Lethal and sublethal effects of essential oils from Piper capitarianum Yunck and Piper krukoffi i Yunck on Plutella xylostella L.

: Plutella xylostella (L.) is responsible for considerable vegetable crop losses in the metropolitan region of Manaus, Brazil. In recent decades, essential oils have been investigated as an alternative to synthetic insecticides. The genus Piper is widely distributed in Amazonia and essential oils from these plants have insecticidal properties. This study describes the chemical composition of the essential oils from Piper capiterianum and Piper krukoffi i as well as the lethal and sublethal effects on P. xylostella . The phytotoxicity of the oils on the host plant was also evaluated. Globulol was the major constituent of the P. krukoffi i oil and o -cymene was the major constituent of the P. capitarianum oil. The oil from P. capiterianum exhibited greater toxicity to larvae and eggs. This oil also presented greater repellant action, feeding deterrence and mild phytotoxicity to the host plant ( Brassicae oleraceae ). The fi ndings suggest that this oil can be used in the preparation of a formulated insecticide for the management of P. xylostella in different development phases. However, further studies are needed to evaluate the effect of this oil on crops under fi eld conditions as well as non-target organisms and determine the cost-benefi t ratio of a product formulated with P. capitarianum oil.


INTRODUCTION
The diamondback moth, Plutella xylostella (L.) (Lepidoptera: Plutellidae), is the major pest of Brassica (cruciferous) crops throughout the world (Zalucki et al. 2012).This cosmopolitan pest is distributed from the cold mountains of Himalaya to hot, dry regions of Ethiopia (Mohan et al. 2009).It also occurs throughout the entire country of Brazil (Castelo Branco & França 2015).In 2012, the total annual cost related to the management of P. xylostella for the protection of cruciferous crops was US$ 4 to 5 billion and this fi gure was US$ 17 million in Brazil alone (Zalucki et al. 2012).
According to the Brazilian Seed and Seedling Association, the Brazilian production of cabbage surpassed 1.4 million tons in 2017/2018.However, crop losses are quite high in some locations of the northern region of the country due to attacks from agricultural pests, especially P. xylostella in the São Francisco/ Terra Nova farming community, which is located in the metropolitan region of Manaus in the state of Amazonas, Brazil.The main form of pest control in this community consists of the application of synthetic insecticides, such as chloranthraniliprole, cyantraniliprole, chlorfenapyr and deltamethrin, the latter of which is less costly for small farmers.However, the indiscriminate use of these products has caused serious harm to non-target organisms, such as natural enemies, and has led to the emergence of resistant pest populations (Moraes & Marinho-Prado 2016).The insecticides used in Brazil include chlorantraniliprole, cyantraniliprole, chlorfenapyr and deltamethrin, for which there are reports of resistant populations of P. xylostella (Ribeiro et al. 2017, Lima Neto et al. 2016, Oliveira et al. 2011).In recent decades, the use of plant-based insecticides has become an ecologically viable alternative to synthetic products.Such products can be obtained from the leaves, flowers and stems of plants and used in the form of powders, extracts and essential oils, the effectiveness of which has been proved in several studies (de Melo & da Camara 2019, Bandeira et al. 2013, Silva et al. 2018).
Brazil is the country with the greatest vegetal genetic diversity and is home to 30% of all the tropical forests on the planet (Lewinsohn & Prado 2005).According to Maia & Andrade (2009), among the 280 medicinal plant species cataloged from the Amazon, approximately 40% belong to the family Piperaceae.The genus Piper is one of the largest in the family, with 2000 species encountered in tropical and temperate regions in both hemispheres (Quijano-Abril et al. 2006).Among the 290 species of Piper found in Brazil, 137 have been recorded for the state of Amazonas (AM) (Guimarães et al. 2015).Plants of this genus stand out for their production of essential oils, amides and phenylpropanoids, which have insecticidal properties that affect hemipterans (Piton et al. 2014), lepidopterans (Lima et al. 2009), dipterans (Santana et al. 2015) and coleopterans (Pereira et al. 2008).Among the species that occur in the Amazon, Piper krukoffii Yunck and Piper capitarianum Yunck stand out by its broad distribution and biological properties, such as antioxidant activity and larvicidal action against the mosquito Aedes aegypti (L.) (Diptera: Culicidae) (da Silva et al. 2011, França 2015).However, no previous study has evaluated the lethal and sublethal effects of essential oils from the leaves of these species on P. xylostella, which is an important agricultural pest that affects cruciferous crops in the community of São Francisco/Terra Nova in metropolitan Manaus, Brazil.
Giving continuity to the chemical and biological study of essential oils from aromatic species with insecticidal potential, the aim of the present study was to determine the chemical composition of the essential oils from the leaves of P. krukoffii and P. capitarianum collected from the Amazon biome and evaluate the effects on P. xylostella in terms of mortality (eggs and larvae), feeding deterrence and repellent action to enable the formulation of a plant-based insecticide containing these Piper oils as the main ingredient.The phytotoxicity of the oils to the host plant was also investigated.The results were compared to those obtained with commercial plant-based (Azadirachtin) and synthetic (Deltamethrin) insecticides used as positive controls.

Isolation of essential oils
Essential oils from the leaves of P. krukoffii (100 g) and P. capitarianum (100 g) were obtained by hydrodistillation using a modified Clevenger apparatus for 4 h.The oil layers were separated and dried over anhydrous sodium sulfate, stored in hermetically sealed glass containers and kept at a low temperature (-5 °C) until analysis and the assays.Total oil yields were expressed as percentages (g/100 g of fresh plant material).All experiments were carried out in triplicate.

Chemicals
The chemicals used as standards for the identification of volatile compounds in the oils were purchased from Sigma-Aldrich (Brazil).Deltamethrin (Decis ® 25 g i.a./L EC Bayer CropScience) and azadirachtin (Azamax ® 12 g i.a./L EC E.I.D. Parry) were acquired from the local market and used as positive controls.

Gas chromatography fid analysis
Gas chromatography (GC) was performed using a Hewlett-Packard 5890 Series II GC apparatus equipped with a flame ionization detector (FID) and a non-polar DB-5 fused silica capillary column (30 m x 0.25 mm x 0.25 μm film thickness) (J and W Scientific).The oven temperature was programmed from 60 to 240 °C at a rate 3 °C min -1 .Injector and detector temperatures were 260 °C.Hydrogen was used as the carrier gas at a flow rate of 1 ml min -1 in split mode (1:30).The injection volume was 0.5 μL of diluted solution (1/100) of oil in n-hexane.The percentage of each compound was obtained from GC-FID peak areas in the order of the DB-5 column elution and expressed as the relative percentage of the area of the chromatograms.The analysis were performed in triplicate.

Gas chromatography-mass spectrometry (gcms) analysis
The GC-MS analysis of the essential oils was carried out using a Varian 220-MS IT GC system with a mass selective detector mass spectrometer in EI 70 eV with a scan interval of 0.5 s and fragments from 40 to 550 Da. fitted with the same column and temperature program as used for GC-FID with the following parameters: carrier gas = helium; flow rate = 1 ml min -1 ; split mode (1:30); injected volume = 1 μL of diluted solution (1/100) of oil in n-hexane.

Identification of components
Identification of the components was based on GC-MS retention indices with reference to a homologous series of C 8 -C 40 n-alkanes calculated using the van Den Dool and Kratz equation (van Den Dool & Kratz 1963) and by computer matching against the mass spectral library of the GC-MS data system (NIST version 14 and WILEY version 11), co-injection with authentic standards and other published mass spectra (Adams 2007).Area percentages were obtained from the GC-FID response without the use of an internal standard or correction factors.

Acquisition and rearing of Plutella xylostella
Specimens of P. xylostella were originally collected from collard greens (Brassica oleracea var.acephala) in 2015 in the municipality of Recife, state of Pernambuco, Brazil (08º 01'08.3"S 34º 56' 45.5" W) and maintained at the Laboratory for the Chemical Investigation of Natural Insecticides of UFRPE, Brazil, with approximately 60 generations having occurred by 2019.The moths were reared at a temperature of 25 ± 1 ºC, relative humidity of 65 ± 5% and a 12-h photoperiod and without any exposure to insecticides.The breeding method was adapted from Bandeira et al. (2013).

Larvicidal assay
The residual effect bioassays were based on the method described by Bandeira et al. (2013).Experiments were performed with open Petri dishes (10 cm diameter).Leaf discs (2.5 cm diameter) cut from collard greens were immersed for 30 seconds in the solutions prepared with essential oil, diluted in the solvent to dissolve (WPDA = distilled Water + 1.0% Polyoxyethylene sorbitan monolaurate + 0.1% Dodecylbenzenesulfonic Acid), using the immersion method and allowed to dry on a paper towel at room temperature for 30 minutes.Ten third instar P. xylostella larvae were placed on each dish.The experimental design was entirely randomized, totaling 120 larvae per treatment.The concentrations ranged from 0.0035 to 1.90 mg ml -1 (P.capitarianum), 1.02 to 24.50 mg mL -1 (P.krukoffii), 0.003 to 0.200 mg mL -1 (Deltamethrin) and 0.003 to 0.300 mg mL -1 (Azadirachtin).Mortality was recorded after 48 hours of exposure.Specimens with no sign of movement were considered dead.Negative control disks were only immersed in the WPDA solvent.

Ovicidal assay
The ovicidal bioassay was the same as that employed by Zago et al. (2010).Ten newly emerged adult males and females in pairs were placed in screened recipients containing leaf disks (2.5 cm diameter) of collard greens for oviposition.At six-hour intervals, the leaf disks were removed from the recipients.Thirty eggs were counted and the remaining eggs were removed.Leaf disks with 30 eggs were immersed for 30 seconds in different concentrations of the essential oils and positive controls (Azamax ® and Deltamethrin) diluted in WPDA solvent.The concentrations ranged from 0.01 to 0.25 mg ml -1 (P.capitarianum), 0.5 to 6.0 mg mL -1 (P.krukoffii), 0.005 to 0.25 mg mL -1 (Azadirachtin) and 0.003 to 1.5 mg mL -1 (Deltamethrin).Negative control disks were only immersed in the WPDA solvent.After drying at room temperature for 30 minutes, the leaf disks with the eggs were placed on filter paper on sponge saturated with water in plastic trays and kept in a climatic chamber (BOD MA 403) at 25 ± 1 o C and 70 ± 10% relative humidity.Egg viability was evaluated 96 hours after exposure to the substances by counting the number of hatched larvae.

Antifeedant bioassays
The feeding deterrence method was adapted from Akhtar et al. (2012).Third instar P. xylostella larvae were transferred to Petri dishes and deprived of food for four hours prior to the experiments.Collard leaf disks (2.0 cm diameter) were immersed for 30 seconds in the solutions prepared with essential oil and positive control, diluted in WPDA solvent and allowed to dry on a paper towel at room temperature.Control disks were only immersed in distilled water.The concentrations ranged from 0.01 to 0.35 mg ml -1 (P.capitarianum), 1.2 to 9.59 mg mL -1 (P.krukoffii) and 0.01 to 0.97 mg mL -1 (Azadirachtin).After drying, a treated disk and control disk were placed at a distance of 2.0 cm in each Petri dish.A larva was placed in the center of the Petri dish between the two leaf disks and allowed to feed for 24 h.Thirty repetitions were used for each treatment, with each repetition consisting of one larvae.After 24 h of exposure, the larvae were removed.The areas of the leaves consumed in the control and treatment disks were determined with the aid of the Licor-3100 leaf area meter, which has high accuracy and repeatability, with reading resolution ranging from 0.1 to 1 mm 2 .The feeding deterrence index (FDI) was calculated as follows: FDI = 100{(C -T) / (C + T)}, in which C and T are the areas consumed on the control and treated disks, respectively.Cienc (2022) 94(2) e20200072 5 | 19 The results were compared to those obtained with the positive control (Azadirachtin).

Larval repellency bioassay
The larval repellency bioassay was adapted from Lobo et al. (2019).Collard leaf disks (2.0 cm diameter) were immersed for 30 seconds in the solutions prepared with sublethal concentrations of the essential oils and positive control diluted in WPDA solvent.Control disks were only immersed in distilled water.The material was set to dry on paper towels at room temperature for 30 minutes.After drying, a treated disk and control disk were placed at a distance of 2.0 cm in each Petri dish.A third instar P. xylostella larvae was placed in the center of the Petri dish between the two leaf disks.Thirty repetitions were used for each treatment, with each repetition consisting of one Petri dish containing one larva.The repellent effect was evaluated 1, 2, 4, 6, 12 and 24 hours after the onset of the experiment, with the recording of the number of P. xylostella larvae on the treatment and control disk leaves.The sublethal concentrations for the evaluation of the repellency index were 0.02, 0.04, 0.06 and 0.07 mg ml -1 (P.capitarianum), 1.20, 1.74, 2.23 and 2.72 mg ml -1 (P.krukoffii) and 0.007, 0.010, 0.015 and 0.020 mg ml -1 (Azadirachtin).
The repellency index (RI) was calculated as follows: RI = 2G / (G + P), in which G is the % of larvae found on the leaf disks treated with the essential oil or positive control and P is the % of larvae on the control leaf disks.The RI ranges from 0 to 2. RI = 1 denotes neutral action, RI > 1 denotes attraction and RI < 1 denotes repellency.As the margin of safety for this classification, the standard error (SE) of each treatment was added to or subtracted from 1.00 (index indicative of neutrality).Thus, each treatment was only considered repellent or attractive when the RI was outside the 1.00 ± SE range (Mazzonetto & Vendramim 2003).A repellency intensity scale based on Bustos et al. (2017) was used for the classification of the degree of repellency of the essential oils and positive control to P. xylostella larvae (0.76-0.99 = weak; 0.51-0.75= moderate; 0.26-0.50strong; 0.00-0.25 very strong).

Phytotoxicity test
The method for the phytotoxicity test was adapted from Torres et al. (2006).Collard leaf disks (5 cm diameter) were immersed for 10 s in the essential oils diluted in WPDA solvent and set to dry at room temperature.After 48 h, the phytotoxicity index (PI) of each leaf disk was evaluated with the aid of the AFSoft program.The images were analyzed using criteria of the phytotoxicity scale proposed by Alvez et al. (1974): 0.00 to 4.90% = slight; 5.00 to 14.99% = mild; 15.00 to 29.99% = acceptable; 30.00 to 39.99% = borderline acceptable; 40.00 to 100.00% = severe.The PI was calculated using the following formula: PI = TA% -SA%, in which TA is total area and SA is the area of sound (unaffected) leaf.The phytotoxic assessment was performed with the greatest concentration of essential oil and positive control (Azadirachtin and Deltamethrin) used in the toxicity bioassays.

Statistical analysis
To estimate the curve slopes, the results of the larvicidal and ovicidal assays, LC 50 (lethal concentration) and FDI 50 (lethal concentration) of each Piper oil and positive control were submitted to PROBIT analysis (Finney 1971) using SAS software (version 9.0) (SAS 2002).The concentrations were calculated based on the logarithmic series proposed by Robertson et al. (2017).The data from the repellency bioassays were submitted to analysis t-test using PROC TTEST SAS, with the means compared by the X 2 estimated using the Statistical Analysis System software (SAS 2002).

Chemical analysis and identification of constituents of essential oils
The yields and percentages of the chemical constituents identified in the Piper oils are displayed in Table I.Hydrodistillation of the leaves of the two species analyzed provided yellowish oils with a citric aroma.The yields were 0.49 ± 0.05% for P. krukoffii and 0.23 ± 0.02% for P. capitarianum.

P. Xylostella larvicidal and ovicidal bioassay
Table II displays the mean lethal concentrations (LC 50 ) of the essential oils from the leaves of P. krukoffii and P. capitarianum and the positive controls (Azadirachtin and Deltamethrin) for P. xylostella larvae and eggs.The susceptibility of the pest varied in accordance with the plant species from which the oil was extracted and the development phase of the pest.The P. capitarianum oil (LC 50 for larvae = 0.21 mg mL -1 and eggs = 0.079 mg mL -1 ) was respectively 30.3-fold and 33.9-fold more toxic to larvae and eggs than the P. krukoffii.Moreover, based on the LC 50 , eggs were respectively 4.60-fold and 3.44-fold more susceptible to the P. capitarianum and P. krukoffi oils than third instar larvae.These results show that the essential oils from P. capitarianum and P. krukoffi are promising as active ingredients in the formulation of a natural insecticide for the control of the different developmental forms of P. xylostella.However, it is necessary to test of the constituents, found in the oils, separately or in the form of mixtures.
In the comparison of relative toxicity, both Piper oils were less toxic than the positive controls (Azadirachtin and Deltamethrin) to the P. xylostella larvae.Regarding ovicidal activity, only the P. capitarianum oil had the same effectiveness as Deltamethrin, whereas Azadirachtin had greater ovicidal action than both Piper oils.

Feeding deterrence and larval repellency bioassay
The P. capitarianum and P. krukoffii oils applied to the collard leaves were capable of reducing the feeding of the P. xylostella larvae at sublethal concentrations (Table III).The P. capitarianum oil exhibited greater deterrent action, reducing the feeding of the P. xylostella larvae 46-fold more than the P. krukoffii oil.Moreover, the P. capitarianum oil was approximately twofold more efficient than the plant-based commercial Regarding the repellent action, the P. capitarianum oil exhibited high to moderate degrees of repellency at 0.02, 0.04, 0.06 and 0.07 mg mL -1 to 3 rd instar larvae of P. xylostella in the first 12 hours after exposure to the oil.After 24 hours, the larvae began to be attracted to the P. capitarianum oil (RI > 1) (Figure 1).The P. krukoffii oil at 1.20, 1.74 and 2.23 mg mL -1 was repellent only in the first hour after application.At 2.72 mg mL -1 , however, this oil was repellent for 12 h (Figure 2).The positive control Azadirachtin was attractive to the larvae throughout the evaluation period at all concentrations tested (0.007, 0.010, 0.015 and 0.020 mg ml -1 ) (Figure 3).

Phytotoxicity bioassay
Table IV displays the phytotoxicity of the essential oils to collard greens (Brassica oleraceae var.acephala).Based on the phytotoxicity scale proposed by Alvez et al. (1974), the P. capitarianum and P. krukoffii respectively exhibited mild (9.83%) and acceptable (16.68%) toxicity to the host plant.Considering the positive controls, the essential oils from P. capitarianum and P. krukoffii were less phytotoxic than the synthetic insecticide.The commercial insecticides Deltamethrin (synthetic) and Azadirachtin (plantbased) were respectively 6.93-fold and 3.72fold more phytotoxic than the P. capitarianum oil.Moreover, the P. krukoffii was respectively 4.01-fold and 2.12-fold less phytotoxic than Deltamethrin and Azadirachtin.

DISCUSSION
The yield for the fresh leaves of P. krukoffi i oil investigated herein was much lower than that reported by da Silva et al. (2011) for leaves of this species collected in the state of Pará (collection in February, with leaves air dried and three hours of hydrodistillation).This difference may be related the method and other factors involved in the production and accumulation of essential oils in plants (Lima et al. 2003).
The greater toxicity to the eggs and larvae found for the P. capitarianum oil compared to the P. krukoffii oil may be explained by qualitative and quantitative chemical differences between the two oils, as demonstrated by GC-MS.Moreover, P. xylostella was more susceptible to both oils in the egg phase than in the larval phase.In contrast, Sangha et al. ( 2017) found that the oil from P. nigrum was more toxic to the larval phase than the egg phase of P. xylostella.The greater susceptibility of the egg phase in the present investigation may be explained by the physical effect on the eggs.While the oils acted on the larvae through residual contact (affecting target sites after the penetration of the larval tegument), the action on eggs was through direct contact, as the eggs were immersed in an aqueous solution of the oil, affecting not only target sites in the embryo, but also forming an oily layer on the surface of the egg that served as a barrier impeding the exchange of gases between the embryo and the external environment (Krinski et al. 2018).However, other factors should be considered when evaluating differences in susceptibility between the larval and egg phases of P. xylostella, such as the chemical profile of the oil, volatility, degree of lipophilicity and the capacity to form a film on the egg surface.
The comparison of the LC 50 estimated for the Piper oils investigated herein on 3 rd instar larvae of P. xylostella and values reported for oils from other plants reveals that the oil from P. capitarianum is more efficient that oils from Mentha longifolia L. Huds., Mentha piperita L., Mentha spicata L., Cymbopogon flexuosus Steud.and Curcuma aromatica Salisb (Koundal et al. 2018).In studies on larvicidal action against P. xylostella, the toxicity of the essential oil from Allium tuberosum L. (LC 50 = 0.56 µl mL -1 ) was 2.66fold lower (Gao et al. 2019) and the toxicity of the essential oil from Zingiber officinale Roscoe (LC 50 = 6176.31mg L -1 ) was 29.41-fold lower (Babu et al. 2018) than that found for the P. capitarianum oil.Sangha et al. (2017) found that the oil from Piper nigrum L. was toxic to the eggs and larvae of P. xylostella, but the comparison of the results reveals that the P. capitarianum oil is respectively 160-fold and 34.78-fold more toxic to P. xylostella eggs and larvae than the P. nigrum oil.
There are few reports in the literature addressing the ovicidal effect of essential oils on P. xylostella.However, ovicidal action has been investigated for other lepidopterans.Krinski et al. (2018) reported the ovicidal action of 21 essential oils from species of Piper against Anticarsia gemmatalis Hübner (Lepidoptera: Eribidae), highlighting the oils from P. fuligineum Kunth.and P. mollicomum Kunth., which exhibited the same level of toxicity (LC 50 = 0.4%).Lourenço et al. (2018) found that the viability of eggs from Spodoptera frugiperda J.E.Smith (Lepidoptera: Noctuidae) was reduced by up to 80% when exposed to 3.3 µl ml -1 of the essential oil from Siparuna guianensis Aublet.
The present results on the lethal action of the P. capitarianum and P. krukoffii oils suggest that these oils affect the larval phase, which causes damage to crops, as well as the egg phase, impeding the development into the larval stage.
The insecticidal potential of essential oils not only causes the death of insects, but also repels, deters feeding, inhibits growth, causes the deformation of pupae and reduces both the longevity and fecundity of insects (Mossa 2016).The antifeedant effect and repellency are important properties of an insecticide for integrated pest management.These properties affect the behavior of the pest, keeping it away from the host and minimizing crop damage (da Camara et al. 2015).The greater antifeedant and repellent properties found for the P. capitarianum oil compared to the P. krukoffii oil may be attributed to differences in the chemical profile of these oils.These behavioral effects of the Piper oils on P. xylostella larvae may stem from the monoterpenes and sesquiterpenes that compose the oils, as terpenes are known to have such effects as part of the defense of plants against herbivory (Singh & Sharma 2015, Pichersky & Raguso 2018, Block et al. 2019).
Although a significant number of essential oils from other botanical genera have been evaluated with regards to their effects on P. xylostella (Reddy et al. 2016, Wei et al. 2015), this is the first report of the antifeedant effect of oils from species of the genus Piper on the larvae of this important pest of cruciferous vegetables.On the other hand, essential oils from other species of Piper have been investigated with regards to their antifeedant effect on other arthropod pests.For instance, the oil from P. hispidinervum C. DC. exhibited antifeedant activity against the caterpillars Spodoptera frugiperda (Lima et al. 2009) and S. littoralis Boisduval (Lepidoptera: Noctuidae) (Andrés et al. 2017) at a concentration of 0.81 mg mL -1 and 100 µL cm 2 , respectively.
The feeding deterrence found for the Piper oils investigated herein, especially the P. capitarianum oil, which is composed mainly of monoterpenes and sesquiterpenes, is in agreement with studies conducted by Koul et al. (2008), who state that terpenes are the chemical class with the greatest known antifeedant diversity.
A large number of essential oils extracted from different families have been shown to be highly repellent to arthropod species (Nerio et al. 2010).While investigations evaluating the repellant effect of essential oils on larvae of the order Lepidoptera are scarce, the essential oils from Zanthoxylum armatum DC (Kumar et al. 2016), Tagetes minuta L., Mentha spicata and Hedychium spicatum Herm.have been found to be repellent to 3 rd instar larvae of P. xylostella (Reddy et al. 2016).Although there are no previous reports of the repellent action of essential oils from species of Piper against P. xylostella larvae, the effects of oils from the genus on other arthropod pests have been investigated.For example, Santana (2018) found that the oil from P. divaricatum G. Mey.exhibited repellent activity against the aphid Lipaphis pseudobrassicae Davis (Hemiptera: Aphididae) for 24 h.The oil from P. nigrum exhibited significant repellency activity against the beetle Tribolium castaneum Herbst (Coleoptera: Tenebrionidae) (Upadhyay & Jaiswal 2007) as well as the cockroach species Periplaneta americana L. (Blattaria: Blattidae) and Blatella germanica L. (Blattodea: Blattellidae) (Thavara et al. 2007).
Due to their high volatility, essential oils form a vapor barrier that avoids contact between the arthropod and the surface of the host plant (Brown & Hebert 1997).In the present study, greater repellent activity was found for the P. capitarianum oil, which suggests a better interaction between the vapor formed by the volatile constituents of the oil and olfactory receptors in the pest (Tyagi 2016).
Phytotoxicity is a concern when formulating new pest control products (Correia & Durigan 2007), as it can cause irreversible damage to the structure and physiology of the host plant (Carvalho et al. 2009).The most common symptom of phytotoxicity is leaf necrosis.The mild and acceptable levels of phytotoxicity respectively found for the P. capitarianum and P. krukoffii oils to the host plant (Brassica oleracea var.acephala) did not cause necrosis to the point of compromising the quality of the product.In contrast, the degrees of necrosis found after the application of the positive controls Deltamethrin and Azadirachtin indicated severe phytotoxicity, compromising the quality of the product to be sold.
Few studies have investigated the phytotoxicity of essential oils from species of Piper to host plants of agricultural pests.However, several studies have demonstrated high phytotoxicity of essential oils (Jaramillo-Colorado et al. 2019, Souza Filho et al. 2009, Andrés et al. 2017) and ethanolic extracts (Lustosa et al. 2007, Pukclai & Kato-Noguchi 2011, Huang et al. 2010) from species of this genus on weeds.The phytotoxic effects of essential oils from other plant species on host plants of agricultural pests have been investigated.For instance, oils from Achillea millefolium Afan., Santolina chamaecyparissus L. and Tanacetum vulgare L. presented phytotoxicity at a concentration of 0.8%, with accentuated necrosis on the leaves of the pea (Pisum sativum L.), which is a host plant for the aphid Myzus persicae (Czerniewicz et al. 2018).In another study, Sertkaya et al. (2010) found no evidence of phytotoxicity of oils from Origanum onites L., Thryptomene spicata Rye and Trudgen, Lavandula stoechas L. and Mentha spicata at a concentration of 15 g mL -1 on the leaves of different host plants of the pest Tetranychus urticae Koch (Acari: Tetranychidae) (tomato, bell pepper, cucumber and eggplant).
The chemical investigation of the P. capitarianum and P. krukoffii oils using GC-MS enabled the identification of two new chemotypes: globulol/4-epi-cis-dihydroagarofuran/γmuurolene for P. krukoffii and o-cymene/ dehydro-aromadendrene for P. capitarianum.This is the first report of the lethal (larvicidal and ovicidal) and sublethal (antifeedant and repellency) effects of the essential oils from P. krukoffii and P. capitarianum on P. xylostella as well as phytotoxicity to the host plant, Brassicae oleraceae var.acephala.
The present findings reveal that the P. capitarianum oil was more efficient than the P. xylostella oil and also when compared to the results of the positive controls (Deltamethrin and Azadirachtin).Moreover, the considerable availability of this plant in the São Francisco/Terra Nova agricultural community of metropolitan Manaus, Brazil, makes it a promising candidate for the preparation of an insecticidal formula containing the essential oil from the leaves.The characterization of the chemical composition of these oils allows them to be used as a standard in the preparation of formulations in laboratories, based on their chemical composition, so that they can be used against the pest.Essential oils have advantages over synthetic insecticides, such as biodegradability, obtainment from renewable sources and generally lower toxicity to mammals.However, due to their high volatility, essential oils are susceptible to degradation by physical (light and temperature) and chemical (air and humidity) agents (Pavela & Sedlák 2018), therefore requiring formulations on a nanometric scale that preserves their physicochemical properties (Pavela et al. 2019).Thus, further studies are needed to evaluate post-application conditions, especially temperature, that may significantly affect the efficacy of Piper oils.
The essential oils investigated herein are promising alternatives to synthetic pesticides for the control of P. xylostella.As these substances have a natural origin and are generally safer, further studies should be conducted to evaluate possible environmental impacts, especially on non-target organisms, as well as determine the cost-benefit ratio for the formulation of a plant-based insecticide for use in the integrated management of P. xylostella.Also, it is necessary to carry out future studies that separately evaluate the compounds found here, as well as mixtures of these, since biotic and abiotic factors can cause qualitatives and quantitatives changes in the chemical composition of the essential oils of the plants.

Table I .
Chemical composition of essential oils from leaves of Piper krukoffi and Piper capitarianum.

Table I .
Continuation.

Table II .
Toxicity of the Piper essential oil against Plutellla xylostella.

Table III .
Antifeedant activity of the Piper essential oil against Plutella xylostella.

Table IV .
Percentage of injury (phytotoxicity) promoted by Piper essential oils on collard greens leaf discs.
SE=Standard Error; The darker area on leaf disc is the injury.β