| Soil acidification occurs in major Chinese croplands caused of excessive use of nitrogen fertilizer,which impact the crop production and result in related environmental problems.Based on a long-term greenhouse experiment with a sharp gradient of decreasing soil pH by excessive use of nitrogen fertilizer under different planting years(1,3,5,7 and 9 years),this study analyzed the evolution characteristics of key microbial community structure of nitrification and denitrification by real-time PCR and high-throughput sequencing techniques,combined with the nitrification potential and denitrification potential and observed N2O emissions in situ to determine the impact of structure changes of the key microbial communities on its function.The results help to develop the rational nitrogen management measures,improve nitrogen use efficiency and reduce the negative environmental effects.The main results and conclusions are as follows:(1)All the treatments were obviously acidified with the increased of planting period.The soil pH was decreased to 7.00,6.33,5.69,4.88 and 4.31 respectively after cultivated 1,3,5,7 and 9 years.Besides causing the reduction in soil pH,soil acidification also led to changes in other soil chemical properties.Total N,C and soil dissolved organic carbon(DOC)varied from 0.08 to 0.22%,0.69 to 1.50%and 25.11 to 110.87 ?g C g-1 in soils with different cropping years,respectively.The lowest values in total N,C and DOC were found in the 5-year cropping history.With acidity increasing,the contents of NH4+-N in soils markedly increased and the highest value was up to 166.11 mg kg-1 at pH 4.3.In contrast,NH3 content(0.55-9.07 ?M)showed a decreasing trend along increasing cropping years.Soil nitrate-N contents ranged from 18.13 to 618.75 mg kg-1,and its contents in the 5 and 9-year cropping histories were higher than those in the others.(2)The abundance and structure of ammonia-oxidizing microorganisms were changed by soil acidification.The abundance of ammonia-oxidizing bacteria(AOB)in each treatment was significantly increased,and then sharply decreased with the decreased of soil pH.The abundance of AOB with the pH 6.33 was the highest,and there was no difference between 5-9 years(pH<5.69)which was significantly less than the neutral treatment.Compared with AOB,ammonia-oxidized archaea(AOA)had a strong adaptability to soil pH,and the abundance of AOA was significantly higher 1-3 magnitude orders than AOB.Soil acidification significantly reduced the abundance and diversity of AOB,but had no significant effect on AOA.The soil pH and NH3 contents were the main factors affecting the abundance of AOB.The ammonia oxidizer community structure was significantly affected by soil acidification,but the acid adaptability among different populations was significantly different.For example,OTU1,OTU4 and OTU6 of AO A were widely distributed in each treatment,but OTU3 was only having higher relative abundance under neutral condition.The CCA showed that soil pH,NH3 content and DOC were critical factors shaping ammonia oxidizer community structure.According to phylogenetic analysis,most of the AOB sequences were affiliated with Nitrosospira,the AOA sequences belonged to Nitrosopumius,Nitrosotalea and Nitrososphaera.(3)With the increased of acidification,the function of ammonia-oxidizing bacteria and ammonia-oxidizing bacteria was changed in different planting period.The results of AOA and AOB to the relative contribution rate of RNP showed that AOB was predominated in 1 and 3-year treatment(pH>6.33)which contributed 59.44%and 61.99%of RNP,respectively.The role of AOB and AOA to RNP began to change at pH 5.69,the contribution rate of AOA was 63.22%.When pH<4.88,AOA accounted for absolute position in soil nitrification,contributed 94.72-98.27%of the RNP.The correlation analysis showed that there was no correlation between NP and abundance of AOA,but there was a significant positive correlation with AOB.(4)Soil acidification significantly reduced the abundance and diversity of denitrifying microorganisms,but different denitrifying microorganisms responded differently to pH.The abundance of nirK-denitrifiers gradually decreased with the increased of acidification.The abundance of nirS-denitrifiers was the highest in 3-year treatment,the pH below 4.88 treatments was significantly lower than neutral treatment,but nosZ-denitrifiers were sensitive to pH,and only had higher abundance under neutral conditions.The abundance of nirS-denitrifiers was higher compared with the nirK-denitrifiers in each treatment.The correlation analysis showed that soil pH was significant positive correlation with nirK-,nirS-and nosZ-denitrifiers abundance and diversity.The results showed that soil acidification had obvious effect on microbial community and structure of nirK-,nirS-and nosZ-denitrifiers,but each population had its own unique niche,such as OTU43 and OTU44 of nirK-denitrifying population only had higher relative abundance at pH 5.69 treatment.The results of CCA showed that soil pH and DOC were critical factors shaping denitrifiers community structure.According to phylogenetic analysis,it was found that most of the nirK-,nirS-and nosZ-denitrifiers OTUs belonged to the uncultured bacteria.(5)Soil acidification significantly decreased soil denitrification potential increased N2O/(N2O+N2)and promoted N2O emission.The results showed that soil pH was significant positive correlation with DP but significant negative correlation with N2O/(N2O+N2).Meanwhile,the abundance of nirK-denitrifiers was significant positive correlation with soil denitrification,nosZ-denitrifiers abundance was significant negative correlation with N2O/(N2O+N2).In addition,the diversity of denitrifying microbial population was positively correlated with soil DP,but was significantly negative correlated with N2O/(N2O+N2).The cumulative emission of N2O was significantly higher than that in the conventional wheat and maize rotation soils at the same period.There was no correlation between cumulative emission of N2O and DP,but had a significant positive correlation with soil NP by the correlation analysis.(6)In addition,the nitrate content were as high as 379.59 and 618.75 mg N kg-1 for 3 and 9-year treatment respectively,while the NP was significantly higher than neutral treatment,which had a higher risk of negative environmental effects by nitrate leaching. |
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