Microbial Community Structure Of Potato-cultivated Soils And Its Response To Fertilization Managements In The Semi-arid Area Of Central Gansu Province

Gansu Province is an important potato seed and commodity potato production area in China.The potato industry has become a strategic leading industry to promote the development of agriculture and rural economy,agricultural efficiency and increase farmers'income in Gansu Province.In this study,high-throughput sequencing technique based on 16S rRNA gene was used to study the characteristics of bacterial community structure and its relationship with geographic and main chemical variables by multi-field sampling in typical potato farmland in an arid area of Central Gansu Province.In addition,based on potato experimental station located in Anding District,Dingxi City,Gansu Province,we used real-time fluorescence quantitative PCR,high-throughput sequencing and DNA-based stable isotope probing?DNA-SIP?technique based on 16S rRNA gene and amoA gene to reveal the effects of chronic nitrogen application rate and different fertilization managements on the communities of soil bacteria and microbial ammonia-oxidizers under both in the field trial and laboratory microcosmic culture experiments.The results are as follows:1.Both geographic variables and main soil chemical variables affected the bacterial community structure in potato-cultivated soils of Central Gansu Province.20 potato-cultivated plots were selected to reveal the geographic variables?latitude,longitude and altitude?and main soil chemical variables?organic matter,total nitrogen,moisture,pH and carbon/nitrogen rate?,we found that both geographic and main soil chemical variables affected the soil bacterial community structure in the main potato production area of Central Gansu Province.1.1 The geographical distribution of main soil chemical variables?organic matter,total nitrogen and moisture?showed the law of latitude gradient distribution in the sampling area.Soil pH was positively correlated with latitude,while organic matter,total nitrogen and water content were negatively correlated with latitude;1.2 A total of 22 phyla were observed,among that,Acidobacteria,Proteobacteria,Bacteroidetes,Chloroflexi,Planctomycetes,Gemmatimonadetes,Actinobacteria,Nitrospirae,Armatimonadetes and Firmicutes were the dominant bacterial phyla and accounted for a total of 96.3%of the total bacterial sequences.The relative abundance of Acidobacteria was positively correlated with soil organic matter,total nitrogen and moisture,while negative correlation relationship between these soil chemical variables and Actinobacteria,Gemmatimonadete and Armatimonadetes were observed.The relative abundance of Acidobacteria Subgroup 4,Sphingobacterials and Xanthomonadales were positively correlated with soil organic matter,total nitrogen and moisture at the order level,while negative correlation relationship between these soil chemical variables and Gemmatimonadales,Cytophagales and Planctomycetales were observed.In addition,the relative abundance of Blastocatella showed positive correlation relationship with soil organic matter,total nitrogen and moisture at genus level;1.3 At the OTU taxonomic level,geographic variables may be the driving forces for bacterial community structure variations.Among them,Phylogenetic abundance and phylogenetic diversity were negatively correlated with longitude,but positively correlated with altitude;1.4 The difference of soil bacterial community was influenced by both geographic variables and main soil chemical variables,and the effect of soil chemical variables on the community structure variations was more significant than that of geographic variables.Non-metric multidimensional scaling analysis?NMDS?indicated that the main soil chemical variables?soil organic matter,total nitrogen and moisture?were positively correlated with NMDS1 and NMDS2,NMDS1 have a significant positive or negative correlation with latitude,longitude and altitude,respectively,while no relationship was observed between NMDS2 and geographic variables.The main chemical variables?soil organic matter,total nitrogen,moisture?were positively correlated with NMDS1,but negatively correlated with NMDS2.Redundancy analysis?RDA?also showed that organic matter content,total nitrogen,latitude,altitude and longitude could affect the bacterial community structure in the potato planting area.Geographic variables explained 14.2%of bacterial community variations,while soil chemical variables explained 31.4%of bacterial community variations.In this study,soil organic matter,total nitrogen and moisture were the most important environmental factors affecting the bacterial community structure.1.5 The potato yield of all sampled farmlands ranged from 6.0 t ha^-1 to 69.8 t ha^-1,the highest yield was about 11.6-fold compared with the lowest.In this study,the relative abundance of Chloroflexi?Anaerolineales and Firmicutes in high-yield fields,which had the characteristics of organic matter degradation and drought resistance,was higher than that in low-yield soils.2 Chronic nitrogen application rates significantly changed the community structure of bacteria and microbial ammonia oxidizers in potato-cultivated soils.Chronic nitrogen application rates field trial began in 2013 with six different nitrogen treatments:?1?N0:no nitrogen application control;?2?N75:75 kg N ha^-1;?3?N150:150 kg N ha^-1;?4?N225:225 kg N ha^-1;?5?N300:300 kg N ha^-1;?6?N375:375 kg N ha^-1.The communities of bacteria and microbial ammonia oxidizers in 0²0 cm and 20⁴0 cm soil profile at potato tuber expansion period in August 2016?the 3rd year after the experiment started?were analyzed by using high-throughput sequencing,real-time fluorescence quantitative PCR and other molecular techniques.The communities of bacteria and microbial ammonia oxidizers in potato soils were significantly affected by different chemical nitrogen application rates.2.1 The soil bacterial community structure was changed with chronic nitrogen application rates.Nitrate and pH were the main environmental factors for the change of soil bacterial community structure.Proteobacteria,Actinobacteria,Acidobacteria,Chloroflexi,Bacteroidetes,Planctomycetes,Firmicutes,Gemmatimonadetes and Nitrospira were the dominant bacterial phyla in this study.The bacterial copy numbers increased with the increase of nitrogen application rate,and reached the maximum in N225 soil,while decreased in excess nitrogen application rate.Soil nitrate and pH explained the differences in bacterial communities of 16.9%and 16.0%,respectively.The relative abundance of Bacteroides was increased by32.0%?0²0 cm?and 46.1%?20⁴0 cm?,respectively;2.2 A significant variation of soil microbial ammonia-oxidizers were found under chronic nitrogen application rates.The relative abundance of soil AOA was not affected by chronic nitrogen application rates,while the relative abundance of soil AOB in N375 soil increased significantly as the nitrogen amount goes up,the relative abundance of soil AOB in 0²0 cm and 20⁴0 cm were 11.9-fold and 3.7-fold compared with N0.QPCR results indicated that the copy number of soil AOB increased significantly with nitrogen amount goes up,the AOB amoA gene copy number were 11.8-fold and 7.8-fold compared with N0 in 0²0 cm and 20⁴0 cm soil profiles.AOB was more sensitive to urea and dominated the soil nitrification process in this study;2.3 Phylogenetic relationship of AOB and AOA was analyzed by high-throughput sequencing technique.Results showed that Nitrosospira Cluster 3 was the dominant AOB group in N0 soil,while 54d9-like cluster dominated soil AOA communities.Chronic nitrogen application rate changed the community structure of AOB and AOA,the relative abundance of29i4-like cluster increased and became the dominant group of AOA in N375 soil.2.4 Chronic nitrogen application rate significantly increased the soil nitrate and soil potential nitrification rate?PNR?,respectively.PNR in N375 soil was significantly higher than that in low nitrogen?N75,N150?and medium nitrogen?N225?soils,representing 2.0-fold and2.8-fold compared with N0 in 0²0 cm and 20⁴0 cm soil profile,with nitrate content of13.7-fold and 17.6-fold compared with N0 soils in 0²0 cm and 20⁴0 cm soil profiles,respectively;2.5 Chronic nitrogen application rates changed the potato yield.The highest tuber yield was found in N75 soil,representing 15.1%higher compared with N0.N150,N225,N300 and N375 decreased potato tuber yield by 4.1%,6.0%,1.9%and 10.8%,respectively.The potato tuber yield of potato with excessive nitrogen application decreased.In this study,the relative abundance of potentially pathogenic bacteria in high-yield soils,such as Bacteroides-like and Streptomyces-like,was lower than that in low-yield soils.3 Effects of different fertilization managements on the community structures of soil bacteria and microbial ammonia oxidizers.Different fertilization managements field trial began in 2015 with four different nitrogen treatments:?1?CK:no application control;?2?NPK:chemical fertilization;?3?M:organic fertilization;?4?NPKM:combined application of chemical and organic fertilization.The communities of bacteria and microbial ammonia oxidizers in 0²0 cm and 20⁴0 cm soil profile at potato maturation period in October 2016?the 2nd year after the experiment started?were analyzed by using high-throughput sequencing,real-time fluorescence quantitative PCR and other molecular techniques.The communities of bacteria and microbial ammonia oxidizers in potato soils were significantly affected by different fertilization managements.3.1 The soil bacterial community was altered with different fertilization treatments.Proteobacteria,Acidobacteria,Actinobacteria,Chloroflexi,Bacteroidetes,Planctomycetes,Firmicutes,Gemmatimonadetes,Nitrospira and Armatimonadetes were the dominant bacterial phyla in this study.Fertilization significantly increased the number of bacteria in soil,and the copy number of bacteria in 0²0 cm and 20⁴0 cm M soil was 40.9%and 50.0%higher than that in CK soil,respectively.Soil pH explained 30.2%of the bacterial community differences,leading to the variation of soil bacterial community structure under short-term different fertilization treatments.The relative abundance of soil Proteobacteria,Actinobacteria,Bacteroidetes,Gemmatimonadetes,Armatimonadetes and Chloroflexi were increased,representing 10.4%,7.9%,7.9%,3.2%,2.1%and 0.5%compared with CK soil,respectively;3.2 The response patterns of soil AOA and AOB were different under different fertilization treatment.The relative abundance of soil AOA in M and NPKM soils decreased by 9.0%and9.3 in 0²0 cm soil profile,and 5.2%and 4.4%lower in 20⁴0 cm soil profile compared with CK,respectively.On the contrary,the abundance of soil AOB increased after chemical and organic fertilization.Results showed that the response ability of AOB to fertilizer was higher than that of soil AOA,is was the soil AOB that dominated the soil nitrifying process under different fertilization treatments;3.3 Different fertilization managements altered the microbial community structure of soil microbial ammonia oxidizers.The relative abundance of 29i4-like AOA cluster in M and NPKM soils were higher than that in CK and NPK soils.The relative abundance of Nitrososphaera viennensis-like AOA branch was the highest in NPK soil,although it was not the dominant AOA branch,indicating that the branch is more susceptible to the effect of soil nitrogen content.3.4 NPK significantly increased soil nitrate content compared with treatments with the addition of organic fertilizer?M and NPKM?,representing 46.9%and 73.8%higher compared with CK soil in 0²0 cm and 20⁴0 cm soil;3.5 The potato tuber yield was affected by different fertilization managements.Tuber yield of NPK,M and MPKM soils increased by 0.3%,14.9%and 7.0%compared of CK,respectively,and increased significantly in M treatment?P<0.05?.In this study,the relative abundance of Steroidobacter in high-yield fields,which had the characteristics of organic matter degradation,was higher than that in low-yield soils.4 DNA-SIP technique revealed that the diversity of soil active microbial ammonia-oxidizers decreased under chronic application rateThree treatments,CK?no application control?,LN(low nitrogen,75 kg N ha^-1)and HN(high nitrogen,375 kg N ha^-1)were applied to the soil sampled in August 2015?tuber expansion stage,the 3rd year after experiment start?.The stable isotope ¹³C was used to trace the nucleic acid DNA of ammonia-oxidizing microorganisms,together with cloning library and real-time fluorescence quantitative PCR,three isotope marker groups were used to label soil active microbial ammonia oxidizers:?1?¹²CO₂ control,?2?¹³CO₂ labeled,?3?¹³CO₂+C₂H₂,to explore the relative contribution of active AOA and AOB to soil nitrification in potato soils.4.1 Chronic nitrogen application rate increased ¹³C-labeled soil active AOA and AOB.¹³C-amoA gene of AOA and AOB were 2.58×10⁴ copies and 1.55×10⁶ copies,respectively,representing 1.6-fold and 16.2-fold higher than those of N0,respectively.The number of active cells was 2.4-fold?AOA?and 24.0-fold?AOB?compared with CK,respectively.It indicated that soil AOB dominated the soil ammonia oxidation progress;4.2 The relative abundance of soil dominant ¹³C-labeled AOA and AOB changed under chronic nitrogen rates.Nitrogen application significantly increased the proportion of 54d9-like AOA up to 90%in HN soil,while the Nitrososphaera gargensis-like and Nitrososphaera viennensis-like AOA were inhibited and completely disappeared.Nitrogen application also resulted in the community shift of active AOB-dominant group from Nitrosospira briensis-like cluster to Nitrosospira sp.TCH711-like cluster.