Rice Field Soil Research Articles

Molecular analysis of the major capsid genes (g23) of T4-type bacteriophages in an upland black soil in Northeast China

Bacteriophages (phages) are the most abundant biological entities on the planet and are important as the greatest genomic reservoirs in both marine and terrestrial environments. In this study, we analysed T4-type phage communities in an upland black soil by monitoring g23 clones in DNA extracted from seasonal soil samples with no fertilizer, chemical fertilizers, chemical fertilizers plus manure, and natural restoration treatments. PCR products with degenerate primers MZIA1bis and MZIA6 were subjected to denaturing gradient gel electrophoresis. In total, 46 clones with different g23 sequences were obtained. Phylogenetic analyses indicated that T4-type phage communities in the upland black soil were distinctly different from those in marine environments and in an Antarctic lake, which strongly suggested that T4-type phage communities in soil differed from those in aquatic environments. Among 46 clones, 18 clones formed clusters with the clones from rice field soils, 14 clones formed three new clusters, and 13 clones were left as ungrouped, which indicated that T4-type phage communities in the upland black soil were relatively similar to those in rice field soils but that specific communities also inhabit in the upland black soil exclusively.

Transcriptional response of methanogen mcrA genes to oxygen exposure of rice field soil

Methane production in paddy soil is substantially suppressed after even a brief exposure of soil to oxygen. We hypothesized that the strong response of methanogen activity is reflected in the transcription of functional genes rather than in the composition of the community of methanogens. Therefore, we determined the community composition and the transcriptional response of methanogens in a rice field soil by targeting the mcrA gene (encoding the α subunit of methyl-coenzyme M reductase). Transcription of mcrA genes measured by quantitative PCR decreased by an order of magnitude after brief exposure to O2 . Terminal restriction fragment length polymorphism of mcrA genes and gene transcripts showed that although the community structure of methanogens did not change, the composition of transcripts dramatically responded to O2 exposure. In the beginning, transcripts of Methanocellales were the relatively most abundant, indicating resistance of these hydrogenotrophic methanogens against O2 stress. Later on, mcrA transcripts of acetoclastic methanogens became relatively more abundant coinciding with the turnover of acetate. The transcription of Methanosarcinaceae was relatively greater when acetate accumulated while Methanosaetaceae became more active when acetate concentrations decreased. In the presence of methyl fluoride, a specific inhibitor of acetoclastic methanogenesis, mcrA transcription by Methanosaetaceae was greatly suppressed while that of Methanosarcinaceae was less affected. Our study showed that in contrast to constant community structure as revealed by DNA-based fingerprinting the transcription of functional mcrA genes strongly responded to O2 stress and the presence of inhibitor CH3 F. The response patterns reflected the genomic and physiological traits of individual methanogens.

Stable carbon isotope discrimination in rice field soil during acetate turnover by syntrophic acetate oxidation or acetoclastic methanogenesis

Rice fields are an important source for the greenhouse gas methane. In Italian rice field soil CH 4 is produced either by hydrogenotrophic and acetoclastic methanogenesis, or by hydrogenotrophic methanogenesis and syntrophic acetate oxidation when temperatures are below and above about 40–45 °C, respectively. In order to see whether these acetate consumption pathways differently discriminate the stable carbon isotopes of acetate, we measured the δ 13C of total acetate and acetate-methyl as well as the δ 13C of CO 2 and CH 4 in rice field soil that had been pre-incubated at 45 °C and then shifted to different temperatures between 25 and 50 °C. Acetate transiently accumulated to about 6 mM, which is about one-third of the amount of CH 4 produced, irrespective of the incubation temperature and the CH 4 production pathway involved. However, the patterns of δ 13C of the CH 4 and CO 2 produced were different at low (25, 30, 35 °C) versus high (40, 45, 50 °C) temperatures. These patterns were consistent with CH 4 being exclusively formed by hydrogenotrophic methanogenesis at high temperatures, and by a combination of acetoclastic and hydrogenotrophic methanogenesis at low temperatures. The patterns of δ 13C of total acetate and acetate-methyl were also different at high versus low temperatures, indicating the involvement of different pathways of production and consumption of acetate at the two temperature regimes. Isotope fractionation during consumption of the methyl group of acetate was more pronounced at low ( α = 1.010–1.025) than at high ( α = 1.0–1.01) temperatures indicating that acetoclastic methanogenesis exhibits a stronger isotope effect than syntrophic acetate oxidation. Small amounts of propionate also transiently accumulated and were analyzed for δ 13C. The δ 13C values slightly increased (by about 10‰) during production and consumption of propionate, but were not affected by incubation temperature. Collectively, our results showed distinct isotope discrimination for different paths of acetate (and propionate) production and consumption, albeit differences were only small, and discrimination between methanogenic and syntrophic acetate consumption in nature may be difficult to detect.

Regulation of microbial methane production and oxidation by intermittent drainage in rice field soil

Soil drainage is one of the most promising approaches to mitigate methane (CH(4) ) emission from paddy fields. The microbial mechanism for the drainage effect on CH(4) emission, however, remains poorly understood. In the present study, we determined the effect of short (four drainages of 5-6 days each) and long drainage cycles (two drainages of 10-11 days each) on CH(4) emission and analyzed the response of the structure and abundance of methanogens and methanotrophs in a Chinese rice field soil at the DNA level. Rice biomass production was similar between drainage and the practice of continuous flooding. The rate of CH(4) emission, however, was reduced by 59% and 85% for the long and short drainage cycles, respectively. Quantitative (real-time) PCR analysis revealed that the total abundance of archaeal populations decreased by 40% after multiple drainages, indicating the inhibitory effects on methanogen growth. The structure of the methanogen community as determined by terminal restriction fragment length polymorphism analysis, however, remained unaffected by drainages, although it varied among rhizosphere, bulk and surface soils. Quantitative PCR analysis of the methanotrophic functional pmoA genes revealed that the total abundance of methanotrophs in rhizosphere soil increased two to three times after soil drainages, indicating a stimulation of methanotroph growth. The CH(4) oxidation potential in the rhizosphere soil also increased significantly. Furthermore, drainages caused a shift of the methanotrophic community, with a significantly increase of type II methanotrophic bacteria in the rhizosphere and surface soil. Thus, both inhibition of methanogens and stimulation of methanotrophs were partly responsible for the reduction of CH(4) emissions. The methanotroph community, however, appeared to react more sensitively to soil drainage compared with the methanogen community.

Strain-specific incorporation of methanotrophic biomass into eukaryotic grazers in a rice field soil revealed by PLFA-SIP

In wetland ecosystems, methane is actively utilized by methanotrophs. The immobilized methane carbon is then passed on to other organisms such as grazers. Here, we traced the incorporation of methanotrophic biomass into eukaryotes in a rice field soil using phospholipid fatty acid stable-isotope probing (PLFA-SIP). Addition of (13)C-labeled cells of five methanotrophs to soil (5 × 10(7) cells g(-1) soil) did not affect the CO(2) release rate, but significantly increased the carbon isotopic ratio within 24 h. In 48 h, 2-7% of the added bacterial biomass carbon was detected as (13)CO(2) . The soil with Methylobacter luteus released the highest amount of (13)CO(2) , comparable to that with Escherichia coli. The amount of polyunsaturated PLFAs (C18:3ω6c and C20:4ω6c) was not affected by the addition of bacterial cells to soil, but their carbon isotopic ratio increased significantly within 24-48 h. The extent of (13)C-enrichment in PLFAs differed between the added methanotrophs, with the highest labeling upon addition of M. luteus. The relative abundance of (13) C-labeled C18:3ω6c to C20:4ω6C also differed between the strains. The results indicated that the eukaryotes in soil, probably protozoa, preferentially graze on specific methanotrophs and immediately incorporate their biomass.

Methyl halide and chloroform emissions from a subsiding Sacramento–San Joaquin Delta island converted to rice fields

Net fluxes of methyl halides and chloroform were measured from recently converted rice fields on Twitchell Island, California, using field- and laboratory-based incubations. A stable isotope tracer method was used to demonstrate that the net emissions of CH3Cl and CH3Br during the growing season were predominantly the result of large gross production rates, with gross consumption rates being relatively minor. In agreement with prior studies, the production rates for methyl halides differed significantly at different rice plant growth phases. The Twitchell Island rice field, however, had production rates of CH3Cl during the growth phases that were higher than rates reported at commercial rice fields, presumably because of higher soil chloride concentrations. Laboratory soil incubations showed that the non-flooded rice field soils acted as a small net sink for CH3Cl and as small net sources for CH3Br, CH3I and CHCl3. Despite the higher CH3Cl emissions during the growing season, the overall emissions of halomethanes from the conversion of all potential islands in the San Joaquin Delta to rice paddies is predicted to be an insignificant source of halomethanes.

Thermoanaerobacteriaceae oxidize acetate in methanogenic rice field soil at 50°C

Rice field soils contain a thermophilic microbial community. Incubation of Italian rice field soil at 50°C resulted in transient accumulation of acetate, but the microorganisms responsible for methane production from acetate are unknown. Without addition of exogenous acetate, the δ(13)C of CH(4) and CO(2) indicated that CH(4) was exclusively produced by hydrogenotrophic methanogenesis. When exogenous acetate was added, acetoclastic methanogenesis apparently also operated. Nevertheless, addition of [2-(13)C]acetate (99% (13)C) resulted in the production not only of (13)C-labelled CH(4) but also of CO(2), which contained up to 27% (13)C, demonstrating that the methyl group of acetate was also oxidized. Part of the (13)C-labelled acetate was also converted to propionate which contained up to 14% (13)C. The microorganisms capable of assimilating acetate at 50°C were targeted by stable isotope probing (SIP) of ribosomal RNA and rRNA genes using [U-(13)C] acetate. Using quantitative PCR, (13)C-labelled bacterial ribosomal RNA and DNA was detected after 21 and 32 days of incubation with [U-(13)C]acetate respectively. In the heavy fractions of the (13)C treatment, terminal restriction fragments (T-RFs) of 140, 120 and 171 bp length predominated. Cloning and sequencing of 16S rRNA showed that these T-RFs were affiliated with the bacterial genera Thermacetogenium and Symbiobacterium and with members of the Thermoanaerobacteriaceae. Similar experiments targeting archaeal RNA and DNA showed that Methanocellales were the dominant methanogens being consistent with the operation of syntrophic bacterial acetate oxidation coupled to hydrogenotrophic methanogenesis. After 17 days, however, Methanosarcinacea increasingly contributed to the synthesis of rRNA from [U-(13)C]acetate indicating that acetoclastic methanogens were also active in methanogenic Italian rice field soil under thermal conditions.

Measurement and analysis of precompression stress of soil in rice field.

Measurement and analysis of precompression stress of soil in rice field.

Methanobacterium kanagiense sp. nov., a hydrogenotrophic methanogen, isolated from rice-field soil

A pure culture of an obligately anaerobic, hydrogenotrophic, methanogenic archaeon, designated strain 169(T), which grows with hydrogen and carbon dioxide as the sole energy and carbon sources, was isolated from an anaerobic propionate-oxidizing enrichment culture originally obtained as an inoculant from rice-field soil in Japan. Cells of strain 169(T) were non-motile, Gram-reaction-variable and rod-shaped or slightly curved rods with rounded ends (1.6-5.0 × 0.35-0.5 µm). Strain 169(T) had fimbriae at both ends of the cell (up to ~10 per cell) but did not possess flagella. Ultrathin sections showed a single-layered, electron-dense cell wall about 6 nm thick, which is typical of Gram-positive bacteria. Growth was observed at 15 °C-45 °C (optimum 40 °C), at pH 6.5-9.6 (optimum pH 7.5-8.5) and in 0-70 g NaCl l(-1) (0-1.2 M) (optimum 5 g NaCl l(-1); 0.086 M). Strain 169(T) utilized only hydrogen and carbon dioxide as energy and carbon sources. The DNA G+C content was 39.3 mol%. The results of 16S rRNA gene sequence analysis indicated that strain 169(T) was most closely related to Methanobacterium subterraneum DSM 11074(T) (96.8 % sequence similarity) and Methanobacterium formicicum DSM 1535(T) (96.4 %). On the basis of its morphological, physiological and phylogenetic characteristics, strain 169(T) ( = DSM 22026(T) = JCM 15797(T)) represents a novel species of the genus Methanobacterium, for which the name Methanobacterium kanagiense sp. nov. is proposed.

Isolation of a new heterolobosean amoeba from a rice field soil: Vrihiamoeba italica gen. nov., sp. nov.

A heterolobosean amoeba strain 6_5F was isolated from an Italian rice field soil. Although 18S rRNA gene sequence analysis demonstrated that the new isolate was closely related to Stachyamoeba sp. ATCC 50324, further molecular analysis and morphological observation showed distinct differences amongst the two. The 5.8S rRNA gene was successfully amplified and sequenced for strain 6_5F but not for strain ATCC 50324. Trophozoites of strain ATCC 50324 transform into flagellate forms in the late stage of incubation before encystment, while strain 6_5F do not show flagellate forms under different conditions of the flagellation test. Light and electron microscopic observation showed the structural difference of cysts of strain 6_5F from strain ATCC 50324 and also from the type strain Stachyamoeba lipophora. The results show that the strain 6_5F is distinct from Stachyamoeba spp. and we propose a new genus and species for this isolate, Vrihiamoeba italica gen. nov., sp. nov.

Assessment of the methane mitigation potentials of alternative water regimes in rice fields using a process‐based biogeochemistry model

AbstractRice production is a substantial source of atmospheric CH4, which is second only to CO2 as a contributor to global warming. Since CH4 is produced in anaerobic soil environments, water management is expected to be a practical measure to mitigate CH4 emissions. In this study, we used a process‐based biogeochemistry model (DNDC‐Rice) to assess the CH4 mitigation potentials of alternative water regimes (AWR) for rice fields at a regional scale. Before regional application, we tested DNDC‐Rice using site‐scale data from three rice fields in Japan with different water regimes. The observed CH4 emissions were reduced by drainage of the fields, but were enhanced by organic amendments. DNDC‐Rice gave acceptable predictions of variation in daily CH4 fluxes and seasonal CH4 emissions due to changes in the water regime. For regional application, we constructed a GIS database at a 1 × 1 km mesh scale that contained data on rice field area, soil properties, daily weather, and farming management of each cell in the mesh, covering 3.2% of the rice fields in Japan's Hokkaido region. We ran DNDC‐Rice to simulate CH4 emissions under five simulated water regimes: the conventional water regime and four AWR scenarios with gradually increasing drainage. We found that AWR can reduce CH4 emission by up to 41% compared with the emission under conventional water regime. Including the changes in CO2 and nitrous oxide emissions, potential mitigation of greenhouse gas (GHG) was 2.6 Mg CO2 Eq. ha−1 yr−1. If this estimate is expanded to Japan's total rice fields, expected GHG mitigation is 4.3 Tg CO2 Eq. yr−1, which accounts for 0.32% of total GHG emissions from Japan. For a reliable national‐scale assessment, however, databases on soil, weather, and farming management must be constructed at a national scale, as these factors are widely variable between regions in Japan.

Erratum for “Isolation and Characterization of a Novel Facultative Anaerobic Filamentous Fungus from Japanese Rice Field Soil”

A novel filamentous fungus strain designated RB-1 was isolated into pure culture from Japanese rice field soil through an anaerobic role tube technique. The strain is a mitosporic fungus that grows in both aerobic and strict anaerobic conditions using various mono-, di-, tri-, and polysaccharides with acetate and ethanol productions. The amount of acetate produced was higher than that of ethanol in both aerobic and anaerobic cultures. The characteristic verrucose or punctuate conidia of RB-1 closely resembled those of some strains of the genus Thermomyces, a thermophilic or mesophilic anamorphic ascomycete. However, based on phylogenetic analysis with the small subunit (SSU) and large subunit (LSU) rDNA sequences, RB-1 was characterized as a member of the class Lecanoromycetes of the phylum Ascomycota. Currently, RB-1 is designated as an anamorphic ascomycete and is phylogenetically considered an incertae sedis within the class Lecanoromycetes.

Effect of temperature change on the composition of the bacterial and archaeal community potentially involved in the turnover of acetate and propionate in methanogenic rice field soil

The microbial community structure was investigated together with the path of methane production in Italian rice field soil incubated at moderate (35 degrees C) and high (45 degrees C) temperature using terminal restriction fragment length polymorphism and stable isotope fractionation. The structure of both the archaeal and bacterial communities differed at 35 degrees C compared with 45 degrees C, and acetoclastic and hydrogenotrophic methanogenesis dominated, respectively. Changing the incubation of the 45 degrees C soil to different temperatures (25, 30, 35, 40, 45, 50 degrees C) resulted in a dynamic change of both microbial community structure and stable isotope fractionation. In all treatments, acetate first accumulated and then decreased. Propionate was also transiently produced and consumed. It is noteworthy that acetate was also consumed at thermophilic conditions, although archaeal community composition and stable isotope fractionation indicated that acetoclastic methanogenesis did not operate. Instead, acetate must have been consumed by syntrophic acetate oxidizers. The transient accumulation and subsequent consumption of acetate at thermophilic conditions was specifically paralleled by terminal restriction fragments characteristic for clostridial cluster I, whereas those of clostridial clusters I and III, Acidaminococcaceae and Heliobacteraceae, paralleled the thermophilic turnover of both acetate and propionate.

Methanocella arvoryzae sp. nov., a hydrogenotrophic methanogen isolated from rice field soil

A novel hydrogenotrophic methanogen, designated strain MRE50(T), was isolated from a methanogenic consortium, which was originally established from an Italian rice field soil. Cells were non-motile rods, 1.3-2.8 μm long and 0.4-0.7 μm wide. Coccoid cells were also observed in cultures at the late-exponential phase of growth. Strain MRE50(T) grew at 37-55 °C (optimally at 45 °C), at pH 6-7.8 (optimally at pH 7.0) and in the presence of 0-20 g NaCl l(-1). The isolate utilized H(2)/CO(2) and formate for growth and methane production. Phylogenetic analyses of the 16S rRNA gene and the methanogen-specific marker gene mcrA showed that strain MRE50(T) is affiliated with the order Methanocellales, previously known as uncultured archaeal group Rice Cluster I. Based on both 16S rRNA gene and mcrA gene sequences, strain MRE50(T) was related most closely to Methanocella paludicola SANAE(T). Levels of sequence similarity were 92.5 and 86.1 %, respectively, indicating that strains MRE50(T) and Methanocella paludicola SANAE(T) represent different species within the genus Methanocella. In addition, although these strains shared phenotypic properties including cell morphology and substrate utilization, they differed with respect to susceptibility to antibiotics, and temperature and NaCl ranges for growth. Given the phenotypic differences and the distinct phylogenetic placement of the new isolate relative to the type species of the genus Methanocella, strain MRE50(T) is considered to represent a novel species of the genus Methanocella, for which the name Methanocella arvoryzae sp. nov. is proposed. The type strain is MRE50(T) (=NBRC 105507(T) =DSM 22066(T)).

Seasonal Change in Methanotrophic Diversity and Populations in a Rice Field Soil Assessed by DNA-Stable Isotope Probing and Quantitative Real-Time PCR

The community structure of methane-oxidizing bacteria (methanotrophs) is affected by concentrations of methane and oxygen. In rice fields, concentrations of both gases differ significantly between the flooded and drained seasons. We investigated the active methanotrophic community structures in flooded and drained soils by DNA-based stable isotope probing. Active methanotrophic diversity was assessed with clone library-based analyses of the 16S rRNA gene and the particulate methane monooxygenase gene (pmoA). The active methanotrophic populations were also estimated by group-specific quantitative real-time PCR assays targeting the 16S rRNA gene and the pmoA gene in (13)C-labeled DNA. These molecular biological analyses showed that the flooded rice field soil was dominated by Type II methanotrophs closely related to the genera Methylocystis and Methylosinus, whereas the drained rice field soil was dominated by Type I methanotrophs closely related to the genera Methylomonas, Methylosarcina, and Methylomicrobium. The alternating conditions in a rice field select for methanotrophs adapted to each environment, resulting in a dramatic change in methanotrophic community structure from one season to another.

Molecular Characterization of Fungal Communities in Non-Tilled, Cover-Cropped Upland Rice Field Soils

This study aimed to characterize soil fungal communities in upland rice fields managed with tillage/non-tillage and winter cover-cropping (hairy vetch and cereal rye) practices, using PCR-based molecular methods. The study plots were maintained as upland fields for 5 years and the soils sampled in the second and fifth years were analyzed using T-RFLP (terminal restriction fragment length polymorphism) profiling and clone libraries with the internal transcribed spacer (ITS) region and domain 1 (D1) of the fungal large-subunit (fLSU) rRNA (D1(fLSU)) as the target DNA sequence. From the 2nd-year-sample, 372 cloned sequences of fungal ITS-D1(fLSU) were obtained and clustered into 80 nonredundant fungal OTUs (operational taxonomic units) in 4 fungal phyla. The T-RFLP profiling was performed with the 2nd- and 5th-year-samples and the major T-RFs (terminal restriction fragments) were identified using a theoretical fragment analysis of the ITS-D1(fLSU) clones. These molecular analyses showed that the fungal community was influenced more strongly by the cover-cropping than tillage practices. Moreover, the non-tilled, cover-cropped soil was characterized by a predominance of Cryptococcus sp. in the phylum Basidiomycota. We provided a genetic database of the fungal ITS-D1(fLSU)s in the differently managed soils of upland rice fields.

Infiltration Process in Rainfed Rice Field Soil of Ghanaian Inland Valley

Infiltration Process in Rainfed Rice Field Soil of Ghanaian Inland Valley

Microbial mechanism for rice variety control on methane emission from rice field soil

AbstractRice variety is one of the key factors regulating methane (CH4) production and emission from the paddy fields. However, the relationships between rice varieties and populations of microorganisms involved in CH4 dynamics are poorly understood. Here we investigated CH4 dynamics and the composition and abundance of CH4‐producing archaea and CH4‐oxidizing bacteria in a Chinese rice field soil planted with three types of rice. Hybrid rice produced 50–60% more of shoot biomass than Indica and Japonica cultivars. However, the emission rate of CH4 was similar to Japonica and lower than Indica. Furthermore, the dissolved CH4 concentration in the rhizosphere of hybrid rice was markedly lower than Indica and Japonica cultivars. The rhizosphere soil of hybrid rice showed a similar CH4 production potential but a higher CH4 oxidation potential compared with the conventional varieties. Terminal restriction fragment length polymorphism analysis of the archaeal 16S rRNA genes showed that the hydrogenotrophic methanogens dominated in the rhizosphere whereas acetoclastic methanogens mainly inhabited the bulk soil. The abundance of total archaea as determined by quantitative (real‐time) PCR increased in the later stage of rice growth. However, rice variety did not significantly influence the structure and abundance of methanogenic archaea. The analysis of pmoA gene fragments (encoding the α‐subunit of particulate methane monooxygenase) revealed that rice variety also did not influence the structure of methanotrophic proteobacteria, though variable effects of soil layer and sampling time were observed. However, the total copy number of pmoA genes in the rhizosphere of hybrid rice was approximately one order of magnitude greater than the two conventional cultivars. The results suggest that hybrid rice stimulates the growth of methanotrophs in the rice rhizosphere, and hence enhances CH4 oxidation which attenuates CH4 emissions from the paddy soil. Hybrid rice is becoming more and more popular in Asian countries. The present study demonstrated that planting of hybrid rice will not enhance CH4 emissions albeit a higher grain production than the conventional varieties.

Responses of methanogenic archaeal community to oxygen exposure in rice field soil

Methanogens are regarded as strict anaerobes and hence sensitive to O2 exposure. It has been demonstrated that CH4 production and emission from rice field soil are substantially reduced when soil is drained or aerated even shortly. However, the response of different methanogenic populations to O2 stress remains unclear. Therefore, we determined CH4 production and structure of the methanogenic community in a Chinese rice field soil after short-term (24 h) and long-term (72 h) exposure to O2 under laboratory conditions. O2 stress strongly inhibited CH4 production, and the inhibitory effect increased with the duration of O2 exposure. O2 exposure also resulted in dramatic increase of ferric iron and sulfate concentrations. H2 partial pressures were significantly reduced, most probably due to the competitive consumption by iron and sulfate-reducing bacteria. However, substrate competition could not explain the inhibition of acetoclastic methanogenesis, since acetate accumulated after O2 exposure compared with the control. Quantitative (real-time) PCR analyses of both archaeal 16S rRNA and mcrA genes (coding for alpha subunit of the methyl coenzyme M reductase) revealed that growth of the methanogenic populations was suppressed after O2 exposure. However, terminal restriction fragment length polymorphism (T-RFLP) analyses of both 16S rDNA and 16S rRNA showed that the structure of the methanogenic archaeal community remained remarkably stable, and that acetoclastic Methanosarcinaceae were always dominant whether with or without O2 exposure. Thus, O2 stress apparently did not differentially affect the various methanogenic populations, but instead inhibited CH4 production by enabling competition, generally suppressing growth and differentially affecting existing enzyme activity.

Spatial heterogeneity of methanotrophs: a geostatistical analysis of pmoA‐based T‐RFLP patterns in a paddy soil

Despite numerous studies on methanotrophs, virtually nothing is known about their spatial heterogeneity in nature. These patterns, however, have strong influences on the interpretations made from analysing microbial processes and community structure. Here we report the first use of geostatistics to analyse the spatial heterogeneity of methanotrophs in a rice field soil (Vercelli, Italy). We used the gene encoding the particulate methane monooxygenase, pmoA, for terminal restriction fragment length polymorphism (T-RFLP) analysis. The profiles obtained were compared using a pseudo-variogram analysis to study autocorrelation as a function of distance. We demonstrated that there was no large-scale spatial structure at this study site, but a micro-scale spatial structure could not be excluded. A species accumulation curve with all terminal restriction fragments revealed that even 75 samples were insufficient to cover the diversity of methanotrophs in a rice field. However, a species accumulation curve of methanotrophs defined as operational taxonomic units validated from a clone library with 90% coverage demonstrated saturation after approximately 15 samples. The results of this study have consequences for studying the diversity and function of methanotrophs. In this agroecosystem population structure showed no spatial pattern implying that both a systematic and random sampling design would be adequate.