Seagrasses worldwide are commonly infected by endophytic protists of the genus Labyrinthula. To date, the nature of the interaction of endophyte and host is not well understood. In eelgrass Zostera marina, the endophyte Labyrinthula zosterae may become virulent (pathogenic) and lead to the loss of entire seagrass beds. One of the best known examples of any marine epidemic was a series of outbreaks of the 'wasting disease' on both sides of the Atlantic in the 1930s, but smaller infestations have been reported more recently. Until now, detection of infection by Labyrinthula spp. was based on the wasting index (i.e. the relative area of leaf lesions) or microscopy, while genetic data were virtually absent. We characterized a ~1400 bp portion of the 18S small subunit rDNA in L. zosterae isolates (N = 41) from 6 northern European sites and 1 southern location (Adriatic Sea) in order to assess the identity and potential diversity of endophytic protists. Because there are indications that low salinity impedes Labyrinthula growth, sampling sites included a wide range of salinities from 5 to 34 psu. A search against the non-redundant GenBank database revealed that most isolates are 99% similar to the only L. zosterae 18S sequence available from the database at all but the Finnish site (salinity values 5 to 7 psu). At this site, a different Labyrinthula species occurred, which was also found in fully marine Wadden Sea cultures. A third species was detected in Skagerrak, south-western Baltic and North Sea samples (20 to 25 psu). We conclude that L. zosterae is widespread among northern European eelgrass sites across wide ranges of salinity. KEY WORDS: Labyrinthula infection · Eelgrass · 18S sequence analysis · Salinity · Wasting diseaseResale or republication not permitted without written consent of the publisher

The protist Labyrinthula zosterae (Phylum Bigyra, sensu Tsui et al. 2009) has been identified as a causative agent of wasting disease in eelgrass (Zostera marina), of which the most intense outbreak led to the destruction of 90% of eelgrass beds in eastern North America and western Europe in the 1930s. Outbreaks still occur today, albeit at a smaller scale. Traditionally, L. zosterae has been quantified by measuring the necrotic area of Z. marina leaf tissue. This indirect method can however only lead to a very rough estimate of pathogen load. Here, we present a quantitative real-time polymerase chain reaction (qPCR) approach to directly detect and quantify L. zosterae in eelgrass tissue. Based on the internal transcribed spacer (ITS) sequences of rRNA genes, species-specific primers were designed. Using our qPCR, we were able to quantify accurately and specifically L. zosterae load both from culture and eelgrass leaves using material from Europe and North America. Our detection limit was less than one L. zosterae cell. Our results demonstrate the potential of this qPCR assay to provide rapid, accurate and sensitive molecular identification and quantification of L. zosterae. In view of declining seagrass populations worldwide, this method will provide a valuable tool for seagrass ecologists and conservation projects.