Effects Of Buried White Clover On Nitrification And Denitrification Microbial Diversity In Apple Orchard Soil

The Loess Plateau is the main apple producing area in China.The long-term over-application of nitrogen fertilizer in the pursuit of high yield leads to a large loss of soil N,causing a series of environmental problems,such as global warming caused by N2 O,the ground water pollution by NO3-,as well as the eutrophication of NH3.On the side,the excessive application of fertilizer will lead to the decline of soil quality,apple production and quality.Mowing grass and buried in the orchard is an effective way that to adapt to the special climatic conditions in the area.It could improve the soil organic matter and soil fertility and fruit quality.At present,the research on the degradation of apple orchard has just started,and mainly focused on the soil organic matter,fertility,enzyme activity,and the abundances of soil bacteria and fungi.There were litter studies on the nitrification and denitrification of microorganisms in the nitrogen cycle of orchard soil.In this study,stems and leaves of white clover(Trifolium repens L.),the best suitable for the apple orchard in the apple orchard,were buried in the apple orchard.The treatments were BW1(buried 1 kg of white clover),BW3(buried 3 kg of white clover)BW5(buried 5 kg of white clover).The control was not buried soil.The soil samples were collected at 1,2,3,6 and 12 months after the degradation of white clover.PCR(qPCR),restriction enzyme digestion polymorphism(T-RFLP)and high-throughput sequencing were used to study the diversity of soil nitrification and denitrification microbial communities.The main results are as follows:(1)The contents of soil nutrient,available nutrient,ammonium nitrogen,nitrate nitrogen and organic matter increased significantly after buried white clover,and increased with the increase of buried amount.(2)The changes of abundance of functional genes were same in 0 ~ 10 cm and 10 ~ 20 cm soil layers.The abundance of AOB and AOA increased in the early stage and the later period of embedding,the abundance of nirK-type,nirS-type and nosZ-type denitrifying bacteria decreased in the early stage of embedding,and there was no change in the middle of the embedding.In the early stage of embedding,the abundance of AOB and AOA increased significantly in the amount of 3 kg.The abundance of nirK-type denitrifying bacteria increased with the increase of buried amount at most of buried time.The correlation analysis showed that the effect of available potassium content on thr abundance of AOB and nirK-type denitrifying bacteria.The abundance of AOA and nosZ-type denitrifying bacteria was positively correlated with ammonium nitrogen content and pH.The correlation between abundance of nirS-type denitrifying bacteria and soil properties was not significant.(3)The relative abundance of ammonia oxidizing bacteria(AOB)and ammonia-oxidizing archaeal(AOA)and nirK-type,nirS-type and nosZ-type denitrifying bacteria changed,which changed the community structure.The community structure of Ammonia-oxidizing Bacteria(AOB)in 0~10 cm soil layer and 10~20 cm soil layer is different.The community structure of ammonia-oxidized archaea(AOA)and nosZ-type denitrifying bacteria was different at 12 months from that of other embedding sites.The community structure of AOB had a significant response to soil potassium content,and soil moisture was an important factor affecting the community structure of AOA and nirK-type and nosZ-type denitrifying bacteria.(4)The community composition of ammonia-oxidizing bacteria and denitrifying bacteria after buried 3 kg of white clover at 1 month was studied by Illumina Miseq high-throughput sequencing platform.The research indicates that Shannon diversity index and Chao index of AOB community,nirS-type and nosZ-type denitrifying bacteria communities increased,while the Simpson uniformity index decreased.These quences of AOB include Nitrosospira,Nitrosomonas and Nitrosovibrio of ?-proteobacteria,while the Nitrosospira was the dominated genus.The relative abundance of nitrospira increased after buried white clover.The dominated genuses of nirS-type denitrifying bacteria were Cupriavidus,Ideonella,Rubrivivax and Sulfuritalea.The abundance of the dominant species of Cupriavidus increased by 60.90% after buried white clover,while the abundance of Ideonella,Rubulvivax,Sulfuritalea reduced.NosZ-type denitrifying bacteria mianly contain Alphaproteobacteria and Betaproteobacteria,in which the abundance of Alphaproteobacteria was high.The abundance of dominant species of azospirillum,Sinorhizobium and Mesorhizobium were increased after buried white clover,while the abundance of Bradyrhizobium reduced.