To extract the EO, fresh flowers (350 g) were collected from each harvesting point at each crop site, weighed and put into a two-liter flask. The EO was extracted by distilled water using a Clevenger-type apparatus for 5 h. The extract was dehydrated using sodium sulfate, separated, and kept in the dark in a dark-colored bottle at 4°C until later use. The EO yield for each harvesting point was expressed based on weight/weight percent (% w/w) at each site. The EO yield for each site was reported as the mean± SD of three replications (harvesting points).

A GC-MS instrument was employed to determine the chemical composition of the EO samples. The instrument consisted of a 6890 gas chromatograph (GC) coupled to an Agilent 5973 N mass spectrometer (MS) with a capillary column of HP-5 MS and 5% methyl phenyl of the stagnant phase (30 m length, 0.25 mm internal diameter, the thickness of stagnant layer 0.25 μm) and the ionization energy of 70 eV.

The oven temperature program for the analysis was as follows: it was set at 60°C and raised by 3°C per minute to reach 246°C. The injector and the detector temperatures were set at 250°C. The injection volume of sample was 1μL, split mode (1.50). The helium carrier gas flow was 1.5 mL/min.

The chemical constituents of the EOs were determined based on the GC-MC analysis of each EO sample in relation to the retention indices (RI) and standard mixtures of n-alkane (C8-C20) mixtures and mass spectral data of each peak using spectral libraries (NIST-14 and Wiley-14) and comparing the obtained results with those in the literature [51].

2.5.3.1. Microbial strains. Eleven standard microbial strains were used to assess the antimicrobial activities of the EOs. These strains were four Gram-positive bacteria (Staphylococcus epidermidis CIP 81.55, S. aureus ATCC 29737, Streptococcus pyogenes ATCC 19615, and Bacillus subtilis ATCC 6633), five Gram-negative bacteria (Klebsiella pneumoniae ATCC 10031, Escherichia coli ATCC 10536, Pseudomonas aeruginosa ATCC 27853, Salmonella paratyphi A serotype ATCC 5702, and Shigella dysenteriae PTCC 1188, and the two fungal strains of Candida albicans ATCC 10231 and Aspergillus brasiliensis ATCC 16404. The microorganisms were obtained from the Iranian Research Organization for Science and Technology (IROST). The bacterial strains were cultured on nutrient agar and the fungal strains on Sabouraud dextrose agar (SDA). The plates inoculated with the bacteria and fungi cultures were incubated overnight at 37°C and 30°C, respectively.

2.5.3.2. Agar well-diffusion method. The protocol of the Clinical and Laboratory Standards Institute [52] was employed in using this method. Plates containing Mueller-Hinton agar and SDA were first prepared to culture the bacterial and fungal strains, respectively. The EO was dissolved in dimethyl sulfoxide (DMSO) and the concentration was raised to 300 μg/mL. The turbidity of each microbial suspension (100 μL) was adjusted to that of a 0.5 McFarland standard and the suspensions were cultured on the culture media under identical conditions. A number of wells with 6 mm diameter and 4 mm thickness were made in the culture plates and 10 μL EO (at concentration 300μg/mL) was added to each well. The plates were kept at 4°C for 2 h. The plates inoculated with the bacterial strains were then incubated at 37°C for 24 h and those incubated with the A. brasiliensis ATCC 16404 and C. albicans ATCC 10231 were incubated at 30°C for 72 and 48 hours, respectively. Gentamicin (10 μg/disc) and rifampin (5μg/disc) were used as positive controls for the bacterial strains and nystatin (100,000 units/mL) as the positive control for the fungal strains under the same conditions as those for the EO tests. IZD was measured using a ruler (that measures in millimeters) to generate the antibiogram. The test was performed in triplicate for each EO to assess reproducibility and become certain of the reliability of the results. The IZDs were reported as mean ± SD.

2.5.3.3. MIC. To determine the MICs for the bacterial and yeast strains, sterile 96-well microtiter plates and the broth microdilution method were used according to the CLSI protocol [52]. Various dilutions of the EO were prepared first. A certain volume of the EO was weighed, dissolved in the culture medium and DMSO at a suitable ratio to prepare the initial stock at a concentration of 2000 μg/mL. This stock was used to prepare the 1000, 500, 250, 125, 62.5, 31.25, and 15.63 μg/mL dilutions. Each well in the microplate was filled with 200 μL of a solution containing 95 μL of brain heart infusion (BHI) broth for bacteria or with 95 μL of SD broth for yeast, and 5 μL of the microbial suspension with the turbidity adjusted to that of a 0.5 McFarland standard and 100 μL of one of the EO dilutions were added. The wells intended as negative controls were filled with the culture medium instead of the essential oil and for the wells intended as positive controls antibiotic powders (gentamicin and rifampin) were used instead of the EO for the bacterial strains and nystatin powder for the yeast. Plates inoculated with bacterial and fungal plates were incubated at 37°C for 24 h and at 30°C for 48 h, respectively. MIC was determined taking into account the turbidity and the change in the color in each well of the microplate. The test was performed in triplicate for each EO sample and the mean was reported as the minimum concentration of the EO that inhibited the growth of bacteria or yeast.

The agar dilution assay was used to determine the MICs for the fungal strains based on the protocol introduced by of [53]. The suitable amounts of EO at different concentrations (2000, 1000, 500, 250, 125, 62.5, 31.25 and 15.63 μg/mL) were added to SDA containing 50% (v/v) Tween 20. Nystatin powder was used as the positive control, and the negative control was the plate with SDA containing 50% (v/v) Tween 20 without any EO. The culture media were spot inoculated with 4 ml of spores (104 spores /mL). The inoculated plates were incubated at 30°C for 72 h, the test was performed in triplicate for each essential oil, and the minimal concentration of the essential oil that inhibited the growth of the fungi was reported as the MIC.

2.5.3.4. MBC. The broth microdilution method based on CLSI protocol (2012) was used as described above to determine MBC. Following 24 h of incubation, the nutrient agar medium was inoculated with 5 μL of each well that exhibited no trace of bacterial growth (light well) and incubated at 37°C for 24 h. MBC refers to the minimal concentration of the EO that kills 99.9% of the inoculated bacteria.

Soil pH, EC and phosphorous, potassium, organic carbon, nitrogen, lime and gypsum contents and its texture components were measured using a pH meter, an EC meter, the Olson method, the ammonium acetate extraction method, the Walkley and Black method (1934), the Kjeldahl method, the acid-base titration method, the acetone method, and the hydrometric method, respectively (Zargoosh et al., 2019).

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