The endophytic colonization, nitrogen fixation, and plant growth-promoting abilities of Herbaspirillum sp. strain B501 gfp1, which is a diazotrophic endophyte isolated from wild rice, were studied after infection (at 10² and 10⁸ cells ml^-1) of seedlings of cultivated rice Oryza sativa cv. Nipponbare. Both doses resulted in colonization of the roots and stem (basal stem and leaf sheath). No colonization of leaves was observed. Higher bacterial populations were observed in the roots than stems. The bacteria colonized the intercellular spaces of the root epidermis and the spaces at the junctions of the lateral roots. They also colonized the epidermis and pericycle of the basal stem and the sub-epidermal tissues of the dermal tissue system of the leaf sheath at later stages. The colonizing bacteria incorporated significant amounts of ¹⁵N₂ into the infected plants. The inoculated plants also had higher dry weights and fresh weights than the control (uninoculated) plants.

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To develop a real-time PCR method for quantification of the abundance of cyanophages infecting Microcystis aeruginosa in aquatic environments, we characterized three cyanophage clones infecting M. aeruginosa, and compared them to the cyanophage Ma-LMM01 which was isolated previously. The clones were similar to Ma-LMM01 in morphological features and genome size. Further, the nucleotide sequences of the putative genes coding for the alpha- and beta-subunits of ribonucleotide reductase and the sheath protein from the three isolates were identical to those of Ma-LMM01. The isolates were closely related to Ma-LMM01 and designated Ma-LMM01-type phages. We designed a real-time PCR primer set to amplify a conserved region of the gene encoding the sheath protein, and quantified Ma-LMM01-type phages in environmental samples. The phages were detected when Microcystis blooms occurred, however, the amino acid sequence deduced from the nucleotide sequence of the PCR products was relatively diverse. This will be a useful tool for studies of the ecological impact of cyanophages on the Microcystis bloom. However, throughout these experiments, we did not detect any phages lytic to M. aeruginosa strain NIES298. This suggests three hypotheses: 1) diversity of host specificity in phages, 2) dominance of defective cyanophages in nature, and 3) lysogeny in the examined host strain NIES298.

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The efficiency with which lysis of five strictly anaerobic and six facultatively anaerobic bacterial species, all well-known human colonic commensals, were lysed was tested using a reference method for general metagenomic analysis and an improved method that involves higher levels of lysozyme and proteinase K, as well as the addition of achromopeptidase. Ten species were lysed with an efficiency of >80% by the reference method, while the lytic efficiency for Clostridium ramosum JCM 1298^T was <50%. The lytic efficiency of the improved method for C. ramosum JCM 1298^T was 82.5%. Similarly, five samples of human feces were tested with these methods, as well as with the QIAamp DNA stool mini kit. Although the efficiency of lysis of the microbes recovered from the fecal samples fluctuated depending on the sample in the cases of the reference method (13.3-84.6%) and QIAamp DNA stool mini kit (38.8-69.2%), the improved method gave stable and high-level lysis (>90%) for all the fecal samples. Accordingly, since the DNA samples isolated by the improved method can reflect nearly true genomic information in the microbial flora, our improved method should be applicable to metagenomic analyses, not only for bacteria in the human intestine but also for bacteria in other environments.

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Koi herpes virus (KHV) has been recognized as a worldwide cause of mortality among populations of koi Cyprinus carpio koi and common carp. KHV disease can be diagnosed by the isolation of the virus, by anatomical assessment, and by the polymerase chain reaction (PCR) assay with agarose gel electrophoresis. Among these methods, the PCR assay has advantages over other methods in accuracy, but it retains a disadvantage with respect to quantification of the KHV genome. The TaqMan real-time PCR assay (TaqMan PCR assay) using the TaqMan-Probe has recently become more commonly used for the quantification of the KHV genome. However, it remains difficult to determine whether or not an amplified sequence originated from the KHV genome, because the TaqMan-Probe is degraded after PCR amplification. To overcome this problem, we utilized a novel quenching probe (QProbe) for quantitative PCR. This probe can be used to perform a melting curve analysis, thus eliminating the likelihood of obtaining false positives for the amplified product after PCR amplification. Two QProbes were designed to target SphI-5 and thymidine kinase, and these probes successfully detected the KHV genome; the amplified sequences could thus be evaluated by melting curve analysis.

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A first assessment of anammox activity, which is a unique N₂ emission process, was conducted in samples of coastal marine sediment from Japan with a ¹⁵N tracer. The occurrence and diversity of bacteria possibly responsible for the anammox process were also evaluated by selective PCR-amplification of the 16S rRNA gene for known anammox bacteria. Anammox activity, detected by measuring ¹⁴N¹⁵N gas production, was only found in samples collected at the intertidal sand bank located at the Yodo River estuary. In the Yodo River samples, 16S rRNA gene fragments affiliated with the known anammox bacterial lineage were also recovered, and the two major phylotypes were both "Candidatus Scalindua wagneri" relatives with 95% and 98% sequence similarity. Even from the other samples in which no recognizable anammox activity was detected, 16S rRNA gene fragments related to known anammox bacteria, but not to "Ca. S. wagneri", were detected. This is the first report of anammox-mediated N₂ emission in coastal marine environments in Japan. Notably, the PCR-based analysis allowed us to discover unexpected phylogenetic diversity of anammox bacteria-related 16S rRNA gene sequences. The selective PCR primer set developed in this study could be a powerful tool to unveil the ecology of anammox bacteria in natural environments.

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Diverse 2,4-D and 2,4,5-T-degrading bacteria were isolated country-wide from ten Vietnamese soils, with or without a history of exposure to Agent Orange. The 353 degraders were phylogenetically grouped into three major categories; Burkholderias spp. (43.3% of all degraders), Sphingomonas spp. (40.2%), and Ralstonia spp. (15.3%) and two minor ones; Bradyrhizobium sp. (0.8%) and Nocardioides sp. (0.3%). The 2,4,5-T degraders, 65% of all degraders, were isolated from all soil samples and their 16S rRNA genes were the most homologous with that of Sphingomonas spp., Burkholderia spp. or Bradyrhizobium sp. The following four degradative genes were found by PCR: tfdA (tfdAα) in the Burkholderia spp., Ralstonia spp., Bradyrhizobium sp., and Nocardioides sp.; tfdB in all degraders; tftA (cadA) in the Sphingomonas spp., Burkholderia sp. and Bradyrhizobium sp.; tftC only in the Burkholderia sp. The degraders among Burkholderia spp. were isolated only from the central and southern sites, while those among Ralstonia spp. were found only at the north sites with one exception. The Sphingomonas spp. were isolated country-wide, but four phylogenetically different groups were found at one site, while only one group was found at the other five sites. At least three different plasmids that carried the tfd genes were found in the Burkholderia spp. and Ralstonia spp. without relation to the sites and the phylogenetic groups. These results suggest that the 2,4-D- and 2,4,5-T-degrading microbial consortia have spread countrywide and are diverse on a genetic as well as geographic basis.

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We studied planktonic bacterial population dynamics in response to the changing environment in a coastal system during an observation period of over 5 years using fluorescence in situ hybridization. To estimate the environmental constraint on the bacterial community, we focused on temperature, salinity, abundance of photoplankton (chlorophyll a), and dissolved organic carbon (DOC). The total number of bacteria (TDC) amounted to 3.0×10⁵ to 5.0×10⁶ cells mL^-1, with 1.0×10⁵ to 1.0×10⁶ cells mL^-1 for Bacteria, accounting for 11.8 to 74.8% of TDC, and 1.0×10⁴ to 1.0×10⁵ cells mL^-1 for Gammaproteobacteria, 1.0 to 20.8% of TDC. The abundance of Archaea, which contributed from 0.1 to 12% to TDC, ranged from 2.0×10³ to 3.0×10⁴ cells mL^-1. We found a positive relationship between environmental parameters such as temperature, salinity, chlorophyll a, and DOC and the abundance of total bacteria and Bacteria. The number of Gammaproteobacteria correlated with temperature, salinity, and chlorophyll a, but not with DOC. We suggest that increasing the temperature under eutrophic conditions will lead to high bacterial abundance and probably a change in the bacterial community.

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The growing biogeochemical and economic importance of biogenic methane contained in gas hydrates necessitates a better understanding of the microorganisms involved in the last phase of organic matter degradation. Here, the distribution and relative abundance of methylotrophic methane-producing Archaea (mMPA) were studied in an upwelling area off central Chile by rRNA dot blot hybridization. The mMPA were detected mostly during active upwelling periods, and were more abundant in the deeper layer of the water column (>50 m), where they represented ~10% of the prokaryote rRNA extractable from seawater samples in the oxygen minimum zone (OMZ). Significant correlations were found between the concentration of rRNA from mMPA and (i) nitrate concentration (r=-0.54, p=0.0392) and (ii) temperature (r=-0.51, p=0.0267). Enrichment experiments with water samples from the OMZ were carried out to evaluate the cellular viability of mMPA. These experiments showed that some of these Archaea remain viable in the planktonic environment although not essentially associated with fecal pellets or any type of compact macroaggregate. The results suggest that mMPA in the water column come mostly from sediment and that a fraction correspond to psychrophilic varieties of facultative methylotrophs.

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The effects of water availability defined as water activity (a_w) or matric water potential (ψ_m) on microbial community dynamics during the start-up operation of mesophilic fed-batch composting (FBC) of household biowaste were studied using commercially available personal composters. The community changes were monitored for 2 months by direct cell counting, quinone profiling, and 16S rRNA gene sequencing of culturable predominant bacteria. The a_w level lowered linearly with operation time and reached around 0.95 at the end of operation. During the steady-state period, a_w or ψ_m had a strong positive correlation with moisture content and ubiquinone content and negative correlation with pH, electric conductivity, and partially saturated menaquinone content. Results of 16S rRNA gene-based phylogenetic identification of the predominant isolates as well as of culture-independent quinone profiling indicated that a drastic population change from ubiquinone-containing members of Proteobacteria to Actinobacteria took place during the overall period of operation. Most of the actinobacterial isolates grew on nutrient agar with an a_w of less than 0.98, whereas none of the proteobacterial isolates did. These results suggest that water availability is an important determinant of the population shift from the Proteobacteria to Actinobacteria during the start-up operation of mesophilic fed-batch garbage composting.

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Different sugars and amino acids were added to a conducive soil to study their effects on bacterial wilt of tomato caused by Ralstonia solanacearum YU1Rif43. The compounds were selected mainly based on the utilization ability of the pathogen and added to the soil at application rates of 0.5 to 5 mg g^-1, together with an inoculation of the pathogen. While tomato seeds germinated and grew healthy in soils amended with carbohydrates at a rate of 5 mg g^-1, most of the seeds failed to germinate in soils to which serine, glycine and alanine were applied at the same rate. At 2.5 mg g^-1, the inhibitory effect on tomato germination disappeared except for methionine. Most of the compounds decreased the incidence of wilt during 30 days of cultivation, while sucrose, fructose, threonine, acetate, and glycerol had no apparent suppressive effect. The compounds that showed the most suppressive effect were glucose, proline, glutamine, serine, arginine, and lysine. The pathogen utilized glucose, proline, and glutamine, but not serine, arginine and lysine. Certain compounds stimulated microbial activity and decreased the survival of the pathogen, and thereby suppressed bacterial wilt of tomato.

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We optimized conditions for cryopreservation of a cyanophage (Ma-LMM01) infecting the toxic cyanobacterium Microcystis aeruginosa. The quality of cryopreservation was estimated by comparing the phage titer before and after preservation at 4, −80, or −196°C and with or without a cryoprotectant: glycerol, dimethyl sulfoxide (DMSO), or Cellbanker. Storage at 4°C for 100 days resulted in ~90% loss of infectivity; whereas cryopreservation at −196°C resulted in stable preservation with or without cryoprotectant. Therefore, we established methods to stably preserve the phage, Ma-LMM01, that may be useful in further studies of cyanophages and may be used in isolating new phages.

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Sixty thousand of deaths among cultured Ezo abalone Haliotis discus hannai occurred within a few days at an abalone farm in Japan in the middle of August, 2002. Dead animals were characterized by a hemolymphatic edema around the major circulatory system. Vibrios showing swarming motility dominated in the edema. The pathogenic vibrios were identified as Vibrio harveyi based on a phylogenetic analysis and a phenotypic characterization. In both immersion and injection experiments, the swarming vibrios fulfilled Koch's postulates as a pathogen for Ezo abalone. Using a GFP-tagged V. harveyi S20, a clump of bacterium was detected on the gills of the abalone within 48 hours after contact with the bacterium. This is the first report of V. harveyi infection in Ezo abalone Haliotis discus hannai.

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