Lethal and sublethal effects of essential oils fromPiper capitarianumYunck andPiper krukoffiiYunck onPlutella xylostellaL

Abstract

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 genusPiperis widely distributed in Amazonia and essential oils from these plants have insecticidal properties. This study describes the chemical composition of the essential oils fromPiper capiterianumandPiper krukoffiias well as the lethal and sublethal effects onP. xylostella. The phytotoxicity of the oils on the host plant was also evaluated. Globulol was the major constituent of theP. krukoffiioil ando-cymene was the major constituent of theP. capitarianumoil. The oil fromP. capiterianumexhibited 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 findings suggest that this oil can be used in the preparation of a formulated insecticide for the management ofP. xylostellain different development phases. However, further studies are needed to evaluate the effect of this oil on crops under field conditions as well as non-target organisms and determine the cost-benefit ratio of a product formulated withP. capitarianumoil.

Key words
Plutella xylostella; larvicide; ovicide; repellency; feeding deterrence; phytotoxicity

INTRODUCTION

The diamondback moth,Plutella xylostella(L.) (Lepidoptera: Plutellidae), is the major pest ofBrassica(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 ofP. xylostellafor the protection of cruciferous crops was US$ 4 to 5 billion and this figure 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, especiallyP. xylostellain 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 ofP. 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 genusPiperis 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 ofPiperfound 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 krukoffiiYunck andPiper capitarianumYunck stand out by its broad distribution and biological properties, such as antioxidant activity and larvicidal action against the mosquitoAedes 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 onP. 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 ofP. krukoffiiandP. capitarianumcollected from the Amazon biome and evaluate the effects onP. xylostellain terms of mortality (eggs and larvae), feeding deterrence and repellent action to enable the formulation of a plant-based insecticide containing thesePiperoils 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.

MATERIALS AND METHODS

Collection of plant material

Leaves fromPiper krukoffiiandPiper capitarianumwere collected from the ISB/UFAM reserve in Coari, AM, Brazil (04°07’20”S 63°04’29”W) and along roadway BR-174 in Manaus, AM, Brazil (02°50’24”S 60°01’58”W), respectively. The plants were identified by botanist M.R. Pereira (National Institute for Amazonian Research). Vouchers of both samples were mounted and deposited in the herbarium of theInstituto Nacional de Pesquisas da Amazônia(INPA) under numbers 685 (P. capitarianum) and 700 (P. krukoffii).

Isolation of essential oils

Essential oils from the leaves ofP. krukoffii(100 g) andP. 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^-1in 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 (gc-ms) 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 C₈-C₄₀n-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 ofP. xylostellawere originally collected from collard greens (Brassica oleraceavar.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 instarP. xylostellalarvae 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^oC 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 instarP. xylostellalarvae 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 mm². 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. 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 instarP. xylostellalarvae 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 ofP. xylostellalarvae 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 toP. xylostellalarvae (0.76-0.99 = weak; 0.51-0.75 = moderate; 0.26-0.50 strong; 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, LC₅₀(lethal concentration) and FDI₅₀(lethal concentration) of eachPiperoil 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 theX ²estimated using the Statistical Analysis System software (SAS 2002).

RESULTS

Chemical analysis and identification of constituents of essential oils

The yields and percentages of the chemical constituents identified in thePiperoils 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% forP. krukoffiiand 0.23 ± 0.02% forP. capitarianum.

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

Compounds	RIL	RIC	P. krukoffii % ± S.E.	P. capitarianum % ± S.E.
Yield (%)±S.E.			0.49 ± 0.05	0.23 ± 0.02
Tricyclene!!	929	921	-	0.92 ± 0.02
β-Pinene‡‡	974	974	-	0.55 ± 0.32
6-methyl-5-Hepten-2-one!!	978	981	-	0.88 ± 0.02
Myrcene‡‡	985	988	-	0.73 ± 0.02
o-Cymene‡‡	1024	1022	-	40.74 ± 0.97
γ-terpinene‡‡	1054	1054	-	2.73 ± 0.06
m-Cymene!!	1086	1082	-	0.45 ± 0.26
Terpinen-4-ol!!	1176	1174	-	0.50 ± 0.29
p-Cymen-8-ol!!	1181	1179	-	0.87 ± 0.02
Linalool formate!!	1218	1214	-	0.58 ± 0.34
trans-Ascaridol glycol!!	1267	1266	-	0.86 ± 0.02
p-Cymen-7-ol!!	1286	1289	-	1.40 ± 0.04
trans-tetrahydro Jasmone!!	1296	1309	-	0.64 ± 0.02
δ-Terpinyl acetate!!	1309	1316	-	1.43 ± 0.03
(E)-Jasmonol!!	1315	1322	-	1.96 ± 0.05
neoiso-Carvomethyl acetate!!	1344	1347	-	0.61 ± 0.35
α-Ylangene!!	1366	1373	1.76 ± 0.04	-
α-Copaene!!	1370	1374	-	0.80 ± 0.02
β-Bourbonene!!	1383	1387	0.87 ± 0.02	-
β-Elemene!!	1385	1389	5.34 ± 0.10	-
methyl Eugenol!!	1400	1403	1.17 ± 0.02	-
Longifolene!!	1401	1407	0.55 ± 0.32	-
(Z)-Caryophyllene!!	1403	1408	6.75 ± 0.13	-
β-Duprezianene!!	1426	1421	-	1.78 ± 0.04
β-Gurjunene!!	1428	1431	0.57 ± 0.33	-
α-Guaiene!!	1431	1437	1.30 ± 0.03	-
α-Himachalene!!	1448	1449	-	1.82 ± 0.04
α-Humulene‡‡	1450	1452	1.56 ± 0.03	-
Allo-Aromadendrene!!	1455	1458	1.21 ± 0.03	-
dehydro-Aromadendrene!!	1458	1460	2.45 ± 0.05	12.32 ± 0.29
9-epi-(E)-Caryophyllene!!	1463	1464	1.00 ± 0.02	-
β-Chamigrene!!	1474	1476	-	9.96 ± 0.24
γ-Muurolene!!	1475	1478	11.03 ± 0.21	-
β-Selimene!!	1485	1489	2.60 ± 0.05	-
δ-Selimene!!	1491	1492	3.02 ± 0.06	-
epi-Cubebol!!	1492	1493	4.95 ± 0.10	-
γ-Amorphene!!	1498	1495	-	0.65 ± 0.02
4-epi-cis-Dihydroagarofuran!!	1498	1499	12.25 ± 0.23	-
α-Muurolene!!	1500	1500	0.54 ± 0.31	-
trans-β-Guaiene!!	1501	1502	1.47 ± 0.03	-
δ-Amorphene!!	1509	1511	-	0.96 ± 0.02
Hedycaryol!!	1547	1546	1.39 ± 0.03	-
(E)-Nerolidol‡‡	1560	1561	1.09 ± 0.03	-
Longipinanol!!	1566	1567	1.94 ± 0.34	-
Caryophyllene oxide‡‡	1580	1582	-	5.96 ± 0.14
Globulol‡‡	1589	1590	17.54 ± 0.07	-
Viridiflorol!!	1593	1592	-	0.87 ± 0.02
Longiborneol!!	1600	1599	-	0.88 ± 0.02
Guaiol!!	1601	1600	3.50 ± 0.09	-
β-Eudesmol!!	1650	1649	4.65 ± 0.07	-
Pogostol!!	1652	1651	3.51 ± 0.08	4.00 ± 0.10
epi-Zizanone!!	1665	1668	3.77 ± 0.34	-
Helifolenol A!!	1675	1674	0.60 ± 0.83	-
Total			98.42 ± 0.83	95.85 ± 0.75
Monoterpenes			-	55.85 ± 0.95
Sesquiterpenes			97.25 ± 0.82	40.00 ± 0.20
Phenylpropanoids			1.17 ± 0.02	-

• SE= Standard Error; RIL =Retention indices from the literature; RIC=Retention indices calculated from retention times in relation to those of a series of C₈ –C₄₀ n-alkanes on a DB-5 capillary column; !!Method of identification: Retention Index and Mass Spectroscopy. ‡‡Method of identification: Retention Index; Mass Spectroscopy; Co-Injection with authentic compounds.

The GC-MS analysis enabled the identification of 28 and 27 compounds in the oils ofP. krukoffiiandP. capitarianum,respectively representing 98.42 ± 0.83% and 96.60 ± 0.75% of the total oil. Among the compounds identified in the oils, only dehydro-aromadendrene [P. krukoffii(2.45 ± 0.05%) andP. capitarianum(12.32 ± 0.29%)] and pogostol [P. krukoffii(3.51 ± 0.08%) andP. capitarianum(4.00 ± 0.10%)] were found in both oils (^{Supplementary Material - Figure S1}). These data suggest qualitative and quantitative differences in the chemical composition of the two oils.

Globulol (17.54 ± 0.07%) followed by 4-epi-cis-dihydroagarofuran (12.25 ± 0.23%) and γ-muurolene (11.03 ± 0.21%) were the major constituents in theP. krukoffiioil, whereas o-cymene (40.74 ± 0.97%) followed by dehydro-aromadendrene (12.32 ± 0.29%) and β-chamigrene (9.96 ± 0.24%) were the major constituents of theP. capitarianumoil. TheP. krukoffiioil was composed mainly of sesquiterpenes (97.25 ± 0.12%), while inP.capitarianumoil the content of monoterpenes and sesquiterpenes was very similar. No phenylpropanoids were identified in theP. capitarianumoil and methyl eugenol (1.17%) was the only compound from this chemical class found in theP. krukoffiioil in the present investigation.

P. Xylostella larvicidal and ovicidal bioassay

Table II displays the mean lethal concentrations (LC₅₀) of the essential oils from the leaves ofP. krukoffiiandP. capitarianumand the positive controls (Azadirachtin and Deltamethrin) forP. xylostellalarvae 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. TheP. capitarianumoil (LC₅₀for larvae = 0.21 mg mL^-1and eggs = 0.079 mg mL^-1) was respectively 30.3-fold and 33.9-fold more toxic to larvae and eggs than theP. krukoffii. Moreover, based on the LC₅₀, eggs were respectively 4.60-fold and 3.44-fold more susceptible to theP. capitarianumandP. krukoffioils than third instar larvae. These results show that the essential oils fromP. capitarianumandP. krukoffiare promising as active ingredients in the formulation of a natural insecticide for the control of the different developmental forms ofP. xylostella. However, it is necessary to test of the constituents, found in the oils, separately or in the form of mixtures.

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

Essential oil	Bioassay	N	LC50 (mg mL-1) (CI‡‡ 95%)	LC90 (mg mL-1) (CI‡‡ 95%)	Slope±SE	χ2	DF
Piper capitarianum	Larvicidal	353	0.21 (0.15-0.29)	1.15 (0.75-2.22)	1.78±0.24	7.01	5
	Ovicidal	356	0.079 (0.068-0.092)	0.25 (0.20-0.33)	2.58±0.23	8.20	5
Piper krukoffi	Larvicidal	382	6.37 (5.10-8.05)	23.38 (16.58-39.83)	2.27±0.29	7.73	5
	Ovicidal	323	2.68 (2.39-3.03)	6.79 (5.54-9.22)	3.18 ± 0.36	1.21	6
Decis ®	Larvicidal	337	0.039 (0.035-0.044)	0.14 (0.12-0.18)	2.27±0.13	6.66	5
	Ovicidal	1198	0.066 (0.054-0.079)	1.13 (0.84-1.61)	1.03±0.04	7.20	6
Azamax ®	Larvicidal	356	0.033 (0.029-0.038)	0.22 (0.17-0.30)	1.54±0.09	3.68	6
	Ovicidal	1050	0.035 (0.031-0.039)	0.16 (0.13-0.19)	1.94±0.09	6.12	5

• N= Number of Plutellla xylostella; LC= Lethal Concentration values; CI= Confidence Interval; SE= Standard Error; χ ² =Chi-square; DF= Degree of Freedom.

In the comparison of relative toxicity, bothPiperoils were less toxic than the positive controls (Azadirachtin and Deltamethrin) to theP. xylostellalarvae. Regarding ovicidal activity, only theP. capitarianumoil had the same effectiveness as Deltamethrin, whereas Azadirachtin had greater ovicidal action than bothPiperoils.

Feeding deterrence and larval repellency bioassay

TheP. capitarianumandP. krukoffiioils applied to the collard leaves were capable of reducing the feeding of theP. xylostellalarvae at sublethal concentrations (Table III). TheP. capitarianumoil exhibited greater deterrent action, reducing the feeding of theP. xylostellalarvae 46-fold more than theP. krukoffiioil. Moreover, theP. capitarianumoil was approximately twofold more efficient than the plant-based commercial insecticide (Azadirachtin) used as the positive control. The results show that these essential oils are promising as a plant-based insecticide for use in the management ofP. xylostellalarvae.

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

Essential oil	AC50 (mg mL-1) (CI‡‡ 95%)	AC90 (mg mL-1) (CI‡‡ 95%)	Slope±SE	χ2	DF
Piper capiterianum	0.07 (0.06-0.08)	0.24 (0.20-0.30)	2.41±0.16	5.85	6
Piper krukoffi	3.32 (3.12-3.55)	7.41 (6.57-8.63)	3.67±0.25	5.78	6
Azamax ®	0.154 (0.13-0.18)	0.87 (0.68-1.19)	1.70±0.11	7.71	5

• AC= Antifeedant Concentration values; CI=Confidence Interval; SE=Standard Error; χ2=chi-square. DF=degree of freedom.

Regarding the repellent action, theP. capitarianumoil exhibited high to moderate degrees of repellency at 0.02, 0.04, 0.06 and 0.07 mg mL^-1to 3^rdinstar larvae ofP. xylostellain the first 12 hours after exposure to the oil. After 24 hours, the larvae began to be attracted to theP. capitarianumoil (RI > 1) (Figure 1). TheP. krukoffiioil at 1.20, 1.74 and 2.23 mg mL^-1was 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).

Figure 1
P. xylostellalarval repellency after exposure toP. capitarianumoil for 24 h.

Figure 2
P. xylostellalarval repellency after exposure toP. krukoffiioil for 24 h.

Figure 3
P. xylostellalarval repellency after exposure to plant-based insecticide Azadirachtin for 24 h.

Phytotoxicity bioassay

Table IV displays the phytotoxicity of the essential oils to collard greens (Brassica oleraceaevar. acephala). Based on the phytotoxicity scale proposed by Alvez et al. (1974), theP. capitarianumandP. krukoffiirespectively exhibited mild (9.83%) and acceptable (16.68%) toxicity to the host plant. Considering the positive controls, the essential oils fromP. capitarianumandP. krukoffiiwere less phytotoxic than the synthetic insecticide. The commercial insecticides Deltamethrin (synthetic) and Azadirachtin (plant-based) were respectively 6.93-fold and 3.72-fold more phytotoxic than theP. capitarianumoil. Moreover, theP. krukoffiiwas respectively 4.01-fold and 2.12-fold less phytotoxic than Deltamethrin and Azadirachtin.

Table IV
Percentage of injury (phytotoxicity) promoted by Piper essential oils on collard greens leaf discs.

	Essential oils		Positive control		Negative control
	P. krukoffii %±SE	P. capitarianum %±SE	Azamax ® %±SE	Decis ® %±SE	Solvent only %±SE
Leaf disc†
Phytotoxicity	16.68 ± 1.41	9.83 ± 1.21	36.58 ± 3.95	68.12 ± 1.39	0.00 ± 0.00

• SE=Standard Error; The darker area on leaf disc is the injury.

DISCUSSION

The yield for the fresh leaves ofP. krukoffiioil 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 to the method and other factors involved in the production and accumulation of essential oils in plants (Lima et al. 2003).

Previous investigations involving GC-MS analyses of the leaf oils of these species reported other chemotypes, such as myristicin/apiole forP. krukoffiifrom the municipality of Parauapebas in the state of Pará, Brazil, and β-caryophyllene/β-myrcene/α-humulene forP. capitarianumfrom the city of Manaus in the state of Amazonas, Brazil (da Silva et al. 2011). While we found mainly sesquiterpenes in theP. krukoffiioil, theP. capitarianumoil had similar quantities of monoterpenes and sesquiterpenes. These data diverge from previous reports for this species collected in other localities of the northern Brazil. For instance, oils fromP. krukoffiicollected in the state of Pará andP. capitarianumcollected in the state of Amazonas were composed predominantly of phenylpropanoids (69.2%) (da Silva et al. 2011) and sesquiterpenes (78.13%) (França 2015), respectively. Methyl eugenol, which was the only phenylpropanoid found in theP. krukoffiioil, was identified by da Silva et al. (2011) at a proportion less than 1% in the oil from a sample collected in the state of Pará. In the present investigation, we found two new chemotypes for the species ofPiperinvestigated: globulol/4-epi-cis-dihydroagarofuran/γ-muurolene forP. krukoffiiando-cymene/dehydro-aromadendrene forP. capitarianum. Differences in the chemical composition of essential oils from the same species that occur in different localities or even within the same region can be explained by variations in environmental and/or geographic conditions (da Camara et al. 2017).

The insecticidal effects of essential oils from different botanical genera on different development phases ofP. xylostellahave been widely investigated (Chaudhary et al. 2011, Purwatiningsih & Hassan 2012, Reddy et al. 2016, Koundal et al. 2018). Comparing toxicity values, the oil fromP. capitarianumwas more toxic toP. xylostellalarvae (LC₅₀= 0.21 mg mL^-1) than oils from other plant species studied in Brazil and other regions of the world [Corymbia citriodora(LC₅₀= 21.53 08 mg mL^-1),Acorus calamus(LC₅₀= = 0.39 mg mL^-1),Cedrus deodara(LC₅₀= 1.08 mg mL^-1),Aegle marmelos(LC₅₀= 8.76 mg mL^-1),Tagetes minuta(LC₅0 = 10.15 mg mL^-1),Murraya koenigii(LC₅₀= 2.98 mg mL^-1),Curcuma aromatic(LC₅₀= 1.35 mg mL^-1),Mentha piperita(LC₅₀= 1.37 mg mL^-1),Mentha spicata(LC₅₀= 1.86 mg mL^-1),Mentha longifolia(LC₅₀= 1.06 mg mL^-1) andCymbopogon flexuosus(LC₅₀= 1.80 mg mL^-1)] (Reddy et al. 2016, Filomeno et al. 2017, Koundal et al. 2018).

Few studies have investigated the effects of essential oils from the genusPiperon this pest (Sangha et al. 2017). However, other derivates fromPiperplants have been investigated, such as extracts of different polarities and fixed constituents. A study conducted with hexane extracts from the leaves of differentPiperspecies [P. sarmentosum(LC₅₀= 2061.29 μg mL^-1),P. interruptum(LC₅₀= 1328.24 μg mL^-1),P. nigrum(LC₅₀= 2800.95 μg mL^-1) andP. retrofractum(LC₅₀= 237.38 μg mL^-1)] collected in Thailand revealed high toxicity toP. xylostellalarvae (Kraikrathok et al. 2013). Park (2012) reported toxicity toP. xylostellalarvae for the hexane extract ofPiper nigrum(100% mortality at 2.5 mg mL^-1) and its major constituents guineensine (LC₅₀= 0.013 mg mL^-1), retrofractamide A (LC₅₀= 0.020 mg mL^-1), pipercide (LC₅₀= 0.033 mg mL^-1) and pellitorine (LC₅₀= 0.046 mg mL^-1).

The greater toxicity to the eggs and larvae found for theP. capitarianumoil compared to theP. krukoffiioil may be explained by qualitative and quantitative chemical differences between the two oils, as demonstrated by GC-MS. Moreover,P. xylostellawas more susceptible to both oils in the egg phase than in the larval phase. In contrast, Sangha et al. (2017) found that the oil fromP. nigrumwas more toxic to the larval phase than the egg phase ofP. 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 ofP. 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 LC₅₀estimated for thePiperoils investigated herein on 3^rdinstar larvae ofP. xylostellaand values reported for oils from other plants reveals that the oil fromP. capitarianumis more efficient that oils fromMentha longifoliaL. Huds.,Mentha piperitaL.,Mentha spicataL.,Cymbopogon flexuosusSteud. andCurcuma aromaticaSalisb (Koundal et al. 2018). In studies on larvicidal action againstP. xylostella,the toxicity of the essential oil fromAllium tuberosumL. (LC₅₀= 0.56 µl mL^-1) was 2.66-fold lower (Gao et al. 2019) and the toxicity of the essential oil fromZingiber officinaleRoscoe (LC₅₀= 6176.31 mg L^-1) was 29.41-fold lower (Babu et al. 2018) than that found for theP. capitarianumoil.

Sangha et al. (2017) found that the oil fromPiper nigrumL. was toxic to the eggs and larvae ofP. xylostella, but the comparison of the results reveals that theP. capitarianumoil is respectively 160-fold and 34.78-fold more toxic toP. xylostellaeggs and larvae than theP. nigrumoil.

There are few reports in the literature addressing the ovicidal effect of essential oils onP. xylostella. However, ovicidal action has been investigated for other lepidopterans. Krinski et al. (2018) reported the ovicidal action of 21 essential oils from species ofPiperagainstAnticarsia gemmatalisHübner (Lepidoptera: Eribidae), highlighting the oils fromP. fuligineumKunth. andP. mollicomumKunth., which exhibited the same level of toxicity (LC₅₀= 0.4%). Lourenço et al. (2018) found that the viability of eggs fromSpodoptera frugiperdaJ.E. Smith (Lepidoptera: Noctuidae) was reduced by up to 80% when exposed to 3.3 µl ml^-1of the essential oil fromSiparuna guianensisAublet.

The present results on the lethal action of theP. capitarianumandP. krukoffiioils 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 theP. capitarianumoil compared to theP. krukoffiioil may be attributed to differences in the chemical profile of these oils. These behavioral effects of thePiperoils onP. xylostellalarvae 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 onP. xylostella(Reddy et al. 2016, Wei et al. 2015), this is the first report of the antifeedant effect of oils from species of the genusPiperon the larvae of this important pest of cruciferous vegetables. On the other hand, essential oils from other species ofPiperhave been investigated with regards to their antifeedant effect on other arthropod pests. For instance, the oil fromP. hispidinervumC. DC. exhibited antifeedant activity against the caterpillarsSpodoptera frugiperda(Lima et al. 2009) andS. littoralisBoisduval (Lepidoptera: Noctuidae) (Andrés et al. 2017) at a concentration of 0.81 mg mL^-1and 100 µL cm², respectively.

The feeding deterrence found for thePiperoils investigated herein, especially theP. capitarianumoil, 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 fromZanthoxylum armatumDC (Kumar et al. 2016),Tagetes minutaL.,Mentha spicataandHedychium spicatumHerm. have been found to be repellent to 3^rdinstar larvae ofP. xylostella(Reddy et al. 2016). Although there are no previous reports of the repellent action of essential oils from species ofPiperagainstP. xylostellalarvae, the effects of oils from the genus on other arthropod pests have been investigated. For example, Santana (2018) found that the oil fromP. divaricatumG. Mey. exhibited repellent activity against the aphidLipaphis pseudobrassicaeDavis (Hemiptera: Aphididae) for 24 h. The oil fromP. nigrumexhibited significant repellency activity against the beetleTribolium castaneumHerbst (Coleoptera: Tenebrionidae) (Upadhyay & Jaiswal 2007) as well as the cockroach speciesPeriplaneta americanaL. (Blattaria: Blattidae) andBlatella germanicaL. (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 theP. capitarianumoil, 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 theP. capitarianumandP. krukoffiioils to the host plant (Brassica oleraceavar. 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 ofPiperto 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 fromAchillea millefoliumAfan.,Santolina chamaecyparissusL. andTanacetum vulgareL. presented phytotoxicity at a concentration of 0.8%, with accentuated necrosis on the leaves of the pea (Pisum sativumL.), which is a host plant for the aphidMyzus persicae(Czerniewicz et al. 2018). In another study, Sertkaya et al. (2010) found no evidence of phytotoxicity of oils fromOriganum onitesL.,Thryptomene spicataRye and Trudgen,Lavandula stoechasL. andMentha spicataat a concentration of 15 g mL^-1on the leaves of different host plants of the pestTetranychus urticaeKoch (Acari: Tetranychidae) (tomato, bell pepper, cucumber and eggplant).

The chemical investigation of theP. capitarianumandP. krukoffiioils using GC-MS enabled the identification of two new chemotypes: globulol/4-epi-cis-dihydroagarofuran/γ-muurolene forP. krukoffiiando-cymene/dehydro-aromadendrene forP. capitarianum. This is the first report of the lethal (larvicidal and ovicidal) and sublethal (antifeedant and repellency) effects of the essential oils fromP. krukoffiiandP. capitarianumonP. xylostellaas well as phytotoxicity to the host plant,Brassicae oleraceaevar. acephala.

The present findings reveal that theP. capitarianumoil was more efficient than theP. xylostellaoil 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 ofPiperoils.

The essential oils investigated herein are promising alternatives to synthetic pesticides for the control ofP. 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 ofP. 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.

ACKNOWLEDGMENTS

This work was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (PQ-2-302860/2016-9), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (88887.368587/2019-00) and Fundação de Amparo à Ciência e Tecnologia do Estado de Pernambuco (FACEPE; grants BCT-0253-1.06/19, APQ-0476-1.06/14, APQ-08601.06/16 and APQ- 10081.06/15).

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SUPPLEMENTARY MATERIAL

Figure S1

Publication Dates

• Publication in this collection
  08 Aug 2022
• Date of issue
  2022

History

• Received
  16 Jan 2020
• Accepted
  06 May 2020