Effect Of Organic Fertilizer With Biochar On Nitrogen Forms And Nitrification And Denitrification Microbial Community In Soil Of Lemon Orchards

Lemon has a great demand for nutrients,in order to improve the yield and quality of lemon,fruit farmers apply a lot of chemical fertilizers for a long time in agricultural production,and the consumption of chemical fertilizers in China accounts for 1/3 of the world,but the utilization rate of nitrogen and phosphorus fertilizer is less than 40%,which is much lower than that of developed countries such as Europe and the United States,especially nitrogen fertilizer.Irrational application of nitrogen fertilizer will not only reduce the utilization rate of nitrogen fertilizer,cause soil degradation and environmental problems such as soil slumping,soil acidification,nutrient loss,heavy metal pollution,but also increase the risk of agricultural surface pollution,which is not conducive to the sustainable development of agriculture.Therefore,the rational application of fertilizers to improve crop yields,maintain soil fertility,and take into account ecological effects is one of the major challenges in agricultural research.China is the world’s highest agricultural waste output countries,the annual production of agricultural waste up to more than 5 billion tons,but the agricultural waste resource utilization rate is not high,which wastes resources at the same time caused serious impact on the ecological environment.Promoting the resource utilization of agricultural waste such as straw and livestock manure,giving full play to the value of agricultural waste,is of great significance to the green and sustainable development of China’s agriculture.Biomass carbonization is one of the main ways of agricultural waste resource utilization,and fertilization is also the main way of agricultural waste resource utilization,among which making organic fertilizer from livestock and poultry manure is an important way.Biochar and organic fertilizers not only improve soil fertility level,increase effective soil nutrient content and improve soil nitrogen availability,but also promote crop growth and improve crop yield,and have positive effects on soil microbial activity and community structure.At present,studies on biochar and organic fertilizers have mainly focused on the effects on soil quality and environmental effects,and relatively few studies have been conducted on the effects of biochar and organic fertilizers on soil nitrogen morphological conversion and soil nitrification and denitrification microbial communities.Therefore,in this study,Yulik lemon was used as the test object,purple tide soil was used as the test soil,biochar made from burned rice husk straw and organic fertilizer（fresh pig manure and rotted pig manure）were used as the test materials,and six treatments of no fertilizer（CK）,conventional fertilizer（F）,organic fertilizer（P）,chemical fertilizer+biochar（FP）,organic fertilizer+biochar（PP）,and fresh organic fertilizer+biochar（NPP）were set up in a pot experiment.Analysis of soil N fugacity patterns using a modified graded leaching and separation method for N in sediments.Determination of soil nitrification and denitrification potentials using the suspension incubation method and the modified nitrate residual method.Quantification of the copy number of AOA amo A,AOB amo A,nir S,nir K and nos Z genes using real-time fluorescence quantitative PCR.Community structure of ammonia-oxidizing microorganisms（AOA,AOB）and nir S,nir K,and nos Z-type denitrifying microorganisms using terminal restriction fragment length polymorphism（T-RFLP）technique.The redundancy analysis（RDA）method was used to investigate the relationship between the microbial community structure of AOA amo A,AOB amo A,nir S,nir K and nos Z genes and environmental parameters.In order to provide a theoretical basis for the rational use of organic fertilizer and biochar in agriculture.The main research results are as follows.（1）The soil physicochemical properties and plant growth and development differed significantly among fertilization treatments.Among the fertilization treatments,soil p H was the lowest in treatment F.The application of biochar was able to significantly increase soil p H,especially in the inter-root soil,and the organic fertilizer with biochar treatment（PP and NPP）was the most effective,and the inter-root soil p H in the PP and NPP treatments increased by 0.54 to 0.63 units compared with the CK treatment.Compared with the F treatment,PP and NPP treatments increased the organic matter content of the inter-rhizosphere soil by 24%and 83%,respectively,and increased the total nitrogen content of the inter-rhizosphere soil by 7%and 29%,respectively.The soil quick-acting nutrient content of PP and NPP treatments was significantly lower than that of F treatment,but organic fertilizer and biochar had long-lasting and slow-release properties,which could continuously provide nutrients to plant roots and facilitate long-term nutrient uptake and utilization by plant roots.PP and NPP treatments increased plant nitrogen accumulation by 14%to 27%,plant phosphorus accumulation by167%to 174%,and plant potassium accumulation by 122%to 123%compared to F treatment.The dosed biochar significantly increased the plant biomass,and the best results were obtained with PP and NPP treatments,which increased the aboveground dry weight of plants by 35%to 48%and the belowground dry weight of plants by 78%to 100%compared with F treatment.（2）The results of soil N grading showed that there were significant differences between the contents of each form of N in different fertilization treatments,and the contents of the four transformable forms of N（TF-N）were,in descending order,IMOF-N>OSF-N>IEF-N>CF-N.The soil IMOF-N content ranged from 157.33 to 317.70 mg·kg^-1,OSF-N content ranged from 116.09 to204.34 mg·kg^-1,IEF-N content ranged from 6.40 to 85.75 mg·kg^-1,and CF-N content ranged from20.12 to 34.29 mg·kg^-1 for each treatment.Among them,OSF-N accounted for 27%～47%of TF-N content,and IMOF-N accounted for 36%～62%of TF-N content.Fertilizer application significantly increased soil ion-exchange state N（IEF-N）content,with the best results obtained with F and FP treatments.The application of organic fertilizer and biochar significantly increased the soil carbonate-bound nitrogen（CF-N）content,and the inter-root soil CF-N content increased by 49%,43%and 12%in the P,PP and NPP treatments,respectively,compared with the F treatment.Biochar application increased soil iron-manganese oxide-bound nitrogen（IMOF-N）and organic sulfide-bound nitrogen（OSF-N）content.The soil IMOF-N content of PP and NPP treatments increased by 6%and 78%,respectively,compared with F treatment,and the OSF-N content of P,PP and NPP treatments increased by 14%to 76%in the inter-root soil compared with F treatment.（3）Compared with CK treatment,F treatment increased soil nitrification potential,organic fertilizer and organic fertilizer with biochar treatment decreased soil nitrification potential,and P,PP and NPP treatments decreased soil nitrification potential by 61%,22%and 56%,respectively,compared with CK treatment.The AOA amo A gene copy number was 3.36×10⁶～4.92×10⁸ copies·μl^-1 and the AOB amo A gene copy number was 3.11×10⁷～5.50×10⁸ copies·μl^-1 in soil and plant roots of each treatment,and the AOB amo A gene abundance was significantly higher than that of AOA.Pearson correlation analysis showed a significant positive correlation between soil nitrification potential and AOB amo A gene abundance（P<0.05）,indicating that ammonia-oxidizing bacteria（AOB）dominate the ammonia oxidation process in acidic purple tidal soils.The abundance of AOA amo A genes in the PP treatment was 1.90～4.11 times higher than that in the CK treatment,the abundance of AOA amo A genes in the NPP treatment was 0.22～0.58 times higher than that in the CK treatment,and the abundance of AOB amo A genes in the roots of the PP and NPP treatments was 0.12～0.67 times higher than that in the CK treatment.Using T-RFLP technique to analyze the structure of AOA and AOB communities,it was found that PP and NPP treatments increased the Shannon index and richness index of soil AOA communities and decreased the Shannon index and richness index of AOB communities in non-rhizosphere soils,and organic fertilizer with biochar increased the diversity of AOA communities and decreased the diversity of AOB communities in soils.The results of RDA analysis showed that total potassium,p H and organic matter were the main drivers of changes in AOA community structure in non-interstitial soils,and carbon to nitrogen ratio,p H,total nitrogen and organic matter were the main drivers of changes in AOA community structure in inter-rhizosphere soils.The variation of AOB community structure in non-rhizosphere soils was mainly influenced by total potassium,alkaline nitrogen and water content,and the variation of AOB community structure in rhizosphere soils was mainly influenced by p H,carbon to nitrogen ratio,water content and organic matter.（4）The application of organic fertilizer significantly increased the soil denitrification potential,and the inter-root soil denitrification potential was 43.82,39.94 and 28.73 times higher in the P,PP and NPP treatments than in the F treatment,respectively.The soil denitrification potential of organic fertilizer with biochar（PP and NPP）was reduced by 9%～67%compared with organic fertilizer（P）,and the application of biochar suppressed soil denitrification,and the suppression effect was more obvious with NPP treatment.The soil nir K gene copy number was 1.33 to 267.44 times higher than the nir S gene copy number,and the nir K gene copy number was 0.02 times higher than the nir S gene copy number in the roots of plants in each treatment,indicating that nir K-type denitrifying microorganisms were the major nitrite reducing microorganisms in the soil and nir S-type denitrifying microorganisms were the major nitrite reducing microorganisms in the roots of plants.The nir S gene copy number of PP and NPP treatments was 1.21～4.72 times higher than that of CK treatment,the nir K gene copy number was 1.10～33.39 times higher than that of CK treatment,and the nos Z gene copy number of NPP treatment was 1.08～2.11 times higher than that of CK treatment.PP and NPP treatments increased the Shannon index,richness index and evenness index of nir S and nos Z-type denitrifying microbial communities,and organic fertilizer with biochar increased the diversity of nir S and nos Z-type denitrifying microbial communities,but had no significant effect on the diversity of nir K-type denitrifying microbial communities.The results of RDA analysis showed that soil p H,organic matter and nitrate nitrogen were the main drivers of changes in the community structure of nir S-type denitrifying microorganisms.Soil organic matter,effective phosphorus,carbon to nitrogen ratio,water content and nitrate nitrogen were the main drivers of changes in nir K-type denitrifying microbial community structure.nos Z-type denitrifying microbial community structure changes were mainly related to soil nitrate nitrogen,p H,carbon to nitrogen ratio,fast-acting potassium and organic matter.In summary,compared with conventional fertilizer treatment（F）,organic fertilizer with biochar treatment（PP and NPP）can increase soil p H,organic matter content and full nitrogen content,increase CF-N,IMOF-N and OSF-N content in soil,promote plant growth and development,it can also inhibit the nitrification of the soil,and reduce the risk of environmental pollution,so it is an environmentally friendly and efficient fertilization method.