Biocidal effects of Piper hispidinervum (Piperaceae) essential oil and synergism among its main components

Accepted Manuscript Biocidal effects of Piper hispidinervum (Piperaceae) essential oil and synergism among its main components M.F. Andrés, G.E. Rossa, E. Cassel, R.M.F. Vargas, O. Santana, C.E. Díaz, A. González-Coloma PII:

S0278-6915(17)30186-2

DOI:

10.1016/j.fct.2017.04.017

Reference:

FCT 9002

To appear in:

Food and Chemical Toxicology

Received Date: 3 March 2017 Revised Date:

11 April 2017

Accepted Date: 13 April 2017

Please cite this article as: Andrés, M.F., Rossa, G.E., Cassel, E., Vargas, R.M.F., Santana, O., Díaz, C.E., González-Coloma, A., Biocidal effects of Piper hispidinervum (Piperaceae) essential oil and synergism among its main components, Food and Chemical Toxicology (2017), doi: 10.1016/ j.fct.2017.04.017. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

SAFROLE

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Essential oil

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Piper hispidinervum

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TERPINOLENE

Insect antifeedant

synergism Nematicidal

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Biocidal effects of Piper hispidinervum (Piperaceae) essential oil and synergism among its main components.

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M.F. Andrésa, G.E. Rossab, E. Casselb, R.M.F. Vargasb, O. Santanac, C.E. Díazd, A. González-Colomaa*

Instituto de Ciencias Agrarias, CSIC, Serrano 115-bis, 28006 Madrid, Spain

b

Faculdade de Engenharia, Departamento de Engenharia Química, PUCRS, Av. Ipiranga,

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6681, 90619-900 Porto Alegre, RS, Brazil

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a

Parque Científico y Tecnológico de Albacete, Paseo de la Innovación 1,02006, Albacete,

Spain

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917452500

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*Corresponding author: A. González-Coloma. e-mail: [email protected]. Phone: 0034

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ABSTRACT In this study we evaluated the effect of a pressure gradient (1-2 atm) in the extraction and composition of the essential oil (EO) of Piper hispidinervum by steam distillation. We also

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evaluated the insect antifeedant effects (Spodoptera littoralis, Leptinotarsa decemlineata, Myzus persicae and Rhopalosiphum padi) and nematicidal activity (Meloidogyne javanica) of the oils, their major components and their synergistic interactions. Safrole was the major

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component (78 - 81 %) followed by terpinolene (5 – 9 %). The EOs tested were effective insect antifeedants. Safrole, explained most of the insect antifeedant action of P.

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hispidinervum EOs. When safrole and terpinolene were tested in binary combinations, low ratios of safrole improved the antifeedant effects of terpinolene. P.hispidinervum EOs caused higher mortality of M. javanica juveniles than their major components. In binary combinations, low ratios of terpinolene increased the nematicidal effects of safrole. The EO

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treatment strongly suppressed nematode egg hatching and juvenile infectivity. P.hispidinervum EOs affected the germination of S. lycopersicum and L. sativa mostly at 24 h of treatment, being L. sativa the most sensitive. Safrole moderately affected

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germination and root growth of L. sativa, S. lycopersicum and L. perenne. Terpinolene only

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affected S. lycopersicum root growth.

Keywords: Piper hispidinervum; oil; safrole; terpinolene; nematicidal; antifeedant.

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1. Introduction

EOs and their constituents can play an important role in crop protection and have been

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proposed as an environmentally friendly alternative to synthetic pesticides, including insecticidal, and nematicidal agents (Isman et al., 2011 Andres et al., 2012, GonzálezColoma et al., 2013).

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Piper is the largest genus of the family Piperaceae including about 1000 species. Essential oils from some Piper species have are commercial importance for the fragrance

piperamides (Scott et al., 2008).

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and pharmaceutical industries and have shown insecticidal properties due to their content in

Piper hispidinervum (pimenta longa in Brazil), is a shrub distributed throughout South America. This species is especially prominent in the state of Acre in Brazil (Rocha and

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Ming, 1999) where a sustainable production system has been implemented (Pacheco et al., 2013). P. hispidinervum oil is an important raw source of safrole, a chemical used to synthetize piperonyl butoxide (PBO), a vital ingredient of pyrethroid insecticides (Pimentel

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et al., 2006). Additionally, the essential oil of P. hispidinervum has biological effects including amoebicidal (Sautier et al. 2012) and insecticidal activity against Sitophilus

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zeamais (Estrela et al., 2006), Tenebrio molitor (Fazolin et al., 2006) and Spodoptera frugiperda (Lima et al., 2009; Cruz et al., 2014, Alves et al., 2014). In this study, we have evaluated the effect of pressure in the extraction of the essential oil of P. hispidinervum by steam distillation and the composition of the different extracts. We also studied the insect antifeedant effects (Leptinotarsa decemlineata, Spodoptera littoralis, Myzus persicae and Rhopalosiphum padi), the nematicidal activity against 3

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Meloidogyne javanica and phytotoxicity (against Lactuca sativa, Lycopersicum solani and Lolium perenne) of these extracts, their major components and their synergistic

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interactions.

2. Materials and methods

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2.1 Plant material

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The aerial parts of the plants were collected in summer season. Cultures were established in the Agricultural Centre of the EMATER in southern Brazil as described by Sauter et al., (2012).

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2.2 Essential oil

A total of 1325 g of plant material (fresh leaves and twigs) was used for each extraction

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in a steam distillation pilot apparatus that was designed to generate experimental data for process scale up. The equipment has industrial sensors: temperature sensor (Pt-100 –

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Novus, Brazil) and pressure transducer (model 0 to 5 bars – Huba Control, Switzerland). This apparatus consists of 1kWh electric boiler (Sulinox, Brazil), 10 L extractor vessel (Sulinox, Brazil), multitubular heat exchanger (Mecânica de Base, Brazil), and glass liquidliquid separator. The liquid-liquid separator was designed for this study, because the P. hispidinervum essential oil is denser than water. Extractions were performed at saturation conditions and three pressures: 1.0 atm, 1.5 atm, and 2.0 atm, respectively. P. 4

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hispidinervum essential oils were collected in liquid-liquid separator and the experiments were finalized when the volatile extract volume did not vary after three consecutive measurements of 5 min. The average moisture content of the aerial plant parts was 80.0 %

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(Halogen Moisture Analyzer – HB43 – Mettler Toledo, Brazil). Three essential oils (EOs) were obtained from P. hispidinervum at pressures of 1.0 atm, 1.5 atm, and 2.0 atm (Ph1,

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Ph1.5 and Ph2).

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2.3 GC and GC–MS analysis

P. hispidinervum EOs were analyzed by gas chromatography (GC) on an Agilent 7890A and gas chromatography-mass spectrometry (GC/MS) equipped with a mass spectrometer Agilent 5975C. The capillary column was a HP-5MS silica capillary column (30 m x 250

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µm i.d.), coated with 5% phenyl methyl silox (0.25 µm phase thickness); column temperature, 60ºC for 8 min, rising to 180˚C at 3˚C/min, 180-250ºC at 20˚C/min, then 250˚C for 10 min. Injector temperature 250˚C; detector temperature 280˚C; injection mode,

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split; split ratio 1:55; volume injected, 0.2 µL of the oil. Carrier gas was Helium, flow rate 1 mL/min; interface temperature 250˚C; acquisition mass range, m/z 40-450.

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The components of the volatile extracts were identified by comparison of their linear retention indexes (LRIs) on the GC column, determined in relation to a homologous series of n-alkanes C8-C20 (Fluka Analytical), with those from pure standards (Adams, 2007). Comparison of fragmentation patterns in the mass spectra with those stored on the GC–MS databases (Adams, 2007) was also performed.

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2.4. Insect bioassays

Insect colonies of S. littoralis, L. decemlineata, M. persicae and R. padi were reared on

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an artificial diet, potato (Solanum tuberosum), bell pepper (Capsicum annuum) and barley (Hordeum vulgare) plants, respectively, and maintained at 22 ± 1 ºC, >70% relative humidity with a photoperiod of 16:8 h (L:D) in a growth chamber. Bioassays were

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conducted with newly emerged S. littoralis sixth instar larvae, L. decemlineata adults (10 replicates with 2 insects each) and adults of the aphids M. persicae and R. padi (20

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replicates with 10 insects each) feeding or settling inhibition (%FI or %SI) were calculated as FI= [1-(T/C)] x 100, where T and C are the consumption of treated and control leaf disks, respectively, or as %SI= [1-10 (%T/%C)] x 100 where %C and %T are percent aphids settled on control and treated leaf disks, respectively, as described in (Santana et al.,

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2014) . The antifeedant effects (FI/SI) were analyzed for significance by the non-parametric Wilcoxon signed-rank test. EC50 values (effective dose to obtain 50% feeding inhibition) were determined for EOs and pure compounds with FI/SI values > 60% from linear

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regression analysis.

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2.5 Nematicidal activity

M. javanica population was maintained on L. esculentum plants (var. Marmande) in pot cultures at 25 ± 1ºC, 70 % relative humidity. Egg masses of M. javanica were handpicked from infected tomato roots. Second-stage juveniles (J2) were obtained from hatched eggs by incubating egg masses in a water suspension at 25 ºC for 24 h. 6

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2.4.1 In vitro effect on juveniles. EOs and their components were dissolved in distilled water containing 5% of a DMSO-

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Tween solution (0.5% Tween 20 in DMSO) and evaluated as described by Andres et al. (2012). The initial concentrations tested were of 1 and 0.5 mg ml-1 for EOs or pure compound respectively. EOs, with mortality rates > 90 %, were further tested to asses J2

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mortality after 24 and 48 h. The nematicidal activity data are presented as percent dead J2 corrected according to Scheider-Orelli’s formula. Effective lethal doses (LC50 and LC90)

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were calculated by Probit Analysis. Five concentrations of selected EOs were used to obtain the LC50 and LC90 and four replicates were used in each concentration.

2.4.2 In vitro effect on egg hatching

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Three egg masses of uniform size were washed with sterilized distilled water and transferred to a 96-well plate (BD Falcon, San Jose, CA, USA) containing the nematicidal solutions. Egg masses placed in sterilized distilled water with 5% DMSO-Tween solution

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were used as controls. Each experiment consisted of four replicates of treatment and control. The plates were covered to prevent evaporation and incubated in darkness at 25 ºC.

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After 5 days the hatched J2 were counted, the test solutions removed and the wells with egg masses washed and filled with sterilized distilled water. The eggs were monitored during 4 weeks, until hatching was complete in the control. Relative hatching percentages (compared with the controls) were calculated (Andrés et al., 2012).

2.4.3 Effect on juvenile infection capacity. 7

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Seeds of tomato susceptible variety, Marmande, were germinated in seed trays and, when three-week old, seedlings were transplanted into 5-cm diameter clay pots filled with 10 mL quartz sand. Seedlings were individually inoculated with 180 J2 treated with P.

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hispidinervum (1 bar) oil at 0.7 mg/mL (LC50). Micropots with inoculated plants were maintained in a growth chamber at 25 ± 2 ºC with a 16 h photoperiod. A week post nematode inoculation, seedlings were removed from the pots and roots were stained in acid

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fuchsine (Byrd et al., 1983). Juveniles within the roots of each individual seedling were counted.by examining the entire root system under a stereomicroscope. The experiment

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was repeated three times. Relative percentages of J2 penetration (treated vs untreated) were calculated to obtain inhibition rates of J2 infectivity (Julio et al., 2017).

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2.5 Phytotoxic Activity.

The experiments were conducted with L. sativa cv. Teresa, L. sculentum cv. Marmande and L. perenne seeds (100 seeds/test) in 12-well microplates as described previously

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(Santana et al., 2014) The organic extracts and pure compounds were tested at initial concentrations of 0.4 and 0.2 mg/mL (final concentration in the well), respectively, and

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diluted serially (1:2 dilutions), if needed. Germination was monitored for 6 (L. sativa and L. sculentum) or 7 days (L. perenne), and the root length measured at the end of the experiment (25 plants were selected randomly for each experiment, digitalized, and measured with the application ImageJ, http//rsb.info. nih.gov./ij/). A nonparametric analysis of variance (ANOVA) was performed on radical length data.

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3. Results and Discussion

P. hispidinervum essential oil yields (% v/w of plant biomass) for each

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extraction/pressure were 0.632%, 0.889% and 0.665% for 1.0 atm, 1.5 atm, and 2.0 atm respectively. Extraction at 1.5 atm resulted in a higher total amount of essential oil. This result may be attributed to increased solubility of the components of the plant material at

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this pressure and temperature. [Table 1]

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The chemical analysis of P. hispidinervum EOs by GC–MS showed a similar composition for the three essential oils, with 24 compounds representing 98% of the total area identified (Table1). Safrole was the major component (77.7 - 81.3 %), followed by terpinolene (4.6 – 8.8 %) (see structures in Fig 1). In terms of selectivity, the extract

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obtained at a pressure of 1.5 atm showed about 4% higher content in safrole than the ones obtained at 1.0 atm or 2.0 atm (Table 1). Safrole has been reported as the main component in the essential oil of P. hispidinervum, with concentrations greater than 90% in the

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Amazon region (Fazolin et al., 2007); or between 77% and 90% in plants cultivated in southern Brazil (Nascimento et al., 2008; Riva et al., 2011; Sauter et al., 2012).

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[Figure 1]

Table 2 shows the insect antifeedant effects of P. hispidinervum EOs against L. decemlineata, S. littoralis, M. persicae and R. padi. The EOs tested were effective antifeedants against L. decemlineata, being Ph2 the most effective. This oil had a composition similar to Ph1; therefore minor changes in composition could explain this increased effect. The oil Ph1.5, with the highest safrole content, was the most effective on 9

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S. littoralis and affected the aphid M. persicae. P. hispidinervum essential oil (80-90% safrole) has been reported as insecticidal against S. zeamais (Estrela et al., 2006), T. molitor (Fazolin et al., 2007) and S. frugiperda (Lima et al., 2009; Cruz et al., 2014, Alves et al.,

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2014), however this is the first report on insect antifeedant effects of P. hispidinervum. [Table 2]

Since safrole could be acting not only as an insecticidal component but as a synergist

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(Scott et al., 2008), major compounds, safrole and terpinolene (S and T), were tested alone and in different combinations (Table 2). Safrole was an effective antiffedant to S. littoralis

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and M. persicae with an effective dose similar (S. littoralis) or lower (M. persicae) than EO Ph1.5, while terpinolene had moderate antifeedant effect on M. persicae. Therefore, safrole alone explained the antifeedant effect of the EOs against these insects. In the case of L. decemlineata, S and T were antifeedant with potencies similar to EOs Ph1 and Ph1.5, and

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less potent than Ph2. Therefore, safrole and terpinolene could explain the antifeedant activity of EOs Ph1 and Ph1.5 but not Ph2 (Table 3). The binary S/T mixtures (1/1-16/1) were also active against S. littoralis with potencies

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within the range of the EOs or S alone. In the case of M. persicae, the S/T mixtures 9/1 and 16/1 (found in the EOs) were as effective as safrole alone and more active than the EO

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Ph1.5 (Table 2). None of the S/T combinations were synergistic against these two insect species. S/T combinations with lower doses of safrole (1/9 and 1/16) were also active on L. decemlineata, but only the 1/16 combination had stronger effects than S and T alone (6-9 times more effective). Therefore, safrole potentiated the antifeedant effects of terpinolene against L. decemlineata at ratios 1/16 or lower.

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Terpinolene acted as a biting deterrent and toxicant against mosquitos (Ali et al., 2015; Conti et al., 2012), reduced larval growth rate of spruce budworm (Chonistoneura fumiferana) (Kumbasli and Bauce, 2013) had fumigant and contact toxicant to stored

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product insects (Triboleum castaneum and Sitophilus zeamais) (Wang et al., 2009) and acted as a synergist of the mountain pine beetle (Dendroctonus ponderosae) aggregation pheromone in combination with myrcene (Borden et al., 2008). Safrole showed contact and

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fumigant toxicity against several species of dipterans (Yi et al., 2015), and stored product insects (S. zeamais and T. castaneum). Safrole also reduced their relative growth rate,

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inhibited T. castaneum α-amylase (Huang et al., 1999) and showed larvicidal effects against S. litura (Bhardwaj et al., 2010). Furthermore, safrole with a methylenedioxyphenyl group in its molecule (Figure 1) inhibits insect P450 microsomal monooxygenases, acting as a natural insecticide synergist (Yu, 2000; Yu et al., 1993). In this work, low ratios of safrole

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potentiated the antifeedant effect of terpinolene on L. decemlineata. The nematicidal activity of P. hispidinervum EOs showed in Tables 3 and 4. The three essential oils (Ph1, Ph1.5 and Ph2) induced high mortality of M. javanica J2 at 1 mg/ml

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after 72 h exposure, with similar LC50 and LC90 values. These results indicate that the nematicidal effects were not influenced by the EO extraction conditions (pressure).

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The two major oil components, safrole (S) and terpinolene (T), and paired mixtures of them were tested on M. javanica (Tables 3 and 4). Safrole was moderately active, causing 50% J2 mortality (at 0.5 µg/µ, 72 h incubation) while terpinolene was not nematicidal. Conversely, some binary mixtures induced strong nematicidal effects, specifically S/T at 9/1 and 16/1 (100% mortality). Thus, the nematicidal effects of P. hispidinervum EO could be attributed to a synergistic interaction of safrole with terpinolene. We previously reported 11

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on the synergistic nematicidal effect of binary mixtures of terpenes from Mentha species essential oils. In particular, carvone showed synergistic interactions with other terpenes (limonene, cineole, menthol) (Andres et al., 2012; Santana et al., 2014).

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[Table 3]

The egg hatchability of M. javanica was strongly inhibited by the essential oil tested (Ph1) after 5 days of incubation (Table 4), inducing a >95% reduction. This effect was

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maintained on egg masses immersed in water over time, with a relative hatchability suppression rate of 89 % at the end of the experiment (28 days). The strong effect on

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nematode egg hatching observed demonstrated the ability of P. hispidinervum EOs to penetrate the gelatinous matrix and act on nematode eggs, even though egg masses are less sensitive to the effects of extracts than J2 (Andrés et al., 2012). Additionally, strong effects on egg hatchability from masses treated with the three binary mixtures of

[Table 4]

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safrole/terpinolene were found (Table 4).

To further test the nematicidal potential of P. hispidinervum on M. javanica, in vivo

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tests on tomato seedlings were carried out. The results showed a high suppression of J2 infection capacity when treated with the EO Ph1 at a sublethal concentration (0.7 mg/ml)

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(Figure 2). The three experiments showed similar effects with a significant decrease of J2 root penetration (78, 71 and 68 % inhibition rate) respect to the control (inoculated with untreated J2). Therefore, P. hispidinervum EO strongly suppressed the invasion capacity of M. javanica J2, lowering their penetration and host-plant root colonization. This study demonstrates for the first time the in vitro and in vivo nematicidal activity of Piper hispidinervum EO against root-knot nematodes. 12

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[Figure 2] Given the promising biocidal effects of P. hispidinervum essential oils, we tested their phytotoxic effects against two dicotyledonous species, including S. littoralis, M. persicae

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and M. javanica host plants (Lactuca sativa and S. lycopersicum) and a monocotyledonous weed (Lolium perenne). The essential oils affected the germination of both dicotyledonous plants mostly at 24 h of treatment, being L. sativa the most sensitive (Fig 3). On the

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contrary, the leaf and root growth of L. perenne and root growth of L. sativa were affected by EOs Ph2 and 1.5 (20-40% inhibition). Safrole affected germination (24 h) and root

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growth of L. sativa, S. lycopersicum and L. perenne (20-30% inhibition) (Figure 3). Terpinolene was less phytotoxic, affecting only S. lycopersicum root growth (25%). The effects of the binary S/T combinations tested with respect to the EOs (Ph2 and Ph1.5) were lower on L. sativa, lower-similar on S. lycopersicum and similar on L. perenne for

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combinations 1/1 and 9/1 (Figure 3).

P. hispidinervum essential oil has been reported as phytotoxic against grassland weeds (Mimosa pudica, Senna obtusifolia) by inhibiting mostly their germination (Souza Filho et

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al., 2009). Therefore, careful dose management must be taken into account when considering P. hispidinervum EOs for the control of insects and nematodes on lettuce and

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tomato plants. [Figure 3]

In this work, we have demonstrated that P. hispidinervum essential oils were insect antifeedants. The main component, safrole, explained most of the insect antifeedant action of P. hispidinervum EOs, and also found that low ratios of safrole improved the antifeedant effects of terpinolene on L. decemlineata, being the first report on the potentiation effects of 13

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safrole on the insect antifeedant action of other natural product. We have also demonstrated that P. hispidinervum essential oil was a strong nematicidal agent with higher nematicidal activity than their major components. The binary S/T combinations tested suggested that

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terpinolene is critical to increase the nematicidal effects of safrole.

Acknowledgements

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This work has been supported by grants 2011BR0081 (CSIC-Spain / CNPq-Brazil cooperation program) and CTQ2015-64049-C3-1-R (Spain).

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DC.) on fall armyworm Spodoptera frugiperda (J. E. Smith, 1797) (Lepidoptera: Noctuidae) Acta Amaz. 39, 377 – 382. Nascimento, F. R., Cardoso, M. G., Souza, P E., Lima, R. K., Salgado, A. P. S. P.,

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Guimarães, L. G. L. 2008. The effect of Long-pepper essential oil (Piper hispidinervum C. DC.) and of Tween®; 80 emulsifier on the mycelial growth of Alternaria alternate (Fungi: Hyphomycetes). Acta Amaz. 38, 503–508.

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Pacheco J. F., Silva J. B., Negreiros, J. R., Silva, M. R. G., Farias, S. B. 2013. Germination and vigor of long-pepper seeds (Piper hispidinervum) as a function of temperature

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1

T

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S

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Figure 1.

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Figure 2 100% 90%

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80%

60% 50%

CONTROL

40%

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J2 penetration %

70%

30%

10% 0%

Exp 2

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Exp 1

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20%

20

Exp 3

Ph1 EO

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Figure 3

24h

72h

Root

Ph2

Ph1

Ph1.5

S

0 -20 -40

-80

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-60

48h 40 20

Ph2

Ph1.5

Ph1

-20 -40 -60

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-80

S/T (9/1)

S/T (16/1)

72h

Root

S

T

S/T (1/1)

S/T (9/1)

S/T (1/1)

S/T (9/1)

S/T (16/1)

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0

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[%] CONTROL (S. lycopersicum)

S/T (1/1)

T

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[%] CONTROL (L. sativa)

20

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40

72h

192h

Root

Leaf

[%] CONTROL (L. perenne)

40 20

Ph2

Ph1.5

Ph1

S

0

-20 -40 -60 -80

21

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S/T (16/1)

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Table 1: Chemical composition of Piper hispidinervum essential oil extracted by steam

distillation. LRIb

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The identification of peaks is based on comparison between the experimental Linear Retention Index (LRI)

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a

theorical experimental P = 1.0 atm P = 1.5 atm P = 2.0 atm 934 931 0.556 0.300 0.444 988 993 0.390 0.183 0.343 1002 1005 0.212 0.085 0.206 1008 1009 0.972 0.514 0.865 1014 1016 0.269 0.114 0.269 1025 1027 0.122 0.179 0.399 1032 1039 0.661 0.245 0.506 1044 1049 1.585 0.604 1.230 1054 1058 0.305 0.142 0.311 1086 1089 8.857 4.690 8.772 1285 1297 77.928 81.347 77.749 1374 1377 0.133 0.331 0.293 1389 1393 0.050 0.160 0.155 1403 1406 0.020 0.138 0.091 1417 1421 1.053 1.672 1.456 1452 1455 0.148 0.278 0.206 1458 1462 0.187 0.314 0.256 1484 1483 0.493 0.522 0.652 1500 1499 3.776 4.386 3.988 1522 1526 0.283 0.496 0.425 1555 1559 0.167 0.302 0.181 1577 1580 0.439 0.725 0.200 1600 1600 0.238 0.253 0.168 1652 1658 0.125 0.326 0.146 98.969 98.306 99.311

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α-Pinene Myrcene α-Phellandrene δ-3-Carene α-Terpinene Sylvestrene Z-β-Ocimene E-β-Ocimene γ-Terpinene Terpinolene Safrole α-Copaene β-Elemene Methyl eugenol E-caryophyllene α-Humulene Allo-Aromadendrene Germacrene-D Bicyclogermacrene δ-Cadinene Elemicin Spathulenol Guaiol α-Cadinol Total identified

Area (%)c

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Compounda

data with those from literature (Adams, 2007). b

Linear retention time calculated in relation to n-hydrocarbons series reported according to their elution order

c

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on HP-5MSseries.

The values correspond to relative proportions of the constituents of essential oils that were expressed as

percentages obtained by normalizing the peak area.

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Table 2: Antifeedant effects of P. hispidinervum essential oils, their major components and

their combinations on S. littoralis larvae, M. persicae and R. padi apterous adults in choice

L. decemlineata S. littoralis a % FI ± SE EC50 (µg/cm2)b

Essential oil Ph2

84.96 ± 11.4 0.4 (0.03, 4.8) 81.01 ± 6.8 18.3 (6.1, 54.4) 84.6 ± 10.1 14.4 (4.3, 44.7)

91.0 ± 6.9 17.7 (9.1, 34.1) 97.7 ± 1.0 3.1 (1.1, 8.4) 93.14 ± 4.1 20.1 (12.5, 31.8)

S/T (1/1)

79.52 ± 10.1 6.1 (2.2, 16.4) 80.36 ± 12.3 9.4 (3.7, 40.4) 67.73 ± 13.4

S/T (9/1)

58.43 ± 16.9

S/T (16/1)

54.41 ± 14.2

80.5 ± 7.2 5.25 (1.3, 20.7) 73.0 ± 12.6 53.80 (26.8, 99.4) 94.7 ± 3.1 41.48 (20.7, 81.4) 97.7 ± 2.2 17.2 (8.1, 36.5) 91.4 ± 7.3 16.4 (8.3, 29.9) 63.6 ± 6.9

Ph1.5 Ph1

Safrole (S) Terpinolene (T)

56.9 ± 5.9

68.5 ± 6.1

81.9 ± 4.9 27.6 (20.7, 36.9) 61.08 ± 6.7

68.00 ± 6.2

70.8 ± 6.7 8.85 (2.5, 31.5) 47.8 ± 8.9

23.7 ± 7.8

47.6 ± 8.8

18.9 ± 6.4

78.5 ± 6.3 8.1 (4.1, 16.4) 85.94 ± 4.2 8.3 (4.9, 13.5) 41.9 ± 9.5

37.4 ± 7.6

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Compounds

M. persicae R. padi a % SI ± SE EC50 (µg/cm2)b

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Sample

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tests.

63.7 ± 8.9

33.4 ± 8.3

26.9 ± 7.2

b

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51.6 ± 7.5 85.63 ± 6.8 5.11 (1.5, 16.4) S/T (1/16) 81.19 ± 6.8 30.1 ± 11.3 23.9 ± 8.1 56.4 ± 8.7 1.11 (0.4, 4.5) a Percent feeding (FI) / setting (SI) inhibition (100 µ/cm2 for EOs and 50 µ/cm2 for pure compounds). S/T (1/9)

EC50 95% confidence limits (lower, upper), concentration needed to produce 50% feeding / setting

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inhibition. * p<0.05, Wilcoxon paired rank test.

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Table 3: Effects of Piper hispidinervum essential oils, obtained at three pressures (Ph1, 1.0

atm; Ph1.5, 1.5 atm; Ph2, 2.0 atm), their major components and combinations, on mortality of second stage juveniles (J2) of Meloidogyne javanica and their effective doses.

Safrole (S) Terpinolene (T) S/T (1/1) S/T (9/1) S/T (16/1) S/T (1/9) S/T (1/16)

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1.19 (1.14-1.26) 1.32 (1.26-1.41) 1.23 (1.18-1.30)

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Corrected according to Scheider-Orelli’s formula. Values are means of four replicates.

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a

48.6 ± 5.5 3.1 ± 0.2 93.3 ± 2.3 100.0 ± 0.0 100 .0 ± 0.0 50,43± 7,07 36,19± 3,65

LC90 µg/µlb (95% CLc)

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Essential oil Ph1 Ph1.5 Ph2 Compound

J2 mortality (%)a LC50 mg/mlb at 72h (95% CLc) (1 µg/µl) 100±0 0.77 (0.75-0.81) 100±0 0.86 (0.82-0.89) 99.3 ±2.6 0.84 (0.81-0.87) (0.5 µg/µl)

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Sample

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Table 4: Effects of Piper. hispidinervum essential oil (Ph1) and mixtures of EO

Sample

Ph1 Safrole (S) Terpinolene (T) S/T (1/1) S/T (9/1) S/T (16/1)

Each value represents the hatch inhibition rate in the respective treatment corrected according to the control

(Scheider-Orelli’s formula). Values are means of four replicates.

Time 0: after 5 days of immersion in test solutions; time 7 and subsequent times: number of days of

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immersion in water after time 0.

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b

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a

%Egg hatch suppressiona with time b 0 7 14 21 28 95.6 86.7 90.2 89.2 88.9 94.8 86.3 88.5 88.6 88.7 94.4 87.0 88.4 88.7 88.8 91.5 84.5 87.6 87.5 87.6 94.4 87.0 88.4 88.7 88.8 91.5 84.5 87.6 87.5 87.6

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components on Meloidogyne javanica egg hatching over time.

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Figure Captions

Figure 1. Chemical structures of safrole (S) and terpinolene (T). Figure 2. Effects of Piper hispidinervum essential oil at sublethal dose on infection

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capacity of Meloidogyne javanica juveniles. Bars represent the penetration relative

percentage of treated J2 at 0.7 mg/mL (Ph1) vs untreated J2 (CONTROL) in tomato

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seedling roots on three independent experiments: Exp 1, Exp2, and Exp3

Figure 3.Phytotoxic activity of Piper hispidinervum essential oils obtained at three

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pressures (Ph1, 1.0 atm; Ph1.5, 1.5 atm; Ph2, 2.0 atm), their major components (Safrole, S and terpinolene, T) and combinations against Lactuca sativa, Lycopersicum sculentum and Lolium perenne. Germination and root length are expressed as % of growth

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inhibition respect to the control..

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The main component, safrole, explained most of the insect antifeedant action

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Low ratios of safrole improved the antifeedant effects of terpinolene

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P. hispidinervum essential oil was a strong nematicidal agent

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The nematicidal activity of essential oil was higher than its major

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•

components.

Terpinolene is critical to increase the nematicidal effects of safrole.

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•