Studies On The Diversity Of Soil Microorganisms In Gaoligongshan National Natural Reserve, Yunnan

Soil microorganism play integral and often unique roles in global material cycle of carbon, nitrogen,and so on,thus it affects the material balance,the composition of the atmosphere and geochemistry procedure in the global scale.Soil microbial diversity may represent the ability of a soil to cope with perturbations,and has been proposed as an indicator for soil quality. Understanding the structure and composition of microbial communities and their responses to environmental perturbations,such as toxic contamination,climate change,and land use changes, is critical for prediction,maintenance,and restoration of desirable ecosystem functions.Due to the extremely high diversity of environmental samples,microbial detection,characterization,and quantification are great challenges.The development and application of nucleic acid-based techniques have largely eliminated the reliance on cultivation-dependent methods for microbial detection and consequently have greatly advanced characterization of microorganisms in natural habitats.Gaoligongshan national natural reserve located in the southwestern part of Yunnan Province with altitude ranging from 760 to 3 100m.It is one of the key areas for biodiversity protection in the globe and was named "The Genebank of Earth" for its various species.The forest destruction and the global change caused the environmental deterioration in Gaoligongshan national natural reserve.Research on the distribution and regional character of soil microorganisms in this area could help to evaluate environment,understand the influence in global change and offer scientific data for making the protected policy.In this study,cultured and uncultured methods combined with advanced molecular technology such as microarray,T-RFLP and sequencing were used to investigate the diversity and community structure of microbe,bacteria,cellulose-decomposing fungi and functional genes. The influences of plant community type,altitude and environment factors were discussed.The key results from these studies are summarized as follows:(1) The quantities of soil microbe were studied in different vegetation types and altitudes in Gaoligongshan national natural reserve.The quantities of microbes,the activities of enzymes and the amount of nutrition were examined in 8 soils,which were collected from 8 fixed monitoring sample plots with different habitats and the elevation ranging from 960 to 2 878 meters.The results showed that the quantities of bacteria,actinomyces and fungi varied with elevation.The highest values occurred at the elevation of about 2 000 meters.The quantities of aerobic and anaerobic cellulose-decomposing fungi were plentiful in the scrub and coffee forest with low altitude,while the quantities were relatively few in the plot of primitive forest with high altitude.The quantities of aerobic and anaerobic nitrogen-fixing bacteria were the most in maize plot and the lowest in coffee plot.Therefore,the quantities of cellulose fungi and nitrogen-fixing bacteria were very different in different plots(P＜0.05).Microbial biomass carbon in plots of maize,coffee and forest with the altitude of 2000m were quite plentiful,and the differences among all plots were significant(P＜0.01).The change of soil microorganism with the altitude was relative to the enzyme and ecological factors in the soil.The bacteria quantity,the quantity of total microbe,and the microbial biomass carbon were significantly correlative to urease and amylase respectively(P＜0.05);the quantity of fungi was correlative to amylase significantly(P＜0.05);the anaerobic nitrogen-fixing bacteria was correlative to urease,protease and amylase significantly(P＜0.05).The bacteria quantity and the quantity of total microbe were correlative to the contents of soil organic matters(P＜0.05), and the quantity of actinomycetes was correlative to total nitrogen,total phosphorus,total potassium,altitude,water content,pH vale and temperature(P＜0.05);cellulose-decomposing fungi was correlative to temperature,altitude and water content(P＜0.05);aerobic nitrogen-fixing bacteria was in positive correlation to total phosphorus(P＜0.05),and anaerobic nitrogen-fixing bacteria was also in positive correlation to organic matters,total nitrogen,total phosphorus and water content(P＜0.05);carbon content was positively correlative to content of organic matters (P＜0.05).All results suggested that the major ecological factors influencing on the quantity of soil microorganism and the vertical distribution of diversity were soil enzyme,temperature,soil organic matters and water content.The human cultivating activities,the change of soil utilization and the vegetation type also influenced the quantity of microorganism with the comprehensive effects.(2) Genetic diversity of 16s rDNA of soil bacteria in 8 plots with different altitudes and vegetations in Gaoligongshan national natural reserve was analyzed by the combination technology of ribosome DNA gene library and T-RFLP.There were 289 OTU restricted by MspⅠ,and 216 OTU by Rsa I in 8 plots.Resulting from the fingerprint map of MspⅠ,the diversity of soil bacteria with the altitude about 2000m was more than other plots with different altitudes and vegetations generally,and the bacteria diversity of plot was significantly correlative to the quantity of cultured microorganism.By random sequencing of 16s rDNA library,there were 132 special sequences from 150 positive clones,which belonged to seven phylums.Phylum Acidobacteria accounted for the 57%of total sequenced,which was the dominant bacteria in Gaoligongshan national natural reserve.The different land use and temperature were the major factors influencing on the diversity of soil bacteria.The geological distance and difference on vegetation type were the factors influencing on the ecological distribution of soil bacteria. Moreover,pH value of soil might play important role on the community structure,while other ecological factors were correlative to the diversity of community structure of soil bacteria and ecological distribution.(3) In order to investigate the community structure and difference of microbes related to nitrogen cycle in soil from Gaoligongshan national natural reserve,T-RFLP technology was applied for the analysis on the diversity and ecological distribution of nifH and nosZ and 8 samples with different altitudes and vegetations were selected for this study.There were four restriction enzymes(HaeⅢ,HhaⅠ,RsaⅠ,MspⅠ) applied on nifH and nosZ genes.The number of OTU was the most by restriction of HhaⅠ,63 OTU in nifH and 156 OTU in nosZ respectively.The diversity of nifH(H') was ranged from 0.454(Plot 10) to 1.200(Plot 5).There were 3-4 dominant floras in each plot.The diversity of nifH was highly correlative to pH value of soil,relative to other ecological factors with different degrees,and was influenced by soil cultivation system and vegetation type.The diversity of nosZ(H') was ranged from 0.760(Plot 10) to 1.464(Plot 13).There were 2-4 dominant floras in each plot.The diversity of nosZ was highly correlative to temperature,altitude,pH value and water content.The geological distance and vegetation type also greatly influenced the distribution of nifH and nosZ.(4) In order to understand the diversity of culturable cellulose-decomposing fungi(CCDF), Carboxymthyl cellulose-congo red(CMC-CR) plate and potato dextrose agar(PDA) plate were used to isolate and culture the cellulose-decomposing fungi from plots.The quantities and diversity of soil culturable cellulose-decomposing fungi are significantly correlated with organic matter and total nitrogen in the soil.Different plant community and human disturbance have effect on the quantities and diversity of CCDF.There were 35 strains with high activities,the CMC activity by 96h of liquid fermentation was ranged from 79.6 IU(strain 6) to 228.9 IU (strain 3).A total of thirty five independent isolates belongs to 15 genus,which are identified using both morphological characteristics and rDNA ITS sequence analysis.Thirty one(88.6%) isolates belongs to 12 genus from phylum Ascomycetes and four(11.4%) isolates belongs to 3 genus from the phylum Basidiomycetes.Aspergillus,Penicillium,Stachybotrys and Chaetomium were the dominant genus.Strain 3 was identified as the Penicillium expansuma,with great developmental potential since of its particular enzyme system and utilization on substrate.(5) GeoChipⅡwas applied in the detection of functional microorganism of 6 plots on the altitude of 960-2 878m of Gaoligongshan national natural reserve,which including 11 kinds of functional genes,such as carbon cycle,nitrogen cycle,phosphor cycle,sulfur cycle,metal resistance,organic degradation.There were 1 515 functional genes and 27 unknown genes detected out in 6 plots.The order of number of detected genes was ORG＞MET＞CDEG＞NRED＞NIT＞DSR＞methane_ox＞CFIX＞NFIX＞methane_gen＞PER.The order of plots according to the number of genes and diversity was Plot 5＞Plot 10＞Plot 7＞Plot 13＞Plot 15＞Plot 8.The diversity of microorganism functional genes was influenced by various factors including human disturbance,vegetation type,altitude and ecological factors,among them,the human disturbance and the change of vegetation type greatly altered the functions of microorganism.Seven kinds of genes related to carbon and nitrogen cycles in microorganism of 6 plots in Gaoligongshan national natural reserve were analyzed,including CDEG,CFIX,methane_ox, methane_gen,NFIX,NIT and NRED.The number of genes detected out,diversity and evenness of these genes were different in different plots,and the number of the same gene detected from different plot are different.Genetic diversity of carbon-nitrogen cycle was relative to soil organic matters,total nitrogen,carbon-nitrogen ratio,available phosphor and water content with different degrees.The human disturbance with different strength and the different vegetation type also influenced the number and distribution of carbon and nitrogen cycle genes.