Novel insights into the rhizosphere and seawater microbiome of Zostera marina in diverse mariculture zones.

BACKGROUND: Seagrasses offer various ecosystem services and possess high levels of primary productivity. However, the development of mariculture has affected the homeostasis of seagrass meadow ecosystems. Plant-microbiome associations are essential for seagrasses health, but little is known about the role of environmental microbiomes and how they affect seagrass in a mariculture environment. In this study, we investigated the influence of mariculture on the rhizosphere and seawater microbiome surrounding Zostera marina and focused on the bacterial, eukaryotic, and fungal components in the composition, diversity, metabolism, and responses to mariculture-related environmental factors. RESULTS: Significant differences in the composition, richness, diversity, and internal relations of the bacterial community between the seawater and rhizosphere sediment surrounding Z. marina were observed, while differences in the eukaryotic and fungal communities were less significant. More complex bacterial and fungal co-occurrence networks were found in the seawater and rhizosphere sediment of the Saccharina japonica (SJ) and sea cucumber (SC) culture zones. The seawater in the SJ zone had higher levels of dissimilatory and assimilatory nitrate reduction, denitrification, and nitrogen fixation processes than the other three zones. The assimilatory sulfate reduction enzymes were higher in the rhizosphere sediments of the SJ zone than in the other three zones. Tetracycline, sulfonamide, and diaminopyrimidine resistance genes were enriched in the mariculture SJ and SC zones. CONCLUSIONS: Our findings might contribute to a better understanding of the effects of mariculture on the seagrass and the meadow ecosystems and thus revealing their potential operating mechanisms. These insights may serve to raise awareness of the effects of human activities on natural ecosystems, regulation of antibiotic usage, and environmental restoration. Video Abstract.

Manipulation of the seagrass-associated microbiome reduces disease severity.

Host-associated microbes influence host health and function and can be a first line of defence against infections. While research increasingly shows that terrestrial plant microbiomes contribute to bacterial, fungal, and oomycete disease resistance, no comparable experimental work has investigated marine plant microbiomes or more diverse disease agents. We test the hypothesis that the eelgrass (Zostera marina) leaf microbiome increases resistance to seagrass wasting disease. From field eelgrass with paired diseased and asymptomatic tissue, 16S rRNA gene amplicon sequencing revealed that bacterial composition and richness varied markedly between diseased and asymptomatic tissue in one of the two years. This suggests that the influence of disease on eelgrass microbial communities may vary with environmental conditions. We next experimentally reduced the eelgrass microbiome with antibiotics and bleach, then inoculated plants with Labyrinthula zosterae, the causative agent of wasting disease. We detected significantly higher disease severity in eelgrass with a native microbiome than an experimentally reduced microbiome. Our results over multiple experiments do not support a protective role of the eelgrass microbiome against L. zosterae. Further studies of these marine host-microbe-pathogen relationships may continue to show new relationships between plant microbiomes and diseases.

Predictable Changes in Eelgrass Microbiomes with Increasing Wasting Disease Prevalence across 23° Latitude in the Northeastern Pacific.

Predicting outcomes of marine disease outbreaks presents a challenge in the face of both global and local stressors. Host-associated microbiomes may play important roles in disease dynamics but remain understudied in marine ecosystems. Host-pathogen-microbiome interactions can vary across host ranges, gradients of disease, and temperature; studying these relationships may aid our ability to forecast disease dynamics. Eelgrass, Zostera marina, is impacted by outbreaks of wasting disease caused by the opportunistic pathogen Labyrinthula zosterae. We investigated how Z. marina phyllosphere microbial communities vary with rising wasting disease lesion prevalence and severity relative to plant and meadow characteristics like shoot density, longest leaf length, and temperature across 23° latitude in the Northeastern Pacific. We detected effects of geography (11%) and smaller, but distinct, effects of temperature (30-day max sea surface temperature, 4%) and disease (lesion prevalence, 3%) on microbiome composition. Declines in alpha diversity on asymptomatic tissue occurred with rising wasting disease prevalence within meadows. However, no change in microbiome variability (dispersion) was detected between asymptomatic and symptomatic tissues. Further, we identified members of Cellvibrionaceae, Colwelliaceae, and Granulosicoccaceae on asymptomatic tissue that are predictive of wasting disease prevalence across the geographic range (3,100 kilometers). Functional roles of Colwelliaceae and Granulosicoccaceae are not known. Cellvibrionaceae, degraders of plant cellulose, were also enriched in lesions and adjacent green tissue relative to nonlesioned leaves. Cellvibrionaceae may play important roles in disease progression by degrading host tissues or overwhelming plant immune responses. Thus, inclusion of microbiomes in wasting disease studies may improve our ability to understand variable rates of infection, disease progression, and plant survival. IMPORTANCE The roles of marine microbiomes in disease remain poorly understood due, in part, to the challenging nature of sampling at appropriate spatiotemporal scales and across natural gradients of disease throughout host ranges. This is especially true for marine vascular plants like eelgrass (Zostera marina) that are vital for ecosystem function and biodiversity but are susceptible to rapid decline and die-off from pathogens like eukaryotic slime-mold Labyrinthula zosterae (wasting disease). We link bacterial members of phyllosphere tissues to the prevalence of wasting disease across the broadest geographic range to date for a marine plant microbiome-disease study (3,100 km). We identify Cellvibrionaceae, plant cell wall degraders, enriched (up to 61% relative abundance) within lesion tissue, which suggests this group may be playing important roles in disease progression. These findings suggest inclusion of microbiomes in marine disease studies will improve our ability to predict ecological outcomes of infection across variable landscapes spanning thousands of kilometers.

Diversity and Composition of Microbial Communities in an Eelgrass (Zostera marina) Bed in Tokyo Bay, Japan.

Zostera marina (eelgrass) is a widespread seagrass species that forms diverse and productive habitats along coast lines throughout much of the northern hemisphere. The present study investigated the microbial consortia of Z. marina growing at Futtsu clam-digging beach, Chiba prefecture, Japan. The following environmental samples were collected: sediment, seawater, plant leaves, and the root-rhizome. Sediment and seawater samples were obtained from three sampling points: inside, outside, and at the marginal point of the eelgrass bed. The microbial composition of each sample was analyzed using 16S ribosomal gene amplicon sequencing. Microbial communities on the dead (withered) leaf surface markedly differed from those in sediment, but were similar to those in seawater. Eelgrass leaves and surrounding seawater were dominated by the bacterial taxa Rhodobacterales (Alphaproteobacteria), whereas Rhodobacterales were a minor group in eelgrass sediment. Additionally, we speculated that the order Sphingomonadales (Alphaproteobacteria) acts as a major degrader during the decomposition process and constantly degrades eelgrass leaves, which then spread into the surrounding seawater. Withered eelgrass leaves did not accumulate on the surface sediment because they were transported out of the eelgrass bed by wind and residual currents unique to the central part of Tokyo Bay.

Global Diversity and Biogeography of the Zostera marina Mycobiome.

Seagrasses are marine flowering plants that provide critical ecosystem services in coastal environments worldwide. Marine fungi are often overlooked in microbiome and seagrass studies, despite terrestrial fungi having critical functional roles as decomposers, pathogens, or endophytes in global ecosystems. Here, we characterize the distribution of fungi associated with the seagrass Zostera marina, using leaves, roots, and rhizosphere sediment from 16 locations across its full biogeographic range. Using high-throughput sequencing of the ribosomal internal transcribed spacer (ITS) region and 18S rRNA gene, we first measured fungal community composition and diversity. We then tested hypotheses of neutral community assembly theory and the degree to which deviations suggested that amplicon sequence variants (ASVs) were plant selected or dispersal limited. Finally, we identified a core mycobiome and investigated the global distribution of differentially abundant ASVs. We found that the fungal community is significantly different between sites and that the leaf mycobiome follows a weak but significant pattern of distance decay in the Pacific Ocean. Generally, there was evidence for both deterministic and stochastic factors contributing to community assembly of the mycobiome, with most taxa assembling through stochastic processes. The Z. marina core leaf and root mycobiomes were dominated by unclassified Sordariomycetes spp., unclassified Chytridiomycota lineages (including Lobulomycetaceae spp.), unclassified Capnodiales spp., and Saccharomyces sp. It is clear from the many unclassified fungal ASVs and fungal functional guilds that knowledge of marine fungi is still rudimentary. Further studies characterizing seagrass-associated fungi are needed to understand the roles of these microorganisms generally and when associated with seagrasses. IMPORTANCE Fungi have important functional roles when associated with land plants, yet very little is known about the roles of fungi associated with marine plants, like seagrasses. In this study, we report the results of a global effort to characterize the fungi associated with the seagrass Zostera marina across its full biogeographic range. Although we defined a putative global core fungal community, it is apparent from the many fungal sequences and predicted functional guilds that had no matches to existing databases that general knowledge of seagrass-associated fungi and marine fungi is lacking. This work serves as an important foundational step toward future work investigating the functional ramifications of fungi in the marine ecosystem.

Microeukaryotic Communities Associated With the Seagrass Zostera marina Are Spatially Structured.

Epibiotic microorganisms link seagrass productivity to higher trophic levels, but little is known about the processes structuring these communities, and which taxa consistently associate with seagrass. We investigated epibiotic microeukaryotes on seagrass (Zostera marina) leaves, substrates, and planktonic microeukaryotes in ten meadows in the Northeast Pacific. Seagrass epibiotic communities are distinct from planktonic and substrate communities. We found sixteen core microeukaryotes, including dinoflagellates, diatoms, and saprotrophic stramenopiles. Some likely use seagrass leaves as a substrate, others for grazing, or they may be saprotrophic organisms involved in seagrass decomposition or parasites; their relatives have been previously reported from marine sediments and in association with other hosts such as seaweeds. Core microeukaryotes were spatially structured, and none were ubiquitous across meadows. Seagrass epibiota were more spatially structured than planktonic communities, mostly due to spatial distance and changes in abiotic conditions across space. Seawater communities were relatively more similar in composition across sites and more influenced by the environmental component, but more variable over time. Core and transient taxa were both mostly structured by spatial distance and the abiotic environment, with little effect of host attributes, further indicating that those core taxa would not show a strong specific association with Z. marina.

Thiosulfativibrio zosterae gen. nov., sp. nov., and Thiosulfatimonas sediminis gen. nov., sp. nov.

Aerobic, Gram-stain-negative, obligately chemolithoautotrophic thiosulfate-oxidizing bacteria, strains AkT22T and aks77T were isolated from a brackish lake in Japan. Strains AkT22T and aks77T were isolated from samples of eelgrass and sediment, respectively. Growth on sulfide, tetrathionate, elemental sulfur, and organic substrates was not observed for both strains. Growth of the strains was observed at 5 °C or higher temperature, with optimum growth at 22 °C. Strain AkT22T grew at a pH range of 5.8-8.0, with optimum growth at pH 6.7-7.8. Strain aks77T grew at a pH range of 5.8-8.5, with optimum growth at pH 7.0-7.9. Major cellular fatty acids (> 10% of total) of strain AkT22T were C16:1, C18:1, and C16:0. The sole respiratory quinone was ubiquinone-8 in both strains. The genome of strain AkT22T consisted of a circular chromosome, with size of approximately 2.6 Mbp and G + C content of 43.2%. Those values of the genome of strain aks77T were ca. 2.7 Mbp and 45.5%, respectively. Among cultured bacteria, Thiomicrorhabdus aquaedulcis HaS4T showed the highest sequence identities of the 16S rRNA gene, to strains AkT22T (94%) and aks77T (95%). On the basis of these results, Thiosulfativibrio zosterae gen. nov., sp. nov. and Thiosulfatimonas sediminis gen. nov., sp. nov. are proposed, with type strains of AkT22T (= BCRC 81184T = NBRC 114012T = DSM 109948T) and aks77T (= BCRC 81183T = NBRC 114013T), respectively.

Recovery and Community Succession of the Zostera marina Rhizobiome after Transplantation.

Seagrasses can form mutualisms with their microbiomes that facilitate the exchange of energy sources, nutrients, and hormones and ultimately impact plant stress resistance. Little is known about community succession within the belowground seagrass microbiome after disturbance and its potential role in the plant's recovery after transplantation. We transplanted Zostera marina shoots with and without an intact rhizosphere and cultivated plants for 4 weeks while characterizing microbiome recovery and effects on plant traits. Rhizosphere and root microbiomes were compositionally distinct, likely representing discrete microbial niches. Furthermore, microbiomes of washed transplants were initially different from those of sod transplants and recovered to resemble an undisturbed state within 14 days. Conspicuously, changes in the microbial communities of washed transplants corresponded with changes in the rhizosphere sediment mass and root biomass, highlighting the strength and responsive nature of the relationship between plants, their microbiome, and the environment. Potential mutualistic microbes that were enriched over time include those that function in the cycling and turnover of sulfur, nitrogen, and plant-derived carbon in the rhizosphere environment. These findings highlight the importance and resilience of the seagrass microbiome after disturbance. Consideration of the microbiome will have meaningful implications for habitat restoration practices.IMPORTANCE Seagrasses are important coastal species that are declining globally, and transplantation can be used to combat these declines. However, the bacterial communities associated with seagrass rhizospheres and roots (the microbiome) are often disturbed or removed completely prior to transplantation. The seagrass microbiome benefits seagrasses through metabolite, nutrient, and phytohormone exchange and contributes to the ecosystem services of seagrass meadows by cycling sulfur, nitrogen, and carbon. This experiment aimed to characterize the importance and resilience of the seagrass belowground microbiome by transplanting Zostera marina with and without intact rhizospheres and tracking microbiome and plant morphological recovery over 4 weeks. We found the seagrass microbiome to be resilient to transplantation disturbance, recovering after 14 days. Additionally, microbiome recovery was linked with seagrass morphology, coinciding with increases in the rhizosphere sediment mass and root biomass. The results of this study can be used to include microbiome responses in informing future restoration work.

Fungi, bacteria and oomycota opportunistically isolated from the seagrass, Zostera marina.

Fungi in the marine environment are often neglected as a research topic, despite that fungi having critical roles on land as decomposers, pathogens or endophytes. Here we used culture-dependent methods to survey the fungi associated with the seagrass, Zostera marina, also obtaining bacteria and oomycete isolates in the process. A total of 108 fungi, 40 bacteria and 2 oomycetes were isolated. These isolates were then taxonomically identified using a combination of molecular and phylogenetic methods. The majority of the fungal isolates were classified as belonging to the classes Eurotiomycetes, Dothideomycetes, and Sordariomycetes. Most fungal isolates were habitat generalists like Penicillium sp. and Cladosporium sp., but we also cultured a diverse set of rare taxa including possible habitat specialists like Colletotrichum sp. which may preferentially associate with Z. marina leaf tissue. Although the bulk of bacterial isolates were identified as being from known ubiquitous marine lineages, we also obtained several Actinomycetes isolates and a Phyllobacterium sp. We identified two oomycetes, another understudied group of marine microbial eukaryotes, as Halophytophthora sp. which may be opportunistic pathogens or saprophytes of Z. marina. Overall, this study generates a culture collection of fungi which adds to knowledge of Z. marina associated fungi and highlights a need for more investigation into the functional and evolutionary roles of microbial eukaryotes associated with seagrasses.

Seed selection and storage with nano-silver and copper as potential antibacterial agents for the seagrass Zostera marina: implications for habitat restoration.

Globally, seagrass meadows are extremely important marine ecosystems that are disappearing at an alarming rate. Therefore, research into seagrass restoration has become increasingly important. Various strategies have been used in Zostera marina L. (eelgrass) restoration, including planting seeds. To improve the efficiency of restoration by planting seeds, it is necessary to select high-quality seeds. In addition, a suitable antibacterial agent is necessary for wet storage of desiccation sensitive seeds to reduce or inhibit microorganism infection and seed decay. In the present study, an efficient method for selecting for high-quality eelgrass seeds using different specific gravities of salt water was developed, and potential antibacterial agents (nano-silver and copper sulfate) for seed storage were assessed. The results showed that the highest proportion of intact seeds (72.91 ± 0.50%) was recorded at specific gravities greater than 1.20. Therefore, specific gravities greater than 1.20 can be used for selecting high-quality eelgrass seeds. During seed storage at 0 °C, the proportion of intact seeds after storage with nano-silver agent was over 90%, and also higher than 80% with copper sulfate agent, which was significantly higher than control treatments. The findings revealed a potential selection method for high-quality seeds and long-term seed storage conditions for Z. marina, which could facilitate conservation and habitat restoration.

Rapid Metabolome and Bioactivity Profiling of Fungi Associated with the Leaf and Rhizosphere of the Baltic Seagrass Zostera marina.

Zostera marina (eelgrass) is a marine foundation species with key ecological roles in coastal habitats. Its bacterial microbiota has been well studied, but very little is known about its mycobiome. In this study, we have isolated and identified 13 fungal strains, dominated by Penicillium species (10 strains), from the leaf and the root rhizosphere of Baltic Z. marina. The organic extracts of the fungi that were cultured by an OSMAC (One-Strain-Many-Compounds) regime using five liquid culture media under both static and shaking conditions were investigated for their chemical and bioactivity profiles. All extracts showed strong anti-quorum sensing activity, and the majority of the Penicillium extracts displayed antimicrobial or anti-biofilm activity against Gram-negative environmental marine and human pathogens. HPLC-DAD-MS-based rapid metabolome analyses of the extracts indicated the high influence of culture conditions on the secondary metabolite (SM) profiles. Among 69 compounds detected in all Penicillium sp. extracts, 46 were successfully dereplicated. Analysis of SM relatedness in culture conditions by Hierarchical Cluster Analysis (HCA) revealed generally low similarity and showed a strong effect of medium selection on chemical profiles of Penicillium sp. This is the first study assessing both the metabolite and bioactivity profile of the fungi associated with Baltic eelgrass Z. marina.

Surface chemical defence of the eelgrass Zostera marina against microbial foulers.

Plants rely on both mechanical and chemical defence mechanisms to protect their surfaces against microorganisms. The recently completed genome of the eelgrass Zostera marina, a marine angiosperm with fundamental importance for coastal ecosystems, showed that its re-adaptation from land to the sea has led to the loss of essential genes (for chemical communication and defence) and structural features (stomata and thick cuticle) that are typical of terrestrial plants. This study was designed to understand the molecular nature of surface protection and fouling-control strategy of eelgrass against marine epiphytic yeasts. Different surface extraction methods and comparative metabolomics by tandem mass spectrometry (LC-MS/MS) were used for targeted and untargeted identification of the metabolite profiles of the leaf surface and the whole tissue extracts. Desorption electrospray ionization-imaging mass spectrometry (DESI-IMS) coupled with traditional bioassays revealed, for the first time, the unique spatial distribution of the eelgrass surface-associated phenolics and fatty acids, as well as their differential bioactivity against the growth and settlement of epiphytic yeasts. This study provides insights into the complex chemical defence system of the eelgrass leaf surface. It suggests that surface-associated metabolites modulate biotic interactions and provide chemical defence and structural protection to eelgrass in its marine environment.

Oxygen loss from seagrass roots coincides with colonisation of sulphide-oxidising cable bacteria and reduces sulphide stress.

Seagrasses thrive in anoxic sediments where sulphide can accumulate to phytotoxic levels. So how do seagrasses persist in this environment? Here, we propose that radial oxygen loss (ROL) from actively growing root tips protects seagrasses from sulphide intrusion not only by abiotically oxidising sulphides in the rhizosphere of young roots, but also by influencing the abundance and spatial distribution of sulphate-reducing and sulphide-oxidising bacteria. We used a novel multifaceted approach combining imaging techniques (confocal fluorescence in situ hybridisation, oxygen planar optodes, and sulphide diffusive gradients in thin films) with microbial community profiling to build a complete picture of the microenvironment of growing roots of the seagrasses Halophila ovalis and Zostera muelleri. ROL was restricted to young root tips, indicating that seagrasses will have limited ability to influence sulphide oxidation in bulk sediments. On the microscale, however, ROL corresponded with decreased abundance of potential sulphate-reducing bacteria and decreased sulphide concentrations in the rhizosphere surrounding young roots. Furthermore, roots leaking oxygen had a higher abundance of sulphide-oxidising cable bacteria; which is the first direct observation of these bacteria on seagrass roots. Thus, ROL may enhance both abiotic and bacterial sulphide oxidation and restrict bacterial sulphide production around vulnerable roots, thereby helping seagrasses to colonise sulphide-rich anoxic sediments.

Eelgrass Sediment Microbiome as a Nitrous Oxide Sink in Brackish Lake Akkeshi, Japan.

Nitrous oxide (N2O) is a powerful greenhouse gas; however, limited information is currently available on the microbiomes involved in its sink and source in seagrass meadow sediments. Using laboratory incubations, a quantitative PCR (qPCR) analysis of N2O reductase (nosZ) and ammonia monooxygenase subunit A (amoA) genes, and a metagenome analysis based on the nosZ gene, we investigated the abundance of N2O-reducing microorganisms and ammonia-oxidizing prokaryotes as well as the community compositions of N2O-reducing microorganisms in in situ and cultivated sediments in the non-eelgrass and eelgrass zones of Lake Akkeshi, Japan. Laboratory incubations showed that N2O was reduced by eelgrass sediments and emitted by non-eelgrass sediments. qPCR analyses revealed that the abundance of nosZ gene clade II in both sediments before and after the incubation as higher in the eelgrass zone than in the non-eelgrass zone. In contrast, the abundance of ammonia-oxidizing archaeal amoA genes increased after incubations in the non-eelgrass zone only. Metagenome analyses of nosZ genes revealed that the lineages Dechloromonas-Magnetospirillum-Thiocapsa and Bacteroidetes (Flavobacteriia) within nosZ gene clade II were the main populations in the N2O-reducing microbiome in the in situ sediments of eelgrass zones. Sulfur-oxidizing Gammaproteobacteria within nosZ gene clade II dominated in the lineage Dechloromonas-Magnetospirillum-Thiocapsa. Alphaproteobacteria within nosZ gene clade I were predominant in both zones. The proportions of Epsilonproteobacteria within nosZ gene clade II increased after incubations in the eelgrass zone microcosm supplemented with N2O only. Collectively, these results suggest that the N2O-reducing microbiome in eelgrass meadows is largely responsible for coastal N2O mitigation.

Environment, dosage, and pathogen isolate moderate virulence in eelgrass wasting disease.

Eelgrass wasting disease, caused by the marine pathogen Labyrinthula zosterae, has the potential to devastate important eelgrass habitats worldwide. Although this host-pathogen interaction may increase under certain environmental conditions, little is known about how disease severity is impacted by multiple components of a changing environment. In this study, we investigated the effects of variation in 3 different L. zosterae isolates, pathogen dosage, temperature, and light on severity of infections. Severity of lesions on eelgrass varied among the 3 different isolates inoculated in laboratory trials. Our methods to control dosage of inoculum showed that disease severity increased with pathogen dosage from 104 to 106 cells ml-1. In a dosage-controlled light and temperature 2-way factorial experiment consisting of 2 light regimes (diel light cycle and complete darkness) and 2 temperatures (11 and 18°C), L. zosterae cell growth rate in vitro was higher at the warmer temperature. In a companion experiment that tested the effects of light and temperature in in vivo inoculations, disease severity was higher in dark treatments and temperature was marginally significant. We suggest that the much greater impact of light in the in vivo inoculation experiment indicates an important role for plant physiology and the need for photosynthesis in slowing severity of infections. Our work with controlled inoculation of distinct L. zosterae isolates shows that pathogen isolate, increasing dosage of inoculum, increasing temperature, and diminishing light increase disease severity, suggesting L. zosterae will cause increased damage to eelgrass beds with changing environmental conditions.

Seagrass rhizosphere microenvironment alters plant-associated microbial community composition.

The seagrass rhizosphere harbors dynamic microenvironments, where plant-driven gradients of O2 and dissolved organic carbon form microhabitats that select for distinct microbial communities. To examine how seagrass-mediated alterations of rhizosphere geochemistry affect microbial communities at the microscale level, we applied 16S rRNA amplicon sequencing of artificial sediments surrounding the meristematic tissues of the seagrass Zostera muelleri together with microsensor measurements of the chemical conditions at the basal leaf meristem (BLM). Radial O2 loss (ROL) from the BLM led to ∼ 300 µm thick oxic microzones, wherein pronounced decreases in H2 S and pH occurred. Significantly higher relative abundances of sulphate-reducing bacteria were observed around the meristematic tissues compared to the bulk sediment, especially around the root apical meristems (RAM; ∼ 57% of sequences). Within oxic microniches, elevated abundances of sulphide-oxidizing bacteria were observed compared to the bulk sediment and around the RAM. However, sulphide oxidisers within the oxic microzone did not enhance sediment detoxification, as rates of H2 S re-oxidation here were similar to those observed in a pre-sterilized root/rhizome environment. Our results provide novel insights into how chemical and microbiological processes in the seagrass rhizosphere modulate plant-microbe interactions potentially affecting seagrass health.

Dokdonia aurantiaca sp. nov., isolated from seaweed Zostera marina.

A non-motile, orange-coloured and rod-shaped bacterial strain, designated strain ZOW29T, was isolated from a seaweed sample collected from the South Sea, Republic of Korea. Cells were Gram-stain-negative, aerobic and non-motile. The isolate required sea salts for growth. Carotenoid pigment was produced. A phylogenetic tree based on 16S rRNA gene sequences showed that strain ZOW29T forms an evolutionary lineage within the radiation enclosing the members of the genus Dokdonia with Dokdonia diaphoros MSKK-32T, Dokdonia eikasta PMA-26Tand Dokdonia donghaensis DSW-1T (97.1 % sequence similarity each) as its nearest neighbours. The DNA-DNA relatedness values between strain ZOW29T and these four type strains were 35-48 %. The major fatty acids were iso-C15:0, iso-C17 : 0 3-OH and iso-C15 : 1 G. Strain ZOW29T contained MK-6 and phosphatidylethanolamine, an unidentified aminolipid and an unidentified polar lipid as the only isoprenoid quinone and the major polar lipids, respectively. The DNA G+C content of strain ZOW29T was 38 mol%. On the basis of polyphasic characterization, it is suggested that the isolate represents a novel species of the genus Dokdonia, for which the name Dokdonia aurantiaca sp. nov. (type strain, ZOW29T=KCTC 52956T=JCM 32295T) is proposed.

Dokdonia flava sp. nov., isolated from the seaweed Zostera marina.

A non-motile, proteorhodopsin-containing, yellow and rod-shaped bacterial strain, designated ZODW10T, was isolated from the seaweed Zostera marina collected from the West Sea, Republic of Korea. Cells were Gram-stain-negative, aerobic and non-motile. The isolate required sea salts for growth. A carotenoid pigment was produced. A phylogenetic tree based on 16S rRNA gene sequences showed that strain ZODW10T forms an evolutionary lineage within the radiation enclosing members of the genus Dokdonia with Dokdoniadiaphoros CIP 108745T (96.7 % sequence similarity) as its nearest neighbour. The major fatty acids were iso-C15:0, iso-C17 : 0 3-OH and iso-C15 : 1 G. Strain ZODW10T contained menaquinone 6 (MK-6) and phosphatidylethanolamine, an unidentified aminolipid and an unidentified polar lipid as the only isoprenoid quinone and the major polar lipids, respectively. The DNA G+C content of strain ZODW10T was 36 mol%. On the basis of the present polyphasic characterization, it is suggested that the isolate represents a novel species of the genus Dokdonia, for which the name Dokdonia flava sp. nov. (type strain, ZODW10T=KCTC 52953T=JCM 32293T) is proposed.

Production of indoleacetic acid by strains of the epiphytic bacteria Neptunomonas spp. isolated from the red alga Pyropia yezoensis and the seagrass Zostera marina.

Neptunomonas sp. BPy-1 is an epiphytic bacterium isolated from in vitro culture of the red alga Pyropia yezoensis. It uses ethanol as a sole carbon source and promotes the growth of host alga. A related bacterium, Neptunomonas sp. BZm-1, was isolated from leaves of Zostera marina found in the Yatsushiro Sea (Japan). BZm-1 showed 99% 16S rRNA sequence identity with Neptunomonas sp. BPy-1. Similar to BPy-1, BZm-1 grew in artificial seawater (ASW) medium containing ethanol or butanol. When thalli were treated with a multi-enzyme cleaner, the growth of treated thalli was retarded, but the addition of BZm-1 to the medium promoted growth. To explore the benefits of epiphytic bacteria, indoleacetic acid (IAA) production by isolated bacteria was examined under conditions of limited nutrients. Salkowski assays and GC-MS analysis revealed that both BZm-1 and BPy-1 excreted IAA during growth in ASW medium containing glucose or ethanol in the presence of tryptophan. In ASW medium containing tryptophan but lacking a carbon source, neither isolate grow, but produced IAA. ASW medium includes nitrate as the sole nitrogen source. In the absence of carbon source, different nitrogen forms in the presence of tryptophan did not affect IAA production by the two isolates. These findings indicate that IAA production by the two isolates is strictly dependent on tryptophan but less affected by carbon and nitrogen sources. Based on the different origins of BPy-1 and BZm-1, this mode of IAA production seems to be conserved among relatives of BPy-1.

Seagrass wasting disease: Nitrate enrichment and exposure to a herbicide (Diuron) increases susceptibility of Zostera marina to infection.

Seagrass meadows suffered large-scale declines in the past century. The 'wasting disease', pathognomonically associated with Labyrinthula zosterae, reduced populations of Zostera marina on both sides of the North Atlantic in, and since, the 1930s, coinciding with intensive agricultural use of artificial fertilizers and herbicides. This study tests the long-standing hypothesis that nutrient enrichment and a herbicide increases vulnerability to pathogens. Z. marina shoots from the Thames Estuary grown in elevated nitrate concentrations had significantly higher rates of infection by L. zosterae than controls, but not by Aplanochytrium sp., another slime-mould like protist. Z. marina shoots grown in 2µg·l-1 Diuron solutions and infected separately by L. zosterae and Aplanochytrium sp. had significantly higher wasting indices than controls. The results identified Aplanochytrium sp. as another opportunistic pathogen causing a seagrass wasting-type disease and support the hypothesis that pollution by herbicides and nitrate increases the susceptibility of Z. marina to infections.