Naturally Occurring Pest Bioregulators - American Chemical Society

Chapter 17

Ovipositional Behavior of Tobacco Budworm and Tobacco Hornworm Effects of Cuticular Components from Nicotiana Species 1

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R. F . Severson , D . M. Jackson , A . W . Johnson , V . A . Sisson , and M . G . Stephenson Downloaded by UNIV MASSACHUSETTS AMHERST on August 2, 2012 | http://pubs.acs.org Publication Date: January 9, 1991 | doi: 10.1021/bk-1991-0449.ch017

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R. B. Russell Agricultural Research Center, U.S. Department of Agriculture, P.O. Box 5677, Athens, G A 30613 Crops Research Laboratory, U.S. Department of Agriculture, P.O. Box 1555, Oxford, N C 27565 Pee Dee Research and Education Center, Clemson University, U.S. Department of Agriculture, Route 1, Box 531, Florence, S C 29501 Nematodes, Weeds and Crops Research, U.S. Department of Agriculture, P.O. Box 748, Tifton, G A 31793 2

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The USDA - Nicotiana species germplasm collection was evaluated for tobacco budworm [Heliothis virescens (F.)] and tobacco hornworm [Manduca sexta (L.)] field infestation levels and for ovipositional responses of moths within screenedfieldcages. Species which did not produce observable trichome exudates did not receive as many budworm or hornworm eggs as a flue-cured tobacco in paired choice or no-choice experiments. Qualitative and quantitative data on the cuticular chemistries were obtained. The cuticular extracts of all the Nicotiana species contained a series of aliphatic hydrocarbons. Major components in the trichome exudates were found to be diterpenes (duvane and/or labdane types) and/or sugar esters (sucrose and/or glucose). Eight different general types of sucrose esters and two types of glucose esters were identified. All sugar ester types contained a complex mixture of C3-C8 fatty acids attached to the 2,3, and 4 positions of the glucose moiety. Cuticular components were isolated and tested for tobacco budworm ovipositional response. Several cuticular diterpenes(α-and ß-4,8,13-duvatriene-l,3-diols, α- and ß4,8,13-duvatrien-1-ols, manool andlabda-13-ene-8α-15-diol)and two sucrose ester types (6-0-acetyl-2,3,4-tri-0-acyl- sucrose and 2,3,4-tri-0-acyl-4'-0-acetyl-sucrose) were found to increase oviposition by tobacco budworm moths when these materials were sprayed onto a leaf devoid of them. We believe that these components are contact ovipositional stimulants. During the 1970's the U S D A Nicotiana tabacum germplasm collection was evaluated in field plots at the Clemson University Pee Dee Research and Education Center, Florence, S C for their resistance to the tobacco hornworm, Manduca sexta (L.) (1), and the tobacco budworm, Heliothis virescens (F.) (2). During this investigation a large variation in leaf trichome types and density was observed. Johnson et al. (3,4) classified the major trichome types from the various tobacco types as simple trichomes without exudates, glandular trichomes without exudates, glandular trichomes with exudates, and This chapter not subject to U.S. copyright Published 1991 American Chemical Society In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

Downloaded by UNIV MASSACHUSETTS AMHERST on August 2, 2012 | http://pubs.acs.org Publication Date: January 9, 1991 | doi: 10.1021/bk-1991-0449.ch017

17. S E V E R S O N E T A L .

Ovipositional Behavior of Budworm & Hornworm

small trichome hydathodes [See Johnson et al. (4) or Severson et al. (5), for trichome photographs]. In general, N tabacum plants which lack glandular trichomes with exudates are resistant to insects (5,6). However, some tobacco types with observable trichome exudates are also resistant to insect damage. Thus, the surface chemistry of insect-resistant and susceptible tobacco types with the different types of trichomes was investigated (7). As shown in Table I (5,6,9), tobacco cultivars and introductions with glandular secreting trichomes, N C 2326, Golden Burley, N F T , TI1223, TI1341, TI165 and T I 1396 produce duvane diterpenes and/or labdane diterpenes and/or sucrose esters (See Figures 1 and 2 for structures and References 5,7 and 8 for capillary gas chromatograms). The tobacco introductions with simple trichomes, T I 1112,1-35, and those with nonsecreting glandular trichomes, T I 1024 and T I 1406, produce low levels of diterpenes and sucrose esters. The cuticular extracts of all N tabacum plants studied, independent of trichome type, contained a series of C - C aliphatic hydrocarbons consisting of a series of straight-chain and iso- and anteiso-methyl-branched-hydrocarbons (5,7). These components apparently are not associated with insect resistance. Controlled larval feeding and opposition tests (Table I) showed that a major mode of resistance to tobacco budworms in T I 1112,1-35, T I 1024, and N F T is ovipositional non-preference (antixenosis). The budworm resistance observed with T I 165 and T I 1396 appears to result from larval antibiosis (5,6,9). Field studies with naturally occurring populations of tobacco hornworms conducted at Oxford, N C ; Florence, SC; and Tifton, G A in 1985 showed that T I 1112 and 1-35 received only 11% and 16%, respectively, of the hornworm eggs relative to those deposited on N C 2326, a commercial flue-cured cultivar (9). Thus the high level of hornworm resistance observed with T I 1112 and 1-35 also appears to be due to ovipositional non-preference. In this report, we will discuss investigations of the cuticular components from Nicotiana species and their effects on tobacco hornworm and tobacco budworm moth oviposition. The response of tobacco budworm moths to specific cuticular isolates from a Nicotiana species also will be discussed. 2 5

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Experimental A l l N. tabacum plants evaluated for insect resistance and cuticular chemistries were grown under field conditions normally used for the production of flue-cured tobacco at the Clemson University Pee Dee Research and Education Center, Florence, SC; the Crops Research Laboratory, Oxford, N C and the University of Georgia Coastal Plain Experiment Station, Tifton, G A . Other Nicotiana species were evaluated at Oxford, N C or Tifton, G A . From 1984-1987 70 accessions of 64 Nicotiana species were planted to evaluate their effects on tobacco budworm and hornworm oviposition in field plots and in choice tests versus N C 2326 in cages (20). In 1985 and 1986 the different Nicotiana species were grown in the field and cuticular chemical extracts were obtained and analyzed in 1985. Field plots were also screened for natural infestations of insect pests. Quantitation, Isolation and Characterization of Cuticular Components About six weeks after transplantation, cuticular components from field-grown plants of each Nicotiana species were extracted by dipping young leaves in 8 oz. wide mouth bottles containing methylene chloride. After removal of the methylene chloride, the extract residue was treated with 1:1

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

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8 13-Duvatrien-l-ols

(a & (J D V T - o l s )

p-4 (a & p D V T - d i o l s )

a & p-4,8,13-Duvatriene-l,3-diols (cfs-ablenol)

(12-Z)-Labda-12,lA-dien-8a-ol

(Labdenediol)

(13-E)-Labda-13-cne-8a,15-diol


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SEVERSON E T AL.


Comparison of the Cuticular Chemistries of Various N. tabacum Types to Resistance Ratings of Tobacco Budworm and Hornworm (5,6,9)

TABLE I

Resistance Ratings

N. tabacum

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Tobacco Hornworm

Tobacco Budworm


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Field Field Laboratory Plant Plant Larval Cage Damage Feeding Oviposition Damage

NC 2326 Golden Burley TI 1112 1-35 TI 1024 TI 1406 NFT TI 1223 TI 1341 TI 165 TI 1396

S S R R MR MR MR S S R MR

S S MR MR MR MR S S S R R

S S R R R R R S S S S

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Sucrose a- & fi- a- & fiDVT-ols DVT-diols Cis-abienol Esters

S S R R MR MR MR S S MR MR

46.0 21.0 0.6 0.2 0.8 0.8 0.6 3.0 34.0 97.0 61.0

0.8 0.4 0.1 0.4 0.1 20.0 59.0 2.0 3.0

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0.3 21.0 23.0 23.0

2.0 1.0 0.9 1.0 9.0 7.0 21.0 31.0 35.0

Relative to NC 2326: R-resistant; MR-moderately resistant; S=susceptible. Six weeks after transplantation, Oxford, NC and Tifton, GA, 1982 and 1983. 6-0-acetyl-2,3,4-tri-0-acyl-sucrose. b c

CHJORJ

CH OR! 2

R = C - C Acyl groups R = H or Acetyl 3

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Sucrose Ester Types A B C D E F G H

Glucose Esters

6-0-acetyl-2,3,4-tri-0-acyl-sucrose 6-0-acetyl-2,3,4-tri-0-acyl-3'-0-acetyl-sucrose 6-0-acetyl-2,3,4-tri-0-acyl-4'-0-acetyl-sucrose 2,3,4-tri-O-acyl-sucrose 2,3,4-tri-0-acyl-3'-0-acetyl-sucrose 2,3,4-tri-0-acyl-4'-0-acetyl-sucrose 2,3,4-tri-0-acyl-3,4-di-0-acetyl-sucrose 2,3,4-tri-O-acyl-l' ,3' ,4'-tri-0-acetyl-sucrose ,

Figure 2

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Cuticular Chemistry (/xg/cm )

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' 6-0-acetyl-2,3,4-tri-0-acyl-glucose D' 2,3,4-tri-O-acyl-glucose

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Cuticular Glucose and Sucrose Esters of Nicotiana Species.


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NATURALLY OCCURRING PEST BIOREGULATORS

N,0-bis(trimethylsily) trifluoroacetamiderdimethylforrnarnide to convert hydroxylated components to trimethylsilylethers. Samples were analyzed by capillary gas chromatography as described by Severson et al. (7). Larger quantities of cuticular components for characterization and bioassay studies were obtained by dipping whole plant tops (upper 1/3) into methylene chloride. Components were isolated from the cuticular extracts using solvent partitioning between hexane and 80% M e O H - H 0 and/or a combination of alumina, silicic acid and Sephadex L H - 2 0 column chromatography. Specific methodology for the isolation of components homN tabacum (10,11), andN glutinosa (12, 13) have been described. The cuticular extracts of the other Nicotiana species were solvent partitioned between hexane and 80% M e O H - H 0 , and resulting fractions were characterized by G C retention and G C / M S data. The sucrose esters (and glucose esters) of N kawakamii (14), N. otophora (15), N setchellii (15), N tomentosa (15), N tomentosiformis (14), and N clevelandii (16) were isolated from M e O H - H 0 soluble fractions using Sephadex LH-20-CHC1 column chromatography and confirmed by G C / M S analyses. The M e O H - H 0 soluble fractions containing the cuticular sugar esters were hydrolyzed and the resulting fatty acids were analyzed as butyl esters as described by Severson et al. (17) 2


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Ovipositional Bioassays Ovipositional bioassays were conducted using potted plants treated with cuticular isolates from N tabacum accessions in 2.4 X 2.4 X 2 m screened cages at Oxford, N C as previously described (18,19). Treatment plants, TI 1112 sprayed with a cuticular component, and control plants, T I 1112 sprayed with solvent blank, were placed at opposite corners of a cage. Cuticular components in 0.5 ml hexane-methylene chloride (3:1) were diluted with 9.5 or 14.5 ml of carrier solution [watenacetone (1:3)] and sprayed onto test plants with an air-brush (Badger Air-Brush Co., Model 250). Before dark, 10 mated females were released into each cage and the following morning plants were examined for eggs. Insects were from a laboratory colony started from larva collected from tobacco near Oxford, N C , and were reared for 7-10 generations on artificial diet before use. Twelve smaller (0.46 x 1.31 m), hemicylindrical cages were used to bioassay cuticular components from N. glutinosa for tobacco budworm ovipositional preference. These experiments were run in a glass greenhouse in which the walls were covered with black plastic film to block extraneous lights. In the center of each end of a cage was a 10.2-cm diam. hole through which was placed a plastic frustum (11.7-cm O D bottom; 9.5-cm O D top; 8.9-cm high) holding a tobacco leaf so that it's abaxial (lower) surface was exposed. Thus, each leaf disk exposed 71 square centimeters of leaf area to the ovipositing moths. The leaf disks at opposite ends of a cage (1.3 m apart) were bioassayed for ovipositional preference in a choice-test situation. The cuticular isolates in 1 ml of acetone were mixed with 1 ml of acetone:water(l:l) and sprayed onto the leaf disks as described. Five female moths were introduced into each cage in the late afternoon, and the number of eggs on each treatment were counted the next morning. The insects were reared and prepared for bioassay as described for the outdoor oviposition cages. RESULTS Cuticular leaf chemistries for the different Nicotiana species are given in Table II. Also included in the table are the percent of tobacco budworm and



17. S E V E R S O N E T A L .


hornworm eggs deposited on each species relative to the tobacco cultivar, N C 2326, in cages in choice tests (20). In the choice tests none of the Nicotiana species were significantly more attractive than N C 2326 to budworm or hornworm oviposition. However, in field evaluations N kawakamii was more susceptible to budworm damage and several species were more susceptible to hornworm damage than N C 2326 (Table III). Excluding N tabacum, 20 and 21 of the Nicotiana species were as attractive as N C 2326 to budworm and hornworm oviposition, respectively. A l l other species which did not produce observable trichome exudates (Nicotiana spp. Nos. 19,23,25,27,31,44,47,52,53,63,64, and 71) were not attractive to budworm oviposition. Excluding tabacum types, only N sylvestris produced significant levels of a- and 0-DVT-diols. Nine of the Nicotiana species produced labdane diterpenes, N tabacum cv Samsun produced cis-abienol and labdenediol. Major cuticular labdanes on N glutinosa 24 are manool, 15-hydroxy manool, sclareol, 13-episclareol and labdenediol. N glutinosa 24A produces only the sclareol and labdenediol The labdane diterpenes of two other Nicotiana species, N raimondii, raimonol and iso-raimonol (22) and N setchelliiy setchelol and iso-setchelol (23), have been characterized. Most of the Nicotiana species with observable trichome exudates produced sugar esters. However, as shown in Figure 2 and Table IV, large variations in sugar ester types and distribution of ester moieties were found. W e identified eight different general types of sucrose esters and two types of glucose esters (Table IV). The glucose esters are further complicated by the presence of a- and 0 forms. A l l types characterized to date have a complex mixture of C to C^ fatty acids attached to the 2,3 and 4 positions of the glucose moiety. These acids consist of normal chains, and iso- and anteiso methyl-branched isomers. For most species the major acyl group on the glucose moiety were methyl-branched C to C isomers. N hesperis was the only Nicotiana species where the major sugar ester acyl groups were normal chain acids. Low levels of unsaturated acyl groups were detected in the sugar ester hydrolysates of several of the species. The unsaturated acyl group, 2-methyl-2-butenoyl, was a major component in the sugar ester isolates from N hesperis. Results of the ovipositional response of tobacco budworm moths to various cuticular isolates from Nicotiana spp. when applied to the leaves of the non-preferred T I 1112 are shown in Table V . The hexane soluble fraction from the cuticular extract from N C 2326 did not stimulate budworm oviposition (18). Previously, we reported that the a- and 0-DVT-diol mixture, a-DVT-diol and a mixture of a- and 0-DVT-ols produced a significant ovipositional response (P

Naturally Occurring Pest Bioregulators - American Chemical Society

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