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Very few studies to date have focused on phosphorus (P) dynamics and microbial P acquisition during anaerobic soil disinfestation (ASD), an alternative to chemical soil fumigation for soil diseases. We evaluated the effects of ASD using diluted ethanol as well as the effects of drying treatments following ASD on available P concentrations, phosphomonoesterase activity, microbial biomass P, and microbial community compositions, including those harboring the alkaline phosphomonoesterase gene (phoD), in two arable Andosols. An increase in the Bray-2P concentration and a decrease in the ratio of phosphomonoesterase/β-D-glucosidase activities were observed during ASD in both soils, indicating an increase in P availability during ASD. A significant negative correlation between the diversity of profile of denaturing gradient gel electrophoresis for phoD-harboring community and alkaline phosphomonoesterase activity was found, suggesting that a less diverse bacterial group might produce alkaline phosphomonoesterase in soils with higher activity. ASD followed by drying and rewetting significantly increased the Bray-2P concentration and microbial biomass P in one Andosol. These results support the potential of ASD to increase P availability in addition to suppressing soil-borne diseases in arable Andosols.

γ-Hexachlorocyclohexane (γ-HCH)-degrading strain, Sphingobium sp. TA15, was newly isolated from an experimental field soil from which the archetypal γ-HCH-degrading strain, S. japonicum UT26, was isolated previously. Comparison of the complete genome sequences of these 2 strains revealed that TA15 shares the same basic genome backbone with UT26, but also has the variable regions that are presumed to have changed either from UT26 or from a putative common ancestor. Organization and localization of lin genes of TA15 were different from those of UT26. It was inferred that transposition of IS6100 had played a crucial role in these genome rearrangements. The accumulation of toxic dead-end products in TA15 was lower than in UT26, suggesting that TA15 utilizes γ-HCH more effectively than UT26. These results suggested that genome evolution related to the γ-HCH metabolic function in the soil microbial population is ongoing.

Recent studies demonstrated that phylogenetically more diverse and abundant bacteria and fungi than previously considered are responsible for denitrification in terrestrial environments. We herein examined the effects of land-use types on the community composition of those denitrifying microbes based on their nitrite reductase gene (nirK and nirS) sequences. These genes can be phylogenetically grouped into several clusters. We used cluster-specific PCR primers to amplify nirK and nirS belonging to each cluster because the most widely used primers only amplify genes belonging to a single cluster. We found that the dominant taxa as well as overall community composition of denitrifying bacteria and fungi, regardless of the cluster they belonged to, differed according to the land-use type. We also identified distinguishing taxa based on individual land-use types, the distribution of which has not previously been characterized, such as denitrifying bacteria or fungi dominant in forest soils, Rhodanobacter having nirK, Penicillium having nirK, and Bradyrhizobium having nirS. These results suggest that land-use management affects the ecological constraints and consequences of denitrification in terrestrial environments through the assembly of distinct communities of denitrifiers.

In southern China, high levels of atmospheric nitrogen (N) are being deposited in forests. Soil acidification and high rates of soil nitrification and subsequent NO 3 − leaching have been observed in the N-saturated forests. We previously did not detect NH 3 -oxidizing bacteria in non-N-saturated or N-saturated forest soils. However, NH 3 -oxidizing archaea were present in both soils, more in the saturated ones. The purpose of this study was to investigate the roles of autotrophic NH 3 -oxidizing archaea and heterotrophic nitrifiers in soil N transformations in N-saturated and non-N-saturated forests in southern China. We investigated the contribution of heterotrophic nitrifiers in the soils by determining the gross nitrification rates with and without an inhibitor of autotrophic nitrification, acetylene (C 2 H 2 ). We also reevaluated nitrification by NH 3 -oxidizing archaea by correlating the C 2 H 2 -inhibited gross nitrification rates with the abundance of the amoA transcripts of NH 3 -oxidizing archaea. We further measured the gross NH 4 + production rates and analyzed the community composition of the NH 3 -oxidizing archaea. The results suggest that NH 3 -oxidizing archaea, rather than heterotrophic nitrifiers and NH 3 -oxidizing bacteria, are responsible for the nitrification in the N-saturated forest soils. NH 3 -oxidizing archaea in the soils could be acidophilic, having low amoA diversity, indicating their strong adaptation to the highly acidified soils. The gross NH 4 + production rate did not differ between N-saturated and non-N-saturated forests; however, the gross nitrification rate was higher in N-saturated forests. Consequently, the high abundance and NH 3 oxidation activity of NH 3 -oxidizing archaea caused the high rates of nitrification and subsequent leaching of NO 3 − in the N-saturated forest. This study suggests that acidophilic NH 3 -oxidizing archaea have a great impact on soil N cycling in N-saturated forests.

Nitrite reductase is a key enzyme for denitrification. There are two types of nitrite reductases: copper-containing NirK and cytochrome cd1-containing NirS. Most denitrifiers possess either nirK or nirS, although a few strains been reported to possess both genes. We herein report the presence of nirK and nirS in the soil-denitrifying bacterium Bradyrhizobium sp. strain TSA1T. Both nirK and nirS were identified and actively transcribed under denitrification conditions. Based on physiological, chemotaxonomic, and genomic properties, strain TSA1T (=JCM 18858T=KCTC 62391T) represents a novel species within the genus Bradyrhizobium, for which we propose the name Bradyrhizobium nitroreducens sp. nov.

Elucidating the soil phosphorus cycle driven by soil microbes is a vital question in soil microbial ecology. The Japanese arable Andisols, occupying half of the Japanese cropland, are known for their high phosphorus sorption capacity. However, limited information is currently available on microbially driven phosphorus mineralization in arable Andisols. We herein report that the phosphorus-mineralizing community in the Japanese arable Andisols showed characteristic distribution and composition patterns, from those in other types of soils. We performed a chemical analysis and microbial community analysis of 43 amble Andisols along the Japanese archipelago. Soil phosphomonoesterase activities measured at pH 11 were approximately 70% of those at pH 6.5, which indicates that alkaline phosphatase contributes to phosphorus cycling, although most soil samples were acidic. Functional gene predictions based on 16S rRNA gene sequencing indicated that the alkaline phosphatase gene phoD was more abundant than other alkaline phosphatase genes and, thus, plays major roles. Hence, amplicon sequencing targeting phoD was performed and the results obtained showed that alphaproteobacterial phoD was dominant. This is in contrast to previously reported phoD compositions in other soils and may be attributed to the nutrient conditions in arable Andisols, which favor copiotrophic Alphaproteobacteria. Furthermore, the composition of phoD correlated with soil pH and bioavailable phosphorus concentrations rather than carbon or nitrogen concentrations. These results were partly different from previous findings, varying in the soil types and geographic ranges of sampling sites. Collectively, the present results indicate that the phosphorus-mineralizing community in the Japanese arable Andisols is regulated differently from those in other soil types., Article, MICROBES AND ENVIRONMENTS. 33(3):282-289 (2018)

To elucidate the factor(s) controlling the concentrations of readily available organic phosphorus (Po) in soils, we assessed the phosphatase-hydrolyzable Po pools in 0.5 M NaHCO3-extracts of arable and forest soils and evaluated their association with soil properties in several soil types collected in Japan. Concentrations of phosphatase-hydrolyzable Po (simple monoester P, phytate like-P, and DNA-like P) in 0.5 M NaHCO3-extracts were determined using three phosphatases with different substrate specificities. Monoester P (i.e., simple monoester P and phytate like-P) was dominant in the phosphatase-hydrolyzable Po of 0.5 M NaHCO3-extracts in all soils. In monoester P, the concentration of phytate-like P was greater than that of simple monoester P in all soil types except Fluvisols. Both phytate-like P and DNA-like P in 0.5 M NaHCO3-extracts significantly increased with decreasing soil pH, suggesting that soil pH would play a crucial role in determining the availability of Po in soils.

According to the resource allocation model for extracellular enzyme synthesis, microorganisms should preferentially allocate their resources to phosphorus (P)-acquiring enzyme synthesis when P availability is low in soils. However, the validity of this model across different soil types and soils differing in their microbial community composition has not been well demonstrated. Here we investigated whether the resource allocation model for phosphatase synthesis is applicable across different soil types (Andosols, Acrisols, Cambisols, and Fluvisols) and land uses (arable and forest), and we examined which soil test P and/or P fraction microorganisms responded to when investing their resources in phosphatase synthesis in the soils. The ratio of alkaline phosphatase (ALP) to β-d-glucosidase (BG) activities in the arable soils and the ratio of acid phosphatase (ACP) to BG activities in the forest soils were significantly negatively related with the available inorganic P concentration. We also observed significant effects of available inorganic P, pH, soil types, and land uses on the (ACP + ALP)/BG ratio when the data for the arable and forest soils were combined and used in a stepwise multiple regression analysis. These results suggest that microbial resource allocation for phosphatase synthesis is primarily controlled by available inorganic P concentration and soil pH, but the effects of soil types and land uses are also significant.

Waterlogged paddy soils possess anoxic zones in which microbes actively induce reductive nitrogen transformation (RNT). In the present study, a shotgun RNA sequencing analysis (metatranscriptomics) of paddy soil samples revealed that most RNT gene transcripts in paddy soils were derived from Deltaproteobacteria, particularly the genera Anaeromyxobacter and Geobacter. Despite the frequent detection of the rRNA of these microbes in paddy soils, their RNT-associated genes have rarely been identified in previous PCR-based studies. This metatranscriptomic analysis provides novel insights into the diversity of RNT microbes present in paddy soils and the ecological function of Deltaproteobacteria predominating in these soils.

The aim of this study was to screen nitrous oxide (N 2 O)-reducing denitrifier strains showing both N 2 O mitigation and plant growth-promoting (PGP) effects in soil systems, and the effects of selected strains were monitored in soil and plant, analyzed, and comparatively evaluated. Forty denitrifier strains affiliated with Azospirillum and Herbaspirillum , previously isolated from three different paddy soils, were evaluated. Of these, 11 produced indole-3-acetic acid (>5 μg mL −1 ), 9 promoted the growth of red clover ( Trifolium pratense L. var. Medium) or timothy ( Phleum pratense L. var. Horizon) on agar plates, and 7 were inoculated into two different soils for cultivating red clover and timothy in a greenhouse. Compared with non-inoculated control, N 2 O flux from red clover soil and from timothy soil were significantly lower 8 and 14 days and 9 days onwards, respectively, after inoculation with these seven strains. Cumulative N 2 O emissions from red clover soil were significantly lowered through inoculation with these seven strains. The growth parameters, including plant height, leaf area, fresh weight or dry weight, of the two pasture plants were significantly greater in soils inoculated with most of these seven strains than in non-inoculated soils. The uptake of C and N by the two pasture plants was significantly greater in soils inoculated with most of these seven strains than in non-inoculated soils. In conclusion, inoculating N 2 O-reducing denitrifiers to pasture soil could mitigate N 2 O emissions and simultaneously promote the growth of pasture plants in a greenhouse. These strains will be invaluable microbiological resources for developing novel biofertilizers.

To evaluate the effects of aluminum (Al) toxicity on microbial processes in acidic forest soils, we determined microbial biomass, soil enzyme activities associated with carbon (C), nitrogen, and phosphorus cycling (β- d -glucosidase and polyphenol oxidase, l -asparaginase, and acid phosphatase, respectively), exchangeable base cations, soluble and exchangeable Al (CaCl2–Al and KCl–Al), and organically bound (Alp and Fep) and both organically bound and noncrystalline forms (Alo and Feo) of Al and iron (Fe) in Japanese forest soils (25 Inceptisols, seven allophanic Andisols, and eight nonallophanic Andisols). The exchangeable calcium (Ca) and magnesium (Mg) concentrations decreased significantly with decreasing soil pH. In contrast, the KCl–Al and CaCl2–Al concentrations exponentially increased with decreasing pH, and the increase was steeper in Andisols than in Inceptisols. The Alp concentration also increased exponentially with decreasing soil pH in Andisols, and the Fep concentration increased in Andisols and Inceptisols, whereas the concentration of the noncrystalline Al pool (Alo − Alp) decreased with decreasing pH in Andisols. We showed that the β- d -glucosidase, polyphenol oxidase, and acid phosphatase activities were mainly suppressed by soluble and exchangeable Al, whereas l -asparaginase activity was depressed by soil pH. In Andisols, a negative effect of organically bound Al and Fe on the biomass C/organic C ratio was observed. These results suggest that Al toxicity and acidity repressed soil enzyme activities, leading to suppressed microbially mediated nutrient cycling, and that Al toxicity and a reduced availability of organic matter, as a result of Al and Fe binding, may protect a substantial pool of organic C from microbial degradation in acidic forest soils.

Long-term fertilization experiments are a useful way to elucidate the impacts of fertilization on soil ecosystems. Here, we report the prokaryotic community structure in experimental field soil after 80 years of successive fertilization. Our 16S rRNA gene sequencing detected 20,996 amplicon sequence variants, including major phyla such as Proteobacteria, Acidobacteria, and Actinobacteria., Article, Microbiol. Resour. Announc. 8:e01551-18 (2019)

A considerable part of soil phosphorus is bound to metal cations or metal oxides, and cannot be used in these forms by soil microbes and plants. Phosphate-solubilizing bacteria (PSB) are abundant in pectin-rich rhizosphere, and their pectin-degrading activity has been reported. Therefore, we hypothesized that pectin activates PSB and promotes soil phosphorus solubilization. To test this hypothesis, we first tested the phosphate-solubilizing activity of pectin-degrading bacteria. PSB were more frequently isolated from pectin-containing medium, compared with other media (P

Soil microbes are drivers of global ecosystem functionality and are continuously subjected to external perturbations. It is fundamental for ecologists and environmental scientists to understand and further predict the microbes’ responses to these perturbations. A major and ubiquitous perturbation is the addition of chemical nutrients, including fertilizers and animal urine, to soil. Recent biogeographical studies suggest that soil nutrient stoichiometry (i.e., nutritional balance) determines microbial community structure and its functions with regard to material circulation. Given this information, here, we show that soil nutrient stoichiometry, or the bioavailable C:P ratio, determines the impact of nutrient addition on the soil’s microbial communities. We sampled two soils with similar carbon and nitrogen concentrations but with a 20-fold difference in phosphorus bioavailability. Soil microcosms with carbon and nitrogen amendments were constructed for both the soils. The phosphorus-depleted soil received prolonged effect from carbon and nitrogen amendments: the phosphatase activity gradually increased over a 24-day incubation period and the microbial community structure did not present recovery to its initial state. In contrast, in the other soil, both phosphatase activity and microbial community structure gradually returned to those of the control samples. Phosphorus depletion mitigated carbon and nitrogen intake; therefore, the effects of carbon and nitrogen amendment lasted longer. Our results demonstrate that nutritional stoichiometry is a strong predictor of microbial community dynamics in response to trophic perturbation, particularly when considering the length of time the trait of perturbation persists in the soil.

Pseudogulbenkiania is a relatively recently characterized genus within the order Neisseriales, class Betaproteobacteria. This genus contains several strains that are capable of anaerobic, nitrate-dependent Fe(II) oxidation (NDFO), a geochemically important reaction for nitrogen and iron cycles. In the present study, we examined denitrification functional gene diversities within this genus, and clarified whether other Pseudogulbenkiania sp. strains perform denitrification and NDFO. Seventy strains were analyzed, including two type strains, a well-characterized NDFO strain, and 67 denitrifying strains isolated from various rice paddy fields and rice-soybean rotation fields in Japan. We also attempted to identify the genes responsible for NDFO by mutagenesis. Our comprehensive analysis showed that all Pseudogulbenkiania strains tested performed denitrification and NDFO; however, we were unable to obtain NDFO-deficient denitrifying mutants in our mutagenesis experiment. This result suggests that Fe(II) oxidation in these strains is not enzymatic, but is caused by reactive N-species that are formed during nitrate reduction. Based on the results of the comparative genome analysis among Pseudogulbenkiania sp. strains, we identified low sequence similarity within the nos gene as well as different gene arrangements within the nos gene cluster, suggesting that nos genes were horizontally transferred. Since Pseudogulbenkiania sp. strains have been isolated from various locations around the world, their denitrification and NDFO abilities may contribute significantly to nitrogen and iron biogeochemical cycles.

Fungal denitrification in soils is receiving considerable attention as one of the dominant N 2 O production processes. However, because of the lack of a methodology to detect fungal denitrification-related genes, the diversity and ecological behavior of denitrifying fungi in soil remains unknown. Thus, we designed a primer set to detect the fungal nitrite reductase gene ( nirK ) and validated its sensitivity and specificity. Through clone library analyses, we identified congruence between phylogenies of the 18S rRNA gene and nirK of denitrifying fungal isolates obtained from the surface-fertilized cropland soil and showed that fungi belonging to Eurotiales, Hypocreales, and Sordariales were primarily responsible for N 2 O emissions in the soil.

A mesophilic, Gram-stain-positive, spore-forming bacterium that formed branched mycelia was isolated from paddy soil in Gunung Salak (Mount Salak), West Java, Indonesia. This strain, designated S-27T, grew at temperatures between 20 and 37 °C; the optimum growth temperature was 25 to 30 °C, and no growth was observed at 15 or 45 °C. The pH range for growth was pH 3.5 to 8.6; the optimum pH was 6.0, and no growth was observed at pH 3.0 or 9.2. Strain S-27T was able to hydrolyse polysaccharides such as starch, cellulose and xylan. The G+C content of the DNA of strain S-27T was 55.7 mol%. The major fatty acids were iso-C17 : 0 and C16 : 1 2-OH, and the major menaquinone was MK-9 (H2). The cell wall of strain S-27T contained d-glutamic acid, glycine, l-alanine, d-alanine, l-ornithine and β-alanine in a molar ratio of 1.0 : 1.6 : 1.4 : 0.6 : 0.9 : 1.1. The polar lipids consisted of phosphatidylglycerol, phosphatidylinositol and two glycolipids. The major cell-wall sugar was arabinose. Detailed phylogenetic analysis based on 16S rRNA gene sequences indicated that strain S-27T belongs to the order Ktedonobacterales and is most closely related to Ktedonobacter racemifer SOSP1-21T (89.6 % sequence identity). On the basis of its chemotaxonomic and phenotypic features and phylogenetic position, we concluded that strain S-27T represents a novel genus and species, for which we propose the name Dictyobacter aurantiacus gen. nov., sp. nov. The type strain of Dictyobacter aurantiacus is strain S-27T (=NBRC 109595T=InaCC B312T). Emendation of the description of the genus Thermosporothrix is also provided.

Thirty-nine denitrifying bacterial strains closely related to one another, represented by strains TSA40T and TSA66T, were isolated from rice paddy soils. Strains TSA40T and TSA66T were Gram-stain-negative, slightly curved rod-shaped, and motile by means of polar flagella. They were able to reduce nitrate, nitrite and nitrous oxide, but unable to fix atmospheric N2. While strain TSA66T was able to grow autotrophically by H2-dependent denitrification, strain TSA40T could not. Phylogenetic analysis suggested that they belong to the family Oxalobacteraceae , the order Burkholderiale s in the class Betaproteobacteria . Major components in the fatty acids (C16 : 0, C17 : 0 cyclo, C18 : 1ω7c and summed feature 3) and quinone (Q-8) also supported the affiliation of strains TSA40T and TSA66T to the family Oxalobacteraceae . Based on 16S rRNA gene sequence comparisons, strains TSA40T and TSA66T showed the greatest degree of similarity to Herbaspirillum massiliense JC206T, Noviherbaspirillum malthae CC-AFH3T, Noviherbaspirillum humi U15T, Herbaspirillum seropedicae Z67T and Paucimonas lemoignei LMG 2207T, and lower similarities to the members of other genera. Average nucleotide identity values between the genomes of strain TSA40T, TSA66T and H. massiliense JC206T were 75–77 %, which was lower than the threshold value for species discrimination (95–96 %). Based on the 16S rRNA gene sequence analysis in combination with physiological, chemotaxonomic and genomic properties, strains TSA40T (=JCM 17722T=ATCC TSD-69T) and TSA66T (=JCM 17723T=DSM 25787T) are the type strains of two novel species within the genus Noviherbaspirillum , for which the names Noviherbaspirillum denitrificans sp. nov. and Noviherbaspirillum autotrophicum sp. nov. are proposed, respectively. We also propose the reclassification of Herbaspirillum massiliense as Noviherbaspirillum massiliense comb. nov.

The mineralization of organic phosphorus (P) in soil is mainly driven by extracellular phosphatases secreted by soil microbes. Previous studies have suggested that microbial phosphatase production is regulated by soil nutrient stoichiometry and, thus, promoted by carbon and nitrogen (CN) amendment. However, the mechanism leading to increased phosphatase activity after nutrient amendment to soil is unclear, and it is not known how long the effect of the nutrient amendment lasts. To address these questions, we performed a 24-day time-series analysis of full-factorial soil microcosms with CN amendment. Phosphatase activity as well as that expressed per unit of β- d -glucosidase activity, increased sharply and significantly in response to the CN amendment. This suggests that the microbial community shifted their resource allocation after the nutrient amendment and preferentially produced phosphatases rather than β- d -glucosidase. This shifted resource allocation pattern was maintained throughout the 24-day incubation period, and copiotrophic microbes dominated soils that received CN amendment. These results indicate that the effect of the CN amendment on soil microbial resource allocation lasted for >24 days. Together, our results highlight the importance of high-resolution time-series observations that complement long-term studies for which frequent observation is often difficult.

Pelleted poultry manure is recommended for use with agricultural soil as a replacement for chemical fertilizers; however, application of the manure stimulates nitrous oxide (N2O) emission from the soil through denitrification. To mitigate the N2O emission caused by application of pelleted poultry manure, soil microcosms were set up; each microcosm was inoculated with one of the following N2-generating denitrifier strains previously been isolated from paddy soil: Azoarcus, Dyella, Dechloromonas, Niastella, and Burkholderia. When pelleted poultry manure was incubated on its own, N2O was produced by denitrification. In contrast, N2O emission was significantly lowered when the manure was inoculated with most of the N2-generating strains. In soil microcosms, N2O was emitted during incubation after application of the pelleted manure, while N2O flux was significantly lowered when the soil was inoculated with Azoarcus sp. KS11B, Niastella sp. KS31B, or Burkholderia sp. TSO47-3 on the 12th day of incubation. In addition, when pelleted manure was inoculated with the strains prior to application in the soil microcosms, the level of N2O emission was significantly lowered to ca. 40–60 % that from the non-inoculated control. Our study provides the prototype of a technique that uses microbial technology to mitigate N2O emission from agricultural soil fertilized with pelleted poultry manure.

Agricultural fields are one of the largest anthropogenic sources of atmospheric nitrous oxide (N2O). Fungi have been suggested to contribute substantially to N2O emission in terrestrial environments; however, the extent of the fungal contribution and the phylogenetic and physiologic nature of related fungal communities in agricultural fields are largely unknown. This study focused on the large N2O emission from cropland soil that occurs after granular organic fertilizers were applied to the surface. The granular organic fertilizers applied were evidently covered by fungal mycelia. An experiment using a soil microcosm was established to imitate the field observations. N2O emissions following surface organic fertilization were suppressed by 84 and 20% after the addition of cycloheximide (a fungal inhibitor) and streptomycin (a bacterial inhibitor), respectively, suggesting that fungi provide the main contribution to the observed N2O emission. The population density and community composition of fungi in the surface-fertilized and non-fertilized soils in the field were determined using colony counting, denaturing gradient gel electrophoresis and subsequent phylogenetic analyses. Thirty-four fungal strains were isolated from the soils, and their N2O producing activities were analyzed. Fungal population density in the surface-fertilized soil (2.6 × 106 CFU/g) was much higher than that in the non-fertilized soil (1.0 × 105 CFU/g). In addition, the fungal community compositions of the soils differed. Actinomucor elegans, Bionectria ochroleuca, Fusarium avenaceum, Fusarium equiseti, Fusarium oxysporum, Fusarium solani and Nectria sp. dominated the surface-fertilized soil, and their activity in producing N2O was confirmed. These results suggested that N2O emission after the surface application of granular organic fertilizers in the cropland field mainly resulted from fungal denitrification.

The taxonomic properties of strain DC2a-G7T, a Gram-negative, ovoid to rod-shaped, gellan gum-lysing bacterium, were examined. The 16S rRNA gene sequence similarity showed that DC2a-G7T is a member of the phylum Verrucomicrobia and the closest type strain of a species with a validly published name is Verrucomicrobium spinosum DSM 4136T, with a sequence similarity of 91.2 %. In addition to this similarity value lower than 95 %, the absence of prostheca, the orangey-red colony colour and the compositions of the major menaquinones and polar lipids also supported the differentiation of this bacterium from the genus Verrucomicrobium . Here, we propose the name Roseimicrobium gellanilyticum gen. nov., sp. nov. for the isolate. The type strain of Roseimicrobium gellanilyticum is DC2a-G7T ( = NBRC 108606T = DSM 25532T).

Denitrification is one of the important nitrogen transformation processes in rice (Oryza sativa L.) paddy soil. We have performed both culture-dependent and -independent studies to investigate the key players in denitrification in rice paddy soil. Our previous works suggested the possibility that community composition of denitrifying populations is different among soils. To confirm this possibility, in the present study, denitrifying bacteria were isolated from rice paddy field soils (Kumamoto and Tokyo) in Japan by the functional single-cell isolation method, and examined for their taxonomic position. Combined with our previously isolated strains from Niigata paddy field soil, our results clearly indicated that the taxonomic composition of the denitrifying bacterial community is different at the genus level among three soils. A significant difference in mean potential denitrification activity of the isolates at the genus level was also found. Soil and/or other environmental factors could shape t...

Paddy soils have an environment in which waterlogging and drainage occur during the rice growing season. Fingerprinting analysis based on soil RNA indicated that active microbial populations changed in response to water management conditions, although the fundamental microbial community was stable as assessed by DNA-based fingerprinting analysis. Comparative clone library analysis based on bacterial and archaeal 16S rRNAs (5,277 and 5,436 clones, respectively) revealed stable and variable members under waterlogged or drained conditions. Clones related to the class Deltaproteobacteria and phylum Euryarchaeota were most frequently obtained from the samples collected under both waterlogged and drained conditions. Clones related to syntrophic hydrogen-producing bacteria, hydrogenotrophic methanogenic archaea, rice cluster III, V, and IV, and uncultured crenarchaeotal group 1.2 appeared in greater proportion in the samples collected under waterlogged conditions than in those collected under drained conditions, while clones belonging to rice cluster VI related to ammonia-oxidizing archaea (AOA) appeared at higher frequency in the samples collected under drained conditions than in those collected under waterlogged conditions. These results suggested that hydrogenotrophic methanogenesis may become active under waterlogged conditions, whereas ammonia oxidation may progress by rice cluster VI becoming active under drained conditions in the paddy field.

Various types of mineral particles in a soil probably provide different microenvironments for microorganisms. The purpose of this study is to investigate whether different types of mineral in a soil harbor different bacterial populations. DNA was extracted from five types (quartz, feldspar, pyroxene, magnetite, iron-coated reddish brown particles) of sand-size mineral particles separated from a sandy soil, and was amplified for partial 16 S rRNA gene by polymerase chain reaction (PCR). Twenty-nine to 69 amplicons per each type of mineral were cloned and sequenced, followed by phylogenetic affiliation of the sequences. As a result, some types of bacteria were detected on all of the types of mineral including the orders Rhizobiales, Bacillales, and Acidobacteriales. In the case of Acidobacteriales, higher percentages were found on magnetite and quartz. Some taxa were restricted to specific types of mineral; the class Actinobacteria was found on pyroxene but not on quartz, and rarely on magnetite and feldspar. Bacterial diversity at the order level estimated by Chao1 value was higher in feldspar and pyroxene than the other three types of mineral. The UniFrac Significance test indicated that the differences in bacterial communitiy structures among the particles were suggestive except that between feldspar and pyroxene. These results support the idea that different communities of bacteria were associated with each of the mineral types., Soil Science and Plant Nutrition, 58(5), pp.562-567; 2012

Nitrification has been believed to be performed only by autotrophic ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) until the recent discovery of ammonia-oxidizing archaea (AOA). Meanwhile, it has been questioned whether AOB are significantly responsible for NH(3) oxidation in acidic forest soils. Here, we investigated nitrifying communities and their activity in highly acidified soils of three subtropical forests in southern China that had received chronic high atmospheric N deposition. Nitrifying communities were analyzed using PCR- and culture (most probable number)-based approaches. Nitrification activity was analyzed by measuring gross soil nitrification rates using a (15) N isotope dilution technique. AOB were not detected in the three forest soils: neither via PCR of 16S rRNA and ammonia monooxygenase (amoA) genes nor via culture-based approaches. In contrast, an extraordinary abundance of the putative archaeal amoA was detected (3.2 × 10(8) -1.2 × 10(9) g soil(-1) ). Moreover, this abundance was correlated with gross soil nitrification rates. This indicates that amoA-possessing archaea rather than bacteria were predominantly responsible for nitrification of the soils. Furthermore, sequences of the genus Nitrospira, a dominant group of soil NOB, were detected. Thus, nitrification of acidified subtropical forest soils in southern China could be performed by a combination of AOA and NOB.

Denitrification occurs markedly in rice paddy fields; however, few microbes that are actively involved in denitrification in these environments have been identified. In this study, we used a laboratory soil microcosm system in which denitrification activity was enhanced. DNA and RNA were extracted from soil at six time points after enhancing denitrification activity, and quantitative PCR and clone library analyses were performed targeting the 16S rRNA gene and denitrification functional genes (nirS, nirK and nosZ) to clarify which microbes are actively involved in denitrification in rice paddy soil. Based on the quantitative PCR results, transcription levels of the functional genes agreed with the denitrification activity, although gene abundance did not change at the DNA level. Diverse denitrifiers were detected in clone library analysis, but comparative analysis suggested that only some of the putative denitrifiers, especially those belonging to the orders Neisseriales, Rhodocyclales and Burkholderiales, were actively involved in denitrification in rice paddy soil.

How nitrogen (N) cycling is regulated and how environmental change affects it are major study questions in forest ecology, because N availability often limits the primary production of plants in many forest ecosystems. These are being extensively highlighted because of growing concerns regarding chronic and elevated N deposition in forest ecosystems on a global scale. Until now, N cycling has been mainly documented in association with various environmental factors other than microbial communities. However, with the recent rapid development in culture-independent molecular-based techniques, microbial ecologists have discovered that alterations in N cycling are highly associated with alternations in microbial communities through changes in either resource supplies or processing rates. In this review, we describe nitrification as a key N cycling process and present general approaches to associate the nitrification process with the nitrifying community in forest soils. Furthermore, we briefly summarize currently available information about the relationship between the process and nitrifying community dynamics in soil. We suppose that linking N cycling processes with microbial community dynamics provides a deeper insight into the mechanisms regulating N cycling in forest ecosystems.

Measurements of gross NH4+ and NO3− production in forest soils were conducted using the 15N pool dilution method. Mineral topsoils (0–10 cm depth) were collected from four forests from northern to southern Japan with a natural climate gradient to elucidate the mechanisms regulating gross nitrification rates in forest soils. Additionally, we attempted to evaluate the relative importance of heterotrophic nitrification in gross total nitrification using acetylene as a specific inhibitor of autotrophic nitrification. Distinct differences were found among sites in the gross rates of NH4+ production (3.1–11.4 mg N kg−1 day−1) and gross total nitrification (0.0–6.1 mg N kg−1 day−1). The rates of gross heterotrophic nitrification were low in this study, indicating that heterotrophic nitrification is of minor importance in most forest mineral topsoils in Japan. Significant relations were found between gross autotrophic nitrification and gross NH4+ production, soil N, and soil C concentrations, but none was...

Oligotrophic denitrifying bacteria, including those belonging to the genera Herbaspirillum , Azospirillum , and Bradyrhizobium , were obtained using a single-cell isolation technique. The taxonomic composition of the denitrifier population was similar to those assessed by previous culture-independent studies. The sequencing of nitrite reductase and N 2 O reductase genes of these strains revealed previously unknown links between 16S rRNA and the denitrification-functional gene phylogenies. In particular, we identified Bradyrhizobium strains that harbor nirS sequences previously detected only in culture-independent studies.

Denitrifiers can produce and consume nitrous oxide (N(2)O). While little N(2)O is emitted from rice paddy soil, the same soil produces N(2)O when the land is drained and used for upland crop cultivation. In this study, we collected soils from two types of fields each at three locations in Japan; one type of field had been used for continuous cultivation of rice and the other for rotational cultivation of rice and soybean. Active denitrifiers were isolated from these soils using a functional single-cell isolation method, and their taxonomy and denitrifying properties were examined. A total of 110 denitrifiers were obtained, including those previously detected by a culture-independent analysis. Strains belonging to the genus Pseudogulbenkiania were dominant at all locations, suggesting that Pseudogulbenkiania denitrifiers are ubiquitous in various rice paddy soils. Potential denitrifying activity was similar among the strains, regardless of the differences in taxonomic position and soil of origin. However, relative amounts of N(2) in denitrification end products varied among strains isolated from different locations. Our results also showed that crop rotation had minimal impact on the functional diversity of the denitrifying strains. These results indicate that soil and other environmental factors, excluding cropping systems, could select for N(2)-producing denitrifiers.

The measurement of (15)N concentrations in environmental samples requires sophisticated pretreatment devices and expensive isotope-ratio mass spectrometry (IRMS). This report describes the use of a gas chromatograph equipped with a quadrupole-type mass spectrometer (GC/MS) to measure (15)N concentrations of ammonium, nitrate, nitrite, and total dissolved nitrogen (TDN) in distilled water, a 2 M KCl solution and a 0.5 M K(2)SO(4) solution. The system measures nitrous oxide (N(2)O) that is ultimately converted from the target N compound, requiring no special apparatus such as a purge-and-trap pretreatment device. It uses a denitrifier lacking N(2)O reductase, which produces N(2)O from nitrate. Persulfate oxidation was applied to convert TDN to nitrate, while additional pretreatment with ammonia diffusion was required for ammonium prior to the persulfate oxidation. Up to 100 samples can be measured daily using the system. We can generally run (15)N measurements with only 1-10 mL of sample for each chemical species of N, a volume 1/10-1/100 times smaller than the amount necessary for conventional methods. Our method is useful for measuring (15)N with GC/MS, offering greater convenience than IRMS.

Rice paddy soil has been shown to have strong denitrifying activity. However, the microbial populations responsible for the denitrification in various rice paddy soils are not well known. In the present study, key denitrifiers were identified in six rice paddy soils by comparing the bacterial community structures in soils with strong denitrifying activity with those in soils without the activity. The effect of crop rotation on the denitrifier populations was also examined. Our study revealed three notable findings: (1) community structures of total bacteria were similar among the six rice paddy soils with different soil characteristics, (2) the populations that responded to denitrification-inducing conditions differed with soil type (Andosol, Gley soil or Gray lowland soil), (3) the populations that responded to denitrification-inducing conditions were similar within each type of soil, regardless of the cropping system (e.g. continuous or rotation).

We examined the abundance and community structure of sphingomonads in the decaying leaf residues of eight plant species as well as the nearby soil, by 16S rRNA gene-based real-time PCR and denaturing gradient gel electrophoresis. In the leaf residues, the sphingomonads generally accumulated to high levels, comprising approximately 15% of the total bacteria, and formed a community structure related to sampling locations. At least within the time period studied, their abundance in leaf residues changed, but their community structure was basically maintained. In soil, sphingomonads made up only 1.7% of total bacteria on average. The community structure of sphingomonads differed between the leaf residues and bulk soil, among plant plots, and among samples collected at different times. The results show that particular sphingomonad populations accumulate in leaf residues compared to the surrounding bulk soil under field conditions.

Rice paddy soil has been shown to have strong denitrifying activity. However, the microbial populations responsible for nitrate respiration and denitrification have not been well characterized. In this study, we performed a clone library analysis of >1,000 clones of the nearly full-length 16S rRNA gene to characterize bacterial community structure in rice paddy soil. We also identified potential key players in nitrate respiration and denitrification by comparing the community structures of soils with strong denitrifying activity to those of soils without denitrifying activity. Clone library analysis showed that bacteria belonging to the phylum Firmicutes , including a unique Symbiobacterium clade, dominated the clones obtained in this study. Using the template match method, several operational taxonomic units (OTUs), most belonging to the orders Burkholderiales and Rhodocyclales , were identified as OTUs that were specifically enriched in the sample with strong denitrifying activity. Almost one-half of these OTUs were classified in the genus Herbaspirillum and appeared >10-fold more frequently in the soils with strong denitrifying activity than in the soils without denitrifying activity. Therefore, OTUs related to Herbaspirillum are potential key players in nitrate respiration and denitrification under the conditions used.

Denitrification is an important part of the nitrogen cycle in the environment, and diverse bacteria, archaea, and fungi are known to have denitrifying ability. Rice paddy field soils have been known to have strong denitrifying activity, but the microbes responsible for denitrification in rice paddy field soils are not well known. Present study analyzed the diversity and quantity of the nitrite reductase genes (nirS and nirK) in a rice paddy field soil, sampled four times in one rice-growing season. Clone library analyses suggested that the denitrifier community composition varied over sampling time. Although many clones were distantly related to the known NirS or NirK, some clones were related to the NirS from Burkholderiales and Rhodocyclales bacteria, and some were related to the NirK from Rhizobiales bacteria. These denitrifiers may play an important role in denitrification in the rice paddy field soil. The quantitative PCR results showed that nirK was more abundant than nirS in all soil samples, but the nirK/nirS ratio decreased after water logging. These results suggest that both diversity and quantity changed over time in the rice paddy field soil, in response to the soil condition.

Rice paddy soil has been shown to have strong denitrifying activity. However, the microbial populations responsible for the denitrification have not been well characterized. In this study, we employed Stable Isotope Probing (SIP) to study succinate-assimilating denitrifiers in soil microcosms amended with nitrate and (13)C-succinate. Microbial populations represented in (12)C- and (13)C-DNA fractions were different based on denaturing gradient gel electrophoresis (DGGE) of the PCR-amplified 16S rRNA gene fragment. A nearly full-length 16S rRNA gene was also amplified, cloned, and sequenced from (13)C-DNA fraction. Both PCR-DGGE and clone library analyses revealed that Burkholderiales and Rhodocyclales dominated the succinate-assimilating population in denitrifying soil after 24 h incubation. Among these, novel Betaproteobacteria, possibly within the order Rhodocyclales, represented 43% of the clones obtained. Nitrite reductase genes, nirS and nirK, were also amplified and cloned from the (13)C-DNA fraction. While most nirK clones in this study were similar to the nirK sequences from Rhizobiales, a majority of the nirS clones were similar to the nirS sequences from Burkholderiales and Rhodocyclales, consistent with the 16S rRNA gene analysis. These groups of bacteria, including the novel Betaproteobacteria, may play an important role in denitrification in rice paddy soil.

The bacterial community structure in soil of a tropical rainforest in East Kalimantan, Indonesia, where forest fires occurred in 1997-1998, was analysed by denaturing gradient gel electrophoresis (DGGE) with soil samples collected from the area in 2001 and 2002. The study sites were composed of a control forest area without fire damage, a lightly-burned forest area, and a heavily-burned forest area. DGGE band patterns showed that there were many common bacterial taxa across the areas although the vegetation is not the same. In addition, it was indicated that a change of vegetation in burned areas brought the change in bacterial community structure during 2001-2002. It was also indicated that, depending on a perspective, community structure of soil bacteria in post-fire non-climax forest several years after fire can be more heterogeneous compared with that in unburned climax forest. The dominant soil bacteria in the field of the present study were Acidobacteria, Actinobaceria, and Alphaproteobacteria based on the DNA sequences of DGGE bands, although they were not dominant among the culturable bacteria from the same soil samples.

With the aim of understanding how diverse bacteria are distributed within a heterogeneous soil, we compared local bacterial communities on individual soil particles. We picked up 11 coarse sand-sized particles (quartz, whitish feldspar, yellow feldspar or unidentified brown particles) from a sandy soil, extracted DNA from each particle, and carried out partial 16S rDNA polymerase chain reaction denaturing gradient gel electrophoresis analysis. Bacterial communities located on soil particles of the same type were more similar in composition than communities located on particles of the other types. Thus, the local structure of a bacterial community is related to the type of soil particle, which suggests that a high diversity of soil bacteria emerges through a combination of local bacterial communities on different types of soil particles.

linA is a gene encoding γ-hexachlorocyclohexane dehydrochlorinase identified in a γ-HCH-degrading Sphingobium japonicum UT26 that had been isolated from soil of an experimental field treated with γ-HCH. The presence of linA-homologous nucleotide sequences and their sequence diversity was investigated by linA-targeted PCR amplification of DNA obtained from different soils in the field and in Japan. linA-homologous sequences were obtained from almost all the soils some of which had never been treated with γ-HCH. Deduced amino acid sequences suggested that they can be regarded as fragments of linA that encode LinA. Some common, natural function of LinA in soil was suggested., Microbes and environments. 2007, 22(4), p.399-404

Characterizing rhizomicrobial community structure is important to our understanding of the mechanisms of suppressive soils towards soilborne plant diseases. We investigated whether the bacterial community structure, i.e., the number of the constituent bacterial strains and the community-level, sole carbon source utilization pattern of rhizobacteria estimated by BIOLOG EcoPlates, affects bacterial wilt of tomato. To assess their impact on the bacterial wilt, we used 15 artificial soil systems into which 5–20 rhizobacterial isolates from a disease-suppressive soil were inoculated. Although most of the tomato plants wilted by 16 days after pathogen inoculation, significant negative relationships were observed between the number of wilted plants, the number of the bacterial strains and the number of carbon sources that the rhizobacterial community was able to utilize. BIOLOGs showed that several carbon sources were utilized by the pathogen but not by the rhizobacterial community from the fastest-wilting treatments, suggesting that competition between the pathogen and the other rhizobacteria for carbon sources played an important role in suppressing the growth of the pathogen.

Phototrophic purple bacteria (PPB) are one group of dinitrogen (N2)-fixing bacteria often found in the anoxic and photic zone of paddy soil. In the present study, the effects of inoculation of PPB on grain yield of rice and changes in their populations and nitrogenase activity were investigated with and without surface application of rice straw in a pot experiment. As an inoculant, Rhodopseudomonas palustris strain KN122 isolated from a paddy soil was selected, and the cell suspension was inoculated into the floodwater once or three times during the cultivation. As a result, the inoculation of PPB increased the grain yield of rice. Compared to the control, the grain yield was 9% higher in the inoculated plot without rice straw application. The treatment was more effective in combination with rice straw application. In the plots where PPB were inoculated with rice straw, grain yields were 21% (single inoculation) and 29% (triple inoculation) higher. Populations of PPB in the plots without rice straw applic...

A pot experiment was conducted to investigate the influences of indigenous phototrophs on methane (CH4) emissions from a paddy soil where rice straw was incorporated or was surface-applied. During the cultiva- tion, half of the pots were covered with aluminum foil, except for the minimum space for rice plants, to prevent ambient light reaching the floodwater or the soil surface. Growth of oxygen-producing phototrophs was hardly observed in the unilluminated plots, whereas intensive growth of algae, duckweed and hydrophytes was found in the illuminated ones. Plant growth was not affected by the different treatments. Seasonal changes in CH4 emission determined by a closed chamber method indicated that illumination had no or only minor effects on CH4 emissions when rice straw was incorporated or was not applied, but significantly reduced CH4 emissions when rice straw was surface-applied. Methanogenesis occurring in the soil-floodwater interface was further investigated in two lab-scale model experiments measuring methanogenic activity. As a result, more activated methanogenesis was found in the surface-applied rice straw and the soil around the straw compared with the soil incubated without rice straw. The magnitude of the methanogenic activity in the rice straw incubated under illuminated conditions was significantly lower than that incubated in the dark. Consequently, this study demonstrates that methanogen- esis in paddy soil occurs even in the soil-floodwater interface if plant residues like rice straw exist, and such methanogenesis is likely to be suppressed by growth of indigenous phototrophs under illumination.

Ensifer adhaerens CSBa was screened as a cobalamin producer. The draft genome sequence revealed that the strain possesses 22 cobalamin biosynthetic genes (cob genes). The cob gene arrangement on the genome of E. adhaerens CSBa was similar to that of other Ensifer species, and most similar to that of Pseudomonas denitrificans SC510. The cobN sequence phylogeny was generally congruent with that of the 16S rRNA gene, and it is suggeted that E. adhaerens CSBa might have inherited the cob genes from common ancestors of the Ensifer species. It was also suggested that the cob genes can be laterally transferred.