Quantitative PCR reveals strong spatial and temporal variation of the wasting disease pathogen, Labyrinthula zosterae in northern European eelgrass (Zostera marina) beds

Seagrass beds are the foundation species of functionally important coastal ecosystems worldwide. The world’s largest losses of the widespread seagrass Zostera marina (eelgrass) have been reported as a consequence of wasting disease, an infection with the endophytic protist Labyrinthula zosterae. During one of the most extended epidemics in the marine realm, ~90% of East and Western Atlantic eelgrass beds died-off between 1932 and 1934. Today, small outbreaks continue to be reported, but the current extent of L. zosterae in European meadows is completely unknown. In this study we quantify the abundance and prevalence of the wasting disease pathogen among 19 Z. marina populations in northern European coastal waters, using quantitative PCR (QPCR) with primers targeting a species specific portion of the internally transcribed spacer (ITS1) of L. zosterae. Spatially, we found marked variation among sites with abundances varying between 0 and 126 cells mg−1 Z. marina dry weight (mean: 5.7 L. zosterae cells mg−1 Z. marina dry weight ±1.9 SE) and prevalences ranged from 0–88.9%. Temporarily, abundances varied between 0 and 271 cells mg−1 Z. marina dry weight (mean: 8.5±2.6 SE), while prevalences ranged from zero in winter and early spring to 96% in summer. Field concentrations accessed via bulk DNA extraction and subsequent QPCR correlated well with prevalence data estimated via isolation and cultivation from live plant tissue. L. zosterae was not only detectable in black lesions, a sign of Labyrinthula-induced necrosis, but also occurred in green, apparently healthy tissue. We conclude that L. zosterae infection is common (84% infected populations) in (northern) European eelgrass populations with highest abundances during the summer months. In the light of global climate change and increasing rate of marine diseases our data provide a baseline for further studies on the causes of pathogenic outbreaks of L. zosterae

Current European Labyrinthula zosterae Are Not Virulent and Modulate Seagrass (Zostera marina) Defense Gene Expression

Pro- and eukaryotic microbes associated with multi-cellular organisms are receiving increasing attention as a driving factor in ecosystems. Endophytes in plants can change host performance by altering nutrient uptake, secondary metabolite production or defense mechanisms. Recent studies detected widespread prevalence of Labyrinthula zosterae in European Zostera marina meadows, a protist that allegedly caused a massive amphi-Atlantic seagrass die-off event in the 1930's, while showing only limited virulence today. As a limiting factor for pathogenicity, we investigated genotype×genotype interactions of host and pathogen from different regions (10–100 km-scale) through reciprocal infection. Although the endophyte rapidly infected Z. marina, we found little evidence that Z. marina was negatively impacted by L. zosterae. Instead Z. marina showed enhanced leaf growth and kept endophyte abundance low. Moreover, we found almost no interaction of protist×eelgrass-origin on different parameters of L. zosterae virulence/Z. marina performance, and also no increase in mortality after experimental infection. In a target gene approach, we identified a significant down-regulation in the expression of 6/11 genes from the defense cascade of Z. marina after real-time quantitative PCR, revealing strong immune modulation of the host's defense by a potential parasite for the first time in a marine plant. Nevertheless, one gene involved in phenol synthesis was strongly up-regulated, indicating that Z. marina plants were probably able to control the level of infection. There was no change in expression in a general stress indicator gene (HSP70). Mean L. zosterae abundances decreased below 10% after 16 days of experimental runtime. We conclude that under non-stress conditions L. zosterae infection in the study region is not associated with substantial virulence

Distribution and Pathogenicity of the Protist Labyrinthula sp. in western Mediterranean Seagrass Meadows

The presence of the pathogenic protist Labyrinthula sp., the causative agent of seagrass wasting disease and mass mortality events, was assessed in 18 seagrass meadows in the Balearic region (western Mediterranean). This protist was found in 70% of seagrass meadows investigated and in all seagrass species present in the region (i. e., Posidonia oceanica, Cymodocea nodosa, and Zostera noltii). Labyrinthula spp. cultures isolated from seven P. oceanica and one Thalassia testudinum meadows were used as inocula to perform cross-infection experiments in order to test seagrass vulnerability to Labyrinthula spp. infection. These isolates produced lesions on P. oceanica and other seagrass species (Zostera marina, Z. noltii, and C. nodosa). P. oceanica and Z. noltii, both species autochthonous to the Mediterranean Sea, were the seagrasses most vulnerable to infection by the tested isolates. One of the P. oceanica isolates of Labyrinthula sp. also infected the Atlantic seagrass Z. marina, and all of the Mediterranean seagrasses were infected by Labyrinthula sp. isolated from the T. testudinum, native to the Caribbean and Gulf of Mexico. This work confirms that Labyrinthula sp. is commonly found on seagrasses of the Mediterranean Sea and demonstrates that Labyrinthula sp. can infect seagrasses in different genera, in contrast to previous studies where Labyrinthula sp. was considered to be genus-specific. This finding points out the broadly pathogenic nature of some Labyrinthula sp. isolates. Finally, this work identifies Labyrinthula sp. as a possible detrimental agent for P. oceanica. © 2011 Coastal and Estuarine Research Federation.This work was funded by the Fundación BBVA (project Praderas), the Spanish Ministry of Environment (project ref: 55/2002), and the Spanish Marine Science and Technology program (project MEDEICG, CTM2009-07013).Peer Reviewe

Impacts of Groundwater Discharge at Myora Springs (North Stradbroke Island, Australia) on the Phenolic Metabolism of Eelgrass, Zostera muelleri, and Grazing by the Juvenile Rabbitfish, Siganus fuscescens

Myora Springs is one of many groundwater discharge sites on North Stradbroke Island (Queensland, Australia). Here spring waters emerge from wetland forests to join Moreton Bay, mixing with seawater over seagrass meadows dominated by eelgrass, Zostera muelleri. We sought to determine how low pH /high CO2 conditions near the spring affect these plants and their interactions with the black rabbitfish (Siganus fuscescens), a co-occurring grazer. In paired-choice feeding trials S. fuscescens preferentially consumed Z. muelleri shoots collected nearest to Myora Springs. Proximity to the spring did not significantly alter the carbon and nitrogen contents of seagrass tissues but did result in the extraordinary loss of soluble phenolics, including Folin-reactive phenolics, condensed tannins, and phenolic acids by >= 87%. Conversely, seagrass lignin contents were, in this and related experiments, unaffected or increased, suggesting a shift in secondary metabolism away from the production of soluble, but not insoluble, (poly)phenolics. We suggest that groundwater discharge sites such as Myora Springs, and other sites characterized by low pH, are likely to be popular feeding grounds for seagrass grazers seeking to reduce their exposure to soluble phenolics

Toxicity of reduced nitrogen in eelgrass (Zostera marina) is highly dependent on shoot density and pH

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