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

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.


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).
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.
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 (Na 2 SO 4 ), 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 βcaryophyllene (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 LC 50 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 (LC 50 and LC 100 ).

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).According to the determined lethal concentrations (LC 50 and LC 100 ), 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 LC 50 values, which resulted in higher toxicities achieved at lower concentrations, leading to 50% mortality.LC 100 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.(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)  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 LC 50 and LC 100 values for mortality of R. dominica.
All studied oils in the fumigation bioassays demonstrate potential to control R. dominica.

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; χ 2 = 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.
Campos et al. (2014) of essential oils of C. pulegiodorus and O. basilicum on C. maculatus and Zabrotes subfasciatus Boh., 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.