| Since the discovery of complete ammonia oxidizing bacteria,which is also called Comammox.The relationship between Comammox and soil pH and nutrient as well as its function and niche differentiation have caused attention.Red soil is one of the important types of cultivated land in China.Its low fertility is a problem for agricultural output.In this study,we examined red soils from Hunan's Quaternary dryland and measured changes to pH and nutrient content across different soil layers and years of different treatments.These treatments included fallow(F),no fertilizer control(CK),a single application of inorganic fertilizer of nitrogen,phosphate,and kalium(NPK),inorganic fertilizer combined with strawreturn(NPKS),inorganic fertilizer combined with lime(NPKL),inorganic fertilizer combined with amendments of organic crushed-bones(NPKA),and inorganic fertilizer combined with commercial bioorganic fertilizer(NPKC).We also compared the activity of enzymes related to carbon,nitrogen,and phosphorus cycles in this soil using the microplate fluorescence method.The effects of red soil improvement on the abundance and community structure of Comammox were investigated by fluorescence quantitative PCR and high-throughput sequencing.The combination of DNA stable isotopes with nitrification inhibitors clarified the function of Comammox and its relative importance to N2 O production in red soil.Through the above studies,the environmental factors affecting the abundance and community structure of Comammox in red soil,the contribution of Comammox in nitrification and the niche differentiation of different branches were clarified.The main outcome measures as follow:(1)Some treatments significantly affected soil nutrients and enzyme activities.Soil organic matter(SOM),total nitrogen(TN),total phosphorus(TP),and available phosphorus(AP)in the 0-20 cm soil layer treated with NPKC in 2020 increased by 73%,29%,61%,and 1847%,respectively,relative to the control.This treatment also significantly increased the enzyme activities of the enzymes that participate in the carbon cycle,including ?-1,4-glucosidase(?G),?-1,4-glucosidase(?G),?-1,4-xylosidase(?X),and cellobiohydrolase(CBH).Also,the activity of ?-1,4-N-acetylglucosamine(NAG);which is involved in the nitrogen cycle,was increased.Correlation analysis showed that SOM was significantly positively correlated with enzyme activities of ?G,?G,?X,CBH,and NAG(P < 0.01).Additionally,the pH value was significantly negatively correlated with acid phosphatase(ACP)activity(P < 0.01).The effect of improvement measures on the enzyme activity of 0-20 cm soil layer was greater than on deeper layers.In2019,compared with the control,NPKA treatment increased CBH enzyme activity in the 0-20 cm soil layer by 352%,but only by 2% in the 20-40 cm soil layer.Besides,ACP enzyme activity in the soil also showed a trend of increasing with years of treatment.The combination of inorganic fertilizer and organic materials can significantly improve the nutrient status and soil enzyme activity in red soils.This can be used to efficiently improve the fertility of barren red soil.(2)The abundance of ammonia-oxidizing microorganisms in red soil was significantly increased by different ameliorative measures.Among them,the abundance of ammonia-oxidizing archaea(AOA)in NPK and NPKS treatments was significantly higher than that in other treatments,being 1.19×108copies·g-1 and 1.11×108 copies·g-1,respectively,an increase of 457.95% and 422.36% over CK treatment.The abundance of ammonia-oxidizing bacteria(AOB)was the highest in NPKL treatment(2.14 × 108copies·g-1),an increase of 9253.17% over CK treatment.The abundance of Comammox Clade A was the highest in the NPKC treatment(1.09 × 107 copies·g-1),an increase of 768.38% over CK treatment.The abundance of Comammox Clade B was the highest in NPKA treatment(1.06 × 108 copies·g-1),an increase of 560.70% over CK treatment.This indicates that the application of inorganic fertilizer and the combination of inorganic fertilizer and straw returning can significantly improve the abundance of AOA in red soil.Adding lime can significantly increase the abundance of AOB in red soil.Adding organic fertilizer could significantly increase the abundance of Comammox in red soil.Moreover,the phylogenetic analysis showed that the proportion of Comammox Clade B in F,CK,and NPK treatments was higher than that of Comammox Clade A2,with the proportion ranging from 61% to 84%.The proportion of Comammox Clade A2 in NPKA,NPKL,NPKS,and NPKC treatments was higher than that of Comammox Clade B,with the proportion ranging from 51% to 93%.It is speculated that Comammox Clade B mainly plays a role in F,CK and NPK treatments.Comammox Clade A2 plays a major role in NPKA,NPKL,NPKKS and NPKC treatments.(3)Based on the analysis of the function of Comammox and its relative importance to N2 O production in red soils,it was found that the content of N2 O in the treatment without ammonium salt fluctuated.However,the abundances of Comammox Clade A and Clade B were significantly reduced under the treatment of nitrification inhibitors.When ammonium salt was added,the content of N2 O remained at the same level,but the inhibitory effect of acetylene on the abundance of Comammox Clade A and Clade B was weakened.In red soil,Comammox Clade A can assimilate 13 C labeled CO2 and all of them are Clade A2,but Clade B cannot assimilate 13 C labeled CO2.These results indicate that Comammox Clade A and Clade B have different niche differentiation and that Clade A2 represents Comammox in red soil.In conclusion,the application of inorganic fertilizer combined with bone meal organic fertilizer and inorganic fertilizer combined with biological organic fertilizer significantly improved the nutrient status of red soil and soil enzyme activities.The abundance and community structure of Comammox in red soil under different amelioration measures were mainly affected by ammonium concentration,and Clade A2 in Comammox clade played a major role. |
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