Journal of Chemical Ecology, Vol. 12, No. 7, 1986
SUSCEPTIBILITY TO VOLE ATTACKS DUE TO BARK PHENOLS A N D TERPENES IN Pinus contorta PROVENANCES INTRODUCED INTO SWEDEN
LENNART
HANSSON, 1 ROLF GREF, 2 LENNART OLOF THEANDER 3
L U N D R E N , 3 and
~Department of Wildlife Ecology Swedish University of Agricultural Sciences S-750 07 Uppsala, Sweden 2Department of Forest Genetics and Plant Physiology Swedish University of Agricultural Sciences S-901 83 UmeiT, Sweden 3Department of Chemistry and Molecular Biology Swedish University of Agricultural Sciences S-750 07 Uppsala, Sweden (Received June 13, 1985; accepted October 29, 1985) Abstract--Seedlings of North American Pinus contorta introduced to Sweden and Finland are severely gnawed by voles, e.g., Microtus agrestis. The level of damage varies between provenances. Chemical analyses of various phenolic compounds, monoterpenes, and resin acids of different provenances and of damaged and undamaged stems showed that some phenolic substances in the bark increased after damage without deterring the animals, that monoterpene differences between provenances were not related to vole damage, and that certain resin acids occurred in larger amounts in lightly than in severely damaged provenances. Levopimaric and neoabietic acid, and possibly abietic acid, may be important for a partial resistance to vole browsing. Key Words--Pinus contorta, vole, damage, microtus agrestis, defense, phenols, monoterpenes, resin acids.
INTRODUCTION P i n u s contorta has b e e n i n t r o d u c e d in S w e d i s h forestry as a fast-growing pine species. H o w e v e r , it has suffered serious d a m a g e f r o m bark c o n s u m p t i o n by voles, chiefly M i c r o t u s agrestis ( H a n s s o n and L a v s u n d , 1982). Seeds or seedlings h a v e b e e n transferred f r o m several localities in western C a n a d a and north1569 0098-0331/86/0700-t569505.00/0 9 1986 Plenum Publishing Corporation
1570
HA~SSON ET AL.
western United States, constituting distinct provenances. These provenances have been tested for growth and survival in field trials on reforestation areas all over Sweden (Lindgren, 1983). Clear differences in bark attacks by voles have been found between the provenances (Hansson, 1983, 1985). Genetic differences in resistance to animal consumption are usually related to chemical defense systems, be they constant or induced (Haukioja, 1980), although mechanical defense or nutritional value may also vary. Two different chemical systems seem to be operating (cf. Rhoades and Cates, 1976), one with digestion-reducing substances (quantitative defense) and one with acutely toxic substances (qualitative defense). The former is assumed to be more important for long-lived and common plants such as pines and is suggested to be based on phenolic or terpenoid compounds, while the latter, based on, e.g., alkaloids, is known mainly in the case of rare or fugitive species. Phenolics have played an important role in the discussion of induced defense of deciduous trees and bushes (Bryant and Kuropat, 1980), while terpenes seem more important in conifers (e.g., Sturgeon, 1979, for bark beetles, and Radwan et al., 1982, regarding rodents). Therefore in the present study, bark of Pinus contorta was analyzed for various phenolic compounds, monoterpenes, and resin acids (diterpenes) in order to reveal any defense systems related to genotypes or provenances.
METHODS AND MATERIALS
Samples were taken from a field trial at Skalet, Str6msund (64~ established in 1971 (IUFRO 1970/71), in northern Sweden on September 23, 1983, after vole damage inflicted during the winters of 1980-1981 and 1981-1982. Frost and snow appear usually in early October in this area, where indigenous pines (Pinus silvestris) are in the winter physiological state by late September. One square decimeter of bark was cut at 0.5 m height, i.e., at places usually attacked by the voles, from six different provenances. Three of them had been damaged severely by voles in various trials in Sweden (the conditions at Str6msund, with very high level of damage, were not completely representative) and the other three only slightly (Hansson, 1983). From the severely damaged provenances, both damaged and undamaged stems were sampled, but only undamaged stems were taken from the slightly damaged provenances. It was planned to analyze four samples in each series, but in two of the severely damaged provenances only three samples of undamaged stems could be examined. With the material obtained, differences could be estimated between all the provenances, between severely and slightly damaged provenances, and between damaged and undamaged stems. All these examinations of differences in chemical contents were statistically tested with one-way analysis of variance. In this
VOLE A T T A C K S ON PINUS CONTORTA
1571
way provenance variation independent of vole resistance could be separated from true defense varying between provenances or individuals. An induced defense should be manifested in a higher level of defense chemicals in damaged plants. The bark was kept deep-frozen until chemical analyses were performed. The methods used for the various substances are described below. Phenolics. The bark, cut into small pieces, (5 g), was refluxed with 2 • 10 ml acetone for 2 x 30 rain. The extract was filtered and evaporated at reduced pressure, and the evaporated residue was fractionated between water (10 ml) and hexane (2 x 10 ml). Glycosides in the water phase were hydrolyzed with pectinase (Sigma, No. P-4625 from Aspergillus niger) in the dark at room temperature for 24 hr. The hydrolysate was acidified with one drop of concentrated hydrochloric acid and extracted with 3 • 10 ml ethyl acetate. The organic phase was dried with anhydrous sodium sulfate, filtered, and evaporated. The sample was dissolved in 10 ml methanol and analyzed with a Waters highperformance liquid chromatography system on a Radia-PAK C 18, 5/~m column (Waters). The mobile phase consisted of a linear gradient of water-methanolformic acid 80 : 20 : 1 to 30 : 70 : 1 for 50 min at a flow rate of 1.5 ml/min. UV absorbance at 280 nm was measured. Monoterpenes. These were analyzed in n-pentane extracts of fresh bark samples by gas chromatography (GC), and absolute amounts of monoterpenes were determined by reference to p-cymene (1-isopropyl-4-methyl-benzene), used as an internal standard. Pentane extracts were analyzed using GC on a 50m • 0.3-mm WCOT column with SP-1000 stationary phase inserted into a glass precolumn of 3% SP-1000 on Chromasorb W, an inlet splitter, flame ionization detector, and chromatographic data system. Operating conditions were: injector 200~ detector 230~ carrier gas N2 at 1.5 ml/min, split ratio 2 : 55, sample vol 1-2/~1, temperature programmed from 50 ~ to 110 ~ at 4~ then isothermally at 150~ Resin Acids. The bark samples were crushed in a mortar with liquid nitrogen and thereafter freeze-dried for 24 hr. A 100-rag sample of the freeze-dried bark meal was extracted with 2 ml of petroleum ether-diethyl ether (1 : 1) containing heptadecanoic acid as an internal standard in an ultrasonic bath for 2 hr. The sample was centrifuged and the extractive solution was transferred to a screw cap test tube. The residue was washed twice with 1 ml of diethyl ether, and the combined extracts were evaporated to dryness under a stream of nitrogen. The dried extract was redissolved in 1 ml of diethyl ether methanol (9:1) and methylated with freshly prepared diazomethane prior to the GC analysis. The samples were analyzed on a Varian 2700 GC equipped with a glass wall, coated capillary column with SE-30 as a stationary phase and equipped with a flame ionization detector (FID). The chromatograph was operated isothermally at 240~ Other operation parameters were as follows; injector and detector temperatures 280~ carrier gas hydrogen at 2.0 ml/min, and a split ratio of
1572
HANSSON ET AL.
1 : 50. Individual resin acids were identified by comparing their relative retention times to published data and with GC-mass spectrometry (Holmbom, 1977; Foster and Zinkel, 1982).
RESULTS
Vole Damage. During 1980-1982, 60% of the still-living P. contorla stems were damaged by voles (Table 1). Damaged appeared often repeatedly on the same stems during the two winters. Earlier, almost half the plants had died, many of them due to vole damage. Examinations of provenances with more than 12 still-living seedlings showed also in this trial significant differences between provenances as regards damage with complete stem girdling (X 2 = 40.00, df = 22, P < 0.05). This difference was still more pronounced in trials with a lower total frequency of vole damage (Hansson, 1983). Provenances with IUFRO Nos. 2003, 2019, and 2027 were used as examples of severely damaged provenances; and Nos. 2017, 2022, and 2034 as lightly damaged ones. Differences between these provenances were greater in other less damaged trials. Phenols. The types of phenolic compounds were generally identified with regard to the retention time in HPLC chromatograms (but see below for specific cases) and the amounts as the height (mm) of the peaks or, in the case of large amounts, in a relative sense as the integrated area below the peaks (Table 2). Analyses of variance showed significant differences for certain compounds in TABLE 1. VOLE DAMAGE AT SKALET PROVENANCE TRIAL ( I U F R O 70/71 SERIES), NEAR STROMSUND IN NORTHERN SWEDENa
Stems
Provenance (IUFRO-No.) 2003 2019 2027 2017 2022 2034 Remaining 77 provenances
Vole attacked 1980-1982
Undamaged 1982
Dead earlier
13 12 6 9 9 8
7 4 4 9 9 10
13 8 13 6 4 2
433
298
612
aSeedlings of 83 North American Pinus contorta provenances were planted as random single-tree plots.
1573
VOLE ATTACKS ON PINUS CONTORTA
TABLE 2. STATISTICAL ANALYSES OF HOMOGENEITY 1N PHENOLIC CONTENTS BETWEEN PROVENANCES AND STEMS a
Probability of homogeneitybetween Substances (retention time, rain) 3.40 5 5.20 6.89 8.07 9.27 9.62 9.93 l 1.61 12.52
Six provenances 0.18 0.15 0.02 0.18 0.88 0.95 0.02 0.02 0.05 0.31
Severely and lightly damaged provenances
Total damaged and undamaged stems
0.29 0.45 0.14 0.45 0.23 0.53 0.89 0.95 0.03 0.05
0.27 0.24 0.008 0.002 0.36 0.39 0.64 0.57 0.01 0.11
15.63
0.20
0.03
0.08
17.93 18.55 19.44 20.63 23.38 27.95 33.63
0.12 0.15 0.0001 0.33 0.20 0.35 0.15
0.08 0.04 0.01 0.19 0.74 0.14 0.08
0.86 0.01 0.006 0.31 0.35 0.89 0.30
"Significant deviations (P _< 0.05) are underlined. The various phenolic substances were distinguished by chromatographic retention times.
at least some of the combinations of damaged/undamaged seedlings and provenances. Peaks with retention times of 9.62 and 9.93 rain showed only differences between the six provenances and were thus not related to the vole damage. Peaks at 5.20, 6.89, 11.61, 18.55, and 19.44 showed a lower probability of being samples from the same population for separate plants than for severely and lightly damaged provenances and thus there was no evidence of a provenance effect in those cases. Only the differences for the peaks at 12.52 and 15.63 min were significant between severely and lightly damaged provenances, and there were no separate stem effects. However, these latter probabilities were just at the border of significance. The means amounts, as defined above, showed a consistently higher level among vole-damaged stems or provenances than among undamaged stems or lightly damaged provenances (Table 3). This demonstrates that either the voles were attracted by high contents of phenolics or that the vole damage had in-
h h a h h a a
5.20 6.89 11.61 12.52 15.63 18.55 19.44
25 18 445 41 38 264 904
-I- 6 + 8 • 174 • 20 + 24 _+ 110 • 118
Severely d a m a g e d provenances (N = 22) 21 16 322 28 21 172 745
_+ 8 • 6 +_ 115 + 16 • 8 • 92 • 238
Lightly damaged provenances (N = 12) 27 22 491 44 41 3t8 959
_+ 6 + 7 _+ 170 _+ 23 +_ 31 _+ 92 + 118
Damaged stems (N = 12)
2l 15 354 28 27 193 786
_+ 7 + 6 +_ 142 _+ 12 • 12 +_ 93 + 186
Undanmged stems (N = 22)
Between stems
"The values refer to either chromatographic peak height (h) or integrated areas under peaks (a) and are given as means + standard deviation.
Measurement
Substance (retention time, min)
Between provenances
TABLE 3. AMOUNTS OF VARIOUS PHENOLIC SUBSTANCES, DIFFERING BETWEEN PROVENANCES OR STEMS a
Z
9
Z
~Z ;>
,-..0
U.
VOLE ATTACKS ON PINUS CONTORTA
1575
duced an increase in the amounts of several phenolic compounds. This higher level in damaged stems was especially pronounced at the retention times of 19.44 (identified as trans-p-coumaric acid), 11.61, and 18.55 min (the flavonoid taxifolin). Monoterpenes. There were significant differences in the percentages of certain monoterpenes (retention times 14.14, 15.30, and 18.41 min) between the six provenances but not between severely and lightly damaged provenances nor between damaged and undamaged stems (Table 4). Thus, the provenance variations were not related to any resistance against vole attacks. Resin Acids. Significant differences appeared between severely and lightly damaged provenances as regards certain resin acids (levopimaric and neoabietic acids, and less clearly so for abietic acid) and for total resin acids (Table 5). No differences appeared when only undamaged and damaged stems were compared, so individual differences were smaller than those between provenances. The amounts of the resin acids mentioned were consistently higher in the slightly damaged provenances (Table 6), which might be of importance in browsing repellence.
TABLE 4. STATISTICAL ANALYSES OF HOMOGENEITY IN MONOTERPENE CONTENTS BETWEEN PROVENANCES AND STEMS a
Probability of homogeneity between Substances (retention time, min)
Six provenances
Severely and lightly damaged provenances
Total damaged and undamaged stems
7.86 10.08 11.23 12.41 12.70 12.93 13.19 13.39 13.61 13.81 14.14 15.30 15.71 18.41
0.58 0.25 0.50 0.28 0.10 0.28 0.47 0.38 0.58 0.50 0.04 0.03 0.28 0.04
0.28 0.55 0.38 0.38 0.27 0.10 0.21 0.11 0.23 0.09 0.66 0.66 0.44 0.31
0.53 0.95 0.36 0.61 0.80 0.82 0.79 0.79 0.74 0.75 0.72 0.58 0.40 0.90
Significant deviations (P
SUSCEPTIBILITY TO VOLE ATTACKS DUE TO BARK PHENOLS A N D TERPENES IN Pinus contorta PROVENANCES INTRODUCED INTO SWEDEN
LENNART
HANSSON, 1 ROLF GREF, 2 LENNART OLOF THEANDER 3
L U N D R E N , 3 and
~Department of Wildlife Ecology Swedish University of Agricultural Sciences S-750 07 Uppsala, Sweden 2Department of Forest Genetics and Plant Physiology Swedish University of Agricultural Sciences S-901 83 UmeiT, Sweden 3Department of Chemistry and Molecular Biology Swedish University of Agricultural Sciences S-750 07 Uppsala, Sweden (Received June 13, 1985; accepted October 29, 1985) Abstract--Seedlings of North American Pinus contorta introduced to Sweden and Finland are severely gnawed by voles, e.g., Microtus agrestis. The level of damage varies between provenances. Chemical analyses of various phenolic compounds, monoterpenes, and resin acids of different provenances and of damaged and undamaged stems showed that some phenolic substances in the bark increased after damage without deterring the animals, that monoterpene differences between provenances were not related to vole damage, and that certain resin acids occurred in larger amounts in lightly than in severely damaged provenances. Levopimaric and neoabietic acid, and possibly abietic acid, may be important for a partial resistance to vole browsing. Key Words--Pinus contorta, vole, damage, microtus agrestis, defense, phenols, monoterpenes, resin acids.
INTRODUCTION P i n u s contorta has b e e n i n t r o d u c e d in S w e d i s h forestry as a fast-growing pine species. H o w e v e r , it has suffered serious d a m a g e f r o m bark c o n s u m p t i o n by voles, chiefly M i c r o t u s agrestis ( H a n s s o n and L a v s u n d , 1982). Seeds or seedlings h a v e b e e n transferred f r o m several localities in western C a n a d a and north1569 0098-0331/86/0700-t569505.00/0 9 1986 Plenum Publishing Corporation
1570
HA~SSON ET AL.
western United States, constituting distinct provenances. These provenances have been tested for growth and survival in field trials on reforestation areas all over Sweden (Lindgren, 1983). Clear differences in bark attacks by voles have been found between the provenances (Hansson, 1983, 1985). Genetic differences in resistance to animal consumption are usually related to chemical defense systems, be they constant or induced (Haukioja, 1980), although mechanical defense or nutritional value may also vary. Two different chemical systems seem to be operating (cf. Rhoades and Cates, 1976), one with digestion-reducing substances (quantitative defense) and one with acutely toxic substances (qualitative defense). The former is assumed to be more important for long-lived and common plants such as pines and is suggested to be based on phenolic or terpenoid compounds, while the latter, based on, e.g., alkaloids, is known mainly in the case of rare or fugitive species. Phenolics have played an important role in the discussion of induced defense of deciduous trees and bushes (Bryant and Kuropat, 1980), while terpenes seem more important in conifers (e.g., Sturgeon, 1979, for bark beetles, and Radwan et al., 1982, regarding rodents). Therefore in the present study, bark of Pinus contorta was analyzed for various phenolic compounds, monoterpenes, and resin acids (diterpenes) in order to reveal any defense systems related to genotypes or provenances.
METHODS AND MATERIALS
Samples were taken from a field trial at Skalet, Str6msund (64~ established in 1971 (IUFRO 1970/71), in northern Sweden on September 23, 1983, after vole damage inflicted during the winters of 1980-1981 and 1981-1982. Frost and snow appear usually in early October in this area, where indigenous pines (Pinus silvestris) are in the winter physiological state by late September. One square decimeter of bark was cut at 0.5 m height, i.e., at places usually attacked by the voles, from six different provenances. Three of them had been damaged severely by voles in various trials in Sweden (the conditions at Str6msund, with very high level of damage, were not completely representative) and the other three only slightly (Hansson, 1983). From the severely damaged provenances, both damaged and undamaged stems were sampled, but only undamaged stems were taken from the slightly damaged provenances. It was planned to analyze four samples in each series, but in two of the severely damaged provenances only three samples of undamaged stems could be examined. With the material obtained, differences could be estimated between all the provenances, between severely and slightly damaged provenances, and between damaged and undamaged stems. All these examinations of differences in chemical contents were statistically tested with one-way analysis of variance. In this
VOLE A T T A C K S ON PINUS CONTORTA
1571
way provenance variation independent of vole resistance could be separated from true defense varying between provenances or individuals. An induced defense should be manifested in a higher level of defense chemicals in damaged plants. The bark was kept deep-frozen until chemical analyses were performed. The methods used for the various substances are described below. Phenolics. The bark, cut into small pieces, (5 g), was refluxed with 2 • 10 ml acetone for 2 x 30 rain. The extract was filtered and evaporated at reduced pressure, and the evaporated residue was fractionated between water (10 ml) and hexane (2 x 10 ml). Glycosides in the water phase were hydrolyzed with pectinase (Sigma, No. P-4625 from Aspergillus niger) in the dark at room temperature for 24 hr. The hydrolysate was acidified with one drop of concentrated hydrochloric acid and extracted with 3 • 10 ml ethyl acetate. The organic phase was dried with anhydrous sodium sulfate, filtered, and evaporated. The sample was dissolved in 10 ml methanol and analyzed with a Waters highperformance liquid chromatography system on a Radia-PAK C 18, 5/~m column (Waters). The mobile phase consisted of a linear gradient of water-methanolformic acid 80 : 20 : 1 to 30 : 70 : 1 for 50 min at a flow rate of 1.5 ml/min. UV absorbance at 280 nm was measured. Monoterpenes. These were analyzed in n-pentane extracts of fresh bark samples by gas chromatography (GC), and absolute amounts of monoterpenes were determined by reference to p-cymene (1-isopropyl-4-methyl-benzene), used as an internal standard. Pentane extracts were analyzed using GC on a 50m • 0.3-mm WCOT column with SP-1000 stationary phase inserted into a glass precolumn of 3% SP-1000 on Chromasorb W, an inlet splitter, flame ionization detector, and chromatographic data system. Operating conditions were: injector 200~ detector 230~ carrier gas N2 at 1.5 ml/min, split ratio 2 : 55, sample vol 1-2/~1, temperature programmed from 50 ~ to 110 ~ at 4~ then isothermally at 150~ Resin Acids. The bark samples were crushed in a mortar with liquid nitrogen and thereafter freeze-dried for 24 hr. A 100-rag sample of the freeze-dried bark meal was extracted with 2 ml of petroleum ether-diethyl ether (1 : 1) containing heptadecanoic acid as an internal standard in an ultrasonic bath for 2 hr. The sample was centrifuged and the extractive solution was transferred to a screw cap test tube. The residue was washed twice with 1 ml of diethyl ether, and the combined extracts were evaporated to dryness under a stream of nitrogen. The dried extract was redissolved in 1 ml of diethyl ether methanol (9:1) and methylated with freshly prepared diazomethane prior to the GC analysis. The samples were analyzed on a Varian 2700 GC equipped with a glass wall, coated capillary column with SE-30 as a stationary phase and equipped with a flame ionization detector (FID). The chromatograph was operated isothermally at 240~ Other operation parameters were as follows; injector and detector temperatures 280~ carrier gas hydrogen at 2.0 ml/min, and a split ratio of
1572
HANSSON ET AL.
1 : 50. Individual resin acids were identified by comparing their relative retention times to published data and with GC-mass spectrometry (Holmbom, 1977; Foster and Zinkel, 1982).
RESULTS
Vole Damage. During 1980-1982, 60% of the still-living P. contorla stems were damaged by voles (Table 1). Damaged appeared often repeatedly on the same stems during the two winters. Earlier, almost half the plants had died, many of them due to vole damage. Examinations of provenances with more than 12 still-living seedlings showed also in this trial significant differences between provenances as regards damage with complete stem girdling (X 2 = 40.00, df = 22, P < 0.05). This difference was still more pronounced in trials with a lower total frequency of vole damage (Hansson, 1983). Provenances with IUFRO Nos. 2003, 2019, and 2027 were used as examples of severely damaged provenances; and Nos. 2017, 2022, and 2034 as lightly damaged ones. Differences between these provenances were greater in other less damaged trials. Phenols. The types of phenolic compounds were generally identified with regard to the retention time in HPLC chromatograms (but see below for specific cases) and the amounts as the height (mm) of the peaks or, in the case of large amounts, in a relative sense as the integrated area below the peaks (Table 2). Analyses of variance showed significant differences for certain compounds in TABLE 1. VOLE DAMAGE AT SKALET PROVENANCE TRIAL ( I U F R O 70/71 SERIES), NEAR STROMSUND IN NORTHERN SWEDENa
Stems
Provenance (IUFRO-No.) 2003 2019 2027 2017 2022 2034 Remaining 77 provenances
Vole attacked 1980-1982
Undamaged 1982
Dead earlier
13 12 6 9 9 8
7 4 4 9 9 10
13 8 13 6 4 2
433
298
612
aSeedlings of 83 North American Pinus contorta provenances were planted as random single-tree plots.
1573
VOLE ATTACKS ON PINUS CONTORTA
TABLE 2. STATISTICAL ANALYSES OF HOMOGENEITY 1N PHENOLIC CONTENTS BETWEEN PROVENANCES AND STEMS a
Probability of homogeneitybetween Substances (retention time, rain) 3.40 5 5.20 6.89 8.07 9.27 9.62 9.93 l 1.61 12.52
Six provenances 0.18 0.15 0.02 0.18 0.88 0.95 0.02 0.02 0.05 0.31
Severely and lightly damaged provenances
Total damaged and undamaged stems
0.29 0.45 0.14 0.45 0.23 0.53 0.89 0.95 0.03 0.05
0.27 0.24 0.008 0.002 0.36 0.39 0.64 0.57 0.01 0.11
15.63
0.20
0.03
0.08
17.93 18.55 19.44 20.63 23.38 27.95 33.63
0.12 0.15 0.0001 0.33 0.20 0.35 0.15
0.08 0.04 0.01 0.19 0.74 0.14 0.08
0.86 0.01 0.006 0.31 0.35 0.89 0.30
"Significant deviations (P _< 0.05) are underlined. The various phenolic substances were distinguished by chromatographic retention times.
at least some of the combinations of damaged/undamaged seedlings and provenances. Peaks with retention times of 9.62 and 9.93 rain showed only differences between the six provenances and were thus not related to the vole damage. Peaks at 5.20, 6.89, 11.61, 18.55, and 19.44 showed a lower probability of being samples from the same population for separate plants than for severely and lightly damaged provenances and thus there was no evidence of a provenance effect in those cases. Only the differences for the peaks at 12.52 and 15.63 min were significant between severely and lightly damaged provenances, and there were no separate stem effects. However, these latter probabilities were just at the border of significance. The means amounts, as defined above, showed a consistently higher level among vole-damaged stems or provenances than among undamaged stems or lightly damaged provenances (Table 3). This demonstrates that either the voles were attracted by high contents of phenolics or that the vole damage had in-
h h a h h a a
5.20 6.89 11.61 12.52 15.63 18.55 19.44
25 18 445 41 38 264 904
-I- 6 + 8 • 174 • 20 + 24 _+ 110 • 118
Severely d a m a g e d provenances (N = 22) 21 16 322 28 21 172 745
_+ 8 • 6 +_ 115 + 16 • 8 • 92 • 238
Lightly damaged provenances (N = 12) 27 22 491 44 41 3t8 959
_+ 6 + 7 _+ 170 _+ 23 +_ 31 _+ 92 + 118
Damaged stems (N = 12)
2l 15 354 28 27 193 786
_+ 7 + 6 +_ 142 _+ 12 • 12 +_ 93 + 186
Undanmged stems (N = 22)
Between stems
"The values refer to either chromatographic peak height (h) or integrated areas under peaks (a) and are given as means + standard deviation.
Measurement
Substance (retention time, min)
Between provenances
TABLE 3. AMOUNTS OF VARIOUS PHENOLIC SUBSTANCES, DIFFERING BETWEEN PROVENANCES OR STEMS a
Z
9
Z
~Z ;>
,-..0
U.
VOLE ATTACKS ON PINUS CONTORTA
1575
duced an increase in the amounts of several phenolic compounds. This higher level in damaged stems was especially pronounced at the retention times of 19.44 (identified as trans-p-coumaric acid), 11.61, and 18.55 min (the flavonoid taxifolin). Monoterpenes. There were significant differences in the percentages of certain monoterpenes (retention times 14.14, 15.30, and 18.41 min) between the six provenances but not between severely and lightly damaged provenances nor between damaged and undamaged stems (Table 4). Thus, the provenance variations were not related to any resistance against vole attacks. Resin Acids. Significant differences appeared between severely and lightly damaged provenances as regards certain resin acids (levopimaric and neoabietic acids, and less clearly so for abietic acid) and for total resin acids (Table 5). No differences appeared when only undamaged and damaged stems were compared, so individual differences were smaller than those between provenances. The amounts of the resin acids mentioned were consistently higher in the slightly damaged provenances (Table 6), which might be of importance in browsing repellence.
TABLE 4. STATISTICAL ANALYSES OF HOMOGENEITY IN MONOTERPENE CONTENTS BETWEEN PROVENANCES AND STEMS a
Probability of homogeneity between Substances (retention time, min)
Six provenances
Severely and lightly damaged provenances
Total damaged and undamaged stems
7.86 10.08 11.23 12.41 12.70 12.93 13.19 13.39 13.61 13.81 14.14 15.30 15.71 18.41
0.58 0.25 0.50 0.28 0.10 0.28 0.47 0.38 0.58 0.50 0.04 0.03 0.28 0.04
0.28 0.55 0.38 0.38 0.27 0.10 0.21 0.11 0.23 0.09 0.66 0.66 0.44 0.31
0.53 0.95 0.36 0.61 0.80 0.82 0.79 0.79 0.74 0.75 0.72 0.58 0.40 0.90
Significant deviations (P
