Effectiveness of Mexican oregano essential oil from the Dominican Republic ( Lippia graveolens ) against maize pests ( Sitophilus zeamais and Fusarium verticillioides )

The insecticidal and antifungal properties of Mexican oregano (Lippia graveolens) essential oil from the Dominican Republic were investigated under laboratory conditions against two main pests of stored grains: Sitophilus zeamais and Fusarium verticillioides. Although oregano essential oil at 600 μl/l air did not result in a significant mortality of S. zeamais after 24 hours of exposure by fumigation, this essential oil was a powerful acetylcholinesterase (AChE) inhibitor in vitro. One of the main components of oregano essential oil, p-cymene, presented fumigant toxicity and AChE inhibition activity against the maize weevil. The L. graveolens essential oil antifungal activity against F. verticillioides was evaluated at 50, 100 and 200 μl/l, and it was found that growth parameters were affected by the presence of oregano essential oil in the media, whereas FB1 production was not inhibited. The results demonstrate that oregano essential oil and p-cymene can be used as alternatives to synthetic pesticides against F. verticillioides and S. zeamais, respectively.


IntRodUctIon
The maize weevil Sitophilus zeamais Motschulsky (Coleoptera: Curculionidae) is a serious pest of stored grains, especially corn.It consumes the grains and creates the conditions that favor the development of fungi, resulting in major losses, both quantitative and qualitative (Caneppele et al., 2003;Trematerra et al., 2013).Fusarium verticillioides (Sacc,) Nirenberg (= F. moniliforme Sheldon teleomorph Giberella fujikuroi [Sawada] Ito in Ito & Kimura) is a frequent fungal pathogen in stored maize.In addition, F. verticillioides is one of the most important mycotoxin fumonisin B 1 (FB 1 ) producers.It can be produced during maize storage and represents a major problem due to its toxicological implications for humans and farm animals (Stockmann-Juvala & Savolainen, 2008;Theumer et al., 2010).The use of synthetic fumigants, such as methyl bromide, phosphine and sulfuryl fluoride, is the most widespread method to control stored product pests.However, the use of these compounds has led to a variety of problems including the development of insecticide resistance (Pimentel et al., 2009), toxic residual effects in food grains, toxicity to non-target organisms and environmental pollution (Isman, 2006).Moreover, the increasing public awareness of pesticide safety and possible damage to the environment has resulted in greater attention being given to the control of stored food pests by means of natural products (Rajendran & Sriranjini, 2008), of which essential oils (EOs) are considered to be an interesting alternative because of their effectiveness and versatility.In fact, their volatility and chemical diversity makes them excellent fumigants, insecticides, fungicides and repellents.The use of EOs as low-risk biopesticides has increased considerably owing to their popularity with organic farmers and environmentally conscious consumers (Rajendran & Sriranjini, 2008;Gleiser & Zygadlo, 2009;Regnault-Roger et al., 2012;Hernández-Lambraño et al., 2014;Sousa et al., 2015), with this demand for naturally active compounds such as EOs having stimulated the search for these chemicals in biodiversity hotspots.
Lippia graveolens Kunth (Lamiales: Verbenaceae), one of the most commercial species within the genus Lippia, is an aromatic plant commonly known as Mexican oregano or oreganillo.Interest in this species is increasing due to its insecticidal and antimicrobial activities (Cavalcanti et al., 2004;Gleiser & Zygadlo, 2007;Bueno-Durán et al., 2013) as a result of its chemical constituents, such as thy-mol and carvacrol.In this context, the aim of this study was to analyze the EO composition of L. graveolens growing in the Dominican Republic, and to evaluate the insecticidal activity and acetylcholinesterase (AChE) inhibition of L. graveolens EO and its main components against S. zeamais.The antifungal properties and FB 1 inhibition of Mexican oregano EO from the Dominican Republic against the F. verticillioides strain M3125 were measured.

Insects
Adults of S. zeamais were obtained from Metan, Salta, Argentina, and were maintained in sealed containers (10 l) under controlled conditions (26 °C and 60% relative humidity) with a lighting regime of 12:12 hours (D:L).The insects were reared on whole maize grains, and all bioassays were carried out under the same environmental conditions and in complete darkness (FAO, 1974).The colony was kept in the laboratory for two years without being exposed to insecticides before testing.The unsexed adult weevils used in all the experiments were approximately two weeks old.

essential oil and pure compounds
Lippia graveolens Kunth samples were collected in commercial crops in Santo Domingo, Dominican Republic.The aerial parts of the plants were air dried, chopped into small pieces using a mill with rotary knives, and extracted by hydro-distillation for 2 hours in a Clevenger's apparatus in order to collect their vaporized EO.The EO obtained was stored in dark glass tubes under refrigeration (4 °C) until evaluation.
The density of EO was measured by gravimetric method and found to be 0.93 g/ml at room temperature (± 2 o C).Identification of the constituents in EO was determined using electron impact mass spectra (EI-MS) obtained from gas chromatography-mass spectrometry (GC-MS) and by co-injection of standards (Sigma Aldrich Co. Buenos Aires, Argentina), with the mass spectra libraries Adams, NIST and a homemade library being utilized.Compound concentrations were expressed as a percentage of the peaks area, and the retention index (RI) of each compound was obtained for a homologous series of n-alkanes C 9 -C 20 (Sigma Aldrich Co. Buenos Aires, Argentina).GC-MS was performed on a GC-MS Perkin Elmer 600 with the chromatography conditions being as follows: DB-5 capillary column, which was programmed: temperature profile of 60 °C for 5 minutes, ramped up to 170 °C at 4 °C/minute, and then to 240 °C at 20 °C/minute; injector temperature 250 °C; detector temperature 250 °C; carrier gas, H 2 at 45 cm/second, split into 50 ml/minute.The identification and quantification of limonene, b-phellandrene and 1,8-cineole were carried out using Carbowax capillary columns, at the same conditions mentioned above for the DB-5 column.

Fumigation toxicity assay
Insecticidal activity against S. zeamais was evaluated using a fumigant toxicity assay previously described by Herrera et al. (2015a), with modifications.Different amounts of oregano EO and its main pure compounds (p-cymene, thymol and carvacrol) were applied to Whatman filter paper disks (2 cm diameter) placed on the underside of the screw cap of fumigation chambers (30 ml-glass vials) at doses from 75 to 600 µl/l air.A piece of voile was also placed under the screw cap to avoid direct contact of the weevils with the EOs.Ten adults of S. zeamais and 5 g of whole maize grains were placed in each vial, with the inclusion of corn allowing the natural conditions in a silo to be mimicked.Five replicates per dose were carried out, and control treatments were performed under the same conditions without EOs or pure compounds (negative control) and with 2,2-dichlorovinyl dimethyl phosphate (DDVP) compound 0.06 µl/l air (positive control).This latter compound was used as a positive control due to its high vapor pressure and its known insecticidal activity.Insect mortality was evaluated after 24 hours, and the mortality percentages and LC 50 and LC 95 values were then calculated.

In vitro Ache inhibition tests
The effects of oregano EO and its main components (p-cymene, thymol and carvacrol) on ace-tylcholinesterase (AChE) activities were examined at different concentrations (0.09, 0.19, 0.46, 1.39, 4.65, 13.95 and 23.25 mg/ml for oregano EO, and 0.025, 0.05, 0.1 and 0.5 mM for its components).Untreated whole S. zeamais adults (0.1 g) were homogenized with 1 ml phosphate buffer (pH 7.4), and then the homogenate was centrifuged (6500 rpm for 20 minutes at 0°C) and the supernatant was used as crude AChE.Inhibition of AChE was then determined by the colorimetric method of Ellman et al. (1961) using acetylthiocholine iodide (ATChI) at 2.5 mM (Sigma Aldrich Co., St. Louis, MO USA) as the substrate.Enzyme aliquots (20 µl) and 5,5-dithio-bis (2-nitrobenzoic) acid (DTNB) (20 µl of 4 mM) were added to 0.1M phosphate buffer (pH 7.4; 120 µl), and the EO active compounds (20 µl) prepared in absolute ethanol were added to this mixture.Control treatments used the addition of absolute ethanol (20 µl) instead of an active compound.All mixtures were incubated at 35 °C for 15 minutes, and the reactions were started by adding ATChI (20 µl), with absorbance being measured at 412 nm using a spectrophotometer (Model 680 Microplate Reader, Bio-Rad).
AChE activity was examined, with each treatment being corrected by blanks for nonenzymic hydrolysis.The inhibition percentage of AChE activity was calculated as follows: AChE inhibition% = (ODC-ODT)/ODC x 100, where ODC is the optical density of control and ODT is the optical density of the treatment.Each concentration was analyzed for triplicate assays, and the mid-point inhibitive concentration (IC 50 ) values were determined graphically from the inhibition curves (log inhibitor concentration vs percentage of inhibition) (Mohammadi-Farani et al., 2013).

In vivo Ache inhibition tests
For in vivo AChE inhibition tests, S. zeamais adults were cultured for 24 hours in the absence or presence of Mexican oregano EO (150 µl/l air), with the experimental conditions and procedures carried out as described above.Homogenates were prepared in phosphate buffer (0.1 M, pH 7.4) and kept on ice during the homogenization, with approximately fifty insects (0.1 g) per ml of buffer being used for each sample and the activity of AChE being determined immediately after the preparation of the homogenates.

effect of Lippia graveolens eo on fungal growth and fumonisin production
Fusarium verticillioides M3125 inoculum was grown on Czapek-dox agar Petri plates for 7 days at 28 ºC in the dark to allow profuse sporulation.Then, sterile distilled water was added to each plate, and a conidia suspension was obtained by scraping the colony surface with a sterile Drigalsky spatula and filtering it through a cheesecloth.The suspension was then counted using a Neubauer chamber and adjusted to 10 6 conidia/ml.The antifungal activity was tested at different concentrations of L. graveolens EO using Czapek-Dox agar in Petri dishes (90 mm).The culture medium was autoclaved at 120 °C for 15 minutes, and before cooling at 45 °C, an appropriate volume of EO was added to the media to obtain concentrations of 50; 100 and 200 µl/l.Czapek-Dox agar plates were inoculated centrally with 10 µl of the conidia suspension and incubated in the dark at 28 °C.Czapek-Dox Agar plates without the addition of EO were used as control.
The amount of radial mycelial growth was determined by periodical measurement of two rightangled diameters of the colonies, until the colonies reached the edge of the plate.Colony diameters versus time were plotted, and radial growth rates (mm/day) were evaluated from the slope by linear regression.Lag phase was determined as the abscissa from these growth rate curves.
To evaluate FB 1 biosynthesis, the inoculated plates were incubated in the dark at 28 °C for 28 days.After this incubation, the agar in the experimental plates was dried for 96 hours at 60 °C in a forced-air oven before being ground to a fine dry powder.Finally, 10 ml of water was added to the dried agar, and FB 1 was extracted by shaking the dried dishes with water for 120 minutes on an orbital shaker, after which the mixture was centrifuged at 5000 rpm for 15 minutes.All experiments were performed in triplicate.

Fumonisin B 1 quantification
FB 1 quantification was carried out according to a methodology described by Shephard et al.(2000).Briefly, samples (1000 μl) from the FB 1 extracts were diluted with acetonitrile (1:1), and then an aliquot (50 µl) was derivatized with 200 µl of a solution prepared by adding 5 ml of 0.1 M sodium tetraborate and 50 µl of 2-mercaptoethanol to 1 ml of methanol containing 40 mg of o-phthaldialdehyde.The analysis of the derivatized samples was performed using a Perkin Elmer HPLC equipped with a fluorescence detector with the wavelengths used for excitation and emission being found to be 335 nm and 440 nm, respectively, and with an analytical reverse phase column C 18 (150 mm × 4.6 mm in-ternal diameter and 5 µm particle size) connected to a precolumn C 18 (20 mm × 4.6 mm and 5 µm particle size).Methanol and NaH 2 PO 4 0.1 M (75:25) were used as the mobile phase, with the pH being set at 3.35 ± 0.2 with orthophosphoric acid and a flow rate of 1.5 ml/min.The quantification of FB 1 was carried out by comparing the peak areas obtained from the samples with those corresponding to the analytical standards of FB 1 using PROMEC (Program on mycotoxins and experimental carcinogenesis, Tygerberg, Republic of South Africa).

Statistical analysis
The concentration-mortality data were subjected to Probit analysis (Finney, 1971) to obtain the LC 50 and LC 95 values using the SPSS 21.0 software program.In the AChE inhibition, antifungal activity and anti-mycotoxicogenic activity assays, the data were analyzed by one-way analysis of variance (ANOVA).The mean values were compared using Fisher's LSD posteriori test.

ReSUltS And dIScUSSIon
The hydro-distillated EO of L. graveolens from the Dominican Republic was analyzed by GC-MS, identifying thirty-seven different components in the oil (Table 1).According to this analysis, thymol (22.8%), carvacrol (22.7%) and p-cymene (18.8%) were the major volatile compounds of Mexican oregano EO, similar to the results reported by Calvo-Irabién et al. (2014) and Rodriguez-Garcia et al. (2016).
In the present study, the EO from oregano was evaluated for use as an insecticide against S. zeamais adults.Although pure EO obtained from L. graveolens leaves did not show significant mortality of S. zeamais after 24 hours of exposure by fumigation at 600.0 µl/l air (LC 50 > 600.0 µl/l air), p-cymene, a principal compound of oregano EO, presented a higher insecticidal activity and had LC 50 and LC 95 values of 237.9 µl/l air and 390.3 µl/l air, respectively.Similarly, in another study, Lee et al. (2001) found that p-cymene was an important toxic fumigant against the rice weevil (LC 50 =25.0µl/l air).The present results revealed higher LC 50 values, possibly due to differences in the experimental procedure causing effects such as interactions taking place between the EO and corn added in the vials.In contrast with the present findings, some authors have reported that EOs of Lippia species present insecticidal (Gleiser & Zygadlo, 2007;Silva et al., 2008), acaricidal and repellent activities (Ruffinengo et al., 2005;Martinez- et al., 2011;Gomes et al., 2012).These differences in biological activities, with respect to the present study, may have been due to differences in the chemical composition between the EOs of the plant species (Gomes et al., 2012).According to some authors, the main chemical constituents of Mexican oregano EO, thymol and carvacrol, are the ones responsible for insecticide activity (Martinez-Velazquez et al., 2011;Gomes et al., 2012), but in the present study, these compounds did not show insecticidal activity at 300.0 µl/l air (LC 50 > 300 µl/l air).Similarly to the findings of this study, Phillips & Appel (2010) and Yeom et al. (2012) demonstrated that the fumigant toxicities of carvacrol and thymol were lower than those of other compounds, such as a-pinene, b-pinene and limonene against adult German cockroaches.
Inhibition of AChE activity is one of the most important mechanisms of insecticidal action.The compounds that inhibit or inactivate AChE cause acetylcholine to accumulate at the cholinergic site, which brings about continuous stimulation of the cholinergic nerve fibers throughout the central and peripheral nervous system, resulting in paralysis and death (Siramon et al., 2009).In this study, AChE inhibition in insects exposed to Mexican oregano EO was not observed, but enzyme inactivity was important in vitro (IC 50 = 7.38 ± 3.06 mg/ ml).Although carvacrol and thymol did not reveal insecticidal activity, they produced AChE inhibition.The IC 50 of carvacrol could not be determined, but it showed the highest AChE inhibition percentages of all tested concentrations.On the other hand, pcymene showed insecticide activity and also produced AChE inhibition (IC 50 =0.048± 0.015 mg/ml) (Figure 1).Similar results to those of the present study were obtained by Herrera et al. (2015b) using camphor against S. zeamais.It was found that this compound produced a strong inhibitory activity on AChE, but did not show any insecticidal activity, probably because of its inability to reach its target site due to a hindrance caused by its topological and physicochemical properties.Related to this, several authors did not find a direct correlation between insect toxicity and AChE inhibition (Lee et al., 2001;Yeom et al., 2012;Yeom et al., 2013;Herrera et al., 2015b) suggesting that there may be different modes of action of monoterpene toxicity to the insects, and consequently, mortality may be caused by other factors (Martinez-Velazquez et al., 2011).
The antifungal activity of Mexican oregano EO was evaluated against F. verticillioides M 3125, and the 100 and 200 µl/l concentrations of EO were observed to affect the growth and lag phase, while the growth rate showed a significant difference with the control at all the evaluated concentrations with a dose dependent response being found (Table 2).Antimicrobial properties of Mexican oregano have also been reported in other studies, and the present results are in agreement with Portillo-Ruiz et al. (2012), who observed that Lippia berlandieri EO inhibits Aspergillus, Penicillium, and Rhizopus sp.growth and with Mendez et al. (2012) also reporting antibacterial activity of oregano EO against Enterobacter aerogenes, Escherichia coli, Salmonella typhi and Staphylococcus aureus.In the present study, although FB 1 production did not show a significant difference with the control, differences among treatments were detected at concentrations of 200 µl/l of EO (Table 2).
Several authors have attributed the antimicrobial activity of the EOs to their phenolic compound contents (Dambolena et al., 2012;Abbaszadeh et al., 2014;Gallucci et al., 2014).In this sense, previous studies have shown that thymol and carvacrol present antifungal activity against F. verticillioides, thereby reducing the radial growth, fungal sporulation and FB 1 production (Dambolena et al., 2011;Dambolena et al., 2012).Lipophilicity is the main property involved in the antifungal activity of phenolic compounds with this property indicating the possibility that the compounds reach the target site (Dambolena et al., 2012).
Summing up, the present results should encourage research for new active natural compounds in commercial plants, in order to offer an alternative to synthetic pesticides.Mexican oregano EO is an interesting source of compounds which has grain-protectant properties against F. verticillioides and S. zeamais, and should be analyzed in future studies in order to develop new biopesticides to be used in integrated pest management plans of stored grains.Due to the practical use of biopesticides developed from EOs further studies are necessary to elucidate their mode of action, side effects, and formulation development in order to improve their efficacy and stability.Moreover, toxicity and in vivo efficacy studies using formulated products are clearly required, and the economic feasibility of these products developed from EOs has still to be demonstrated.

table 1 .
Chemical composition of essential oil extracted from Lippia graveolens leaves.RI: identification based on Retention indices; GC-MS: identification based on mass spectra; Co: coinjection with standard.The compounds are listed by elution order in the DB-5 column.(*) Limonene, b-phellandrene and 1,8-cineole were also separated on the Carbowax column.Relative contents are expressed as percentages.