F. oxysporum f. sp. fragariae isolate SFW-10 used in the present study was isolated from naturally infected strawberry plants (cv. Akihime) grown in Hadong-gun, Gyeongnam, South Korea in 2010. This fungus was cultured on 1/2 potato dextrose agar (PDA) media at 25°C. To obtain conidial suspension, mycelia were streaked onto culture media and incubated for 14 days. Quarterly diluted sterile potato dextrose broth (PDB) was poured onto the 14 day-old fungal culture. The concentration of conidial suspension was adjusted to 5 × 10⁵ conidia/ml. To prepare mycelial plugs, mycelial discs (5 mm in diameter) were inoculated at the center of the media. Fresh mycelial discs (5 mm in diameter) were cut from the growing edge of 7 day-old PDA cultures.

Cinnamon oil and thyme oil were purchased from Sigma-Aldrich Co., Ltd (St. Louis, MO, USA). Fennel oil and origanum oil were purchased from Tokyo Chemical Industry Co., Ltd (Tokyo, Japan). Each viscous plant essential oil concentrate was diluted to 5 or 20% (v/v) solution in 95% ethanol for easy handling. Increasing doses of each plant essential oil (0, 0.1, 0.2, 0.5, 1, 2 and 5 μl/disc) were dropped onto sterile paper disc (8 mm in diameter) (Toyo Roshi Kaisha, Ltd, Tokyo, Japan) and 95% ethanol was additionally laid to make the same loading volume (50 μl) in each paper disc. These paper discs were air-dried for 20 min at room temperature to evaporate ethanol on a clean bench.

Volatile antifungal activities of these four plant essential oils against in vitro conidial germination and mycelial growth were evaluated using method described in our previous study (Hong et al., 2015) with slight modifications. Briefly, for conidial germination-inhibiting assays, four conidial suspension drops (20 μl each) prepared in 1/4 PDB on glass slides were treated with plant essential oils for 10 h at 25°C in plastic square dishes (100 mm × 100 mm × 13 mm). Conidia were stained with 1 μl of lactophenol-trypan blue solution and conidial suspensions were covered by coverslips before observation under a microscope. For mycelial growth-inhibiting assays, fungal mycelial plugs (5 mm in diameter) were inoculated onto the centre of 10 ml of PDA media in Petri dishes (85 mm in diameter, 13 mm in height). Fungal colony diameters were measured at 6 days after inoculation onto PDA media at 25°C. Diameters of fungal colonies treated with plant essential oils were compared to those of untreated colonies and expressed as percentage (%).

Different plant essential oils were applied simultaneously to determine their synergistic efficacies against in vitro conidial germination and mycelial growth of F. oxysporum f. sp. fragariae. Paper discs containing 0.5 and 1 μl/disc of each plant essential oil were attached to the inside plate cover singly or concurrently with other paper disc(s) for conidial germination and mycelial growth inhibition assays, respectively. Conidial germination and mycelial growth were evaluated at 10 h and 6 days after incubation at 25°C with different combinations of these plant essential oils described above.

Data were subjected to analysis of variance using SAS version 9.1 (SAS Institute, Inc., Cary, NC, USA). Means were separated by least significant difference (LSD) test at P < 0.05. Graphing was prepared using SigmaPlot 10.0 (Systat Software, Inc., San Jose, CA, USA).

The four plant essential oils tested in this study showed different antifungal efficacies against in vitro conidial germination of F. oxysporum f. sp. fragariae in dose-dependent manners (Fig. 1). Treatment with cinnamon oil drastically suppressed conidial germination at the lowest dose used (0.1 μl/disc). Conidial germination was completely inhibited by cinnamon oil at 0.2 μl/disc. Fennel oil was less effective in reducing conidial germination compared to cinnamon oil. It failed to completely suppress conidial germination even at the highest dose used in this study (5 μl/disc). Origanum oil and thyme oil demonstrated moderate antifungal activities against conidial germination at similar levels. Both of them inhibited conidial germination in a dose-dependent manner. Increasing concentrations of origanum oil and thyme oil concentrations resulted in gradual decreases of conidial germination. No germination was found in conidia treated with 5 μl/disc of thyme oil.

All four plant essential oils also possessed antifungal activities against mycelial growth of F. oxysporum f. sp. fragariae in a dose-dependent manner (Fig. 2). Cinnamon oil, origanum oil and thyme oil showed similarly elevated mycelial growth-inhibiting activities with increasing doses. Antifungal efficacy of cinnamon oil at 0.5 μl/disc was approximately 89.4% compared to untreated control. Increasing its dose to 1 μl/disc did not augment its antifungal activity. However, relatively higher doses of cinnamon oil at 2 μl/disc and 5 μl/disc resulted in significant reduction in mycelial growth (to approximately 60.0% and 6.9%, respectively). At least 0.2 μl/disc of origanum oil and thyme oil was needed to inhibit mycelial growth. Higher concentrations reduced mycelial growth more effectively. At the highest dose (5 μl/disc), origanum oil and thyme oil completely inhibited mycelial growth. By contrast, fennel oil had relatively lower antifungal activity with limited ability to arrest mycelial growth with increasing doses. Even at the highest dose (5 μl/disc), it only slightly reduced mycelial growth (approximately 84.4% compared to the untreated control).

Different combinations of these four plant essential oils were applied to investigate whether they might have synergism in suppressing conidial germination and mycelial growth of F. oxysporum f. sp. fragariae (Fig. 3, 4). Several combinations of these plant essential oils synergistically suppressed conidial germination (Fig. 3A). Treatment with cinnamon oil alone almost completely suppressed conidial germination. Additional treatment with other plant essential oils did not significantly increase the high suppressive activity of cinnamon oil. Three different combinations of two different essential oils (fennel oil + origanum oil, fennel oil + thyme oil, origanum oil + thyme oil) significantly increased their antifungal activities against conidial germination compared to any single plant essential oil treatment. No additional increase in antifungal activity against conidial germination was found by mixing three or four plant essential oils (Fig. 3B).

Enhanced suppressive activities against fungal mycelial growth were demonstrated by combined treatments with different plant essential oils (Fig. 4). After treatments with different combinations of essential oils, colony formation on PDA was demonstrated at 6 days after culture (Fig. 4A). Cinnamon oil treatment alone slightly reduced mycelial growth. Additional application with fennel oil failed to enhance its antifungal activity. However, addition application with origanum oil or thyme oil accelerated mycelial growth restriction caused by cinnamon oil. Single treatment with origanum oil or thyme oil drastically suppressed mycelial growth. Combined treatment with fennel oil and origanum oil showed synergistic effect on the suppression of mycelial growth compared to single treatment. Triple treatment with cinnamon oil, fennel oil and origanum oil resulted in more reduction of mycelial growth. Antifungal activity against mycelial growths by triple treatment with cinnamon oil, origanum oil and thyme oil was similar to that by dual treatment with origanum oil and thyme oil. Triple treatment with cinnamon oil, fennel oil and thyme oil exerted additive antifungal activity compared to treatment with a combination of any two plant essential oils. Fennel oil treatment did not enhance the antifungal activity of dual treatment with origanum oil and thyme oil. Antifungal activity with a combination of all four essential oils was not significantly different from that with a combination of origanum and thyme oil (Fig. 4B).