Pesticide Discovery: Screening Euonymus americana L. Seed Coat Extracts and Fractions to control Whi

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 10 to 11 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 Duncans 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 p0.0001) 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
• 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 pwhiteflies 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. Cooks 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.