Study On Seasonal Changes Of Abundance And Community Structure Of Denitrifiers In Tibetan Alpine Meadow Soil Under Long-term Fertilization

Nitrogen (N), as one of the indispensable nutrient elements of organisms, is often the limiting factor for aboveground biomass and primary producer, therefore, nitrogen cycle are the main research hotspot in ecology. A great deal of studies had demonstrated that microorganisms take an important part in the nitrogen cycle. Many researchers had focused on the denitrification drived by denitrifiers, because it was one of the significant contributors to greenhouse. The Tibetan plateau is less disturbed and is sensitive to global climate change. However, in the past few years, natural disasters (rodents) and human-induced destructions had led to pasture degradation in alpine meadow, which not only hindered the development of local stocking farm, but also affected the stability and sustainable development of Tibetan alpine meadow ecosystem. Therefore, fertilization was taken as the main strategy to recover the degradation grassland and sustain the stability of alpine meadow ecosystem. Several previous studies about the Tibetan plateau had revealed that fertilization had significant effects on the diversity and community composition of plants, whereas, how the microorganisms response to fertilization in this alpine meadow soil is poorly understood. In order to explore the response of denitrifying bacteria community to fertilization regimes as well as the relationships between plant community, soil properties and the abundance and community structure of denitrifiers in this alpine meadow soil, we investigated the community structure and abundance of denitrifiers by using clone library analysis and quantitative polymerase chain reaction (qPCR) under different fertilizer inputs (0,30,60,90,120 g m-2 year-1) on three sampling dates, respectively. The results are as follows:1. The effects of long-term fertilization on the abundance of denitrifiersThe results based on nirK gene, estimated by real-time quantitative PCR (qPCR), showed that fertilization had no significant effects on the abundance of denitrifiers in alpine meadow soil (p>0.05). The gene copy numbers of nirK gene ranged from 7.43 (±1.08) ×106 to 2.33 (±0.15) ×107 copies g-1 dry soil among different fertilization treatments. Although fertilization increased the gene copy numbers of nirK gene compared to the unfertilized control, no significant changes among different fertilization treatments were observed with the increasing fertilizer inputs.2. The effects of long-term fertilization on the community composition of denitrifiersClone library results based on nirK gene showed that long-term fertilization altered the community structure of denitrifiers significantly (r=0.5212, p=0.001).1) The total of 508 sequences were grouped into 9 Clusters, Cluster Ⅰ (59.97～94.96%) dominated among them, followed by Cluster Ⅳ and Cluster Ⅳ, and with little clones distributed in the Clusters of Ⅱ、Ⅲ、 Ⅴ、Ⅶ、Ⅸ.2) The community structure of denitrifiers differed among different fertilization treatments. In May, Cluster Ⅰ decreased with the increasing fertilizer inputs; Cluster Ⅴ was only occurred in the fertilization treatments of F30, F60, F90 and increased with the increasing fertilizer inputs. In July, Cluster Ⅳ decreased with the increasing fertilizer inputs; Cluster Ⅴ and Cluster Ⅵ were distributed only in fertilization treatments, whereas, Cluster Ⅸ was only observed in control plot. In September, Cluster Ⅰ had no significant changes among different fertilization treatments; Cluster Ⅷ was only detected in the unfertilized treatments.3. Relationships between the abundance and community structure of denitrifiers and environmental factorsPearson’s correlation analysis showed that no significant relationships were obtained between the abundance of nirK gene and environmental parameters under different fertilization treatments in our study (p>0.05). In contrast to nirK gene abundance, NMDS analysis indicated that soil moisture, organic C, and nitrogen contents had significant effects on the community structure of denitrifiers (p<0.05). In May, soil NH4+-N, total N, available N, and NO3--N were the main influencing factors for the changes of nirK community structure (p<0.05). Soil organic C, total N, available N, and NO3--N were important in governing distinct community structure of denitrifiers in July (p<0.05). However, soil moisture (r2=0.4137, p= 0.03) was the only influencing factors for the changes of denitrifiers in September.4. Seasonal changes of the abundance and community structure of denitrifiersThe copy numbers of nirK gene had significant seasonal changes in unfertilized treatments (F4=9.367, p=0.004). The copy numbers of nirK gene in July were 2.6 and 2.2 times higher than that in May and September. Pearson’s correlation analysis showed that the abundance of nirK gene was positively correlated to soil moisture (r=0.604, p=0.022) and negatively related to soil organic C (r=-0.838, p<0.001) and total N (r=-0.783, p=0.001). In addition, the community structure of nirK gene also varied significantly over the sampling time. NMDS analysis indicated that Soil moisture, organic C and nitrogen contents were important in governing seasonal changes of community structure of denitrifiers (p<0.05).5. Relationships between community structure of denitrifiers and plant community structureThe results based on the mantel’s test of community structure of denitrifiers and plant community structure showed that plant diversity had significant effect on the community composition of denitrifiers (r=0.3631, p=0.003), however, the changes of plant community composition had no significant effects on the community composition of denitrifiers, no significant corresponding relationships were observed (p>0.05). These results indicated that the community structure of denitrifiers was influenced by plant diversity, but not plant community composition in this study.In conclusion, long-term fertilization significantly altered the community composition of denitrifiers, but not abundance; furthermore, the abundance and community composition had significant seasonal changes; Plant community had no significant effects on the community structure of denitrifiers. however, plant diversity、soil moisture、organic C and nitrogen contents had significant effects on the nirK community structure (p<0.05).