Soil Phosphorus Forms In Different Forest Age And Density Of Chinese Fir And Study On The Screening And Mechanism Of Phosphate Solubilizing Endophytes

Chinese fir（Cunninghamia lanceolata）is mainly distributed in the Yangtze River Basin and the area south of the Qinling Mountains in my country.Soil acidification,low phosphorus utilization rate and serious shortage of soil available phosphorus content directly affect the productivity of forest ecosystems.Soil phosphate solubilizing microorganisms are closely related to the transformation of phosphorus,but their application effect is unstable due to the influence of colonization and competitiveness.The endophytic phosphate solubilizing bacteria isolated and screened from Chinese fir tissues are facultative endophytes,which have strong colonization and competition ability and can effectively convert the ineffective phosphorus in the soil into effective phosphorus.This study is based on the study of the changes in soil phosphorus forms in different forest ages and different densities of Chinese fir plantations.Use high-throughput sequencing technology to first determine the presence of endophytic bacteria in the roots,stems,and leaves of robust Chinese fir seedlings in different regions and their composition is rich.Then use selective media to isolate and screen high-efficiency and multifunctional phosphorus-dissolving endophytic strains,the screened high-efficiency phosphorus-solubilizing endophytes are used to study the phosphorus solubilization effect of different phosphorus forms of invalid phosphorus and the mechanism of metabolic acid and phosphorus solubilization.It will provide a theoretical basis for the effective utilization of soil phosphorus in Chinese fir plantations in different regions and the sustainable management of Chinese fir by phosphate-solubilizing endophytes.The main research results of this paper are as follows:（1）The soil inorganic phosphorus components of Chinese fir plantations ofdifferent ages accounted for 55.37%～74.22%of the total phosphorus（TP）content,and the inorganic phosphorus components had the highest occluded P（O-P）,followed by iron-bound P（Fe-P）and calcium-bound P（Ca-P）,and the lowest was aluminum-bound P（Al-P）.The soil total phosphorus,available phosphorus,Al-P,O-P content showed a trend of first decreasing and then increasing with the increase of forest age,and reached the minimum at 12a or 18a.The Fe-P content gradually increased but the difference was not significant.The content of Ca-P increased first and then decreased.Correlation analysis of various phosphorus elements proves that soil available phosphorus（AP）is positively correlated with Al-P,Fe-P,and O-P content,and O-P is positively correlated with Al-P,Fe-P content.（2）Based on the research findings of soil phosphorus in 35-year-old mature forests with initial planting densities of 1667（D1）,3333（D2）,5000（D3）,6667（D4）and 10,000（D5）plants·hm^-2,the soil inorganic phosphorus content of different densities of Chinese fir plantations accountedfor 66.67%-72.89%of the total phosphorus content,which proved that inorganic phosphorus is a huge soil phosphorus source pool.With the increase of stand density,the content of soil TP,O-P and Ca-P showed a trend of decreasing first and then increasing,reaching the minimum at D3.The content of soil AP,Al-P and Fe-P showed positive feedback from D1 to D4,and negative feedback from D4 to D5.Correlation analysis showed that different phosphorus forms were significantly positively correlated with organic matter（OM）content（P<0.01）.In addition to p H,Al-P and Fe-P are positively correlated with other soil indicators.In addition,Al-P,Fe-P,O-P and TN have a significant positive correlation.（3）The microbial diversity of the roots,stems and leaves of Chinese fir in the four production areas is significantly different.The bacterial community diversity of roots and leaves is Jiangxi>Sichuan>Hubei≈Guangxi,the stems showed the order of Sichuan>Jiangxi>Guangxi>Hubei.The fungal community diversity of roots,stems and leaves is shown as follows:Sichuan>Jiangxi>Hubei>Guangxi.The composition of the microbial community in the roots,stems and leaves of Chinese fir in different habitats is similar.The dominant bacterial phyla are Proteobacteria,Actinobacteria,Firmicutes,and the dominant fungal phyla are Ascomycota and Basidiomycota,but their relative abundance and quantity are significantly different.From the genus level,there are significant differences in the genus and composition of the dominant microorganisms in the roots,stems and leaves of fir in different habitats.（4）Determine the best surface disinfection time for separating different tissues and organs of Chinese fir endophytes.20 strains of phosphate solubilizing bacteria were isolated from the roots,stems and leaves of Chinese fir using PVK inorganic phosphorus medium,of which 15were from roots,3 were from stems,and 2 were from leaves.Combining physiology and biochemistry,16Sr DNA identification and phylogenetic tree construction,it was determined that the selected phosphorus-solubilizing bacteria belonged to 9 genera,namely Pseudomonas,Enterobacter,Klebsiella,Paraburkholderia,Pantoea,Novosphingobium,Ochrobactrum,Alcaligenes,Allorhizobium.Among the 20 strains,HRP2（123.733mg·L-1）had the highest phosphate solubilizing ability.And HRP2,HRP3,SSP2,SRP2,GRP2 and JRP22 all possess various characteristics of promoting plant growth,such as nitrogenase,ACC deaminase,IAA production and siderophore.（5）Taking high-efficiency and multifunctional phosphate solubilizing bacteria HRP2,HRP3,SSP2,SRP2,GRP2 and JRP22 as the research objects,it was found that their ability to dissolve different forms of inorganic phosphorus is significantly different,which is expressed as calcium phosphate>aluminum phosphate>iron phosphate.Among them,the ability of SSP2to dissolve phosphorus is at a relatively high level.The phosphate solubilizing ability of phosphate solubilizing bacteria differs significantly between different inoculation treatments.The phosphate solubilizing ability is expressed as inoculation phosphate solubilizing bacteria fermentation stock>centrifugal sediment>centrifugal supernatant>sterilized supernatant.The significant difference in the ability of solubilizing phosphorus between inoculation treatments indicates that the ability of solubilizing phosphorus to solubilize phosphorus is determined by complex metabolites such as strains,enzyme activities,organic acids,protons,and so on.（6）Taking high-efficiency phosphate solubilizing bacteria HRP2,HRP3,SSP2,SRP2,GRP2 and JRP22 as the research objects,it was found that the types and contents of organic acids secreted by phosphate solubilizing bacteria under different phosphorus sources are significantly different.When calcium phosphate is used as the phosphorus source,the phosphorus solubilizing strains can all produce oxalic acid,tartaric acid,and citric acid.The maximum concentration is 180.52mg·L^-1,220.71mg·L^-1,294.02mg·L^-1.But HRP2 and JRP22can also secrete lactic acid and acetic acid,HRP3 can secrete lactic acid,and GRP2 can produce malic acid and lactic acid.;When iron phosphate is used as the phosphorus source,the phosphorus solubilizing strains can all produce oxalic acid,lactic acid,and acetic acid.The maximum concentration is 253.33mg·L^-1,246.38mg·L^-1,343.43mg·L^-1.Except SRP2,other strains can secrete tartaric acid,SSP2,JRP22,HRP3,JRP2 can secrete malic acid,SSP2,HRP2,JRP22,SRP2 can secrete citric acid,and the secretion content of each acid is significantly different;When aluminum phosphate is used as the source of phosphorus,all the phosphorus solubilizing strains can produce oxalic acid,tartaric acid,and lactic acid.The maximum concentration is 166.25mg·L^-1,301.18mg·L^-1,368.25mg·L^-1.But SSP2 and HRP2can also secrete lactic acid,citric acid,and acetic acid,SRP2 can secrete citric acid and acetic acid,and JRP22 and GRP2 can secrete malic acid,indicating that the secretion of organic acids and types of strains are significantly different.（7）Exogenous single organic acid dissolves calcium phosphate as tartaric acid>acetic acid>citric acid>malic acid>oxalic acid,dissolves iron phosphate as tartaric acid>lactic acid>acetic acid>citric acid,and dissolves aluminum phosphate as tartaric acid>lactic acid>citric acid>Acetic acid>malic acid>oxalic acid.The phosphate solubilizing capacity of combined acid and single organic acid is significantly lower than that of phosphate solubilizing bacteria,which proves that the phosphate solubilizing ability of phosphate solubilizing bacteria is the result of the combined effect of organic acid,phosphatase,proton and other factors.