Celite

Ethanol (99%), acetone (≥99%), sulfuric acid (96%), oxygen peroxide (50%), and Nessler’s reagent were obtained from VWR chemicals (Leuven, Belgium). Celite, sodium hydroxide (1N solution), and hydrochloric acid (≥32%) were acquired from Sigma-Aldrich (Steinheim, Germany). MES hydrate (>99%) and ammonium sulfate were provided by Alfa Aesar (Kandel, Germany). TRIS (>99.8%) was obtained from Acros Organics (Geel, Belgium). Petroleum ether (60‒80 °C) was supplied by LAB-SCAN analytical sciences (Gliwice, Poland). Amylase thermostable (3000 U/mL), protease (9 tyrosine equivalent units/mg), and amyloglucosidase (3260 U/mL), suitable for AOAC International total dietary fiber and starch analytical procedures, were obtained from Megazyme (Te Huissen, The Netherlands). Ultrapure water was prepared in a Simplicity UV water purification system (Millipore, Molsheim, France).

Seeds of Cucurbita pepo L. subsp. pepo var. styriaca GREB. (Saatzucht Gleisdorf Ges.mbH, Gleisdorf, Austria) were germinated in seed trays containing perlite. Seedlings were cultivated in growth chambers with a photoperiod of 12 h and a light intensity of 700 μmol m⁻² s⁻¹. The temperature was kept at 22 °C during the day and 20 °C during the night at a relative humidity of 60%. One week old seedlings were potted in soil and grown under the same conditions. Two weeks after germination cotyledons of one plant group were inoculated by rubbing a homogenate containing ZYMV-infected leaf material (strain id.: DSMZ PV-0466; obtained from DSMZ Plant Virus Collection, Braunschweig, Germany) and Celite (Sigma–Aldrich, Vienna, Austria) onto the cotyledons before the first foliage leaves fully emerged. The sap was obtained by homogenization of ZYMV-infected leaves in Sørensen phosphate buffer (pH 6.8). Mock inoculation was performed with control plants by rubbing Sørensen phosphate buffer without ZYMV-infected leaf material onto the cotyledons.

To determine the molecular weight by GPC, polymeric chains were detached from the surface of DGO-Br through a reverse cation exchange process. In brief, the resulting graphene-PMMA nanocomposite (0.5 g) was dissolved in 50 mL of tetrahydrofuran (THF), and lithium chloride (0.05 g) was added to the reaction mixture. The solution was refluxed for 24 h and filtered through Celite (Sigma-Aldrich). The free polymer was recovered by adding the filtrate into methanol and was then filtered and dried in a vacuum oven.

Plant leaves were air-dried and ground to a fine powder using an electric grinder (Cambridge Food Processor, FP 235, Cambridge Electric Company, Cambridge, UK). Powdered plant material (50 g) was wrapped in Whatman filter paper and fixed in the extractor glass tube of a Soxhelt apparatus. Methanol (500 mL) was used as the extraction solvent and poured into the round-bottomed flask of the apparatus. The apparatus was run at 60 °C, with running water used as cooling agent for the condenser. The plant material-to-solvent ratio was kept as 1:10 (w/v). Extraction was run for five cycles for a total of 4 h. Crude extract collected within the round-bottomed flask at the end of extraction was cooled to room temperature and sieved through Whatman filter paper by funneling into a conical flask. The volume of methanol solvent was reduced using a Rotary Evaporator (4000 Efficient, Heidolph Instruments, Schwabach, Germany). The extracts were then freeze-dried (Alpha 2–4 LD Lyophilizer, Martin Christ, Osterode am Harz, Germany) after adding 50% celite (w/w) (Sigma-Aldrich, Poole, UK) to generate a fine powder. The extracted (lyophilized) compounds were used for experimentation.