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ajor compounds and insecticidal activities of two Tunisian Artemisiassential oils toward two major coleopteran pests
lfa Bachrouch a, Nadhem Ferjani b, Soumaya Haouel c, Jouda Mediouni Ben Jemâa c,∗
Laboratoire de Protection des Végétaux, Institut National de la Recherche Agronomique de Tunisie (INRAT), Université de Carthage, Rue Hedi Karray,unis, Ariana 2049, TunisiaInstitut Supérieur des Sciences Biologiques Appliquées de Tunis, Université de Tunis El Manar, TunisiaLaboratoire de Biotechnologie Appliquée à l’Agriculture, Institut National de la Recherche Agronomique de Tunisie (INRAT), Université de Carthage, Rueedi Karray, Tunis, Ariana 2049, Tunisia
r t i c l e i n f o
rticle history:eceived 6 August 2014eceived in revised form 1 December 2014ccepted 2 December 2014
eywords:ssential oilumigantPM
a b s t r a c t
The aim of this research was to investigate the chemical composition and evaluate the insecticidal activ-ities of Artemisia herba-alba and Artemisia absinthium areal parts essential oil from Tunisia. Fumigantand contact toxicities were assessed toward two major stored product beetles: Orysaephilus surinamensisand Tribolium castaneum. The chemical composition of the two oils was characterized by qualitative andquantitative differences. The major common compounds were camphor, 1,8 cineole, camphene, and bor-neol, whereas beta-thujone was the characteristic component of A. absinthium oil. Results indicated thatboth oils exhibited fumigant and contact toxicity potential. The fumigant bioessays showed A. herba-albaessential oil to be more toxic and O. surinamensis to be more susceptible. The corresponding LC50 and
opical application LC95 values were respectively 30.22 and 132.11 �L/L air. The contact bioessays using topical applicationsrevealed that A. absinthium essential oil was more efficient and again the saw-toothed grain beetle to bemore sensitive. The corresponding LD50 and LD95 values were 0.209 and 1.963 �L. Our work indicatesconcern in the use of Artemisia essential oils from Tunisia, both as fumigant and contact bioinsecticidesagainst stored product pests of economic importance.
The genus Artemisia (Asteracae family) belongs to the tribenthemideae, and includes over 500 species, found mainly insia, Europe, and North America (Bora and Sharma, 2011). Theyre known to produce essential oils valorised in folk and mod-rn medicine, cosmetics and pharmaceutical industry (Teixeira dailva, 2004; Abad et al., 2012)
Generally, essential oils exhibited wide range of bioactivity,ue to the presence of secondary metabolites, acting throughiverse modes of action (Abad et al., 2012). Artemisia species
solated compounds have been known mainly for their antidepres-ant (Mahmoudi et al., 2009), hepatoprotective (Gilani and Janbaz,
995), acaricidal (Chiasson et al., 2001), antimicrobial (Juteau et al.,003), antifungal (Saban et al., 2005), neuroprotective (Bora andharma, 2010), antehelmintic (Tariq et al., 2009), antiprotozoal
effects (Tariku et al., 2011), antitumoral (Bhakuni et al., 2001), andinsecticidal (Kordali et al., 2005, 2006) proprieties.
In Tunisia, five species of the genus Artemisia were recorded(Nabli, 1989). Artemisia herba-alba (Asso., 1779) known as “desertwormwood” is a prominent plant of the Irano-Turanien steppes ofSpain, North Africa, and the Middle East (Quezel and Santa, 1962; LeFloc’h et al., 1989). It’s a spontaneous herb named “Shih” in Tunisia(Haouari and Ferchichi, 2009). Moreover, Artemisia absinthium (L.,1753) known as wormwood, is an aromatic bitter perennial smallshrub. It is also locally known as “chajret mariem” in Tunisia.
Aromatic plants were traditionally used as stored product pro-tectants against pest infestation. Their essential oils have beenappraised as alternatives to chemical pesticides (Isman et al., 2011).
Numerous studies have reported the insecticidal proprieties ofArtemisia species essential oils versus a wild range of pests. In thiscontext, repellent and fumigant activities of essential oils from var-ious Artemisia species have been reported toward the red flourbeetle Tribolium castaneum (Herbst, 1797): Artemisia vulgaris (L.,
1753) (Wang et al., 2006), Artemisia sieberi (Besser) (Negahbanet al., 2007), Artemisia scoparia (Waldst. & Kit) (Negahban et al.,2004) and A. herba-alba (Asso.) (Moumni et al., 2014). Similarly,
phene (2.63–2.37%), and borneol (2.29–1.77%) were the commonmajor compounds for both oils, whereas �-thujone (22.72%) wasthe major component of A. absinthium essential oil.
Table 1Essential oil yields (%) of A. herba-alba and A. absinthium.
Artemisia Oil yields (%)
28 O. Bachrouch et al. / Industrial C
nsecticidal activities from different Artemisia essentioil oils wereecorded against the saw-toothed grain beetle Oryzaephilus surina-ensis (L., 1758); Artemisia judaica (L.) (Kostyukovsky et al., 2002);
rtemisia argyi (H. Lév. & Vaniot) (Lü et al., 2011); A. absinthiumMediouni Ben Jemâa, 2014).
Considering the interesting insecticidal potential of Artemisiapecies essential oils, in this study, we investigate composition,umigant and contact toxicities of A. herba-alba and A. absinthiumssential oils collected from North Tunisia against laboratory rearedtrains of O. surinamensis and T. castaneum as a component in acreening program to find new agrochemicals from aromatic plants.
. Materials and methods
.1. Plant material
Leaves of A. herba-alba and A. absinthium were procured duringhe vegetative period in July 2011 respectively from the botani-al garden of National Agricultural Research Institute of Tunisia10◦15′E, 36◦862′N) and the region of Saouaf, Zagouhan (10◦15′E;6◦217′N) both situated in North Tunisia. The plant materialas botanically characterized by Professor Smaoui (Borj Cedria
iotechnology Center, Tunisia) according to the morphologicalescription presented in Tunisian flora (Pottier–Alapetite 1979).oucher specimens (P.l.01 and P.l.02) were deposited in the Labora-
ory of Biotechnology Applied to Agriculture (National Agriculturalesearch Institute of Tunisie, INRAT). The harvest material was air-ried at 20–25 ◦C (room temperature) for two weeks.
.2. Essential oil extraction and chemical analysis
The essential oils were extracted from the air dried samples byydrodistillation (50 g of each sample in 500 ml of distilled water)sing a modified clevenger-type apparatus for 3 h. The oils wereried over anhydrous sodium sulphate and stored in sealed glassials in a refrigerator at 4 ◦C prior to analysis.
Four experiments were designed to determine essential oilields. These latest were calculated according to dry weight ofhe plant materials. Chemical composition was analyzed usingn Agilent-Technologies 6890 N Network GC system equippedith a flame ionization detector and HP-5MS capillary column
30 m × 0.25 mm, film thickness 0.25 �m; Agilent-Technologies,ittle Falls, CA, USA). Temperatures of injector and detector wereet respectively at 220 ◦C and 290 ◦C. The temperature of columnas programmed from 80 ◦C to 220 ◦C at a rate of 4 ◦C/min, with
he lower and upper temperatures being held for 3 and 10 min,espectively.
Helium was used as a carrier gas with a flow rate of 1.0 ml/min.n oil sample of 1.0 �l was injected, using split mode (split ratio,:100). A built-in data-handling program provided by the manufac-urer of the gas chromatograph was used in all quantifications. Theomposition was reported as a relative percentage of the total peakrea. The identification of the essential oil constituents was basedn a comparison of their retention times to n-alkanes, compared toublished data and spectra of authentic compounds. Compoundsere further identified and authenticated using their mass spec-
ra compared to the Wiley version 7.0 library. Major compounds inach group were marked in bold form.
.3. Insect rearing
Insects used in this study were taken from laboratory rearingolonies initiated from infested flour samples since 2009. O. suri-amensis and T. castaneum were reared on wheat flour. The culturesere maintained in the dark in growth chamber set at 25 ± 1 ◦C and
nd Products 65 (2015) 127–133
65% ± 5% relative humidity. Adult insects, 7 days old, were used forfumigant and contact toxicities tests.
2.4. Fumigant toxicity bioassays
A whatman filter paper (diameter 2.0 cm), impregned with oil,was attached and hanged up to the scraw caps of a 44 ml plexi-glass bottle with a support (2 cm length wire). Caps were screwedtightly on the vials, each of which contained separately 10 adultsof each species. The tested oil doses were 2.5, 5, 7.5, 10, 12.5,and 15 �L providing equivalent concentrations of 56.82, 113. 64,170.45, 227.27, 284.09, and 340.90 �L/L air. Each treatment andcheck were repeated four times. Mortality was recorded hourly.Abbott correction formula (Abbott, 1925) was applied to assessinsect mortality. Results from all replicates were submitted to pro-bit analysis (Finney, 1971) to determine lethal concentrations (LC50,LC95) and lethal times (LT50, LT95).
2.5. Contact toxicity
Essential oils contact toxicity towards O. surinamensis and T. cas-taneum adults was measured according to Liu and Ho (1999). Aserial dilution of the essential oil was prepared in acetone (0.1 ml).1 �l of aliquots dilutions were deposited topically to dorsal thoraxof beetles. Acetone was used as a check trial. After that, treated andcontrol insects with culture media (2 g) were placed into Plexiglassbottles (10 beetles/bottle). Each concentration was repeated fourtimes. Mortality was observed each hour til end-point. The LD50values were estimated using probit analysis (Finney, 1971).
2.6. Statistical analysis
All data of insecticidal activities were expressed as mean ± SD.They were analyzed by one-way analysis of variance (ANOVA) usingstatistica (Statsoft, 1998). Duncan test was applied to the means todetect significant differences at the 0.05 percent level. The SPSSpackage software (Version 11.5) was used for the analyses.
3. Results
3.1. Essential oil yield and composition
Essential oil yields of A. herba-alba and A. absinthium are illus-trated in Table 1. Results demonstrated that essential oil yieldsvaried according to Artemisia species. Highest yield was obtainedfrom leaves of A. herba-alba compared to A. absinthium. Moreover,significant statistical differences were observed (Table 1).
GC and GC–MS analysis of A. herba-alba and A. absinthium essen-tial oils showed that 22 and 31 compounds were identified, whichrepresent respectively 49.03% and 56.26% of total constituents(Table 2). Camphor (11.48–16.71%); 1,8 cineol (19.59–5.47%); cam-
A. herba-alba 2 ± 0.2 aA. absinthium 0.6 ± 0.05 b
Mean values followed by different letters are significantly different by Duncan test(p < 5%).
: Not detected. RI: Retention Index calculated on HP.5MS cappillary column (30 m
.2. Fumigant toxicity
Results related to fumigant toxicity bioessays were shown inig. 1. Essential oil from A. herba-alba was more toxic for bothnsects at all tested concentrations. Susceptibility of adult pestopulation strains of O. surinamensis was greater compared to pop-lation strains of T. castaneum (Fig. 1). This led to that fumigantctivity changed with insect strain, plant species, oil concentra-ion, and exposure duration. These results were proved by lethaloncentration values. The corresponding LC50 and LC95 for A. herba-
lba were respectively 3.05 and 13.41 �L/L air for O. surinemensisgainst 5.22 and 45.18 �L/L air for T. castaneum. Concerning A.bsinthium, the corresponding LC50 and LC95 were respectively
able 3C50 and LC95 values (�L/L air) of fumigant bioessay with A. herba-alba and A. absinthium
Artemisia oils Insect species CL50a CL95
a
A. herba-alba O. surinemensis 3.05b, A 13.41b,
T. castaneum 27.76a, B 37.63a,
A. absinthium O. surinemensis 5.22b, A 45.18b,
T. castaneum 35.18a, A 59.87a,
or each oil, comparaison were made between LC50 and LC95 values for both insects. Meauncan test at p < 0.05. Between oils, comparaisons were made between LC50 and LC95 valot statistically different by Duncan test at p < 0.05.a Units LC50 and LC95 �L/L air, applied for 24 h at 25 ◦C.
49.03 56.26
mn × 0.25 �m).
27.76 and 37.63 �L/L air for O. surinemensis against 35.18 and59.87 �L/L air for T. castaneum. Statistical analysis showed signifi-cant differences between oils and insects (Table 3).
The lethal time bioassay results again showed the saw-toothedgrain beetle to be more sensitive to both essential oils than thered flour beetle. The LT50 and LT95 values for O. surinamensis wererespectively 3.725 and 7.119 h for A. herba-alba essential oil and10.750 and 18.258 h for A. absinthium essential oil versus 17.308 and35.57 h and 75.576 and 98.167 h, respectively, regarding A. herba-alba and A. absinthium essential oils for T. castaneum. Moreover,
significant differences between means were observed for each oiland each insect (Table 4).
ns followed by same letter (letter in lowercase) were not statistically different byues of each insect species. Means followed by same letter (letter in uppercase) were
130 O. Bachrouch et al. / Industrial Crops and Products 65 (2015) 127–133
expos
3
tabcmc
TLa
FMaLu
Fig. 1. Percentage of corrected mortality of O. surinamensis and T. castaneum
.3. Contact toxicity
Results related to contact toxicity bioessays by topical applica-ion was described in Fig. 2. Mortality recordings revealed that A.bsinthium essential oil to be more efficient and the saw-toothedeetle to be more sensitive. Moreover, toxicity depends upon oils
oncentrations. Although at the lowest concentration (0.025 �L), allortalities did not exceed 22.5%, they reached 55% at the highest
oncentration (0.1 �L) (Fig. 2).
able 4T50 and LT95 (h) values of A. herba-alba and A. absinthium essential oils calculatedt the lowest concentration 56.82 �L/L air.
Artemisia oils Insect species LT50a LT95
a
A. herba-alba O. surinemensisT. castaneum
3.725b, B17.30a, B
7.119b, B35.57a, A
A. absinthium O. surinemensisT. castaneum
10.750b, A75.57a, A
18.258b, A98.16a, B
or each oil, comparaison were made between LC50 and LC95 values for both insects.eans followed by same letter (letter in lowercase) were not statistically different
ccording to Duncan test at p < 0.05. Between oils, comparaisons were made betweenC50 and LC95 values of each insect species. Means followed by same letter (letter inppercase) were not statistically different according to Duncan test at p < 0.05.a Units LT50 and LT95 (h), applied for 24 h at 25 ◦C.
ed for variousdurations to essential oil from A. Herba-alba and A. Absinthium.
The results depicted in Table 5 again showed that the saw-toothed grain beetle was more sensitive to both essential oilsmainly to A. absinthium oil. The corresponding LD50 and LD95 val-ues were 0.209 and 1.963 �L. Whereas, the red flour beetle wasmore tolerant to both oils, mainly A. herba-alba oil with corre-sponding LD50 and LD95 values of 7.432 and 133.323 �L. Indeed,
results revealed significant statistical differences between lethaldoses means of oils (Table 5).
Table 5LD50 and LD95 values (�L) of contact bioessay with A. herba-alba and A. absinthiumessential oils.
Artemisia oil Insect species LD50a LD95
a
A. herba-alba O. surinemensisT. castaneum
2.242b, A7.432a, A
16.864b, A133.323a, A
A. absinthium O. surinemensisT. castaneum
0.209b, B2.261a, B
1.963b, B5.291a, B
For each oil, comparaison were made between LC50 and LC95 values for both insects.Means followed by same letter (letter in lowercase) were not statistically differentaccording to Duncan test at p < 0.05. Between oils, comparaisons were made betweenLC50 and LC95 values of each insect species. Means followed by same letter (letter inuppercase) were not statistically different according to Duncan test at p < 0.05.
a Units LD50 and LD95 (�L), applied for 24 h at 25 ◦C.
O. Bachrouch et al. / Industrial Crops and Products 65 (2015) 127–133 131
sed fo
4
Gp�chMecAtfl2
e1rca2lgT
iw�tpetsetw
Fig. 2. Percentage of mortality of O. surinamensis and T. castaneum expo
. Discussion
A. herba-alba essential oil composition investigated using bothC and GC–MS techniques showed that 1,8 cineol (19.59%), cam-hor (11.48%), �-pinene (5.4%), camphene (2.63%), borneol (2.29%),-pinene (1.63%), and �-terpineol (1.09%) are considered as majorompounds and being reported in previous studies on Tunisian A.erba-alba oils (Haouari and Ferchichi, 2009; Mighri et al., 2010).oreover, our results were in accordance with those of Feuerstein
t al. (1988); Salido et al. (2004); Bakkali et al. (2008) reporting 1,8ineol, camphor, �-terpineol and borneol as major constituents of. herba-alba essential oil. Nevertheless, changes in the composi-ion of desert wormwood from different regions and bioclimaticoors have been widely studied (Segal et al., 1987; Salido et al.,004; Belhattab et al., 2014).
On the other hand, our results showed that A. absinthiumssential oil was rich on �-thujone (22.72%), camphor (16.71%),,8 cineole (5.47%), and camphene (2.37%). Previous studiesevealed that A. absinthium essential oils from Tunisia wereharacterized by chamazulene, �-thujone, bornan-2-one, bornylcetate, 1-terpinen-4-ol, p-cymene, and �-myrcene (Riahi et al.,013). Recently, Dhen et al. (2014) reported that chamazu-
ene, camphor, bornyl-acetate, myrcene, 1-4 terpineol, camphene,amma-terpinene, and �-terpinène were the major compounds ofunisian wormwood oil.
Chemical composition of A. abinthium essential oil was widelynvestigated. In this context, Lawrence, (1992) reported that worm-
ood essential oil extracted from plants grown in USA showed-thujone and cis-sabinyl acetate as the main components. Besides,
he Algerian oil was characterized by the predominance of cam-hor and borneol (Erdogan-Orhan et al., 2010). Moreover, Oravt al. (2006) reported that four chemotype of A. absinthium essen-ial oil were identified from different geographical areas in Europe:abinene and myrcene rich oil, �- and �-thujone rich oil,/break
poxyocimene rich oil, and (E)-sabinyl acetate rich oil. Investiga-ions on Artemisia species also revealed that A. absinthium oil fromestern Canada was distinguished by high amounts of myrcene,
r various durations toessential oil from A. Herba-alba and A. Absinthium.
trans-thujone and trans-sabinyl acetate (Lopez-Lutz et al., 2008).In addition, �-pinene and �-thujone were the main compoundsof Iranian wormwood essential oil (Khangholi and Rezaeinodehi,2008). The above results clearly demonstrated that both essentialoils content bioactive insecticidal coompouds.
Results of the present study indicated that Tunisian A. herba-albaand A. absinthium essential oils have fumigant and contact toxicityproprities against adults of O. surinamensis and T. castaneum withA. herba-alba oil being more toxic in fumigant assay against T. cas-taneum with median lethal concentration of 278.66 �L/L air. Suchactivity seems to be moderate compared to Morrocan oils: LC50values of 17.4 and 43.3 �L/L air (Abbad et al., 2014; Moumni et al.,2014). While, fumigant activity of A. herba-alba seems to be strongercompared with the Iranian oil: LC50 = 564.4 �L/L air (Sharifian et al.,2012). Regarding A. absinthium essential oil insecticidal activity,few previous works were recorded on stored products pests. Avail-able data showed the interesting potential of this oil in controllingAcanthoscelides obtectus (Say 1831), Rhizoperta dominica (Fabricius,1792) and O. surinamensis (Derwich et al., 2009; Dhen et al., 2014;Mediouni Ben Jemâa, 2014).
Our work related to contact toxicity bioessays determinedusing topical application showed that both Artemisia essential oilsexhibited an intersting potentiel. As reported elsewhere, Artemisiaessential oil revealed a contact toxicity activity. In this context,Abbad et al. (2014) reported the contact toxicity of A. herba-albaagainst T. castaneum. Moreover, essential oils from various speciesof Artemisia exhibited contact toxicity to several major stored prod-uct pests: Sitophilus zeamais (Motschulky, 1855) (Artemisia vestita,(Chu et al., 2010); Artemisia capillaris and Artemisia mongolica (Fisch.Ex Besser) (Nakai, 1917; Liu et al., 2010); Artemisia frigida Willd. (Liuet al., 2014)), Acanthoscelides obtectus, (A. herba-alba, (Mohamedet al., 2010)).
The insecticidal propriety of many essential oils are mainlyattributed to monoterpenoids which are typically volatile and
rather lipophilic compounds that can penetrate into insects rapidlyand interfere with their physiological functions (Isman, 2000;Bakkali et al., 2008; Coloma et al., 2010). Due to their high volatility,
hey have fumigant and gaseous action which are very important inontrolling the stored-product insects. Artemisia species essentialils exhibited important insecticidal activities that depend uponhe type and nature of the constituents and their individual concen-ration levels (Salido et al., 2004; Paolini et al., 2010; Sharifian et al.,012; Liu et al., 2014). In the same context, Coloma et al. (2010)eported that the toxic effects of A. absinthium could be attributedo its major components. However, the insecticidal effects of thessential oils cannot be explained by the action of their majoromponents only, suggesting that these actions are the result of
synergistic interaction between all components. Indeed, Zhangt al. (2014) reported that the bioactivity properties of A. argyiH. Lév. & Vaniot) essential oil can be attributed to the synergisticffects of its diverse major and minor components.
. Conclusion
This study provides comparative investigation on the chemi-al composition and insecticidal potential of A. herba-alba (Desertormwood) and A. absinthium L. (Wormwood) essential oils from
orth Tunisia. Quantitative and qualitative differences in chemicalomposition were detected between both oils. The strongest fumi-ant potential of A. herba-alba essential oil seems to be attributedo its richness on compounds with insecticidal activity (� and-pinene, camphene, 1,8 cineole, camphor, borneol, �-terpineol).urthermore, high rate of �-thujone conferred the toxicity to A.bsinthium essential oil which makes these oils and their com-ounds of choice for industrially source of bioactive substances.
The study indicated that the essential oils of A. herba-alba and. absinthium had a potential to be developped to a natural fumi-ant/contact insecticide for the management of the grain storagensects. All those results valorize Tunisian A. herba-alba and A.bsinthium as medicinal and aromatic plants which can be a sourcef biological active compounds. Thus, these species might be goodandidates for further investigation in developing new botanicalnsecticide and can be used as a component in an IPM strategynstead of toxic synthetic pesticides. However, to develop a practi-al application for these essential oils as novel fumigant/insecticide,urther research into their safety in humans is needed. Additionaltudies on the development of formulations are also necessary tomprove efficacy and stability as well as to reduce cost.
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