son, and Bill Randle for their excellent technical assistance. We would also like to thank the Sakata Seed Company for prosiding a cross-section of their culdvars ...
Reprinted from: Proc. Ha. State Hort. Soc. 107: 163-167. 1994.
P O S S I B L E B I O P E S T I C I D E F R O M P E T U N I A F O R T H E C O N T R O L O F T H E S W E E T P O TAT O
WHITEFLY {BEMISIA TABACI) ON VEGETABLE CROPS. crops in Florida (Anon., 1991; Faust, 1992). These outbreaks
StanlltJ. Kays', RavF. Se\^rso\-,
were attributed to a new strain of B. tabaci, called the B strain,
Stephen F. Nottingham^ Richard B. Chalfant',
poinsettia strain, or more colloquially, the "Superbug"
AND Orestes Chortvk-^
(Anon., 1991). Recently, B. tabaci strain B has been described as a new species, the silverleaf whitefly, Bemisia argentifolli N. (Bellows et al., 1994)''. Annual economic losses in the United States due to this insect now exceed $200 million (Faust, 1992). The sweetpotato whitefly is extremely polyphagous, hav
'Department of Horticulture, The University of Georgia, Athens, OA 30602-7273
^Fhytochemical Research Unit, Russell Research Center, U.S. Dej)t. Agr., Athens, OA 30613
^Department of Entomology, The University of Georgia, Coastal Plain Expt. Sta., Tifton, GA 31793-0748
Additional index words, phytochemical, insecticide, Bemisia argentifolli Abstract. Plant species represent a potentially abundant source of blopestlcldes that can be used to address serious agricul tural pest control problems. It was shown that Petunia x hybrida Hort. Is a rich source of blopestlclde compounds In the form of sugar esters Isolated from leaves and flowers. Individ
ual Petunia cultlvars exhibited substantial quantitative differ ences In the level of sugar esters present. Using a crosssection of Petunia cultlvars, sugar esters were extracted with MeClj and purified by partitioning between hexane and acetonltrlle. The sugar ester extract was derlvatlzed and analyzed by gas capillary chromatography. An aliquot of the sugar esters was hydrolyzed and the fatty acids were converted to butyl es ters and analyzed by gas chromatography/mass spectrosco py. The acyl components of Petunia sugar esters differed quantitatively and qualitatively from Nicotiana gossei L. Domln. sugar esters. A composite sample of sugar esters was bloassayed using aqueous sprays applied to sweetpotato whitefly {Bemisia tabaci Gennadlus, B strain) nymph Infested sweetpotato leaves [ipomoea batatas (L.) Lam.] and Immobi lized adults. Treatment concentrations of 1 and 0.5 mg • ml'^ gave excellent whitefly control. In field tests with whitefly In fested summer squash {Cucurbits pepo L.), Petunia sugar es ter treatments gave significantly lower egg and adult counts than controls.
ing been collected from more than 506 plant species belong ing to 74 plant families (Greathead, 1986) and is spread quickly on transported plants (Broadbent et al., 1989). The B strain is now a major pest on a diverse range of field crops, in
cluding tomato, cole, cucurbits, lettuce, peanut, sugar beet, cotton and ornamentals, all over the U.S.A. (Faust, 1992; Gill,
1992; Natwickand Zalom, 1984, Price etal., 1987). The insect can also develop on a variety of weed species, thus maintain ing itself in areas during the absence of crop hosts (Coudriet et al., 1985). In addition to feeding damage, B. tabaci is a vec tor of a complex of virus diseases (Duffus and Flock, 1982). In general, the control of agricultural insect pests has largely been with synthetic chemicals and traditional plant
breeding systems. Increasing environmental concerns and re sistance of pests to many pesticides has increased the necessity for finding new pesticides that exhibit superior control poten tial and are biologically safe. Control of the sweetpotato
whitefly has also become more difficult due to the develop
ment of resistance to a number of insecticides (Prabhaker et
al., 1985). Insect pests can be controlled through the use of one or more of the follo\ving means: 1) pheromones; 2) allelochem-
icals; 3) toxicants; 4) hormonal compounds; 5) microbial or viral agents; 6) phytoalexins; and 7) induced autointoxicants (Hedin, 1991). Often multiple forms of control are required to decrease the rapidity of resistance developing within the in sect population. Plants represent a rich source of biologically active chemicals that have the potential for control of insects pests and there has been an increased interest in identifying
The sweetpotato whitefly, Bemisia tabaci (Gennadiiis), also known as the cotton, poinsettia, cassava, tobacco, and silverleaf whitefly, was first described on tobacco in Greece as Aleyrodes tabaci (Gennadius, 1889). It has been a pest of tropical crops for many years (Greathead, 1986), but was not of major agricultural significance in North America until the 1980's,
when massive outbreaks occurred in greenhouse poinsettia
and exploiting these natural products.
In nature, a cross-section of plant species exist that exhibit little to no insect damage. For example, a number of tropical
and desert plants (Gutler, 1988; Kubo and Nakanishi, 1977; Mabry et al., 1977; Morgan and Mandava, 1985) have evolved
toxic compounds due to the high insect selection pressure in
those environments. The pyrethroids are an example of a nat ural phytochemical that have led to the development of syn thetic forms for insect control (Elliott, 1977). More recently, the neem plant {Azadirachta indica A. ]uss.), which possesses
Acknowledgment. The authors would like to thank the Fred C. Gloeckner
an allelochemical, azadirachtin (Stone, 1992), that acts as an
son, and Bill Randle for their excellent technical assistance. We would also
antifeedant, has generated considerable interest. The plant grows wild in Africa and Asia (Koul, et al., 1990) and its ability
Foundation for financial support of the research and Susan Wilson, Patsy Ma
like to thank the Sakata Seed Company for prosiding a cross-section of their culdvars.
""Polymerase chain reaction experiments suggested that the poinsettia or
B strain of B. tabaci may in fact be a separate species (Perring et al., 1993). This conclusion is not universally accepted at present in that ribosomal RNA genes do not appear to differ between the A and B strains. Regardless of the
final taxonomic treatment, strain B has \irtually replaced strain A and repre sents the strain of primary agricultural concern. The B strain was the bioas.say organism in the following experiments. Proc. Ela. State Hort. Soc. 107: 1994.
t o w a r d o ff i n s e c t s h a s b e e n k n o w n f o r s e v e r a l c e n t u r i e s i n I n
dia. Phytochemicals from the neem deter a cross-section of in sect species and appear to be much safer than most synthetic insecticides.
Sugar esters represent another group of phytochemicals that exhibit insecticidal properties. They are a class of chemi cals in which a variety of aliphatic acids are esterified to one 163
biopesticide potential of the Petunia is to identify the funda mental chemical components present in a diverse cross-sec tion of the germplasm and to determine the potential of specific compounds in controlling selected insect pests. In an initial series of experiments, the levels of sugar esters on se lected Petunia cultivars and the chemistry of the sugar esters
OTjOR' 6
/'
C
o
OR
Hi
from a composite sample from a cross-section of Petunia lines H
were analyzed, and the toxicity of the composite extract was tested on the whitefly in greenhouse and field experiments.
OR
GLUCOSE ESTERS R = C, - C, Acyl groups R' = H or Acetyl
Materials and Methods
Plant material and isolation of sugar esters. A cross-section of Petunia cultivars ['Falcon Mid-Blue', 'Highlight Carmine', 'Crimson Titan' (SakataSeed Co.), 'Recoverer White', 'Rose/
CH^OR
White Flash' (Sluis & Croot), 'Postilion Salmon' (Royal Sluis), 'Primetime Blue Star', and 'Pink Cloud' (Goldsmith)]
were grown at the University of Georgia Horticulture farm in 1992 and 1994. Three replicates of each line were collected and weighed on an analytical balance. The leaves were dipped 10 times each into a scintillation vial containing 10 ml of methylene chloride. Surface areas were obtained by plac ing the dipped leaves onto thermal paper and tracing the out line of each leaf. The outlines were subsequendy cut out and run through a leaf area meter (LICOR 1000). The vials were SUCROSE ESTERS Figure 1. Base structures of sucrose and glucose esters isolated from
Nicotiana species.
or more hydroxyl groups on the various carbons of the sugar molecule. Sucrose and glucose esters were first reported as a
sealed with a teflon lined cap and stored in the freezer (-18C) until analysis. After warming to room temperature, extracts were reduced to dryness under nitrogen. The residue was then partitioned between hexane (1 ml) and acetonitrile (1 ml) in the scintillation vial.
A composite sample from the entire above-ground part of
a wide cross-section of cultivars (i.e. ~ 114) was collected for
component of the cuticular waxes of green tobacco, Nicotiana tabacum L., leaves by our research group (Severson et al., 1985). The base structures are presented in Figure 1 (glucose carbons are designated Cj to Cg; fructose C,' to C2'). In Nicoti
preliminary bioassays and field treatments. The composite sample was extracted with methylene chloride and the cutic ular extract was fractionated into sugar ester components as follows. The sample was taken to dryness and then parti
ana, 19 different structural types of sucrose esters and 5 glu
tioned between hexane and acetonitrile, 180 ml and 100 ml
2,3,4-tri-O-acyl-glucose) (Severson et al., 1992b). For each sug
per 5 gm sample, respectively. The acetonitrile fraction con tained the sugar esters and was used in the appropriate con centrations for bioassays and field tests. Analysis of sugar ester isolate. A 100 pi aliquot of the acetoni trile layer was transferred to a micro vial and blown to dryness
cose esters have been reported (for example: 2,3,4-tri-O-acylsucrose, 2,3,4-tri-0-acyl-3'-0-acetyl-sucrose, and 6-O-acetylar ester type, a series of homologs, differing in the acyl groups present, are found. Sucrose and/or glucose esters have been identified to date only from members of the Solanaceae fam ily, e.g. Datura metel L., Lycopersicon hirsutum f. and glabratum.
Petuniax hybrida Wort., SolanumberthaultiiWdM^iGS, S. neocarde-
nasii, S. poluademium, and S. tarijense (King et al., 1987), and most of the Nicotiana species (Severson et al., 1992b). Their importance to the plant stems from an apparent role as sur face components that modulate the interaction between the
plant and certain insect species. For example, sugar esters ap pear to be involved in resistance to tomato (Goffreda et al.,
1989), potato (King et al., 1987), and tobacco aphids (Sever son et al., 1992b) and confer to Solanum berthaultii Hawkes en
hanced resistance to the Colorado potato beede, Leptinotarsa decemlineata Say (Neal et al., 1989). Recent evidence (Severson et al., 1992b) indicates that
the application of sugar esters from Nicotiana has a pro nounced effect on the B or silver leaf strain of the sweetpotato whitefly and may, as a consequence, have value as an insecti
cide. Petunia was found to also display high levels of sugar es
ters (Son, et al., unpublished data), the chemistry of which may differ sufficiently from that of Nicotiana to present addi
tional biopesticide uses. A realistic approach to exploiting the 164
under nitrogen. C,, alcohol was used as an internal standard,
11 pg was added to dried sample and blown to dryness under nitrogen. A mixture of 50 pi of 1:1 N,0-bis(trimethylsilyl)-trifluoroacetamide:dimethyl-formamide (BSTFA:DMF) derivatizing reagent was added and the capped vial was heated at
76C for 45 minutes to convert the sugar esters to volatile trimethylsilyl ethers, suitable for gas chromatography. The sam
ple was analyzed on a Hewlett Packard 5890 gas chromatograph equipped with an autosampler, 0.3 mm i.d. x 20m bonded SE-54 fused silica column (O.lp film thickness) and flame ionization detector. The sample was injected in the splitless mode (purge activation time 1 min.) at a column temperature of lOOC and then temperature programmed at
a rate of 8C • min"' to 330C. The amounts of glucose and su
crose esters were determined using internal standard meth ods with response data obtained from previously isolated glucose ester and sucrose ester fractions.
Fatty acid butyl ester preparation, analysis and characterization. Approximately 450 pg of sugar ester isolate was saponified with 120 pi of 1.0 N KOH in 80% methanol-water at room
temperature for 24 to 48 hours. Three replicates of 30 pi aliProc. Fla. State Hort. Soc. 107: 1994.
quots each of the saponificates were transferred to micro vials and taken to dryness under nitrogen, 30 pi of butanol con taining 1 N j&-toluenesulfonic acid was added to each capped sample vial, heated for 45 min at 76C and then cooled to
room temperature. After addition of 25 pi of 5% NasCO, to neutralize the excess acid, 50 pi of distilled isooctane was add
ed. The fatty acid butyl esters were partitioned into the isooctane-butanol phase. Analyses of butyl esters were conducted with a HP 5890
gas chromatograph equipped with 0.3 mm x 30 m bonded SE54 fused silica capillary column (0.5p film thickness) pre pared by the method of Arrendale and Martin (1988). The oven temperature was held at 35C for 2 min, then increased
at 4C/min to 220C. Injection port and detector temperatures were 200C and 275C, respectively. The carrier gas was hydro gen with a linear velocity of 60 cm • sec '. One pi of each sam ple was injected in the splitless mode using a 0.5 min purge activation time. Relative levels of the acids were determined
from the chromatographic data using response data obtained from a mixture of fatty acid standards.
Figure 2. Capillary gas chromatographic separation of glucose and sucrose esters from Petunia leaves.
The butyl esters were identified using GC/MS and GC re tention time data of an authentic standard (Cg - C,o acids) which were treated in the same manner as the samples. The butyl esters were characterized by GC/MS on a HP 5989A
GC/MS/Unix system.
Insect bioassay and field tests. The toxicity of the sugar esters was tested on adult B. tabaci by knocking them from sweetpo-
tato plants onto 3 by 14 cm Sticky Strips (Olson Products, Inc., Medina, OH), placed on damp paper towels in flat plas tic boxes (Pittarelli et al., in preparation). Each strip had two 3-cm-square areas of sticky surface exposed, onto which ap proximately 30 adults per square adhered. Two strips were used per treatment with applications replicated on different dates. The whiteflies adhering to the strips were sprayed with test compound solutions, using an airbrush (Badger 2000), with fine-mist nozzle setting, in a laboratory fume hood. Two
ml of each test compound was sprayed with counts for mortal ity made 2 hr after spraying using a binocular microscope. Whitefly nymphs were established on young sweetpotato plants [Ipomoea batatas (L.) Lam.] in a muslin-tented area, with a clear polyethylene roof, within a greenhouse. Four to
six leaf plants were placed in the culture cage for two to three days, to be infested with eggs, and then removed to a whiteflyfree section of the greenhouse. After two to three weeks, when nymphal stages were present, the undersides of leaves were sprayed using Spritzer applicators (Bel-Art Products, Pequannock, N.J.). Mortality was scored 6 days later on the underside of the leaf within areas of 3.15 ± 0.09 cm^ (n = 15
leaf disc area) marked using a number 14 cork borer. Three leaf discs per treatment were scored and percentage mortality obtained. Treatments were replicated on different dates. Field tests were conducted at the Coastal Plain Experi
Results and Discussion
The composition of the sugar esters (as trimethylsilyl de rivatives) from Petunia is illustrated by the capillary gas chromatogram in Figure 2. The predominate components were sucrose esters. The levels of sugar esters were determined in
eight cultivars (Table 1), representing genetic materi
al from four seed companies. There was substantial variation
in the concentration of sugar esters, indicating distinct genet ic control over expression of the trait. The levels ranged from
22.6 pg • mg ' in 'Falcon Mid-Blue' to 3.8 pg • mg ' in 'Crimson Titan'. The fatty acid distribution from the 1994 composite isolate was determined to characterize the acid components
of the sugar esters (Table 2). Acid group chain lengths ranged from acetic (Cg) to octanoic (Cg) acids, with malonic acid being the second most abundant acid. As malonic acid (methane dicarboxylic acid) has not been found in any of the sugar esters of the Nicotiana species, this is the first report of
this unusual acid being a component of plant sugar esters. At this time, it is not known whether only one of the carboxyl
groups is attached to the sucrose molecule with the other groups being free to form an acidic sucrose ester or whether both acid groups are esterified to the same or two different su crose or glucose molecules. 2-Methyl butyric and hexanoic ac ids were the primary fatty acids present, accounting for over 30% of the total. Intermediate levels of acetic, 4-methyl valer-
Table I. Concentrations of sugar esters from the leaves of selected Petunia cultivars (1992).
ment Station in Tifton, GA. Summer squash, cultivar 'Pavo',
C u l t i v a r To t a l s u g a r e s t e r s ( j i g • m g ' ) '
was seeded on 4 Aug. 1994 in 91 cm rows with an in-row spac ing of 30 cm. Treatments were replicated four times in a ran
'Falcon 'Recoverer
and counting eggs and nymphs in three 1 cm^ fields per plot
'Rose/White 'Postilion 'Premature 'Pink
domized complete block design. Insecticidal efficacy was determined by recording adult whiteflies on 5 leaves per plot
at four dates approximately one week apart. Applications were made using a COg pressured backpack sprayer (R&D
Sprayers, Obelousas, LA) at 374 liter of water • ha '. Treat
ment concentrations were 0, 0.5, 1.0, and 2.0 mg sugar esters
• gm' Hp.
Proc. Fla. State Hort. Soc. 107: 1994.
Mid-Blue* White'
± ±
2.05* 0.79
± ±
1.32 2.01 1.03 ±0.94
Flesh* 21.2 Salmon* 13.8 Blue Star* 12.8 Cloud* 9.8
'Highlight 'Crimson
22.6 21.7
±
Carmine* Titan*
3.8
4.610.81 ±
0.41
'Micrograms of sugar ester isolate per milligram of crude extract. Mean ± standard error.
165
Table 2. Acid distribution on petunia
hybrida Hort. sugar ester isolate
(1994).
Table 4. Number of adults and eggs {Bemisia tabaci) on summer squash {Cucurbita pepo L., cv. 'Pavo') sprayed with Petunia » hybrida Hort. sugar ester isolate (1994) (mean of four dates, total of five leaves).
Acid group
Number of carbon atoms Relative distribution
Extract conc. (mg • ml ') Adults Eggs
Acet)'l Propionyl Malonyl iso-Butyiyl n-But)'ryl 2-Metliyl butyiyl 3-Meth\i butyryl
'Mean separation at P= 0.05 using Duncan's Multiple Range Test.
Valeiyl 4-Methyl valeiyl Hexanoyl
5-Methyl hexanoyl
Literature Cited
4-Methyl hexanoyl Heptanoyl 6-Methyl heptanoyl
Anon. 1991. "Superbug" atutcks California crops. Science 257:1445. Arrendale, R. P., R. M. Martin. 1988. The preparation of immobilized stationai7 pha.se fused silica capillary columns with OV-1701-vinyl deacth'ator. J.
Octanovl
High Re.soIution Chrom. and Chrom. Comm. 11:157-161. Bellows, T. S., Jr., T. M. Perring, K. Arakawa, and C. A. Farrar. 1988. Patterns
ic, and heptanoic were detected, while propionic acid was not
detected. There were distinct quantitative and qualitative dif ferences in fatt)' acids between the composite Petunia sample and N. gossei (Severson et al., 1992a). For example, in Nicotiana, 2-methyl butyric acid was a relatively minor component; in Petunia, it was the most abundant of the acid groups present.
in diel flight actirity oi' Bemisia tabaci (Homoptera: Aleyrodidae) in crop ping s>'stems in southern Calif. Environ. Entomol. 17:225-228.
Broadbent, A. B., R. G. Foottit, and G. D. Murphy. 1989. Sweetpotato whitefly Bemisia tabaci (Gennadius)(Homoptera: Aleyrodidae), a potential insect pe.st of Canada. Can. Entomol. 121:1027-1028.
Coudriet, D., N. Prabhaker, A. N. Kishaba, and D. E. Meyerdirk. 1985. Varia tion in developmental rate on different hosts and ovenvintering of the sweetpotato whitefly, Bemisia tabaci (Homoptera: Aleyrodidae). Environ. Entomol. 64:1333-1334.
Application of the composite sugar ester isolate from
1994 on whitefly adults and nymphs gave substantial mortality
at the 1.0 and 0.5 mg • ml ' concentration range (Table 3) in greenhouse bioassays. Application of the composite sugar es ter isolate (1994) on whitefly populations on field grown sum mer squash gave a significant reduction in the populations when compared to the untreated plants (Table 4). Both the number of adults and eggs were reduced. From greenhouse and field experiments it was evident that sugar esters exhibit a significant level of toxicity to the whitefly, the mode of ac tion of which remains to be ascertained. The results indicate that the trichomes on the surface of
Petunia leaves produce a complex mixture of sucrose and glu cose esters, the concentration of which differed quantitatively among cultivars. Fourteen different acids, ranging in chain length from Cjto C^, were found esterified to the sugar mole cules. When composite samples of sugar esters were sprayed in greenhouse bioassays of adults and nymphs, significant mortality was achieved. Likewise, field application to whitefly populations on summer squash resulted in reduced adult and egg counts. Subsequent work will focus on the isolation of in
dividual types of glucose and sucrose esters to ascertain struc ture/activity relationships and identifying compounds with superior toxicity. The fact that there are distinct quantitative
and qualitadve differences between Nicotiana and Petunia, as
well as significant variation among cultivars, opens the possibility of identifying specific sugar esters or types of esters with greater toxicity.
Cutler, H. G. (ed.) 1988. Biologically active natural products: Potential uses in agriculture. Amer. Chem. Soc. Symp. Ser. No. 380, Amer. Chem. Soc., Washington, DC.
Dufrus,J. E., and R. A. Flock. 1982. Whitefly-transmitted disease complex of the desert .southwest. Calif. Agr. 36(11-12):4-6. Elliott, M. (ed.) 1977. Synthetic pyrethroids. Amer. Chem. Soc. Symp. Ser. No. 42, Amer. Chem. Soc., Washington DC. Faust, R. M. 1992. Conference report and action plan for development of management and control methodology for the sweetpotato whitefly. Houston, Texas Feb. 18-21, U.S. Dept. Agr. - Agr. Res. Ser. Gennadius, P. 1889. Disease of the tobacco plantations in the Trikonia. The aleurodidd of tobacco. Ellenike Georgia 5:1-3. Gill, R.J. 1992. A reriew of the sweetpotato whitefly in Southern California. Pan-Pacific Entomologist 68: 144-152. GofiVeda, J. C., M. A. Mutschler, D. A. Ave, W. M. Tingey, and J. C. Stephens. 1989. Aphid deterrence by gluco.se esters in the glandular exudate of the wild tomato, Lycopersicon pennetti]. Chem. Ecol. 15:2135-2147. Greathead, A. H. 1986. Host plants, p. 17-25. In: M.J. W. Cock (ed.). Bemisia tabaci- A literature survey on the cotton whitefly with an annotated bibli ography. CAB Intern. Inst. Biol. Control, Silwood Park, Ascot, England. Hedin, P. A. 1991. Use of natural products in pest control, p. 1-11. In: P.A. Hedin (ed.). Naturally occurring pest bioregulators. Amer. Chem. Soc. S>mp. Series 449, Washington DC.
King, R. R., R. P. Singh, and A. Boucher. 1987. Variation in sucrose esters from the type b glandular trichomes of certain wild potato species. Amer. Pouto. J. 64: 529-534.
Kubo, I., and K. Nakanishi. 1977. Insect anitfeedants and repellents from Af rican plants, p. 165-178. In: P.A. Hedin, (ed.). Host plant resistance to pests. Amer. Chem. Soc. Ser. No. 62, Amer. Chem. Soc., Washington, DC. Kuol, O., M. B. Isman, and C. M. Ketkar. 1990. Properties and uses of neem, Azadirachta indica. Can. J. Bot. 68:1-11. Mabry, T. J., W. C. Burnett, S. B.Jones, and J. E. Gill. 1977. Antifeedant ses quiterpene lactones in the Compositae. p. 179-184. In: P. A. Hedin, (ed.). Host plant resistance to pests. ASC Symp. Ser. No. 62, Amer. Chem. Soc., Washington, DC.
Table 3. Percent mortality of adult (Bemisia tabaci) sprayed with Petunia
