Insecticidal activity of two formulations of essential oils against the cereal leaf beetle

ABSTRACT

Purpose: Plant-derived essential oils and their encapsulations have recently received increasing interest as an alternative to synthetic biopesticides suitable for integrated pest management and organic farming. In this study, combinations of essential oils (EO) from Rosmarinus officinalis with Cymbopogon citratus and Pelargonium graveolens with Thymus vulgaris in two formulations including encapsulate were tested for their potential for wheat protection against adults and larvae of Oulema melanopus (L.).

Materials and methods: The chemical composition of the essential oils was analyzed by gas chromatography-mass spectrometry (GC-MS). Testing of insecticidal activity of the evaluated substances was conducted with 2.5 ml of tested combinations of EOs sprayed on the paper. Ten adults or larvae of O. melanopus were placed into a vessel. Control of mortality of O. melanopus specimens was assessed after 24 h after establishment of the experiment.

Results and conclusions: Tarsal contact toxicity assay showed the effectiveness of EO in concentration against O. melanopus larvae and adults in both formulations (essential oil and encapsulation) causing 100% mortality within 24 h. These results indicate the great potential of these essential oils and their encapsulations for future use in crop protection against insect pests.

KEYWORDS:

• Biopesticides
• crop protection
• encapsulation
• organic agriculture
• Triticum aestivum

Introduction

The cereal leaf beetle, Oulema melanopus (L.) (Coleoptera: Chrysomelidae), is a well-known and important pest of cereals throughout its geographical distribution (Bezděk and Baselga 2015). This pest is native to Europe and Asia (Kostov 2001), and in 1962, it was found in North America in the state of Michigan (Dysart et al. 1973). This species is univoltine and active in the field, with a peak oviposition period from late May to mid-June (Kher et al. 2011). A female is able to lay between 50 and 275 eggs on the upper surfaces of leaves, either singly or in clusters (Miller 1956; Schmitt 1988; Piesik and Piesik 1998). Larvae have a distinct fecal shield on the dorsum and have four instars that feed on adaxial leaf surfaces (Smith et al. 1971). Larvae are pupated in the soil (Dysart et al. 1973). The new-generation adults emerge in approximately three weeks and feed on various monocotyledonous plants before overwintering (Grant and Patrick 1993; Kher et al. 2011).

Adults and larvae of O. melanopus attack many wild but primarily domesticated members of the family Poaceae (Gutierrez et al. 1974). Wilson and Shade (1966) divided the possible host plants within the Poaceae into two categories: (1) the most preferred host plants, including wheat (Triticum aestivum L.), oat (Avena sativa L.) and barley (Hordeum vulgare L.), and (2) acceptable but less-preferred plants, such as corn or grasses like Sudan grass (Sorghum × drummondi) and green foxtail (Setaria italica). It is, however, well known that these host preferences of O. melanopus are variable in different eco-regions (Wilson and Shade 1966; Philips et al. 2011). Both adult and larval stages of this pest feed on leaves, but the majority of damage on cereals is caused by larvae (Grant and Patrick 1993; Buntin, et al. 2004). The larvae of the final instar cause significant yield losses (ca. 75% of the total quantity of damage throughout larval development) (Zarubova et al. 2015).

Recently, a large group of potential biopesticides, primarily including essential oils, have been studied for their wide spectrum of biological effects, including various pest controlling and repellent properties (Regnault-Roger et al. 2012). Some of the oils exhibit certain degrees of phytotoxicity, usually inhibiting seed germination and root and seedling growth, and thus precluding their utilisation as pest control agents; however, many other plant substances have no or very low phytotoxicity (Singh and Upadhyay 1993), suggesting their potential for direct application against pests and diseases (Benelli et al. 2017; Pavela et al. 2017; Skuhrovec et al. 2017). There is only one recent report on the use of essential oil against the serious cereal crop pest O. melanopus. Zarubova et al. (2015) found that the Citrus sinensis essential oil was not active against O. melanopus adults but exerted an interesting larvicidal effect.

In this study, we tested the effect of combinations of essential oils (EO) from Rosmarinus officinalis with oil from Cymbopogon citratus on adults and Pelargonium graveolens with Thymus vulgaris on larvae of O. melanopus under laboratory conditions. Conventional formulations of EO were tested, as well as formulations utilising encapsulation of EO into microparticles. This will be the first report on the acute toxicity of EO from R. officinalis with oil from C. citratus on the adults as well as P. graveolens with T. vulgaris to the larvae of this important cereal pest using tarsal tests.

Material and methods

Insects

Adults and larvae of O. melanopus (Coleoptera: Chrysomelidae) used in experiments were obtained by sweeping in field with wheat growth in anthesis stage localised in Praha Ruzyně (N 50.0878500, E 14.2946111). All specimens were reared in fabric cages (60 × 60 × 90 cm; Entosphinx, Czech Republic) together with wheat plants. Cages were housed in a greenhouse at 21 ± 5°C, a relative humidity of 40–60%, and a 16:8 (L:D) photoperiod, and plants inside were irrigated regularly. Adults and larvae needed for experiments were directly withdrawn from cages.

The cereal leaf beetle belongs to the taxonomically difficult O. melanopus group, with five sibling species: O. melanopus, O. duftschmidi (Redtenbacher), O. rufocyanea (Suffrian), O. mauroi Bezděk and Baselga, and O. verae Bezděk and Baselga (Bezděk and Baselga 2015). The first author of the taxonomic revision studied a large quantity of material for two years around the location used in this project and found only adult specimens of O. melanopus.

Chemicals

Combinations of essential oils (EO) from Rosmarinus officinalis with oil from Cymbopogon citratus and from Pelargonium graveolens with Thymus vulgaris were used in our trials. Plant oils were chosen according to authors’ previous unpublished results. Conventional formulations of EO were prepared in the following way: the essential oils (Saloos, Czech Republic) were diluted with commercially available rapeseed oil in a 1:1 ratio, and a 40% water solution of Tween 20 (Sigma Aldrich) was used for final dilution. Tested concentrations of 0.12; 0.204; 0.36; 0.58 and 1% were prepared in this manner. Concentration setup was based on the highest non-phytotoxic concentration (1%, unpublished results) and with regards to the encapsulate application, which proved to be difficult at concentrations over 1%.

Biopolymer set up

A total of 8 g of gelatine (Sigma) was diluted in 200 ml of distilled water and 2 g of chitosan (Sigma) in 60 ml of 0.15 M acetic acid (Lachner). Resulting solutions were mixed together; 1.2 ml of glycerol (Lachner) and 1.2 g of D-Sorbitol (Sigma) were subsequently added, and the resulting solution was mixed thoroughly. All dilutions were conducted at 45°C, and solutions were mixed continuously.

Encapsulate formulation

A total of 15 ml of 10% plant EO (Saloos) diluted in rapeseed oil was poured into a 500 ml beaker. Then, 300 µl of Tween®80 (Sigma) was added, and the resulting solution was emulsified with the use of YELLOW^LINE DI 25 basic homogenizer (IKA) at 9500 rpm. Afterwards, 20 ml of biopolymer was added, and the solution was emulsified using IKA homogenizer at 9500 rpm. Then, 114.7 ml of 0.5% sodium tripolyphosphate (Sigma) solution was prepared in another beaker, and the contents of the first beaker were slowly poured into it. The mixture was emulsified at 9500 rpm during the process. The same concentrations as in the previous case study were used in trials. Encapsulate formulation was designated EN.

Tarsal experiment

Tarsal experiments were placed in transparent plastic vessels (11 × 9 × 5 cm). Wet filter paper (11 × 9 cm) was placed on the bottom of each vessel, and 2.5 ml of tested combinations of EO from R. officinalis with oil from C. citratus (on adults) and P. graveolens with T. vulgaris (on larvae) was sprayed on the paper using an air brush spraying pistol. All experiments were done in the laboratory at 21 ± 1°C, a relative humidity of 40%–60%, and a 16:8 (L:D) photoperiod. Five replications per concentration were tested with 10 adults in each box, and one replication per concentration was tested with 10 larvae in each box. However, five replications were tested with the lowest concentration of 0.12%. Ten adults or larvae of O. melanopus were placed into a vessel that was covered by nylon mesh fabric afterwards. Control of mortality of O. melanopus specimens was assessed after 24 h after establishment of the experiment.

Chemical analysis of essential oils composition

The components of EO were identified by gas chromatography-mass spectrometry (GC-MS) using an Agilent 7890A GC coupled to an Agilent 5975C single-quadrupole mass detector whereas their content was measured using Agilent 7890A GC with flame ionisation detector (GC-FID). Both chromatographs were equipped with an Agilent HP-5MS capillary column (30 m × 0.25 mm, 0.25 µm film) (Agilent, Santa Clara, CA, USA). The samples diluted at a ratio of 5:100 in hexane were injected at volumes of 1 µl in splitless mode into the injector and heated to 250°C. The oven programme started at 60°C for 3 min; then the temperature was raised to 231°C at a rate of 3°C min⁻¹ and kept constant for 10 min. Helium was used as a carrier gas, with a flow rate of 1 ml min⁻¹. GC-MS analysis was carried out in full-scan mode, and the electron ionisation energy was set at 70 eV. The EO constituents were identified by comparing their mass spectra and Kovats retention indices with the National Institute of Standards and Technology Library (NIST, USA) as well as with literature (Adams 2007). The identification of 29 compounds was further confirmed by comparison with authentic standards (indicated by ^b index in Table 1; all obtained from Sigma-Aldrich, CZ). The calculation of percentage compositions was based on the peak areas without using correction factors.

Table 1. Essential oils percentage composition, as identified by GC–MS and quantified by GC-FID.

RI^a	Compound	Cymbopogon citratus	Pelargonium graveolens	Rosmarinus officinalis	Thymus vulgaris
926	Tricyclene	0.16	–	–	–
931	α-Thujene	–	–	0.37	0.35
939	α-Pinene^b	0.35	0.66	10.27	1.27
953	Camphene	0.74	–	4.53	1.51
978	1-Octen-3-ol	–	–	–	0.88
980	β-Pinene^b	–	–	7.03	–
987	6-methyl-5-Hepten-2-one	1.49	–	–	–
991	Myrcene^b	–	–	0.92	1.34
1011	3-Carene^b	–	–	0.29	–
1018	α-Terpinene^b	–	–	–	1.09
1026	p-Cymene^b	–	–	2.81	16.40
1031	Limonene^b	3.87	–	–	0.33
1033	Eucalyptol^b	–	–	43.35	1.29
1040	cis-β-Ocimene	0.55	–	–	–
1050	trans-β-Ocimene	0.40	–	–	–
1062	γ-Terpinene^b	–	–	–	5.74
1098	Linalool^b	0.72	5.22	0.77	4.68
1111	cis-Rose oxide^b	–	1.57	–	–
1127	trans-Rose oxide^b	–	0.60	–	–
1143	Camphor^b	–	–	14.01	1.87
1151	Menthone^b	–	2.35	–	–
1153	β-Citronellal^b	0.85	–	–	–
1164	Isomenthone	–	6.34	–	–
1165	Borneol^b	1.13	–	4.88	1.76
1177	Terpinen-4-ol	–	–	0.94	1.78
1177	Verbenol^t	0.89	–	–	–
1189	α-Terpineol	0.30	–	2.20	0.23
1204	Decanal	0.37	–	–	–
1228	Citronellol^b	–	32.56	–
1235	Thymol methyl ether	–	–	–	0.58
1240	Neral	29.59	–	–	–
1244	Carvacrol methyl ether	–	–	–	0.92
1255	Geraniol^b	3.94	14.92	–	–
1270	Geranial^b	39.68	0.78	–	–
1279	Citronellyl formate	–	8.86	–	–
1285	Bornyl acetate^b	0.33	–	1.94
1290	Thymol^b	–	–	–	48.07
1298	Carvacrol^b	–	–	–	2.75
1300	Geranyl formate	–	3.59	–	–
1356	Eugenol^b	0.31	–	–	0.23
1376	α-Copaene	0.24	0.79	0.29	–
1384	β-Bourbonene	–	2.26	–	–
1385	Geranyl acetate^b	5.41	–	–	–
1391	β-Elemene	0.27	–	–	–
1404	Methyleugenol	0.30	–	–	–
1418	β-Caryophyllene^b	2.88	1.81	3.85	5.91
1444	Citronellyl propionate^b	–	0.58	–	–
1447	trans-Isoeugenol	0.34	–	–	–
1454	α-Caryophyllene^b	0.31	–	0.47	–
1475	Geranyl propionate	–	1.06	–	–
1480	Germacrene D	–	1.47	–	–
1524	δ-Cadinene	0.46	2.18	–	0.26
1529	Citronellyl butyrate	–	0.64	–	–
1549	Elemol	0.30	–	–	–
1564	Geraniol butyrate	–	1.36	–	–
1581	Caryophyllene oxide^b	0.85	–	1.07	0.33
1584	Phenethyl tiglate	–	1.24	–	–
1630	γ-Eudesmol	–	6.87	–	–
1700	Geranyl tiglate	–	1.65	–	–
	Total identified compounds	97.06	99.37	100.00	99.59

^aRI: identification based on Kovat's retention indices (HP-5MS capillary column) and mass spectra.

^bIdentification confirmed by co-injection of authentic standard.

^tTentative identification.

The chemical standards used for the identification of EO components were purchased from Sigma–Aldrich (Prague, Czech Republic), and hexane (Merck, Prague, Czech Republic) was used as a solvent for chemical analysis.

Statistical analysis

Acquired data of Oulema mortality was corrected by transformation according to Abbot with regard to mortality in untreated control variants, and LC₅₀ and LC₉₀ values were established with the use of probit analysis (Skuhrovec et al. 2017) if possible.

Results

Essential oil composition

The volatile components of EO analyzed by GC-MS are listed in Table 1. A total of 28 different compounds were identified in the case of EO from Cymbopogon citratus, representing 97.06% of total peak areas. Geranial was the most prevailing component of this oil, followed by neral. Twenty two compounds were identified in EO from Pelargonium graveolens (99.37% of the total peak areas), with primary components of citronellol and geraniol. A total of 13 components were identified in the case of EO from Rosmarinus officinalis (100% of the total peak areas), with major compounds including eucalyptol, camphor and α-pinene. Finally, 22 compounds were identified in EO from Thymus vulgaris (99.59% of the total peak areas), with thymol and p-cymene as major components.

The effect of essential oil on O. melanopus adults and larvae

In the case of adult specimens, the slightly higher mortality was observed in variants treated with plant EO formulated conventionally using rapeseed oil and Tween 20, but results achieved with EN were almost identical (Table 2). Low mortality in control variants enabled researchers to establish LC₅₀ and LC₉₀ values for both tested formulations; these values were 5105 and 11,220 ppm, respectively, for standard formulations, as well as 4570 and 11,482 for encapsulate.

Table 2. Mortality of Oulema melanopus adults after tarsal experiment with the essential oils from Rosmarinus officinalis with Cymbopogon citratus.

	Mortality (after 24 h) (%)
Applied concentration (%)	Essential oil	Encapsulation
0.12	0	0
0.204	8	14
0.36	30	32
0.58	46	66
1	90	86
Untreated control	0.1

Considering results obtained from larvae, the first set of experiments was negatively influenced by high mortality in control variants, which ranged between 50% and 60%. Despite this negative effect, 90%–100% mortality was observed in concentrations from 0.36% to 1% (Table 3). The tarsal experiment with larvae was repeated with five replicates and a concentration of only 0.12% for confirmation of the previous results. In this case, the larvae mortality in the control variant was substantially lower, with an average of 28%, but was still high to calculate LC₅₀ and LC₉₀ values reliably (Table 4).

Table 3. Mortality of Oulema melanopus larvae after tarsal experiment in five concentrations with the essential oils from Pelargonium graveolens with Thymus vulgaris.

	Mortality (after 24 h) (%)
Applied concentration (%)	Essential oil	Encapsulation
0.12	50	70
0.204	90	80
0.36	100	80
0.58	100	90
1	100	90
Untreated control	60

Table 4. Mortality of Oulema melanopus larvae after tarsal experiment in one low concentration with the essential oils from Pelargonium graveolens with Thymus vulgaris.

	Mortality (after 24 h) (%)
Applied concentration (%)	Essential oil	Encapsulation
0.12	48	46
Control (water)	28
Control (rapeseed oil and Tween 20)	52

Discussion

EO application represents the new strategy of integrated pest management aimed at decreasing traditional chemical plant protection with regards to food quality and safety, and there have been many successful recent examples (e.g. Bakkali et al. 2008; El Asbahani et al. 2015; Pavela and Benelli 2016). We examined the combinations of EO from R. officinalis with oil from C. citratus and from P. graveolens with T. vulgaris for the use against one of the most serious insect pest of wheat, O. melanopus, under laboratory conditions. Until now, there has been only one study about the effect of EO on the cereal leaf beetle: Zarubova et al. (2015) studied the effect of the Citrus sinensis EO on O. melanopus. The direct contact toxicity assay showed no effect of the EO on the O. melanopus adults, but it showed great larvicidal potential, with 42.5% larvae mortality observed 24 h after topical application.

The EO composition obtained by GC-MS shown in Table 1 is basically in accordance with the literature. Essential oil from Thymus vulgaris contained thymol as a major compound, but its relative quantity was higher than that reported by Manou et al. (1998) and Šegvić Klarić et al. (2007), and the quantity of the second major compound, p-cymene, was substantially lower. Most recent attention to the use of EO has focused on Thymus vulgaris, which demonstrated larvicidal (Pavela et al. 2009) and repellent (Park et al. 2005) effects on mosquitoes. Its toxic effect was also demonstrated on the Coleoptera pest Acanthoscelides obtectus (Regnault-Roger and Hamraoui 1994). Higher contents of the major compound eucalyptol (1,8-Cineole) as shown in Table 1 was detected in EO from Rosmarinus officinalis when compared with results obtained by Miresmailli et al. (2006) and Wang et al. (2012). Ovicidal activity of EO from Rosmarinus officinalis was previously detected on stored-product insects (Tunc et al. 2000). Possible synergistic effects of several chemical constituents of EO from Rosmarinus officinalis were also described in the armyworm Pseudaletia unipuncta and the cabbage looper Trichoplusia ni (Isman et al. 2008), as well as in Lymantria dispar with Rosmarinus oil delivered in microcapsules (Moretti et al. 2002).

The composition of EO from Pelargonium graveolens presented in our study (Table 1) is in accordance with results of Fayed (2009), with the highest amount of citronellol, though a different composition with half the volume of this compound was recently published by Boukhris et al. (2012). With regards to EO from Cymbopogon citratus, our results shown in Table 1, which include the major compounds geranial (citral α) and neral (citral β), correspond well to the composition of this oil described by Shah et al. (2011) and Tyagi and Malik (2010). Substantially less research has been conducted on EO from Cymbopogon citratus. However, in the case of this plant species, biological activity of its EO was described in Tribolium castaneum (Olivero-Verbel et al. 2010) and Anopheles arabiensis (Karunamoorthi and Ilango 2010), whereas the insecticidal effects of EO from Pelargonium graveolens were detected on the sweet potato whitefly Bemisia tabaci (Baldin et al. 2015) along with repellent effects detected on mosquitoes (Barnard and Xue 2004; Amer and Mehlhorn 2006).

Our results of tests of the combination of EO and EN from P. graveolens with T. vulgaris on the mortality of larvae have been negatively influenced by high mortality in the control group – almost up to 60% as shown in Table 3. This negative effect could be partially caused by the larvae dehydration and surface distortion, as was described by Zarubova et al. (2015) or observed by scanning electron microscopy on housefly larvae (Kumar et al. 2012). In the second experiment, there were distinct differences in mortality between two variants of control (see Table 4): (1) water and (2) rapeseed oil and Tween 20, which are used for dilution of EO. The mortality of larvae in rapeseed oil and Tween 20 was identical to the combinations that used EO, and twice as high as that which used water. Therefore, the mortality of larvae in low concentrations of EO may also be affected by mechanical obstructions of their tracheal systems by rapeseed oil. The difference between EO and EN was not observed. The effect of EN should be observed for a longer time (El Asbahani et al. 2015), but this was not the main purpose of this study.

In the case of adult specimens, the results as shown in Table 2 were distinctly more positive due to low mortality in the control and to the possibility of establishing LC₅₀ and LC₉₀ values for both tested formulations (EO: LC₅₀ = 5105 ppm and LC₉₀ = 11,220 ppm; EN: LC₅₀ = 4570 and LC₉₀ = 11,482). The harmfulness of adults on crops is distinctly smaller than that of larvae, and treatment against adults is often not recommended (Zarubova et al. 2015), but a suitable treatment against females before oviposition should be very useful for reducing the larval population within cereal growth. The results in both formulations show relatively high acute mortality in low concentrations (Table 1). In the case of EN with long-lasting effects, this should be a very effective combination against the adults in dispersal time, when new adult insects arrive every day.

This is the first report on the use of EO and EN against the serious cereal crop pest O. melanopus, and the tested formulations were effective against both active stages of this important pest. Future research should study the persistence of EO and EN in treated plants, as well as the chronic mortality of both stages in low concentrations. The larvicidal activity of rapeseed oil and Tween 20 should also be studied. Essential oils are a promising and effective alternative to synthetic pesticides and appear to be suitable for use in integrated pest management and organic farming.

Disclosure statement

No potential conflict of interest was reported by the authors.

Notes on contributors

Jiří Skuhrovec works as researcher/entomologist at the Crop Research Institute, Prague (Czech Republic). His studies concern biological control, invasion ecology, ecological entomology (temperature effects on development), biology and morphology of immature insect stages and taxonomy of Curculionidae.

Ondřej Douda works at the Crop Research Institute Prague (Czech Republic). His research interests cover alternative methods of crop protection against invertebrate pests including research and development of new diagnostic tools.

Miloslav Zouhar is associated professor at the Department of Plant Protection at the Czech University of Life Sciences Prague. His research work focuses on all aspects of plant protection but his attention was dedicated especially to the development of alternative crop protection against plant pests or diseases.

Marie Maňasová is a researcher at the Department of Plant Protection at the Czech University of Life Sciences Prague. Her research interests cover alternative methods of crop protection against invertebrate pests and fungi pathogens including research and development of new diagnostic technique.

Pavel Nový is senior researcher at the Department of Quality of Agricultural Products at CULS. His research is dedicated to plant essential oils, their analyses, development and evaluation of bioactivity assays.

Matěj Božik is a PhD candidate at the Czech University of Life Sciences Prague (CULS). His research is focused on plant antimicrobials and their use as food preservatives, including analysis of composition and residues detection.

Pavel Klouček is associated professor and head of the Department of Quality of Agricultural Products at the Czech University of Life Sciences Prague (CULS). His main research interests are natural compounds – their bioactivity, chemistry and their use in agricultural and food industry.

Additional information

Funding

This work was supported by the Ministry of Agriculture of the Czech Republic [grant numbers QJ1310226 and RO0416] and by the Czech University of Life Sciences [grant number 20162004].