Congo red

The Fusarium spp. isolates were tested for their growth on plant-derived polysaccharides by growing them on cellobiose (Fluka Honeywell Specialty Chemicals, Seelze, Germany), trehalose (Carl Roth GmbH), Avicel (50 µm size cellulose, Sigma-Aldrich, Darmstadt, Germany), pectin (Sigma-Aldrich), or starch (neoLab, Heidelberg, Germany). The media contained 0.5% of respective polysaccharides, 0.1% yeast nitrogen base (Carl Roth GmbH), 0.05% congo red (Carl Roth GmbH) and 1.5% agar (VWR, Darmstadt, Germany). The plates (Ø 9 cm) were inoculated by placing, at the centre of the plate, an agar plug that was taken from the rim of 10-day-old actively growing colonies of each isolate on PDA (Table 1). Isolates were incubated at 25 °C for 4 (cellobiose, trehalose, and starch) or 7 days (cellulose, pectin) on the respective agar media. Seven days of incubation on cellulose and pectin was chosen because of the slower growth of these media. The mycelial diameter was measured as an indicator for carbon utilisation, as well as the diameter of congo red discoloration as an indicator of the production of extracellular enzymes. Eight replicate plates were tested per isolate and per carbon source.

For the colony-forming assay, 250 hTCEpi cells were plated on 100 × 20 mm cell culture dishes (Saarstedt AG, Nümbrecht, Germany) and cultured in KGM-2 at 37 °C in a humidified atmosphere. On day 2, the cells were treated with 1% and 5% PVI or 0.04% PHMB for different periods of time, washed three times with BSS each for 1 min, and cultured for an additional 12 days. An untreated control group and a BSS-rinsing control group were also used. Cell colonies were fixed with 4% PFA for 30 min (Thermo Fisher Scientific, Germany) and were irrigated with PBS. Before colony counting, cells were stained with Congo red for 15 min (Carl Roth, Karlsruhe, Germany) at room temperature. To obtain the colony-forming efficiency (CFE), the following equation was used: CFE = number of colonies per plate/number of seeded cells × 100. This experiment was performed in three different biological replicates.

After reaching the respective instar, animals were fixed, stained and processed as described previously [29 (link)]. In short, we fixed the animals with 4% formaldehyde (J.T. Baker, Germany) diluted in phosphate buffered saline (PBS; pH 7.4, 0.1 M), stained them with 0.1% Congo red (Carl Roth GmbH + Co. KG, Karlsruhe, Germany) and scanned them with confocal microscopes (Leica TCS SP5II (Leica Microsystems, Wetzlar, Germany), Zeiss LSM 710 (Carl Zeiss Ltd., Cambridge, UK) and Nikon A1R (Nikon Instruments Europe B.V., Amsterdam, Netherlands)). We used the automatic image stitching when necessary for large specimens.