Aquatic Botany 122 (2015) 1–8 Contents lists available at ScienceDirect Aquatic Botany jou rn al hom ep age: www.elsevier.com/locate/aquabot Metabolites derived from the tropical seagrass Thalassia testudinum are bioactive against pathogenic Labyrinthula sp Stacey M. Trevathan-Tackett a,c , Amy L. Lane b , Nichole Bishop a , Cliff Ross a,∗ a Department of Biology, University of North Florida, 1 UNF Drive, Jacksonville, FL 32224, USA b Department of Chemistry, University of North Florida, 1 UNF Drive, Jacksonville, FL 32224, USA c University of Technology Sydney, Ultimo, NSW 2007, Australia a r t i c l e i n f o Article history: Received 15 October 2014 Received in revised form 11 December 2014 Accepted 21 December 2014 Available online 26 December 2014 Keywords: Seagrass defense Thalassia testudinum Labyrinthula Secondary metabolites Wasting disease a b s t r a c t Temperate and tropical seagrasses are susceptible to wasting disease outbreaks caused by pathogenic protists of the genus Labyrinthula. Even though there is an increasing awareness of the environmental conditions that influence the etiology of seagrass-Labyrinthula disease dynamics, the biochemical basis of seagrass defense responses, in particular chemical defenses, is still vastly understudied. Using an in vitro bioassay, we provide evidence that previously characterized phenolic and potentially novel, undescribed non-phenolic metabolites derived from Thalassia testudinum Banks ex Konig exhibit anti-labyrinthulid activity. All phenolic compounds tested displayed dose-dependent behavior and selected combinations interacted synergistically. The flavone glycoside thalassiolin B was roughly 20–100 times more active than any phenolic acid tested. Based upon values reported in the literature, it was calculated that infected specimens of T. testudinum contain natural concentrations of phenolic acids that are consistently greater than what is required to inhibit Labyrinthula growth. This suggests that while there may be an ample supply of phenolic-based derivatives available to inhibit Labyrinthula growth, they may not be readily bio-accessible. Using a bioactivity-guided approach, a semi-purified chemical fraction from T. testudinum was found to contain anti-labyrinthulid activity. 1 H NMR spectra for this fraction lacked aromatic hydrogen signals, suggesting that the bioactive compound was non-aromatic in nature. Furthermore, the LC–MS fragmen- tation patterns were suggestive of the presence of glycosylated natural products of an unknown structural class. This has the potential to provide a foundation for future chemical investigations. © 2014 Elsevier B.V. All rights reserved. 1. Introduction Seagrass meadows form the underpinning of numerous tropical and temperate coastal ecosystems. Aside from serving as impor- tant nursery grounds for commercial fisheries, these plant-based ecosystems perform a multitude of services that include primary production, carbon storage and biogeochemical cycling (Heck et al., 2003; Barbier et al., 2011). Over the past 80 years, seagrass beds have experienced significant declines due to environmental stress- ors that operate at both regional (e.g. nutrient loading) and global Abbreviations: 1 H-NMR, proton nuclear magnetic resonance; 3,4-dihyd, 3,4-dihydroxybenzoic acid; ACN, acetonitrile; H2O, water; LC–MS, liquid chromatography-mass spectrometry; IC50, half maximal inhibitory concentra- tion; MeOH, methanol; p-coum, p-coumaric acid; p-hyd, p-hydroxybenzoic acid; RP-HPLC, reverse-phase high performanceliquid chromatography; SA/PP, shikimic acid and phenylpropanoid. ∗ Corresponding author. Tel.: +1 904 620 1853; fax: +1 904 620 3885. E-mail address: cliff.ross@unf.edu (C. Ross). (e.g. elevated sea surface temperatures) scales (Waycott et al., 2009). One such cause of decline is the incidence of disease in which pathogenic protists of the genus Labyrinthula sp. (Protistan supergroup Chromoalveolata, phylum Labyrinthulomycota) cause significant ‘wasting’ epidemics (Muehlstein, 1989). These wasting disease outbreaks are typically observed as patchy mortality events yet there are long-lasting ramifications as selected populations of dominant seagrass species (e.g. Thalassia testudinum Banks ex Konig, which is found in tropics and subtropics and Zostera marina Linnaeus, which is found in temperate regions) can be driven to local extinction (Short et al., 1988). As seagrass cover continues to decline at a rapid pace, the need exists to better understand the biological and physical factors that regulate host-pathogen inter- actions in the marine environment. Even though there is an increasing awareness in the environmental conditions that influence the etiology of seagrass- Labyrinthula disease dynamics (reviewed in Sullivan et al., 2013), the biochemical basis of seagrass defense responses, in particu- lar antimicrobial chemical defenses, is still vastly understudied http://dx.doi.org/10.1016/j.aquabot.2014.12.005 0304-3770/© 2014 Elsevier B.V. All rights reserved.