石澤 秀紘

ABSTRACT We report the complete genome sequences of six bacterial strains isolated from a floating macrophyte, duckweed. These six strains, representing the six dominant families of the natural duckweed microbiome, establish a simple model ecosystem when inoculated onto sterilized duckweed. Their genomes would provide insights into community assembly in plant microbiome.

Understanding the assembly of multispecies microbial communities represents a significant challenge in ecology and has wide applications in agriculture, wastewater treatment, and human healthcare domains. Traditionally, studies on the microbial community assembly focused on analyzing pairwise relationships among species; however, neglecting higher-order interactions, i.e., the change of pairwise relationships in the community context, may lead to substantial deviation from reality. Herein, we have proposed a simple framework that incorporates higher-order interactions into a bottom–up prediction of the microbial community assembly and examined its accuracy using a seven-member synthetic bacterial community on a host plant, duckweed. Although the synthetic community exhibited emergent properties that cannot be predicted from pairwise coculturing results, our results demonstrated that incorporating information from three-member combinations allows the acceptable prediction of the community structure and actual interaction forces within it. This reflects that the occurrence of higher-order effects follows consistent patterns, which can be predicted even from trio combinations, the smallest unit of higher-order interactions. These results highlight the possibility of predicting, explaining, and understanding the microbial community structure from the bottom–up by learning interspecies interactions from simple beyond-pairwise combinations.

We have completed a system that can achieve both deep x-ray lithography and submicron x-ray lithography with a single beamline by introducing the combination of x-ray plane and cylindrical mirrors. This x-ray lithography system can provide a large-scale microfabrication processing with 210 × 300 mm2 (A4 size). To exploit multiscale lithography, the beamline has a beam transport vacuum duct with a two-stage stacked structure and a 5-axis stage. This two-stage stacked structure allows us to fabricate both micron scale structures with high aspect ratios and submicron scale structures using the same beamline. In addition, x-ray imaging and computer tomography (CT) system are connected to the x-ray lithography system for nondestructive inspection and evaluation of the fabricated microstructures. The x-ray imaging system constructed this study has a relatively low energy range of x-ray energy in the beamline, which is in the range of 2–15 keV or less. Therefore, relatively good absorption contrast can be obtained for plastic materials, biomaterials, and the like. Since nondestructive imaging of the processed shape by x-ray lithography is possible, it is a very useful system in processing and evaluation can be performed simultaneously. This system also enables us to obtain the live images with keeping the creature alive in liquid using an indirect x-ray imaging system which converts x-ray images to visible light images through the fluorescent plate.

We report the complete genome sequences of two predatory bacterial strains, Bacteriovorax sp. HI3 and Myxococcus sp. MH1, which were isolated from a freshwater pond. These two strains are grouped with the Bdellovibrio and like organisms and myxobacteria, respectively. Their genomes expand our knowledge of the characteristics of predatory bacteria.

Predatory bacteria, which prey on other bacteria, have significant functions in microbial ecosystems and have attracted increasing attention for their biotechnological use. However, knowledge of the characteristics of wild-type environmental predatory bacteria remains limited. This study isolated two predatory bacteria, Bacteriovorax stolpii HI3 and Myxococcus sp. MH1, from a freshwater pond and characterized their predation capabilities. Determination of the prey range using 53 potential prey strains, including 52 environmental strains, revealed that B. stolpii HI3 and Myxococcus sp. MH1 could prey on a wide spectrum of Gram-negative bacteria and a broader range of bacteria, irrespective of phylogeny, in accordance with the common characteristics of Bdellovibrio and like organisms and myxobacteria, respectively. Liquid culture assays also found that although predation by B. stolpii HI3 rapidly and largely occurred, the prey bacteria regrew, possibly through plastic phenotypic resistance to predation. In contrast, predation by Myxococcus sp. MH1 occurred at relatively low efficiency but was longer lasting. The two strains exhibited slightly distinct temperature preferences but commonly preferred slightly alkaline pH. The novel findings of this study provide evidence for the coexistence of predatory bacteria with diverse predation capabilities in the natural aquatic environment.

<title>Abstract</title>Bacterial communities associated with aquatic macrophytes largely influence host primary production and nutrient cycling in freshwater environments; however, little is known about how specific bacteria migrate to and proliferate at this unique habitat. Here, we separately identified bacterial genes involved in the initial colonization and overall fitness on plant surface, using the genome-wide transposon sequencing (Tn-seq) of <italic>Aquitalea magnusonii</italic> H3, a plant growth-promoting bacterium of the floating macrophyte, duckweed. Functional annotation of identified genes indicated that initial colonization efficiency might be simply explained by motility and cell surface structure, while overall fitness was associated with diverse metabolic and regulatory functions. Genes involved in lipopolysaccharides and type-IV pili biosynthesis showed different contributions to colonization and fitness, reflecting their metabolic cost and profound roles in host association. These results provide a comprehensive genetic perspective on aquatic-plant-bacterial interactions, and highlight the potential trade-off between bacterial colonization and proliferation abilities on plant surface.

Plant growth-promoting bacteria (PGPB) have recently been demonstrated as a promising agent to improve wastewater treatment and biomass production efficiency of duckweed hydrocultures. For their reliable use in aqueous environments, this study analyzed the plant colonization dynamics of PGPB and its ecological consequence on the entire duckweed-associated bacterial communities. A PGPB strain, Aquitalea magnusonii H3, was inoculated to duckweed at different cell densities or timings in the presence of three environmental bacterial communities. The results showed that strain H3 improved duckweed growth by 11.7-32.1% in five out of nine experiments. Quantitative-PCR and amplicon sequencing analyses showed that strain H3 successfully colonized duckweed after 1 and 3 d of inoculations in all cultivation tests. However, it significantly decreased in numbers after 7 d, and similar bacterial communities were observed on duckweed regardless of H3 inoculation. Predicted metagenome analysis suggested that genes related to bacterial chemotactic motility and surface attachment system are consistently enriched through community assembly on duckweed. Taken together, strain H3 dominantly colonized duckweed for a short period and improved duckweed growth. However, the inoculation of the PGPB did not have a lasting impact due to the strong resilience of natural duckweed microbiome.

© 2019 Elsevier Ltd Plant growth-promoting bacteria (PGPB) are considered a promising tool to improve biomass production and water remediation by the aquatic plant, duckweed; however, no effective methodology is available to utilize PGPB in large hydroponic systems. In this study, we proposed a two-step cultivation process, which comprised of a “colonization step” and a “mass cultivation step,” and examined its efficacy in both bucket-scale and flask-scale cultivation experiments. We showed that in the outdoor bucket-scale experiments using three kinds of environmental water, plants cultured through the two-step cultivation method with the PGPB strain, Acinetobacter calcoaceticus P23, yielded 1.9 to 2.3 times more biomass than the control (without PGPB inoculation). The greater nitrogen and phosphorus removals compared to control were also attained, indicating that this strategy is useful for accelerating nutrient removal by duckweed. Flask-scale experiments using non-sterile pond water revealed that inoculation of strain P23 altered duckweed surface microbial community structures, and the beneficial effects of the inoculated strain P23 could last for 5–10 d. The loss of the duckweed growth-promoting effect was noticeable when the colonization of strain P23 decreased in the plant. These observations suggest that the stable colonization of the plant with PGPB is the key for maintaining the accelerated duckweed growth and nutrient removal in this cultivation method. Overall, our results suggest the possibility of an improved duckweed production using a two-step cultivation process with PGPB.

© 2019, Springer Nature Switzerland AG. Aims: We aimed to compare uptake and litter flux of silicon (Si) across tropical tree species and sites on Mt. Kinabalu, Borneo. Methods: Si flux components were measured at eight plots in tropical forests at four altitudes (700–3,100 m above sea level) on two types of parent materials (acidic sedimentary/granite rock and ultramafic igneous rock, paired at each elevation). Results: Leaf Si concentration differed substantially among tree species (0.24–13.6 mg g−1). Species with high leaf Si concentrations occurred mostly in the lowest elevation plots on both parent materials. The abundance-weighted community means of live-leaf Si concentrations, as well as Si concentration in leaf litter, declined with increasing elevation. Annual leaf-litter Si flux and water extractable Si per unit air-dried soil also decreased with increasing elevation, whereas parent material type had little influence despite consistently higher pH by 0.5–1.0 unit in ultramafic soil. Conclusions: Si uptake and litter Si flux were greater in lower elevation forests regardless of parent material types, most likely because Si accumulating species are more abundant in lowland tropical forests. These results suggest a stronger role of biota than the geochemistry of parent material and rock weathering in Si cycling in the forests on Mt. Kinabalu.

© 2019 The Ecological Society of Japan Tropical tree species differ widely in their silicon (Si) accumulation patterns, but the implications of such species differences for ecosystem Si flux remain unexplored. We analyzed how biogenic Si flux via leaf litter and soil Si availability in the upper soil vary spatially within a 2 ha area in a lowland Dipterocarp forest in Peninsular Malaysia. Silicon concentration per unit leaf dry mass of 86 dominant tree species in this plot ranged from 0.4 to 126 mg/g. Soil Si availability at 100 sampling points and Si flux via leaf litter at 66 litter traps showed large variations up to 3.3- and 17.9-folds, respectively. However, they correlated neither with each other nor with the expected Si flux from tree species composition in the neighborhood. When we classified tree species to four groups by their leaf Si concentrations (negligible, low, moderate and high), tree species with moderate leaf Si concentrations were significantly more abundant on Si-rich soils. In contrast, the other three groups did not show any significant spatial relationships with soil Si availability. These results indicate that soil Si availability, Si accumulation by vegetation and Si flux via leaf litter show wide spatial variations in a lowland tropical forest, without a simple relationship with each other.

© 2019 Elsevier Ltd The effectiveness of a plant growth-promoting bacterium (PGPB) of duckweed, Aquitalea magnusonii H3, was studied under treatment with various abiotic stress factors repressing host plant growth (copper, zinc, salinity, weak light, low temperature, and nutrient scarcity). In the absence of stress factors, strain H3 stably improved the weekly yield of duckweed (Lemna minor) by 5.2–8.1% relative to aseptic control plants. However, in the presence of stress factors that reduced the duckweed weekly yield by approximately 40%, the performance of strain H3 changed both positively and negatively depending on the stress factor. Specifically, the growth promotion of strain H3 increased to 17.2% and 14.3% when the plant was subjected to copper and zinc stresses, respectively. Further evidence of increased plant accumulation of copper and zinc in the presence of strain H3 suggested that strain H3 enhanced the plant's capacity to both accumulate and tolerate these heavy metals. Conversely, the effects of strain H3 were inhibitory for duckweed growth under high salinity and nutrient-poor conditions. Additionally, other stress factors (weak light and low temperature) had no significant influence on PGPB performance. These results suggest that notably higher or lower performance of PGPB can be expressed under different kinds of plant stress factors.

© 2019, Springer Science+Business Media, LLC, part of Springer Nature. Despite the considerable role of aquatic plant-associated bacteria in host plant growth and nutrient cycling in aquatic environments, the mode of their plant colonization has hardly been understood. This study examined the colonization and competition dynamics of a plant growth-promoting bacterium (PGPB) and two plant growth-inhibiting bacteria (PGIB) in the aquatic plant Lemna minor (common duckweed). When inoculated separately to L. minor, each bacterial strain quickly colonized at approximately 10 6 cells per milligram (plant fresh weight) and kept similar populations throughout the 7-day cultivation time. The results of two-membered co-inoculation assays revealed that the PGPB strain Aquitalea magnusonii H3 consistently competitively excluded the PGIB strain Acinetobacter ursingii M3, and strain H3 co-existed at almost 1:1 proportion with another PGIB strain, Asticcacaulis excentricus M6, regardless of the inoculation ratios (99:1–1:99) and inoculation order. We also found that A. magnusonii H3 exerted its growth-promoting effect over the negative effects of the two PGIB strains even when only a small amount was inoculated, probably due to its excellent competitive colonization ability. These experimental results demonstrate that there is a constant ecological equilibrium state involved in the bacterial colonization of aquatic plants.

Copyright © 2018 Ishizawa et al. Acinetobacter ursingii M3 and Asticcacaulis excentricus M6 are plant growth-inhibiting bacteria that reduce the yield of the duckweed Lemna minor. We report here the complete genome sequences of A. ursingii M3 and A. excentricus M6, sequenced using the PacBio RS II platform.

© 2017 Kuroda et al. Sphingobium fuliginis OMI is a bacterium that can degrade a variety of recalcitrant alkylphenols and bisphenols. This study reports the draft genome sequence of S. fuliginis OMI.

© 2017 Elsevier Masson SAS Bacteria colonizing the plant rhizosphere are believed to positively or negatively affect the host plant productivity. This feature has inspired researchers to engineer such interactions to enhance crop production. However, it remains to be elucidated whether rhizobacteria influences plant oxidative stress vis-a-vis other environmental stressors, and whether such influence is associated with their growth promoting/inhibiting ability. In this study, two plant growth-promoting bacteria (PGPB) and two plant growth-inhibiting bacteria (PGIB) were separately inoculated into axenic duckweed (Lemna minor) culture under laboratory conditions for 4 and 8 days in order to investigate their effects on plant oxidative stress and antioxidant activities. As previously characterized, the inoculation of PGPB and PGIB strains accelerated and reduced the growth of L. minor, respectively. After 4 and 8 days of cultivation, compared to the PGPB strains, the PGIB strains induced larger amounts of O2•−, H2O2, and malondialdehyde (MDA) in duckweed, although all bacterial strains consistently increased O2•− content by two times more than that in the aseptic control plants. Activities of five antioxidant enzymes were also elevated by the inoculation of PGIB, confirming the severe oxidative stress condition in plants. These results suggest that the surface attached bacteria affect differently on host oxidative stress and its response, which degree correlates negatively to their effects on plant growth.

Copyright © 2017 Ishizawa et al. Aquitalea magnusonii strain H3 is a promising plant growth-promoting bacterium for duckweed. Here, we report the draft genome sequence of strain H3 comprising 4,750,601 bp in 73 contigs. Several genes associated with plant root colonization were identified.

© 2017 The Author(s). Background: Duckweed (family Lemnaceae) has recently been recognized as an ideal biomass feedstock for biofuel production due to its rapid growth and high starch content, which inspired interest in improving their productivity. Since microbes that co-exist with plants are known to have significant effects on their growth according to the previous studies for terrestrial plants, this study has attempted to understand the plant-microbial interactions of a duckweed, Lemna minor, focusing on the growth promotion/inhibition effects so as to assess the possibility of accelerated duckweed production by modifying co-existing bacterial community. Results: Co-cultivation of aseptic L. minor and bacterial communities collected from various aquatic environments resulted in changes in duckweed growth ranging from -24 to +14% compared to aseptic control. A number of bacterial strains were isolated from both growth-promoting and growth-inhibitory communities, and examined for their co-existing effects on duckweed growth. Irrespective of the source, each strain showed promotive, inhibitory, or neutral effects when individually co-cultured with L. minor. To further analyze the interactions among these bacterial strains in a community, binary combinations of promotive and inhibitory strains were co-cultured with aseptic L. minor, resulting in that combinations of promotive-promotive or inhibitory-inhibitory strains generally showed effects similar to those of individual strains. However, combinations of promotive-inhibitory strains tended to show inhibitory effects while only Aquitalea magnusonii H3 exerted its plant growth-promoting effect in all combinations tested. Conclusion: Significant change in biomass production was observed when duckweed was co-cultivated with environmental bacterial communities. Promotive, neutral, and inhibitory bacteria in the community would synergistically determine the effects. The results indicate the possibility of improving duckweed biomass production via regulation of co-existing bacterial communities.