Phytotoxic Effect of Essential Oil from Hyssop (Hyssopus officinalis L.) against Spring Wheat and White Mustard ^†

Abstract

Hyssop essential oil is a rich source of biologically active compounds. This study aimed to characterize the chemical composition of essential oil from the hyssop herb and its phytotoxicity against germination and initial growth of wheat and mustard. The main compounds of the oil were identified as isopinocamphone and pinocamphone. In a Petri dish experiment, the oil mainly inhibited the germination and initial growth of wheat, whereas mustard was less affected. In conclusion, hyssop oil displays phytotoxic potential against the studied species and should be tested further.

1. Introduction

Hyssop (Hyssopus officinalis L.) essential oil is one of the most valuable oils mentioned in the European Pharmacopeia [1]. It is rich in valuable active compounds that have antimicrobial and antioxidant activities [2]. This oil can also be used to control plant fungal diseases [3,4]. Moreover, it has been shown that hyssop oil also displays herbicidal effects on the germination of some weeds and crops [5,6,7]. There has been growing interest in using essential oils (EO) as natural, botanical herbicides [8] to replace the synthetic products that cause environmental pollution [9]. However, as many authors have showed, EOs also display phytotoxic effects against crops [6,10,11], which is why studying the response of crops to EOs is justified. Hence, this study aimed to assess the phytotoxic potential of the essential oil obtained from the hyssop herb (Oleum hyssopi officinalis) on the germination and initial growth of seedlings of two crops: spring wheat (Triticum aestivum L.) and white mustard (Sinapis alba L.).

2. Materials and Methods

The essential oil was hydro-distilled from the hyssop herb collected from central Poland. The EO was analyzed by gas chromatography coupled with mass spectrometry (GC-FID-MS), using a Trace GC Ultra gas chromatograph coupled with DSQ II mass spectrometer (Thermo Electron Corporation). The percentages of the constituents were computed from the GC peak area without using a correction factor [10]. Identification of the components was based on comparing their mass spectra and linear retention indices (RI, non-polar column) with those in [12] and computer libraries: NIST 2011 and MassFinder 4.1.

The hyssop EO was stored in a dark glass in a cool place. The biological tests were performed in three replications, two series and two seasons, 2018 and 2020. Seeds of spring wheat cv. Harenda (breeder: MHR, PL) and Blondynka (breeder: IHAR, ZD Grodkowice, PL) were used in 2018 and 2020, respectively, and white mustard cv. Borowska (breeder: MHR, PL) was used. Oil in water (o/w) emulsions with five EO doses: 0.6 g, 1.0 g, 1.4 g, 2.8 g, and 4.3 g L⁻¹ were prepared. A 2.0% aqueous solution of acetone was used as a carrier. The EO and water with acetone were weighed out (w/w). Dry and clean Petri dishes (11 cm diameter) were lined with two autoclaved filter paper pieces. Seven grams of o/w emulsion per dish was poured evenly. The emulsions contained: 0.004; 0.007; 0.01; 0.02 and 0.03 g EO per dish. The seeds of the tested plants were surface-sterilized with 5% ethanol solution and abundantly rinsed with water. Next, 20 seeds of each species, separately, were placed in each dish. The dishes were put in thin polypropylene bags to reduce the EO evaporation and placed in a shaded place at a room temperature of 22 ± 3 °C. After seven days, the seedlings were counted, and their leaves/shoots and roots measured with a ruler. A dose–response test, using a “drc” package in the R program ver. 3.5.3 [13] was performed. The ED50 value, i.e., a dose causing a 50% reduction in a plant trait, was calculated for the percentage of germination and seedlings’ leaves/shoots and root length. One-way ANOVA for a randomized design was applied to test differences between the EO concentrations, and means were separated using the Tukey test. Since the series in 2018 and 2020 were significantly different, they were analyzed separately.

3. Results and Discussion

In the analyzed hyssop EO, 57 compounds were identified by GC-MS (

Table 1. Chemical composition of hyssop oil with the average content of main compounds (%).

Compound	RIexp 1	RIlit 2	[%]
α-Pinene	926	926	0.4
Camphene	948	950	0.1
Sabinene	972	973	1.6
β-Pinene	976	978	8.8
Myrcene	986	987	1.4
β-Phellandrene	1023	1024	2.4
Limonene	1024	1025	0.8
(E)-β-Ocimene	1038	1041	0.4
γ-Terpinene	1049	1051	0.1
trans-Sabinene hydrate	1051	1053	0.2
Terpinolene	1080	1082	tr 3
α-Thujone	1087	1089	0.3
Linalool	1084	1086	0.4
β-Thujone	1099	1103	0.2
Pinocamphone	1138	1139	10.6
Myrtenyl methyl ether	1144	1145	3.6
Isopinocamphone	1153	1151	42.1
Terpinen-4-ol	1161	1164	0.1
Myrtenal	1170	1172	0.1
α-Terpineol	1174	1176	0.1
Myrtenol	1176	1178	0.9
Carvotanacetone	1218	1220	tr
Methyl myrtenate	1271	1275	0.1
Myrtenyl acetate	1303	1306	tr
δ-Elemene	1338	1340	1.2
Methyl eugenol	1370	1369	0.1
α-Copaene	1376	1379	0.1
α-Bourbonene	1380	1378	0.9
β-Bourbonene	1386	1386	0.3
α-Gurjunene	1409	1413	0.5
(E)-β-Caryophyllene	1418	1421	2.6
β-Copaene	1428	1430	0.2
Calarene	1439	1437	0.1
(E)-β-Farnesene	1446	1446	0.2
α-Humulene	1451	1455	0.5
Alloromadendrene	1456	1462	1.7
γ-Muurolene	1472	1474	tr
Germacrene D	1477	1479	5.4
Alloaromadendr-9-ene	1488	1489	0.2
Bicyclogermacrene	1491	1494	2.7
α-Muurolene	1495	1496	0.1
γ-Cadinene	1505	1507	0.2
cis-Calamenene	1512	1517	tr
δ-Cadinene	1516	1520	0.2
α-Cadinene	1531	1534	0.1
Elemol	1538	1541	3.9
(E)-Nerolidol	1549	1553	0.5
Spathulenol	1565	1572	0.4
Caryophylene oxide	1571	1578	0.3
Viridiflorol	1590	1592	tr
Ledol	1596	1600	0.3
10-epi-γ-Eudesmol	1607	1609	0.1
γ-Eudesmol	1616	1618	0.8
τ-Cadinol	1628	1633	0.2
β-Eudesmol	1637	1641	0.4
α-Cadinol	1640	1643	0.2
α-Eudesmol	1649	1653	0.2

¹ RI_exp—experimental retention index; ² RI_lit—standard retention index; ³ tr—trace content < 0.05%.

). The essential oil was rich in oxygenated terpene compounds. The main compounds were monoterpene ketones: isopinocamphone (42.1%) and pinocamphone (10.6%). Other important constituents were mononeterpene and sesquiterpene hydrocarbons e.g., β-pinene (8.8%), germacrene D (5.4%), bicyclogermacrene (2.7%), and (E)-β-caryophyllene (2.6%) and oxygenated constituents e.g., elemol (3.9%), myrtenyl methyl ether (3.6%).

The qualitative composition matched previous reports for isopinocamphone-rich hyssop EO [14,15,16,17,18]. However, myrtenyl methyl ether was relatively rarely found [16,17,18,19]. The main constituents were within the requirements of the ISO 9841:2013 standard.

Table 2. Germination and seedlings growth (mean value ± standard error) of wheat cv. Harenda (an experiment in 2018) and cv. Blondynka (an experiment in 2020) in the presence of increasing doses of hyssop essential oil.

Dose of EO (g per Dish)	cv. Harenda			cv. Blondynka
	Germinated [%]	Leaf [mm]	Root [mm]	Germinated [%]	Leaf [mm]	Root [mm]
0	100 ± 0 a 1	62.0 ± 2.91 a	90.2 ± 5.57 a	90.0 ± 2.36 a	14.0 ± 1.42 a	45.0 ± 2.39 a
0.004	98.3 ± 1.36 a	52.8 ± 1.05 ab	66.1 ± 1.86 ab	90.0 ± 6.24 a	10.8 ± 0.67 ab	34.4 ± 1.15 ab
0.007	93.3 ± 3.6 a	49.7 ± 3.08 ab	60.8 ± 5.55 b	91.7 ± 1.36 a	7.31 ± 1.18 bc	28.9 ± 2.66 bc
0.01	93.3 ± 1.36 a	35.4 ± 10.36 b	42.3 ± 8.37 b	58.3 ± 11.9 b	4.97 ± 0.22 c	18.2 ± 1.23 cd
0.02	21.7 ± 9.53 b	3.41 ± 1.24 c	7.98 ± 2.72 c	41.7 ± 18.0 b	2.40 ± 1.02 c	11.5 ± 1.89 d
0.03	0 ± 0 c	0 ± 0 c	0 ± 0 c	11.7 ± 4.91 c	1.53 ± 0.62 c	5.40 ± 2.21 d
ED50	0.017	0.011	0.009	0.016	0.008	0.009

¹ diverse letters in the column denote a significant difference between means, according to the Tukey test at p < 0.05.

presents the results of one-way ANOVA for germination and growth of wheat seedlings in the presence of hyssop EO in 2018 and 2020. The germination of wheat was visibly inhibited by hyssop EO at a dose of 0.02 g of EO per dish (equal to 2.8 g EO L⁻¹) and 0.01 g per dish (equal to 1.4 g EO L⁻¹) for cv. Harenda and cv. Blondynka, respectively. The ED50 dose for germination for each of those two cultivars was similar and equal to 0.017 and 0.016 g EO per dish. Seedlings of both wheat cultivars displayed different susceptibility to the hyssop EO, with wheat cv. Harenda being more susceptible. The growth of the roots of both cultivars was more inhibited by the EO than the leaves. In case of wheat cv. Harenda, a visible drop in the elongation of the leaf and root was visible at doses of 0.01 and 0.007 g of EO per dish, respectively. At the highest dose of hyssop EO, wheat cv. Harenda did not germinate. On the contrary, wheat cv. Blondynka germinated, and seedlings grew even at the highest dose of hyssop EO; however, its growth was strongly inhibited. A visible inhibition of the growth of cv. Blondynka seedlings was observed at an EO dose of 0.007 g per dish (equal to 1 g of EO per L⁻¹) for both leaves and roots.

The germination and seedling growth of mustard was less affected by the hyssop EO than wheat (

Table 3. Germination and seedlings growth of white mustard cv. Borowska during the experiments performed in 2018 and 2020 in the presence of increasing doses of hyssop essential oil.

Dose of EO (g per Dish)	2018			2020
	Germinated [%]	Shoot [mm]	Root [mm]	Germinated [%]	Shoot [mm]	Root [mm]
0	61.7 ± 5.44 a 1	22.3 ± 3.56 a	12.2 ± 1.54 a	93.3 ± 2.72 a	23.7 ± 1.14 a	32.9 ± 1.81 a
0.004	60.0 ± 2.36 a	18.3 ± 3.09 a	11.6 ± 3.11 a	81.7 ± 3.60 a	21.9 ± 1.89 a	24.9 ± 2.54 a
0.007	50.0 ± 4.71 ab	22.9 ± 6.47 a	15.2 ± 6.04 a	86.7 ± 3.60 a	22.1 ± 2.02 a	23.3 ± 0.85 ab
0.01	66.7 ± 7.20 a	23.2 ± 3.49 a	21.7 ± 2.14 a	81.7 ± 5.93 a	14.3 ± 1.17 ab	15.0 ± 1.58 bc
0.02	36.7 ± 8.28 bc	8.31 ± 1.32 b	5.35 ± 0.86 b	76.7 ± 9.53 ab	10.6 ± 0.48 b	10.9 ± 2.07 c
0.03	28.3 ± 3.60 c	5.87 ± 0.77 b	3.17 ± 0.52 b	65.0 ± 4.08 b	6.72 ± 0.16 b	8.56 ± 0.82 c
ED50	0.03	0.02	0.02	0.09	0.02	0.01

¹ diverse letters in the column denote a significant difference between means, according to the Tukey test at p < 0.05.

). The EO doses of 0.02 and 0.03 g of EO per dish significantly affected the germination of mustard in 2018 and 2019, respectively, which is why the resulting ED50 doses were high, especially in 2020. As for the growth of mustard seedlings, similar values of ED50 doses point to similarities in their response to the EO in both study periods. In 2018, only the two highest doses of the EO caused a significant inhibition of mustard seedling growth. In contrast, in 2020, a significant decrease in shoots and root growth was observed already at 0.01 and 0.007 g of EO per dish, respectively.

Hyssop oil displays herbicidal effects against germination and the initial growth of some weed species, e.g., Lepidium sativum [5], which could be correlated with the high monoterpenes content in the oil [10,20]. In another study, the hyssop oil displayed a low herbicidal effect against rapeseed germination (Brassica napus L.) [6]. That result is compatible with our finding that hyssop oil is less phytotoxic against the initial growth of Sinapis alba. Perhaps, this phenomenon could be connected with a higher content of oils in the seeds of both mustard and rapeseed, as the authors of [21] suggest.

4. Conclusions

The tested hyssop oil was rich in monoterpene ketones, e.g., isopinocamphone (42.1%) and pinocamphone (10.6%). The oil inhibits wheat and mustard germination, with wheat being more inhibited than mustard. The hyssop oil also inhibits the elongation of seedlings of both crops in a dose–response manner. The visible inhibition of wheat seedlings occurs at a dose of oil equal to 1.0 g per L⁻¹, whereas in mustard it occurs at a dose of 2.8 g per L⁻¹. Further testing of the phytotoxicity of hyssop essential oil should be carried out to assess the physiological background of different wheat and mustard seedlings’ susceptibility to this oil.