Chemical composition and herbicidal activity of Oregano (Origanum onites) essential oil on weeds and wheat

Oregano essential oil (EO) is known as one of the EOs with the highest biological activity on target plant species. In this study, the inhibitory effects of different doses of oregano (Origanum onites L.) EO on the germination and seedling growth of some wheat cultivars and some problem weed species in wheat fields was investigated. For this purpose, both laboratory and pot experiments were carried out. Oregano EO obtained by steam distillation method and component analysis was made via GS/MS. Germination tests and pot experiments were carried out under controlled laboratory and greenhouse conditions. In the study, five wheat varieties, four for bread and one for durum, and five different weed species were used. The effects of different doses of oregano EO on the germination rate, viable plant rate, seedling length, and biomass of test plants were analyzed. According to the results, it was observed that oregano EO showed bioactivity on all species and cultivars included in the experiment. In terms of all parameters, it was concluded that wheat varieties showed higher resistance to oregano EO than weed species. These results show that oregano EO can be used to control some weed species that are a problem in wheat.

Keywords: Oregano; essential oil; bioherbicide; allelopathy; weed control; wheat cultivars

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Weeds are an important biotic factor that causes various problems in both agricultural and non-agricultural areas. At the beginning of the problems caused by weeds in agricultural areas is the loss of yield in crops due to competition. In addition, they cause a decrease in the quality of products (Zimdahl [64]). Weeds are more of a problem in field crops. Especially wheat is a sensitive plant to weed competition (Das and Yaduraju [11]; Wenda-Piesik et al. [59]). Wheat is one of the most cultivated plants in the world and has great importance in the nutrition of humans (Igrejas et al. [27]). Therefore, one of the most studied crop plants is wheat. Wheat is also one of the cultivated plants in which herbicides are used most intensively. Accordingly, the highest number of weed species that develop resistance to herbicides is in wheat cultivation (Heap [24]). On the other hand, nearly half of the pesticides used in the world are herbicides (De et al. [12]). Due to this intensive use of herbicides, there are many other health and environmental problems in addition to the weed resistance problem. This situation has increased the interest in alternative methods to herbicides. One of these alternative methods is the use of some phytotoxic compounds obtained from plants as bioherbicides against weeds. These phytochemicals exert various effects on the target plant. For instance, they induce changes in the micro and ultrastructure of cells (Wu et al. [60]), inhibit cell growth and proliferation (Sa´nchez-Moreiras et al. [47]), disrupt the antioxidant system balance (Batish et al. [7]), enhance cell membrane permeability (Farhoudi and Lee [19]), influence the plant growth regulator system (Lin et al. [35]), affect enzymatic activity (Cheng [9]), modify respiration (Hejl and Koster [25]), impact photosynthesis (Uddin et al. [57]), influence water and nutrient uptake (Yu and Matsui [63]), and alter protein and nucleic acid metabolism (Guo et al. [23]). The idea of using plant extracts in weed control is not new (Duke [16]; Muller and Muller [39]). This issue is seen as an environmentally friendly alternative to synthetic herbicides. Because plant compounds have a shorter half-life period and their toxicity to non-target organisms is lower (Duke [16]). In particular, the degradation of EOs is quite rapid (Mahanta et al. [36]). The growing problems caused by the widespread use of synthetic herbicides also support these approaches. There are many studies on the effect of plant compounds on weeds. However, many of these have not gone beyond laboratory or in-vitro trials. On the other hand, most of the studies were built on the effects of plant extracts only on weed species, and their effects on crops were often ignored. Whereas it is very important to evaluate the effects of plant EOs on weed species as well as to reveal their effects on crop species and cultivars. As the tolerance of each crop species is not the same (Tursun et al. [55]), there may be differences in sensitivity between cultivars belonging to the same species (Atak et al. [4]). It is known that essential volatile components are the group whose bio-herbicidal effect is the most studied among plant chemicals due to their high phytotoxic effects (Dudai et al. [15]; Tworkoski [56]). Essential oils are complex mixtures of volatile compounds, mainly terpenes and phenylpropanoids, particularly abundant in aromatic plants (Christaki et al. [10]; Nikolova and Berkov [42]; Sadgrove et al. [46]). One of the most studied plant oils regarding its herbicidal effect is oregano (Origanum spp.) EO. It is seen that the most effective results are obtained from oregano among the aromatic plants tested in many studies (Atak et al. [4]; Dudai et al. [15]; Frabboni et al. [21]; Mirmostafaee et al. [38]). Unlike rare plants, oregano is one of the aromatic plants produced and traded in many parts of the world. This is important in terms of the supply and widespread use of oregano EO. In this study, the phytotoxic effects of oregano (O. onites) EO were investigated against wheat, and some weed species with laboratory and pot experiments. Our hypothesis is that oregano EO is safe for wheat varieties at certain doses but can have an inhibitory effect on the germination and/or development of weed species. In this context, the aim of the study is to determine whether an oregano EO application can be made to control weeds without harming the wheat.

This study was carried out with five different wheat (Triticum spp.) cultivars and five different weed species that are a problem in wheat fields. The used wheat cultivars are 'cv. Bayraktar-2000', 'cv. Demir-2000', 'cv. Esperia' 'cv. Kızıltan-91', and 'cv. Tosunbey' which are commonly grown in Turkey. Among these cultivars, 'Kızıltan-91' is for durum and the others are for bread. Wheat seeds were provided from The Central Research Institute of Field Crops in Ankara. The seeds of the weed species (Festuca arundinacea Schreb., Lolium perenne L., Poa pratensis L., Secale cereale L., Vicia sativa L.) used in the study were collected from grain fields which were different locations. The plant materials of oregano (O. onites) whose EO was used in the study, were obtained from a farmer in the Denizli province where is southwest of Turkey. Oregano plants were collected during the maximum blooming period in field condition. Plant materials were first air-dried in the shade at room temperature for a week and then dried in a drying cabinet at 40°C for 72 h.

The EO of oregano was obtained by using the hydrodistillation method. Herbal material (100 g) was cut into small pieces and put into the Clavenger type distillation apparatus according to the standard procedures of the European Pharmacopoeia (de l'Europe [13]) and extracted with 2 L of pure water for 3.5 h (until no more EO was obtained). The EO was collected, dried under anhydrous sodium sulphate and stored in an opaque bottle at 2–4°C until used. Oregano EO component analysis was performed with Gas Chromatography/Mass Spectrometry (GC–MS). Analysis was carried out in the Central Laboratory of Süleyman Demirel University. Analytical conditions of the GC-MS system were presented in Table 1.

Table 1. GC–MS analytical conditions.

Determination of each essential oil ingredient was performed with the aid of retention times of analytical standards by comparison of mass spectrums produced by Wiley and NIST Library (Stein [53]).

An in-vitro study was carried out under controlled conditions in the laboratory to determine germination rates. For this purpose, double-layered filter paper (Whatman no.1) was placed at the base of the 9 cm diameter sterile petri dishes and 25 seeds for wheat cultivars, rye and common vetch and 100 seeds for other species were put into each petri dish. In the preliminary studies, since doses of 5 μl and above of oregano EO inhibited the germination of all wheat cultivars, 0 (control), 1, 2, 3 and 4 µl doses were tested in the study. Oregano EO was applied at the determined doses by absorbing it onto the filter paper at the bottom of the petri dish with the help of a micropipette, and 10 ml of distilled water was added to it. Petri dishes were wrapped with parafilm and sealed in plastic bags immediately after application. The petri dishes were placed in the germination chamber, which was then set to a constant 22°C temperature in the dark. After ten days, the seeds whose radicula reached 0.5 cm were counted as germinated, and the germination rate was calculated with following formula.

Germination Rate = (ng /nt) × 100ng: The number of germinated seeds

nt: Total number of seeds

The germination bioassay was repeated two times with the same procedure.

An equal amount of soil, sand and peat (1 + 1 + 1) mixture was placed in 1.5-liter plastic pots. 25 seeds for wheat cultivars, rye and common vetch and 100 seeds for other species were sown into each pot and oregano EO at the rate of 0 (control), 0.5, 1.0, 1.5 and 2.0 ml kg−1 was applied with 500 ml water. During the study, equal amounts of water were given to the pots based on their needs. In the greenhouse, air temperature and relative humidity were recorded hourly with the RH/Temp data logger (Hobo© H08-003-02) during the experiment (Figure 1). The 30-day average air temperature in the greenhouse was recorded at 19.5°C, and the air humidity was 51.8%. Wheats and weeds in pots were observed daily in the greenhouse for 30 days. Germinating, surviving, and dying plants were recorded. At the end of 30 days, the number of live plants, their height and dry weight were determined. In order to determine the dry weight of the plants, the above-ground parts were dried at 65°C for 72 h and weighed on a precision scale.

PHOTO (COLOR): Figure 1. Greenhouse experimental conditions.

Both germination and pot experiments were established in a completely randomized plot design with 5 replications for the germination bioassay and 4 replications for the pot trials. After arcsine transformation, the data were submitted for analysis of variance, and the means were separated using the LSD test at a P 0.05 significance level. The statistical analysis were done using SAS version 9.2 software (SAS [49]).

The yield of the EO obtained by hydrodistillation from the dried oregano plants was found to be 2.36%. Chemical composition of oregano (O. onites) EO is given Table 2. A total of 10 components were identified in oregano EO. The ratio of these 10 components in the essential oil was determined as 94.43%. All the peaks in the chromatogram were tried to determine by using related analytical standards. However, some peaks could not be determined due to a lack of analytical standards. Therefore, these peaks were classified as 'unknown components'. The total percentage of the unknown components was found to be 5.57%. Carvacrol (69.4%) was found to be major component. p-Cymene (6.2%), γ-Terpinene (5.0%) and linalool (3.9%) were detected as other prominent components.

Table 2. Chemical composition of O.onites essential oil by GC/MS.

The effect of the first three doses (1, 2 and 3 μl) of oregano EO on the germination rate of wheat cultivars was found to be very limited. On the other hand, except for S. cereale, L. perenne and V. sativa, the germination rate of the other two species (F. arundinacea and P. pratensis) decreased significantly even at the first dose (1 μl). In the 2 μl dose of oregano EO, the germination rate of all weed species was significantly reduced compared to the wheat cultivars (Table 3). While the average germination rate of wheat cultivars was only 1.3% lower at a 2 μl dose compared to the control, it was 41.9% in weed species at the same dose. While the germination rate of wheat cultivars decreased by 17.7% at 3 μl dose, the average germination rate of weed species decreased by 52.3%. The 4 μl dose of oregano EO suppressed the average germination rate of all species tested in the study by more than 85%. According to petri studies, cv. Demir-2000 is the most resistant wheat variety to oregano EO, while cv. Kızıltan-91 is the most sensitive. Meanwhile, cv. Kızıltan-91 is the only durum cultivar in the experiment. Among the weed species, it was observed that the most sensitive species was P. pratensis, followed by F. arundinacea. V. sativa was the weed species that was least affected by oregano EO. On the other hand, it was clearly seen that grassy weed species were more affected by oregano EO than wheat cultivars.

Table 3. Effect of oregano EO on the germination rate (%) of wheat cultivars and weed species (n = 5).

1 Means within a row with the same lower-case letter and a column with the same upper-case letter are not significantly different at 0.5% level by LSD test.

According to the data, the rate (%) of living plants in the pot dropped at all doses for both wheat cultivars and weed species. However, as in the germination studies, the rate of this reduction was less in wheat cultivars compared to weed species. For instance, while the number of live plants in wheat cultivars decreased by 69.9% at a 0.5 ml dose compared to the control, this rate was 75.6% in weed species. Similarly, it is observed that other doses of oregano EO have a greater impact on weed species. Like the germination study, cv. Demir-2000 is the least affected wheat cultivar by oregano EO. It was found that cv. Esperia is the most sensitive wheat cultivar. Again, among the weed species, V. sativa was observed to be the most resistant. In comparison to other grassy weeds, S. cereale is more resistant. L. perenne was observed to be the most sensitive weed species to oregano EO in pot experiments, and this species did not germinate or develop at any concentration of oregano EO (Table 4).

Table 4. Effect of oregano EO on the rate (%) of alive plants in the pot experiment (n = 4).

2 Means within a row with the same lower-case letter and a column with the same upper-case letter are not significantly different at 0.5% level by LSD test.

Considering the effect of oregano EO on the dry weight of the test plants, a reduction was observed in all species from the first dose (0.5 ml) (Table 5). This decrease in dry weight increased with increasing dose. However, this decrease was lower in wheat varieties compared to weed species. When weed species were evaluated together, the decrease in dry weight was 75.5%, 95.3%, 98.5%, and 100%, respectively, according to the doses. In wheat varieties, this decrease was 66.1%, 82.7%, 83.4%, and 96.6%, respectively, according to the applied doses. Among the wheat cultivars, cv. Demir-2000 and cv. Kızıltan-91 were found to be more tolerant to oregano EO in terms of dry weight. The most sensitive cultivar was found to be cv. Esperia. Among the weed species, V. sativa was found to be the most durable in terms of dry weight. The most sensitive species were determined as L. perenne (100%), P. pratensis (99.7%), and F. arundinacea (99.2%), respectively. Since the tested plants are of different genotypes and naturally their phenotypic characteristics are different, no statistical comparison was made between different genotypes in terms of biomass and seedling length parameters.

Table 5. Effect of oregano EO on dry weight (g) of the test plants at the pot experiment (n = 4).

3 Means within a row with the same letter are not significantly different at 0.5% level by LSD test. No comparison was made between genotypes as their phenotypes and habitus were different from each other.

There was a decrease in seedling length from the first dose in all test plants. However, this decrease was at a much lower level in wheat varieties than in weed species, as in other investigated parameters. Depending on the applied oregano EO dose, the decrease in seedling lengths of the viable wheat cultivars was 12.0%, 20.6%, 12.8%, and 16.0%, respectively, while this decrease in the viable weed species was 32.9%, 33.1%, 58.2% and 100% (no plant), respectively. Among the wheat cultivars, the seedling length decreased significantly in cv. Esperia depending on the increasing dose. However, the other wheat cultivars showed variation in terms of seedling length reduction between doses. This may be due to the decrease in the number of plants that remain viable as the dose increases, and therefore the low number of individuals measured. While no live plants were found in L. perenne among weed species, the seedling length of F. arundinacea and P. pratensis decreased dramatically (Table 6).

Table 6. Effect of oregano EO on seedling length (cm) of the test plants at the pot experiment (n = 4).

4 Means within a row with the same letter are not significantly different at 0.5% level by LSD test. No comparison was made between genotypes as their phenotypes and habitus were different from each other.

In the study the major component obtained from O. onites EO was carvacrol (69.4%) (Table 2). In many other studies, it has been reported that the most abundant phytochemical in oregano EO is carvacrol (Arslan et al. [3]; Atak et al. [4]; Becer et al. [8]; Erbas et al. [17]; Erenler et al. [18]; Karan et al. [28]; Kordali et al. [32]; Kutlular and Ozel [34]; Ozdemir et al. [44]; Stefanaki et al. [52]; Vokou et al. [58]). In previous studies, carvacrol rates of O. onites species collected in the Mediterranean basin were determined at 57.0% (Atak et al. [4]), 58.7% (Kordali et al. [32]), 59.9% (Yilar et al. [62]), and 91.4% (Erbas et al. [17]). Many studies have demonstrated the inhibitory effect of carvacrol on seed germination but there is no study on the mechanism that causes it. However, few studies demonstrated that the EOs prevent seed germination by the disruption of mitochondrial respiration and oxidative pentose phosphate pathway (Podestá and Plaxton [45]; Abrahim et al. [1]; Muscolo et al. [40]). In our study, other prominent components p-Cymene (Becer et al. [8]; Demirel and Erdogan [14]; Erenler et al. [18]; Kordali et al. [32]; Ozdemir et al. [43]; Sarıkaya [48]; Spyridopoulou et al. [51]; Stefanaki et al. [52]), γ-Terpinene (Arslan et al. [3]; Erenler et al. [18]; Karan et al. [28]; Kizil et al. [30]; Kordali et al. [32]; Sarıkaya [48]) and linalool (Becer et al. [8]; Demirel and Erdogan [14]; Sarıkaya [48]; Yigit and Kocaayan [61]) are compatible with other many previous study. However, it is considered that the difference between the components and proportions of the EO is caused by factors such as geography, climate, soil properties, collection time, and analysis method (Figuérédo et al. [20]; Hussain et al. [26]; McGimpsey et al. [37]; Sefidkon et al. [50]; Toncer et al. [54]).

In the study, according to the effect of oregano EO on the germination rate of the test plants, it was observed that wheat cultivars were not affected much by the other doses except for the highest dose (4 μl) tested, but the germination rate of other weed species except V. sativa decreased significantly. While there was no statistical difference between the germination rates of wheat cultivars in the first three doses tested (except for cv. Kızıltan-91 at 3 μl dose), especially in small-seeded monocot weed species, significant differences occurred from the first dose (Table 3). Among the weed species, it was observed that the most sensitive species was P. pratensis, followed by F. arundinacea. Among the monocot weed species, S. cereale and L. perenne, respectively, showed higher resistance to oregano EO compared to the other two species (Table 3). At this point, the difference in seed size between these species draws attention. Kohler et al. ([33]) showed that small-sized seeds are more susceptible to the phytotoxic effect of essential oil than large seeds, and the germination rate is more reduced. Therefore, it can be stated that small-sized seeds will be more affected by exposure to a chemical under equal conditions. V. sativa was the weed species that was least affected by oregano EO in the study. In fact, among all tested plants, V. sativa was the least susceptible to oregano EO. It was able to achieve 46.4% germination even at the highest (4 μl) dose (Table 3). It is believed that this is the reason that V. sativa is the only non-Poaceae and dicotyledon species included in the study and that its seeds have a different structure. Thus, Grul'ova et al. ([22]) reported that the phytotoxic effect of Origanum vulgare EO on monocotyledon species is greater than on dicotyledon species. In a previous study, the EO of O. onites strongly suppressed the germination of Amaranthus retroflexus, while its effect on V. sativa was relatively limited (Kitis and Ozkan [29]). On the other hand, it was clearly seen that grassy weed species were more affected by oregano oil than wheat cultivars. It implies that specific oregano EO dosages can control some weed species while being selective for wheat. According to Atak et al. ([4]), the germination of different wheat cultivars was inhibited by Origanum onites EO at varying rates, but more significantly, the seeds of Avena sterilis and Sinapis arvensis displayed more sensitivity than wheat. Erbas et al. ([17]) found that oregano (O. onites) EO did not affect wheat germination up to 3 μl as in our study (Table 3); however, at the same dose, S. arvensis and A. retroflexus seeds did not germinate at all, while Rumex crispus could only germinate 45%. Similarly, Tursun et al. ([55]) found in their study; that while wheat seeds germinated 100% in 1 μl dose of oregano (Origanum syriacum) EO, Glycyrrhiza glabra, Rumex crispus, and Physalis angulata did not germinate at the same dose, and A. retroflexus could only germinate 3.3%. However, the germination of wheat was completely inhibited at a dose of 5 μl of the same EO. In our study, while the germination rate of wheat varieties at a dose of 4 μl decreased by more than 85% (Table 3), almost none of the wheat seeds germinated in the preliminary trials with 5 μl of oregano EO. These results closely resemble the outcomes of our own investigation. Monoterpenes such as carvacrol, which are commonly found in EOs, are known to inhibit the germination of plant seeds (Aviv et al. [5]; Azirak and Karaman [6]; Dudai et al. [15]; Kordali et al. [31]). However, the sensitivity of each species is different, which creates selectivity.

In the pot experiment, the rate of alive plants decreased with the increasing dose of oregano EO. However, this decrease was found to be lower in wheat varieties (Table 4). In the pot experiments, V. sativa was the least affected by oregano EO in terms of the number of live plants, while no live plants were seen in L. perenne. Among monocot weeds, S. cereale was found to be slightly more resistant, while F. arundinaceae and P. pratensis were also highly sensitive, and there were no live plants of these two species at all doses except the first dose. Seed size and the structure of the seed coat are thought to be important factors in this difference in sensitivity between species. According to the pot experiments of Erbas et al. ([17]), while the rate of live wheat plants at a dose of 2 mg kg−1 of oregano (O. onites) EO was 90% and above, there was almost no live weed species (A. retroflexus, S. arevensis, Rumex crispus) at the same dose. Pot experiments gave similar results in both studies.

Since the dry weights of the test plants in the pot experiment were directly correlated with the quantity of living plants, the results were similar. Oregano EO had an impact on wheat varieties as well, however the reduction in plant biomass was less than that of weeds. In terms of plant biomass among wheat varieties, cv. Kızıltan-91 performed better than the others. However, a statistical comparison was not made between wheat cultivars because their phenotypic characteristics were different. Among weed species, V. sativa was the least affected by oregano EO in terms of dry weight, while L. perenne was the most affected. In a different pot experiment, it was found that the oregano EO significantly inhibited the dry matter ratio of Amaranthus retroflexus more than Sinapis arvensis (Erbas et al. [17]). Again, the difference in seed size stands out as an important factor.

The results of the seedling length of the test plants in the pot experiment were evaluated based on the length of only viable seedlings. Accordingly, as in other parameters, the decrease in seedling length in wheat varieties was much less than in weeds. While the decrease in seedling length in wheat varieties was 16% on average, this rate was 50% in weeds. Erbas et al. ([17]) determined that while O. onites EO slightly decreased the stem length of wheat, the decrease in stem length was much higher in weeds. The inhibition of plant growth, attributed to phytochemicals such as EOs, is anticipated to arise from the suppression of mitosis and disruption of cell organelle structure (Sa´nchez-Moreiras et al. [47]; Mushtaq et al. [41]). Araniti et al. ([2]) demonstrated that treatment with Origanum vulgare EOs resulted in the inhibition of glutamate and aspartate metabolism, thereby affecting the photorespiratory pathway and the photosynthetic machinery.

As a result of a series of experiments to investigate the effect of oregano EO on the germination and development of wheat and some weed species that are problems in wheat fields, it was observed that all species were affected depending on the amount of oregano EO, but wheat varieties were more resistant than especially grassy weed species. This shows that oregano EO can be used to control some weeds if a suitable dose adjustment is made. The results need to be supported by field trials. However, the biggest problem in such essential oil applications is that the material applied is not permanent and has a short-term effect, even if its effect is high. For this reason, it is important to formulate EOs by slow-release encapsulation or similar methods to have a residual effect in order to be used as a bioherbicide, and studies on this subject will fill an important gap.

I would like to thank the Field Crops Central Research Institute of Turkish Agricultural Ministry for providing the wheat cultivars and Ms. Burcu Tazegül and Mr. Ender Gümüş for their help.

No potential conflict of interest was reported by the author(s).