Chemical compositions and antifungal activity against Botrytis cinerea of the essential oils from the leaves of three conifer species

Figures & data

Table 1. Effect of pretreatment on essential oil yield of leaves.

Species	Cutting	Grinding (crushing)
C. obtusa	2.6 ± 0.17^a*	3.7 ± 0.25^a
C. pisifera	2.3 ± 0.09^a	3.5 ± 0.15^a
T. occidentalis	1.2 ± 0.11^b	1.3 ± 0.10^b

*Values represent the mean ± SD (n = 5). Means followed by the same letters are not significantly different by Duncan’s test (p < 0.05).

Figure 1. Antifungal activity against B. cinerea on various concentration of essential oils. B. cinerea cultured on PDB within 6 days and plates were incubated in controlled environment champers maintained at 25 °C. Values represent the mean ± SD (n = 3). Means followed by the same letters are not significantly different by Duncan’s test (p < 0.05).

Figure 2. Impacts on various concentrations of three essential oils. (A) T. occidentalis. (B) C. obtuse. (C) C. pisifera. Colony growth (mm) of B. cinerea raised on PDB within 9 days. And plates were incubated in controlled environment champers maintained at 25 °C. Values represent the mean ± SD (n = 3). Means followed by the same letters are not significantly different by Duncan’s test (p < 0.05).

Figure 3. Capillary gas chromatogram of three species leaves volatiles obtained by steam distillation. (A) C. cbtusa, (B) C. pisifera, and (C) T. occidentalis. The GC column was a 60 mm × 0.25 mm × 0.25 μm i. d. HP-1 fused silica capillary column. The peak numbers correspond to the numbers in Table 1. 1: I-Phellandrene, 2: Tricyclene, 3: α-Pinene, 4: Camphene, 5: Sabinene, 6: β-Pinene, 7: β-myrcene, 8: Myrcene, 9: 3-Carene, 10: Benzene, 11: Bicyclo, 12: Thujone, 13: α-Thujone, 14: Camphor, 15: 3-Cyclohexen-1-1-ol, 16: Verbenol, 17: Benzenemethanol, 18: 3-Cyclohexen-1-methan, 19: 1,4-Methanoazulene, 20: Caryophyllene, 21: Bornyl acetate, 22: 2-Cyclohexen-1, 23: Terpinyl acetate, 24: trans-2-Caren, 25: α-Cedrane, 26: 1-methyl-4, 27: Widdrene, 28: methyl, 29: Naphthalene, 30: Cyclohexanemethanol, 31: Phenanthrene, 32: α-cedrol, 33: 2-Naphthalenemethanol, 34: 7-Methanoazulen, 35: β-Eudesmol, 36: Humulen, 37: Norkaur, 38: Limonene

Table 2. Volatile compounds in steam-distilled essential oil of the leaves from C. obtuse and C. pisifera identified by gas chromatography-mass spectrometry^a.

Compounds	MF^b	MW^c	RT^d	RI^e	Essential oil composition (%)^f
					C. cbtusa	C. pisifera	T. occidentalis
Phellandrene	C10H16	136	5.17	1014	1.32*	–	1.64
Tricyclene	C10H16	136	5.21	915	–	1.88	–
α-Pinene	C10H16	136	5.33	934	4.02	10.79	9.21
Camphene	C10H16	136	5.6	970	–	4.46	2.52
Sabinene	C10H16	163	5.67	973	6.44	–	–
β-Pinene	C10H16	136	6.12	970	–	1.29	–
β-myrcene	C10H16	136	6.44	991	2.76	10.55	14.62
3-Carene	C10H16	136	6.83	1005	–	9.25	–
Bicyclo	C10H16O	152	8.21	1124	–	8.57	2.31
Thujone	C10H16O	152	8.52	1105	–	–	20.10
Camphor	C10H16O	152	9.16	1129	–	–	1.97
Verbenol	C10H16O	152	9.71	1144	–	2.46	–
Caryophyllene	C15H2	204	10.24	1409	–	1.25	1.53
Bornyl acetate	C12H20O2	196	11.31	1283	14.45	20.43	9.31
Terpinyl acetate	C12H20O2	196	12.18	1327	12.04	4.61	3.29
α-Cedrane	C15H2	206	13.01	1633	2.24	–	1.06
Widdrene	C15H24	204	13.51	1429	1.37	–	–
Naphthalene	C10H8	128	14.34	1735	1.52	–	–
Phenanthrene	C14H10	178	15.29	1792	–	2.16	3.14
α-Cedrol	C15H26O	222	15.47	1596	1.78	–	1.93
β-Eudesmol	C15H26O	222	16.12	1649	4.24	–	–
Humulen	C15H24	204	16.75	1666	2.85	–	–
Norkaur	C20H32	272	19.13	2241	3.99	3.63	10.82
Limonene	C10H16	136	19.78	1198	–	6.07	5.05

^aThis table summarizes major terpenes excluding benzyl alcohol among volatile substances. ^bMF: molecular formula. ^cMW: molecular weight; ^dRT: retention time. ^eRetention index (RI) was obtained by referring to analysis data (Van Den Dool and Kraftz 1963; National Institute of Standards and Technology (NIST 2021) Chemistry Webbook). ^fThe content of essential oil components is expressed in %, and the numerical value is an average value for three repeated experiments.

Figure 4. Putative bioactive compound profiled by GC-MS analysis. Terpinyl acetate (L); Bornyl acetate (R).

Table 3. Antifungal activity of terpinyl acetate, bornyl acetate, a mixture of two monoterpenes Antifungal activity according to each treatment concentration.

Treatments	Antifungal activity according to each treatment concentration (μl/l)
	1	2	4	8	16	32
Terpinyl acetate (TA)	45.05 ± 0.91^d*	64.92 ± 1.71^c	67.34 ± 0.7^c	72.81 ± 2.51^b	75.28 ± 2.33^ab	76.98 ± 0.16^a
Bornyl acetate (BA)	76.48 ± 1.46^a	78.14 ± 1.11^a	77.95 ± 1.39^a	79.43 ± 1.69^a	80.37 ± 1.94^a	80.39 ± 2.61^a
TA + BA	77.18 ± 0.63^c	78.04 ± 1.25^c	80.46 ± 1.12^b	77.07 ± 0.8^c	78.1 ± 1.38^c	83.04 ± 1.85^a
Essential oil (EO)	75 ± 2.12^c	77.17 ± 1.59^bc	79.57 ± 2.07^b	80.03 ± 2.6^b	88.24 ± 0.9^a	91.14 ± 0.64^a
TA/EO	0.601	0.841	0.846	0.910	0.853	0.845
BA/EO	1.020	1.013	0.980	0.993	0.911	0.882
TA + BA/EO	1.029	1.011	1.011	0.963	0.885	0.911

*Values represent the mean ± SD (n = 5). Means followed by the same letters are not significantly different by Duncan’s test (p < 0.05).

Figure 5. Antifungal activity of B. cinerea on various concentration of six individual monoterpene. B. cinerea raised on PDB within 6 days and plates were incubated in controlled environment champers maintained at 25 °C. Values represent the mean ± SD (n = 3). Means followed by the same letters are not significantly different by Duncan’s test (p < 0.05).

Figure 6. Antifungal activity of B. cinerea on various concentration of six individual monoterpene. A: Distribution divided into first principal component (PC1) and second principal component (PC2). B: Location of each species.

Van Den Dool H, Kratz PD. 1963. A generalization of the retention index system including linear temperature programmed gas-liquid partition chromatography. J Chromatogr Aug. 11:463–471.

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