DITERPENE RESIN ACIDS: MAJOR ACTIVE. PRINCIPLES IN TALL OIL AGAINST. VARIEGATED CUTWORM, Peridroma saucia. (LEPIDOPTERA: NOCTUIDAE).
Journal of Chemical Ecology, Vol. 19, No. 6, 1993
DITERPENE RESIN ACIDS: MAJOR ACTIVE PRINCIPLES IN TALL OIL AGAINST VARIEGATED CUTWORM, Peridroma saucia (LEPIDOPTERA: NOCTUIDAE)
YONGSHOU
XIE, 1 MURRAY and ALFRED
B. I S M A N , I ' * Y I F E N G , 2 WONG 2
tDepartment of Plant Science University of British Columbia Vancouver, British Columbia, Canada V6T IZ4 2Arbokem Inc. Suite 101,358 East Kent Ave. Vancouver, British Columbia, Canada V5X 4W6 (Received April t3, 1992; accepted January 25, 1993)
Abstract--Tall oil, a by-product of the kraft process for pulping softwood, has been shown to have insecticidal properties. In the present study, the active principles in tall oil against the variegated cutworm, Peridroma saucia Hfibher, were investigated. GC-MS analysis showed that abietic, dehydroabietic, and isopimaric acids were major resin acid components of crude tall oil and depitched tall oil. When crude tall oil samples of differing resin acid composition were incorporated into artificial diet at a concentration of 2.0 % fresh weight, they suppressed larval growth by 45-60% compared to controls. This suppression was significantly (P N 0.05) correlated with the equivalent contents of abietic, dehydroabietic, isopimaric, and total resin acids. These results were also evident from a diet choice test, showing that the second-instar larvae obviously selected diets with low levels of resin acids when different diets were randomly arranged in a Petri dish. Bioassays with pure resin acids (abietic, dehydroabietic, and isopimaric acids) demonstrated that all individual chemicals have similar bioactivity against this insect. Comparison of the bioactivities of depitched tall oil and an equivalent mixture of pure resin acids in the Peridroma chronic growth bioassay indicated that pure resin acids and depitched tall oil share a common mode of action to this insect. This study confirms that resin acids are major active principles in tall oil against the variegated cutworm, but other chemicals likely also contribute to the bioactivity of tall oil.
*To whom correspondence should be addressed. 1075 0098-0331/93/0600-1075507.00/0 9 1993PlenumPublishingCorporation
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Key Words--Tall oil, resin acids, abietic acid, dehydroabietic acid, isopimaric acid, Peridroma saucia, Lepidoptera, Noctuidae, variegated cutworm, bioactivity, natural insecticide.
INTRODUCTION
It is well known that higher plants are a potential source of new insecticides (Arnason et al., 1989), and a few natural products, such as rotenone from Derris species, and pyrethrins from Chrysanthemum species, are commercially used as insecticides (Isman et al., 1991). In a recent exploration for new sources of natural pesticides, tall oil, a by-product of the kraft process for pulping softwood (~ is the Swedish word for pine), has attracted attention. Because tall oil is produced abundantly in the softwood kraft pulp industry, the concept of developing a pest control product from this precursor is very attractive. Cousin (1989) first reported that tall oil neutrals have insecticidal activities. More recently, crude tall oil and two derivatives were found to have toxic and feeding deterrent activities against the variegated cutworm, Peridroma saucia Hfibner. These materials significantly reduced growth, feeding, and dietary utilization by variegated cutworm larvae in chronic larval growth bioassays, choice and no-choice feeding tests, and nutritional experiments (Xie and Isman, 1992). However, the active principles associated with the insecticidal properties of tall oil are unknown. The objectives of the present study are to determine the major chemical constituents in typical crude tall oil and depitched tall oil collected in central British Columbia, Canada, to assess the impact of chemical variation in tall oil on bioactivity to the variegated cutworm, and to evaluate the role of several pure commercial resin acids as deterrents to feeding and growth of the variegated cutworm. Our study aims to identify the active principles in tall oil associated with toxic and feeding deterrent activities against the variegated cutworm. METHODS AND MATERIALS
Insects. The variegated cutworms used in this study were obtained from a laboratory culture maintained at 25 + 1 ~ and a photopefiod of 16L: 8D, and reared on artificial diet (No. 9795, BioServ Inc., Frenchtown, New Jersey). Test Materials. Dehydroabietic and isopimaric acids were obtained from Helix Biotech Corporation, Richmond, British Columbia, Canada. Their purities were stated to be >99%. Abietic acid was purchased from Sigma Chemical Company, St. Louis, Missouri (purity 85%), and purified by crystallization. The purified chemical was analyzed by gas chromatography-mass spectrometry (GC-MS), and the purity was >90%. Crude tall oil and depitched tall oil
DITERPENE RESIN ACIDS
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supplied by B.C. Chemicals Limited, Prince George, British Columbia, Canada, were used for chemical analysis and bioassay. Chemical Analysis of Tall Oil Samples. Crude tall oil and depitched tall oil samples were analyzed by GC-MS. GC-MS analyses were performed on a Hewlett-Packard 5890-II gas chromatograph equipped with a HP 5971 Mass Selective Detector. The column employed was a 30-m • 0.25-ram DB-I (J&W Scientific, Inc., Folsom, California). Ultra high pure (UHP) helium was used as the carrier gas at a head pressure of 15 psi. The injector part was maintained at 325~ and the oven temperatures were programmed from 160~ to 350~ at a rate of 7~ The temperature of the GC-MS interface was 320~ Samples and reference chemicals were dissolved in ether-methanol (9 : 1) solution, and methylated with diazomethane. The resin acid esters were identified by comparing GC retention times and mass spectra to those of reference chemicals purchased from Helix Biotech Corporation. The acid concentrations were quantified by an internal standard, tricosanoic acid methyl ester.
Bioassays of Crude Tall Oil Samples of Differing Diterpene Resin Acid Composition. Several dozen crude tall oil samples, collected from four different pulp mills on a monthly basis over 13 months in central British Columbia, Canada, were analyzed for their chemical composition by GC-MS. Based on these results, six crude tall oil samples, two with high, two with medium, and two with low total resin acid contents, were used for bioassay via incorporation of the test materials into artificial diet. The test materials were dissolved in solvent (isopropyl alcohol) and added to the dry diet constituents at a concentration of 2.0% fresh weight of diet. Control diets were treated with the cartier solvent alone. Bioassays performed were a chronic larval growth test and a diet choice test. In the former case, the bioassay was conducted by placing 20 neonate larvae individually in compartments in a plastic tray with approximately 1 g of treated or control diet. Larvae were maintained in a growth chamber at 25 ~ and a photoperiod of 16L:8D. After seven days, all larvae were individually weighed, and the mean weights for each treatment group were expressed as a percentage of controls. The diet choice test was performed to determine insect response to diets of differing chemical contents by randomly placing six pieces of treated artificial diet with the six different crude tall oil samples (2.0% fresh weight of diet) in a plastic Petri dish (9 cm diameter). Twenty second-instar larvae were introduced into the center of each dish. Ten replicates were prepared. After 24 hr in complete darkness (25~ the number of larvae on each diet was recorded. Bioassay of Commercial Resin Acids. Bioassays with commercial resin acids (abietic, dehydroabietic, and isopimaric acids) were conducted to verify the importance of individual resin acids on bioactivity of tall oil. The individual test chemicals, dissolved in methanol, were added to the dry diet constituents at concentrations of 0.5-2.0% fresh weight of diet. A tertiary mixture, main-
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taining total resin acid concentrations of 0.5-2.0% fresh weight, was incorporated into artificial diet to determine if any additive o r synergistic action is involved. Chronic larval growth bioassays were performed as described above.
Comparative Bioassays of Depitched Tall Oil and Commercial Resin Acids. A further set of bioassays was conducted to compare the action of depitched tall oil and a mixture of commercial resin acids on the variegated cutworm. Depitched tall oil was incorporated into artificial diet at concentrations of 0.5-2.5% fresh weight. The three diterpene resin acids, which were mixed together based on their proportions present in the depitched tall oil sample, were incorporated into artificial diet at a total concentration of 0.27-1.09 % fresh weight. This concentration range represents the equivalent contents of the three resin acids present in depitched tall oil at concentrations of 1.0-4.0% fresh weight. Chronic larval growth bioassays were performed as described before. Data Analysis. Linear regression analysis was applied to define all doseresponse relationships (Figures 1-4 below) when correlation was found to be significant. Analysis of covariance (ANCOVA) was used to test equality of regression coefficients in Figure 3, and a t test was used to compare regression coefficients (slope and intercept) of depitched tall oil and the mixture of pure resin acids in Figure 4 (Zar, 1984). An arcsin transformation was performed for percentage data before analysis because this transformation would make small or large percentages nearly normal in distribution (Zar, 1984).
RESULTS
GC-MS analyses revealed that representative samples of crude tall oil and depitched tall oil typically contained 34.3% and 40.4% fatty acids and 27.0% and 37.2% diterpene resin acids, respectively. Abietic, dehydroabietic, and isopimaric acids were the major resin acid components of crude tall oil and depitched tall oil. These three resin acids account for 66.1% and 72.8 % of total diterpene resin acids in crude tall oil and depitched tall oil, respectively (Table 1). When the test materials (six selected crude tall oil samples) were incorporated into artificial diet at a concentration of 2.0% fresh weight, they suppressed larval growth by 45-60% compared with controls (treated with carrier solvent alone). This suppression was significantly (P _< 0.05) correlated with the equivalent contents of abietic (r = - 0 . 9 6 , P = 0.0017), dehydroabietic (r = - 0 . 9 7 , P = 0.0010), isopimaric (r = - 0 . 8 0 , P = 0.0507), and total resin acids (r = - 0 . 9 6 , P = 0.0019) (Figure 1). The diet choice test revealed that the second-instar larvae selected diets with low levels of resin acids when different diets were randomly arranged in a Petri dish, showing that the number of larvae on diet was significantly (P < 0.05) and negatively correlated with the equivalent dietary concentrations of
DITERPENE RESIN ACIDS
1079
TABLE 1. RESIN ACID COMPOSITION OF CRUDE TALL OIL AND DEPITCHED TALL O1La
Concentration (weight %) Component
Crude tall oil
Depitched tall oil
Abietic acid Dehydroabietic acid Isopimaric acid Palustric and levopimaric acids Neoabietic acid Pimafic acid Sandaracopimaric acid Dehydrodehydroabietic acid Other resin acids Total resin acids
9.1 4.7 4.0 3.7 2.1 1.7 0.6 0.2 0.9 27.0
15.4 6.7 5.0 3.5 2.2 2. l 0.7 0.5 1.1 37.2
aData represent results from GC-MS analysis for typical crude tall oil and depitched tall oil samples. abietic (r = - 0 . 8 6 , P = 0.0272), dehydroabietic (r = - 0 . 8 6 , P = 0.0272), and total resin acids (r = - 0 . 9 4 , P = 0.0051) (Figure 2). Although our results (Figures 1 and 2) suggest that resin acids may in some instances be responsible for antifeedant and growth inhibitory action of tall oil against the variegated cutworm, bioassays with commercial pure resin acids (abietic, dehydroabietic, and isopimaric acids) could verify the importance of individual resin acids to the bioactivity of tall oil. Each of the test chemicals inhibited larval growth in a dose-dependent manner. Significant (P < 0.05) negative correlations between larval growth and resin acid concentration in diet were found by regression analysis (Figure 3). A N C O V A indicated that regression slopes of all individual chemicals and their tertiary mixture were not significantly different (P > 0.05)(F = 0.15, Fo.o5o),3 ,8 = 4.07), suggesting that each of the test chemicals inhibits larval growth in a similar fashion, and no synergistic action was involved. These conclusions received further support from another experiment aimed at comparing the action of a mixture of pure resin acids and depitched tall oil on bioactivity to the variegated cutworm. Our results clearly indicated that the resin acid mixture and depitched tall oil share a common mode of action to the variegated cutworm because no significant (P > 0.05) difference in slopes of the growth curves was found (Figure 4). However, the regression analyses indicate that the intercepts differ significantly (P < 0.05), with depitched tall oil showing as much as a 50% stronger growth suppression than the pure resin acid mixture at the same concentrations (Figure 4), suggesting that chemicals other than resin acids substantially contribute to the bioactivity of depitched tall oil to this insect.
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DISCUSSION
It is well known that terpenoids are one of the largest groups of plant secondary compounds known to have insecticidal and repellent properties (Beek and de Groot, 1986). Among the terpenoids, diterpenes have been demonstrated to have antifeedant and/or deterrent action against the pink bollworm, Pectinophora gossypiella (Elliger et al., 1976), and to deter larval feeding, growth, and dietary utilization of the sawflies, Pristiphora erichsonii, Neodiprion dubiosus, and N. rugifrons (Wagner et al., 1983; Schuh and Benjamin, 1984). Depitched tall oil, in which diterpenic acids (i.e., resin acids) account for over 37% of weight, has been demonstrated to be toxic to neonate P. saucia and inhibitory to larval growth (Xie and Isman, 1992). In the present study, we have
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confirmed that these diterpenic acids are the major active principles in tall oil against P. saucia. The major basis for this conclusion is the strong correlation between resin acid content in the samples and their bioactivity on larval growth of P. saucia. Correlation coefficients for bioactivity versus resin acid contents in tall oil samples indicate that 60-94% of the variation in bioactivity of tall oil can be accounted for by individual or total resin acid contents. This fact clearly suggests that resin acids are largely responsible for the growth-deterrence (physiological) activity of tall oil. This conclusion is further supported by the choice tests, which are useful in detecting small difference in food acceptability (Schoonhoven, 1982). Food acceptability of P. saucia larvae was reduced possibly by chemosensory effects. This reduction was also correlated with resin acid contents in tall oil samples, indicating that resin acids likely account for the antifeedant (behavioral) effects of tall oil. Isman et al. (1990) similarly reported that bioac-
1082
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tivities o f n e e m oils with various azadirachtin levels on P. saucia w e r e significantly (P < 0.05) correlated to their azadirachtin content, confirming that azadirachtin was the m a j o r active c o m p o n e n t in n e e m oil against this insect. B e r e n b a u m (1985) reported that a natural mixture o f six furanocoumarins f r o m parsnip was m o r e toxic to the corn e a r w o r m (Heliothis zea) than an e q u i v alent dose o f xanthotoxin, the m o s t active o f the six c o m p o u n d s w h e n tested individually. This indicated that synergistic action was involved. H o w e v e r , our results for resin acids with the variegated c u t w o r m r e v e a l e d that the tertiary mixture has bioactivity similar to each o f the individual c h e m i c a l s , suggesting a lack o f synergistic action. D e p i t c h e d tall oil and pure resin acids share a c o m m o n m o d e o f action to P. saucia. The significantly greater bioactivity o f depitched tall oil to P. saucia (cf. pure resin acids) indicates that resin acids are not the only c h e m i c a l s in depitched tall oil that contribute substantially to bioactivity. The role o f other c o m p o n e n t s o f tall oil in suppression o f larval growth o f P. saucia is under investigation.
Acknowledgments--We thank Yanfen Zheng and Nancy Brard for technical assistance and BC Chemicals Ltd., Prince George, British Columbia, Canada for supplying test materials. Supported by grants from NSERC (CRD 112262), Forestry Canada, and BC Chemicals Ltd. to M.B.I.
REFERENCES
ARNASON,J.T., PH~LOG~NE,B.J.R., and MORAND,P. (eds.). 1989. Insecticides of Plant Origin. ACS Symposium Series 387. American Chemical Society, Washington, D.C. BEEK, T.A. VAN,and DE GROOT,A. 1986. Terpenoid antifeedants, part I. An overview of terpenoid antifeedants of natural origin. Recl. Trav. Chim. Pays-Bas. 105:513-527. BERENBAUM,M. 1985. Brementown revisited: Interactions among allelochemicals in plants: Recent Adv. Phytochem. 19:139-169. COUSIN, M.J. 1989. Tall oil neutrals to protect plants from insects and the like. U.S. Patent No. 4874610. ELL1GER,C.A., ZINKEL,D.F., CHAN, B.G., and WMss, A.C., JR. 1976. Diterpene acids as larval growth inhibitors. Experientia 32:1364-1366. ISMAN, M.B., KOUL, O., LUCZYNSKI,A., and KAMINSKI, J. 1990. Insecticidal and antifeedant bioactivities of neem oils and their relationship to azadirachtin content. J. Agric. Food Chem. 38:1046-1411. ISMAN, M.B., KOUL, O., ARNASON,J.T., STEWART,J., and SALLOUM,G.S. 1991. Developing a neem-based insecticide for Canada. Mem. Entomol. Soc. Can. 159:39-47. SCHOONlaOVEN,L.M. 1982. Biological aspects of antifeedants. Entomol. Exp. Appl. 31:57-69. SCmJH, B.A., and BENJAMIN,D.M. 1984. Evaluation of commercial resin acids as feeding deterrents against Neodiprion dubiosus, N. lecontei, and N. rugifrons (Hymenoptera: Diprionidae). J. Econ. Entomol. 77:802-805. WAGNER,M.R., BENJAMIN,D.M., CLANCY,K.M., and Sc,tJn, B.A. 1983. Influence of diterpene
1084
X m ET AL.
resin acids on feeding and growth of larch sawfly, Pristiphora erichsonii (Hartig). J. Chem. Ecol. 9:119-127. X~E, Y.S., and ISMAN, M.B. 1992. Antifeedant and growth inhibitory effects of tall oil and derivatives against the variegated cutworm, Peridroma saucia Hiibner (Lepidoptera: Noctuidae). Can. Entomol. 124:861-869. ZAR, J.H. 1984. Biostatistical Analysis. Prentice-Hall, Englewood Cliffs, New Jersey.
