Phosphorus (P) is an essential macronutrient for plant growth and is mainly present in agricultural soil in unavailable forms. Phosphate-solubilizing microorganisms (PSMs) increase soil P availability. The objective of the present study was to assess the population and type of PSMs and their relationships with soil characteristics in the agricultural soil of Manokwari. Twenty-one composite soil samples (0–20‍ ‍cm) were collected at the rhizospheres of plants in the Prafi and Masni Districts. A dilution technique and plate count method on Pikovskayas agar medium were used to examine the PSM population, phosphate-solubilizing index (PSI), and various soil properties. The results obtained showed that the total population of phosphate-solubilizing bacteria ranged between 25×10³ and 550×10³ CFU g^–1 of soil, while that of phosphate-solubilizing fungi was between 2.0×10³ and 5.0×10³ CFU g^–1 of soil at all locations. The PSI of the isolates ranged between 1.1 to 3.6‍ ‍mm, with the most efficient and highest PSI being obtained for Bacillus sp. (strain 8) and the lowest for Pseudomonas sp. (strain 15). Six isolates found at all locations were identified at the genus level: Chromobacterium sp., Pseudomonas sp., Bacillus sp., Micrococcus sp., Caulobacter sp., and Aspergillus sp. A correlation was observed between the number of PSMs and the level of soil P availability and moisture content, indicating an increase in soil P availability with a greater abundance of PSMs in soil.

Methane-oxidizing bacteria (MOB) are ubiquitous and play an important role in the mitigation of global warming by reducing methane. MOB are commonly classified into Type I and Type II, belonging to Gammaproteobacteria and Alphaproteobacteria, respectively, and the diversity of MOB has been examined. However, limited information is currently available on favorable environments for the respective MOB. To investigate the environmental factors affecting the dominant type in the MOB community, we performed MOB enrichment using down-flow hanging sponge reactors under 38 different environmental conditions with a wide range of methane (0.01–80%) and ammonium concentrations (0.001–2,000‍ ‍mg N L^–1) and pH 4–7. Enrichment results revealed that pH was a crucial factor influencing the MOB type enriched. Type II was dominantly enriched at low pH (4–5), whereas Type I was dominant around neutral pH (6–7). However, there were some unusual cultivated biomass samples. Even though high methane oxidation activity was observed, very few or zero conventional MOB were detected using common FISH probes and primer sets for the 16S rRNA gene and pmoA gene amplification. Mycobacterium mostly dominated the microbial community in the biomass cultivated at very high NH₄⁺ concentrations, strongly implying that it exhibits methane oxidation activity. Collectively, the present results revealed the presence of many unknown phylogenetic groups with the capacity for methane oxidation other than the reported MOB.

In premise plumbing, microbial water quality may deteriorate under certain conditions, such as stagnation. Stagnation results in a loss of disinfectant residual, which may lead to the regrowth of microorganisms, including opportunistic pathogens. In the present study, microbial regrowth was investigated at eight faucets in a building over four seasons in one year. Water samples were obtained before and after 24 h of stagnation. In the first 100‍ ‍mL after stagnation, total cell counts measured by flow cytometry increased 14- to 220-fold with a simultaneous decrease in free chlorine from 0.17–0.36‍ ‍mg L^–1 to <0.02‍ ‍mg L^–1. After stagnation, total cell counts were not significantly different among seasons; however, the composition of the microbial community varied seasonally. The relative abundance of Pseudomonas spp. was dominant in winter, whereas Sphingomonas spp. were dominant in most faucets after stagnation in other seasons. Opportunistic pathogens, such as Legionella pneumophila, Mycobacterium avium, Pseudomonas aeruginosa, and Acanthamoeba spp., were below the quantification limit for real-time quantitative PCR in all samples. However, sequences related to other opportunistic pathogens, including L. feeleii, L. maceachernii, L. micdadei, M. paragordonae, M. gordonae, and M. haemophilum, were detected. These results indicate that health risks may increase after stagnation due to the regrowth of opportunistic pathogens.

Halomonas species, which are aerobic, alkaliphilic, and moderately halophilic bacteria, produce diverse biochemicals. To identify food-related Halomonas strains for bioremediation and the industrial production of biochemicals, 20 strains were isolated from edible seashells, shrimp, and umeboshi (pickled Japanese plum) factory effluents. All isolates were phylogenetically classified into a large clade of Halomonas species. Most isolates, which grew in wide pH (6–13) and salt concentration (0–14%) ranges, exhibited the intracellular accumulation of poly(3-hydroxybutyrate) granules. The characteristics of these isolates varied. A020 isolated from umeboshi factory effluents exhibited enhanced stress tolerance and proliferation and comprised two plasmids. IMZ03 and A020 grew to more than 200 OD₆₀₀, while IMZ03 produced 3.5% 3-hydroxybutyrate in inorganic medium supplemented with 10% sucrose. The mucus of TK1-1 cultured on agar medium comprised approximately 64‍ ‍mM of ectoine. Whole-genome sequencing of A020 was performed to elucidate its origin and genomic characteristics. The genome ana­lysis revealed a region exhibiting synteny with a large virus genome isolated from the ocean, but did not identify any predictable pathogenic genes. Therefore, saline foods and related materials may be suitable resources for isolating Halomonas strains exhibiting unique, useful, and innocuous features.

In legume–rhizobia symbiosis, partner recognition and the initiation of symbiosis processes require the mutual exchange of chemical signals. Chemicals, generally (iso)flavonoids, in the root exudates of the host plant induce the expression of nod genes in rhizobia, and, thus, are called nod gene inducers. The expression of nod genes leads to the production of lipochitooligosaccharides (LCOs) called Nod factors. Natural nod gene inducer(s) in Lotus japonicus–Mesorhizobium symbiosis remain unknown. Therefore, we developed an LCO detection method based on ultra-high-performance liquid chromatography–tandem-quadrupole mass spectrometry (UPLC-TQMS) to identify these inducers and used it herein to screen 40 phenolic compounds and aldonic acids for their ability to induce LCOs in Mesorhizobium japonicum MAFF303099. We identified five phenolic acids with LCO-inducing activities, including p-coumaric, caffeic, and ferulic acids. The induced LCOs caused root hair deformation, and nodule numbers in L. japonicus inoculated with M. japonicum were increased by these phenolic acids. The three phenolic acids listed above induced the expression of the nodA, nodB, and ttsI genes in a strain harboring a multicopy plasmid encoding NodD1, but not that encoding NodD2. The presence of p-coumaric and ferulic acids in the root exudates of L. japonicus was confirmed by UPLC-TQMS, and the induction of ttsI::lacZ in the strain harboring the nodD1 plasmid was detected in the rhizosphere of L. japonicus. Based on these results, we propose that phenolic acids are a novel type of nod gene inducer in L. japonicus–Mesorhizobium symbiosis.

Pseudomonas syringae pv. tabaci 6605 (Pta6605) is a foliar plant pathogen that causes wildfire disease on tobacco plants. It requires chemotaxis to enter plants and establish infection. While chemotactic signals appear to be the main mechanism by which Pta6605 performs directional movement, the involvement of aerotaxis or energy taxis by this foliar pathogen is currently unknown. Based on domain structures and similarity with more than 50 previously identified putative methyl-accepting chemotaxis proteins (MCPs), the genome of Pta6605 encodes three potential aerotaxis transducers. We identified AerA as the main aerotaxis transducer and found that it possesses a taxis-to-serine-and-repellent (Tsr)-like domain structure that supports a periplasmic 4HB-type ligand-binding domain (LBD). The secondary aerotaxis transducer, AerB, possesses a cytosolic PAS-type LBD, similar to the Aer of Escherichia coli and Pseudomonas aeruginosa. Aerotaxis ability by single and double mutant strains of aerA and aerB was weaker than that by wild-type Pta6605. On the other hand, another cytosolic PAS-type LBD containing MCP did not make a major contribution to Pta6605 aerotaxis in our assay system. Furthermore, mutations in aerotaxis transducer genes did not affect surface motility or chemotactic attraction to yeast extract. Single and double mutant strains of aerA and aerB showed less colonization in the early stage of host plant infection and lower biofilm production than wild-type Pta6605. These results demonstrate the presence of aerotaxis transducers and their contribution to host plant infection by Pta6605.

An endpoint recombination amplification reaction (RPA) assay for assessing the abundance of the gene encoding thiocyanate dehydrogenase (TcDH) in Thiohalobacter has been developed. The RPA reaction was performed at 37°C for 30‍ ‍min, terminated by the addition of sodium dodecyl sulfate (SDS) solution, and the DNA concentration of the RPA product was fluorometrically measured. The abundance of TcDH in 22 activated sludge samples and 7 thiocyanate-degrading enrichment cultures ranged between 2.5×10³ and 1.5×10⁶ copies μL^–1, showing a linear relationship (R²=0.83) with those measured using a conventional quantitative PCR assay.

Indole-3-acetic acid (IAA) is an exogenous growth regulatory signal that is produced by plants and various microorganisms. Microorganisms have been suggested to cross-communicate with each other through IAA-mediated signaling mechanisms. The IAA-induced tolerance response has been reported in several microorganisms, but has not yet been described in Saccharomycetales yeasts. In the present study, three common stressors (heat, osmotic pressure, and ethanol) were examined in relation to the influence of a pretreatment with IAA on stress tolerance in 12 different lineages of Saccharomyces cerevisiae. The pretreatment with IAA had a significant effect on the induction of ethanol tolerance by reducing the doubling time of S. cerevisiae growth without the pretreatment. However, the pretreatment did not significantly affect the induction of thermo- or osmotolerance. The IAA pretreatment decreased the lethal effects of ethanol on S. cerevisiae cells. Although yeasts produce ethanol to outcompete sympatric microorganisms, IAA is not a byproduct of this process. Nevertheless, the accumulation of IAA indicates an increasing number of microorganisms, and, thus, greater competition for resources. Since the “wine trait” is shared by both phylogenetically related and distinct lineages in Saccharomycetales, we conclude that IAA-induced ethanol tolerance is not specific to S. cerevisiae; it may be widely detected in both pre-whole genome duplication (WGD) and post-WGD yeasts belonging to several genera of Saccharomycetales.

Spores are a stress-resistant form of Bacillus spp., which include species that are plant growth-promoting rhizobacteria (PGPR). Previous studies showed that the inoculation of plants with vegetative cells or spores exerted different plant growth-promoting effects. To elucidate the spore-specific mechanism, we compared the effects of viable vegetative cells, autoclaved dead spores, and viable spores of Bacillus pumilus TUAT1 inoculated at 10⁷ CFU plant^–1 on the growth of the C4 model plant, Setaria viridis A10.1. B. pumilus TUAT1 spores exerted stronger growth-promoting effects on Setaria than on control plants 14 days after the inoculation. Viable spores increased shoot weight, root weight, shoot length, root length, and nitrogen uptake efficiency 21 days after the inoculation. These increases involved primary and crown root formation. Additionally, autoclaved dead spores inoculated at 10⁸ or 10⁹ CFU plant^–1 had a positive impact on crown root differentiation, which increased total lateral root length, resulting in a greater biomass and more efficient nitrogen uptake. The present results indicate that an inoculation with viable spores of B. pumilus TUAT1 is more effective at enhancing the growth of Setaria than that with vegetative cells. The plant response to dead spores suggests that the spore-specific plant growth-promoting mechanism is at least partly independent of symbiotic colonization.

Membrane vesicles (MVs) released from the bacterium Paracoccus denitrificans Pd1222 are enriched with the quorum sensing (QS) signaling molecule N-hexadecanoyl-l-homoserine lactone (C16-HSL). However, the biogenesis of MVs in Pd1222 remains unclear. Investigations on MV formation are crucial for obtaining a more detailed understanding of the dynamics of MV-assisted signaling. In the present study, live-cell imaging showed that P. denitrificans Pd1222 produced MVs through cell lysis under DNA-damaging conditions. DNA sequencing of MVs and a transcriptome ana­lysis of cells indicated that the expression of a prophage region was up-regulated at the onset of MV formation under DNA-damaging conditions. A further sequence ana­lysis identified a putative endolysin (Pden_0381) and holin (Pden_0382) in the prophage region. The expression of these genes was regulated by RecA. Using gene knockout mutants, we showed that prophage-encoded endolysin was critical for MV formation by P. denitrificans Pd1222 under DNA-damaging conditions. MV triggering by endolysin was dependent on the putative holin, which presumably transported endolysin to the periplasmic space. C16-HSL quantification revealed that more signals were released into the milieu as a consequence of the effects of endolysin. Using a QS reporter strain, we found that the QS response in P. denitrificans was stimulated by inducing the expression of endolysin. Collectively, these results provide novel insights into the mechanisms by which a bacterial cell-to-cell communication system is manipulated by phage genes.

Pea wilt disease, caused by the soilborne and seedborne fungal pathogen Fusarium oxysporum f. sp. pisi (Fop), first appeared in Japan in 2002. We herein investigated the molecular characteristics of 16 Fop isolates sampled from multiple locations and at different times in Japan. The 16 isolates were divided into three clades in molecular phylogenic ana­lyses based on both the TEF1α gene and the rDNA-IGS region. All of the Fop isolates harbored a PDA1 gene, which encodes the cytochrome P450 pisatin demethylase (Pda1), and also carried one or both of the SIX6 and SIX13 genes, which encode secreted in xylem (Six) proteins. Other forms of F. oxysporum and other species of Fusarium did not carry these sets of genes. Based on these results, a PCR method was developed to identify Fop and differentiate it from other forms and non-pathogenic isolates of Fusarium spp. We also demonstrated that the PCR method effectively detected Fop in infected pea plants and infested soils.