Sputter coater

JM109-Thy^r and original JM109 strains were observed by scanning electron microscopy (SEM). After overnight incubation in LB broth at 37 °C, bacterial cells were suspended to OD 600 = 0.5 in LB broth and divided into two sterile Eppendorf tubes to which thymol was added to one tube at a concentration of 100 μg l^− 1, whilst the other was left untreated as a control. Samples were incubated in a rotary shaker set at 200 rpm and 37 °C. After 2 h, the cells were harvested by centrifugation at 14,000x g for 2 min, washed twice and suspended in phosphate buffer saline (PBS). Each suspension (200 μl) was placed on poly-L-lysine-coated glass cover slips for 15 min on both sides. Adhered bacteria were fixed with a solution of 2.5% glutaraldehyde pH 7 for 15 min. After fixation, samples were washed with water for 15 min, dehydrated by increasing serial dilution of ethanol (30, 50, 70, 80, 90%) immersions for 10 min each and for 1 h in 100%. Samples were dried in a Balzers critical point dryer CPD 030 (Bal-Tec, Germany), and metal coated in a sputter coater (Edwards, UK). All samples were observed with a field emission Quanta SEM equipped with a cold stage and a cryo-preparation chamber (Thermo Fisher Scientific, MA, USA). The experiment was performed in triplicate.

Experiment J. The OP assay with cellophane agar was used for SEM observations. The mycelium of H. abietinum strain 10 exposed to the VOCs of P. protegens strain CHA0 grown on LBA was compared to the mycelium of the same fungal strain exposed to the VOCs of CHA0 grown on PDA (i.e., LBA vs. PDA). CHA0 was grown on LBA or PDA for 2 days, and H. abietinum strain 10 was grown on PDA for 5 days, both at 27°C in the dark. Then, the bacterium and fungal plates were joined without the lid and sealed, so the exposure to VOCs started. SEM inspections were made at 15 and 30 min, 1, 3, 6, 12, 24, 48, and 168 h of exposure to VOCs. Three replicates (plates) per treatment and time point were used, and three cellophane squares per plate were sampled at each time point. Cellophane squares were taken from the fungal colony edge for observing the actively growing hyphae. Cellophane squares were placed firmly onto aluminum stubs for SEM by double-sided carbon tape and directly subjected to gold/palladium sputter coating (Edwards Sputter Coater) under 10^–1 mbar gas pressure and 20 mA current for 1 min. Coated samples were placed in the chamber of an SEM Hitachi TM 3000 Tabletop Microscope and observed in Standard Mode Observation with 15KV voltage supply and 5 × 10^–3 mbar gas pressure.

TEM images were obtained using a JEOL JEM-2100 electron microscope at an accelerating voltage of 100 kV. SEM images were obtained using a field-emission scanning electron microscopy (model JEOL 7600F) at an acceleration voltage of 5 kV. Prior to analysis, the samples were coated with gold layer using an Edwards Sputter Coater to enhance their conductivity. To prepare the sample, 0.4 mL of the sample solution was taken and centrifuged at the speed of 12g for 8 min (for bowls, bottles, and cucurbits), or 1.5g for 12 min (for more complex interconnected structures). The supernatant was removed, and the precipitate solution was dropped on a copper grid, which was pre-treated by a Harrick Plasma cleaner for 30 s to remove oxidations. For DLS measurement, 1 mL of the sample solution was injected to a four sides transparent glass cuvette, and then capped to avoid volatilize of solvent. A model ZEN 5600 from Malvern, and a back-scattering mode with an angle of 173° was used for all the measurements. SAED and HR-TEM were performed on a JEOL JEM-3010 TEM at 300 kV. HAADF-STEM imaging and EELs spectrum were carried out on a FEI-Titan ST electron microscope operated at 300 kV.