Non-selective microbiota reduction after the elicitation of a seaweed's immune response.

Pattern-triggered immunity (PTI) is an integral part of the innate immune system of many eukaryotic hosts, assisting in the defence against pathogen invasions. In plants and animals, PTI exerts a selective pressure on the microbiota that can alter community composition. However, the effect of PTI on the microbiota for non-model hosts, including seaweeds, remains unknown. Using quantitative polymerase chain reaction complemented with 16S rRNA gene and transcript amplicon sequencing, this study profiled the impact that PTI of the red seaweed Gracilaria gracilis has on its microbiota. PTI elicitation with agar oligosaccharides resulted in a significant reduction in the number of bacteria (by >75% within 72 h after treatment). However, the PTI elicitation did not cause any significant difference in the community diversity or structure. These findings demonstrated that PTI can be non-selective, and this might help to maintain a stable microbiota by uniformly reducing bacterial loads.

Current approaches to genetic modification of marine bacteria and considerations for improved transformation efficiency.

Marine bacteria play vital roles in symbiosis, biogeochemical cycles and produce novel bioactive compounds and enzymes of interest for the pharmaceutical, biofuel and biotechnology industries. At present, investigations into marine bacterial functions and their products are primarily based on phenotypic observations, -omic type approaches and heterologous gene expression. To advance our understanding of marine bacteria and harness their full potential for industry application, it is critical that we have the appropriate tools and resources to genetically manipulate them in situ. However, current genetic tools that are largely designed for model organisms such as E. coli, produce low transformation efficiencies or have no transfer ability in marine bacteria. To improve genetic manipulation applications for marine bacteria, we need to improve transformation methods such as conjugation and electroporation in addition to identifying more marine broad host range plasmids. In this review, we aim to outline the reported methods of transformation for marine bacteria and discuss the considerations for each approach in the context of improving efficiency. In addition, we further discuss marine plasmids and future research areas including CRISPR tools and their potential applications for marine bacteria.

Disentangling direct vs indirect effects of microbiome manipulations in a habitat-forming marine holobiont.

Host-associated microbiota are critical for eukaryotic host functioning, to the extent that hosts and their associated microbial communities are often considered "holobionts". Most studies of holobionts have focused on descriptive approaches or have used model systems, usually in the laboratory, to understand host-microbiome interactions. To advance our understanding of host-microbiota interactions and their wider ecological impacts, we need experimental frameworks that can explore causation in non-model hosts, which often have highly diverse microbiota, and in their natural ecological setting (i.e. in the field). We used a dominant habitat-forming seaweed, Hormosira banksii, to explore these issues and to experimentally test host-microbiota interactions in a non-model holobiont. The experimental protocols were aimed at trying to disentangle microbially mediated effects on hosts from direct effects on hosts associated with the methods employed to manipulate host-microbiota. This was done by disrupting the microbiome, either through removal/disruption using a combination of antimicrobial treatments, or additions of specific taxa via inoculations, or a combination of thew two. The experiments were done in mesocosms and in the field. Three different antibiotic treatments were used to disrupt seaweed-associated microbiota to test whether disturbances of microbiota, particularly bacteria, would negatively affect host performance. Responses of bacteria to these disturbances were complex and differed substantially among treatments, with some antibacterial treatments having little discernible effect. However, the temporal sequence of responses antibiotic treatments, changes in bacterial diversity and subsequent decreases in host performance, strongly suggested an effect of the microbiota on host performance in some treatments, as opposed to direct effects of the antibiotics. To further test these effects, we used 16S-rRNA-gene sequencing to identify bacterial taxa that were either correlated, or uncorrelated, with poor host performance following antibiotic treatment. These were then isolated and used in inoculation experiments, independently or in combination with the previously used antibiotic treatments. Negative effects on host performance were strongest where specific microbial antimicrobials treatments were combined with inoculations of strains that were correlated with poor host performance. For these treatments, negative host effects persisted the entire experimental period (12 days), even though treatments were only applied at the beginning of the experiment. Host performance recovered in all other treatments. These experiments provide a framework for exploring causation and disentangling microbially mediated vs. direct effects on hosts for ecologically important, non-model holobionts in the field. This should allow for better predictions of how these systems will respond to, and potentially mitigate, environmental disturbances in their natural context.

Functional guilds and drivers of diversity in seaweed-associated bacteria.

Comparisons of functional and taxonomic profiles from bacterial communities in different habitats have suggested the existence of functional guilds composed of taxonomically or phylogenetically distinct members. Such guild membership is, however, rarely defined and the factors that drive functional diversity in bacteria remain poorly understood. We used seaweed-associated bacteria as a model to shed light on these important aspects of community ecology. Using a large dataset of over 1300 metagenome-assembled genomes from 13 seaweed species we found substantial overlap in the functionality of bacteria coming from distinct taxa, thus supporting the existence of functional guilds. This functional equivalence between different taxa was particularly pronounced when only functions involved in carbohydrate degradation were considered. We further found that bacterial taxonomy is the dominant driver of functional differences between bacteria and that seaweed species or seaweed type (i.e. brown, red and green) had relatively stronger impacts on genome functionality for carbohydrate-degradation functions when compared to all other cellular functions. This study provides new insight into the factors underpinning the functional diversity of bacteria and contributes to our understanding how community function is generated from individual members.

Marine diseases and the Anthropocene: Understanding microbial pathogenesis in a rapidly changing world.

Healthy marine ecosystems are paramount for Earth's biodiversity and are key to sustaining the global economy and human health. The effects of anthropogenic activity represent a pervasive threat to the productivity of marine ecosystems, with intensifying environmental stressors such as climate change and pollution driving the occurrence and severity of microbial diseases that can devastate marine ecosystems and jeopardise food security. Despite the potentially catastrophic outcomes of marine diseases, our understanding of host-pathogen interactions remains an understudied aspect of both microbiology and environmental research, especially when compared to the depth of information available for human and agricultural systems. Here, we identify three avenues of research in which we can advance our understanding of marine disease in the context of global change, and make positive steps towards safeguarding marine communities for future generations.

The impact of interspecific competition on the genomic evolution of Phaeobacter inhibens and Pseudoalteromonas tunicata during biofilm growth.

Interspecific interactions in biofilms have been shown to cause the emergence of community-level properties. To understand the impact of interspecific competition on evolution, we deep-sequenced the dispersal population of mono- and co-culture biofilms of two antagonistic marine bacteria (Phaeobacter inhibens 2.10 and Pseudoalteromononas tunicata D2). Enhanced phenotypic and genomic diversification was observed in the P. tunicata D2 populations under both mono- and co-culture biofilms in comparison to P. inhibens 2.10. The genetic variation was exclusively due to single nucleotide variants and small deletions, and showed high variability between replicates, indicating their random emergence. Interspecific competition exerted an apparent strong positive selection on a subset of P. inhibens 2.10 genes (e.g., luxR, cobC, argH, and sinR) that could facilitate competition, while the P. tunicata D2 population was genetically constrained under competition conditions. In the absence of interspecific competition, the P. tunicata D2 replicate populations displayed high levels of mutations affecting the same genes involved in cell motility and biofilm formation. Our results show that interspecific biofilm competition has a complex impact on genomic diversification, which likely depends on the nature of the competing strains and their ability to generate genetic variants due to their genomic constraints.

Not all parents are the same: Diverse strategies of symbiont transmission in seaweeds.

Different marine seaweed species have been shown to harbour specific bacterial communities, however, the extent to which vertical symbiont transmission from parents to offspring contributes to host-specificity is unclear. Here we use fluorescence and electron microscopy as well as 16S rRNA gene-based community analysis to investigate symbiont transmission in members of the three major seaweed groups (green Chlorophyta, red Rhodophyta and brown Phaeophyceae). We found seaweeds employ diverse strategies to transfer symbionts to their progeny. For instance, the green Ulva australis does not appear to have the capacity for vertical transmission. In contrast, the brown Phyllospora comosa adopts a non-selective vertical transmission. The red Delisea pulchra demonstrates weak selectivity in symbiont transmission, while the brown Hormosira banksii exhibits a strongly selective symbiont transfer. Mucilage on the gametes appears to facilitate vertical transmission and transferred bacteria have predicted properties that could support early development of the seaweeds. Previous meta-analysis has indicated that vertical transmission is rare in aquatic compared to terrestrial environments, however, our results contribute to the growing evidence that this might not be the case and that instead vertical transmission with various degrees of symbiont selection occurs in the ecologically important group of seaweeds.

Naturally occurring beneficial bacteria Vibrio alginolyticus X-2 protects seaweed from bleaching disease.

Microbiome manipulation is gaining fresh attention as a way to mitigate diseases in aquaculture. The commercially farmed seaweed Saccharina japonica suffers from a bacterial-induced bleaching disease, which has major implications for the reliable supply of healthy sporelings. Here, we identify a beneficial bacterium, Vibrio alginolyticus X-2 that significantly reduces the risk of bleaching disease. By combining infection assays and multi-omic analyses, we provide evidence to suggest that the underlying protective mechanisms of V. alginolyticus X-2 involve maintaining epibacterial communities, increasing the gene expression of S. japonica related to immune and stress protection pathways, and stimulating betaine concentrations in S. japonica holobionts. Thus, V. alginolyticus X-2 can elicit a suite of microbial and host responses to mitigate the bleaching disease. Our study provides insights into disease control in farmed S. japonica through the application of beneficial bacteria. IMPORTANCE Beneficial bacteria can elicit a suite of microbial and host responses to enhance the resistance to bleaching disease.

The Reemergence of Phycopathology: When Algal Biology Meets Ecology and Biosecurity.

Viruses, bacteria, and eukaryotic symbionts interact with algae in a variety of ways to cause disease complexes, often shaping marine and freshwater ecosystems. The advent of phyconomy (a.k.a. seaweed agronomy) represents a need for a greater understanding of algal disease interactions, where underestimated cryptic diversity and lack of phycopathological basis are prospective constraints for algal domestication. Here, we highlight the limited yet increasing knowledge of algal pathogen biodiversity and the ecological interaction with their algal hosts. Finally, we discuss how ecology and cultivation experience contribute to and reinforce aquaculture practice, with the potential to reshape biosecurity policies of seaweed cultivation worldwide.

A coral disease outbreak highlights vulnerability of remote high-latitude lagoons to global and local stressors.

Outbreaks of coral disease are often associated with global and local stressors like changes in temperature and poor water quality. A severe coral disease outbreak was recorded in the primary reef-building taxa Montipora spp. in a high-latitude lagoon at Norfolk Island following heat stress and pollution events in 2020. Disease signs suggest the occurrence of a Montiporid White Syndrome with four distinct phases and maximum measured tissue loss of 329 mm-2 day-1. In December 2020 and April 2021, 60% of the Montipora community were impacted and disease severity increased by 54% over this period. Spatial patterns in prevalence indicate the disease is associated with exposure to poor water quality in addition to size class of coral colonies. High prevalence levels make this event comparable to some of the most severe coral disease outbreaks recorded to date demonstrating the vulnerability of this system to combined impacts of warming and pollution.

Differential priority effects impact taxonomy and functionality of host-associated microbiomes.

Most multicellular eukaryotes host complex communities of microorganisms, but the factors that govern their assembly are poorly understood. The settlement of specific microorganisms may have a lasting impact on community composition, a phenomenon known as the priority effect. Priority effects of individual bacterial strains on a host's microbiome are, however, rarely studied and their impact on microbiome functionality remains unknown. We experimentally tested the effect of two bacterial strains (Pseudoalteromonas tunicata D2 and Pseudovibrio sp. D323) on the assembly and succession of the microbial communities associated with the green macroalga Ulva australis. Using 16S rRNA gene sequencing and qPCR, we found that both strains exert a priority effect, with strain D2 causing initially strong but temporary taxonomic changes and strain D323 causing weaker but consistent changes. Consistent changes were predominately facilitatory and included taxa that may benefit the algal host. Metagenome analyses revealed that the strains elicited both shared (e.g., depletion of type III secretion system genes) and unique (e.g., enrichment of antibiotic resistance genes) effects on the predicted microbiome functionality. These findings indicate strong idiosyncratic effects of colonizing bacteria on the structure and function of host-associated microbial communities. Understanding the idiosyncrasies in priority effects is key for the development of novel probiotics to improve host condition.

A pathway to improve seaweed aquaculture through microbiota manipulation.

Eukaryotic hosts are associated with microbial communities that are critical to their function. Microbiota manipulation using beneficial microorganisms, for example, in the form of animal probiotics or plant growth-promoting microorganisms (PGPMs), can enhance host performance and health. Recently, seaweed beneficial microorganisms (SBMs) have been identified that promote the growth and development and/or improve disease resistance of seaweeds. This knowledge coincides with global initiatives seeking to expand and intensify seaweed aquaculture. Here, we provide a pathway with the potential to improve commercial cultivation of seaweeds through microbiota manipulation, highlighting that seaweed restoration practices can also benefit from further understanding SBMs and their modes of action. The challenges and opportunities of different approaches to identify and apply SBMs to seaweed aquaculture are discussed.

Opportunistic diseases in marine eukaryotes: Could Bacteroidota be the next threat to ocean life?

Bacteria within the phylum Bacteroidota (Bacteroidetes) are known to cause devastating and widespread disease outbreaks in marine eukaryotic hosts. However, with few pathogens described in detail, their prevalence and virulence strategies remain largely unknown. Here, we systematically reviewed the literature to evaluate the current understanding of Bacteroidota that cause disease in marine hosts. Isolates affiliated with the genera Tenacibaculum and Aquimarina (Flavobacteriaceae) were the most widely reported and characterized pathogens. Although cultured isolates were predominantly Flavobacteriia, culture-independent studies also found classes Bacteroidia, Cytophagia and Sphingobacteriia associated with disease. We found that pathogenic marine Bacteroidota largely conformed to an opportunistic lifestyle but could also act as secondary pathogens or were involved in polymicrobial diseases. Many diseases were also associated with an environmental stressor, especially those affecting coral, macroalgae and fish. Key virulence traits included the production of adhesins and host tissue-degrading enzymes. Overall, the nature of disease involving Bacteroidota pathogens appears to be an outcome of complex host-pathogen-environment interactions; however, our understanding of virulence remains limited by the lack of functional characterization studies. This is concerning as Bacteroidota have the potential to emerge as a serious threat to marine ecosystems and aquaculture industries, driven by global changes in ocean conditions.

Pathogen exposure leads to a transcriptional downregulation of core cellular functions that may dampen the immune response in a macroalga.

Diseases in marine eukaryotic organisms caused by opportunistic pathogens represent a serious threat to our oceans with potential downstream consequences for ecosystem functioning. Disease outbreaks affecting macroalgae are of particular concern due to their critical role as habitat-forming organisms. However, there is limited understanding of the molecular strategies used by macroalgae to respond to opportunistic pathogens. In this study, we used mRNA-sequencing analysis to investigate the early antipathogen response of the model macroalga Delisea pulchra (Rhodophyta) under the environmental conditions that are known to promote the onset of disease. Using de novo assembly methods, 27,586 unique transcripts belonging to D. pulchra were identified that were mostly affiliated with stress response and signal transduction processes. Differential gene expression analysis between a treatment with the known opportunistic pathogen, Aquimarina sp. AD1 (Bacteroidota), and a closely related benign strain (Aquimarina sp. AD10) revealed a downregulation of genes coding for predicted protein metabolism, stress response, energy generation and photosynthesis functions. The rapid repression of genes coding for core cellular processes is likely to interfere with the macroalgal antipathogen response, later leading to infection, tissue damage and bleaching symptoms. Overall, this study provides valuable insight into the genetic features of D. pulchra, highlighting potential antipathogen response mechanisms of macroalgae and contributing to an improved understanding of host-pathogen interactions in a changing environment.

Arbuscular Mycorrhizal Fungi Contribute to Phosphorous Uptake and Allocation Strategies of Solidago canadensis in a Phosphorous-Deficient Environment.

Arbuscular mycorrhizal fungi (AMF) can facilitate the uptake of limiting or inaccessible nutrients by plants. However, the importance of AMF for invasive plants under phosphorus (P) limitation is poorly well understood because of the presence of non-focal microorganisms, such as endophytes or rhizosphere bacteria. In this study, we investigated how an invasive clonal plant Solidago canadensis benefits from the AMF Glomus intraradices by using a completely sterile culturing system, which is composed of aseptic seedlings, a pure AMF strain, and a sterile growth environment. We found that the colonization rate, abundance, and spore production of AMF in the insoluble P treatment was more than twice as much as in the available P treatment. Plant above-ground growth was enhanced almost 50% by AMF in the insoluble P treatment. Importantly, AMF were able to facilitate P acquisition by the plant in insoluble P conditions, allowing plants to have lower investment into below-ground biomass and higher benefit/return for above-ground biomass. This study demonstrated the important contribution that AMF make to plants in phosphate-deficient environments eliminating interference from non-focal microorganisms. Our results also suggest that interaction with AMF could contribute to the invasiveness of clonal plant S. canadensis in a resource-deficient environment.

Cross-Host Protection of Marine Bacteria Against Macroalgal Disease.

Despite an increasing awareness of disease impacts on both cultivated and native seaweed populations, the development of marine probiotics has been limited and predominately focused on farmed animals. Bleaching (loss of thallus pigmentation) is one of the most prevalent diseases observed in marine macroalgae. Endemic probiotic bacteria have been characterized to prevent bleaching disease in red macroalgae Agarophyton vermiculophyllum and Delisea pulchra; however, the extent to which probiotic strains provide cross-protection to non-endemic hosts and the influence of native microbiota remain unknown. Using A. vermiculophyllum as a model, we demonstrate that co-inoculation with the pathogen Pseudoalteromonas arctica G-MAN6 and D. pulchra probiotic strain Phaeobacter sp. BS52 or Pseudoalteromonas sp. PB2-1 reduced the disease risks compared to the pathogen only treatment. Moreover, non-endemic probiotics outperformed the endemic probiotic strain Ralstonia sp. G-NY6 in the presence of the host natural microbiota. This study highlights how the native microbiota can impact the effectiveness of marine probiotics and illustrates the potential of harnessing probiotics that can function across different hosts to mitigate the impact of emerging marine diseases.

Bacterial controlled mitigation of dysbiosis in a seaweed disease.

Disease in the marine environment is predicted to increase with anthropogenic stressors and already affects major habitat-formers, such as corals and seaweeds. Solutions to address this issue are urgently needed. The seaweed Delisea pulchra is prone to a bleaching disease, which is caused by opportunistic pathogens and involves bacterial dysbiosis. Bacteria that can inhibit these pathogens and/or counteract dysbiosis are therefore hypothesised to reduce disease. This study aimed to identify such disease-protective bacteria and investigate their protective action. One strain, Phaeobacter sp. BS52, isolated from healthy D. pulchra, was antagonistic towards bleaching pathogens and significantly increased the proportion of healthy individuals when applied before the pathogen challenge (pathogen-only vs. BS52 + pathogen: 41-80%), and to a level similar to the control. However, no significant negative correlations between the relative abundances of pathogens and BS52 on D. pulchra were detected. Instead, inoculation of BS52 mitigated pathogen-induced changes in the epibacterial community. These observations suggest that the protective activity of BS52 was due to its ability to prevent dysbiosis, rather than direct pathogen inhibition. This study demonstrates the feasibility of manipulating bacterial communities in seaweeds to reduce disease and that mitigation of dysbiosis can have positive health outcomes.

Novel Nematode-Killing Protein-1 (Nkp-1) from a Marine Epiphytic Bacterium Pseudoalteromonas tunicata.

Drug resistance among parasitic nematodes has resulted in an urgent need for the development of new therapies. However, the high re-discovery rate of anti-nematode compounds from terrestrial environments necessitates a new repository for future drug research. Marine epiphytes are hypothesised to produce nematicidal compounds as a defence against bacterivorous predators, thus representing a promising yet underexplored source for anti-nematode drug discovery. The marine epiphytic bacterium Pseudoalteromonas tunicata is known to produce several bioactive compounds. Screening heterologously expressed genomic libraries of P. tunicata against the nematode Caenorhabditis elegans, identified as an E. coli clone (HG8), shows fast-killing activity. Here we show that clone HG8 produces a novel nematode-killing protein-1 (Nkp-1) harbouring a predicted carbohydrate-binding domain with weak homology to known bacterial pore-forming toxins. We found bacteria expressing Nkp-1 were able to colonise the C. elegans intestine, with exposure to both live bacteria and protein extracts resulting in physical damage and necrosis, leading to nematode death within 24 h of exposure. Furthermore, this study revealed C. elegans dar (deformed anal region) and internal hatching may act as a nematode defence strategy against Nkp-1 toxicity. The characterisation of this novel protein and putative mode of action not only contributes to the development of novel anti-nematode applications in the future but reaffirms the potential of marine epiphytic bacteria as a new source of novel biomolecules.

Genomic Evolution of the Marine Bacterium Phaeobacter inhibens during Biofilm Growth.

Phaeobacter inhibens 2.10 is an effective biofilm former on marine surfaces and has the ability to outcompete other microorganisms, possibly due to the production of the plasmid-encoded secondary metabolite tropodithietic acid (TDA). P. inhibens 2.10 biofilms produce phenotypic variants with reduced competitiveness compared to the wild type. In the present study, we used longitudinal, genome-wide deep sequencing to uncover the genetic foundation that contributes to the emergent phenotypic diversity in P. inhibens 2.10 biofilm dispersants. Our results show that phenotypic variation is not due to the loss of the plasmid that carries the genes for TDA synthesis but instead show that P. inhibens 2.10 biofilm populations become rapidly enriched in single nucleotide variations in genes involved in the synthesis of TDA. While variants in genes previously linked to other phenotypes, such as lipopolysaccharide production (i.e., rfbA) and cellular persistence (i.e., metG), also appear to be selected for during biofilm dispersal, the number and consistency of variations found for genes involved in TDA production suggest that this metabolite imposes a burden on P. inhibens 2.10 cells. Our results indicate a strong selection pressure for the loss of TDA in monospecies biofilm populations and provide insight into how competition (or a lack thereof) in biofilms might shape genome evolution in bacteria. IMPORTANCE Biofilm formation and dispersal are important survival strategies for environmental bacteria. During biofilm dispersal, cells often display stable and heritable variants from the parental biofilm. Phaeobacter inhibens is an effective colonizer of marine surfaces, in which a subpopulation of its biofilm dispersal cells displays a noncompetitive phenotype. This study aimed to elucidate the genetic basis of these phenotypic changes. Despite the progress made to date in characterizing the dispersal variants in P. inhibens, little is understood about the underlying genetic changes that result in the development of the specific variants. Here, P. inhibens phenotypic variation was linked to single nucleotide polymorphisms (SNPs), in particular in genes affecting the competitive ability of P. inhibens, including genes related to the production of the antibiotic tropodithietic acid (TDA) and bacterial cell-cell communication (e.g., quorum sensing). This work is significant as it reveals how the biofilm lifestyle might shape genome evolution in a cosmopolitan bacterium.