Nutrient agar na

The Eijkman method was used to detect the presence of E. coli in the samples. All positive bottles from the previous test were subcultured into fresh double strength and single strength MacConkey broth and peptone water and incubated at 37 °C for 24 h. The MacConkey bottles were checked after incubation for lactose fermentation (yellow colouration) and gas production (presence of a bubble in the Durham tubes). All positive MacConkey bottles were noted and three drops of Kovac’s reagent were added to their corresponding peptone water bottles to detect indole (indicated by a red coloured ring). All positive samples were cultured on Eosin Methylene Blue Agar plates and incubated aerobically at 37 °C for 24 h. Up to three distinct colonies showing green metallic sheen were aseptically picked and streaked onto Nutrient agar (NA) (Oxoid Ltd., Basingstoke Hampshire, England) plates which were, in turn, incubated aerobically at 37 °C for 24 h [14 (link)]. All suspected E. coli isolates were stored at -20 °C in glycerol broths for further examination.

For isolation of bacteria, wastewater and river water samples were serially diluted in 10-fold physiological saline and 1.0 mL aliquots of appropriate dilutions (10⁻²–10⁻⁶) were inoculated and plated on non-selective agar media, tryptone soya agar (TSA) and plate count agar (PCA) (Oxoid Ltd., Basingstoke, Hampshire, UK). Duplicate plates were incubated under aerobic condition at 35 °C for up to 48 h. Bacterial counts were taken every 24 h of incubation. Morphologically distinct colonies were subcultured onto fresh plates of nutrient Agar (NA) (Oxoid Ltd., Basingstoke, Hampshire, UK). Up to eight colonies with different morphologies were taken from each plate. Isolates were restreaked up to three times and purity was verified by Grams reaction and microscopy. Pure colonies of each isolated bacteria strain were stored on NA slants at 4 °C, and for prolonged storage, at −20 °C in tryptic soy broth (TSB) containing 15% glycerol.

Bacterial strains (Supplementary Table 1) were routinely grown on Nutrient Agar (NA; Oxoid, Basingstoke, United Kingdom) at 27°C. The phytopathogenic oomycete Pythium ultimum was maintained on Potato Dextrose Agar (Oxoid) at 25°C.
LeDSF3 was obtained from Avanti Polar Lipids (Alabaster, AL, United States). IND, GLY, BUT, 3HBA, 4BHA, N-(3-hexanoyl)-L-homoserine lactone, N-(3-oxooctanoyl)-L-homoserine lactone, and N-(3-oxododecanoyl)-L-homoserine lactone were purchased from Merck (Sigma-Aldrich, Darmstadt, Germany). Aqueous stock solutions were prepared, except for LeDSF3 and the mixture of AHLs, which were prepared in pure methanol.

Split Petri dishes (Sarstedt, diameter 92 mm), with ventilation cams and two physically separated compartments were used. 5 mL of PDA medium were poured in one compartment and Nutrient Agar (NA, Oxoid) in the other. After solidification of the media, 50 μL of AZ78 cell suspension were spread on NA using sterile spatulas. Subsequently, Petri dishes were sealed with Parafilm (Bemis, Neenah, United States) and incubated for 72 h at 25°C. Petri dishes containing uninoculated NA medium were used as control. In all the experiments, a plug (5 mm in diameter) was cut from the border of 7 days old fungal or oomycete colony using a sterile cork borer. Subsequently, the mycelium plugs were placed on PDA-containing side and the Petri dishes were incubated at 25°C in the dark. After 96 h post-inoculation (hpi), the mycelial growth of the tested pathogens was determined by measuring the mycelium colony diameter (mm) parallel to the separating barrier of the two compartments. It is worth noting that the 96 hpi time point of the measurements corresponded to a total AZ78 incubation time of 168 h, as AZ78 was incubated for 72 h prior to pathogens inoculation. Thus, 168 h was subsequently chosen as one of the sampling time points for the analysis of AZ78 VOCs profile described below. Four replicates (Petri dishes) were analyzed for each treatment and the experiment was carried out twice.

Swabs were broken into tubes containing 2 mL Buffered Peptone Water (BPW, Oxoid, Basingstoke, United Kingdom), vortexed for 2 min, and later incubated for 24 h at 37 °C. A loop-full of the bacteria suspension was streaked on MacConkey Agar (MA, Oxoid, Basingstoke, United Kingdom) plates and incubated at 37 °C for 24 h. Putative positive E. coli colonies usually pink to red were identified, selected randomly, then sub-cultured on Nutrient Agar (NA, Oxoid, Basingstoke, United Kingdom) plates and incubated at 37 °C for 24 h under aerobic conditions for the isolation of pure isolates.