Hydroxygeranyllinalool Glycosides from Tobacco Exhibit Antibiosis Activity in the Tobacco Budworm [Heliothis virescens (F.)] Maurice E. Snook,* ,† Albert W. Johnson, ‡ Ray F. Severson, †,§ Quincy Teng, | Robert A. White, Jr., † Verne A. Sisson, ⊥ and D. Michael Jackson # Russell Research Center, USDA-ARS, P.O. Box 5677, Athens, Georgia 30604; Pee Dee Research and Education Center, Clemson University, Florence, South Carolina 29501; Chemistry Department, University of Georgia, Athens, Georgia 30602; Department of Crop Science, North Carolina State University, P.O. Box 1555, Oxford, North Carolina 27565; and U.S. Vegetable Laboratory, USDA-ARS, 2875 Savannah Highway, Charleston, South Carolina 29414 Leaves of Tobacco Introduction TI-165 were found to be resistant to tobacco budworm [Heliothis virescens (F.)] attack. HPLC profiles of leaf extracts showed that TI-165 had relatively high levels of two components (A and B) that were absent in susceptible varieties. Compounds A and B were isolated from TI-165 by a combination of preparative C 18 , silicic acid column, and centrifugal thin- layer chromatography. They were identified as diterpene glycosides: compound A, 16-hydroxy- geranyllinalyl-3-O-[R-L-rhamnopyranosyl(1f4)]--D-glucopyranoside-16-O-[R-L-rhamnopyranosyl- (1f6)]--D-glucopyranoside; compound B, 16-hydroxygeranyllinalyl-3-O-[R-L-rhamnopyranosyl(1f4)]- - D-glucopyranoside-16- O-[ R- L-rhamnopyranosyl(1 f6)]-[ R- L-rhamnopyranosyl(1 f4)]- - D- glucopyranoside (hydroxygeranyllinalool glycosides). Budworm bioassays with whole tobacco leaves and purified mixtures of A and B showed significant correlation between larval weights and levels of A and B. HPLC analyses of freeze-dried leaves of 68 Nicotiana species indicated that 26 species had high levels of diterpene glycosides identical to or related to A and B. Keywords: Nicotiana; Heliothis virescens (F.); antibiosis; 16-hydroxygeranyllinalool glycosides; diterpene glycosides INTRODUCTION Tobacco budworm [Heliothis virescens (F.)] infests tobacco, cotton, and other crops. The economic cost to South Carolina tobacco farmers (leaf damage and control) in 1994 was >$6 million (Gooden et al., 1996). Budworm-resistant tobacco genotypes have been identi- fied; these exhibit various modes of resistance. One mode of resistance to budworm attack in tobacco is ovipositional nonpreference. Chemicals, such as R- and -4,8,13-duvatriene-1,3-diols and their corresponding mono-ols and sucrose esters, associated with the leaf cuticular waxes, have been shown to be ovipositional stimuli (Jackson et al., 1984, 1986; Johnson and Sev- erson, 1982, 1984; Severson et al., 1984, 1985). These compounds were found to be absent from the resistant TI-1112 cultivar (Severson et al., 1984). A second mode of resistance involves moth flight orientation due to certain volatile chemicals emitted by tobacco leaves. Mitchell et al. (1991) and Tingle et al. (1990) demon- strated that susceptible tobacco cultivars (e.g., NC2326) emit attracting volatiles, while Tingle et al. (1990) showed that budworms do not fly toward the resistant variety, TI-1112, which apparently lacks the attracting compounds. A third mode of budworm resistance is antibiosis. Cultivars identified with this type of resis- tance were TI-163, TI-165, TI-168, and TI-170 (Johnson and Severson, 1984; Johnson et al., 1992, 1995). How- ever, the antibiosis mode of resistance in these lines was unknown. This paper reports the isolation from TI-165 and the subsequent identification and HPLC analyses of two diterpene glycosides (designated compounds A and B, Figure 1) that have been shown to correlate with H. virescens resistance both in field-grown tobacco and in laboratory dietary bioassays. We also report the results of an HPLC survey for related diterpene glycosides in the leaves of the Nicotiana species. † Russell Research Center. ‡ Clemson University. § Deceased. | University of Georgia. ⊥ North Carolina State University. # U.S. Vegetable Laboratory. Figure 1. Structures of compounds A and B (16-hydroxygera- nyllinalool glycosides). 2299 J. Agric. Food Chem. 1997, 45, 2299-2308 S0021-8561(96)00748-0 CCC: $14.00 © 1997 American Chemical Society