Journal of Chemical Ecology, Vol. 18, No. 7, 1992

INFLUENCE OF THE PLANT ANTIFEEDANT, PINOSYLVIN, ON SUPPRESSION OF FEEDING BY SNOWSHOE HARES

THOMAS

P. S U L L I V A N , l ' * D O U G L A S R. C R U M P , 2 H A L and ELISABETH A. DIXON 3

WlESER, 3

~Applied Mammal Research Institute 23523 47th Avenue, R.R. 7 Langley, B.C., Canada V3A 4R1 2Chemistry Division Department of Scientific and Industrial Research Private Bag, Petone, New Zealand 3Department of Chemistry University of Calgary Calgary, Alberta, Canada T2N 1N4 (Received December 16, 1991; accepted March 3, 1992) Abstract--This study investigated the influence of the plant antifeedants, pinosylvin and pinosylvin methyl ether (PME), on suppression of feeding by snowshoe hares (Lepus americanus). Screening bioassays showed clearly that pinosylvin suppressed feeding by hares when sprayed directly on coniferous tree seedlings. Pinosylvin odor, when associated with food (but not mixed with it), also significantly reduced consumption of rabbit chow by hares. Large pen bioassays indicated that both pinosylvin and PME significantly reduced feeding on tree seedlings by hares when the antifeedants were sprayed directly on trees. In field bioassays near Prince George, British Columbia, Canada, pinosylvin sprayed on trees and encapsulated in controlled-release devices of PVC plastic, with an internal wire as a twist-tie for attachment to tree, significantly reduced feeding by hares. Thus, pinosylvin will generate an "avoidance response" in terms of feeding by snowshoe hares. This response is likely triggered by an olfactory pathway based on positive results with controlledrelease devices attached to seedlings. Our study reports the first practical utilization of plant antifeedants for forest crop protection and wildlife management. Key Words--Plant

antifeedants, pinosylvin, pinosylvin methyl ether,

*To whom correspondence should be addressed at Department of Forest Sciences, Faculty of Forestry, University of British Columbia, Vancouver, B.C., Canada V6T 1W5. 1151 0098-0331/92/0700-1151506.50/09 1992PlenumPublishingCorporation

1152

SULLIVAN ET AL. snowshoe hare, Lepus americanus, herbivore, lodgepole pine, forest plantation, release devices, feeding suppression, crop protection.

INTRODUCTION

The Snowshoe hare (Lepus americanus) is widely distributed across the boreal and subboreal forests of North America, This rabbit population fluctuates dramatically in abundance, with peaks occurring every 9-10 years (Keith, 1963). Hares prefer habitats dominated by early and midsuccessional deciduous and coniferous trees and shrubs which provide both food and cover (Conroy et al., 1979; Wolff, 1980; Pietz and Tester, 1983; Sullivan and Sullivan, 1983). During peak years of the cycle, intense browsing may severely damage this natural food supply (Fox, 1978; Pease et al., 1979; Wolff, 1980). A consequence of this damage is the production of adventitious shoots which are very unpalatable to snowshoe hares (Bryant and Kuropat, 1980). These adventitious shoots of several common boreal forest trees contain significantly higher concentrations of terpene and phenolic resins than the mature growth form twigs of the same species (Bryant, 1981). In general, these resins are repellent to snowshoe hares and likely explain the avoidance of adventitious shoots by hares (Bryant, 1981). One such boreal tree species associated with snowshoe hares is Alaskan green alder (Alnus crispa). Hares will feed on intemodes but avoid juvenile growth forms of this species. This feeding preference is govemed by concentrations of two deterrent secondary metabolites, pinosylvin and pinosylvin methyl ether; laboratory bioassays have confirmed the repellency of these two compounds to hares (Bryant et al., 1983; Clausen et al., 1986). Feeding damage to coniferous plantations by the snowshoe hare (Lepus americanus) is a serious problem in many areas of forest regeneration in boreal and subboreal forests of North America (Radvanyi, 1987; Bergeron and Tardiff, 1988). Hares damage seedlings by clipping terminal and lateral shoots and by girdling stems. The most severe damage occurs in the 2-3 years during each peak in the 10-year cycle (Sullivan and Sullivan, 1982, 1988). Repellents have been advocated as a potential means to reduce this damage (Sullivan and Cramp, 1984; Radvanyi 1987). Some success has been achieved with predator odor repellents at the field level (Sullivan and Cramp, 1984, 1986), but there have been no investigations of plant antifeedants as repellents for tree seedlings and snowshoe hares. This paper reports on the influence of two plant antifeedants, pinosylvin and pinosylvin methyl ether, in suppressing snowshoe hare feeding on coniferous tree seedlings. The study was designed to test the hypothesis that these phenolic resins, when associated with tree seedlings, will elicit an "avoidance response" in terms of feeding by snowshoe hares.

| 153

PLANT ANTIFEEDANT

METHODS AND MATERIALS

Synthesis of Compounds. Pinosylvin (3,4-dihydroxystilbene) (5) (Figure 1) was prepared in the following manner: the bistetrahydropyranyl ether of ethyl 3,5-dihydroxy benzoate (1) was reduced with lithium aluminium hydride in diethyl ether to the benyl alcohol (2), which was subsequently oxidized with pyridinium chlorochromate in dichloromethane to the benzaldehyde derivative (3). IH NMR (CDC13, 60 MHz): 3.2-3.9 (2H, bm, Ht"~ and HI), 7.0 (H, bs, H4) , 7.3 (2H, bs, H2, H6), 9.9 (1H, S, CHO). Benzyl bromide (11 ml) and triethylphosphite (29 ml) were heated under reflux for 2 hr and then excess reagents were distilled out (70 ~ water pump vacuum). The crude diethylbenzyl phosphonate was added to sodium hydride (3.4 g) in dimethyl formamide at 0~ followed by the benzaldehyde derivative (3) (21.8 g). The mixture was stirred for 1 hr at 15~ heated at 100~ for 1 hr, and then stirred at 15~ overnight. Workup and purification by silica gel chromatography gave the protected stilbene (4), 17.6 g. Deprotection was effected by heating the crude product in aqueous methanoic oxalic acid on a steam bath for 2 hr. Neutralization with NaHCO 3 followed by ether extraction gave pinosylvin, 9.4 g; mp 55-57~ (lit. 55-56~ IH NMR (CDC13 60 MHz): 6.45 (1, m, H4), 6.90 (2, m, H 2, H6) , 7.10 (2, S, alkene), 7.10-7.50 (5, m, phenyl). Pinosylvin methyl ether (3-hydroxy-5-methoxystilbene) (6) was similarly prepared from ethyl 3-hydroxy-5-methoxybenzoate. IH NMR (CDC13, 90 MHz): 3.78 (3, s, OCH3), 6.35 (1, m, H4) , 6.40 (2, m, H 2, H6) , 6.67 (2, s, alkene), 7.24-7.44 (5, m, phenyl); M + 226. Purities of pinosylvin and PME were greater than 95 %. The vanillin PME analogue, 4-hydroxy-3-methoxystilbene (7), was similarly prepared from vanillin (4-hydroxy-3-methoxybenzaldehyde). IH NMR (CDC13, 60 MHz): 3.8 (3, s, OCH3) , 7.0 (2, bs, alkene), 6.8-7.6 (8, bm, aromatic); M + 226. Compounds and Preparations. With respect to compounds and preparations, pinosylvin was tested as a 20% solution in methanol at 50 mg/tree. Seedlings were dipped in the solution and allowed to dry prior to the start of a given trial. As a prelude to the testing of controlled-release devices, a 5-cmwide cloth band was impregnated with pinosylvin and attached around a 500ml plastic bowl containing commercial rabbit chow. This compound was also tested in a clay pellet (activated alumina, Catalog No. H026-00-050, Edwards High Vacuum, Oakville, Ontario, Canada) which acted as a controlled-release device, in Wheaton vials, and on paper sohxlet extraction thimbles. Four or five pellets (or an extraction thimble) were attached to each seedling in a collar of Scotch tape. Untreated pellets (or thimbles) were attached to control trees. A final screening bioassay assessed the influence of a pinosylvin methyl ether (7) analogue on feeding. Feeding by hares on seedlings or chow (amount eaten)

HO

THPO

(5)

(1)

~

OH

OTHP

t

(e)

(2)

CH3

OTHP THPO

(T)

~ ~'~OCH3 HO

(3)

(4)

~1~ ~~OTHP OTHP THPO

FIG. 1. Structural formulas of compounds.

HO

THPO

CH2OH

>Z >

PLANT ANTIFEEDANT

1155

was recorded daily in a given trial. Three bioassays were conducted with pinosylvin directly on trees, and two bioassays were done for each of the clay pellets, Wheaton vials, and thimbles. Screening Bioassays. Bioassays were conducted at the Applied Mammal Research Institute, Langley, B.C., Canada. The bioassay enclosure (9.15 • 18.3 rn) was composed of three sections or pens set in a natural environment (see Sullivan and Crump, 1984). The entire enclosure was covered by a fiberglass roof to eliminate effects of adverse weather. The control and experimental pens (A and B) were enclosed by polyethylene on the walls to keep odors within the respective treatment section. These pens had all natural vegetation cleared prior to start of trials. Douglas fir (Pseudotsuga menziesii) bark mulch covered the ground of each pen to a depth of 20 cm. Each pen had three experimental units, each composed of 10 Styrofoam blocks to anchor coniferous seedlings. One-year-old (nursery stock) lodgepole pine seedlings were used in all trials with one seedling per block. Thus, there was a maximum of 30 seedlings in each of the control and treatment pens (A or B) at the start of a given trial. The natural habitat (pen C) was not cleared of native vegetation and provided cover and some grasses and forbs for hares. Thus, this pen served as a potential refuge area for hares. Access of hares to pens A or B from pen C was through 30 • 30-cm openings in each adjoining wall. Hares moved freely among all three pens and new animals were always allowed at least 5 to 7 days to acclimate to this experimental configuration. To exert adequate feeding pressure on the experimental material and keep social interactions at a minimum, one or two hares were used in each trial. All hares used were from our northern study areas at Prince George, B.C. Hares were replaced at regular intervals to overcome potential variability and habituation among animals. Rabbit pellets, grass hay, and water were available ad libitum throughout these trials. Large Pen Bioassays. Two trials were conducted in a 4.5-ha enclosure at the Pineridge Forest Nursery, 130 km northeast of Edmonton, Alberta. The pen was composed of 2-m-high steel mesh and was buried in the ground to a depth of 30 cm. This study area was located in the boreal mixed wood zone (Strong and Leggat, 1981), with jack pine (Pinus banksiana) and aspen (Populus tremuloides) being the main tree species. Wild rose (Rosa spp.) and saskatoon berry (Amelanchieralnifolia) were the dominant shrub species. Within this area, four blocks (30 m • 30 m) were established and separated by at least 100 m. In July 1984, each block was cleared of trees and scarified in a manner similar to a typical logging and site preparation prescription. After planting, these blocks acted as plantations of lodgepole pine (P. contorta) seedlings in trials. Lodgepole pine is preferred by snowshoe hares over all other coniferous species (see Sullivan et al., 1985). Each block had 100 (10 • 10) seedlings for a given trial. The enclosure was stocked with snowshoe hares captured locally and from Prince George. This stocking allowed us to approach a peak population [4-5

1156

SULLIVAN ET AL.

hares/ha (Keith and Windberg, 1978)] of hares, thereby providing a rigorous test of various predator odor compounds in a simulated "field" situation. Both spring and fall represent periods when coniferous seedlings are particularly vulnerable to snowshoe hare feeding attacks. Snow cover is not adequate to protect seedlings and alternative summer herbaceous foods are not available. In the first trial, pinosylvin and pinosylvin methyl ether (PME) were each made into 20% solutions with methyl alcohol. Fifty seedlings were dipped in the pinosylvin solution and another 50 seedlings were dipped in the PME solution. The 100 seedlings were then planted in a random manner in the experimental block. Methanol was applied to 50 trees in a control block which also had a total of 100 seedlings. The trial commenced on October 9, 1985, with browsing of seedlings checked on October 18 and 29 and November 7 (snow had essentially covered the seedlings by this time). Feeding (clipping) on the terminal was considered as mortality to a given seedling. All trees were completely covered with snow by mid-November and remained so until the spring assessment on May 2, 1986. In the second trial, a solution of pinosylvin (5 g/37.7 ml methanol) was sprayed/dripped by syringe onto 50 (5 x 10) planted trees. A second set of 50 trees was treated with a similar solution of PME (5 g/37.7 ml methanol). The pinosylvin-treated trees were planted in one-half of the experimental block and the PME-treated trees in the other half. A second block acted as a control and had methanol only applied to 50 (5 x 10) of 100 trees. The trial began on May 2, 1986, and hare browsing of seedlings was checked on May 9, 22, and 28 and June 18. A common observation in the pen trials at Pineridge Nursery was the apparent preference by hares to feed on seedlings adjacent to the forest edge within each block. Therefore, separate analyses have been conducted which exclude one row of perimeter trees from each block in the overwinter and spring trials. These adjusted results are compared with the standard results for each trial. Field Bioassays. Two trials were conducted in central British Columbia during a snowshoe hare peak in population in 1988-1990. In the first trial, a plantation was located both at Shelley, 30 km northeast of Prince George, and near the Salmon River, approximately 60 km northwest of Prince George. Both sites were planted in the spring of 1988, interior spruce (Picea glauca x Picea engelmannii) at Shelley and lodgepole pine at Salmon River. In each plantation, all control and treatment blocks were located adjacent to areas with heavy vegetation cover within or on the perimeter of the plantations. Feeding damage by snowshoe hares tends to be most severe near such cover. Three blocks of 200 trees each were chosen at each plantation. A 20% solution of pinosylvin in methanol was sprayed directly on each of the 200 trees in one treatment block at each plantation. The same concentration of pinosylvin encapsulated in a

PLANT ANTIFEEDANT

1157

6-cm-long PVC plastic rope with internal copper wire as a twist-tie (PheroTech Inc., Delta, B.C., Canada) was attached to each tree in the second treatment block at each plantation. The third block acted as a control. Prior to application, 10% of the trees on each respective treatment and control block were flagged as sample trees. The applications were installed on October 30-31, 1988, and efficacy of treatments was evaluated on May 15-16, 1989. As for the large pen trials, feeding on the terminal was considered as mortality to a given seedling. In the second trial, three plantations were located near the Salmon River, and one large plantation was located 80 km east of Quesnel. Again, control and treatment blocks were established near heavy vegetation cover or on the perimeter of the plantations. At each study site, pinosylvin was dispensed via a spray application (50 mg/tree) directly onto trees (1-year-old lodgepole pine and interior spruce seedlings planted in spring 1989), in controlled-release devices (25 mg/tree) of PVC plastic with the intemal wire, plastic bubble caps, and C-flex rubber tubing. Each treatment was applied to a total of 400 trees with three replicate blocks at the Salmon River site and four replicates at the Quesnel site. Replicate control blocks completed the experimental design at each site. This trial was installed during October 15-30, 1989, and feeding damage to seedlings was assessed during May 21-31, 1990. Statistical Analysis. Comparison of the number of seedlings surviving in control and treatment blocks was analyzed by chi-square, with significance levels of P < 0.05 and P < 0.01.

RESULTS

Screening Bioassays. In the initial two bioassays, pinosylvin sprayed directly on trees reduced feeding by hares over a 2-day period with respect to percentage of seedlings surviving (trial 1--control, 0.0 and 0.0, treatment, 66.7 and 33.3; trial 2--control, 13.3 and 0.0, treatment, 90.0 and 90.0). A more intensive assessment was conducted in trial 3 (Figure 2). Pinosylvin clearly suppressed feeding by hares compared with the control block and declined in efficacy only after control trees stopped being replaced at day 8. The excellent results of trial 3 set the stage for testing controlled-release devices for pinosylvin. A contact repellent will not protect new tree growth and is not durable to prevailing weather conditions. Some type of encapsulation is necessary since an effective repellent should be present as an odor in the plantation environment but not necessarily attached to tree foliage. Results of four replicate trials assessing the influence of pinosylvin odor (soaked in cloth) on feeding by hares indicated a significant reduction in percentage of chow consumed after each 24-hr trial: (1) control, 39.0%, and treatment, 0.0%; (2) control, 54.3%, and treatment, 8.4%; (3) control, 68.2%, and treatment 7.2%;

1158

SULLIVAN ET AL.

Control O

Treatment (No. r e m e i n i n g 1 3 0 ) X 1 0 0

&

Treatment (No. remaining/No, remaining on previous d a y ) X 1 0 0 Control not replaced on previous day

100

t E

80

@

t 0

60 @ | O

~

4O

@ t

t~

20

0 1

2

3

4

5

6

7

8

9

10

Days

FIG. 2. Screening bioassay of the efficacy of pinosylvin in suppressing feeding on lodgepole pine seedlings by snowshoe hares.

(4) control, 46.0%, and treatment, 16.9%. The Wheaton vials and extraction thimbles attached to trees were not effective as controlled-release devices for pinosylvin. However, the clay pellets, when soaked in the pinosylvin for 24 hr prior to a trial, did significantly reduce feeding over a 2-day period with respect to the percentage of seedlings surviving (control, 10.0 and 0.0; treatment, 87.0 and 53.0). A lower concentration of pinosylvin in the pellets or fewer pellets per tree did not reduce feeding.

PLANT ANTIFEEDANT

i 159

A final screening bioassay on the pinosylvin methyl ether analogue (7) showed that this compound had no effect on feeding by hares with respect to the percentage of seedings surviving over a 2-day period: control, 50.0 and 3.3; treatment, 43.3 and 10.3. Large Pen Bioassays. Pinosylvin and PME significantly reduced hare feeding on pine seedlings throughout the fall trial of 1985 and, again, in the spring of 1986 (Figure 3A). There was no difference between pinosylvin and PME in reduction of feeding, and so these data were combined. In addition, survival of nonperimeter trees was significantly higher than that recorded when perimeter trees were included in the analysis. This trial was terminated on May 2. The spring 1986 trial with pinosylvin and PME, in separate halves of the block, also indicated that these compounds significantly reduced hare feeding (Figure 3B). These results provide temporal replication and, when combined with the screening bioassays, clearly show the impressive potential of pinosylvin as an antifeedant for snowshoe hares. Field Bioassays. In the first trial, pinosylvin sprayed directly on trees and as an antifeedant odor from a plastic controlled-release device significantly reduced feeding damage to lodgepole pine seedlings (Table 1). There was insufficient browsing on spruce seedlings but the tendency was for less feeding in the pinosylvin-treated blocks. In the second trial, one of three plantation replicates had sufficient feeding pressure to make comparisons among control and treatment blocks at the Salmon River site. The pinosylvin spray and PVC + wire and plastic bubble cap significantly reduced feeding compared with the control and C-flex tubing treatment blocks (Table 2). There was insufficient browsing on seedlings throughout all control and treatment blocks to make any statistical comparisons at the Quesnel site.

DISCUSSION This study has clearly demonstrated that the plant antifeedant, pinosylvin (and PME), will suppress feeding by snowshoe hares. This feeding suppression corroborates those results obtained by Bryant et al. (1983) and Clausen et al. (1986) for bioassays with pinosylvin and laboratory foods. Our study reports the first test and use of pinosylvin associated with tree seedlings as a plant antifeedant repellent. Therefore, we accept the hypothesis that pinosylvin will generate an "avoidance response" in terms of feeding by snowshoe hares. This response is likely triggered by an olfactory pathway based on our results with the controlled-release devices attached to seedlings. The odor of pinosylvin was clearly an effective means of discouraging hares from feeding. There are several studies of other plant antifeedants deterring snowshoe

1985

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May 22 1986

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81:181

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Oct. 29

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excluded

Perimeter

~

i May 2 1986

Control

.....

Treatment

FIG. 3. Efficacy of pinosylvin + pinosylvin methyl ether (PME) in suppressing feeding on lodgepole pine seedlings by snowshoe hares during: (A) fall (October-early November) and overwinter to the spring (early May); (B) spring (May-June). Analyses were conducted with and without one row of perimeter trees in each block. (*) P < 0.05; (**) P < 0.01; significant difference by chi-square.

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PLANTANTIFEEDANT

1161

TABLE 1. PERCENTAGESURVIVALOF TREE SEEDLINGSFROMFEEDINGDAMAGEBY SNOWSHOEHARESAT PRINCEGEORGESTUDYAREAS, OVERWINTER1988-1989~ Lodgepole pine

Release device Spray application

Spruce

Pinosylvin

Control

Pinosylvin

Control

100.0b (20) 100.~ (20)

25.0 (20) --

90.0 (20) 100.0 (20)

75.0 (20) --

"Sample size in parentheses. bp < 0.01; significantdifference by chi-square. TABLE2. EFFICACYOF PINOSYLVIN(PLANTANTIFEEDANT)IN TERMSOF PERCENTAGE SURVIVALOF TREE SEEDLINGSFROMFEEDINGDAMAGEBY SNOWSHOEHARESAT THE PRINCE GEORGE(SALMONRIVER) STUDYAREA, OVERWINTER1989-1990a

Plantation 1

2 3 Total

Control 91.1 (79)

NB 60.6 (94y'b" 74.6 (173)a~f

Spray

PVC + wire

Plastic bubble

C-flex tubing

91.5 (130) NB 86.5 (111)~g 89.2 (241)~j

92.1 (38) NB 84.9 (86)bh 87.1 (124)ek

91.9 (62) NB 97.6 (85)"~ 95.2 (147):

NB NB 68.7 (99)~hi 68.7 (99)jk/

aSample size in parentheses. Superscripts a-a, b-b, c-c, d-d, e-e, and f-f, P < 0.01; g-g, h-h, i-i, j-j, k-k, and 1-1, P < 0.05; significantdifference by chi-square. NB, no browsingby hares.

hares from feeding. These include papyriferic acid from Alaska paper birch (Betula resinifera) (Reichardt et al., 1984), phenolglycoside and catechin fractions of willow (Salix spp.) bark (Tahvanainen et al., 1985), camphor from white spruce (Sinclair et al., 1988), germacrone from labrador tea (Ledum groenlandicum) (Reichardt et al., 1990a), and several compounds from balsam poplar (Populus balsamifera) (Jogia et al., 1989; Reichardt et al., 1990b). An intriguing follow-up to these studies would be testing of the various antifeedants on tree seedlings to determine their repellency to snowshoe hares. In particular, camphor from juvenile white spruce suppresses feeding on rabbit chow by hares in a laboratory situation (Sinclair et al., 1988). Plantations of nursery-raised white and interior spruce seedlings on cutover forest land are fed on by snowshoe hares during peak populations. Therefore, a critical test for camphor as an antifeedant is to apply the compound to tree seedlings in a field situation as we have done in our study. Camphor is presumably at a low concentration in nursery-raised spruce, or other metabolic constituents may mask the antifeedant effect of this compound.

1162

SULLIVAN ET AL.

Similar feeding damage to tree seedlings is often caused by high populations of voles of the genus Microtus (Green, 1978; Roy and Bergeron, 1990a; Sullivan and Martin, 1991). Preference for certain coniferous species and rejection of others was related to concentration of phenolic compounds (Hansson et al., 1986; Roy and Bergeron, 1990a). In addition, voles will manipulate their food resource by cutting branches of young seedlings, and after 3 days, the cut branches have significantly less phenolics and condensed tannins (Roy and Bergeron, i990b). By this manipulation, a low-quality resource is transformed into one compatible with vole feeding patterns and detoxification mechanisms. The senior author has also observed this behavior in snowshoe hares, which may cut and leave seedlings in plantations. Thus, mammals are able to evaluate the palatability of potential foods on the basis of secondary metabolites. Pinosylvin clearly plays a role in this selection for snowshoe hares and, along with other plant antifeedant compounds, may regulate the 10-year cycle of abundance (Bryant and Kuropat, 1980; Bryant et al., 1983; Coley et al., 1985). The specificity of chemical structure of pinosylvin appears to be of critical importance to the snowshoe hare. Presumably pinosylvin and other repellent antifeedants have specific receptor sites within the olfactory system of the snowshoe hare and perhaps other small mammal herbivores. The ineffectiveness of the PME analogue (7) as an analogous compound to pinosylvin supports this specificity. This result is similar to that recorded for mustelid scent gland compounds and their analogues (Sullivan and Crump, 1984). The use of avoidance-inducing compounds as area repellents to reduce feeding damage to tree seedlings is clearly important to forest protection and management. A plant-derived repellent, pine oil, which is a mixture of terpene alcohols and monoterpenes has reduced feeding by snowshoe hares and voles (M. townsendii) when mixed with laboratory rations (Bell and Harestad, 1987). Similarly, red deer calves (Cervus elaphus) consumed less laboratory rations when this food was mixed with odors of crushed lodgepole pine and various monoterpenes (Elliot and Loudon, 1987). Again, as discussed previously, the critical test for plant antifeedants, as repellents to reduce feeding by herbivores, is application of the synthetic compounds to tree seedlings in field situations. Our study reports the first practical utilization of plant antifeedants for crop protection and wildlife management.

Acknowledgments--Wethank the Forest Research Branchof the Alberta Forest Service(AFS) and the ForestResourceDevelopmentAgreement(BritishColumbiaMinistryof Forests and Forestry Canada) for financialsupportand provisionof seedlings. We thank LakelandMills Ltd. for logistical support and A. Chadderton, V. Craig, N. Daintith, and C. Sutherland for field assistance. We are grateful to Pineridge Forest Nursery, AFS, for the enclosure facility and assistance of P. Au and B. Court. Special thanks to J. Soos, former director of the Research Branch, AFS, for his vision and support of this work. We thank S. Bloor, DSIR Chemistry, for preparing the PME analogue.

PLANT ANTIFEEDANT

1163 REFERENCES

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