In Situ Ammonia Oxidation Activity And Spatial Distribution Of Nitrogen-transforming Microorganisms In Potted Rice Soil

Nitrogen(N)fertilizers(usually as urea and ammonia)have been applied intensively in paddy soils to obtain high yields over the last decades,especially in China,which is the largest rice producing nation in the world.It is theoretically and practically significant to understand accurately and deeply the spatial distribution of ammonia oxidizing(a key step in nitrogen fertilizers conversion)activity and functional microorganisms involved in nitrogen transformation in paddy ecosystems.Long-term flooding and rice growth have resulted in the formation of three typical microzones including soil-water interface,rhizosphere soil and bulk soil in paddy soil.Most of the current studies on the ammonia oxidizing activities of paddy soils involve incubation under completely aerobic or anaerobic conditions based on destructive sampling.The results originated from such studies cannot accurately reflect the real conditions of paddy soils.This study intends to study in-situ the aerobic and anaerobic ammonia oxidizing activities,and functional microorganisms in the soil-water interface,rhizosphere soil and bulk soil.We thought this study can more accurately reveal the nitrogen transformation and spatial distribution of functional microorganisms in paddy soil than ever before.In this study,rice pot experiments were set up,and metagenomic sequencing technology was used to determine the relative abundances of genes and related microorganisms involved in nitrogen transformation in the three microzones;the aerobic and anaerobic ammonia oxidizing activities in different microzones were characterized in-situ by using 15N stable isotope labeling technology,and the related ammonia-oxidizing microorganisms were analyzed by using quantitative reversetranscript PCR(RT-qPCR)and metagenomic sequencing;a pot experiment was set up to study in-situ the effect of pH on aerobic and anaerobic ammonia oxidizing activities and functional microorganisms in different microzones with soil pH being adjusted to different levels including 5,6 and 7.The main results are shown as follows:(1)There were significant differences between relative abundances of nitrogentransforming genes and between related microorganisms involved in nitrogen transformation.①Aerobic ammonia oxidation process:The relative abundance(0.08%)of ammonia-oxidizing archaeal(AOA)and ammonia-oxidizing bacterial(AOB)amoABC genes at the soil-water interface was significantly higher than that in the rhizosphere soil(0.02%)and the bulk soil(0.02%).There were no significant differences in the aerobic ammonia-oxidizing microorganisms between the three microzones except the relative abundance of Nitrosospira(0.13%)at the soil-water interface was significantly higher than that in the rhizosphere soil(0.05%)and the bulk soil(0.04%).②Anammox process:There is no significant difference in functional genes and microbial groups between the three microzones.③ Denitrification process:The relative abundance of napABC(1.92%),narGHJI(5.05%),and norBC(3.01%)at the soil-water interface was significantly lower than that in the rhizosphere soil and the bulk soil.The relative abundance of Pseudomonans(0.22%)and Cytophaga(0.01%)at the soil-water interface was significantly higher than that in the rhizosphere soil and the bulk soil,and there is no significant difference in the relative abundance of other related microorganisms.④Nitrogen fixation process:The relative abundance of the nifWKD genes at the soil-water interface(2.34%)was significantly lower than that in the rhizosphere soil(3.65%).Symbiotic nitrogen-fixing bacteria in the three microzones were dominated by nitrogen-fixing microorganisms,and their relative abundance(3.40%)was significantly higher than associative nitrogen-fixing bacteria and abiogenous nitrogen-fixing bacteria.⑤Assimilation nitrate reduction ammonium(ANRA):The relative abundances of nirA(1.54%),nasAB(2.14%),and narBC(1.55%)at the soil-water interface were significantly higher than that in the rhizosphere soil.Dissimilation nitrate reduction to ammonium(DNRA):The relative abundance of nrfABCD(5.41%)and napABC(1.92%)at the soil-water interface was significantly lower than that in the rhizosphere soil,and the abundance of Desulfovibrio(0.41%)in the rhizosphere soil was significantly higher than that in the water-soil interface and the rhizosphere soil.The abundance of Clostridium(0.12%)at the water-soil interface was significantly higher than that in the rhizosphere soil and the bulk soil.(2)There were significant differences between in-situ ammonia oxidizing activities and between microbial community composition in different microzones of potted paddy soil.The rate of total ammonia oxidation(Rtotal)at the soil-water interface(6.06 mmol N L-1 d-1)was significantly higher than that in the rhizosphere soil(2.17 mmol N L-1 d-1)and the bulk soil(2.71 mmol N L-1 d-1).There was only aerobic ammonia oxidation at the water-soil interface,and its aerobic ammonia oxidizing rate(RA,6.06 mmol NL-1 d-1)was significantly higher than that in the rhizosphere soil(1.39 mmol N L-1 d-1)and the bulk soil(1.42 mmol NL-1 d-1).Aerobic ammonia oxidation and anammox coexisted in the rhizosphere soil and the bulk soil.The rate of anammox(RAn)in the bulk soil(1.33 mmol N L-1 d-1)was significantly higher than that in the rhizosphere soil(0.75 mmol N L-1 d-1).There was no significant difference in RA between the three microzones.The transcript abundances of AOA and AOB amoA genes in the three microzones were significantly positively correlated with RA(AOA p=0.005,R2=0.47;AOB p=0.027,R2=0.32),suggesting that both AOA and AOB were active in the three microzones.The transcript abundance of amA genes of AOA and AOB was consistent in the three microzones.The transcript abundance at the soil-water interface was the highest,followed by that in the rhizosphere soil,and that in the bulk soil was the lowest.The dominant genera of AOB were Nitrosospira and Nitrosococcus,which accounted for 51.0%and 33.1%of all AOB groups,respectively.The dominant genus of AOA was Nitrososphaera,which accounted for 69.0%of all AOA groups.The transcript abundance of AnAOB hzsB gene in the three microzones was significantly and positively correlated with RAn(p<0.001,R2=0.98).Its spatial distribution was contrary to that of amoA genes.Three genera of AnAOB were identified,including Kuenenia,Brocadia,and Scalindua,but the relative abundance of each genus was not significantly different from each other.Unlike transcript abundance,the abundance of amoA and hzsB genes was not related to RA or RAn.Stepwise regression analysis showed that DO,pH,and DOC were the key environmental factors affecting in-situ aerobic and anaerobic ammonia oxidation activities and functional microorganisms.(3)There were significantly differences between in-situ RA,RAn and between the transcript abundance of functional genes in the rhizosphere soil and the bulk soil of paddy soils at different pH levels,and the effects of pH on those in the rhizosphere soil and the bulk soil were different.In the rhizosphere soil,RA at pH 6 and pH 7 were 3.86 N kg-1 d-1 and 3.05 mmol N kg-1 d-1,respectively,significantly higher than the rate at pH 5(0.92 N kg-1 d-1).RAn(2.69 mmol N kg-1 d-1)was the highest at pH 7,followed by 2.28 mmol N kg-1 d-1 at pH 6,and 1.92 N kg-1 d-1 at pH 5.The transcript abundance of AOA amoA gene was the highest at pH 6,which was significantly higher than that at pH 5 and pH 7.The transcript abundance of hzsB gene was the highest at pH 7,which was significantly higher than that at pH 5 and pH 6.In bulk soil,there was no significant difference in RA(0.60～1.11 mmol N kg-1 d-1)at each pH level,and RAn at pH 6(2.41 mmol N kg-1 d-1)and pH 7(2.56 mmol N kg-1 d-1)level was significantly higher than that at pH 5(0.47 mmol N kg-1 d-1).The transcript abundance of AOA amoA gene was not significantly different between all pH levels.The transcript abundance of hzsB gene was the highest at pH 7 while it was the lowest at pH 5.No transcript abundance of AOB amoA genes was detected in the rhizosphere soil and the bulk soil at the three pH levels.At pH 5,RAn in the bulk soil were significantly lower than that in the rhizosphere soil,and there was no significant difference between RA in the bulk and the rhizosphere soil.RAn was significantly lower than RA in the rhizosphere soil.There was no significant difference between RAn and RA in the bulk soil.At pH 6,RA in the bulk soil were significantly lower than those in the rhizosphere soil,RAn was not significantly different between the bulk and the rhizosphere soil.RA were significantly higher than RAn.At pH 7,RA in the bulk soil were significantly lower than that in the rhizosphere soil,and there was no significant difference in RAn between the bulk and the rhizosphere soil.RAn was significantly higher than RA in the bulk soil.Linear regression analysis showed a significant and positive correlation between in-situ ammonia oxidizing activities and transcript abundance of functional genes.Stepwise regression analysis showed that RA were significantly affected by pH and DOC,RAn was significantly affected by NH4+-N and pH,while AOA amoA gene transcript abundance was significantly affected by TOC,and hzsB gene transcript abundance was significantly affected by pH,TOC.Two-way analysis of variance showed that the increase of pH could significantly stimulate the in-situ activities of anammox and the transcript abundance of hzsB gene,but had no significant effect on the in-situ activities of aerobic ammonia oxidation and the transcript activities of AOA amoA genes;compared with pH,root activity had stronger and more significant effects on aerobic ammonia oxidizing in-situ activities and AOA gene.This study revealed the in-situ aerobic and anaerobic ammonia oxidizing activities and the spatial distribution of nitrogen-transforming microorganisms in different microzones.It was proved that bulk soil was the most important ammonia oxidizing microzones,the contribution of anaerobic and aerobic ammonia oxidation was 9.8%and 9.4%in potted soil,respectively.It was found that the anammox was strongly affected by soil pH and the aerobic ammonia oxidation was strongly affected by rhizosphere effects.This study would provide a reference for the response of in-situ ammonia oxidizing activities to pH changes in different microzones,and further deepen the understanding of in-situ nitrogen transformation,in-situ ammonia oxidation activities and spatial distribution of nitrogen-transforming microorganisms.