Detoxifying symbiosis: microbe-mediated detoxification of phytotoxins and pesticides in insects.

Covering: up to 2018 Insects live in a world full of toxic compounds such as plant toxins and manmade pesticides. To overcome the effects of these toxins, herbivorous insects have evolved diverse, elaborate mechanisms of resistance, such as toxin avoidance, target-site alteration, and detoxification. These resistance mechanisms are thought to be encoded by the insects' own genomes, and in many cases, this holds true. However, recent omics analyses, in conjunction with classic culture-dependent analyses, have revealed that a number of insects possess specific gut microorganisms, some of which significantly contribute to resistance against phytotoxins and pesticides by degrading such chemical compounds. Here, we review recent advances in our understanding on the symbiont-mediated degradation of natural and artificial toxins, with a special emphasis on their underlying genetic basis, focus on the importance of environmental microbiota as a resource of toxin-degrading microorganisms, and discuss the ecological and evolutionary significance of these symbiotic associations.

Insect's intestinal organ for symbiont sorting.

Symbiosis has significantly contributed to organismal adaptation and diversification. For establishment and maintenance of such host-symbiont associations, host organisms must have evolved mechanisms for selective incorporation, accommodation, and maintenance of their specific microbial partners. Here we report the discovery of a previously unrecognized type of animal organ for symbiont sorting. In the bean bug Riptortus pedestris, the posterior midgut is morphologically differentiated for harboring specific symbiotic bacteria of a beneficial nature. The sorting organ lies in the middle of the intestine as a constricted region, which partitions the midgut into an anterior nonsymbiotic region and a posterior symbiotic region. Oral administration of GFP-labeled Burkholderia symbionts to nymphal stinkbugs showed that the symbionts pass through the constricted region and colonize the posterior midgut. However, administration of food colorings revealed that food fluid enters neither the constricted region nor the posterior midgut, indicating selective symbiont passage at the constricted region and functional isolation of the posterior midgut for symbiosis. Coadministration of the GFP-labeled symbiont and red fluorescent protein-labeled Escherichia coli unveiled selective passage of the symbiont and blockage of E. coli at the constricted region, demonstrating the organ's ability to discriminate the specific bacterial symbiont from nonsymbiotic bacteria. Transposon mutagenesis and screening revealed that symbiont mutants in flagella-related genes fail to pass through the constricted region, highlighting that both host's control and symbiont's motility are involved in the sorting process. The blocking of food flow at the constricted region is conserved among diverse stinkbug groups, suggesting the evolutionary origin of the intestinal organ in their common ancestor.

Insecticide applications to soil contribute to the development of Burkholderia mediating insecticide resistance in stinkbugs.

Some soil Burkholderia strains are capable of degrading the organophosphorus insecticide, fenitrothion, and establish symbiosis with stinkbugs, making the host insects fenitrothion-resistant. However, the ecology of the symbiotic degrading Burkholderia adapting to fenitrothion in the free-living environment is unknown. We hypothesized that fenitrothion applications affect the dynamics of fenitrothion-degrading Burkholderia, thereby controlling the transmission of symbiotic degrading Burkholderia from the soil to stinkbugs. We investigated changes in the density and diversity of culturable Burkholderia (i.e. symbiotic and nonsymbiotic fenitrothion degraders and nondegraders) in fenitrothion-treated soil using microcosms. During the incubation with five applications of pesticide, the density of the degraders increased from less than the detection limit to around 10(6)/g of soil. The number of dominant species among the degraders declined with the increasing density of degraders; eventually, one species predominated. This process can be explained according to the competitive exclusion principle using V(max) and K(m) values for fenitrothion metabolism by the degraders. We performed a phylogenetic analysis of representative strains isolated from the microcosms and evaluated their ability to establish symbiosis with the stinkbug Riptortus pedestris. The strains that established symbiosis with R. pedestris were assigned to a cluster including symbionts commonly isolated from stinkbugs. The strains outside the cluster could not necessarily associate with the host. The degraders in the cluster predominated during the initial phase of degrader dynamics in the soil. Therefore, only a few applications of fenitrothion could allow symbiotic degraders to associate with their hosts and may cause the emergence of symbiont-mediated insecticide resistance.

Symbiont-mediated insecticide resistance.

Development of insecticide resistance has been a serious concern worldwide, whose mechanisms have been attributed to evolutionary changes in pest insect genomes such as alteration of drug target sites, up-regulation of degrading enzymes, and enhancement of drug excretion. Here, we report a previously unknown mechanism of insecticide resistance: Infection with an insecticide-degrading bacterial symbiont immediately establishes insecticide resistance in pest insects. The bean bug Riptortus pedestris and allied stinkbugs harbor mutualistic gut symbiotic bacteria of the genus Burkholderia, which are acquired by nymphal insects from environmental soil every generation. In agricultural fields, fenitrothion-degrading Burkolderia strains are present at very low densities. We demonstrated that the fenitrothion-degrading Burkholderia strains establish a specific and beneficial symbiosis with the stinkbugs and confer a resistance of the host insects against fenitrothion. Experimental applications of fenitrothion to field soils drastically enriched fenitrothion-degrading bacteria from undetectable levels to >80% of total culturable bacterial counts in the field soils, and >90% of stinkbugs reared with the enriched soil established symbiosis with fenitrothion-degrading Burkholderia. In a Japanese island where fenitrothion has been constantly applied to sugarcane fields, we identified a stinkbug population wherein the insects live on sugarcane and ≈8% of them host fenitrothion-degrading Burkholderia. Our finding suggests the possibility that the symbiont-mediated insecticide resistance may develop even in the absence of pest insects, quickly establish within a single insect generation, and potentially move around horizontally between different pest insects and other organisms.

Diversity of the Hydroxylamine Oxidoreductase (HAO) Gene and Its Enzyme Active Site in Agricultural Field Soils.

Nitrification is a key process in the biogeochemical nitrogen cycle and a major emission source of the greenhouse gas nitrous oxide (N2O). The periplasmic enzyme hydroxylamine oxidoreductase (HAO) is involved in the oxidation of hydroxylamine to nitric oxide in the second step of nitrification, producing N2O as a byproduct. Its three-dimensional structure demonstrates that slight differences in HAO active site residues have inhibitor effects. Therefore, a more detailed understanding of the diversity of HAO active site residues in soil microorganisms is important for the development of novel nitrification inhibitors using structure-guided drug design. However, this has not yet been examined. In the present study, we investigated hao gene diversity in beta-proteobacterial ammonia-oxidizing bacteria (ß-AOB) and complete ammonia-oxidizing (comammox; Nitrospira spp.) bacteria in agricultural fields using a clone library ana-lysis. A total of 1,949 hao gene sequences revealed that hao gene diversity in ß-AOB and comammox bacteria was affected by the fertilizer treatment and field type, respectively. Moreover, hao sequences showed the almost complete conservation of the six HAO active site residues in both ß-AOB and comammox bacteria. The diversity of nitrifying bacteria showed similarity between hao and amoA genes. The nxrB amplicon sequence revealed the dominance of Nitrospira cluster II in tea field soils. The present study is the first to reveal hao gene diversity in agricultural soils, which will accelerate the efficient screening of HAO inhibitors and evaluations of their suppressive effects on nitrification in agricultural soils.

A novel method for RNA extraction from Andosols using casein and its application to amoA gene expression study in soil.

The lack of a universal method to extract RNA from soil hinders the progress of studies related to nitrification in soil, which is an important step in the nitrogen cycle. It is particularly difficult to extract RNA from certain types of soils such as Andosols (volcanic ash soils), which is the dominant agricultural soil in Japan, because of RNA adsorption by soil. To obtain RNA from these challenging soils to study the bacteria involved in nitrification, we developed a soil RNA extraction method for gene expression analysis. Autoclaved casein was added to an RNA extraction buffer to recover RNA from soil, and high-quality RNA was successfully extracted from eight types of agricultural soils that were significantly different in their physicochemical characteristics. To detect bacterial ammonia monooxygenase subunit A gene (amoA) transcripts, bacterial genomic DNA and messenger RNA were co-extracted from two different types of Andosols during incubation with ammonium sulfate. Polymerase chain reaction-denaturing gradient gel electrophoresis and reverse transcription polymerase chain reaction-denaturing gradient gel electrophoresis analyses of amoA in soil microcosms revealed that only few amoA, which had the highest similarities to those in Nitrosospira multiformis, were expressed in these soils after treatment with ammonium sulfate, although multiple amoA genes were present in the soil microcosms examined.

Insecticide resistance by a host-symbiont reciprocal detoxification.

Insecticide resistance is one of the most serious problems in contemporary agriculture and public health. Although recent studies revealed that insect gut symbionts contribute to resistance, the symbiont-mediated detoxification process remains unclear. Here we report the in vivo detoxification process of an organophosphorus insecticide, fenitrothion, in the bean bug Riptortus pedestris. Using transcriptomics and reverse genetics, we reveal that gut symbiotic bacteria degrade this insecticide through a horizontally acquired insecticide-degrading enzyme into the non-insecticidal but bactericidal compound 3-methyl-4-nitrophenol, which is subsequently excreted by the host insect. This integrated "host-symbiont reciprocal detoxification relay" enables the simultaneous maintenance of symbiosis and efficient insecticide degradation. We also find that the symbiont-mediated detoxification process is analogous to the insect genome-encoded fenitrothion detoxification system present in other insects. Our findings highlight the capacity of symbiosis, combined with horizontal gene transfer in the environment, as a powerful strategy for an insect to instantly eliminate a toxic chemical compound, which could play a critical role in the human-pest arms race.

Ammonia Oxidation Potentials and Ammonia Oxidizers of Lichen-Moss Vegetated Soils at Two Ice-free Areas in East Antarctica.

The maximum ammonia oxidation potential (AOP) of a topsoil in Langhovde, East Antarctica was 22.1±2.4| |ng N g-1 dry soil h-1 (2| |mM ammonium, 10°C, n=3). This topsoil exhibited twin AOP peaks (1 and 2| |mM ammonium) at 10°C, but not at 20°C. Six and ten operational taxonomic units (OTUs) were identified for ammonia-oxidizing bacteria (AOB) and archaea (AOA) amoA, respectively. AOB were classified into Nitrosospira; the two dominant OTUs corresponded to the Mount Everest cluster. AOA were classified into three clusters; Nitrososphaera and Nitrosocosmicus were the two dominant clusters.

Enrichment of Comammox and Nitrite-Oxidizing Nitrospira From Acidic Soils.

In agricultural soils fertilized with a high amount of ammonium nitrogen, the pH decreases because of the oxidation of ammonia by nitrifiers. Molecular-based analyses have revealed that members of the genus Nitrospira dominate over other nitrifiers in some acidic soils. However, terrestrial Nitrospira are rarely cultivated and little is known about their ecophysiology. In addition, recent studies discovered a single microbe with the potential to oxidize both ammonia and nitrite (complete ammonia oxidizer; comammox) within Nitrospira, which had been previously recognized as a nitrite oxidizer. Despite their broad distribution, there are no enrichment samples of comammox from terrestrial or acidic environments. Here, we report the selective enrichment of both comammox and nitrite-oxidizing Nitrospira from the acidic soil of a heavily fertilized tea field. Long-term enrichment was performed with two individual continuous-feeding bioreactors capable of controlling ammonia or nitrite concentration and pH. We found that excessive ammonium supply was a key factor to enhance the growth of comammox Nitrospira under acidic conditions. Additionally, a low concentration of nitrite was fed to prevent the accumulation of free nitrous acid and inhibition of cell growth under low pH, resulting in the selective enrichment of nitrite-oxidizing Nitrospira. Based on 16S rRNA gene analysis, Nitrospira accounting for only 1.2% in an initial soil increased to approximately 80% of the total microorganisms in both ammonia- and nitrite-fed bioreactors. Furthermore, amoA amplicon sequencing revealed that two phylotypes belonging to comammox clade A were enriched in an ammonia-fed bioreactor. One group was closely related to previously cultivated strains, and the other was classified into a different cluster consisting of only uncultivated representatives. These two groups coexisted in the bioreactor controlled at pH 6.0, but the latter became dominant after the pH decreased to 5.5. Additionally, a physiological experiment revealed that the enrichment sample oxidizes ammonia at pH <4, which is in accordance with the strongly acidic tea field soil; this value is lower than the active pH range of isolated acid-adapted nitrifiers. In conclusion, we successfully enriched multiple phylotypes of comammox and nitrite-oxidizing Nitrospira and revealed that the pH and concentrations of protonated N-compounds were potential niche determinants.

Effect of low C/N crop residue input on N2O, NO, and CH4 fluxes from Andosol and Fluvisol fields.

Crop residues are produced from agriculture in large amounts globally. Crop residues are known to be a source of nitrous oxide (N2O); however, contrasting results have been reported. Furthermore, the effect of crop residues on nitric oxide (NO) and methane (CH4) fluxes has not been well studied. We investigated N2O, NO, and CH4 fluxes after low C/N crop residue (cabbages and potatoes) inputs to lysimeter fields for two years using with automated flux monitoring system. Lysimeters were filled with two contrasting soil types, Andosol (total C: 33.1 g kg-1; clay: 18%) and Fluvisol (17.7 g kg-1; 36%). Nitrogen application rates were 250 kg N ha-1 of synthetic fertilizer and 272 kg N ha-1 of cow manure compost for cabbage, and 120 kg N ha-1 of synthetic fertilizer and 136 kg N ha-1 of cow manure compost for potato, respectively. Large N2O peaks were observed after crop residues were left on the surface of the soil for 1 to 2 weeks in summer, but not in winter. The annual N2O emission factors (EFs) for cabbage residues were 3.02% and 5.37% for Andosol and Fluvisol, respectively. Those for potatoes were 7.51% and 5.10% for Andosol and Fluvisol, respectively. The EFs were much higher than the mean EFs of synthetic fertilizers from Japan's agricultural fields (0.62%). Moreover, the EFs were much higher than the Intergovernmental Panel on Climate Change (IPCC) default N2O EFs for synthetic fertilizers and crop residues (1%). The annual NO EFs for potatoes were 1.35% and 2.44% for Andosol and Fluvisol, respectively, while no emission was observed after cabbage residue input. Crop residues did not affect CH4 uptake by soil. Our results suggest that low C/N crop residue input to soils can create a hotspot of N2O emission, when temperature and water conditions are not limiting factors for microbial activity.

Complementary cooperation between two syntrophic bacteria in pesticide degradation.

Interactions between microbial species, including competition and mutualism, influence the abundance and distribution of the related species. For example, metabolic cooperation among multiple bacteria plays a major role in the maintenance of consortia. This study aims to clarify how two bacterial species coexist in a syntrophic association involving the degradation of the pesticide fenitrothion. To elucidate essential mechanisms for maintaining a syntrophic association, we employed a mathematical model based on an experimental study, because experiment cannot elucidate various conditions for two bacterial coexistence. We isolated fenitrothion-degrading Sphingomonas sp. TFEE and its metabolite of 3-methyl-4-nitrophenol (3M4N)-degrading Burkholderia sp. MN1 from a fenitrothion-treated soil microcosm. Neither bacterium can completely degrade fenitrothion alone, but they can utilize the second intermediate, methylhydroquinone (MHQ). Burkholderia sp. MN1 excretes a portion of MHQ during the degradation of 3M4N, from which Sphingomonas sp. TFEE carries out degradation to obtain carbon and energy. Based on experimental findings, we developed mathematical models that represent the syntrophic association involving the two bacteria. We found that the two bacteria are characterized by the mutualistic degradation of fenitrothion. Dynamics of two bacteria are determined by the degree of cooperation between two bacteria (i.e., supply of 3M4N by Sphingomonas sp. TFEE and excretion of MHQ by Burkholderia sp. MN1) and the initial population sizes. The syntrophic association mediates the coexistence of the two bacteria under the possibility of resource competition for MHQ, and robustly facilitates the maintenance of ecosystem function in terms of degrading xenobiotics. Thus, the mathematical analysis and numerical computations based on the experiment indicate the key mechanisms for coexistence of Sphingomonas sp. TFEE and Burkholderia sp. MN1 in syntrophic association involving fenitrothion degradation.

Infection dynamics of insecticide-degrading symbionts from soil to insects in response to insecticide spraying.

Insecticide resistance is a serious concern in modern agriculture, and an understanding of the underlying evolutionary processes is pivotal to prevent the problem. The bean bug Riptortus pedestris, a notorious pest of leguminous crops, acquires a specific Burkholderia symbiont from the environment every generation, and harbors the symbiont in the midgut crypts. The symbiont's natural role is to promote insect development but the insect host can also obtain resistance against the insecticide fenitrothion (MEP) by acquiring MEP-degrading Burkholderia from the environment. To understand the developing process of the symbiont-mediated MEP resistance in response to the application of the insecticide, we investigated here in parallel the soil bacterial dynamics and the infected gut symbionts under different MEP-spraying conditions by culture-dependent and culture-independent analyses, in conjunction with stinkbug rearing experiments. We demonstrate that MEP application did not affect the total bacterial soil population but significantly decreased its diversity while it dramatically increased the proportion of MEP-degrading bacteria, mostly Burkholderia. Moreover, we found that the infection of stinkbug hosts with MEP-degrading Burkholderia is highly specific and efficient, and is established after only a few times of insecticide spraying at least in a field soil with spraying history, suggesting that insecticide resistance could evolve in a pest bug population more quickly than was thought before.

Seabird-affected taluses are denitrification hotspots and potential N2O emitters in the High Arctic.

In High Arctic tundra ecosystems, seabird colonies create nitrogen cycling hotspots because of bird-derived labile organic matter. However, knowledge about the nitrogen cycle in such ornithocoprophilous tundra is limited. Here, we determined denitrification potentials and in-situ nitrous oxide (N2O) emissions of surface soils on plant-covered taluses under piscivorous seabird cliffs at two sites (BL and ST) near Ny-Ålesund, Svalbard, in the European High Arctic. Talus soils at both locations had very high denitrification potentials at 10 °C (2.62-4.88 mg N kg-1 dry soil h-1), near the mean daily maximum air temperature in July in Ny-Ålesund, with positive temperature responses at 20 °C (Q10 values, 1.6-2.3). The talus soils contained abundant denitrification genes, suggesting that they are denitrification hotspots. However, high in-situ N2O emissions, indicating the presence of both active aerobic nitrification and anaerobic denitrification, were observed only at BL (max. 16.6 µg N m-2 h-1). Rapid nitrogen turnover at BL was supported by lower carbon-to-nitrogen ratios, higher nitrate content, and higher δ15N values in the soils at BL compared with those at ST. These are attributed to the 30-fold larger seabird density at BL than at ST, providing the larger organic matter input.

Responses of denitrifying bacterial communities to short-term waterlogging of soils.

Agricultural soil is often subjected to waterlogging after heavy rainfalls, resulting in sharp and explosive increases in the emission of nitrous oxide (N2O), an important greenhouse gas primarily released from agricultural soil ecosystems. Previous studies on waterlogged soil examined the abundance of denitrifiers but not the composition of denitrifier communities in soil. Also, the PCR primers used in those studies could only detect partial groups of denitrifiers. Here, we performed pyrosequencing analyses with the aid of recently developed PCR primers exhibiting high coverage for three denitrification genes, nirK, nirS, and nosZ to examine the effect of short-term waterlogging on denitrifier communities in soil. We found that microbial communities harboring denitrification genes in the top 5 cm of soil distributed according to soil depth, water-soluble carbon, and nitrate nitrogen. Short-term waterlogging scarcely affected abundance, richness, or the alpha-diversities of microbial communities harboring nirK, nirS, and nosZ genes, but significantly affected their composition, particularly in microbial communities at soil depths of 0 to 1 cm. Our results indicated that the composition of denitrifying microbial communities but not the abundance of denitrifiers in soil was responsive to short-term waterlogging of an agricultural soil ecosystem.

Comparison of the Effects of Phenylhydrazine Hydrochloride and Dicyandiamide on Ammonia-Oxidizing Bacteria and Archaea in Andosols.

Dicyandiamide, a routinely used commercial nitrification inhibitor (NI), inhibits ammonia oxidation catalyzed by ammonia monooxygenase (AMO). Phenylhydrazine hydrochloride has shown considerable potential for the development of next-generation NIs targeting hydroxylamine dehydrogenase (HAO). The effects of the AMO inhibitor and the HAO inhibitor on ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA) present in agricultural soils have not been compared thus far. In the present study, the effects of the two inhibitors on soil nitrification and the abundance of AOA and AOB as well as their community structure were investigated in a soil microcosm using quantitative polymerase chain reaction and pyrosequencing. The net nitrification rates and the growth of AOA and AOB in this soil microcosm were inhibited by both NIs. Both NIs had limited effect on the community structure of AOB and no effect on that of AOA in this soil microcosm. The effects of phenylhydrazine hydrochloride were similar to those of dicyandiamide. These results indicated that organohydrazine-based NIs have potential for the development of next-generation NIs targeting HAO in the future.

An acid-tolerant ammonia-oxidizing γ-proteobacterium from soil.

Nitrification, the microbial oxidation of ammonia to nitrate via nitrite, occurs in a wide range of acidic soils. However, the ammonia-oxidizing bacteria (AOB) that have been isolated from soil to date are acid-sensitive. Here we report the isolation and characterization of an acid-adapted AOB from an acidic agricultural soil. The isolated AOB, strain TAO100, is classified within the Gammaproteobacteria based on phylogenetic characteristics. TAO100 can grow in the pH range of 5-7.5 and survive in highly acidic conditions until pH 2 by forming cell aggregates. Whereas all known gammaproteobacterial AOB (γ-AOB) species, which have been isolated from marine and saline aquatic environments, are halophiles, TAO100 is not phenotypically halophilic. Thus, TAO100 represents the first soil-originated and non-halophilic γ-AOB. The TAO100 genome is considerably smaller than those of other γ-AOB and lacks several genes associated with salt tolerance which are unnecessary for survival in soil. The ammonia monooxygenase subunit A gene of TAO100 and its transcript are higher in abundance than those of ammonia-oxidizing archaea and betaproteobacterial AOB in the strongly acidic soil. These results indicate that TAO100 plays an important role in the nitrification of acidic soils. Based on these results, we propose TAO100 as a novel species of a new genus, Candidatus Nitrosoglobus terrae.

Purification and characterization of fenitrothion hydrolase from Burkholderia sp. NF100.

The organophosphorus pesticide hydrolase was purified to homogeneity from Burkholderia sp. NF100 by detergent extraction of the cell membrane fraction, anion-exchange, chromatofocusing, and gel filtration chromatographies. The purified enzyme had a molecular mass of 55 kDa and a pI 5.8, and the hydrolase activity was strongly inhibited by EDTA, dithiothreitol (DTT), Hg2+ and 1,10-phenanthroline. The optimum pH and temperature for the enzyme activity were 8.0 and 40 degrees C, respectively. The enzyme hydrolyzed five organophosphorus pesticides.

Cloning and nucleotide sequence of carbaryl hydrolase gene (cahA) from Arthrobacter sp. RC100.

A carbaryl hydrolase gene (cahA) encoded on the plasmid pRC1 in Arthrobacter sp. RC100 was cloned and sequenced. The entire region of the deduced amino acid sequence was found to be homologous to that of an amidase family. Parts of the consensus sequences of the amidase gene have been identified in CahA from strain RC100. CahA was overexpressed in Escherichia coli JM109, and the enzyme was purified to homogeneity by protamine sulfate treatment, ammonium sulfate precipitation, and hydrophobic and anion-exchange chromatographies. The purified enzyme showed hydrolase activity toward 1-naphthylacetamide and isobutyramide but showed no activity toward 1-naphthylacetate. This is the first report of an amidase that is able to hydrolyze N-methylcarbamate pesticides.

Mitigation of soil N2O emission by inoculation with a mixed culture of indigenous Bradyrhizobium diazoefficiens.

Agricultural soil is the largest source of nitrous oxide (N2O), a greenhouse gas. Soybean is an important leguminous crop worldwide. Soybean hosts symbiotic nitrogen-fixing soil bacteria (rhizobia) in root nodules. In soybean ecosystems, N2O emissions often increase during decomposition of the root nodules. Our previous study showed that N2O reductase can be used to mitigate N2O emission from soybean fields during nodule decomposition by inoculation with nosZ++ strains [mutants with increased N2O reductase (N2OR) activity] of Bradyrhizobium diazoefficiens. Here, we show that N2O emission can be reduced at the field scale by inoculation with a mixed culture of indigenous nosZ+ strains of B. diazoefficiens USDA110 group isolated from Japanese agricultural fields. Our results also suggested that nodule nitrogen is the main source of N2O production during nodule decomposition. Isolating nosZ+ strains from local soybean fields would be more applicable and feasible for many soybean-producing countries than generating mutants.

Effect of Elevated CO2 Concentration, Elevated Temperature and No Nitrogen Fertilization on Methanogenic Archaeal and Methane-Oxidizing Bacterial Community Structures in Paddy Soil.

Elevated concentrations of atmospheric CO2 ([CO2]) enhance the production and emission of methane in paddy fields. In the present study, the effects of elevated [CO2], elevated temperature (ET), and no nitrogen fertilization (LN) on methanogenic archaeal and methane-oxidizing bacterial community structures in a free-air CO2 enrichment (FACE) experimental paddy field were investigated by PCR-DGGE and real-time quantitative PCR. Soil samples were collected from the upper and lower soil layers at the rice panicle initiation (PI) and mid-ripening (MR) stages. The composition of the methanogenic archaeal community in the upper and lower soil layers was not markedly affected by the elevated [CO2], ET, or LN condition. The abundance of the methanogenic archaeal community in the upper and lower soil layers was also not affected by elevated [CO2] or ET, but was significantly increased at the rice PI stage and significantly decreased by LN in the lower soil layer. In contrast, the composition of the methane-oxidizing bacterial community was affected by rice-growing stages in the upper soil layer. The abundance of methane-oxidizing bacteria was significantly decreased by elevated [CO2] and LN in both soil layers at the rice MR stage and by ET in the upper soil layer. The ratio of mcrA/pmoA genes correlated with methane emission from ambient and FACE paddy plots at the PI stage. These results indicate that the decrease observed in the abundance of methane-oxidizing bacteria was related to increased methane emission from the paddy field under the elevated [CO2], ET, and LN conditions.