Vacuum pump

Plasmopara viticola sporangia were collected from infected plants from the vineyard at the Portuguese Ampelographic Grapevine Collection (CAN, international code PRT051, established in 1988) at INIAV-Estação Vitivinícola Nacional (Dois Portos), using a vacuum pump (Millipore) at 10 kPa and stored at -20°C. Inoculum was propagated in V. vinifera ‘Carignan’ (susceptible cultivar) leaves by spreading a sporangia solution on the lower side surface of the leaf using a sterilized laboratory spreader, with an undefined concentration of P. viticola sporangia. After infection, leaves were kept in the dark for the first 8 to 12 hours and then kept in a climate chamber with controlled conditions (25°C; 16/8 hours with light/dark; 90% humidity) until sporulation appeared in all leaf surface. Sporangiophores with sporangia were collected with a vacuum pump (Millipore) at 10 kPa and stored at -20°C until further use.

The bioreactors were sampled regularly, whereby for each sampling time point, triplicate samples were withdrawn from the bioreactor. The triplicate samples (5 mL each) were filtered with a vacuum pump (Millipore, Darmstadt, Germany) through a preweighed cellulose acetate filter (Braun Sartorius, Germany) with a pore size of 0.45 µm. Afterward, the obtained filtrate was aliquoted into 1.5 mL tubes and stored at -20 °C until later nutrient analytics. In addition to the triplicate samples, 10–15 mL of the culture broth was filled in 15 mL falcons and also stored at -20 °C until later analysis. Residual nutrient concentrations were measured as described elsewhere [47 (link)]. The filters with the biomass were dried at 105 °C to estimate the dry weight.
After five days of cultivation (approximately 92 h), the bioreactor batch cultivations were stopped. The culture broth was harvested and filtered through a dish towel or fabric sheet into a bucket. The resulting mycelium was wrung by hand to remove most liquid, then laid out flat on a tablet and dried at 40 °C overnight. The dried mycelium and the filtrate were stored separately at -20 °C until further use.

A sterile culture medium based on lyophilized powder of P. palmata at 80% humidity was prepared for fungal enzyme production. Similar fungal culture media at 80% humidity have already been used for enzymatic production, with various carbon sources [73 (link),74 (link)]. For fungal growth and enzymatic production, 1.5 mL of conidial suspension was added on all the surface of culture medium prepared with 20 g of lyophilized powder of P. palmata. Such culture was performed in triplicate for each strain and incubated 7 days at 27 °C. Biological negative control was performed with the incubation of culture medium without any conidia suspension. After incubation, 35 mL of sterile distilled water was added in the fungal cultures and homogenization was performed using a blender (Virtis 23, New York, NY, USA). The mash was then centrifuged 15 min at 9500 rpm (SL 8R, Thermo Scientific^TM, Waltham, MA, USA) and the supernatant was filtered through a glass microfiber filter at 1.2 µm retention porosity (Whatman^TM, Buckinghamshire, United Kingdom) using a vacuum pump (Millipore, Burlington, VT, USA). Glycerol 87% w/w (PlusOne, St George, UT, USA) was added to reach a final concentration of 10% in the filtrate which is then stored at −80 °C. This filtrate corresponded to the enzymatic extract used for the following tests.