Effects Of Different Long-term Fertilizations On Community Properties And Functions Of Methanotrophs Of Black Soil In Northeastern China

It is uncertain that the microbial mechanism of different long-term fertilizations regimes change methane oxidation of Chinese upland arable soil. In the present study, we used the long-term fertilization experiments in Heihe and Harbin to investigate whether different fertilizations regimes changed methane oxidizing rate of black soil by altering soil properties and thereafter the community properties of methanotrophs. Four treatments were selected to conduct this research project, i.e. 1) neither organic nor mineral fertilizer (CK); 2) organic manure (M); 3) mineral fertilizer (Heihe: NP; Harbin: NPK) and 4) both mineral fertilizers and organic manures (Heihe: MNP; Harbin: MNPK), which represented low input, organic, mineral and conventional managements. Soil methane oxidizing rate was measured with gas chromatography (GC). Community structures and abundances of methanotrophs were monitored with polymerase chain reaction/denaturing gradient gel electrophoresis (PCR-DGGE) and quantitative real time PCR (QPCR), respectively. The following results were obtained in this study:1. Compared with CK, NP showed no effect on methane oxidation rates of Heihe soils, whereas NPK reduced methane oxidation rates by 79.1% in Harbin soils. M did not change the methane oxidation rates both Heihe and Harbin soils. Combined use of mineral fertilizer and compost (MNP or MNPK) reduced soil methane oxidation rates obviously, with a reduction by 61.2% in Heihe soils and by 52.9% in Harbin soils, respectively.2. Specific activity of methanotrophs was calculated via dividing methane oxidation rates by abundances of pmoA gene. The results indicated that M addition (M or MNP) significantly decreased specific activity of methanotrophs compared with no M addition (CK or NP) in Heihe soils. Compared with CK, M significantly reduced specific activity of methanotrophs, whereas NPK and MNPK had no effect in Harbin soils.3. DGGE profiling showed that M addition (M or MNP) significantly increased the bands numbers but NP showed no effect compared with CK in Heihe soils. In Harbin soils, M addition (M or MNPK) significantly increased bands numbers while NPK significantly reduced them. Digitations of DGGE profiles showed that M addition (M or MNP) significantly increased Shannon index of methanotrophs, whereas NP had no effect in Heihe soils. In Harbin soils, MNPK significantly increased Shannon index of methanotrophs, while NPK significantly decreased them relative to CK. M did not changed the Shannon index of methanotrophs compared with CK in Harbin soils..4. QPCR results showed that M addition (M or MNP) significantly increased pmoA gene abundances of methanotrophs, whereas NP had no effect in Heihe soils. In Harbin soils, M significantly increased pmoA gene abundances, NPK and MNPK did not changed pmoA gene abundances of methanotrophs compared with CK. 5. Methane oxidation rates in Heihe dark brown soils were significantly correlated with specific activities of methanotrophs, with a correlation coefficient of 0.685 (p = 0.014). Methane oxidation rates in Harbin soils were significantly correlated with pmoA gene abundances and community structures of methanotrophs, with correlation coefficients of 0.648 (p = 0.032) and 0.304 (p = 0.020), respectively. These results indicated that methane oxidation rates were closely coupled with community properties of methanotrophs in the soils investigated.6. Redundancy analysis results showed that community structures of methanotrophs were significantly correlated with pH, TN and OM of Heihe soils, with correlation coefficients of 0.825 (p = 0.002), 0.620 (p = 0.002) and 0.500 (p = 0.006), respectively. Community structures of methanotrophs were significantly correlated with soil moisture and pH of Harbin, with correlation coefficients of 0.359 (p = 0.006) and 0.303 (p = 0.010), respectively. These results indicated a clear influence of soil properties on the community structure methanotrophs.7. Phylogenetic analysis of pmoA gene in Heihe soils showed that Cluster 2 was replaced with Methylocaldum which belong to type I methanotrophs under organic manure fertilizer. However, organic manure had no effect on type II methanotrophs. In conclusion, the results obtained in the present study showed that different fertilizations regimes could change methane oxidizing rates of black soils by altering soil properties and thereafter the community properties of methanotrophs. In particular, the changes in methanotrophs community in Heihe soils by organic manure was via the replacement of Cluster 2 with Methylocaldum which belong to type I methanotrophs rather than a change in type II methanotrophs. It should be noted that the methane oxidation rates were decoupled with methanotrophs abundance in Heihe soils, which suggests only parts of the methanotrophs in M treated soils were active in Heihe. Future researches on how to activate these"inactive"methanotrophs will considerably increase the potential of methane oxidation by organic manure application.