| Pinus koraiensis is a preciously coniferious tree species in Northeast China and the Pinus koraiensis-dominated mixed forest is a zonal vegetation type in this area,which has high productivity and ecological status.In the processes of of seedling regenerations of Pinus koraiensis,secondary succession,and reaching the top community through competition,the soil microorganisms as decomposers play an extremely important role in regulating nutrient circulation,participating material circulation and energy flow,decomposing litter,transforming organic matter,and improving the soil environment.However,the studies on the characteristics in community composition and diversity of soil microorganisms and their relationship with soil physicochemical properties in different forest types of Pinus koraiensis forest have not been reported,and especially the studies on the microbial communities involved in nitrogen circulation were relatively scarce.The analysis on the community composition,diversity characteristics and influencing factors of soil microorganisms in different forest types of Pinus koraiensis forest,especially for the microbial communities involved in nitrogen circulation,will be helpful to understand the process and rules of material circulation and energy flow in Pinus koraiensis forest,and to analyze the adaptability of Pinus koraiensis to soil environment.Therefore,the soil under the three forest types of primitive Pinus koraiensis forest(Picea koraiensis-Abies nephrolepis-Pinus koraiensis forest,Tilia tuan Szyszyl-Pinus koraiensis forest and Betula costata-Pinus koraiensis forest),Pinus koraiensis plantation and natural Pinus koraiensis secondary forest in Liangshui national nature reserve was as research objects,on the basis of the comprehensive determination of the soil physicochemical properties,marker genes of related soil microbial functional groups(bacterial 16S rDNA,fungal ITS1,nitrogen-fixing bacteria nifH,denitrifying functional genes nirK and nosZ)were amplified as molecular targets.The community structure,diversity characteristics of these soil microbial groups and their relationship with soil physicochemical properties and influencing factors of related microbial groups were thoroughly analyzed and discussed by high-throughput sequencing and bioinformatics analysis.The main results are as follows:(1)Soil physicochemical properties and community composition and diversity characteristics of soil bacteria:there was a significant difference in soil ammonium nitrogen(NH4+-N)content between the five forest types(P<0.05)and there was also significant difference in nitrate nitrogen(NO3--N)content between the five forest types((P<0.05),except between the Pinus koraiensis plantation and Picea koraiensis-Abies nephrolepis-Pinus koraiensis forest and natural Pinus koraiensis secondary forest.Most of the physicochemical properties between the primitive Pinus koraiensis forest and the secondary forest and the natural Pinus koraiensis secondary forest were significant,but there was no significant difference in soil pH,total organic carbon and soil bulk density between the three primitive Pinus koraiensis forest.The dominant phylum of soil bacteria in the five forest types of Pinus koraiensis forest were Proteobacteria,Actinobacteria,Verrucomicrobia and Acidobacteria.The soil bacterial Simpson index of the primitive Pinus koraiensis forest and the Pinus koraiensis plantation was higher than that of the natural Pinus koraiensis secondary forest(but the difference between the Betula costata-Pinus koraiensis forest and natural Pinus koraiensis secondary forest was not significant).There was no significant difference in the other alpha diversity index between the five forest types and the alpha diversity index was not also significantly correlated with physicochemical factors.There was significant difference in community composition of five types of Pinus koraiensis forest based on bacterial species classification(R=0.210,P=0.037,the R value reflects the degree of difference between groups and within groups),but there was no significant difference in community composition based on phylogenetic relationship(R=0.106,P=0.131).The soil total porosity,total organic carbon and pH vale are significantly correlated with bacterial community composition(P<0.05),and the soil total porosity may be the main physicochemical factor affecting the bacterial community composition.(2)Community composition and diversity characteristics of soil fungi:the alpha diversity index(Shannon index)and richness index(Ace index and Chaol index)of soil fungi in the three types of primitive Pinus koraiensis forest were higher than those of the Pinus koraiensis plantation and the natural Pinus koraiensis secondary forest.The difference of the alpha diversity index between other forest types was not significant.Soil pH was negatively correlated with the alpha diversity index of soil fungi in five forest types,however,soil total nitrogen content were positively correlated with it,including that total nitrogen content were significantly positively correlated with Chaol and Ace index.The dominant soil fungal phylum are Ascomycota and Basidiomycota.There was significant difference in fungal community composition of five types of Pinus koraiensis forest based on bacterial species classification(R=0.261,P=0.005),but there was no significant difference in fungal community composition based on phylogenetic relationship(R=0.053,P=0.252).The soil total nitrogen,total organic carbon and soil total porosity are significantly correlated with fungal community composition(P<0.05),and soil total organic carbon may be the main physicochemical factor affecting the fungal community composition.(3)The community composition and diversity characteristics of soil nitrogen-fixing bacteria:there was no significant difference in four alpha diversity index of soil nitrogen-fixing bacteria in five forest types,and there was no significant correlation with soil physicochemical properties.The dominant soil nitrogen-fixing bacterial phylum are Proteobacteria,Cyanobacteria,Firmicutes and Verrucomicrobia(composition proportion is above 1%overall).There was significant difference in nitrogen-fixing bacterial community composition of five types of Pinus koraiensis forest based on species classification(R=0.243,P=0.005),but there was also significant difference in nitrogen-fixing bacterial community composition based on phylogenetic relationship(R=0.245,P=0.002).Soil nitrate nitrogen,ammonia nitrogen content and pH vale were significantly correlated with nitrogen-fixing bacterial community composition(P<0.05).Soil nitrate nitrogen content may be the main physicochemical factor affecting the nitrogen-fixing bacterial community composition,followed by soil ammonium nitrogen content and pH.(4)Community composition and diversity characteristics of nirK denitrifying microorganisms:the Ace index and Chaol index of nirK denitrifying microorganisms in Tilia tuan Szyszzyl-Pinus koraiensis forest were significantly higher than that in Pinus koraiensis plantation and natural Pinus koraiensis secondary forest,but the difference of alpha diversity index between the three forest types of primitive Pinus koraiensis forest,between the Pinus koraiensis plantation and natural Pinus koraiensis secondary forest was not significant.Richness indexs(Ace index and Chaol index)of nirK denitrifying microorganisms were significant correlation with soil total porosity,nitrate nitrogen and ammonia nitrogen content,including that Ace index and Chaol index were very significant correlation with soil total porosity.The dominant phylum of soil nirK denitrifying microorganisms are proteobacteria(composition proportion is 30.7%-54.0%).There was significant difference in community composition of nirK denitrifying microorganisms in the five forest types of Pinus koraiensis forest based on species classification(R=0.448,P=0.001)and there was also significant difference in community composition of nirK denitrifying microorganisms based on phylogenetic relationship(R=0.434,P=0.001).Soil nitrate nitrogen and water content were significantly correlated with community composition of nirK denitrifying microorganisms(P<0.05).Soil nitrate nitrogen content may be the main physicochemical factor affecting nirK denitrifying microbial community composition,followed by water content.(5)Community composition and diversity characteristics of nosZ denitrifying microorganisms:there was no significant difference in four alpha diversity indexs of soil nosZ denitrifying microorganisms in five forest types,except that the Shannon and Simpson index in Tilia tuan Szyszyl-Pinus koraiensis forest were significantly higher than that of natural Pinus koraiensis secondary forest.Simpson index of nosZ denitrifying microorganisms were negatively significant correlation with soil pH,however,Ace index and Chaol index of nirK denitrifying microorganisms were positively significant correlation with soil nitrate nitrogen conttent.The dominant phylum of soil nosZ denitrifying microorganisms are proteobacteria(composition proportion is 41.28%-65.17%)and dominant genus of soil nosZ denitrifying microorganisms are Burkholderia,Flavobacterium,Bradyrhizobium,Pseudomonas,Dechloromonas,Gemmatimonas,Achromobacter and Sinorhizobium.There was significant difference in community composition of soil nosZ denitrifying microorganisms in the five forest types of Pinus koraiensis forest(R=0.387,P=0.006)and there was also significant difference in community composition of nirK denitrifying microorganisms based on phylogenetic relationship(R=0.391,P=0.004).Soil ammonia nitrogen and total nitrogen content were significantly correlated with nosZ denitrifying microbial community composition(P<0.05).Soil ammonia nitrogen content may be the main physicochemical factor affecting nosZ denitrifying microbial community composition,followed by total nitrogen content.There were a large number of undetermined soil bacteria,fungi,nitrogen-fixing bacteria,nirK and nosZ denitrifying microorganisms in the soil of five forest types of Pinu.s koraiensis forest by high-throughput sequencing and bioinformatics analysis.However,in the microbial communities with annotated information,Proteobacteria,Actinobacteria,Verrucomicrobia and Acidobacteria were dominant phylum of soil bacteria,the dominant soil fungal phylum are Ascomycota and Basidiomycota,The dominant soil nitrogen-fixing bacterial phylum are Proteobacteria,Cyanobacteria,Firmicutes and Verrucomicrobia.And Proteobacteria is the dominant phylum of soil nirK and nosZ denitrifying microorganisms.The difference of soil physicochemical properties in five forest types of Pinus koraiensis forest did not significantly affect the alpha diversity index of related soil microbial communities,but affected significant difference in the beta-diversity of these microbial communities,but there was no significant difference in soil bacterial and fungal community composition based on phylogenetic relationship analysis.Whether it is based on species classification or phylogenetic relationship,soil microbial community composition of the natural Pinus koraiensis secondary forest is quite different from that of primitive Pinus koraiensis forest and Pinus koraiensi.s plantation,but the difference between the three types of primitive Pinus koraiensis forest is small.Soil total porosity may be the main physicochemical factor affecting bacterial community composition,soil total organic carbon may be the main physicochemical factor affecting fungal community composition,soil nitrate nitrogen and ammonia nitrogen content may be the main physicochemical factor affecting microbial community composition related to nitrogen cycle.Community composition,diversity characteristics,and key influencing factors of related soil microbial functional groups of Pinus koraiensis forest were comprehensively revealed by high-throughput sequencing techniques based on the coding genes of molecular targets of related soil microbial functional groups in the first time.The relationship between stand composition,soil physicochemical properties,microbial community composition and diversity in Pinus koraiensis forest ecosystem was demonstrated.These results will lay the scientific foundation for understanding of soil material circulation in Pinus koraiensis forest,especially the processes and rules related to nitrogen circulation,also provide basic data and scientific evidences for the protection,artificial restoration and sustainable management of Pinus koraiensis forest resources. |
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