Study On The Growth-promoting Effect Of Rhizosphere Phosphate-solubilizing Bacteria And Endophytic Phosphate-solubilizing Fungi On Masson Pine Seedling

In order to obtain a compound phosphate（P）solubilizing microbial agent that has an effective growth-promoting effect on P.massoniana seedlings,this study was conducted to screened both endophytic P solubilizing fungi（PSF）and rhizosphere P solubilizing bacteria（PSB）of P.massoniana,and to test the P solubilizing characteristics of the strains and their growth-promoting effect on P.massoniana seedlings to obtain PSF and PSB with obvious growth-promoting effects.On this basis,the compatibility of PSF and PSB was tested by the antagonism method,and then the combination of PSF and PSB with good compatibility was inoculated into P.massoniana seedlings,and finally the growth,physiology,nutrients and transcriptional regulation of P.massoniana seedlings as well as the nutrients,enzyme activity and microbial community of the rhizosphere soil were determined,to clarify the growth-promoting effect and mechanism of the combination on P.massoniana seedlings.The main results were as follows:（1）A total of five PSF were screened out of sixty-nine endophytic fungi of P.massoniana isolated in the early stage of the research group.The P solubilizing index（PSI）of five PSF ranged from 2.17 to 2.87,and the P solubilizing capacity（PSC）ranged from 238.97 to 354.38 mg/L.After identification of morphology and molecular biology,the strains Y1-1,Y2,Y7,G-BP and G-17 were Colletotrichum godetiae,Pestalotiopsis grevilleae,Paraphaeosphaeria verruculosa,Peronospora viciae and Penicillum oxalicum,respectively.The PSC of five PSF to four kinds of phosphates was shown as:Ca₃（PO₄）₂（238.96～348.01mg/L）>Fe PO₄·2H₂O（160.01～262.73 mg/L）>Ca HPO₄·2H₂O（120.29～225.36 mg/L）>Al PO₄（55.17～101.09 mg/L）.After inoculating five PSF into P.massoniana seedlings for 60 days,all strains could colonize in the roots of P.massoniana seedlings and show a certain growth-promoting effect,especially strains G-BP and G-17.Compared with the control,the inoculation of strains G-BP and G-17 could significantly increase seedling height（85.87%,45.11%）,root length（119.01%,102.11%）,lateral roots number（65.00%,55.00%）,fresh weight（75.80%,47.13%）,dry weight（212.61%,100.00%）,total chlorophyll（9.09%,18.18%）,soluble protein（31.06%,42.98%）,soluble sugar（254.05%,76.41%）,total P（65.91%,54.55%）and total K（31.04%,26.64%）of P.massoniana seedlings,as well as available P（47.00%,34.99%）,available N（23.00%,13.00%）and available K（22.62%,44.69%）in the rhizosphere soil（P<0.05）.（2）A total of seventeen PSB were isolated from the rhizosphere soil of healthy P.massoniana.The PSI of each PSB ranged from 2.83 to 5.97,and the PSC ranged from 47.69 to 411.98 mg/L.Among them,strains WJ2 and WJ27 had the strongest PSC,which were 411.98 mg/L and 375.15 mg/L,respectively,which were significantly higher than the PSC of other strains（P<0.05）.Strains WJ2 and WJ27 were identified as Burkholderia ginsengisoli and B.arboris by phenotype observation,physiological and biochemical identification and phylogenetic tree analysis.The results of single factor experiment showed that strain WJ2had the best PSC when the carbon source was lactose,the nitrogen source was urea,the C/N was 10:1,the Na Cl content was 0 g/L and the initial p H was 8.The strain WJ27 had the highest PSC when the carbon source was galactose,the nitrogen source was urea,the C/N was 25:1,the Na Cl content was 5 g/L,and the initial p H was 5.Both strains WJ2 and WJ27 had certain ability to solubilize various insoluble phosphates,as Ca₃（PO₄）₂（204.42 mg/L,220.85 mg/L）>Al PO₄（177.70mg/L,182.33 mg/L）>Fe PO₄·2H₂O（151.82mg/L,129.19 mg/L）>Ca HPO₄·2H₂O（108.19 mg/L,115.23 mg/L）.There were significantly negative correlations between the PSC of strain WJ2 to Ca₃（PO₄）₂,Al PO₄and the p H of culture mediums but no significantly correlations between the PSC of strain WJ2 to Fe PO₄·2H₂O,Ca HPO₄·2H₂O and the p H of culture mediums;while there were significantly negative correlations between the PSC of strain WJ27 to four phosphates and the p H of culture mediums.HPLC-MS analysis showed that the main organic acids secreted by strain WJ2 were trifluoroacetic acid,trans-aconitic acid,D-pipecolinic acid and citric acid,and the main organic acids secreted by strain WJ27 were citric acid,malonic acid and D-pantothenic acid.In the pot experiment,compared with the control,the inoculation of strains WJ2 and WJ27 could significantly promote seedling height（11.74%,14.27%）,root length（45.73%,36.95%）,lateral roots number（39.87%,56.08%）,root fresh weight（116.67%,158.33%）,root dry weight（50.00%,100.00%）,shoot（stem,leaf and branch）soluble protein（32.40%,23.21%）,root soluble protein（85.70%,31.57%）,shoot soluble sugar（57.22%,59.77%）,total N（24.70%,32.50%）,total P（13.82%,16.26%）and total K（21.48%,50.42%）of P.massoniana seedlings,as well as available P（18.32%,11.94%）and available K（10.68%,17.01%）in rhizosphere soil（P<0.05）.In addition,WJ27 also could significantly promote the content of available N in rhizosphere soil,which increased by 18.11%（P<0.05）.（3）In vitro,two combinations of bacteria and fungi with strong compatibility were screened out after pair culture of PSF and PSB,which were WJ27+G-17 and WJ2+G-BP.Compared with single inoculation of strain WJ27 or G-17,the PSC of WJ27+G-17 significantly increased 74.67%and 44.42%,respectively（P<0.05）.Compared with single inoculation of strain WJ2 or G-BP,the PSC of WJ2+G-BP significantly increased 98.88%and 71.20%,respectively（P<0.05）.The results showed that there was a certain synergistic effect between the strains in the two combinations on P dissolution.In addition,after inoculation of the composite and single strains for 60 days,compared with the control,all inoculations could significantly promote seedling height,root length,shoot fresh weight,root fresh weight,shoot dry weight and root dry weight of P.massoniana seedlings,especially with WJ27+G-7 after processing plant growth is most obvious,were increased by 37.85%,87.89%,57.36%,108.00%,40.69%and 68.16%（P<0.05）.The physiology of P.massoniana seedlings were determined and found that all inoculations could promote multiple physiological indicators of seedlings to varying degrees compared with the control treatment.Among them,the number of physiological indicators increased the most after treatment with WJ27+G-17,especially root activity,chlorophyll b,total chlorophyll,shoot soluble protein,root soluble protein,shoot soluble sugar,root soluble sugar,shoot SOD,root SOD,shoot POD,root POD,shoot CAT and root CAT,which significantly increased by 114.45%,64.06%,35.41%,32.87%,44.44%,49.12%,127.02%,176.47%,43.24%,131.71%,58.08%,40.19%and 35.79%,respectively（P<0.05）.The nutrient content of P.massoniana seedlings was tested and found that compared with the control,all inoculations could increase the total N,total P and total K content of the plant to varying degrees,especially the seedlings under WJ27+G-17 treatment,which significantly increased by 28.37%,103.17%and 44.44%,respectively（P<0.05）.In addition,compared with the control,the available N,available P,available K,urease,phosphatase and catalase in rhizosphere soil of P.massoniana seedlings inoculated by WJ27+G-17 were significantly increased by 81.62%,115.25%,43.87%,132.19%,37.31%and 100.00%,respectively（P<0.05）.In summary,the combined treatment of strains WJ27 and G-17 had better growth-promoting effects on P.massoniana seedlings than treatment with a single strain.（4）The effects of single and combined treatments of strains WJ27 and G-17 on the rhizosphere soil microbial community of P.massoniana seedlings showed that there were 706875 effective fungal sequences and 828066 bacterial effective sequences in the rhizosphere soil of P.massoniana seedlings under different treatments.All sequences were clustered at a similarity level of 97%,and the numbers of fungal OTUs obtained were 635（CK）,597（WJ27）,619（G-17）and 562（WJ27+G-17）;bacterial OTU numbers were1814（CK）,2114（WJ27）,1645（G-17）and 1454（WJ27+G-17）.Alpha diversity analysis showed that compared with control,inoculation with bacteria WJ27 significantly reduced fungal diversity of rhizosphere soil,but had no significant effect on bacterial diversity.In contrast,inoculation with fungal G-17significantly reduced bacterial diversity,but had no significant effect on fungal diversity.When combined inoculation of strains WJ27 and G-17,both bacterial and fungal diversity decreased significantly（P<0.05）.Microbial community structure showed that the dominant fungal phyla in the rhizosphere soil of P.massoniana seedlings under different treatments were Ascomycota and Basidiomycota,and the dominant bacteria phyla were Proteobacteria,Actinomycetes,Acidobacteria,Bacteroidetes,Chloroflexi,and Chlamydiae.Among them,at the genus level,compared with the control,the relative abundance of functional microflora such as Trichophaea,Tomentella,Burkholderia,and Sphingomonas in the rhizosphere soil of P.massoniana seedlings treated by WJ27+G-17 significantly increased,while the relative abundance of plant-pathogenic microflora such as Ustilaginoidea,Fusarium,and Gliocladiopsis significantly decreased（P<0.05）.（5）A total of 194960 Unigene were obtained from the transcriptome sequencing of P.massoniana seedlings in two treatment groups（WJ27+G-17 treatment and clear water control）.Through comparative expression profile analysis,compared with the control,combined inoculation of strain WJ27 and G-17caused a total of 7115 significantly differentially expressed genes（DEGs）in P.massoniana seedlings,including 2418 up-regulated genes and 4697 down-regulated genes.GO enrichment analysis showed that the DEGs mainly involved response to stimulus,signaling,multi-organism process,extracellular region,membrane part,catalytic activity,etc.;KEGG analysis showed that the DEGs were significantly enriched in ribosome,carbon metabolism,glyoxylate and dicarboxylate metabolism,plant-pathogen interaction,glycolysis,citrate cycle,photosynthesis pathway and other pathways.Among them,the DEGs of photosynthetic system I（psa B,psa C,psa L）,photosynthetic system II（psb B,psb E,psb P,psb27,psb28）,cytochrome b6f（pet B）,photosynthetic system electron transport（pet E）and f-ATPase（ATPF1A,ATPF1G,ATPF0A）in the photosynthesis pathway were significantly up-regulated by WJ27+G-17.