Journal of Entomology and Zoology Studies 2016; 4(2): 230-235

E-ISSN: 2320-7078 P-ISSN: 2349-6800 Insecticidal effect of visnaga L. (: JEZS 2016; 4(2): 230-235 © 2016 JEZS Apial) methanolic extract against a citrus pest, Received: 22-01-2016 Accepted: 26-02-2016 Toxoptera aurantii (Aphididae: Homoptera) under

Brahmi Fairouz controlled conditions Laboratory of research: Naturals products, Nutritional aspects and Biologic activity, University of Brahmi Fairouz, Chahbar Nora, Kebbouche Gana Salima Tlemcen, Algeria.

Abstract Chahbar Nora Department of biology, faculty L. (Apiaceae) is an endemic Algerian aromatic , largely used in traditional medicine, of sciences, University M’hamed and mostly for renal problems with a very few reported studies on its insecticidal activity. Current work BOUGARA of Boumerdès, is a contribution to the insecticidal effect of methanolic extract of aerial parts of Ammi visnaga collected Algeria at various stages of growth against Toxoptera aurantii. Eight different extract of six different doses were tested which showed a proportional relation between mortality and the applied doses. The maximum of Kebbouche Gana Salima mortality (98.33%) was obtained for the highest dose (30000 ng/aphids) after 24h for steams extract Department of biology, faculty collected before flowering. Cumulative mortality of the treated aphids occurred in the second day with a of sciences, University M’hamed rate of 100%. The LD50 value determined using the linear regression was 310.53 ng/ aphids < [D5-D6] for BOUGARA of Boumerdès, the said dose. Algeria Keywords: Apiaceae, Ammi visnaga L., Toxoptera aurantii, methanolic extract, controlled conditions

Introduction Aphids cause extensive damages on reducing the quality and the quantity of Agricultural products [1]. Because of its efficacy and the simplicity of its application, the chemical control (Foliar insecticides) constitute the most rapid and practical way to kill aphids [2]. Abusive use

of insecticides causes several problems like pests’ resistance, significant damages on human health and on the environment. As a response to this situation, the research of biocide was more than necessary [3]. Sevral of Apiaceae are known for their powerful acaricidal and insecticidal effect against desert locust, tick and mosquito [4-6]. Ammi visnaga. is an endemic Algerian aromatic plant. It’s a biannual herb measured between 0.8-1m high with

robust stem all covered with leaves, white 5 to 11.4 cm diameter appears around June. [7] Its ovoid small seeds (2 - 2.5mm) are brownish to greenish brown with violet tinge . This Mediterranean plant is rich in coumarins, flavonoid, essential oil and fixed oil [8]. The major

active components of the plant are and visnagin (furanochromones) which have been both reported from the fruits of A. visnaga [9-11]. This plant is used in the traditional medicine [11-13] [8] [14] for the treatment of urinary problems , cardiovascular problems , diabetes , vitiligo [15-17], psoriasis [11], painful menstruation [16], menstruation regulation [16], teeth problems [8] and [10-16] [16] asthma . Khella have several properties including: diuretic , antispasmodic, [10-12] [15] [19] [19] antilithiastic , antioxidant , antibacterial and fungicidal . In addition, essential oil and of extract of A. visnaga have been reported to be a powerful pesticide against Mayetiola [4.20] destructor and Culex quinquefasciatus . The aim of this study was to investigate the insecticidal effect of methanolic extract of aerial

parts of A. visnaga at different stage of growth collected from Boumerdes (center of Algeria) against the black citrus aphid Toxoptera aurantii under laboratory conditions.

Materials and Methods Biological materials Correspondence Chahbar Nora Pests Department of biology, faculty Aphides adults (T. aurantii, Aphididae: Homoptera) used in this study were deduct from of sciences, University M’hamed Citrus sinensis, Thomson variety from an experimental private station located at Boumerdes BOUGARA of Boumerdès, (45 Km east of Algiers). The infestation of plants with T. aurantii was natural. Algeria ~ 230 ~ Journal of Entomology and Zoology Studies

Vegetal material the northern Algeria) respectively in April, June and July 2014 The vegetal material constituted of the aerial parts of the plant (Table 1). After that, the plant samples were dried in the shade A. visnaga. (Leaves, stems, flowers and seeds) which were for 20 days at room temperature. A voucher specimen was collected at different period of growth stage: before flowering, deposited for identification at the laboratory of agricultural flowering and fructification period from Bordj-Menaiel zoology and forestry in the national school of agronomy (Latitude: 33°44´30´´; Longitude: 3°43´23´´; Altitude: 33 m, in (INA), Algiers, Algeria (BRAH FAI, Av 15/012).

Table 1: Harvest period, used parts and doses of A. visnaga.

Plant Harvest period Used parts Doses ng /aphids Abreviation 3. 104 D1 April 2014 (before flowering) Leaves, stems 1. 104 D2 5.103 D3 A. visnaga. 3.103 D4 June 2014 (flowering) Leaves, stems, flowers 5.102 D5 250 D6 July 2014 (after flowering) Leaves, stems, seeds

Methanolic extract preparation of 5%. The LD50 values are determined from the regression The vegetal pulverized powder (1g) representing each part of lines obtained by transformations in Probit percentage of the plant during the three different stage of growth was corrected mortality, and changes in the logarithm of the macerated in methanol (20 ml) at room temperature for 48 administered doses and were expressed as nanograms (ng) of followed by filtration and evaporation of the solvent at 60 °C. methanolic extract per treated aphids. The standard method for The left residue was weighed and dissolved in methanol (3 ml) evaluating the toxicity of insecticides involves the calculation for further analysis. Eventually, the solutions were stored at - of toxicity data (LD50) following standard guidelines such as 20 °C [21]. European Council Directive 91/414 on the one hand and that of the Federal Insecticides and fungicides rodenticides in the Experimental Conditions United States on the other [23.24]. The bioassay was realized in the laboratory conditions using random dispositive. The experimental unit constituted of Petri Results and Discussion dish which contained twenty adult aphids on fresh leaves of The results of toxicity of the eight methanolic extract (Table 1) Citrus sinensis serving as a support for the pests. Hydrophilic of A. visnaga against T. aurantii are represented in figure 1. wet cotton was put down under the fresh leaves to keep the Each extract was tested at five different doses (ng /aphids): leaves fresh during the test period (96h). 250, 500, 3x103, 5x103, 104 and 3x104. The control mortality Eight methanolic extract of A. visnaga were tested against was below 10%. We noted that the number of individual death Toxoptera aurantii. Five doses of 250, 500, 3000, 5000, increased with the eight extract administered dose, however, 10000, 30000 nanograms /aphids were prepared from each there was a proportional relationship between doses and the methanolic extract by simple dilution method with methanol. observed mortality. 1µl of each extract was deposed on aphids’ thorax using Indeed for the first extract (leaves before flowering period), micro-syringe type Hamilton. Control individuals received 1µl 4 the highest dose D1 (3x10 ng/ aphid) caused 58.33% of of methanol. The Petri dishes were maintained under mortality after 24 hours and 94.55% after 96h. After 24h, the laboratory conditions (temperature: 28 °C; relative humidity lowest dose D6 (250 ng / aphid) caused 11.8% of mortality 80%). All experiments were conducted in triplicate. 4 3 while the doses between D2 (10 ng/aphid) and D4 (5.10 ng / aphid) recorded mortalities that are closer to the order of 46% Determination of mortality rate (Fig.1A). To follow the chronological evolution of mortality of aphids Concerning the second extract (leaves during the flowering submitted to different concentrations of each extract, period), the highest dose (30000ng / aphid) has caused 61.67% successive observations were done after treating the aphids of mortality, while the lowest dose D6 (250 ng / aphid) caused with the different extract, after 1, 3, 24, 48, 72 and 96h. The 33.33% of mortality after 24 hours (Fig. 1B). 100% mortality count of the dead individuals was realized with binocular is reached after 96h for all the administered doses. magnifying glass. While the third extract (A. visnaga leaves collected at the The mortality rate was expressed by percentage, taking in fructification period), a mortality of 88.33% was caused by the consideration the initial population after correction. highest dose (3.104 ng/aphid) after 24h. This mortality reached The mortality rate was calculated as follows [22]: 100% after 72 hours. While the lowest dose D6 (250 ng /  P T  aphid) caused 43.33% of mortality after 24 hours, the mortality M(%)  x100 rate continues to increase to 82.27% after 96 h (Fig. 1C).   4  S  We note that the highest dose (3.10 ng/aphid) of the fourth extract (Flowers) caused a mortality of 88.33% after 24h. This M : Corrected Mortality expressed by percentage. mortality reached 100% after 96h (Fig. 1D). While doses D2 4 3 P : Mortality caused by the methanolic extract substance and D3 (10 and 5. 10 ng / aphid) caused a mortality of 86.67% T : Mortality of control. and 73.33 respectively after 24 hours. These mortalities S : Number of surviving individual control increased to reach 100% after 72 and 96 hours. It was observed for the fifth extract (stems collected before Statistical Analysis flowering period), that the highest dose (3x104ng / aphid) The data obtained were processed by analysis of variance caused a mortality of 98.33% after 24h. This mortality reached 4 (ANOVA) and comparisons were made at a probability level 100% after only 48 hours. While the dose D2 (10 ng/aphid)

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has caused 85% of mortality after 24 hours, the mortality generates the highest mortality (88.33%) after 24h (Fig.1G). reached 100% after 96h (Fig. 1E). A mortality of 55% was the second and the third dose (D2 and D3) caused 70% of recorded by the lower dose after 24 hours, while 72.73% was mortality while the doses D4, D5 and D6 generate mortality recorded after 96h. close to 55%. For doses D1, D2, D3 and D4 100% of mortality Closed mortalities of 86% were recorded for the highest doses are achieved after only 48h (so, 100% of mortality are noted at 4 4 D1 (3.10 ng/aphid) and D2 (10 ng /aphid) caused by the sixth 96h). While D5 and D6 reached approximately 90% after 96h. extract (stems collected at the flowering period) after 24 hours, Indeed, the highest dose (3.104 ng / aphid) of the eighth extract the mortality rate reached approximately 98% after 96h (Fig. (Seeds) generated 86.67% of mortality after 24 hours and 3 1F). In addition, the average doses D3 (5.10 ng/aphid) and D4 100% mortality after 96 hours (Fig. 1H). The lowest dose D6 (3x103 ng / aphid) cause about 80% of mortality after 24h and (250 ng / aphid) caused only 35% of mortality after 24 hours, reached approximately 98% after 96h. The lowest doses D5 and the mortality rate continues to increase to 70.91% after 2 (5x10 ng / aphid) and D6 (250 ng / aphid) cause 96h. approximately 57% of mortality after 24h, this mortalities The results of the ANOVA (p<0.00001) showed that the increase to reach 78% and 90% of mortality after 96 hours. treatment effect is highly significant after 24h, 48h, 72h and For the seventh extract (steams collected at the fructification 96h in terms of sensitivity to all tested extract to T. aurantii. period), it was noted that the highest dose (3x104 ng/aphid)

Fig.1 Doses-mortality relation after topic contact with different methanolic extract of A. visnaga

Mortality kinetics A proportional relationship was noted between doses and the The results of the mortality kinetics show that the mortality observed mortality (Figure 2 A.B.C.D.E.F and G). Mortality rate increases with the augmentation of A. visnaga methanolic increases with the administered dose for the eight extract. extract doses (figure 2). Aphid’s mortality was observed for Indeed for the first extract (leaves collected before flowering six doses (ng/aphids): 250, 500, 3x103, 5x103, 104 and 3x104. period), the maximum of mortality is reached after 96 h for

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doses that are between D1 and D4 (Fig. 2 A). Moreover, the ng / aphid) reaches 100% of mortality after 96h. Doses D2, D3, second extract (leaves collected during the flowering period) D4, D5 and D6 reached their maximum of mortality after 96h generated 100% of mortality after 96 hours for all (Fig. 2F). administered doses (Fig. 2 B). Indeed, on the eighth extract (seed), all administered doses While for the third extract (leaves collected during the recorded the highest mortalities after 96 hours with a fructification period), the maximum of mortality (100%) is percentage equal to 98.18% for D1, D2 and D3 and 94.55%, reached after 96 hours for the doses D1 and D4 and after 72 92.73%, 90.91% for doses D4, D5 and D6, respectively (Fig. hours for D2 and D3 (Fig. 2C). For the lowest doses D5 and D6, 2G). the maximum of mortality (92.73 and 87.27%) was reached Note that the mortality of the aphids increases with the after 96h. Furthermore, 100 % of mortality was achieved after elevation of the dose administered for all the methanol extract 72 h for dose D1 for the fourth extract (flowers).Whereas, the tested. Indeed, there is a direct proportional relationship maximum of mortality was reached after 96 h for the doses D2, between the dose of administered extract and observed D3, D4, D5 and D6 (Fig. 2D). mortality. The maximum of mortality is the most time reached We record 100% of mortality after 72 hours caused by the after 96 hours with the exception of the extract of the flowers sixth extract (steams collected during the flowering period) for with dose D1, the extract of leaves collected during the doses D1, D2, D3 and D4 (Fig. 2E). The two lower doses D5 and fructification period with D2 and D3 where 100 % of mortality D6 reach their maximum mortalities after 72 hours (91.07% was reached after 72 hours. The highest dose D1 of stem and 76.79% respectively). collected before flowering period extract caused 100 % of For the seventh extract (stems collected during the mortality after 48 hours. 4 fructification period), we note that the highest dose D1 (3.10

Fig. 2 Mortality Kinetic

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With: LD50 values A: Leaves collected before flowering, B : Leaves collected After drunkenness intoxication of aphids topically with the during the flowering period, C : Leaves collected during the eight methanolic extract of A. visnaga representing different fructification period, D :Flowers, E : Steams collected before aerial parts collected during different periods of growth, flowering, F : Steams collected during the flowering period, mortality is monitored for 96 hours. The LD50 values for the G : Steams collected during the fructification period, H :Seeds. eight extract tested for aphid are summarized in Table 2.

Table 2: Change in LD50 values of A. visnaga extract administered to T. aurantii.

DL (ng /aphids) 50 1 2 3 4 5 6 7 8 24h 11265.64 [D1 - D2] 3249.92 [D3-D4] 171.56< D6 706.24 [D4-D5] 310.53 [D5-D6] 259.53 [D5-D6] 281.58 [D5-D6] 0.054 < D6 48h 7.12

LD50 values of methanolic extract of the plant A. visnaga L. Conclusion obtained for T. aurantii aphids are small. Indeed, after 24 The toxicity of methanolic extract of A. visnaga was evaluated hours the lowest LD50 values are respectively equal to against T. aurantii and presented by the IC50 value. The lower approximately 171.65 ng / aphid for the extract of the leaves values of LD50 were obtained after 96h. The most effective collected at the fructification period and 0.054 ng / aphid for extract of A. visnaga is the extract of seeds with LD50 is equal seed extract topically (Table 2). However, after 48 hours the to 0.153 ng/aphids. So, we concluded the effectiveness of lowest LD50 values are respectively equal to 1.345 ng / aphid methanolic extract and their LD50 values as follows: LD50 for seed extract, 7.12 ng / aphid for leaves collected before Seeds (0.054ng/aphids) > LD50 leaves of the flowering period flowering period extract, 11 ng / aphid the extract of stem (8.09 ng/aphids) > DL50 leaves of the fructification period collected during the flowering period and 16.87 ng / aphid the (13.09 ng/aphids) > DL50 leaves of before flowering period (22 extract of leaves collected during the fructification period ng/aphids) > DL50 stems of the flowering period (30.15 topically. Whereas after 72 hours the lowest LD50 values are ng/aphids) > DL50 stems of the fructification period (54.43 respectively equal to 1.258, 11.19, 13.97, 30.77, 70.17 and ng/aphids) > LD50 stems of before flowering period (113.24 48.65 ng / aphids representing respectively the extract of ng/aphids) > LD50 Flowers (238.53 ng/aphids). seeds, leaves of flowering period, leaves before flowering period, stems of flowering period, leaves and stems of References fructification period. Thus, at the end of 96h, the LD50 values 1. Wanga XZ, Liua DY, Zhanga W, Wangb CJ, Cakmakc I, are equal to 0.153 ng/aphid for seed extract, 8 ng/aphid for the Zoua CQ. An effective strategy to improve grain zinc extract of leaves of flowering period, 13.09 ng/aphid for the concentration of winterwheat, Aphids prevention and extract of leaves of fructification period, 22 ng/aphid for farmers’ income. Field Crops Research. 2015; 184:74-79. extract of leaves collected before flowering period, 30.15 2. 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The plant Acanthoscelides obtectus (Say) (Coleoptera: Bruchidae). extract seems also to provide good protection for stored grain Journal Stored Products Research. 2002; 38:117-128. against granary weevil Sitophilus granarius [26] and rice weevil 6. Evergetis E, Michaelakis A, Haroutounian SA. Essential Sitophilus oryzae [28]. A. visnaga essential oils showed the oils of Umbelliferae (Apiaceae) Family Taxa as Emerging stronger ovicidal effect against Mayetiola destructor [20] and a Potent Agents for Mosquito. Control Integrated Pest very interesting larvicidal effect against Culex mosquitoes [29]. Management and Pest Control. Current and Future Tactics In their whose comparative study, Zekri et al. [30] showed a 2012; 613-638. high insecticidal effect with the Mentha suaveolens hydrosol 7. Brahmi F, Kebbouche-Gana S, Antibacterial Activity and which is more effective against citrus pests than that of Chemical Composition of A. visnaga. Essential Oil Mentha pulegium L. So, both hydrosols were toxic toward T. Collected from Boumerdes (Algeria) During Three aurantii, but M. suaveolens hydrosol has the highest Periods of the Plant Growth. Journal of essential oil- insecticidal activity. According to the Zekri et al. [30], the bearing plants 2014 v.17 no.6 pp. 1317-1328. degree of insecticidal effect depended significantly on applied http://www.tandfonline.com/doi/abs/10.1080/0972060X.2 doses and the age of tested aphids [30]. 014.912164.

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