Impact Of Green Manure And Straw Return On Microbial Functional Genes In Rice Rhizosphere

Green manure is an important agricultural organic fertilizer source.The effects of green manure on the improvement of soil conditions and promotion of crop growth are closely related to its effect on soil microorganisms.The effects of microorganisms on soil nutrient cycling and phytohormone regulation have gained great attention,but the involved microbial mechanism of green manure affecting soil nutrient cycling and other functions is largely unknown.In order to clarify the microbial mechanism of the effects of green manure on soil nutrient cycle and phytohormone related functions,and to provide a basis for the scientific utilization of green manure,we comprehensively analyzed the effects of green manure on soil nutrient cycling and phytohormone related functional microorganisms by metagenomics.The rice rhizosphere soils in plots with or without green manure in combination with or without rice straw returning were collected from a long-term green manure field experiment in Qiyang,Hunan,China.Shotgun metagenomic sequencing was performed on an Illumina NovaSeq platform with a PE150 strategy.The sequences were aligned with SWISS-PROT databases using MetaPhlAn2 and Diamond BLASTX after quality-controlled and assembled by read?qc and megahit moules of METARWAP respectively;mean while,we checked the name,function and encoding protein of each single gene one by one based on related references,and classify the relevant functional genes according to carbon,nitrogen,phosphorus,iron,sulfur,heavy metals and hormones,and focused on 768 functional genes;we finally combined the sequence alignment results with the collected functional genes list,and extracted the relative abundance information of each gene.Then R was used to further analyze the difference of functional genes relative abundance between green manure and none green manure and the relationship between genes and soil properties in rice rhizosphere soil,thus the effect of green manure on soil relative functional microorganisms was expounded.We also extracted the relative abundance of TOP1000 KOs,named homologous functional gene,from the results of sequences aligned with KEGG database,and analyzed the effects of green manure on microbial functional processes in the rice rhizosphere.The main results are as follows:1?The results show that 9 conventional indicators including the soil pH,available phosphorus and NO₃-N had no significant difference between green manure and no green manure.The straw significantly improved the relative abundance of the archaea and reduced the relative abundance of the bacteria in the soil,whereas the green manure significantly inhibited the effect of straw on the microbacteria of methane in archaea.2?The carbon-cycle genes malAEFKH,xylAHS,cbbM,and the nitrogen-cycle genes aniA,nifC,ntrB,and the phosphorus-cycle genes phoAPU,phnAPILN,pitA,and the iron-and the sulfur-cycle genes cbrB,frdB,dsrFA and the phytohormone related gene log and other related genes upto 80functional genes,which involves all the functional groups of carbon,nitrogen,phosphorus and others,and upto 140 homologous functional genes in the relative abundance TOP1000,which involves 5metabolic processes including carbohydrate metabolism,lipid metabolism and amino acid metabolism,and 4 microbial life processes including genetic information transmission and cell signal transmission,were significantly influenced by green manure,indicated that green fertilizer had a wide impact on soil microorganisms.3?Under straw conditions,the results showed that the carbon cycle gene diversity index and the carbon-fixed acetyl-COA pathway gene acsA,with the relative abundance upto 0.4%,both were significantly lower in the green manure than in the non-green manure treatment,indicated that straw may cause the accumulation of organic matter in green manure treatment and thus affect the release of organic nutrients.The related abundance of Methanoregulaceae significantly lower in the treatment of green manure than no green manure under the straw condition,may indicate that green manure can inhibit the methane release caused by straw.The nitrogen cycle genes,such as eutA,glnA,tdcB and ureG,which significantly affected by green manure,were mainly included in the function group of organic nitrogen degradation and synthesis.The relative abundance of ntrB,the gene belongs to the two-components system of the nitrogen limiting response,was significantly lower in the green manure than that in no green manure under the condition of straw,may indicate that the straw reduced the nitrogen level in the soil treated with green manure to a certain extent.Twelve genes such as pitA,glpBO,phnAILNP,phoAPU,included in five functional groups of phosphorus cycle,were significantly influenced by green manure;And there upto 35 homologous genes,from the 140 homologous functional genes significantly influenced by green manure,were phosphorus cycle related;Indicated that green manure may have the greatest influence on phosphorus cycling genes of microorganisms in this study.None of the 11 genes,which showed a significant difference in relative abundance between treatments with or without green manure under the absence of straw,showed significant difference between treatments with or without green manure under the straw condition,indicated that the straw may weakened the influence of green manure on phosphorus cycle genes.4?The genes fbpC and feoB in the iron related functional group,which respectively encoding the Fe?3+?and the Fe?2+?transport proteins,were relatively high,whereas the relative abundance of genes encoding proteins of siderophore transport and synthesis were relatively low,and greem manure has less influence on the common genes such as fhuABCD,fepBDEG,which related with siderophore synthesis and transport.However,there were many ferrithioredoxin related functional genes,such as dmsB,fdxD and frdB,were affected by green manure,indicating that green manure had a significant influence on cell processes involved in REDOX and other related processes.5?We found that the relative abundance of the cytokinin related gene log was significantly higher in green manure than that in no green manure under the condition of straw,indicating that green manure may have regulating effect on plant growth.Correlation and redundancy analysis results showed that pH was significantly correlated with the relative abundance of carbon,nitrogen,phosphorus,iron,sulfur and phytohormone related functional genes respectively,indicating that pH could be an important factor affecting microbial functional genes in green manure treatment.In conclusion,metagenomics is an effective method to study the effect of green manure on functional genes of soil microorganisms.It can simultaneously detect the changes of a large number of relative genes in a wide range.The effects of green manure on microbial nutrient cycling and phytohormone related genes in rice rhizosphere were extensive.The returning of early rice straw may weaken the effect of green manure on related functional genes.