Diversity Of Nitrogen Cycle Core Function Microbial In Sugarcane//Soybean Intercropping System Based On The DNA-Stable Isotope Probing

The sugarcane is the main sugar crop in China.There are many problems in the production of sugarcane,such as excessive fertilization,partial application of nitrogen fertilizer and single planting.Sugarcane-soybean intercropping can not only improve the land use efficiency,but also strengthen the ecosystem service function of sugarcane field.Reduce the input of chemical nitrogen fertilizer.The effects of nitrogen reduction and soybean intercropping on yield,agronomic shape,land productivity and soil fertility were studied on the basis of 9 years field location experiment.The pot experiment was carried out using isotope in situ labeling,stable isotope nucleic acid probe?DNA-SIP?,Quantitative real-time PCR?qPCR?and high-throughput sequencing to study the N shifts in the sugarcane//soybean intercropping system from the perspectives of inter-crop nitrogen transfer,key functional genes in the nitrogen cycle,and microbiological functions of the nitrogen cycle.The main results are as follows:1.The results of field location test for 9 years showed that intercropping soybean could improve land productivity and land use efficiency.The intercropping pattern of sugarcane//soybean 1:2 intercropping pattern of reducing nitrogen application(300 kg·hm^-2)can meet the nitrogen requirement of sugarcane growth.Through competitive absorption of nitrogen in soybean rhizosphere soil,intercropping sugarcane can relieve or reduce the“nitrogen repressor”effect and promote nitrogen fixation of soybean.And with the increase of planting years,the intercropping model can effectively alleviate the continuous cropping barriers.Through analysis,it is concluded that the intercropping pattern of sugarcane//soybean can effectively utilize land resources,improve system productivity,keep crop yield stable,save production cost and increase economic benefits.It is a kind of structure optimization and reasonable agronomic planting measure,and it is worth popularizing in practice.2.The ¹⁵N isotopic abundance of soybean and sugarcane in different parts and soil were detected by means of(¹⁵NH₄)₂SO₄ solution.Higher ¹⁵N abundance was detected in the aboveground part of soybean,soybean root and soybean root circumjacent soil samples treated with marker monoculture and intercropping treatment,and are significantly higher than that of the control group.It was proved that ¹⁵N was successfully labeled to crop,soil and soil microbes and enriched by crop,soil and soil microbes.The relative abundance distributions of six nitrogen cycling functional genes in DNA of different buoyant density centrifugation fluids were detected by real-time PCR?qPCR?.Through the analysis of the relative abundance of nitrogen cycling functional genes,the gene abundance peaks of the nifH and amoA genes in sugarcane-soybean intercropping and soybean monoculture were shifted in the ¹⁵N marker group and the control group,the gene abundance peaks of ChiA were only shifted in soybean monocropping mode,andthe abundance peaks of nirS,nirK and nosZ did not shift in both planting patterns.The results showed that nifH and amoA genes can be used as indicator genes to effectively identify DNA-SIP technology ¹⁵N-DNA positions in sugarcane-soybean intercropping system.3.Quantitative determination of nifH,chiA,amoA,nirK,nirS,and nosZ genes that regulate soil nitrogen cycling in sugarcane//soybean intercropping systems under potting conditions using qPCR.The results showed that the relative abundance of nifH gene and amoA gene in intercropping treatment was significantly higher than that in monoculture treatment.The results showed that sugarcane//soybean intercropping could effectively promote the nitrogen fixation potential and nitrification process of soybean and change the community structure of microbial bacteria in rhizosphere soil.4.The results of microbial sequencing in pot soybean rhizosphere showed that the abundance of bacteria in soil nitrogen cycle of sugarcane//soybean intercropping system was significantly higher than that of ancient bacteria.Bacteria are the main drivers of soil nitrogen cycling in sugarcane//soybean intercropping system.The dominant phylums of microorganisms directly involved in the soil nitrogen cycle are Proteobacteria,Acidobacteria,Actinobacteria,Chloroflexi,Planctomycetes,Gemmatimonadetes,Nitrospirae,and Armatimonadetes.The advantages genera are Burkholderia,Candidatus Koribacter,Nitrospira,Ralstonia,Rhodoplanes,Candidatus Solibacter and Pseudoxanthomonas.The number of unique OTUs that directly participate in the nitrogen cycle microbial species in the same planting pattern is significantly higher than the number of non-direct.Cluster analysis showed that all microbial species labeled with ¹⁵N were clustered into one group,and all unlabeled species were clustered into another group.The results showed that there were significant differences between soil nitrogen cycling and microbial species which were not directly involved in sugarcane//soybean intercropping system.5.Redundancy analysis?RDA?showed that the contents of NO₃^--N,AN,TN in soil was the main factor affecting the community structure of soil nitrogen cycling core microbes directly involved in sugarcane//soybean intercropping system.But pH,SOM and TK were main factor that affected the structure of microbial community without participating in the core function of soil nitrogen cycle.In conclusion,the intercropping pattern SB2-N1 of sugarcane//soybean at reduced nitrogen application level increased the total crop yield and system productivity on the basis of no reduction of sugarcane yield.It is a scientific and reasonable agronomic measure.The qPCR of nifH gene and amoA gene can be used as indicator genes to accurately identify the¹⁵N-DNA enrichment sites of DNA-SIP in sugarcane//soybean intercropping system.The pattern of intercropping could promotes the expression of nitrogen cycle genes.For ancient bacteria,bacteria are the main drivers of soil nitrogen cycling in sugarcane//soybean intercropping systems.There were significant differences in the distribution of soil nitrogen cycling between the microorganisms labeled with ¹⁵N and the unlabeled microbe communities,and the distribution of the former communities was affected by the nitrogen content in the soil.