Introduction

1. Pesticide Discovery: Screening Euonymus americana L. Seed Coat Extracts and Fractions to control Whitefly Françoise Favi and Mark Kraemer, Agricultural Research Station, Virginia State University, Petersburg, VA 23806 Introduction Euonymus americana L (Celastraceae), commom burning-bush or strawberry bush (fig. 1) is a perennial shrub native to North America. It is found in the middle and eastern part of the United States, including Virginia, and is distinguished by its thick and almost sessile leaves, greenish-purple flowers and the five divisions of its ripe capsules. The bark and the root of Euonymus atropurpureus and E. americana has been in marketed indiscriminately under the commom name of wahoo. It is used as expectorant, laxative and tonic (Foster and Duke, 1990). The bark can be used as tea to treat malaria, liver congestion and constipation (Moerman, 1998; Foster and Duke, 1990). Tea made from the roots is used in case of uterine prolapse, vomiting of blood and stomach aches (Moerman, 1998; Foster and Duke, 1990). An infusion of the bark has been used to stimulate menstruation and should not be used by pregnant women (Moerman, 1998). The seed extract is a strong laxative (Foster and Duke, 1990). The active ingredients are extracted better in alcohol than water. Greenhouse whitefly (Trialeurodes vaporariorum West.) and sweetpotato whitefly (Bemisia tabaci (Hadelman)) are crop pests, cosmopolitan in distribution and extremely polyphagous, having been recorded from over 60 different plant families (van Lenteren et al., 1995). Whiteflies cause millions of dollars worth of crop damage. Greenhouse whitefly vectors closteroviruses whereas sweetpotato whitefly is a vector of geminiviruses. Honeydew deposition on leaves produces a shiny, sticky sheen, and provides an ideal substrate for sooty mold growth (e.g., Cladosporium and Alternaria spp.). Declines in the aesthetic and marketable qualities of ornamental plants in greenhouses due to honeydew, black sooty mold contamination, or flying adults are also problems associated with high whitefly densities. Disfigurement of plants by whiteflies results in consumers rejection of both vegetables and ornamentals. Because whiteflies are usually found on the underside of plant leaves and are sap feeders they are best controlled by systemic pesticides. The objective of our research is to discover botanical compounds with insecticidal activity towards whitefly from a plant that was used for centuries to cure diseases. Table 1: Insect mortality from silica gel fractions of dichloromethane extract Table 2: Insect mortality from silica gel fractions of ethyl alcohol extract Table 3: Comparison of insect mortality with crude extracts and active fractionst Fig. 6: Behavior of non-treated adult whiteflies Fig. 5: Behavior of whiteflies treated with fraction 3 Figs. 7 & 8: Behavior of whiteflies treated with fraction 3B Figure 1: Seed capsule, ripe fruit and seeds of Euonymus americana. Figs. 2: Seeds with & without fleshy coat. Fig. 3: Chromatograph of fraction 3 showing further partitioning. Fig. 4: Black box for collecting whitefly adults. • Materials and Methods • 1.36 g of dried seed coat (fig. 2), air dried, room temperature for seven days) was extracted and fractioned as follows: • 1) extracted with 500 ml of dichloromethane (x 3) • 2) extracted thereafter with 1000 ml ethyl alcohol (x 3) • Extracts were concentrate using rotary evaporator to 15 ml and 25 ml respectively. • Both extracts were partitioned individually using individual silical gel columns (70-150 mesh; 300 cm x 1.5 cm). Fractions (250 ml) were obtained with an elution solvent gradient of hexane and ethyl acetate; from 1:0 to 1:1 mixture of hexane and EtOAc. Fractions 1-5 were from the dichloromethane crude extract and fractions 6-10 from the EtOAc crude extract column. Fraction 11 was obtained with 100% EtOAc. • Fractions were compared using thin layer chromatography and High Performance Liquid Chromatography (HPLC). • Fraction 3 was further partitioned into three fractions (A, B and C) using HPLC (Fig. 3). Solvent removed by freeze drying. • Mortality generated by each set of fractions was assessed on adult whiteflies using the following procedure: 1) 200µl applied to Whatman paper no.2 (3.5 cm x 4.5 cm) and dried under purified nitrogen for 30 min, 2) adult whiteflies harvested from black box (Fig. 4) were added to 40 ml vial containing treated paper, and 3) tomato leaf disc (10 mm dia.) was added to the vial one hour post treatment. • Percentage of dead whiteflies was determined at intervals from 45 min to 24 hours post treatment. • Proc GLM procedure of SAS statistical package (2005) was used to analyze data. Means were separated according to Duncan’s Least Significant Difference test. • Light, transmission and scanning electron microscopes were used to study seed coat fine structure. Results continues…. • It was observed that whitefly exposed to fraction 9 did not settle down and feed whereas fraction 11 made them defecate profusely. A laxative effect was also reported in humans (Moerman, 1998; Foster and Duke, 1990). • Fraction 3 agitated whiteflies and they appeared more sensitive to light. They raised their wings, possibly for maximum ventilation, compared to normal whiteflies (Figs. 5 and 6). A lesser laxative effect was also observed. • Fractions 3A, 3B & 3C obtained by partitioning fraction 3 (fig. 3) were tested in a separate experiment. Fraction 3B was the most potent, killing all whitefly within an hour. The adults raised their wings high before dying (figs. 7 and 8). • Results of microscopy study of cells • Sequential development of seed coats from both immature green and mature red fruits was observed using light and electron microscopy (figs. 9-16). Tissue samples from maturing seed coats showed that epidermal cells contained carbohydrate compound presumably mucilage, three rows of large parenchyma cells with scattered secreting ducts (fig. 10). A few secreting structures were also observed (fig. 11). Transmission electron microscopy showed numerous plasmodesmata between these cells along with numerous mitochondria and smooth endoplasmic reticulum within each cell. The presence of these structures suggest biosynthesis with active transport of oily products across cells. • Light microscopy of the mature seed coat showed parenchyma cells producing a dense compound (fig. 14) and later filled up with a milky mucilage secretion (fig. 15). Fine structure from electron-microscopy showed a mucilage (unstained) and a dense black oily substance stained with osmiun tetraoxide. (figs. 16-19). This mucilage was observed within the chloroplasts (figs.17 & 18) and the dense dark product was found within the cell lumen (figs. 17-19). Between cell transportation of the dark substance was observed (figs. 18-19). This could be the defense component of the seed coat being mobilized for storage. Figs. 9-12: Light and electron microscopy of immature seed coat (dots) of Euonymus americana. 9, light microscopy of fruit split into two sections and showing the green seed coat. 10, light microscopy of cross section of seed coat shows development of duct surround by secreting cells 11, light microscopy shows developing secreting cells and lumen. The seed coat also develop oil gland; this type of gland grows by recruitment: a small set of cells begins producing oil, and they stimulate their neighbors to begin doing the same thing. As the neighbor cells become active, they in turn activate the ones just to the exterior of themselves to produce and oil gland.12 transmission electron microscope picture showing mitochondria smooth endoplasmic reticulum and numerous plasmodesmata between adjacent cells within the oil gland. EC, epidermal cells; Cu, cuticle; CW, cell wall; ISC, inner seed coat; L, lumen; M, mitochondrion; PD, pasmodesmata; S, seed SC, secreting cells; • Results • Bioassay Results • Bioassays of the dichloromethane extract fractions (1-5) showed fraction 3 had significantly (F = infinity, DF = 9 and p< 0.0001) higher mortality after 45 min (table 1). • Whitefly mortality from the EtOAc crude extract was greater than any of its fractions (6-10) after 30 minutes treatment (F = infinity, DF = 9 and p< 0.0001) (table 2). • Comparative lethality analysis of fractions 3, 9 & 11 showed crude ethyl alcohol extract, ½ diluted extract, and fraction 3 significantly (F = 15.93, DF = 9 and p< 0.0001) more toxic to whiteflies than any other fractions after 120 min. and 24 hours (F = 41.95, DF = 9, p ,0.0001) post treatment (table 3). • Conclusions • Fraction 3b showed showed rapid and significant insecticidal activity with an unique behavioral changes. The mode of action (preventing insect from flying) of fraction 3b is different from any pesticide currently use to control insects. It was selected for further study. • Other fractions (9 and 11) showed insecticidal activity over a longer time period and likely contain other, more polar insecticial components. • Natural product secretion structures were already present in the green seed coat and was found within the rip seed coat along with flavonoids and may be responsible for the production of insecticidal compounds. Figs. 13-19: Light (13-15) and electron microscopy (16-19) studies of mature cell coat of Euonymus americana. 13, mature seed split into two to show orange seed coat around the whitish seed. 14, Seed coat showing cuticle, epidermal cells and parenchyma cells begin to fill up with mucilage. 15, Fully developed seed coat with mucilage and colored substance in parenchyma cells. 16, Picture of three adjacent cells showing secreted mucilage contained within each cell. 17, enlargement of part of figure 15 shows chroloplast surrounded by mucilage and osmophilic dark substance. 18, Osmiophilic substance seems to cross cell wall of adjacent cells through plasmodesmata. 19, Mucilage secretions were observed in the chloroplast. E, embryo; CW, cell wall; PD, plasmodesmata; PL, plastids; OS, osmiophilic substance; Sco, seed coat. 1,2 & 3, number of adjacent cells. References Cited Foster, S and J A Duke. 1990. A field guide to medecinal plants. Eastern and Central N. America. Houghton Mifflin Co. Moerman D. 1998. Native American ethnobotany. Timber Press. Oregon. ISBn 0-88192-453-9. William Cook. 1869. Cook’s physiomedical dispensatory Van Lenteren, J C and LPJJ Noldus. 1990. Whitefly-Plant Relationships: Behavioural and Ecological Aspects. In Whiteflies: Their Bionomics, Pest Status and Management (Dan Gerling, Ed.), pp. 47-89. Antheneum Press, Great Britian. Acknowledgments Thanks to Ms. Edwina Westbrook for her technical assistance with the microscopy part of this study.