Characteristics Of Soil Components Stoichiometry And Microbial Community After The Transition Of Primary Korean Pine Broad-leaved Forests To Secondary Forests

Forests are remarkably important for the maintenance of global C balance and mitigation of climate change.Because of long-term anthropogenic and natural disturbances,primary forests have almost disappeared,and the restoration characteristics and ecological services of secondary forests,the world's major forest resources,have been a hot topic of concern.Primary Korean pine broad-leaved forests(PF)are one of the important representative communities of temperate coniferous and broad-leaved mixed forests and the top zonal vegetation in the eastern part of northeast China.The secondary forests(SF)formed after the clear-cutting of PF have problems such as low function and slow recovery,and the establishment of the edificator Korean pine(Pinus koraiensis Siebold&Zucc.)seedlings is difficult.In this study,PF were used as controls to explore the characteristics of stoichiometric variation of different soil components throughout the growing season in SF,in order to assess the dynamic balance of elements C,N,and P and microbial resource limitations;The level of recovery of soil microbial community structure and functions was evaluated from multiple perspectives of community diversity,assembly processes,species connectivity,and abundance of functional gene.The main results were as follows:1.Among the stoichiometric ratios of soil resources,C:N ratio explained 84.41%of the variation in forest biomass between the two forest types,and the soil C:N ratio of the SF was remarkably lower,indicating a relatively faster rate of microbial decomposition.Compared with PF,soil microbial biomass in SF exhibited obvious competitive patterns with plants and sensitive homeostasis regulation,suggesting that,in SF,soil microbes competed more strongly with plants for nutrients,and microbial metabolism was more susceptible to the changes in soil resources.Compared with PF,soil microbial biomass in SF exhibited obvious competitive patterns with plants and sensitive homeostasis regulation(Homeostasis Index H:0.59-1.25),suggesting that,in SF,soil microbes competed more strongly with plants for nutrients,and microbial metabolism was more susceptible to the changes in soil resources.Furthermore,the results of eco-enzymatic stoichiometry indicated that the enzymatic vector angles of the two forest types are both greater than 45°,and the vector angles of the SF is remarkably larger than that of the PF,indicating soil microbes in the two forest types were restricted by P,but the P limitation was more obvious in SF.Compared with the PF,the longer enzymatic vector lengths of the SF suggested that the soil microbes in SF were more limited by C.Stoichiometric ratios of the different soil components formed a complex network.2.There were significant differences in most soil physical and chemical properties in different sampling periods during the growing season and different forest types(all p<0.05).Two-way ANOVA showed that all indicators,except DOC and TC,were significantly affected by the forest type,sampling period,and their interaction(all p<0.01).Throughout the growing season,the PF showed high ammonium(NH4+)and low nitrate(NO3-),and the NH4+ content of the SF was significantly lower than that of the PF.In addition,the soil net ammonification rates,net nitrification rates,and net N mineralization rates were all significantly affected by the forest type,sampling period,and their interaction(all p<0.001),throughout the growing season,the net N mineralization rates of the SF were significantly higher than that of the PF.Stepwise regression analysis revealed that TP,DTN,and pH were important driving factors of soil N turnover rates.3.There were no significant differences in the alpha diversity of soil bacterial community(including phylogenetic diversity:PD,NTI,and NRI)between the two forest types(all p>0.05);while differences in bacterial community structure based on Bray curtis and Weighted unifrac distances were significant(all p<0.05).The results of LEfSe analysis revealed that Nitrospira was significantly enriched in the SF and was the most important indicator species(largest LDA value,4.09),involved in nitrification process in the soil N cycle.The analysis of the soil bacterial ecological network showed the higher average node degree,clustering coefficient,and connectivity,as well as the shorter average path length in the SF,indicating that soil bacterial community had the higher degree of interaction/coupling,while relatively low modularity implied a weak resistance to environmental changes.Furthermore,both NTI and NRI were greater than 2,and two forest types showed significant aggregation of soil bacterial phylogeny compared to the null model,suggesting that environmental filtering was the main driver of spatial variation in the bacterial community.4.There were no significant differences(except for nifH)in alpha diversity of N cycle functional microbial community AO A,AOB,nirK,and nirS between the two forest types(all p>0.05),while all functional microbial community structure(Bray curtis and Weighted unifrac),except for AOB(Weighted unifrac),were significantly different(all p<0.05),and the degree of differences(? diversity)in the composition of the different functional microbial communities was significantly different(all p<0.001).Community assembly of functional microbial communities varied.Except for AOB,which was mainly affected by stochastic processes(-2<(NTI and NRI)<2),other functional microbial communities were mainly influenced by environmental filtering(deterministic processes)(NTI and NRI>2).Ecological network analysis showed that the network topology characteristics of each functional microbial group did not change consistently between the two forest types,indicating that they adopted different survival strategies.Soil NO3-and NH4+had the high average degree in the ecological networks and they were key factors driving the recovery of N cycle functional microbial communities.5.Metagenomic analysis of soil microbes from two forest types revealed that there were 14 phyla with relative abundance greater than 1%,13 of which were significantly different in the relative abundance between PF and SF.Proteobacteria and Actinobacteria were significantly enriched in the PF;however,Acidobacteria was significantly enriched in the SF.At the genus level,the fold-changes of relative abundance in the dominant genera and the common genera between the two forest types were smaller(-1<fold-change<1),while 96 of rare genera had larger fold-changes(fold-change<-2).There were significant differences in 18 COG functional classifications and 13 metabolic pathways(KEGG level3>1%)between the two forest types.Genes related to the microbial core metabolic functions,virulence factors,stress and defence were significantly enriched in the SF.Analysis of the differences in N cycle pathways between the two forest types revealed that,in the PF,the stronger ammoniation and dissimilatory nitrate reduction(DNRA),as well as the weaker nitrification provided a genetic explanation for PF dominated by NH4+ rather than NO3-.The DNRA decreased significantly in the SF,which indicated that the ability of the SF to alleviate the N limitation of the ecosystem and maintain the balance of N cycling declined,while enhanced of nitrification and denitrification exacerbate soil N loss through nitrate leaching and N2O production.In summary,the above results explained the reasons for low functions and productivity of the SF and the difficulties in regeneration of constructive species(Korean pine)from the perspectives of ecosystem nutrient balance and microbial community structure and functions,and providing new insights on soil microbial community role in maintaining the dynamic balance of soil C and nutrients and driving the forest restoration succession process.