Sugarcane for traditional Wasanbon sugar has long been cultivated in Higashi-Kagawa. Three sugarcane cultivars were cultivated at three sites in Kagawa Prefecture, and root-associated bacterial communities were analyzed using 16S rRNA gene amplicon sequencing. Bacterial community compositions varied primarily according to the cultivation site rather than the cultivar, and soil bacterial communities showed similar site-dependent clustering patterns. These results indicate that the cultivation site affects the root-associated bacterial community structure.
The plant microbiota plays important roles in plant responses to biotic and abiotic stresses. Wasabi (Eutrema japonicum), a traditional crop indigenous to Japan, remains poorly characterized in terms of its associated bacterial community. In the present study, 16S rRNA gene amplicon sequencing revealed distinct bacterial community structures across plant and soil compartments. The rhizome microbiota, the edible plant organ used as a spice, exhibited intermediate characteristics between those of the root and leaf microbiota. These results provide fundamental insights into the wasabi-associated bacterial microbiota and identify the rhizome as a distinct microbial niche.
Individuals who regularly handle specific chemicals may harbor microorganisms capable of metabolizing those compounds. Such hands may therefore provide a practical route for obtaining functional microorganisms from easily accessible human-associated environments. In this study, we found that nearly 90% of professional dyers harbored dye-decolorizing bacteria, which were efficiently isolated from their fingertips, with 20% of isolates exhibiting decolorizing activity. These strains showed distinct genus-level profiles. These findings indicate that occupational exposure to dyes strongly enriches dye-decolorizing bacteria, suggesting that the hands of textile workers may serve as a source for isolating microorganisms with potential applications in dye bioremediation.
Since 2018, foot rot disease caused by Diaporthe destruens has emerged as a serious threat to sweet potato production in Japan. Although plant-associated bacteria have been suggested to contribute to disease suppression, the bacterial community structure of asymptomatic sweet potato tubers in foot rot-infested fields remains unclear. In the present study, we analyzed the bacterial communities of asymptomatic tubers collected adjacent to infected tubers from four foot rot-infested fields. Thirteen bacterial taxa with >1% relative abundance were detected, among which Enterobacter (ASV-129 sequence; 427 bp) was dominant, accounting for an average of 49.4% of total 16S rRNA gene amplicon reads. Forty bacterial strains were isolated from asymptomatic sweet potato tubers using R2A agar plates. Among them, strain 12CK showed 100% sequence identity to ASV-129 and was identified as Enterobacter hormaechei based on a genome anal-ysis (genome size: 4,832,483 bp). In the present study, D. destruens 2YO was also isolated from infected plants, and its pathogenicity was confirmed. In growth inhibition assays (dual culture assay), E. hormaechei 12CK strongly inhibited the growth of D. destruens 2YO. Pot and field trials will be required to examine the applicability of strain 12CK as a microbial biocontrol agent.
Pseudomonas protegens, a member of the P. fluorescens complex, is a key biocontrol bacterium with well-documented potential to protect plants against diverse pathogens. Although P. protegens strains have been widely examined globally, those originating from Japan have not been well described. In this study, we isolated and characterized a new P. protegens strain, GSF-73, from the rhizosphere of Allium fistulosum in Gifu, Japan, and assessed its performance against several major plant diseases. Draft genome sequencing produced a 7.13-Mbp assembly, and average nucleotide identity values of 97.81-98.26% with reference strains (Cab75, CHA0, and Pf-5) confirmed its species identity. A comparative genomic analysis showed that GSF-73 possessed a larger genome than the reference strains, containing eight conserved biosynthetic gene clusters for antimicrobial compounds and an expanded set of strain-specific genes related to metabolism, regulation, mobilome functions, and secretion. GSF-73 exhibited broadspectrum antagonistic activity in vitro against fungal, oomycete, and bacterial pathogens. In biocontrol assays, GSF-73 significantly suppressed spinach Fusarium wilt, cucumber anthracnose on detached cotyledons, tomato bacterial wilt, and cucumber downy mildew. In contrast, root and/or seed treatments enhanced Pythium root rot in spinach and anthracnose in pot-grown cucumbers, indicating pathosystem-dependent efficacy. Despite these contrasting outcomes, GSF-73 shows strong biocontrol potential and merits further study to elucidate the mechanisms underlying both beneficial and adverse effects for its optimized use as a locally adapted biocontrol agent for Japanese agriculture.
The taxonomic composition of Daphnia microbiota is affected not only by external environmental conditions, but also by the host's internal physiological state, which is partly governed by genetic factors. However, the extent to which host genetics constrain the composition of associated bacterial communities remains unclear. In the present study, we conducted mixed-culture experiments using obligately parthenogenetic Daphnia cf. pulex individuals from genetically distinct lineages. The results obtained showed that the taxonomic composition of host-associated microbiota significantly differed between genotypes, both within and across lineages, with certain bacterial taxa being exclusive to specific genotypes. When genetically distinct hosts were co-cultured, some bacterial taxa initially exclusive to one genotype appeared in the microbiota of another, indicating the horizontal transmission of microbiota between hosts. Nevertheless, the overall taxonomic composition of microbiota was largely unaffected by the presence of genetically different hosts. These results suggest that although the horizontal transfer of microbiota occurs between different Daphnia genotypes, it is not extensive enough to override genotype-specific microbiota compositions. Therefore, in D. cf. pulex, host genetics play a major role in shaping the composition of the associated microbiota.
To investigate the NO3- tolerance of Candidatus Scalindua sp., a continuous reactor was gradually exposed to increasing NO3- concentrations up to 3,200 mg N L-1. High NH4+ and NO2- removal efficiencies were maintained up to 2,600 mg N L-1, above which performance declined and Ca. Scalindua relative abundance decreased to 0.8%. After one year of recovery, removal efficiencies exceeded 97%, whereas Ca. Scalindua relative abundance only reached 6.5%. EC50 values for NH4+ and NO2- were both 3,000 mg N L-1. We demonstrated that our enriched Ca. Scalindua population tolerated NO3- up to 2,600 mg N L-1, far exceeding the levels typically encountered in most human-derived wastewaters.
Fusarium crown and root rot (FCRR), caused by the fungus Fusarium oxysporum f. sp. asparagi, is one of the most destructive diseases affecting asparagus worldwide. Despite evidence of the suppression of FCRR by several nonpathogenic F. oxysporum sensu lato strains in laboratory experiments, biological control trials in FCRR-infested fields have not consistently demonstrated the effectiveness of these strains. Therefore, we screened nonpathogenic Fusarium spp. strains for the biological control of FCRR with the modified inoculation method considering the colonization to soil of biocontrol agents (BCAs). Two strains from different lineages (SM007 and FAoc5), inoculated by irrigating a bud-cell suspension and applying cultured wheat bran medium around the root of the transplants, effectively suppressed FCRR on young asparagus seedlings. These strains also suppressed FCRR on mature second-year plants to some extent by the application of cultured wheat bran medium as an inoculation carrier. Both strains exhibited the capacity to impede chlamydospore germination of the pathogen when glucose was incorporated into soil. Although SM007 sometimes failed to show biocontrol effects, differences in efficiency appeared to be related to the fungal density colonizing wheat bran medium. These results suggest that the screened strains interact with the pathogen and also that the amount of BCAs introduced into soil is closely related to their biocontrol efficacy.
Bacterial communities often originate from physical encounters between distinct species on solid surfaces; however, the mechanisms underlying these initial interactions remain unclear. The γ-hexachlorocyclohexane (γ-HCH; a toxic recalcitrant insecticide)-degrading consortium includes the surface-motile bacterium Cupriavidus sp. strain TKC and the γ-HCH-degrading Sphingobium sp. strain TKS. During a co-culture on R2A agar, TKC colonies exhibited directional colony growth (DCG), characterized by asymmetric expansion toward neighboring TKS colonies. When TKC colonies eventually overgrew TKS colonies, non-motile TKS cells were passively carried along the expanding TKC front, indicating that DCG facilitates the physical association and surface dispersal of Sphingobium cells. DCG activity was also observed against various strains of Sphingobium, Sphingomonas, and Novosphingobium, whereas extremely weak or no DCG activity was observed toward other bacterial groups, including representatives of α-, β-, and γ-proteobacteria as well as actinobacteria. These results indicate that TKC exhibits DCG selectively toward sphingomonads, recognizing taxon-specific cues rather than responding indiscriminately to neighboring colonies. Among the components tested, sphingosine reproducibly triggered DCG in a dose-dependent manner, and the inhibition of sphingolipid biosynthesis in Sphingobium with myriocin markedly suppressed the inducing effect. These results highlight the sphingosine-mediated induction of DCG as a molecular mechanism underlying the initial step in the spatial organization of bacterial communities on solid surfaces.
Horizontal chromosome transfer (HCT) has been demonstrated in Fusarium oxysporum. Several pathogenic F. oxysporum strains have been used as donors in HCT experiments, while the non-pathogenic strain Fo47 has mainly been employed as a recipient. It currently remains unknown whether other non-pathogenic F. oxysporum strains are recipients of mobile chromosomes. In the present study, we investigated whether the non-pathogenic strain 08C-3B, obtained from cabbage, acquired the mobile chromosomes of F. oxysporum f. sp. conglutinans strain Cong:1-1, which infects cabbage. We detected HCT between Cong:1-1 and 08C-3B in a conidial anastomosis tube (CAT) fusion-inductive medium, yielding HCT progeny strains that carried scaffolds (SCs) 8 and 9 of Cong:1-1. These progeny strains exhibited reduced colony growth on potato dextrose agar plates and produced no symptoms on cabbage. These results suggest that SC8 and/or SC9 hinder vegetative growth, but do not confer virulence to 08C-3B. We then conducted HCT experiments to assess whether the HCT progeny strain transfers the acquired chromosomes to other strains. However, no progeny strains were obtained, suggesting that 08C-3B does not function as a donor for mobile chromosomes.
Giant viruses are distinguished not only by their large particle size, but also by their extensive genomes, often reaching megabase levels. Many sequences within these genomes are considered to have been introduced by hosts, surrounding organisms, or other viruses. Since the natural hosts of many giant viruses remain unidentified, analyzing sequences potentially derived from other organisms may aid in clarifying their hosts. In the present study, we identified eukaryote-homologous sequences by isolating those not shared among viruses, an aspect previously overlooked. Our primary focus was on pandoravirus, which, with a genome size of ~2 Mb, is the largest among giant viruses. We obtained 375 BLAST hits with an average sequence identity of ~90%. Among the 102 detected species, those with higher hits included Mus musculus, Lampetra planeri, Melanogrammus aeglefinus, Lampetra fluviatilis, Scylla paramamosain, Cardiocondyla obscurior, Monodelphis domestica, Vespula pensylvanica, Micromonas pusilla, Physcomitrium patens, and Peromyscus californicus. Similar anal-yses of Cedratvirus and Pithovirus, which share an amphora-shaped particle structure with pandoraviruses, yielded fewer data (48 and 5 hits, respectively), with no common taxa at the order level. Thirteen BLAST hits exceeded 100 bp, including conserved non-coding elements (CNEs) in fish and other taxa, along with sequences of unknown functions. These results indicate the presence of short regions with sequence similarity in non-shared sequences, although direct host identification proved difficult.
Electric field-assisted (EFA) technology has been extensively employed in the realm of biodegradation. Candida tropicalis, with a high capability for degrading thiophene, has been successfully screened through electric field-assisted screening (EFAS). The present study investigated the cell density distribution of C. tropicalis at various locations within an electric field. Under an electric field, cells migrated and accumulated toward the cathode plate. Under a loading electric field intensity of 0.6 V cm-1, the concentration of cells peaked near the cathode plate and remained stable with a loading time of 10 min. Furthermore, the thiophene degradation efficiency of strain CZ60, which was screened under optimal loading voltage and time conditions, reached 91.4% after a 4-h reaction. These results establish a solid theoretical foundation for utilizing EFAS to identify biodegradable microorganisms, with potential implications for environmental remediation strategies.
We exami-ned the effects of soil moisture changes on soybean growth, yield, and the structure of soybean-nodulating bradyrhizobial communities in cultivars with different Rj genotypes. The experiment was conducted using cultivation pots with soybean cultivars Bragg (non-Rj), CNS (Rj2Rj3), D-51 (Rj3), and Fukuyutaka (Rj4). Test strains included Bradyrhizobium diazoefficiens USDA 110T, B. japonicum USDA 6T and USDA 123, and B. elkanii USDA 31. Cultivation pots were built with 15-cm ridges, and three soil moisture conditions were generated by varying the presence and placement of drainage holes on the pots. Declining soil moisture significantly reduced shoot length, shoot dry weight, root dry weight, root length, nodule number, pod number, pod dry weight, and seed number. An occupancy anal-ysis showed that USDA 110 dominated Fukuyutaka only; across treatments, it was the most abundant under high soil moisture, but significantly declined with reductions in soil moisture, where USDA 31 became dominant. A non-metric multidimensional scaling anal-ysis revealed shifts in community compositions in response to soil moisture and cultivar. Collectively, these results indicate that soybean growth, yield, and symbiosis with bradyrhizobia are strongly affected by soil moisture and also that these effects vary among cultivars.
Bradyrhizobium ottawaense has prospects as an environmentally friendly inoculant for soybean farming because of its higher N2O reductase (N2OR) activity than that of B. diazoefficiens. To examine high N2O-reducing B. ottawaense, we performed a PCR anal-ysis of nosZ genes in 8,640 soybean nodules from 68 fields in Japan. Of 384 PCR-positive nodules, we obtained 90 isolates of bradyrhizobia with B. ottawaense-type nosZ, derived exclusively from 18 fields in Gunma and Osaka prefectures. Of 77 monophyletic isolates, 73 had significantly higher N2OR activity than B. diazoefficiens USDA110. Another 13 isolates from Osaka were phylogenetically placed outside of the B. ottawaense clade with B. liaoningense or B. betae, 8 of which also exhibited significantly higher N2OR activity than B. diazoefficiens USDA110. An anal-ysis of nopP gene sequences revealed amino acid sequence variations in the NopP effector protein among these high N2O-reducing isolates, with the NopPUSDA122 type being one of the variations identified. The NopP-mediated symbiotic incompatibility of soybean host plants may eliminate nodulation by indigenous bradyrhizobia and facilitate inoculant nodulation to reduce N2O emissions. Therefore, 90 isolates and their observed NopP types are potentially important resources for N2O mitigation. Furthermore, the dense geographical map of Bradyrhizobium species based on Internal Transcribed Spacer-Restriction Fragment Length Polymorphisms (ITS-RFLP) of the 16S-23S rRNA gene from 8,640 nodules revealed the recent northward expansion of B. elkanii to central Japan potentially due to global warming. This change in indigenous soybean bradyrhizobia is important for application strategies of bradyrhizobial inoculants under field conditions.
Nitrous oxide (N2O) is a potent greenhouse gas, and the enzyme Nos catalyzes its reduction to dinitrogen (N2). Bradyrhizobium ottawaense exhibits strong N2O-reducing activity with high nosZ expression. To investigate whether promoter sequences affect nosZ expression, we constructed reciprocal promoter-swapped mutants between B. ottawaense and B. diazoefficiens. The swapping of promoters did not significantly affect expression levels. B. ottawaense mutants maintained approximately 200-fold higher expression levels than B. diazoefficiens, and the introduction of the B. ottawaense promoter into B. diazoefficiens did not increase expression levels. Therefore, the present results indicate that promoter sequence differences are not the primary factor affecting nosZ expression, suggesting regulation by other factors.
Symbiotic N2-fixing bradyrhizobia nodulate various leguminous plants and possess a large symbiosis island (SI) encoding symbiotic functions in their genomes. We obtained 30 rhizobial isolates from root nodules of the tribe Desmodieae of native leguminous plants in northern Japan. Based on their 16S rRNA gene sequences, most isolates (24/30=80%) phylogenetically belonged to Bradyrhizobium. Two isolates (LCT1 and LCT2) from Lespedeza cuneata were placed phylogenetically with Bradyrhizobium diazoefficiens USDA110T, a well-studied soybean (Glycine max [L.] Merr.) symbiont. Genomic comparisons revealed different SIs in the Met-tRNA and Val-tRNA genes on the respective genomes. In contrast, core genomic regions outside the SI regions showed strong collinearity between strains LCT2 and USDA110. Phenotypically, LCT2 formed N2-fixing root nodules on L. cuneata, an original host plant, but not on soybean, whereas USDA110 formed N2-fixing root nodules on soybean, but not on L. cuneata. Therefore, the SI variants were expected to contain the genes responsible for this host specificity. Genes relevant to the type III secretion system (T3SS) showed less homology between LCT2 and USDA110 than nod genes encoding Nod factor biosynthesis. Host plant inoculations with T3SS mutants suggested the involvement of T3SS effectors in differential host specificity. Therefore, the acquisition of distinct SI variants may confer strong host specificity through symbiotic interactions between Bradyrhizobium and host legumes. We discuss the possible pathway of symbiotic bradyrhizobial evolution and its application to the mitigation of greenhouse gas emissions.
Insertion sequences (ISs) are major drivers of genomic plasticity in rhizobia, frequently promoting local recombination events. To quantitatively compare the stability of genomic regions inside and outside of the symbiosis island, we engineered Bradyrhizobium diazoefficiens USDA122 mutants carrying a sacB/aadA counter-selectable cassette at four distinct loci-three on symbiosis island A (SymA) and one in the core genome. During 5 days of saprophytic growth, cassette deletion occurred at frequencies of up to 1.77×10-3 within SymA, whereas the deletion rate in core genomic regions was markedly lower (3.29×10-6). Within SymA, cassettes inserted adjacent to the nif and rhc clusters, where IS copies with the same orientation were enriched, were lost more frequently than those placed in other SymA regions, indicating marked intra-island variability in genomic stability. Similar yet overall lower deletion frequencies were observed in B. diazoefficiens USDA110. These results demonstrate that SymA contains genomic loci with greater susceptibility to IS-mediated rearrangements and also that such recombination events may contribute to the diversification of Bradyrhizobium symbiosis islands. Based on our comparative IS mapping in B. japonicum and B. ottawaense, we discuss the potential for the IS-mediated deletion of genome regions harboring nod genes.
The biological reduction of N2O, a potent greenhouse gas, is crucial for environmental sustainability. We developed an automated system for continuous N2O monitoring in the gas phase of a flask containing an anaerobic bradyrhizobial culture, and then exami-ned the kinetic parameters of bacterial N2O reduction. The maximum reaction rate (Vmax) was approximately 61-fold higher for Bradyrhizobium ottawaense SG09 (1,471 nmol h-1 109 cells-1) than for B. diazoefficiens USDA110 (24 nmol h-1 109 cells-1). Our kinetics anal-ysis confirmed that SG09 maintained higher N2O-reducing activity than USDA110 even at the atmospheric concentration of N2O (0.34 ppm).
A genome anal-ysis is essential for identifying valuable microbial resources for future applications. In the present study, we exami-ned potential CO2-fixing microorganisms based on the presence of the Calvin-Benson-Bassham (CBB) cycle using 6,262 bacterial and 487 archaeal genomes from available cultures in the Japan Collection of Microorganisms (JCM), a well-established culture collection, in October 2023. A total of 306 strains (147 genera, eight phyla) carried CBB cycle genes, and a literature survey showed that 74 genera had experimental evidence of autotrophic growth while 73 lacked supporting information. A phylogenetic anal-ysis of the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (RbcL) identified diverse forms (IA, IB, IC, IE, I+α, II, and III) with distinct metabolic associations; IA was associated with sulfur species oxidation and formed IC with hydrogen oxidation. Genome-based metabolic predictions identified the potential for CO2 fixation in numerous species lacking experimental evidence. Our anal-yses indicate that members of Actinomycetota harboring IE RbcL were generally associated with hydrogen oxidation, possibly by using oxygen or nitrate as an electron acceptor. Additionally, 12 species in Pseudomonadota contained photosystem II reaction center proteins (PufL and PufM), suggesting phototrophic capabilities. However, the prediction of electron donors failed in some of these species. They may use the CBB cycle to regulate the intracellular redox balance under photoheterotrophic growth. The present results reveal unrecognized autotrophic potential in JCM strains and broaden our knowledge of the diversity of CO2-fixing microorganisms. Experimental validation will clarify their roles in the global carbon cycle and their potential for biotechnological applications towards environmental sustainability.
Lettuce black root rot caused by Berkeleyomyces rouxiae occurs during the hot season in Japan, whereas black root rot in other crops often develops during cooler seasons. The present study investigated the relationship between temperature and symptom severity in lettuce and other plant species. Inoculation tests conducted with different isolates revealed that symptoms on lettuce were the most severe at 25°C, whereas those on cotton, okra, and cowpea were the most severe at 15-20°C. These results align with the seasonal occurrence of lettuce black root rot in Japan. The present study provides valuable insights for predicting and managing this disease.
