Streptomycin sulphate salt

The broth microdilution test was carried out to study the effect of varying concentrations of twelve selected essential oils on phytopathogenic bacteria P. syringae pv. pisi. A two-fold dilution series of essential oils ranging from 1% to 0.016% was prepared by dissolving the essential oils in ethanol to increase solubility at a ratio of 1:3 (for the highest essential oil concentration, 1%, the concentration of ethanol was 3%) and then further dissolving with MHB containing 0.05% (w/v) 2,3,5-Triphenyltetrazolium chloride (TTC; Sigma Aldrich, UK) as a growth indicator. One hundred microlitres of essential oil dilutions was added into 96-well plates (Corning Incorporated, USA) and each well was inoculated with 100 µl of bacterial suspension (OD600=0.132). Three microlitres of bacterial suspension (diluted 1/20 in sterile water) was plated in MHA and incubated at 28°C for 48h. Streptomycin sulphate salt (Sigma-Aldrich, UK) was used as a positive control at 1 mg/ml and 10 mg/ml concentrations, and MHB as negative control.
Absorbance (OD600) was measured at t=0h and plates were incubated at 28°C. Absorbance was measured at 20, 24 and 48h using an Infinite M200 TECAN plate reader. Three microlitres from each clear well was plated into MHA and incubated at 28°C for 48h. All tests were performed in triplicate. MIC and MBC were obtained from this test.

The disk diffusion assay was performed as described by the European Committee on Antimicrobial Susceptibility Testing (EUCAST, 2017) with a few modifications, to evaluate the antimicrobial activity of 41 essential oils against P. syringae pv. pisi, P. fluorescens and P. carotovorum. Bacterial suspensions (OD600=0.132) were homogenously streaked onto MHA plates using sterile cotton swabs. Whatman antibiotic assay discs (6mm diameter; GE Healthcare, UK) were saturated with 10 µl of each essential oil (100%) and placed on the agar surface using sterile tweezers. Plates were left at room temperature for 15 minutes before incubating at 30°C (28°C for P. syringae pv. pisi) overnight. streptomycin sulphate salt (Sigma-Aldrich, UK) was used as a positive control at concentrations of 1 mg/ml and 10 mg/ml (a calibration curve on the effect of streptomycin at increasing concentrations on P. syringae pv. pisi was obtained using the disk diffusion assay) and sterile water and empty disks were used as negative controls. Inhibition zones were measured to the nearest millimetre after 24h of incubation. Essential oils with the largest inhibition zones as well as oils with specificity for pathogenic strains were selected for further testing. All tests were performed in triplicate.