Characterization And Plant Growth-promoting Mechanism Of Cold-adapted Pgpb From Dominant Herbage In The Alpine Grassland Of The Qilian Mountains

The degradation of alpine grassland has seriously threatened the sustainable development of animal husbandry and ecology in western China.Although the protection and restoration measures have been taken from a national strategic height,it takes a long time to fully restore the degraded grassland to a state of natural balance due to its ecological fragility.Plant growth-promoting bacteria（PGPB）are a group of beneficial microorganisms that widely exist in nature.Due to the abilities of growth promotion,biocontrol,and soil ecological regulation,PGPB are considered to be the potential strain resources to reduce fertilization while increase efficiency in agriculture,as well as in the near-natural restoration of degraded ecology.In this study,culturable cold-adapted PGPB strains with growth-promoting characteristics were screened by selected medium from dominant plants in alpine grasslands of the Qilian Mountains,China.Their growth-promoting characteristics such as nitrogen fixation,phosphorus solubilization,IAA secretion,ACC deaminase and siderophore production were further studied.The genetic diversity and identity of the PGPB strains were revealed by 16S r DNA sequencing.Moreover,excellent strains were used to prepare compound microbial agent,and the effects of compound microbial agent combined with chemical fertilizer reduced on mixed pasture growth were analyzed using high-throughput absolute quantification sequencing.The inorganic phosphate solubilization mechanism of Pseudomonas canadensis MYp En8 was studied based on the integrated analysis of genomics,transcriptomics and metabolomics.The mains results are as follows:（1）A total of 556 and 265 PGPB strains were screened from the rhizosphere and root samples of ten grass species in the alpine grasslands of the Qilian Mountains.Among them,there were 286,147,and 123 strains of nitrogen-fixing,inorganic,and organic phosphorus-dissolving bacteria in the rhizosphere,respectively,and 163,29,and 73 strains in the roots,respectively.Among all isolates,there were 19,104,and 115 strains of ACC deaminase-,siderophore-and IAA-producing capacity.Moreover,the distribution of PGPB varied with different geographical habitats and host plant species,showing rhizosphere effects.（2）A total of 172 cold-adapted PGPB with potential applications at low temperatures were screened,and more than 83.14%of them had two or more growth-promoting functions.Their nitrogenase activity was between 22.51-907.31 nmol/mg·h.And the inorganic phosphate solubility ranged from 23.18 to 538.13μg·m L^-1,which was negatively correlated with p H.The organic phosphate solubility ranged from 1.81 to 85.96μg·m L^-1,which had no significant correlation with p H.Sixty-two strains could secrete IAA,with contents between3.24-48.95μg·m L^-1.Nineteen strains had ACC deaminase activity,and the enzyme activity ranged from 0.0087 to 3.24μmol/（mg·protein·h）.The siderophore units（SU）of seventy-five strains that effectively inhibited Fusarium oxysporum,Rhizoctonia solani,Alternaria solani,and Helminthosporium tritici-vulgaris were between 0.06-0.739.All isolates were divided into 9 genera by 16S r RNA gene sequencing and phylogenetic analysis,of which Pseudomonas was the dominant genus,Serratia and Erwinia were the subdominant genera.（3）The PGPB possessing different growth-promoting characteristics made different contributions to the development of Elymus nutans seedlings.The abilities of nitrogen fixation,inorganic phosphate solubilization and ACC deaminase activity were positive for aboveground parts,while the phosphate solubility and siderophore concentrations had a positive effect on roots.Six compound bacterial agents were constructed by excellent PGPB strains according to equal biomass,all of which could significantly promote plant growth.Their root colonization trended to increase firstly and then decline.The ¹⁵N isotopic dilution method showed that their nitrogen fixing efficiency（%）ranged from 16.40 to 25.05.In general,the optimal combination was Pseudomonas sp.MYp En8,Pseudomonas sp.MYn D1,Acinetobacter sp.TZn Hn8,Pseudomonas sp.MYm G4,and Flavobacterium sp.MYp Jn1.（4）The growth of mixed pasture can be greatly affected by different fertilizer methods.The optimized inoculation treatment was 15%chemical fertilizers reduced combined with compound bacterial agents.Chemical fertilizer application could not only significantly increase soil nutrients such as available nitrogen（AN）and available phosphorus（AP）,but also increase soil electrical conductivity（EC）.Inoculation with microbial fertilizers not only increased the contents of AN and AP but also increased the activities of soil catalase（S-CAT）,soil sucrase（S-SC）,and soil alkaline phosphatase（S-ALP）.Different fertilizer applications sharply affected the community structure and ecological function of soil bacteria and fungi.Actinobacteria,Proteobacteria,Firmicutes,and Acidobacteria were the dominant bacterial phylum,while Bacillus,Gaiella,and Gp6 were the dominant genera.And the abundance of the aerobic ammonia oxidation group significantly increased,but methanol oxidation group decreased under all treatments.Ascomycota and Mortierellomycota were the dominant fungal phylum.However,there were significant differences at the genus level,and Mortierella was the only common dominant genus.The abundance of saprotroph groups such as dung saprotroph,wood saprotrop,soil saprotroph,and undefined saprotroph increased significantly under different fertilization treatments,whereas the abundance of algal parasite undefined saprotroph,plant pathogen,ectomycorrhizal-wood saprotroph decreased.The amount of chemical fertilizer and microbial fertilizer disturbed bacterial community structure and ecological function.Soil fungi were more sensitive to chemical fertilizer compared of microbial fertilizer.EC,AN,AP,available potassium（AK）,and solid-urease（S-UE）were the key factors driving the structures of bacterial and fungal communities.（5）The draft genome sequence of the cold-adapted phosphorus-solubilizing strain Pseudomonas canadensis MYp En8 contains 6171758 bp with 60.33%GC.A total of 5664coding genes were predicted,with an average length of 975 bp,and the total length of coding DNA region accounted for 89.48%of the whole genome.Additionally,the number of interspersed nuclear elements,tandem repeats,non-coding RNAs,genomic islands,prophage sequences,secreted proteins,and secondary metabolic gene clusters were 69,156,103,10,6,486,and 11,respectively.A total of 5591 genes were annotated by GO,KEGG,and COG databases,which accounted for 98.71%of coding genes.In terms of the Go cellular component,genes were mainly enriched in cell and cell part.In terms of molecular function,genes were mainly enriched in catalytic activity and binding.And in terms of biological process,genes were mainly enriched in cellular process and metabolic process.KEGG annotation revealed that the abundance of metabolism-related genes was active,and amino acid metabolism was the main pathway at the level 2 of classification.Several organic acid synthase genes,including gluconic acid,citric acid,malic acid,and fumaric acid were also identified.In addition,phosphate metabolism-related genes,such as pho regulon（pho）,phosphate-specific transport（Pst）system,and polyphosphate kinase（ppk）,as well as eight homologous sequences of cold shock protein Csp A.All of them may related to the phosphate-solubilizing effect of MYp En8 at low temperatures.（6）The transcriptomics of MYp En8 under different incubation time using Ca₃（PO₄）₂ as the phosphorus source was analyzed using RNA-seq technology.The differentially expressed genes（DEGs）among samples cultured for 6 h（A）,24 h（C）,and 48 h（D）were compared with 12 h（B）.There were 1,994 common DEGs for groups A vs B,C vs B,and D vs B,while the specific DEGs were 657,116,and 366,respectively,and all DEGs were divided into four clusters by H-Cluster.GO enrichment showed that the terms most enriched involved translation,ribosome,and structural constituent of ribosome.KEGG enrichment analysis showed that the DEGs within significant up-regulated trends were enriched in biosynthesis of secondary metabolites,biosynthesis of amino acids,ribosome and biosynthesis of cofactors,and biosynthesis of secondary metabolites,among which biosynthesis of secondary metabolites was detected at the highest abundance.And the DEGs within significant down-regulated trends were enriched in quorum sensing and biofilm formation-Pseudomonas aeruginosa.In general,the expression patterns of phosphate solubilizing genes in MYp En8 were different in all comparison groups,involving multiple metabolic pathways and different organic acid.（7）The transcriptomics of MYp En8 under different incubation time using Ca₃（PO₄）₂ as the phosphorus source was analyzed based on untargeted metabolomics.The differential metabolites cultured for 6 h（A）,24 h（C）,and 48 h（D）were compared with 12 h（B）.The number of metabolites detected under negative and positive ionization modes was 257,337,respectively,which mainly enriched in the metabolism pathway.And the secondary pathway mainly involved global and overview maps,and amino acid metabolism.In the human metabolome database（HMDB）,the metabolites all involved organic acids and derivatives.There were 7 of the top10 differential metabolites in all samples.And groups B,C,and D shared 13 common metabolites,while groups C and D shared 17 common metabolites,involving various small organic acids such as glucuronic acid,galacturonic acid,glutaric acid,malonic acid,D-glucuronic acid,D-（-）-quinic acid,succinic acid,5-Keto-D-gluconate,and pantothenic acid.KEGG enrichment showed that several metabolites,including pantothenate and Co A biosynthesis,pyrimidine metabolism,propanoate metabolism,ubiquinone and other terpenoid-quinone biosynthesis,and biosynthesis of amino acids were significantly enriched in groups C vs B,and D vs B,indicating that the longer the incubation time,the more similar the expression patterns and the metabolic pathways of differential metabolites involved.The correlation analysis between metabolome and transcriptome showed that there was a significant correlation between top 10 DEGs and top5 differential metabolites.The carbon metabolism pathway was significantly enriched in all groups.And oxidative phosphorylation,sulfur metabolism,microbial metabolism in diverse environments,biosynthesis of secondary metabolites,pyrimidine metabolism,and aminoacyl-t RNA biosynthesis were significantly enriched in comparison groups C vs B and D vs B,which indicating that the main DEGs and differential metabolism involved in the same biochemical metabolic pathway at the late phage of fermentation.In summary,the accumulation of low-molecular-weight organic acids was the main reason of p H decline and insoluble inorganic phosphate solubilization for strain MYp En8.However,it was not the result of single organic acid,but the synergistic action of various organic acids.