Chemical composition and insecticidal activity of the essential oils of Piper marginatum , Piper callosum and Vitex agnus-castus

: During grain storage, a considerable amount of product is lost because of insects, such as Zabrotes subfasciatus . Currently, to mitigate these risks, studies are searching for plants with potential for the control of agricultural pests, also known as botanical insecticides. In this study, the fumigant toxicity of the essential oils of Piper callosum (PC-EO), Piper marginatum (PM-EO) and Vitex agnus-castus (VA-EO) against Zabrotes subfasciatus was investigated. The essential oils of PC-EO, PM-EO and VA-EO were analysed by gas chromatography (GC-MS), and the major components were 3,4-methylenedioxypropiophenone (10.4%), bicyclogermacrene (10.1%) and germacrene D (9.9%) for PM-EO; safrol (29.3%) for PC-EO; and 1,8-cineol (23.8%) for VA-EO. In fumigation tests, VA-EO killed 100% Zabrotes subfasciatus at a concentration of 0.004 µL/L air after 24 h of treatment, whereas PC-EO and PM-EO at 0.01 µL/L air caused 100% Z. subfasciatus mortality after 48 h. The VA-EO sample provided the lowest LD 50 after 24 h (0.17 µL/L air), followed by PC-EO (0.78 µL/L air) and PM-EO (1.17 µL/L air). These results demonstrate that the essential oils of these species can be an alternative to control pests in stored products. This is the fi rst report of the fumigant potential of these species against Z. subfasciatus .

The essential oils of these medicinal species have not yet been evaluated for their insecticidal potential against Zabrotes subfasciatus (Boheman, 1833) (Coleoptera: Bruchidae), the main pest of the bean Phaseolus vulgaris L. This species is one of the most produced and consumed beans in Brazil and is considered an important protein source in the diet of Brazilians (Oliveira et al. 1979). However, during grain storage, a considerable amount of product is lost because of insects, such as Z. subfasciatus.
Several populations of insects are resistant to the currently registered synthetic insecticides (Pimentel et al. 2010, Boyer et al. 2012) that consist largely of phosphines for fumigation and pyrethroids and organophosphates for preventive control. In Brazil, phosphine and pyrethroids are allowed to control Zabrotes subfasciatus in stored beans seeds and grains, according to the Ministério da Agricultura, Pecuária e Abastecimento (2020). However, they are toxic and dangerous to the environment. Phosphine is very toxic to all forms of animal life, hence exposure of human beings even to small amounts should be avoided. The effectiveness of phosphine can be reduced considerably by development of resistance in insects (Bond 1984).
Currently, studies are searching for chemical compounds in plants with potential for the control of agricultural pests, also known as botanical insecticides (Mazzonetto & Vendramim 2003, Nakano et al. 1981. The advantages of botanical insecticides compared with conventional insecticides are related to their lower mammalian toxicity and decreased health risk to applicators and their rapid degradability, which reduces residues in the environment and in the treated products (Isman 2006). In this respect, essential oils have been shown to be potentially active as insecticides of plant origin against Z. subfasciatus. After 12-h treatment, essential oils extracted from Chenopodium ambrosioides L. and Ocimum gratissimum L. at 20.0 mL/L of air killed 100% Z.

Extraction of essential oil
Fresh leaves (500 g) of P. marginatum and V. agnus-castus and aerial parts (leaves, branches and inflorescences) of P. callosum (500 g) were subject to hydrodistillation for a period of 6 h in a Clevenger-type apparatus. The essential oils were centrifuged for 10 minutes at 3500 rpm to separate the water. After centrifugation, the water was removed with a pipette. The samples were stored in an amber bottle and kept under refrigeration until GC-MS analysis and insecticidal assays. The yields of the extractions were calculated on the basis of the oil volume and the weight of the plant material used.

GC-MS analysis
One microlitre of the extracted oils, dissolved in hexane (5.0 µL/mL), was injected and analysed by gas chromatography/mass spectrometry (GC-MS) on a Shimadzu QP-2010. The analyses were performed using a DB-5MS column (30 m x 0.25 mm, with an internal film thickness of 0.25 µm). The analysis was performed in electron impact ionization mode. The injector and interface were set at a temperature of 250ºC while the oven was programmed with a temperature range from 60º to 240ºC (3ºC/min). Helium (99.999% purity) was used as the carrier gas at a flow rate of 1.3 ml/min. The identification of the constituents was based on the interpretation of mass spectra by comparison with the library database Wiley 7, NIST 08 and FFNSC1.2, calculation of the linear retention index and comparison with the literature (Adams 2007). The linear retention indices were calculated using a homologous series of n-alkanes (Van Den Dool & Kratz 1963). The structures were computer-matched with the spectral libraries, and their fragmentation patterns were compared with literature data.
The relative concentrations of the chemical components of essential oils were obtained by normalization of the peak areas (%).

Fumigant toxicity
The evaluation of insecticide activity was performed at FCA/UNESP-Botucatu under the supervision of Prof. Dr. Edson Luiz Lopes Baldin. The essential oils (PM-EO, PC-EO and VA-EO) were applied to a filter paper attached to the bottom of the screw cap of a 50-mL glass vial (fumigation chamber) at concentrations of 0, 0.002, 0.004, 0.006, 0.008 and 0.01 µL/L air. Four replications were performed for each treatment. Five adult one-day-old Z. subfasciatus couples and 10 g of beans were transferred to the vials. The percent adult mortality was recorded 24, 48, and 72 h after treatment.

Statistical analysis
The results were subjected to analysis of variance by the F Test, and the means were compared by the Tukey test (P≤0.05) using the statistical software SISVAR version 5.6. The LD 50 was determined by Probit analysis using Stat Plus 2007 Professional Build 4.7.5.0. The Abbott formula was used to calculate the control efficiencies (Abbott 1987).
The essential oils were obtained and analyzed by GC-MS to determine its composition. In the PM-EO sample, twentyfive compounds were identified, representing 81.8% of the total essential oil obtained, with predominance of hydrocarbon sesquiterpenes, followed by phenylpropanoids, hydrocarbon monoterpenes and oxygenated sesquiterpenes. The major compounds of PM-EO were 3,4-methylenedioxypropiophenone (10.4%), bicyclogermacrene (10.1%) and germacrene D (9.9%) ( Table I).

Fumigant toxicity of PM-EO, PC-EO and VA-EO
The VA-EO sample killed 100% of the insects at a concentration of 0.004 µL/L air after 24 h of treatment (Table II). However, PC-EO killed 100% of insects after 48 h of treatment at a concentration of 0.01 µL/L air. The PM-EO sample was the least toxic against Z. subfasciatus. At a concentration of 0.01 µL/L air, PM-EO killed 55% of the insects after 24 h of treatment and caused 97.5% mortality after 72 h. Essential oils of the three species showed toxicity against Z.  subfasciatus. After 24 h of treatment, the VA-EO sample had the lowest LD 50 (0.17 µL/L air), followed by PC-EO with an LD 50 of 0.78 and PM-EO with an LD 50 of 1.17 µL/L air (Table III).
The results obtained in the toxicity test against Z. subfasciatus were similar to those obtained with the essential oil of Chenopodium ambrosioides L., which killed 100% Z. subfasciatus   Houghton et al. 2006). Besides that, trans-cinnamaldehyde, eugenol, (-)-menthone and (-)-terpinen-4-ol monoterpenes were evaluated and showed a reduction in the progeny of Sitophilus oryzae after 45 and 90 days of treatment (Saad & Abdelgaleil 2018). Some of the purified terpenoid constituents of essential oils are moderately toxic to mammals, but, with few exceptions, the oils themselves or products based on oils are mostly nontoxic to mammals, birds, and fish. In addition, due to their volatility, essential oils have limited persistence in field conditions (Isman 2006). In turn, PM-EO and PC-EO contain the compounds 3,4-methylenedioxypropiophenone and safrole, respectively. These substances have the methylenedioxyphenyl group, characteristic of many other compounds derived from secondary metabolism of plants, as dillapiole occurring in P. aduncum, as well as piperine and piperolein existing in P. nigrum, traditionally used as botanical insecticides (Mukerjee et al. 1979, Scott et al. 2008). In addition, the phenylpropanoid safrole, major constituent of PC-EO, was responsible for the fumigant action on Sitophilus zeamais Motschulsky and Tribolium castaneum, stored grain pests (Huang et al. 1999). In addition, the complex mixture of chemical substances present in the studied essential oils can be directly related to the insecticidal activity, as they can act synergistically. Therefore, interest in plant products such as essential oils and their compounds has increased in recent years due to their fumigant action, as it is believed that natural compounds from plant sources may have advantages over conventional fumigants in terms of low toxicity in mammals, rapid degradation and regional availability. (Rajendran & Sriranjini 2008).

CONCLUSIONS
Essential oils of Piper marginatum, Piper callosum and Vitex agnus-castus showed fumigant toxicity on Z. subfasciatus, with VA-EO being the most toxic, followed by PC-EO and PM-EO. There was no oviposition in the treatments and exposure times evaluated. The results suggest that essential oils can be used as an environmentally friendly alternative to control pests in stored products. However, further studies are needed to determine which compounds may be responsible for such activities.