Filter paper

All HPLC measurements were performed on Dionex Ultimate 3000 (Thermo Scientific, Waltham, MA, USA) HPLC equipment. Dried samples (25 g) were homogenized with 2.5 g sodium chloride (VWR) and 50 mL of 80% methanol (HPLC, Sigma-Aldrich, St. Louis, MO, USA) at high-speed mixing. The extract was diluted in a 1:4 ratio with 40 mL of distilled water. The homogenized sample was filtered into spherical flasks through filter paper (Macherey-Nagel). The diluted extract was filtered, and 10 mL of it was loaded onto the aflatoxin immunoaffinity column (VICAM AflaTest WB HPLC Columns, Weber Consulting Ltd., Göd, Hungary). The column was washed with 10 mL of distilled water, and the toxin was eluted with methanol (5 mL) and was evaporated in Rotavapor R114 (Büchi). After the addition of 1 mL mobile phase (methanol: water, 45:55), the solute was filtered through Millex-GV 0.22 μm filter (Merck-Millipore) and applied to HPLC. Phenomenex (Torrance, CA, USA) RP-C18 column (150 × 4.6 mm, 5 µm) was used with Romer UV derivatization unit (Romer Labs Ltd., Tulln, Austria) and a fluorescence detector ex360 nm, em440 nm, with methanol: water (45:55) eluent. Biopure Aflatoxin Mix 1 standard solution (Romer Labs, Tulln, Austria) was applied to the column.

To determine the actual dosage of BA applied to the plants, a filter paper (MACHEREY-NAGEL, Duren, Germany) was used. The filter paper was cut to different shapes and sizes according to leaves sourced from the top, middle and bottom of plants from both varieties of rose plants. The filter papers were weighed before used and then placed at the top, middle and bottom of the rose plants. After application of BA, as described above, the filter papers were removed and weighed again. This method was repeated twice and the average was taken (Table 1). Methods to quantify spray retention by leaves and the actual dosage, following the application of PGRs are still lacking, despite growing interest by researchers.

We estimated the biofilm formation of the Janthinobacterium sp. SLB01 strain grown in 12-well plates (TPP Techno Plastic Products AG, Trasadingen, Switzerland) in LB broth at temperatures of 3, 22, and 30 °C. The biofilm amount was determined at regular time intervals according to a previously described method [11 (link)]. The wells of the plate were carefully emptied and washed twice with sterile 0.01 M phosphate-buffered saline (PBS) (Sigma-Aldrich, St. Louis, MO, USA) every 24 h. Then, 5 mL of an aqueous solution of 0.1% safranin (Sigma-Aldrich, St. Louis, MO, USA) was added to each well and incubated at room temperature for 20 min. The dye was removed with 0.01 M PBS buffer. Then, the plates were inverted onto filter paper (Macherey-Nagel, Düren, Germany) and dried for 3 h. Safranin was extracted with a mixture of ethanol 96% and acetone (80:20 v/v). The biofilm formation was quantified by measuring the optical density (OD₄₈₄) of each sample using a spectrophotometer (GBC Scientific Equipment Ltd.—Cintra 20, Melbourne, Australia).