Tunisian Journal of Plant Protection Vol. 9, No. 1, 2014


Tunisian Journalof

Plant Protection

Volume 9Number 1June 4 201

Barbouche, NaïmaEntomology, I N A T

Ben Hamouda, M. Habib Entomology, I N A T

Ben Jamâa, M. LahbibEntomology, I N R G R E F

Chérif, Mohamed Phytopathology, I N A T / C T A

Hajlaoui, M. Rabeh Phytopathology, I N A TR

Hamada Walid, Disease Resistance, E S A KHorrigue-Raouani, Najet Nematology, I S A Ch-MKsantini, MohieddineEntomology, I OMekki, MounirWeed & Pesticide Sc., I S A Ch-MRhouma, AliPhytopathology, OI / IRESA

Editorial Board

Evaluation Board

Editor-in-ChiefNasraoui, Bouzid

Phytopathology, INAT

Abou-Haider, Mounir G., Phytopathology, CanadaBayaa, Bassam, Phytopathology, Syria

Benazoun, Abdessalam, Entomolgy, MoroccoBernier, Louis, Phytopathology, Canada

Besri, Mohamed, Phytopathology, MoroccoBouzned, Zouaoui, Phytopathology, Algeria

Dellagi, Alia, Phytopathology, FranceDu Jardin, Patrick, Biotechnology, Belgium

Duncan, Larry, Nematology, Florida USAFargette, Mireille, Nematology, France

Fuchs, Jacques G., Biol. Control, SwitzerlandHullé, Maurice, Entomology, France

Ippolito, Antonio, Phytopathology, ItalyKamoun, Sophien, Phytopathology, Ohio USA

Kema, Gert, Phytopathology, NetherlandsKerlan, Camille, Phytopathology, France

Kreiter, Serge, Acarology, FranceKremer, Robert J., Weed Sc., Missouri USALepoivre, Philippe, Phytopathology, Belgium Lieutier, François, Entomology, FranceMakkouk, Khaled, Phytopathology, LebanonMateille, Thierry, Nematology, FranceMugniery, Didier, Nematology, FrancePeever, Tobin, Phytopathol., Washington St., USA Ramade, François, Eco-Toxicology, FranceReignault, Philippe, Phytopathology, FranceRusso, Agatino, Entomology, ItalySchiffers, Bruno, Pesticide Sc., BelgiumSchoelz, James, Phytopathology, Missouri USASimpson, Stephen J., Entomology, AustraliaTirilly, Yves, Phytopathology, FranceZiedan, El Sayed, Phytopathology, Egypt

Sustaining Members

Institution de la Recherche et de l’Enseignement Supérieur Agricoles (IRESA)Institut National Agronomique de Tunisie (INAT)Ecole Supérieure d’Agriculture du Kef (ESAK)

Visa no. 2872/04-04-2005

Tunisian Scientists and :

Created and from 2006 to 2012 Hosted in the TunisiaEcole Supérieure d’ du , Agriculture Kef ( )

Assistant Editor-in-ChiefDaami-Remadi, Mejda

Phytopathology, C R R H A B ChM-

Scientific Journal Hosted in the TunisiaInstitution de la Recherche et de l’Enseignement Supérieur Agricoles( )


Tunisian Journal of Plant ProtectionTunisian Journal of Plant ProtectionTunisian Journal of Plant ProtectionTunisian Journal of Plant Protection

http://www.iresa.tn/tjpp

Volume 9, Number 1, June 2014

Contents ABIOTIC ASPECTS 01. Allelopathic potential of ferulic acid on tomato. N.B. Singh and Sunaina. (India)

11. Allelopathic effects of aqueous extracts of Eucalyptus occidentalis, Acacia ampliceps and Prosopis juliflora on the germination of three cultivated species. E. Saadaoui, N. Ghazel, Ch. Ben Romdhane, N. Tlili, and A. Khaldi. (Tunisia)

MYCOLOGICAL ASPECTS 17. Antifungal activity of culture filtrates and organi c extracts of Aspergillus spp.

against Pythium ultimum. R. Aydi-Ben Abdallah, M. Hassine, H. Jabnoun-Khiareddine, R. Haouala and M. Daami-Remadi. (Tunisia)

31. Chitosan and Trichoderma harzianum as fungicide alternatives for controlling Fusarium crown and root rot of tomato. R.S.R. El-Mohamedy, F. Abdel-Kareem, H. Jabnoun-Khiareddine, and M. Daami-Remadi. (Egypt/Tunisia)

45. Control of root rot diseases of tomato plants caused by Fusarium solani, Rhizoctonia solani and Sclerotium rolfsii using different chemical plant resistance inducers. R.S.R. El-Mohamedy, H. Jabnoun-Khiareddine, and M. Daami-Remadi. (Egypt/Tunisia)

ENTOMOLOGICAL ASPECTS

57. Chemical composition and fumigant toxicity of Artemisia absinthium essential oil against Rhyzopertha dominica and Spodoptera littoralis. N. Dhen, O. Majdoub, S. Souguir, W. Tayeb, A. Laarif and I. Chaieb. (Tunisia)

67. Chemical constituents and toxicity of essential oils from three Asteraceae plants against Tribolium confusum. D. Haouas, P.L. Cioni, M. Ben Halima-Kamel, G. Flamini, and M.H. Ben Hamouda,. (Tunisia/Italy)

83. Chemical composition of Ruta chalepensis essential oils and their insecticidal activity against Tribolium castaneum. O. Majdoub, N. Dhen, S. Souguir, D. Haouas, M. Baouandi, A. Laarif, and I. Chaieb. (Tunisia)

91. Insecticidal activities of fruit peel extracts of pomegranate (Punica granatum) against the red flour beetle Tribolium castaneum. A. Ben Hamouda, A. Mechi, K. Zarred, I. Chaieb, and A. Laarif. (Tunisia)

Photo of the cover page: Pythium leak of potato (Courtesy Rania Aydi-Ben Abdallah)


Guest Editorial

Application of Allelopathy in Weed Control Weeds are the cause of serious

yield reduction problems in crop production worldwide. Losses caused by weeds vary from one country to another, depending on the predominant weed flora and on the control methods practiced by farmers. Chemical control is an efficient method to control weeds and herbicides account for two thirds of total pesticide usage in the world. However, increasing thrust for organically produced commodities, public awareness about health, evolution of weed resistance to herbicides and other environmental concerns drive scientists to concentrate on research to find out some natural practice or method to control this menace, thereby minimizing or avoiding the frequent use of herbicides in future. In this regard, allelopathy has been found to offer eco-friendly practice which can be used for weed control.

The use of allelopathy for controlling weeds could be either by using allelochemicals as natural herbicides or through directly utilizing natural allelopathic interactions. Although diverse classes of Allelochemicals have been identified in plant kingdom, only few chemicals have been identified as natural herbicides. Some plant compounds have served as structural templates for herbicides. For example, many phenoxy herbicides are auxin analogs and the halogenated derivatives of benzoic acid (TBA, TIBA, dicamba), which is frequently known as

allelopathic compound, are commercially used as herbicides. Cinmethylin (a product of Shell Co.) is a cineole (natural plant compounds in several plant species) alcohol with the addition of a substituted benzyl group, which represents a new class of herbicide in the market.

With respect with the second case, there are several weed control strategies in which allelopathy can be involved such as (a) using allelopathic crop in crop rotations; (b) using allelopathic crop residues as cover crops and mulches; (c) using allelopathic crop as a smother crop; (d) using allelopathic crops in crop mixtures and intercropping; and (e) using allelopathic extract and residues in combination with lower rates of herbicides.

Of all the above strategies, using of plant residues is the most effective and promising. Soil incorporation or surface application as mulch (dead or living mulch) of allelopathic crop residues affects weed dynamics by reducing/delaying seed germination and suppressing weed growth, hence contributing to overall decline in the density and vigor of the weed community (Gallandt et al., 1999). In most cases, allelopathic extracts or crop residues provide limited weed suppression, and most often reductions in weed growth are not comparable to those observed with labeled herbicides. Therefore, other methods to increase the efficacy of allelopathic extracts or residues may be


critical to enhance weed suppression while at the same time reducing our reliance on herbicides.

Substantial scope to reduce the herbicide rate has been observed by various investigators when herbicides are applied together with aqueous extracts of different allelopathic crops. The results of the research conducted in recent years has revealed that herbicides use can be reduced by 50-70 % when herbicides are used in combination with aqueous sorghum extracts for weed control in field crops such as wheat, cotton, mung bean and maize (Cheema et al., 2003a, b, c; Iqbal et al., 2009). Although successful results have been obtained from sorghum extracts applied two to three times with low herbicide rates, the following steps of research work need to be conducted before definite recommendation can be made: (a) The large volumes of extracts and the numbers of sprays should be reduced in order to apply for large scale field operation; (b) Formulation and stabilization of allelopathic crop extract over relatively long period of time under normal storage conditions need to be done and; (c) Appropriate concentration for each crop should be determined to provide data base for each crop and its companion weeds. Due to these limitations, an alternative practical and feasible approach has been developed where the residues of some tested allelopathic crops, such as sorghum and sunflower, have been left to dry under field conditions and then promptly incorporated into production sites for weed management and low herbicide were applied along with residue incorporation (Alsaadawi et al., 2011, 2013). By using this approach with Vicia faba, it was found that combinations of

reduced (50% of the labeled rate) rate of trifluralin herbicide with sunflower residue, incorporated at 600 and 1400 g/m2, suppressed weed population and biomass better than the labeled rate of trifluralin herbicide and provided similar yield advantage as was noticed with 100% herbicide rate. Almost, similar trend was observed when residues of sorghum were used with the pre emergence herbicide in Vicia faba field. In a two year trial, it was found that application of half of the labeled rate of trifluralin in plots amended with sorghum residue at 3.5 and 5.3 t/ha suppressed weed population and dry biomass and generated seed yields similar to that of the labeled dose of herbicide applied alone (Alsaadawi et al., 2013).

When this method was applied on weeds of cereal crops using post emergence herbicide, the trends of weed inhibition and crop stimulation remained almost consistent. Foliar spray of reduced dose (50% of the labeled rate) of 2,4-D and Topic herbicides on plots amended with sunflower residues at 600 and 1400 g/m2 suppressed weed population and provided seed yield similar to the labeled rate of the herbicides. Similarly, foliar spray of reduced (50% of labeled rate) rate of Chevalier on wheat plants grown in plots amended with sorghum residue at 3 t/ha recorded least suppression to weeds’ density and dry biomass and this suppression was statistically similar to the labeled rate of Chevalier herbicide.

Field observations on the above experiments indicated that low percentage of weeds emerged in plots, which contained residues of sunflower or sorghum, at the beginning of sowing time and continued for about two months.


After that, the emergence and growth of weeds started to increase rapidly. Chemical analysis of samples taken periodically from field soil revealed that phenolic acids were highly increased after residue incorporation into soil until reaching maximum at 4 weeks from sowing time then starting to decline until vanished at 6 weeks from sowing. Thus, it appears that increase in concentration of total phenolic acids in soil coincided with high suppressive activity of weeds in the field and vice versa and these results suggest that phenolics are the main cause of the suppressive activity of weeds.

Interestingly, this method not only suppressed weeds and enhanced yield of crops, but it also improved the beneficial soil microflora. Greenhouse experiment revealed that incorporation of sorghum residues at 10g/kg significantly increased nodulation and nitrogen fixation over control (soil + Rhizobium vignae) in mung bean. In another field experiment, sporulation and mycorrhiza incidence were significantly increased in wheat roots due to incorporation of sunflower residues in soil of wheat field.

In conclusion, the efficacy allelopathic sorghum and sunflower residues can be improved when combined with half dose of test herbicides. Besides

suppressing weeds, these residues could also have a positive bearing on the soil physical and biological characteristics, and add nutrients to soil. Investigations into the allelopathic potential of other residues along with different herbicides under varying crop and environmental conditions need to be carried out further.

Dr. Ibrahim S. Alsaadawi, Professor of Physiological Plant

Ecology, Baghdad University, Baghdad, Iraq

Selected references Alsaadawi, I.S., Khaliq, A., Lahmood, N.R., and

Matllob, A. 2013. Allelopathy Journal 32: 203-212.

Alsaadawi, I.S., Khaliq, A., Al-Temimi, A.A., and Matloob, A. 2011. Planta Daninha. 29: 849-859.

Cheema, Z.A., Farid, M.S., and Khaliq, A. 2003a. J. Anim. Plant. Sci. 13:48-51.

Cheema, Z.A., Hussain, S., and Khaliq, A. 2003b. Ind. J. Plant. Sci. 2: 21-25.

Cheema, Z.A., Jaffar, I., and Khaliq, A. 2003c. Pak. J. Weed Sci. Res. 9: 153-160.

Gallandt, E.R., Libeman, M. and Huggins, D.R. 1999. J. Crop Prod. 2: 95-121.

Iqbal, J., Cheema, Z.A., and Mushtaq, N. 2009. Int. J. Agric. Biol. 11: 360-366.

Tunisian Journal of Plant Protection 1 Vol. 9, No. 1, 2014

Allelopathic Potential of Ferulic Acid on Tomato

Narsingh Bahadur Singh and Sunaina, Plant Physiology Laboratory, Department of Botany, University of Allahabad, Allahabad, India __________________________________________________________________________ ABSTRACT Singh, N.B. and Sunaina. 2014. Allelopathic potential of ferulic acid on tomato. Tunisian Journal of Plant Protection 9: 1-9. This study deals with allelopathic effects of exogenous application of ferulic acid (FA) on biophysical and biochemical parameters of tomato (cv. Pusa ruby) seedlings grown in hydroponic culture. FA exhibited phytotoxic effects on tomato seedlings grown in a Hoagland solution. After 7 days of seedling growth, FA was added to the growth medium at concentrations of 0.1, 0.5, 1.0, and 1.5 mM. The Hoagland solution without FA was used for experimental units. The root and shoot length, fresh and dry weight of seedlings, chlorophyll a (chl a), chlorophyll b (chl b), total chlorophyll (chl t), carotenoids, protein and sugar content, nitrate reductase (NR) activity and antioxidant enzymes accumulation were examined in 25 days time period. FA exhibited significant inhibitory effects on some biophysical and biochemical traits. A gradual decrease in all studied traits in FA based-treatments was observed. The root and shoot length, the fresh and dry weight of the seedlings significantly decreased in a dose dependent manner under FA allelopathic potential. The chl a, chl b, chl t and carotenoids contents decreased with the increase of FA concentrations. A significant reduction in sugar and protein contents and in NR activity was recorded especially with the highest concentrations of FA used. Higher FA concentrations significantly enhanced the activities of antioxidant enzymes [superoxide dismutase (SOD), catalase (CAT) and peroxidase (POX)] which appeared as a form of tolerance and/or a defense to allelochemicals used. Keywords: Allelopathy, antioxidants, ferulic acid, Hoagland solution, hydroponic culture, tomato _________________________________________________________________________ Plants may affect the growth and productivity of neighboring ones directly through secondary metabolites (51). This phenomenon is known as allelopathy and involved secondary metabolites in the process are called allelochemicals (42) which are bioactive compounds that influence germination, growth and development of receptor plants. Allelochemicals are released through the leaching, evaporation and decomposing crop residues which have either inhibitory or stimulatory effects (46). Alleloche-

Corresponding author: Narsingh Bahadur Singh Email: [email protected]

Accepted for publication 30 April 2014

micals may affect physiological processes in plants, such as, stomatal closure (8), plant water balance (7), cell division (4), membrane permeability (24), nutrient uptake (6), photosynthesis (11) respiration (3) and many other metabolic processes.

Ferulic acid (FA) considered as allelopathic agent is one of the secondary metabolites which is a derivative of cinnamic acid (21). FA was extracted from soil with a range (0.01-0.1 mM) of concentrations (54, 55). Luthria et al. (37) found FA in tomato and this finding was also reported in other works (9, 25, 27, 48). Leaf leachates (2), root exudates (49, 50), plant debris (15, 18) and soil extracts (26, 43) were frequently investigated for


allelopathic potential. Exposure to FA adversely affected various biophysical and biochemical processes in plants by reducing water utilization (30), foliar expansion and root elongation (14), nutrient uptake (12, 38), photosynthesis, leaf water potential, turgor and osmotic pressure and growth (23, 41). The inhibitory effects of FA on Lemna minor (20, 41), Abutilon theophrasti (40), soybean (19) and soybean and sorghum (22) were often reported in allelopathy studies.

FA was selected as allelochemical because it was frequently reported as an inhibitory substance to many plant species. This study was conducted to investigate the effect of FA on tomato growth and development.

MATERIALS AND METHODS Plant material and treatments.

Certified seeds of tomato (Lycopersicon esculentum, cv. Pusa ruby) were purchased from Seed Company of Allahabad, Uttar Pradesh, India. Seeds were sown in nursery beds (1 m × 1 m) at the experimental station, Allahabad University, Allahabad (24o47’, 50o47’ N latitude; 81o91’, 82o21’ E longitude; 78 m-asl). The seed bed was watered as required. After 15 days, seedlings were uprooted and washed with tap water followed by distilled water, then transferred at the rate of 10 seedlings per pot in transparent plastic pot [(23×17×9) =3519 cm3] each containing 2 liters of Hoagland solution (29). FA at 0.1(T1), 0.5 (T2), 1.0 (T3) and 1.5 (T4) mM concentrations were prepared in distilled water and used for treatment. After 1 week of establishment, the FA was added to Hoagland solution according to the treatment. The Hoagland solution free of FA was taken as control. Pots were covered with black papers to avoid the algal growth in the growth medium, then

fitted with aerating tubes, and any pot opening was plugged with cotton to hold the seedlings in a vertical position. The seedlings growth experiment was conducted in a glass house, and replicated 3 times. Pots were continuously aerated, and data was collected after 72 h for biochemical and biophysical analyses.

Measurements.

Root and shoot lengths (RL, SL) of tomato seedlings were measured with a metric scale, as was the case for fresh and dry matters. Fresh plant materials were weighted (FW) and then oven dried at 70oC for 72 h to get the dry weight (DW).

Estimation of chlorophyll, carotenoids and protein contents.

Chlorophyll and carotenoids from plant materials were extracted with 80% acetone. The amount of photosynthetic pigments was determined by Lichtenthaler (34). A fresh leaf sample of 10 mg was homogenized in 10 ml of 80% acetone and then centrifuged. Supernatant was taken and optical density was measured at 663, 645 and 470 ηm.

Protein content was determined following the method of Lowry et al. (36). The amount of protein was calculated with reference to standard curve obtained from bovine serum albumin. Nitrate reductase activity.

Nitrate reductase (NR) activity was assayed by modified procedure of Jaworski (31) based on incubation of fresh tissue (0.25 g) in 4.5 ml medium containing 100 mM phosphate buffer (pH = 7.5), 3% KNO3 and 5% propanol. About 0.4 ml aliquot was treated with 0.3 ml 3% sulphanilamide in 3-N HCl and 0.3 ml 0.02% N-1-naphthyl ethylene diamine dihydrochloride (NEDD). The absorbance was measured at 540 ηm. NR


activity was calculated with a standard curve prepared from NaNO2 and expressed in µmol NO2.g

-1FW.h-1.

Sugar content. Sugar content was estimated

following Hedge and Hofreiter method (28). About 0.25 g of the plant leaves were homogenized in 2.5 ml of 95% ethanol. After centrifugation, the sugar content was determined in 0.1 ml of the supernatant, mixed with 4 ml of anthrone reagent and heated in a boiling water bath for 8 min. Absorbance was taken at 620 ηm after rapid cooling. Standard curve was prepared with glucose.

Antioxidant enzyme assay.

Enzyme extract was prepared by homogenizing 500 mg of leaves tissue in 10 ml of 0.1 M sodium phosphate buffer (pH = 7.0). A homogenate was filtered and centrifuged at 15000 g at for 30 min and temperature was maintained at 4oC. The supernatant was collected and used for analysis of superoxide dismutase, catalase and peroxidase.

Superoxide dismutase (SOD) activity was determined by the nitroblue tetrazolium (NBT) photochemical assay method described by Beyer and Fridovich (13). The reaction mixture (4 ml) contained 63 µM NBT, 13 mM methionine, 0.1 mM ethylene diamintetra acetic acid (EDTA), 13 µM riboflavin, 0.5 M sodium carbonate and 0.5 ml clear supernatant. Test tubes were placed under fluorescent lamps for 30 min and absorbance was recorded at 560 ηm. One unit of enzyme was defined as the amount of enzyme which caused 50% inhibition of NBT reduction.

Catalase (CAT) activity was assayed following the method of Cakmak and Marschner (17). The reaction mixture (2 ml) contained 25 mM sodium

phosphate buffer (pH = 7.0), 10 mM H2O2 and 0.2 ml enzyme extract. The activity was determined by measuring the rate of disappearance of H2O2 for 1 min at 240 ηm and estimated using extinction coefficient of 39.4 mM-1.cm-1 and expressed as enzyme unit/g fresh weight. One unit of CAT was defined as the amount of enzyme required to oxidize 1 µM H2O2/min.

Peroxidase (POX) activity was assayed following the method of Mc Cune and Galston (39). Reaction mixture contained 2 ml enzyme extract, 2 ml sodium phosphate buffer, 1 ml 0.1-N pyrogallol and 0.2 ml 0.02% H2O2 and determined spectrophotometrically at 430 ηm. One unit of enzyme activity was defined as the amount which produced an increase of 0.1 optical density per minute.

Statistical analysis.

Standard errors of means were calculated in triplicates. In addition, a one way ANOVA was carried out for all the data generated from this experiment test using GPIS package (GRAPHPAD, California, USA).

RESULTS

The growth of tomato seedlings were measured in terms of RL, SL, FW and DW with respect to treated and untreated seedlings by FA. The growth was significantly affected for treated plants when compared to the control. The reduction in the RL, SL, FW and DW of seedlings was 35.43, 43.79, 40.43, and 67.76%, respectively, when compared with control. The maximum growth was recorded in the seedlings under control. Plant treated with 1.5 mM concentration exhibited the maximum reduction in growth (Table 1).


Table 1. Effects of ferulic acid (FA) applied at different concentrations on the shoot and root length and on the fresh and dry weight of tomato seedlings

Treatment Shoot length (cm)

Root length (cm)

Fresh weight (g/plant)

Dry weight (g/plant)

C 15.3 ± 0.115 6.35 ± 0.028 13.85 ± 0.028 2.84 ± 0.051 T1 12.4 ± 0.057 a 5.9 ± 0.057 13.05 ± 0.086 a 2.23 ± 0.049 a T2 10.2 ± 0.057 a 5.25 ± 0.202 a 10.35 ± 0.259 a 1.24 ± 0.008 a T3 9.35 ± 0.202 a 4.9 ± 0.346 a 8.95 ± 0.202 a 1.03 ± 0.026 a T4 8.6 ± 0.635 a 4.1 ± 0.230 a 8.25 ± 0.028 a 0.92 ± 0.019 a

Data are means of 3 replicates ± SEM, a Treatment significantly (P < 0.001) different to the control(C), C: Control, T1:0.1 mM, T2: 0.5 mM, T3,:1.0 mM, and T4:1.5 mM concentrations of FA.

The chlorophyll content decreased under FA treatment. The maximum decrease in chlorophyll and carotenoids content were decreased by 50.95, 35.53,

45.81, and 52.08%, respectively, under the highest concentration of FA as compared with control (Table 2).

Table 2. Effects of ferulic acid (FA) applied at different concentrations on chlorophyll a, chlorophyll b, chlorophyll t and carotenoids contents of tomato seedlings

Treatment Chlorophyll a (mg/g FW)

Chlorophyll b (mg/g FW)

Total Chlorophyll (mg/g FW)

Carotenoids (mg/g FW)

C 2.61 ± 0.040 1.21 ± 0.076 3.82 ± 0.036 2.40 ± 0.012 T1 2.45 ± 0.002b 1.16 ± 0.004 3.61 ± 0.007 a 2.28 ± 0.005 T2 2.13 ± 0.041a 1.06 ± 0.033 3.20 ± 0.007 a 1.87 ± 0.322 T3 1.46 ± 0.040a 0.92 ± 0.076a 2.38 ± 0.036 a 1.19 ± 0.501 b T4 1.28 ± 0.005a 0.78 ± 0.018a 2.07 ± 0.012 a 1.15 ± 0.001 b

Data are means of 3 replicates ± SEM, a Treatment significantly (P < 0.001) different to the control, b Treatment significantly (P < 0.01) different to the control (C). C: Control, T1: 0.1 mM, T2: 0.5 mM, T3:,1.0 mM, and T4:1.5 mM concentrations of FA.

A significant reduction in protein content was also recorded under FA treatment. The maximum decrease (16.55%) in protein was related to 1.5 mM of FA as compared with control, while the other concentration of FA decreased significantly the protein content too. The inhibitory effect of FA showed a maximum 17.96% reduction in sugar content under the highest

concentration of FA while the reduction was concentration dependent. The NR activity in the leaves of the treated tomato seedlings was adversely affected by FA treatment as compared with control. NR activity significantly decreased in a dose dependent manner. The maximum inhibition (44.63%) was recorded with 1.5 mM of FA as compared with control (Table 3).


Table 3. Effects of ferulic acid (FA) applied at different concentrations on protein and sugar content and nitrate reductase activity of tomato seedlings

Treatment Protein (mg/g FW)

Sugar (mg/g FW)

NR (µmol NO2 g-1

FW h-1) C 61.91 ± 0.028 91.47 ± 0.583 20.23 ± 0.631

T1 59.28 ± 0.072 a 86.92 ± 0.291 a 19.02 ± 0.288 b

T2 59.06 ± 0.028 a 85.15 ± 0.145 a 15.86 ± 0.126 a

T3 56.20 ± 0.028 a 79.84 ± 0.291 a 12.17 ± 0.090 a

T4 51.65 ± 0.202 a 75.04 ± 0.729 a 11.20 ± 0.072 a

Data are means of 3 replicates ± SEM, a Treatment significantly (P < 0.001) different to the control, b. Treatment significantly (P < 0.01) different to the control. C: Control, T1: 0.1 mM, T2: 0.5 mM, T3,:1.0 mM, and T4:1.5 mM concentrations of FA.

Activities of antioxidant enzymes (SOD, CAT, POX) increased significantly (P < 0.001) in response to FA. A maximum 60, 33 and 19%

stimulation were recorded for SOD, CAT and POX activities with concentration of 1.5 mM treatment, respectively (Table 4).

Table 4. Effects of ferulic acid (FA) applied at different concentrations on antioxidant enzyme activities of tomato seedlings

Treatment SOD

(Enzyme Unit/g FW)

CAT (Enzyme

Unit/g FW)

POX (Enzyme

Unit/g FW) C 22.31 ± 0.076 6.27 ± 0.816 70.35 ± 0.317 T1 23.17 ± 0.038 7.15 ± 0.034 74.05 ± 0.692 a T2 27.87 ± 0.903 a 7.75 ± 0.035 b 74.95 ± 0.115 a T3 33.3 ± 0.653 a 7.87 ± 0.035 b 79.57 ± 0.158 a T4 35.79 ± 0.057 a 8.36 ± 0.035 a 83.97 ± 0.793 a

Data are means of 3 replicates ± SEM, a Treatment significantly (P < 0.001) different to the control, b Treatment significantly (P < 0.01) different to the control. C: Control, T1: 0.1 mM, T2: 0.5 mM, T3,: 1.0 mM, and T4:1.5 mM concentrations of FA.

DISCUSSION

Allelochemicals released into the surrounding by evaporation, exudation, leaching, and residues decomposition affect the neighboring plants. Allelochemicals accumulate in the soil (47, 52) and affect the growth and metabolism of neighboring plants (47). Exposed plants to allelochemical undergo a drastic change in their biophysical and biochemical processes. Reduction in plant

growth was common feature under allelopathic conditions as reported in literature (10, 44, 45, 46, 56). Decrease in RL and SL, FW and DW was the manifestation of impaired metabolic activities due to allelochemicals (41, 47).

The observed significant reduction in photosynthetic pigment contents (chl a, chl b, chl t) and carotenoids of tomato seedlings treated by FA are in complete agreement with the results reported by


Kanchan and Jayachandra (33) and Singh et al. (46). It is known that photosynthetic rate is dependent of pigment contents and the plant dry matter is closely related to chlorophyll content (16, 47).

Protein and sugar contents decreased significantly in response to FA application in to the growth medium of the tomato seedlings. This result is similar to what has been reported about the effect of allelochemicals with protein biosynthesis (47).

As it was the case for the protein content, the activity of NR decreased dependently of FA concentrations applied in the experiment. The reduced tomato plant growth and NR activity could be attributed to a decreased nitrate absorption by tomato plant roots as reported in previous researches (1, 47). It has been also reported that

allelochemicals caused oxidative damage which induces antioxidant enzymes (5, 53). The prior findings are in support of the results relative to SOD, CAT and POX accumulation as form of tolerance to FA.

In the present study, FA has shown an inhibitory activity on tomato seedlings growth. Decreased pigment content, protein, sugar and NR activity indicated the allelopathic potential of FA. However, tomato seedlings induced antioxidative enzymes (SOD, CAT, POX) accumulation to mitigate the adverse effects of allelochemicals. ACKNOWLEDGMENTS

The authors are thankful to the University Grants Commission, New Delhi, India for sponsoring this particular research.

_________________________________________________________________________ RESUME Singh N.B. et Sunaina. 2014. Potentiel allélopathique de l’acide of férulique sur la tomate. Tunisian Journal of Plant Protection 9: 1-9. Cette étude porte sur les effets allélopathiques d’une application exogène d’acide férulique (FA) sur les paramètres biophysiques et biochimiques de plantules de tomate (cv. Pusa ruby) cultivées hydroponiquement. FA a montré des effets phytotoxiques sur les plantules de tomate cultivées dans une solution Hoagland. Après 7 jours de croissance, FA a été ajouté au milieu de culture à des concentrations de 0,1, 0,5, 1,0 et 1,5 mM. Une solution Hoagland sans FA a été utilisée pour les unités expérimentales. La longueur de la tige et des racines, les poids frais et sec des plantules, les teneurs en chlorophylle a (chl a), chlorophylle b (chl b), chlorophylle totale (chl t), caroténoïdes, protéines et sucre, l’activité nitrate réductase (NR) et l’accumulation d’enzymes antioxidantes ont été examinées en une période de temps de 25 jours. FA a montré des effets inhibiteurs significatifs sur certaines caractéristiques biophysiques et biochimiques. Une chute graduelle de toutes les caractéristiques étudiées a été observée avec les traitements à base de FA. La longueur des racines et de la tige ainsi que les poids frais et secs des plantules ont été significativement réduits d’une manière dépendante de la dose sous le potentiel allélopathique de FA. Les teneurs en chl a, chl b, chl t et caroténoïdes ont diminué avec l’augmentation des concentrations de FA. Une réduction significative des teneurs en sucre et en protéines et dans l’activité NR a été enregistrée surtout avec les concentrations de FA les plus élevées utilisées. Les concentrations de FA les plus élevées ont significativement augmenté les activités des enzymes antioxidantes [superoxide dismutase (SOD), catalase (CAT) et péroxidase (POX)] qui semble être une forme de tolérance et/ou de défense aux allélochimiques utilisés. Keywords: Acide férulique, allélopathie, antioxidants, culture hydroponique, solution Hoagland, tomate. __________________________________________________________________________


__________________________________________________________________________ ��

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� ��ر�� ھ�ه ا �را�� ات ا ��ھ�� " ا !��و�# (FA) ��$�%&�'(�) -, ا �+�%�* ا

� 0(��ت ا /��ط ��$��1(�) +� أظ�� . ��$5ا �&رو-� '5 ا )�4 ا Pusa ruby) 203( واFA ,- �� ات��> إ:�'� � 7��Hoagland .ا /��ط� ا �&رو-� '5 #)ل 8(��ت ،(�0 ��$5 ��1&ات ا )�4 ا إ , FAأ%�م < ا

� ا��ل #)ل . ��)ل 1.5و 1.0و 0.5 و 0.1�Hoagland �'�:ون إ��FA رب�� (C و. '5 و�Bات ا �� ا ��I)ر و (chl b) ا ��I)ر بو (chl a) أ 1��ت ا ��I)روا ��ف 0(��ت و Gط�وا )زن ا ط)ل ا �Eق وا ��ور

J�K ��ة وذ � ا ��دة �E1Oة ا8M&%��ت ��ا1��ات و�وا �Kرو��0ت وا (�و��0ت وا �KE و�L8ط ا��&ال ا (chl t) 0ا25 �(% . ��#)ظ� -, ا �+�%�* ا (�)'�&%�$�� وا (�)FA ��$��1أظ �/)R )UB اI8!�ض ��رج 'J1 5 و. ��ات

*�%�+� �(��ا � �� Vت ا�� . FA ـ وا ��ف 0(��ت GEB ا ��G &�1ط�+X ط)ل ا ��ور وا �Eق وا )زن ا�#> ا +�رة -, ا ��ھ�� ى FA .�#�)ىو X+ chl a و chl bو chl t �1&ات و��ا �Kرو��0ت -�0 ا &%�دة '5

FA . �ُ 5' ظ(# ��L8ط ا��&ال ا 0ا �KE وا (�و��0ت و 1��ت�J اI8!�ض �� &ت B!ّ و. FA ـ��ات C ا ��1&ات ا#)ظ� �L8ط�ت �!\� �� [زو superoxide dismutase (SOD) [ ا ��دة �E1Oة ا8M&%��تا ��1&ات ا�K ا

(CAT) و��E1و��) �(�و (POX) [ 1 زاا 5� �)اد ا ��ھ�� ا ��E��ا أو /< ا �#�J و 0)عا � . �+�و

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Short Communication

Allelopathic Effects of Aqueous Extracts of Eucalyptus occidentalis, Acacia ampliceps and Prosopis juliflora on the Germination of Three Cultivated Species

Ezzeddine Saadaoui, Naziha Ghazel, Chokri Ben Romdhane, Station Régionale de Gabès, Institut National des Recherches en Génie Rural, Eau et Forêt (INRGREF), Université de Carthage, Tunis, Tunisia, Nizar Tlili , Faculté des Sciences de Gafsa, Université de Gafsa, Tunisia, and Abdelhamid Khaldi,

INRGREF, Université de Carthage, Tunis, Tunisia __________________________________________________________________________ ABSTRACT Saadaoui, E., Ghazel, N., Ben Romdhane, Ch., Tlili, N., and Khaldi, A. 2014. Allelopathic effects of aqueous extracts of Eucalyptus occidentalis, Acacia ampliceps and Prosopis juliflora on the germination of three cultivated species. Tunisian Journal of Plant Protection 9: 11-16. This study concerns the effect of the aqueous extracts of Eucalyptus occidentalis, Acacia ampliceps and Prosopis juliflora on the germination of three species frequently cultivated in the South of Tunisia: Barley (Hordeum vulgare), annual lucerne (Medicago sativa) and jew's mallow (Corchorus olitorius). Aqueous extracts were obtained after a maceration of the dry plant material in distilled water (90 g/l) during 48 h at 60°C. The extraction was made from three vegetative organs (roots, twigs and leaves) for each species. The results showed a variable behavior between the species according to the origin of the extract. Barley is the most sensitive species, showing decrease of germination rate essentially with the leaf extracts of P. juliflora (52.5 ± 15.86%), E. occidentalis (61.5 ± 7.89%) and A. ampliceps (65.5 ± 5.7%). The annual lucerne showed a moderate tolerance; its germination rate was 76 ± 11.61, 81.5 ± 5.74 and 96 ± 2.82%, respectively, for the leaf extracts of A. ampliceps, E. occidentalis and P. juliflora. C. olitorius was found to be the most tolerant species to all extracts; only leaf extracts of E. occidentalis resulted in a lower germination than the control; it was 90 ± 4.32%. The extracts of the studied species affected the root system length; a reduction of the length was essentially observed for M. sativa and C. olitorius. Keywords: Acacia ampliceps, allelopathy, aqueous extract, Eucalyptus occidentalis, germination, Prosopis juliflora __________________________________________________________________________ Allelopathy, from the latin words allelon of each other and pathos to suffer refers to the chemical inhibition of one Corresponding author: Ezzeddine Saadaoui Email: [email protected] Accepted for publication 11 May 2014

species by another. Although, the term allelopathy is most commonly used to describe the chemical interaction between two plants, it has also been used to describe microbe-microbe, plant-microbe and plant-insect or plant-herbivore chemical communication (9). The chemical substances, collectively known as allelochemicals, are usually secondary


plant products or waste products of main metabolic pathways of plants (10). In plants, allelochemicals can be present in leaves, bark, roots, root exudates, flowers and fruits. The delivery of allechemicals into the rhizosphere is often thought to occur through leaching from leaves and other aerial plant parts, through volatile emissions by roots exudation and by the breakdown of bark and leaf litter (12). Indeed, plants use up to 30% of their photosynthate in the production of root exudates, which affect the local soil environment, termed the rhizosphere. Each of these processes may release chemicals that mediate allelopathic interactions between plants. Media containing exudates inhibited the growth and germination of several plant species, suggesting the presence of phytotoxins (3). Eucalyptus sp., Acacia sp., and Prosopis sp. have an allelopathic effect on seed germination of other species (4, 5, 6, 7). The purpose of this study is to elucidate the effect of the aqueous extraction of three species (Eucalyptus occidentalis, Acacia ampliceps and Prosopis juliflora) and three organs from each species (leaves, twigs and roots) on the germination of three other species (Hordeum vulgare, Medicago sativa and Corchorus olitorius) cultivated frequently in the region of Gabes, in the south of Tunisia.

The aqueous extracts were prepared from dry plant material. A mass of 90 g of each plant material was soaked into 1 liter of distilled water and kept at 60°C for 48 h before filtration to prepare extract of 90 g/l. The seeds were thoroughly washed with distilled water and surface sterilized with sodium hypochlorite (12%) for 2-3 min. In each Petri dish, 50 seeds were placed and 2 ml of aqueous water were added (leaf, twig and root), and then kept inside the

incubator at 28°C. The control was treated with 2 ml of distilled water. Each treatment had four replicates. Seeds belong to three cultivated species in the south of Tunisia (H. vulgare, M. sativa and C. olitorius). Seed germination was investigated every day and radicle length was recorded. Data were analyzed by the analysis of variance (ANOVA), using the XLstat, 2012 software package and differences were considered statistically significant at P < 0.05.

The leaf extract of E. occidentalis reduced significantly the germination for the three studied species (P < 0.05). However, the twig extract affected only on the germination of H. vulgare seeds (Fig. 1). The aqueous extract of the leaves showed the lowest rate of germination for the three studied species. The extracts of all organs of E. occidentalis reduced only the extension of M. sativa and C. olitorius roots (Fig. 1). For A. ampliceps extracts, the lowest germination percentage was obtained for H. vulgare seeds treated with root, twig and leaf extracts and with leaf extract for M. sativa seeds. C. olitorius showed a constant rate of germination for all treatments. The leaf extract of A. ampliceps affected negatively the germinating potential of M. sativa and H. vulgare seeds. A. ampliceps extracts had also reduced the extension of M. sativa and C. olitorius roots (Fig. 2). The extracts of the roots, twigs and leaves of P. juliflora decreased significantly the germination percentage of H. vulgare (P < 0.05). Indeed, the lowest rate of germination was noted on barley seeds treated with leaf extract (52.5 ± 15.86%) as indicated by a decrease of about 40% compared to the untreated control. The inhibitory effect was not significant for M. sativa and C. olitorius seeds. This decrease was not observed for the root extension (Fig. 3).


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Fig. 1. Effects of aqueous extracts (root, twig and leaf) of Eucalyptus occidentalis on germination (A) and root length (B) of Hordeum vulgare, Medicago sativa and Corchorus olitorius. For each cultivated species, bars affected by the same letters are not significantly different at P < 0.05.

Fig. 2. Effects of aqueous extracts (root, twig and leaf) of Acacia ampliceps on germination (A) and root length (B) of Hordeum vulgare, Medicago sativa and Corchorus olitorius. For each cultivated species, bars affected by the same letters are not significantly different at P < 0.05.

Fig. 3. Effects of aqueous extracts (root, twig and leaf) of Prosopis juliflora on germination (A) and root length (B) of Hordeum vulgare, Medicago sativa and Corchorus olitorius. For each cultivated species, bars affected by the same letters are not significantly different at P < 0.05.

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The aqueous extracts of the three studied species (E. occidentalis, A. ampliceps and P. juliflora) decreased the germination rate of seeds of three plants tested i.e H. vulgare, M. sativa and C. olitorius. This decrease was more significant for leaf extract for the three species. These findings agreed with those obtained by other authors (4, 5, 6). The high inhibitory effect of Triticum aestivum germination was recorded with P. juliflora leaf extract (10). High susceptibility of H. vulgare seeds to Eucalyptus (8), Ocimum basilicum (12), Armoracia rusticana (11), Salvia officinalis and Juglans regia extracts (1, 2) was reported. The decrease of germination also depended on the species studied. H. vulgare was found to be the

most sensitive species to all extracts tested while M. sativa and C. olitorius exhibited a tolerance to the water extracts. Indeed, tolerance of M. sativa seeds to aqueous extracts from Eucalyptus leaves was also reported by Yu (13). The decrease of root system length was highest for M. sativa and C. olitorius and lowest for H. vulgare. No positive correlation was observed between the decrease in the percentage of germination and that of the root length. This phenomenon can be understandable by a different specific physiological behavior. The study of the allelopathic effect on the germination and the production of the cultivated species needs to be more elucidated in arid regions.

__________________________________________________________________________ RESUME Saadaoui E., Ghazel N., Ben Romdhane Ch., Tlili N. et Khaldi A. 2014. Effet allélopathique des extraits aqueux d’Eucalyptus occidentalis, Acacia ampliceps et Prosopis juliflora sur la germination de trois espèces cultivées. Tunisian Journal of Plant Protection 9: 11-16. Cette étude concerne l’effet des extraits aqueux d’Eucalyptus occidentalis, Acacia ampliceps et Prosopis juliflora sur la germination de trois espèces fréquemment cultivées au sud tunisien: l’orge (Hordeum vulgare), la luzerne annuelle (Medicago sativa) et la corète (Corchorus olitorius). Les extraits aqueux ont été obtenus après une macération du matériel végétal sec (90 g/l) dans de l’eau distillée pendant 48 h à 60°C. Pour chaque espèce étudiée, les extraits ont été obtenus à partir de trois organes (racines, rameaux et feuilles). Les résultats ont montré un comportement variable entre les espèces selon l’origine de l’extrait. L’orge est l’espèce la plus sensible, montrant une chute du taux de germination et surtout avec les extraits des feuilles de P. juliflora (52,5 ± 15,86%), E. occidentalis (61,5 ± 7,89%) et A. ampliceps (65,5 ± 5,7%). La luzerne annuelle a montré une tolérante modérée, son pourcentage de germination a été de 76 ± 11,61, 81,5 ± 5,74 et 96 ± 2,82% avec les extraits de feuilles d’A. ampliceps, E. occidentalis et P. juliflora, respectivement. La corète s’est montrée l’espèce la plus tolérance à tous les extraits; seul l’extrait de feuilles d’E. occidentalis a induit une faible germination comparé au témoin qui a été de 90 ± 4,32%. Les extraits des espèces étudiées ont affecté la longueur de la racine principale; une réduction de la longueur a été observée surtout chez M. sativa et C. olitorius. Mots clés : Acacia ampliceps, allélopathie, Eucalyptus occidentalis, extrait aqueux, germination, Prosopis juliflora __________________________________________________________________________


__________________________________________________________________________ ��

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Tunisian Journal of Plant Protection 9: 11-16.

ھ�� 0/ ھ"ه ا -را(� #�� ا�� س ا�� )Eucalyptus occidentalis (�) Acacia( وا+*ampliceps ( '�#وزو�� 2�ب ) Prosopis juliflora(وا# �3�� رو�� 4�#��ت #"ور �!�� أ��اع ��& إ

�� وھ�� وا 78 (Hordeum vulgare) ا ��56 ا�9 Corchorus) وا ��:�� (Medicago sativa) اolitorius) ./� �� ا 79�ل ��& ھ"ا ا� ت 5�90 �� إ ا�2 ف �3غ �> ا� ء �-ة 1 ا� 48ل �> ا

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��8ض �3 ��تا��Oا ��وزو#�' ا(�5�ل �� أوراق�Q :7�ص ��سوا (52,5 ± 15,86%) ا� (%7,89 ± 61,5) �)� .(65,5 ± 5,7%) وا+*Rا �� ST�# M�A � -�5� D�9� �� 78 ت*� أظ�0ت ا� ± 76

ل ��7ت أوراق 2,82% 96 ±و 81,5 ± 5,74% و %11,61 �5�)# �)وا ا+*� ��وزو#�' ��س ��& وا� 7ت � �-ى �� أوراق ،أ� ا ��:��. ا ��ا�� S ا+*�4 �9�! �D ا* -W3سا�� ا "ي أظ�0

Hت أ��A �3 S-ودإ* �� 6ھ- وا ��& ط�ل ا �"ور ا+��اع ا �-رو(�*� أ ��ت ��7ت . D %4,32 ± 90 �> ا �LA ��اQB ا Z�ل :ص� -ى M�A �� ا 78ا�9 . وا ��:�� ا

��Aت ��8��*: �تإ�ھ��، �� ،��، Acacia ampliceps، Eucalyptus occidentalis، Prosopis

juliflora ___________________________________________________________________________ LITERATURE CITED 1. Bajalan, I., Zand, M., and Rezaee S. 2013a.

Allelopathic effects of different organs aqueous extracts of walnut (Juglans regia) on seed germination of barley (Hordeum vulgare). Int. J. Agron. Plant Prod. 4: 324-328.

2. Bajalan, I., Zand, M., and Rezaee, S. 2013b. Allelopathic effects of aqueous extract from Salvia officinalis L. on seed germination of barley and purslane. Int. J. Agric. Crop Sci. 5: 802-805.

3. Broz, A.K., Vivanco, J.M., Schultz, M.J., Perry, L.G., and Paschke M.W. 2006. Secondary metabolites and allelopathy in plant invasions: A case study of Centaurea maculosa. Plant Physiology, Fifth Edition. Chapter 13, Essay 13.2.

4. Fatunbi, A.O., Dube, S., Yakubu, M.T., and Tshabalala T. 2009. Allelopathic potential of Acacia mearnsii De Wild. World Appl. Sci. J. 7: 1488-1493

5. Fikreyesus, S., Kebebew, Z., Nebiyu, A., Zeleke, N., and Bogale S. 2011 Allelopathic effects of Eucalyptus camaldulensis Dehnh. on germination and growth of tomato. American-Eurasian J. Agric. & Environ. Sci. 11: 600-608.

6. Getachew, S., Demissew, S., and Woldemariam, T. 2012. Allelopathic effects of the invasive Prosopis juliflora (Sw.) DC. on selected native plant species in Middle Awash, Southern Afar

Rift of Ethiopia. Management of Biological Invasions 3: 105-114.

7. Jeddi, J., Cortina, J., and Chaieb M. 2009. Acacia salicina, Pinus halepensis and Eucalyptus occidentalis improve soil surface conditions in arid southern Tunisia. Journal of Arid Environments 73: 1005-1013.

8. Kohli, R.K. and Singh, D. 1991. Allelopathic impact of volatile components from Eucalyptus on crop plants. Biologia Plantarum 33: 475-483.

9. Mohamadi, N. and Rajaie, P. 2009. Effects of aqueous Eucalyptus (E. camaldulensis Labill) extracts on seed germination, seedling growth and physiological responses of Phaseolus vulgaris and Sorghum bicolor. Res. J. Biol. Sci. 4: 1292-1296.

10. Siddiqui, S., Ruchi Yadav, R., Yadav, K., Wani, F.A., Meghvansi, M.K., Sharma, S., and Jabeen, F. 2009. Allelopathic potentialities of different concentration of aqueous leaf extracts of some arable trees on germination and radicule growth of Cicer arietinum var. – C-235. Global Journal of Molecular Sciences 4: 91-95.

11. Şipoş, M., Blidar, C.F., and Bunta D. 2012. Allelopathic effects of aqueous extracts from horderadish (Armoracia risticana L.) metamorphosed roots on several cereals. Romanian Agricultural Research 29: 169-173.


12. Verma, S.K., Kumar, S., Pandey, V., Verma, R.K., and Patra, D.D. 2012. Phytotoxic effects of sweet basil (Ocimum basilicum L.) extracts on germination and seedling growth of commercial crop plants. Euro. J. Exp. Biol. 2: 2310-2316.

13. Yu, Z., Jian, Z., WanQin, Y., FuZhong, W., MaoSong, F., and XiaoHong, Ch. 2009. Allelopathic effects of Eucalyptus grandis on Medicago sativa growing in different soil water conditions. Acta Prataculturae Sinica 10: 81-86.

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Antifungal Activity of Culture Filtrates and Organi c Extracts of Aspergillus spp. against Pythium ultimum

Rania Aydi-Ben Abdallah, Marwa Hassine, Hayfa Jabnoun-Khiareddine, UR13AGR09, Centre Régional des Recherches en Horticulture et Agriculture Biologique de Chott-Meriem (CRRHABCh-M), Université de Sousse, 4042, Chott-Meriem, Tunisia, Rabiaa Haouala, UR13AGR05, Institut Supérieur Agronomique de Chott-Mariem, Université de Sousse, 4042, Chott-Mariem, Tunisia, and Mejda Daami-Remadi, UR13AGR09, CRRHABCh-M, Université de Sousse, 4042, Chott-Meriem, Tunisia. __________________________________________________________________________ ABSTRACT Aydi-Ben Abdallah, R., Hassine, M., Jabnoun-Khiareddine, H., Haouala R., and Daami-Remadi M. 2014. Antifungal activity of culture filtrates and organic extracts of Aspergillus spp. against Pythium ultimum. Tunisian Journal of Plant Protection 9: 17-30. Culture filtrates, chloroform and ethyl acetate extracts of nine isolates of Aspergillus spp. (A. niger, A. terreus, A. flavus and Aspergillus sp.), isolated from soil and compost, were tested for antifungal activity against Pythium ultimum the causal agent of the potato Pythium leak. Culture filtrates showed a significant antifungal activity at the different tested concentrations. Total inhibition of the pathogen was induced by the filtrate of CH8 of Aspergillus sp., used at 10% (v/v) and those of CH12 (A. niger) and MC8 (A. terreus), applied at 20% (v/v). Chloroform and ethyl acetate extracts of isolates CH12 of A. niger, CH2 and MC8 of A. terreus and CH8 of Aspergillus sp., tested at 5% (v/v), had completely inhibited the in vitro growth of P. ultimum. Tested as tuber treatment, culture filtrates and both organic extracts significantly reduced Pythium leak severity compared to the inoculated and untreated control tubers. Rot lesion diameter and average penetration of the pathogen decrease, recorded after 48 h of incubation at 25°C, induced by the culture filtrates ranged from 71 to 84% and 42 to 85%, respectively. All chloroform and ethyl acetate extracts had also limited both disease severity parameters by 78 to 84% and 72 to 80%, respectively. This study revealed the presence of bioactive metabolites against P. ultimum in the culture filtrates and organic extracts of Aspergillus species used. Keywords: Aspergillus spp., culture filtrates, inhibition, organic extracts, potato leak, Pythium ultimum __________________________________________________________________________

Potato is one of the most important vegetable crops in the world (45) and is one of the strategic crop in Tunisia (20). It is an important nutrient source in human nutrition (32) and even considered as a cereal substitute (23). Corresponding author: Rania Aydi-Ben Abdallah Email: [email protected]

Accepted for publication 27 March 2014

Fungal diseases are the major constraints to agricultural production. About 25 to 50% of total yield losses recorded worldwide were attributed to plant diseases and the third of them caused by fungal infections (43). Potato tubers may develop during storage different types of wet and dry rots such as Pythium leak caused by Pythium spp. (33, 41), pink rot incited by Phytophthora erythroseptica (40) and dry rot induced


by different Fusarium species (13, 17, 18).

Pythium leak or watery wound rot caused by Pythium aphanidermatum and P. ultimum is a serious disease in Tunisia that may cause significant losses early after harvest and/or during storage (40, 41). This disease is difficult to control due to the lack of cultivar resistance, the rapid development and spread of the watery rot (41), and the absence of registered fungicides for controlling the pathogen in storage (8). Therefore, there is an increasing interest in the development of control alternatives. Biological control was considered as an environmentally safe alternative. Biocontrol agents can inhibit pathogen growth via competition (14), antibiosis (19) and mycoparasitism (21) and/or indirectly through the induction of plant defense systems (44). Applied as spore suspension (15) or as cell-free culture filtrates, satisfactory results were obtained against some plant pathogenic fungi (38, 46) with Trichoderma species. The direct action of the biocontrol agents and the antifungal activity of their active biomolecules have been extensively studied in several pathosystems (2, 8, 10, 27, 36, 42, 46).

Soilborne fungi that exhibit antagonism towards plant pathogens have attracted increasing interest. Among the antagonistic agents explored, Aspergillus spp. were used due to their ability to control many fungal plant pathogens through mycoparasitism, mycelial lysis and antibiosis via the synthesis of volatile and/or non volatile metabolites (10, 18). Aspergillus species have successfully controlled many plant pathogenic fungi such as Phytophthora palmivora (3), Sclerotinia sclerotiorum (28), Rhizoctonia solani (31, 35, 42), Fusarium

oxysporum f. sp. lycopersici (4), Colletotrichum gloeosporioides (22), Peleospora herbarum (2) and wood decay fungi (39).

The present study was conducted to evaluate in vitro and in vivo, the antifungal potential of culture filtrates and organic extracts (ethyl acetate and chloroform extracts) from Aspergillus spp. against P. ultimum, the causal agent of potato Pythium leak. MATERIALS AND METHODS Plant material.

Apparently healthy and undamaged potato tubers (cv. Spunta) were used in this study. This cultivar was known by its susceptibility to Pythium watery rot (33).

Pathogen.

The isolate of P. ultimum used in this study was originally isolated from potato tubers showing typical symptoms of Pythium leak and belonged to the fungus culture collection of the Laboratory of Plant Pathology at the Centre Régional des Recherches en Horticulture et Agriculture Biologique de Chott-Mariem. It was cultured on Potato Dextrose Agar (PDA) and incubated at 25°C for 48 h before use.

Biocontrol agents.

Four Aspergillus species namely A. niger, A. terreus, A. flavus and Aspergillus sp., isolated from soil and compost, were used in this study (Table 1). They were identified and proved to be effective in controlling Pythium leak in a previous work (16). They were cultured on PDA medium and incubated at 25°C for five days before being used in the bioassays.


Table 1. Aspergillus species tested against Pythium ultimum and isolates origin. Isolate Species Origin Year of isolation CH1 Aspergillus niger Solarized soil 2006 CH2 A. terreus Solarized soil 2006 CH3 Aspergillus sp. Compost 2006 CH4 Aspergillus sp. Compost 2006 CH8 Aspergillus sp. Non-solarized soil 2006 CH12 A. niger Non-solarized soil 2006 MC2 A. niger Compost 2002 MC5 A. flavus Compost 2002 MC8 A. terreus Compost 2002

Assessment of the in vitro antifungal activity of Aspergillus spp. culture filtrates against P. ultimum. Each antagonistic agent tested was cultured in Potato Dextrose Broth (PDB) for 28 days at room temperature (25-27°C) and under continuous stirring at 150 rpm. Liquid cultures obtained were filtered through Whatman No.1 filter paper and the filtrate was then centrifuged for 10 min at 10,000 rpm. The centrifugation was repeated three times. Supernatant fluids were sterilized by filtration through a 0.45 µm pore size filter. The control was the PDB medium filtrate (42). The filtrates were added aseptically to Petri dishes containing the culture medium (PDA) amended with streptomycin sulfate (300 mg/l) and tested at three concentrations (10, 15 and 20%, v/v). After solidification of the mixture, three agar plugs of the same pathogen (6 mm diameter) were placed equidistantly in each Petri plate. Fungal cultures were incubated at 25°C for two days.

The colony diameter of the pathogen (treated and untreated control) was measured daily. The mycelial growth inhibition rate was calculated using the following formula:

I ℅ = [(C2-C1) / C2] × 100 with C2: Mean diameter of the control colony and C1: Mean pathogen colony diameter in the presence of the antagonist (30).

Assessment of the in vivo antifungal activity of Aspergillus spp. culture filtrates against P. ultimum. Potato tubers were washed under running tap water to remove excess soil, dipped in 10% sodium hypochlorite for 5 min, rinsed twice with sterile distilled water (5 min each) and air-dried.

Tubers were wounded (6 mm in diameter and in depth) at two sites along the tuber longitudinal axis by a 6 mm diameter Pasteur pipette. Each wound was then inoculated with a mycelium agar disc (6 mm diameter) colonized by the pathogen removed from a 7-day-old cultures at 25°C.

Culture filtrates, prepared as described above, were tested individually and applied by inoculating each wound with 200 µl, simultaneously by pathogen inoculation. The positive control was inoculated with the pathogen only and treated with sterile distilled water and the negative control was inoculated with an agar plug and treated with sterile distilled water.

All inoculated and treated or untreated tubers were placed in plastic boxes covered at their base by a filter paper moistened with sterile distilled water and incubated at 25°C for two days.

The lesion diameter, width (l) and depth (p) of the Pythium leak rot were measured. These parameters were noted after making a longitudinal section on


potato tubers. The penetration of the pathogen was calculated using the following formula:

P (mm) = [(l / 2) + (P-6)] / 2, (16). The rate of reduction of the lesion diameter and the penetration were calculated using the following formula:

I ℅ = [(C2-C1) / C2] × 100, with C2: lesion diameter of the rot or penetration of the pathogen in the untreated control tubers, and C1: lesion diameter of the rot or penetration of the pathogen in the treated tubers (30). Assessment of the in vitro antifungal activity of Aspergillus spp. organic extracts against P. ultimum. Only the isolates CH2 and MC8 of A. terreus, CH8 of Aspergillus sp. and CH12 of A. niger were used for this test as their culture filtrates were found to possess an interesting inhibitory activity against the pathogen. Two types of extraction were carried out: an extraction with chloroform and a second with ethyl acetate. Ten milliliters of the culture filtrate of the antagonist studied, prepared as described above, were placed in a separating funnel. Then, 10 ml of the solvent (chloroform or ethyl acetate) were added carefully. The funnel was reversed several times by degassing from time to time. The mixture was allowed to settle for few minutes with the cap open. The organic phase (the lower phase for extraction with chloroform and the upper one with ethyl acetate) were collected. The aqueous phase is replaced in the funnel and the extraction is repeated two other times by adding 10 ml of solvent for each step. The solvent was evaporated in a rotary evaporator at 90°C with a slight rotation of 150 rpm (6). Extract of the organic phase was taken up by 2 ml of methanol (6). A volume of 500 µl of each type of extract

was added to Petri dishes containing 10 ml PDA culture medium amended with streptomycin sulfate (300 mg/l). After solidification of the mixture, three discs (6 mm diameter) of the same pathogen were arranged in an equidistant manner. The culture medium PDB is used as a control for both types of extracts. Fungal cultures were incubated for two days at 25°C.

The colony diameter of the treated and control pathogen was measured and the percentage of inhibition of the mycelial growth was calculated as described above.

Assessment of the in vivo antifungal activity of Aspergillus spp. organic extracts against P. ultimum.

Organic extracts of Aspergillus spp., prepared as described above, were tested individually and applied by inoculating each wound with 100 µl. The positive control was inoculated with the pathogen and treated with sterile distilled water only and the negative control was inoculated with agar plug and treated with sterile distilled water. Incubation conditions and parameters are the same as in the in vivo essay described above. Statistical analysis. Data were subjected to a one-way analysis of variance (ANOVA) by using SPSS 16.0. For all the tests conducted in vitro, each individual treatment was repeated three times. The in vitro essay of culture filtrates was analyzed according to a completely randomized factorial model with two factors (culture filtrates tested and concentrations used). The in vitro essay of organic extracts and the in vivo essays were analyzed in a completely randomized model. For all the tests conducted in vivo, each elementary treatment was replicated six times. Means

Tunisian Journal of Plant Protection 21

were separated using Student-Newman-Keul’s (SNK) test (P ≤ 0.05). RESULTS Effect of Aspergillus spp. culture filtrates on the mycelial growth of P. ultimum.

Results shown in Fig. 1 revealed that the culture filtrates tested exhibited a significant inhibitory effect (P ≤ 0.05) against P. ultimum. In fact, the colony diameter of the pathogen was reduced by 12 to 88% as compared to the untreated control. The culture filtrates of Aspergillus spp. tested were more active at the concentration of 20% than at 15 or 10% (v/v). In fact, the filtrate of MC2 of A. niger decreased pathogen growth by 77.40% when applied at 20% compared

to 59.22% and 39.22% recorded at 15 and 10%, respectively. In addition, CH12 of A. nigerpathogen by 90.65 % whereas 74.28% and 41.99% were noted witconcentrations 15 and 10%, respectively. The culture filtrate of MC8 of reduced pathogen growth by 87.53% when used at 20% compared to 83.38% and 17.68% recorded with the concentrations 15 and 10%, respectively (Fig. 1).

Complete inhibitioby the culture filtrates of CH8 of AspergillusMC8 of were found to be more active at 20% than at 15 or 10% (Figs. 1

Fig. 1. Effect of Aspergillus spp. culture filtrates tested at different concentrations on growth noted after 48 h of incubation at 25°C. FCH1, FCH2, FCH3, FCH4, FCH8, FCH12, FMC2, FMC5 and FMC8: Culture filtrates of isolates CH1 of CH3, CH4 and CH8 of Aspergillus sp., CH12 and MC2 of A. niger, MC5 of respectively; Control: PDB culture medium. LSD (Culture filtrates tested 0.05.

Fig. 2. Effect of the culture filtrate of CH12 of Aspergillus niger tested at different concentrations on the mycelial growth of Pythium ultimum noted after 48 h of incubation at 25°C.

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to 59.22% and 39.22% recorded at 15 and 10%, respectively. In addition, CH12 of A. niger, used at 20%, inhibited the pathogen by 90.65 % whereas 74.28% and 41.99% were noted with the concentrations 15 and 10%, respectively. The culture filtrate of MC8 of A. terreus reduced pathogen growth by 87.53% when used at 20% compared to 83.38% and 17.68% recorded with the concentrations 15 and 10%, respectively (Fig. 1).

Complete inhibition was induced by the culture filtrates of CH8 of Aspergillus sp., CH12 of A. niger and MC8 of A. terreus, used at 20%. They were found to be more active at 20% than at 15 or 10% (Figs. 1, 2).

spp. culture filtrates tested at different concentrations on Pythium ultimum mycelial

FCH1, FCH2, FCH3, FCH4, FCH8, FCH12, FMC2, FMC5 and FMC8: Culture filtrates of isolates CH1 of A. niger, , MC5 of A. flavus, CH2 and MC8 of A. terreus,

respectively; Control: PDB culture medium. LSD (Culture filtrates tested × concentrations used): 0.44 cm at P ≤

tested at different concentrations on noted after 48 h of incubation at 25°C.


Pathogen colonies treated with the culture filtrate of MC8 (A. terreus) used at 10% (Figs 1 and 3A) showed reduction of mycelium density as compared to the untreated control. However, use of cell-

free culture filtrates of Aspergillus resulted in maximum growth ipathogen in dual culture

Fig. 3. Effect of the culture filtrate of MC8 of Aspergillus terreus (A) and CH8 of 10% (v/v) on the mycelial growth of Pythium ultimum observed after 48 h of incubation at 25°C.

Effect of Aspergillus spp. culture filtrates on Pythium leak severity. Analysis of variance showed a significant variation of the rot lesion diameter and the average penetration of the pathogen by the tested culture filtrates. Indeed, the rot lesion diameter of the Pythium leak recorded after 48 h of incubation at 25°C was significantly reduced by 71 to 84% with all the tested culture filtrates, compared to the inoculated and untreated control (Fig. 4A). Except for the culture filtrate of CH3

(Aspergillusfiltrates have significantly limited the average penetration of the pathto 85% as compared to the inoculated and untreated control (Fig. 4B).

A reduction of 83.90% of the rot lesion diameter, compared to the inoculated and untreated control, was recorded with the culture filtrate of CH8 of Aspergilluspenetration of the pathogen was limited by 84.94 % (Figs. 4

Fig. 4. Effect of Aspergillus spp. culture filtrates tested on the rot lesion diameter (A) and the average penetration of Pythium ultimum (B), noted after 48 h of incubation at 25°C. FCH1, FCH2, FCH3, FCH4, FCH8, FCH12, FMC2, FMC5 and FMC8: Culture filtrates of isolates CH1 of A. niger, CH3, CH4 and CH8 of MC2 of A. niger, MC5 of A. flavus, CH2 and MC8 of A. terreus, respectively; Control inoculated and untreated), Control +: Positive control (inoculated and untreated). Bars affected bnot significantly different according to Student–Newman–Keuls SNK test at

P. ultimumP. ultimum P.ultimum+FMC8

A B

B A

A B

B

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free culture filtrates of CH8 of Aspergillus sp. at the same concentration resulted in maximum growth inhibition of pathogen in dual culture (Figs. 1 and 3B).

(A) and CH8 of Aspergillus sp. (B) applied at observed after 48 h of incubation at 25°C.

Aspergillus sp.), all the other tested filtrates have significantly limited the average penetration of the pathogen by 42 to 85% as compared to the inoculated and untreated control (Fig. 4B).

A reduction of 83.90% of the rot lesion diameter, compared to the inoculated and untreated control, was recorded with the culture filtrate of CH8

Aspergillus sp.; also, the average penetration of the pathogen was limited by 84.94 % (Figs. 4, 5).

spp. culture filtrates tested on the rot lesion diameter (A) and the average penetration of FCH1, FCH2, FCH3, FCH4, FCH8, FCH12, FMC2, , CH3, CH4 and CH8 of Aspergillus sp., CH12 and

, respectively; Control -: negative control (non-Control +: Positive control (inoculated and untreated). Bars affected by the same letter are

test at P ≤ 0.05.

P.ultimum+FCH8 P. ultimum


Fig. 5. Decreased Pythium leak severity induced by the culture filtrate of the isolate CH8 of Aspergillus sp. recorded after 48 h of incubation at 25°C.

Effect of Aspergillus spp. organic extracts on the in vitro growth of P. ultimum.

Analysis of variance revealed a significant (at P ≤ 0.05) variation in the average diameter of P. ultimum colonies with the two types of organic extracts (chloroform extracts and ethyl-acetate

extracts) from (CH2 and MC8 of AspergillusAll chloroform (Figs. 6A and 7A) and ethyl-acetate extracts (Figs. 6Bthe tested isolates, used at 5% (v/v), have completely inhibited compared to the controls.

Fig. 6. Effect of chloroform (A) and ethyl acetate extracts (B) from Aspergillus growth noted after 48 h of incubation at 25°C as compared to the controls. ECH2, ECH8, ECH12 and EMC8: Organic extracts of isolates CH2 and MC8 of A. terreus, CH8 of AspergillusControl (A) and (B): PDB culture medium. Organic extracts were used at the concentration of 5%by the same letter are not significantly different according to Student–Newman

Effect of Aspergillus spp. organic extracts on Pythium leak severity.

Pythium leak severity on potato tubers, as estimated by the lesion rot diameter and mean pathogen penetration, varied significantly (P ≤ 0.05) depending on organic extracts tested (chloroform and ethyl acetate extracts). Fig. 8A shows

that the decrease in the rot lesion diameter induced by extracts, as compared to inoculated and untreated control, ranged between 78 and 84% whereas with ethyl acetate extracts (Fig. 8B), this paramet80 to 83%, respectively.

P.ultimum

P.ultimum P.ultimum

P.ultimum

A B

Vol. 9, No. 1, 2014

leak severity induced by the culture sp. recorded after 48 h of

extracts) from Aspergillus spp. tested (CH2 and MC8 of A. terreus, CH8 of Aspergillus sp. and CH12 of A. niger). All chloroform (Figs. 6A and 7A) and

acetate extracts (Figs. 6B, 7B) of the tested isolates, used at 5% (v/v), have completely inhibited P. ultimum growth compared to the controls.

Aspergillus spp. on Pythium ultimum mycelial growth noted after 48 h of incubation at 25°C as compared to the controls. ECH2, ECH8, ECH12 and EMC8:

Aspergillus sp. and CH12 of A. niger, respectively; ture medium. Organic extracts were used at the concentration of 5%, v/v. Bars affected

Newman–Keuls SNK test at P ≤ 0.05.

that the decrease in the rot lesion diameter induced by Aspergillus spp. chloroform extracts, as compared to inoculated and untreated control, ranged between 78 and 84% whereas with ethyl acetate extracts (Fig. 8B), this parameter was reduced by 80 to 83%, respectively.

P.ultimum +

P.ultimum +


Fig. 7. Effect of chloroform (A) and ethyl acetate extracts (B) from Aspergillusniger) on Pythium ultimum mycelial growth noted after 48 h of incubation at 25°C.

Fig. 8. Effect of chloroform (A) and ethyl acetate (B) Aspergillus spp. extracts on the rot lesion diameter induced by Pythium ultimum after 48 h of incubation at 25°C as compared to controls. ECH2, ECH8, ECH12 and EMC8: Organic extracts of isolates CH2 and MC8 of A. terreus, CH8 of AspergillusControl - (A) and (B): negative control (non-inoculated and untreated),(inoculated and untreated). Bars affected by the same letter are not significantly different according to Newman–Keuls SNK test at P ≤ 0.05.

The chloroform (Fig. 9A) and ethyl acetate (Fig. 9B) extracts of Aspergillus spp. significantly (P ≤ 0.05) reduced the average penetration of the pathogen from

75 to 80% and from 72 to 79%, as compared to the inoculated and untreated control, respectively.

A

B

A

B

P. ultimum+ECH12

P. ultimum

P. ultimum

P. ultimum+ECH12

P. ultimum

P. ultimum

B A

Vol. 9, No. 1, 2014

Aspergillus spp. (CH8: Aspergillus sp. CH12: A. mycelial growth noted after 48 h of incubation at 25°C.

spp. extracts on the rot lesion diameter induced by after 48 h of incubation at 25°C as compared to controls. ECH2, ECH8, ECH12 and EMC8:

Aspergillus sp. and CH12 of A. niger, respectively; , Control + (A) and (B): Positive control

(inoculated and untreated). Bars affected by the same letter are not significantly different according to Student–

75 to 80% and from 72 to 79%, as compared to the inoculated and untreated control, respectively.

P. ultimum+ECH8

P. ultimum+ECH8

P. ultimum

P. ultimum


Fig. 9. Effect of chloroform (A) and ethyl acetate extracts (B) from AspergillusPythium ultimum noted after 48 h of incubation at 25°C as compared to the controls. ECH2, ECH8, ECH12 and EMC8: Organic extracts of isolates CH2 and MC8 of A. terreus, CH8 of respectively; Control – (A) and (B): negative control (non-inoculated and untreated) Control + (A) and (B): Positive control (inoculated and untreated). Bars affected by the same letter are not significantly different according to Student–Newman–Keuls SNK test at P ≤ 0.05.

The chloroform extract from the isolate CH12 of A. niger has reduced the rot lesion diameter by 83.84 % and the average penetration of the pathogen by 19.62% compared to the inoculated and untreated controls (Figs. 8A, 9A, 10A).

The ethyl acetate extract fCH8 of rot lesion diameter by 83.30% and the pathogen8B, 9B,

Fig.10. Effect of chloroform extract from Aspergillus niger(A) and ethyl acetate extract from AspergillusPythium leak severity noted 48 h after incubation at 25°C as compared to the untreated controls.

P. ultimum + ECH8 P. ultimum

P. ultimum + ECH8

P. ultimum + ECH12 P. ultimum

P. ultimum + ECH12 A

B

A B

Vol. 9, No. 1, 2014

Aspergillus spp. on the average penetration of noted after 48 h of incubation at 25°C as compared to the controls. ECH2, ECH8, ECH12 and

, CH8 of Aspergillus sp. and CH12 of A. niger, inoculated and untreated) Control + (A) and (B): Positive

control (inoculated and untreated). Bars affected by the same letter are not significantly different according to

The ethyl acetate extract from the isolate CH8 of Aspergillus sp. also limited the rot lesion diameter by 83.30% and the pathogen penetration by 79.21% (Figs.

10B).

Aspergillus niger (CH12) Aspergillus sp. (CH8) (B) on

Pythium leak severity noted 48 h after incubation at 25°C as

P. ultimum

P. ultimum

P. ultimum

P. ultimum


DISCUSSION Culture filtrates of Aspergillus spp.

(A. niger, A. terreus, A. flavus, and Aspergillus sp.) were tested for their antifungal activity against P. ultimum. The study revealed that the secondary metabolites of Aspergillus spp. contained some bioactive compounds that inhibited the in vitro and in vivo pathogen growth and that this inhibitory effect varied depending on the concentrations tested (10, 15 and 20%, v/v). The inhibitory effect of Aspergillus culture filtrates are in accordance with those of Vibha (42) conducted on Rhizoctonia solani biocontrol by using A. niger isolates.

Findings from in vitro experiments concerning the inhibitory effects of the filtrates used are in agreement with several previous works (14, 19, 30, 34, 37, 38). The biomolecules have an important effect on the germination of spores of pathogens and on destruction of host cells (22, 34).The antifungal activity of culture filtrates may be attributed either to the production of antibiotics (9, 14) or to the production of lytic enzymes (12, 30, 38).

All the culture filtrates of Aspergillus spp. tested were more active at the concentration of 20% than 15 and 10% (v/v). Total inhibition of P. ultimum was recorded with the culture filtrate of isolate CH8 of Aspergillus sp. with the concentration of 10%. In addition, the mycelial density of this pathogen has been greatly reduced by the culture filtrate of isolate MC8 of A. terreus applied to this concentration. This alteration is probably induced by enzymes present in the culture filtrate of A. terreus, such as cellulases causing the degradation of the cell walls of P. ultimum (30).

In this study, a chloroform and ethyl acetate extraction was carried out to test, in vitro and in vivo the antifungal activity of secondary metabolites of the

liquid cultures of the isolates CH12 of A. niger, CH8 of Aspergillus sp., MC8 and CH2 of A. terreus. The study showed that all of the chloroform extracts and ethyl acetate extracts, tested at 5% (v/v), have totally inhibited the development of P. ultimum in vitro. Abdel-Motaal et al. 2010 (1) also evaluated the inhibitory ability of ethyl acetate extracts from Aspergillus spp. (A. candidus, A. flavus, A. fumigatus, A. nidulans, A. niger, A. ochraeous, A. oryzae, A. sydowi, A. terreus, A. ustus, A. versicolor) against Gibberella zeae and Thanatephorus cladosporioides. The ethyl acetate extract of A. niger inhibited Rigidoporus microporus by 0, 19, 24, 28, and 33% at the concentrations 10, 50, 100, 500 and 1000 mg/l, respectively. However, the hexane extracts of this antagonist had no inhibitory effect. A methanolic extract from A. niger inhibited R. microporus by 20 and 25% at 500 and 1000 mg/l, respectively (29). Indeed, for the same antagonist, the inhibitory effect of various extracts seems to vary depending on the concentrations, temperature and time of incubation, culture medium and pH (26).

The extracts of Aspergillus spp. tested may also contain antibiotics, toxins and/or lytic enzymes against the studied pathogens. Different types of active organic metabolites have been produced by these antagonists (25). In fact, Sen et al. (36) reported that A. niger, an effective biocontrol agent against R. solani, acted by antibiosis, overgrowth and hyperparasitism.

In this study, potato Pythium leak caused by P. ultimum was controlled by culture filtrates and organic extracts from Aspergillus spp. Disease severity was significantly reduced by all the culture filtrate of tested Aspergillus. These biocontrol agents isolated from compost were able to suppress P. ultimum in vivo by 69.4% (30). Abdel-Sater (2) also


demonstrated the effectiveness of A. niger culture filtrates in reducing the severity of the disease caused by Peleospora herbarum infecting onion leaves. All chloroform and ethyl acetate extracts from Aspergillus spp. significantly reduced Pythium leak severity. Daami-Remadi et al. (16) and Aydi et al. (7) also showed that Aspergillus spp., applied as spore suspension, were able to reduce the severity of potato Pythium leak.

Chloroform extracts and ethyl acetate extracts from Aspergillus spp. were also found to be active against Fusarium sambucinum involved in the potato dry rot disease (8).

The chemical nature of the metabolites involved in these inhibitory effects recorded against P. ultimum will give additional information about the potential use of Aspergillus species as sources of bioactives molecules.

__________________________________________________________________________ RESUME Aydi-Ben Abdallah R., Hassine M., Jabnoun-Khiareddine H., Haouala R. et Daami-Remadi M. 2014. Activité antifongique des filtrats de culture et des extraits organiques d’Aspergillus spp. contre Pythium ultimum. Tunisian Journal of Plant Protection 9: 17-30. Les filtrats de culture, les extraits de chloroforme et d'acétate d'éthyle de neuf isolats d’Aspergillus spp. (A. niger, A. terreus, A. flavus et Aspergillus sp.), isolés du sol et du compost, ont été testés pour leur activité antifongique contre Pythium ultimum, l'agent causal de la pourriture aqueuse des tubercules de pomme de terre. Les filtrats de culture ont montré une activité antifongique significative aux différentes concentrations testées. Une inhibition totale de l'agent pathogène a été induite par le filtrat de culture de CH8 d’Aspergillus sp., utilisé à 10% (v/v) et ceux de CH12 d’A. niger et MC8 d’A. terreus, appliqués à 20% (v/v). Les extraits de chloroforme et d'acétate d'éthyle des isolats CH12 d’A. niger, CH2 et MC8 d’A. terreus et CH8 d'Aspergillus sp., testés à 5% (v/v), ont totalement inhibé la croissance in vitro de P. ultimum. Testés en traitement des tubercules, les filtrats de culture et les extraits organiques ont significativement réduit la sévérité de la pourriture aqueuse comparés aux tubercules témoins inoculés et non traités. La réduction du diamètre externe de la pourriture et de la pénétration moyenne du pathogène, enregistrés après 48 h d'incubation à 25°C, induite par les filtrats de culture a varié de 71 à 84% et de 42 à 85%, respectivement. Tous les extraits chloroformiques et d'acétate d'éthyle ont également limité les deux paramètres de sévérité de la maladie par 78 à 84% et 72 à 80%, respectivement. Cette étude a révélé la présence des métabolites bioactifs contre P. ultimum dans les filtrats de culture et les extraits organiques des espèces d'Aspergillus utilisées. Mots clés: Aspergillus spp, extraits organiques, filtrats de culture, inhibition, pourriture aqueuse, Pythium ultimum __________________________________________________________________________

�� 2014..�� ة ا��&% ا��دي ر�� ا��، وو ھ�"�ء ��ن ��ر ا��و ��وى ��ورا�� ا��ي �� الله،

� �� Pythium ض� .Aspergillus spp ـا��1ط ا�&)�د ��"/��ت ��ى ا��وا.- ا�,را�� وا�&�+��*�ت ا��)Tunisian Journal of Plant Protection 9: 17-30. ultimum.

�وا�� ا �را�� و��ت ا ��رورم وأ� � ا�� & ��%ت �$ #�"�ت ا! �� '�Aspergillus spp.

A.terreus, A. flavus, Aspergillus sp.) A. niger,( ، $و� �� (� �$ ا�� *" +� �12ط(� ا -/�د ، ا -��-,ا45� �ت 3,� �45 � 67�P. ultimum ا 87�- �وا�� ا �را�� ;�1ط� . ��> در;�ت ا �47ط� -�:+ا 59$ �ا 2� ا��

��ت��/�دا �� = 2� روا�� ا �9%ت . ��9-�ل ��< ا��Aspergillus sp. CH8 ��@�� -9��- 10(v/v)، ا�@�� ، ا A. terreus MC8و A. niger CH12و ،%�� =-20(v/v) %، �45 ��5 @�� $ ;- �67 ا ,A �$ ا

87��- + ا �ا $59 �@�� #�"�ت ا! �@-� B,ت ��ت ا ��رورم وأ� .ا �47ط� ,ر;�ت -�:+ا� ��-�د ا ،'�5%


(v/v)، -2 ��5 @�� $ ا ,A �+�$ ا7;Cا �وا�� ا �را�� . P. ultimumـ ،��2� ا� 9/ �2, وا -��ت ا��29� �5 � �B $,ة ,A -,اواة در;�ت ا �47ط�، �$ ا �) � -�:+ا 59$ ا �ا��9-��Fو �AG�- ,ر;�ت ا �1ھ, ا��Gر;�

�9 =�ا- . ُ� ,G� 4� ا ��ر�+ G� 45� �=' ا;��5ض ھ�م �وا�� ا �را��، �59$ و -9,ل �-K ا ��> ا ,ر;�ت ا -,اواة ,9��ار �� 48A � ا ,ر�� اA" �;�/A �اوح ��$ س°25 $ ا" N�B ،71 85و 42و %84و% + . ��> ا �ا

� ��ت ا ��رورم وأ��O �-@� !ت ا�"�# ْ+��G� $� '� $� ض�- %80إ > 72و�$ %84إ > B78,ة ا+ 2� ھSه ا ,را�� و�د .��> ا �ا��9 � G�� ,3��7ت �� P. ultimum ت��- �وا�� ا �را�� وا + ا

. ا -��-9�� Aspergillusا /9 � C;اع

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Chitosan and Trichoderma harzianum as Fungicide Alternatives for Controlling Fusarium Crown and Root Rot of Tomato

Riad S.R. El-Mohamedy and Farid Abdel-Kareem, Plant Pathology Department, National Research Center, Dokki, Giza, Egypt, Hayfa Jabnoun-Khiareddine, and Mejda Daami-Remadi, UR13AGR09, Centre Régional des Recherches en Horticulture et Agriculture Biologique de Chott-Meriem, Université de Sousse, 4042, Chott-Mariem, Tunisia. _______________________________________________________________________ ABSRACT El-Mohamedy, R.S.R., Abdel-Kareem, F., Jabnoun-Khiareddine, H., and Daami-Remadi, M. 2014. Chitosan and Trichoderma harzianum as fungicide alternatives for controlling Fusarium crown and root rot of tomato. Tunisian Journal of Plant Protection 9: 31-43. Tomato is one of the most important vegetable crops in Egypt and Tunisia. Fusarium crown and root rot (FCRR), caused by Fusarium oxysporum f. sp. radicis-lycopersici (Forl), is one of the most damaging soilborne disease of tomato and is becoming more common in commercial greenhouses. In the present study, effect of individual or combined application of Trichoderma harzianum and chitosan against Forl was assessed in vitro and in vivo. T. harzianum had significantly reduced the mycelial growth of the five Forl tested isolates. Chitosan applied at different concentrations (from 0.5 to 4 g/l) had also significantly decreased the mycelial growth of the pathogen and a total inhibition was obtained at the concentration 4 g/l. Under greenhouse conditions, application of T. harzianum and chitosan (1 g/l) as root dipping treatment combined with chitosan (0.5 g/l) as foliar spray has reduced FCRR incidence and severity by 66.6 and 47.6%, respectively. Treatments based on T. harzianum alone or in combination with chitosan led to an increase in the total phenols and to an enhancement of chitinase and β,1-3-glucanase activities in leaves of treated tomato plants compared with the untreated ones. The results from this study showed the possibility of using combined treatments based on T. harzianum and chitosan commercially as an approach for controlling FCRR on tomato. Keywords: Chitosan, disease severity, Fusarium oxysporum f. sp. radicis-lycopersici, tomato, Trichoderma harzianum. __________________________________________________________________________

Tomato is one of most important

vegetable crops in the world and particularly in Egypt and Tunisia. Fusarium crown and root rot (FCRR) caused by Fusarium oxysporum f. sp. radicis-lycopersici (Forl) is becoming the Corresponding author: Riad S.R. El-Mohamedy Email: [email protected]


most damaging soilborne disease of tomato in both countries. The disease occurs in both greenhouse and open field tomato crops and causes significant yield losses (22, 29, 34). FCRR of tomato is responsible of about 70-83% loss of tomato plants attributed to rot and basal stem decays and eventual death of heavily infected plants (34). Although application of fungicides is far the most effective method to control tomato wilt and FCRR


disease, it can be involved in many problems due to health risk concerns and environmental pollution. Thus, there is a growing need to develop alternative approaches for the management of this pathogen. An acceptable approach that is being actively investigated involves the use of bio-agents and bio-active substances such chitosan in controlling soilborne fungi (1, 2, 7, 11, 21, 30, 40, 42, 47).

Trichoderma spp. are effective biocontrol agents against different pathogens and some isolates are also known for their ability to induce systemic resistance in plants (26). The fungus Trichoderma, a natural soil-inhabiting genus, has been used successfully to control FCRR of tomato (28, 29, 30, 42). The mechanisms of action of Trichoderma spp. include competition for space and nutrients, antibiosis, antagonism, inhibition of pathogen enzymes and plant growth enhancement (8, 27). T. harzianum was applied as a peat-bran preparation to the rooting medium at the transplanting time of tomato plants. Such an application resulted in significant decrease in FCRR throughout the growing season (12, 32).

Chitosan is a partly de-acetylated form of chitin, and consists of polymers of β-1,4-glucosamine subunits, with molecular weight up to 400 kDa. It is environmentally safe and non-toxic to the majority of organisms (35). Chitosan and its derivatives display antibiotic activity against microorganisms including bacteria (38, 47, 53) and fungi (40, 47, 53, 54). Chitosan can also enhance plant resistance in seeds (14, 15, 16, 36), fruits (14, 54) or leaves (54) and reduce disease caused by fungal pathogens (21, 47). Oligomers of chitosan, which are likely to be released by the action of plant encoded-chitinases from walls of invading fungi, can protect tomato roots

against Forl when applied to seeds or roots (15, 36, 42). Chitosan derivatives were applied to decapitated tomato plants and evaluated for their potential to induce defense mechanisms in root tissues infected by Forl (27, 42). Field application of chitosan for inducing resistance against late and early blight diseases of potato and root rot diseases of tomato plants was also largely reported (3, 4, 5, 6).

Management of many fungal pathogens in different pathosystems through the application of Trichoderma or chitosan individually or in combination is well documented (5, 6, 15, 19, 42). The present study was carried out to control FCRR of tomato and to assess the induction of phenolic compounds and defense enzymes in Forl-infected tomato plants in response to the application of T. harzianum individually or in combination with chitosan.

The objective of the present study is to assess the effect in vitro and in vivo of individual or combined applications of Trichoderma harzianum and chitosan against Forl.

MATERIALS AND METHODS Fungal and plant material.

F. oxysporum f. sp. radicis-lycopersici (Forl), the causal agent of tomato FCRR disease, was isolated and identified in Plant Pathology Department, National Research Center, Giza, Egypt. Five isolates were used in the present study.

One isolate of T. harzianum was used in this experiment and was procured from the Plant Pathology Department, National Research Center, Giza, Egypt. Stock cultures were stored on Potato Dextrose Agar (PDA) medium at 4°C.

Tomato seeds cv. Kastel rock were obtained from Vegetable Crops Research Department, Agricultural Research


Center, Giza, Egypt. This cultivar is known by its susceptibility to FCRR.

Study of the effect of T. harzianum and chitosan on Forl mycelial growth.

Antagonistic ability of T. harzianum against Forl was carried out on PDA medium using the dual culture technique (24). Five Petri dishes were used as replicates for each treatment. All plates were incubated at 25°C for 4 and 8 days. Antagonistic ability of T. harzianum was expressed as percent inhibition of Forl mycelial growth as compared to the untreated control.

The inhibitory effect of chitosan (Sigma Company) against Forl was tested in vitro by using five concentrations i.e 0.5, 1.0, 2.0, 3.0, and 4.0 g/l. Chitosan concentrations were prepared according to Benhamou et al. (15). Chitosan was added to conical flasks containing sterilized PDA before solidification and rotated gently then poured into sterilized Petri plates (9 cm diameter). Plates were individually challenged at the center with equal agar plugs (5 mm diameter) taken from Forl 10 days-old cultures then incubated at 25 ± 2°C. Mean colony diameter was measured when the control plates (PDA free of chitosan) reached full growth. Five plates were used for each elementary treatment.

Preparation of Forl inoculum.

A highly aggressive isolate of Forl was grown on sand maize medium. Sand and ground maize seeds were mixed in the ratio of 2:1 (w/w) and moistened to 40% moisture content. After preparation, 200 g of the medium was filled into 500 ml Erlenmeyer conical flasks and autoclaved for two hours. One ml spore suspension of Forl (106 conidia/ml) was added to the sand maize medium and incubated at room temperature (25-27°C) for 14 days before use.

Production of T. harzianum inoculum. T. harzianum was grown in 250 ml

Erlenmeyer conical flasks containing Potato Dextrose Broth at 28 ± 1°C for 8 days. Liquid culture of T. harzianum was homogenized in a blender (5×106 conidia/ml) and used for root dipping of tomato seedlings.

Management of Fusarium crown and root rot.

Two weeks after soil infestation with pathogen inoculum (5%, w/w) prepared as mentioned above, the following treatments of tomato seedlings were carried out:

(a) Root dipping (RD) 1- T. harzianum (RD); 2- Chitosan

0.5 g/l (RD); 3- Chitosan 1.0 g/l (RD); 4- T. harzianum + chitosan at 0.5 g/l (RD); 5- T. harzianum + chitosan 1.0 g/l (RD).

(b) Root dipping (RD) + foliar application (FA)

1- T. harzianum (RD) + Chitosan 0.5g/l (FA); 2- Chitosan 0.5 g/l (RD) + Chitosan 0.5 g/l (FA); 3- Chitosan 1.0 g/l (RD) + Chitosan 0.5 g/l (FA); 4- T. harzianum + chitosan at 0.5 g/l (RD) + Chitosan 0.5 g/l (FA); 5- T. harzianum + chitosan 1.0 g/l (RD) + Chitosan 0.5 g/l (FA).

(c) Control Control 1 (artificially infected and

untreated plants); Control 2 (Healthy plants uninfected untreated plants).

Tomato transplants cv. Kastle rock

grown in free soil treatments were dipped for 30 min in homogenized growth culture of T. harzianum (5×106 conidia/ ml) or water emulsion containing 0.5 or 1.0 g/l of chitosan. The treated seedlings were transplanted into plastic pots (20 cm diameter) filled with Forl infested soil. Ten pots with five seedlings each were used as replicates for each treatment. Foliar application of chitosan was


applied, after transplanting; three times (100 ml/pot) at 10 days interval.

Disease assessment.

FCRR incidence and severity were evaluated 45 days post-planting (DPP). Disease severity was scored by using a modified scale of Rowe (46) where 0 = no internal or external browning, 1 = no internal browning, discrete superficial lesions on tap root or stem base and root lesions at the points of emergence of lateral roots, 2 = brown tap root with slight internal browning at the tip of the tap root, 3 = moderate internal browning of the entire tap root, 4 = severe internal browning extending from tap root into lower stem above soil surface, abundant lesions on distal roots, 5 = dead plants (46).

Study of the effect of T. harzianum and chitosan on physiological activities of tomato plants.

Determination of total phenolic content. Total phenol content of leaves of treated and untreated as well as healthy tomato plants was determined using the Folin-Ciocalteau reagent (49). Freshly collected leaves (2 g) were homogenized in 80% aqueous ethanol with a pinch of neutral sand to facilitate crushing and the mixture was passed through a clean cloth to filter the debris. The filtered extract was centrifuged at 10,000 g for 15 min and the supernatant was saved. The residue was re-extracted twice with 80% ethanol and supernatants were collected, put into evaporating dishes and evaporated to dryness at room temperature. Following evaporation, the residue was dissolved in 5 ml of distilled water. Extract was diluted to 3 ml with water and 0.5 ml of Folin-Ciocalteau reagent was added. After 3 min, 2 ml of 20% of sodium carbonate were added and the contents were mixed thoroughly. The

color was developed and absorbance was measured at 650 ηm in a spectrophotometer (Systronics, uv-vis, 117) after 60 min. Catechol was used as a standard. The phenolic content was expressed as mg catechol/100 g of fresh weight of tomato leaves.

Determination of ββββ-1,3glucanase

and chitinase activities. Extraction of enzymes. One gram

of leaf tissue was homogenized with 0.2 M tris HCl buffer (pH 7.2) containing 14 mM of b-mercaptoethanol at the rate of 1/3 (w/v). The homogenate was centrifuged at 300 rpm for 15 min, the supernatant was used to determine the enzyme activity (51)

β-1,3glucanase activity. The method of Abeles and Forrence (9) was used to determine the β-1,3glucanase activity. Laminarin was used as substrate and dinitrosalicylic acid as reagent to measure reducing sugar. The optical density was determined at 500 ηm using a spectrophotometer (TM, Spectronic Educatar). The β-1,3glucanase activity was expressed as mM glucose equivalent released in gram fresh weight tissue/60 min.

Chitinase activity. Colloidal chitin was used as substrate and dinitrosalicylic acid as reagent to measure reducing sugar (41). The optical density was determined at 540 ηm. Chitinase activity was expressed as mM N-acetylglucose amine equivalent released in gram fresh weight tissues/60 min.

Statistical analyses.

All experiments were set up according to a completely randomized design. One-way ANOVA was used to analyze differences between treatments. A general linear model option of the analysis system SAS (48) was used to perform the ANOVA. Duncan’s multiple


range test at P < 0.05 was used for mean separation (57).

RESULTS Effect of T. harzianum on the mycelial growth of Forl. Antagonistic ability of T. harzianum against Forl was carried out on PDA medium using the dual culture technique. Results presented in Table 1 indicate that T. harzianum significantly reduced the mycelial growth of all the Forl tested isolates. T. harzianum reduced the in vitro growth of Forl by more than 77.7%. The highest antagonistic potential of T. harzianum was recorded against Forl-2, Forl-3 and Forl-5 isolates where

the mycelial growth was reduced by 93.3, 90.0 and 87.7%, respectively.

Effect of chitosan on the radial growth of Forl.

The inhibitory effect of five chitosan concentrations (0.5, 1.0, 2.0, 3.0, and 4.0 g/l) against Forl was tested in vitro. Results presented in Table 2 indicate that all tested concentrations have significantly reduced the mycelial growth of Forl. Complete inhibition of the pathogen was obtained with chitosan applied at 4 g/l. The highest reduction was obtained with chitosan used at 3 g/l where the mycelial growth was reduced by up to 88.8% for all the Forl tested isolates. Used at 1.0 and 2.0 g/l, chitosan induced moderate antifungal activity.

Table 1. Effect of Trichoderma harzianum on the mycelial growth of Fusarium oxysporum f. sp. radicis-lycopersici isolates on PDA medium noted after 8 days of incubation at 25°C

Forl isolate Colony diameter (mm) Inhibition rate* (%) Forl-1 16.0 bc 82.2 Forl-2 6.0 d 93.3 Forl-3 9.0 d 90.0 Forl-4 20.0 b 77.7 Forl-5 11.0 cd 87.7 Control 90.0 a -

* Growth reduction as compared to the untreated control. Means followed by the same letters are not significantly different according to Duncan’s multiple range test (P ≤ 0.05).

Table 2. Effect of chitosan concentrations on the mycelial growth (mm) of Fusarium oxysporum f. sp. radicis-lycopersici isolates on PDA medium noted after 8 days of incubation at 25°C

D= Colony diameter (mm); I = Inhibition percentage (%) as compared to the untreated control. Means followed by the same letters are not significantly different according to Duncan’s multiple range test (P ≤ 0.05).

Forl isolate

Chitosan concentration (g/l) 0.5 1.0 2.0 3.0 4.0

D I D I D I D I D I Forl-1 81 c 10.0 68 c 24.4 55 b 38.8 25 b 72.2 0 b 100 Forl-2 84 b 6.6 74 b 17.7 52 bc 42.2 30 b 66.3 0 b 100 Forl-3 80 cd 11.1 66 c 26.6 47 cd 47.7 27 b 70.0 0 b 100 Forl-4 76 de 15.5 65 cd 27.7 45d 50.0 10 c 88.8 0 b 100 Forl-5 70 e 22.2 60 d 33.3 33 e 63.3 8 c 91.1 0 b 100 Control 90 a - 90 a - 90 a - 90 a - 90 a -


Management of tomato Fusarium crown and root rot disease.

T. harzianum and chitosan (concentrations 0.5 and 1.0 g/l) applied individually or as combined treatments were tested against Forl on tomato plants. Results shown in Table 3 indicate that all the tested treatments had significantly reduced FCRR incidence, noted 45 DPP, as compared to the untreated tomato plants (Control 1). The most effective treatments were T. harzianum + chitosan (0.5 and/or 1.0 g/l) applied as root dipping combined with chitosan (0.5 g/l) as foliar spray application where the FCRR incidence was reduced by 60.0 and

66.6%, and disease severity decreased by 47.8 and 47.6%, respectively. Root dipping treatment based on T. harzianum + chitosan (0.5 or 1.0 g/l) led to reduced FCRR incidence (50.0%) and severity (28.5 and 47.8%, respectively for both tested concentrations). T. harzianum and chitosan applied as individual treatments (without chitosan foliar sprays) caused lesser reduction in disease incidence as compared to combined treatments together with chitosan foliar sprays. Chitosan treatments were the least effective in reducing disease incidence and severity (Table 3).

Table 3. Fusarium crown and root rot of tomato in response to Trichoderma harzianum and chitosan based-treatments applied as root dipping and/or foliar sprays with chitosan under greenhouse conditions noted 45 days post-planting

Treatment Foliar spray

Disease incidence and severity

Incidence (%)

Incidence reduction (%)

Severity Severity reduction (%)

T. harzianum

Chitosan

0 g/l

45.0 d 40 3.2c 23.8

Chitosan 0.5 g/l 62.5 b 16.6 3.8 b 9.5

Chitosan 1.0 g/l 45.0 cd 40 3.5 b 16.6

T. harzianum + Chitosan 0.5 g/l 37.5 d 50 3.0 c 28.5

T. harzianum + Chitosan 1.0 g/l 37.5 d 50 2.4 d 42.8

T. harzianum

Chitosan 0.5 g/l

37.5 d 50 2.7 c 35.7

Chitosan 0.5 g/l 55.0 c 26.6 3.5 b 16.6

Chitosan 1.0 g/l 42.5 d 43.3 3.0 c 28.5

T. harzianum + Chitosan 0.5 g/l 30.0 eg 60 2.4 d 42.8

T. harzianum + Chitosan 1.0 g/l 25.0 g 66.6 2.2 d 47.6

Control 1 (infested soil and untreated plants) 75.0 a - 4.2 a -

Control 2 (non infected and healthy plants) 0 - 0 -

In each column, means followed by the same letter are not significantly different according to Duncan’s multiple range test (at P ≤ 0.05).

Effects of T. harzianum and chitosan on physiological activities of tomato plants.

Effects of T. harzianum and chitosan on phenol content. T.

harzianum and chitosan were applied individually or as combined treatments onto tomato plants inoculated with Forl. The effects of these treatments on total phenol contents in both treated and


untreated plants were presented in Table 4. Obtained results showed that all T. harzianum and chitosan based-treatments had increased phenol production in tomato plants infected with Forl and treated with T. harzianum + chitosan as root dipping followed with foliar sprays with chitosan (0.5 g/l). Meanwhile, untreated plants showed no evident changes in their phenol content level. Dipping root system of tomato seedlings in chitosan (at 0.5 or 1.0 g/l) then their transplanting in Forl infested soil led to an increase of the total phenols in leaves of treated seedlings. This content increase reached 114.8 and 124.0 mg/100 g of fresh weight at 30 DPP as compared to 85.6 and 74.2 mg/100 g of fresh weight noted on infected and untreated plants (Table 4). The highest levels in phenol content were recorded in tomato seedlings treated with T. harzianum + chitosan (at 0.5 or 1.0 g/l) combined with foliar spray with chitosan (at 0.5 g/l) with 134.2 and 148.2 mg/100 g of fresh weight noted 30 DPP, respectively. The total phenol content was significantly higher in Forl infected seedlings following the application of T. harzianum + chitosan (at 0.5 or 1.0 g/l) combined with foliar application with chitosan (at 0.5 g/l). In our investigation, the highest reduction in disease incidence and severity was

accompanied by the accumulation of maximum amounts of phenolic compounds in infected tomato leaves treated with T. harzianum and chitosan as root dipping in combination with chitosan as foliar spray.

Effects of T. harzianum and

chitosan on β-1,3glucanase and chitinase activities. Results shown in Table 5 indicate that all treatments tested enhanced enzyme activity in leaves of Forl-infected tomato seedlings as compared to the control plants. T. harzianum + chitosan (0.5 and 1.0 g/l) applied as root dipping individually or in combination induced significant increase in chitinase and β-1,3 glucanase activities in treated plants as compared with the untreated control ones. The most effective treatments is the combination between T. harzianum + chitosan (0.5 or 1.0 g/l) as foliar spray where the chitinase activity increase was of about 111.1 and 122.2%, respectively. The same treatments have also increased the β-1,3glucanase activity by 146.2 and 146.2%, respectively. These combined treatments applied without additional chitosan foliar spray had also enhanced chitinase and β-1,3glucanase activities by 88.9 and 122.2% and by 76.9 and 107.7%, respectively.

Table 4. Total phenolic compounds in leaves of tomato plants inoculated with Fusarium oxysporum f. sp. radicis-lycopersici and treated with Trichoderma harzianum and chitosan as root dipping and/or foliar application of chitosan.


Total phenol [mg/100 g fresh weight of tomato leaves

0 DPP 10 DPP 20 DPP 30 DPP T. harzianum

Chitosan 0 g/l

72.8 a 88.1 d 92.2 e 96.1 e T. harzianum + Chitosan 0.5 g/l 69.4 a 94.8 c 107. 2 d 114. 8 d T. harzianum + Chitosan 1.0 g/l 71.4 a 102.4 b 116.2 c 124.4 c

T. harzianum Chitosan 0.5 g/l

72.2 a 92.4 c 95.6 e 102.0 e T. harzianum + Chitosan 0.5 g/l 71.0 a 118.2 a 128.2 b 134.2 b T. harzianum + Chitosan 1.0 g/l 72.8 a 123.6 a 140.0 a 148.2 a Control (untreated plant + infested soil) 71.2 a 74.8 e 77.2 f 85.6 f Control (healthy plant) 68.0 a 72.2 e 73.0 f 74.2 g For each column, means followed by the same letter are not significantly different according to Duncan’s multiple range test (P ≤ 0.05). DPP: Days post-planting.


Table 5. Chitinase and β-1,3glucanase activities in tomato leaves inoculated with Fusarium oxysporum f. sp. radicis-lycopersici affected by Trichoderma harzianum and chitosan based-treatments applied alone or in combination


Chitinase β-1,3 glucanase

Activity 20 DPP

Increase * (%)

Activity 20 DPP

Increase* (%)

T. harzianum Chitosan 0.0 g/l

1.6 b 77.8 2.1 d 61.5

T. harzianum + Chitosan 0.5 g/l 1.7 b 88.9 2.3 d 76.9

T. harzianum + Chitosan 1.0 g/l 2.0 a 122.2 2.7 bc 107.7 T. harzianum

Chitosan 0.5 g/l

1.7 b 88.9 2.2 d 69.2

T. harzianum + Chitosan 0.5 g/l 1.9 a 111.1 3.2 a 146.2

T. harzianum + Chitosan 1.0 g/l 2.0 a 122.2 3.2 a 146.2

Control (infested soil) 0.9 c - 1.3 e -

* Increase as compared to the control. For each column, means followed by the same letter are not significantly different according to Duncan’s multiple range test (P ≤ 0.05). DPP: Days post-planting.

DISCUSSION

Tomato is an economically important crop and may be infected by different pathogens. FCRR is a serious infecting tomato and is becoming more common in commercial productions (22,

29, 34). Farmers tend to use huge amounts of chemicals leading to human, animal and environmental problems. Therefore, effective and environmentally safe management approaches are increasingly needed.

In this study, the biocontrol agent T. harzianum and chitosan were used individually or as combined treatments for controlling FCRR on tomato as well as to assess their potential to induce phe-nolic compounds and defense enzymes production in Forl-infected tomato plants. The results clearly demonstrated that T. harzianum has significantly reduced the in vitro growth of all Forl isolates. This inhibition is attributed to the multiple mechanisms of action of Trichoderma that acts as mycoparasite. In fact, this biocontrol agent defects its host by sugar-lectin linkage and excrete extracellular lytic enzymes such as β-1,3glucanases, chitinase, protease and/or lipase (55, 56). Trichoderma has a substantial ability to suppress a wide range of pathogenic fungi by various mechanisms including the production of cell wall degrading enzymes (27, 33). On the other hand, complete inhibition of the pathogen was also obtained with chitosan applied at 4 g/l. The inhibitory effect of chitosan

against root rot and wilt pathogens was reported by many authors (1, 3, 15, 16, 25, 21, 42, 47). Furthermore, chitosan was reported to induce resistance in tomato plants against root rot diseases when applied as seed treatment, root dipping, foliar application or soil amendment (6, 16, 20, 42). In this respect, two models have been proposed to explain the antifungal activity of chitosan; firstly, the activity of chitosan is related to its stability while interfering with the plasma membrane function (39) and secondly, the interaction of chitosan with fungal DNA and RNA (31).

In the greenhouse trial, more promising results were obtained by using T. harzianum and chitosan (0.5 and 1.0 g/l) as combined treatments against Forl. The results indicated that treating inoculated tomato plants with chitosan and T. harzianum had significantly reduced FCRR incidence as compared to the control. The most effective treatments in reducing both disease incidence and


severity were T. harzianum + chitosan (0.5 and/or 1.0 g/l) applied as root dipping combined with foliar spray with chitosan (0.5 g/l). These results are in agreement with other findings (7, 11, 20, 42, 23). It is interesting to note that present findings clearly demonstrated that using chitosan in combination with T. harzianum to control FCRR enhanced more the ability of T. harzianum to inhibit Forl and consequently disease especially when chitosan was applied as foliar additional spray. The efficiency of T. harzianum and/or chitosan in controlling the disease was also reported in other references (28, 29, 30, 42). T. harzianum and chitosan activated host defense genes leading to physical and biochemical changes in plant cells involved directly or indirectly in disease suppression. These changes included accumulation of phenol compounds and increasing activity of host defense enzymes (13, 14, 15, 16, 31, 50, 52). The present results also indicated that all T. harzianum and chitosan based-treatments increased phenol production in tomato plants infected with Forl and treated with T. harzianum + chitosan applied as root dipping combined with foliar sprays with chitosan (at 0.5 g/l). Meanwhile, untreated plants showed no noticeable changes in their phenol content. In this respect, Benhamou et al. (15) reported that chitosan induced systemic resistance to Forl in tomato seedlings by triggering a hypersensitive-like response at sites of fungal entrance and stimulating rapid accumulation of newly formed macromolecules such as 1,3glucans, phenols, and lignin like compounds. Ojha and Chatterjee (45) reported that application of T. harzianum and salicylic acid stimulated the formation of total soluble phenols in host tissues. It was concluded that the increase

in phenolic content was positively proportional to the degree of plant resistance against pathogens (10). Phenolics seem to inhibit disease development through different mechanisms involving accumulation of phenolics at the infection site to isolate the pathogen, inhibition of extracellular fungal enzymes, inhibition of fungal oxidative phosphorylation, nutrient deprivation (metal complexation, protein insolubilisation), and antioxidant activity in plant tissues (18). Beckman (13) concluded that the efficiency of phenolic compounds in reducing diseases may be attributed to their effect on host defense pathways and in signaling for host defenses more than the direct toxic effect on the pathogen. In addition, T. harzianum + chitosan (at 0.5 and 1.0 g/l) applied as root dipping individually or in combination are found to increase chitinase and β-1,3glucanase activities in treated plants as compared to the untreated control ones. These results are in accordance with many other findings highlighting that T. harzianum and chitosan based-treatments enhance these enzymatic activities in many plants since chitinase and β-1,3glucanase are involved in the hydrolysis of the fungal cell walls (27, 37).

The current study revealed that inducing plant defense mechanisms by applying T. harzianum and chitosan particularly in combination could provide protection of tomato plants against FCRR disease.

ACKOWLEDGEMENTS This work was carried out during a Collaborative Project Tunisia-Egypt Funded jointly by the Ministry of Scientific Research in Egypt (grand no.4/10/4) and the Ministry of Higher Education and Scientific Research of Tunisia.


__________________________________________________________________________ RESUME El-Mohamedy R.S.R., Abdel-Kareem F., Jabnoun-Khiareddine H. et Daami-Remadi, M. 2014. Le chitosane et Trichoderma harzianum comme alternatives aux fongicides pour lutter contre la fusariose des racines et du collet de la tomate. Tunisian Journal of Plant Protection 9: 31-43.

La tomate est l’une des plus importantes cultures légumières en Egypte et en Tunisie. La fusariose des racines et du collet de la tomate, causée par Fusarium oxysporum f. sp. radicis-lycopersici (Forl) est l’une des maladies telluriques les plus destructives de la tomate et elle est devenue très commune dans les cultures sous serre. Dans la présente étude, l’effet de Trichoderma harzianum appliqué d’une façon individuelle ou combiné au chitosane contre Forl a été évalué in vitro et in vivo. T. harzianum a significativement réduit la croissance mycélienne des cinq isolats de Forl testés. Le chitosane appliqué à différentes concentrations (de 0,5 à 4,0 g/l) a aussi significativement réduit la croissance mycélienne du pathogène et une inhibition totale a été obtenue à la concentration 4 g/l. Sous les conditions de serre, l’application de T. harzianum et du chitosane (1,0 g/l) par trempage racinaire combiné avec le chitosane (0,5 g/l) par pulvérisation foliaire a réduit l’incidence et la sévérité de FCRR par 66.6% et 47.6%, respectivement. Le traitement à base de T. harzianum appliqué seul ou en combinaison avec le chitosane a entraîné une augmentation des teneurs en phénols totaux et une stimulation des activités chitinase et β,1-3-glucanase dans les feuilles des plants de tomate traités comparés aux non traités. Les résultats de cette étude ont montré la possibilité d’utiliser des traitements combinés à base de T. harzianum et de chitosane à l’échelle commerciale comme approche pour lutter contre la fusariose des racines et du collet de la tomate. Mots clés: Chitosane, Fusarium oxysporum f. sp. radicis-lycopersici, tomate, Trichoderma harzianum, sévérité de la maladie. __________________________________________________________________________

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دة أو �T. harzianum .�0<9ة &?5 اN"#��ل �ا��,J� رة��ت ا�<?�� 4G�J�,ى ا��"(� �Y ا�<�"�زان �� ا��9دة �� ا�"�$�> � إ�از أ��9ت ا��0ع ا� !�#�� $�� @�-Oو chitinase و β,1-3-glucanase أوراق �!��ت ا� ��ط� �

�Y� �F ا�<�" T. harzianumا�, اN"#��ل ا�0را�N إ�5 إ�<�� د�[ھ-ه �\� �"�^[ . ��4\�ھ�F�� 0ر� ا��#��?� O زان�� ا�"�ج وا�.-ور ا�,��زاري &?5 ا� ��ط��04?� ��<�� ض ,#.

،�O، Fusarium oxysporum f. sp. radicis-lycopersici"�زان، ط��ط�، 0Tة ا��ض :O?��ت �,"��1

Trichoderma harzianum. __________________________________________________________________________


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Control of Root Rot Diseases of Tomato Plants Caused by Fusarium solani, Rhizoctonia solani and Sclerotium rolfsii Using Different Chemical Plant Resistance Inducers

Riad S.R. El-Mohamedy, Plant Pathology Department, National Research Center, Dokki, Giza, Egypt, Hayfa Jabnoun-Khiareddine, and Mejda Daami-Remadi, UR13AGR09, Centre Régional des Recherches en Horticulture et Agriculture Biologique de Chott-Meriem, Université de Sousse, 4042, Chott-Mariem, Tunisia. ________________________________________________________________________ ABSRACT El-Mohamedy, R.S.R., Jabnoun-Khiareddine, H., and Daami-Remadi, M. 2014. Control of root rot diseases of tomato plants caused by Fusarium solani, Rhizoctonia solani and Sclerotium rolfsii using different chemical plant resistance inducers. Tunisian Journal of Plant Protection 9: 45-55. Root rots of tomato plants caused by Rhizoctonia solani, Fusarium solani and Sclerotium rolfsii are serious diseases leading to delayed growth and subsequent death of severely infected plants. Effect of some chemical inducers such as potassium salts, salicylic acid and sorbic acid on control of root rot pathogens and their impact on growth, quantity and quality parameters of tomato cv. Super Strain B were investigated. All the tested chemical inducers significantly reduced severity of root rots under greenhouse and field conditions. Potassium salts based-treatments, followed by salicylic acid, were the most effective in decreasing incidence of root rots induced by all tested pathogens. However, sorbic acid was found to be the least effective treatment. In field trials, the highest reductions of root rot incidence and disease severity were recorded on tomato plants treated with potassium sorbate used at 7.5% and dipotassium hydrogen phosphate (K2HPO4) 400 mM followed by salicylic acid 100 mM treatment. Disease incidence and severity were reduced by 65.4 and 62.5% in 2012, and by 63.2 and 53.8% in 2013 cropping seasons, respectively. Application of potassium salts followed by salicylic acid was the most efficient for the increase of growth parameters, yield and quality of tomato fruits while compared to control. Therefore, it could be suggested that application of plant chemical resistance inducers could be commercially used for controlling tomato root rot diseases and increasing both quality and quantity of tomato since they are safe, less expensive and effective against these diseases even under field conditions. Keywords: Chemical resistance inducers, disease control, root rot, tomato, yield quality _________________________________________________________________________

Tomato is an important vegetable

crop not only for its economic importance but also for its nutritional value (19). It is essentially present in all countries either

Corresponding author: Riad S.R. El-Mohamedy Email: [email protected]


as open field or protected crops. As other countries, it is one of the most important vegetable crops in Egypt and used for food and industrial purpose (16).

Tomato plants are infected by several soilborne fungal pathogens such as Fusarium spp., Rhizoctonia solani, and Sclerotium rolfsii which cause serious diseases as root rots and wilt and finally reduced crop yield and quality (6, 28).


Control of such diseases mainly depends on fungicide treatments and grafting (17). However, intensive application of fungicides causes hazards to human health and environmental degradation and is not always satisfactory. Therefore, alternative approaches for the control of plant diseases should be emphasized (24).

Induction of resistance in plants to overcome pathogen infection is a promising approach for controlling plant diseases. Exogenous or endogenous factors could substantially affect host physiology, lead to rapid and coordinated activation of defense-gene in plants normally expressing susceptibility to pathogen infection (24, 25). This induced resistance to pathogens can be achieved by the application of various abiotic agents (chemical inducers) such as salicylic acid, potassium salts and sorbic acid (6, 8, 13, 15). Conversely, application of these chemical inducers under field conditions have increased growth parameters, yield components and quality of fruits in many vegetable plants (16, 17, 22, 31).

The present study was conducted to investigate the effects of some chemicals as resistance inducers in tomato plants against root rot pathogens under greenhouse and field conditions and to more elucidate their impacts on growth parameters, yield and fruit quality.

MATERIALS AND METHODS Root rot pathogens.

Rhizoctonia solani RsG1, Fusarium solani FsG1, and Sclerotium rolfsii Sr Mn2 isolates obtained from Plant Pathology Department, National Research Center, Egypt, were used in this study. These isolates were proved to be aggressive for inducing root rot of tomato plants in previous studies (15, 16).

Preparation of fungal inocula. Inoculum of F. solani was

prepared by culturing pathogen on Potato Dextrose Broth (PDB) medium in 250 ml Erlenmeyer flasks for 10 days at 25 ± 2°C. The culture was filtered to remove mycelia and the resulting conidial suspension was adjusted to 106 conidia/ml by using a haemocytometer. Soil infestation was carried out by adding 50 ml of the conidial suspension (106 conidia/ml) to 1 kg of soil (12).

Inoculum of R. solani and S. rolfsii was the upper solid mycelium layers grown on Potato Dextrose Agar (PDA) medium which was washed and air-dried with sterilized filter paper layers. The air-dried mycelium was blended in distilled water to obtain inoculum pieces of 1-2 mm. Soil inoculation was carried out at the rate of 2.0 g of dry mycelium/kg of soil (9).

Greenhouse experiment.

The efficacy of four chemical resistance inducers i.e. salicylic acid, dipotassium hydrogen phosphate (K2HPO4), potassium sorbate and sorbic acid, used at different concentrations, was tested against tomato root rot pathogens. These chemicals were applied as seedling root dipping for 2 h before transplanting, followed by foliar spray application after transplanting, applied as follows:

(a) Seedlings root dipping 1 - Salicylic acid 25, 50 and 100

mM, 2 - Dipotassium hydrogen

phosphate (K2HPO4) 100, 200 and 400 mM,

3 - Potassium sorbate 2.5, 5.0 and 7.5%,

4 - Sorbic acid 2.5, 5.0 and 7.5%, 5 - Control untreated seedlings. (b) Foliar spray 1 - Salicylic acid 25 mM,


2 - Dipotassium hydrogen phosphate (K2HPO4) 100 mM,

3 - Potassium sorbate 2.5%, 4 - Sorbic acid 2.5%, 5 - Control untreated plants. Plastic pots (25 cm diameter, 5.0

kg of soil) were filled with soil artificially inoculated with each of the tested pathogens. Healthy tomato seedlings (40 day-old, cv. Super Strain B) were sown in plastic pots at the rate of 4 seedlings/pot, following five replicates for each treatment along with check treatment (non-inoculated soil).

Root rot disease incidence and severity were evaluated 45 days post-planting. Disease incidence was recorded as the number of root rot diseased plants relative to the number of planted seedlings in each treatment.

Disease severity was scored based on a modified Rowe (27) scale where: 0 = no internal or external browning, 1 = no internal browning, discrete superficial lesions on tap root or stem base and root lesions at the points of emergence of lateral roots, 2 = brown tap root with slight internal browning at the tip of the tap root, 3 = moderate internal browning of the entire tap root, 4 = severe internal browning extending from tap root into lower stem above soil surface, abundant lesions on distal roots and 5 = dead plants.

The percentages of reduction of disease incidence and severity were also calculated.

Open field experiment.

The most promising treatments found to be effective against tomato root rot diseases based on pot experiments were applied under field conditions. Four different chemical resistance inducers i.e., salicylic acid 100 mM, dipotassium hydrogen phosphate (K2HPO4) 400 mM,

potassium sorbate 7.5%, and sorbic acid 7.5% were applied as seedling root dipping for 2 h + foliar application treatments (as mentioned above for the greenhouse experiment). This experiment was conducted in two successive growing seasons 2012 and 2013 in a field naturally infected with the causal organisms of root rot disease of tomato located at the private farm of El-Minia Governorate, Egypt.

Tomato seedlings (cv. Super Strain B, 40 days-old) were soaked for 2 h at the rate of 100 seedlings per 250 ml of the tested chemical resistance inducers. Two seedlings/hill were sown with 50 cm apart between hills. Untreated seedlings were used as control. Disease incidence and severity were recorded at 25, 50 and 75 days post-planting (DPP) and the percentages of their reductions as compared to the control were also calculated. Plant height, number of branches, number of fruits per plant, fruit weight per plant (kg), mean fruit weight (g) and fruit yield (T/ha) were noted at the end of the growing season. Total soluble solids of tomato fruits from each treatment were measured using a refractometer.


All experiments were set up according to a complete randomized block design. One-way ANOVA was used to analyze differences between treatments. A general linear model option of the analysis system SAS (SAS Institute Inc.) (29) was used to perform the ANOVA. Duncan’s multiple range test at P < 0.05 was used for mean separation (30).

RESULTS Management of the disease under greenhouse.

Data shown in Tables 1 and 2 clearly indicate that all chemical inducers


tested have significantly (P ≤ 0.05) reduced tomato root rot incidence (Table 1) and severity (Table 2) caused by F. solani, R. solani and S. rolfsii compared with the untreated control plants. Root rot incidence and severity on tomato plants were decreased by all tested concentrations and reached their minimum records at the highest concentration of potassium sorbate 7.5%, dipotassium hydrogen phosphate (K2HPO4) 400 mM, salicylic acid 100 mM and sorbic acid 7.5%. The most effective treatments were potassium sorbate 7.5% followed by dipotassium hydrogen phosphate (K2HPO4) 400 mM. In fact, they decreased F. solani and R. solani root rot incidence by 80.0 and 76.3% and by 74.1 and 63.1%; while S. rolfsii incidence was reduced by 71.0 and

61.7%, respectively (Table 1). For root rot severity, these treatments decreased F. solani by 67.6 and 61.7%, R. solani by 61.9 and 52.3% and S. rolfsii by 58.3 and 50.0%, respectively (Table 2). Meanwhile, plants treated with salicylic acid and sorbic acid showed reduced root rot incidence (72 and 60.2%, respectively) and severity (55.8 and 50.0%, respectively).

Tomato plants treated with salicylic acid 25 mM, dipotassium hydrogen phosphate (K2HPO4) 100 mM, potassium sorbate 2.5%, sorbic acid 2.5% combined with foliar spray with the same concentrations of the same chemicals gave rise to the lowest protection against all tested pathogens as compared with the other tested concentrations (Table 1).

Table 1. Effect of chemical resistance inducers tested at different concentrations on the incidence of tomato root rots caused by Fusarium solani, Rhizoctonia solani and Sclerotium rolfsii (greenhouse conditions)

Chemical resistance inducer

Treatment / Concentration

Incidence of root rot pathogens

F. solani R. solani S. rolfsii

SRD FA Incidence (%)

Reduction (%)

Incidence (%)

Reduction (%)

Incidence (%)

Reduction (%)

K 2HPO4 100 mM

100 mM

26.1 b 55.0 21.9 b 48.0 23.6 b 42.2 200 mM 17.3 c 70.1 18.9 c 55.2 19.0 c 53.4 400 mM 13.7 d 76.3 15.5 c 63.1 15.6 c 61.7

SA 25 mM

25 mM

27.8 b 52.1 23.5 b 44.1 24.4 b 40.1 50 mM 19.8 c 65.8 17.7 c 58.0 18.0 c 55.8 100 mM 16.2 cd 72.0 15.5 c 63.1 16.2 c 60.2

PSO 2.5%

2.5% 22.5 c 61.1 17.6 c 58.3 18.0 c 55.8

5.0% 15.1 cd 73.9 13.5 d 67.9 14.4 c 64.7 7.5% 11.6 d 80.0 10.9 d 74.1 11.8 d 71.0

SOA 2.5%

2.5% 29.0 b 50.0 24.8 b 41.2 26.2 b 35.7

5.0% 23.0 c 60.2 21.1 b 50.0 21.2 bc 48.0 7.5% 25.0 b 56.8 18.2 c 56.8 8.8 d 54.0

Control 58.0 a - 42.2 a - 40.8 a - For each column, means followed by the same letter are not significantly different according to Duncan’s multiple range test (P ≤ 0.05). SA: Salicylic acid, PSO: Potassium sorbate, SOA: Sorbic acid, SRD: Seedling root dipping, FA: foliar application.


Table 2. Effect of chemical resistance inducers tested at different concentrations on root rot severity on tomato plants inoculated with Fusarium solani, Rhizoctonia solani and Sclerotium rolfsii (greenhouse conditions)



Root rot severity

F. solani Rhizoctonia R. solani S. rolfsii

SRD FA Severity Reduction

(%) Severity Reduction


(%)

K 2HPO4 100 mM

100 mM

1.9 b 44.1 1.3 b 38.0 1.6 b 33.3 200 mM 1.5 c 55.8 1.2 b 42.8 1.3 c 45.8 400 mM 1.3 c 61.7 1.0 c 52.3 1.2 c 50.0

SA 25 mM

25 mM

2.0 b 41.1 1.4 b 33.3 1.7 b 29.1 50 mM 1.9 b 50.0 1.2 b 42.8 1.5 bc 37.5 100 mM 1.5 c 55.8 1.1 cd 47.6 1.3 c 45.8

PSO 2.5%

2.5% 1.6 c 52.9 1.1 cd 47.6 1.4 c 41.6

5.0% 1.4 cd 58.8 0.9 de 59.1 1.1 cd 54.2 7.5% 1.1 d 67.6 0.8 de 61.9 1.0 d 58.3

SOA 2.5%

2.5% 2.1 b 38.2 1.5 b 28.0 1.7 b 29.1

5.0% 1.9 b 44.1 1.3 b 38.0 1.6 b 33.3 7.5% 1.7 b 50.0 1.2 b 42.8 1.4 c 41.6

Control 3.4 a - 2.1 a - 2.4 a - For each column, means followed by the same letter are not significantly different according to Duncan’s multiple range test (P ≤ 0.05). SA: Salicylic acid, PSO: Potassium sorbate, SOA: Sorbic acid, SRD: Seedling root dipping, FA: foliar application.

Management of the disease in open field.

Effect of chemical resistance inducers on root rot incidence and severity. Data presented in Tables 3 and 4 indicate that all chemical inducers had significantly protected tomato plants against root rot pathogens as compared to the untreated control in both growing seasons (2012 and 2013). In fact, all tested chemicals had significantly reduced the percentages of root rot incidence on tomato plants noted 25, 50 and 75 DPP. The highest reductions of root rot incidence (Table 3) and severity (Table 4) were obtained with potassium sorbate 7.5% and dipotassium hydrogen phosphate (K2HPO4) 400 mM based-treatments followed by salicylic acid 100 mM. Indeed, these both parameters were decreased by 65.4 and 62.5% in 2012 cropping season, as compared to 63.2 and 53.8% recorded in 2013. Treatment of tomato seedlings by root dipping with potassium sorbate 7.5% and potassium hydrogen phosphate (K2HPO4) 400 mM combined with foliar spray with 2.5% and

100 mM of the same inducers led to high decrease in tomato root rot disease incidence and severity after 25, 50 and 75 DPP in the two cropping seasons. At 75 DPP, the same treatments reduced, respectively, disease incidence records by 65.4 and 60.0% in 2012 and by 63.2% and 57.4% in 2013, respectively (Table 3). Disease severity, noted 75 DPP, was also decreased by 62.5 and 54.1% in 2012 and by 53.8 and 50.0% in 2013 cropping season (Table 4). Salicylic acid 100 mM based-treatment caused considerable reduction in tomato root rot during the two seasons where incidence and severity decreased up to 51.7 and 50.0%, respectively. Meanwhile, sorbic acid 7.5% treatment exhibited the least inhibitory effect with 50.0% reduction recorded at 75 DPP during both cropping seasons. The most effective chemical inducers in decreasing root rot incidence and severity at 25 and 50 DPP were potassium sorbate 7.5% and dipotassium hydrogen phosphate (K2HPO4) 400 mM, whereas salicylic acid 100 mM and sorbic acid 7.5% based-treatments showed the lowest efficacy.


Table 3. Effect of chemical resistance inducers tested at different concentrations on incidence of root rot diseases on tomato plants grown in a naturally infested soil (open field conditions)



Incidence

25 DPP 50 DPP 75 DPP

SRD FA Incidence

(%) Reduction

(%) Incidence

(%) Reduction

(%) Incidence

(%) Reduction

(%) Season 2012

K 2HPO4 400 mM 100 mM 17.8 cd 52.2 19.2 c 58.6 16.4 d 60.0

SA 100 mM 25 mM 20.0 b 46.2 22.0 b 52.6 19.8 c 51.7

PSO 7.5% 2.5% 15.6 d 58.1 16.6 c 64.2 14.2 d 65.4

SOA 7.5% 2.5% 21.4 b 42.4 23.8 b 48.7 22.2 b 45.8

Control 37.2 a - 46.4 a - 41.0 a -

Season 2013

K 2HPO4 400 mM 100 mM 19.6 c 51.0 21.1 c 53.1 18.8 de 57.4

SA 100 mM 25 mM 22.0 b 46.0 22.4 c 50.2 21.4 c 51.6

PSO 7.5% 2.5% 18.0 c 55.0 17.4 d 61.3 16.2 e 63.2

SOA 7.5% 2.5% 24.0 b 40.0 25.2 b 44.0 25.2 b 43.0

Control 40.0 a - 45.0 a - 44.2 a -

For each column, means followed by the same letter are not significantly different according to Duncan’s multiple range test (P ≤ 0.05). SA: Salicylic acid, PSO: Potassium sorbate, SOA: Sorbic acid, SRD: Seedling root dipping, FA: foliar application, DPP: days post-planting.

Table 4. Effect of chemical resistance inducers tested at different concentrations on root rot severity on tomato plants grown in a naturally infested soil (open field conditions)



Severity

25 DPP 50 DPP 75 DPP

SRD FA Severity Reduction



(%) Season 2012

K 2HPO4 400 mM 100 mM 0.8 c 50.0 1.0 c 54.5 1.1 c 54.1 SA 100 mM 25 mM 1.0 b 44.4 1.2 b 43.4 1.2 bc 50.0

PSO 7.5% 2.5% 0.8 c 55.5 0.9 c 59.1 0.9 c 62.5 SOA 7.5% 2.5% 1.1 b 38.8 1.3 b 40.4 1.4 b 41.6

Control 1.8 a - 2.2 a - 2.4 a - Season 2013

K 2HPO4 400 mM 100 mM 1.1 c 45.0 1.2 c 50.0 1.3 c 50.0 SA 100 mM 25 mM 1.2 bc 40.0 1.4 b 41.6 1.4 b 46.2

PSO 7.5% 2.5% 1.0 c 50.0 1.1 c 54.2 1.2 c 53.8 SOA 7.5% 2.5% 1.4 b 30.0 1.4 b 41.6 1.5 b 42.2

Control 2.0 a - 2.4 a - 2.6 a - For each column and each cropping season, means followed by the same letter are not significantly different according to Duncan’s multiple range test (P ≤ 0.05). SA: Salicylic acid, PSO: Potassium sorbate, SOA: Sorbic acid SRD: Seedling root dipping, FA: foliar application, DPP: days post-planting.

Effect of chemical resistance inducers on tomato growth parameters. All of the tested chemical inducers significantly increased growth parameters in treated tomato plants i.e. plant height and number of branches per plant compared with the untreated control in

both growing seasons (Table 5). The most effective chemical inducers in enhancing plant height (68.4 and 60.4 cm ), was potassium sorbate 7.5% followed by dipotassium hydrogen phosphate (K2HPO4) 400 mM (66.4 and 65.2 cm) in


2012 and 2013 growing seasons, respectively.

The same trend was also observed in the case of the number of branches per plant. In fact, tomato seedlings treated with potassium sorbate 7.5% exhibited the highest branch number per plant (6.2 and 5.8 during 2012 and 2013 cropping seasons, respectively) followed by dipotassium hydrogen phosphate

(K2HPO4) 400 mM (5.8 and 5.4, respectively). Salicylic acid 100 mM based-treatment led to significant increase in plant height during the two seasons. Meanwhile, sorbic acid 7.5% treatment showed the least effect, as there are no significant difference in plant height between treated and untreated (control) plants during the 2013 season.

Table 5. Effect of different chemical resistance inducers on growth parameters of tomato plants grown in naturally infested soil (open field conditions)


Treatment /Concentration Plant height (cm) No. of branches/plant

SRD FA 2012 2013 2012 2013 K 2HPO4 400 mM 100 mM 66.4 a 65.2 a 5.8 b 5.4 b

SA 100 mM 25 mM 58.0 b 55.8 b 5.0 c 4.8 c PSO 7.5% 2.5% 68.4 a 60.4 b 6.2 a 5.8 a SOA 7.5% 2.5% 50.4 c 52.0 c 4.6 d 4.4 d

Control 42.8 d 48.2 c 3.8 e 4.0 c For each column, means followed by the same letter are not significantly different according to Duncan’s multiple range test (P ≤ 0.05). SA: Salicylic acid, PSO: Potassium sorbate, SOA: Sorbic acid, SRD: Seedling root dipping, FA: foliar application.

Effect of chemical inducers on tomato yield and fruit quality. Reduction in disease incidence during two seasons, 2012 and 2013, means increasing in plant stand and growth parameters, which reflect on the obtained tomato fruit yield. There was a significant effect of the chemical inducers on the tested quantitative parameters i. e., number of fruits/plant, fruit weight per plant, mean fruit weight, total soluble solids and total fruit yield. The obtained data in Table 6 show that all tested chemicals significantly improve the quality parameters of tomato compared with the untreated control. The most efficient inducers were potassium sorbate (PSO) 7.5% and potassium hydrogen phosphate (K2HPO4) 400 mM where the highest numbers of fruits per plant recorded were

75 and 68, and 71 and 65 during 2012 and 2013 cropping seasons, respectively. Fruit weight per plant noted on tomato plants treated with potassium hydrogen phosphate (K2HPO4) 400 mM and potassium sorbate (PSO) 7.5% was 4.62 and 5.28 kg, and 4.35 and 5.12 kg for each treatment in 2012 and 2013, respectively. Mean fruit weight (70 and 62, 6.2 and 5.5 g) and total yield increase, compared to the untreated control (63.2 and 56.4%, 53.6 and 50.3%), were higher in tomato plants treated with potassium sorbate (PSO) 7.5% and potassium hydrogen phosphate (K2HPO4) 400 mM in 2012 and 2013 cropping seasons, respectively. Salicylic acid 100 mM and sorbic acid 7.5% based-treatments also increased all yield parameters as compared to the control.


Table 6. Effect of different chemical resistance inducers on yield and fruit quality of tomato plant grown in a naturally infested soil (open field conditions)


Treatment /Concentration Quality parameters Total yield SRD FA NF FWP

(kg) FW (g)

TSS Yield (T/ha)

Increase (%)

Season 2012 K 2HPO4 400 mM 100 mM 68 a 5.28 a 62 b 5.2 b 62.38 b 56.4 SA 100 mM 25 mM 55 b 4.4 a 57 a 4.8 b 49.52 c 45.1 PSO 7.5% 2.5% 75 a 4.62 a 70 a 6.2 a 73.80 a 63.2 SOA 7.5% 2.5% 46 b 3.15 b 60 b 4.2 b 44.76 c 39.3

Control 32 c 1.52 c 58 a 4.0 b 27.14 d 0.0 Season 2013

K 2HPO4 400 mM 100 mM 65 a 5.12 a 61 b 5.5 a 67.14 a 50.3 SA 100 mM 25 mM 52 b 4.25 a 50 c 5.0 ab 55.71 b 40.1 PSO 7.5% 2.5% 71 a 4.25 a 72 a 6.2 a 71.90 a 53.6 SOA 7.5% 2.5% 42 b 2.88 b 44 d 4.4 ab 48.09 c 30.6

Control 30 c 1.25 c 50 c 4.1 b 33.33 d 0.0 For each column and each cropping season, means followed by the same letter are not significantly different according to Duncan’s multiple range test (P ≤ 0.05). SA: Salicylic acid, PSO: Potassium sorbate, SOA: Sorbic acid, SRD: Seedling root dipping, FA: foliar application, NF: Number of fruits per plant, FWP: fruit weight per plant, FW: fruit weight, TSS: Total soluble solids.

DISCUSSION

Soilborne diseases including root rots are involved in considerable losses of the most important vegetable crops including tomato. Chemical resistance inducers are largely used as bioactive substances in controlling soilborne as well as foliar plant pathogens (2, 3, 4, 6, 7, 11, 14, 15, 17, 26). In the present study, different concentrations of salicylic acid, dipotassium hydrogen phosphate (K2HPO4), potassium sorbate, sorbic acid were used as seedlings root dipping combined with foliar application of the same chemical inducers in order to evaluate their efficacy in controlling tomato root rot caused by F. solani, R. solani and S. rolfsii in artificially infested soil under greenhouse conditions as well as in naturally infested soil under open field conditions.

Our data in pot experiment clearly show that root rot incidence and severity on tomato plants were reduced at all tested chemicals concentrations. The lowest records of these two parameters were noted at the highest concentration of potassium sorbate 7.5%, dipotassium hydrogen phosphate (K2HPO4) 400 mM,

salicylic acid 100 mM and sorbic acid 7.5%. The most effective treatments were potassium sorbate 7.5% followed by dipotassium hydrogen phosphate (K2HPO4) 400 mM. They reduced both incidence and severity of root rot. These results are in agreement with previous findings (1, 2, 14, 17, 26) where many chemical resistance inducers were successfully used against root rot pathogens infecting many crops. Chemically induced resistance in plants against pathogens is a widespread phenomenon that has been investigated with respect to the underling signaling pathways as well as to its potential use in plant protection. Elicited by a local infection, plants respond with a salicylic acid dependent signaling cascade that leads to the systemic expression of a broad spectrum and long-lasting disease resistance that is efficient against fungi, bacteria and viruses (21). The tested chemical inducers might stimulate some defense mechanisms such as phenolic compounds, oxidative enzymes and other metabolites (7, 10, 13).

Under open field conditions, our results indicate that the highest reduction


of root rot incidence and severity was obtained with potassium sorbate 7.5% and dipotassium hydrogen phosphate (K2HPO4) 400 mM treatments followed by salicylic acid 100 mM combined with foliar application with 2.50%, 100 mM, 25 mM of the same chemical inducers, respectively. Many research studies have been conducted on chemical resistance inducers used for controlling root rot and wilt diseases under greenhouse and field conditions (2, 5, 10). It should be mentioned that some chemical inducers may also have a direct antimicrobial effect and are, thus, involved in cross-linking in cell walls, induction of gene expression, phytoalexin production and induction of systemic resistance (6).

On the other hand, an important finding from this study revealed that all tested chemical inducers had positive effects on plant growth, yield and fruit quality of tomato plants grown under field conditions during two cropping seasons. These increases in growth, yield quantity

and quality may be attributed to elicitors effect on physiological processes in plant such as ion uptake, cell elongation, cell division, enzymatic activation and protein synthesis (11, 18). Gunes et al. (20) also reported that salicylic acid acts as endogenous signal molecule involved in induction of tolerance to abiotic stresses in plants. They emphasized that exogenous application of salicylic acid increased plant growth significantly both under saline and non saline conditions. Some chemical inducers are also endogenous growth regulators of phenolic nature, which influence a range of diverse processes in plants, including seed germination (6, 18), ion uptake and transport, membrane permeability, photosynthetic and growth rate (23).

ACKNOWLEDGEMENT This work was carried out during a Collaborative Project Tunisia-Egypt Funded by the Ministry of Scientific Research, in Egypt (Grand no.4/10/4) and the Ministry of Higher Education and Scientific Research of Tunisia.

______________________________________________________________________ RESUME El-Mohamedy R.S.R., Jabnoun-Khiareddine H. et Daami-Remadi, M. 2014. Contrôle des pourritures racinaires des plants de tomate causées par Fusarium solani, Rhizoctonia solani et Sclerotium rolfsii en utilisant différents inducteurs chimiques de résistance. Tunisian Journal of Plant Protection 9: 45-55.

Les pourritures racinaires des plants de tomate causées par Rhizoctonia solani, Fusarium solani et Sclerotium rolfsii sont des maladies graves conduisant à un retard de croissance et à la mort des plants sévèrement infectés. L’effet de certains inducteurs chimiques, à savoir, les sels de potassium, l'acide salicylique et l'acide sorbique, dans la lutte contre les pathogènes causant les pourritures racinaires et leur impact sur les paramètres de croissance, de la quantité et de la qualité de tomate cv. Super Strain B a été étudié. Tous les inducteurs chimiques testés ont réduit significativement la sévérité des pourritures racinaires sous serre et en plein champ. Les traitements à base de sels de potassium, suivi par l'acide salicylique, ont été les plus efficaces dans la réduction de l'incidence des pourritures des racines induites par tous les agents pathogènes testés. Cependant, le traitement par l'acide sorbique s'est montré le moins efficace. Pour les essais au champ, les réductions les plus importantes de l’incidence et de la sévérité des pourritures racinaires ont été enregistrées sur les plants de tomate traités avec du sorbate de potassium utilisé à 7,5% et le phosphate d'hydrogène dipotassique (K2HPO4) 400 mM, suivi par le traitement par l'acide salicylique 100 mM. L'incidence et la sévérité des maladies ont été réduites de 65,4 et 62,5% en 2012, et de 63,2 et 53,8% en 2013, respectivement. L’application des sels de potassium, suivie par de l'acide salicylique a été la plus efficace pour l'augmentation des paramètres de croissance, de rendement et de la qualité de la tomate par rapport au témoin. Par conséquent, il pourrait être suggéré que l'application des inducteurs chimiques de résistance à la plante peut être utilisée dans


le commerce pour le contrôle des pourritures des racines de la tomate et pour l’augmentation de la qualité et de la quantité de tomate à la fois, car ils sont sains, moins coûteux et efficaces contre ces maladies, même dans les conditions du champ. Mots clés: Contrôle de la maladie, inducteurs chimiques de résistance, pourriture racinaire, tomate, qualité, rendement. _______________________________________________________________________

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��ت�B !�9��: �� دة، ا��ور، ت�V ض ، ��دود�� ، ط��ط#�"��8ت ا��6و�! ا��7! ،��8! ا�________________________________________________________________________ LITERATURE CITED 1. Abdel-Kader, M.M., El-Mougy, N.S., El-

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Chemical Composition and Fumigant Toxicity of Artemisia absinthium Essential Oil Against Rhyzopertha dominica and

Spodoptera littoralis Najla Dhen, Ons Majdoub, Slaheddine Souguir, UR13AGR09, Centre Régional des Recherches en Horticulture et Agriculture Biologique de Chott-Mariem (CRRHAB Ch-M), Université de Sousse, 4042 Chott-Mariem, Tunisia, Wafa Tayeb, Laboratoire de Biochimie, Faculté de Medicine de Monastir, Université de Monastir, 5000 Monastir, Tunisia, Asma Laarif and Ikbal Chaieb, UR13AGR09, CRRHAB Ch-M, Université de Sousse, 4042 Chott-Mariem, Tunisia __________________________________________________________________________ ABSTRACT Dhen, N., Majdoub, O., Souguir S., Tayeb, W., Laarif A., and Chaieb I. 2014. Chemical composition and fumigant toxicity of Artemisia absinthium essential oil against Rhyzopertha dominica and Spodoptera littoralis. Tunisian Journal of Plant Protection 9: 57-65. Natural products are excellent alternative to synthetic pesticides due to their reduced harmful impacts on human health and environment. Pesticides based on plant essential oils or their constituents have demonstrated efficacy against a range of pests and pre- and postharvest diseases. In this study, the pesticide potentiality of the essential oils from the absinthe wormwood Artemisia absinthium (Asteraceae) was investigated against two insect pests i.e. Rhyzopertha dominica and Spodoptera littoralis. Essential oil of the aerial parts was obtained by hydrodistillation and was analyzed by GC–MS in order to determine its chemical composition. The major components identified were: camphor (24.81%), camazulene (13.17%), bronylacetate (5.89%), and myrcene (5.83%). The essential oil of A. absinthium exhibited strong fumigant toxicity against R. dominica adults, a stored product pest, with a LC50 value of 18.23 µl/l air and LC90 value of 41.74 µl/l air. The wormwood essential oil showed high fumigant activity against S. littoralis, one of the most dangerous pests of protected crops, with a LC50 value of 10.59 µl/l air and a LC90 value of 17.12 µl/l air. Keywords: Artemisia absinthium, chemical composition, essential oil, fumigant toxicity, Rhyzopertha dominica, Spodoptera littoralis __________________________________________________________________________

The widespread use of synthetic insecticides has led to severe damage on human health and on environmental equilibrium. There is an urgent need to develop safer, more eco-friendly and efficient alternatives (18). The

Corresponding author: Najla Dhen Email: [email protected]


valorization of natural resources such as plant extracts and secondary metabolites, particularly those used by the popular tradition and readily available, acquire a great interest due to their biologically active compounds. Among plant secondary metabolites, essential oils extracted from aromatic plants have been widely investigated and were found to have a broad spectrum of activity against insect and mite pests, plant pathogens and


nematodes (5). They have considerable potential as crop protectants against pests of stored products and protected crops.

Plant essential oils are produced from different plant organs (leaves, flowers, buds, wood…). The richest plants belong mainly to Apiaceae, Lamiaceae, Lauraceae, and Myrtaceae families. Essential oils are complex mixtures of monoterpenes, sesquiterpenes, and aromatic compounds (4). Due to these compounds, essential oils possess fumigant, antifeedant and repellent properties against various insect species (1). These plant-originated insecticides may have the advantage over synthetic ones in terms of low mammalian toxicity, rapid degradation and local availability (5, 6). The lack of persistence of essential oils under field conditions could provide some measure of temporal selectivity favoring non-target species, especially natural enemies.

Essential oils have been largely employed for their properties already observed in nature. Thus, it was shown that essential oils might constitute new alternatives to currently used insecticides not only against stored product pests but also against protected crops pests (19, 20) such as aphids, moth or others. Essential oils of cumin, anise, oregano and eucalyptus were effective as fumigants against the cotton aphid (Aphis gossypii) (19). Pavela (14) reported on fumigant toxicity of essential oils from Mentha citrata, Nepeta cataria, Salvia sclarea, Origanum vulgare, Origanum compactum, Melissa officinalis, Thymus mastichina, and Lavandula angustifolia against S. littoralis.

Insecticidal activities of essential oils against stored product pests were cited in several studies. In fact, Yoon et al. (21) reported a repellent activity of six

essential oils from Carum carvi, Salvia sclarea, Fragaria vesca, Thymus satureoïdes, and Cananga odorata against Sitophilus oryzae. Contact and fumigant activities of essential oils of Pinus sylvestris, Eucalyptus globulus, Coriandrum sativum against the rice weevil S. oryzae and adzuki bean weevil (Callosobruchus chinensis) and the rice moth (Corcyra cephalonica) were demonstrated by Pathipati (13). Also, the essential oils of the aerial parts of three Artemisia species (A. absinthium, A. santonicum and A. spicigera) were found to be toxic to adults of Sitophilus granarius (7).

The Artemisia genus, small herbs and shrubs, is one of the largest and most widely distributed aromatic plants. Members of this genus are found growing naturally in large areas or can be cultivated, thus, readily available. Among them, A. absinthium, is a yellow-flowering perennial plant which has been used as an herbal medicine throughout Europe, the Middle East, North Africa, and Asia (18). This plant is found to be of botanical and pharmaceutical interest (7, 8, 15). Medicinal value of plants is related to their phytochemical components and their secondary metabolites such as essential oils. The aim of this study was to assess the chemical composition and the insecticidal potential of the essential oils isolated from the aerial parts of A. absinthium against a stored product pest (R. dominica) and a greenhouse pest (S. littoralis). MATERIALS AND METHODS Plant material and essential oil extraction.

This study was performed using the aerial part of the wormwood A. absinthium. This plant was cultivated in


an organic parcel belonging to the Technical Center of Organic Agriculture of Chott-Mariem (CTAB). The samples were used immediately after their harvest (March, 2013) for the extraction of essential oils.

Essential oils were obtained by hydrodistillation method using a Clevenger-type apparatus. About 200 g of the aerial parts of the plant were mixed in a flask with 400 ml distilled water. The mixture was boiled for 4 h at 350°C. The extract was condensed in cooling vapor to collect the essential oil. The volatile distillate was collected until no oil drop out. The obtained oil was weighed in order to calculate its extraction yield and refrigerated prior to analysis.

GC-MS analysis of the essential oil.

The essential oils were analyzed by gas chromatography (GC) (model: HP 6890N) coupled with mass spectrometry (MS) (HP model 5975B). They are equipped with a flame ionization detector and capillary column with HP-5MS 5% phenylmethyl siloxane (30 m _ 0.25 mm _ 0.25 µm). The GC settings were as follows: the initial oven temperature was held at 50°C for 2 min and ramped at 7°C/min to 250°C for 2 min. The injector temperature was maintained at 240°C. The samples (1 µl) were injected neat, with a split ratio of 1: 50. Helium gas (100% pure) was used as carrier gas at a constant flow rate of 1.2 ml/min. Mass transfer line and ion source temperatures were set at 150 and 230°C, respectively. Most constituents were identified by comparison of their retention indices and their mass spectra, in the stationary phase, with those of the bank Willey data 175.L.

Insect rearing. Third instar larvae of S. littoralis

were obtained from a laboratory colony maintained on artificial diet (150 g chickpea powder, 20 g agar, 750 ml water, 5 g ascorbic acid, 1 g benzoic acid, 1 g nipagin, 30 g yeast). The colony came from laboratory rearing at 28 ± 2°C and 60 ± 5% of relative humidity (RH), in the Regional Center of Research on Horticulture and Organic Agriculture (Chott-Mariem, Sousse-Tunisia).

R. dominica adults were provided with maize grains as food in a plastic container and were maintained in the dark in growth chamber set at 28 ± 2°C and 50-60% humidity. All experiments were carried out under the same environmental conditions. Fumigant toxicity .

In order to assess the A. absinthium essential oil fumigant toxicity, different concentrations 25, 50, 100 and 200 µl/l air and 5, 12.5, 25 and 50 µl/l air were tested respectively against the third instar larvae of S. littoralis and newly hatched unsexed adults of R. dominica. Mortalities in both cases were compared to untreated control individuals maintained under the same conditions.

Ten pests were put in a 40 cm3 container. A Whatman filter paper (3 cm in diameter), impregnated with essential oil, was placed on the underside of the screw cap. Control insects were kept under the same conditions without any essential oil. Each concentration and control was replicated five times. Mortality was assessed after 24 h of exposure to the essential oil.

These bioassays were designed to determine the median effective dose causing 50% of mortality (LC50) and the


effective dose causing 90% of mortality (LC90) using Probit analyses. Statistical analyses.

Statistical analyses were performed using SPSS program (version 20) (17). Mean’s differentiations were carried out using Duncan’s multirange test at P < 0.05. RESULTS Extraction yield and chemical composition of A. absinthium essential oil. The hydrodistillation of A. absinthium aerial parts yielded dark blue colored essential oil with a strong odor. In general, essential oils are very complex natural mixtures which can contain about 20-60 components at quite different

concentrations (5). They are characterized by two or three major components at fairly high concentrations (20-70%) compared to the other components present in trace amounts (2). Table 1 lists the composition of wormwood essential oils. According to the GC-MS results, 47 compounds have been identified, in which camphor (24.81%), camazulene (13.17%), bornyl acetate (5.89%), myrcene (5.83%), trimethylnaphthalene (5.09%), 1,4-terpeniol (4.97%), camphene (3.74%), γ-terpinene (3.59%) and linalool (3.53%) were major components. Qualitative similarities were found in chemical composition of Tunisian wormwood investigated by Riahi et al. (15) but there were some notable quantitative differences.

Table 1. Volatile constituents identified in the essential oils of Artemisia absinthium aerial parts.

Compound Peak N°

TR (min)*

Percentage (%)

Tricyclene 1 6.11 0.12

α-thujene 2 6.21 0.43

α-pinene 3 6.37 3.02

Camphene C10 4 6.9 3.74

Sabinene 5 7.24 0.27

β-pinene 6 7.32 0.19

6-methyl-5-hepten-2-one 7 7.55 0.23

Myrcene C10 8 7.63 5.83

α-phellandrene 9 7.93 0.45

α-trpinene 10 8.2 2.09

Ortho-cymene 11 8.39 0.57

Limonene C10 12 8.48 1.16 1,8-cineole 13 8.55 0.31

γ-Terpinene 14 9.15 3.59

Trans-sabinene hydrate C10 15 9.37 1.52

α-terpinolene 16 9.81 0.67

2-nonanone 17 9.87 0.53


Linalool 18 10.07 3.53 butanoic Acid 19 10.12 0.56 P-menth-2-en-1-ol 20 10.58 0.27

δ-terpinene 21 10.98 0.18

Camphor C10 22 11.13 24.81 Borneol 23 11.55 0.26 1,4-Terpeniol 24 11.78 4.97 Myrcenol 25 11.93 0.53 α-terpineol 26 12.06 0.33 2-methylheptyl acetate 27 12.9 0.8 Perilla aldehyde 28 13.77 0.17

Bornyl-acetate C12 29 13.97 5.89

2-undecanone C11 30 14.06 1.27

Methyl eugenol 31 16.21 0.16

β-caryophyllene 32 16.58 0.71

α-dodecylene 33 16.65 0.2

Germacene 34 17.69 1.53

Trimethyl-dihydronaphtalene 35 18.25 1.03

Trimethyl-dihydronaphtalene 36 18.33 0.10 Caryophyllene oxide 37 19.49 0.33 Geranyl isovalerate 38 19.73 0.23 Diethyl-dimethyl-tricyclo-hexane 39 20.19 0.27 Trimethylnaphtalene 40 20.45 5.09 (2S,5E)-caryophyll-5-en-12-al 41 20.45 0.6

1,2-dimethyl-4-methylene-3-phenyl-cyclopentene

42 20.58 0.25

Trimethyl-dihydrocyclo-propainden-6(6a)-one

43 20.64 3.12

Camazulene 44 21.47 13.71 (8) paracyclophane-2.4-diene 45 21.18 0.51 2,4-dimethylfuran 46 25.48 0.22 Bromoacetonitrile 47 25.79 0.99 Total - - 97.34

*RT: retention times

A. absinthium is characterized by a

wide range of phytochemical variability (12) associated with different factors (geographical origins of the samples, extraction method used, seasonal fluctuations or plant parts used for extraction). Fumigant toxicity .

Experiments were conducted to assess the fumigant activity of A.

absinthium essential oil against R. dominica adults and S. littoralis third instar larvae. In all cases, considerable differences in insect’s mortality exposed to essential oil vapor were observed with the different concentrations.

Results of the bioassay (Table 2) were recorded after 24 h of exposure. According to this study, significant differences among all treated concentrations of the essential oils were


obtained (P < 0.01). The mortality increased with the increase in the concentrations of the essential oils. In fact, high fumigant toxicity against the third instar larvae of S. littoralis was noted with a LC50 value of 10.59 µl/l air and a LC90 value of 17.12 µl/l air.

As shown in Table 2, essential oil of A. absinthium exhibited fumigant toxicity against R. dominica adults with a LC50 value of 18.23 µl/l air and LC90 value of 41.74 µl/l air. The essential oil causes quick knock down followed by mortality. DISCUSSION

The main aim of this research was to investigate A. absinthium essential oils composition and insecticide potentialities. Essential oil of the aerial plant parts was obtained by hydrodistillation and analyzed by gas chromatography-mass spectrometry (GC-MS). A total of 47 components of the essential oil were identified. Several studies have been reported on the chemical composition of the essential oils from A. absinthium belonging to different regions in the

world. Analysis of the chemical composition of A. absinthium oils extracted from plants grown in USA showed beta-thujone (17.5-42.3%) and cis-sabinyl acetate (15.1-53.4%) as the main components (9). Other investigation from Turkey (7) indicated chamazulene (17.8%), nuciferol butanoate (8.2%), nuciferol propionate (5.1%), and caryophyllene oxide (4.3%) as the main components in A. absinthium oil. The Canadian A. absinthium oil was characterized by high amounts of myrcene (10.8%), trans-thujone (10.1%) and trans-sabinyl acetate (26.4%) (10). Those from Tajikistan were markedly different from those from European, Middle Eastern, or other Asian locations and likely represent new chemotypes (18). This phytochemical variability in A. absinthium is normally associated with different geographical origins of the samples (12). Jointly or independently, these identified compounds are involved in the bioefficacy of the essential oils used with a range of effects i.e. insecticidal, repellent, antifeeding, and ovicidal activities.

Table 2. LC50 and LC90 values of Artemisia absinthium essential oil against adults of Rhyzopertha dominica and larvae of Spodoptera littoralis.

Insect Concentration (µl/l air)

Mortality (%) after 24 h

LC 50 (µl/l air) LC 90 (µl/l air)

R. dominica

0 0 a

18.23 41.74 5 35 ± 12.91 b 12.5 45 ± 12.91 b 25 67.5 ± 17.08 c 50 92 ± 9.57 d

S. littoralis

0 0a

10.593 17.122 2.5 2 ± 4.47 a 5 18 ± 19.24 a 10 52 ± 32.71 b 20 94 ± 8.94 c

* For each insect, mortality values followed by the same letter are not significantly different based on Duncan's multirange test at P < 0.05.


The insecticidal propriety of many essential oils are mainly attributed to monoterpenoids (4) which are typically volatile and rather lipophilic compounds that can penetrate into insects rapidly and interfere with their physiological functions (2, 4, 5). Due to their high volatility, they have fumigant and gaseous action which are very important in controlling the stored-product insects.

In vitro experiments undertaken to assess the fumigant activity of A. absinthium essential oil that they possessed high fumigant toxicity against a stored product insect, as well as a greenhouse pest and its activity varied with insect species and concentrations of the oil used.

The toxic effects of A. absinthium could be attributed to its major components. However, the insecticidal effects of the essential oils cannot be explained by the action of their major components only, suggesting that these actions are the result of a synergistic interaction between all components (4).

Previous studies demonstrated that Artemisia species essential oils have very important insecticidal activities (7). A recent investigation by Martin et al. (11)

highlighted the insecticidal effect of Artemisia essential oils against three greenhouse pests, S. littoralis, Myzus persicae and Rhopalosiphum padi via an antifeedant action. Concerning stored products pests, the essential oil of A. vestita showed strong fumigant toxicity against Sitophilus zeamais adults with a LC50 value of 13.42 mg/l air. Further, it possessed also contact toxicity against this pest with a LD50 value of 50.62 mg/adult (21).

Pavela (14) reported the high fumigant toxicity of Mentha citrata, Nepeta cataria, Salvia sclarea, Origanum vulgare, O. compactum, Melilotus officinalis, Thymus mastichina, and Lavandula angustifolia essential oils against S. littoralis. Moreover, the essential oils of aerial parts of three Artemisia species (A. absinthium, A. santonicum and A. spicigera) were found to be toxic to adults of Sitophilus granarius (7).

The above findings suggest that the essential oil of A. absinthium can play an important role in pest control and reduce the need for synthetic insecticides and also the risks associated with their use.

__________________________________________________________________________ RESUME Dhen N., Majdoub O., Souguir S., Tayeb W., Laarif A. et Chaieb I. 2014. Composition chimique et toxicité par fumigation des huiles essentielles d’Artemisia absinthium contre Rhyzopertha dominica et Spodoptera littoralis. Tunisian Journal of Plant Protection 9: 57-65. Les produits naturels constituent une excellente alternative aux pesticides de synthèse à cause de leurs impacts réduits sur la santé humaine et l’environnement. Les pesticides à base d’huiles essentielles ont montré une efficacité contre une large gamme d’insectes et de maladies de pré- et de post-récolte. Dans ce travail, la potentialité insecticide de l’huile essentielle de l’armoise absinthe Artemisia absinthium a été investiguée contre deux insectes ravageurs à savoir Rhyzopertha dominica et Spodoptera littoralis. L’huile essentielle des parties aériennes a été extraite par hydrodistillation et analysée par GC-MS pour déterminer sa composition chimique. Les constituants majoritaires identifiés sont le camphre (24,81%), le camazulène (13,17%), le bornylacétate (5,89%), le myrcène (5,83%) et le trimèthylnaphtalène (5,09%). L’huile essentielle d’A. absinthium a montré une forte toxicité par fumigation contre les


adultes de R. dominica, un insecte des denrées stockées, avec des concentrations létales CL50 de 18,23 µl/l d’air et CL90 de 41,74 µl/l d’air. Cette huile a aussi montré une forte activité fumigène contre S. littoralis, un des insectes les plus dangereux des cultures protégées, avec des concentrations létales CL50 de 10,593 µl/l d’air et CL90 de 17,122 µl/l d’air. Mots clés: Activité fumigène, Artemisia absinthium, composition chimique, huile essentielle, Rhyzopertha dominica, Spodoptera littoralis __________________________________________________________________________

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�P6/90و ل CL 17.122 �P6/ل .

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__________________________________________________________________________ LITERATURE CITED 1. Ayvaz, A., Sagdic, O., Karaborklu, S., and

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Chemical Constituents and Toxicity of Essential Oils from Three Asteraceae Plants against Tribolium confusum

Dalila Haouas, UR06AGR05, Ecole Supérieure d’Agriculture du Kef, Université de Jendouba, 7119, Le Kef, Tunisia, Cioni Pier Luigi, Dipartimento di Farmacia, Università di Pisa, Via Bonanno 33, 56126 Pisa, Italy, Monia Ben Halima-Kamel, Institut Supérieur Agronomique de Chott Mariem, Université de Sousse, 4042 Sousse, Tunisia, Flamini Guido, Dipartimento di Farmacia, Università di Pisa, Via Bonanno 33, 56126 Pisa, Italy, and Ben Hamouda Mohamed Habib, Institut Supérieur Agronomique de Chott Mariem, Université de Sousse, 4042 Sousse, Tunisia __________________________________________________________________________ ABSTRACT Haouas, D., Cioni, P.L., Ben Halima-Kamel, M., Flamini, G., and Ben Hamouda, M.H. 2014. Chemical constituents and toxicity of essential oils from three Asteraceae plants against Tribolium confusum. Tunisian Journal of Plant Protection 9: 67-82. Plants produce a high diversity of secondary metabolites with a prominent function in the protection against pests and pathogens. In this work, we studied the chemical composition and the effect of six essential oils extracted from three Asteraceae species (Heteromera fuscata, Coleostephus myconis and Mauranthemum paludosum) on nutritional indexes, adult emergence and larva and adult toxicity of Tribolium confusum. Flower and leaf essential oils were obtained by hydrodistillation. The study of their chemical composition was carried out by GC-MS. The results showed that they are rich on mono- and sesquiterpens. The consumption of essential oils mixed with artificial diet of T. confusum larvae showed that H. fuscata leaf essential oil delayed the insect growth, reduced the food consumption and exhibited significant food deterrent index (0.02 ± 0.01 mg/mg/j, 0.05 ± 0.02 mg/mg/j, and 71.20 ± 19.22%, respectively) on treated larvae. Topical application of Asteraceae essential oils on pupae less than 24 h of age caused malformation on emerged adults. The highest level of malformation (18%) was induced by C. myconis leaf essential. Topical application of those essential oils on T. confusum adults (10 and 14 days-old) showed higher toxicity. The highest mortality of about 41% was recorded 7 days post-treatment on adults treated with essential oils from M. paludosum leaves. These preliminary results could represent the basis for further investigations on the susceptibility of the other developmental stages and elucidation of the mode of action of mono- and sesquiterpenoids against these insect pests. Keywords: Asteraceae, essential oil, ingestion, topical application, Tribolium confusum __________________________________________________________________________

Over 60 species insect feed on

stored grains. Care must be taken to protect the stored products against

Corresponding author: Dalila Haouas Email: [email protected] Accepted for publication 21 June 2014

deterioration, especially loss of quality and weight during storage. Control of stored product insects relies heavily on the use of synthetic insecticides, which has led to problems such as environmental disturbances, increasing costs of application, pest resistance to


pesticides and consequent resurgence and lethal effects on non-target organisms, in addition to direct toxicity to users (15, 40). In recent years, research has focused on the use of plant oils as possible alternatives to synthetic chemical insecticides. Plant essential oils and their components have been shown to possess potential for development as new insecticides and they may have advantages in terms of low mammalian toxicity, rapid degradation and local availability (16). Botanical pesticides have the advantage of providing novel modes of action against insects that can reduce the risk of cross-resistance as well as offering new leads for design of target-specific molecules (15, 16). In stored-product insect pest control, essential oils may have numerous types of effect: they may have a fumigant activity, penetrate inside the insect body as contact insecticides, act as repellents or as antifeedants or they may affect some biological parameters such as growth rate (5, 14, 18, 21, 24, 27, 36).

The Asteraceae family is very common in Mediterranean basin countries (19, 26, 29). Some species of these plants are characterized by their insecticidal propriety such as Glebionis coronaria, Glebionis segetum and C. cinerareafolium (6, 10, 37). Recent new

research on other Tunisian species highlights the insecticidal activity of Plagius grandiflorus, G. segetum, Heteromera fuscata, Chrysanthoglossum trifurcatum, and Coleostephus macrotus methanolic extracts against Spodoptera littoralis caterpillars (12, 13). H. fuscata, P. grandiflorus and G. coronaria essentials oils composition was also determined and studied for their insecticidal activity against Tribolium confusum (11). In this work, we investigate the chemical composition and the insecticidal activity of H. fuscata, Coleostephus myconis and Mauranthemum paludosum essential oils against T. confusum, a serious pest in stored grain and related products (3, 31) and resistant to several traditional insecticides (4). MATERIALS AND METHODS Plant material.

Chrysanthemum species were collected from three different regions of Tunisia characterized by different climates (Table 1). Plant identity was confirmed by experts of the Plant Biology Department of the University of Monastir. Voucher specimens were deposited in the National Gene Bank of Tunisia.

Table 1. Former and new classification, sampling dates and climate conditions of sites of Chrysanthemum species used in this study

Former classification (26) New classification (20) Sampling

date Site Site climate

Chrysanthemum fuscatum Heteromera fuscata April 2007

Gafsa (South)

Arid

Chrysanthemum myconis Coleostephus myconis March 2007

Beja (North)

Sub-humid

Chrysanthemum paludosum ssp. glabrum

Mauranthemum paludosum May 2007

Rades (North)

Semi-arid


Essential oil extraction. Essential oils were extracted from

fresh leaves and flowers by steam distillation using a Clevenger apparatus for 4 h. The essential oils were stored at 4°C until use. Chemical analysis.

The GC analyses were accomplished with a HP-5890 Series II instrument equipped with HP-WAX and HP-5 capillary columns (30 m × 0.25 mm, 0.25 mm film thickness), working with the following temperature program: 60°C for 10 min, ramp of 5°C/min up to 220°C; injector and detector temperatures 250°C; carrier gas nitrogen (2 ml/min); detector dual FID; split ratio 1:30; injection of 0.5 µl). The identification of the components was performed, for both columns, by comparison of their retention times with those of pure authentic samples and by mean of their linear retention indices (l.r.i.) relative to the series of n-hydrocarbons.

GC/EIMS (Electron Impact Ionization Mass Spectrometry) analyses were performed with a Varian CP-3800 gas chromatograph equipped with a HP-5 capillary column (30 m × 0.25 mm, 0.25 mm film thickness) and a Varian Saturn 2000 ion-trap mass detector. Analytical conditions: injector and transfer, line temperatures 220 and 240°C, respectively; oven temperature was programmed from 60 to 240°C at 3°C/min; carrier gas was helium at 1 ml/min; injection volume was 0.2 ml (10% hexane solution); split ratio was 1:30. Identification of the constituents was based on comparison of the retention times with those of authentic samples comparing their linear retention indices (l.r.i.) relative to the series of n-hydrocarbons, and on computer matching against commercial (2) and home-made

library mass spectra built up from pure substances and components of known oils and MS literature data (2, 7, 17, 23, 33, 34). Moreover, all the molecular masses of the identified substances were confirmed by GC/CIMS (Chemical Ionization Mass Spectrometry), using MeOH as ionizing gas. Insects.

Tribolium confusum larvae and adults were obtained from laboratory cultures maintained in the laboratory of Entomology of the Higher Agronomic Institute of Chott-Mariem in the dark in incubators at 30 ± 1°C and 70-80% RH. The two stages were reared on wheat flour mixed with yeast (10/1, w/w).

Study of the effect of essential oils on nutritional indexes and mortality.

Flour discs were prepared using a previously described method (38). The weights of the discs ranged from 35 to 39 mg. Each flour disc was treated with 5 µl of 1% acetone solution of each essential oil tested. Control discs were treated with 5 µl of acetone only. The discs were left at room temperature for 15 min to allow the solvent to evaporate. Pre-weighted discs were placed in glass vials (5 cm diameter). Each glass vial contained either two untreated discs (control), or one treated disc and one untreated disc (choice test), or two treated discs (no-choice test). Ten group-weighed, 14 days- old T. confusum larvae were added separately to each vial. The larvae were starved for 24 h before starting the experiment. Five replicates were used per elementary treatment. The weights of the flour discs and the number of live insects were determined after 7 days of exposure. Nutritional indices were calculated according to the formulae of Manuwoto and Scriber (22) and Farrar et al. (8) with


modifications as follows: relative growth rate (RGR) = (A - B)/B × day-1, where A = weight of live insects on the 7th day (mg)/number of live insects on the 7th day and B = initial weight of insects (mg)/initial number of insects; relative consumption rate (RCR) = D/B x day-1, where D = biomass ingested (mg)/number of live insects on the 7th day; efficiency of conversion of ingested food (ECI) (%) = (RGR)/(RCR) × 100. Using the means of the amount of flour in the control and treated discs consumed by the insects, the percentage feeding deterrence index (FDI) was calculated: FDI (%) = (C - T)/C × 100, where C = consumption of control discs and T = consumption of treated discs. The insect mortality (%) was recorded each seven days during three weeks. Contact toxicity by topical application on adults.

A 1% acetone solution of essential oils was prepared and 1 µl was topically applied to the ventral surface of the thoracic segments of the insects using a Hamilton microsyringe. Controls were treated with the solvent alone. After treatment, insects were placed in an incubator into plastic vials containing food. Five replicates of 10 adults (10-14 days-old) were prepared. The mortality (%) of insects was noted daily during seven days (28).

Contact toxicity by topical application on pupae.

20 pupae of T. confusum, less than 24 h of age, were selected from breeding. A volume of 1 µl of the same dilution of the essential oils was applied directly on each pupa. Pupae were placed in Petri dishes and glass uncovered. The mortality of pupae and the emergence of new adults were recorded daily for 7 days (total

emerged adults from all live nymphs) (25). The percentage of malformed new emerged adults was calculated.


In the whole experiment, the essays were repeated 5 times to ensure the reproducibility of the obtained results as well as to make a correct statistical analysis of each treatment in each bioassay. Antifeedant and nutritional indexes for the different treatments tested were compared using analysis of variance (ANOVA) followed by Duncan test for multiple-comparison when significant differences were observed at P < 0.05.

The recorded mortality data in ingestion and topical toxicity tests were adjusted for mortality in the control using Abbott’s (1) formula then analysed by one-way analysis of variance (ANOVA) and means were compared using Duncan multirange test at P < 0.05 using an SPSS v.16.0 software package in Microsoft Windows 7 operating system.

Insects were considered death when tactile stimuli elicited no visible normal reaction.

Mc = (Mo-Me)/(100-Me) × 100 with Mo: Mortality rate of treated adults, Me: Mortality rate of control, Mc: Adjusted mortality rate. RESULTS Essential oil composition.

The chemical composition of all used essential oils is listed in Table 2 and a total of 159 compounds were counted. The essential oil yield (v/w, volume/fresh weight) was of 0.07% and 0.25% for C. myconis leaves and flowers, respectively, of 0.06% and 0.25% for M. paludosum, respectively, and of 0.08% and 0.22% for H. fuscata organs, respectively.


Table 2. Chemical composition of the essential oils from leaves and flowers of Heteromera fuscata, Coleostephus myconis and Mauranthemum paludosum and percentage content of components

No Compounda RIb H. fuscata C. myconis M. paludosum

Leaves Flowers Leaves Flowers Leaves Flower

s 1 n-hexanol 871 tr - - - - 0.2 2 santolina triene 910 - - - - 0.2 - 3 α-thujene 933 0.1 0.3 - - - - 4 α-pinene 941 2.4 2.8 tr tr 0.5 0.1 5 camphene 955 0.5 0.1 tr 0.8 - - 6 benzaldehyde 963 tr 1.0 tr tr - - 7 sabinene 978 2.2 0.8 - - - - 8 β-pinene 981 4.4 - tr - 0.3 -

9 6-methyl-5-hepten-2-one

986 - - 0.9 tr 1.4 0.7

10 myrcene 992 7.1 2.2 0.2 - - - 11 yomogi alcohol 999 - 9.6 - tr - - 12 limonene 1032 20.6 12.9 0.3 - 0.6 - 13 β-phellandrene 1033 6.5 - - - - - 14 lavender lactone 1040 - 3.6 tr - - -

15 phenyl acetaldehydede

1049 - - tr tr 0.4 tr-

16 (E)-β-ocimene 1051 1.5 1.2 - - - - 17 γ-terpinene 1064 tr 0.1 - - - - 18 cis -linalool oxyde 1073 - - 0.2 - 0.7 -

19 trans -linalool oxyde

1087 - - tr - 0.5 -

20 terpinolene 1089 0.2 0.4 - - - - 21 linalool 1101 tr 0.8 tr tr 0.5 - 22 nonanal 1104 tr 0.4 - - - 0.1

23 1-octen-3-yl-acetate

1113 tr - 1.2 1.0 0.8 1.1

24 cis-p-menth-2-en-1-ol

1123 - 0.3 - - 0.3 tr

25 (E)-myroxide 1145 - tr 0.4 - - - 26 camphor 1145 - - - 0.2 - - 27 isopulegol 1150 - 0.3 - - - - 28 borneol 1167 - 0.3 - tr - - 29 pinocarvone 1168 0.2 tr - - - - 30 3-thujanol 1169 - 0.9 - - - -

31 p-mentha-1,5-dien-8-ol

1170 - - - - - 0.2

32 4-terpineol 1179 0.3 - - - - - 33 isopinocamphone 1181 0.5 - - - - - 34 cryptone 1186 1.1 0.6 - - - - 35 α-terpineol 1189 tr 0.2 - tr - - 36 n-decanal 1206 - tr tr - - - 37 trans-piperitol 1212 0.3 0.3 - - - 0.2 38 cumin aldehyde 1241 0.1 tr - - - - 39 neral 1243 - tr tr - - -

40 benzene acetic acid, ethyle ester

1247 - - - - - 0.2

41 geraniol 1256 - 0.7 - - 0.4 -

42 cis-chrysanthenyl acetate

1265 - 0.4 - - 0.4 0.3


43 geranial 1272 - - tr tr 0.3 - 44 p-menth-1-en-7-al 1276 0.9 - - - - - 45 isobornyl acetate 1286 1.3 0.9 tr - 0.2 0.4 46 γ-terpinen-7-al 1291 0.2 tr - - - - 47 cumin alcohol 1291 0.3 - - - - - 48 silphiperfol-5-ene 1329 0.3 tr tr - - -

49 7-epi-silphiperfol-5-ene

1348 - 0.2 0.2 - - -

50 α-terpinyl acetate 1349 0.5 - tr - - - 51 citronellyl acetate 1353 - 0.1 - - - - 52 neryl acetate 1362 1.6 - - tr - - 53 α-copaene 1377 1.9 0.8 tr 0.3 0.4 0.5 54 β-maaliene 1382 1.6 0.4 0.3 tr - - 55 β-cubebene 1388 1.7 0.7 tr - - - 56 β-caryophyllene 1420 4.2 3.5 1.2 tr - - 57 β-cedrene 1421 0.5 0.1 - - - - 58 β-gurjunene 1432 tr 0.1 - - - - 59 β-copaene 1432 - - 0.2 - - -

60 (2Z,6E)-dodeca-2,6-dien-1-al

1447 - - - - 0.3 -

61 α-humulene 1455 2.7 1.7 - - - - 62 (E)-β-farnesene 1460 - - 5.6 1.0 - -

63 allo-aromadendrene

1461 - - tr 0.3 - -

64 dehydro-aromadendrane

1463 - 0.5 - - - -

65 dehydro-sesquicineole

1472 - - - 0.2 - -

66 geranyl n-propanoate

1478 - 0.5 - - - -

67 γ-muurolene 1480 tr tr 0.2 tr tr 0.3 68 germacrene D 1481 10.4 2.9 0.3 - - - 69 a-himachalene 1483 - - - 0.3 - - 70 β-selinene 1487 - tr 1.4 0.3 - -

71 trans-muurola-4(14),5-diene

1493 0.1 tr - - - -

72 valencene 1493 - - 0.3 0.2 - - 73 bicyclogermacrene 1495 1.5 0.3 - - - - 74 α-muurolene 1499 0.5 - - tr - - 75 (E,E)-α-farnesene 1507 0.3 - 5.5 - 0.2 - 76 trans-γ-cadinene 1513 0.3 tr tr 0.6 - -

77 geranyl isobutyrate

1515 0.2 2.2 - - - -

78 cubenol 1515 0.4 - - - - - 79 δ-cadinene 1524 1.4 1.0 0.5 - - 0.3

80 (Z)-nerolidol (=cis nerolidol)

1533 - - 0.6 - 1.3 -

81 Cis-calamenene 1540 - - 0.2 - 82 elemol 1550 - - 1.8 5.1 - - 83 (E)-nerolidol 1563 0.2 0.2 0.7 - tr 0.4 84 geranyl n-butyrate 1564 - 0.4 - - - - 85 ledol 1567 - - - - - 1.2 86 trans-longipinanol 1569 - 0.5 - - - - 87 dendrolasin 1570 - - 0.7 0.3 - - 88 (Z)-3-hexenyl 1572 - - - - - 0.6


benzoate 89 α-cedrene epoxide 1575 - - - - - 1.1 90 spathulenol 1577 2.5 0.3 3.2 1.4 0.9 1.8

91 caryophyllene oxide

1582 0.9 3.6 1.6 4.3 1.6 7.6

92 globulol 1585 0.6 0.4 0.8 0.4 - 0.4 93 viridiflorol 1593 - - 0.3 0.4 - -

94 geranyl-2-methyl butanoate

1601 2.2 7.0 - - - -

95 geranyl-isovalerate 1607 - 9.3 - - - -

96 (Z)-sesquilavandulol

1607 - - 0.7 - 0.2 -

97 5-epi-7-epi-α-eudesmol

1608 - - - 0.1 - -

98 humulune epoxide II

1608 - 2.5 0.5 0.4 - 0.7

99 trans-arteannuic alcohol

1613 - 3.8 - - 1.3

100 1,10-di-epi-cubenol

1615 0.4 - - 1.3 - -

101 humulane-1,6-dien-3-ol

1620 - - - - - 0.4

102 10-epi- γ-eudesmol

1624 - - 0.3 - - -

103 1-epi-cubenol 1629 0.3 0.3 0.2 - - - 104 10-epi-g-eudesmol 1631 - - - 2.1 - - 105 γ-eudesmol 1632 - - 1.3 3.1 - - 106 α-acorenol 1633 - 0.3 - - - - 107 β-acorenol 1637 - 0.9 - - - -

108 6-methyl-6-(3-methylphenyl)-heptan-2-one

1641 1.6 - - - - -

109 T-cadinol 1642 0.7 - 0.5 - 0.2 0.8 110 T-muurolol 1643 0.7 - 0.6 18.4 0.3 0.6 111 cubenol 1647 0.9 - 0.5 1.7 - 1.0 112 α-muurolol 1647 - 5.5 - - - - 113 α- eudesmol 1647 - - 1.6 - - - 114 himachalol 1654 - 0.2 - - - - 115 α-cadinol 1655 1.4 1.6 - 9.4 1.0 2.6 116 3-thujopsanone 1655 - - - - 1.0 - 117 valerianol 1657 0.3 - - 3.8 - -

118 α-bisabolol oxide B

1658 - - 3.1 6.7 - -

119 valeranone 1675 - 0.7 - 1.1 - - 120 Heilifolenol B 1679 - - - 1.2 - - 121 epi-α-bisabolol 1685 - - 4.8 18.0 - -

122 α-bisabolone oxide A

1686 - - - 0.2 - -

123 Z-trans- α -bergamotol

1691 - - - 0.5 - -

124 2-pentadecanone 1695 - - - 0.2 - -

125 (Z,E)-farnesyl acetate

1701 - - 3.5 - - -

126 14-hydroxy- α-humulène

1714 - - 0.5 - - -

127 pentadecanal 1716 - - - - - 0.4


128 pentadecanal 1717 - 0.3 - - - 0.2

129 methyl tetradecanoate

1724 0.2 - 0.5 - 0.2 0.2

130 (E,E)-farnésol 1725 - 0.5 0.9 - - - 131 oplopanone 1740 tr - 0.4 - - - 132 (E,Z)-farnesol 1746 - 0.5 - - - -

133 α-bisabolol oxide A

1749 - - - 0.6 - -

134 benzyl benzoate 1760 - 0.1 - - - - 135 tetradecanoic acid 1764 - - - 0.8 - 1.1

136 14-oxy-α-muurolene

1769 - - 1.6 - 1.5 1.5

137 β-bisabolenal 1770 - - - 0.4 - -

138 α-bisabolyle acetate

1798 - - 1.1 - -

139 14-hydroxy-δ-cadinene

1804 0.2 - - - - -

140 2-ethylhexyl- salicylate

1807 - - - - 0.5 -

141 hexahydrofarnesylacetone

1843 0.2 - - 0.7 - 1.7

142 6,10,14-triméthyl pentadécanone

1843 0.5 0.7

143 Z-lanceol 1856 - - - 0.2 - - 144 α-chenopodiol 1857 - - - 1.4 - - 145 benzyl salicylate 1866 - - 0.4 - - -

146 oplopanonyl acetate

1888 - - - 0.5 - -

147 11-hydroxy-eudesm-4-en-3-one

1928 - - - 0.2 - -

148 n-hexadecanoic acid

1940 - - 0.7 1.0 - 19.1

149 11-acétoxyeudésman-4-α-ol

1940 - - - - 1.4 -

150 Phytol 1943 - - 0.3 - - -

151 hexadecanoate ethyle

1993 - - 0.2 - 0.3 -

152 methyl linoleate 2096 - tr - 0.2 - - 153 n-heineicosane 2100 tr tr - 0.1 0.6 1.7 154 linoleic acid 2141 - - - - 0.8 2.0 155 1-docosene 2190 - - - 0.2 - - 156 n-docosane 2200 - - - - 0.3 - 157 n-tricosane 2300 0.6 0.7 tr 3.4 0.8 5.2 158 n-tetracosane 2400 tr 0.3 tr 0.3 2.1 3.7 159 n-pentacosane 2500 0.8 0.7 - 1.4 2.5 26.9

- Mononterpene hydrocarbons

45.5 20.8 0.5 0.8 1.6 -

- Oxygenated mononterpenes

8.6 37.8 0.6 - 3.3 0.5

- Sesquiterpene hydrocarbons

27.4 12.2 15.9 1.9 1.9 0.9

- Oxygenated sesquiterpenes

9.7 21.8 32.3 68.4 7.4 16.5


- Non-terpene hydrocarbons

1.4 1.7 3.0 3.1 9.2 52.0

- Oxygenated non-terpenes

2.9 2.4 0.9 0.3 3.2 0.6 a Compounds listed in order of elution. b RI: retention index relative to n-alkane under conditions listed in the experimental section. tr = traces < 0.1.

Chemical analyses of the six samples shared a similar qualitative composition but with some quantitative differences. Substantially, all the essential oils were mainly constituted by mono- and sesquiterpenoids. Non-terpene derivatives varied from 0.3 to 52.0%. These compounds were almost equally distributed between flowers and leaves in H. fuscata (4.1% vs. 4.3%, respectively) and C. myconis (3.3% vs 3.9%, respectively), while they were consistently higher in flowers than leaves in M. paludosum samples (52.0% vs. 9.2%, respectively). Monoterpene hydrocarbons were not detected in M. paludosum flowers (0.0% vs. 1.6%) whereas they were detected in higher amounts in H. fuscata leaves (45.5% vs. 20.8%, respectively), and were almost equal in C. myconis ones (0.5% vs. 0.8%, respectively). Oxygenated monoterpenes were minor compounds in C. myconis (0.0% and 0.6% in flowers and leaves, respectively) and M. paludosum (0.5% and 3.3% in flowers and leaves, respectively). In contrast, they reached important percentages in H. fuscata, particularly in flowers (8.6% vs. 37.8%, respectively). Sesquiterpene hydro-carbons were minor in all essential oil where the highest amount was detected in H. fuscata leaves (27.4%). Oxygenated sesquiterpenes followed an opposite trend in C. myconis (68.4% and 32.3% in flowers and leaves, respectively), H. fuscata (9.7% and 21.8%, respectively) and M. paludosum (16.5% and 7.4%, respectively). The main volatiles were different, both for the three species and

the used organs. The leaves of H. fuscata gave an essential oil especially rich in limonene (20.6%), germacrene D (10.4%) and myrcene (7.1%). In the case of C. myconis, T-muurolol (18.4%), epi-α-bisabolol (18.0%) and α-cadinol (9.4%) were the main compounds detected in flowers and (E)-β-farnesene (5.6%) and epi-α-bisabolol (4.8%) were the most detected in leaves. The essential oil of the flowers of M. paludosum was particularly rich in n-pentacosane (26.9%) and n-hexadecanoic acid (19.1%) while the oil from the leaves mainly contained n-pentacosane (2.5%) and n-tetracosane (2.1%) (Table 2).

Effect of essential oils on nutritional indexes and insect mortality.

All the studied essential oils had negative effects on nutritional indexes of T. confusum larvae. Statistical analysis showed that the oils obtained from the leaves affected significantly the relative growth rate (RGR), more than those obtained from the flowers, except for those from C. myconis that exhibited the same effect. Concerning their activity on relative consumption rate (RCR), the H. fuscata leave essential oil has significantly (P < 0.05) the highest inhibitory activity (0.05 mg/mg/d) and the highest inhibition level on efficiency of conversion of ingested food (ECI). Nevertheless the most significant antifeedant effect on T. confusum larvae was recorded on individuals treated with H. fuscata leaf essential oils (71.20%) while the flower essential oils exhibited the lowest feeding deterrence index (Table 3).


Table 3. Effect of six Asteraceae essential oils on nutritional indexes of during 7 days at concentration of 1% †

Species Organ RGR (mg/mg/d)

RCR (mg/mg/d)

Control - 0.12 ± 0.02 a 0.21 ± 0.00 a

Heteromera fuscata

Flowers 0.09 ± 0.03 b 0.15 ± 0.03 b Leaves 0.02 ± 0.01 f 0.05 ± 0.02 g

Coleostephus myconis

Flowers 0.06 ± 0.01 d 0.14 ± 0.05 c Leaves 0.06 ± 0.03 d 0.13 ± 0.04 d

Mauranthemum paludosum

Flowers 0.07 ± 0.02 c 0.10 ± 0.05 e Leaves 0.05 ± 0.01 e 0.09 ± 0.01 f

RGR: relative growth rate, RCR: relative consumption rate, ECI: efficiency of conversion of ingested food, FDI: feeding deterrence index, †Means in the same column followed by the same letters are not significantly (determined by Duncan’s multirange test.

Mortality of T. confusum larvae

was affected by exposure duration and the origin of the essential oil. Seven days after consumption of flour discs treated with essential oil from C. myconis leaves, they showed statistically significant (P < 0.05) mortality (49.6%) (Fig. 1). At the same time, essential oil extracted from H. fuscata flowers caused the lowest mortality and reached -6.0% after mortality correction using Abbott’s

formula (Fig. 1). In the second week, the percentage of mortality caused by fuscata significantly from 8.7 to 65.0% in comparison with the other essential oils tested. After three weeks of treatment, larvae fed on flour discs treated with essential oils from showed the highest mortality records (85.3%) (Fig. 1).

Fig. 1. Corrected mortality (%) of treated Tribolium confusum discs with six Asteraceae essential oils used at a concentration of 1%.

Vol. 9, No. 1, 2014

es of Tribolium confusum larvae treated

ECI (%) FDI (%)

73.09 ± 1.33 a -

58.15 ± 11.62 c 15.91 ± 8.87 g 39.99 ± 9.77 f 71.20 ± 19.22 a 40.91 ± 10.96 e 41.63 ± 23.93 e 41.53 ± 17.71 d 55.33 ± 22.53 b 67.77 ± 16.73 b 52.69 ± 19.50 c 58.12 ± 13.83 c 40.33 ± 17.65 f

RGR: relative growth rate, RCR: relative consumption rate, ECI: efficiency of conversion of ingested food,

Means in the same column followed by the same letters are not significantly (P < 0.05) different as

formula (Fig. 1). In the second week, the percentage of mortality caused by H.

leaf essential oil increased significantly from 8.7 to 65.0% in comparison with the other essential oils tested. After three weeks of treatment, larvae fed on flour discs treated with essential oils from C. myconis leaves showed the highest mortality records (85.3%) (Fig. 1).

larvae after consumption of treated

essential oils used at a concentration of 1%.


Contact toxicity by topical application on pupae.

T. confusum pupae (less than 24 h-old) were treated with topical applications of essential oils at 1% and then daily followed-up for toxicity for seven days. The results showed that all essential oils exhibited variable insecticidal effets (Fig. 2j). In fact, the essential oil from M.

paludosummortality in treated pupae (23.26%) noted seven days postsignificant dbetween and C. myconisFurthermore, oil induced the lowest toxicity (6.34%) on T. confusum

Fig. 2. Corrected mortality (j) and malformation (k) level (%) of treated Asteraceae essential oils used at a concentration of 1%. HFF: Heteromera fuscata Flowers; MPL: Mauranthemum paludosumLeaves; CMF: Coleostephus myconis Flowers; MPF: Mauranthemum paludosumColeostephus myconis Leaves

After emergence of the new adults, an occurrence of fatal aberation was recorded where the highest level was registred on T. confusum pupae treated with C. myconis leaf essential oils (18%). These results increased the effect of this essential oil and the new mortality calculated was of about 35%. The essential oil extracted from M. paludosum flowers exibited similar toxicity against

T. confusum(23%) added to the deadly aberation (12%) (Fig. 2k). Among these anomalies, it is necessary to note the persistence of nymphal characters with apparition of legs (Fig. 3b) and the presence of new adults with elytra malformation (Fig. 3c). These anenhanced the inhibitory potential of the essential oils by topical application.

j k

e

Vol. 9, No. 1, 2014

paludosum flowers induced the highest mortality in treated pupae (23.26%) noted seven days post-treatment (Fig. 2j). No significant differences were observed between C. myconis and H. fuscata leaf

C. myconis flower essential oils. Furthermore, M. paludosum leaf essential oil induced the lowest toxicity (6.34%) on T. confusum pupae (Fig. 2j).

Corrected mortality (j) and malformation (k) level (%) of treated Tribolium confusum pupae with six

paludosum Leaves; HFL: Heteromera fuscata Mauranthemum paludosum Flowers; CML:

T. confusum once the the pupae mortality (23%) added to the deadly aberation (12%) (Fig. 2k). Among these anomalies, it is necessary to note the persistence of nymphal characters with apparition of legs (Fig. 3b) and the presence of new adults with elytra malformation (Fig. 3c). These anomalies were also fatal and enhanced the inhibitory potential of the essential oils by topical application.

c

d

b c

c

a


Fig. 3. Morphological effect of Asteraceae essential oils topically applied against confusum pupae at a concentration of 1% as compared to the untreated control.(a) Control, (b) Persistence of nymphal characters, (c) Elytra malformation

T. confusum adults (10-14 days-

old) were treated with topical applications of essential oils at 1% and then daily surveyed for toxicity for seven days. The results showed that all essential oils have a variable insecticidal activity (Fig. 4). In fact, essential oil from M. paludosum

leaves induced the highest mortality in treated adults (41%) followed by the those from (23 and 20%, respectively). The lowest toxicity (10%) was recored on individuals treated with essential oil (Fig. 4).

Fig. 4. Corrected mortality level (%) of treated Tribolium confusumoils applied at a concentration of 1%.

a b

Vol. 9, No. 1, 2014

essential oils topically applied against Tribolium

pupae at a concentration of 1% as compared to the untreated control. lytra malformation (arrows).

induced the highest mortality in treated adults (41%) followed by the those from C. myconis flowers and leaves (23 and 20%, respectively). The lowest toxicity (10%) was recored on individuals treated with M. paludosum flower essential oil (Fig. 4).

Tribolium confusum adults with studied essential

c


DISCUSSION Plant essential oils are considered

to be an alternative means of controlling many insect pests (35). In integrated stored product protection, phytochemicals may be used for pest prevention, early pest detection or pest control (21).

This study reported the chemical composition of six essential oils extracted from three species belonging to the Asteraceae family (H. fuscata, C. myconis and M. paludosum) collected from different region in Tunisia. All these essential oils were screened for their potential insecticidal activity against T. confusum larvae and pupae. Based on these findings, these three species presented a similar qualitative composition but a very different distribution of the main volatiles in their essential oils depending on used organs. Chemical analysis revealed that these essential oils were rich on mono- and sesquiterpenes at different levels. The distribution of the main volatiles compounds depended on species tested and organs used. Few studies are reported concerning the essential oil composition of H. fuscata, C. myconis and M. paludosum, however, analyzes reported in a previous study by Haouas et al. (11) showed a similar qualitative composition between G. coronaria, H. fuscata, and P. grandiflorus samples and a variation in the percentage of each volatile compound. Nevertheless, the percentage of volatile compounds can vary for the same species depending on sampling sites. This hypothesis was confirmed by Flamini et al. (9) who demonstrated that essential oil extracted from G. coronaria flowers growing in Italy contained more camphor (22.1%) and cis-chrysanthenyl acetate (19.9%) than that extracted from the same species and collected from Tunisia (camphor 12.6%, 13.4%) (11).

Yang et al. (2005) also highlighted that essential oil composition can vary with geographical distribution, harvesting time, growing conditions and method of extraction (39).

Concerning the insecticidal activity of the essential oils used against T. confusum in this study, it should be noted that these oils exhibited a negative effect on nutritional indices which was more accentuated when diet was treated with essential oil extracted from H. fuscata leaves. Based on chemical analysis, this last essential oil was found to be rich in monoterpens especially in limonene (20.6%). According to Raina et al. (30), this compound can significantly reduce the insect feeding (30). Moreover, this compound is the most toxic one for all T. confusum stages and particularly for the larval stage (32).

In contact method (topical application), this study also demonstrated that M. paludosum leaf essential oils had a lethal effect on T. confusum. Its chemical analyses showed that there are no major compounds in this essential oil, so this effect can be attributed to their high lipophilicity that permits them to rapidly penetrate into insect bodies and interfere with their physiological functions.

In conclusion, this research revealed that each essential oil of the three Asteraceae species has its specific mode of action on the same beetle, T. confusum. In fact, the essential oils extracted from H. fuscata can affect the nutritional behavior and led to the death of insect by starvation. Those from M. paludosum induced an insecticidal activity by contact whereas those of C. myconis can contribute to the death of the confused beetle by hormone perturbation (lethal malformation). Thus, these essential oils may be used as sources of


actives biomolecules for developing environmentally friendly and safe biopesticides.

ACKNOWLEDGMENTS The authors are grateful to the International

Foundation for Science (IFS grant N°. F/3968-1) and to the Organization for the Prohibition of Chemical Weapons (OPCW) for financial support. They also thank Prof. Fethia Skhiri-Harzallah for her help in the identification of Chrysanthemum species.

__________________________________________________________________________ RESUME Haouas D., Cioni P.L., Ben Halima-Kamel M., Flamini G. et Ben Hamouda M.H. 2014. Constituants chimiques et toxicité des huiles essentielles de trois plantes Asteraceae contre Tribolium confusum. Tunisian Journal of Plant Protection 9: 67-82. Les plantes produisent une grande diversité de métabolites secondaires qui les protègent contre les ravageurs et les agents pathogènes. Dans ce travail, nous avons étudié la composition chimique et l’effet de six huiles essentielles extraites de trois espèces d’Asteraceae (Heteromera fuscata, Coleostephus myconis et Mauranthemum paludosum) sur l'indice nutritionnel, l'émergence des adultes et la toxicité contre les larves et des adultes de Tribolium confusum. Les huiles essentielles des fleurs et des feuilles ont été obtenues par hydrodistillation. L'étude de leur composition chimique a été réalisée par CG-SM. Les résultats ont montré qu'elles sont riches en mono-et sesquiterpènes. La consommation des huiles essentielles mélangées avec le milieu artificiel des larves de T. confusum a montré que l’huile essentielle de H. fuscata a retardé la croissance des insectes, réduit la prise de la nourriture et a présenté l'indice d’anti-appétence le plus important contre les larves traitées (respectivement de 0,02 ± 0,01 mg/mg/j, 0,05 ± 0,01 mg/mg/j et 71,20 ± 19,22). L'application topique des huiles essentielles de ces trois Asteraceae contre les nymphes âgées de moins de 24 heures a provoqué des malformations chez les nouveaux adultes émergés. Le plus haut taux de malformation (18%) a été induit par l’huile essentielle des feuilles de C. myconis. L'application topique de ces huiles essentielles sur les adultes de T. confusum (âgés de 10 et 14 jours) a montré une toxicité importante. La plus forte mortalité d’environ 41% a été enregistrée, 7 jours après le traitement, chez les insectes traités par l’huile essentielle des feuilles de M. paludosum. Ces résultats préliminaires pourraient constituer la base pour d'autres investigations sur la sensibilité des autres stades de développement et pour l’élucidation du mode d'action des mono- et sesquiterpènes contre ces insectes. Mots clés: Application topique, Asteraceae, huile essentielle, ingestion, Tribolium confusum ___________________________________________________________________________

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Chemical Composition of Ruta chalepensis Essential Oils and their Insecticidal Activity against Tribolium castaneum

Ons Majdoub, Najla Dhen, Salaheddine Souguir, UR13AGR09, Centre Régional des Recherches en Horticulture et Agriculture Biologique de Chott-Mariem (CRRHAB ChM), Université de Sousse, 4042, Chott-Mariem, Tunisia, Dalila Haouas, UR06AGR05, Ecole Supérieure d’Agriculture du Kef, Université de Jendouba, 7119, Le Kef, Tunisia, Mariem Baouandi, Asma Laarif, and Ikbal Chaieb, UR13AGR09, CRRHAB ChM, Université de Sousse, 4042, Chott-Mariem, Tunisia __________________________________________________________________________ ABSTRACT Majdoub, O., Dhen, N., Souguir, S., Haouas, D., Baouandi, M., Laarif, A., and Chaieb, I. 2014. Chemical composition of Ruta chalepensis essential oils and their insecticidal activity against Tribolium castaneum.Tunisian Journal of Plant Protection 9: 83-90. Essential oils are secondary plant metabolites well known for their defensive role in plants. Many essential oils were described as having potent insecticidal activity. In the present work, the chemical composition and the insecticidal activity of Ruta chalepensis essential oils against stored product pest Tribolium castaneum (adults and larvae) were investigated. The determination of their chemical composition was carried out by using GC-MS technique. Twenty compounds were identified and results showed that essential oil of R. chalepensis are rich on 2-undecanon (48.28%) and 2-nonanon (27%). The insecticidal activity of the indicated volatile fractions was screened against adults and larvae of T. castaneum. R. chalepensis essential oil was found to be more active against adults (LC50 = 176.075 µl/l air and LC90 = 291.9 µl/l air) than larvae (LC50= 415.348 µl/l air and LC90=685.907 µl/l air). After 24 h of exposure at the dose of 200 µl/l air, 14% and 60% of mortality were recorded for larvae and adults, respectively. These preliminary findings may be useful for further studies of R. chalepensis essential oil use against other food storage pests and for deeper investigations on their mode of action. Keywords: Chemical composition, essential oils, insecticidal activity, LC50, LC90, Ruta chalepensis, Tribolium castaneum. __________________________________________________________________________

The major cause of post-harvest losses in storage is due to insect pests. These pests are able to destroy a whole stock in a very short period of time during

Corresponding author: Ikbal Chaieb Email: [email protected] Accepted for publication 10 March 2014

their development (13). Synthetic insecticides are currently the primary means used to protect stored grains from insects. However, chemical control has been associated with dangers like environmental pollution, human toxicity, development of insecticide resistance and adverse effects on non target organisms (23, 22, 17). The use of natural plant compounds, having insecticidal activity


and little environmental effect, is one of promising alternative to protect post-harvest products (7, 19). Among botanicals, the Rutaceae species have attracted a lot of attention due to the range of biological activities induced by their secondary metabolites including antifungal, antioxidant, and anti-inflammatory properties (5, 6, 8, 9, 12, 15). One of the widely diffused species in the Mediterranean area is Ruta chalepensis commonly known as fringed rue (6). This perennial herb is characterized by oval, large, pinnate and blue-green leaves that have many oblong lanceolate lobes. The inflorescence of this species is in the form of cyme (3).

The main purpose of this work is to determine the chemical composition and to study, under laboratory conditions, the insecticidal effects of the essential oils from R. chalepensis leaves against an important pest of stored grains Tribolium castaneum. MATERIALS AND METHODS Insect rearing.

Adults and larvae of T. castaneum used for testing were obtained from laboratory rearing, maintained in growth chamber in total obscurity at 28 ± 1°C and 60% RH in the Laboratory of Entomology of the Regional Center of Research on Horticulture and Organic Agriculture of Chott-Mariem (CRRHAB ChM), Tunisia. Plant material.

R. chalepensis leaves were collected from the southern Tunisia (Mednine) where it grows spontaneously. Essential oil extraction.

Essential oils were extracted from fresh leaves by hydrodistillation using a

Clevenger apparatus for 4 h at 350°C. The extracted oils were weighed in order to calculate their extraction yield and stored at 4°C prior to analysis. Chemical analysis.

The essential oils were analyzed by gas chromatography coupled to mass spectrometry (GC–MS) using a HP 6890N GC Mass spectrometer with electron impact ionization (HP 5975B). A HP-5MS capillary column (30 m × 250 µm, 0.25 µm film thickness) was used. Oven temperature was programmed to rise from 60 to 220°C at a rate of 40°C/min; transfer line temperature was 230°C. The carrier gas was helium with a flow rate of 0.8 ml/min and a split ratio of 50:1. Scan time and mass range were 1 sec and 50-550 m/z, respectively. The essential oils constituents were identified by comparing their relative retention times. The determination of the percentage composition was based on peak area normalization without using correction factors. Fumigant test.

This bioassay was determined by using closer container method, which consists in isolating groups of ten insects, and then, fixing paper discs, treated by different doses of oil (25, 50,100 and 200 µl/l air), on the top of containers. Five replicates were used for tested oils and control. The mortality of insects (%) was observed and determined after 3, 6, and 24 h of exposure. Data analysis.

The estimation of LD50 and LD90 values of R. chalepensis leaf essential oils against adults and larvae of T. castaneum were determined by probit analysis. Mean mortality separation were done using


Duncan’s multirange test at the 5% level through SPSS (Version 19) (18). RESULTS Essential oil composition.

Table 1 summarizes the composition of R. chalepensis leaf essential oils. In total, 22 compounds

were identified. The 2-undecanone is the most predominant compound (48.28%) followed by the 2-nonanone (27.15%). The terpenic compounds are present in low proportion in comparison to the aromatic ones. In fact, the 1,8-cineole (1.87%) and the viridiflorol (1.74%) are the most frequent compounds in this oil.

Table 1. Chemical composition of Ruta chalepensis leaf essential oils

N° Compound Peak area (%)

Retention time (min)

1 α-pipene 0.71 6.38 2 Camphene 0.65 6.70 3 β-Pinene 0.22 7.33 4 2-Octanone 0.47 7.64 5 Limonene 0.24 8.49 6 1,8-Cineole 1.63 8.56 7

Ǵ-terpinene 0.17 9.16

8 2-Nonanone 23.70 10.10 9 2-Nonanol 1.71 10.17 10 Nonanal 0.24 10.23 11 Geyrene 1.33 11.02 12 Camphor 0.85 11.11 13 Borneol 0.30 11.58 14 2-Decanone 2.58 12.07 15 Octyl acetate 0.11 12.41 16 1-Nonene 14.20 13.04 17 Pregeijerene 0.12 13.31 18 2-Undecanone 42.15 14.14 19 2-Dodecanone 1.34 15.47 20 2-Tridecanone 0.49 17.83 21 Viridiflorol 1.52 19.52 22 α-eudesmol 0.19 20.65

Fumigant activity.

Mortality. Fumigant toxicity of essential oils of R. chalepensis on adults and larvae of T. castaneum noted after 3, 6 and 24 h of exposure is shown in Table 2. Statistical analyses showed that mortality rate is proportional to the dose and time of exposure. Results also indicated that adults were more susceptible to R. chalepensis essential oils

than larvae with 60 and 14% of mortality, respectively, noted after 24 h post-treatment.

Determination of LC50 and LC90

values. Calculated LC50 and LC90 values makes out that fumigant toxicity of R. chalepensis leaf oil was more active against adults than larvae of T. castaneum (Table 3).


Table 2. Mortality rate of adults and larvae of Tribolium castaneum noted depending on doses of Ruta chalepensis essential oils and exposure time.

Insect instar

Dose (µl/l air)

Mortality per exposure time (%) 3 h 6 h 24 h

Adult

0 0 a 0 a 0 a

25 2 ± 4.47 a 2 ± 4.47 ab 2 ± 4.47 ab 50 4 ± 5.48 a 18 ± 10.95 c 18 ± 10.95 b

100 4 ± 5.48 a 12 ± 10.95 bc 16 ± 13.42 ab 200 12 ± 8.37 b 18 ± 8.37 c 60 ± 18.71 c

Larva

0 0 a 0 a 0 a 25 0 a 4 ± 5.48 a 4 ± 5.48 a 50 0 a 4 ± 5.48 a 6 ± 5.48 ab 100 0 a 6 ± 5.48 a 8 ± 4.47 ab 200 2 ± 4.47 a 8 ± 4.47 a 14 ± 5.48 b

For each developmental stage et exposure time, values followed by the same letter are not significantly different based on Duncan's multirange test at P < 0.05.

Table 3. Determination of LC50 and LC90 values of Ruta chalepensis leaf oil against Tribolium castaneum depending on target instars.

T. castaneum C50 (µl/l air) C90 (µl/l air)

Adult 176.075 291.9

Larva 415.348 685.907

DISCUSSION

R. chalepensis essential oil was obtained by hydrodistillation. The obtained essential oil yield (0.34%) is comparable to those reported in the literature (4, 10). Moreover, the abundance of 2-undecanone in oils is also observed in the works done on R. chalepensis essential oils but with some variation of the percentage mentioned as those from Algeria (68.95%)(11), Argentina (38.1%) (20), Turkey (66.5%) (2), Iran (52.5%) (16) and Tunisia (77.18%) (10). The nature and proportions of other compounds present in the oils investigated in the current study are not always the same in comparison with those reported in previous studies. These variations can be attributed to climatic, seasonal and geographic conditions, harvest period,

chemotypes and/or extraction procedures (14). Concerning the fumigant toxicity of the obtained essential oil, insecticidal activity was highest against adults of T. castaneum than larvae. This result can be explained by the relationship that may exist between the insect physiology and the mode of action of essential oils. In fact, these volatile compounds act directly on the nervous system of the insect which is primitive for larvae and explains their resistance to essential oils. Unfortunately, in the literature we did not found studies that highlight the potential bio-insecticide of R. chalepensis essential oils against stored products pests. However, several biological activities of R. chalepensis essential oils were reported such as antifungal, antimicrobial, antioxidant, anti-inflammatory, phytotoxic activities (3, 5, 6, 8, 9, 12, 15). In addition, LC50


and LC90 values were lower for adults than for larvae. Similar effect was observed by Wang et al. (21) where Artemisia vulgaris oil had strong fumigant activity against T. castaneum adults and larvae but adults were much more susceptible than larvae. Moreover, Bachrouch et al. (1) highlighted that the essential oil of Pistacia lentiscus have fumigant toxicity against the red flour beetle T. castaneum. This toxicity potential also depended on developmental stages, oil concentrations and exposure times. In fact, the fumigant toxicity of P.

lentiscus was higher against adults (LC50 = 28.03 µl/l, LC95 = 63.46 µl/l) than against the third instar larvae (LC50 = 112.12 µl/l, LC95 = 253.53 µl/l).

The results from study revealed an interesting potential of R. chalepensis essential oils in controlling the food storage pest T. castaneum. Further studies are needed to better explain the observed phenomenon and to determine a relationship between the chemical composition of the essential oils and their mode of action against the target insect pests.

__________________________________________________________________________ RESUME Majdoub O., Dhen N., Souguir S., Haouas D., Baouandi M., Laarif A. et Chaieb I. 2014. Composition chimique des huiles essentielles de Ruta chalepensis et leur activité insecticide contre Tribolium castaneum. Tunisian Journal of Plant Protection 9: 83-90. Les huiles essentielles sont des métabolites secondaires des plantes, connues pour leur rôle défensif chez les plantes. De nombreuses huiles essentielles ayant une activité insecticide puissante ont été décrites. Dans le présent travail, la composition chimique et l'activité insecticide des huiles essentielles de Ruta chalepensis contre un ravageur des denrées stockées, Tribolium castaneum (adultes et larves) ont été étudiées. La détermination de leur composition chimique a été réalisée moyennant la technique GC-MS. Vingt composés ont été identifiés et les résultats ont montré que les huiles essentielles de R. chalepensis sont riches en 2-undecanone (48,28%) et en 2-nonanone (27%). L'activité insecticide de ces huiles a été criblée contre des adultes et des larves de T. castaneum. Les huiles essentielles de R. chalepensis se sont montrées plus actives contre les adultes (CL50 = 176,075 µl/l air et CL90 = 291,9 µl/l d'air) que les larves (CL50 = 415,348 µl/l air et CL90 = 685,907 µl/l d'air). Après 24 h d'exposition à la dose de 200 µl/l air, 14% et 60% de mortalité ont été enregistrés chez les larves et les adultes, respectivement. Ces résultats préliminaires peuvent être utiles pour mener des études ultérieures sur l’utilisation des huiles essentielles de R. chalepensis contre d'autres ravageurs des denrées stockées et des investigations plus profondes sur leur mode d'action. Mots clés: Activité insecticide, composition chimique, huile essentielle, LC50, LC90, Ruta chalepensis, Tribolium castaneum __________________________________________________________________________

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___________________________________________________________________________ LITERATURE CITED 1. Bachrouch, O., Mediouni-Ben Jemâa, J., Chaieb,

I., Talou, T., Marzouk, B., and Abderraba M. 2010. Activité insecticide de l’huile essentielle du pistachier lentisque Pistacia lentiscus contre Tribolium castaneum comme alternative au traitement chimique en stockage. Tunisian J. Plant Prot. 5: 63-70.

2. Başer, K.H.C., Özek, T., and Beis, S.H. 1996. Constituents of the essential oil of Ruta chalepensis L. from Turkey. J. Essent. Oil Res. 8: 413 -414.

3. Ben Bnina, E., Hammami, S., Daami-Remadi, M., Ben Jannet, H., and Mighri, Z. 2010. Chemical composition and antimicrobial effects of Tunisian Ruta chalepensis L. essential oils. Journal de la Société Chimique de Tunisie 12: 1-9.

4. Dob, T., Dahmane, D., Gauriat-Desrdy, B., and Daligault, V. 2008. Volatile constituents of the essential oil of Ruta chalepensis L. subsp. angustifolia (Pers.) P. Cout. J. Essent. Oil Res. 20: 306-309.

5. Gonzalez-Trujano, M.E., Carrera, D., Ventura-Martinez, R., Cedillo-Portugal, E., and Navarrete, A. 2006. Neuropharmacological profile of an ethanol extract of Ruta chalepensis L. in mice. J. Ethnopharm. 106: 129-135.

6. Iauk, L., Mangano, K., Rapisarda, A., Ragusa, S., Maiolino, L., Musumeci, R., Costanzo, R., and Serra, A. 2004. J. Ethnopharm. 90: 267-272.

7. Isman, M.B. 1994. Botanical insecticides. Pesticide Outlook 5: 26-31.

8. Kabouche, Z., Benkiki, N., Seguin, E., and Bruneau, C. 2003. A new dicoumarinyl ether and two rare furocoumarins from Ruta montana, Fitoterapia 74: 194-196.

9. Meepagala, K.M., Schrader, K.K., Wedge, D.E., and Duke, S.O. 2005. Algicidal and antifungal compounds from the roots of Ruta graveolens and synthesis of their analogs. Phytochemistry 66: 2689-2695.

10. Mejri, J., Abderrabba, M., and Mejri, M. 2010. Chemical composition of the essential oil of Ruta chalepensis L.: Influence of drying, hydro-distillation duration and plant parts. Indust. Crop. Prod. 32: 671-673.

11. Merghache, S., Hamza, M., and Tabti B. 2009. Etude physicochimique de l’huile essentielle de Ruta chalepensis L. de Tlemcen, Algérie. Afrique Science 5: 67-81.

12. Milesi, S., Massot, B., Gontier, E., Bourgaud, F., and Guckert, A. 2001. Ruta graveolens L.: a promising species for the production of furanocoumarins. Plant Science 161: 189-199.

13. Ngamo, L.S.T. and Hance, T. 2007. Diversité des ravageurs des denrées et méthodes alternatives de lutte en milieu tropical. Tropicultura 25: 215-220.

14. Palá-Paúl, J., Pérez-Alonso, M., Velasco-Negueruela, J.A., Varadé J., Ma Villa, A., and Sanz, J. 1993. Composition and antimicrobial properties of essential oils of four Mediterranean Lamiaceae. J. Ethnopharm. 39: 167-170.

15. Raghav, S.K., Gupta B., Agrawal C., Goswami K., and Das H.R. 2006. Anti-inflammatory effect of Ruta graveolens L. in murine macrophage cells. J. Ethnopharm. 104: 234-239.

16. Rustaiyan, A., Khossravi, M., Sultani-Lotfabadi, F., Yari, M., Masoudi, S., and Monfared, A. 2002. Constituents of the essential oil of Ruta chalepensis L. from Iran. J. Essent. Oil Res. 14: 378-379.

17. Sharma, D.R. and Kalra, R.L. 1998. Phosphine resistance during different developmental stages of Trogoderma granarium (Everts). Annals of Plant Protection Science 6: 198-200.

18. SPSS. SPSS Version 20. SPSS Inc, 233 S. Wacker Drive; Chicago, Illinois. 2011.


19. Taylor, R.W.D.1994. Methyl bromide: Is there any future for this fumigant?, J. Stored Prod. Res. 30: 253-260.

20. Verzera A., Mondello L., Ragusa S., and Dugo G. 2000. Essential oil of the leaves of a typical mediterranean plant: Note II. Ruta chalepensis L. (Rutaceae). Essenze e derivati agrumari 70: 207-210.

21. Wang, J., Zhu, F., Zhou, X.M., Niu, C.Y., and Lei, C.L. 2006. Repellent and fumigant activity of essential oil from Artemisia vulgaris to

Tribolium castaneum (Herbst). J. Stored Prod. Res. 42: 339-347.

22. White, N.D.G. 1995. Insects, mites and insecticides in stored grain ecosystems. Pages 123-168. In: Stored Grain Ecosystem. M. Dekker (Ed.), New York, USA.

23. Zettler, J.L. and Cuperus, G.W. 1990. Pesticide resistance in Tribolium castaneum (Coleoptera: Tenebrionidae) and Rhyzopertha dominica (Coleoptera: Bostrichidae) in wheat. J. Econ. Entomol. 83: 1677-1681.

--------------------------


Insecticidal Activities of Fruit Peel Extracts of Pomegranate (Punica granatum) against the red flour beetle Tribolium castaneum Amel Ben Hamouda, Atika Mechi, Khaoula Zarred, Ikbal Chaieb, and Asma Laarif, UR13AGR09, Centre Régional des Recherches en Horticulture et Agriculture Biologique de Chott-Mariem, Université de Sousse, 4042, Chott-Mariem, Tunisia __________________________________________________________________________ ABSTRACT Ben Hamouda, A., Mechi, A., Zarred, K., Chaieb, I., and Laarif, A. 2014. Insecticidal activities of fruit peel extracts of pomegranate (Punica granatum) against the red flour beetle Tribolium castaneum. Tunisian Journal of Plant Protection 9: 91-100. Aqueous, ethanol and methanol fruit peel extracts of Punica granatum were tested under laboratory conditions for their insecticidal activities against larvae and adults of the red flour beetle, Tribolium castaneum. The beetles were exposed to the different extracts by topical application and ingestion treatment. Highest mortalities were recorded on larvae treated with ethanol extract with mortalities of 72 and 56% for topical application and ingestion treatment respectively. The three extracts exhibited anti-feeding effects (70% > AFI ≥ 50%) against T. castaneum larvae whereas only methanol extract exhibited a low anti-feeding activity (50% > AFI ≥ 20%) against adults. Additionally, only ethanol extract showed repellent activity. Results suggested the presence of toxic active components in the ethanol fruit peel extract acting by mainly ingestion and topical application. The treatment with this botanical insecticide may be promising in protecting stored grains from coleopteran pest infections. Keywords: Fruit peel extracts, insecticidal activity, method of treatment, Punica granatum, Tribolium castaneum. __________________________________________________________________________

Plants may provide potential alternatives to currently used insect-control tools because they constitute a rich source of bioactive chemicals (31). These plant-derived compounds are active against a limited number of species including specific target insects, biodegradable to non-toxic products, and are potentially suitable for use in integrated pest management. Terrestrial plants produce natural substances.

Corresponding author: Amel Ben Hamouda Email: [email protected] Accepted for publication 27 March 2014

Insecticidal activity of many plants against several insect pests has been demonstrated by many researchers (4, 9, 10). The deleterious effects of phytochemcials or crude plant extracts against insects are expressed in several ways, including suppression of calling behavior (11), growth retardation (3), toxicity (7), oviposition deterrence (33), feeding inhibition (30) and reduction of fecundity and fertility (23). Tribolium species are considered as major pests of stored grains (29). The red flour beetle, T. castaneum, is one of the most severe secondary insect pests that feeds on a widespread range of long-lasting stored grain products including cereals and


derived products and other high value products such as dried fruits cocoa and beans (13). Management of this insect pest and the other insect pests of stored grains mainly depends on the use of residual insecticides and gaseous fumigants (21). However, their widespread use has led to serious problems including development of resistance, toxic residues in stored grains, and increasing costs of application (24). Yet, there is an urgent need to develop low cost safe control alternatives and environmental-friendly. Considerable efforts have been focused on plant-derived materials, potentially useful as commercial bioinsecticides. Punica granatum, commonly known as pomegranate belongs to family Punicaceae. It is a native shrub of central Asia, especially parts of Iran in the Transcaucasia-Caspian region (6) from where it has spread to the rest of the world (14, 25). Pomegranate is an important crop known by its taste and nutritional and medicinal properties (17, 18, 22). Several studies have reported the antimicrobial and antifungal (2, 28), molluscicidal (27) and insecticidal (5) activities of extracts from different tree parts, such as bark, leaves, fruit, and fruit peel. The aim of the present study is to evaluate the insecticidal activity of the aqueous, ethanol and methanol fruit peel extracts from P. granatum against larvae and adults of T. castaneum. Length of larvae, mortality of larvae and adults, anti-feeding and repellent activities are assessed.

MATERIALS AND METHODS Preparation of pomegranate peel extracts.

Pomegranates, P. granatum cv. Kalaii were obtained from local market. The fruits were washed and the peels were manually removed, dried at room

temperature (20 to 25°C) and powdered to get 0.5 mm size. About 100 g of the powder was extracted by stirring using a magnetic stirrer with 300 ml of ethanol, methanol and water for 24 h each at 25°C. The extract was sieved through Whatman filter paper to remove peel particles. After filtration, the ethanol and methanol extracts were let to evaporate at room temperature during 48 h and the aqueous extract was evaporated under vacuum at -100°C.

Insect rearing. The red floor beetle T. castaneum

was reared on artificial diet of semolina mixed with corn flour and beer yeast (100/50/5, w/w/w) at a constant temperature of 30 ± 1°C in the dark. Adult insects of 10 to 14 days old and third instar larvae were used for toxicity tests.

Toxicity bioassays. Topical application bioassay.

Twenty mg of each crude extract was dissolved in distilled water to obtain the final concentration of 2%. One micro-liter of each solution (ethanol, methanol and aqueous) was applied on the abdomen of 10 larvae and 10 adults. The control received 1 µl of distilled water only (five replications). The mortality rate was recorded after 2, 7, 14 and 21 days. The assessment of mortality rate was corrected for control mortality according to Abbott’s correction formula (1):

Mc = (Mo - Me / 100 - Me) ×100 with Mc = corrected mortality rate (%), Mo = mortality rate of treated adults (%), Me = mortality rate of control (%).

Semolina disk bioassay. Semolina disks were prepared according to methods of Xie et al. (32) and Huang et al. (8). In brief, 10 g of artificial diet was mixed with 50 ml of distilled water. The dough was cut into small discs of 1 cm diameter and 20 mg weight. The disks were left in


the fume hood overnight to dry. Volumes of 50 µl of each extract (ethanol, methanol, aqueous) prepared at a concentration of 2% were applied on the semolina disks. Control disks receive only 5 µl of ethanol. After evaporation of the solvent, the disks were weighed and placed each one in a Petri dish containing 5 third instars larvae whose length has been measured. Twenty one days after, the weight and length of larvae, the weight of adult, the mortality and the feeding deterrence index were determined. Formula described by Simmonds et al. (26) was used for calculating the feeding deterrence index,

FDI = (C-T / C+T) × 100 where C = the food consumption in control disks and T = food consumption in treated disks.

The following criteria were adopted to categorize the feeding deterrence degree: FDI% < 20%: (-) No feeding deterrence, 50% > FDI% ≥ 20%: (+) Weak feeding deterrence, 70% > FDI% ≥ 50%: (++) Moderate feeding deterrence, FDI% ≥ 70%: (+++) Strong feeding deterrence.

Repellent activity bioassay. The repellency was tested

according to McDonald et al. (16). Half filter paper discs (Whatman No. 40, 9 cm diam.) were prepared and 20 mg of each extract was diluted in 1 ml of methanol at the concentration of 2%. A volume of 200 µl of each concentration was applied separately to one half of the filter paper as uniformly as possible with a micropipette. The other half (control) was treated with 200 µl of methanol. Both the treated and the control halves were allowed to dry out as exposed in the air for 10 min. Each treated half-disc was then attached lengthwise, edge-to-edge, to a control half disc with adhesive tape and placed in a

Petri dish (9 cm diameter). Twenty adult insects were released in the middle of each filter-paper circle. Each concentration was replicated five times. Insects that settled on each half of the filter paper disc were counted after 15 min, 30 min and 2 h. The average of the counts was converted to percentage repellency (PR) using the formula of McDonald et al. (16):

PR = [(Nc - Nt) / (Nc + Nt)] × 100 where Nc = number of insects in control test; Nt = number of insects in treated test.

The mean repellency value of each extract was calculated and assigned to repellency classes from 0 to V: class 0 (PR ≤ 0.1%), class I (PR = 0.1 - 20%), class II (PR = 20.1 - 40%), class III (40.1 - 60%), class IV (60.1 - 80%) and class V (80.1 - 100%).

Statistical analyses. The mortality data were adjusted

for mortality in the water (Topical application test) and ethanol control (Ingestion test) using Abbott’s correction (1). Five replications were performed for each test. For statistical comparison among several means, all the data on larval length gain and mortality were subjected to a one-way analysis of variance (ANOVA) followed by mean comparisons (at P = 0.05) and Student-Newman-Keuls (SPSS 11.0).

RESULTS Toxicity by topical application.

During the experiment, mortality rate increased constantly with increasing time until reaching 72% for larvae and 44% for adults at the third week post-treatment (Figs. 1, 2). Statistical analyses showed significant effect of ethanol extract on pest mortality. The comparison activity between different extracts showed that ethanol was more toxic than the other extracts.


Fig. 1. Mortality rate of Tribolium castaneum larvae treated by topical application with ethanol, methanol and aqueous fruit peel extracts of pomegranate. Mortality rate was corrected using Abbott's formula (1).

After 21 days, a significant mortality rate was recorded with ethanol extract reaching 72% for larvae and 44%

for adults, followed by methanol (14% for larvae and adults) and aqueous extracts (10% for larvae and only 6% for adults).

Fig. 2. Mortality rate of Tribolium castaneum adults treated by topical application with ethanol, methanol and aqueous fruit peel extracts of pomegranate. Mortality rate was corrected using Abbott's formula (1).

-20

-10

0

10

20

30

40

50

60

70

80

0 2 7 14 21

Mo

rtal

ity

rate

(%

)

Exposure time (day)

Ethanol extract

Methanol extract

Aqueous extract

0

5

10

15

20

25

30

35

40

45

50

0 2 7 14 21

Mo

rtal

ity

rate

(%

)

Exposure time (day)

Ethanol extract

Methanol extract

Aqueous extract


Toxicity by ingestion.

According to Fig. 3, it appears that the two lowest toxicity records were induced by methanol and aqueous

extracts whereas the ethanol extract led to the highest mortality (56%) in T. castaneum individuals.

Fig. 3. Mortality rate of Tribolium castaneum larvae treated by ingestion with ethanol (EE), methanol (ME) and aqueous (AE) fruit peel extracts of pomegranate as compared to the untreated control (C). Bars attributed by the same letter are not significantly different according to the Student-Newman-Keuls test (P ≤ 0.05).

Feeding deterrent activity.

Results related to the feeding deterrent activity of the three extracts tested against larvae and adults of the red flour beetle are shown in Table 1. The three extracts tested (ethanol, methanol and aqueous extracts) exhibited a significant feeding deterrent activity against T. castaneum larvae at a concentration of 2%. This effect was weaker and only the methanol extract has a low effect against adults 50% > 30.66 ≥ 20%.

Larval length.

Fig. 4 shows that the three extracts significantly (P < 0.05) limited the larvae length gain as compared to the untreated control. This inhibition was mainly due to the feeding-deterrent activity. The ethanol extract showed the most inhibitory effect with an average of -1.82 ± 0.67 mm, while the methanol and aqueous extracts have the lower inhibitory effects.

0

10

20

30

40

50

60

C EE ME AE

Mo

rta

lity

ra

te (

%)

Extract tested

a

a

b

a


Table 1. Feeding deterrent activity of pomegranate fruit peel extracts against Tribolium castaneum larvae and adults

Extract Feeding deterrence index (FDI) %

Larvae Adults

Ethanol 61.56 ± 23.29 (++) 10.59 ± 13.94 (-)

Methanol 55.14 ± 28.02 (++) 30.66 ± 17.56 (+)

Aqueous 53.26 ± 41.50 (++) 3.54 ± 10.57 (-)

Fig. 4. Length gain of Tribolium castaneum larvae treated by ingestion with ethanol (EE), methanol (ME) and aqueous (AE) fruit peel extracts of pomegranate as compared to the control (C). C: Larvae were fed on disks treated with ethanol. Bars attributed by the same letter are not significantly different according to the Student-Newman-Keuls test (P ≤ 0.05).

Repellent activity.

The average repellency values for the tested extracts on T. castaneum adults noted after 15, 30 and 120 min are reported in Table 2. The lowest level of repellency was recorded after 15 and 30 min of exposure of adults to ethanol extract. Other extracts exhibited an attractive effect.

DISCUSSION

The results revealed insecticidal effects of the three extracts of

pomegranate fruit peel. However, ethanol extract was found to be the most toxic. Toxicity of ethanol extract on T. castaneum was expressed by 56% larval mortality after ingestion. In this respect, Koide et al. (12) reported that toxicity caused by P. granatum is due to the astringent properties of tannins contained in the peel fruit which stop insect’s infestation.

-3 -2,5 -2 -1,5 -1 -0,5 0 0,5 1 1,5

C

EE

ME

AE

Larvae length gain (mm)

Ex

tra

ct t

est

ed

a

b

b

c


Table 2. Repellent effects of different fruit peel extracts from pomegranate against Tribolium castaneum adults noted after 2 h of exposure.

In addition to their toxicity, use of the three extracts led to a significant feeding deterrent effect against T. castaneum larvae at the concentration of 2% expressed by the reduction of larval length. Ethanol extract showed the real effect by a reduction of -1.82 mm, followed by aqueous (-0.62 mm) and methanol extract (-0.08 mm). The toxic effect of ethanol extract was also observed in topical application test. Similar findings were obtained by Mohammad (19) where the ethanol extracts also induced mortality of T. confusum larvae and adults. Mortality caused by methanol extract was low; not exceeding 14%. In this context, Liu et al. (15) denied the contact toxicity effects of these extracts. Regarding feeding deterrent test, the current study revealed a weak effect against T. castaneum adults, another species of stored grains, while Liu et al. (15) indicated moderate effect of this extract.

Among the three extracts of pomegranate fruit peel tested, only the ethanol extract exhibited a repellent activity (Table 2); this effect did not exceed 20% and was reduced after 30 min of exposure. Recently, Mohammad (20) reported a strong repellent effect (86.7%) caused by ethanol extract of pomegranate fruit peel after two hours of exposure at a concentration of 2.5% for T. confusum. The leaf powder of P. granatum also showed an insecticidal activity against T. castaneum. Indeed, Gandhi et al. (5) indicated that spraying of this powder led to 40% to 85% of adult mortality. Besides, the powder caused a delay in the development of this insect.

These preliminary data suggest that the ethanol extract of the pomegranate fruit peel should be further investigated in order to determine its chemical composition and to elucidate more its insecticidal potential.

__________________________________________________________________________ RESUME Ben Hamouda A., Mechi A., Zarred K., Chaieb I. et Laarif A. 2014. Activités insecticides des extraits de l’écorce de la grenade (Punica granatum) contre le Tribolium rouge de la farine Tribolium castaneum. Tunisian Journal of Plant Protection 9: 91-100.

Extract Exposure time (min)

Mean Repellency (%)

Repellency Class

Ethanol 15 30 120

20 16 -4

I I 0

Methanol 15 30 120

-28 -28 -40

0 0 0

Distilled water 15 30 120

-14 -20 -10

0 0 0


Les extraits aqueux, éthanoliques et méthanoliques de l’écorce de Punica granatum ont été testés dans les conditions de laboratoire pour leurs activités insecticides contre les larves et les adultes du Tribolium rouge de la farine, Tribolium castaneum. Les insectes ont été exposés aux différents extraits par application topique et par ingestion. Les mortalités les plus élevées ont été enregistrées sur les larves traitées avec l’extrait éthanolique avec des mortalités de 72 et 56% pour les traitements par application topique et par ingestion, respectivement. Les trois extraits ont montré des effets anti-appétants (70% > IAA ≥ 50%) contre les larves alors que l’extrait méthanolique a présenté une faible activité anti-appétante (50% > IAA ≥ 20%) contre les adultes. De plus, seul l’extrait éthanolique a montré une activité répulsive. Les résultats obtenus suggèrent la présence de composés toxiques actifs dans l’extrait éthanolique, l’écorce des grenades agissant principalement par ingestion et application topique. L’application de cet insecticide botanique pourrait être prometteuse pour la protection des grains stockés contre les infections par les insectes coléoptères. Mots clés: Activité insecticide, extraits d’écorce de grenade, méthode de traitement, Punica granatum, Tribolium castaneum. __________________________________________________________________________

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__________________________________________________________________________ LITERATURE CITED 1. Abbott, W.S. 1925. A method of computing

effectiveness of an insecticide. J. Econ. Entomol. 18: 265-267.

2. Al-Zoreky, N.S. 2009. Antimicrobial activity of pomegranate (Punica granatum L.) fruit peels. Intern. J. Food Microbiol. 134: 244-248.

3. Breuer, M. and Schmidt, G.H. 1995. Influence of a short period treatment with Melia azedarach extract on food intake and growth of the larvae of Spodoptera frugiperda (Lepidoptera; Noctuidae). J. Plant Dis. Prot. 102: 633-654.

4. Carlini, C.R. and Grossi-de-Sá, M.F. 2002. Plant toxic proteins with insecticidal

properties. A review on their potentialities as bioinsecticides. Toxicon 40: 1515-1539.

5. Gandhi, N., Pillai, S., and Patel, P. 2010. Efficacy of pulverized leaves of Punica granatum (Lythraceae) and Murraya koenigii (Rutaceae) against stored grain pest, Tribolium castaneum (Herbst.) (Coleoptera: Tenebrionidae). Int. J. Agric. Biol. 12: 616-620.

6. Harlan, J.R. 1992. Crops and Man. 2nd Edition, American Society of Agronomy and Crop Science Society of America, Madison, WI, 289 pp.

7. Hiremath, I.G., Youngjoon, A., Soonll, K., Ahn, Y.J., and Kim, S.I. 1997. Insecticidal activity of Indian plant extracts against Nilaparvata


lugens (Homoptera: Delphacidae). Appl. Entomol. Zool. 32: 159-166.

8. Huang, Y., Tan, J.M.W.L., Kini, R.M., and Ho, S.H. 1997. Toxic and antifeedant action of nutmeg oil against Tribolium castaneum (Herbst) and Sitophilus zeamais Motsch. J. Stored Prod. Res. 33: 289-298.

9. Isman, M.B. 2000. Plant essential oils for pest diseases management. Crop Prot. 19: 603-608.

10. Jilani, G. and Su, H.C.F. 1983. Laboratory studies on several plant materials as insect repellents for protection of stored grains. J. Econ. Entomol. 76: 154-157.

11. Khan, Z.R. and Saxena, R.C. 1986. Effect of steam distillate extracts of resistant and susceptible rice cultivars on behaviour of Sogatella furcifera (Homoptera: Delphacidae). J. Econ. Entomol. 79: 928-935,

12. Koide, T., Nose, M., Inoue, M., Ogihara, Y., Yabu, Y., and Ohta, N. 1998. Trypanocidal effects of gallic acid and related compounds. Planta Medica 64: 27-30.

13. Lee, B.H., Lee, S.E., Annis, P.C., Pratt, S.J., Part, B.S., and Tumaalii, F. 2002. Fumigant toxicity of essential oils and monoterpenes against the red flour beetle, Tribolium castaneum Herbst. J. Asia Pac. Entomol. 5: 237-240.

14. Levin, G.M. 1994. Pomegranate (Punica granatum) plant genetic resources in Turkmenistan. Plant Genetic Resource Newsletters 97: 31-36.

15. Liu, Z.L., Goh, S.H., and Ho, S.H. 2007. Screening of Chinese medicinal herbs for bioactivity against Sitophilus zeamais Motschulsky and Tribolium castaneum (Herbst). J. Stored Prod. Res. 43: 290-296.

16. McDonald, L.L, Guy, R.H., and Speirs, R.D. 1970. Preliminary evaluation of new candidate materials as toxicants, repellents and attractants against stored-product insects. Agricultural Research Service, U.S. Department of Agriculture, Washington D.C., Marketing Research Report No. 882.

17. Melgarejo, P., Martínez, J., Hernández, F., Martínez, F.R., Barrows, P., and Erez, A. 2004. Kaolin treatment to reduce pomegranate sunburn. Sci. Hortic. 100: 349-353.

18. Melgarejo, P., Salazar, D.M., and Artés, F. 2000. Organic acids and sugars composition of harvested pomegranate fruits. Eur. Food Res. Technol. 211: 185-190.

19. Mohammad, H.H. 2012. Insecticidal Effect of Different Plant Extracts against Tribolium confusum (du val) (Coleoptera:Tenebrionidae). Journal of

Agricultural Science and Technology A 2: 1175-1181.

20. Mohammad, H.H. 2013. Repellency of Ethanolic Extract of Some Indigenous Plants against Tribolium confusum (du val) (Coleoptera: Tenebrionidae). Journal of Agriculture and Veterinary Science 2: 27-31.

21. Mondal, M. and Khalequzzaman, M. 2006. Toxicity of essential oils against red flour beetle, Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae). Journal of Bio-Science 14: 43-48.

22. Mutahar, S., Shiban M., Al-Otaibi, M., Najeeb, S., and Al-Zoreky, N. 2012. Antioxidant activity of pomegranate (Punica granatum L.) fruit peels. Food and Nutrition Sciences 3: 991-996.

23. Muthukrishnan, J. and Pushpalatha, E. 2001. Effects of plant extracts on fecundity and fertility of mosquitoes. J. Appl. Entomol. 125: 31-35.

24. Riebeiro, B., Guedes, R., Oliveira, E., and Santos, J. 2003. Insecticide resistance and synergism in Brasilian populations of Sitophilus zeamais (Coleoptera: Curculionidae). J. Stored Prod. Res. 39: 21-31.

25. Simmonds, N.W. 1976. Evolution of Crop Plants. Longman Editions, London, UK, 340 pp.

26. Simmonds, M.S.J., Blaney, W.M., Ley, S.V., Savona, G., Bruno, M., and Rodríguez, B. 1989. The antifeedant activity of clerodane diterpenoids from Teucrium. Phytochemistry 28: 1069-1071.

27. Tripathi, S.M. and Singh, D.K. 2000. Molluscicidal activity of Punica granatum bark, Canna indica root. Braz. J. Med. Biol. Res. 33: 1351-1355.

28. Voravuthikunchai, S.P., Limsuwan, S., Supapol, O., and Subhadhirasakul, S. 2006. Antibacterial activity of extract from family Zingiberaceae against food borne pathogens. Journal of Food Safety 26: 325-334.

29. Weston, P.A. and Rattlingourd, P.L. 2000. Progeny production by Tribolium castaneum (Coleoptera: Tenebrionidae) and Oryzaephilus surinamensis (Coleoptera: Silvanidae) on maize previously infested by Sitotroga cerealla (Lepidoptera: Gelechiidae). J. Econ. Entomol. 93: 533-536.

30. Wheeler, D.A. and Isman, M. 2001. Antifeedant and toxic activity of Trichilia americana extract against the larvae of Spodoptera litura. Entomol. Exp. Appl. 98: 9-16.

31. Wink, M. 1993. Production and application of phytochemicals from an agricultural perspective. Pages 171-213. In: Phytochemistry and Agriculture. T.A. Van


Beek, and H. Breteler, Ed. Editions Clarendon, Oxford, UK.

32. Xie, Y.S., Bodnaryk, R.P., and Fields, P.G. 1996. A rapid and simple flour-disk bioassay for testing substances active against stored product insects. The Canadian Entomologist 128: 865-875.

33. Zhao, B., Grant, G.G., Langevin, D., and MacDonald, L. 1998. Deterring and inhibiting effects of quinolizidine alkaloids on the spruce budworm (Lepidoptera: Tortricidae) oviposition. Environ. Entomol. 27: 984-992.

----------------------

Tunisian Journal of Plant Protection

Plant Protection News

Report

on

The First Africa- International Allelopathy Congress(AIAC-2014)

Sousse, Tunisia, February 6

The First Africa-International

Allelopathy Congress (AIAC-2014) took place under the title “Allelopathy: Looking Ahead” on February 6–9, 2014 at Sousse, Tunisia. This theme is meant to convey the realization that allelopathy is safe alternative to sustain development in agriculture and forestry and to maintain

a clean environment for our future generations.organized by the TunisSustainable Agriculture Tunisienne pour une Agriculture Durable, ATAD) , the Agronomic Higher Institute of ChottAgronomique de Chott

Tunisian Society for Sustainable

Agriculture (ATAD)

Agronomic Higher

of Chott

Vol. 9, No. 1, 2014

Plant Protection News

International Allelopathy Congress

Sousse, Tunisia, February 6 - 9, 2014

a clean environment for our future generations. This congress was jointly organized by the Tunisian Society for Sustainable Agriculture (Association Tunisienne pour une Agriculture Durable,

, the Agronomic Higher Institute of Chott-Mariem (Institut Supérieur Agronomique de Chott-Mariem, ISA-

Agronomic Higher Institute

of Chott-Mariem (ISAChM)


ChM) and the Regional Center of Research on Horticulture and Organic Agriculture of Chott-Mariem (Centre Régional des Recherches en Horticulture et Agriculture Biologique de Chott-Mariem, CRRHAB-ChM).

It was sponsored by the Ministry of Agriculture, the Institution of Agricultural Research and Higher Education (Institution de la Recherche et de l’Enseignement Supérieur Agricoles, IRESA), the Ministry of Higher Education and Scientific Research, the University of Sousse, ISA-ChM, The Doctoral School Agronomy and Environment of ISA-ChM, CRRHAB-ChM, the Technical Center of Organic Farming, the Research Unit “ Agrobiodiversité” in ISA-ChM, the Faculty of Sciences of Bizerte, the Olive Institute, The Center of Biotechnology of Borj Cedria, the Istituto Italiano di Cultura Tunisi, and the company Adiegio Hermanos S.A (CIF A50024181)-DIV. Catsaigner.

The opening ceremony was inaugurated by Dr. Mejda Daami-Remadi, the President of ATAD who welcomed all the participants and addressed a special welcome to the honorable guests namely Mr The governor of Sousse, Prof. Fayçal Mansouri, the president of the University of Sousse, Prof. Shamsher Sam Narwal, the representative of the International Allelopathy Foundation, Dr. Abdelhamid Boujelbene, the Director General of ISA-ChM, Dr. Messaoud Mars, the Director General of CRRHAB-ChM, the official representative of IRESA, and Dr. Rabiaa Haouala, the convener of AIAC-2014.

In the opening ceremony, three speakers have presented their special speeches devoted to this event namely Dr.

R. Haouala who has introduced the congress, Prof. F. Mansouri who has highlighted the interest of organizing such events with this high number of partipants from different nationalities and Dr. M. Mars who has officially opened the congress as the official representative of IRESA.

About 146 participants, from abroad and Tunisia, have attended this first African congress: Tunisia (77 participants), Pakistan (10 participants), India (11 participants), Algeria (17 participants), Iraq (3 participants), Turkey (6 participants), China (3 participants), Hungary (5 participants), Italy (4 participants), Nigeria (4 participants), Egypt (2 participants), Japan (1 participant), South Africa (1 participant) and Belgium (2 participants).

The organizers succeeded in compiling an attractive scientific program which included 12 plenary lectures and conferences, presented by the most relevant scientists worldwide known by their original and reference work on allelopathy or allelopathy related topics namely Prof. Shamsher Sam Narwal (India), Prof. Ibrahim S. Alsaadawi (Iraq), Prof. Moncef Ben-Hammouda (Tunisia), Prof. Zahid Ata Cheema (Pakistan), Prof. Mounir Mekki (Tunisia), Prof. Shiming Luo (China), Prof. Ombir Singh (India), Prof. Marina Della Greca (Italy), Prof. Hisashi Kato-noguchi (Japan), Dr. Rabiaa Haouala (Tunisia) and Prof. U.K. Sahoo (India). These key lectures represented an update and evolution of allelopathy definitions, concepts, and applications mainly for weed management and increase of crop yields.

The scientific programme included 35 Oral Presentations and 78 Posters


dealing with the seven themes treated in the congress: • Allelopathy in sustainable and

organic agriculture • Allelopathy in natural ecosystems • Allelopathy in soil sickness • Chemistry of allelochemicals • Molecular biology and genetics of

allelopathy • Physiology and biochemistry of

allelopathy • Allelopathy mechanisms and

interactions Summaries of all contributions

have been compiled in an abstract book distributed to all participants and some of the most relevant participations were published in Allelopathy Journal and The Tunisian Journal of Plant Protection.

It should be highlighted that during this congress, a committee has been elected to plan the foundation of the African Allelopathy Society (AAS) and the coming African Allelopathy Congresses. It was agreed during the meeting of the present African participants that the next

Africa-International Allelopathy congress will be organized in Egypt on 2016.

This symposium offered a great opportunity for all participants to promote the cooperation and the collaboration between Tunisian and foreign scientists working in the field of allelopathy, to share their experiences, findings and issue ideas on allelopathy research. The last day of the congress was dedicated to an excursion to Mahdia and Monastir.

At the closing ceremony, all the participants congratulated the organizing and the scientific staff and expressed their satisfaction with the content of the congress, the scientific organization and promised to participate to similar events that will be organized in this excellent welcoming country, Tunisia.

Dr. Mejda Daami-Remadi President of ATAD (CRRHAB ChM),

& Dr. Rabiaa Haouala General Secretary of ATAD (ISA ChM)

and Convener of the Congress


Ministère de

l’Enseignement

Supérieur et de

la Recherche

Scientifique

ا�� وزارة�� ا� وا��ا�

Association Tunisienne

pour une Agriculture

Durable

Institut Supérieur

Agronomique de

Chott-Mariem

Ecole Doctorale

"Agronomie et

Environnement" de

l'ISA Chott-Mariem"

Gouvernorat de Sousse

Ministère de

l’Agriculture

وزارة ا��

I S Ac h o t t M é r i e m

Vol. 9, No. 1, 2014

Ministère de

Sponsoring Institutions:

Ministry of Agriculture

Institution of Agricultural

Research and Higher Education

Ministry of Higher Education

and Scientific Research

University of Sousse

Agronomic Higher

Institute of Chott-

Mariem

Regional Center of

Research on Horticulture

and Organic Agriculture of

Chott-Mariem

Technical Center of

Organic Farming

Research Unit

“AGROBIODIVERSITE”

Faculty of Sciences of Bizerte

Olive Institute

Center of Biotechnology of Borj

Cedria

Istituto Italiano di Cultura

Tunisi

Governorate of Sousse

Sponsoring Company

Adiegio Hermanos SA (CIF

A50024181)-DIV. Catsaigner


Report

on

The International Short Course on Allelopathy (ISCA)

The Agronomic Higher Institute of ChottSousse, Sousse, Tunisia, February 10

Allelopathy is a new and potential

field of research, it provides basis to sustainable agriculture. Hence, currently, allelopathy research is being undertaken in many countries worldwide. The

indiscriminate use of pesticides for pest management (Weeds, Insand Pathogens) has resulted in serious ecological and environmental problems. Some agricultural commodities may

Tunisian Society for Sustainable

Agriculture (ATAD)


of Chott

Vol. 9, No. 1, 2014

The International Short Course on Allelopathy (ISCA) - 2014

of Chott-Mariem, University of Sousse, Sousse, Tunisia, February 10-15, 2014

indiscriminate use of pesticides for pest management (Weeds, Insects, Nematodes, and Pathogens) has resulted in serious ecological and environmental problems. Some agricultural commodities may


of Chott-Mariem (ISAChM)


contain minute quantities of pesticides residues, with long term adverse effects on human and livestock health. Thus, serious ecological questions about the reliance on pesticides for pest control have been raised. As allelopathy is a new field of science, there is less awareness among agricultural scientists about the scope and input of allelopathy in agricultural and biological sciences.

An International Short Course on Allelopathy (ISCA) - 2014 was jointly organized by the Tunisian Society for Sustainable Agriculture (Association Tunisienne pour une Agriculture Durable, ATAD) and the Agronomic Higher Institute of Chott-Mariem (Institut Supérieur Agronomique de Chott-Mariem, ISA-ChM).

This course aimed to make strong foundations of young future allelopathy researchers with (i) background knowledge, (ii) recent advances in allelopathy and (iii) methods of allelopathy research. This course was designed to impart (a) theoretical knowledge, (b) practical experience of allelopathy research and (c) how to describe the research results effectively in research papers.

The coordinator was Prof. S.S. Narwal, the Chief Editor of Allelopathy Journal and a member of International Allelopathy Foundation, and the co-coordinator was Dr. Rabiaa Haouala, Assistant Professor in Eco-Physiology at the ISA-ChM.

A number of 31 participants from Hungary, Nigeria, Algeria and Tunisia have attended this international course in addition to ATAD staff. This course included theoretical and practical sessions.

The lecture of Prof. Shamsher S. Narwal began with an introduction on the history of allelopathy. Then, the scientist presented the allelopathy in multiple cropping systems with main focus on crop rotations and intercropping/crop mixtures and also presented an overview on the application of allelopathy for weed management with limited or no use of herbicides.

The course of Prof. Ibrahim S. Al Saadawi (Department of Biology, College of Science, Baghdad University, Baghdad, Iraq) was more focused on the methods of collection, extraction, isolation and identification of root exudates with allelopathic potential. Prof. Al Saadawi also presented a review on the role of allelopathy in weed management by non-herbicidal methods.

The next conference was presented by Prof. Ibrahim I. Ozigit (Biology Department, Faculty of Arts & Science, Marmara University, Goztepe, Istanbul, Turkey) and was more focused on application of biotechnology by using plant tissue cultures and plant gene transfer systems for enhancing the mass production of allelochemicals.

Prof. Moncef Ben Hammouda (Higher School of Agriculture of Kef, University of Jendouba, Tunisia) also participated to this forming session by presenting a key conference on allelopathy in crop production based on practical findings.

The practical formation was animated by Prof. Narwal, Prof. Al Saadawi and Prof. Ozigit and was divided in different sessions i.e. Practical-I: Fallow lands, Practical-II: Crop fields, Practical-III: Natural forests, Practical-IV: Planted forest, Samples’ collection, Processing, Storage, Lab. bioassays,


Preparation of extracts/leachates, Conducting of lab bioassays, Preparation of plant tissue cultures, Installation of pot culture and field experiments.

At the closing ceremony of the course, all participants were evaluated based on data collecting, analysis and

interpretation before receiving their certificates of participation.

Dr. Rabiaa Haouala

General Secretary of ATAD (ISA-ChM) and the Coordinator of the Congress

& Dr. Mejda Daami-Remadi President of ATAD (CRRHAB-ChM)


Recent Doctorate Theses in Plant Protection (2013/14)

Mdellel, Lassaad. 2013. Bioecology of the brown peach aphid Pterochloroides persicae Cholodovsky 1899 (Hemiptera, Aphididae) and the biotic potential of its parasitoid Pauesia antennata Mukerji 1950 (Hymenoptera, Braconidae). Doctorate Thesis in Agronomic Sciences (Plant Protection and Environment), ISA Chott Mariem, University of Sousse, Tunisia, 158 pp. (Public Defense: 12 October 2013)

This work deals with the

bioecology of the brown peach aphid Pterochloroides persicae Cholodovsky 1899 and the biotic potential of its parasitoid Pauesia antennata Mukerji 1950. Hence, four aspects have been investigated to carry out such a task. The first part is a P. persicae morphometric study and its genetic diversity in terms of its geographical distribution and host plants. Indeed, the morphometric study of different instars of aphid showed an allometric growth depending on the age and 5 antenna articles for the two first instars and 6 for the other stages. Our results showed that morphometry is slightly affected during the dispersal of the insect from a geographical site to another and from a host plant to another. The analysis of the mitochondrial DNA of P. persicae on different host plants and various geographic sites revealed the presence of two haplotypes in Tunisia.

The second part focuses on the biological particularities of P. persicae in Tunisia. Results revealed the presence of the aphid on peach, almond, plum, apricot and apple. The study of its biological features in laboratory conditions showed that the larval development duration of P. persicae is about 22.09 days at 20 ± 1°C, a humidity rate up to 70 ± 10% and a photoperiod of 14L/10D. Data concerning the mean relative growth rate and the generation mean doubling time revealed that peach and temperature of 20 ± 1°C are favorable to P. persicae multiplication. As for irrigated peach, aphid was observed on the aerial parts during winter, spring and summer and on the roots in autumn. However, it was observed on the aerial parts of different Prunus cultivated in dry conditions. The analysis of the effect of the phloem sap pressure and the sap mineral composition showed that the pest needs a pressure higher than (-7 bars) and a nitrogen


concentration more than 0.50% which required to install aphid.

The third part deals with the prospecting and the identification of the natural enemies of P. persicae and evaluation of their efficiency. Results showed the presence of Coccinella algerica Kovar 1977 (Coccinellidae), Episyrphus balteatus De Geer 1776 and Metasyrphus carollae Fabricus 1794 (Syrphidae) and Chrysoperla carnea Stephans 1836 (Chrysopidae), and two entomopathogenic fungi Beauveria bassiana and Metacordyceps lianshanensis.

E. balteatus and M. carollae instars predatory efficiency on P. persicae study showed a daily predation rate of three individuals. C. algerica is able to consume 30 individuals during its larval development period, whereas an

adult consumes only 9.18 individuals per day.

The fourth part of this work is about biotic potential of P. antennata parasitoid. Parasitic activity study showed longevity of adult of 3.90 days, a fertility rate of 26.73, a larval development average of 14.48 days, a parasitism and emergence rate and sexual rate which vary with aphid population size and numeric importance of parasitoid.

The overall results are promising and incite us to explore the possibility of P. antennata mass rearing and also the possibility of its use in the field in a biological control program against P. persicae.

--------------------

Harbaoui, Kalthoum. 2013. Genetic diversity and population structure of Phytophthora infestans: The key to understand late blight in Tunisia. Doctorate Thesis in Agronomic Sciences (Plant Science), INAT, University of Carthage, Tunisia, 124 pp. (Public Defense: 28 November 2013)

In Tunisia, late blight caused by

Phytophthora infestans is a serious threat in potato and tomato. The Mediterranean weather conditions can be conducive for infection in all seasons when host crops, tomato and potato, are grown year round. Potato is planted and harvested in two to four overlapping intervals from August to June and tomato is grown yearly both in open fields and in greenhouse. In order to characterize the population of P. infestans

in Tunisia, we conducted a survey in which a total of 165 P. infestans isolates were collected from five sub-regions in Tunisia (Bizerte, Nabeul, North-West, Tunis area and Chott-Mariem) between 2006 and 2008 which were studied for (i) mating types distribution; (ii) genotypic diversity through nuclear (SSR) and mitochondrial (haplotypes) markers; ii) metalaxyl sensitivity; (iii) virulence and aggressiveness, and (iv) host specificity.


The mating type of 165 P. infestans isolates collected from five regions in Tunisia was identified using CAPS markers in order to distinguish between A1 and A2 genotypes. Of the 165 isolates tested, 141 (85%) were of the A1 mating type and 24 (15%) were of the A2 mating type. The A2 genotype was detected in both North (Bizerte) and North-East (Korba) only. All tested isolates from tomato (29 isolates) had the A1 mating type while both A1 and A2 strains were detected in potato.

Furthermore, a representative set of Tunisian P. infestans isolates selected according to geographic diversity was used to analyze either virulence or aggressiveness diversity. Thirty-one isolates were tested for virulence onto detached leaves of a differential set of Solanum demissum having 11 late blight resistance R genes. Of these isolates, twenty-one races were characterized. P. infestans isolates showed different virulence patterns depending on sampling regions. Thirty-six isolates were also tested for aggressiveness onto detached leaves of cultivar ‘Bintje’.

The presence of high level of aggressiveness in potato isolates but not in tomato could inform that P. infestans population in Tunisia undergoes changes in potato crops but not in tomato. On the other hand, the most effective R genes recorded in Tunisia are R5, R9 and R8 that withstood more than 90% of tested isolates. These R genes could be useful in breeding programs by their introgression in crop species. To diagnose virulence in

P. infestans, we need a useful and high throughput technique reliable and linked to the bioassay screening that could replace neutral markers (allozymes, SSR markers, mtDNA). Combining phenotypic (disease bioassay) and genotypic techniques (virulence markers) could be a very interesting tool to identify the pathogen population structure and find a rapid solution for the break-down of resistance of potato cultivars.

Elsewhere, we studied the specificity of the pathogen-host interaction by analyzing the expression patterns of epic1 gene using RT-PCR technique. The epic1 gene is involved in degradation of plant defense metabolites against P. infestans. The expression of epic1 gene proved the specificity of Tunisian strains to their hosts. This could explain any preference or specificity of the strains for their interaction with one plant versus the other. Such result has an important impact on the crop rotation and the infection from tomato fields to potato ones.

By the use of SSR markers, genotypic diversity of the population collected from the North regions of Tunisia seems to be highly important. Phylogenetic analysis revealed the presence of a major clonal lineage (NA-01, A1 mating type, Ia mtDNA haplotype). Isolates belonging to this clonal lineage showed a relatively simple virulence pattern on a potato differential set carrying different S. demissum R-genes. Next to this clonal lineage, a group of isolates was found that showed a high


genetic diversity, comprising both mating types and a more complex race structure and also highly resistant to metalaxyl.

The population on potato and tomato seems to be under different selection pressures. Isolates collected from tomato showed a low genetic diversity even though potato isolates collected simultaneously from the same location showed a high genetic diversity. Based on the SSR profile comparison, we could demonstrate that the four major clonal lineages found in the Netherlands and also in other European countries could not be found in Tunisia. Despite the massive import of potato seeds from Europe, the P. infestans population in Tunisia is still clearly distinct.

The uncovered dynamics of the P. infestans population in Tunisia compel continued monitoring of this pathogen and may require adaptation of the control strategies for late blight in Tunisia. This full survey of the diversity, structure, behavior of the highly damaging pathogen in Tunisia could be considered as a scope on the situation of this disease in a North African area still for long time ambiguous. Based on what is reported in this research, it could be highly interesting to follow these reliable surveys in order to peel annual genetic changes of this pathogen population.

------------------ Ladhari, Afef. 2013. Cytological, physiological and biochemical aspects of plant-plant interactions and plant microorganisms. Study of some species of the Capparidaceae family. Doctorate Thesis in Biological Sciences, Faculty of Sciences of Bizerte, University of Carthage, Tunisia, 185 pp. (Public Defense: 17 December 2013)

The evaluation of two

Capparidaceae, growing in Tunisia (Cleome arabica and Capparis spinosa), allelopathic potential and isolation, purification and identification of their active molecules are the aims of this study. A screening of the aqueous and the organic extracts of all their organs was carried out for their herbicidal and insecticidal activities. Two crops (lettuce and radish) and two weeds (peganum and thistle) were chosen as target species to test the first activity and the larvae of

Spodoptera littoralis, for the second. The isolation and purification of the active molecules were based on bio-guided chromatographic techniques, and the pure compound identification was provided by NMR 1H and 13C. The mode of action of allelochemicals was determined on lettuce. The extracts induced a percentage inhibition varying between 2 and 100% for germination and between 4 and 100% for seedling growth, according to the concentration, the organs and the target species. C. arabica siliquae and C.


spinosa leaves, exhibited the strongest phytotoxicity, hence they were chosen to isolate and identify their active molecules. Three flavonoids were identified from C. spinosa leaves:

quercetine-3-O-β-D-glucopyranoside, which was the most toxic, kaempferol-3-O-β-D-glucopyranoside and quercetin. For C. arabica siliquae, five compounds

were identified: 11-α-acetylbrachy-

carpone-22(23)-ene, β-sitosterol, 17-α-hydroxycabraleactone, amblyone, and calycopterin. The first one was the most toxic. The phytotoxicity seems to be due to the cytotoxic effect, which revealed a significant decrease in the mitotic index of root cells coupled with morphological changes and necrosis. In addition, inhibition of germination seems to be linked to the membrane deterioration, revealed by a strong electrolyte leakage and increased content of malondialdehyde (MDA). An increase in production of

proline (antistress substance) and secondary metabolites, having antioxidant properties such as polyphenols and flavonoids, was registered in lettuce seedlings. This accumulation was the result of the registered lyase (PAL and TAL) activity stimulation, involved in their biosynthesis. A reduction in carotenoid content was also recorded, which would be involved in the lettuce growth reduction. Finally, all extracts showed a strong insecticidal activity toward S. littoralis larvae. Results demonstrated the richness of these two plants on biomolecules with herbicide and insecticide potential, which encourages their consideration as a renewable source of substances that can be used in the agro-ecosystems management to reduce our dependence on the very harmful industrial inputs.

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Omezzine, Faten. 2013. Colchicine effect on the allelopathic potential of Trigonella foenum-graecum L. Doctorate Thesis in Biological Sciences, Faculty of Sciences of Bizerte, University of Carthage, Tunisia, 195 pp. (Public Defense: 17 December 2013)

The present study was conducted

to evaluate the effect of developmental stage and the genetic load on the chemical composition and allelopathic potential of fenugreek. The genetic load enhancement was obtained by immersing pre-germinated fenugreek seed in 0.05% colchicines, giving mixoploid plants (M),

which will be compared to those from non-treated seeds: Diploid (D) plants. Plants (D and M) were harvested at vegetative, floral and fruiting stage. The difference between genetic load of the two types of plants was confirmed by the flow cytometry analysis, which provided profiles with a single peak (2n) for plants


D and with four peaks (2n), (4n), (6n) and (8n) for plants M. In addition, mixoploid plants have shown increased rates of photosynthesis, stomatal conductance, transpiration and secondary metabolite production, particularly at the flowering stage, where the compound groups (polyphenols, flavonoids, alkaloids, tanin) concentrations are doubled. This production increase could be a result of the activity stimulation of enzymes involved in their biosynthesis: phenylalanine ammonia-lyase (PAL) and tyrosine ammonia lyase (TAL). Chemical analysis by LC-MS/MS detected eleven flavonol glycosides compounds, including five, identified for the first time in fenugreek aerial parts. This analysis revealed, also, differences between the composition of plants D and M, under the influence of mixoploidy: some compounds have gone others have appeared. Allelopathic activities were evaluated on Lactuca sativa and two phytopathogenic fungi: Fusarium oxysporum f. sp. radicis-lycopersici (FORL) and Fusarium oxysporum f. sp. lycopersici (FOL). All extracts showed a highly significant toxicity for lettuce and the highest aqueous extract toxicity was recorded with the material collected at the vegetative stage for the two types of plants. For organic extracts, ethyl acetate and methanol fractions were the harmful, especially those corresponding to the fruiting stage for D and vegetative one for M. For antifungal potential, these same

extracts of plants D harvested at fruiting stage, were the most effective in reducing the mycelial growth. For plants M, the hexane fractions corresponding to the vegetative and flowering stages exhibited the highest inhibitory effect. To determine their mode of action, the aqueous extracts were used as biotic stress factor on lettuce. It appears that germination inhibition was due to membrane deterioration, proved by a strong electrolyte leakage and increased content of malondialdehyde (MDA), and a mitochondrial respiration disruption due to a decrease in dehydrogenases activity. For seedling growth, the roots showed the same disturbances and in aerial part a reduction in the levels of pigments (chlorophyll b and carotenoids) was registered. The lettuce seedlings have circumvented this allelochemical stress, by increasing the PAL and TAL activity, accumulating the proline (anti-stress substance) and producing secondary metabolites with antioxidant power, such as polyphenols, flavonoids and alkaloids. It seems that mixoploidy could be an easy and effective biotechnological tool to improve the production of molecules of interest in high demand. Also, the recognition of the most productive developmental stage, is a great contribution to optimize the operation of a given plant allelopathic potential.

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El-Imem, Mohamed. 2014. Study of host-plant-thrips-predator interactions in the Tunisian Sahel region and different control ways evaluation: particular case of Frankliniella occidentalis Pergande 1895 (Thysanoptera; Thripidae) on pepper crop. Doctorate Thesis in Agronomic Sciences (Plant Protection and Environment), ISA Chott Mariem, University of Sousse, Tunisia, 259 pp. (Public Defense: 26 June 2014)

The taxonomic study of thrips

collected from pepper crops in the Sahel region showed the presence of one phytophagous species; Frankliniella occidentalis Pergande (1895) (Thysanoptera, Thripidae) commonly known as the Western Flower Thrips (WFT).

The monitoring of the populations of this species on pepper using different appropriate and complementary sampling methods allowed identifying the optimal development periods in relationship with the climatic conditions and the phenology of the host plant. The population growth was recorded during the months of April, May and early June.

The assessment of the density of males and females showed that the number of males decreases towards the summer. The sex ratio follows the same pattern.

The populations of F. occidentalis are a complex of three distinct morphs: yellow, brown and intermediate. Their abundance is highly dependent on climatic conditions, including temperature.

The vertical distribution of populations of F. occidentalis on pepper plants is homogeneous during the cold period, but becomes heterogeneous from

March with a concentration of individuals at the apex of plants compared to middle and basal regions.

We showed that the availability of Chlorophylls a (Ca), Chlorophylls b (Cb) and Xanthophylls in the leaves and the fruits facilitates thrips infestations. It is the same for the richness of flowers in pollen. Also, severely attacked plant organs showed a decrease in Ca and Cb Chlorophylls, Xanthophylls.

Three species of Hemipteran Anthocorid species predators of thrips on Chrysanthemum coronarium Linnaeus (Asterales; Asteraceae) were identified in the Sahel region of Tunisia: Orius laevigatus Fieber (1860), O. albidipennis Reuter (1884) and O. majusculus Reuter (1879). O. laevigatus is the most abundant species, its populations increase with the summer season.

Local strain O. laevigatus is more effective in controlling the populations of F. occidentalis on pepper that the commercial strain of Koppert ® Holland. Use of the predatory mite Amblyseius swirskii Atthias-Henriot (1962) (Acari; Phytoseiidae) Koppert ® Hollande showed good results.

The use of the sex pheromone of F. occidentalis showed that the traps provided with pheromone lures capture


more adults than those without pheromone and they catch as many females as males, which improves their efficiency. Moreover, the attractiveness of blue sticky traps equipped with kairomone capsules is more important than blue sticky traps without kairomone.

The study of the impact of Spinetoram and Oxymatrin (plant extract

based products) did not reveal significant differences between the three applied doses that cause mortality rates between 40 and 80% for the first product and 30 to 60% for the second one.

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Instructions to Authors

The is published in English twice a year. Tunisian Journal of Plant ProtectionContributions on all aspects of plant protection are considered. Papers are evaluated by highly qualified national and international scientists of the related disciplines. The original manuscript should be ubmitted to the Editor-in-Chief :s Prof. Bouzid NasraouiInstitut National Agronomique de Tunisie, 43 Avenue Charles Nicolle, 1082 Tunis-Mahrajène, TunisiaEmail: @iresa.tntjpp Phone: (+216) 98 29 29 17 Submitted manuscript must not be published or submitted for publication elsewhere. Manuscripts should be emailed on Microsoft Word and Microsoft Excel. It should be typed on one side page (A4 format) in double space (Times New Roman, 12), starting by the title in the center, followed by author names and their addresses. Three abstracts are required : English, Arabic and French. The two latter come at the end of the manuscript. When necessary, the may translate Journalthe English abstract into Arabic and/or French. Abstracts start with the title of the manuscript and finish by the keywords with seven words at most. The other sections of the manuscript are : Introduction, Materials and Methods, Results, Discussion, Acknowledgement (if necessary), French and Arabic abstracts, and Literature Cited. Not divided short communications are also published.and review articles Tables should be cited in numeric order in the manuscript. They must be intelligible without reference to the text. The title should summarize the information presented in the table. Subheadings should be brief. Abbreviations are accepted but those which are not standard must be explained in footnotes. Figures should also be presented in numeric order. Captions should describe the contents, so each illustration is understandable when considered apart from the text. Color photos are published only on-line; they are black and white on the paper version. References in the text should be cited by their numbers. In the Literature Cited, references must be reported in their original language when latin alphabet is used, otherwise, they have to be translated to English between square brackets and their original language mentioned between brackets. They should be numbered and arranged in alphabetical order, with author last names, their initials, publication year, title, journal or other source, volume and page numbers. Examples below must be followed.Article in a journal :Haddar, T. 1989. Check list of weeds naturally infesting agricultural fields in northern Tunisia. Arab

Journal of Plant Protection 7: 174-178.Chapter in a book :Nasraoui, B. and Lepoivre, P. 2003. Les champignons phytopathogènes. Pages 111-143. In:

Phytopathologie, bases moléculaires et biologiques des pathosystèmes et fondements des stratégies de lutte. P. Lepoivre, Ed. Editions De Boeck Université. Belgium.

Proceedings :Medini, M., Hamza, S., Harrabi, M., and Lamari, L. 2003. Virulence of Mycosphaerella

graminicola from Tunisia, Algeria and Canada. Pages 14-44. In: Proceedings of the 6 th

International Symposium on and Diseases of Cereals. December 8-Septoria Stagonospora12, 2003, Tunis, Tunisia.

Book :Jarraya, A. 2003. Principaux nuisibles des plantes cultivées et des denrées stockées en Afrique du

Nord: leur biologie, leurs ennemis naturels, leurs dégâts et leur contrôle. Maghreb Editions, Tunisia, 415 pp.

Thesis :Horrigue-Raouani, N. 2003. Variabilité de la relation hôtes-parasites dans le cas des Meloidogyne

spp. (Tylenchida, Meloidogynidae). Doctorat Es-Sciences Naturelles. Faculté des Sciences de Tunis, Université Al-Manar, Tunisia, 222 pp.


CONTENTS

Vol. 9 No. 1 JUNE 2014

TUN ISIA N JOU RNA LOF PLANT PROTECTION

Plantae Senae in Terra Sena

ABIOTIC ASPECTS01. Allelopathic potential of fe rulic acid on tomato. N.B. Singh and Sunaina.(Ind ia)

11. Alle lopath ic effects of aqueous extrac ts ofEucalyptus occ identalis, Acacia amplicepsand Prosopis juliflora on the germ ination of three cultivated spec ies.E. Saadaoui, N.Ghazel, Ch. Ben Romdhane, N. Tlili, and A. Khaldi.(Tunisia)

MYCOLOGIC AL ASPECT S17. Antifungal activity of culture filtrates and organic extra cts of Aspergillus spp.

against Pythium ultimum. R. A ydi-Ben Abdallah, M. Hassine, H. Jabnoun-Khiareddine,R. Haouala and M. Daam i-Remadi.(Tunisia)

31. Chitosan and Trichoderma harzianum as fungicide alternat ives for controllingFusar ium crown and root rot of tomato. R .S.R. El-Mohamedy, F. Abdel-Kareem, H.Jabnoun-Khiareddine, and M. Daami-Remadi.(Egypt/Tunisia)

45. Control of root rot disea ses of tomato plants caused byFusarium solan i, Rhizoctoniasolani and Sclerotium rolfsii using dif ferent chemical plan t resistance inducers.R.S.R. El-M ohamedy, H. Jabnoun-Khiareddine , and M . Daami-Rem adi.(Egypt/Tunisia)

ENTOMOLOGICAL ASPECT S57. Chemical composition and fumigant toxicity of Artemisia absinthium essent ial oil

against Rhyzopertha dominica and Spodoptera littoralis. N. Dhen, O. Majdoub, S.Souguir, W. Tayeb, A. Laarif an d I. Chaieb. (Tunisia)

67. Chemical constituents and toxicity of essential oils from three Asteraceae plantsagainst Tribolium confusum . D. Haouas, P.L. Cioni, M. Ben Halima-Kamel, G. Flamini,and M.H. Ben Hamouda,. (Tunisia/Italy)

83. Chem ical composition of Ruta chalepensis essent ial oils and their insectic idalactivity against Tribolium castaneum. O. Majdoub, N. Dhen, S. Souguir, D. Haouas, M.Baouandi, A. Laarif, and I. Chaieb.(Tun isia )

91. Insecticidal act ivit ies of fruit pee l extrac ts of pom egranate (Punica granatum)against the red flour beetle Tribolium castaneum . A. Ben Ham ouda, A. Mechi, K.Zarred, I. Chaieb, and A. Laarif.(Tunisia)

Photo of the cover pa ge: Pythium lea k of pota to (Cou rtesy R ania Ayd i-Ben Abdallah)

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