The phytosphere is an attractive habitat for microorganisms due to an abundance of nutrients and relative environmental stability. The microorganisms that occupy this habitat assist in the uptake of nutrients from soils and can exert considerable influence upon the overall health of the plant. Recent technical advances in environmental microbiology have enabled the tracing and assessment of these microorganisms using rapid and simple molecular techniques without any culture-dependent bias. We herein review the current status of these modern molecular techniques in the study of plant-associated microbes, and summarize the issues relevant to the phytosphere from the aspect of both basic and applied science.

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Quenching probe (QProbe) PCR-based methods for quantifying the abundance of Rhodocyclus-related and actinobacterial polyphosphate-accumulating organisms (PAOs) in an enhanced biological phosphorus removal (EBPR) process are described. PCR primer-QProbe sets specific for the 16S rRNA genes of Rhodocyclus-related PAOs (RPAOs) and actinobacterial PAOs (APAOs) were newly designed, and their specificity tested using database searches, a selection of pure cultures, and sludge DNA. The sets were found to be specific for most of the target sequences. QProbe PCR with the newly designed sets was used to enumerate the 16S rRNA genes of RPAOs and APAOs in a laboratory-scale EBPR sludge. The copy number of 16S rRNA genes of RPAOs was larger than that of APAOs before the phosphorus removal performance of the reactor deteriorated, and the amount of both PAOs decreased in the deterioration period. The approaches developed in this study may become a powerful tool for a high-throughput analysis of PAO abundance in EBPR processes.

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Fifteen oligonucleotide probes, targeting a broad range of microorganisms, were spotted on a DNA microarray. Specificity and reproducibility were evaluated using fluorescently labeled rRNA from 16 bacterial and eukaryal strains. The oligonucleotide microarray was then used to analyze activated sludge samples of saline industrial wastewater in which microbial communities had been characterized previously using 16S rRNA clone libraries. The results obtained were partially consistent with the results of cloning, and demonstrate the possibility of using oligonucleotide microarrays for the monitoring and characterization of saline industrial wastewater populations.

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The efficacy of a scab disease control technique that combined an antagonistic microorganism (Trichoderma asperellum strain F-288) and fertilization amelioration was examined by determining the incidence of scab disease and by using quantitative competitive quenching probe PCR (QCQP-PCR) to determine the copy number of the nec1 gene in soil. We conducted the nec1 gene analysis and a planter experiment with red-yellow soil specimens collected from the Shimabara Peninsula of Nagasaki Prefecture, Japan. The results of the planter experiment suggested the possibility of inhibiting the development of potato scab disease using an antagonistic microorganism alone for soil with a low initial copy number of the nec1 gene, and using a combination of an antagonistic microorganism and fertilization amelioration for soil with a high initial copy number. We conducted research on the relationship between the copy number of nec1 in the soil and the concentration in soil of aqueous aluminum ions, an inhibitory agent of common scab disease in fertilization amelioration. The result suggested that the incidence of the disease could be reduced if the concentration of aqueous aluminum ions in the soil was 0.1 mg/L or more.

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A No.22 consortium was isolated from contaminated soils at oilfields by repeated transfer on a mineral salts medium with the aromatic hydrocarbon fraction from crude petroleum. The consortium was able to degrade aromatic hydrocarbons at high rates. Fourteen major DNA bands were detected in the consortium by PCR-DGGE. Three strains were capable of forming colonies on agarose plates containing mineral salts and aromatic hydrocarbons, and identified as Pandoraea sp. Y1, Hyphomicrobium facile Y3, and Burkholderia multivorans Y4 by 16S rRNA gene sequencing. The consortia of three cultures; an aromatic subculture, a saturated culture transferred from the subculture, and an aromatic obtained culture from the transferred culture, were compared using PCR-DGGE, a clone library, and quantitative real-time PCR. Burkholderia species containing strain Y4 became the dominant species in the saturated culture whereas its abundance decreased in the aromatic culture. Pandoraea sp. Y1, which was of low abundance in the saturated culture, and an unisolated bacterium, Brachymonas sp. F, in contrast, increased in the aromatic culture. The aromatic hydrocarbons were degraded by Pandoraea sp. Y1 and B. multivorans Y4, whereas the saturates were only degraded by B. multivorans Y4. The results indicate that the No.22 consortium adapts well to different carbon sources through a change in its predominant species. This study provides some basic information regarding bioremediation using the consortium.

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Oligobrachia mashikoi, a frenulata, is a marine invertebrate living in an unusual habitat in Tsukumo Bay (20-25 m deep), Japan. It lacks a mouth and a gut, and instead possesses specialized cells called bacteriocytes, in which endosymbiotic bacterial cells are kept. The endosymbiosis involves specific interactions and obligatory metabolic exchanges between the host and endosymbiotic bacterial cells. Analyses of 16S rRNA genes from the endosymbiotic cells indicated that there are at least seven phylotypes of endosymbionts in O. mashikoi, and these phylotypes are closely related Gammaproteobacteria among which sequence homology was more than 97.6%. The analyses suggested that an adult worm predominantly accommodates one of the seven phylotypes. In situ hybridization targeting 16S rRNA demonstrated that the distribution patterns of the bacterial cells in adult worms were very similar among at least two phylotypes of the endosymbionts. The findings suggest that O. mashikoi has a strict selection mechanism for its endosymbiont.

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Fourteen strains of 2,4-dichrolophenoxyacetic acid (2,4-D)-degrading bacteria were isolated from soil samples from eight different sites in Japan and identified as members of the genera Burkholderia, Cupriavidus, and Sphingobium. To characterize them in view of the 2,4-D-degrading genes that they had, the strains were divided into three groups based on similarities of PCR-amplified partial nucleotide sequences of their 2,4-D-degrading genes to the sequences of well-characterized 2,4-D-degrading genes, tfdA, tfdB, and tfdC in Cupriavidus necator strain JMP134, Achromobacter xylosoxidans strain EST4002, and Sphingomonas sp. strain TFD44. The nucleotide sequences of the tfdA, tfdB, and tfdC gene homologs in eight of the 14 strains were highly similar to those in Burkholderia cepacia strain RASC. The eight strains were classified into the RASC subgroup in the EST4002 group. Southern hybridization indicated that at least one set of the putative tfdA, tfdB, and tfdC genes in each of the 14 strains was located on a replicon (ca. 90-600 kb). In particular, the RASC-type 2,4-D-degrading genes of the eight strains were located on plasmids of similar size (ca. 600 kb), and the hybridization experiments suggested that the 2,4-D-degrading genes of seven strains among the eight were located on restriction fragments of the same length. In view of the fact that the seven strains originated from six distant sites in Japan and had different 16S rRNA gene sequences, our results suggest that the RASC-type 2,4-D-degrading genes on similar plasmids were horizontally transferred among bacteria and were distributed throughout Japan.

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The phylogenetic relationship of archaea in the gut of the wood-feeding higher termite Nasutitermes takasagoensis fed with various carbon sources was investigated using a clonal analysis based on 16S rRNA gene sequences. The representative phylotypes were affiliated with the Methanomicrobiales, Methanosarcinales, Methanobacteriales, and Thermoplasmatales of the Euryarchaeota, and with the Crenarchaeota. The majority of clones obtained in this study were closely related to those obtained from other termite species, regardless of the carbon source in the feed. The relative clone frequencies of the libraries from wood powder-, xylose-, cellobiose-, and glucose-fed termites were similar to those from the library of wood-fed termites. On the other hand, the relative clone frequency of the library from xylan-fed termites in which the Thermoplasmatales predominated differed from that of the library from the wood-fed termites. These results suggest that, except for xylan, the carbohydrate components in feeds have a limited influence on archaeal community structures.

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Microbial diversity in milled rice was examined using culture-independent methods. Environmental DNA samples were extracted from commercial products of milled rice, and the intergenic spacer regions between the small and large subunit ribosomal RNA genes were amplified by ribosomal intergenic spacer analysis (RISA). The results indicated the presence of microbial communities in milled rice and subsequent sequencing of RISA amplicons has identified 17 unique microbial sequences. These include several sequences highly similar to known plant-associated microbes, including Pseudomonas fluorescens, Xanthomonas sacchari, and Pseudozyma antarctica, as well as to several uncultured bacteria. In addition, one sequence shows significant similarity to the sequence of Fusarium graminearum, a potent pathogenic fungus in foodstuffs that produces trichothecene mycotoxins. The potential presence of a pathogenic Fusarium fungus in milled rice was also suggested by a diagnostic PCR procedure that detects the genes involved in the production of trichothecene mycotoxins.

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The culturable endophytic bacteria in the leaves and roots of rice plants (Oryza sativa, cultivar Kinuhikari) cultivated on an experimental plot adjacent to a paddy field were studied during the maturation process, and a comparison with the bacteria inside rice seeds reported in a previous study was conducted. Culturable epiphytic bacteria were also isolated from the leaves and compared with the endophytic bacteria. The isolates were identified based on their 16S rRNA gene sequences. Many endophytes closely related to Bacillus, Curtobacterium, Methylobacterium, Sphingomonas and Pantoea from the leaves, and to Bacillus, Brevibacillus, Mycobacterium, Enterobacter, and Chryseobacterium from the roots were isolated. The endophytic bacterial flora of the leaves was similar to that of the seeds, but differed from that of the roots. As the rice plant matured, the ratio of Gram-negative strains among the isolates increased in the leaves (the isolates related to Curtobacterium decreased in number and those related to Methylobacterium and Pantoea increased), whereas the ratio of Gram-negative strains decreased in the roots (the isolates related to the order Rhizobiales decreased in number and were replaced by strains related to Bacillus and Brevibacillus capable of forming endospores). The isolates from the inside and surface of the leaf were closely related to 13 and 19 species, respectively. Among these isolates, the following 6 species related to Bacillus pumilus, Curtobacterium citreum (or C. flaccumfaciens, C. pusillum as the closest species), Methylobacterium aquaticum, Methylobacterium fujisawaense (or M. radiotolerans), Sphingomonas melonis and Sphingomonas yabuuchiae were common to both sites and constituted the majority at both sites.

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Pseudomonas aeruginosa strain PAO1 was attracted by ethylene. Assays of a set of mutants containing deletions in 26 potential methyl-accepting chemotaxis protein genes revealed that tlpQ (PA2654) encodes the chemoreceptor for positive chemotaxis to ethylene. Pseudomonas fluorescens, Pseudomonas putida, and Pseudomonas syringae strains also exhibited positive chemotaxis toward ethylene.

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To distinguish subgroups of Defluvicoccus-related glycogen-accumulating organisms (Defluvicoccus-related G-bacteria), new oligonucleotide probes were designed. Two of these probes, DF636 and DF827, were used successfully to detect each subgroup of Defluvicoccus-related G-bacteria. The morphological differences in clusters and cells between the DF636- and DF827-binding cells were confirmed. Interestingly, DF636-binding cells showed autofluorescence under UV light. Additionally, autofluorescence was observed in some alphaproteobacterial G-bacteria, which did not bind to any of the probes designed in this study. The results suggested that the Defluvicoccus-related G-bacteria consist of several physiologically different subspecies.

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