We revisited the applicability of two fungal cell wall markers, Calcofluor White (CFW) and wheat germ agglutinin (WGA), for the direct visualization of chytrid infections on phytoplankton in laboratory-maintained isolates and field-sampled communities. Using a comprehensive set of chytrid–phytoplankton model pathosystems, we verified the staining pattern on diverse morphological structures of chytrids via fluorescence microscopy. Empty sporangia were stained most effectively, followed by encysted zoospores and im-/mature sporangia, while the staining success was more variable for rhizoids, stalks, and resting spores. In a few instances, the staining was unsuccessful (mostly with WGA), presumably due to insufficient cell fixation, gelatinous cell coatings, and multilayered cell walls. CFW and WGA staining could be done in Utermöhl chambers or on polycarbonate filters, but CFW staining on filters seemed less advisable due to high background fluorescence. To visualize chytrids, 1 μg dye mL−1 was sufficient (but 5 μg mL−1 are recommended). Using a dual CFW–WGA staining protocol, we detected multiple, mostly undescribed chytrids in two natural systems (freshwater and coastal), while falsely positive or negative stained cells were well detectable. As a proof-of-concept, we moreover conducted imaging flow cytometry, as a potential high-throughput technology for quantifying chytrid infections. Our guidelines and recommendations are expected to facilitate the detection of chytrid epidemics and to unveil their ecological and economical imprint in natural and engineered aquatic systems. The uploaded data included complementary information for Figure 2 and Figure S6, and image analyses (data of fluorescence intensity shown in Figure 4)

Study on laboratory-maintained co-cultures (in April 2018), consisting of parasitic fungi (chytrids) and phytoplankton hosts. Method: Cell co-culturing, Inverted fluorescence microscopy, Imaging flow cytometry.File Fig2_Data_Pathosystems.csv: Raw count data of CFW (Calcofluor White) and WGA (Wheat Germ Agglutinin) stained morphologies in various phytoplankton‒chytrid model systems. Staining was distinguished as Y – yes, entirely stained, P -partly stained, and N – not stained. Of each morphological feature, mostly 25 events (in a few instances 5–20 events) were evaluated in parallel with both fluorescence channels. Data are plotted in Figure 2 in Klawonn et al. n.p. feature not present.File Fig4_FigS6_ImageJ_Macro.txt and Fig4_FigS6_ImageJ_Macro.imj: Macro used to process microscopy and flow cytometry images in ImageJ (v. 1.51).