Soil Microbial Community And The Regulation Mechanisms Of Nitrification In Winter Green Manure-rice Cropping System

In South China,the utilization of green manure in rice cropping system is an effective practice to guarantee the stable and high yield of rice.The plantation of winter green manure has profound influences on soil nitrogen transformation,which is a process controlled by microbes.However,the characteristics of soil microbes involved in nitrogen transformation in the green manure-rice cropping system are lack of understanding,and the mechanisms of the soil nitrogen transformation affected by green manure are still unclear.In this study,by using joint experiments in different provinces,long-term experiment and pot experiment,as well as the technologies of Roche 454 and Illumina MiSeq sequencing,and real-time quantitative PCR,we investigated the characteristics of soil microbes after multi-years' winter plantation of green manures in South China,and explored the responses of nitrification process in paddy soil to different green manures,and to the regulation of nitrogen fertilizer and rice straw return.1.The soil microbial community and the abundance of functional genes involved in nitrogen transformation in winter green manure-rice cropping system.The joint experiments are located in Hunan,Jiangxi,Anhui,Henan,Hubei and Fujian provinces.All the experiments have four treatments,i.e.,winter fallow and no chemical fertilizer as control(NF),milk vetch as green manure(GM),winter fallow and chemical fertilizer(CF),and the combination of chemical fertilizer and milk vetch(GMCF).The soil microbial community had significant difference among different sites.The soil microbial community in Anhui,Henan and Hubei province were similar,while the microbial community of Hunan province were different from all the other 5 provinces.The Weighted UniFrac distances between GM and NF treatments,and between GMCF and NF treatments were large,indicating that plantation of winter milk vetch changed soil microbial community.The function microbes involved in carbon,nitrogen,sulfur cycling of soils changed after years' utilization of milk vetch.The abundance of methane-oxidizing bacteria and sulfur reducing bacteria increased,and the microbes involved in nitrogen fixation,nitrification and denitrification also increased in some provinces.The abundance of function genes in nitrogen fixation(nifH),nitrification(archaeal and bacterial amoA),and denitrification(nirK and nosZ)varied among sites,and their responses to GM and CF treatments also changed.In Jiangxi and Henan provinces,the GMCF treatment increased nifH gene copies.Compared with ammonia oxidizing bacteria(AOB),ammonia oxidizing archaea(AOA)were predominant in all the six provinces.In Hunan,Jiangxi and Fujian provinces,the GMCF treatment increased the AOA to AOB ratio significantly.For denitrifizer,the GMCF treatment increased the abundance of nirK genes in Hunan,while the GM treatment increased the nosZ genes in Jiangxi,the highest nosZ/nirK ratio was found in Hunan province.2.The characteristics of ammonia oxidizers in paddy soil under long-term application of green manures.Long-term experiment which was started in 1982 includes four treatments,i.e.rice-rice-winter fallow,rice-rice-ryegrass,rice-rice-rape and rice-rice-milk vetch.The tested soil is typical red paddy soil in South China.The AOA amoA gene copies were more abundant than AOB,and the ratios of AOA to AOB ranged from 36 to 1686.Winter green manures,especially milk vetch,increased the abundance of both AOA and AOB amoA genes after the manures were turned over.The most abundant OTUs of AOA changed in response to the application of green manures,while most abundant OTUs of AOB remained unaffected.Phylogenetic analysis showed that Nitrososphaera and Nitrosospira were dominant in AOA and AOB,respectively.In conclusion,in red paddy soil,AOA were more abundant in red paddy soil and more responsive to changes in soil conditions compared to AOB.3.Effects of different green manures on nitrification in paddy soil.The pot experiment had four treatments,i.e.,winter fallow-double rice,milk vetch-double rice,rape-double rice and ryegrass-double rice.Aplication of different winter green manures increased the amount of soil microbial biomass carbon(SMBC)and soil microbial biomass nitrogen(SMBN)at the tillering and jointing stages of early rice.There were obvious seasonal various in soil microbial biomass.Plantation of 3 kinds of winter green manures decreased soil nitrification potential(NP)at the stages of before transplantation of early rice and the tillering and jointing stages of early rice.NH4+-N increased while NO3--N decreased at most of the sampling stages by winter green manuring.The recovery nitrification potential(RNP)and the relative contribution of AOB(RNPAOB)were increased by different green manuring at the tillering stage of early rice.The relative contribution of AOA(RNPAOA)were decreased by green manuring before transplantation and at the jointing stage of early rice.AOB contributed more to RNP at all the sapling stages.The contribution rates of AOB to RNP ranged from 61.02%to 82.37%.4.Responses of nitrification to nitrogen fertilizer levels under application of green manure in paddy soil.Pot experiment include two factors,i.e.,winter fallow and milk vetch,and three nitrogen fertilizer levels(0%,60%,100%).At the same level of nitrogen fertilizer,application of milk vetch increased soil organic matter.Soil NP and NO3--N decreased,while NH4+-N increased after the application of milk vetch.After application of winter green manure,the RNP and RNPAOB increased at the tillering stage of early rice,while the RNPAOA decreased at jointing and mature stages of early rice.The contribution rate of RNPAOB to RNP decreased at the full-bloom stage of green manure and increased at rhe tillering and jointing stages of early rice in the green manure treatments.5.Responses of nitrification to co-incorporation of milk vetch and rice straw in paddy soil.Pot experiment include four factors,i.e.,no addition control,milk vetch only,rice straw only,and co-incorporation of milk vetch and rice straw,and two nitrogen fertilizer levels(with and without nitrogen fertilizer).Soil NP varied a lot among sampling stages and treatments.Co-incorporation of milk vetch and rice straw significantly increased soil NP at the early stage of rice growth period,while decreased at the late stage of rice growth period.At the mature stage of early rice,the treatments with milk vetch,rice straw and co-return of milk vetch and rice straw all decreased the RNP compared with control and nitrogen fertilizer only.The contribution rates of AOA and AOB to RNP varied a lot among sampling stages.AOB contribute more to RNP at the tillering and jointing stages of early rice,and the contribution rates of AOB were 81.35%and 82.95,respectively.While AOA contribute more to RNP at the harvest stages of early and late rice,and the contribution rates of AOA was 60.56%and 62.76%,respectively.In conclusion,the effects of winter green manuring differed among sites.Application of milk vetch in paddy soil changed soil microbial community,and influenced the processes of soil nutrient cycling,especially nitrogen cycling.In the regulation of soil nitrification,winter plantation of green manure roughly decreased soil nitrification potential,thus reduced the risk of nitrate leaching.In winter green manure-rice cropping system,reasonable application of chemical fertilizer and co-utilization of milk vetch and rice straw are important factors that ensure the potential of saving fertilizer and improving efficiency by green manure application.