Effects Of Continuous Nitrogen Application On Community Structure Of Ammonia-oxidizing Flora And Bacteria On Potato-planting Soil

Gansu Province is an important potato seed potato and commercial potato production and starch processing base,potato industry is to promote agricultural and rural economic development,promote agricultural efficiency,increase farmers’income strategic leading industry.However,the phenomenon of excessive fertilizer input,especially the excessive use of nitrogen fertilizer,is very serious in potato planting process.For this reason,this study was conducted in Dingxi City,Gansu Province,using conventional soil chemical analysis methods combined with molecular ecology methods such as cloning,sequencing and fluorescence quantitative PCR,through the nitrogen fertilizer localization test in the main potato growing area of Dingxi City,Gansu Province.The effects of different nitrogen application rates on soil nitrification and community structure of bacteria and ammonia-oxidizing microorganisms were studied in order to reveal the response mechanism of soil bacteria and ammonia-oxidizing microorganisms to different nitrogen rates under long-term continuous nitrogen application.The field trial began in 2013 with six different nitrogen treatments:N0:control,no nitrogen fertilizer;N75:The amount of nitrogen applied was 75 kg N/hm²;N150:The amount of nitrogen applied was 150 kg N/hm²;N225:The amount of nitrogen applied was 225 kg N/hm²;N300:The amount of nitrogen applied was 300 kg N/hm²;N375:The amount of nitrogen applied was 375 kg N/hm².The main results are as follows:1.Nitrogen application significantly increased the nitrification potential and the nitrate nitrogen content in the soil profile,especially in the 0-20cm soil,and increased the nitrification potential of the soil,especially the nitrification potential of the soil.（1）The nitrification potential of soil increased significantly with the increase of nitrogen application rate.The nitrification potential increased by 79%,95%,69%,11.3%and 13.9%respectively in 0-20cm soil layer compared with that of N0 without nitrogenous fertilizer.The increase of nitrification potential is the direct reason for the increase of nitrate nitrogen content in soil profile.（2）Nitrogen application significantly increased nitrate nitrogen content in soil profile for four consecutive years,and with the increase of nitrogen application rate,nitrate nitrogen content in each layer of soil profile increased,especially in 0-20 cm soil layer,and the nitrate-N content in soil profile increased with the increase of nitrogen application rate,especially in 0-20 cm soil.The contents of NO₃^--N in the treatments were 4.3 mg/kg,6.7 mg/kg,9.2 mg/kg,11.3 mg/kg,20.0mg/kg,46.8 mg/kg,which increased by 55.8%,113.9%,162.8%,365.1%and 988.4%,respectively.When N application rate exceeded 300kg N/hm2,the NO₃^--N content in 0-20cm and20-40cm soil layers exceeded the environmental risk threshold of 20mg/kg,which brought potential risks to soil and groundwater environment.2.Nitrogen application significantly changed the structure of bacterial community and decreased the diversity of bacterial community in the surface soil.（1）The abundance of actinomycetes and Bacteroides increased by 6.1%-17.4%and 16.4%-46.8%respectively compared with N0 treatment,and the abundance of Acidobacterium decreased 7.8%-26.4%compared with N0,and the abundance of actinomycetes and Bacteroides increased by 6.1%-17.4%and 16.4%-46.8%,respectively,compared with that of N0,and the abundance of Acidobacterium decreased by 7.8%-26.4%compared with that of N0.The abundance of Bacteroides increased by 33.8%,44.8%,83.3%,109.2%and 173.3%,respectively,with the increase of nitrogen application rate compared with N0 in the dominant soil bacteria groups at the class level.The relative abundance of AcidobacterGp4 and Anaerolineae decreased by 16.3%-33.8%and 8.1%-22.7%respectively in AcidobacterGp4 and Anaerolineae treatments compared with that in N0 treatment with the increase of N application rate.The relative abundance of AcidobacterGp4 and Anaerolineae decreased by 16.3%-33.8%and 8.1%-22.7%,respectively,with the increase of N application rate.The abundance of Ohtaekwangia and Streptomyces increased 33.6%-173.1%and 3.4%-66.4%compared with N0,while the abundance of Gaiella and Solirubrobacter decreased 6.9%-30.8%and 4.5%-27.6%compared with N0,respectively.（2）Continuous nitrogen application decreased soil bacterial community diversity and bacterial/fungal ratio.The results showed that the Shannon index was significantly lower and the simpson index was significantly higher in N375 treatments than that in other treatments,although there was no significant difference between OTU and Chao1 indexes in N375 treatments,with the increase of N application rate,but no significant differences in OTU and Chao1 indexes were observed between N375 treatments and N375 treatments,but no significant differences were found in OTU and Chao1 indices.But the values of OTU and Chao1 of soil samples treated with N375 were the lowest compared with other treatments,which indicated that excessive N application could reduce bacterial diversity,and continuous N application significantly changed the number of microorganisms in dryland soil.The abundance of bacteria and fungi in the soil increased with the increase of nitrogen application rate,and the B/F value of the treatments decreased by 14%,19%,28%and 44%,respectively,compared with that of the control,which resulted in the transition from"bacterial"to"fungal"type of soil.The results showed that the B/F ratio of the two treatments decreased significantly with the increase of nitrogen application rate,but decreased by 14%,19%,28%and 44%,respectively.3.Nitrogen application significantly increased the number of AOB（ammonia-oxidizing bacteria）in soil.Under the condition of this experiment,AOB dominated the process of soil ammonia oxidation.（1）The main results were as follows:1)the community structure and abundance of AOA did not change significantly in all treatments,but the abundance of AOA was significantly increased by continuous nitrogen application,and increased with the increase of nitrogen application rate.The abundance of AOB in 0-20 cm soil was 74.8%,131.1%,176.1%,412.1%and 489.8%higher than that of N0,respectively.Similarly,the abundance of AOB in the 20-40 cm soil layer was also increased with the increase of nitrogen application rate.There was no obvious change trend of AOA in the soil layer between 20 cm and 40 cm.（2）The results of AOB phylogenetic tree analysis showed that soil AOB was mainly distributed in Nitrosomonas,Nitrosospira and Nitrosococcus,and with the increase of nitrogen application rate,the amount of Nitrosococcus in soil increased significantly,compared with that of N0 treatment,the amount of Nitrosococcus in soil increased significantly.The amount of Nitrosococcus increased by 4.9%,51.8%,164.7%,504.9%,383.5%respectively,but the abundance of Nitrosomonas and Nitrosospira did not change significantly with the increase of nitrogen application rate.4.The decrease of soil pH and the increase of nitrate nitrogen content were the main reasons for the change of community structure of bacteria and ammonia-oxidizing microorganisms under the condition of continuous application of large amounts of nitrogen.（1）The results showed that with the increase of nitrogen application rate,the soil pH decreased significantly,and reached the lowest value of 8.02 in N375,which was 0.27 units lower than that of N0 treatment（pH 8.29）.The results showed that the soil pH decreased significantly with the increase of nitrogen application rate,and reached the lowest value of 8.02 in N375.Therefore,the long-term use of nitrogen fertilizer can lead to a significant decrease in soil p H,and the higher the amount of nitrogen applied,the greater the decrease.（2）Correlation analysis showed that the abundance of soil AOB was significantly positively correlated with soil nitrification potential and NO₃^--N content.The results of redundancy analysis and correlation analysis showed that the abundance of actinomycetes,Bacteroides,Ohtaekwangia,Streptomyces and Lysobacter were positively correlated with soil NO₃^--N content,and negatively correlated with soil pH.The results of RDA and correlation analysis showed that the abundance of Actinomycetes,Bacteroides,Ohtaekwangia,Streptomyces and Lysobacter were positively correlated with soil NO₃^--N content and negatively correlated with soil pH.The abundance of Acidobacteria,Gaiella,Solirubrobacter and Gemmatimonas was negatively correlated with soil NO₃^--N content and positively correlated with soil pH.To sum up,the continuous application of large amounts of nitrogen accelerated the transformation process of fertilizer nitrogen in the soil by increasing the nitrification potential of the soil,resulting in the accumulation of nitrate nitrogen in the soil profile,and changed the community structure of soil bacteria by applying a large amount of nitrogen continuously.The diversity of soil bacterial community and the ratio of bacteria to fungi were decreased,which led to the transition of soil from"bacterial"to"fungal",and ammonia oxidizing bacteria（AOB）dominated the ammonia oxidation of dryland soil.The results of correlation analysis showed that:the decrease of soil pH and the increase of nitrate-N content were the main reasons for the change of community structure of bacteria and ammonia-oxidizing microorganisms.