The lesser grain borer, Rhyzopertha dominica (Fabricius, 1792) (Coleoptera: Bostrichidae), is the main post-harvest wheat insect pest and has broad distribution in Brazil. However, a number of crops are attacked by this insect, such as wheat, barley, triticale, rice and oats. Both the adults and larvae of this beetle bore into the tegument of seeds, producing large amounts of powder and causing direct damage to the kernels (Faroni et al., 2004).
The preventive control of this agricultural pest is performed with inert powders, such as diatomaceous earth (Athanassioua et al., 2014), or conventional insecticides, such as deltametrin. Curative control is performed with fumigant agents, especially phosphine. However, the indiscriminate use of these forms of pest control are the main reason for the emergence of resistant populations, especially the use of deltametrin (Shahid et al., 2014) and phosphine (Pimentel et al., 2010).
Natural products, such as those derived from plants (powders, extracts and essential oils), constitute an alternative to conventional insecticides and have demonstrated repellent, anti-feeding and sterilizing activities as well as causing the death of the immature or adult phases of different arthropods (Cruz et al., 2014).
The broad-leafed pepper tree, Schinus terebinthifolius Raddi (Anarcadiaceae), is a medicinal plant native to Brazil, Paraguay, Uruguay and Argentina. This plant occurs in Brazil between the states of Pernambuco (Northeastern region) and Rio Grande do Sul (Southern region) (Lorenzi and Matos, 2002). In Pernambuco, this plant is widely distributed in fragments of the Atlantic Rainforest, especially forests on the coastal plains. The fruit is rich in essential oil and is used in local culinary practices as a substitute spice for black pepper (Bertoldi, 2006). There are reports in the literature of the broad biological activities of the essential oil from the broad-leafed peppertree, such as antibiotic (Silva et al., 2010), anti-inflammatory (Jain et al., 1995), antioxidant and anti-cancer (Bendaoud et al., 2010) properties. A previous study on the insecticidal potential of this essential oil demonstrated toxicity to mosquitoes that transmit malaria [Anopheles gambiae, A. Arabiensisand and Culex quinquefasciatus (Kweka et al., 2010)], dengue fever [Aedes aegypti (Silva et al., 2010; Santos et al., 2010)] and pests that attack stored grains [Hypothenemus hampei (Santos et al., 2013), Acanthoscelides obtectus, Zabrotes subfasciatus (Santos et al., 2007), Sitophilus oryzae and Tribolium castaneum (Mohamed and Abdelgaleil, 2008)]. Nascimento et al. (2012) report that limonene is the main component of oils from ripened and unripened S. terebinthifolius fruit and found that the unripened fruit oil was more active in fumigation tests than the ripened fruit oil against the two-spotted spider mite (Tetranychus urticae). However, there are no previous reports of the effect of the essential oil from this plant on R. dominica.
Continuing the systematic study on the insecticidal potential of aromatic plants that occur in fragments of the Atlantic Rainforest in the state of Pernambuco, Brazil, the aim of the present investigation was to determine the insecticidal action of essential oils from ripened and unripened S. terebinthifolius fruit against R. dominica. The insecticidal activity of the chemical constituents of these oils [limonene, (E)-nerolidol, α-terpineol, β-terpineol, terpinolene, α-pinene and β-pinene] was tested for each compound individually as well as in the form of blends.
MATERIALS AND METHODS
Collection of Plant Material
Fresh fruits were collected from a fragment of the Atlantic forest at an altitude of around 66.60 m, in the city of Recife state of Pernambuco (7°57’7.494”S - 34°53’39.708”W), Brazil, in June 2010. The plants were identified by Dr. Maria Rita Cabral Sales de Melo of the Biology Department of the Universidade Federal Rural de Pernambuco. A voucher specimen of the species was deposited in the Vasconcelos Sobrinho Herbarium of the Universidade Federal Rural de Pernambuco under number 49.259.
Isolation of the Essential Oil
The essential oils from fresh unripened (UFr; green color) and ripened (RFr; red color) fruits (100 g) were separately isolated using a modified Clevenger-type apparatus and hydrodistillation for 2 h. The oil layers were separated and dried over anhydrous sodium sulfate, stored in hermetically sealed amber glass containers and kept at low temperature (-5 °C) until the insecticide assays and analysis.
Chemicals
Monoterpenes and sesquiterpene used in the bioassay and to make up artificial blends of Schinus oils are showed in Table 1 and Figure 1. All compounds including eugenol used as positive control were purchased from Sigma-Aldrich (Brazil).
Table 1 Selected constituents selected identified in ripened and unripened fresh S. terenbinthifolius oils of the reported by Nascimento et al. (2012).
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Figure 1 Structure of selected constituents identified in S. terenbinthifolius oils reported by Nascimento et al. (2012).
Comparative toxicity of compounds
In order to evaluate the relationship between chemical structure and insect pest activity, six monoterpenes and one sesquiterpene present in the essential oil of S. terebinthifolius fruits were selected with the objective of evaluating their activities on R. dominica. The monoterpenes were initially divided into two groups of constitutional isomers. The first group consists of four isomers with the molecular formula C10H16, two monocyclic monoterpenes (Limonene and terpinolene) and two bicyclic monoterpenes (α-pinene and β-pinene). The second group of constitutional isomers consisted of α-terpineol and β-terpineol (C10H18O). In addition to these, the sesquiterpene (E)-nerolidol was selected.
Insecticide Assay
The adult specimens of Rhyzopertha dominica were taken from a research colony in continuous culture on wheat grain in the Agronomy Department of the Universidade Federal Rural de Pernambuco without previous exposure to any pesticide. The insects used for all experimental procedures were reared on Triticum monococcum grain at a temperature of 25 ± 5 °C, relative humidity of 65 ± 10% and a 12-h photophase.
Fumigant Assay
The fumigant method was the same used by Pontes et al. (2007). Glass recipients with a capacity of 1 L were used as test chambers, whit three replicates. One replicate consisted of 10 specimens of R. dominica. The oils were applied with an automatic pipette on a piece of filter paper (5 × 3 cm) attached to the underside of the recipient lid. The oil amounted ranged from 5 to 90 µL. Mortality was determined after 24, 48 and 72 h. Following exposure, the insects were then removed from the recipients, and were lightly touched with a brush in order to determine mortality. Those with no sign of movement were considered dead. The mortality data for each of the RFr and UFr oils were also analyzed with the Probit model using the POLO-PC software (LeOra, 1987) for the determination of the lethal concentration average (LC50) values, with 95% confidence levels determined for all experiments.
Fumigation Bioessays of Individual Compounds and Artificial Blends
To investigate the relative toxicity of monoterpenes and sesquiterpenes found in the S. terebinthifolius oils, fumigation bioessay was performed with the compounds described in Table 1. Tests were also performed with artificial blends comprising compounds selected on the bases of the ripened and unripened fruit oils at the same proportions identified through gas chromatography-mass spectrometry (Nascimento et al., 2012). Other blends were then prepared by the removal of one component at a time from the artificial blend to evaluate the role of each component in toxicity to R. dominica (incomplete artificial blend). The concentration used for each of the constituents was that led to ≥ 95% mortality of R. dominica (50 μL L-1 of air) for the oils from the ripened and unripened S. terenbinthifolius fruit after 72 h of exposure. The mortality data were submitted to analysis of variance and mean values were compared using Tukey’s test with 5% probability with the aid of the SAS program (SAS Institute, 2002).
RESULTS AND DISCUSSIONS
The Table 2 displays the estimated LC50 of R. dominica for the vapors of the ripened and unripened S. terenbinthifolius fruit as well as eugenol, which was used as the positive control. The susceptibility of the pest to the ripened and unripened fruit oils increased over time, with no significant differences between oils at each evaluation period (24, 48 and 72 h). Similar behavior was found when the pest was submitted to the vapors of the positive control, the toxicity of which increased over time. The ripened and unripened fruit oils were more effective than the positive control in the shortest evaluation time (24 h). This finding suggests that the greater activity of S. terenbinthifolius can be attributed to the chemical diversity of the oils, which may act through different active sites in the pest at the same time, leading to a faster, more effective response in comparison to the action of only one compound (eugenol) (Isman et al., 2011; Araújo et al., 2012). A possible explanation for the greater toxicity of the positive control against R. dominica beginning at 48 h resides in the polar nature of eugenol, which has slower evaporation, in comparison to the nonpolar nature of the oils, which are comprised mainly of monoterpene hydrocarbons. The findings suggest that the same toxicity found for the oils in each evaluation period (24, 48 and 72 h) can be attributed to the similarity in the chemical profile of the different oils (Nascimento et al., 2012).
Toxicity due to fumigation of the constituents selected from the S. terebinthifolius and artificial blends
A large portion of studies on the insecticidal activity of essential oils report only the action of the oil without establishing a relationship between toxicity and the chemical composition or attributing the activity of the oil only to the relative toxicity of its isolated constituents (Araújo et al., 2012; Born et al., 2012; Santos et al., 2013; Furtado et al., 2005). The scarcity of information on interactions among the chemical constituents and the proportions at which these compounds are found in essential oil has motivated our research group to investigate the degree of interaction among the chemical constituents individually at the same proportion at which the compounds are found in the S. terebinthifolius oil against R. dominica (Moraes et al., 2012). Figure 2 A displays the toxicity to R. dominica of the individual compounds selected from the ripened and unripened fruit oils of S. terebinthifolius at a concentration of 50 μL/L of air after 72 h of exposure.
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The numbers indicate full mixture missing the constituent noted: (1) limonene, (2) (E)-nerolidol, (3) α-terpineol, (4) β-terpineol, (5) terpinolene, (6) α-pinene, (7) β-pinene. Means corresponding to each treatment with different letters are significantly different from each other by the Tukey’s test (P≤0.05).
Figure 2 A. Mean toxicity caused by the oil and constituents of S. terebinthifolius ripened (RFr) and unripened (UFr) fruits to R. dominica. B. Mean toxicity caused by the RFr oil, Full Artificial Mixture (FAM) and selected blends of constituents. C. Mean toxicity caused by the UFr oil, FAM and selected blends. Concentrations were applied at levels equivalent to the fruit oils that showed mortality ≥ 95% (72h).
All compounds tested were toxic, except the sesquiterpene (E)-nerolidol, which did not differ significantly from the negative control. The non-oxygenated monoterpenes limonene, terpinolene, α-pinene and β-pinene were the most toxic, followed by the oxygenated monoterpenes α-terpineol and β-terpineol. Based on the relative toxicity of these terpenes, the presence of the hydroxyl group in the aliphatic ring for monocyclic monoterpenes resulted in a reduction in insecticidal activity in comparison to the other monoterpenes. This finding may be related to the greater volatility of non-oxygenated monoterpenes due to the lower molecular weight and weaker inter-molecular interactions in comparison to oxygenated monoterpenes (Moraes et al., 2012). Moreover, the lipophilic nature of the cuticular outer layer of the insect is more selective regarding substances with polar groups, such as the hydroxyl group found in α-terpineol and β-terpineol (López et al., 2005). The influence of hydroxyl in monoterpenes on the reduction of insecticidal activity has been reported for other groups of insects. Investigating the insecticidal effects of different oxygenated and non-oxygenated monoterpenes on larvae from the mosquito Aedes aegypt, Santos et al. (2010) found that the presence of hydroxyl was related to less activity.
The presence of dual bonds in monoterpenes seems to have considerable importance for certain groups of insects, such as dipterans (Santos et al., 2010). The toxicity of four monoterpenes with three insaturations (terpinolene, limonene, α-pinene and β-pinene) against R. dominica did not differ significantly. These findings suggest that the position of the exocyclic dual bond in the terpinolene and limonene isomers did not affect insecticidal activity against R. dominica. The same was found for the α-pinene and β-pinene isomers, as the activity of these compounds was independent of the position of the dual bond (endocyclic or exocyclic).
The results suggest that the insecticidal properties of the S. terenbinthifolius fruit oils against R. dominica may be attributed to the toxicity of the individual compounds. However, in a previous investigation, Moraes et al. (2012) found that the activity of an essential oil is not necessarily attributable to only the relative toxicity of each constituent, as it is also necessary to take into account possible interactions among the constituents and the proportions at which such constituents are found in essential oils. Thus, the fumigation bioassays were repeated with artificial blends prepared with the selected components at the proportions found in the ripened and unripened S. terenbinthifolius fruit oils (Nascimento et al., 2012).
Figure 2B displays the insecticidal activity of the complete artificial blends with all constituents of the ripened fruit oil (complete blend) as well as with the removal of one constituent at a time (incomplete oils). The toxicity differed between the complete and incomplete artificial blends. Considering the individual activity of the constituents used in the complete artificial blend, the same degree of insecticidal activity as that found for the ripened fruit oil may be explained by the synergic interaction among constituents with lower activity [(E)-nerolidol, α-terpineol and β-terpineol] and those with greater activity (limonene, terpinolene, α-pinene and β-pinene). Miresmailli and Isman (2006), report similar findings regarding the acaricidal activity of an artificial blend prepared with selected constituents of Rosmarinus officinalis oil. Figure 2 also shows that the absence of limonene and α-terpineol, followed by terpinolene and β-pinene, in the incomplete artificial blends led to a considerable reduction in the mortality rate of the agricultural pest, thereby suggesting that these constituents contribute significantly to the toxicity of the essential oil from the ripened fruit. Moreover, the individual role of these constituents in the activity of the ripened fruit oil did not depend on either the proportion of these compounds in the oil or individual activity. For instance, limonene, which is a major component of the ripened fruit oil (31.8%), and α-terpineol, which is a minor component of this oil (0.6%), exerted the same degree of insecticidal activity in the essential oil. Regarding the individual activity of the selected constituents, α-pinene, limonene, terpinolene and β-pinene demonstrated the greatest insecticidal activity against R. dominica, but the individual removal of these compounds did not alter the degree of activity of the incomplete artificial blend.
Figure 2C displays the toxicity of the complete and incomplete artificial blends prepared with the chemical constituents selected from the unripened fruit oil. Unlike what was found with the ripened fruit oil and its artificial blends, which had the same degree of toxicity against R. dominica, the complete artificial blend of the unripened oil did not reach the same degree of toxicity as the essential oil. This finding may be attributed to the different proportions of the compounds found in the ripened and unripened fruit oils as well as the absence of α-pinene in the complete artificial blend of the unripened fruit oil (Table 2). As found for the complete artificial blend of the ripened fruit oil, the absence of some constituents led to a considerable reduction in the mortality of the pest. The absence of limonene, followed by β-terpineol and terpinolene, suggests that these constituents contribute significantly to the toxicity of the unripened fruit oil. In contrast, the absence of (E)-nerolidol enhanced the degree of toxicity of the incomplete artificial blend in comparison to the unripened fruit oil, which suggests an antagonistic interaction between this component and other constituents of the complete artificial blend, as the absence of (E)-nerolidol led to the same toxicity found in the essential oil.
CONCLUSIONS
Independently of the qualitative and quantitative differences in the chemical constituents of the ripened and unripened fruits oils from the plant S. terenbinthifolius tested, no significant differences were found between the two oils regarding the insecticidal activity against the lesser grain borer (R. dominica). The results of the bioassays employed to evaluate the insecticidal activity of these oils suggest that such oils affect the respiratory system of this agricultural pest. However, the fumigant properties of the pure oils and artificial blends suggest that the action of these oils cannot be attributed only to the individual toxicity of each constituent, but rather that proportion and possible interactions (synergistic and antagonist) among the constituents. The present study demonstrates that it is possible to create a blend with different chemical constituents of essential oils for use in the control of agricultural pests.
ACKNOWLEDGMENTS
Our thanks to Conselho Nacional de Desenvolvimento Científico e Tecnológico and Fundação de Amparo à Ciência e Tecnologia do Estado de Pernambuco for scholarship and financial support (FACEPE; APQ-0476-1.06/14 and APQ- 10081.06/15, CNPq - universal 477778/2013-5, CNPq - PQ-2 302860/2016-9, APQ - 0860-1.06/16) and Dr Irenaeus Lorini and Dr. Paulo Roberto Valle da Silva Pereira, Embrapa, for giving insects sample to the start of the insect colony.