Bacteriophage Diversity, Abundence And Its Role In The Production Of Disolved Organic Carbon In Napahai Plateau Wetland

Wetland is one of the most active places of energy flow and material circulation on the earth, and is the biggest carbon reserve pool of terrestrial organic carbon. The carbon reservoir of wetlands is up to 30% of terrestrial soil carbon content. Due to higher productivity and lower decomposition rate, plateau wetlands become the richest reserve pools of organic carbon, which play important roles in regulating the balance of the global carbon cycle. Napahai plateau wetland is located in the northwest of Yunnan province of China. She is also a typical quaternary water erosion and moraine lake, and is a unique low-latitude, low-temperature, high-altitude and seasonal plateau marsh wetland. Napahai wetland contributed as a water source of upper tributaries of the Yangtze River and lies beside the centre of the Three Parallel Rivers. Napahai wetland has been considered as an important niche to explore various geological and biology research due to its rich geological and biological resources. There are many researches on diversity of plant community, patterns and mechanism of wetland degradation, evolution patterns and driving mechanism of lakeside zone, and wintering Black Stork habitat selection until now. Current researches mainly focus on the revealing of the background value of soil carbon, space distribution, response of soil organic matters to water contents, impacts of hydrological periods on soil carbon, which are only considered from physical and chemical factors. Obviously, until now little is known about the roles of biological factors on carbon cycle in Napahai wetland.As the simplest life style, bacteriophages distributed every corner of our planet and played important roles in regulating bacterial community structure and horizontal gene transfer. More researches have demonstrated that they also played essential roles in global biogeochemical cycles, especially in organic carbon cycle. The roles of bacteriophages in organic carbon cycle, and even the distribution, abundance of them and their host bacteria in Napahai wetland have not been received any attention. Therefore, it would be interesting to study the ecological distribution, abundance of bacteriophages and their host bacteria in this area. In addition, exploration of functional roles of bacteriophage in organic carbon cycle in Napahai wetland is also a challenging work.At first, the ecological distribution and abundance of bacterioplankton and virioplankton that most of them are baceriophages in different seasons was investigated. The average values of bacterioplankton abundance were 3.52×105/mL and 2.02×106/mL in dry and rainy season by using Epifluorescence Microscopy (EFM) technique, while the average values of virioplankton abundance were 3.63×106 /mL and 3.71×107/mL, respectively. The abundance of virioplankton was 10 folds higher than that of planktonic bacteria in all seasons, and both abundance of virioplankton and bacterioplankton in rainy season are significantly higher than that of in dry season. The correlation of the abundance between virioplankton and bacterioplankton and environmental factors were analyzed. Virioplankton abundance has a significant positive correlation (P<0.01) with bacterioplankton in dry season, but has only a very weak negative correlation with chlorophyll-a (Chl-a) (P<0.05), which indicating that bacteriophages instead of cyanophage or phytoplankton are the dominant species of phytoplankton in dry season. Virioplankton abundance has a significant positive correlation with bacterioplankton abundance (P<0.01) and Chl-a (P<0.05), suggesting a significant impact by Chl-a content in rainy season. It was suggested that phytoplankton viruses and cyanophage have become the dominant species of virioplankton, due to the main host cells changed. The research showed that virioplankton abundance has a significant negative correlation with temperature in dry season, while negative but not significantly correlation with temperature in rainy season. Virioplankton abundance was negatively correlated with pH in rainy season, due to the ionic composition significant change during rainy season. From the average value of all samples, the virioplankton abundance showed no significant difference (P>0.05) between rainy and dry season, but significant difference in several individual sampling spots were observed, which indicated that the distribution of bacteriophages in Napahai wetland was not a continuous pattern. Viruses-to-Bacterium Ratio (VBR) in Napahai wetland was between 4.41 and 40.11, and the average value is 14.88. Virioplankton and bacterioplankton abundance showed a significant positive correlation with VBR and this value was not varied in different seasons. These result indicated that ratio of distribution of bacteriophages and their host has become stable and keep balance under a long evolutionary history, suggesting this ecosystem has been highly developed.Secondly, the roles of virioplankton which mainly occupied by bacteriophages, on the dissolve organic carbon cycle in Napahai wetland were studied in this work. It showed that bacterial carbon production is 8.01 μgC/L·h and 10.3 μg C/L·h in dry and rainy season. The fraction of bacterial mortality caused by viral lysis (FMVL) is 54.9% and 27% in dry and rainy season, respectively. All data were analysis by Binder model, and the results showed that the potential Dissolved Organic Carbon (DOC) input from viral lysis to the DOC pool occupied the dominant position in Napahai wetland and varied significantly in dry and rainy seasons with value of 84.37% and 72.9%. It was the first report on the relationship between phages, bacteria and DOC in Napahai wetland.In order to investigate the distribution of different kinds of baceriophages in Napahai wetland, the distribution of T4-type bacteriophage communities in Napahai wetland was detected by using the highly conserved biomarker gene of g23, which encodes the major capsid protein of T4-type bacteriophage and can be amplified with primers of MZIA1 and MZIA6. Total of 36 and 18 clones were obtained from samples of YW and YN, respectively. The UniFrac analysis of g23 assemblages indicated that T4-type phage community compositions were significantly varied between samples of YW and YN, which demonstrated that diversity of bacteriophage is very popular in Napahai wetland area. Phylogenetic analysis showed that two unique groups of NPHI and II are distributed in Napahai wetland, while the rest of them are similar to other isolates all around the world. This result also can be confirmed by using circular phylogenetic analysis. According to the results, the sequences of g23 come from different sampling spots in wetland varied significantly with those from marine water samples, and the samples from water and mud are fallen in individual clusters respectively. Only a few samples can be distributed into same cluster with those samples from Donghu and Beikal lakes. All samples can be distributed into a unique cluster, except one sequence is fallen into same cluster with Japan paddy fields sample. Therefore, the phylogenetic analysis based on g23 of T4 phage has demonstrated that the bacteriophage diversity and uniqueness were significant in Napahai wetland due to its special environment.At the end, the ecological distributions of bacteriophages and their host bacteia were investigated in this area. Total of 380 cold-adapted cultivatable bacterial strains were isolated and identified, and 19 cold-active bacteriophages were obtained through typical two-layer technique. All 19 cold-active bactriohages have a tail structure and are classified as members of family of Myoviridae, Podoviridae and Siphoviridae. A new phage with Zoogloea sp. strain as host was identified, which has not been reported previously world wide. Three bacteriophages that isolated from Pseudomonas, Flavobacterium, Aeromonas sobria bacteria respectively were identified as group of Myoviridae phage based on their biological characteristics. Isolation, identification and characterization of three bacteriophags were performed. In this work, due to the limitation of the techniques in cultivation and isolation, no T4-type cultivatable phage has been isolated from all samples. According to morphological analysis, it was showed that Napahai wetland has been inhabited with very rich cold-active bacteriophages and cold-adapted bacteria, and should be an ideal research base to perform scientific research and a treasure for exploiting biological resource.This is the first report on the viroplankton abundance, ecological distribution and cold-active bacteriophages isolation in Napahai which is a unique low-latitude, low-temperature, high-altitude and seasonal plateau marsh wetland. The relationship between the DOC and bacteriophages also has been studied, while a batch of culturable cold-active bacteriophages were isolated and their biological characteristics were investigated thoroughly. It was an important complementary ecological research and a full report about ecological function of bacteriophages in this area until now. Two novel T4 group bacteriophages were identified, and several new methods and techniques for studying phages in Napahai wetland were established. The results also demonstrated that the lysis through bacteriophage played a significant role in organic carbon recycle and varied at different seasons in this area, which provide a new insight on further microbial ecology research. A batch of culturable cold-active bacteriophages and their host bacterial strains have been isolated, which provided more opportunities for further qualitative or quantitative analysis of ecological function of phages in this wetland. New isolates were a valuable bioresource for further studying on cold-adaptation mechanism, co-evolution and interaction between phage and its host. All experiments are well designed and performed systematicly, and much valuable fundamental data and experience for further microbial ecology and biogeochemistry research are accumulated. The microbial resource collected in this area can be further studied and exploited in future.