Active Methanotrophs in Two Contrasting North American Peatland Ecosystems Revealed Using DNA-SIP

The active methanotroph community was investigated in two contrasting North American peatlands, a nutrient-rich sedge fen and nutrient-poor Sphagnum bog using in vitro incubations and ¹³C-DNA stable-isotope probing (SIP) to measure methane (CH₄) oxidation rates and label active microbes followed by fingerprinting and sequencing of bacterial and archaeal 16S rDNA and methane monooxygenase (pmoA and mmoX) genes. Rates of CH₄ oxidation were slightly, but significantly, faster in the bog and methanotrophs belonged to the class Alphaproteobacteria and were similar to other methanotrophs of the genera Methylocystis, Methylosinus, and Methylocapsa or Methylocella detected in, or isolated from, European bogs. The fen had a greater phylogenetic diversity of organisms that had assimilated ¹³C, including methanotrophs from both the Alpha- and Gammaproteobacteria classes and other potentially non-methanotrophic organisms that were similar to bacteria detected in a UK and Finnish fen. Based on similarities between bacteria in our sites and those in Europe, including Russia, we conclude that site physicochemical characteristics rather than biogeography controlled the phylogenetic diversity of active methanotrophs and that differences in phylogenetic diversity between the bog and fen did not relate to measured CH₄ oxidation rates. A single crenarchaeon in the bog site appeared to be assimilating ¹³C in 16S rDNA; however, its phylogenetic similarity to other CO₂-utilizing archaea probably indicates that this organism is not directly involved in CH₄ oxidation in peat.     

A Multi-Year Record of Methane Flux at the Mer Bleue Bog,Southern Canada

The Mer Bleue peatland is a large ombrotrophic bog with hummock-lawn microtopography, poor fen sections and beaver ponds at the margin. Average growing-season (May–October) fluxes of methane (CH₄) measured in 2002–2003 across the bog ranged from less than 5 mg m⁻² d⁻¹ in hummocks, to greater than 100 mg m⁻² d⁻¹ in lawns and ponds. The average position of the water table explained about half of the variation in the season average CH₄ fluxes, similar to that observed in many other peatlands in Canada and elsewhere. The flux varied most when the water table position ranged between −15 and −40 cm. To better establish the factors that influence this variability, we measured CH₄ flux at approximately weekly intervals from May to November for 5 years (2004–2008) at 12 collars representing the water table and vegetation variations typical of the peatland. Over the snow-free season, peat temperature is the dominant correlate and the difference among the collars’ seasonal average CH₄ flux is partially dependent on water table position. A third important correlate on CH₄ flux is vegetation, particularly the presence of Eriophorum vaginatum, which increases CH₄ flux, as well as differences in the potential of the peat profile to produce and consume CH₄ under anaerobic and aerobic conditions. The combination of peat temperature and water table position with vegetation cover was able to explain approximately 44% of the variation in daily CH₄ flux, based on 1097 individual measurements. There was considerable inter-annual variation in fluxes, associated with varying peat thermal and water table regimes in response to variations in weather, but also by variations in the water level in peripheral ponds, associated with beaver dam activity. Raised water level in the beaver ponds led to higher water tables and increased CH₄ emission in the peatland.     

The Impact of Nitrogen Placement and Tillage on NO, N2O, CH4 and CO2 Fluxes from a Clay Loam Soil

To evaluate the impact of N placement depth and no-till (NT) practice on the emissions of NO, N₂O, CH₄ and CO₂ from soils, we conducted two N placement experiments in a long-term tillage experiment site in northeastern Colorado in 2004. Trace gas flux measurements were made 2–3 times per week, in zero-N fertilizer plots that were cropped continuously to corn (Zea mays L.) under conventional-till (CT) and NT. Three N placement depths, replicated four times (5, 10 and 15 cm in Exp. 1 and 0, 5 and 10 cm in Exp. 2, respectively) were used. Liquid urea–ammonium nitrate (UAN, 224 kg N ha⁻¹) was injected to the desired depth in the CT- or NT-soils in each experiment. Mean flux rates of NO, N₂O, CH₄ and CO₂ ranged from 3.9 to 5.2 μg N m⁻² h⁻¹, 60.5 to 92.4 μg N m⁻² h⁻¹, −0.8 to 0.5 μg C m⁻² h⁻¹, and 42.1 to 81.7 mg C m⁻² h⁻¹ in both experiments, respectively. Deep N placement (10 and 15 cm) resulted in lower NO and N₂O emissions compared with shallow N placement (0 and 5 cm) while CH₄ and CO₂ emissions were not affected by N placement in either experiment. Compared with N placement at 5 cm, for instance, averaged N₂O emissions from N placement at 10 cm were reduced by more than 50% in both experiments. Generally, NT decreased NO emission and CH₄ oxidation but increased N₂O emissions compared with CT irrespective of N placement depths. Total net global warming potential (GWP) for N₂O, CH₄ and CO₂ was reduced by deep N placement only in Exp. 1 but was increased by NT in both experiments. The study results suggest that deep N placement (e.g., 10 cm) will be an effective option for reducing N oxide emissions and GWP from both fertilized CT- and NT-soils.     

Vegetation heterogeneity and ditches create spatial variability in methane fluxes from peatlands drained for forestry

Drainage of peatlands for forestry starts a succession of ground vegetation in which mire species are gradually replaced by forest species. Some mire plant communities vanish quickly following the water-level drawdown; some may prevail longer in the moister patches of peatland. Drainage ditches, as a new kind of surface, introduce another component of spatial variation in drained peatlands. These variations were hypothesized to affect methane (CH₄) fluxes from drained peatlands. Methane fluxes from different plant communities and unvegetated surfaces, including ditches, were measured at the drained part of Lakkasuo mire, Central Finland. The fluxes were found to be related to peatland site type, plant community, water-table position and soil temperature. At nutrient-rich fen sites fluxes between plant communities differed only a little: almost all plots acted as CH₄ sinks (−0.9 to −0.4 mg CH₄ m⁻² d⁻¹), with the exception of Eriophorum angustifolium Honck. communities, which emitted 0.9 g CH₄ m⁻² d⁻¹. At nutrient-poor bog site the differences between plant communities were clearer. The highest emissions were measured from Eriophorum vaginatum L. communities (29.7 mg CH₄ m⁻² d⁻¹), with a decreasing trend to Sphagna (10.0 mg CH₄ m⁻² d⁻¹) and forest moss communities (2.6 mg CH₄ m⁻² d⁻¹). CH₄ emissions from different kinds of ditches were highly variable, and extremely high emissions (summertime averages 182–600 mg CH₄ m⁻² d⁻¹) were measured from continuously water-covered ditches at the drained fen. Variability in the emissions was caused by differences in the origin and movement of water in the ditches, as well as differences in vegetation communities in the ditches. While drainage on average greatly decreases CH₄ emissions from peatlands, a great spatial variability in fluxes is emerged. Emissions from ditches constantly covered with water, may in some cases have a great impact on the overall CH₄ emissions from drained peatlands.     

Response of methanotrophs and methane oxidation on ammonium application in landfill soils

To test the dose effect of ammonium (NH₄ ⁺) fertilization on soil methane (CH₄) oxidation by methanotrophic communities, batch incubations were conducted at a wide scale of NH₄ ⁺ amendments: 0, 100, 250, 500, and 1,000 mg N kg_dry soil ⁻¹. Denaturing gradient gel electrophoresis and real-time quantitative PCR analysis were conducted to investigate the correlation between the CH₄ oxidation capacity and methanotrophic communities. Immediately after the addition of NH₄ ⁺, temporal inhibition of CH₄ oxidation occurred, and this might have been due to the non-specific salt effect (osmotic stress). After a lag phase, the CH₄ oxidation rates of the soils with NH₄ ⁺ fertilization were promoted to levels higher than those of the controls. More than 100 mg N kg_dry soil ⁻¹ of NH₄ ⁺ addition resulted in the reduction of type II/type I MOB ratios and an obvious evolution of type II MOB communities, while less than 100 mg N kg_dry soil ⁻¹ of NH₄ ⁺ addition induced nearly no change of methanotrophic community compositions. The NH₄ ⁺-derived stimulation after the lag phase was attributed to the improvement of N availability for type I MOB. Compared with the controls, 100 mg N kg_dry soil ⁻¹ of NH₄ ⁺ addition doubled the CH₄ oxidation peak value to more than 20 mg CH₄ kg_dry soil ⁻¹ h⁻¹. Therefore, an appropriate amount of leachate irrigation on the landfill cover layer might efficiently mitigate the CH₄ emissions.     

Spatiotemporal variations in the abundances of the prokaryotic rRNA genes, <Emphasis Type="Italic">pmoA</Emphasis>, and <Emphasis Type="Italic">mcrA</Emphasis> in the deep layers of a peat bog in Sarobetsu-genya wetland,Japan

Spatiotemporal variations in microbial gene abundances were investigated to identify potential zones of methanotroph and methanogen biomass in a peat bog in Sarobetsu-genya wetland. The abundances of the bacterial and archaeal 16S rRNA genes, pmoA, and mcrA were 10⁷–10⁹, 10⁷–10⁸, 10⁴–10⁶, and 10⁴–10⁷ copies g⁻¹ dry peat, respectively. Correlation analysis based on microbial gene abundances and environmental factors showed that the spatiotemporal distributions of the abundances of the four microbial genes in peat layers were similar. The mcrA abundance showed a significant negative correlation with the dissolved organic carbon content and a significant positive correlation with the peat temperature. The pmoA abundance was not detectable during the spring thaw when the lowest peat temperature at a depth of 50 cm was recorded. At a depth of 200 cm, the peat temperature exceeded 6°C throughout the year, and the mcrA abundance exceeded 10⁴ copies g⁻¹ dry peat. These results indicate that the seasonal microbial activity related to methane should be evaluated in not only the shallow but also the deep peat layers in order to elucidate the methane dynamics in boreal wetlands.     

Species-specific Effects of Vascular Plants on Carbon Turnover and Methane Emissions from Wetlands

Species composition affects the carbon turnover and the formation and emission of the greenhouse gas methane (CH₄) in wetlands. Here we investigate the individual effects of vascular plant species on the carbon cycling in a wetland ecosystem. We used a novel combination of laboratory methods and controlled environment facilities and studied three different vascular plant species (Eriophorum vaginatum, Carex rostrata and Juncus effusus) collected from the same wetland in southern Sweden. We found distinct differences in the functioning of these wetland sedges in terms of their effects on carbon dioxide (CO₂) and CH₄ fluxes, bubble emission of CH₄, decomposition of ¹⁴C-labelled acetate into ¹⁴CH₄ and ¹⁴CO₂, rhizospheric oxidation of CH₄ to CO₂ and stimulation of methanogenesis through root exudation of substrate (e.g., acetate). The results show that the emission of CH₄ from peat–plant monoliths was highest when the vegetation was dominated by Carex (6.76 mg CH₄ m⁻² h⁻¹) than when it was dominated by Eriophorum (2.38 mg CH₄ m⁻² h⁻¹) or Juncus (2.68 mg CH₄ m⁻² h⁻¹). Furthermore, the CH₄ emission seemed controlled primarily by the degree of rhizospheric CH₄ oxidation which was between 20 and 40% for Carex but >90% for both the other species. Our results point toward a direct and very important linkage between the plant species composition and the functioning of wetland ecosystems and indicate that changes in the species composition may alter important processes relating to controls of and interactions between greenhouse gas fluxes with significant implications for feedback mechanisms in a changing climate as a result.     

Ecophysiological mechanisms characterising fen and bog species: focus on variations in nitrogen uptake traits under different soil–water pH

Although the productivity and nitrogen (N)-use traits of mire plants differ dramatically between fens and bogs, soil N richness does not necessarily differ, whereas the soil–water pH is distinctly lower in bogs than in fens. The ecophysiological mechanisms underlying these relations are unclear. To assess the relative availability of N forms in relation to soil–water pH, we focused on the net N uptake rate per unit root weight (NNUR), glutamine synthetase activity and nitrate reductase activity, and performed reciprocal transplant experiments with the seedlings of fen (Carex lyngbyei) and bog (C. middendorffii) sedge species in intact habitat sites. The soil–water pH was clearly lower at the bog site, but the NH₄ ⁺, NO₃ ⁻ or dissolved organic-N concentrations did not differ between the fen and bog sites. The activity of both enzymes for inorganic-N assimilation did not differ among the sites and species. However, the fen species grown at bog sites showed a drastic decrease in the NNUR, suggesting a suppression of organic-N uptake. The bog species showed no NNUR difference between the sites. These results indicate that inorganic-N availability does not differ between the two habitats, but organic-N availability is lowered in a low-pH bog, particularly in the case of fen species. Therefore, the relative availability of N forms shows species-specific variations that depend on the differences in the soil–water pH of root zone, even at similar N richness, which would play a key role in plant distribution strategies in relation to the fen-bog gradient.     

CH4 production and oxidation processes in a boreal fen ecosystem after long‐term water table drawdown

Fens, which extend over vast areas in the Northern hemisphere, are sources of the greenhouse gas CH₄. Climate change scenarios predict a lowering water table (WT) in mires. To study the effect of WT drawdown on CH₄ dynamics in a fen ecosystem, we took advantage of a WT drawdown gradient near a ground water extraction plant. Methane fluxes and CH₄ production and oxidation potentials were related to microbial communities responsible for the processes in four mire locations (wet, semiwet, semidry, and dry). Principal component analyses performed on the vegetation, pH, CH₄, and WT results clearly separated the four sampling locations in the gradient. Long‐term lowering of WT was associated with decreased coverage of Sphagnum and aerenchymatic plants, decreased CH₄ field emissions and CH₄ production potential. Based on mcrA terminal restriction fragment length polymorphism the methanogen community structure correlated best with the methane production and coverage of aerenchymatic plants along the gradient. Methanosarcinaceae and Methanocellales were found at the pristine wet end of the gradient, whereas the Fen cluster characterized the dry end. The methane‐oxidizing bacterial community consisted exclusively of Methylocystis bacteria, but interestingly of five different alleles (T, S, R, M, and O) of the particulate methane monooxygenase marker gene pmoA. The M allele was dominant in the wet locations, and the occurrence of alleles O, S, and T increased with drainage. The occurrence of the R allele that characterized the upper peat layer correlated with CH₄ oxidation potential. These results advance our understanding of mire dynamics after long‐term WT drawdown and of the microbiological bases of methane emissions from mires.     

Effects of short-term drying and irrigation on CO2 and CH4 production and emission from mesocosms of a northern bog and an alpine fen

Atmospheric CO₂ and CH₄ exchange in peatlands is controlled by water table levels and soil moisture, but impacts of short periods of dryness and rainfall are poorly known. We conducted drying-rewetting experiments with mesocosms from an ombrotrophic northern bog and an alpine, minerotrophic fen. Efflux of CO₂ and CH₄ was measured using static chambers and turnover and diffusion rates were calculated from depth profiles of gas concentrations. Due to a much lower macroporosity in the fen compared to the bog peat, water table fluctuated more strongly when irrigation was stopped and resumed, about 11 cm in the fen and 5 cm in the bog peat. Small changes in air filled porosity caused CO₂ and CH₄ concentrations in the fen peat to be insensitive to changes in water table position. CO₂ emission was by a factor of 5 higher in the fen than in the bog mesocosms and changed little with water table position in both peats. This was probably caused by the importance of the uppermost, permanently unsaturated zone for auto- and heterotrophic CO₂ production, and a decoupling of air filled porosity from water table position. CH₄ emission was <0.4 mmol m^?2 day^?1 in the bog peat, and up to >12.6 mmol m^?2 day^?1 in the fen peat, where it was lowered by water table fluctuations. CH₄ production was limited to the saturated zone in the bog peat but proceeded in the capillary fringe of the fen peat. Water table drawdown partly led to inhibition of methanogenesis in the newly unsaturated zone, but CH₄ production appeared to continue after irrigation without time-lag. The identified effects of irrigation on soil moisture and respiration highlight the importance of peat physical properties for respiratory dynamics; but the atmospheric carbon exchange was fairly insensitive to the small-scale fluctuations induced.     

<Emphasis Type="Italic">In vitro</Emphasis> multiplication of heavy metals hyperaccumulator <Emphasis Type="Italic">Thlaspi caerulescens</Emphasis>

A micropropagation protocol through multiple shoot formation was developed for Thlaspi caerulescens L., one of the most important heavy metals hyperaccumulator plants. In vitro seed-derived young seedlings were used for the initiation of multiple shoots on Murashige and Skoog (MS) medium with combinations of benzylaminopurine (BA; 0.5–1.0 mg dm⁻³), naphthaleneacetic acid (NAA; 0–0.2 mg dm⁻³), gibberellic acid (GA₃; 0–1.0 mg dm⁻³) and riboflavin (0–3.0 mg dm⁻³). The maximum number of shoots was developed on medium containing 1.0 mg dm⁻³ BA and 0.2 mg dm⁻³ NAA. GA₃ (0.5 mg dm⁻³) in combination with BA significantly increased shoot length. In view of shoot numbers, shoot length and further rooting rate, the best combination was 1.0 mg dm⁻³ BA + 0.5 mg dm⁻³ GA₃ + 1.0 mg dm⁻³ riboflavin. Well-developed shoots (35–50 mm) were successfully rooted at approximately 95 % on MS medium containing 20 g dm⁻³ sucrose, 8 g dm⁻³ agar and 1.0 mg dm⁻³ indolebutyric acid. Almost all in vitro plantlets survived when transferred to pots.     

Methane flux dynamics in an Irish lowland blanket bog

Pristine peatlands are a significant source of atmospheric methane (CH₄). Large spatio–temporal variation has been observed in flux rates within and between peatlands. Variation is commonly associated with water level, vegetation structure, soil chemistry and climatic variability. We measured spatial and temporal variation in CH₄ fluxes in a blanket bog during the period 2003–2005. The surface of the bog was composed of different vegetation communities (hummocks, lawns and hollows) along a water level gradient. CH₄ fluxes were measured in each community using a chamber method. Regression modelling was used to relate the fluxes with environmental variables and to integrate fluxes over the study period. Water level was the strongest controller of spatial variation; the average flux rate was lowest in hummocks and highest in hollows, ranging from 3 to 53 mg CH₄ m⁻² day⁻¹. In vegetation communities with a permanently high water level, the amount and species composition of vegetation was also a good indicator of flux rate. We observed a clear seasonal variation in flux that was chiefly controlled by temperature. The annual average flux (6.2 g CH₄ m⁻² year⁻¹) was similar to previous estimates from blanket bogs and continental raised bogs. No interannual variation was observed.     

Microplankton respiratory activity and CO2 production rates in the Otranto Strait (Mediterranean Sea)

Respiratory activity and metabolic CO₂production of the microplankton in the Otranto Strait (Mediterranean Sea) were determined by monitoring the Electron Transport System activity. Ten stations were repeatedly investigated during two oceanographic surveys in February–March and August 1994. Respiratory activity and CO₂ production, estimated from the surface to the bottom, were higher in the euphotic layers (0-200 m) during summer (mean values: Winter = 0.024 μg C h⁻¹ dm⁻³; Summer = 0.042 μg C h⁻¹ dm⁻³); in the aphotic zone (deeper than 200 m), the rates were similar throughout different seasons (0.013 and 0.014 μg C h⁻¹ dm⁻³, respectively). A comparison with data collected by other authors from the euphotic layers of the Mediterranean Sea was made. Respiratory activities decreased from Western to Eastern Mediterranean Basins. The values of CO₂ production, integrated between 200 and 1000 m in the Otranto Strait (mean value 237.7 mg C m⁻² d⁻¹), were compared with other data collected from the Mediterranean Sea as well as from the Pacific, Atlantic and Indian Oceans. The comparison showed the Otranto Strait to be a site of organic matter oxidation. This revised version was published online in August 2006 with corrections to the Cover Date.     

Micropropagation of <Emphasis Type="Italic">Zingiber rubens</Emphasis> and assessment of genetic stability through RAPD and ISSR markers

Protocol was developed for high frequency in vitro multiplication of an endemic species, Zingiber rubens Roxb. The sprouted buds of the rhizomes were cultured on Murashige and Skoog (MS) medium supplemented with 6-benzyladenine (BA; 0.5–5.0 mg dm⁻³), indole-3-acetic acid (IAA; 0.5–2.0 mg dm⁻³), kinetin (KIN; 1.0–3.0 mg dm⁻³), naphthaleneacetic acid (NAA; 0.5–1.0 mg dm⁻³) and adenine sulphate (ADS; 80–100 mg dm⁻³). MS basal medium supplemented with 3 mg dm⁻³ BA and 0.5 mg dm⁻³ IAA was optimum for shoot elongation. The elongated shoots (1–2 cm) were transferred to multiplication medium containing 2 mg dm⁻³ BA, 1 mg dm⁻³ IAA and 100 mg dm⁻³ ADS. The multiplication rate remained unchanged in subsequent subcultures. Upon ex vitro transfer, 85 % of plants survived. Genetic stability of micropropagated clones were periodically evaluated at an interval of 6 months up to 30 months in culture using random amplified polymorphic DNA (RAPD) and inter simple sequence repeat (ISSR) analysis and genetic uniformity in all regenerants was confirmed.     

Methane Budget of a Black Forest Spruce Ecosystem Considering Soil Pattern

Study included seven soils, an adjacent spring and brook and was conducted to estimate CH₄ source and sink strengths of forest soils along a wetness gradient, i.e. their exchange with atmosphere (direct emission), and hydrosphere (indirect emission). Soils are represented by anaerobic Histosol, oxic Cambisols, Histosol with degraded peatlayers and Gleysols having intermediate redox state. They could be separated into three emission groups: CH₄ emitting (248–318 kg C ha⁻¹ a⁻¹), CH₄ uptake (−0.1 to −5 kg C ha⁻¹ a⁻¹), and soils on the edge of CH₄ uptake and release (−0.2–20 kg C ha⁻¹ a⁻¹). Although soils with CH₄ uptake were dominant (75%), the soil specific CH₄ budget identified the study field (6.53 ha) as CH₄ source (40.9 kg C ha⁻¹ a⁻¹). Not only CH₄ emissions, but also dissolved CH₄ in soil solution varied regularly with soil type. Individual soil solutions contained 0.008–151 μmol CH₄ l⁻¹. CH₄ vanished to negligible loads, when dissolved CH₄ passed an oxidative downslope soil zone, but promoted CH₄ uptake was measured at this soil. In turn, CH₄ was discharged to the atmosphere, when the soil solution left the pedosphere across an anaerobic soil zone. These measured indirect emissions were low (34 g C a⁻¹), but the values of individual soil solution indicate possible higher discharges (3.9 kg a⁻¹) at a different soil pattern. The results suggest that CH₄ uptake rates of temperate forests are overestimated.     

Methane uptake rates in Japanese forest soils depend on the oxidation ability of topsoil, with a new estimate for global methane uptake in temperate forest

To clarify the reason for the higher CH₄ uptake rate in Japanese forest soils, twenty-seven sites were established for CH₄ flux measurement. The first order rate constant for CH₄ uptake was also determined using soil core incubation at 14 sites. The CH₄ uptake rate had a seasonal fluctuation, high in summer and low in winter, and the rate correlated with soil temperature at 17 sites. The annual CH₄ uptake rates ranged from 2.7 to 24.8 kg CH₄ ha⁻¹ y⁻¹ (the average of these rates was 9.7 or 10.9 kg CH₄ ha⁻¹ y⁻¹, depending on method of calculation), which is somewhat higher than the uptake rates reported in previous literature. The averaged CH₄ uptake rate correlated closely with the CH₄ oxidation rate of the topsoil (0–5 cm) in the study sites. The CH₄ oxidation constant of the topsoil was explained by a multiple regression model using total pore volume of the soil, nitrate content, and C/N ratio (p < 0.05, R ² = 0.684). This result and comparison with literature data suggest that the high CH₄ uptake rate in Japanese forest soils depends on the high porosity probably due to volcanic ash parent materials. According to our review of the literature, the CH₄ uptake rate in temperate forests in Europe is significantly different from that in Asia and North America. A new global CH₄ uptake rate in temperate forests was estimated to be 5.4 Tg y⁻¹ (1 SE is 1.1 Tg y⁻¹) on a continental basis.     

Quantification of methane emissions from Chinese rice fields (Zhejiang Province) as influenced by fertilizer treatment

Methane emissions from rice paddies were quantified by using an automatic field system stationed in Zhejiang Province, one of the centres for rice cultivation in China. The data set showed pronouned interannual variations over 5 consecutive vegetation periods; by computing average values of all experimental plots the annual emissions were 177 g CH₄ m⁻² yr⁻¹ in 1987, 50 g CH₄ m⁻² yr⁻¹ in 1988, and 187 g CH₄ m⁻² yr⁻¹ in 1989. The field preparations encompassed 4 different treatments: (1) no fertilizers, (2) mineral fertilizer (KCl, K₂SO₄), (3) organic manure (rape seeed cake, animal manure), (4) mineral fertilizer plus organic manure. The methane emission rates of the different fertilizer treatments did not show significant differences. The mean emission rates, calculated over the entire observation period of 5 seasons, were 30.4 mg CH₄ m⁻² h⁻¹ (non-fertilized plot) and 28.3 mg CH₄ m⁻² h⁻¹ (mineral fertilizers). These values indicate a high level of methane production even without additional input of organic material into the rice-soils. In the other plots, the organic fertilizers were added once per vegetation period at app. 1 t fresh weight per ha, a relatively low application rate by agronomical standards. The mean emission rates were 35.1 mg CH₄ m⁻² h⁻¹ when manure was applied as sole fertilizer and 27.5 mg CH₄ m⁻² h⁻¹ when applied jointly with potassium fertilizers. Based on the results of this study we estimate a range of 18–28 Tg CH₄ yr⁻¹ as the total methane emission from Chinese rice fields. However, more field data from representative sites in China are needed to reduce the uncertainties in this estimate.     

An efficient in vitro propagation of Aristolochia indica

A rapid and efficient in vitro plant regeneration method was developed for Aristolochia indica. Multiple shoot formation was induced from shoot tip and nodal explants on Murashige and Skoog (MS) medium with 1 – 6 mg dm⁻³ 2-isopentenyl-adenine (2-iP) or 1 – 4 mg dm⁻³ 6-benzyladenine (BA). Maximum number of shoots were induced with 5 mg dm⁻³ 2-iP alone (about 12 – 14 shoots). Shoot differentiation occurred directly from the leaf bases as well as from the internodes when cultured on 1 – 4 mg dm⁻³ BA and 0.8 – 2 mg dm⁻³ α-naphthaleneacetic acid (NAA) containing medium. Regeneration from the callus occurred when the calli initiated on MS medium containing 0.6 – 4 mg dm⁻³ NAA in combination with 0.8 – 3 mg dm⁻³ BA were transferred to 1 – 6 mg dm⁻³ BA alone containing medium. Elongated shoots were separated and rooted in MS medium containing 1 mg dm⁻³ indole-3-butyric acid. These were then transferred to soil after gradual acclimatization.     

Methanotrophic community structure and activity under warming and grazing of alpine meadow on the Tibetan Plateau

Knowledge about methanotrophs and their activities is important to understand the microbial mediation of the greenhouse gas CH₄ under climate change and human activities in terrestrial ecosystems. The effects of simulated warming and sheep grazing on methanotrophic abundance, community composition, and activity were studied in an alpine meadow soil on the Tibetan Plateau. There was high abundance of methanotrophs (1.2–3.4 × 10⁸ pmoA gene copies per gram of dry weight soil) assessed by real-time PCR, and warming significantly increased the abundance regardless of grazing. A total of 64 methanotrophic operational taxonomic units (OTUs) were obtained from 1,439 clone sequences, of these OTUs; 63 OTUs (98.4%) belonged to type I methanotrophs, and only one OTU was Methylocystis of type II methanotrophs. The methanotroph community composition and diversity were not apparently affected by the treatments. Warming and grazing significantly enhanced the potential CH₄ oxidation activity. There were significantly negative correlations between methanotrophic abundance and soil moisture and between methanotrophic abundance and NH₄–N content. The study suggests that type I methanotrophs, as the dominance, may play a key role in CH₄ oxidation, and the alpine meadow has great potential to consume more CH₄ under future warmer and grazing conditions on the Tibetan Plateau.