FUMIGATION TOXICITY OF ESSENTIAL OILS AGAINST Rhyzopertha dominica (F.) IN STORED MAIZE GRAIN

TOXICIDADE POR FUMIGAÇÃO DE ÓLEOS ESSENCIAIS SOBRE Rhyzopertha dominica (F.) EM GRÃOS DE MILHO ARMAZENADOS

ABSTRACT:

The Rhyzopertha dominica F. (Coleoptera: Bostrichidae) is a primary pest of stored grains in many regions of the world. In this work we evaluated the fumigant activity of essential oils of Ocimum basilicum L., Citrus aurantium L., Mentha spicata L. and Croton pulegiodorus Baill on adult R. dominica in stored maize. Tests were conducted to determine lethals concentrations (CL₅₀ and CL₁₀₀) and mortality (fumigation).The fumigation test was done in containers made of glass containing 10 individuals of R. dominica, where essential oils were applied at different concentrations: O. basilicum and M. spicata (5, 10, 15, 20, 30 and 40µL/L of air), C. aurantium (10, 20, 30, 40, 50 and 60µL/L of air) and C. pulegiodorus (0, 20, 30, 50, 70 and 90µL/L of air). After 48 hours of exposure to the oils the percentage of insect mortality was evaluated. According to LC₅₀ and LC₁₀₀ the toxicity of essential oils decreased in the following order: O. basilicum> M. spicata> C. pulegiodorus> C. aurantium. The essential oil of O. basilicum exhibited strong fumigant toxicity against R. dominica adults, with a LC₅₀ value of 17.67 µL/L air and LC₁₀₀ value of 27.15 µL/L air. The C. aurantium essential oil required higher concentrations than O. basilicum, M. spicata and C. pulegiodorus to kill insects. However, all oils evaluated presented fumigating property to promote the control of R. dominica and demonstrated potential use in the management of this coleoptera.

Keywords:
Botanical insecticides; Fumigant effect; Bostrichidae; Stored grains

RESUMO:

Rhyzopertha dominica F. (Coleoptera: Bostrichidae) é uma praga primária de grãos armazenados em muitas regiões do mundo. Este trabalho teve como objetivo avaliar a atividade fumigante dos óleos essenciais de Ocimum basilicum L., Citrus aurantium L., Mentha spicata L. e Croton pulegiodorus Baill. sobre adultos de R. dominica em grãos de milho armazenados. Para isso, testes para determinação das concentrações letais (CL₅₀ e CL₁₀₀) e mortalidade (fumigação) foram realizados em câmaras constituídas por recipientes de vidro, contendo 10 indivíduos de R. dominica, com óleos essenciais em diferentes concentrações: O. basilicum e M. spicata (5, 10, 15, 20, 30 e 40 µL/L de ar), C. aurantium (10, 20, 30, 40, 50 e 60 µL/L de ar) e C. pulegiodorus (0, 20, 30, 50, 70 e 90 µL/L de ar). Após 48 h de exposição aos óleos, avaliou-se a percentagem de mortalidade dos insetos. Com base nas CL₅₀ e CL₁₀₀, a toxicidade dos óleos essenciais decresceu na seguinte ordem: O. basilicum > M. spicata > C. pulegiodorus > C. aurantium. O óleo essencial de O. basilicum exibiu forte toxicidade por fumigação sobre adultos de R. dominica, com valores de CL₅₀ de 17,67 µL/L de ar e de CL₁₀₀ de 27,15 µL/L de ar. O óleo essencial de C. aurantium requereu concentrações mais altas que os óleos de O. basilicum, M. spicata e C. pulegiodorus para matar os insetos. Contudo, todos os óleos avaliados apresentaram propriedades fumigantes para controlar a R. dominica e demonstraram potencial de utilização no manejo deste coleóptero.

Palavras-chave:
Inseticidas botânicos; Efeito fumigante; Bostrichidae; Grãos armazenados

INTRODUCTION

Maize is very important as it constitutes a staple food for humans and animals, and is the most consumed cereal worldwide. Maize production is among the main agricultural activities practiced in Brazil (CONAB, 2012). However, many maize farmers are small-scale farmers who grow low-input crops by planting seeds saved in one year during the next, usually intercropped with other species. In this sense, it favors the incidence of pests that cause large losses by attacking at all stages of their cycle (NÉRI et al., 2005; OLIVEIRA et al., 2007).

Damage incurred by stored grain is definitive and unrecoverable, since pest feeding causes qualitative and quantitative losses, such as grain weight loss, decrease in nutritional value and germination capacity of seeds, and product devaluation (GALLO et al., 2002; LORINI, 2008; SCHEEPENS et al., 2011). Of the insect pests that affect maize during storage, Sitophilus zeamais Motsch. (Coleoptera: Curculionidae), Sitophilus oryzae L. (Coleoptera: Curculionidae), Rhyzopertha dominica F. (Coleoptera: Bostrichidae), and Ephestia kuehniella Zeller (Lepidoptera: Pyralidae) are responsible for physical, physiological, and sanitary deterioration (LORINI, 2008).

The R. dominica beetle is notable because it attacks various cereals and is considered one of the most destructive corn pests owing to its high incidence and the great difficulty in avoiding the losses that it causes to grains (LORINI, 2008). This insect is mainly controlled using synthetic insecticides (organophosphates or pyrethroids) and fumigation, phosphine being remarkable as the main fumigant used (LORINI; GALLEY, 1999; GONÇALVES et al., 2007). However, studies revealed phosphine resistance in Brazilian R. dominica populations (ATHIÉ et al., 2001; PIMENTEL et al., 2008), which could render the use of these products unviable if alternative methods are not implemented.

Recently, studies with powders, essential oils, and plant-based extracts have been developed in integrated pest management programs, mainly for the control of Callosobruchus maculatus (Coleoptera: Chrysomelidae), R. dominica, and S. zeamais (ALMEIDA et al., 2005; BRITO et al., 2006; NAIMA et al., 2013). In Brazil, there are various plant families with insecticidal action that can be used for their essential oils, such as, Piperaceae, Myrtaceae, Lauraceae, Euphorbiaceae, Verbenaceae, Lamiaceae, Rutaceae, and Poaceae (COITINHO et al., 2011).

The genera Ocimum and Mentha, belonging to the family Lamiaceae, have recently been investigated with regard to their insecticidal properties against diverse insect pests (KÉITA et al., 2001; KHALFI et al., 2006; LOPEZ et al., 2008; OGENDO et al., 2008; BENAYAD et al., 2012; ESMAILI et al., 2013). Similarly, essential oils of the genus Citrus, family Rutaceae, have been described to have insecticidal properties against insect pests of stored grains (PRATES et al., 1998; FOROUZAN et al., 2013; RINGUELET et al., 2014). Several studies involving the family Euphorbiaceae have been also conducted to evaluate its potential in essential oil production, the genus Croton being the most tested in controlling insect pests (BRITO, 2014; MAGALHÃES, 2014). However, there is still no information about the use of these essential oils against R. dominica in Brazil.

In this context, the present study aims to evaluate the insecticidal effect of essential oils of basil (Ocimum basilicum), bitter orange (Citrus aurantium), spearmint (Mentha spicata), and "velaminho" (Croton pulegiodorus) on R. dominica adults by fumigation of stored maize grains.

MATERIAL AND METHODS

The study was performed at the Entomology Laboratory of the Serra Talhada Academic Unit (UAST)/Federal Rural University of Pernambuco (UFRPE), Brazil, and the following steps were conducted:

a) Elimination of infestation and balance of grain moisture content - maize grains, 2013/14 crop season, from farmers of Serra Talhada, PE (07° 59′ 31″ S, 38° 17′ 54″ W, and 429 m), were used for rearing and bioassays with R. dominica. For this, the grains were cleaned (sieved), dried, and placed in plastic bags and kept in a freezer (-10 °C) for 7 days, to eliminate subsequent infestations of insects from the field. After this period, the grains were placed in glass flasks and kept at room temperature (28 ± 2 °C and 65 ± 5% moisture content).

b) R. dominica rearing - the insects were reared in maize grains at 30 ± 2 °C, 60% relative humidity, and 12-hour photoperiod, in biochemical oxygen demand (BOD) climatic chambers, placed in closed containers with perforated plastic lids and the inside lined with thin material to allow gas exchange. They remained confined for 7 days to induce oviposition and were then removed and the containers stored until emergence of the next generation. This procedure was performed for five generations to ensure the number of adults necessary to execute the experiments.

c) Essential oil obtainment - essential oils of basil (O. basilicum), bitter orange (C. aurantium), and spearmint (M. spicata) were acquired from the company Terra-Flor Indústria e Comércio de Aromaterápicos Ltda. Basil essential oil was obtained by the vapor distillation extraction method using the leaves; spearmint essential oil was obtained by vapor distillation using the stems and leaves; bitter orange essential oil was obtained by cold pressing the peels. The plant material used to produce essential oil of "velaminho" (C. pulegiodorus) was harvested at Triunfo, PE (07° 50′ 17″ S, 38° 06′ 06″ W, and 1004 m), Brazil. The species was identified by a specialist of the Brazilian Semiarid Herbarium (HESBRA), which was deposited under Voucher - S.S. Matos 104. "Velaminho" essential oil was extracted from fresh leaves by hydrodistillation using a modified Clevenger equipment, with 200 g in 3 L of distilled water for 2 hours. The obtained fractions were separated from water by dichloromethane and dried with anhydrous sodium sulfate (Na₂SO₄), and then placed in a rota-evaporator, where dichloromethane was removed, obtaining the essential oil.

Chromatography provided by the Terra-Flor company indicates that the basil essential oil exhibited estragole (72.67%) and linalool (22.41%) as major compounds. The spearmint essential oil mainly comprised menthol (35.20%), isomenthone (18.71%), and menthyl acetate (6.22%). Bitter orange essential oil exhibited limonene (90.78%) and myrcene (3.87%) as main compounds. According to Dória et al. (2010), "velaminho" essential oil had β-caryophyllene (20.96%), bicyclogermacrene (16.89%), and germacrene D (10.55%) as major components.

d) Fumigation - to evaluate the fumigant effect, glass containers (bomboniere) were used as fumigation chambers (1 L), where 10 R. dominica adults were confined. Preliminary tests were conducted with all essential oils, in which several concentrations were evaluated, including intermediaries, with less replicates, and the most promising were used in the definitive tests. Based on the preliminary tests, essential oils were tested at the following concentrations: basil (5, 10, 15, 20, 30, and 40 µL/L of air); bitter orange (10, 20, 30, 40, 50, and 60 µL/L of air); spearmint (5, 10, 15, 20, 30, and 40 µL/L of air), and "velaminho" (10, 20, 30, 50, 70, and 90 µL/L of air). A porous fabric (tulle) was placed between the lids and respective containers themselves to avoid direct contact of the insects with the oils. The containers were sealed with plastic film (PVC) and adhesive tape in order to prevent vapors from escaping. The experiment was conducted in completely randomized design, with six treatments (concentrations in μL/L of air) and 10 replicates, in which insect mortality was evaluated after 48 hours, following the method proposed by Coitinho et al. (2011). The toxicity ratio (TR) was obtained using the quotient between the LC₅₀ of the essential oil that exhibited lowest toxicity and the LC_50s of the remaining essential oils. The results were subjected to analysis of variance using the ASSISTAT program and probit analysis using the STATPLUS program to obtain the lethal concentrations (LC₅₀ and LC₁₀₀).

RESULTS AND DISCUSSION

Basil essential oil led to an increase in mortality with increasing concentration, with 100% mortality observed for 30 and 40 µL/L of air (Figure 1A). Bitter orange essential oil caused up to 45% mortality at the highest concentration (Figure 1B). Spearmint led to a linear increase in mortality until the 20 µL/L of air concentration, with 70% insect mortality (Figure 1C). "Velaminho" essential oil also showed an increase in mortality with increasing concentrations, with 74 and 80% mortality at the higher concentrations (Figure 1D).

Figure 1:
Mortality of Rhyzopertha dominica following fumigation with different concentrations of the essential oil of Ocimum basilicum: y = 3.0262x - 7.4043 and R² = 0.9131 (A); Citrus aurantium: y = 0.6393x - 0.4643 and R² = 0.7673 (B); Mentha spicata: y = 1.2683x - 15.401 and R² = 0.6097 (C); and Croton pulegiodorus: y = 0.9009x + 5.5374 and R² = 0.9716 (D).

According to the determined lethal concentrations (LC₅₀ and LC₁₀₀), toxicity of the essential oils decreased in the following order: O. basilicum > M. spicata > C. pulegiodorus > C. aurantium (Table 1). The essential oils of O. basilicum and M. spicata exhibited the lowest LC₅₀ values, which resulted in higher toxicities achieved at lower concentrations, leading to 50% mortality. LC₁₀₀ followed the same principle, with the highest toxicities achieved for the essential oils of O. basilicum and M. spicata, exhibiting the lowest concentrations, leading to 100% mortality. The toxicity ratio (TR) values were between 3.93 and 1.42 (Table 1). All tested essential oils exhibited potential to cause mortality in R. dominica; however, the essential oils of C. pulegiodorus and C. aurantium required higher concentrations to reach this goal.

Table 1:
Toxicity (fumigation) of essential oils of Citrus aurantium, Croton pulegiodorus, Mentha spicata, and Ocimum basilicum against Rhyzopertha dominica in stored maize grains.

• N = number of individuals; SEM = standard error of the mean; CI = confidence interval; TR = toxicity ratio; χ² = chisquare; P = probability.

Several studies with essential oils from species of the genus Ocimum exhibited satisfactory results regarding their insecticidal effect against insect pests. Kéita et al. (2001) evaluated the fumigant effect of O. basilicum and O. gratissimum in controlling C. maculatus and obtained 80 and 70% mortality with 25 µL, respectively. Rozman et al. (2007) reported toxicity against Tribolium castaneum Herbst (Coleoptera: Tenebrionidae), R. dominica, and S. oryzae in fumigation with linalool. For R. dominica, linalool was highly effective and caused 100% mortality at the lowest used concentration (0.1 mL/720 mL of volume). Ogendo et al. (2008) obtained 98, 99, and 100% mortality, respectively, against R. dominica, Oryzaephilus surinamensis L. (Coleoptera: Silvanidae), and Callosobruchus chinensis L. (Coleoptera: Chrysomelidae), in using 1 μL/L of air of O. gratissimum essential oil.

Relevant results have been described for Mentha essential oils in controlling diverse pests of stored products. Khalfi et al. (2006) observed an insecticidal effect of the composition of M. spicata essential oil in different proportions of 1.8 cineol and carvone against R. dominica. The results indicated that insecticidal activity of this essential oil led to a synergistic effect of both compounds, since composition of the essential oil differed depending on the extraction time.Benayad et al. (2012) evaluated the chemical composition and insecticidal effect of essential oils of M. suaveolens and M. pulegium against S. oryzae and R. dominica. The essential oils were very toxic for the two Coleoptera species within the first 24 hours, with 100% mortalities when 50 μL and 12 μL were used, respectively.

The essential oils of bitter orange and "velaminho" exhibited higher concentrations than did the other oils used to cause mortality in R. dominica. Essential oils from fruit peels of some citrus species were reported to have insecticidal properties against insect pests of stored grains. Prates et al. (1998) evaluated cineol (from Eucalyptus spp.) and limonene (from Citrus spp.) to determine possible fumigant activity, contact, and ingestion against R. dominica and T. castaneum. These authors observed that limonene was more effective against T. castaneum than R. dominica. The toxic effects of these substances are mediated by penetrating the insect body through the respiratory system (via a fumigant), the cuticle (by contact), or the digestive system (via ingestion). Forouzan et al. (2013) evaluated the potential of essential oil from Citrus reticulata peels against R. dominica during different exposure periods and concluded that this essential oil was indicated as a good fumigant against adults of this insect.

Brito (2014) evaluated the effect of essential oils of C. pulegiodorus and O. basilicum on C. maculatus and Zabrotes subfasciatus Boh. (Coleoptera: Chrysomelidae) at different concentrations (0.0, 5, 10, 15, and 20 μL/L of air). According to this author, the fumigant effect of O. basilicum caused 100% mortality in C. maculatus, but there was no significant difference for the concentrations of C. pulegiodorus essential oil. According to the same author, 100% mortality in Z. subfasciatus was observed for all used concentrations of both essential oils. Using essential oils of C. pulegiodorus and O. basilicum against S. zeamais and T. castaneum, Magalhães (2014) observed 90% mortality in S. zeamais at 20 μL concentration of C. pulegiodorus essential oil. On the other hand, there was no significant difference between the tested concentrations against T. castaneum. Campos et al. (2014) observed 100% mortality in C. maculatus adults using essential oil of Baccharis articulata.

Insect mortality is only one of the effects to be achieved in pest control with insecticidal plants (HUANG et al., 2002; KETOH et al., 2005). According to these authors, these plants also affect feeding, oviposition, and growth, and lead to decrease in the emergence of adults, among other effects not occurring with conventional insecticides, which are usually more toxic and cause insect mortality only.

CONCLUSIONS

Essential oils of O. basilicum, C. pulegiodorus, and M. spicata, at the highest concentrations, cause mortality higher than 70% in R. dominica, whereas the essential oil of C. aurantium causes mortality lower than 45%.

Oils of O. basilicum and M. spicata exhibit the lowest LC₅₀ and LC₁₀₀ values for mortality of R. dominica.

All studied oils in the fumigation bioassays demonstrate potential to control R. dominica.

ACKNOWLEDGEMENTS

The authors would like to thank UFRPE, FACEPE and CNPq for financial support.

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• 2
  Paper extracted from the masters thesis of the first author.

Publication Dates

• Publication in this collection
  Apr-Jun 2016

History

• Received
  09 May 2015
• Accepted
  06 Feb 2016