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Chilean journal of agricultural research

versión On-line ISSN 0718-5839

Chilean J. Agric. Res. vol.72 no.3 Chillán set. 2012 

Agricultural Research 72(3) July - September 2012


Toxicity of Boldo Peumus boldus Molina for Sitophilus zeamais Motschulsky and Tribolium castaneum Herbst

Toxicidad del Boldo, Peumus boldus Molina, sobre Sitophilus zeamais Motschulsky y Tribolium castaneum Herbst.


Margarita Ortiz U.1, Gonzalo Silva A.1*, Maritza Tapia V.1, J. Concepción Rodriguez M.2, Angel Lagunes T.2, Candelario Santillán-Ortega3, Agustín Robles-Bermúdez3, and Sotero Aguilar-Medel4

1Universidad de Concepción, Facultad de Agronomía, Av. Vicente Méndez 595, Chillán, Chile. Corresponding author (
2Colegio de Postgraduados, Programa de Entomología y Acarología, km 36,5 Carretera México-Texcoco, Montecillo, Estado de México, México.
3Universidad Autónoma de Nayarit, Unidad Académica de Agricultura, km 9 Carr. Fed. Tepic-Compostela, Xalisco, Nayarit México.
4Universidad Autónoma del Estado de México, Centro Universitario Tenancingo, Tenancingo, Estado de México, México.

The maize weevil (Sitophilus zeamais Motschulsky) and the red flour beetle (Tribolium castaneum Herbst) are two key pests of stored-grain products worldwide. The insecticidal activity of boldo (Peumus boldus Molina) powder, liquid ethanolic and hexanic extracts against S. zeamais and T. castaneum were evaluated under laboratory conditions. The evaluated variables were mortality, emergence of adult insects (F1), and grain weight loss. The experimental design was completely randomized. The mortality in S. zeamais was 100% even at the lowest powder concentration (0.5% w/w), whereas emergence of F1 adult insects was 0% and grain weight loss was < 0.08%. For T. castaneum, only 8 and 16% w/w powder concentrations reached 100% mortality. The liquid ethanolic and hexanic extracts caused 100% mortality of S. zeamais, whereas only the ethanolic extract reached this value for T. castaneum. Therefore, the powder and the evaluated extracts of P. boldus were toxic for S. zeamais and T. castaneum and are promising against these and other stored-grain pests.

Key words: Botanical insecticides, stored grains, maize weevil, red flour beetle.

El gorgojo del maíz (Sitophilus zeamais Motschulsky) y el gorgojo castano de la harina (Tribolium castaneum Herbst) son plagas primarias de productos almacenados a nivel mundial. Se evaluó en laboratorio la actividad insecticida de polvo y extractos líquidos etanólicos y hexánicos del boldo (Peumus boldus Molina) sobre S. zeamais y T. castaneum. Las variables evaluadas fueron mortalidad y emergencia de insectos adultos (F1) y pérdida de peso de los granos con un diseno experimental completamente al azar. La mortalidad en S. zeamais fue 100%, incluso con la concentración menor (0,5% p/p) mientras que la emergencia de insectos adultos y la pérdida de peso de granos de maíz fue < 0,08%. Para T. castaneum sólo las concentraciones de 8 y 16% p/p de polvo causaron una mortalidad de 100%. Los extractos en agua, etanol, y hexano tuvieron un efecto insecticida de 100% en S. zeamais, mientras que en T. castaneum sólo el extracto en etanol alcanzó este valor. Por lo tanto, el polvo y los extractos evaluados de P. boldus presentan actividad insecticida contra S. zeamais y T. castaneum y son promisorios para utilizarse contra éstas y otras plagas de granos almacenados.

Palabras clave: insecticidas vegetales, granos almacenados, gorgojo del maíz, gorgojo castano de la harina.

Stored grains are affected by several insect pests causing significant losses that may amount up to 50% of the harvest. In addition, pest damage to grain allows the entrance of pathogenic organisms such as fungus or bacteria (Regnault, 1997). The maize weevil (Sitophilus zeamais Motschulsky, Coleoptera: Curculionidae), rice weevil (Sitophilus oryzae Linnaeus, Coleoptera: Curculionidae), and grain moth (Ephestia kuehniella Zeller, Lepidoptera: Pyralidae) are three of the most important stored-grain pests in Chile (Larraín, 1994). Synthetic pesticides have been considered to be the most effective and easy to use tools against these insect pests (Huang and Subramanyam, 2005). However, their misuse has caused several problems such as the presence of high or illegal pesticide residues, human intoxication, and development of insect resistance (Roel and Vendramim, 2006).

The search for alternative methods includes using natural products that are both effective and environmentally friendly, such as botanical insecticides (Roel and Vendramim, 2006). These compounds have been traditionally used in developing countries against stored-grain pests such as Sitophilus weevils (De Oliveira et al., 2003). Many of the secondary metabolites of plants act as insecticides, ovicidal, ovipositional, and feeding deterrents, and growth retardants (Pugazhvendan et al., 2009). Currently, research about their use as grain protectants involves tropical plant species; therefore, the search for Chilean flora with promising activity is needed.

The perennial boldo tree Peumus boldus Molina (Monimiaceae) is native to Chile and in powder form has insecticidal activity against S. zeamais (Páez et al., 1990; Silva et al., 2003; 2005; 2006; Pérez et al., 2007), third instar larvae of Spodoptera littoralis Boisduval (Lepidoptera: Noctuidae) (Zapata et al., 2006), Spodoptera frugiperda J.E. Smith (Lepidoptera: Noctuidae), Helicoverpa zea (Boddie) (Lepidoptera: Noctuidae) (Silva et al., 2010), as an extract against Xanthogaleruca luteola Muller (Coleoptera: Chrysomelidae) (Chiffelle et al, 2011), and as fungicide properties against Penicillum spp., Fusarium spp., Aspergillus niger Thieg., and A. flavus Link (Leite de Souza et al., 2005). The leaves of P. boldus contain a group of boldine alkaloids that have antioxidant, anti-inflammatory, and antipyretic properties (Vogel et al., 1999). However, the insecticidal properties of P. boldus extracts against S. zeamais and T. castaneum are not documented, so the aim of this research was to assess the insecticidal properties of P. boldus leaf powder and water, hexane, and ethanol extracts against S. zeamais and T. castaneum adults in the laboratory.


Study site, plant material, and insects
The study was carried out at the Laboratory of Entomology, Faculty of Agronomy, Universidad de Concepción in Chillán, Bío Bío Region, Chile.

Peumus boldus foliage was collected from trees in the park of the Universidad de Concepción, Chillán by following the criteria set out by Vogel et al. (1997).

Wheat (Triticum aestivum L.) flour was employed to rear T. castaneum, whereas maize (Zea mays L.) grains with 14% moisture were used as food substrate for S. zeamais. To avoid any previous infestation, grains were washed and dried in an oven (Memmert Gmbh, UNB 500, Schwabach, Germany) at 25 °C for 12 h and then frozen at -4 ± 1 °C for 48 h prior to use.

Insects for the bioassays were provided by the Laboratory of Entomology of the Universidad de Concepción, Chillán. They were reared in 1-L glass flasks containing maize for S. zeamais or wheat flour for T. castaneum as food, respectively. Insects were maintained in total darkness at a temperature of 25 ± 1 °C in a bioclimatic chamber (Memmert Gmbh, IPS 749, Schwabach, Germany).

Boldo leaves were washed and dried during 48 h at 40 °C. Then, they were ground in an electric coffee grinder (Moulinex ultra 505) and passed through a 20 mesh (0.841 mm) (Dual Manufacturing, Chicago, Illinois, USA) sieve to produce fine powder.

For S. zeamais, 100 g of maize were placed in 250mL jars (Silva et al., 2003) before adding the respective treatment and hand shaken 1 min to homogenously cover the grain prior to infestation with 20 insect couples not older than 10 d. Sex was determined with criteria proposed by Halstead (1963). After infestation, jars were transferred to a bioclimatic chamber (25 ± 1 °C, 60% HR). The evaluated treatments were 0.5, 1.0 and 2.0% w/w. All insects were removed 15 d after infestation (DAI) and percentage of mortality was registered and corrected for statistical analysis by Abbott's formula (Abbott, 1925). The maximum mortality level accepted for the untreated control was 10%. Insects were considered dead when they failed to move after being prodded gently with a needle for 30 s. Afterward, the percentage of adult emergence (F1) was determined at 55 DAI. Grain weight loss was determined based on the difference between initial (100 g) and final weight.

For T. castaneum, 10 g of wheat flour were placed in 7-cm Petri dishes, mixing the flour with boldo foliage powder at 0.5, 1.0, 2.0, 4.0, 8.0, and 16.0% w/w following the methodology by Chavez et al. (1992), and infesting each dish with 20 T. castaneum adults (unsexed 48-h-old). All dishes were located in a bioclimatic chamber at the same temperature as for S. zeamais. Mortality was recorded at 12 and 24 h and 3, 7, 14, and 21 DAI using the same criteria described above and corrected by Abbott's formula (Abbott, 1925).

Extracts. The aqueous extract was obtained with the methodology of Prates et al. (2003) by placing 10 g boldo powder in a jar with 100 mL distilled water at boiling point, covering, filtering after 24 h through Whatman N°10 filter paper, and discarding the solid part. The extract was considered as the 100% stock compound. The ethanol and hexane extracts were obtained via Soxhlet extraction during 12 h (Kamaraj et al., 2008) and concentrated with a rotary evaporator (Fisatom, Sao Paulo, Brazil).

For S. zeamais, 6-mL test tubes with a 1 mL solution of each extract in distilled water and 0.2 mL of Tween® 20 surfactant (Kouninki et al., 2007) were employed at 0.25, 0.5, 1.0, and 2.0% v/v; an untreated control with 1 mL distilled water and the surfactant was covered with plastic film. Tubes were manually agitated for 1 min to properly cover the inner surface. The aqueous content was eliminated and test tubes were allowed to dry at room temperature for 1 h. Then, 10 unsexed 48-h-old adult insects were placed in each tube. Treatments were kept in a bioclimatic chamber. Mortality was recorded at 12, 24, 48, and 72 h. In this bioassay every extract had a reference control consisting of a tube with its inner surface covered with the respective solvent and infested with insects.

For T. castaneum, 10 g wheat flour were mixed in 7-cm Petri dishes with 2 mL of each extract solution at 0.5, 1.0, 2.0, 4.0, 8.0, and 16.0% w/v and an untreated control with 1 mL distilled water; 2 mL Tween® 20 surfactant was added to each treatment (Jbilou et al., 2006). Each dish was infested with 20 unsexed 48-h-old T. castaneum adults. Mortality was recorded at 12, 24, 48, and 72 h after infestation and corrected by Abbott's formula (Abbott, 1925). As in the S. zeamais bioassay, every extract had a reference control consisting of a Petri dish with wheat flour mixed only with the respective solvent and infested with insects.

Experimental design and statistical analysis
Treatments had 10 replicates and bioassays were replicated three times on separate days with a completely randomized experimental design. The response variables were transformed to arcsine Vx/100 prior to ANOVA (a = 0.05) analysis with the SAS program (SAS Institute, 1990). Statistical differences were determined through Tukey tests (P < 0.05).


Effect of powder on S. zeamais
All powder treatments caused 100% mortality (Table 1). At 1.0 and 2.0% concentrations mortality was similar to the results obtained by Páez et al. (1990), Silva et al. (2003; 2005; 2006), and Cruzat et al. (2009). However, at 0.5%, we observed 100% mortality as compared to < 30% reported by these researchers. According to Pérez et al. (2007), differences in toxic potency are probably related to the field-collection date of plant material, and there was no emergence of F1 adults. In all treatments with powder, grain weight loss was < 0.5%, which is significantly lower (P < 0.05) than the untreated control (Table 1).

Table 1. Mortality, adult insect emergence (F1), and grain weight loss in maize treated with Peumus boldus powder at concentrations of 0.5, 1.0, and 2.0% (w/w) to control Sitophilus zeamais under laboratory conditions.

1Values within a column with the same letter are not significantly different according to Tukey test (P < 0.05).
2Mortality in control was corrected by Abbott's formula (Abbott, 1925).

Effect of powder on T. castaneum

The insecticidal effect of P. boldus powder against T. castaneum adults increased over time (Table 2). However, this species is less affected than S. zeamais for which concentrations of 8.0 and 16.0% lead to 100% mortality at 14 DAI. The reduced sensibility of T. castaneum to vegetable powders agrees with data found by Denloye et al. (2010), who obtained higher mortality with extracts (LC50 = 0.04 g L-1) than with powders (LC50 = 250 g kg-1) using Chenopodium ambrosioides L. (Chenopodiaceae). Peumus boldus powder is more potent against T. castaneum than other vegetable powders derived from plants such as Khaya senegalensis (Bamaiyi and Bolanta, 2006) or Punica granatum, and Murraya koenigii (Gandhi et al., 2010). Furthermore, plant powder gives a greenish color to the treated flour, thus affecting its quality for human consumption. This is why some researchers suggest not mixing powder with flour (Kordan et al., 2003; Husain and Hasan, 2008; Pugazhvendan et al., 2009).

Table 2. Mortality of Tribolium castaneum adults in flour wheat mixed with Peumus boldus powder at concentrations of 0.5, 1.0, 2.0, 4.0, 8.0, and 16.0% (w/w) under laboratory conditions.

1Values within a column with the same letter are not significantly different according to Tukey test (P < 0.05).
2Mortality in control was corrected by Abbott's formula (Abbott, 1925).

Sitophilus zeamais. The hexanic extract was the most toxic after 24 h exposure in all evaluated concentrations and caused 100% mortality (Table 3). The aqueous and ethanolic extracts reached 100% mortality at 72 h. Considering the polarity of used solvents (water is polar, ethanol has intermediate polarity, and hexane is non-polar), we inferred that the insecticidal properties of P. boldus extracts are due to polar and non-polar molecules because all extracts reached 100% mortality. It is probable that insecticidal properties of boldo extracts are due to a group of molecules with joint action similar to neem (Azadirachta indica J. [Meliaceae]) (Schmutterer, 1990). The highest toxicity of hexanic extract as compared to ethanolic extract agrees with Hincapie et al. (2008), who evaluated Annona muricata L. (Annonaceae) extracts at a 5% concentration and obtained 100% S. zeamais mortality with the hexanic extract, while the ethanolic extract under the same conditions cause no mortality (0%). We estimated that P. boldus has a higher insecticidal potency than other plant extracts such as Brassica napus (L.) (Brassicaceae) (Salem et al., 2007), Clerodendrum inerme L. (Verbenaceae), Withania somnifera L. (Solanaceae), Gliricidia sepia L. (Fabaceae), Cassia tora L. (Caesalpiniaceae), and Eupatorium odoratum L. (Asteraceae) (Yankanchi and Gadache, 2010). In these cases, at least a 5% concentration was needed to reach 80% mortality. Using Jatropa curcas L. (Euphorbiaceae) extracts, a 20% concentration was required to reach a 70 to 90% mortality (Asmanizar and Idris, 2012).

Table 3. Mortality of Sitophilus zeamais adults treated with Peumus boldus extracts in water, hexane, and ethanol at concentrations of 0.5, 1.0, and 2.0% (w/v) under laboratory conditions.

1Values within a column with the same letter are not significantly different according to Tukey test (P < 0.05).
2Mortality in control was corrected by Abbott's formula (Abbott, 1925).

Tribolium castaneum. Plant powder extracts also gave a greenish color to the treated flour. The aqueous extracts caused no mortality (0.0%) in all the evaluated concentrations. The hexanic extract killed 6.7% of treated individuals at 16.0% concentration (Table 4). The highest mortality (100%) was achieved by the ethanolic extract during 12 h exposure at concentrations > 2%. It seems that P. boldus is more toxic than other plant extracts such as Manilkara zapota (L.) (Sapotaceae) (Osman et al., 2011), Chrysanthemum spp. (Asteraceae) (Haouas et al., 2008), Hyptis spicigera Lam. (Labiatae) (Othira et al., 2009), Peganum harmala L. (Nitrariaceae) (Jbilou and Sayah, 2008), and Mantis alcaduriaei (Spach) Briq. & Cavill (Asteraceae) (Boussaada et al., 2008) in which only larval mortality was documented. Some researchers suggest that plant extracts are powerful repellents but not promising contact insecticides against T. castaneum (Kanvil et al., 2006; Saidana et al., 2007).

Table 4. Mortality of Tribolium castaneum adults treated with wheat flour mixed with Peumus boldus extracts in water1, hexane, and ethanol at concentrations of 0.5, 1.0, 2.0, 4.0, 8.0, and 16.0% (w/v) under laboratory conditions.

1The aqueous extracts caused no mortality (0.0%) in all the evaluated concentrations.
2Values within a column with the same letter are not significantly different according to
3Mortality in control was corrected by Abbott's formula (Abbott, 1925).


The foliage powder of Peumus boldus is more effective against Sitophilus zeamais as compared to Tribolium castaneum under laboratory conditions. The hexanic and ethanolic extracts are more effective against S. zeamais and T. castaneum than the aqueous extract. These results must be field-validated in order to evaluate their usefulness as a chemical tool against these insect pest species.



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Received: 2 February 2012.
Accepted: 3 June 2012.

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