||Biological dinitrogen (N2) fixation, the reduction of atmospheric N2 gas to ammonium, is a process carried out by certain nifH gene possessing microorganisms known as diazotrophs. N2 fixation is active in the oligotrophic ocean like South China Sea (SCS). In this study highly specific primers and probes were designed and used to quantitatively detect the nifH gene copies of α- and γ-proteobacteria in the northern SCS. We established the nifH PCR clone library and derived according to the phylogenetic diversity analysis two subgroups of γ-proteobacteria (γ-24774A11 and Gamma 4) and α-proteobacteria (α-Group1 and α-Group13), respectively. γ-24774A11 has been reported in the SCS. Gamma 4 has been reported in the south Pacific but was the first time recovered in the SCS. α-Group1 has been reported in the north Atlantic and north Pacific but was the first time recovered in the SCS. The need to design suitable primers and probes for detecting α-Group1 and α-Group13 became obvious as existing primers and probes are absent (α-Group1) or in low specificity (α-Group13).|
In the abundance study, primes and probes of 8 different unicellular diazotrophs were used to detect their biomass in the northern SCS. Water samples were collected generally at depths ranging 0-200 m from 3-shelf, 2-slope and 6-basin stations in 20o-22oN, 116o-123oE. The sampling were carried out during three cruises of different seasons (late spring, fall and winter). Deep sea water samples (400-1500m) were collected in basin stations. Results showed that the maximum integrated (0-200 m) nifH gene copies of γ-24774A11 was 105 nifH gene copies m-2. The nifH gene copies of γ-24774A11 ranged between 102-104 nifH gene copies L-1 was detected in all samples. In contrast, Gamma 4 was not detectable with a limit of 42 gene copies L-1. γ-24774A11 distributed in upper 200 m and its abundance were higher in late spring and fall than in winter. Its nifH gene was detectable in the deep sea water samples but the gene copies was all below the quantitative limitation (Ct value=40). The abundance of γ-24774A11 was similar to those of unicellular cyanobacteria A, UCYN-A but higher than those of UCYN-B and UCYN-C. The abundance of γ-24774A11 was significantly positively correlated with surface temperature and the abundances of UCYN-A and UCYN-C, and was significantly negatively correlated with the concentration of surface nitrate and nitrite, Chl. a, density and mixing layer depth. Suggesting the need for DOC and light to support its growth. On the other hand, high concentrations of nitrate and nitrite might inhibit the growth of γ-24774A11.
Similar to γ-proteobacteria, the maximum integrated nifH gene copies of α-proteobacteria was 105 nifH gene copies m-2. The nifH gene copies of α-proteobacteria was not detectable in all samples but many in deeper water samples. The nifH gene copies of α-Group1 was only detectable in the winter samples (103-104 nifH gene copies L-1) from as deep as 400 m. The abundance of α-Group13 was higher in fall and winter than in late spring. It was detectable at 102 nifH gene copies L-1 as deep as 1500 m. There was no significant correlation between the abundance of α-proteobacteria and environment factors.
Our results revealed that unlike α-proteobacteria, the abundance of γ-proteobacteria in the northern SCS were related with many environmental factors. α-proteobacteria was distributed in deeper water than γ-proteobacteria.
Keywords：South China Sea, α-, γ-proteobacteria, nifH gene, phylogenetic analysis, primer and probe, qPCR