| Alpine meadow is one of vegetation types in Gannan grassland, which accounts for11.8%of the total grassland area. It is well known for its high economic value with quality forage and great influence on the global ecosystem. However, alpine meadow has suffered serious degradation due to the dual role of anthropogenic activeties and naturer itself. A long-term fertilization experimental station was established at Walaka, Gansu province, being used for estimate the effects of long-term fertilization and soil fertility on the agricultural production. Whilst there is considerable information available concerning the impact of inorganic fertiliser application on communities of macrobiota, the effects on microbial community composition in soils, especially prokaryotic microorganisms and ammonia oxidizers, have received little attention, even though they represent a major portion of living biomass in terrestrial ecosystems. To better understand how N-fertilization impact soil prokaryotic and ammonia oxidizers community composition and diversity, this study was investigated the long-term fertilization experiment at an alpine meadow using real time PCR and clone-library sequencing. The objectives were to (1) assess the response of prpkaryotic microbial community and ammonia oxidizers to different fertilizer additions in alpine meadow soil, and (2) to reveal the environmental factors affecting the soil prpkaryotic microbial community and ammonia oxidizers. We hope that the study could contribute to the restoration of degraded grasslands and the maintenance of biodiversity on the Tibetan Plateau. The main results of this study are as follows:1. The impact of long-term fertilization on the prokaryotic diversity and community structureBy aligning with GenBank database, all retrieved bacterial sequences were classified into eight groups:Acidobacteria, Proteobacteria(a,β,δ-), Bacteroidetes, Actinomycetes, Verrucomicrobia, Gemmatimonadetes, Chloroflexi and Unclassified. Acidobacteria and Proteobacteria were significantly dominant groups. Almost all the archaeal sequences appeared to be composed solely of members of the Crenarchaeota. OTUs aligning to the Crenarchaeota were assigned to three groups:group1.1b, group1.1a and group1.3, based on shared similarity to sequences in the GenBank database. Seeing from the relative abundance of the archaeal groups, the vast majority of the sequences were affiliated with the group1.1b.At the fencing field, the N fertilization significantly affected the relative abundances of bacterial groups. Response to N-fertilization, Acidobacteria decreased since the highest percentage was shown by the FO treatment. And a significant decrease was noted with Actinomycetes. Conversely, Bacteroidetes significantly increased from1.79%to4.92%and β-Proteobacteria ranged from5.98%to8.74%.With different from the above lower fertilizer concentration, the effect of F-120on bacteria community significantly enhanced. At the grazing field, the relative abundances of some bacterial groups were also different with the fertilization rates. From low to high fertilization, Acidobacteria (26.70%-18.88%) and Actinomycetes (4.54%-0.69%) decreased. There are also significant increases in some group response to fertilization. The abundance of β-Proteobacteria ranged from10.79to16.78%,δ-Proteobacteria from5.68%to15.28%, Verrucomicrobia from1.13to9.94%. Moreover, the higher abundance of Bacteroidetes and Actinomycetes were also observed in the fencing field. In contrast, the Chloroflexi and β-Proteobacteria were more abundant in grazing field.Responses of archaeal composition to N fertilization were inconsistent between the fencing field and the grazing field. At the fencing field, the relative abundances of group1.1b, group1.1a and group1.3differed clearly with N fertilization amendments, and the grazing field displayed no obvious trends in the composition of archaea with N fertilization amendments. The fencing field was richer in group1.1b abundance than the grazing field. In contrast, group1.3was greater in grazing field than fencing field.From a diversity point of view, archaeal community population was generally smaller than that of the bacterial community in all treatments. Analysis of the Richness Index、Shannon-Weaver Index and Simpson index indicated that there were significant decrease between treatments for bacterial communities. In contrast to the bacterial community, the archaeal diversity, measured as the Shannon-Weaver Index and Simpson index which takes into account OTU richness and eveness, was quite lower. In addition, no significant differences were found between treatments for the Shannon-Weaver Index and Simpson index in the grazing field, but significant differences with Shannon-Weaver Index and Simpson index were noted between F-60and F-120treatments.2. The impact of long-term fertilization on the abundance and community structure of ammonia oxidizersOur results showed that abundances of AOA amoA genes (ranging from1.48×109to2.00×109copies per gram of dry soil) were significantly higher than those of AOB amoA genes (1.25x107to2.62×108copies per gram of dry soil). With the increase of nitrogen fertilization, the copy number of AOB amoA genes increased and that AOA didn’t respond to fertilization. Phylogenetic tree of the bacterial amoA gene sequences was shown that all AOB sequences fell within Nitrosospira amoA clusters4,3a,3b,9,1and2. AOB communities were dominated by representatives of clusters4, constituting48.94-64.44%in each clone library. The representatives of Clusters3a was the second dominated group. Sequences falling within Clusters9and1were exclusively from soils with the N fertilization of more than treatment F30and increased with6.38-17.3%of the total clones in each clone library. Cluster2sequences were detected only in some high N fertilized soils (F90, F120), accounting for2.27%and14.63%of the total clones. All archaeal amoA sequences recovered were affiliated with the soil/sediment clade and marine sediment clade, and no significant effect was observed on variations of community structure under different fertilization treatments.Through the CCA analysis, it was found that AOB community structure and abundance were strongly correlated with NH4-N and pH. In addition, the diversity of AOB amoA sequences was significantly correlated with the NH4-N and NO3-N. However, all the soil properties investigated did not significantly correlate with community structure and abundance of AOA (p>0.05). In a word, we conclude thatFertilization was found to have a significant effect on the prokaryotic community structure, and their response to bacteria seems more obvious than archaea.The AOA amoA gene abundance higher than that of AOB of AOB either abundance or community composition changed significantly in the fertilization treatments, while AOA remained stable. |
PDF Full Text Request