| In the agro-ecosystem,different fertilization management methods can affect the soil physical and chemical functions,changing the abundance of functional microorganisms and the composition of the community structure.Many studies have shown that compared with single application of chemical fertilizers,the farmland management model of inorganic organic nitrogen fertilizers helps to optimize soil microbial community structure,improve soil physical and chemical properties and nutrient supply,and maintain and improve soil quality.Studying the seasonal and vertical distribution characteristics of soil nitrogen metabolism microbial gene abundance under long-term fertilization treatment.It is helpful to understand the microbial regulation mechanism of nitrogen transformation in brown earth under different fertilization treatments,which can be used for scientific fertilization in this area.The ground force provides a theoretical basis.Using the long-term positioning test field of Shenyang Agricultural University as the platform,chemical analysis,PCR amplification,and real-time PCR(q PCR)were used to detect different sampling periods(April,July,November).Nitrogen metabolism functional gene abundance under different soil depths(0~20cm,20~40cm,40~60cm)for different fertilization treatments(no fertilization CK,low inorganic nitrogen fertilizer N2,high inorganic nitrogen fertilizer N4,organic inorganic nitrogen fertilizer M2N2).By analyzing the relationship between soil physical and chemical properties,fertilization treatment and sampling period on the abundance of nitrogen metabolism functional genes,the possibility of functional genes for early warning of soil environmental changes was discussed.The research results are as follows:(1)The gene abundance of bacteria and archaea in each gram of fresh soil was between4.3×10~9-1.76×10~12and 4.09×10~6-4.0×10~9 respectively.Inorganic nitrogen fertilizer treatment significantly reduced the number of microorganisms,while the application of organic fertilizer showed the opposite result;the number of bacteria in different sampling periods was:November>July>April,the number of archaea showed: April>July> November.The amount of soil microorganisms decreased significantly as the soil layer deepened.(2)The nitrogen metabolism gene in the soil nitrification process showed that the amoA gene copy number of AOB and AOA in each gram of fresh soil was between 5.2×10~3-2.8×10~6and 1.41×10~6-5.73×10~7.Inorganic nitrogen fertilizer treatment significantly reduced AOA abundance and increased AOB abundance.The abundance of AOB in different sampling periods was: April>July>November,AOA was: November>April>July.The copy number of nxr gene and hao gene per gram of fresh soil is between 1.56×10~4-1.53×10~7 and4.36×10~5-2.99×10~8.Inorganic nitrogen fertilizer significantly reduces the gene abundance of both,while the application of organic fertilizer significantly increases its Abundance.The hao gene expression in different sampling periods was: July>November>April,but the nxr gene did not change significantly.As the soil layer deepens,the number of soil nitrifying microorganisms is significantly reduced.(3)The nitrogen metabolism gene in the soil denitrification process showed that the abundance of nap gene and nar G gene per gram of fresh soil was between 1.65×10~6-2.46×10~9and 1.91×10~3-1.12×10~8respectively.High amounts of inorganic nitrogen fertilizer significantly reduce the abundance of both.The copy number of nir K and nir S genes per gram of fresh soil is between 1.09×10~6-9.64×10~7and 4.03×10~5-7.53 × 10~6.The abundance of nir K and nir S genes was significantly affected by fertilization treatment and sampling period,respectively.The copy number of nor B and nos Z genes per gram of fresh soil was between1.95 ×10~5-1.88 ×10~5 and 6.78 ×10~4-3.44 ×10~8,respectively,and inorganic nitrogen fertilizer significantly reduces the abundance of both.The nor B gene expression in different sampling periods was: November>April>July,the nos Z gene abundance was not sensitive to the sampling period.As the soil layer deepens,the number of soil denitrifying microorganisms tends to decrease.(4)The nitrogen metabolism genes in soil nitrogen fixation and ammoniation showed that the abundance of nitrogen-fixing nif H and ammoniated gdh genes per gram of fresh soil was between 1.27×10~4-1.24×10~9and 1.81×10~2-7.85×10~5,respectively.Fertilization reduces the abundance of nif H gene,and low inorganic nitrogen fertilizer can increase the abundance of gdh gene.The gdh gene was expressed in different sampling periods: July>November>April.There were no significant differences between nif H and gdh genes in different soil profiles.(5)Soil fertilization treatment,sampling period and soil depth have different effects on soil physical and chemical properties.Correlation analysis between different environmental factors and nitrogen metabolism genes showed that the number of bacteria,AOA-amo A and denitrification gene nap,nir K,nos Z and nor B were most sensitive to environmental factors in the 0~20cm topsoil.The nitrifying gene AOA-amo A is a sensitive gene in the subsurface soil of 20~40 cm.The RDA results indicate that the sampling period is an important environmental factor that causes the abundance of nitrogen metabolism genes in the tested soil.Correlation analysis showed that p H and ammonium nitrogen were the main factors affecting the abundance of nitrogen metabolism genes in surface and subsurface soils,respectively.The results of VPA analysis showed that the change of soil physical and chemical properties was an important factor affecting the changes of soil nitrogen metabolism functional genes compared with fertilization treatment and sampling period.Changes in soil physical and chemical properties caused by sampling period and soil fertilization treatment are the key factors causing changes in the abundance of nitrogen metabolism functional genes in the tested soil.Among them,p H and ammonium nitrogen are the main factors affecting the abundance of nitrogen metabolism genes in soils of 0-20 cm and20-60 cm,respectively.Among the 14 nitrogen metabolism functional genes,the total bacterial 16 S r DNA and the AOA in the nitrification pathway,the nap,nir K,nor B and nos Z genes in the denitrification pathway were most sensitive to changes in surface soil environmental factors. |
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