Catalase assay

Quantification of the cellular catalase activity in Bd and Bsal isolates was carried out following the protocol of Iwase et al. [21]. A mixture of zoospores and zoosporangia was obtained by flooding 6-day-old broth cultures of Bd isolate JEL 423 and Bsal isolate AMFP 13/1 (both at passage 13) with sterile distilled water. Cell suspensions were centrifuged for 5 minutes at 2500 rpm, rinsed in sterile distilled water, centrifuged and suspended to a final concentration of 10 mg/100 μl (≈ 1x10⁷ cells/ml). Commercial catalase (from bovine liver, Sigma Aldrich) was dissolved in ultra-pure distilled water and diluted to obtain catalase standards containing 0 to 200 catalase units. Hundred μl of each catalase standard was mixed with 100 μl of a 1% Triton^™X-100 solution (Sigma Aldrich) and 100 μl of a 30% H₂O₂ solution, in a borosilicate reagent tube (13mm x 10mm, VWR). Hundred μl of the Bd or Bsal cell suspensions was mixed with equal volumes of Triton^™X-100 and H₂O₂. Samples were incubated at room temperature for 15 min. During incubation, present catalases break H₂O₂ down into H₂O and O₂; O₂-bubbles are trapped by the surfactant Triton^™X-100 and visualized as a foam column in the test tube. The height of the foam column in each reagent tube was measured with a ruler. The catalase activity for each sample was calculated from the standard curve obtained by plotting the defined units of catalase activity. Mean catalase activity was measured in three independent assays.