Journal of Chemical Ecology, Vol. 19, No.

12, 1993

2,3-DIHYDROFARNESOIC ACID, A UNIQUE TERPENE FROM TRICHOMES OF Lycopersicon hirsutum, REPELS SPIDER MITES

JOHN

C . S N Y D E R , 1'* Z H E N H U A JACK P. GOODMAN,

G U O 1"3 R I C H A R D

THACKER,

2 and JAN

ST. PYREK 2

~ Department of Horticulture and Landscape Architecture N318 Ag. Sci. N., University of Kentucky Lexington, Kentucky 40546-0091 2Life Science Mass Spectrometry Facility, College of Pharmacy University of Kentucky, Lexington, Kentucky 40536-0082
(Received June 24, 1993; accepted August 12, 1993)

Abstract--Lycopersicon hirsutum, a wild relative of the tomato, is highly resistant to arthropod herbivores. Both botanic forms of L. hirsutum, L. hirsutum f. glabratum (C.H. Mull.) and L. hirsutum f. typicum (Humb. & Bonpl.), are resistant to two-spotted spider mites, Tetranychus urticae Koch. However, leaves and trichome secretions from f. typicum repel mites more so than those from f. glabratum. We have previously demonstrated that trichome secretions from LA 1363 and LA 1927, accessions of f. typicum,
repelled mites. In this paper we report the identification of the primary component of trichome secretions responsible for repellency. Leaflet washes having compositions similar to trichome secretions were collected and separated into neutral and acid fractions; repellency was mainly associated with the acid fraction, which, when applied to nonrepellent leaflets of f. glabratum, rendered them repellent. Separation of leaflet washes by HPLC allowed purification and subsequent identification by gas chromatography-mass spectrometry and nuclear magnetic resonance of 2,3-dihydrofarnesoic acid (3,7,11-trimethyl-6,10-dodecadienoic acid) as the primary chemical component responsible for repellency. Application of this acid to leaflets of L. esculentum rendered them repellent. Other volatile compounds present in minor amounts in the acid fractions were farnesoic acid and 16:0, 16:3, 18:0, 18:2, and 18:3 fatty acids. This is the first report of the natural occurrence of 2,3dihydrofamesoic acid. * To whom correspondence should be addressed. 3Current address: Department of Agronomy, University of Kentucky, Lexington, Kentucky 405460091. 2981

0098-0331/9311200-2981507.00/0 9 1993PlenumPublishingCorporation

2982

SNYDER ET AL. Key Words--Tomato, Lycopersicon esculentum, host resistance, Tetranychus urticae, Acari, antixenosis, sesquiterpene, trichomes, allomone, 3,7,11-trimethyl-6,10-dodecadienoic acid, farnesoic acid, 2,3-dihydrofarnesoic acid.

INTRODUCTION

Tomatoes (Lycopersicon esculentum Mill.), fill aesthetic and nutritive needs in the human diet worldwide. Insects and mites attack tomato plants and fruit and may cause complete crop loss (Berlinger, 1986). L. hirsutum Humb. & Bonpl., a wild, green-fruited relative of the tomato, is genetically diverse and nearly immune to infestation by arthropods (Rick, 1982). Even though the species L. hirsutum possesses high levels of resistance to insects and mites, this valuable genetic resource has not been used successfully to improve resistance of tomato (Tigchelaar and Foley, 1991). Understanding the intricacies of arthropod herbivore resistance in L. hirsutum may lead to genetic improvement of resistance in tomato. Plant resistance to arthropods often results from the complex interplay of several plant-based characters (Ortman and Peters, 1980). Resistance of L. hirsutum to arthropods has been associated with long-chain methyl ketones, sesquiterpenoids, alkaloids, phenols, and unidentified chemical factors, many trichome borne (Farrar and Kennedy, 1992). Trichome density per se can also alter the interaction between plant and arthropod (Good and Snyder, 1988). Reports of immunity of L. hirsutum to insects suggests that antixenosis underlies its resistance to arthropods. Neither of the two botanical forms of L. hirsutum, f. typicum or f. glabratum, supports high levels of mite infestation. Accessions of f. typicum have sesquiterpenes in their trichome secretions and are generally more resistant, or are resistant in a different manner, to two-spotted spider mites, Tetranychus urticae Koch, than are accessions of f. glabratum, which have long-chain methyl ketones in their secretions (Weston et al., 1989). In terms of mite behavior, this difference between botanic forms is manifested by greater movement of mites onto leaflets of f. glabratum compared with movement onto leaves of f. typicum (Weston et al., 1989). Trichome secretions from f. typicum are more repellent to mites than are those from f. glabratum (Guo et al., 1993). However, the compositions of trichome secretions among accessions of f. typicum are chemically diverse, implying that more than one factor or chemical is responsible for repellency. Previously we identified two accessions of f. typicum, LA 1363 and LA 1927, having repellent trichome secretions with similar, simple composition (Guo, 1992; Guo et al., 1993). Consequently, the objectives of this research were to: (1) isolate the compounds present in trichome secretions from leaves of LA 1363 and LA 1927 that are repellent to spider mites; (2) identify the

TERPENE REPELLENT TO SPIDER MITES

2983

major compound(s) responsible for repellency; and (3) demonstrate that isolated chemicals repel two-spotted spider mites ex planta and in planta.

METHODS AND MATERIALS

Plant Material and Leaflet Hexane Washes. Seed of L. hirsutum accessions were kindly provided by the Tomato Genetic Stocks Center at the University of California Davis, Davis, California. Seed of L. esculentum Mountain Delight were provided by Dr. R. Gardner, North Carolina State University. Plants were raised in the greenhouse. Leaflet washes were prepared by steeping fully expanded, healthy leaflets from plants of L. hirsutum f. typicum accessions LA 1927 and LA 1363 in hexane (1 ml/leaflet) for 60 min, with agitation every 15 min. After decantation, the leaflets were briefly steeped in hexane twice (0.3 ml hexane/leaflet). The combined washes were filtered through Whatman No. 1 filter paper and dried with anhydrous Na2SO4. After evaporating the solvent, the concentration was brought to 1 ml of bexane per leaflet. This concentration is referred to as 1 Washes were stored in glass vials at - 8 0 ~ Compositions of these washes are very similar to those taken directly from trichomes (Guo et al., 1993). Gas Chromatography-Mass Spectrometry (GC-MS). Most of the GC-MS analyses were performed with a Finnigan INCOS 50 instrument equipped with a DB-5ms column (15 m 0.25 mm, 0.1/zm stationary phase). Electron impact mass spectra were acquired at 20 eV in the range of 40-440 amu for samples in the form of either methyl or trimethylsilyl esters. These esters, in turn, were prepared, respectively, by the treatment of the acid fraction with diazomethaneethyl ether solution or bistrimethylsilyltrifluoroacetamide (BSFTA)-pyridine (1 : 1). GC oven temperature was programmed from 100~ for 1 min and then to 280~ at 10~ Additional GC-MS analyses and high-resolution MS measurements were performed using a Kratos Concept IH two-sector instrument. Nuclear Magnetic Resonance (NMR). All 1H and 13C NMR spectra were obtained with a Varian 300 MHz VXR instrument using CDC13 as solvent. Partitioning Leaflet Washes. Washes were extracted three times with an equal volume of 0.05 N NaOH. The upper (neutral) phase was collected; the lower phases were pooled, acidified with 1 N HC1 to pH 5 and then partitioned three times against a 1/3 volume of hexane. Combined hexane layers were reduced to the original volume of the leaflet wash. The leaflet wash and the acid and neutral fractions were analyzed by GC-FID (GC-flame ionization detection) and GC-MS and those from LA 1363 were assayed for repellent activity to mites using the quantitative choice bioassay (see below). Gas Chromatography. Fractionation of leaflet washes and purification were monitored by GC-FID on a Hewlett Packard 5890A gas chromatograph equipped

2984

SNYDERETAL.

with a flame ionization detector and a 10-m 0.54-mm RSL-150 column (Alltech Associates). Helium was the cartier gas (3 ml/min). Injector temperature was 240~ and detector temperature was 300~ The oven temperature was held at 100~ for 1 min and then programmed at 6~ to 170~ and 10~ to 250~ Mite Colony. Mites (Tetranychus urticae Koch) were maintained and handled as described previously (Weston et al., 1989). Female adult mites from bean seedlings moderately damaged by mites were used for bioassays. Spring-Board Bioassay. This bioassay, a choice bioassay, was conducted according to the methods reported by Guo et al. (1993). Quantitative Choice Bioassay. The concentration of repellents on filter paper in the spring-board bioassay could not be calculated precisely because the area wetted by the sample solution could only be roughly estimated. The quantitative choice bioassay was developed to overcome this lack of precision. The bioassay consisted of two filter paper rectangles (1.5 0.5 cm) held parallel to each other and 1.5 cm apart in a clamp (Guo, 1992). Ten microliters of test solution was loaded on one rectangle and, usually, 10 #1 of solvent only was loaded on the other as a control. Ten microliters of solvent just wet the 0.75 cm 2 of filter paper. After the solvent evaporated, a small strip of filter paper (1.5 m m 2.5 cm) was used to bridge the rectangles. A mite was then transferred to the middle of the bridge and its movement was observed. The rectangle over 'which the mite exited was recorded. A mite was replaced by another immediately after it left the bridge or if it was inactive for more than 2 min. Generally 15-20 adult female mites were assayed on a bridge, and two such bridges with the test sample loaded on opposing rectangles (left and right) constituted an assay. Thus, a total of 30-40 mites were usually assayed for a sample. Data were submitted to probit analysis.

Relative Repellency of Leaflet Washes and Acid and Neutral Fractions from LA 1363 and LA 192Z Leaflet washes and fractions were prepared as outlined
above from 25 leaflets of each accession. Leaflet area was determined after extraction. Solvent was removed from each fraction, residues were weighed, and the amount of each fraction per square centimeter of leaflet surface and recoveries were calculated. Residues were redissolved in hexane (15 mg/ml), and serial dilutions (I : 1) were tested for repellency with the quantitative choice bioassay. Data were submitted to probit analysis, and the ECs0 (effective concentration) was determined for each fraction. Because data were obtained from a choice bioassay having an expected choice ratio of 1:1 in the presence of equivalent stimuli, calculation of an ECso from raw data predicts the threshold concentration, the concentration at which mites begin to perceive the stimulus. In order to predict an ECs0 representing a response by 50% of the mites tested, a response index (RI) for the data was first calculated (RI = [(No. of mites exiting over the control - No. of mites exiting over the suspected repellent) +

T E R P E N E R E P E L L E N T T O SPIDER MITES

2985

No. of mites tested]. The data submitted for probit analysis were concentration of the test solution, total number of mites tested - 2, and RI x total number of mites tested 2.

Repellency of Acid Fraction from LA 1363 Applied to Leaflets of Nonrepellent L. hirsutum f glabratum accession, LA 2144. The acid fraction from
LA 1363 was dissolved in 50% ethanol and two-, four-, and eight-fold dilutions of this solution in 50% ethanol allowed application at varying rates to leaflets of LA 2144, a f. glabratum accession having low concentrations of nonrepellent, hexane-soluble volatiles on its leaflet surfaces (Guo et al., 1993). Application was accomplished by dipping cotton balls in a solution and then dabbing them on the adaxial surface of the leaflet, distributing the solution as evenly as possible. Adaxial surfaces of leaflets so treated were assayed for 2 hr with the thumbtack bioassay (Weston and Snyder, 1990). Numbers of mites on the thumbtack were counted and recorded at 30-min intervals. After bioassay, each leaflet was extracted with hexane as outlined previously and leaflet area was determined with a planimeter. The amount of 2,3-dihydrofamesoic acid (see below) applied to the leaflet was quantified by GC-FID and expressed as micrograms per square centimeter of leaflet area. The number of mites remaining on the thumbtack at each observation time and the dosage on the leaflet were submitted to probit analysis to estimate an ECso. Isolation of Repellent Activity. In March 1991 7.6 mg of oleoresin was obtained from a leaflet wash prepared from 75 cm z of leaflets from LA 1927 plants grown in the greenhouse. Aliquots (1 rag) were separated by HPLC. Separation was accomplished with a 10-/zm, Cl8 reverse-phase column (Waters) and a step gradient using acetonitrile-H20-phosphoric acid (60:40: 1, vvv) at 1 ml/min for the first 15 min and 1% phosphoric acid in acetonitrile (vv) at 1 ml/min for the remaining time of each run. The separation was monitored by UV absorbance (218 nm). Detected peaks were collected from each mn and pooled across runs. Pooled fractions were reduced to 2/3 of their original volume on a rotary evaporator and then extracted with 10 ml of hexane three times. The three hexane phases were combined and solvent was evaporated. The residue was weighed and resuspended in 0.5 ml hexane. Composition of each fraction was determined by GC-FID, and each fraction was then bioassayed using the quantitative choice bioassay at dosages equal to the quantity of material in that fraction recovered from 2 cm 2 leaflet area applied to 1 cm 2 of filter paper, assuming 100% recovery (dosage 2 x ) . When a fraction was not repellent at this dose, it was retested at a dosage twice as high (dosage 4 x ) to further ensure that repellent compounds were not overlooked because of low concentration after separation by HPLC. Leaflet wash from LA 1363 was also separated by HPLC, but not bioassayed. Purification of 2,3-Dihydrofarnesoic Acid. 2,3-Dihydrofarnesoic acid, the repellent component in greatest abundance as judged by GC-FID, was purified

2986

SNYDERET AL.

by HPLC. Hexane was evaporated from the acid fraction of LA 1927 with an N2 stream. Five milligrams of residue was dissolved in 1 ml of the carrier solvent (acetonitrile-H20-phosphoric acid 60:40: 1, vvv). Aliquots of 100 /~1 were separated on a 10-#m, C~8 reverse-phase HPLC column (Waters). The isocratic separation was monitored at 218 nm and took place for 40 min at a flow rate of 1.0 ml of sample solvent per minute. Collected fractions were reduced to 2/3 of their volume by evaporation on a rotary evaporator and then extracted with hexane. Purity, as judged by GC, exceeded 97 %. This sample was used for bioassay, to construct a standard curve for quantitation by GC-FID, and for identification by GC-MS and 1H and 13C NMR. Repellency of 2,3-Dihydrofarnesoie Acid Applied to Leaflets of L. esculenturn Mountain Delight. Pure 2,3-dihydrofamesoic acid (98.2%), isolated as outlined above, was diluted in acetone to provide concentrations of 0, 0.2, 0.5, 1.0, 1.5, and 2.0 mg/ml. Leaves of Mountain Delight were removed from 70day-old plants raised in the greenhouse in summer 1991. To maintain turgor, petioles were inserted into glass vials filled with water. A square (1.4 x 1.4 cm) was marked on the adaxial surface of a leaflet by punching a pin hole at each comer. One-tenth milliliter of solution containing 2,3-dihydrofamesoic acid was spread evenly over the square with a pipet. Rates of application were 0, 10, 25, 50, 75, and 100 /zg/cm2. For each rate, four squares on four leaflets provided replications. Thumbtack bioassays were conducted on the treated squares. Number of mites detained on each tack at 1, 2, 4, and 5 hr was recorded. Data were submitted to analysis of variance and probit analysis.

RESULTS

Identification of Components Present in Leaflet Washes. The leaflet washes from LA t363 and LA 1927 and the corresponding neutral and acid fractions were analyzed by GC and, after treatment with diazomethane (to produce methyl esters of carboxylic acids) and BSTFA and pyridine (to produce trimethylsilyl esters), by GC-MS. A typical GC-MS analysis of methylated samples, illustrated by Figure 1, demonstrated the presence of nine distinct components in washes of LA 1363 and 11 components in those of LA 1927. The two early-eluting, neutral components (A and B), sesquiterpenoid hydrocarbons with the same molecular weight of 204, were abundant only in LA 1927 washes. Based on characteristic mass spectra, trace components C and H were phthalate impurities and component C was identified as diethylphthalate (the major plasticizer of Scotch Tape). Each of the other seven components, D - G and I-K, were common to both accessions, having identical Rts and mass spectra. These compounds were methyl esters of sesquiterpenoid and aliphatic fatty acids. Specifically,

TERPENE REPELLENTTO SPIDERMITES

I00-

2987

LA1363
J

_.o n,

E /K

LA 1927
E
(3 B

s ~ ~0
Time

~o
7:~3

~Q
9:37

t0~0
12:01

12b0
14:26

4:49

FIe. 1. GC-MS comparison of the methylated leaflet washes from two accessions of L. hirsutum f. typicum, LA 1927 and LA 1363. The vertical scale (RIC: reconstructed ion curren0 was multiplied by 10. Compounds identified are as follows: A,B--sesqniterpenold hydrocarbons; C,H--phthalate impurities; D--methyl 2,3-dihydrofarnesoic acid; E - methyl farnesoic acid; F, G, I, J, and K--16 : 3, 16 : 0, 18 : 2, 18 : 3, and 18 : 0 fatty acids, respectively.

CH C ~

CH

CH cOOH

2,3-Dihydrofamesoic Acid
CH

CH 3

OH

3 GOOH

Famesoic Acid

FIG. 2. Sesquiterpene acids identified in leaflet washes from two accessions of L. hirsuture f. typicum, LA 1927 and LA 1363.

2988

SNYDER El" AL.

esters of sesquiterpenoid acids included the major component D, identified as methyl 2,3-dihydrofamesoic acid, and E, identified as methyl farnesoic acid (Figure 2). The other five methyl esters (components F, G, I, J, and K) were identified as 16: 3, 16 : 0, 18:2, 18 : 3, and 18 : 0 fatty acids, respectively, with 18 : 3 predominating. This identification was supported by the direct Rt and MS comparisons with the authentic standards of methyl esters of 16:0, 18:3, and 18 : 0 fatty acids. 2,3-Dihydrofarnesoic acid (3,7,11-trimethyl-6,10-dodecadienoic acid) was identified based on the characteristic electron impact mass spectra of the free acid [70 eV spectrum not shown: m/z (relative intensity) at 238 (M +, 0.7), 223 (M-Me, 1.2), 195 (M-C3H7, 17), 123 (16), 109 (35), 95 (8), 81 (9), 69 (100)] and methyl ester and trimethylsilyl ester (Figure 3A and B). Molecular ions (M + at m/z 238, 252, and 310, respectively) were weak but clearly detected, and abundant pseudomolecular ions (MH +) were present in the isobutane chemical ionization spectra of the free acid and trimethylsilyl esters at m/z 239 and 311, respectively. Moreover, the precise measurement performed for the molecular ion of the methyl ester was consistent with the expected elemental composition (measured 252.2088, expected for C15Hz602 252.2089). This acid was isolated by HPLC in an amount enabling its definitive IH and 13C NMR characterization. Table 1 presents full spectral assignments obtained for the free acid and its methyl ester. The infrared spectrum of the methyl ester showed a major band at 1745 cm -1, a saturated ester. Farnesoic acid represented the minor component E ( < 5 %). Although only mass spectral data were available (Figure 3C), this identification was fully confirmed by oxidation of farnesol with Jones' reagent (chromic acid) followed by methytation with diazomethane. Apart from the unsaturated aldehyde, the major product of this reaction sequence, methyl farnesoic acid with the same Rt and mass spectrum as the compound identified in washes from both accessions, was present as the minor oxidation product. Repellency of Fractions from LA 1363. The leaflet wash and neutral and acid fractions from LA 1363 were repellent when assayed against hexane with the spring-board bioassay (Table 2). When tested against each other, the acid fraction was almost as repellent as the leaflet wash, and it took ca. 80 min to assay the 22 mites, an indication of strong repellency. The neutral fraction was less repellent than the acid and leaflet wash. Relative Repellency of Leaflet Wash and Acid and Neutral Fractions. For each accession, the acid fraction comprised about 50-60% of weight of the leaflet wash (Table 3). The main differences between accessions were the weight and composition of the neutral fractions. Repellency of leaflet washes and acid fractions were similar regardless of source (Table 4). The neutral fractions were less repellent. Positive synergism for repellency between neutral and acid frac-

TERPENE REPELLENT TO SPIDER MITES

2989

RIC
100.069 10g

I'- 5ox

A
252

50.0
209

123

,,I I,

237

55

I
173

85
16

2~35
.

177
151 163

M/Z 50
', .

100
, .

150
i .

200
,

250
i

300
i

RIC

109

r-60X

100"01
310

B
I
73 296 173'

123

267

135 213

MIZ 50

100

150

200

250

300

FIG. 3. Electron impact mass spectra (20 eV) of methyl 2,3-dihydrofarnesoic acid (A), trimethylsilyl 2,3-dihydrofarnesoic acid (B), and methyl farnesoic acid (C) identified in the accession LA 1927. Identical spectra were obtained for the accession LA 1363.

2990

SNYDER ET AL.

RIO 100t

r--20X 147

C
207

6ot
175 81 114

219

250

M/Z 50

, ,

100

' ' ' ' '

150

,I tr,,I, ,, ,,,
. . . '

200

,,,I],,I,, I I,II ,htlllI

. . . . . .

250

300

'

FIG. 3. Continued tions did not seem likely because the ECs0 values for acid fractions were less than those for the respective leaflet washes. Repellency of Acid Fraction Applied to Leaflets of L. hirsutum f. glabratum LA 2144. ECso values predicted from thumbtack bioassays of LA 2144 leaflets treated with the acid fraction from LA 1363 containing 2,3-dihydrofarnesoic acid and farnesoic acid were between 1 and 6/zg/cm 2, depending on the time after initiation of the bioassay (Table 5). These values were comparable to the ECso obtained in the quantitative choice bioassay of this fraction, 10/~g/cm 2 (Table 4). Repellency of HPLC Fractions and Pure 2,3-Dihydrofarnesoic Acid. HPLC provided adequate separation of compounds present in leaflet washes (Figure 4). Farnesoic acid eluted at = 2 0 min and 2,3-dihydrofamsoic acid, at =21 rain. The compositions of leaflet washes of LA 1363 and LA 1927 were very similar except that LA 1927 contained more late-eluting hydrocarbons than did LA 1363 (Figure 4). Fraction 5 was highly repellent (Table 6) and was more than 90% 2,3-dihydrofarnesoic acid, as judged by GC-FID. The 3.2 mg recovered from this fraction (Table 6) was equivalent to 42.7 /~g from 1 cm 2 leaflet area of LA 1927. The two other repellent fractions, fractions 4 and 6, also contained mainly 2,3-dihydrofarnesoic acid. Fraction 4 contained a significant amount of farnesoic acid as well, but the repellency of farnesoic acid could not be determined because no fraction contained just this compound. The abundances of the early eluting components were low. Their early elution from HPLC and nondetectability on GC-FID indicated that these components were less lipophilic and less volatile than famesoic acid. The small amounts of these com-

TERPENE REPELLENT TO SPIDER MITES

2991

TABLE 1. 13C AND 1H N M R DATA OF 2,3-DIHYDROFARNESOIC ACID (in CDC13) MEASURED AT 75.43 AND 300 MHz, RESPECTIVELY; FOR FREE ACID MULTIPLICITY ASSIGNED BY APT; AND Me ESTER ASSIGNMENT BY

APT, COSI, AND HETCOR 6 t3C Free acid C-1 C-2 C-3 C-3' C-4 C-5 C-6 C-7 C-7' C-8 C-9 C-10 C-11 C-11' C- 12 Me 178.33 41.34 29.91 19.68 36.74 25.376 124.09" 135.35 16.06 39.80 26.75 ~ 124.37" 131.45 17.78 25.79 Me ester 173.83 41.69 30.13 19.73 36.82 25.40 # 124.20" 135.23 16.05 39.80 26.76 # 124.38" 131.42 17.79 25.80 51.47 Aba -4.50 +0.35 +0.22 +0.05 + 0.08 +0.03 +0.11 -0.12 -0.01 0 +0.01 +0.01 -0.03 +0.01 +0.01 Free acid IH Me ester

2.370 ddc 2.150 dde 0.981 de

5.085 tm 1.598 bq

2.31 ddd 2.12 ddf (1.86 m) 0.943 dg (1.2, 1.4 m) (1.90 m) 5.095 tm 1.597 bq (1.86 m) (1.97 m) 5.095 tm 1.597 bq 1.677 bqh 3.667 s

5.100 tm 1.598 bq 1.676 bqh

a 13 Chemical shift difference induced by methylation. C b-e, denotes uncertain, interchangeable assignments. "JAB = 15.0 HZ, JAX = 6.1 HZ. aJAB = 14.6 HZ, JAX = 5.9 HZ. e J A B = 15.0 Hz, JBx = 8.2 Hz. f JAB = 14.6 Hz; JBx = 8.2 Hz. uJ = 6.6 Hz. hj = 1.2 Hz.

p o n e n t s in t h e leaflet w a s h m a y b e related to l o w a b u n d a n c e o r l i m i t e d solubility in h e x a n e ; t h e i r a b u n d a n c e in o r o n t h e l e a v e s m a y b e h i g h . B i o a s s a y s o f t h e s e f r a c t i o n s at t h e 4 d o s e i n d i c a t e d n o r e p e l l e n c y . F r a c t i o n 8 c o n t a i n e d sesquit e r p e n e h y d r o c a r b o n s t h a t w e r e not w e l l s e p a r a t e d , a n d this f r a c t i o n w a s n o t repellent. N o e v i d e n c e w a s o b t a i n e d i n d i c a t i n g that c o m p o u n d s o t h e r t h a n 2 , 3 d i h y d r o f a r n e s o i c acid o r f a r n e s o i c acid w e r e h i g h l y r e p e l l e n t to mites. T h e ECs0 f o r p u r e 2 , 3 - d i h y d r o f a m e s o i c acid in t h e q u a n t i t a t i v e c h o i c e b i o a s s a y w a s 9 # g / c m 2 w i t h 95 % fiducial l i m i t s o f 6 a n d 1 2 / ~ g / c m z. T h i s v a l u e c o r r e s p o n d e d w i t h the ECso for t h e acid f r a c t i o n t e s t e d s i m i l a r l y , 10 tzg/cm 2 ( T a b l e 3), a n d t h e ECs0 v a l u e s f o r t h e acid f r a c t i o n a p p l i e d to leaflets o f L A 2 1 4 4 , 1 - 6 / ~ g / c m 2 ( T a b l e 5).

2992

SNYDER ET AL.

TABLE 2. REPELLENCY TO MITES OF LEAFLET WASHES FROM LA 1363 AND FRACTIONS DERIVED FROM THEM MEASURED BY SPRING-BOARD BIOASSAY

Test fractions~ A Wash Acid Neutral Wash Wash Acid B Hexane Hexane Hexane Acid Neutral Neutral Exit ratio b A:B 0:30 5 : 25 10 : 30 6 : 16 9:31 6:34 X2 15.0 **C 6.7"* 5.0" 2.3 ns 6.0* 9.8**

aTest solution was loaded on one postion (A) of the spring-board and a control or another test solution was loaded at the other position (B) on the same spring-board. bThe exit ratio is the ratio of the number of mites exiting over position A and position B. c,,** significant at 5% and 1%, respectively; n~ not significant.

TABLE 3. QUANTITY OF RESIDUES IN LEAFLET WASHES AND NEUTRAL AND ACID FRACTIONS AND RECOVERY OF TRICHOME SECRETIONS FROM TWO ACCESSIONS OF L. hirsutum f. typicum

Accession LA 1363 LA 1927

Leaflet wash (t~g/cm 2 of leaflet surface) 81 116

Neutral fraction ( ~zg/cm2 of leaflet surface) 32 48

Acid fraction (t~g/cm 2 of leaflet surface) 50 57

Recovery (%) 101 91

TABLE 4. REPELLENCY TO MITES AS INDICATED BY EC50 a OF LEAFLET WASH AND NEUTRAL AND ACID FRACTIONS FROM TWO ACCESSIONS OF L. hirsutum f. typicum

Leaflet wash 95% fiducial limits 24-38 14-33

Neutral fraction 95% fiducial limits 91-372 46-117

Acid fraction 95% fiducial limits 7-15 8-15

Accession LA 1363 LA 1927

ECso (/zg/cm2) 30 22

ECso (/~g/cm 2) 145 69

ECso (#g/cm 2) 10 10

a Predicted concentration at which 75 % of the spider mites respond to the stimulus.

TERPENE REPELLENT TO SPIDER MITES

2993

TABLE 5. REPELLENCY MITES AS INDICATEDBY ECso~ IN THUMBTACKBIOASSAYOF TO LEAFLETSFROMNONREFELLENTACCESSIONOF L. hirsutum f. glabratura (LA 2144) TREATEDWITHACID FRACTIONOBTAINEDFROMLEAFLETSOF L. hirsutum f. typicum ACCESSIONLA 1363 Time after initiation of bioassay (rain) 30 60 90 120

ECso (#g/cme) 0.7 2.9 4.5 5.7

95% fiducial limits 0.1-1.8 1.4-4.8 1.2-7.9 3.5-8.8

~Predicted concentration at which 50% of the spider mites respond to the stimulus.

I 2 I 3 1415161
Fraction

7 18191

FIG. 4. HPLC fractionation of leaflet washes from two accessions of L. hirsutum f. typicum, LA 1363 (upper) and LA 1927 (lower).

Repellency of 2,3-Dihydrofarnesoic Acid Applied to Leaflet Surfaces of Tomato. Application of pure 2,3-dihydrofarnesoic acid on abaxial leaflet surfaces of Mr. Delight rendered them repellent (Figure 5). Repellency was related to dose, and the ECso at 1 hr, 1.5 ~g/cm 2, was similar to that obtained at 1 hr for the acid fraction applied to LA 2144 (2.9/zg/cm 2, Table 5). However, at 2 hr the predicted EC50 (33 #g/era 2) was about fivefold greater than that obtained at 2 hr for the acid fraction applied to leaflets of LA 2144 (5.7/zg/cm 2, Table 5). The differences in the EC5o values at the 2-hr sampling periods in the bioassays may relate to the different plant material used for the two bioassays. LA 2144 has a trichome vesture distinct from that of Mt. Delight, which probably contributed to the greater durability of repellency on L A 2144. Nevertheless, both bioassays demonstrated that response to applied chemical repellents varied with dose.

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SNYDER ET AL.

TABLE 6. QUANTITY OF RESIDUE RECOVERED FROM AND REPELLENT ACTIVITY TO MITES ASSOCIATED WITH H P L C FRACTIONS OF LEAFLET WASHES FROM LA 1927 a

Fraction
1 2 3 4 5 6 7 8 9

Weight of residue in fraction (mg)

0.1 0.2 0.2 0.9 3.2 0.9 0.4 1.1 1.4

Exit ratio (sample : control) Weight of residue (ttg/cm2 leaflet area)

1 2 2 7 43 7 3 8 10

2 x dose 16:14 20:10 12:18 5 : 25 b 0 : 30 b 5 : 25 b 15:15 19:11 17:13

4 x dose 18:12 18:12 12:18 NT c NT NT 14:16 8:22 13:17

aSee Figure 4. The 2 x and 4 x dose indicate the concentration of the residue tested with the quantitative choice bioassay, assuming 100% recovery. Calculated recovery was 110%. bRatios significantly deviate from 1 : 1 at P < 1% as determined by X2. CNT: the 4 x dose was not tested because the 2 x dose was repellent.

10

pg.cm"~ y

100

75
5o

~
~

25
10

HOURS

FIc. 5. Repellency to mites o f 2,3-dihydrofamesoic acid applied to leaves o f L. esculentum Mt. Delight as measured by the thumbtack bioassay. Ten mites were initially placed on each tack.

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DISCUSSION

Previously we reported that leaves and trichome secretions of LA 1927 and LA 1363 repelled mites and that removal of trichomes and secretions from leaflets by wiping reduced their repellency (Guo et al., 1993). Evidence presented herein substantiates that 2,3-dihydrofarnesoic acid is present in hexane washes of leaflets and is repellent to spider mites when encountered on filter paper or on leaflets treated with the acid. Because most, if not all, of the 2,3dihydrofarnesoic acid is associated with trichomes (Guo, 1992) and because the concentration of the acid on the leaflet is well above its ECs0 for repellency, it is likely that the presence of this acid in trichomes is responsible for repellency to mites of these accessions of L. hirsuturn f. typicum. The presence of 2,3-dihydrofarnesoic acid may confer resistance to other arthropods. Fatty acids, especially C8-C12, are toxic to arthropods (Siegler and Popenoe, 1925), and dodecanoic acid and (E)-/3-farnesene are aphid alarm pheromones, which act by deterring settling of aphids (Bowers et al., 1972; Greenway et al., 1978). 2,3-Dihydrofarnesoic acid is structurally similar to (E)-/3farnesene and has physical and chemical properties similar to Cl2 fatty acids. Thus, it is reasonable to anticipate that the presence of 2,3-dihydrofarnesoic acid on plants may confer resistance to arthropods other than mites via toxicity and action similar to an alarm pheromone. Furthermore, farnesol serves as a precursor to juvenile hormone III, methyl farnesoic acid, a hormone regulating larval development of some insect species (Judy et al., 1973). It is possible that farnesoic acid or 2.,3-dihydrofarnesoic acid may also modify plant-arthropod interactions by altering larval development. Terpenes are known to affect mite behavior. Monoterpenes have been reported as attractive to predatory mites (Takabayashi et al., 1991) and to act as sex attractants for male two-spotted mites (Regev and Cone, 1980). Farnesol has been reported as a male sex attractant for two-spotted spider mites (Regev and Cone, 1975). Patterson et al. (1975) reported the presence of an unidentified sesquiterpene alcohol in plants of f. hirsutum PI 251303 that repelled female two-spotted spider mites. Our documentation that 2,3-dihydrofarnesoic acid repels mites adds to the list of terpenes reported to modify mite behavior. The relationships among chemical structure, response, mite species and sex, however, need additional research for greater delineation. The presence of sesquiterpene acids has been reported in other accessions of L. hirsutum f. typicurn. (+)-(E)-a-Santanlen-12-oic acid and (+)-(E)-endofl-bergamoten-12-oic acid, present in trichomes, are ovipositional attractants for Heliothis zea in LA 1777 (Coates et al., 1988). These acids are also repellent to mites as are acids of zingiberene and curcumene in trichome secretions of f. typicum PI 251303 (unpublished). Thus, the presence of sesquiterpene acids in trichome secretions of L. hirsutum f. typicum appears to be relatively common,

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but structures may be diverse. To the best of our knowledge this is the first demonstration of the natural occurrence of 2,3-dihydrofarnesoic acid, the compound known as a synthetic intermediate (Ahlquist and Stallberg-Stenhagen, 1971). interestingly, the corresponding alcohol, terrestrol, has been found to occur in the bumble bee, B o m b u s terrestris (Stallberg-Stenhagen, 1970). The evidence presented here strongly supports the idea that the presence of 2,3-dihydrofarnesoic acid on leaves of plants of LA 1927 and LA 1363 causes mites to avoid them. It is also reasonable to expect that the presence of 2,3dihydrofarnesoic acid may alter the interaction of these accessions with other arthropods. We must emphasize, though, that 2,3-dihydrofamesoic acid can not explain the repellency of all accessions of f. typicum, because this acid does not occur or occurs at very low levels in some highly repellent f. typicum accessions. Furthermore, the presence of 2,3-dihydrofamesoic acid on leaves of LA 1363 and LA 1927 is likely just a single facet of a resistance complex that operates in these accessions, rendering them immune to mites.
Acknowledgments--The investigationreported in this paper (No. 93-10-100) is in connection with a project of the KentuckyAgricultureExperimentStation and is publishedwith the approval of the Director.

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