Labyrinthula Zosterae Research Articles

Pathogenicity and phylogeny of Labyrinthula spp. isolated in Washington and Oregon, USA.

The class Labyrinthulomycetes constitutes a multitude of species found ubiquitously in the environment, and includes pathogens of corals, hard clams, turfgrasses, and seagrasses. Labyrinthula zosterae, the causative agent of seagrass wasting disease, has been associated with declines in seagrass coverage since the 1930s. However, pathogenic and nonpathogenic Labyrinthula spp. have been isolated from seagrass tissue. These isolates are difficult to distinguish morphologically, and the diversity of isolates where seagrass wasting disease is present is often unknown. This study aimed to increase knowledge on the pathogenicity and phylogeny of Labyrinthula spp. in Washington and Oregon, USA where a high prevalence of seagrass wasting disease has been associated with eelgrass, Zostera marina, declines. We tested the pathogenicity of 14 Labyrinthula isolates and compared partial 18S rRNA gene sequences of 12 isolates to sequences from around the world through the NCBI database. We found that pathogenic isolates could be identified as Labyrinthula zosterae, while nonpathogenic isolates did not form a clade with any previously identified SSU ribotypes. These results add to the growing data on Labyrinthula and seagrass wasting disease and can improve our understanding of pathogen evolution and spread in the future.

Seagrass wasting disease prevalence and lesion area increase with invertebrate grazing across the northeastern Pacific.

Disease is a key driver of community and ecosystem structure, especially when it strikes foundation species. In the widespread marine foundation species eelgrass (Zostera marina), outbreaks of wasting disease have caused large-scale meadow collapse in the past, and the causative pathogen, Labyrinthula zosterae, is commonly found in meadows globally. Research to date has mainly focused on abiotic environmental drivers of seagrass wasting disease, but there is strong evidence from other systems that biotic interactions such as herbivory can facilitate plant diseases. How biotic interactions influence seagrass wasting disease in the field is unknown but is potentially important for understanding dynamics of this globally valuable and declining habitat. Here, we investigated links between epifaunal grazers and seagrass wasting disease using a latitudinal field study across 32 eelgrass meadows distributed from southeastern Alaska to southern California. From 2019 to 2021, we conducted annual surveys to assess eelgrass shoot density, morphology, epifauna community, and the prevalence and lesion area of wasting disease infections. We integrated field data with satellite measurements of sea surface temperature and used structural equation modeling to test the magnitude and direction of possible drivers of wasting disease. Our results show that grazing by small invertebrates was associated with a 29% increase in prevalence of wasting disease infections and that both the prevalence and lesion area of disease increased with total epifauna abundances. Furthermore, these relationships differed among taxa; disease levels increased with snail (Lacuna spp.) and idoteid isopod abundances but were not related to abundance of ampithoid amphipods. This field study across 23° of latitude suggests a prominent role for invertebrate consumers in facilitating disease outbreaks with potentially large impacts on coastal seagrass ecosystems.

Invertebrate herbivores influence seagrass wasting disease dynamics.

Although invertebrate herbivores commonly impact terrestrial plant diseases by facilitating transmission of plant pathogens and increasing host susceptibility to infection via wounding, less is known about the role of herbivores in marine plant disease dynamics. Importantly, transmission via herbivores may not be required in the ocean since saline ocean waters support pathogen survival and transmission. Through laboratory experiments with eelgrass (Zostera marina), we showed that isopods (Pentidotea wosnesenskii) and snails (Lacuna spp.) created grazing scars that increased disease severity and thus indirectly facilitated transmission of Labyrinthula zosterae (Lz), a protist that causes seagrass wasting disease. Experiments also quantified different feeding preferences among herbivores: Amphipods (Ampithoe lacertosa) selectively consumed diseased eelgrass, while isopods and snails selectively grazed asymptomatic leaves, suggesting different herbivore taxa may have contrasting impacts on disease dynamics. Our experiments show no sign that herbivores directly vector Lz from diseased to asymptomatic eelgrass. However, we isolated live Lz from isopod, amphipod, and snail feces and detected Lz with quantitative polymerase chain reactionin amphipods and snails, suggesting that herbivores eating diseased eelgrass could pass the live pathogen. Finally, field surveys demonstrated a close association between seagrass wasting disease and invertebrate grazing scars; disease prevalence was 29 ± 4.7% (95% CI) higher on eelgrass leaves with herbivore scars. Collectively, these findings show that some herbivores can increase eelgrass disease risk by facilitating the spread of an important pathogen via wounding, but not via direct transmission. Thus, herbivores may play different roles in plant disease dynamics in terrestrial versus marine ecosystems depending on the pathogen's ability to survive and transmit without a vector.

Marine heatwaves and disease alter community metabolism and DOC fluxes on a widespread habitat-forming seagrass species (Zostera marina)

Climate change and disease are two major threats to maintaining healthy seagrass habitats. Seagrasses, and the ecosystems they support, play a critical ecological role in global carbon (C) cycles, providing key ecosystem services, such as blue carbon storage. Zostera marina (eelgrass), the most widespread seagrass species globally, is increasingly affected by warming and is also regularly infected by the endophytic pathogen Labyrinthula zosterae. Both stressors negatively impact plant physiology and population distributions, yet the effects of these stressors on C cycling, and particularly on C metabolism and dissolved organic carbon (DOC) fluxes in eelgrass, remain largely unexplored. Through a mesocosm experiment simulating a marine heatwave (MHW) followed by pathogen challenge with L. zosterae, it was observed that the simulated MHW initially decreased daily community DOC fluxes and Net Production Rates (NPR), while not changing Respiration Rates. DOC released into the water column at the end of the MHW also was less bioavailable than DOC from the control treatment. Importantly, community NPR recovered to control levels after the simulated MHW was over, demonstrating the community's resilience to warming. On the other hand, plants challenged with L. zosterae, which caused a significant decrease in aboveground biomass, exhibited significant decreases in DOC and NPR up to 20 days after the infection. These results have important implications in blue carbon processes, given that both stressors significantly impact the quantity and quality of DOC produced by Z. marina communities. These findings also highlight the differing levels of resilience of C cycling in this system by showing that the impacts of the simulated heat wave may be more transient when compared to the effects of disease.

High infectivity and waterborne transmission of seagrass wasting disease.

Pathogen transmission pathways are fundamental to understanding the epidemiology of infectious diseases yet are challenging to estimate in nature, particularly in the ocean. Seagrass wasting disease (SWD), caused by Labyrinthula zosterae, impacts seagrass beds worldwide and is thought to be a contributing factor to declines; however, little is known about natural transmission of SWD. In this study, we used field and laboratory experiments to test SWD transmission pathways and temperature sensitivity. To test transmission modes in nature, we conducted three field experiments out-planting sentinel Zostera marina shoots within and adjacent to natural Z. marina beds (20 ± 5 and 110 ± 5 m from bed edge). Infection rates and severity did not differ among outplant locations, implicating waterborne transmission. The infectious dose of L. zosterae through waterborne exposure was assessed in a controlled laboratory experiment. The dose to 50% disease was 6 cells ml-1 and did not differ with the temperatures tested (7.5°C and 15°C). Our results show L. zosterae is transmissible through water without direct contact with infected plants. Understanding the transmission dynamics of this disease in the context of changing ocean conditions will improve Z. marina protection and restoration in critical coastal habitats worldwide.

Taking the Pulse of Resilience in Conserving Seagrass Meadows.

Foundational habitats such as seagrasses and coral reefs are at severe risk globally from climate warming. Infectious disease associated with warming events is both a cause of decline and an indicator of stress in both habitats. Since new approaches are needed to detect refugia and design climate-smart networks of marine protected areas, we test the hypothesis that the health of eelgrass (Zostera marina) in temperate ecosystems can serve as a proxy indicative of higher resilience and help pinpoint refugia. Eelgrass meadows worldwide are at risk from environmental stressors, including climate warming and disease. Disease outbreaks of Labyrinthula zosterae are associated with recent, widespread declines in eelgrass meadows throughout the San Juan Islands, Washington, USA. Machine language learning, drone surveys, and molecular diagnostics reveal climate impacts on seagrass wasting disease prevalence (proportion of infected individuals) and severity (proportion of infected leaf area) from San Diego, California, to Alaska. Given that warmer temperatures favor many pathogens such as L. zosterae, we hypothesize that absent or low disease severity in meadows could indicate eelgrass resilience to climate and pathogenic stressors. Regional surveys showed the San Juan Islands as a hotspot for both high disease prevalence and severity, and surveys throughout the Northeast Pacific indicated higher prevalence and severity in intertidal, rather than subtidal, meadows. Further, among sites with eelgrass declines, losses were more pronounced at sites with shallower eelgrass meadows. We suggest that deeper meadows with the lowest disease severity will be refuges from future warming and pathogenic stressors in the Northeast Pacific. Disease monitoring may be a useful conservation approach for marine foundation species, as low or absent disease severity can pinpoint resilient refugia that should be prioritized for future conservation efforts. Even in declining or at-risk habitats, disease surveys can help identify meadows that may contain especially resilient individuals for future restoration efforts. Our approach of using disease as a pulse point for eelgrass resilience to multiple stressors could be applied to other habitats such as coral reefs to inform conservation and management decisions.

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.

Characterizing host-pathogen interactions between Zostera marina and Labyrinthula zosterae

IntroductionSeagrass meadows serve as an integral component of coastal ecosystems but are declining rapidly due to numerous anthropogenic stressors including climate change. Eelgrass wasting disease, caused by opportunistic Labyrinthula spp., is an increasing concern with rising seawater temperature. To better understand the host-pathogen interaction, we paired whole organism physiological assays with dual transcriptomic analysis of the infected host and parasite. MethodsEelgrass (Zostera marina) shoots were placed in one of two temperature treatments, 11° C or 18° C, acclimated for 10 days, and exposed to a waterborne inoculation containing infectious Labyrinthula zosterae (Lz) or sterile seawater. At two- and five-days post-exposure, pathogen load, visible disease signs, whole leaf phenolic content, and both host- and pathogen- transcriptomes were characterized. ResultsTwo days after exposure, more than 90% of plants had visible lesions and Lz DNA was detectable in 100% percent of sampled plants in the Lz exposed treatment. Concentrations of total phenolic compounds were lower after 5 days of combined exposure to warmer temperatures and Lz, but were unaffected in other treatments. Concentrations of condensed tannins were not affected by Lz or temperature, and did not change over time. Analysis of the eelgrass transcriptome revealed 540 differentially expressed genes in response to Lz exposure, but not temperature. Lz-exposed plants had gene expression patterns consistent with increased defense responses through altered regulation of phytohormone biosynthesis, stress response, and immune function pathways. Analysis of the pathogen transcriptome revealed up-regulation of genes potentially involved in breakdown of host defense, chemotaxis, phagocytosis, and metabolism. DiscussionThe lack of a significant temperature signal was unexpected but suggests a more pronounced physiological response to Lz infection as compared to temperature. Pre-acclimation of eelgrass plants to the temperature treatments may have contributed to the limited physiological responses to temperature. Collectively, these data characterize a widespread physiological response to pathogen attack and demonstrate the value of paired transcriptomics to understand infections in a host-pathogen system.

The effect of warming on seagrass wasting disease depends on host genotypic identity and diversity.

Temperature increases due to climate change have affected the distribution and severity of diseases in natural systems, causing outbreaks that can destroy host populations. Host identity, diversity, and the associated microbiome can affect host responses to both infection and temperature, but little is known about how they could function as important mediators of disease in altered thermal environments. We conducted an 8-week warming experiment to test the independent and interactive effects of warming, host genotypic identity, and host genotypic diversity on the prevalence and intensity of infections of seagrass (Zostera marina) by the wasting disease parasite (Labyrinthula zosterae). At elevated temperatures, we found that genotypically diverse host assemblages had reduced infection intensity, but not reduced prevalence, relative to less diverse assemblages. This dilution effect on parasite intensity was the result of both host composition effects as well as emergent properties of biodiversity. In contrast with the benefits of genotypic diversity under warming, diversity actually increased parasite intensity slightly in ambient temperatures. We found mixed support for the hypothesis that a growth-defense trade-off contributed to elevated disease intensity under warming. Changes in the abundance (but not composition) of a few taxa in the host microbiome were correlated with genotype-specific responses to wasting disease infections under warming, consistent with the emerging evidence linking changes in the host microbiome to the outcome of host-parasite interactions. This work emphasizes the context dependence of biodiversity-disease relationships and highlights the potential importance of interactions among biodiversity loss, climate change, and disease outbreaks in a key foundation species.

Disease surveillance by artificial intelligence links eelgrass wasting disease to ocean warming across latitudes

AbstractOcean warming endangers coastal ecosystems through increased risk of infectious disease, yet detection, surveillance, and forecasting of marine diseases remain limited. Eelgrass (Zostera marina) meadows provide essential coastal habitat and are vulnerable to a temperature‐sensitive wasting disease caused by the protist Labyrinthula zosterae. We assessed wasting disease sensitivity to warming temperatures across a 3500 km study range by combining long‐term satellite remote sensing of ocean temperature with field surveys from 32 meadows along the Pacific coast of North America in 2019. Between 11% and 99% of plants were infected in individual meadows, with up to 35% of plant tissue damaged. Disease prevalence was 3× higher in locations with warm temperature anomalies in summer, indicating that the risk of wasting disease will increase with climate warming throughout the geographic range for eelgrass. Large‐scale surveys were made possible for the first time by the Eelgrass Lesion Image Segmentation Application, an artificial intelligence (AI) system that quantifies eelgrass wasting disease 5000× faster and with comparable accuracy to a human expert. This study highlights the value of AI in marine biological observing specifically for detecting widespread climate‐driven disease outbreaks.

Pacific oysters are a sink and a potential source of the eelgrass pathogen, Labyrinthula zosterae

Oyster aquaculture and seagrasses often co-occur and are each vital to the ecological and economic value of coastal ecosystems. Global declines in seagrasses, including Zostera marina, have recently been observed in association with multiple factors, including infection with diseases such as seagrass wasting disease (SWD), caused by the protist Labyrinthula zosterae. Protection of seagrasses has led to restrictions on oyster aquaculture due to perceived negative impacts on seagrass beds; however, positive impacts may also occur. An important aquaculture species, the Pacific oyster Crassostrea gigas, can filter L. zosterae from the water, potentially reducing pathogen transmission, although oysters may vector infection if they accumulate and release live L. zosterae into the water. We investigated whether oyster presence decreases lesion severity and infection intensity in eelgrass, or acts as a vector of L. zosterae, via laboratory and field experiments. In the laboratory, oysters and eelgrass were exposed to L. zosterae for 24 h and kept at 11°C or 18°C for 13 d. In the field, eelgrass ramets were deployed with and without oysters for 28 d adjacent to eelgrass known to have SWD. In the laboratory experiment, the presence of oysters significantly decreased lesion severity and infection intensity, but oysters previously exposed to L. zosterae did transmit the pathogen to naïve eelgrass. Temperature did not affect oyster ability to mitigate SWD; however, increased temperature significantly increased lesion severity. Oysters had no effect on SWD in the field. Further research is needed regarding the potential for oysters to vector L. zosterae and to quantify when oysters reduce SWD in the field.

Effects of Seagrass Wasting Disease on Eelgrass Growth and Belowground Sugar in Natural Meadows

Seagrass meadows provide valuable ecosystem benefits but are at risk from disease. Eelgrass (Zostera marina) is a temperate species threatened by seagrass wasting disease (SWD), caused by the protist Labyrinthula zosterae. The pathogen is sensitive to warming ocean temperatures, prompting a need for greater understanding of the impacts on host health under climate change. Previous work demonstrates pathogen cultures grow faster under warmer laboratory conditions and documents positive correlations between warmer ocean temperatures and disease levels in nature. However, the consequences of disease outbreaks on eelgrass growth remain poorly understood. Here, we examined the effect of disease on eelgrass productivity in the field. We coupled in situ shoot marking with high-resolution imagery of eelgrass blades and used an artificial intelligence application to determine disease prevalence and severity from digital images. Comparisons of eelgrass growth and disease metrics showed that SWD impaired eelgrass growth and accumulation of non-structural carbon in the field. Blades with more severe disease had reduced growth rates, indicating that disease severity can limit plant growth. Disease severity and rhizome sugar content were also inversely related, suggesting that disease reduced belowground carbon accumulation. Finally, repeated measurements of diseased blades indicated that lesions can grow faster than healthy tissue in situ. This is the first study to demonstrate the negative impact of wasting disease on eelgrass health in a natural meadow. These results emphasize the importance of considering disease alongside other stressors to better predict the health and functioning of seagrass meadows in the Anthropocene.

First report of Labyrinthula zosterae (Labyrinthulomycetes) as the causal pathogen of wasting disease in the seagrass Zostera marina in Korea.

Zostera marina L. plants have been seriously impacted by wasting disease along the Atlantic coasts of North America and Europe since the 1930s (Muehlstein 1989). Sudden declines in the population sizes of Zostera marina affect primary and secondary producers of different trophic levels in blue carbon ecosystems (Gleason et al. 2013). Muehlstein et al. (1991) first identified Labyrinthula zosterae (Labyrinthulomycetes) as the pathogen causing wasting disease in Zostera marina. However, there have been no reports of wasting disease pathogens affecting seagrass in Korea. In this study, we collected leaves of Z. marina showing symptoms of wasting disease in the southern region of South Korea (Dongdaeman, Namhae, Gyeongnam Province) during field monitoring (from April to September 2013). The pathogens of wasting disease, Labyrinthula zosterae has been isolated from the infected leaves of Z. marina and established as a culture strain (Supplementary Figure 1). Samples of Z. marina and L. zosterae were deposited at the Fisheries Seed and Breeding Research Institute (previous Seaweed Research Center, National Institute of Fisheries Science, South Korea). Microscopic examination of the infected leaf tissues revealed fusiform or spindle-shaped vegetative Labyrinthula cells (4-5 × 15-20 μm). These were similar in size and shape to those previously described for Labyrinthula species. The fusiform cells were cultured in 1% serum seawater agar medium, and they formed colonies and showed gliding motility along a network of hyaline slime filaments. To validate the pathogenicity, re-inoculation tests by L. zosterae were performed with the isolated strains in accordance with Koch's postulates. Healthy leaves of Z. marina collected from the field were used in the re-inoculation tests and were cultured at 15°C under white fluorescent irradiation of approximately 20 μmol·photons·m-2·s-1 and a 12:12-h light:dark cycle (Supplementary Figure 1). Labyrinthula zosterae re-isolated from artificially infected leaves of Z. marina was confirmed by DNA sequence similarity analysis. Total genomic DNA from the infected leaf cells and the culture strains was extracted using the QIAamp DNA Stool Mini Kit (Qiagen, Germany). Internal transcribed spacer (ITS) sequences of nuclear ribosomal DNA were determined to identify Labyrinthula species. L. zosterae-specific primers (Lz2forward (5'- CTAAGACTAAACGAGGCGAAAGCCTAC-3') and Lz2reverse (5'-AGGTTTACAAAACACACTCGTCCACA-3') in Bergmann et al. (2011)) were used to confirm the infection of L. zosterae in the leaves from the field samples and the re-inoculation test samples. Next, PCR products were cloned using a pLUG-Prime® TA-cloning Vector (iNtRON Biotechnology, Korea) and commercially sequenced (SolGent, Korea). The ITS sequence of Korean L. zosterae (accession number MW357748) showed high sequence similarity (99.3-100%) with that of L. zosterae deposited in GenBank (National Center for Biotechnology Information) from BLAST searches. These findings confirm that this is the first report of L. zosterae as the causal pathogen of wasting disease in Z. marina in Korea.

Metabarcoding of environmental samples suggest wide distribution of eelgrass (Zostera marina) pathogens in the north Pacific

Seagrass meadows provide important ecological services to the marine environment but are declining worldwide. Although eelgrass meadows in the north Pacific are thought to be relatively healthy, few studies have assessed the presence of known disease pathogens in these meadows. In a pilot study to test the efficacy of the methods and to provide foundational disease biodiversity data in the north Pacific, we leveraged metabarcoding of environmental DNA extracted from water, sediment, and eelgrass tissue samples collected from five widely distributed eelgrass meadows in Alaska and one in Japan and uncovered wide prevalence of two classes of pathogenic organisms – Labyrinthula zosterae and other associated strains of Labyrinthula, and the Phytophthora/Halophytophthora blight species complex – known to have caused decline in eelgrass (Zostera marina) elsewhere in the species’ global distribution. Although the distribution of these disease organisms is not well understood in the north Pacific, we uncovered the presence of at least one eelgrass pathogen at every locality sampled.

Shifts in the seagrass leaf microbiome associated with wasting disease in

Seagrass wasting disease (SWD), an infection believed to be caused by Labyrinthula zosterae, has been linked to seagrass declines in several places around the world. However, there is uncertainty about the mechanisms of disease and the potential involvement of opportunistic colonising microorganisms. Using 16S rRNA gene amplicon sequencing, we compared the microbiome of SWD lesions in leaves of Zostera muelleri with communities in adjacent asymptomatic tissues and healthy leaves. The microbiome of healthy leaf tissues was dominated by Pseudomonas and Burkholderia, whereas the most predominant taxa within adjacent tissues were Pseudomonas and Rubidimonas. Members of the Saprospiraceae, potential macroalgal pathogens, were over-represented within SWD lesions. These pronounced changes in microbiome structure were also apparent when we examined the core microbiome of different tissue types. Although the core microbiome associated with healthy leaves included three operational taxonomic units (OTUs) classified as Burkholderia, Cryomorphaceae and the SAR11 clade, a single core OTU from the Arenicella was found within adjacent tissues. Burkholderia are diazotrophic microorganisms and may play an important role in seagrass nitrogen acquisition. In contrast, some members of the Arenicella have been implicated in necrotic disease in other benthic animals. Moreover, microbiome structure was maintained across sites within healthy tissues, but not within SWD lesions or the tissues immediately adjacent to lesions. Predicted functional profiles revealed increased photoautotrophic functions in SWD tissues relative to healthy leaves, but no increase in pathogenicity or virulence. Notably, we demonstrated the presence of L. zosterae in SWD lesions by polymerase chain reaction, but only in one of the two sampled locations, which indicates that other microbiological factors may be involved in the initiation or development of SWD-like symptoms. This study suggests that the dynamics of the seagrass microbiome should be considered within the diagnosis and management of SWD.

Complex Interactions of Temperature, Light and Tissue Damage on Seagrass Wasting Disease in Zostera marina

The temperate seagrass species eelgrass Zostera marina can be infected by the wasting disease pathogen Labyrinthula zosterae, which is believed to have killed about 90% of the seagrass in the Atlantic Ocean in the 1930s. It is not known why this opportunistic pathogen sometimes becomes virulent, but the recurrent outbreaks may be due to a weakening of the Z. marina plants from adverse environmental changes. This study investigated the individual and interactive effects of multiple extrinsic factors (temperature, light, and tissue damage) on the host-pathogen interaction between Z. marina and L. zosterae in a fully crossed infection experiment. The degree of infection was measured as both lesion coverage and L. zosterae cell concentration. We also investigated if the treatment factors affect the chemical defense of the host, measured as the inhibitory capacity of seagrass extracts in bioassays with L. zosterae. Finally, gene expression of a set of targeted genes was quantified in order to investigate how the treatments change Z. marina’s response to infection. Light had a pronounced effect on L. zosterae infection measured as lesion coverage, where reduced light conditions increased lesions by 35%. The response to light on L. zosterae cell concentration was more complex and showed significant interaction with the temperature treatment. Cell concentration was also significantly affected by physical damage, where damage surprisingly resulted in a reduced cell concentration of the pathogen. No treatment factor caused detectable decrease in the inhibitory capacity of the seagrass extracts. There were several interactive effects between L. zosterae infection and the treatment factors on Z. marina growth, and on the expression of genes associated with immune defense, phenol synthesis and primary metabolism, showing that the molecular reaction towards L. zosterae infection depends on prevailing environmental conditions. Our study shows that individual or interactive effects of light, temperature and tissue damage can affect multiple aspects of host-pathogen interactions in seagrass. These results highlights the complexity of marine host-pathogen systems, showing that more multi-factorial investigations are needed to gain a better understanding of disease in marine plants under different environmental conditions.

Eelgrass pathogen Labyrinthula zosterae synthesizes essential fatty acids

Negative consequences of parasites and disease on hosts are usually better understood than their multifaceted ecosystem effects. The pathogen Labyrinthula zosterae (Lz) causes eelgrass wasting disease but has relatives that produce large quantities of nutritionally valuable long-chain polyunsaturated fatty acids (LCPUFA) such as docosahexaenoic acid (DHA). Here we quantify the fatty acids (FA) of Lz cultured on artificial media, eelgrass-based media, and eelgrass segments to investigate whether Lz may similarly produce LCPUFA. We also assess whether field-collected lesions show similar FA patterns to laboratory-inoculated eelgrass. We find that Lz produces DHA as its dominant FA along with other essential FA on both artificial and eelgrass-based media. DHA content was greater in both laboratory-inoculated and field-collected diseased eelgrass relative to their respective controls. If Lz's production scales in situ, it may present an unrecognized source of LCPUFA in eelgrass ecosystems.

Modulation of the Eelgrass – Labyrinthula zosterae Interaction Under Predicted Ocean Warming, Salinity Change and Light Limitation

Marine infectious diseases can have large-scale impacts when they affect foundation species such as seagrasses and corals. Interactions between host and disease, in turn, may be modulated by multiple perturbations associated with global change. A case in point is the infection of the foundation species Zostera marina (eelgrass) with endophytic net slime molds (Labyrinthula zosterae), the putative agent of eelgrass wasting disease that caused one of the most severe marine pandemics across the North-Atlantic in the 1930s. The contemporary presence of L. zosterae in many eelgrass meadows throughout Europe raises the question whether such a pandemic may re-appear if coastal waters become more eutrophic, warmer and less saline. Accordingly, we exposed uninfected Baltic Sea Z. marina plants raised from seeds to full factorial combinations of controlled L. zosterae inoculation, heat stress, light limitation (mimicking one consequence of eutrophication) and two salinity levels. We followed eelgrass wasting disease dynamics, along with several eelgrass responses such as leaf growth, mortality and carbohydrate storage, as well as the ability of plants to chemically inhibit L. zosterae growth. Contrary to our expectation, inoculation with L. zosterae reduced leaf growth and survival only under the most adverse condition to eelgrass (reduced light and warm temperatures). We detected a strong interaction between salinity and temperature on L. zosterae abundance and pathogenicity. The protist was unable to infect eelgrass under high temperature (27°C) in combination with low salinity (12 psu). With the exception of a small positive effect of temperature alone, no further effects of any of the treatment combinations on the defense capacity of eelgrass against L. zosterae were detectable. This work supports the idea that contemporary L. zosterae isolates neither represent an immediate risk for eelgrass beds in the Baltic Sea, nor a future one under the predicted salinity decrease and warming of the Baltic Sea.

Seagrass wasting disease along a naturally occurring salinity gradient

In the 1930s, outbreaks of the wasting disease pathogen Labyrinthula zosterae caused a severe reduction of the eelgrass Zostera marina meadows in the Atlantic Ocean. Many surviving populations were found in low-salinity environments, and low-salinity environments have therefore been hypothesized to act as a refuge for eelgrass against L. zosterae infection. Here, we investigated L. zosterae pathogen load and wasting disease symptoms in eelgrass over a similar to 970 km salinity gradient (6-25 PSU) along the Swedish coast. Furthermore, laboratory infection experiments and studies of inhibitory compounds were carried out to investigated whether resistance against the pathogen is correlated to differences in natural pathogen pressure among eelgrass populations. The degree of L. zosterae infection was positively correlated to salinity and the pathogen was absent in several of the eelgrass meadows in lower salinity (7-8 PSU). However, a low L. zosterae pathogen load was also found in some eelgrass populations in the lowest salinity (6 PSU). No correlation between resistance and pathogen pressure in situ was detected, and all eelgrass shoots produced chemical compounds that inhibited L. zosterae growth. These results imply that positive correlations between L. zosterae and salinity are not due to eelgrass resistance, but rather to the poor ability of L. zosterae to cope with low salinity. However, our results also indicate that some strains of L. zosterae may adapt to low salinity, and therefore there may also be a risk of wasting disease outbreaks in low-salinity eelgrass meadows, in contrast to what so far has been the general hypothesis.

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.