Susumu Asakawa

DGGE method for analyzing 16S rDNA of methanogenic archaeal community in paddy field soil.

A denaturing gradient gel electrophoresis (DGGE) method for analyzing 16S rDNA of methanogenic archaeal community in paddy field soil is presented. Five specific primers for 16S rDNA of methanogenic archaea, which were modified from the primers for archaea, were first evaluated by polymerase chain reaction and DGGE using genomic DNAs of 13 pure culture strains of methanogenic archaea. The DGGE analysis was possible with two primer pairs (0348aF-GC and 0691R; 0357F-GC and 0691R) of the five pairs tested although 16S rDNA of some non-methanogenic archaea was amplified with 0348aF-GC and 0691R. These two primer pairs were further evaluated for use in analysis of methanogenic archaeal community in Japanese paddy field soil. Good separation and quality of patterns were obtained in DGGE analysis with both primer pairs. A total of 41 DNA fragments were excised from the DGGE gels and their sequences were determined. All fragments belonged to methanogenic archaea. These results indicate that the procedure of DGGE analysis with the primer pair 0357F-GC and 0691R is suitable for investigating methanogenic archaeal community in paddy field soil.

Succession and phylogenetic composition of bacterial communities responsible for the composting process of rice straw estimated by PCR-DGGE analysis

Abstract PCR-DGGE analysis followed by sequencing of 16S rDNA was applied to reveal the succession and the phylogenetic composition of the bacterial communities responsible for the composting process of rice straw (RS). RS under the composting process exhibited more complex DGGE band patterns with more numerous bands and more diversification in intensity than the initial RS materials. The DGGE patterns of the bacterial communities in the initial RS materials and RS under the composting process were statistically divided into four groups, namely those characterizing the communities associated with the pre-composting stage, and thermophilic, middle, and curing stages of the compost, which corresponded exactly to the same grouping obtained from the PLFA pattern analysis for the same samples (Cahyani et al. 2002: Soil Sci. Plant Nutr., 48, 735-743). Different bacterial members characterized the respective stages as follows: a-Proteobacteria for the initial RS materials, thermophilic Bacillus spp. and actinomycetes at the thermophilic stage, and Cytophaga and clostridial members at the middle and curing stages, respectively. In addition, mesophilic Bacillus members were always present throughout the composting process. Temperature and substrates available to bacteria seemed mainly to determine the composition of the bacterial members at the respective stages.

Global change pressures on soils from land use and management

Soils are subject to varying degrees of direct or indirect human disturbance, constituting a major global change driver. Factoring out natural from direct and indirect human influence is not always straightforward, but some human activities have clear impacts. These include land-use change, land management and land degradation (erosion, compaction, sealing and salinization). The intensity of land use also exerts a great impact on soils, and soils are also subject to indirect impacts arising from human activity, such as acid deposition (sulphur and nitrogen) and heavy metal pollution. In this critical review, we report the state-of-the-art understanding of these global change pressures on soils, identify knowledge gaps and research challenges and highlight actions and policies to minimize adverse environmental impacts arising from these global change drivers. Soils are central to considerations of what constitutes sustainable intensification. Therefore, ensuring that vulnerable and high environmental value soils are considered when protecting important habitats and ecosystems, will help to reduce the pressure on land from global change drivers. To ensure that soils are protected as part of wider environmental efforts, a global soil resilience programme should be considered, to monitor, recover or sustain soil fertility and function, and to enhance the ecosystem services provided by soils. Soils cannot, and should not, be considered in isolation of the ecosystems that they underpin and vice versa. The role of soils in supporting ecosystems and natural capital needs greater recognition. The lasting legacy of the International Year of Soils in 2015 should be to put soils at the centre of policy supporting environmental protection and sustainable development.

Ecology of viruses in soils: Past, present and future perspectives

Abstract Viruses play important roles in biogeochemical nutrient cycles and act as genomic reservoirs in marine and freshwater environments, the understanding of which brought about the so-called ‘third age’ of virus ecology in aquatic environments. Unfortunately, the third age is in oceanography and limnology and outside the soil world. The main reason why virus ecology in soils has shown less progress is that agronomical and epidemiological interests were the primary motivation of viral studies by soil microbiologists. In this review, past research on viruses in soils is summarized after the introduction of the ecological traits of viruses, which are the effects of viruses on beneficial bacteria and soil-borne plant pathogens, adsorption of viruses to soils, and soil factors affecting viral inactivation and survival in soils. Horizontal gene transfer (transduction) in soils is also reviewed. Second, the abundance of viruses and their roles in biogeochemical nutrient cycles are summarized in aquatic environments. Five to 25% of the carbon fixed by primary producers is estimated to enter into the microbial loop via virus-induced lysis at different trophic levels in aquatic environments. The diversity of virus communities in aquatic environments estimated from analyses of the frequency distribution of capsid sizes and the morphology of virus populations are reviewed, and recent findings on the genomic diversity of viruses and their roles as the greatest genomic reservoirs in aquatic environments follow in the subsequent section. Viral genomics is elucidating the viral diversity and phylogenetic relationships among viruses in different environments. As the soil environment is a more diverse habitat for viruses than aquatic environments, viruses in soils have great potential to play roles comparable in quantity, which are unique in quality, to those in aquatic environments. Therefore, the potentiality and characteristics of viruses in soils are discussed in the final section for future research on virus ecology in soils from the viewpoints of biogeochemistry and genomic diversity. Synecological approaches to viruses in soils may open up a new era of soil virus ecology.

Populations of methanogenic bacteria in paddy field soil under double cropping conditions (rice-wheat)

The methanogenic populations able to use H2−CO2, methanol, and acetate were investigated in paddy field soil in situ under double cropping conditions [rice (Oryza sativa L.) as a summer crop under flooded conditions and wheat (Triticum aestivum L.) as an upland winter crop] over 2 years approximately bimonthly by the most probable number method. Three fields, one without fertilizer, one treated with inorganic fertilizer (mixed fertilizer including urea, ammonium phosphate, and potassium sulfate), and one treated with wheat straw plus inorganic fertilizer, were examined. The population of H2−CO2, methanol, and acetate utilizers in the paddy field soil at a depth of 1–6 cm was 103–104, 104–105, and 104–105 g-1 dry soil, respectively. These values were almost constant during the 2 years irrespective of moisture regime (flooded or nonflooded), crop (rice or wheat), fertilizer treatment, and soil depth (0–1, 1–10, and 10–20 cm).

Characterization of Methanobrevibacter arboriphilicus SA isolated from a paddy field soil and DNA-DNA hybridization among M. arboriphilicus strains

We isolated a methanogenic strain, designated strain SA (= DSM 7056), from an enrichment culture inoculated with a Japanese paddy field soil. Cells of this strain were strictly anaerobic, nonmotile, short rods and stained gram positive. The strain was able to use H2-CO2or formate as a methanogenic substrate. It required vitamins, but not acetate, for growth. Growth was fastest at 35 to 40°C. Methane was produced most rapidly at pH 6.0 to 7.5. The cellular lipid composition of strain SA was similar to that of M. arboriphilicus A2 (= DSM 2462). The G+C content of the DNA was 26.4 mol%. Strain SA had DNA-DNA hybridization values of more than 70% with M. arboriphilicus DH1T(= DSM 1125T). On the basis of phenotypic and genotypic characteristics, we identified strain SA as M. arboriphilicus. In the course of our identification work, the genetic heterogeneity of M. arboriphilicus was revealed by the results of DNA-DNA hybridization experiments. Although strain AZ (= DSM 744) should be classified as a member of a species distinct from the species containing the other four strains studied (DH1T, A2, DC [= DSM 1536], and SA), further phenotypic characterization will be required before a new species can be proposed.

Methanoculleus chikugoensis sp. nov., a novel methanogenic archaeon isolated from paddy field soil in Japan, and DNA-DNA hybridization among Methanoculleus species

A strictly anaerobic, irregularly coccoid, methanogenic archaeon, strain MG62T (= JCM 10825T = DSM 13459T), was isolated from paddy field soil in Chikugo, Fukuoka, Japan. The cells stained gram-negative, were 1.0-2.0 microm in diameter, were lysed by SDS and hypotonic solutions and were flagellated. Motility was not observed. The strain was able to use H2/CO2, 2-propanol/CO2, formate, 2-butanol/CO2 and cyclopentanol/CO2 as substrates for methanogenesis, but did not utilize acetate, ethanol, methanol or methylamines. The optimum temperature and pH were 25-30 degrees C and 6.7-7.2. Analysis of lipid component parts (core lipids, phospholipid polar head groups and glycolipid sugar moieties) showed the characteristic pattern of members of the family Methanomicrobiaceae except for the absence of glucose as a glycolipid sugar moiety. The G+C content of the DNA was 62.2 mol %. Sequence analysis of the 16S rDNA revealed that the strain belonged to the genus Methanoculleus. The strain had DNA-DNA hybridization values of less than 50% with type strains of Methanoculleus species. On the basis of phenotypic, genotypic and phylogenetic characteristics, the name Methanoculleus chikugoensis sp. nov. is proposed for strain MG62T (= JCM 10825T = DSM 13459T). The DNA hybridization study also revealed the close relationships of three species, Methanoculleus olentangyi, Methanoculleus bourgensis and Methanoculleus oldenburgensis, among Methanoculleus species.

Bacterial communities associated with nodal roots of rice plants along with the growth stages: Estimation by PCR-DGGE and sequence analyses

Abstract Bacterial communities in rice roots that developed from different nodes and at different growth stages were compared by using polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis (DGGE) analysis of 16S rDNA. Rice root samples were collected at three stages, namely tillering (July 2), maximum tillering (July 21), and ripening (September 12). The bacterial diversity in rice roots was found to increase along with the growth stages of the rice plants as well as the root age from the numbers of DGGE bands. The community structure of the bacteria was also found to change with the growth stages and root age from cluster analysis. Sequence analysis of the DGGE bands indicated that the dominant bacteria associated with rice roots were Gram-negative bacteria, especially β-Proteobacteria irrespective of the growth stages and root age. DGGE bands related to Janthinobacterium agaricidamnosum W1r3T and Clostridium sp. FCB90-3 were ubiquitous in many roots irrespective to the sampling date. Principal component analysis enabled to characterize the DGGE bands related to nitrogen-fixing Azoarcus spp., and Azovibrio sp. BS20-3 in the samples collected on July 2 and on July 21, and the myxobacteria collected on September 12, respectively, as representative bacteria in the bacterial communities. The habitat around older rice roots at every sampling date was more reductive than that around younger rice roots, and the DGGE bands related to Spirochaeta spp. were specific in older roots at every sampling date. Some specific bacteria that were most closely related to the DGGE bands were found from principal component analysis to characterize young and old. roots at each growth stage as follows: aerobes Flavobacterium sp. 90 clone 2 and Janthinobacterium agaricidamnosus W1r3T in young roots and facultative anaerobes Dechloromonas sp. MissR and Anaeromyxobacter dehalogenans 2CP-3 in old nodal roots on July 2, strict anaerobe Geobacter pelophilus Dfr2 and aerobes Nitrosospira sp. Nsp17 and uncultured Nitrospira sp. clone 4-1 in old roots on July 21, and different Clostridium spp. in both young and old roots and Desulfovibrio magneticus RS-1 in old roots on September 12, respectively. A larger number of the closest relatives of anaerobic bacteria grew at the late stage than at the early stages, and in old roots than in younger roots. Thus, the environment of paddy roots was remarkably heterogeneous as a bacterial habitat, where not only the whole root system but also a root may create oxic and anoxic environments.

Distribution of Archaea in Japanese patients with periodontitis and humoral immune response to the components

There is controversy regarding the existence of archaeal pathogens. Periodontitis is one of the human diseases in which Archaea have been suggested to have roles as pathogens. This study was performed to investigate the distribution of Archaea in Japanese patients with periodontitis and to examine the serum IgG responses to archaeal components. Subgingival plaque samples were collected from 111 periodontal pockets of 49 patients (17 with aggressive periodontitis and 32 with chronic periodontitis), and 30 subgingival plaque samples were collected from 17 healthy subjects. By PCR targeting the 16S rRNA gene, Archaea were detected in 15 plaque samples (13.5% of total samples) from 11 patients (29.4% of patients with aggressive periodontitis and 18.8% of patients with chronic periodontitis). Archaea were detected mostly (14/15) in severe diseased sites (pocket depth > or =6 mm), while no amplicons were observed in any samples from healthy controls. Sequence analysis of the PCR products revealed that the majority of Archaea in periodontal pockets were a Methanobrevibacter oralis-like phylotype. Western immunoblotting detected IgG antibodies against M. oralis in eight of the 11 sera from patients. These results suggest the potential of Archaea (M. oralis) as an antigenic pathogen of periodontitis.

Characterization of Methanosarcina mazeii TMA Isolated from a Paddy Field Soil

We isolated a methanogenic strain, designated as strain TMA (=DSM 9195), from an enrichment culture inoculated with a Japanese paddy field soil. Strain TMA was Gram positive and strictly anaerobic. Cell shape was pseudosarcina-like, and cells were nonmotile. The strain was able to use methylamines, methanol, H2−CO2, and acetate as substrates for methanogenesis, but did not utilize formate. The optimum temperature and optimum pH were 30–37°C and 6.5–7.5 respectively. The G+C content of the DNA was 42.1 mol %. Strain TMA had DNA-DNA hybridization values of more than 80% with Methanosarcina mazeii S-6T (T = type strain). On the basis of phenotypic and genotypic characteristics, we identified strain TMA as M. mazeii. This is the first methylotrophic methanogen isolated from a paddy field soil and identified to the species level.