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Neotropical Entomology

Print version ISSN 1519-566X

Neotrop. entomol. vol.40 no.3 Londrina May/June 2011 



Toxicity of ethanolic extracts from Lippia origanoides and Gliricidia sepium to Tetranychus cinnabarinus (Boisduval) (Acari: Tetranychidae)



A SiviraI; ME SanabriaII; N ValeraI; C VásquezI

IDepto de Ciencias Biológicas. Univ Centroccidental Lisandro Alvarado, Barquisimeto, Venezuela
IIUnidad de Microtecnia, Decanato de Agronomía, Univ Centroccidental Lisandro Alvarado, Barquisimeto, Venezuela





Botanical compounds with insecticidal and acaricidal activities have been used in pest management with different levels of success. Toxicity of ethanolic extracts obtained from wild oregano (Lippia origanoides) and gliricidia (Gliricidia sepium) to Tetranychus cinnabarinus (Boisduval) were evaluated. Mite population was collected from black bean plants growing in Urachiche Municipality, Yaracuy State, Venezuela. Ethanolic extracts of wild oregano and gliricidia leaves were evaluated at different concentrations (5, 10, 15, and 20%) using the leaf disk immersion technique. The presence of alkaloids, flavonoids, phenols and tannins, essential oils and saponins was verified in the plant material used in our study. Tetranychus cinnabarinus oviposition decreased at a rate of 43.7% or 57% when 5% oregano or gliricidia extracts were used, respectively. Also, 10% oregano or gliricidia extracts caused 42.2% or 72.5% of mortality to T. cinnabarinus, respectively. Ethanolic extracts showed acaricidal effects on T. cinnabarinus, as evidenced by maximum mortality (96.6% and 100% caused by wild oregano and gliricidia, respectively) when used at a concentration of 20%. Our results showed that gliricidia and wild oregano are promising for the management of T. cinnabarinus, although their field efficacy remains to be evaluated.

Keywords: Botanical acaricide, carmine spider mite, secondary metabolite




The pesticide activity from secondary metabolites occurring in plants is based on plant-pest or plant-pathogen interactions (Hilker & Meiners 2002, Praslička & Huszár 2004), hence most of these natural substances show insecticidal, nematicidal, fungicidal and bactericidal properties. Previous studies have proved growth inhibition in ticks (Alvarez et al 2008) and in the brown avocado mite, Oligonychus punicae (Hirts) (Vásquez et al 2008). Thus, plant-derived molecules with acaricidal activity have emerged as an ecological strategy for pest management (Castiglioni et al 2002, Araújo et al 2010, Cavalcanti et al 2010).

As botanical pesticides can provide mite control at a low cost, and with a low risk to human and to the environment, their recommendation as a control strategy of tetranychids have been increased (Gencsoylu 2007). Powders and ethanolic or aqueous extracts obtained from different plant species have been used for population pest management. There are several plant species which were investigated and shown to produce compounds which can cause mortality in several Tetranychus species either by contact on by systemic exposure (Chiasson et al 2001, Shi et al 2004). In the case of T. urticae, 67 plant species belonging to six subfamilies from Lamiaceae were shown to produce compounds with acaricidal properties, from which Plecthratus was the genus to show the highest diversity of species (14) producing highly toxic compounds against tetranychids (Rasikari et al 2005).

The carmine spider mite Tetranychus cinnabarinus (Boisduval) is reported in more than 130 plant species of economic importance, including vegetables, fruit-trees, and ornamentals (Bolland et al 1998, Guo et al 1998). Synthetic acaricides have been used as the main strategy for Tetranychus population management resulting in an increased cost for production and environmental impacts (Argüelles et al 2006). Intensive pesticide use has raised resistance development to dicofol, amitraz, propargite, and even to the newly synthesized molecules such a hexythiazox, pyridaben, and tebufenpyrad as for Tetrancyhus urticae Koch (Ramasubramanian et al 2005) and to abamectin as for T. cinnabarinus (Lin et al 2009).

Wild oregano (Lippia origanoides) and gliricidia (Gliricidia sepium) are commonly found in arid environmental conditions in Lara State, Venezuela, representing a valuable source to obtain extracts with pesticidal activity. Biological activity of essential oils from oregano in controlling several microorganisms such as Staphylococus aureus (MRSA), S. aureus (ATCC25923), Escherichia coli (ATCC25922), Candida albicans and C. tropicales was already demonstrated (Dos Santos & Arimatera 2004), but no data is available on its potential or of gliricidia to produce molecules with pesticide activity.

The carmine spider mite is an important pest of various economically important crops often requiring acaricidal treatment (Çakmak et al 2005), although the chemical spraying have frequently failed to properly control this pest (Guo et al 1998). Thus, the toxicity of ethanolic extracts of L. origanoides and G. sepium was evaluated in this study based on biological parameters of T. cinnabarinus, aiming to provide an ecological alternative control method against this mite species.


Material and Methods

Plant extracts

Extracts of L. origanoides and G. sepium were obtained from natural populations (Marcano & Hasegawa 2002). Mature leaves were dried and macerated and soaked in 96% ethanol for one to two weeks. The liquid was filtered using a four-layered gauzed and concentrated in a rotary vacuum drier system (roto-vapor BrinkmamMR, Mod. RE111) at 100ºC. The concentrated crude extract obtained after water and ethanol evaporation was recovered from the rotary drier system and stored at 8ºC until qualitative determination of secondary metabolites and evaluation of the acaricidal properties.

Secondary metabolites

Qualitative determination of secondary metabolites (alkaloids, essential oils, polyphenol and tannin, saponins, antraquinones and flavonoids) in ethanolic extract followed the methods in Marcano & Hasegawa (2002). After dehydration, the ethanolic extract was mixed with 2.5 ml 10% hydrochloric acid and 5 ml chloroform in a separatory funnel to determine alkaloids. This procedure yielded three phases, with the weak-basic alkaloids being collected in the first phase. The intermediary phase was alkalinized with ammonium hydroxide (NH4OH) and re-extracted with chloroform in order to obtain the basic alkaloids. The third phase aimed to detect the quaternary ammonium salts. One microlitre aliquot from each of phases was placed on a silica-gel cromatoplate and treated with Meyer's reagent. Alkaloids presence was confirmed by formation of a brown-reddish coloration. Results were confirmed by adding Dragendorff's reagent.

Essential oils were determined by organoleptic procedures using characteristic odor as indicative (Marcano & Hasegawa 2002). Total polyphenols and tannins were evidenced by characteristic brown coloration in ethanolic extract when added 1% FeCl. The extract was evaporated and added 1% agar in a 1% NaCl solution. The presence of polyphenols and tannins was indicated by the formation of a precipitate (Marcano & Hasegawa 2002).

Saponins were evidenced by the formation of a spume which remained more than 20 min when ethanolic extract was strongly shaken with distilled water (Marcano & Hasegawa 2002). For the determination of the presence of antraquinone, the ethanolic extract was neutralized with 5 ml 0.5% KOH, acidified with 0.5 ml acetic acid (pH 4.8) and then agitated with 5 ml benzene. Finally, NH4OH was added and antraquinone presence was indicated by the development of a reddish coloration.

Flavonoids were detected by a reddish coloration after 20 min 0.5 ml 10% HCl and magnesium (0.35 g) were added to the ethanolic extract. Flavonoids were confirmed by the yellow color development of sample spots in 1% AlCl3-treated filter papers, when observed under 450 nm UVL.

Mite collection and rearing

Mites were collected on 40 d-old black bean plants Phaseolus vulgaris growing under irrigation conditions during a dry season in Fundo Zamorano Bella Vista, Urachiche Municipality, Yaracuy State, Venezuela. In the laboratory, females and males were mounted on slides for microscope observations using the Hoyer´s medium. Taxonomical identification was made by using the taxonomical keys of Gutierrez (1985), and species identification was confirmed by comparisons with voucher specimens from the Acarological Collection from Universidad Central de Venezuela, especially by comparing the morphology of the aedeagus (Ochoa et al 1994).

A laboratory mite culture was initiated with 100 females and males from a field population using rearing units or arenas (Helle & Overmeer 1985) maintained at 27 ± 2ºC, 70 ± 10% RH and 12:12 photoperiod. Males and females were transferred to P. vulgaris leaf disks (6 cm diameter) on each rearing unit (9 cm diameter and 1.5 cm high). They were maintained for 24h period to lay eggs. After this period, males and females were discarded and the eggs were reared until adult emergence.

Toxicity of ethanolic extracts to T. cinnabarinus

Toxicity of ethanolic extracts to T. cinnabarinus was evaluated following Cowles et al (2000). Leaf disks (6 cm diameter) were soaked during five seconds in ethanolic extract (5, 10, 15, and 20%) from both plant species evaluated or into distilled water as a control treatment (0%). Afterwards, the excess of liquid was eliminated and leaves were left to dry at room temperature (25 ± 2 ºC) during 0.5h, before being offered as a substratum into the rearing units. Then, 10 1 d-old females were randomly selected from the laboratory culture and placed on each of the rearing units containing leaf disks from the respective plant species and extract concentration. Ten replicates were run for each treatment. The evaluations were conducted at 24, 48 and 72h after treatment to determine female mortality and oviposition. A female was considered dead when not responding to touch.

The experiments were carried out in a completely randomized design with treatment arranged in a divided plot in time, with the plant extract as the main plot and concentrations as the subplots. Oviposition rate was calculated as the mean number of eggs laid per female per day. Data were corrected for control mortality (Abbott 1925). Linear regression analysis was performed to describe oviposition rate or cumulative mortality against metabolite concentration using Statistix version 8.0


Results and Discussion

Secondary metabolites

The groups of secondary metabolites were similar in extracts from wild oregano (L. origanoides) and gliricidia (G. sepium) (Table 1). Conversely to our results, alkaloids and saponins and lower concentrations of phenols were not found in gliricidia by Galindo et al (1989). These variations could be related to environmental conditions in which plants were cultivated, which could evoke secondary metabolites production as an adaptive strategy (Zhao et al 2005). Also, genetic polymorphism in plant taxa could result in differences in chemical composition in secondary metabolites (Izco 1997).

Toxicity of ethanolic extracts to T. cinnabarinus

Significant differences were detected in T. cinnabarinus oviposition when this mite was exposed to the different ethanolic extract concentrations and plant species (P < 0.01). According to regression lines correlating extract concentration and oviposition rates, higher oviposition rates were observed when leaves were treated with 5% and 10% gliricidia extracts compared to wild oregano at the same concentrations (Fig 1). However, similar oviposition rates were observed at 15% and 20%, regardless the plant species. The lowest concentration of the oregano crude extract (5%) caused 43.7% reduction in the oviposition of T. cinnabarinus, with a little higher reduction (57%) being induced by the gliricidia extract. No oviposition was observed when leaf discs were treated with 20% ethanolic extract solutions.



Tetranychus cinnabarinus mortality increased with concentration of oregano and gliricidia ethanolic extracts (Fig 2). Mortality was 45.7, 55.9, 69.5, and 96.6% higher than control when wild oregano extract was applied at 5, 10, 15, and 20%, respectively. Meanwhile, similar gliricidia extract concentrations caused 41.7, 80.6, 88.9, and 100% mortality.



Botanical products with activity against mites have been shown promising results in field or in laboratory conditions. High mortality levels (> 78.8%) for T. urticae, T. cinnabarinus and T. viennensis were observed with extracts of Kochia scoparia (Shi et al 2006) and for Mononychellus tanajoa (Bondar) treated with neem extracts (Gonçalves et al 2001a, b). Tetranychus urticae females treated with leaf and fruit extracts from Capparis aegyptia showed high mortality and reduction in oviposition rate 15 days after applications (Hussein et al 2006). More recent studies have devoted to identify the active ingredients in plant extracts causing mite mortality. The metil-palmitate (MP), a fatty acid ester from Juglans regia seeds showed to be efficient, causing between 62.3% and 79.0% mortality of T. cinnabarinus and Tetranychus viennensis Zacher at 1 mg/ml MP, in a 24h interval (Wang et al 2007, Wang et al 2009). Furthermore, although higher MP concentration (10 mg/ml MP) caused 97.9% mortality to T. cinnabarinus adults, it showed lower efficacy (57.2%) when used on eggs.

Based on our results, wild oregano and gliricidia ethanolic extracts seems to be a promising alternative for pest management, which could be readily available for small growers. Thus, this natural based product could be included among the practices of carmine mite control by farmers, reducing the dependence on synthetic pesticides, and the risks of environmental pollution. Although in our study alkaloids, flavonoids, phenols and tannins, saponins and essential oils showed to be present, both in wild oregano and gliricidia ethanolic extracts, more comprehensive studies should be addressed in order to evaluate the concentration of each of these secondary metabolites in order to determine their potential use in pest control.



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Carlos Vásquez
Depto de Ciencias Biológicas, Decanato de Agronomía, Univ Centroccidental Lisandro Alvarado
Barquisimeto, Estado Lara, Venezuela

Received 10 December 2009 and accepted 30 November 2010



Edited by Jorge B Torres - UFRPE

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