In order to understand the relationships between understory bamboo and soil properties, we compared microbial community structures in the soil of a Betula ermanii boreal forest with Sasa kurilensis present and removed using high-throughput DNA sequencing. The presence of understory S. kurilensis strongly affected soil properties, including total carbon, total nitrogen, nitrate, and the C:N ratio as well as relative soil moisture. Marked differences were also noted in fungal and bacterial communities between plots. The relative abundance of the fungal phylum Ascomycota was 13.9% in the Sasa-intact plot and only 0.54% in the Sasa-removed plot. Among the Ascomycota fungi identified, the most prevalent were members of the family Pezizaceae. We found that the abundance of Pezizaceae, known to act as mycorrhizal fungi, was related to the amount of total carbon in the Sasa-intact plot. The relative abundance of Proteobacteria was significantly higher, whereas those of Planctomycetes and Actinobacteria were lower in the Sasa-intact plot than in the Sasa-removed plot. Furthermore, the results obtained suggest that some species of the phylum Planctomycetes are more likely to occur in the presence of S. kurilensis. Collectively, these results indicate that the presence of S. kurilensis affects microbial communities and soil properties in a B. ermanii boreal forest.
Termites depend nutritionally on their gut microbes, and protistan, bacterial, and archaeal gut communities have been extensively studied. However, limited information is available on viruses in the termite gut. We herein report the complete genome sequence (99,517 bp) of a phage obtained during a genome analysis of “Candidatus Azobacteroides pseudotrichonymphae” phylotype ProJPt-1, which is an obligate intracellular symbiont of the cellulolytic protist Pseudotrichonympha sp. in the gut of the termite Prorhinotermes japonicus. The genome of the phage, designated ProJPt-Bp1, was circular or circularly permuted, and was not integrated into the two circular chromosomes or five circular plasmids composing the host ProJPt-1 genome. The phage was putatively affiliated with the order Caudovirales based on sequence similarities with several phage-related genes; however, most of the 52 protein-coding sequences had no significant homology to sequences in the databases. The phage genome contained a tRNA-Gln (CAG) gene, which showed the highest sequence similarity to the tRNA-Gln (CAA) gene of the host “Ca. A. pseudotrichonymphae” phylotype ProJPt-1. Since the host genome lacked a tRNA-Gln (CAG) gene, the phage tRNA gene may compensate for differences in codon usage bias between the phage and host genomes. The phage genome also contained a non-coding region with high nucleotide sequence similarity to a region in one of the host plasmids. No other phage-related sequences were found in the host ProJPt-1 genome. To the best of our knowledge, this is the first report of a phage from an obligate, mutualistic endosymbiont permanently associated with eukaryotic cells.
The characterization of microbial communities that promote or suppress soil-borne pathogens is important for controlling plant diseases. We compared prokaryotic communities in soil with or without the signs of tomato bacterial wilt caused by Ralstonia solanacearum. Soil samples were collected from a greenhouse at two different depths because this pathogen is present in deep soil. We used samples from sites in which we detected phcA, a key gene regulating R. solanacearum pathogenicity. The pyrosequencing of prokaryotic 16S rRNA sequences in four soil samples without disease symptoms but with phcA and in two soil samples with disease symptoms indicated that community richness was not significantly different between these two soils; however, microbial diversity in the lower soil layer was higher in soil samples without disease symptoms but with phcA. A difference in prokaryotic community structures between soil samples with and without bacterial wilt was only observed in the upper soil layer despite apparent similarities in the communities at the phylum level. Proteobacteria, Acidobacteria, Chloroflexi, Verrucomicrobia, and several Archaea were more abundant in soil samples without disease symptoms, whereas taxa in another eight phyla were more abundant in soil samples with disease symptoms. Furthermore, some prokaryotic taxa were abundant specifically in the lower layer of soil, regardless of whether disease was present. These prokaryotic taxa may suppress or accelerate the pathogenesis of bacterial wilt and are good targets for future studies on disease control.
Changes in the relative abundances of the transcripts of hydA gene paralogs for [FeFe]-hydrogenase in Clostridium sp. strain H2 and Desulfovibrio sp. strain A1 isolated from paddy field soil were analyzed during H2 production. Strains H2 and A1 had at least five and two phylogenetically different hydA genes, respectively. The relative abundances of their hydA transcripts differed among the paralogs and H2 production activity changed in a manner that depended on the growth phase and conditions. Increases or decreases in the relative abundances of the transcripts of two out of five hydA genes in strain H2 correlated with changes in H2 production rates, whereas those of the others remained unchanged or decreased. In strain A1, the relative abundances of the transcripts of two hydA genes differed between monoculture, sulfate-reducing, and syntrophic, methanogenic conditions. The relative abundance of the transcripts of one hydA gene, predicted to encode a cytosolic [FeFe]-hydrogenase, was higher under syntrophic, methanogenic conditions than sulfate-reducing conditions, while that of the transcripts of the other hydA gene decreased with time under both conditions. This study showed that the transcription of the hydA gene during growth with active H2 production was differently regulated among the paralogs in H2 producers isolated from paddy field soil.
In the present study, 77 strains of endophytic actinomycetes isolated from cabbage were screened in order to assess their biocontrol potential against Alternaria brassicicola on cabbage seedlings. In the first and second screening trials, cabbage seedlings pretreated with mycelial suspensions of each isolate were spray-inoculated with A. brassicicola. Strain MBCN152-1, which exhibited the best protection in screening trials and had no adverse effects on seedling growth, was selected for the greenhouse trial. In the greenhouse trial, cabbage seedlings, which had been grown in plug trays filled with soil mix containing spores of MBCN152-1 (1×108 spores g−1 of soil mix), were spray-inoculated with A. brassicicola and grown in greenhouse conditions. MBCN152-1 reduced disease incidence and significantly increased the number of viable seedlings. The efficacy of MBCN152-1 against damping-off caused by seed-borne A. brassicicola was then evaluated. Cabbage seeds, artificially infested with A. brassicicola, were sown in soil mix containing MBCN152-1 spores. The disease was completely suppressed when infested seeds were sown in a soil mix blended with MBCN152-1 at 1.5×107 spores g−1 of soil mix. These results strongly suggest that MBCN152-1 has the potential to control A. brassicicola on cabbage plug seedlings. MBCN152-1 was identified as a Streptomyces humidus-related species based on 16S rDNA sequencing. Scanning electron microscopy showed that the hyphae of MBCN152-1 multiplied on the surface of the seedlings and penetrated their epidermal cells. In conclusion, strain MBCN152-1 is a promising biocontrol agent against A. brassicicola on cabbage plug seedlings.
Fluorescence in situ hybridization (FISH) has been employed to identify microorganisms at the single cell level under a microscope. Extensive efforts have been made to improve and extend the FISH technique; however, the development of a widely applicable protocol is a continuing challenge. The present study evaluated the effects of divalent cations in the hybridization solution on the FISH-based detection of various species of bacteria and archaea with rRNA-targeted probes. A flow cytometric analysis after FISH with a standard hybridization buffer detected positive signals from less than 30% of Escherichia coli IAM 1264 cells. However, the number of cells with positive signals increased to more than 90% after the addition of calcium chloride to the hybridization buffer. Mn2+ also had positive effects, whereas Mg2+ did not. The positive effects of Ca2+ were similarly observed for bacteria belonging to Enterobacteriaceae, including Enterobacter sakazakii IAM 12660T, E. aerogenes IAM 12348, Klebsiella planticola IAM 14202, and Salmonella enterica subsp. enterica serovar Typhimurium strain LT2. These results indicate that the supplementation of Ca2+ to the hybridization buffer for FISH contributes to the efficient detection of Enterobacteriaceae cells.
Dwarf shrubs of the family Ericaceae are common in arctic and alpine regions. Many of these plants are associated with ericoid mycorrhizal (ERM) fungi, which allow them to take nutrients and water from the soil under harsh environmental conditions and, thus, affect host plant survival. Despite the importance of ERM fungi to alpine plant communities, limited information is available on the effects of microhabitat and host identity on ERM fungal communities. We investigated the communities of putative ERM fungi isolated from five dwarf shrub species (Arcterica nana, Diapensia lapponica, Empetrum nigrum, Loiseleuria procumbens, and Vaccinium vitis-idaea) that co-occur in an alpine region of Japan, with reference to distinct microhabitats provided by large stone pine (Pinus pumila) shrubs (i.e. bare ground, the edge of stone pine shrubs, and the inside of stone pine shrubs). We obtained 703 fungal isolates from 222 individual plants. These isolates were classified into 55 operational taxonomic units (OTUs) based on the sequencing of internal transcribed spacer regions in ribosomal DNA. These putative ERM fungal communities were dominated by Helotiales fungi for all host species. Cistella and Trimmatostroma species, which have rarely been detected in ERM roots in previous studies, were abundant. ERM fungal communities were significantly different among microhabitats (R2=0.28), while the host effect explained less variance in the fungal communities after excluding the microhabitat effect (R2=0.17). Our results suggest that the host effect on ERM fungal communities is minor and the distributions of hosts and fungal communities may be assessed based on microhabitat conditions.
We investigated the effects of the water status (flooded or non-flooded) and presence of the nosZ gene in bradyrhizobia on the bradyrhizobial community structure in a factorial experiment that examined three temperature levels (20°C, 25°C, and 30°C) and two soil types (andosol and gray lowland soil) using microcosm incubations. All microcosms were inoculated with Bradyrhizobium japonicum USDA6T, B. japonicum USDA123, and B. elkanii USDA76T, which do not possess the nosZ gene, and then half received B. diazoefficiens USDA110Twt (wt for the wild-type) and the other half received B. diazoefficiens USDA110ΔnosZ. USDA110Twt possesses the nosZ gene, which encodes N2O reductase; 110ΔnosZ, a mutant variant, does not. Changes in the community structure after 30- and 60-d incubations were investigated by denaturing-gradient gel electrophoresis and an image analysis. USDA6T and 76T strains slightly increased in non-flooded soil regardless of which USDA110T strain was present. In flooded microcosms with the USDA110Twt strain, USDA110Twt became dominant, whereas in microcosms with the USDA110ΔnosZ, a similar change in the community structure occurred to that in non-flooded microcosms. These results suggest that possession of the nosZ gene confers a competitive advantage to B. diazoefficiens USDA110T in flooded soil. We herein demonstrated that the dominance of B. diazoefficiens USDA110Twt within the soil bradyrhizobial population may be enhanced by periods of flooding or waterlogging systems such as paddy–soybean rotations because it appears to have the ability to thrive in moderately anaerobic soil.
The aim of the present study was to obtain insights into the relationship between the chemical (salt content and pH) and physico-mechanical (humidity and compressive strength) properties of mineral-based materials from historic buildings with salt efflorescence and the growth and biodiversity of halophilic microorganisms. Samples were mainly characterized by pH 6.5–8.5 and a moisture content of between 0.12 and 3.3%. Significant variations were also found in the salt content (sulfates, chlorides, and nitrates) of the materials. An SEM/EDS analysis of material surfaces revealed the presence of halite, calcite, gypsum, sodium sulfate, and potassium-sodium sulfate. Culture-dependent and culture-independent (clone library construction) approaches were both applied to detect halophilic microorganisms. Results derived from culturable methods and the materials analysis revealed a correlation between the total halophile count and pH value as well as sulfate content. A correlation was not observed between the concentration of chlorides or nitrates and the number of halophilic microorganisms. The materials studied were inhabited by the culturable halophilic bacteria Halobacillus sp., Virgibacillus sp., and Marinococcus sp. as well as the yeast Sterigmatomyces sp., which was isolated for the first time from mineral materials. Culture-independent techniques revealed the following bacterial species: Salinibacterium, Salinisphaera, Rubrobacter, Rubricoccus, Halomonas, Halorhodospira, Solirubrobacter, Salinicoccus, and Salinibacter. Biodiversity was the highest in materials with high or moderate salinity.
Extracellular DNA (exDNA) is released from bacterial cells through various processes. The antibiotic resistance genes (ARGs) coded on exDNA may be horizontally transferred among bacterial communities by natural transformation. We quantitated the released/leaked tetracycline resistance gene, tet(M) over time under grazing stress by ciliates and heterotrophic nanoflagellates (HNFs), and found that extracellular tet(M) (ex-tetM) increased with bacterial grazing. Separate microcosms containing tet(M)-possessing bacteria with ciliates or HNFs were prepared. The copy number of ex-tetM in seawater in the ciliate microcosm rapidly increased until 3 d after the incubation, whereas that in the HNF microcosm showed a slower increase until 20 d. The copy number of ex-tetM was stable in both cases throughout the incubation period, suggesting that extracellular ARGs are preserved in the environment, even in the presence of grazers. Additionally, ARGs in bacterial cells were constant in the presence of grazers. These results suggest that ARGs are not rapidly extinguished in a marine environment under grazing stress.
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.
Fig. 4. Growth degree of Fusarium oxysporum f. sp. spinaciae for organic fertilizers at each dilution based on an estimation of the ellipse area (A) and extension length (B) of the colony. Cont, compound inorganic fertilizer; SBM, steamed bone meal; CDC, cow dung compost; MI, microbial inoculant. Values show mean of medians of degrees with SE (n=3). The ellipse area and extension length for control plates were 4,475 mm2 and 36.5 mm, respectively.
Fig. 4. Growth degree of Fusarium oxysporum f. sp. spinaciae for organic fertilizers at each dilution based on an estimation of the ellipse area (A) and extension length (B) of the colony. Cont, compound inorganic fertilizer; SBM, steamed bone meal; CDC, cow dung compost; MI, microbial inoculant. Values show mean of growth degrees with SE (n=3). The ellipse area and extension length for control plates were 4,475 mm2 and 36.5 mm, respectively.
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Fig. 5. Growth degree of Fusarium oxysporum f. sp. spinaciae for soil applied with organic fertilizers at each dilution based on an estimation of the ellipse area (A) and extension length (B) of the colony. Cont, compound inorganic fertilizer; SBM, steamed bone meal; CDC, cow dung compost; MI, microbial inoculant. Values show the mean of medians of degrees with SE (n=3). The ellipse area and extension length for control plates were 5,473 mm2 and 40.5 mm, respectively.
Fig. 5. Growth degree of Fusarium oxysporum f. sp. spinaciae for soil applied with organic fertilizers at each dilution based on an estimation of the ellipse area (A) and extension length (B) of the colony. Cont, compound inorganic fertilizer; SBM, steamed bone meal; CDC, cow dung compost; MI, microbial inoculant. Values show the mean of growth degrees with SE (n=3). The ellipse area and extension length for control plates were 5,473 mm2 and 40.5 mm, respectively.
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Fig. 7. Growth degree of Fusarium oxysporum f. sp. spinaciae at each dilution based on an estimation of the ellipse area (A) and extension length (B) of the colony. Cont, compound inorganic fertilizer; SBM, steamed bone meal; CDC, cow dung compost; MI2, microbial inoculant applied with 2,000 kg ha−1; MI10, microbial inoculant applied with 10,000 kg ha−1. Values show the mean of medians of degrees with SE (n=3). The ellipse area and extension length for control plates were 3,404 mm2 and 29.7 mm, respectively.
Fig. 7. Growth degree of Fusarium oxysporum f. sp. spinaciae at each dilution based on an estimation of the ellipse area (A) and extension length (B) of the colony. Cont, compound inorganic fertilizer; SBM, steamed bone meal; CDC, cow dung compost; MI2, microbial inoculant applied with 2,000 kg ha−1; MI10, microbial inoculant applied with 10,000 kg ha−1. Values show the mean of growth degrees with SE (n=3). The ellipse area and extension length for control plates were 3,404 mm2 and 29.7 mm, respectively.
The authors would like to apologize for these corrections and any inconvenience caused.