title: A quantitative real-time polymerase chain reaction assay for the seagrass pathogen Labyrinthula zosterae.
abstract: 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.
authors.affiliation: Leibniz-Institute of Marine Sciences (IFM-GEOMAR), Experimental Ecology - Food Webs, Düsternbrooker Weg 20, 24105 Kiel, Germany.
authors.forename: Nina
authors.initials: N
authors.lastname: Bergmann
authors.forename: Birgit
authors.initials: B
authors.lastname: Fricke
authors.forename: Martina C
authors.initials: MC
authors.lastname: Schmidt
authors.forename: Verena
authors.initials: V
authors.lastname: Tams
authors.forename: Katrin
authors.initials: K
authors.lastname: Beining
authors.forename: Hildegard
authors.initials: H
authors.lastname: Schwitte
authors.forename: Anne A
authors.initials: AA
authors.lastname: Boettcher
authors.forename: Daniel L
authors.initials: DL
authors.lastname: Martin
authors.forename: Anna-Christina
authors.initials: AC
authors.lastname: Bockelmann
authors.forename: Thorsten B H
authors.initials: TB
authors.lastname: Reusch
authors.forename: Gisep
authors.initials: G
authors.lastname: Rauch
mesh_terms: D017931:DNA Primers / Q000235:genetics; D021903:DNA, Ribosomal Spacer / Q000235:genetics; D018515:Plant Leaves / Q000382:microbiology; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D013045:Species Specificity; D058009:Stramenopiles / Q000235:genetics* / Q000502:physiology; D035962:Zosteraceae / Q000382:microbiology*
chemical_list: D017931:DNA Primers; D021903:DNA, Ribosomal Spacer