Community Diversity Of Phosphorus Solubilizing Bacteria In Agricultural Soils Of South China And Their Succession Patterns Under Different Phosphorus Sources Regulation

Phosphorus（P）is one of the three essential mineral nutrients for crop growth and development,and is closely related to crop yield and quality.South China is rich in light,heat,and water resources,making it a major grain and crop producing region in China.Due to the low p H and other soil characteristics of the acidic red soil in this region,the phosphorus element in the soil is extremely easy to be fixed,forming insoluble organic and inorganic phosphorus that is difficult for plants to utilize.At the same time,excessive application of phosphorus fertilizer in agricultural production in pursuit of high yield leads to high total phosphorus content and low available phosphorus content in the soil,and low utilization efficiency of phosphorus fertilizer.This not only exacerbates soil degradation and environmental pollution,but also accelerates the depletion of phosphate rock resources.The soil in South China is rich in beneficial microbial resources,including a large number of phosphorus solubilizing bacteria（PSB）.PSB is a class of functional bacteria that can activate insoluble phosphorus in soil through enzymatic hydrolysis of insoluble organic phosphorus and dissolution of insoluble inorganic phosphorus.Making full use of PSB to activate insoluble phosphorus in soil is an important means to develop sustainable agriculture.However,we are not clear about the PSB resources in agricultural soils in South China,and how soil chemistry affects the composition and distribution of PSB communities;Secondly,different phosphorus sources,including readily available phosphorus,insoluble organic phosphorus,and insoluble inorganic phosphorus,have unclear regulatory mechanisms for PSB community succession.In order to make better use of PSB resources in South China,we first need to fully understand the community composition of PSB in agricultural soils in South China and the response model to soil chemistry;The second step was to explore the response characteristics of PSB communities to different phosphorus sources on a time scale through a microcosmic culture experiment with different phosphorus sources,including control,KH₂PO₄,organic phosphorus（Lecithin）,Ca₃（PO₄）₂,and Fe PO₄;Finally,we will explore new PSB resources in the agricultural soil of South China,providing efficient strains of new resources for the PSB resource pool.In order to answer the above scientific questions,this study designed three experiments to clarify,the main conclusions as follows:（1）Using amplified sequencing and absolute quantitative techniques of functional genes（pho D gene responsible for the hydrolysis of organic phosphorus and pqq C gene responsible for the dissolution of inorganic phosphorus）,the characteristics and influencing factors of PSB community composition in agricultural,vegetable,and orchard soils in Guangdong Province were studied.The results showed that the composition of PSB community in soil was very rich,and the pho D-harbouring bacteria came from 9 phyla,16 classes,41 orders,68 families,111 genera,and 2398OTUs;pqq C-harbouring bacteria come from 6 phyla,10 classes,24 orders,41 families,63 genera,and 1880 OTUs.In general,the species diversity of pho D-harbouring bacteria in soil is higher than that of pqq C-harbouring bacteria.The dominant phyla（relative abundance>1%）of pho D-harbouring bacteria are Proteobacteria（22.80%）and Actinobacteria（8.50%）;The dominant genera（relative abundance>1%）are Bradyrhizobium（7.98%）,Streptomyces（6.24%）,Burkholderia（3.95%）,and Ralstonia（1.95%）.The dominant phyla（relative abundance>1%）of pqq C-harbouring bacteria are Proteobacteria（38.60%）and Actinobacteria（5.07%）;The dominant genera（relative abundance>1%）are Variovorax（6.89%）,Azospirillum（5.76%）,Pseudomonas（3.76%）,Methlibium（2.76%）,Sorangium（2.43%）,Burkholderia（2.42%）,Mycobacterium（1.85%）,Leptothrix（1.67%）Cupriafidus,1.26%,Streptomyces,1.22%,Xanthomonas,1.16%.Redundancy analysis（RDA）showed that soil chemistry properties had a significant impact on the composition of pho D-and pqq C-harbouring bacterial community.The most important soil chemistry properties affecting the composition of pho D-harbouring bacterial community are:p H,OP（organic phosphorus）and TN（total nitrogen）;The most important soil chemistry properties affecting the composition of pqq C-harbouring bacterial community are p H,IP（inorganic phosphorus）,AP（available phosphorus）,and OP（organic phosphorus）.To sum up,the agricultural soil in South China is rich in PSB resources,and the species diversity of pho D-harbouring bacteria is higher than that of pqq C-harbouring bacteria.Proteobacteria and Actinobacteria are the dominant gates of PSB,with Bradyrhizobium dominating among pho D-harbouring bacteria and Variovorax dominating among pqq C-harbouring bacteria.p H is a key factor affecting the structure of PSB communities.Compared to pho D-harbouring bacteria,pqq C-harbouring bacterial community are more susceptible to the impact of phosphorus in soil.（2）The effects of different phosphorus source treatments on the absolute abundance,community composition,and phosphorus composition of PSB at a time scale were studied using techniques such as amplified sequencing,absolute quantification,and phosphorus fractionation of functional genes（pho D and pqq C）.The absolute quantitative results showed that compared with the control group,the addition of KH₂PO₄ and Ca₃（PO₄）₂ significantly increased the abundance of the pho C gene（P<0.05）,and the abundance of the pho C gene increased with the increase in culture time,reaching the highest level on the 10th day（1.72×10⁶ copies/g dry soil）;The addition of KH₂PO₄,Lecithin,and Ca₃（PO₄）₂ significantly increased the abundance of the pho D gene（P<0.05）.The abundance of the pho D gene increased with the increase in culture time,reaching the highest level on the 30th day,respectively（1.57×10⁸、2.03×10⁸、1.37×10⁸ copies/g dry soil）;In the treatment group supplemented with four phosphorus sources,the abundance of pqq C gene increased with the increase of culture time,and reached the highest level on the 30th day（2.99×10⁸ copies/g dry soil）,and there was a significant difference compared to the control group（P<0.05）.The addition of four phosphorus sources had no significant impact on the abundance of gcd genes.Sequencing analysis of functional gene（pho D）amplifiers showed that adding Lecithin decreased the Shannon Wiener index and Simpson’s diversity index of pho D-harbouring bacteria,and increased the Chao1 index and ACE index,with a significant difference between the control group（P<0.05）,while adding KH₂PO₄,Fe PO₄,Ca₃（PO₄）₂ had no significant difference compared to the control group.Principal co-ordinates analysis showed that the addition of Lecithin resulted in changes in the pho D-harbouring bacterial community between day 3 and day 10,and there was a significant difference（P<0.001）compared to the control group.In the treatment group supplemented with Lecithin,there was a significant positive correlation between the absolute abundance of Bradyrhizobium and Afipia and the culture time.In the treatment group supplemented with Fe PO₄,there was a significant positive correlation between the absolute abundance of Gemmatirosa and Herbaspirillum and the culture time;There was a significant positive correlation between the absolute abundance of Gemmatirosa and culture time in the treatment group supplemented with Ca₃（PO₄）₂;There was a significant positive correlation between the absolute abundance of Burkholderia and Gemmatirosa in the treatment group supplemented with KH₂PO₄ and the incubation time.After 30 days of culture,the addition of Lecithin increased the relative abundance of Bradyrhizobium by 69.32%,and the addition of KH₂PO₄ increased the relative abundance of Streptomyces and Burkholderia by 5.43%and 4.66%,respectively,with significant differences compared to the control group（P<0.05）.Sequencing analysis of functional gene（pqq C）amplifiers showed that adding Lecithin reduced the Shannon Wiener index,Chao1 index,and ACE index of the pqq C bacterial community,while adding Fe PO₄ decreased the Shannon Wiener index,which was significantly different from the control group（P<0.05）.Principal co-ordinates analysis showed that the addition of Lecithin resulted in significant changes in the pqq C-harbouring bacterial community from the 3rd day of cultivation,and remained stable on the 10th and 30th days,with a significant difference compared to the control group（P<0.001）.In the treatment group supplemented with Lecithin,there was a significant positive correlation between the absolute abundance of Burkholderia and culture time;There was a significant negative correlation between Burkholderia and culture time in the treatment group supplemented with Ca₃（PO₄）₂.There was a significant negative correlation between Burkholderia and culture time in the treatment group supplemented with KH₂PO₄.After 30 days of cultivation,adding Lecithin increased the relative abundance of Burkholderia by 46.11%,which was significantly different from the control group（P<0.05）.Adding Ca₃（PO₄）₂ increased the relative abundance of Bradyrhizobium by 8.34%,and adding KH₂PO₄,Fe PO₄,and Ca₃（PO₄）₂ increased the relative abundance of Paraburkholderia by 12.33%,11.80%,and 6.21%,respectively,which were significantly different from the control group（P<0.05）.The phosphorus fractionation experiment showed that the addition of KH₂PO₄significantly increased the soil microbial biomass phosphorus（MBP）content compared to other treatment groups（P<0.05）;Compared with the control group,adding Ca₃（PO₄）₂ significantly increased the content of MBP and Ca-P in soil（P<0.05）;Compared with the control group,the addition of Lecithin significantly increased the soil MBP content（P<0.05）and the moderately active organic phosphorus（MLPo）content（P<0.05）.In summary,different phosphorus sources can affect the absolute abundance and community composition of PSB in soil,thereby changing the soil phosphorus composition.In the Lecithin Formation,Bradyrhizobium and Burkholderia were significantly enriched,while the contents of MBP and MLPo in soil increased significantly,which may be related to the enrichment of mineralized insoluble organic phosphorus in bacteria;Bradyrhizobium and Paraburkholderia enriched with Ca₃（PO₄）₂ can activate the absorption of insoluble phosphorus,resulting in a significant increase in MBP content;Adding KH₂PO₄ will enrich Streptomyces and Burkholderia and significantly increase MBP content.（3）A total of 112 strains of PSB were isolated from agricultural soil in South China through the coating plate separation method.Among them,Pseudomonas suspected new species OPS13^T with strong phosphorus solubilizing ability was selected for polyphasic classification and identification,its taxonomy status was clarified,and it was identified as a new species of Pseudomonas and named Pseudomonas citri sp.nov.