Journal of Chemical Ecology, Vol. 10, No. 12, 1984
R O L E OF G L A N D U L A R S C A L E S OF L E P I D O T E R H O D O D E N D R O N S IN I N S E C T RESISTANCE 1
ROBERT E DOSS USDA-ARS, Horticultural Crops Research Laboratory 3420 N. W. Orchard Avenue Corvallis, Oregon 97330
(Received December 28, 1983; revised April 23, 1984) Abstract--Glandular scales on selected lepidote rhododendron species varied in density from 109 _+ 13 to 4180 • 60/cm 2of leaf surface. Globules contained within the scales stained with Sudan IV, a lipophilic dye. Essential oil contents of the scales varied with species from 24 + 8 to 151 _+ 35 ng/scale. Black vine weevil [(Otiorhynchus sulcatus (E)] feeding on leaves from a sample of rhododendron species was inversely related to leaf essential oil content, and weevilfeeding on membrane filters was inhibited by application of essential oil extracts from leaves of most lepidote rhododendrons tested. Results suggest that the glandular scales of the lepidote rhododendrons function, at least in part, in plant defense against insects.
Key Words-Coleoptera, Curculionidae, Otiorhynchus sulcatus (E), black vine weevil, Ericaceae, Rhododendron, trichomes, glandular scales, essential oils, volatiles, plant resistance. INTRODUCTION G l a n d u l a r scales of the lepidote r h o d o d e n d r o n s were described by D e B a r y (1884). I n 1950, C o w a n (p. 107) speculated that in the genus R h o d o d e n d r o n " . . . a n efficient p r o t e c t i o n against desiccation is furnished by d e n d r o i d , r a m i f o r m , rosulate, f u n n e l - s h a p e d , loriform, l o n g rayed, a n d radiate hairs a n d the most c o m p l e t e p r o t e c t i o n by a dense covering of scales." M o r e o v e r , C o w a n believed that scales could a b s o r b m o i s t u r e f r o m the a t m o s p h e r e a n d ~Mention of a trade name or proprietary product does not constitute an endorsement by the U.S. Department of Agriculture, Agriculture Research Service. 1787 0098-0331/84/1200-178753.50/0 @ 1984 Plenum Publishing Corporation
1788
Doss
could secrete excess water under conditions of reduced transpiration. Despite the fact that there is no direct evidence to support the contention that rhododendron leaf scales function primarily in regulating water passage into and out of the leaf, this idea has generally been accepted without question (e.g., Leach, 1961, pp. 25-28), although Seithe (1980) stated that scales do not absorb water. Bell and Clarke (1978) recently published data showing that rhododendron species differed in their resistance to foliar feeding by adult obscure root weevils (Sciopithes obscurus Horn), a serious pest of rhododendrons in the Pacific Northwest. Analysis of these data showed that lepidote species (those with leaf scales) were more resistant than elepidote species (Doss, 1980). Hexane-soluble materials from resistant lepidote species inhibited obscure root weevil feeding (Doss, 1980) and, in the case of R. edgeworthii Hook., a particulary resistant species, a steam volatile fraction containing, among other compounds, the sesquiterpene germacrone, acted as a weevil feeding deterrent (Doss et al., 1980). These findings suggest that glandular scales of lepidote rhododendrons may function in plant defense by containing or secreting volatile materials that discourage insect feeding. The study described below was carried out to test this hypothesis. METHODS AND MATERIALS Table 1 lists the rhododendrons used in this study. These comprise a geographically and taxonomically diverse sample, including 11 lepidotes, all of which are reported to be resistant to obscure root weevil feeding (Bell and Clarke, 1978; Antonelli and Campbell, 1980), two elepidotes, and an elepidote hybrid. Among the elepidotes, R. smirnowii has been reported as resistant to black vine weevil [Otiorhynchus sulcatus (F.)] feeding (Valla, 1980; Nielsen and Dunlap, 1981). R. "Cynthia" was found to be susceptible to feeding by obscure root weevil (Doss, 1980), and R. catawbiense, to feeding by black vine weevil (Nielsen and Dunlap, 1981). Leaves used in this study, with the exception of those from R. "Cynthia," were obtained from the Rhododendron Species Foundation, Federal Way, Washington. "Cynthia" leaves were obtained from a hedge planting at Western Washington Research and Extension Center, Puyallup, Washington. Glandular scales from a measured area of leaf were removed with a fine-tipped scalpel and placed into a small amount of diethyl either (Harborne, 1973, p. 103). After 30 min, the ether was evaporated under nitrogen and the extracted materials were redissolved in hexane. Leaf volatiles were extracted from small leaf disks into diethyl ether (see above)
1789
GLANDULARSCALESOF RHODODENDRON
TABLE 1. RHODODENDRONSPECIES(AND CULTIVAR) USED FOR ESSENTIALOIL STUDY
Species (Cultivar)
Clonea
1. R. campylogynum
74.76
Franch. 2. R. carolinianum
Rehd. 3. R. catawbiense
Michx. 4. R. chryseum e
Balf. f. et Wand 5. R. "Cynthia ''f 6. R. dauricum
Linn. 7. R. edgeworthii
Hook. f. 8. R. ferrugineum
Linn. 9. R. hanceanum
Hemsl. 10. R. heliolepis Franch. 1I. R. lepidotum
G. Don 12. R. rigidum Franch. 13. R. smirnowii Trautv. 14. R. xanthocodon h Hutch.
Seriesb (subsection)c
Campylogynum (Campylogyna) 7 5 . 1 3 3 Carolinianum (Caroliniana) 76.12 Ponticum (Pontica) 75.28 Lapponicum (Lapponica) 66.590
Dauricum (Rhodorasta) 65.383 Edgeworthii (Edgeworthia) 76.381 Ferrugineum (Rhododendron) 76.34 Triflorum (Tephropepla) 65.374 Heliolepis (Heliolepida) 79.53 Lepidotum (Lepidota) 73.353 Triflorum (Triflora) 77.319 Ponticum (Pontica) 73.305 Cinnabrinum (Cinnabarina)
Geographical distrubution b'c WesternYunnan, Tibet, Burma Southeastern U.S.
Scale typed vesicular (L) entire (U, L)
Southeastern U.S. Yunnan to Tibet
undulate (U, L)
Altai, Korea, Japan
entire (U, L)
Sikkim, Bhutan
entire (L)g
Pyreneeseast to Austrian Alps Szechwan
entire (L) entire (U, L)
Yunnan
entire (U, L)
India, Nepal, Bhutan Burma, China Yunnan
entire to undulate (U, L) entire (L)
Caucasus Mountains entire (U, L) Sikkim
aClone number of Rhododendron Species Foundation, Federal Way, Washington. bSee Leach (1961). 'See Cullen and Chamberlain (1978) and Cullen (1980). aSee Cowan (1960) and Seithe (1980). U = scales on upper surface of leaf, L = scales on lower surface of leaf. eConsidered by some as a subspecies of R. rupicola W.W. Sm. (Cullen and Chamberlain, 1978). YR."Cynthia"is a hybrid of R. catawbiense Michx. and R. griffithianum Wight. R. griffithianum is a member of the Fortunei series (Fortunea subsection). gR. edgeworthii possesses a few vestigal scales on the upper leaf surface. h Considered by some as a subspecies of R. cinnabarinum Hook (Cullen 1980).
or o b t a i n e d by steam distillation f r o m leaves u s i n g a modified NielsenKryger a p p a r a t u s with h e x a n e as the o r g a n i c solvent (Veith and Kiwus, 1977). Leaf disks or leaves of lepidote r h o d o d e n d r o n s used for e x t r a c t i o n bore scales. Scale densities were d e t e r m i n e d by c o u n t i n g t h e m o n 5 . 5 - m m d i a m e t e r leaf disks cut f r o m f o u r separate leaves. The a m o u n t of essential oils p r e s e n t in the scales was estimated by gas c h r o m a t o g r a p h y f r o m ether extracts m a d e u s i n g small leaf disks. A 1.9-m X 2 - m m (ID) glass c o l u m n packed with 3% ( w / w ) SP2100| o n 100-120 mesh
1790
Doss
Supelcoport| was used. Nitrogen was employed as the carrier gas (30 cm3/ rain). The flame ionization detector and the injector were maintained at 260 ~ C. Column temperature was increased from 100 to 200 ~ at a rate of 5~C / m i n with initial and final temperatures held for 2 rain. A nerolidol standard allowed estimation of the amount of essential oils present in the leaf extract. Gas chromatographic estimates were checked against estimates obtained by weighing materials obtained by steam distillation. Gas chromatographic traces obtained with extracts prepared from detached scales were compared with traces obtained with extracts of leaf disks. Leaf cross-sections (50 /~m) cut using a vibrating microtome (Vibratome| were stained using the lipophilic dye Sudan IV (Jensen, 1962, pp. 264-265) and mounted in glycerine jelly (Sass, 1958, pp. 102-103). Detached scales were mounted without staining. Black vine weevil colonies were maintained at 20~ under either continous darkness or photoperiodic cycles consisting of 16 hr of fluorescent light and 8 hr of darkness. Weevil colonies were fed leaves from either "Alpine" strawberry (Fragaria vesca L.) or " T o t e m " strawberry (F. X ananassa Duch.). To determine the feeding response of black vine weevil adults to the rhododendron species tested, leaf disks 14.4 mm in diameter were cut using a notched cork borer that left the petiole attached to the leaf disk. Petioles were inserted through a serum stopper into vials containing water. A moist dental wick, four black vine weevils, and a vial with a leaf disk were placed into each of the cylindrical carboard containers (about 250 c m 3 in volume) used as bioassay arenas (Bristow et al., 1979). Bioassays were run for 24 hr at 20 ~ after which time the areas eaten from the leaves were measured. With R. chryseum (leaf area = 93 ___ 6 mm 2, X • SE for four leaves), R ferrugineum (leaf area = 146 • 10 mm 2, for five leaves ), and R. lepidotum (leaf area = 188 • mm 2, for five leaves), intact leaves, instead of leaf disks, were used because leaves of these species were small. With other species, leaf disks rather than intact leaves were used to eliminate any effect of leaf size on weevil feeding. The influence of essential oils on black vine weevil feeding was determined by using a membrane filter bioassay procedure (Doss, 1980). Membrane filters were first pretreated with 50 rag-equivalents of an ethanolic extract of "Alpine" strawberry leaves that stimulated black vine weevil feeding (Doss and Shanks, unpublished). Then l disk treated with the strawberry extract and t disk treated with the extract plus a rhododendron essential oil extract (from a portion of leaf equivalent in area to a 13-mmdiameter membrane filter) were placed into a bioassay arena along with a moist dental wick and four adult black vine weevils. Bioassays (choice tests)
1791
GLANDULAR SCALES OF RHODODENDRON
were run at 20 ~C for 24 hr. Weevil feeding on the filters was compared using the paired t-test. RESULTS Histological examination indicated that rhododendron leaf scales contained lipophilic globules (Figure 1). Gas-liquid chromatographic traces obtained using extracts made from scales and from leaf disks were qualitatively identical. Figure 2 shows such a trace obtained with an extract of R. dauricum leaves. Extracts made using detached scales contained less volatile material (on an area basis) than leaf disks. Some oil was contained in the scale stalk which remained attached to the leaf upon scale removal (Figure 1). Of course, the leaves, exclusive of the scales, also contained volatiles. The 11 lepidotes yielded much larger volatile fractions, averaging about 100,000 ng/cm 2 of leaf, than the elepidotes which averaged about 12,000 ng/cm< R. smirnowii, a heavily indumented elepidote, yielded more volatiles than three of the lepidote species. Essential oil contents varied with species from about 25 to 150 ng/scale for the lepidotes examined (Table 2). The values reported here were estimated gas chromatographically and are, on average 60 +_ 10% (X _ SE) of the values obtained by weighing steam distillate, except for R. "Cynthia," The estimate obtained for this cultivar by weighing steam distillate was 1.3 mg/g fresh wt. In an earlier study (Doss et al., 1980), a value of 0.64 mg/g fresh wt was obtained for R. "Cynthia," suggesting that the gas chromatographic value reported in Table 2 (equivalent to 0.008 rag/g) could be a serious underestimate. Note that values were calculated assuming that all essential oils were contained within the scales even though some volatiles are present on nonscale-bearing leaves. There was a significant negative correlation between the areas eaten from leaf disks (or small leaves) and the volatile oil content (Figure 3). This was also true when only the lepidote species were considered (r = -0.76 with 8 df). The estimates of essential oil contents used in this analysis were obtained by weighing steam distillate, but using estimates obtained gas chromatographically resulted in a similar relationship and correlation coefficient (r = --0.70). The species R. heliolepis was omitted from this study through oversight. In choice tests with membrane filters, nine of the 11 lepidote species yielded extracts that inhibited weevil feeding (Table 3). None of the elepidote species yielded inhibitory extracts and, with the lepidotes, noninhibitory extracts came fron species with relatively small amounts of essential oils. In no-choice tests (not discussed in Materials and Methods), using only 1 membrane filter disk per arena, there was large variation in feeding. The
1792
Doss
Fio. 1. (top) Cross-section through a Rhododendron edgeworthii leaf showing glandular scale. Scales are about 110/xm in diameter. (bottom) Detached scale (300 t~m in diameter) from R. chryseum. Oil droplets are indicated by arrows.
correlation between areas eaten by black vine weevils from phagostimulantbearing membrane filters treated with essential oil extracts and the essential oil contents of the leaves was not significant with the no-choice tests (data not shown).
1793
GLANDULAR SCALES OF RHODODENDRON
8
3
4
I
o-
5
I
10 15 Retention Time (rain)
I
20
25
FIG.2. Gas chromatographic trace of Rhododendron dauricum essential oil extract. The probable identities of the materials giving rise to the number peaks are as follows: 1. fl-caryopheyllene, 2. c~-humulene, 3. cis-nerolidol, 4. trans-nerolidol, 5.8elemenone, 6. 7-eudesmol, 7. a/fl-eudesmol, 8. germacrone. DISCUSSION The role of various types of trichomes in conferring insect resistance upon plants is well documented (Levin, 1973; Webster, 1975; Stipanovic, 1983). In fact, it has been suggested that the presence of trichomes is more often associated with defense against insect attack than with reduction of water loss, a commonly suggested function of plant trichomes (Fitter and Hay, 1981, p. 283). With Rhododendron, Valla (1980) noted that indumented and lepidote types suffered less adult black vine weevil feeding damage than did glabrous leaf types. Data presented by Bell and Clarke (1978) suggested that the lepidote rhododendrons were more ofter resistant to obscure root weevil feeding than elepidotes (Doss, 1980). With respect to the lepidote rhododendrons, results presented here
campylogynum carolinianum catawbiense chryseum
21.8 22.7 27.3 21.4 44.1 22.3 34.5 29.0 34.9 26.9 16.0 21.7 32.4 22.7
4• • • + • • + • • • • • •
0.8 0.8 1.3 0.8 3.4 0.4 1.3 0.8 0.4 0.8 0.8 1.7 0.4 1.3
Fresh 12.6 • 10.9 • 13.9 • 11.4 • 22.7 • 11.4 • 18.9 • 13.9 • 15.6 • 10.9 • 6.7 • 11.4 • 16.8 • 11.4 •
Dry 0.4 0.8 0.4 0.4 1.7 0.4 0.4 0.8 0.4 0.4 0.4 0.8 0.4 1.3 43 + 9
46 128 • 151 • 70 • 128 • 26 • 25 • 32 •
71 5 : 8
63 25 101 4 17 25 13 50
30 35 7 25 3 8 5
65 • 8 8 6 • 12
ng/scale
21
Lower surface
315 • 1206• no scales 1916 • 39 2273 • no scales 122 • 29 798 • 0 2168 • 0 1409 • 59 • 4 219 • 399 • 29 412 • 614 • 17 672 • 0 109 • no scales 538 • 118 1370 •
0 172•
Upper surface
Scales per cm 2
20600 • 2100 118000 • 15000 1656 • 221 180000 • 39000 357 • 72 118000 • 25000 328150 • 75000 98900 • 5900 35600 • 7000 21400 • 2000 311000 • 10100 3430 • 400 27400 • 5000 136000 • 12000
ng/cm2
Essential oils ~
aMeans • standard errors for five samples. bMeans + standard for four samples. CMeans + s t a n d a r d errors for four extracts. Note that ng/scale values ignore volatiles present in the leaf l a m i n a and are thus overestimates (see Results).
R. dauricum R. edgeworthii R.ferrugineum R. hanceanum R. heliolepis R. lepidotum R. rigidum R. smirnowii R. xanthocodon
R. ' C y n t h i a '
R. R. R. R.
Species (cultivar)
Leafweight (mg/cm2) a
TABLE 2. ESSENTIAL OIL CONTENTS OF 1 1 LEPIDOTE AND 3 ELEPIDOTE RHODODENDRON SPECIES (AND CULT1VAR) MEASURED FROM EXTRACTS OF LEAF DISKS
1795
GLANDULARSCALESOFRHODODENDRON
2OO
(~)
160
12
c~
E120 ~z 1~14
6
c
w
~ 80
1_
R O L E OF G L A N D U L A R S C A L E S OF L E P I D O T E R H O D O D E N D R O N S IN I N S E C T RESISTANCE 1
ROBERT E DOSS USDA-ARS, Horticultural Crops Research Laboratory 3420 N. W. Orchard Avenue Corvallis, Oregon 97330
(Received December 28, 1983; revised April 23, 1984) Abstract--Glandular scales on selected lepidote rhododendron species varied in density from 109 _+ 13 to 4180 • 60/cm 2of leaf surface. Globules contained within the scales stained with Sudan IV, a lipophilic dye. Essential oil contents of the scales varied with species from 24 + 8 to 151 _+ 35 ng/scale. Black vine weevil [(Otiorhynchus sulcatus (E)] feeding on leaves from a sample of rhododendron species was inversely related to leaf essential oil content, and weevilfeeding on membrane filters was inhibited by application of essential oil extracts from leaves of most lepidote rhododendrons tested. Results suggest that the glandular scales of the lepidote rhododendrons function, at least in part, in plant defense against insects.
Key Words-Coleoptera, Curculionidae, Otiorhynchus sulcatus (E), black vine weevil, Ericaceae, Rhododendron, trichomes, glandular scales, essential oils, volatiles, plant resistance. INTRODUCTION G l a n d u l a r scales of the lepidote r h o d o d e n d r o n s were described by D e B a r y (1884). I n 1950, C o w a n (p. 107) speculated that in the genus R h o d o d e n d r o n " . . . a n efficient p r o t e c t i o n against desiccation is furnished by d e n d r o i d , r a m i f o r m , rosulate, f u n n e l - s h a p e d , loriform, l o n g rayed, a n d radiate hairs a n d the most c o m p l e t e p r o t e c t i o n by a dense covering of scales." M o r e o v e r , C o w a n believed that scales could a b s o r b m o i s t u r e f r o m the a t m o s p h e r e a n d ~Mention of a trade name or proprietary product does not constitute an endorsement by the U.S. Department of Agriculture, Agriculture Research Service. 1787 0098-0331/84/1200-178753.50/0 @ 1984 Plenum Publishing Corporation
1788
Doss
could secrete excess water under conditions of reduced transpiration. Despite the fact that there is no direct evidence to support the contention that rhododendron leaf scales function primarily in regulating water passage into and out of the leaf, this idea has generally been accepted without question (e.g., Leach, 1961, pp. 25-28), although Seithe (1980) stated that scales do not absorb water. Bell and Clarke (1978) recently published data showing that rhododendron species differed in their resistance to foliar feeding by adult obscure root weevils (Sciopithes obscurus Horn), a serious pest of rhododendrons in the Pacific Northwest. Analysis of these data showed that lepidote species (those with leaf scales) were more resistant than elepidote species (Doss, 1980). Hexane-soluble materials from resistant lepidote species inhibited obscure root weevil feeding (Doss, 1980) and, in the case of R. edgeworthii Hook., a particulary resistant species, a steam volatile fraction containing, among other compounds, the sesquiterpene germacrone, acted as a weevil feeding deterrent (Doss et al., 1980). These findings suggest that glandular scales of lepidote rhododendrons may function in plant defense by containing or secreting volatile materials that discourage insect feeding. The study described below was carried out to test this hypothesis. METHODS AND MATERIALS Table 1 lists the rhododendrons used in this study. These comprise a geographically and taxonomically diverse sample, including 11 lepidotes, all of which are reported to be resistant to obscure root weevil feeding (Bell and Clarke, 1978; Antonelli and Campbell, 1980), two elepidotes, and an elepidote hybrid. Among the elepidotes, R. smirnowii has been reported as resistant to black vine weevil [Otiorhynchus sulcatus (F.)] feeding (Valla, 1980; Nielsen and Dunlap, 1981). R. "Cynthia" was found to be susceptible to feeding by obscure root weevil (Doss, 1980), and R. catawbiense, to feeding by black vine weevil (Nielsen and Dunlap, 1981). Leaves used in this study, with the exception of those from R. "Cynthia," were obtained from the Rhododendron Species Foundation, Federal Way, Washington. "Cynthia" leaves were obtained from a hedge planting at Western Washington Research and Extension Center, Puyallup, Washington. Glandular scales from a measured area of leaf were removed with a fine-tipped scalpel and placed into a small amount of diethyl either (Harborne, 1973, p. 103). After 30 min, the ether was evaporated under nitrogen and the extracted materials were redissolved in hexane. Leaf volatiles were extracted from small leaf disks into diethyl ether (see above)
1789
GLANDULARSCALESOF RHODODENDRON
TABLE 1. RHODODENDRONSPECIES(AND CULTIVAR) USED FOR ESSENTIALOIL STUDY
Species (Cultivar)
Clonea
1. R. campylogynum
74.76
Franch. 2. R. carolinianum
Rehd. 3. R. catawbiense
Michx. 4. R. chryseum e
Balf. f. et Wand 5. R. "Cynthia ''f 6. R. dauricum
Linn. 7. R. edgeworthii
Hook. f. 8. R. ferrugineum
Linn. 9. R. hanceanum
Hemsl. 10. R. heliolepis Franch. 1I. R. lepidotum
G. Don 12. R. rigidum Franch. 13. R. smirnowii Trautv. 14. R. xanthocodon h Hutch.
Seriesb (subsection)c
Campylogynum (Campylogyna) 7 5 . 1 3 3 Carolinianum (Caroliniana) 76.12 Ponticum (Pontica) 75.28 Lapponicum (Lapponica) 66.590
Dauricum (Rhodorasta) 65.383 Edgeworthii (Edgeworthia) 76.381 Ferrugineum (Rhododendron) 76.34 Triflorum (Tephropepla) 65.374 Heliolepis (Heliolepida) 79.53 Lepidotum (Lepidota) 73.353 Triflorum (Triflora) 77.319 Ponticum (Pontica) 73.305 Cinnabrinum (Cinnabarina)
Geographical distrubution b'c WesternYunnan, Tibet, Burma Southeastern U.S.
Scale typed vesicular (L) entire (U, L)
Southeastern U.S. Yunnan to Tibet
undulate (U, L)
Altai, Korea, Japan
entire (U, L)
Sikkim, Bhutan
entire (L)g
Pyreneeseast to Austrian Alps Szechwan
entire (L) entire (U, L)
Yunnan
entire (U, L)
India, Nepal, Bhutan Burma, China Yunnan
entire to undulate (U, L) entire (L)
Caucasus Mountains entire (U, L) Sikkim
aClone number of Rhododendron Species Foundation, Federal Way, Washington. bSee Leach (1961). 'See Cullen and Chamberlain (1978) and Cullen (1980). aSee Cowan (1960) and Seithe (1980). U = scales on upper surface of leaf, L = scales on lower surface of leaf. eConsidered by some as a subspecies of R. rupicola W.W. Sm. (Cullen and Chamberlain, 1978). YR."Cynthia"is a hybrid of R. catawbiense Michx. and R. griffithianum Wight. R. griffithianum is a member of the Fortunei series (Fortunea subsection). gR. edgeworthii possesses a few vestigal scales on the upper leaf surface. h Considered by some as a subspecies of R. cinnabarinum Hook (Cullen 1980).
or o b t a i n e d by steam distillation f r o m leaves u s i n g a modified NielsenKryger a p p a r a t u s with h e x a n e as the o r g a n i c solvent (Veith and Kiwus, 1977). Leaf disks or leaves of lepidote r h o d o d e n d r o n s used for e x t r a c t i o n bore scales. Scale densities were d e t e r m i n e d by c o u n t i n g t h e m o n 5 . 5 - m m d i a m e t e r leaf disks cut f r o m f o u r separate leaves. The a m o u n t of essential oils p r e s e n t in the scales was estimated by gas c h r o m a t o g r a p h y f r o m ether extracts m a d e u s i n g small leaf disks. A 1.9-m X 2 - m m (ID) glass c o l u m n packed with 3% ( w / w ) SP2100| o n 100-120 mesh
1790
Doss
Supelcoport| was used. Nitrogen was employed as the carrier gas (30 cm3/ rain). The flame ionization detector and the injector were maintained at 260 ~ C. Column temperature was increased from 100 to 200 ~ at a rate of 5~C / m i n with initial and final temperatures held for 2 rain. A nerolidol standard allowed estimation of the amount of essential oils present in the leaf extract. Gas chromatographic estimates were checked against estimates obtained by weighing materials obtained by steam distillation. Gas chromatographic traces obtained with extracts prepared from detached scales were compared with traces obtained with extracts of leaf disks. Leaf cross-sections (50 /~m) cut using a vibrating microtome (Vibratome| were stained using the lipophilic dye Sudan IV (Jensen, 1962, pp. 264-265) and mounted in glycerine jelly (Sass, 1958, pp. 102-103). Detached scales were mounted without staining. Black vine weevil colonies were maintained at 20~ under either continous darkness or photoperiodic cycles consisting of 16 hr of fluorescent light and 8 hr of darkness. Weevil colonies were fed leaves from either "Alpine" strawberry (Fragaria vesca L.) or " T o t e m " strawberry (F. X ananassa Duch.). To determine the feeding response of black vine weevil adults to the rhododendron species tested, leaf disks 14.4 mm in diameter were cut using a notched cork borer that left the petiole attached to the leaf disk. Petioles were inserted through a serum stopper into vials containing water. A moist dental wick, four black vine weevils, and a vial with a leaf disk were placed into each of the cylindrical carboard containers (about 250 c m 3 in volume) used as bioassay arenas (Bristow et al., 1979). Bioassays were run for 24 hr at 20 ~ after which time the areas eaten from the leaves were measured. With R. chryseum (leaf area = 93 ___ 6 mm 2, X • SE for four leaves), R ferrugineum (leaf area = 146 • 10 mm 2, for five leaves ), and R. lepidotum (leaf area = 188 • mm 2, for five leaves), intact leaves, instead of leaf disks, were used because leaves of these species were small. With other species, leaf disks rather than intact leaves were used to eliminate any effect of leaf size on weevil feeding. The influence of essential oils on black vine weevil feeding was determined by using a membrane filter bioassay procedure (Doss, 1980). Membrane filters were first pretreated with 50 rag-equivalents of an ethanolic extract of "Alpine" strawberry leaves that stimulated black vine weevil feeding (Doss and Shanks, unpublished). Then l disk treated with the strawberry extract and t disk treated with the extract plus a rhododendron essential oil extract (from a portion of leaf equivalent in area to a 13-mmdiameter membrane filter) were placed into a bioassay arena along with a moist dental wick and four adult black vine weevils. Bioassays (choice tests)
1791
GLANDULAR SCALES OF RHODODENDRON
were run at 20 ~C for 24 hr. Weevil feeding on the filters was compared using the paired t-test. RESULTS Histological examination indicated that rhododendron leaf scales contained lipophilic globules (Figure 1). Gas-liquid chromatographic traces obtained using extracts made from scales and from leaf disks were qualitatively identical. Figure 2 shows such a trace obtained with an extract of R. dauricum leaves. Extracts made using detached scales contained less volatile material (on an area basis) than leaf disks. Some oil was contained in the scale stalk which remained attached to the leaf upon scale removal (Figure 1). Of course, the leaves, exclusive of the scales, also contained volatiles. The 11 lepidotes yielded much larger volatile fractions, averaging about 100,000 ng/cm 2 of leaf, than the elepidotes which averaged about 12,000 ng/cm< R. smirnowii, a heavily indumented elepidote, yielded more volatiles than three of the lepidote species. Essential oil contents varied with species from about 25 to 150 ng/scale for the lepidotes examined (Table 2). The values reported here were estimated gas chromatographically and are, on average 60 +_ 10% (X _ SE) of the values obtained by weighing steam distillate, except for R. "Cynthia," The estimate obtained for this cultivar by weighing steam distillate was 1.3 mg/g fresh wt. In an earlier study (Doss et al., 1980), a value of 0.64 mg/g fresh wt was obtained for R. "Cynthia," suggesting that the gas chromatographic value reported in Table 2 (equivalent to 0.008 rag/g) could be a serious underestimate. Note that values were calculated assuming that all essential oils were contained within the scales even though some volatiles are present on nonscale-bearing leaves. There was a significant negative correlation between the areas eaten from leaf disks (or small leaves) and the volatile oil content (Figure 3). This was also true when only the lepidote species were considered (r = -0.76 with 8 df). The estimates of essential oil contents used in this analysis were obtained by weighing steam distillate, but using estimates obtained gas chromatographically resulted in a similar relationship and correlation coefficient (r = --0.70). The species R. heliolepis was omitted from this study through oversight. In choice tests with membrane filters, nine of the 11 lepidote species yielded extracts that inhibited weevil feeding (Table 3). None of the elepidote species yielded inhibitory extracts and, with the lepidotes, noninhibitory extracts came fron species with relatively small amounts of essential oils. In no-choice tests (not discussed in Materials and Methods), using only 1 membrane filter disk per arena, there was large variation in feeding. The
1792
Doss
Fio. 1. (top) Cross-section through a Rhododendron edgeworthii leaf showing glandular scale. Scales are about 110/xm in diameter. (bottom) Detached scale (300 t~m in diameter) from R. chryseum. Oil droplets are indicated by arrows.
correlation between areas eaten by black vine weevils from phagostimulantbearing membrane filters treated with essential oil extracts and the essential oil contents of the leaves was not significant with the no-choice tests (data not shown).
1793
GLANDULAR SCALES OF RHODODENDRON
8
3
4
I
o-
5
I
10 15 Retention Time (rain)
I
20
25
FIG.2. Gas chromatographic trace of Rhododendron dauricum essential oil extract. The probable identities of the materials giving rise to the number peaks are as follows: 1. fl-caryopheyllene, 2. c~-humulene, 3. cis-nerolidol, 4. trans-nerolidol, 5.8elemenone, 6. 7-eudesmol, 7. a/fl-eudesmol, 8. germacrone. DISCUSSION The role of various types of trichomes in conferring insect resistance upon plants is well documented (Levin, 1973; Webster, 1975; Stipanovic, 1983). In fact, it has been suggested that the presence of trichomes is more often associated with defense against insect attack than with reduction of water loss, a commonly suggested function of plant trichomes (Fitter and Hay, 1981, p. 283). With Rhododendron, Valla (1980) noted that indumented and lepidote types suffered less adult black vine weevil feeding damage than did glabrous leaf types. Data presented by Bell and Clarke (1978) suggested that the lepidote rhododendrons were more ofter resistant to obscure root weevil feeding than elepidotes (Doss, 1980). With respect to the lepidote rhododendrons, results presented here
campylogynum carolinianum catawbiense chryseum
21.8 22.7 27.3 21.4 44.1 22.3 34.5 29.0 34.9 26.9 16.0 21.7 32.4 22.7
4• • • + • • + • • • • • •
0.8 0.8 1.3 0.8 3.4 0.4 1.3 0.8 0.4 0.8 0.8 1.7 0.4 1.3
Fresh 12.6 • 10.9 • 13.9 • 11.4 • 22.7 • 11.4 • 18.9 • 13.9 • 15.6 • 10.9 • 6.7 • 11.4 • 16.8 • 11.4 •
Dry 0.4 0.8 0.4 0.4 1.7 0.4 0.4 0.8 0.4 0.4 0.4 0.8 0.4 1.3 43 + 9
46 128 • 151 • 70 • 128 • 26 • 25 • 32 •
71 5 : 8
63 25 101 4 17 25 13 50
30 35 7 25 3 8 5
65 • 8 8 6 • 12
ng/scale
21
Lower surface
315 • 1206• no scales 1916 • 39 2273 • no scales 122 • 29 798 • 0 2168 • 0 1409 • 59 • 4 219 • 399 • 29 412 • 614 • 17 672 • 0 109 • no scales 538 • 118 1370 •
0 172•
Upper surface
Scales per cm 2
20600 • 2100 118000 • 15000 1656 • 221 180000 • 39000 357 • 72 118000 • 25000 328150 • 75000 98900 • 5900 35600 • 7000 21400 • 2000 311000 • 10100 3430 • 400 27400 • 5000 136000 • 12000
ng/cm2
Essential oils ~
aMeans • standard errors for five samples. bMeans + standard for four samples. CMeans + s t a n d a r d errors for four extracts. Note that ng/scale values ignore volatiles present in the leaf l a m i n a and are thus overestimates (see Results).
R. dauricum R. edgeworthii R.ferrugineum R. hanceanum R. heliolepis R. lepidotum R. rigidum R. smirnowii R. xanthocodon
R. ' C y n t h i a '
R. R. R. R.
Species (cultivar)
Leafweight (mg/cm2) a
TABLE 2. ESSENTIAL OIL CONTENTS OF 1 1 LEPIDOTE AND 3 ELEPIDOTE RHODODENDRON SPECIES (AND CULT1VAR) MEASURED FROM EXTRACTS OF LEAF DISKS
1795
GLANDULARSCALESOFRHODODENDRON
2OO
(~)
160
12
c~
E120 ~z 1~14
6
c
w
~ 80
1_
