Study On Driving Mechanism Of Nitrogen Transformation By Soil Microorganisms In Pinus Massoniana Plantation

Pinus massoniana Lamd.(P.massoniana)is one of the native tree species with a wide distribution area in the subtropical region of China.It is crucial in the construction of ecological environment and development of forestry industry in China.However,the phenomenon such as a large number of low-yielding forests and declining soil fertility exists due to unreasonable management of pinus massoniana plantation in the afforestation process.Soil nitrogen-related form has very important effect on the growth and fertility maintenance of plantations.Soil microorganisms mainly drive soil nitrogen transformation.However,there is a lack of relevant research reports on the microbial driving mechanism of soil nitrogen transformation in pinus massoniana plantation.Forest age,parent rock development,soil and nitrogen application must be considered seriously in the construction of fast-growing and high-yield forest and maintenance of soil fertility stability of masson pine.In this paper,the pinus massoniana plantation in subtropical zone was taken as research object for field and pond experiments.Molecular biological techniques such as q PCR,Phospholipid fatty acid(PLFA),BIOLOG and Illumina Mi Seq high throughput sequencing were used,and the study was made on the forest age,parent rock developed soil and nitrogen application on soil nitrogen transformation and related functional gene abundance and microbial community structure.It aims to analyze soil nitrogen conversion in pinus massoniana plantation and its microbial driving mechanism,and provides scientific basis for directional regulation of soil nitrogen transformation and efficient utilization of available nitrogen.The results of this study are shown below:(1)In the growth process of pinus massoniana plantation,significant changes take place in soil characteristics,nitrogen conversion functional gene abundance and microbial community structure.There is a highly significant difference in stand age,month and soil layer for soil physical and chemical properties,microbial biomass carbon,and nitrogen and enzyme activities,with distinct spatial and temporal distribution.The content of soil characteristic index(except total potassium)shows to be 0-10cm,and the soil layer is higher than 10-20cm.Bacteria is dominant in soil microbial groups in different ages,and the total PLFAs of soil microorganisms,fungi and fungi/bacteria showed a“v”trend with the increase of age.The PLFAs of soil bacteria,Gram-positive bacteria(G~+),Gram-negative bacteria(G~-)and actinomycetes are the highest in 25 years.The general performance for average well color development(AWCD)and diversity index(Shannon index,Simpson index and Mc Intosh index)is 25 years>13 years>58 years>38 years.The plantations of Pinus massoniana grow over 25 years,their soil microbial community structures lower in stability and their metabolic function does in activity,too significantly,which in turn exacerbates microecological imbalance in the soils of Pinus massoniana plantations.The gene abundances of AOA amo A,AOB amo A,nir S and nir K reach the maximum at 25 years;the abundances of nif H and nos Z first reduce and then increase with the increase of forest age,and reach to minimum in 38 years.The dominant groups of soil bacteria are Acidobacteria,Proteobacteria,Actinobacteria and Chloroflexi.There is a close relationship between Acidobacteria,Planctomycetes,Gemmatimonadetes,Nitrospirae and Firmicutes and AOA amo A,AOB amo A,nir K and nir S.It may be the main group that regulates nitrification and de-nitrification.At the generic level,there is a very significant positive correlation between the gene abundance of Bradyrhizobium and nif H,which plays an important role in regulating soil nitrogen fixation.Candidatus?Solibacter,Bryobacter and H16 are significantly correlated with gene abundance of AOA amo A and AOB amo A,which may be the main microbial group driving the soil nitrification process.The gene abundance between Candidatus?Solibacter,Variibacter and Acidibacter and nir K,nirs and nos Z has a significant relationship,which plays an important role in controlling soil denitrification.(2)Soil parent materials have distinct impact on nitrogen mineralization rate,gene abundance of nitrogen transformation function and community distribution of nitrogen fixing bacteria.Soil organic carbon,total nitrogen,alkali-hydrolyzed nitrogen,urease and nitrate reductase activities are the highest in Basalt.The net mineralization rate of soil is mainly net nitrification rate,mainly occurs in 0-10cm soil layer,and has the largest change range in June and reaches to maximum in July.Basalt(BA)has the highest soil development.The gene abundance of nif H,AOA amo A and nir S is the highest in basalt,and gene abundance of AOB amo A and nir K are the highest in the Blastopsammite the gene abundance of nos Z is highest in Quartz sandstone.Nif H and AOA amo A gene abundance showed a significant positive correlation with plant height and ground diameter,respectively.The gene abundance of AOA in the soils of the four types of parent materials is higher than AOB,and the quantity of AOA is sensitive to the change of soil environmental factors,which may play an important role in the ammoxidation process.Basalt-developed soils have a higher content of soil nutrients(organic carbon,total nitrogen and alkali-hydrolyzed nitrogen)than that in other parent rocks,causing soil nitrogen fixing bacteria richness index(Chao1 index,ACE index)and diversity index(Shannon index and Simpson index)higher than those of other soil parent materials.At the phylum level,Proteobacteria and Canobacteria are dominant groups,and Proteobacteria has the highest relative abundance in Basalt,and Cyanobacteria have the highest relative abundance in Blastopsammite.At the genus level,Bradyrhizobium,Calothrix,Rhizobium and Azospirillum are the dominant groups,which may be the main microbial group that drives nitrogen fixation in soil.Bradyrhizobium was significantly positively correlated with tree height and ground diameter.Rhizobium is significantly negatively related to tree height and ground diameter.Basalt(BA)soil is conducive to the growth of pinus massoniana.(3)Soil characteristics,nitrogen conversion rate and microbial community structure have evident time difference on response of nitrogen fertilizer.With nitrogen fertilizer treatment,soil p H decreases with the increase of nitrogen application and there is no significant difference among different treatments.With the increase of fertilization time,soil organic carbon and total nitrogen content increases first and then decrease;with the increase of fertilizer application,ammonium nitrogen and nitrate nitrogen increase.Nitrogen application time,nitrogen application quantity and their interaction have significant effects on the net nitrogen mineralization rate of soil,which is dominant by effect of fertilization time.The net nitrogen mineralization rate of soil is mainly net nitrification rate.In December 2017 and June 2018,the application of N3(high nitrogen)significantly increases total PLFAs of soil microorganisms,bacteria,fungi,G~+,AMF and PLFAs of actinomycetes;after 2 years of nitrogen application(December 2018),N3(high nitrogen)application has an inhibitory effect on total PLFAs of soil microorganisms,bacteria,fungi,G~+,AMF and PLFAs of actinomycetes.Through cluster analysis and principal component analysis,it can be shown that the separation effect between N3(high nitrogen)treatment and other treatments is obvious,and NO(control)and N1 express aggregation effect.Soil organic carbon,alkali-hydrolyzed nitrogen,ammonium nitrogen,nitrate nitrogen and microbial biomass nitrogen have an evident correlation with microbial community structure.In the early stage of nitrogen application,appropriate amount of nitrogen can increase soil nitrogen content and promote soil nitrogen mineralization and increase microbial biomass.As the nitrogen application rate and time increase,the excessive accumulation of nitrogen in soil is not conducive to soil nitrogen mineralization and increase of microbial biomass.(4)The gene abundance and community structure of AOA amo A and AOB amo A take significant changes under nitrogen application.The genes copy numbers of AOA amo A and AOB amo A are 0.86×10~7-0.93×10~7and 0.43×10~5-1.6×10~5copies/g soil respectively.The number of AOA amo A is higher than that of AOB amo A and AOA/AOB ratio is in the range of 60-199.Nitrogen application is conductive to the increase of gene abundance of AOA amo A and AOB amo A,and the gene abundance of AOA amo A and AOB amo A in N2 treatment is higher than that in other treatments.Nitrogen application has significant effects on Chao1 index and ACE index of AOA and AOB communities.Thaumarchaeota is the dominant group in AOA community,and Proteobacteria dominants in AOB community.High nitrogen(N3)application significantly increases soil nitrogen content,which has promoted the relative abundance of Thaumarchaeota and Proteeobacteria.The nitrification rate and mineralization rate is significantly correlated with AOB gene abundance and community structure.Thus,it can be known that ammonia oxidation is mainly driven by AOB.(5)Nitrogen application has remarkable effect on gene abundance and community structure of nir K and nos Z.The gene abundance of different nitrogen fertilizers treatment soils nir K,nir S and nos Z are 1.12×10~6-3.67×10~6,2.39×10~6-3.04×10~6and 2.95×10~6-6.55×10~6copies/g soil,and the gene abundance of nos Z is higher than that of nir K and nir S.Compared with other treatments,the gene abundance of nir K and nir S in N2 treatment is higher.Nitrogen application has an inhibitory effect on nos Z gene abundance,and the inhibition effect of N3(high nitrogen)treatment is significant.Nitrogen application has remarkable impact on?diversity index of nir K and nos Z communities,as well as Shannon index and Simpson index of nir S community.Through the changes in soil ammonium nitrogen,nitrate nitrogen,microbial biomass nitrogen content and ammoniation rate,nitrogen application enables changes in the microbial community of soil denitrification.In the communities of nir K,nir S and nos Z,Proteobacteria is the dominant group,with relative abundance of 96.41%-98.7%,96.75%-99.39%and 59.25%-68.06%.Nitrogen application decreases relative abundance of Proteobacteria in nir K community,and the inhibition decreases gradually with the increase of nitrogen application.Nitrogen application is beneficial to the increase of relative abundance of Proteobacteria in nir S community,which increases with the increase of nitrogen application.The relative abundance of Proteobacteria in nos Z community expresses the“v”trend with the increase of nitrogen application.The nitrogen application rate has a stronger effect on nir K denitrification microbial community structure than that of nir S and nos Z.In the denitrifying microbial groups,Proteobacteria(phylum level)and Bradyrhizobium(genus level)are common and dominant groups.They have a closely related relation with soil nitrogen content.By affecting Proteobacteria and Bradyrhizobium,nitrogen application may regulate denitrification.