| As the most potent greenhouse gas contributing to global warming, nitrous oxide (N2O) contributes to the destruction of the stratospheric ozone layer and has become the most dominant ozone-depleting substance. However, as an important source of greenhouse gas N2O emission, denitrification has been receiving more and more attention. As the source and sink of N2O gas, the Tibetan Plateau swamp meadow has an important influence on the global ecosystem. Furthermore, as the human extremely important environment resources and the habitats of many rare wild animals, the Tibetan Plateau swamp meadow has great and unique influence in the protection of livestock production and regulation on the Yellow River runoff. However, in recent years, the Tibetan Plateau swamp meadow has suffered serious degeneration due to anthropogenic activities and nature itself. To better understand how grazing impact on the abundance and community structure of denitrifiers(nirK and nirS) in the Tibetan Plateau swamp meadow, this study investigated the long-term grazing experiment in the Tibetan Plateau swamp meadow using real time PCR and clone-library sequencing. The objectives were to assess the response of denitrifiers to different grazing intensity in the Tibetan Plateau swamp meadow under different months, and to reveal the environmental factors affecting the soil denitrifiers. We hope that the study could contribute to the restoration of degraded swamp meadow and the maintenance of biodiversity in the Tibetan Plateau. Three treatments were tested, prohibition-grazing (F1), seasonal-grazing (F2) and annual-grazing (F3). Results are as follows:1. The impact of long-term grazing on the abundance of denitrifiers under different monthsThe results showed that the abundance of nirS genes (ranging from 3.86×107 to 9.67×107 per gram of dry soil) were significantly higher than those of nirK genes (ranging from 2.08×105 to 1.32x106 per gram of dry soil). With the increase of grazing intensity, the copy number of nirK genes increased and the copy number of nirS genes decreased. In May field sample, available P and pH had significant effects on the abundance of nirK genes in the Tibetan Plateau swamp meadow (P<0.05), Total C, total P and NO3--N had significant effects on the abundance of nirS genes in the Tibetan Plateau swamp meadow (P<0.05). However, in July field sample, total C and total N had significant effects on the abundance of nirS genes in the Tibetan Plateau swamp meadow (P<0.05), all of environmental parameters had no significant effects on the abundance of nirK genes (P>0.05). In addition, the copy numbers of nirK gene had significant seasonal changes in grazing treatments. The copy numbers of nirK gene in May (ranging from 1.01×106 to 1.32×106 copies g-1 dry soil) were higher than that in July (ranging from 2.08×05 to 5.82×105 copies g-1 dry soil), while no significant differences were found between May (ranging from 5.57×107 to 8.32×107 copies g-1 dry soil) and July (ranging from 3.86×107 to 9.67×107 copies g-1 dry soil) for the abundance of nirS genes.2. The impact of long-term grazing on the community structure of denitrifiers under different monthsClone library results based on nirK and nirS genes shown that long-term grazing altered the community structure of nirK and nirS-type denitrifiers. From a diversity point of view, the community diversity of nirS-type denitrifers was significantly higher than those of mrK-type denitrifers. With the increase of grazing intensity, the community diversity of nirS-type denitrifers increased. In contrast to the nirS-type denitrifers community, nirK -type denitrifers diversity had no significant differences between treatments in May field sample, while nirK-type denitrifers diversity of annual-grazing (F3) treatment significantly higher than prohibition-grazing (F1) and seasonal-grazing (F2) treatments (P<0.05) in July field sample. In addition, our result showed that the community diversity of nirK and nirS-type denitrifers gene in May were higher than that in July.Phylogenetic analysises indicated that most of sequences showed low similarity to the cultured bacteria, but showed high similarity to the uncultured bacteria from soils. Our results showed that different long-term grazing intensities affect the community structure of nirK and nirS-type denitrifiers being month-specific. The nirK genes were grouped into 5 Clusters, Cluster I (74.76%～97.31%) dominated among them. In May field sample, Cluster I which is the dominant groups of nirK-type denitrifiers increased with the increasing grazing intensities, the proportion of the dominant groups in annual-grazing (F3) treatment significantly higher than prohibition-grazing (F1) and seasonal-grazing (F2) treatments (P<0.05), while the proportion of the dominant group in seasonal-grazing (F2) treatment significantly higher than prohibition-grazing (F1) and annual-grazing (F3) treatments in July (P<0.05). Furthermore, the nirS genes were grouped into 7 Clusters, in May field sample, the dominant groups of nirS-type denitrifiers is Cluster V (46.65%) in prohibition-grazing (F1) treatment, while that is Cluster I (28.73%～34.62%) in seasonal-grazing (F2) and annual-grazing (F3) treatments. In July field sample, the dominant groups of nirS-type denitrifiers is Cluster V (39.87%～61.48%) in prohibition-grazing (F1) and seasonal-grazing (F2) treatments, while that is Cluster I (35.23%) in annual-grazing (F3) treatment. In addition, our result showed that the proportion of Cluster V in prohibition-grazing (F1) treatment significantly higher than seasonal-grazing (F2) and annual-grazing (F3) treatments in July (P<0.05).In conclusion, the results indicate that different long-term grazing intensities affect both community structure and abundance of nirK and nirS-type denitrifers. Furthermore, the abundance and community structure of nirK and nirS-type denitrifers had significant seasonal changes. However, the community diversity and abundance of nirS-typs denitrifers was significantly higher than those of nirK-type denitrifers. |
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