Bulletin S.R.B.E./K.B. V.E., 147 (2011) : 71-79

Assessment of the acaricidal activity of several plant extracts on thephytophagous mite Tetranychus urticae (Tetranychidae)

in Tunisian cltrus orchards

Sabrine ATTlAl, Kaouthar Lebdi GRISSA2,Ghrabi-Gammar ZEINEB3,Anne Catherine MAILLEUX1,Georges LOGNAy4 & Thierry RANCEl

1 Earth and Life Institute, Biodiversity Research Centre, Université Catholique de Louvain, Place croix de sud 4-,B-1348 Louvain-la-Neuve, Belgium (e-mail: sabine_bio5@yahoo.fr)

2 Laboratoire d'Bntomologie-acarologie. Institut National Agronomique de Tunisie, 1082 Cité Mahrajène, Tunis,Tunisia.

3 Banque Nationale de Gènes, Tunisia. Av. du Leader Yasser Arafat, ZI Charguia 11080, Tunis, Tunisia,4 Université de Liège Gembloux Agro-Bio Tech Unité de Chimie analytique, Passage de Déportés, 2, B-5030

Gembloux, Belgique.

Abstract

To develop sustainable pest control in Tunisien citrus orchards, the present work aimed to evaluatethe toxicity of 31 plant extracts obtained from Tunisia and two synthetic acaricides (spirodiclofen andfenbutatin oxide) on the phytophagous mite species Tetranychus urticae (Koch). Field experimentsshowed that the extracts of seven plant species (Haplophyllum tuberculatum, Deverra scoparia,Mentha pulegium, Chrysanthemum coronarium, Hertia cheirifolia, Citrus aurantium and Santolinaafricana) are effective and the population density of T. urticae was reduced at 0.30, 0.36, 0.37, 0.46,0.48, 0.50, and 0.53 mites per leaf respectively for more than 21 days compared with the untreatedcontrol (3.7 mites per leaf). They also showed a comparable activity to classical synthetic acaricides(0.50 mites per leaf for spirodiclofen and 0.53 mites per leaf for fenbutatin oxide). The evaluation ofthe potential of biologically active plant volatiles against T. urticae might provide a new approach tothe development of natural acaricides to be used both in biological and integrated pest managementstrategies for controlling two-spotted spider mites in Tunisian citrus orchards.

Keywords: Tunisia, plant extracts, acaricidal activity, Tetranychus urticae, essential oil, distillates.

Introduction

The two-spotted spider mite Tetranychusurticae Koch, 1Sone of the mast important pestsof fruit, vegetable and ornamental plantsworldwide (JOHNSON & LYON, 1991). Theeconomie impact of this mite has increasedrecently in Tunisia, mainly because of itsresistance to acaricides, which hampers pestcontrol in citrus nurseries (LEBDI & DHOUIBI,2002). The main problem with the developmentof pesticide resistance and the resurgence of mitepopulations is the use of non-selective syntheticpesticides that also eliminate natural enemiessuch as predatory mites and spiders (CRANHAM& HELLE,1985). Spider mites have developed aresistance to more than 80 acaricides in morethan 60 countries (ANONYME,2005). For these

reasons, essential oils are realistic alternatives tosynthetic acaricides because of their selectivity,biodegradability and few side effects on non-target organisms and the environment (HAy &WATERMAN,1993; SINGH& UPADHYYAY,1993;ISMAN, 2000, 2001; CHlASSON et al., 2001;BASTA& SPOONER-HART,2002; RAslKARlet al.,2005). They can be applied to field andgreenhouse crops in the same manner as currentacaricides. They also contain numeroussecondary metabolites that deter attack frominsect and generalist herbivores (HARBORNE,1988) and provide an alternative for resistancemanagement because sorne plant phytochemicalpreparations can be highly effective againstinsecticide-resistant pests (LINDQUISTLet al.,1990; SCHMUTTERER,1992; AHNet al., 1997; YIet al., 2007).

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Both BOYD & ALVERSON (2000) andCHIASSONet al. (2004) have investigated the useof plant extraets as alternative aearicides foradult Tetranychus urticae with promising results.They reported the repellent effect of garlicextracts against Tetranychus urticae. The oiltoxic effeet of Chenopodium ambrosioides wasassessed on T. urticae and Panonychus citri(CHIASSONet al., 2004). Other experimentsshowed the acaricidal efficacy of neem(Azadirachta indica) against Tarsonemus latusand revealed that the population growth ratebecame negative when mites were exposed toplants treated with this extract (VENZONet al.,2008). Other studies pointed out that theessential oil of Satureja hortensis L., Ocimumbasi/icum L. and Thymus vulgaris L. wereeffective as fumigants against Bemisia tabaci andTetranychus urticae (ASLANet al., 2004).

ln this study, our aim was to characterise theplant extracts of 31 plant species in citrusorchards in Tunisia and analyse their potentialacaricidal effects against T urticae comparedwith that of spirodiclofen and fenbutatin oxide,two commercialacaricides,

Materials and Metbods

SeJected plants and extraction technique

Thirty-one aromatic and medicinal plantspecies, representing seventeen families werecollected from different Tunisian localities,South (Saddine, Mednine), South-West(Téjrouine), North-West (Kef), North-East(Hammamet, Mraissa, Tunis) during the years of2006 to 2008 (Tablel). The selection of plantspecies was based on previous work but also onthe use of plant products in traditional medicinein Tunisia (BEN HAl JILANIet al., 2007). Thefresh material was free of any pre-harvestchemical treatment (organic products). Thesesamples were freshly harvested and sorted foruniformity and absence of defects before beingstored at _2°C until analysis.

Extracts were obtained from fresh material(Table 1) by hydre-distillation (essential oils anddistillates) during three hours using a Clevenger-type apparatus. A few drops of adjuvant wereadded (Agral 90) for gluing the extracts at theleaf surface.

Toxicity test

Extracts obtained by hydre-distillation werefirst diluted 100x in ethanol then diluted again100x in water.

Field experiment for T urticae control wereestablished in a three hectares lemon (var.Lunari) orchard situated at Birbouragba, Tunisiawhere an infection of T. urticae developednaturally. The orchard was divided into 34 plots,each one containing 4 plants. Two untreatedrows separated each plots. Plant extracts andinsecticide were applied using a knapsacksprayer equipped with a solid-cone nozzle thatoperated at a pressure of 2-3 bar. Specialattention was paid to ensure spraying underneaththe leaves. Each treatment was applied to 3replicates. Spirodiclofen and Fenbutatin oxidewere used as a reference ta be compared betweenwith the treatments using plant extracts.

Sampling took place on 4 occasions: 3, 7, 14and 21 days after the application. At eachsampling, one hundred leaves were takenrandomly from the four different plants in eachplot. Leaves were immediately examined in thelaboratory under a binocular microscope toassess the density of mite species. Leaves werealso observed for phytotoxicity symptoms causedby acaricides or plant extracts.

Data analysis

A three way Anova with Newman-Keuls testof variance, and proc GLM of the SAS instituteine. including Scheffe's and Tukey's tests wereused to compare a11 treatments with control.Tests were performed using Graph Pad Prismversion 5.01 for windows, Graph Pad Software(San Diego, California, USA). Ali tests wereapplied under two-tailed hypotheses and thesignificance levelP was set at 0.05.

Results

Essential oil yields

The yield of essential oil in the current studyvaried greatly from a minimum of 0.01% for A.sativum, to a maximum of 0.5% for D. scoparia.Nine plant extracts did not yield essential oils(Table 2).

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Table l: List of plant species tested for their acaricidal activity, plant part used for extraction, site and date ofsamplings.

Plant familv Seientifle Dame Plant organ Sampled site Samplin2 dateAnacardiaceae Pistacia lentiscus Aerial part Saddine June 2007

Cotin us coggyra Aerial part Tunis March2008Apiaceae Daucus carota Aerialpart Hammamet October 2006

Deverra seoparia Aerial part Téjrouine May 2008Asteraceae Hertia cheirifolia Aerial part Saddine February 2008

Seriphidium herba-album Aerial part Saddine June 2007Chrysantemum coronarium Flowers Tunis March 2008Santolina africana Aerial part Téjrouine Avril 2007

Cupressaceae Juniperus phoenica Green female cones Maraissa March2008Fabaceae Acacia cyanophylla flowers Tunis January 2008

Sophora secundiflora Pods and seeds Tunis March2008Geraniaceae Pelargonium graveolens Aerial part Hammamet March2008Globulariaceae Globularia alypum leaves Saddine June 2007Lamiaceae Salvia officinalis Aerial part Tunis March2008

Thymbra capitata Aerial part Saddine June 2007Rosmarinus officinalis Aerial part Saddine June 2007Mentha pulegium Aerial part Saddine June 2007Lavandula officinalis Aerial part Saddine June 2007

Lauraceae Laurus nobilis leaves Hammamet January 2007Liliaceae Allium sativum bulbs Hanunamet March2008

Allium cepa bulbs Hammamet March2008Meliaceae Melia azedarach fruit Tunis January 2007Myrtaceae Eucalyptus gomphocephala leaves Saddine September 2006

Myrtus communis Aerial part Saddine June 2007Papaveraceae Papaver rhoeas Aerial part Saddine June 2007

Lan/ana camara Aerial part Tunis June 2007Rutaceae Ruta chalepensis Aerial part Saddine June 2007

Citrus aurantium leaves Tunis March2008Haplophyllum tuberculatum Aerial part Mednine Septembre 2007

Urticaceae Urtica pilulufera Aerial part Saddine October 2006Zvgophyllaceae Peganum harmala Aerial part Saddine June 2007

Field experiments

Colonization patterns of phytophagous miteson Lunari variety (control plots)

Toxicity tests on field

The acaricidal effects in citrus orchards of the31 plants extracts is surnmarized in Table 4 and5.

According to the ANOV A test, white theeffect of plots and exposure time interactions ofextracts on T. urticae were not significantlydifferent (P= 0.5593), the effect oftreatment andexposure duration interactions were verysignificant at P

Table 2. Essential oil yields according to plant species

Plant species Fresch weil!bt (g) Volume oit obtained (ml) % yieldPistacia lentiscus 1500 1 0.06Cotinus coggyra 3000 0 0Peganum harmala 1500 0 0Juniperus phoenicea 3500 2.5 0.07Globularia alypum 1500 0 0Laurus nobilis 800 2 0.25Melia azedarach 526 0 0Pelargonium graveolens 450 1.5 0.33Urtica pilu/ufera 700 0 0Daucus caro ta 423 0 0Deverra seoparia 1166 6 0.51Hertia cheirifolia 1705 2 0.11Seriphidium herba-album 3614 10 0.28Chrysantemum coronarium 7300 5 0.07Santolina africana 500 1 0.02Papaver rhoeas 359 0 0Lantana camara 1732 0 0Allium sativum 9000 1 0.01Allium cepa 9000 1.2 0.013Acacia cyanophylla 12000 0.5 0.0041Sophora secundiflora 8500 0 0Eucalyptus gomphocephala 5300 2 0.037Myrtus communis 6200 3 0.048Ruta chalepensis 3000 0.5 0.016Haplophyllum tuberculatum 2000 2.5 0.1Citrus aurantium 1500 6 0.4Salvia officinalis 1250 5.5 0.45Thymbra capitata 7339 35 0.48Rosmarinus officinalis 4203 15 0.36Mentha pulegium 3325 6 0.18Lavandu/a officinalis 2000 2.5 0.20

Active fonns per 100 leaves50045040035030025020015010050O~~~T--.--~-r-----.--.--------.-----.--,-~--,-~-,-----.--~~#####~~~~~~~~§§§§~~~~~-v 1).' " R:! \t('$ ,,~ '0''$ r-:? 'V Of ~ r'f ~: -,,($ ,,($ ,,($ ,,($ !ô~' 'ô~ qf 'ô~ tP

0,; '), ')) ,,0,; ')) -c '\i '!l r•.X" ,,-" ,," •.•." ff ~ ~ ff • .o'U ,,, ')," '),' c!' c.;JlÎ 9>'li c§' .,

\t( ••••••: •••'0 {:>'__ Tetranychus urticae

Fig. 1. Population dynamics of T urticae on Lunari variety in control plots during 2008.

Table 3. Acaricidal effects ID citrus orchards ID function of different interactions (treatments, Plots, and exposureduration)

Source Mean square F value Pr>FTreatments 43899.791 136.95

The second group showed number of mites per100 leaves after 21 days ranging from 97 to244.67. ln the third group, more than 300individuals per 100 Ieaves recorded that indicatesa quasi-absence of activity (Table 4). ln general,the acaricidal activity was relatively enhancedwith adjuvant (Agral 90) and exposure times forall extracts (Table 3). During the experiments,any phytotoxic effects of extracts were noted.

Discussion and Conclusion

To develop sustainable pest control inTunisian citrus orchards, we have evaluate thetoxicity of 31 plant extracts obtained fromTunisia and two synthetic acaricides(spirodiclofen and fenbutatin oxide) on onephytophagous mite specie: T. urticae. Altogether,31 extracts (essential oils and distillates) from 31plants were tested in field trials. Sorne plants areendemie to North African Sahara such as D.scoparia, S. africana and H. cheirifolia (ALA-PETITE1981; OBERPRIEDER,2002; DJERIDANEetal., 2007), sorne ean be found throughout thernediterranean circurnference such as Thymbracapitata, Rosmarinus officinalis, S. herba-album, Pelargonium graveolens (ALAPETITE,1981).

Contact acaricidal activity of plant extractshave been weIl demonstrated against mites(ISMAN, 2000, 2001; MlRESMAILLJ& ISMAN2006). Differences of toxicity between extractsand plant species were recorded in fieldexperiment. AlI selected extracts caused mortalitystatistically similar to that obtained withsynthetic products Spirodiclofen and Fenbutatinoxide except extracts from P. rlzoeas, R. chale-pensis, L. camara P. harmala, S. secundiflora, E.ghomphocephala and L. nobilis that did notcause significant T. urticae adult mortality incomparison to the control after 21 days aftertreatment.

Efficiency of essential oils

The yield of essential oil varied considerablydepending upon plant species. Maximum yieldsof 0.5% were achieved with the essential oil ofD. seoparia. Nine plant extracts do not containessential oils at aIl. This quite poor yield limits

the large scale application possibility of sorne ofthe plant extraet and further development will beneeded to implement these applications and tolook toward a more efficient method of oilextraction and plant culture.

Here we show that the most effective ofextracts are S. africana, C. aurantium, C.coronarium, H. cheirifo/ia M. pulegium D.seoparia and H. tuberculatum, the number ofmites recorded per 100 leaves never overpasses60 individuals after 21 days compared to twosynthetic acaricides. Three of these plant areendernic to north Afriea (POTTIERALAPETITE,1981; LE FLOC'K, 1983; OBERPRJEDER,2002;DJERIDANEet al., 2008).

Other studies showed the acaricidal propertiesof essential oils. PHANANJAy et al. (2005)showed the effeet of sorne rhizome oil on T.urticae. MANSOURet al. (1986) pointed out thatbesides the toxicity, the residues of essential oilsof sorne Labiatae species are repellent andstrongly reduee the fecundity of T. cinnabarinusfemales. Others studies indicate that purerosemary oil and Eco/Trol (rosemary oil basedpesticide) cause complete mortality of spidermites Tetranychus urticae at concentrations thatare not phytotoxic to the host plant and that didnot cause any mortality in Phytoseii/us persimilis(phytoseiidae) neither affected their eggs(MJRESMAILLI& ISMAN 2006). Sorne of theprevious studies (l'uNc & SAHINKAyA, 1998;ISMAN et al., 2001) have been reported thatacaricidal effects of plant essential oils arerelated to their chemieal compositions. Essentialoils can affect animals from different ordersincluding mites. For instance, ANTHONYet al.(2008) showed the biocidal properties of theessential oil of H. tuberculatum and Plectranthuscylindraceus against Meloidogyne javanica(Nematoda).

Efficiency of distillates

Our work is the first testing the efficiency ofdistillates on T. urticae. One main technicaldifficulty is the quantification of the molecules inthe distillates. Here we show that the mosteffective are H tuberculatum and H. cheirifolia,the latter is endernic in North Africa.

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Among 31 plants tested as acaricides against thephytophagous mite T. urticae (Boisduval) in fieldtrials, the most toxie were: H tuberculatum, D.scoparia, M. pulegium, C. eoronarium, Hcheirifolia, C. aurantium and S. africana. Threeof these highly effective plants are endemie toNorth Africa (see Table 4). ln vivo experiments,the acaricidal activity was relatively enhancedwith adjuvant Agral 90 and exposure time for aUextraets: distillates or essential oils. However,except C. coronarium and M. pulegium, this isthe first study demonstrating that D. scoparia, Htubereulatum, H. cheirifolia, C. aurantium, S.africana, have acaricidal activity against T.urticae. Toxicity effect of D. seoparia and H

tuberculatum may be explained by higher alpha-pinene and sabinene contents (MAsoUDI et al.,2004). According to MIRESMAILLI et al. (2006),the major chemical components of essential oilswith acaricidal activity have been identified as1,8 cineole, alpha-pinene, and myrcene.Difference in activity between plant species isthat clearly related to difference in chernicalcomposition and proportion of main componentspresents in extracts (KOUNINKI et al., 2007). Ourresults suggest that these seven plants may havegreat potential for effective rnanagements of T.urticae. Furthermore, plant distillates are widelyavailable and sorne are re1atively inexpensive, as

Table 4. Tests toxicity bioassays of extracts and reference products ta T. urticae on field (results after 1 application,smallletters indicate significant differences at p

Active forms per 100 leafDay 14 (13 July 2008) Day 21 (19 July 2008)

Extracts Mean value ± standard deviation Extracts Mean value ± standard deviationControl 388.67±18.82 a Control 379.33±16.92 aP. harmala D 388±18.68 ab P. rhoeas D 379.33±17.24 aG.alypum D 387.33±13.50 ab R. chalepensis E 367.33±29.67 aP. rhoeas D 377±8 abc L. camaraD 358.67±11.26 aD. carota D 369±12.34 abcd Piharmala D 357.67± 10.59 aS. secundiflora D 349.33±5.85 abcde S. secundiflora D 357.33±11.23 aL. camara D 345±20.66 abcde E. ghomphocephala E 355±12.48 aR. chalepensis E 331±30.80 abcdef L. nobilis E 350.67±44.04 aT. capitata E 288.33±20.30 abcdef M. azedarach D 341±16.70 abU pilulifera D 284±17.57 abcdefgh S herbe-album E 328.67±24.41 abC. aurantium E 282.33± 12.66 bcdefgh D. carota H 322.33±37.16 abC. coggyraD 273.67±24.S8 cdefgh G. alypumD 232.33±60.92 bcL. officinalis E 270.67±15.56 defgh T. capitata E J80.67± 14.57 cdR. officinalis E 267.67±19.50 defgh J. phoenicea E 174.33±JO.50 cdS. herba-album E 262.33±25.54 efghi C. coggyra D 162.67±16.62 cdeA. cyanophylla D 2SO±IS.39 efghij A. cyanophylla D 128.33±32 cdefM. azedarach D 238.67±20.74 fghijk M. communis E 126±6.5S cdefL. nobilis E 237.33±24.94 fghijk L. officinalis E 117.33± 17.24 cdefJ. phoenicea E 232.67± 1l.3 7 fghijk U pilulifera D 115.33±8.08 cdefE. ghomphocephala E 227.67±22.67 fghijkl A. cepa D 112.33±8.32 defA. cepa D 187±II ghijklm P. lentiscus D I06.67±12.50 defS. officinalis E 184.33±7.63 ghijklm P. graveolens E I06.67±12.50 defP. lentiscus D 180±14.10 hijklm R. officinalis E I06±IO.14 defP. graveolens E 180±14.IO hijklm A. sativum D lO5.33±7.02 defA. sativum D 179±16.S2 hijklm S. officina/is E 97±4 defH. cheirifolia D 160.67±15.53 ijklm Fenbutatin oxyde 53.67±8.60 efM. eommunis E 155.33±7.02 jklm S. african« E 53.33±9.29 efM. pulegium E ISI±29.05 jklm C. aurantium E 50.67±8.50 efSpirodiclofen 142.67±18.71 klm Spirodiclofen SO.33±5.50 efFenbutatin oxide 139.33±14.97 klm H. cheirifolia D 48.33±5.68 efC. coronarium E 122±18.15.39 lm C. coronarium E 46.67±7.76 efS. africana E ll9±18.73 m M. pulegium E 37.67±9.60 fD. scoparia E 95.67±12.34 m D. seoparia E 36.67±7.09 fH. tuberculatum D 85.33±8.50 m H. tubercukuium D 30.67±5.50 f

Acknowledgernentscompared with essential oils and distillates. lnthat context, D. scoparia and H. tubercu/atumneed further studies from an economical point ofview. Distillates are especially interesting as theyare not very expensive. Moreover, theirextraction is very easy even for farmers havingno laboratory materials and does not require highquantity of plants on the contrary to essentialoils.

Another further study is also necessary todetermine the toxicity of these extracts onpredatory mites and on other economicallyimportant pests in citrus orchards where pestmanagement depends on chemical applications,which is causing environmental pollution andresistance in pest population (LAMIRI et a/.,2001).

We are very grateful to George van Impe andGuillaume Le Goff for the useful discussions about Turticae. The authors are also indebted to theWallonies-Bruxelles international (wbi). This ispublication BRC 177 of the Biodiversity ResearchCentre at DCL.

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Bulletin S.R.B.E./K.B. V.E., 147 (2011): 79-83

A new myrmecophilousAllochernes from ant nests in the higbaltitude of the eastern Spanish Pyrenees

(Arachnida: Pseudoscorpiones: Chernetidae)

HANSHENDERICKX1,2

1 Department of Biology, Universiteit Antwerpen, Groenenborglaan 171, B-2020 Antwerpen, 2 Royal BelgianInstitute of Natural Sciences, Departrnent of Entomology, Vautierstraat 29, B-1OO0 Brussels (Address forcorrespondence: HemeIrijkstraat 4, B-2400 Mol. E-mail: cavexplorer@gmail.com).

Abstract

Allochernes struyvei sp.n., a new myrmecophilous pseudoscorpion from the Spanish Pyrenees, isdescribed.

Keywords : Pseudoscorpion,Allochernes struyvei sp.n., myrmecophilous,Pyrenees, Spain

Introduction

Some species of the pseudoscorpion genusAllochernes BEIER occur occasionally with ants(BEIER, 1963), other species of this genus haveonly been found in the nests of a particular antspecies (HENDERICKX& VETS, 2003) and seemrestricted to this host.

During collection trips in May and September2009 an unidentified pseudoscorpion was foundin heaps of the ant Formica paralugubrisSEIFERT, 1996 near Setcases, Spain by TimSTRUYVE(Muizen, Belgium), who was searching

for Myrmecophilous Staphylinidae. The pseudo-scorpions were kindly donated to the author andthe new species is described in this publication.

MateriaJ and methods

AIl specimens were hand captured by siftingmaterial from the ant-heaps and fixed in 70%ethanol.

Microscopical examination was performedwith a Leitz microscope and optics, measure-ments with a Zeiss calibration grid. A FEIQuanta-200 was used for scanning electronmicroscopy.

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