Effects Of Fertilization Types On Gene Diversity Of Microorganisms Related To Nitrogen Cycle In Soybean Rotation And Continuous Cropping

The long-term application of chemical fertilizers in agricultural production not only reduces the efficiency of nitrogen fertilizer utilization by crops,but also causes nitrogen overload in the soil,leading to a series of environmental problems.Organic materials contain various nutrients and organic matter,which can not only improve soil fertility and nitrogen use efficiency,but also improve the quality of agricultural products and protect the agricultural environment when applied in combination with chemical fertilizers.On the basis of long-term location experiment,both metagenome sequencing and quantitative PCR were employed to study the effects of fertilization types（NPK fertilizer,NPK fertilizer combined with biochar,NPK fertilizer combined with organic fertilizer and NPK fertilizer combined with biochar+organic fertilizer）on soil physicochemical properties,soil enzyme activities,nitrogen cycle microbial community structure,and nitrogen cycle functional gene abundance under soybean maize rotation and soybean continuous cropping,revealing the process and mechanism of nitrogen cycling driven by microbes in farmland soil.The main results of the paper are as follows:（1）Under soybean corn rotation and soybean continuous cropping modes,compared with NPK application,the combination of organic fertilizer and biochar+organic fertilizer increased soil nutrient content.Under the rotation mode,the combination of organic fertilizer significantly increased TN content（47.06%）（P<0.05）,biochar+organic fertilizer significantly increased AP content（82.62%）（P<0.05）,and the combination of organic fertilizer and biochar+organic fertilizer significantly increased AK（178.41%and 216.83%）and nitrate nitrogen content（288.28%and305.00%）（P<0.05）.Under the continuous cropping mode,the combination of organic fertilizer and biochar+organic fertilizer significantly reduced soil pH（P<0.05）,the combination of organic fertilizer significantly increased OM content（36.17%）（P<0.05）,and the combination of biochar+organic fertilizer significantly reduced ammonium nitrogen content（26.89%）（P<0.05）.（2）Under soybean corn rotation and soybean continuous cropping modes,different fertilization treatments have different effects on soil enzyme activity.Under the rotation mode,the combination of biochar and organic fertilizer reduced soil urease activity（12.36%and 5.37%）,while the combination of biochar and organic fertilizer increased urease activity by 6.57%;the three treatments significantly increased nitrite reductase activity（46.15%～174.45%）（P<0.05）.Under continuous cropping mode,the combination of biochar,organic fertilizer,and biochar+organic fertilizer increased urease activity（53.06%and 61.92%）.The combination of organic fertilizer and biochar+organic fertilizer also significantly increased nitrite reductase activity（89.39%and 93.96%）（P<0.05）,while biochar reduces its activity（18.28%）.（3）Under soybean maize rotation and soybean continuous cropping,different fertilization treatments showed different structural characteristics in nitrogen cycle microbial community composition.Under the rotation mode,the relative abundance of Actinobacteria and Proteobacteria in each treatment exceeded 64.60%,occupying the dominant position.The species significance analysis showed that under both modes,the dominant bacterial genus in the combination of biochar and NPK treatments was Rhodococcus,while genus Afipia belonged to the Proteobacteria in the combination of organic and biochar+organic fertilizer.Restrictive Principal Coordinate Analysis（CPCoA）showed that there was no significant difference in the composition and structure of microbial communities among the four treatments.The continuous cropping mode is similar to the rotation mode,with the sum of relative abundance of Actinobacteria and Proteobacteria exceeding60.40%,still occupying the dominant position.The dominant bacterial genus of the three combination treatments is Mycolicibacterium in the phylum Actinobacteria.CPCoA analysis found that there was a significant difference（P<0.01）between the combination of organic fertilizer,biochar+organic fertilizer,and NPK treatment,while there was no significant difference between the combination of biochar and NPK treatment.（4）Under soybean maize rotation and soybean continuous cropping,the composition and structural characteristics of key genes in denitrification process were significantly different under different fertilization treatments.Under the rotation mode,the TPM（Transcripts Per Kilobase of exon model per Million mapped reads）values of narG and nosZ significantly increased（P<0.05）in the combination of organic fertilizer and biochar+organic fertilizer,while the TPM values of nap A genes significantly decreased（P<0.05）.The TPM values of nirK in the combination of biochar+organic fertilizer significantly increased（P<0.05）.Under continuous cropping mode,all three fertilizations significantly increased the TPM value of nosZ,while the combination of biochar and organic fertilizer also increased the TPM value of narG,nirK,and norB（P<0.05）.CPCoA analysis showed that there was a significant difference in the composition of key genes in the denitrification process between the combination of organic fertilizer and biochar+organic fertilizer and NPK（P<0.05）,but there was no significant difference between the combination of biochar and NPK.The contribution of Actinobacteria phylum is between 56.08%～66.72%,while the contribution of Proteobacteria to nar G and nor B is between 38.97%～72.76%.The planting mode has the greatest impact on the contribution to nirK.unclassified＿P＿Acidobacterium,Bradyrhizobium,and Nocardioides have the highest contribution to the five main functional genes.The planting mode has the greatest impact on the composition of the contribution of bacterial genera to the nirK and narG.Cropping mode has a significant impact on denitrification and nitrogen fixation pathways,and fertilization mode has a significant impact on nitrification and denitrification pathways.（5）Under soybean maize rotation and soybean continuous cropping,different fertilization treatments led to different correlations between environmental factors and species and denitrification functional genes.Under the two modes,the treatment groups of biochar+organic fertilizer and organic fertilizer were more closely related to environmental factors,and the key functional genes of denitrification,except nap A,were significantly negatively related to pH.Under the rotation mode,species were significantly negatively correlated with environmental factors such as pH,OM,NO₃^--N,and NH₄⁺-N,while they were significantly positively correlated with TP（P<0.05）.Under the continuous cropping mode,species had a significant positive correlation with AK（P<0.05）,and the key function genes of denitrification had a stronger correlation with environmental factors.（6）There was no correlation between N₂O emission from soil and the abundance of key genes in denitrification process,as well as the number of copies of nirK,nirS and nosZ genes.Compared to the application of NPK,all three combinations increased soil N₂O emission,with the highest increase in organic fertilizer.The abundance of the key gene nosZ in the process of denitrification from high to low is biochar+organic fertilizer,organic fertilizer biochar and NPK,but there is no significant difference between the four treatments of nirK,nirS and the number of copies of nosZ.In conclusion,soybean continuous cropping had more positive effects on soil physical and chemical properties,enzyme activities,microbial community structure of nitrogen cycle and key functional genes.Rotation promoted denitrification,while continuous cropping enhanced nitrogen fixation.The combined application of biochar and organic fertilizer enhanced the nitrification and denitrification processes,and the combined application of biochar enhanced nitrogen fixation.The combination of biochar,biochar,and organic fertilizer reduced N₂O emissions.