Effects Of Long-term Different Fertilization Regimes On Soil Bacterial And Nitrogen-cycling Related Communities In A Rice-wheat Rotation System

In our present studies,four different fertilization regimes were investigated to study the influence of fertilizations on soil bacterial community and nitrogen-cycling related communities based on a 8-year long term experimental field in Changshu,Jiangsu.Four fertilization regimes were as follows:no nitrogen fertilization treatment(NNF),chemical fertilization(CF),organic-inorganic mixed fertilization treatment(OIMF)and organic fertilization treatment(OF).Soil samples were collected throughout different soil depths and across different plant growth stages of rice-wheat rotation system.Our purposes were to study the effects of different fertilization regimes on soil bacterial and nitrogen-cycling related communities,and we want to understand the relationships between soil microflora and crop yields.Our results showed that:1)Compared with NNF,CF increased rice yields by 43%and wheat yields by 120%.CF treatment also had significantly higher rice and wheat yields than that of organic fertilization treatment(OF).In addition,OIMF treatment had the highest contents of soil NH4+and NO3-,in different soil profiles(1.4 mg kg-1 and 2.86 mg kg-1,respectively),CF treatment had the highest contents of soil NH4+ and NO3-,in different plant growth stages(2.4 mg kg-1 and 19.06 mg kg-1,respectively).In different soil profiles and different plant growth stages,OF treatment had the highest soil pH(7.75 and 7.88,respectively),soil organic matter(SOM,9.5 g kg-1 and 20.5 g kg-1,respectively)and total nitrogen(TN,125.5 mg kg-1 and 69.9 mg kg-1,respectively)contents.NNF treatment had the highest contents of soil available P(AP)and K(AK)(125.5 mg kg-1 and 69.9 mg kg-1,respectively).2)High throughput sequencing was used to analyze the community compositions of soil bacterial communities based on 16S rRNA gene.We collected soil samples from eight different plant growth stages(plant growth stages)under four fertilization regimes.The results showed that plant growth stages rather than different fertilization regime was the key factor influencing the bacterial community compositions and abundance.h Bacterial community compositions were significantly different between wheat and rice soils,in which rice soils contained more anaerobic or facultative anaerobic bacteria compared with that of wheat soils.With respect to fertilization regime,NNF plots had a higher abundance of nitrogen-fixing Cyanobacteria.OIMF had a lower abundance of ammonia-oxidizing Thaumarchaeota compared with CF.Application of chemical fertilizers(CF and OIMF treatments)significantly increased the abundance of some generally oligotrophic bacteria such those belonging to the Acidobacteria,while more copiotrophic of the phylum Protcobacteria increased with OF application.3)Real-time quantitative PCR(qPCR)and terminal restriction fragment length polymorphism(T-RFLP)of nifH gene was used to analyze the changes of diazotrophic community abundance and compositions,respectively.Results revealed that the abundance of nifH declined with increased soil depth regardless of fertilization regimes,although considerably high abundance was also observed in subsoils of 80-100 cm soil layer.By investigating nifH gene across eight representive growing stages,results from qPCR showed that the abundance of nifH gene in rice rotation was higher than that in wheat rotation.Fertilization had no significant influence on nifH gene abundance.Results of T-RFLP showed that the compositions of diazotrophic communities were distinct between wheat and rice growth stages,different plant growth stages but not fertilization regimes determined diazotrophic community composition.4)The abundance and composition of ammonia oxidizers were analyzed using real-time quantitative PCR(qPCR)and terminal restriction fragment length polymorphism(T-RFLP)based on amoA genes,respectively.Results showed that fertilization significantly influenced soil AOB community abundance and composition.CF had the highest abundance of AOB-amoA gene abundance in both different soil profiles and different plant growth stages.Although the abundance and composition of the AOA community was also significantly impacted by soil depth,fertilization and plant growth stage,but not as strong as the effects on AOB community.In addition,across the whole wheat-rice rotation stages,results of multiple stepwise linear regression revealed that AOB played a more important role in ammonia oxidizing process than AOA,AOB could explain 48.7%of soil nitrification,while AOA only explained 6.7%of soil nitrification.5)QPCR were used to determine the abundance of nosZ,nirK and nirS genes in denitrifying microorganisms.By studying nirS and nirK gene throughout soil profiles,we found that topsoil had the highest abundance of nirS and nirK genes.OIMF treatment had the highest nirS and nirK gene copies in 0-20 cm topsoil layer,while in the deeper soil layers,CF had the highest abundance of nirS and nirK genes.Our results also showed that nosZ gene abundance was stable in the first seven stages,while in rice harvesting stage in October,nosZ increased dramatically.In terms of different fertilizations,OF treatment had the highest nosZ gene abundance across the whole wheat-rice rotation system.T-RFLP was used to analysis the community compositions of nosZ contained denitrifiers,results showed that denitrifier compositions were determined by fertilization but not plant growth stage,OF treatment had the significantly distinct compositions compared with other three treatments.6)By analyzing the relationships between crops yields and soil bacterial and nitrogen-cycling related communities,our results revealed that the abundances of bacterial,diazotrophic and AOA communities had no significant relationships with wheat and rice yields.AOB had significantly positively relationships with both wheat and rice yields.Negative relationships were found between denitrifying bacterial abundance and crops yields,while significant difference was only detected with the yield of wheat.We made conclusions that fertilizations significantly influenced the abundances and compositions of soil bacterial and nitrogen-cycling communities.The influence of fertilizations were stronger than that of different plant growth stages on the compositions and abundances of ammonia oxidizers and denitrifier.On the contrary,plant growth stages determined the bacterial and diazotrophic communities abundances and compositions,which indicated that study the response of them should also concern the effects of plant growth.We also analyzed the relationships between the crops yields and the abundances of soil bacteria and nitrogen-cycling related communities,and the results supplied evidence that we could change soil microflora to improve crops yields in the future.