere microbial resources of dominant plants between the two regions.There possess excellent PSB strains in the rhizosphere of H.ammodendron and outstanding PGPR resources in the rhizosphere of spruce.The molecular mechanism of phosphate solubilization of Serratia sp.AWH-NS6 is elucidated.The findings will provide the theoretical basis for the investigation of phosphate solubilization mechanism of PGPR in extreme environments,and explore a new pathway for efficient use of phosphorus in soil-plant system.