Identification of rosmarinic acid and sulfated flavonoids as inhibitors of microfouling on the surface of eelgrass <i>Zostera marina</i>

<p>A bioassay-guided approach was used to identify defense compounds that are present on the surface of <i>Zostera marina</i> and which inhibit settlement of microfoulers at natural concentrations. Moderately polar eelgrass surface extracts inhibited the settlement of seven marine bacteria and one yeast that originated from non-living substrata. In contrast, five other bacterial strains that had been directly isolated from eelgrass surfaces were all insensitive, which suggested a selective effect of surface metabolites on the microbial communities present on eelgrass. Bioassay-guided isolation of active compounds from the extracts in combination with UPLC-MS and ¹H-NMR spectroscopy resulted in the identification of rosmarinic acid, luteolin-7-sulfate and diosmetin-7-sulfate or its isomer chrysoeriol-7-sulfate. All three compounds are nontoxic repellents, as they did not inhibit bacterial growth, but prevented bacterial settlement in a dose-dependent manner. Between 15.6 and 106.8 μg ml⁻¹ of rosmarinic acid were present on the eelgrass surface, enough for half maximal settlement inhibition of bacteria.