Diversities Of Microbes In No-tillage Soils Revealed By Stable Isotope Probing

The emissions of greenhouse gases cause the global warming and the sea level rise,causing a wide range of attention.Soil is the main source of greenhouse gases,about 5%～20%of CO₂ from the soil.Autotrophic microbes has a strong environmental adaptability and potential for CO₂ fixation,but the effect of no-tillage on the number and diversity of autotrophic CO₂-fixing bacteria in paddy soil remains limited.This project selects no-tillage paddy soils in southem China as typical study area,and the CO₂-fixation autotrophic microbial assamblage as the research object,stable isotope-coded affinity tag approach will be adopted to track the behavior of CO₂ assimilation by soil microorganisms,so the the CO₂-fixing rate can be calculated.The aim of this study was to investigate the diversity and community structure of autotrophic CO₂-fixing bacteria in no-till paddy soil in South China,by using DNA-based stable isotope probing（DNA-SIP）technology and PCR-RFLP analysis and high throughput sequencing technology.The results showed that,after 10 weeks of culture,the content of soil 13C-SOC was 70.88～350.15 mg·kg-1,where FG-PL is 350.15 mg·kg-1,FG-PP 133.05 mg·kg-1,FG-PH 70.88mg·kg-1,TF-PL,328.76mg·kg-1,TF-PP 171.62 mg·kg-1,TF-PH 92.01 mg·kg-1.The difference of the visible different profiles of soil 13C-SOC content significantly（P<0.05）,expressed as PL>PP>PH.The difference of soil microbial fixation CO₂ flux in different soil profiles was significant,and it was PL>PP>PH.Two kind of soil 13C-CO₂ fixed rate ranged from 0.10 to 0.49 μg13Cg-1d-1 which FG-PL for 0.49 μg13Cg-1d-1,FG-PP is 0.19 μg13Cg-1d-1,FG-PH is 0.10 μg13Cg-1d-1,TF-PL is 0.46 μg13Cg-1d-1,TF-PP is 0.23 μg13Cg-1d-1 and TF-PH is 0.13 μg13Cg-1d-1.Real time fluorescent quantitative PCR and quantitative cbbLR results showed that the number of red-like cbbLR gene copies in the 8th layers separated from soil microbial total DNAs by CsCl density-gradient centrifugation were the highest after an 80-day incubation,the abundance of cbbLR gene in soil FG and TF were 1.36 × 105 and 2.21 × 105 copies g-1 dry soil respectively.High throughput sequencing results show that Proteobacteria（42.4%）for soil microbial dominant phyla,betaproteobacteria（25.20%）and Alphaproteobacteria（10.00%）as the dominant classes of soil microbial,burkholderiales（20.60%）rhizobiales（5.10%）,Rhodospirillales（3.00%）for soil microbial dominant orders,burkholderiaceae（20.1%）and Rhodospirillaceae（2.40%）as the dominant families of soil microbial.To construct eight cbbLR gene libraries,the cbbLR genes of 13CO₂-labeled and 12CO₂-controled soils,as well as soil FG and TF were amplified separately as tamplates.A total of 30 OTUs were obtained from 672 clones,which belongs to 5 orders、9 families and 12 known genera.Bradyrhizobium and Rubrivivax were the main microorganisms in soil FG,accounted for 60.40%and 13.86%of all clones.The community composition of CO₂-fixing bacteria in soil TF was relatively uniform,Rhodopseudomonas、Bradyrhizobium、Methylibium and Variovorax accounted for 20.92%、11.94%、16.42%and 10.45%of the total clones,respectively.The number of OTUs and diversity index of 13C-CO₂ stable isotope labeled cbbLR library were higher than that of the12C-CO₂ labelled control library,and the community structure also shows significant differences.Thus,we can concluded that there are diverse autotrophic bacteria capable of fixing CO₂ in the no-tillage paddy soil,and it have an important role in the carbon cycling for farmland soils.