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Composition of essential oils extracted from six aromatic plants of Kabylian origin (Algeria) and evaluation of their bioactivity on Callosobruchus maculatus (Fabricius, 1775) (Coleoptera: Bruchidae) 1 1 2 3 1 K. Toudert-Taleb , M. Hedjal-Chebheb , H. Hami , J-F. Debras & A. Kellouche * 1

Faculté des Sciences Biologiques et des Sciences Agronomiques, Université Mouloud Mammeri, 15000 Tizi-Ouzou, Algeria 2 Faculty of Letters and Languages, Mouloud Mammeri University of Tizi-Ouzou, Algeria 3 INRA, UR 1115 Plantes et systèmes de cultures horticoles, F-84000 Avignon, France The essential oils extracted from six plants from Kabylia (Algeria), namely Eucalyptus globulus, Eucalyptus radiata, Myrtus communis, Salvia officinalis, Laurus nobilis and Pistacia lentiscus, were analysed by gas chromatography (GC/MS). Their biological activity was assessed on a pest destructive of stored products, Callosobruchus maculatus on cowpea (Vigna unguicultata). The results showed that these oils have two monoterpene compounds in common: a-pinene and b-pinene in different proportions. The bio-tests were conducted through contact, fumigation and repellency, under laboratory conditions. All the oils tested proved to be active, and the most significant action was the inhibition of oviposition during the tests through contact at a dose of 12 µl/50 g of bean seed. The essential oil of E. globulus caused 50 % mortality in adult C. maculatus at 24 hours at a dose of 4 µl/l of air in fumigation tests. In addition, the essential oils tested were highly repellent towards adult C. maculatus at a dose of 16 µl. Key words: activity, essential oils, cowpea weevil, Vigna unguicultata, Algeria.

INTRODUCTION Cowpea, Vigna unguiculata L., 1753 plays an important role in the lives of people in African countries, including Algeria, and more especially in Kabylia. Unfortunately, this legume is under severe attack during storage of seeds by a destructive pest commonly called the cowpea weevil, Callosobruchus maculatus Fabricius, 1775 (Coleoptera: Bruchidae). Ouedraogo et al. (1996) estimate that more than 800 g/kg are lost after seven months of storage. This insect can destroy 50 to 100 % of the quantity stored and causes a significant decrease in the germination potential of seeds (Elhag 2000). Intensive agriculture imposes constraints that are increasingly burdensome for natural resources, with the disappearance of many plant species and the pollution of the environment through improper use of synthetic chemicals. This has challenged many researchers around the world to attempt to develop sustainable control techniques for proper management of natural resources and the control of populations of different pests, while respecting the environment. Among these techniques, mention can be made of the use *Author for correspondence. E-mail: [email protected]

of plants traditionally used by rural people for their bio-insecticidal properties. The bio-insecticidal activity of many plants against various pests of stored products has been widely reported by many authors (Tripathie et al. 2000; Kellouche et al. 2004; Ayvaz et al. 2010; Nyamador et al. 2010; Karabörklu et al. 2011; Sümer Ercan et al. 2013). Among the products derived from plants, the essential oils could replace conventional fumigants such as methyl bromide after the discovery of its adverse effect on the ozone layer (Elhag 2000). Some aromatic substances extracted from plants have indeed shown insecticidal activity against some species of Bruchidae, such as C. maculatus (Keita et al. 2000; Tapondjou et al. 2002). The biological activity of these natural substances is influenced by their chemical composition and the development stage of the insect pest to be controlled (Tunç et al. 2000; Sahaf et al. 2008). In this study, we investigated the effects of essential oils of six plants from the region of Kabylia (Algeria), using three different techAfrican Entomology 22(2): 417–427 (2014)

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African Entomology Vol. 22, No. 2, 2014

niques: testing by contact, repellency and fumigation.

for about 20 days in order to prevent any prior contamination.

MATERIAL AND METHODS

Bioactivity tests Ten pairs of adult weevils (aged 0 to 24 h) were introduced into glass Petri dishes (14 cm of diameter and 2 cm high) containing 50 g of V. unguiculata seeds treated beforehand with each of the six essential oils at various doses (4, 8, 12 and 16 µl/50 g of V. unguiculata seeds). The evaluation of the bioactivity of these natural substances was carried out using four replicates for each dose and for the control groups (without treatment). The effects of the essential oils, in the contact test, were evaluated using different biological parameters of the weevil such as the number of eggs laid on the V. unguiculata seeds, the hatching rate, and the viability of the eggs. The dead individuals were removed daily from the boxes until the death of all the individuals tested. The counting of eggs laid (hatched and unhatched eggs) on the seeds was done 15 days after being laid. Viable eggs hatched are recognized either by the presence of the visible larva through the chorion, or by their opaque white appearance due to the accumulation of a white powder resulting from the activity of the neonate larva which digging its gallery in the seed. The enumeration of the descendants of the first generation was conducted from the 25th to 45th day after the beginning of the treatment. Thus, adult individuals were removed from the boxes as soon as they emerged from the treated seeds. The effect of the various treatments on the V. unguiculata seeds was evaluated by means of a germination test. The evaluation of the impact of the essential oils was performed on batches of 50 treated seeds and batches of 50 non-treated seeds. They were then germinated in Petri dishes by covering them with cotton wool soaked with water. After 4 days, the seeds germinated in the control batches and those in the treated batches were counted.

Plant material The plant material used in this study included six plant species belonging to four different families. Twenty kilograms of fresh leaves of each plant species were collected in the region of Tizi-Ouzou, some 100 km from Algiers. They were air-dried and protected from light for a week before extraction of essential oils. Analysis of the chemical composition of essential oils The essential oils were extracted from the leaves of the six plants under study by means of the technique of water-steam conduction (Peyron 1992), in the development and research centre of the SAIDAL group (El-Mohamadia, Algiers) in June 2011. The oils obtained were stored in opaque glass flasks, protected from light, at a temperature of 4 °C. For the identification of volatile components of the oils extracted, we used the technique of gas chromatography coupled to mass spectrometry (GC/MS), at INRAP (National Institute for Research and Physicochemical Analyses, Technopole, Sidi Thabet, Tunisia). The GC/MS used was an Agilent, and the injection system is the splitless type. The column length was 30 m, with a diameter of 25 mm and a thickness of 0.25 mm. The initial temperature of 40 °C was maintained for 60 s. It gradually increased at the rate of 2 °C per min to a peak of 240 °C that was maintained for 20 min. The temperature in the injection and the interface was 250 °C, and that of the source is 230 °C. The total ion chromatogram was recorded by means of an electron-impact source, and the kinetic energy of the ions was 70 eV. The standards were identified with spectral masses by the U.S. National Institute of Standards and Technology. Insects The population of C. maculatus used in our tests resulted from mass rearing under laboratory conditions at 28 ± 2 °C and 75 ± 10 % RH inside a dark incubator. Vigna unguiculata seeds, used as spawning substrate for the cowpea weevil, came from a private farm in Tirmitine, a village located in the region of Tizi-Ouzou. The seeds were stored in a refrigerator

Repellency tests Discs of filter paper, 11 cm in diameter, were cut into two equal parts. One of the half-discs was treated with a dose of essential oil diluted in 1 ml of acetone (solvent), and the other part was sprayed with acetone.

Toudert-Taleb et al.: Bioactivity of essential oils on Callosobruchus maculatus (Coleoptera)

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Table 1. Concentrations (%) of volatile organic compounds of the six essential oils from Kabylia (Algeria) analysed.

E. globulus

E. radiata

M. communis

S. officinalis

L. nobilis

Monoterpenes Sesquiterpenes Ketones Diterpenes

40 27.5 2.27 2.5

45.23 23.8 – 2.38

48.14 11.11 – –

66.66 19.04 – –

28.57 32.14 – –

Alcohols Esters Ethers Aldehydes Phenols Phenolic ethers

10 – 2.5 2.5 2.5 –

9.52 – 2.38 4.75 – –

11.11 3.7 3.7 – – 3.7

4.76 – – – 4.75 –

– – – – 3.57 –

Plant species

Compounds

Terpenes

Oxygenate compounds

The box thus prepared was left in the open for 15 min to achieve total evaporation of the solvent. The two half-discs of filter paper were then joined by adhesive tape and deposited in the bottom of Petri dishes. Ten pairs of C. maculatus (0–24 h old) were deposited in the middle of the boxes thus prepared. The doses of oils used, to treat a half-disc, were: 4, 8, 12 and 16 µl. Four replicates were made for each dose. After one hour, a count of weevils present on both parts was conducted. The repellency rate induced by the essential oils against adult weevils was calculated with the formula suggested by Mc Donald et al. (1970): PR (%) = [(Nac – Neo) ¸ (Nac – Neo)] × 100 , where Nac = number of individuals present in the part treated with acetone only, and Neo = number of individuals present in the part treated with essential oil diluted in acetone.

Fumigation tests These tests investigated the effect of essential oils through fumigation on the longevity of adult C. maculatus. In 1-l glass jars, a dose of essential oil was deposited on a piece of Whatman No. 2 filter paper, 2 cm diameter and suspended by a thread on the internal surface of the lid. The dose levels for all six pure oils were: 4, 8, 12 and 16 µl/l of air. In parallel, the control consisted of filter paper without essential oil. Ten pairs of C. maculatus (0–24 h old) were put into each jar, which was then quickly sealed. A count of dead individuals was performed after variable periods of exposure: 24, 48, 72 and 96 h.

Statistical analysis The results of the different tests were subjected to a variance analysis test (ANOVA) according to several criteria of classification, followed by the Newman-Keuls test at 5 %, using the StatBox Version 5 software program, when there was a significant difference among the various treatments. RESULTS Chemical composition of the essential oils The six essential oils extracted are composed of hydrocarbons represented by terpenes, diterpenes and sesquiterpenes as well as oxygenate compounds, such as alcohols, esters, ethers, aldehydes, ketones, phenols and phenolic ethers. Monoterpenes corresponded to the main components of the six natural substances analysed, and the sesquiterpenes were second (Table 1). a-Pinene and b-pinene were the most common monoterpene compounds of the oils extracted. The a-pinene concentrations varied from 2 to 19 % and those of b-pinene from 0.21 to about 3.71 % in the six plant species tested (Table 2). Eucalyptol was the main constituent in E. globulus, E. radiata, M. communis and S. officinalis. Globulol (sesquiterpene) was present at a proportion of 8.65 % in E. globulus while it was absent in E. radiata. The essential oil of S. officinalis was the richest in sabinene with 7.88 % (Table 2). The essential oil of L. nobilis was rich in terpinolene at 13.75 %, while this compound occurred in very low proportions (0.24 %) in E. globulus and E. radiata. Some components were present only in a single

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Table 2. Chemical composition of six essential oils from Kabylia (Algeria) analysed by means of GC/MS. Constituents a-Pinene Camphene b-Pinene b-Myrcene a-Terpene Eucalyptol 1,4-Cyclohexadiene p-Cymene Terpinolene Isopentyl-isovalerate Thujone a-Campholenal a-Gurjunene b-Citral 3-Cyclohexene-1-citral Citral Methylcinnamate Isobutyl isobutanoate 3-Careen Limonene a-Pinene oxide Linalyl butyrate Caryophyllene 3-Cyclohexane-1-ol Mertynal Pulegone a-Caryophyllene Estragol Thujol 1,3-Cyclohexadiene Acetate Geraniolacetate Myrtenol Isobutanoate Geraniol Caryophyllene oxide Eugenol methyl 1H-cycloprop [e] azulene Naphthalene Borneol Naphthalene cis D-fenchyl alcohol Aromadendrene 4-Carbommethanol Alloaromadendrene Viridiflorene a-Terpinene a-Terpineol Cyclohexanol, 2-methylene5-Isopropenyl 2,3-D-hydro-1,8 cineol Palustrol

E. globulus

E. radiata

M. communis

S. officinalis

L. nobilis

P. lentiscus

7.69 0.08 0.39 0.36 0.18 47.05 1.00 3.48 0.24 0.17 0.15 0.15 0.17 – – – – – – – – – – – – – – – – – – 0.29 0.17 – 0.15 – – – – – – 0.23 1.38 0.41 0.42 0.18 3.58 1.93

2.98 – 0.86 1.03 – 66.34 0.98 3.44 0.24 3.64 0.15 0.15 0.11 0.85 0.23 1.11 0.32 – – – – – – – – – – – – – – 0.20 0.17 – 1.04 – – – – – – 0.23 1.34 – 3.01 – 0.21 14.42

18.96 – 0.49 – – 26.2 – 1.33 – – – – – – – – – 0.38 0.14 11.12 0.18 0.26 0.91 0.41 0.18 0.22 0.34 0.42 0.27 0.52 0.36 3.48 1.08 0.15 0.18 0.26 1.57 – – – – – – – – – – 4.84

2.05 4.14 2.67 2.16 – – – – – – – – – – – – – – – – – – – 0.49 – – 2.83 – – – – – – – – 0.67 – – 0.34 1.40 0.46 – 0.20 – – – – –

4.14 0.33 3.71 1.42 – 28.6 – – 13.75 – – – – – – – – – 0.57 1.61 – – – – – – – – – 0.50 – – – – – 0.44 – 0.65 0.37 – – – – – – – – –

2.22 0.24 0.21 – – – – 14.85 – – 0.19 – 0.17 – – – – – – – – – 2.66 3.41 – – 1.08 – – – – – – – – – – – 0.82 – – – 1.88 – – 0.22 – –

0.72 0.32 0.13

0.72 0.25 0.13

– – –

– – –

– – –

– – – Continued on p. 421


421

Table 2 (continued) Constituents Epiglobulol Ledol Globulol g-Gurjunene Eromophilene Spatulenol g-Eudesmol Thymol a-Eudesmol b-Eudesmol Biosol 5-Epi-neointermedeol b-Phellandrene p-Cymol Linalol Terpineol Azulene Thuyene Sabinene 1-Phellandrene g-Terpinene Cis-ocimene Cyclohexene 1-methyl-4 Lepidozene Cis-geranyl acetate b-Bergamotene b-Cadinene Cis-geraniol Caryophyllene oxide Methyl eugenol ether Spathulenol Eugenol Isospathulenol Elemicine Benzyl isopentyl ether a-Phellandrene b-Myrcene 4-Carene D-limonene a-Terpinolene 2-Nonanone Isopentyl hexanoate a-Cubebene Camphor b-Linalool L-bornyl acetate Phellandral Bormylene 2-Carene Piperitone a-Murolene g-Murolene

E. globulus

E. radiata

1.65 0.38 8.65 1.45 0.41 0.63 0.21 1.49 0.58 0.62 1.83 0.16 1.63 1.55 0.55 2.35 – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – –

1.60 0.30 6.13 1.45 0.38 0.25 0.18 0.98 0.60 0.64 1.83 0.13 1.63 1.55 0.53 2.32 – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – –

M. communis – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – –

S. officinalis

L. nobilis

P. lentiscus

– – 0.64 – – – – – – – – – 0.20 1.67 – – – 0.63 7.88 0.13 0.43 0.51 0.27 – – – – – – – – – – – – – – – – – – – – – – – – – – – – –

– – – 0.20 – – – – – 0.21 – – – – – – – – – – 0.92 – – 0.58 0.21 0.27 0.37 0.18 0.44 4.38 2.59 2.59 0.49 0.51 – – – – – – – – – – – – – – – – – –

0.12 0.86 0.75 1.99 0.13 13.09 – – – – – – 5.61 – – – – 0.28 – – 1.65 – – – – – – – – – – – – – 0.05 1.82 1.91 0.85 2.47 0.82 0.11 0.14 0.27 0.08 0.57 0.99 2.39 0.28 1.52 0.28 0.64 0.60 Continued on p. 422

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Table 2 (continued) Constituents d-Cadinene Cuminal a-phellandrene epoxide Calamene p-Cymenol 1,5-Menthadien-7-ol Isoaromadendrene g-cadinene Cuminol a-Cadinol Allospathulenol b-Selinenol Carvacrol Dehydroaromaden drene Lepidozenal Phytol Manool Butyric acide L-4-terpineol Isovaleraldehyde Pentane Phenol

E. globulus – – – – – – – – – – – – – – – – – – – – – –

E. radiata – – – – – – – – – – – – – – – – – – – – – –

plant species, 17 in S. officinalis (eugenol, for example) and 38 in P. lentiscus (D-limonene, camphor, etc.) (Table 2). Bioactivity of the essential oils tested The results showed a significant change in the bioactivity of the essential oils tested by contact, fumigation and repellency against C. maculatus. Effect of the oils on the number of eggs laid on cowpea seeds by C. maculatus The analysis of the variance with two classification criteria showed a highly significant difference for the oil (F = 170.9246, P = 0.000, d.f. = 5), the dose (F = 3537.223, P = 0.000, d.f. = 4), and their interaction (F = 77.2441, P = 0.000, d.f. = 20). The essential oils of L. nobilis and S. officinalis completely inhibited egg-laying of C. maculatus at the lowest dose (4 µl/50 g), that of P. lentiscus is least toxic. At the 12 µl/50 g dose, all the tested essential oils prevented egg laying (Table 3). In untreated plots, the average number of eggs laid by 10 females was 740 ± 43. The reduction of oviposition is inversely proportional to the increase in the oil dose used (Table 3).

M. communis – – – – – – – – – – – – – – – – – – – – – –

S. officinalis – – – – – – – – – – – – – – – – – – – – – –

L. nobilis – – – – – – – – – – – – – – – – – – – – – –

P. lentiscus 1.45 2.37 0.13 0.17 0.29 0.15 0.69 0.42 0.66 0.45 1.03 0.12 2.73 0.17 0.35 0.22 1.43 – – – – –

Effects of essential oils on the hatching rate of eggs in C. maculatus The analysis of variance on two criteria of classification revealed a highly significant difference for oil (F = 866.691, P = 0.000, d.f. = 5), dose (F = 5877.224, P = 0.000, d.f. = 4) and their interaction (F = 330.1008, P = 0.000, d.f. = 20). The results showed a significant reduction in the number of hatched eggs in the treated groups. The most significant effects were recorded with E. globulus and E. radiata essential oils, which inhibited the hatching of some eggs laid on seeds treated with a 4 µl/50 g dose, in comparison to the control groups in which 93.5 % of the eggs hatched (Table 4). Effect of essential oils on the viability rate of eggs laid by C. maculatus The analysis of the variance of two criteria of classification revealed a highly significant difference for the dose factor (F = 835.1902, P = 0.000, d.f. = 4), oil factor (F = 35.550, P = 0.000, d.f. = 5) and for their interaction (F = 14.6512, P = 0.000, d.f. = 20). The viability rate of eggs laid by C. maculatus was higher than 94 % in the controls; this rate decreased


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Table 3. Average fecundity of 10 Callosobruchus maculatus females on Vigna unguiculata seeds treated with the different oils. Doses (µ) 0 4 8 12 16

E. globulus

E. radiata

740.0 ± 43.20 a 1.5 ± 0.57 f 0±0f 0±0f 0±0f

740.0 ± 43.20 a 1.5 ± 0.57 f 0±0f 0±0f 0±0f

M. communis

L. nobilis

740.0 ± 43.20 a 740.0 ± 43.20 a 362.5 ± 9.57 c 0±0g 125.8 ± 57.84 e 0±0f 0±0f 0±0f 0±0f 0±0f

S. officinalis

P. lentiscus

740.0 ± 43.20 a 740.0 ± 43.20 a 0±0f 572.0 ± 66.75 b 0±0f 323.8 ± 27.50 d 0±0f 0±0f 0±0f 0±0f

Means followed by different letters are significantly different (P £ 0.5) according to the Newman-Keuls test.

Table 4. Hatching rate (%) of Callosobruchus maculatus eggs laid on seeds of Vigna unguiculata treated with essential oils. Doses (µ) 0 4 8 12 16

E. globulus

E. radiata

M. communis

L. nobilis

S. officinalis

93.5 ± 2.38 a 0±0e 0±0e 0±0e 0±0e

93.5 ± 2.38 a 0±0e 0±0e 0±0e 0±0e

93.5 ± 2.38 a 87.5 ± 2.88 b 73.8 ± 4.78 c 0±0e 0±0e

93.5 ± 2.38 a 0±0e 0±0e 0±0e 0±0e

93.5 ± 2.38 a 0±0e 0±0e 0±0e 0±0e

P. lentiscus 93.5 ± 2.38 a 85.0 ± 4.08 b 67.5 ± 6.45 c 0±0e 0±0e


progressively as the oil dose increased in the batches treated. No offspring were observed in the groups treated with the essential oils of E. globulus, E. radiata, L. nobilis and S. officinalis, at the 4 µl/50 g dose. The same result was obtained with an 8 µl dose of essential oils in M. communis and P. lentiscus (Table 5). Effect of treatments with the essential oils on the germination faculty of V. unguiculata seeds The analysis of variance on two criteria of classification showed no significant effect of the treatments with the essential oils of E. globulus, E. radiata, L. nobilis and S. officinalis on seed germination of cowpea.

In the treatments with the essential oils of M. communis (4 µl/50 g) and P. lentiscus (all doses), the germination faculty of V. unguiculata seeds was reduced in a highly significant way (less than 34 %) in comparison to seeds from the control group (91.25 %) (Table 6). Fumigation tests The essential oils of E. globulus and S. officinalis proved to be the most toxic after 24 h of exposure to a dose 4 µl/l of air; the adult mortality of C. maculatus reached 50 % and 60 %, respectively. The essential oil of E. globulus induced 100 % mortality in C. maculatus adults after 96 h of exposure to a 4 µl/l dose (Table 7). All the essential oils tested, except that of P.

Table 5. Viability rate (%) of Callosobruchus maculatus hatched eggs in treatments with the different oils tested. Doses (µl) 0 4 8 12 16

E. globulus

E. radiata

M. communis

L. nobilis

S. officinalis

P. lentiscus

94.75 ± 4.11 a 0±0d 0±0d 0±0d 0±0d

94.75 ± 4.11 a 0±0d 0±0d 0±0d 0±0d

94.75 ± 4,11 a 68.75 ± 6.29 b 33.75± 2.5 c 0±0d 0±0d

94.75 ± 4.11 a 0±0d 0± 0 d 0±0d 0±0d

94.75 ± 4.11 a 0±0d 0±0d 0±0d 0±0d

94.75 ± 4.11 a 71.25 ± 6.29 b 30 ± 4.08 c 0±0d 0±0d


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Table 6. Germination faculty rate of Vigna unguiculata seeds (%) treated with the different oils tested. Doses (µl) 0 4 8 12 16

E. globulus

E. radiata

M. communis

91.25 ± 8.53 a 95 ± 4.08 a 100 ± 0 a 100 ± 0 a 100 ± 0 a

91.25 ± 8.53 a 95 ± 4.08 a 100 ± 0 a 100 ± 0 a 100 ± 0 a

91.25 ± 8.53 a 31.25 ± 6.29 b 100 ± 0 a 100 ± 0 a 100 ± 0 a

L. nobilis 91.25 ± 8.53 a 100 ± 0 a 100 ± 0 a 100 ± 0 a 100 ± 0 a

S. officinalis

P. lentiscus

91.25 ± 8.53 a 100 ± 0 a 100 ± 0 a 100 ± 0 a 100 ± 0 a

91.25 ± 8.53 a 31.25 ± 6.29 b 31.25 ± 6.29 b 33.75 ± 4.78 b 33.75 ± 4.78 b


lentiscus, caused 100 % mortality after 24 h exposure to the highest dose (16 µl/l). To achieve the same result with the essential oil of P. lentiscus, adults had to be exposed to the same dose for 48 h. In the controls, there was no mortality even after 96 h (Table 7). Repellency tests All the essential oils showed a highly significant repellent effect towards C. maculatus adults. E. globulus, E. radiata and S. officinalis proved to be the most repellent reaching a rate of 100 % with a 12 µl dose. This rate was obtained with all of the oils at the highest dose tested (16 µl) (Table 8).

tatively, the L. nobilis essential oils from three North African countries (Algeria, Morocco and Tunisia) have similar chemical compositions and t h a t t h e m a j o r c o mmo n c o m p o u n d s a r e 1.8-cineole, linalool and isovaleraldehyde. They Table 7. Mortality rate (%) of adult Callosobruchus maculatus (inhalation test) with six essential oils. Plant species

Exposure time (h) 24

48

72

96

E. globulus

0 4 8 12 16

0 50 70 90 100

0 92 100 100

0 100 – –

0 – – –

E. radiata

0 4 8 12 16

0 42 68 85 100

0 71 100 100 –

0 100 – – –

0 – – – –

M. communis

0 4 8 12 16

0 35 45 70 100

0 74 100 100 –

0 100 – – –

0 – – – –

L. nobilis

0 4 8 12 16

0 75 90 100 100

0 100 100 – –

0 – – – –

0 – – – –

S. officinalis

0 4 8 12 16

0 60 75 87 100

0 90 100 100 –

0 100 – – –

0 – – – –

P. lentiscus

0 4 8 12 16

0 30 60 77 90

0 45 90 94 100

0 60 97 98 –

0 70 100 100 –

DISCUSSION The present study showed that the six essential oils characterized by GC/MS differ from one another in their composition, even though some compounds are present in one or the other species, whereas some are specific to a given species. The P. lentiscus essential oil of Kabylian origin proved to be particularly rich in monoterpenic compounds at a proportion of 43.33 %, followed by sesquiterpenes with 16.16 %. Our result was quite different from that of Benyoucef et al. (2005), who analysed by GC/MS some P. lentiscus essential oils from two different Algerian localities: Baïnem forest in the region of Algiers and El Kala, a region located some 780 km east of Algiers. These authors observed the predominance of sesquiterpenic compounds in contrast to some monoterpenes. This result confirms once again that the geographical origin and the environment in which a plant grows have a considerable influence on the chemical composition of its essential oil (Marotti et al. 1994). The composition of the L. nobilis essential oil is close to those analysed by Mediouni-Ben Jamâa et al. (2012).These researchers maintain that, quali-

Doses (µl/l of air)


425

Table 8. Average repellency rate (%) (McDonald et al. 1970) of the different essential oils from the region of Kabylia (Algeria), towards Callosobruchus maculatus adults. Doses (µl) 4 8 12 16

E. globulus

E. radiata

53.75 ± 4.78 e,f 53.75 ± 4.78 e,f 67.5 ± 2.88 c,d 67.5 ± 2.88 c,d 100 ± 0 a 100 ± 0 a 100 ± 0 a 100 ± 0 a

Repellency rate %)

81.31

Repellency V class (very repellent)

M. communis

L. nobilis

56.25 ± 4,78 e,f 50 ± 4.08 f 70.25 ± 2.5 b,e 62.5 ± 15 c,d,e 72.5 ± 8.66 b,c 78.75 ± 6.29 b 100 ± 0 a 100 ± 0 a

S. officinalis 50 ± 4.08 f 78.74 ± 8.16 b,c,d 100 ± 0 a 100 ± 0 a

P. lentiscus 55 ± 7.07 e,f 61.25 ± 2.5 d,e 71.25 ± 4.78 b,c,d 100 ± 0 a

81.31

74.75

72.75

82.18

71.87

V (very repellent)

IV (repellent)

IV (repellent)

V (very repellent)

IV (repellent)


also noted that some compounds have been detected only in the one or the other of the three oils considered. Accordingly, 2-careen (5.62 %), 4-terpineol (1.52 %) and 1-bornyl acetate (0.52 %) are characteristic of the Moroccan oil only, while pentane (2.14 %), phenol (1.73 %) and terpinene (0.92 %) are found in the Algerian oil. The Tunisian oil is characterized by the presence of camphor (2.66 %), terpinene 1-ol (1.7 %), 2-norbornanone (1.20 %) and eremophil (0.67 %). The authors concluded that geographical location (country) has a direct bearing on the chemical composition of these essential oils. The six essential oils analysed in the present study are mainly composed of monoterpenes, such as a-pinene, b-pinene, camphene, limonene, p-cymene and terpinolene; these compounds have often been characterized by their biocidal and repellent activity against numerous insects destructive to stored commodities (Keita et al. 2000; Ketoh et al. 2002; Papachristos & Stamoupolos 2002; Kellouche et al. 2004; Kellouche et al. 2010). The results obtained in our study reveal that all of the six oils tested are bioactive towards the cowpea-weevil in the contact tests by affecting all of its biological parameters (fertility, embryonic and post-embryonic viability of laid eggs). The results obtained in the contact-mediated treatments confirm those reported by Hedjal et al. (2013), who highlighted a biocidal effect of the essential oil of six conifers from Algeria and Tunisia, on the number of laid eggs and on the rate of viability of these eggs in C. maculatus. The reduced emergence of adults recorded in our tests could be partly understandable by the

low hatching rate of laid eggs. It is likely that the vapours of essential oils spread through the egg chorions affect the physiological and biochemical processes related to embryonic development (Raja et al. 2001). Similarly, Regnault-Roger & Hamraoui (1995) observed the effect of binalool, thymol and carvacrol on the fertility of the weevil Acanthoscelides obtectus (Say, 1831) attacking bean. Among the six essential oils tested, only that of P. lentiscus affected the germination of V. unguiculata seeds (33 %). Our result could be explained by considering the fact that numerous botanical species synthesize and release to the environment molecules, such as essential oils, capable of influencing the seeding and growth of plants developing; this is commonly called ‘allelopathy’ (Macias et al. 1999). In our study, the P. lentiscus essential oil may have been able to affect the basic mechanisms of seed-membrane permeability, breathing and seeding by means of the monoterpenic alcohols or phenols which it contains. The effects of these compounds can be either additive or synergic (Blum 1999). In this respect, Rice (1984) admits that the term ‘allelopathy’ is generally accepted to cover at the same time the effects of stimulation and inhibition of a plant on another. In addition, the natural substances we have tested in the present study have had a toxic effect by fumigation on C. maculatus adults. They have all caused the death of the weevils, after a 96-h exposure with a 16 µl/l dose. A ‘knock-down’ effect has been observed with the E. globulus, E. radiata and L. nobilis essential oil. It was evidenced by an immediate immobility of the adult weevils as soon as they came into contact with the vapours of these oils in hermetically sealed jars. This demonstrates the efficiency of these molecules by fumigation,

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which could be the consequence of their richness in monoterpenic compounds. It may be noted that our results match those of Regnault-Roger & Hamraoui (1995), who observed a toxic effect of monoterpenes by fumigation on the weevil A. obtectus. The same observation is true with regard to Tapondjou et al. (2002), who have shown that crushed plant material and a Chenopodium essential oil were able to reduce from 80 to 100 % the densities of the populations of Callosobruchus chinensis (Linnaeus, 1758), A. obtectus, Sitophilus granarius (Schönherr, 1838) and Prostephanus Truncatus (Horn, 1878), 24 h after the application of a 0.2 µl/cm2 dose. The repellent properties of essential oils are often associated with the presence of monoterpenes and sesquiterpenes, though the toxic effects depend on the insect species, on the plant and on the exposure time to the essential oil (Kim et al. 2002). Our results are similar to those of Shaaya et al. (1991), who tested essential oils extracted from some Labiates, such as oregano, basil, marjoram, thyme, sage, rosemary and lavender; these substances caused 100 % of mortality in Rhyzopertha dominica (Fabricius, 1792) (Coleoptera: Bostrychidae), Oryzaephilus surinamensis (Linnaeus, 1758) (Coleoptera: Sylvanidae), Tribolium castaneum (Herbst, 1797) (Coleoptera: Tenebrionidae) and Sitophilus oryzae (Linnaeus, 1763) (Coleoptera: Curculionidae), with doses varying from 10 to 15 µl/l of air. With the dose 16 µl/l of air, all of the six oils tested showed a total repellent activity (100 %) towards C. maculatus adults. Although the mechanisms of action of the oils on devastating pests are not well understood, it has been suggested that octopamine, a biogenetic structurally close to the norepinephrine of vertebrates, acts as a neurotransmitter in invertebrates. The octopamine has a regulating effect on the

heartbeat, motricity, ventilation, flight, and metabolism of invertebrates (Roeder 1999). Essential oils would act by settling on the octopamine receptors that constitute ideal targets for bio-pesticides. They can also reduce the palatability and induce growth inhibition, through altered protein availability, enzyme inhibition and direct toxicity (Harborne 1993). Enan (2001) has shown that the application of eugenol, a-terpineol and cinnamic alcohol, can hinder the receptor sites of octopamine in Stomoxys calcitrans (Linnaeus, 1758) (Diptera: Muscidae). Similarly, Garcia et al. (2005) suggest the hydroxyl groupings contained in a-terpineol, pulegol and germacrol, are mainly responsible for the biocidal activity of these composites towards T. castaneum. These hydroxyl groupings are characterized by an electrostatic specificity which allows them to generate repellent receptors/substance interactions and the induction of the bioactivity of essential oils; this is most often evidenced by an action on the physiology of insects by inducing anti-appetizing effects, thus affecting the growth, fertility and development of insects. The essential oils of E. globulus, E. radiata, L. nobilis and S. officinalis, have proved to be the most toxic substances towards C. maculatus, and this has been observed in the three tests conducted (contact, fumigation and repellency). Taking into account the interesting results obtained in the present study and those reported by numerous authors worldwide, one may conclude that the use of plants and their extracts could become a reliable alternative to the use of chemical pesticides in an attempt to reduce the important losses caused to cereal and leguminous harvests, in developing countries, by major pest insects such as R. dominica, S. oryzae, S. granarius, A. obtectus and C. maculatus.

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