Split petri dishes

To test whether during cultivation on NA, AZ78 VOCs are absorbed into the PDA medium, split Petri dishes (Sarstedt, diameter 92 mm) containing 5 mL NA and 5 mL PDA medium respectively, were used. Bacteria were inoculated in the compartment with NA (50 μL of a 1 × 10⁸ cells/mL suspension), the Petri dishes were sealed with Parafilm (Bemis) and incubated at 27°C for 168 h. The cultivation period of 168 h was used since pilot studies using headspace vials revealed the richest volatile profile of AZ78. PDA and NA, the latter with AZ78 cultures, were then cut into pieces under the laminar flow with the help of a sterile spatula. The pieces of each medium were placed into separate sterile HS vials, which were sealed with sterile metal caps having 1.3 mm silicone/PTFE septa and the VOC profiles were measured by GC-MS according to the parameters specified below. Empty HS vials and HS vials with uninoculated NA and PDA pieces respectively, were used as controls. Four or five replicates (HS vials) were analyzed for each treatment in a randomized block design and the experiment was carried out twice.

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.