Supplement to: Papazian, Stefano; Parrot, Delphine; Burýšková, Barbora; Weinberger, Florian; Tasdemir, Deniz (2019): Surface chemical defence of the eelgrass Zostera marina against microbial foulers. Scientific Reports, 9(1), https://doi.org/10.1038/s41598-019-39212-3
Dose-response experiments for yeast growth or settlement were conducted in 96-well plates impregnated with eelgrass extracts Concentrations of leaf surface extracts were related to naturally occurring concentrations on Z. marina surfaces assuming a leaf area / fresh-weight ratio of 78.99 cm2*g-1. Correspondingly, a 1-fold natural concentration of surface extract was tested when extract obtained from 0.955 cm² leaf surface was impregnated onto 0.955 cm² well surface. Concentrations of tissue extracts were dosed based on the assumption of a dry-weight / fresh-weight proportion of 10%. Correspondingly, a 1-fold natural concentration of tissue extract was tested when extract obtained from 10 mg dry weight was present in the final volume of 100 µl. The yeasts were maintained at 25°C on a shaker in liquid medium containing: 3 g of yeast extract, 3 g of malt extract, 5 g of peptone, 10 g of glucose, and sea salt 3%.
For settlement assays aliquots of yeast liquid cultures were pipetted into the wells. After 2 h, the wells were emptied and cells attached to the walls were stained with Calcofluor white for 10 min. The unattached cells and the excess dye were removed by rinsing with sterile seawater, and the fluorescence of stained cells attached to the wells was measured at 350 nm excitation and 430 nm emission.
For growth bioassays, 100 µl medium containing yeast cells at 0.07-0.17 initial OD610 were pipetted into the wells. Plates were incubated at 25°C on a shaker in darkness and OD610 was repeatedly measured over 20 h. Exponential growth curves were fitted with the GraphPad Prism 5.0 software package to OD data time series obtained for each well allowing determination of division rates. Division rates obtained for single wells with addition of compounds were then related to mean division rates obtained for six control wells without such addition.
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