Ovipositional response of tobacco budworm moths (Lepidoptera ...

Journal of Chemical Ecology, Vol. 17, No. 12, 1991

OVIPOSITIONAL RESPONSE OF TOBACCO BUDWORM MOTHS (LEPIDOPTERA: NOCTUIDAE) TO CUTICULAR LABDANES AND SUCROSE ESTERS FROM THE GREEN LEAVES OF Nicotiana glutinosa L. (SOLANACEAE) I

D. MICHAEL

J A C K S O N , 2"* R . F . M.G.

SEVERSON, 3 V.A.

STEPHENSON

SISSON, 2 and

4

2ARS, USDA Crops Research Laboratory Oxford, North Carolina 27565 3ARS, USDA Phytochemical Research Unit Athens, Georgia 30613 4ARS, USDA Georgia Coastal Plain Experiment Station Tifton, Georgia 31793 (Received June 13, 1991; accepted August 19, 1991) Abstract--Field plots of three accessions of Nicotiana glutinosa L. (Nicotiana species accessions 24, 24A, and 24B) at Oxford, North Carolina and Tifton, Georgia were heavily damaged by natural populations of tobacco budworms, Heliothis virescens (F.), during 1985-1989. Experiments in outdoor screen cages demonstrated that all accessions of N. glutinosa were as prone to oviposition by H. virescens moths as was NC 2326, a commercial cultivar of flue-cured tobacco, N. tabacum L. However, in greenhouse experiments, tobacco budworm larvae did not survive or grow as well when placed on plants of N. glutinosa as they did when placed on plants of NC 2326. Four labdane diterpenes (manool, 2-hydroxymanool, a mixture of sclareols, and labda-13-ene-8a, 15-diol [labdenediol]) and two sucrose ester fractions (2,3,4tri-O-acyl-3'-O-acetyl-sucrose [G-SE-I] and 2,3,4,-tri-O-acyl-sucrose [G-SE*To whom correspondence should be addressed.

This article reports the results of research only. Mention of a proprietary product does not constitute endorsement or recommendation for its use by USDA, ARS, or by the North Carolina Agricultural Research Service.

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24-90

JACKSON ET AL.

II]) were isolated from green leaves of the three accessions of N. glutinosa. These components were bioassayed for their effects on the ovipositional behavior of tobacco budworm moths using small screen cages in a greenhouse at Oxford, North Carolina. Labdenediol, manool, and both sucrose ester fractions stimulated tobacco budworm moths to oviposit on a tobacco budwormresistant Tobacco Introduction, TI 1112 (PI 124166), when these materials were sprayed onto a leaf. Key Words--lnsecta, Lepidoptera, Noctuidae, Heliothis virescens, tobacco budworm, Nicotiana, ovipositional stimulants.

INTRODUCTION

Nicotiana glutinosa L. is a coarse-viscid pubescent annual in the family Solanaceae (Subgenus Tabacum, Section Tomentosae). It is native to Peru and southern Ecuador, where it has adapted to semi-arid areas such as rocky slopes and ditch banks (Goodspeed, 1954; Tatemichi, 1990). There are three accessions of N. glutinosa (accessions 24, 24A, and 24B) in the USDA-National Plant Germplasm System Collection at the Crops Research Laboratory in Oxford. The Section Tomentosae (2n = 24) is the group of Nicotiana species most closely related to cultivated tobacco, N. tabacum L. (Subgenus Tabacum, Section Genuinae; 2n = 48). N. glutinosa is currently used as a source of desirable traits in tobacco-breeding programs, and chromosomes from this species have been incorporated into commercial tobacco cultivars by conventional breeding techniques (Gerstel, 1945). N. glutinosa is resistant to tobacco mosaic virus (TMV), powdery mildew, Erysiphe cichoracearum DC (Cohen et al., 1983), and cyst nematodes, Globodera solanacearum (Miller & Gray) Behrens. It is the source of resistance to TMV in all commercial tobacco cultivars (Stavely, 1979). However, N. glutinosa is susceptible to several other diseases (Stavely, 1979), and it is also attacked by most common insect pests of tobacco in the United States, including the tobacco hornworm, Manduca sexta (L.), the tobacco aphid, Myzus nicotianae Blackman, and the tobacco budworm, Heliothis virescens (F.) (Thurston, 1961; Thurston et al., 1966; Parr, 1967; Parr and Thurston, 1968; Burk and Stewart, 1969, 1971; Greene and Thurston, 1971). N. glutinosa is preferred for oviposition by moths of tobacco hornworms and tobacco budworms over tobacco, most Nicotiana species, and tomato, Lycopersicon esculentum Mill. (Thurston et al., 1966; Parr, 1967; Greene and Thurston, 1971; Jackson and Severson, 1989; Jackson et al., 1988, 1989; Severson et al., 1990). N. glutinosa does not produce the cuticular duvane diterpenes found on N. tabacum and N. sylvestris Spegazzini & Comes, but it does produce labdane diterpenes (Colledge et al., 1974; Reid, 1979). Severson et al. (1990) listed ten

OVIPOSITIONAL RESPONSE

2491

cuticular labdanes produced by the Nicotiana species. The primary labdanes of N. tabacum are (12Z)-labda-12,14-dien-8ot-ol (cis-abienol) and (13E)-labda13-ene-8ot,15-diol (labdenediol) (Reid, 1974; Severson et al., 1985b). One accession of N. glutinosa (acc. 24) produces (13R) -labda-8,14-dien- 13-ol (manool), (13R)-labda-8,14-dien-2-oxy-13-ol (2-oxymanool), (13R)-labda-8,14diene-2ot,13-diol (2-hydroxymanool), (13R)-labda-14-ene-8~,13-diol (sclareol), (13S)-labda-14-ene-8~,13-diol (13-episclareol), and labdenediol. The second accession of N. glutinosa (acc. 24A) produces only the sclareols and labdenediol, while the third accession of N. glutinosa (acc. 24B) does not produce labdanes (Severson et al., 1988a; Figure 1). An epimeric mixture of sclareol and 13-episclareol inhibits the growth of a wide range of fungal species (Bailey et al., 1974, 1975). Cohen et al. (1983) reported that oxymanool suppresses the emergence of conidial germ tubes of E. cichoracearum, and thus inhibits development of powdery mildew. Cis-abienol from N. tabacum, and 2-hydroxymanool, manool, oxymanool, sclareol, and the eutectic mixture of sclareol and 13-episclareol from N. glutinosa inhibit the growth of wheat coleoptiles (Cutler et al., 1977). We previously reported that cis-abienol does not stimulate oviposition by tobacco budworm moths (Jackson et al., 1986). All three accessions of N. glutinosa produce sucrose esters (SE), which were isolated and characterized by Arrendale et al. (1990). They classified the SE from Nicotiana species based upon the position of the acetyl moieties on the sucrose molecule. The SE from N. glutinosa in this paper are designated G-SE-I (2,3,4-tri-O-acyl-3'-O-acetyl-sucrose) and G-SE-II (2,3,4-tri-O-acylsucrose), while the SE from N. tabacum (6-O-acetyl-2,3,4-tri-O-acyl-sucrose; Severson et al., 1985b, 1990) is designated T-SE-I. Severson et al. (1990) listed a total of eight SE types and two types of glucose esters (GE) in a survey of all of the Nicotiana species. The SE from N. tabacum (TI 165, PI 405002) (Cutler et al., 1986) and N. glutinosa (Severson et al., 1990) inhibit growth of wheat coleoptiles and the growth of some gram positive bacteria. We previously reported that the sugar esters from N. tabacum (TI 165) (SE), N. kawakamii Obashi (SE & GE), N. alata Link & Otto (SE), and N. trigonophylla Dunal (SE & GE) stimulate oviposition by tobacco budworm moths (Jackson et al., 1989; Severson et al., 1990). However, oviposition by tobacco budworm moths is not affected by simple sugars such as sucrose, glucose, or fructose (Jackson et al., 1989). Because the cuticular components from N. glutinosa may be transferred to N. tabacum during the development of tobacco breeding lines, it is of interest to know how these components affect the behavior of associated insect pests. In this paper, we describe the effects that cuticular components from N. glutinosa have on the ovipositional behavior of tobacco budworm moths.

N, glutinosa

(24A)

~

!

_ z

2 rr

W

II

110

Hydrocarbons

210

410

310

TIME ( MIN )

Sucrose Esters Hydrocarbons

,'o

2'o

3'0

1

i

40

TIME ( MIN )

N. glutinosa (24B)

Hydrocarbons

Sucrose Esters

t'o

~o

3'o

;o

TIME ( MIN )

FIG. 1. Capillary gas chromatograms of the cuticular components from the green leaves of three accessions of Nicotiana glutinosa grown in field plots at Oxford, North Carolina, 1985.

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OVIPOSITIONAL RESPONSE MATERIALS AND METHODS

Field Plots Seventy accessions of 66 Nicotiana species (including the three accessions of N. glutinosa) were grown in 20-plant plots at Tifton, Georgia and Oxford, North Carolina during 1985 and 1986. Plots were arranged in a randomized complete block design with three replications. Fields were fumigated with methyl bromide before planting to control soil-borne diseases. Napropamide (Devrinol 50WP | ICI Americas Inc., Wilmington, Delaware) and metalaxyl (Ridomil 2E | Ciba-Geigy Corp., Greensboro, North Carolina) were incorporated into the soil 2 weeks before transplantation for control of weeds and blue mold, Peronospora tabacina D. B. Adam. Plots were cultivated, fertilized, and irrigated as needed. The numbers o f tobacco budworm larvae on each plant were counted at peak infestations 1-2 times each year at each location. Field plants were sampled for cuticular components once each season at each location. One leaf disk (2-cm diam) was removed from each of five plants per treatment, with two replications per location per year. Disks were taken from the center (including midrib) of a fully expanded but not senescent leaf near the top of each plant. Leaf disks were briefly dipped ten times in succession into 10 ml of methylene chloride (distilled-in-glass grade, American Burdick & Jackson, Muskegon, Michigan) in scintillation vials. Samples were chilled on dry ice and kept at - 1 7 ~ until analyzed by gas chromatography (GC).

Ovipositional Choice Experiments with Whole Plants Whole plants of N. glutinosa were bioassayed for tobacco budworm ovipositional preference in choice tests vs. NC 2326, a commercial flue-cured tobacco cultivar, using 16 outdoor screen cages (2.4 x 2.4 x 2.0 meters high; Jackson et al., 1983, 1984, 1986). Each cage had a wooden floor and waterproof gabled roof. Three 7.5 watt light bulbs were positioned over the center of each cage and aimed upward under the roof. Lights were shaded from below by 30.5-cm diam. metal pans, and they were controlled by a central rheostat so that the light intensity was equal within all cages. The cages were three meters apart and they were located in an isolated area of the Tobacco Research Station in Oxford, where there were no extraneous light sources within View of moths inside the cages. These experiments were conducted July 3-31, 1984 and May 31-August 15, 1985. Maximum daytime temperatures ranged from 22 to 37~ and minimum nighttime temperatures ranged from 8 to 23~ during these periods. Plants used in these experiments were germinated in the greenhouse (ambient photoperiod, temperature maintained below 35~ and later transplanted to 12.7-cm diam. plastic pots containing sterilized soil and a time-release

2494

JACKSON ET AL.

fertilizer (14-14-14, N-P-K) (Osmocote| Sierra Chem. Co., Milpitas, California). At transplantation, each seedling was treated with 0.04 ml of metalaxyl (Ridomil 2E) in 100 ml water to prevent fungal diseases. Plants were grown in the greenhouse for an additional 2 weeks then moved to a shady area of mowed grass until they were large enough for testing at 5-7 weeks after transplantation. When aphids were a problem, plants were sprayed with pirimicarb (Pirimor 50 W | ICI Americas Inc., Wilmington, Delaware) at 0.01 g per plant. Tobacco budworms were from a laboratory colony started each fall from larvae collected in tobacco fields near Oxford. This colony was cultured for 710 generations on an artificial diet (Baumhover, 1985) before the start of these experiments. Female moths were 3-4 days old after eclosion, and they were caged together as groups of ten female and ten male moths in 3.8-liter paper cartons for two nights before testing to insure mating. The mating containers and colony moths were kept in rooms at ca. 25~ ca. 70% RH, and a photoperiod of 14 : 10 (L: D). For choice experiments, four N. glutinosa plants were placed in one corner of a cage, and four NC 2326 plants of a similar size were placed in the opposite corner of the cage. Plants ranged in height from ca. 15-30 cm. Ten female tobacco budworm moths were placed into each cage a few hours before sunset. Moths were provided water, but no food. The following morning, the total number of tobacco budworm eggs on the four plants of each entry was counted. Each cage-night was treated as one replication. Data were analyzed by paired t-tests (Steel and Torrie, 1960). For no-choice experiments, eight plants of a treatment and eight plants of the control (NC 2326) were placed in separate cages set up as pairs, and the data were analyzed by paired t-tests.

Ovipositional Bioassays of Cuticular Components Manool, hydroxymanool, labdenediol, a sclareol mixture (sclareol plus episclareol), two SE fractions (G-SE-I and G-SE-II), and a mixture of o~-+ ~-4,8,13-duvatriene-l,3-diols (duvatrienediols) were bioassayed for ovipositional response by tobacco budworm moths in "hoop cages" (0.46 • 1.31 • 0.45 meters high). Twelve hoop cages were located inside a glass greenhouse (7.3 x 9.1 • 4.9 meters high). The walls of this greenhouse were covered with black plastic to block extraneous lights, but the gabled roof was left uncovered. No artificial light sources were used for these experiments. The greenhouse was maintained at 25-30~ during the test period, and the humidity was kept high by hosing down the concrete floor several hours before each experiment. Each hoop cage had a rectangular plywood floor (0.46 x 1.31 meters) and two semicircular end pieces (0.46 meters high). It was covered with 0.32-cm hardware cloth. In the center of the floor of each cage was a trap door large enough to


2495

introduce potted plants and moths. Cages were painted flat black. In the center of each plywood end piece was a 10.2-cm diam. hole through which was pushed the end of a frustum (11.7-cm diam. bottom, 9.5-c:n diam. top, and 8.9-cm high) formed from a 12.7-cm black plastic pot with the end removed. A detached leaf was placed over the end of an intact 12.7-cm plastic pot, and the frustum was pushed over the end of the pot to secure the leaf in place so that its abaxial surface was exposed. After the exposed surface of the leaf was treated with a cuticular component, the frustum was pushed through the hole in the end of the cage and held fast with Velcro | fasteners so that the leaf protruded into the cage ca. 2 cm. For a choice test, a frustum with attached leaf was pushed into each end of the cage. Thus, the two excised leaves at opposite ends of the cage each exposed a circular area of 71 cm 2 to ovipositing moths. Leaves from Tobacco Introduction (TI) 1112 (PI 124166) were used in these experiments. TI 1112 has glandless trichomes on its leaves and, in contrast to most tobacco cultivars and Nicotiana species, it has negligible levels of the common leaf surface constituents (Severson et al., 1984). Plants were grown in 12.7-cm diam. plastic pots under high pressure sodium lights in a greenhouse. Plants grown under these lights are similar in physical and chemical characteristics to field-grown plants (Severson et al., 1985b). Tobacco budworm moths were reared and prepared for bioassay in the same manner as those for the outdoor cage experiments described above. Five female moths were introduced into each cage in the late afternoon after temperatures in the greenhouse had fallen below 30~ Eggs on each exposed leaf surface were counted the following morning. Each cuticular component was bioassayed in a choice test vs. a solvent blank (SB) (0.5 mL methylene chloride in 1.5 mL of 3 : 1 acetone : water) and vs. duvatrienediols. We previously reported that duvatrienediols are strong ovipositional stimulants for tobacco budworm moths (Jackson et al., 1986, 1989). Spray applications were made in the late afternoon with a small air brush (Model 250, Badger Air-Brush Co., Franklin Park, Illinois). This apparatus had a 20mL reservoir, a Teflon | delivery tube, and it was attached to an air compressor that would deliver up to 200 kPa. About 35 kPa were optimal for spraying the plants, because that pressure produced a fine mist that did not run off the leaves. One milliliter of a cuticular component was mixed with 1.0 mL of 1 : 1 acetone:distilled water. Control plants were sprayed with 2.0 mL of SB or with 1.0 mL duvatrienediols plus" 1.0 mL of 1 : 1 acetone : water. Thus, 2.0 mL of spray was applied to the surface of each leaf. Leaf disks were allowed to airdry (ca. 30 min) before they were inserted into the oviposition cage. The following morning after tobacco budworm eggs had been removed, four 2-cm diam leaf disks were taken from each excised leaf. These disks were dipped ten times each into scintillation vials containing 10 mL of methylene chloride (dis-

2496

JACKSON ET AL.

tilled-in-glass grade, American Burdick & Jackson). Samples were frozen on dry ice and kept frozen at - 17~ until they were analyzed for chemical components by capillary GC. Before analysis of variance (ANOVA) for each experiment, data were transformed first to a percentage of the total eggs counted per replication (x), and then transformed to arcsine (square root x). Preference for egg laying on a particular treatment vs. the control was determined using a paired t-test for each experiment (SAS Institute, 1985).

Feeding Experiment In order to determine how well tobacco budworm larvae survive and develop on the three N. glutinosa accessions vs. NC 2326, greenhouse-grown plants were artificially infested with second instars. Plants were grown in the greenhouse under supplemental high pressure sodium lights for 6-8 weeks after transplantation from seed pots in a manner similar to that used to produce plants for ovipositional cage bioassays described above. Five-second instars of H. virescens from the laboratory colony described above were placed on the terminal leaf bud of each plant. Larvae were allowed to feed for 1 week, then they were weighed.

Extraction of Cuticular Components for Bioassays Cuticular components were extracted from all three accessions of N. glutinosa from field plots at Oxford and Tiflon. Plant tops (ca. 6-10 weeks old) were cut off and dipped ten times into 4-liter beakers of methylene chloride (Resi-analyzed reagent grade, J.T. Baker Chemical Co., Phillipsburg, New Jersey; Jackson et al., 1984, 1986). Approximately 100 plants were extracted with ca. 3 liters of solvent. These crude extracts were cooled with dry ice and stored at - 1 7 ~ until needed. After warming to room temperature, the extracts were filtered through folded filter paper containing anhydrous Na2SO4, and the solvent was removed on a roto-evaporator under vacuum at 40~

Isolation of Labdanes and Sucrose Esters The cuticular extract from N. glutinosa (acc. 24A) (10.9 g) was dissolved in 1 : 1 hexane:methylene chloride, deposited onto 50 g of silicic acid, and loaded onto a 50-g silicic acid column. The column was eluted with hexane (500 mL), 1 : 3 hexane : methylene chloride (750 mL), methylene chloride (400 mL), 1 : 9 acetone: methylene chloride (400 mL), and 1 : 4 acetone : methylene chloride (1000 mL). Fractions rich in the sclareol mixture (2.1 g, 9 9 + %) eluted in the third 100 mL portion of the 1 : 9 acetone: methylene chloride fraction. Re-crystallization from hexane yielded a constant melting mixture of sclareols


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(1.8 g, melting point Imp] 96-97~ 99+ %) (reported literature mp = 95~ Reid, 1974). Combination of the last 100 mL of the 1:9 acetone:methylene chloride and the first 400 mL of the 1 : 4 acetone : methylene chloride elutants produced 5.8 g of the sclareols plus labdenediol mixture. A portion of this mixture (2.8 g) was chromatographed on a Sephadex LH-20 column (2.54 cm id • 100 cm) using chloroform (0.75% EtOH as preservative) as the eluting solvent, and 5 mL fractions were collected. After solvent removal and re-crystallization, fractions 77-84 produced 0.9 g of the sctareol-episclareol mixture (mp 96-97 ~ 99 + %). Solvent removal and re-crystallization of fractions 95107 produced 9 9 + % labdenediol (mp 131-132~ (reported literature mp = 131-131.5~ Reid, 1974). Cuticutar extract from N. glutinosa (acc. 24) (6.2 g) was deposited onto 50-g silicic acid, loaded into a 50-g silicic acid column, and eluted with hexane (150 mL), 3 : 1 hexane : methylene chloride (200 mL), hexane : methylene chloride (I000 mL), and 1:19 acetone:methylene chloride (1000 mL). The first 200-mL elutant of the 1 : 19 acetone:methylene chloride fraction yielded 1.7 g of a mixture of 2-oxymanool (9%), sclareols (53%), and 2-hydroxymanool (28%). This mixture was re-chromatographed on the Sephadex LH-20 column as described above. After solvent removal and re-crystallization, combinations of fractions 95-100 yielded 9 9 + % 2-hydroxymanool (0.22 g, mp = 131133~ The G-SE-I and G-SE-II fractions were isolated as described by Arrendale et at. (1990). Manool was obtained from Aldrich Chemical Company (Milwaukee, Wisconsin). The duvatrienediols were isolated as described by Severson et al. (1988b).

Gas Chromatography Chemical samples were analyzed by capillary GC and GC/MS as described by Severson et al. (1984, 1985a,b). Cuticular extracts, fractions, and isolates were taken to dryness under N2 in a micro autosampler vial (100/~L), then 50 /zL of 1 : 1 N-O-bis(trimethylsilyl)trifluoroacetamide : dimethylformamide (Pierce Chemical Company, Rockford, Illinois) was added. The vial was capped and heated at 76~ for 45 min to convert hydroxylated components to trimethylsilyl ethers. After cooling, the samples were analyzed on a Hewlett Packard 5880 gas chromatograph equipped with a splitless injector (injector temp. 250~ purge activation time of 1.5 min) and flame ionization detector (detector temp. 350~ using a 0.3-mm id • 30-m bonded SE54 fused silica capillary column (Arrendale and Martin, 1988). A temperature program was run at 100~ for 5 min, followed by a temperature increase of 10~ to 170~ 3~ from 170-220~ and 5~ from 220-290~ The final temperature was held at 290~ until all components had eluted.

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JACKSON ET AL. RESULTS AND DISCUSSION

Results from field plots showed that N. glutinosa was one of the Nicotiana species most frequently infested with tobacco budworm larvae (Table 1). Although numerically lower, the percentage infestations on the three accessions of N. glutinosa were not significantly different from infestation levels seen on N. tabacum cultivars such as NC 2326 and Samsun, an oriental tobacco (Severson et al., 1985b). Nicotiana kawakamii was the only species that had a significantly higher average infestation rate in the field than N. glutinosa (Table TABLE 1. PERCENTAGE INFESTATION BY NATURAL POPULATIONS OF LARVAE OF THE TOBACCO BUDWORM ON SINGLE R O W PLOTS OF 7 0 ACCESSIONS OF 6 6 Nicotiana

SPECIESAT OXFORD, NORTr~CAROLINAANDTIFTON, G~ORGIA, 1985-1986 C u t i c u l a r c o m p o n e n t s ( / ~ g / c m z)b

Nicotiana species N. N. N. N. N. N. N. N. N. N. N. N. N. N. N.

kawakamii (72) e tabacum cv. S a m s u n tabacum cv. N C 2 3 2 6 alata (3) debneyi (17) glutinosa (24) glutinosa (24A) bigelovii (10) clevelandii (14) otophora (38) acuminata (2) nesophila (34A) rustica v. pavonii (44) glutinosa (24B) setchellii (51) 2 4 Nicotiana species 31 Nicotiana species

Percent o f plants ~ with t o b a c c o b u d w o r m larvae

Total duvanes

Total labdanes

SE"

GE~

46.3 aI

--g

38

24

88

3 3 . 9 ab

93

24

57

--

---79 144 -----. -56

5 56 15 85 20 75 66 32 179 --

--2 --15 7 ----

38 67

-10

23.9 abc 21.3 cdef 18.0 b c d e 17.6 b c d 13.7 c d e f g 13.6 c d e f 9.6 cdefg 9.1 c d e f g 8.5 c d e f g 8.1 c d e f g 8.1 defg 7.4 c d e f g 6.5 c d e f g 0.1-6.4 cdefg 0.0g

105 ------l -Trace . ---

.

.

~ A v e r a g e f o r three d a t a sets: O x f o r d - - 1 9 8 5 , T i f t o n - - 1 9 8 5 , a n d T i f t o n - - 1 9 8 6 ; Three replications per location with 12-plant plots. b D a t a f r o m Severson et al. (1990). c Sucrose esters. d G l u c o s e esters. eNicotiana species seed a c c e s s i o n number: f o r species authority see G o o d s p e e d (1954) or Tatem i c h i (1990). f M e a n s followed b y the s a m e letter are not significantly different, W a l l e r - D u n c a n K-ratio t-test (K = 100, ot = 0.05); d a t a t r a n s f o r m e d to square root (X + 0.5) before A N O V A (SAS Institute, 1985). g B e l o w detection limits.


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1). However, data from field infestation rates must be interpreted cautiously since they are only a relative measure o f host suitability (Jackson and Severson, 1989). This is because several mortality factors (predation, abiotic factors, plant antibiosis) may kill eggs or larvae o f H. virescens once they are on a plant. Oviposition by moths o f H. virescens m a y be heavy on certain tobacco types, but they may show little insect damage in the field because o f high larval mortality due to their antibiotic defenses (Jackson et al., 1988, 1989; Jackson and Severson, 1989). However, because larvae o f H. virescens seldom leave the plants upon which they hatched (Jackson et al., 1989), we may assume that a plant type heavily damaged in the field is highly susceptible to oviposition. The three accessions o f N. glutinosa fall in this latter group, making them quite susceptible to oviposition by tobacco budworm moths. The propensity o f female tobacco budworm moths to oviposit on N. glutinosa was confirmed by both choice and no-choice oviposition cage experiments when compared with the highly susceptible N. tabacum cv. NC 2326 (Table 2). In no-choice tests, N. glutinosa (acc. 24) was equally acceptable as an ovipositional site as NC 2326 with no significant difference in the number o f eggs that were laid on either host. Similarly, when given a choice between one o f the accessions o f N. glutinosa and NC 2326, tobacco budworm moths deposited equal numbers o f eggs on each plant type despite their differences in cuticular chemical profiles (Figure 1). This probably indicates that more than one cuticular component is utilized by moths o f H. virescens as ovipositional stimulants. TABLE 2. OVIPOSITIONALRESPONSE OF FEMALE TOBACCO BUDWORM MOTHS TO THREE ACCESSIONS OF Nicotiana glutinosa IN SCREEN FIELD CAGES, OXFORD, NORTH CAROLINA, 1984-1985 No-choice experiments~'

Choice experiments~

Species accession

Percent of eggs on N. glutinosa

Total no. of eggs

No. of reps

Percent of eggs on N. glutinosa

N. glutinosa (24) N. glutinosa (24A) N. glutinosa (24B)

50.6 NS" 45.7 NS 51.6 NS

5489 2624 2749

20 10 10

46.3 NS'" a --

Total no. of - No. of eggs reps 4452 _ --

15 _ --

~Paired choice tests with four plants of N. glutinosa vs. four plants of N. tabacum cv. NC 2326 flue-cured tobacco in the same cage. bPaired no-choice tests with eight plants of N. glutinosa in one cage vs. eight plants of NC 2326 in a separate cage. "Paired t-test; NS = not significant. For no-choice experiments, data from separate cages containing either NC 2326 or an N. glutinosa accession were paired. dNot tested.

2500

JACKSON ET AL.

While there did not appear to be any difference between N. glutinosa and NC 2326 for ovipositional preference, feeding tests using greenhouse-grown plants showed that tobacco budworm larvae survived and grew better on NC 2326 than on any of the accessions of N. glutinosa (Table 3). This result differs from Burk and Stewart (1971) who listed only one of the accessions of N. glutinosa, 24A, as inducing significantly higher mortality on tobacco budworm larvae than those feeding on a flue-cured control. Differences in survival rates of larvae may help explain the lower percentages of N. glutinosa plants infested with tobacco budworm larvae in the field compared to N. tabacum cultivars (Table 1). Reasons for lower survival and developmental rates for tobacco budworm larvae on N. glutinosa have not been studied, but differences in cuticular components or internal leaf components such as flavanols (Jurzysta, 1983; Snook et al., 1986) or pyridine alkaloids (Sisson and Severson, 1990) may be important. These classes of compounds are known to be toxic to larvae of H. virescens (Lukefahr and Martin, 1966; Shaver and Lukefahr, 1969; Granzow et al., 1985; Jackson et al., 1989; Gunasena et al., 1990). Interestingly, N. glutinosa is not resistant to feeding by tobacco hornworm larvae (Parr and Thurston, 1968). Chromatograms of the cuticular components of the three accessions of N. glutinosa are given in Figure 1, and levels of duvanes, labdanes, and SE found on N. glutinosa and N. tabacum in the field are shown in Table 4. Both SE fractions (G-SE-I and G-SE-II), manool, and labdenediol stimulated tobacco budworm oviposition when these compounds were sprayed onto TI 1112 leaves (Table 5). These components stimulated oviposition to a level not significantly TABLE 3. AVERAGE SURVIVAL AND WEIGHT GAIN AFTER 1 WEEK FOR SECOND INSTAR Heliothis virescens LARVAE PLACED ON GREENHOUSE-GRowN Nicotiana glutinosa PLANTS COMPARED TO THOSE PLACED ON FLUE-CURED TOBACCO, NC 2326 AT OXFORD, NORTH CAROLINA, 1985-1986

Species~ accession

N. N. N. N.

glutinosa (24) glutinosa (24A) glutinosa (24B) tabacum (NC 2326)

Number of b replications 8 9 8 17

Average" percent survival 17.0 27.6 19.6 43.3

a a a b

AverageJ weight gain per larva (mg) 18.1 53.2 22.3 91.0

a b a c

~Six- to 8-week-old plants grown in 12.7-cm diam. pots. b Five plants with five larvae per plant made up one replication. "Means in the same column followed by the same letter are not significantly different, WallerDuncan K-ratio t-test (K = 100, o~ = 0.05); SAS Institute, 1985).

OVIPOSlTIONAL RESPONSE

2501

TABLE 4. LEVELS OF MAJOR CUTICULAR COMPONENTS FOUND ON GREEN LEAVESON FmLD-GROWN PLANTS OF THREE ACCESSIONS OF Nicotiana glutinosa (24, 24A, AND 24B) AND TWO N. tabacum CULTIVARSAT OXFORD, NORTH CAROLINA, 1985 /zg/cm2 on green leaves"

Nicotiana tabacum Cuticular component Duvatrienediols t' Labdenediol d

Cis-Abienol" Manool f Hydroxymanool g Oxymanool h Sclareols i Sucrose Esters j

Nicotiana glutinosa

NC 2326

Samsun

24

24A

24B

105.0 ------5.0

93.0 4.0 20.0 ----57.0

--" 6.0 -7.0 23.0 5.0 43.0 85.0

-50.0 ----94.0 18.0

-------38.0

"Average from two replications of composite samples from five plants, Oxford, North Carolina, 1985. l,a- +/3_4,8,13 -duvatriene- 1,3-diols. 'Below detection levels. d(13E)_labda_ 13-ene-8e~,15-diol. " ( 12Z)-labda- 12,14-dien-8o~-ol. f(13R)-labda-8,14-dien- 13-ol. '~'( 13R)-labda-8,14-diene-2o~,13-diol. h(13R)-labda-8,14-dien-2-oxy- 13-ol. i Mixture of (13R)-labda- 14-ene-Sa, 13-diol (sclareol) and (13-S)-labda- 14-ene-Sa, 13-diol (13-episclareol). iN. glutinosa: G-SE-I (2,3,4-tri-O-acyl-3'-O-acetyl-sucrose) and G-SE-II (2,3,4-tri-O-acyl-sucrose); N. tabacum: T-SE-I (6-O-acetyl-2,3,4-tri-O-acyl-sucrose).

d i f f e r e n t f r o m the effects o f the d u v a t r i e n e d i o l s (Table 5), k n o w n o v i p o s i t i o n a l s t i m u l a n t s f o r 11. virescens ( J a c k s o n et al., 1986). T h e m i x t u r e o f s c l a r e o l s g a v e c o n f l i c t i n g results in the t w o t y p e s o f p a i r e d tests. A l t h o u g h c o m p a r i s o n o f s c l a r e o l - t r e a t e d l e a v e s vs. s o l v e n t b l a n k - t r e a t e d l e a v e s i n d i c a t e d n o o v i p o s i tional activity (statistically n o n s i g n i f i c a n t ) , c o m p a r i s o n o f s c l a r e o l - t r e a t e d leaves vs. d u v a t r i e n e d i o l - t r e a t e d l e a v e s i n d i c a t e activity s i m i l a r to that o f the duvatrie n e d i o l s ( T a b l e 5). W e h a v e n o suitable e x p l a n a t i o n f o r this a p p a r e n t d i s c r e p a n c y . H y d r o x y m a n o o l h a d n o effect o n o v i p o s i t i o n b y t o b a c c o b u d w o r m m o t h s , as i n d i c a t e d by the n o n s i g n i f i c a n t d i f f e r e n c e f r o m the s o l v e n t b l a n k and b y the s i g n i f i c a n t l y l o w e r e g g d e p o s i t i o n w h e n c o m p a r e d w i t h the d u v a t r i e n e d i o l treated leaves. In this p a p e r , w e h a v e d e m o n s t r a t e d that t h r e e a c c e s s i o n s o f IV. glutinosa are a t t a c k e d by larvae o f H. virescens in the field, but that i n f e s t a t i o n levels o f t o b a c c o b u d w o r m larvae o n t h e s e a c c e s s i o n s are slightly less t h a n are f o u n d o n

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JACKSON ET AL.

TABLE 5. OVIPOSITIONAL RESPONSE OF TOBACCO BUDWORM MOTHS TO CUTICULAR COMPONENTS ISOLATED FROM Nicotiana glutinosa AND APPLIED TO TI 1112 LEAVES IN CHOICE TESTS IN SMALL SCREEN CAGES IN A GREENHOUSE SECTION, OXFORD, NORTH CAROLINA, 1988

Entry A (TI 1112 with:)

Entry Ba (TI 1112 with:)

Percent of eggs b on entry A

No. of replications

Labdenediol" Labdenediol

SB DVT

67.8 t 50.5 NS

15 14

Manool d Manool

SB DVT

71.9 t 46.3 NS

12 13

2-Hydroxymanool" 2-Hydroxymanool

SB DVT

40.8 NS 24.8 ~

13 12

Sclareols f Sclareols

SB DVT

53.7 NS 46.5 NS

13 10

G-SE-F G-SE-I

SB DVT

72.6 / 44.7 NS

11 12

G-SE-II h G-SE-II

SB DVT

63.7 t 52.5 NS

11 17

T-SE-I I

SB

63.4 "

41

Duvatriendiols J NC 2326 k

SB TI 1112 k

74.4 m 77.8 "

15 42

a TI 1112 leaves sprayed either with a solvent blank (SB) or with ot + B-4,8,13-duvatriene-l,3diols (DVT). bpaired t-test; NS = not significant. 'Applied at 50.0/.tg/cm2; avg. recovery of 62.3 % from 36 samples. dApplied at 50.0/xg/cm2; avg. recovery of 78.7% from 36 samples. eApplied at 50.0/.tm/cm2; avg. recovery of 68.4% from 35 samples. fMixture of sclareol and 13-episclareol applied at 50.0 #g/cm2; avg. recovery of 73.5% from 36 samples. gG-SE-I = 2,3,4,tri-O-acyl-3'-O-acetyl-sucrose applied at 50.0/zg/cm2; avg. recovery of 52.5% from 38 samples. hG-SE-II = 2,3,4,tri-O-acyl-sucrose applied at 50.0/~g/cm2; avg. recovery of 57.2% from 39 samples. IT-SE-I = 6-O-acetyl-2,3,4-tri-O-acyl-sucrose, tested 1983-1985 in field cages (see Jackson et al., 1989). Jt~-+/~-4,8,13-duvatriene-l,3-diols applied at 50.0 /zg/cm2: avg. recovery of 66.9% from four samples. kUnsprayed NC 2326 and TI 1112 leaves. tSignificant difference (P = 0.05). mSignificant difference (P = 0.01).

2503


TABLE 6. ACID COMPOSITION (C3-C 8) OF THE SUCROSE ESTER FRACTIONS FROM

Nicotiana tabacum ANDN. glutinosa Mole percent a N. glutinosa N. tabacurn b

Acid

(T-SE-I)"

G-SE-I'

G-SE-IV

Propanoic Isobutanoic Butanoic 2-Methylbutanoic 3-Methylbutanoic Pentanoic 3-Methylpentanoic 4-Methylpentanoic Hexanoic 4-Methylhexanoic 5-Methylhexanoic Heptanoic Methylheptanoic Octanoic

0.3 2.7 1.0 8.1 13.9 0.7 68.8 1.4 0.7 1.2 0.8 d 0.3 --

0.7 9.9 0.3 20.1 3.1 1.0 3.7 4.7 1.3 27.5 32.0 0.2 1.1 0.3

0.2 2.8 0.5 8.5 1.3 0.7 1.1 2.4 0.7 42.3 30.6 1A 1.7 0.6

Based on total C3-C8 acids as determined by GC analysis of butylesters after hydrolysis of sucrose ester (Severson et al., 1990). blsolated from TI 165 (Severson et al., 1985a). ~T-SE-I = 6-O-acetyl-2,3,4-tri-O-acyl-sucrose;G-SE-I = 2,3,4-tri-O-acyl-3'-O-acetyl-sucrose,and G-SE-II = 2,3,4-tri-O-acyl-sucrose. dBelow detection limits.

oriental or flue-cured tobacco cultivars. W e have also shown that each o f the accessions o f N. glutinosa is equally susceptible to oviposition by tobacco budworm moths as is a standard flue-cured cultivar, NC 2326. Differences in the infestation levels o f tobacco budworm larvae on tobacco and N. glutinosa appear to be due to larvae not surviving or developing as well on the accessions o f N. glutinosa as they do on tobacco cultivars. W e have also shown that two labdane diterpenes and two SE types from the cuticulae o f N. glutinosa accessions stimulate oviposition by tobacco budworm moths in cage bioassays. W e previously reported (Jackson et al., 1989) that G E and SE type compounds from other Nicotiana species also stimulate oviposition by these insects. There are additional labdanes and GE and SE compounds from the Nicotiana species (Severson et al., 1990) that we have not bioassayed for ovipositional activity. Several o f the Nicotiana species possessing these untested components are also susceptible to tobacco budworm damage in the field (Table 1) and to oviposition by tobacco budworm moths (Jackson

2504

JACKSON ET AL.

et al., 1989; Severson et al., 1990). Sucrose esters from the Nicotiana species differ not only in esterification positions on the sucrose molecule, but also in the acid moiety compositions which vary considerably within the genus (Severson et al., 1990). For example, the major acid moieties of the SE from N. tabacum (T-SE-I from TI 165) are methylpentanoics, whereas the major acid moieties of the SEs from N. glutinosa (G-SE-I and G-SE-II) are methylhexanoics (Table 6). Thus, it appears that several different SE fractions from the genus Nicotiana elicit the same behavioral response, oviposition, by tobacco budworm moths. We are investigating how structural differences and variations in the acid compositions of the SE types from the Nicotiana species affect the ovipositional response of tobacco budworm moths. The components tested in this study and those listed by Severson et al. (1990) might also affect pest or beneficial insects other than tobacco budworms which are associated with tobacco culture, and we are investigating these interactions. Acknowledgments--We thank James S. Cheatham, James M. Hobgood, Jr., Linda Smith, Brenda King, and Sara J. Mueller for their excellent technical assistance. REFERENCES ARRENDALE,R.F. and MARTIN,R.M. 1988. The preparation of immobilized stationary phase fused silica capillary columns with OV-1701-vinyl deactivation. J. High Resolut. Chromatogr. Chromatogr. Commun. 11:157-161. ARRENDALE,R.F., SEVERSON,R.F., SISSON,V.A., COSTELLO,C.E., LEARY,J.A., HIMMELSBACH, D.S., and VAN HAEBEEK,H. 1990. Characterization of the sucrose ester fraction from Nicotiana glutinosa. J. Agric. Food Chem. 38:75-85. BAILEY, J.A., VINCENT, G.G., and BURDEN, R.S. 1974. Diterpenes from Nicotiana glutinosa and their effects on fungal growth. J. Gen. Microbiol. 85:57-64. BAILEY, J.A., CARTER, G.A., BURDEN, R.S., and WAIN, R.S. 1975. Control of rust diseases by diterpenes from Nicotiana glutinosa. Nature 255:328-329. BAt;MHOVER,A.H. 1985. Manduca sexta, pp. 387-400, in P. Singh and R.F. Moore (eds.). Handbook of Insect Rearing, Vol. 2. Elsevier, Amsterdam. BURK, L.G., and STEWART,P.A. 1969. Resistance of Nicotiana species to the green peach aphid. J. Econ. Entomol. 62:1115-1117. BURK, L.G., and STEWART, P.A. 1971. Survey of resistance among Nicotiana species to tobacco h0rnworm and budworm larvae. Tobacco Sci. 15:32-34. COHEN, Y., EYAL, H., GOLDSCHMIDT,Z., and SKLARZ,B. 1983. A preformed chemical inhibitor of tobacco powdery mildew on leaves of Nicotiana glutinosa. Physiol. Plant Pathol. 22:143150. COLLEDGE, A., REID, W.W., and RUSSELL,R.A. 1974. A survey of surface diterpenoids of green leaves. Ann. du Tabak, Sec. 2, 11:159-164. CUTLER, H.G., REID, W.W., and DELETANG,J. 1977. Plant growth inhibiting properties of diterpenes from tobacco. Plant Cell Physiol. 18:711-714. CUTLER, H.G., SEVERSON,R.F., COLE, P.D., JACKSON, D.M., and JOHNSON, A.W. 1986. Secondary metabolites from higher plants: Their possible role as biological control agents, pp. 178-196, in M.B. Green and P.A. Hedin (eds.), Natural Resistance of Plants to Pests: Roles of Allelochemicals. Am. Chem. Soc. Symp. Ser. No. 296.


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