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Chilean journal of agricultural research

versión On-line ISSN 0718-5839

Chilean J. Agric. Res. v.70 n.3 Chillán sep. 2010

http://dx.doi.org/10.4067/S0718-58392010000300007 

Chilean Journal of Agricultural Research 70(3):399-407 (July-September 2010)

RESEARCH

Insecticidal Activity of Peumus boldus Molina Essential Oil against Sitophilus zeamais Motschulsky


Actividad Insecticida del Aceite Esencial de Peumus boldus Molina sobre S itophilus zeamais Motschulsky


Jessica Betancur R.1, Gonzalo Silva A.1*, J. Concepción Rodríguez M.2, Susana Fischer G.1, and Nelson Zapata S.M.1

1Universidad de Concepción, Facultad de Agronomía, Av. Vicente Méndez 595, Chillán, Chile. *Corresponding author (gosilva@udec.cl).
2Colegio de Postgraduados, Programa de Entomología y Acarología, km 36.5, Carretera México-Texcoco, Montecillo, Estado de México, México.


ABSTRACT

In stored grains, the main agents diminishing production are insects, which can produce losses between 20% and 80% before harvest or under storage. The insecticidal properties of the essential oil of fresh leaves of Peumus boldus Molina against maize weevil (Sitophilus zeamais Motschulsky) adults were determined under laboratory conditions. The highest mortality (100%) was achieved at 4% concentration by contact with a treated glass surface. The same concentration in impregnated corn (Zea mays L.) grain, resulted in 98.7% mortality. Mortality by fumigant action at 6 h was 100% with 35 µL oil in 0.15 L (air volume). Concentrations 1, 2 and 4% of essential oil produced 0% F1 adult emergence. At 10 d of residual effect, the 4% concentration reached 63.7% mortality. All treatments were repellent to adults of S. zeamais and corn grain germination was not affected by any treatment.

Key words: Maize weevil, boldus, stored grains, botanical insecticides.


RESUMEN

Los principales agentes que disminuyen la producción en los granos almacenados son los insectos, antes de la cosecha y en el almacenamiento pueden causar pérdidas de 20 a 80%. Se evaluaron las propiedades insecticidas del aceite esencial de hojas frescas de Peumus boldus Molina para el control de adultos de gorgojo del maíz (Sitophilus zeamais Motschulsky) en laboratorio. La mayor mortalidad (100%) por contacto con una superficie de vidrio tratada se obtuvo con la concentración de 4%. Esta misma concentración produjo 98,7% de mortalidad en exposición a grano de maíz (Zea mays L.) tratado. El efecto fumigante a las 6 h de exposición fue 100% con 35 µL de aceite en 0,15 L (volumen de aire). Con las concentraciones de 1, 2 y 4% de aceite esencial, el porcentaje de emergencia de la F1 fue 0%. A los 10 d de efecto residual se alcanzó 63,7% de mortalidad con la concentración de 4%. Todos los tratamientos fueron repelentes para adultos de S. zeamais y ningún tratamiento afectó la germinación de los granos.

Palabras clave: gorgojo del maíz, boldo, granos almacenados, insecticidas vegetales.


INTRODUCTION

Cereal grains are staples in the diets of human beings and domestic animals. Consequently, their conservation is essential to have this basic food available on an ongoing basis (Huang and Subramanyam, 2005). Grains in storage are affected by several insect species that can destroy up to 50% of the harvest in 6 months. As well, storage allows the entrance of phytopathogenic organisms, such as fungus or bacteria (Regnault, 1997). Synthetic pesticides have been considered the most effective and accessible means to control insect pests of stored products (Huang and Subramanyam, 2005). However, their use can result in the presence of residues in the grains (Sanna et al., 2004).

The current trend is the search for and use of alternative methods to manage pests, which, in the economic context, are effective without presenting the risks associated with the use of conventional pesticides. One method consists of using plants with insecticidal properties that can be used as powder, extracts or oils (Mazzonetto and Vendramim, 2003). Botanic insecticides have been used traditionally in developing countries to control pests of stored grain, such as coleoptera of the genus Sitophilus (De Oliveira et al., 2003).

Aromatic plants have been used for both their medicinal and insecticidal properties. Their characteristic aroma derives from essential oils, many of which have proven fumigant and contact activity, as well as ovicide, anti-alimentary and repellent activity (Sanna et al., 2004).

One of the native Chilean plants that has been evaluated as an insecticide in powdered form is boldo (Peumus boldus Molina) (Monimiaceae) (Silva et al., 2003a; 2003b; 2005a; 2005b; 2006), a tree that can reach a height of 20 m, with pruning it appears like a densely branched bush (Vogel et al., 2005). The leaves contain alkaloids, known together as boldine, which is attributed to having antioxidant, anti-inflammatory and antipyretic properties (Vogel et al., 1999). The leaves also have essential oils. Some 45 to 53% of the oil is composed of ascaridole, 1,8 cineole (30%), limonene, α-terpinol and terminen-4-ol (Vogel et al., 2005). The compound 1,8 cineole is also isolated from essential oil of plants such as Eucalyptus globulus Labill, Gomortega keule (Molina) I.M. Johnst., Ocimum kilimandscharicum Baker ex Gürke, Ocimum kenyense (Ayobangira), Ocimum basilicum L. and Salvia officinalis L., and is an effective fumigant for stored grain pest control (Bekele and Hassanali, 2001; Asawalam and Hassanali, 2006; Asawalam et al., 2008; Bittner et al., 2008). Consequently, the objective of this research was to evaluate the insecticidal properties of the essential oil of P. boldus against adults of S. zeamais under laboratory conditions.

MATERIALS AND METHODS

The study was carried out at the Insecticide Toxicology Laboratory of the Faculty of Agronomy of the Universidad de Concepción, Chillán Campus, Bío-Bío Region, Chile.

The insects used in the bioassays were obtained from colonies permanently maintained in the laboratory. They were reproduced in 1-L glass flasks containing maize (Zea mays L.) as a source of food. The insects were maintained in total darkness at a temperature of 30 ± 1 ºC.

Maize grains of with 14% moisture were used as an alimentary substrate. The maize was obtained from the fruit and vegetable market in Chillán. Only healthy grain was used to avoid any prior infestation that could affect the results of the bioassay. The grain was washed with potable water and frozen at a temperature of -4 ± 1 ºC for 48 h.

The essential oil was extracted from fresh leaves of P. boldus collected in the park of the Chillán Campus of the Universidad de Concepción. The leaves were washed with potable water to remove any possible detritus and the oil was obtained with steam distillation using distilled water, as suggested by Vogel et al. (1997). Subsequently, the oil was stored at a temperature of 4.5 °C in amber colored glass containers until they were used.

Bioassays

Mortality by contact with a surface of treated glass.
The methodology of Kouninki et al. (2007) was used, with slight modifications that consisted of using 6-mL test tubes and applying 1 mL, instead of 40 mL and 3 mL, respectively, of a solution of essential oil in acetone (99% purity), at the required concentration. The tubes were agitated for 1 min for the oil to cover the interior surface. The excess was eliminated and the acetone was allowed to evaporate at ambient temperature for 1 h. Finally, 10 adult insects 48 h of age, without sexing, were placed in each tube. The evaluated doses of oil were 0.25, 0.5, 1, 2 and 4% and the control with 1 mL of acetone. Ten replicates were made per treatment. The treatments were kept in a bioclimatic chamber at a temperature of 30 ± 1 ºC. Insect mortality was assessed at 24; 48 and 72 h of exposure to the essential oil.

Mortality by contact with treated grain. This bioassay was carried out with the methodology of Obeng-Oferi and Reichmuth (1997). Solutions of 1 mL of essential oil of P. boldus in acetone were applied to 500-mL glass containers with 25 g of maize in concentrations of 0.25, 0.5, 1, 2 and 4%, plus a control with 1 mL of acetone. The flasks were covered and agitated for 15 s to uniformly cover the grains with oil. They were uncovered and left for 2 h at ambient temperature to evaporate acetone. The flasks were then infested with 20 adult insects 48 h of age, without sexing. Each treatment had ten replicates. The experimental units were stored in a bioclimatic chamber at a temperature of 30 ± 1 ºC and mortality was assessed at 24, 48 and 72 h exposure to the toxic.

Residuality
The methodology Obeng-Oferi and Reichmuth (1997) was used, with the difference that once the grain was mixed with the oil in the respective concentrations, it was stored in containers for 1, 5 and 10 d, respectively. With the elapse of these periods, each container (experimental unit) was infested with 20 adult insects 48 h of age, without sexing, and stored in a bioclimatic chamber at a temperature of 30 ± 1 ºC. For each of these storage durations, the concentrations of 0 (control); 0.25, 0.5, 1, 2 and 4% were assessed, with ten replicates for each treatment. Mortality was assessed at 24, 48 and 72 h after infestation.

Fumigant effect
This bioassay employed the methodology of Pires et al. (2006), which consisted of applying 0 (control), 5, 10, 15, 20, 25, 30 and 35 µL of essential oil on circular Whatman N°10 filter paper (Whatman, Maidstone, Kent, UK) 5.5 cm in diameter, which had been adhered to the covers of 150-m containers (air volume equivalent to 0.15 L), with 25 g of maize infested with ten adult insects, without sexing. The same procedure was used for the control with filter paper without treatment. There were ten replicates for each treatment. The experimental units were kept in a bioclimatic chamber at a temperature of 30 ± 1 ºC. Assessments of mortality were made at 6, 12 and 24 h of exposure. As the mortality rate in the control was lower than 5%, this was corrected with the Abbott formula (1925). An insect was considered dead when there was no movement after prodding it with a dissection needle.

Emergence of adults of F1 compared to the control without treatment
Each experimental unit consisted of a 500-mL flask, 25 g of maize and 10 pairs of adult insects 24 h of age, which were allowed to freely reproduce for 21 d. Subsequently, the adult pairs were removed and the grain was mixed with essential oil of P. boldus diluted in acetone at concentrations of 0 (control); 0.125; 0.25; 0.5; 1; 2 and 4%, according to what was described by Obeng-Oferi et al. (1998). The control received only 1 mL of acetone. The flasks were kept in a breeding chamber at a temperature of 30 ± 1 ºC throughout the bioassay. Every treatment had 10 replicates. As a variable response, the percentage of emergence of adults of the F1 generation was assessed weekly for 7 wk in comparison to the control without treatment. The morphology of the proboscis was used for sexual differentiation, that of the male being rougher and of a higher caliber than that of the female, according to what was described by Halstead (1963).

Repellent effect
The methodology proposed by Mazzonetto and Vendramim (2003), with slight modifications, was used to assess the repellent effect of the essential oil of P. boldus. The experimental unit was a plastic Petri dish 5 cm in diameter containing 50 g of maize grains that had been impregnated with the respective concentrations of essential oil. The treatments were placed in a circle around a central Petri dish that contained 20 individuals of S. zeamais of 48 h of age, without sexing. The central Petri dish was connected to the others through tubes 10 cm long and 0.5 cm in diameter (Procopio et al., 2003). The evaluated treatments were 0 (control); 0.125, 0.25, 0.5, 1, 2 and 4% of oil in acetone. The experimental batch was maintained in a bioclimatic chamber for 24 h at a temperature of 30 ± 1 ºC. Subsequently, the number of insects present in each treatment was counted. Each treatment had 10 replicates and in each replicate the treatments were randomly rotated to avoid external factors from interfering. The repellent index is calculated with these results according to what was described by Mazzonetto and Vendramim (2003), in which the oil is classified as neutral if the index is equal to 1, attracting if it is higher than 1 and repellent if it is less than 1.

Germination test of treated grain
The effect of essential oil of boldo on the germinative power of the maize grains was assessed using the methodology described by Pérez et al. (2007). Groups of 30 seeds were randomly selected from seeds without any apparent damage. The seeds were mixed with oil in 150-mL flasks and placed separately in glass Petri dishes containing permanently moistened filter paper on the bottom. The following concentrations were used as treatments: 0 (control); 0.125; 0.25; 0.5; 1; 2 and 4% of oil. In total, 10 replicates were made. The experimental units were kept at a temperature of 30 ± 1 ºC for seven days in a bioclimatic chamber. Subsequently, the percentage of germination was determined in comparison to the control.

Estimation of the equitoxic concentration
Lethal concentrations of 50% (CL50) and 90% (CL90) were estimated in the treatments of mortality by contact with a surface of treated glass, by contact with treated grain and by fumigation. In all cases, there was 24 h of exposure to the toxic and a Probit analysis (Finney, 1971) was used to estimate the equitoxic concentrations, using the PROC PROBIT procedure of the Statistical Analysis System program (SAS Institute, 1998).

Experimental design
The experimental design was completely random and percentage data were transformed to the arcsine function for its ANOVA (α = 0.05) with the Statistical Analysis System program (SAS Institute, 1998) to determine if any treatments differed from the others. In the case that there were differences, a Tukey means comparison test was employed with a significance of 95% (p ≤ 0.05).

RESULTS AND DISCUSSION

Mortality by contact with a treated glass surface
In general, mortality increased with increased exposure time to the essential oil, which concurs with Bittner et al. (2008). The concentrations of 1, 2 and 4% exceeded 50% mortality at 24 h (Table 1) and the concentration of 4% caused 100% mortality. At 48 h, mortality at concentrations of 2 and 4% was 97.5 and 100%, respectively, without significant differences between them (p > 0.05) (Table 1). At 72 h, these same two concentrations provoked higher mortality. Based on this, the essential oil of P. boldus can be considered as effective as a contact insecticide at doses of 2 and 4%, given that at 48 h both showed an efficacy greater than 90%. These values are higher than those observed by Popovic et al. (2006), who reached mortality rates by contact of 26 and 28% with essential oil of O. basilicum and S. officinalis, respectively.

Table 1. Mortality of Sitophilus zeamais adults exposed to a glass surface treated with different concentrations of essential oil of Peumus boldus under laboratory conditions.


Mortality by contact with treated grain
Results close 100% were obtained at 24 h and with a concentration of 4% (Table 2), while at 48 and 72 h the concentration of 2% showed 85% mortality, without statistical difference with the concentration of 4% (p > 0.05). The direct proportionality between concentration and mortality concurs with what was obtained by Silva et al. (2003b; 2005a) and Pérez et al. (2007) using powder of P. boldus for control of S. zeamais. As well, Popovic et al. (2006) and Bittner et al. (2008) indicate that with all the essential oils used in the control of pests of stored grains, mortality increases as the concentration or exposure time increases. The mortality rate that was obtained by contact with treated grain was higher than that documented with essential oil of O. grattissimum and V. amigdalina, given that at a doses of 0.3% the mortality rate was lower than 30% at 48 h and 82% at 7 d of exposure (Asawalam and Hassanali, 2006; Asawalam et al., 2008).

Table 2. Mortality of Sitophilus zeamais adults exposed to maize grain treated with different concentrations of essential oil of Peumus boldus under laboratory conditions.

Residuality
As the time increases between impregnation of the grain and infestation with adults of S. zeamais, mortality decreases in all the treatments and when the exposure time to treated grain increases, the mortality of adults of S. zeamais also increases (Table 3). During the first day of exposure, the concentration of 4% reached a mortality of 97.5%, and this value maintained the same statistical significance at 48 and 72 h (p ≤ 0.05). At 10 d, mortality was less than 64% with the highest concentration. These results could be due to the loss in the concentration of the active compounds are vulnerable to light and temperature. The lost insecticidal power over time concurs with the research of Silva et al. (2005a), who concluded that the residual effect of P. boldus does not exceed 30 d.

Table 3. Mortality over time of Sitophilus zeamais adults exposed to different concentrations of essential oil of Peumus boldus under laboratory conditions.


Fumigant effect
It was observed in the fumigant action bioassay that 35 µL of oil in a volume of 0.15 L has a rapid toxic effect, producing 100% mortality in 6 h (Table 4). At 12 h of exposure, the doses of 15, 20, 25, 30, and 35 µL in 0.15 L-1 also showed 100% mortality (Table 4). At 24 h, the treatments higher than 20 µL of oil in 0.15 L caused 100% mortality. According to Vogel et al. (2005), the essential oil of P. boldus contains 1,8 cineole. This compound is also present in the essential oil of Rosmarinus officinalis L., Eucalyptus blakelyi Maiden and Melaleuca fulgens R. Br., which have demonstrated fumigant action against the mite Tetranychus urticae Koch (Miresmailli et al., 2006) and insects like Sitophilus oryzae L., Tribolium castaneum Herbst. and Rizopertha dominica F. (Lee et al., 2003). The results obtained are superior to those observed with other essential oils of Chilean plants that contain 1,8 cineole, such as G. keule and Laurelia sempervirens (Ruiz & Pav.) Tul., which provoke 100% mortality at 72 h (Bittner et al., 2008). This level of mortality has been reached in other countries at 48 h with the essential oils of Achillea biebersteinii Afan and A. wilhelmsii, and at 96 h with oil of Pistacia spp. (Aslan et al., 2004). As well, Bekele and Hassanali (2001) indicate that there is a synergism among the components of the essential oils of plants such as O. kilimandscharicum and O. kenyense, so that their use to control as an oil would be more effective than their compound in isolated form, given that among the other benefits are reducing the risk of resistance.

Table 4. Mortality of Sitophilus zeamais adults by the fumigant action of Peumus boldus essential oil under laboratory conditions.

Emergence of adults of F1 in comparison to the control
In all evaluated treatments, adult insects were observed to emerge beginning at week 5 of the infestation. The lowest level of emergence of F1, compared to the control without treatment was observed in the concentrations of 1, 2 and 4%, with values of 0% until week 7 (Table 5). However, these three concentrations only showed significant differences from the other treatments (p ≤ 0.05) in week 7. In week 5 all concentrations were statistically equals (p > 0.05), while in week 6, 0.125% differed from the other treatments (p ≤ 0.05). According to Paranagama et al. (2003), the majority of essential oils produce a significant inhibition of oviposture and emergence of F1 of coleoptera associated with stored grain, which concurs with the results of the current research.

Table 5. F1 emergence of Sitophilus zeamais adults exposed to different concentrations of Peumus boldus essential oil under laboratory conditions.


Repellent effect
All the treatments evaluated had a repellent effect. The value of the index decreased as the concentration increased (Table 6). Consequently, the essential oil of P. boldus can be considered a repellent for adults of S. zeamais and the concentration has a direct relationship with the degree of repellence, which concurs with what was documented by Nuñez (2005) for powder of P. boldus. Similar results were obtained with other plants where the major component is 1,8 cineole, such as Vernonia amygdalina (Lam) and O. grattisimum L., whose oils, according to Asawalam and Hassanali (2006) and Asawalam et al. (2008) are highly repellent against S. zeamais.

Table 6. Repellence index of essential oil of Peumus boldus against Sitophilus zeamais adults under laboratory conditions.


Germination test of treated grain
The percentage of germination of the grain varied from 85.19 to 100%, without statistical differences among the treatments (p > 0.05) (Table 7). These data concur with those of Silva et al. (2006), but contradicts those of Pérez et al. (2007), who affirmed that the powder of P. boldus significantly affect maize germination at concentrations higher than 1%. This difference could be because the compound or compounds that affect germination are found in the powder and not in the essential oil of P. boldus.

Table 7. Maize grain germination exposed to different concentrations of Peumus boldus essential oil.


Estimation of equitoxic concentration
The values of CL50 for the mortality bioassays by contact with treated grain, by contact with a surface of treated glass, and by fumigant action were 1.54, 1.06 and 10.26 µL oil 0.15 L-1, respectively. The CL90 values for these bioassays were 2.34, 2.36 and 14.28 µL oil 0.15 L-1, respectively (Table 8). There were confidence limits at both the levels of CL50 and CL90, consequently there were no statistical differences between the bioassays of exposure to treated grain and to a surface of treated glass. However, these values were higher than those of the bioassay evaluating mortality by fumigant action (Table 8), which implies less toxicity of P. boldus by this method.

Table 8. Toxicity of essential oil of Peumus boldus against Sitophilus zeamais adults exposed to maize grain treated or a treated glass surface.


CONCLUSIONS

The essential oil of P. boldus is toxic for adults of S. zeamais, whether by exposure to the surface of treated glass, exposure to treated grain, or by fumigant action. It also significantly reduces the emergence of F1 in comparison to the control and has a repellent effect. The residual effect is significant for 5 days and does not affect germination of the maize grain.

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Received: 25 May 2009.
Accepted: 17 September 2009.

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