Aqueous Extracts of Some Medicinal Plants are as Toxic as Lmidacloprid to the Sweet Potato Whitefly, Bemisia tabaci

Aqueous extracts of nine plants, known to have medicinal activity, were tested for their toxicity against the sweet potato whitefly, Bemisia tabaci Genn. (Homoptera: Aleurodidae) compared to the toxicity of the insecticide, Imidacloprid. Extracts of Lepidiuim sativum L. (Brassicales: Brassicaceae) killed 71 % of early stage nymphs, which was not significantly different from mortality caused by Imidacloprid. Treatment of pupae with three plant extracts, L. sativum, Achillea biebersteinii L. (Asterales: Asteraceae), or Retama raetam (Forssk.) Webb and Berthel (Fabales: Fabaceae) prevented adult development, and treatment with R. raetam extract killed adults, at levels that were not significantly different from Imidacloprid. None of the other plants showed significant toxicity. However extracts of four plants, Pimpinella anisum L. (Apiales: Apiaceae), Galium longifolium (Sibth. and SM.) (Gentianales: Rubiaceae), R. raetam and Ballota undulata Bentham (Lamiales: Lamiaceae) had a repellent effect.


Introduction
Plants may provide an alternative to currently used pesticides for the control of plant pests, as they constitute a rich source of bioactive chemicals (Kim et al. 2005;Daoubi et al. 2005). Recent studies have demonstrated the insecticidal properties of chemicals derived from plants that are active against specific target species, biodegradable to non toxic products and potentially suitable for use in integrated management programs (Markouk et al. 2000;Tare et al. 2004).
The sweet potato whitefly, Bemisia tabaci Gen. (Homoptera: Aleurodidae), is a key pest of vegetables in Jordan (Al-Musa et al. 1987). It is also a serious economic pest of agronomic, horticultural, and ornamental crops throughout warm regions of the world (Byrne et al. 1990;Brown 1994).
In the present study, the toxicity and repellency of aqueous extracts of nine plants known to have medicinal activity, were investigated against the sweet potato whitefly, B. tabaci. Brassicaceae), Pimpinella anisum L. (Apiales: Apiaceae), Phlomis syriaca Boiss. (Lamiales: Lamiaceae) and Retama raetam (Forssk.) Webb and Berthel (Fabales: Fabaceae), were collected from their natural habitats, from different localities of Jordan. The identity of each plant species mentioned was verified and confirmed by Prof. Dawud M. Al-Eisawi (Department of Biology, Faculty of Science, University of Jordan) using live specimens and photographs. The parts of the different plants used in the experiments are given in Table 1.

Preparation of the aqueous extracts
An aqueous extract was prepared by boiling10 % wt/wt of the air-dried powdered plant part in sterile distilled water for 10 min and then cooled to room temperature overnight. The aqueous extracts were filtered using a Millipore filters (Millipore 0.2 mm, www.waters.com) to remove particulate matter. The final volume of each filtrate was completed to 100 ml with distilled water with 0.2% Tween 80 to account for the evaporated water during boiling. The aqueous extracts were prepared shortly before application. Negative controls represented by the distilled water contained the emulsifier Tween 80.

Insecticide
The insecticide Imidacloprid (Confidor® 200SC, Bayer Crop Science, www.bayercropscience.com) was used as positive control treatment at the recommended field application rate of 0.25 ml/l.

Insect culture
The colony of B. tabaci was maintained on tomato plants. To obtain immature whiteflies, 4-week-old greenhousegrown tomato plants grown in a whitefly-free screened cage were trimmed to three fully expanded leaves and were transferred to the whitefly colony for 48 h. Adults were then aspirated from the plants, and the plants were placed in a separate cage. The synchronouslydeveloping, uniformly-aged whitefly populations were then held until they developed to the appropriate stage.

Egg mortality
Immediately after the adult whiteflies were aspirated from the plants, the plants were sprayed with extracts. Water or Imidacloprid were the negative and positive controls Initial number of eggs prior application ranged from 140 to 300 per plant. There were five replicates (plants) for each treatment. Eight days after treatment, the unhatched eggs and newly emerged nymphs were counted and the percent hatch calculated.

Early stage nymphal mortality
Eight days after infestation, when first instar nymphs had emerged and attached to the leaf, the plants were sprayed as before. The number of first instar nymphs per plant ranged from 98 to 190. There were five replicates (plants) per treatment. Ten days after treatment, the number of dead nymphs were counted under a dissecting microscope. A nymph is considered dead if it was shrunken or its color changed. Normally developed nymphs to adult stage were also counted and the percentage of each was calculated.

Late stage pupal mortality
Fourteen days after infestation, when most nymphs were in the red-eye stage, the plants were sprayed as before.
The number of nymphs per plant ranged from 75 to 150. There were five replicates (plants). Seven days after treatment, when most of the pupae had emerged from control plants, the number of empty pupal cases and pupae that failed to emerge were counted and the percent of emergence was calculated.

Adult mortality
A fully expanded leaf was placed in wet moss inside a Blackman box (Blackman 1971). The leaf was dipped into the solution of the required treatment and left overnight. About 30 adults were then introduced inside each box. Distilled water was used as negative treatment and imidacloprid was used as positive treatment. The number of dead whitefly adults was recorded after 48 hr. A whitefly adult was considered dead if it did not move after probing with a camel hair brush. Five replicates were made for each treatment.

Repellency tests
Two fully expanded leaves of tomato were placed individually in vials containing water. One leaf was dipped in the tested plant extract and the other one was dipped in distilled water. The vials were placed in a plastic jar (20 cm diameter, 30 cm high) covered with fine netting material. About 50 immobilized adults were placed between the two vials. Numbers of adults attracted to each leaf was recorded after 3hr and 24hr. Five replicates were made for each treatment.

Statistical analysis
Arcsin-transformed data were analyzed by ANOVA and means were separated by Tukey's Studentized Range test. Data of the repellency tests were analyzed using ttests.

Results
Over 80% of the eggs hatched and nymphs were able to emerge, regardless of treatment and all plant extracts showed no differences with both negative and positive controls (F = 2.62, df = 10, 44; P = 0.0134) ( Table 2). The highest percentage of dead early stage nymphs (71%) was caused by the extract of L. sativum that was not significantly different from the effect of Imidacloprid (F = 31.05, df = 10, 44; P = 0.0001) ( Table 3). Extracts of three plants, R. raetam, P. syriaca and A. inculata were as toxic as the L. sativum extract, but not as toxic as Imidacloprid. Treatment of pupae with three of the plant extracts, L. sativum, A. biebersteinii, or R. raetam prevented adult development as well as Imiacloprid (F = 5.51, df 10, 44; P = 0.0001) ( Table 4). Treatment with R. raetam extract killed adults was as effectively as Imiacloprid (F = 6.68, df 1, 44; P = 0.0001) ( Table 5).  The extracts of P. anisum, G. longifolium, R. raetam and B. undulata showed repellency effect to adults of whitefly compared with untreated plants (Table 6). However, leaves treated with extracts of A. biebersteinii were more attractive to the whitefly than untreated leaves by 24h after application.

Discussion
Whitefly management has traditionally depended on the use of synthetic insecticides. However, the increasing resistance of Bemisia species to insecticides provides an impetus to use integrated pest control measures, including biopesticides and biological control to combat this pest. Biopesticides are based on natural products and synthetic analogs of naturally occurring biochemicals and are more acceptable than conventional pesticides because of their reputation for being less hazardous to humans and other non-target organisms (McCloskey et al. 1993). Among the biopesticides are chemicals derived from a variety of plant families. The biological activity of plant extracts against bacteria, fungi, viruses and insects has been reported (Bozsik 1996;Macedo et al. 1997;Unicini Manganelli et al. 2005).
In the present work, extract of L. sativum had toxicity that was not significantly different from the effect of Imidacloprid against early stage nymphs and pupae of B. tabaci. Treatment of pupae with three plant extracts, L. sativum, A. biebersteinii, or R. raetam prevented adult development, and treatment with R. raetam extract killed adults, at levels that were not significantly different from Imiacloprid. P. anisum, G. longifolium, R. raetam and B. undulata had repellent effects on adults of B. tabaci.
L. sativum belongs to Cruciferae family that contain glucosinolates (Burow et al. 2007). Glucosinolates are a class of thioglycosides found predominantly in plants of the order Brassicales. An anti-herbivore defense has been attributed to the products formed by myrosinase-catalyzed hydrolysis upon plant tissue damage (Burow et al. 2007). The leaves of L. sativum are antiscorbutic, diuretic and stimulant (Uphof 1959;Chopra et al. 1986). The plant is administered in cases of asthma, cough with expectoration and bleeding piles (Chopra et al. 1986). The root is used in the treatment of secondary syphilis and tenesmus (Chopra et al. 1986). A. biebersteinii is rich in camphor, borneol and 1,8-cineole (Esmaeili et al. 2006). It is used as an antispasmotic, for abdominal pain and healing wounds. R. raetam contains flavinoids, has been used as an herbal remedy for diabetes and has been shown to have hypoglycemic activity in rats (Maghrani et al. 2005).
Repellent activity of some non-insecticidal agents could be attributed to the complex mixture of compounds that are detected by the susceptible insect (Schumutterer 1985). Anise is the common name of P. anisum, and is among the more ancient aromatic plants. The fruits of P. anisum are claimed to possess expectorant, stimulant, carminative, diuretic and diaphoretic properties. They are also used in flatulent colic and in some pharmaceutical preparations for asthma (Siddiqui et al. 2002). Phenylpropanoid derivatives from Ballota nigra exhibited a moderate antimicrobial activity against Proteus mirabilis and Staphylococcus aureus (Didry et al. 1999). Acetone extract of Ballota hirsuta leaves produced growth inhibition in larvae of the stored grain pest Tribolium castaneum (Passcual-Villalobos and Robledo 1999).
Considering toxic effects of L. sativum, A. biebersteinii, and R. raetam, and the repellent effects of of P. anisum, G. longifolium, R. raetam and B. undulata against B. tabaci, it is possible that extracts of these plants can be used as natural control agents. Most of these plants are widely distributed and easy grown. Furthermore, the extraction method is simple and cost-effective and the application techniques could be relatively easily designed for on-farm use. Since B. tabaci transmits tomato leaf curl virus, developing new methods of control is obviously important.