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Picein and piceol concentrations in Norway spruce
ECOTOXICOLOGY
AND
ENVIRONMENTAL
SAFETY
19,30 1-309 (1990)
Picein and Piceol Concentrations
in Norway Spruce’
HANS LSKKE Laboratory of Environmental Sciences and Ecology, Technical University of Denmark, Building 224. DK-2800 Lyngby, Denmark Received January 19, I989 The concentrations of the glucoside picein and its aglucone piceol (Chydroxy acetophenone) in the needles of Norway spruce (Picea abies (L.) Karsten) are considered indicators of plant stress. By use of two clones and a natural population of Norway spruce it was shown that the picein concentration was dependent on the site and the provenance of the trees. No effectswere observed by normal treatment with the herbicides atrazine, glyphosate, and hexazinone, or by drought. The significance of different environmental factors is discussed. o 1990 Academic Press, Inc.
INTRODUCTION Stress has been defined as a physical or chemical factor that tends to alter an existing equilibrium and may be a factor in disease causation (Mish, 1984). The content of picein which is produced in the new needles of P. abies during the summer (Dittrich and Kandler, 197 1) and its aglucone 4-hydroxy acetophenone (piceol) (Fig. 1) have been proposed as stress indicators by Hoque (1984a, 1984b, 1985, 1986). Picein is a secondary metabolite which is nonessential to life although it may be important to the organism that produces it. Secondary metabolites have a restricted distribution, being found mostly in plants and microorganisms, and they are often characteristic of individual genera, species, or strains (Herbert, 198 1). Picein may be hydrolyzed to piceol by ,&glucosidase. The aglucone piceol inhibites the vegetative growth of spruce (Schindlebeck, 1978; Hoque, 1984b) and is phytotoxic at 500 mg liter-‘, causing yellowing and browning of needles, needle-fall, inhibition of apical dominance of epicotyl and root, and inhibition of bud-break (Hoque, 1985). It is suggested that piceol can promote the oxidation of indole-3-acetic acid (IAA) in the presence of peroxidase. Thus, the growth inhibitory activity of piceol in Norway spruce may depend partly on its ability to oxidize IAA (Hoque, 1989). Homans and Fuchs (1970) showed that piceol is fungitoxic for Rhizosphaera kalkhofii and Cladosporium cucumerinum.
On the basis of his work, Hoque suggested that the piceol/picein ratio may be used to identify affected trees having ratios > 1, while healthy trees show ratios < 1. As shown in Fig. 2 this theory is in accordance with studies presented in the literature. In a previous study the influence of selected stress factors were studied on P. abies in the laboratory (Jensen and Lskke, 1990). Minor although significant differences in the concentration of picein in the needles were caused by drought, by soil acidification, and by acid rain at pH 3.8 in combination with soil acidification, respectively, ’ Presented at the 1st European Conference on Ecotoxicology, October I7- 19, 1988, Copenhagen, 301
0147-6513190 $3.00 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
302
HANS LBKKE
OH
HO
Plceol
Picein
FIG. 1. Structures of piceol(4-hydroxyacetophenone)
and picein (P-D-glucopyrane-4-oxyacetophenone).
during 2 months of exposure. At 11 kg ha-’ the herbicide atrazine had no effect on the picein or piceol content in the needles. However, fumigation with ozone at 90 ppb from 10 am to 6 pm in open-top chambers resulted in significant reductions
Picea 10.00
1
abies,
Affected
i/ “r* / / / /
literature trees
/
/
data / '
/ /
Healthy
trees q
A
/
i
/
Picein
mg
g-l
FIG. 2. Concentrations of piceol versus concentrations ofpicein given in milligrams per gram. Data from the literature: (*) Hoque (1984a, 1985), affected trees; (0) Hoque (1984a, 1985), healthy trees; (A) Hoque (1986), healthy trees; (17) Esterbauer et al. (1975), healthy trees; (X) Schliemann (1986), healthy trees.
PICEIN AND PICEOL IN NORWAY
303
SPRUCE
TABLE 1 EXPERIMENTALSITES,SOILTYPES,PROVENANCE, ANDAGEOFP. abies Location
Provenance
Langess, Isleof Funen Egekrogen, Northern Zealand Str0dam,” Northern Zealand Copenhagen,” Zealand Klosterhede, Western Jutland
ClonesVS 146and V5189 Clone V5 I89
Age
Soil
(w-4 10
Treatment
Loamy, neutral Reference
7
Loamy, neutral Glyphosateand hexazinone, top shootcutting
CloneVS 189
7
Sandy,acidic
Clone VS 146
7
Foreststand
70
Loamy, neutral Ambient city air, top shoot cutting Sandy,acidic Fertilizer, neutral rain, controls
Atrazine, drought, control, top shootcutting
aPottedtrees.
(P < 0.0004) of the picein concentration compared to that in trees exposed to ambient concentrations of ozone (40 ppb) or to carbon-filtered air (30 ppb ozone). Ozone had no significant effect on the piceol concentration. However, Osswald et al. (1986) found reductions of piceol following ozone fumigation. As a preliminary conclusion of the previous study (Jensen and Lokke, 1990) the ratio of the piceol and picein concentrations in the needles was not suited as an independent indicator of stress in P. abies due to the variation caused by the provenance and the interaction of several environmental factors. In the present study P. abies trees from two clones used in previous laboratory experiments were exposed in the field and compared with trees of the same clones growing in the field on different locations. Further, a natural population of P. abies was studied which is growing under stress conditions. MATERIALS
AND
METHODS
Plant Material The studies were conducted using samples of needles from clones of Norway spruce (Picea abies (L.) Karsten) which were prepared from second-generation cuttings of Roumanian provenance (V5 146) and of Western Continental (V5 189), respectively. The cuttings were rooted in the spring of 1979 or of 1982. Further, a 70-year-old forest stand of P. abies was studied at Klosterhede, Western Jutland. Treatments The study included five sites and treatments with herbicides and drought as shown in Table 1. It was assumed that climatic differences were of minor significance. Trees of the clones V5 146 and V5 189 growing in Langess Plantation, Isle of Funen, were chosen as references. The trees were rooted in 1979 and planted in loamy soil in the
304
HANS LBKKE
spring of 1982. The trees were not treated with pesticides and they were in a healthy condition. The effect of normal treatment with herbicides was studied on trees of the clone V5 189 which were planted for vegetative formation in Egekrogen, Northern Zealand, by the Danish State Forestry Tree Improvement Station. The top shoots were cut and the trees were treated with Round-up (glyphosate, 1.08 kg ha-‘) in the beginning of July 1987 and 1988. In the 1988 treatment Velpar was also included (hexazinone, 0.55 kg ha-‘). The lower parts of the trees were damaged by the herbicides. Trees from the 1982 clone V5 189 were potted in a nutrient-poor and acidic forest soil (pH 4.2) from the Strsdam Reserve, Gribskov Forest, Northern Zealand. The potted trees were used during 1987 for laboratory investigations on the effect of atrazine and drought on the needle concentrations of piceol and picein (Jensen and Lskke, 1990). The surviving experimental trees were placed in the pots in Strodam Reserve in April 1988 to study the same treatments under natural climatic conditions. The experimental trees included four control trees, three trees provided with cuffs of PTFE to simulate drought, and three trees which were treated with atrazine at 11 kg ha-’ in 1987 in the laboratory. The atrazine treatment was repeated on May 30, 1988, in the field. Distilled water was supplied to the pots during dry periods. The effect of ambient air in the city of Copenhagen was tested on two individuals from the clone V5 146. The trees had been used for nonlethal laboratory experiments and exposed to acid rain during 1985. Thereafter the trees were planted in the spring of 1986 in a shady Copenhagen garden about 50 m upwind from a motor road with an average traffic volume of 65.000 cars day-‘. The variation within a forest stand was studied in Klosterhede Plantation, Western Jutland. The soil is a sandy and acidic heath plain soil poor in calcium. As a part of an ongoing research project on atmospheric inputs and soil chemical processes the soil of three plots of about 400 m2 were covered with a roof and irrigated. A control plot was placed outside the roof. The roof was mounted in November 1987. The plots under the roof receive collected through-fall water, artificial rainwater without pollutants, and a nutrient solution, respectively. The supply of nutrients started in April, 1988. Sampling One-year-old needles from the clone trees were sampled from August 24 to September 8, 1988. Additionally, needles from the current year and 2-year-old needles were sampled from the clone V5 146. One-year-old needles were sampled at 6-9 m above the ground at the Klosterhede Plantation on June 7, 1988. The sampling was performed on the southern side of the trees. At the Copenhagen site additional samples were taken on the northern side of the trees. All samples were frozen with dry ice and kept at - 18°C until analysis. Analytical
Techniques
Analytical standards of picein and piceol were of the same purity as those used in a previous study (Jensen and Lokke, 1990). The needles were removed carefully from the shoots and 150 mg were treated in an Ultra-Turrax blender (T 25, Janke and Kunkel Ltd., GFR) with 2 ml distilled water followed by 2 X 5 ml ethanol + water (4 + 1) and by 3 X 5 ml water. The extracts were vacuum-filtrated using Hyflo Super
PICEIN AND PICEOL IN NORWAY TABLE
305
SPRUCE
2
MEAN CONCENTRATIONS OF PICEIN AND PICEOL IN THE NEEDLES OF P. abies (CLONE VS 146) FROM A COPENHAGEN GARDEN AND FROM THE ISLE OF FUNEN No. of trees
Needle position
Needle age
Picein
Piceol
Copenhagen@
2 2 2
South -
0 1 2
0.76 (0.45) 3.7 (3.6) 4.6 (5.0)
0.025 (0.004) 0.042 (0.03 1) 0.044 (0.040)
Copenhagen”
2 2
North -
0
-
:
0.76 (0.04) 2.4 (1.9) 4.4 (5.3)
0.030 (0.028) 0.008 (0.004) 0.04 1 (0.042)
Location
n
L
Langes0 (Isle of Funen)b
3 3 3
South
0 1 2
-
6.9 (0.4) 10.0 (1.1) 10.4 (2.0)
0.012 (0.016) 0.25 (0.13) 0.72 (0.10)
Note. The concentrations are given in mg g-’ for 0-, I-, and 2-year-old needles. SD in parentheses. ’ Rooted in 1982. ’ Rooted in 1979.
Cel (USA type) as a filter aid. Picein and piceol were analyzed by HPLC using a C- 18 column and UV detection at 270 nm. As mobile phases for gradient elution ethanol + water (1 + 9) and (4 + I), respectively, were used. The data were acquired and processed by a Waters 745 data module. The mean recovery for analysis of plant material was 96% (range 74- 116%) for piceol and 10 1% (range 70- 119%) for picein. RESULTS The Roumanian
AND
DISCUSSION
Clone V5146
In Table 2 the results from the study of the Roumanian clone V5 146 are shown. The concentrations of picein increased with the needle age. This finding is in accordance with the results of Esterbauer et al. (1975) and Hoque (1984a, 1984b, 1986). At the Copenhagen site the concentration of picein was higher on the southern side of the trees than on the northern side. However, only two trees formerly used for laboratory experiments were included and large variation was observed between the trees. Significantly higher concentrations of picein and of piceol were found in samples from Langess, Isle of Funen, compared to those found at the Copenhagen site. On both sites the piceol/picein ratio was ~0.1, which might indicate a low stress level (Hoque, 1985). However, a lower concentration of picein might as a single stress variable reflect stress conditions at the Copenhagen site. The Western Continental
Clone V5189
The concentrations of picein and piceol in needles from Lange@ Egekrogen, and the laboratory trees potted in the Strodam Reserve are shown in Table 3. The content of picein was significantly lower in the needles from the potted trees in Strodam compared to that from those from Langese and Egekrogen. By comparison of the trees
306
HANS LBKKE TABLE
3
MEAN CONCENTRATIONS OF PICEIN AND PICEOL IN 1-YEAR-OLD NEEDLES OF P. abies (CLONE V5 189) GIVEN AS MILLIGRAMS PER GRAM Site
No. of trees
Picein
Piceol
Str0dam, control StrQdam, atrazine Str0dam, drought Egekrogen LangesO
5 3 3 6 3
8.4 (1.4) 6.0(1.8) 7.9 (2.0) 14.3(1.5) 15.7 (2.1)
0.02 1 (0.005) 0.019 (0.004) 0.030 (0.024) 0.027 (0.004) 0.049 (0.038)
Note. Experimental trees potted in the Str&am Reserve, Northern Zealand, are compared with trees growing in Egekrogen, Northern Zealand, and in Langess Plantation, Isle of Funen. SD in parentheses. The trees from Str6dam and Egekrogen were rooted in 1982 and the trees from Langes@ were rooted in 1979.
from Egekrogen and Langess the needle concentrations of picein and piceol, respectively, showed no significant differences. This indicates that the climatic conditions, the edaphic factors, the different ages, as well as the cutting of the top shoots and the herbicide treatment in Egekrogen, had no apparent influence on the picein or the piceol concentrations. The experimental trees which were potted in the Strsdam Reserve showed chlorotic bands on the needles. Further, most trees had needles with necrotic tips. Despite the visible stress symptoms only very low concentrations of piceol were found. This is in accordance with earlier findings by analysis of the same needles (Jensen and Lokke, 1990), but not in agreement with the results of Hoque ( 1985), who found high levels of piceol in highly affected trees which, however, were insufficiently described as to growing conditions, provenance, etc., to justify a full comparison. The atrazine-treated trees showed a significantly lower concentration level than that found in the control group. However, this difference appeared already at the beginning of the previous study on October 1987 (Jensen and Lokke, 1990). Compared with earlier findings on November 27, 1987, the picein concentration in the needles had increased by 34% in control trees, 37% in atrazine-treated trees, and 54% in trees exposed to drought. The increase in picein concentration with the needle age is consistent with the study of the Roumanian clone (Table 2) and the findings of Esterbauer et al. ( 1975) and Hoque ( 1984a, 1984b, 1986). In the previous laboratory study a significant reduction ( 17%) of the picein concentration was observed in trees which received the drought treatment. However, in 1987 the trees did not receive rain in the laboratory while they were exposed to natural precipitation in the field, allowing for some water uptake through the needles. This may have reduced the effect of the drought treatment in the field. In general, significantly higher concentrations were found in the Western Continental clone V5 189 compared to those in the Roumanian clone V5 146. The Natural Population
at Klosterhede
The concentrations of piceol in the needles from Klosterhede are plotted versus the concentrations of picein in Fig. 3. Differences were found between the plots as shown in Table 4. By Duncan’s multiple range test no significant differences were
PICEIN AND PICEOL IN NORWAY
Picea d
abies,
1.000
0.100
/, / J /
i
/
0.010
0.001
Klosterhede
Affected
10.0001
/
/
a-, 0.01
/'
Y
/
Healthy
Station
trees
/ /
Y
trees
0.10
*
A #
1.00
100.00
10.00 Picein
Previous Sampling
307
SPRUCE
mg
g-l
year-needles
on June 7th.
1989
FIG. 3. Concentrations of piceol versus concentrations of picein in l-year-old needles of I? abies from a 70-year-old natural population in Klosterhede Plantation. The results are given in milligrams per gram. (0) Control; ( * ) irrigated with deionized water; (Cl) irrigated with collected through-fall water; (A) fertilized.
found between the plots for picein and those for the sum of piceol and picein expressed as piceol (P = 0.05). Significantly higher concentrations of piceol were found for the control plot than for the fertilized plot. However, Osswald et al. (1986) maintain that fertilizing with Mg and Ca has a positive effect on the content of piceol of spruce needles. The interpretation of the present results requires prolonged investigations. The relatively low concentration of picein might reflect that the trees are growing under stress conditions, i.e., a combination of acidic soil, nutrient deficiency, acid rain, impact of the relatively high ozone concentration level in rural areas, impact of airborne NaCl from the North Sea (280 kg ha-’ year-‘), and relatively low water potential. However, large individual variation was observed within the genetic pool which suggests the presence of adaptive properties to the acidic and nutrient-poor soil in the Klosterhede Plantation. The results are consistent with the results obtained from a field study of 29-yearold P. abies conducted in the Gribskov Forest, Northern Zealand, where the trees are growing under similar stress conditions (Jensen and Lokke, 1990). In that study the mean concentration of picein was 1.24 mg g-’ (range 0.62-2.05 mg g-‘) and of piceol 0.95 mg g-l (range 0.47-l .74 mg g-l). CONCLUSIONS On the basis of the present knowledge, the concentrations of picein and of piceol may be used as indicators for stress in P. abies. However, changes in the concentra-
308
HANS
LBKKE
TABLE 4 MEANVALUES ANDRANGE(INBRACKETS)OF CONCENTRATIONS OFPICEINANDPICEOLIN THENEEDLES OF P.abies FROMA NATURALPOPULATION, KLOSTERHEDEPLANTATION, WITHTHE SOIL COVEREDWITHAROOF
Treatment
No. of trees
Control (no roof)
7
Irrigation, rain water
8
Irrigation, neutral Fertilized Note. The concentrations (P > 0.05). * Expressed as piceol.
10 9
Picein 2.60” (0.1 l-7.29) 3.43”
Piceol 2.49’ ( 13y
(1.36-6.07)
(“.0lO;%493)
3.87” (0.62-10.8) 4.64” (0.9 1-9.28)
(0.050-2.53) 1.14’ (0.010-3.16)
are given in mg g-l. Means
Sumof piceol and picein* 3.62d (1.80-5.06) 3.27d (2.22-5.97) 2.96d (1.04-7.20) 3.14d (1.53-4.49)
with the same letter are not significantly
different
tions of picein and of piceol are not causedby all stressfactors and negligible hydroly-
sis of picein to piceol may be observed in trees showing visible damage. The concentrations of picein and piceol in P. abies are highly dependent on the provenance and the ozone level. Apparently, the following single-impact variables are of minor importance: -herbicide treatment with atrazine, glyphosate, or hexazinone -drought -mechanical damage, e.g., cutting of top shoots -soil acidification -acid rain (direct effect). Probably the temperature, the light quality, and the dose are of importance for the concentration level of picein and piceol. These variables should be included in future studies. Nutrient deficiency, infection with fungi and insects, atmospheric deposition of NaCl, and genetic variations with respect to adaptive properties should be investigated further as well as the influence of the age of the trees and the annual endogene variations. ACKNOWLEDGMENTS It is acknowledged that the Danish Natural Science Research Council has funded the HPLC equipment, and thanks are due to Mr. Jakob Maag for valuable technical assistance, to MSChE Jan Steen Jensen, Institute of Organic Chemistry, Technical University of Denmark, for synthesis of picein, and to Dr. Hans Rovlund, The Arboretum of The Danish Royal Veterinary and Agricultural University, for supplying the cloned plant material.
REFERENCES DITTRICH, P., AND K~~NDLER, pem der Fichte (Picea dies
0. (197 1). Einfluss der Jahreszeit auf Bildung [L.] Karst.). Ber. Dfsch. Bot. Ges. 84,465-472.
und Umzats
von PhenolkBr-
PICEIN AND PICEOL IN NORWAY
SPRUCE
309
ESTERBAUER, H., GRILL, D., AND BECK, G. (1974). Spectrophotometric determination of picein and phydroxyacetophenone in needles of Picea abies with 2,4-dinitrophenylhydrazine. Anal. Chem. 46,789791. ESTERBAUER, H., GRILL, D., AND BECK, G. (1975). Untersuchungen iiber Phenole in Nadeln von Picea abies. Phyton (Horn, Austria) 17,87-99. HERBERT, R. B. (I 98 1). The Biosynthesis of Secondary Metabolites. Chapman and Hall, London. HOMANS, D. L., AND FUCHS, S. (1970). Direct bioautrography on thin layer chromatograms as a method for detecting fungitoxic substances. J. Chromatogr. S&327-329. HOQUE, E. (1984a). Norway spruce die-back: Isolation, biological activity, measurement of concentration ofphydroxy acetophenone and its O-ghtcoside (Picein) by gas chromatography. Eur. J. For. Pathol. 14, 377-382.
HOQUE, E. (1984b). Spruce die-back: Isolation of phydroxyacetophenone from diseased shoots of Picea abies. Phytochemistry 23,923-925. HOQUE, E. (1985). Norway spruce die-back: Occurrence, isolation, biological activity of phydroxy acetophenone and phydroxy acetophenone-O-glucoside and their possible roles during stress phenomena. Eur. J. For. Pathol. 15, 129-145. HOQUE, E. (1986). High-performance liquid chromatographic analysis of phydroxyacetophenone and phydroxyacetophenone-P-D-glucopyranoside, two major phenolic compounds in Norway spruce. J. Chromatogr. 360,452-458. HOQUE, E. (1989). Effects of phydroxyacetophenone and its ghrcoside on the enzymatic oxidation of indole-3-acetic acid. Agric. Biol. Chem. 53,239-240. JENSEN,J. S., AND LBKKE, H. ( 1990). 4-Hydroxyacetophenone and its ghtcoside picein as chemical indicators for stressin Pieea abies. Z. Pflanzenkrankh. Pflanzenschutz. 97(3), in press. J~~TTNER,F. (1987). Quantitative Analyse der Terpene und anderer VOC in Fichtennadels von natiirlichen Standorten und aus unterschiedhch begasten (03/S02) Open-Top-Kammer Bestlnden. 3. Statuskolloquium des PEF vom 10. bis 12. MHrz 1987 in Karlsruhe (F. Horsch, W. G. Filby, N. Fund, S. Gross, B. Hanisch, E. Kilz, and A. Seidel, Eds.), pp. 149-I 57. Projekt Europlisches Forschungszentrum fur Massnahmen zur Luftreinhaltung (PEF). Kernforschungszentrum Karlsruhe, GFR. MISH, F. C., Ed. ( 1984). Webster’s Ninth New Collegiate Dictionary. Merriam-Webster, Springfield, MA. OSSWALD, W. F., HEINISCH, H., AND ELSTNER, E. F. (1986). Einfluss von Mineralstoffernahrung, Ozon und saurem Nebel auf den Gehalt der fungitoxischen Substanz p-Hydroxyacetophenon in Fichtennadeln (Picea abies (L.) Karst.). Forstwiss. Centralbl. 105,26 1-264. SCHINDLBECK, E. (1978). Identifizierung eines Wachstuminhibitors (4-Hydroxy-Acetophenonrest) aus mlnnlichen Fichtenbhiten (Picea abies (L.) Karst.). Silvae Genet. 27(l), 42-47. SCHLIEMANN, W. (1986). p-Hydroxyacetophenone and its fl-D-Glucopyranoside from shoots of Picea abies. Pharmazie41. 138-l 39.
AND
ENVIRONMENTAL
SAFETY
19,30 1-309 (1990)
Picein and Piceol Concentrations
in Norway Spruce’
HANS LSKKE Laboratory of Environmental Sciences and Ecology, Technical University of Denmark, Building 224. DK-2800 Lyngby, Denmark Received January 19, I989 The concentrations of the glucoside picein and its aglucone piceol (Chydroxy acetophenone) in the needles of Norway spruce (Picea abies (L.) Karsten) are considered indicators of plant stress. By use of two clones and a natural population of Norway spruce it was shown that the picein concentration was dependent on the site and the provenance of the trees. No effectswere observed by normal treatment with the herbicides atrazine, glyphosate, and hexazinone, or by drought. The significance of different environmental factors is discussed. o 1990 Academic Press, Inc.
INTRODUCTION Stress has been defined as a physical or chemical factor that tends to alter an existing equilibrium and may be a factor in disease causation (Mish, 1984). The content of picein which is produced in the new needles of P. abies during the summer (Dittrich and Kandler, 197 1) and its aglucone 4-hydroxy acetophenone (piceol) (Fig. 1) have been proposed as stress indicators by Hoque (1984a, 1984b, 1985, 1986). Picein is a secondary metabolite which is nonessential to life although it may be important to the organism that produces it. Secondary metabolites have a restricted distribution, being found mostly in plants and microorganisms, and they are often characteristic of individual genera, species, or strains (Herbert, 198 1). Picein may be hydrolyzed to piceol by ,&glucosidase. The aglucone piceol inhibites the vegetative growth of spruce (Schindlebeck, 1978; Hoque, 1984b) and is phytotoxic at 500 mg liter-‘, causing yellowing and browning of needles, needle-fall, inhibition of apical dominance of epicotyl and root, and inhibition of bud-break (Hoque, 1985). It is suggested that piceol can promote the oxidation of indole-3-acetic acid (IAA) in the presence of peroxidase. Thus, the growth inhibitory activity of piceol in Norway spruce may depend partly on its ability to oxidize IAA (Hoque, 1989). Homans and Fuchs (1970) showed that piceol is fungitoxic for Rhizosphaera kalkhofii and Cladosporium cucumerinum.
On the basis of his work, Hoque suggested that the piceol/picein ratio may be used to identify affected trees having ratios > 1, while healthy trees show ratios < 1. As shown in Fig. 2 this theory is in accordance with studies presented in the literature. In a previous study the influence of selected stress factors were studied on P. abies in the laboratory (Jensen and Lskke, 1990). Minor although significant differences in the concentration of picein in the needles were caused by drought, by soil acidification, and by acid rain at pH 3.8 in combination with soil acidification, respectively, ’ Presented at the 1st European Conference on Ecotoxicology, October I7- 19, 1988, Copenhagen, 301
0147-6513190 $3.00 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
302
HANS LBKKE
OH
HO
Plceol
Picein
FIG. 1. Structures of piceol(4-hydroxyacetophenone)
and picein (P-D-glucopyrane-4-oxyacetophenone).
during 2 months of exposure. At 11 kg ha-’ the herbicide atrazine had no effect on the picein or piceol content in the needles. However, fumigation with ozone at 90 ppb from 10 am to 6 pm in open-top chambers resulted in significant reductions
Picea 10.00
1
abies,
Affected
i/ “r* / / / /
literature trees
/
/
data / '
/ /
Healthy
trees q
A
/
i
/
Picein
mg
g-l
FIG. 2. Concentrations of piceol versus concentrations ofpicein given in milligrams per gram. Data from the literature: (*) Hoque (1984a, 1985), affected trees; (0) Hoque (1984a, 1985), healthy trees; (A) Hoque (1986), healthy trees; (17) Esterbauer et al. (1975), healthy trees; (X) Schliemann (1986), healthy trees.
PICEIN AND PICEOL IN NORWAY
303
SPRUCE
TABLE 1 EXPERIMENTALSITES,SOILTYPES,PROVENANCE, ANDAGEOFP. abies Location
Provenance
Langess, Isleof Funen Egekrogen, Northern Zealand Str0dam,” Northern Zealand Copenhagen,” Zealand Klosterhede, Western Jutland
ClonesVS 146and V5189 Clone V5 I89
Age
Soil
(w-4 10
Treatment
Loamy, neutral Reference
7
Loamy, neutral Glyphosateand hexazinone, top shootcutting
CloneVS 189
7
Sandy,acidic
Clone VS 146
7
Foreststand
70
Loamy, neutral Ambient city air, top shoot cutting Sandy,acidic Fertilizer, neutral rain, controls
Atrazine, drought, control, top shootcutting
aPottedtrees.
(P < 0.0004) of the picein concentration compared to that in trees exposed to ambient concentrations of ozone (40 ppb) or to carbon-filtered air (30 ppb ozone). Ozone had no significant effect on the piceol concentration. However, Osswald et al. (1986) found reductions of piceol following ozone fumigation. As a preliminary conclusion of the previous study (Jensen and Lokke, 1990) the ratio of the piceol and picein concentrations in the needles was not suited as an independent indicator of stress in P. abies due to the variation caused by the provenance and the interaction of several environmental factors. In the present study P. abies trees from two clones used in previous laboratory experiments were exposed in the field and compared with trees of the same clones growing in the field on different locations. Further, a natural population of P. abies was studied which is growing under stress conditions. MATERIALS
AND
METHODS
Plant Material The studies were conducted using samples of needles from clones of Norway spruce (Picea abies (L.) Karsten) which were prepared from second-generation cuttings of Roumanian provenance (V5 146) and of Western Continental (V5 189), respectively. The cuttings were rooted in the spring of 1979 or of 1982. Further, a 70-year-old forest stand of P. abies was studied at Klosterhede, Western Jutland. Treatments The study included five sites and treatments with herbicides and drought as shown in Table 1. It was assumed that climatic differences were of minor significance. Trees of the clones V5 146 and V5 189 growing in Langess Plantation, Isle of Funen, were chosen as references. The trees were rooted in 1979 and planted in loamy soil in the
304
HANS LBKKE
spring of 1982. The trees were not treated with pesticides and they were in a healthy condition. The effect of normal treatment with herbicides was studied on trees of the clone V5 189 which were planted for vegetative formation in Egekrogen, Northern Zealand, by the Danish State Forestry Tree Improvement Station. The top shoots were cut and the trees were treated with Round-up (glyphosate, 1.08 kg ha-‘) in the beginning of July 1987 and 1988. In the 1988 treatment Velpar was also included (hexazinone, 0.55 kg ha-‘). The lower parts of the trees were damaged by the herbicides. Trees from the 1982 clone V5 189 were potted in a nutrient-poor and acidic forest soil (pH 4.2) from the Strsdam Reserve, Gribskov Forest, Northern Zealand. The potted trees were used during 1987 for laboratory investigations on the effect of atrazine and drought on the needle concentrations of piceol and picein (Jensen and Lskke, 1990). The surviving experimental trees were placed in the pots in Strodam Reserve in April 1988 to study the same treatments under natural climatic conditions. The experimental trees included four control trees, three trees provided with cuffs of PTFE to simulate drought, and three trees which were treated with atrazine at 11 kg ha-’ in 1987 in the laboratory. The atrazine treatment was repeated on May 30, 1988, in the field. Distilled water was supplied to the pots during dry periods. The effect of ambient air in the city of Copenhagen was tested on two individuals from the clone V5 146. The trees had been used for nonlethal laboratory experiments and exposed to acid rain during 1985. Thereafter the trees were planted in the spring of 1986 in a shady Copenhagen garden about 50 m upwind from a motor road with an average traffic volume of 65.000 cars day-‘. The variation within a forest stand was studied in Klosterhede Plantation, Western Jutland. The soil is a sandy and acidic heath plain soil poor in calcium. As a part of an ongoing research project on atmospheric inputs and soil chemical processes the soil of three plots of about 400 m2 were covered with a roof and irrigated. A control plot was placed outside the roof. The roof was mounted in November 1987. The plots under the roof receive collected through-fall water, artificial rainwater without pollutants, and a nutrient solution, respectively. The supply of nutrients started in April, 1988. Sampling One-year-old needles from the clone trees were sampled from August 24 to September 8, 1988. Additionally, needles from the current year and 2-year-old needles were sampled from the clone V5 146. One-year-old needles were sampled at 6-9 m above the ground at the Klosterhede Plantation on June 7, 1988. The sampling was performed on the southern side of the trees. At the Copenhagen site additional samples were taken on the northern side of the trees. All samples were frozen with dry ice and kept at - 18°C until analysis. Analytical
Techniques
Analytical standards of picein and piceol were of the same purity as those used in a previous study (Jensen and Lokke, 1990). The needles were removed carefully from the shoots and 150 mg were treated in an Ultra-Turrax blender (T 25, Janke and Kunkel Ltd., GFR) with 2 ml distilled water followed by 2 X 5 ml ethanol + water (4 + 1) and by 3 X 5 ml water. The extracts were vacuum-filtrated using Hyflo Super
PICEIN AND PICEOL IN NORWAY TABLE
305
SPRUCE
2
MEAN CONCENTRATIONS OF PICEIN AND PICEOL IN THE NEEDLES OF P. abies (CLONE VS 146) FROM A COPENHAGEN GARDEN AND FROM THE ISLE OF FUNEN No. of trees
Needle position
Needle age
Picein
Piceol
Copenhagen@
2 2 2
South -
0 1 2
0.76 (0.45) 3.7 (3.6) 4.6 (5.0)
0.025 (0.004) 0.042 (0.03 1) 0.044 (0.040)
Copenhagen”
2 2
North -
0
-
:
0.76 (0.04) 2.4 (1.9) 4.4 (5.3)
0.030 (0.028) 0.008 (0.004) 0.04 1 (0.042)
Location
n
L
Langes0 (Isle of Funen)b
3 3 3
South
0 1 2
-
6.9 (0.4) 10.0 (1.1) 10.4 (2.0)
0.012 (0.016) 0.25 (0.13) 0.72 (0.10)
Note. The concentrations are given in mg g-’ for 0-, I-, and 2-year-old needles. SD in parentheses. ’ Rooted in 1982. ’ Rooted in 1979.
Cel (USA type) as a filter aid. Picein and piceol were analyzed by HPLC using a C- 18 column and UV detection at 270 nm. As mobile phases for gradient elution ethanol + water (1 + 9) and (4 + I), respectively, were used. The data were acquired and processed by a Waters 745 data module. The mean recovery for analysis of plant material was 96% (range 74- 116%) for piceol and 10 1% (range 70- 119%) for picein. RESULTS The Roumanian
AND
DISCUSSION
Clone V5146
In Table 2 the results from the study of the Roumanian clone V5 146 are shown. The concentrations of picein increased with the needle age. This finding is in accordance with the results of Esterbauer et al. (1975) and Hoque (1984a, 1984b, 1986). At the Copenhagen site the concentration of picein was higher on the southern side of the trees than on the northern side. However, only two trees formerly used for laboratory experiments were included and large variation was observed between the trees. Significantly higher concentrations of picein and of piceol were found in samples from Langess, Isle of Funen, compared to those found at the Copenhagen site. On both sites the piceol/picein ratio was ~0.1, which might indicate a low stress level (Hoque, 1985). However, a lower concentration of picein might as a single stress variable reflect stress conditions at the Copenhagen site. The Western Continental
Clone V5189
The concentrations of picein and piceol in needles from Lange@ Egekrogen, and the laboratory trees potted in the Strodam Reserve are shown in Table 3. The content of picein was significantly lower in the needles from the potted trees in Strodam compared to that from those from Langese and Egekrogen. By comparison of the trees
306
HANS LBKKE TABLE
3
MEAN CONCENTRATIONS OF PICEIN AND PICEOL IN 1-YEAR-OLD NEEDLES OF P. abies (CLONE V5 189) GIVEN AS MILLIGRAMS PER GRAM Site
No. of trees
Picein
Piceol
Str0dam, control StrQdam, atrazine Str0dam, drought Egekrogen LangesO
5 3 3 6 3
8.4 (1.4) 6.0(1.8) 7.9 (2.0) 14.3(1.5) 15.7 (2.1)
0.02 1 (0.005) 0.019 (0.004) 0.030 (0.024) 0.027 (0.004) 0.049 (0.038)
Note. Experimental trees potted in the Str&am Reserve, Northern Zealand, are compared with trees growing in Egekrogen, Northern Zealand, and in Langess Plantation, Isle of Funen. SD in parentheses. The trees from Str6dam and Egekrogen were rooted in 1982 and the trees from Langes@ were rooted in 1979.
from Egekrogen and Langess the needle concentrations of picein and piceol, respectively, showed no significant differences. This indicates that the climatic conditions, the edaphic factors, the different ages, as well as the cutting of the top shoots and the herbicide treatment in Egekrogen, had no apparent influence on the picein or the piceol concentrations. The experimental trees which were potted in the Strsdam Reserve showed chlorotic bands on the needles. Further, most trees had needles with necrotic tips. Despite the visible stress symptoms only very low concentrations of piceol were found. This is in accordance with earlier findings by analysis of the same needles (Jensen and Lokke, 1990), but not in agreement with the results of Hoque ( 1985), who found high levels of piceol in highly affected trees which, however, were insufficiently described as to growing conditions, provenance, etc., to justify a full comparison. The atrazine-treated trees showed a significantly lower concentration level than that found in the control group. However, this difference appeared already at the beginning of the previous study on October 1987 (Jensen and Lokke, 1990). Compared with earlier findings on November 27, 1987, the picein concentration in the needles had increased by 34% in control trees, 37% in atrazine-treated trees, and 54% in trees exposed to drought. The increase in picein concentration with the needle age is consistent with the study of the Roumanian clone (Table 2) and the findings of Esterbauer et al. ( 1975) and Hoque ( 1984a, 1984b, 1986). In the previous laboratory study a significant reduction ( 17%) of the picein concentration was observed in trees which received the drought treatment. However, in 1987 the trees did not receive rain in the laboratory while they were exposed to natural precipitation in the field, allowing for some water uptake through the needles. This may have reduced the effect of the drought treatment in the field. In general, significantly higher concentrations were found in the Western Continental clone V5 189 compared to those in the Roumanian clone V5 146. The Natural Population
at Klosterhede
The concentrations of piceol in the needles from Klosterhede are plotted versus the concentrations of picein in Fig. 3. Differences were found between the plots as shown in Table 4. By Duncan’s multiple range test no significant differences were
PICEIN AND PICEOL IN NORWAY
Picea d
abies,
1.000
0.100
/, / J /
i
/
0.010
0.001
Klosterhede
Affected
10.0001
/
/
a-, 0.01
/'
Y
/
Healthy
Station
trees
/ /
Y
trees
0.10
*
A #
1.00
100.00
10.00 Picein
Previous Sampling
307
SPRUCE
mg
g-l
year-needles
on June 7th.
1989
FIG. 3. Concentrations of piceol versus concentrations of picein in l-year-old needles of I? abies from a 70-year-old natural population in Klosterhede Plantation. The results are given in milligrams per gram. (0) Control; ( * ) irrigated with deionized water; (Cl) irrigated with collected through-fall water; (A) fertilized.
found between the plots for picein and those for the sum of piceol and picein expressed as piceol (P = 0.05). Significantly higher concentrations of piceol were found for the control plot than for the fertilized plot. However, Osswald et al. (1986) maintain that fertilizing with Mg and Ca has a positive effect on the content of piceol of spruce needles. The interpretation of the present results requires prolonged investigations. The relatively low concentration of picein might reflect that the trees are growing under stress conditions, i.e., a combination of acidic soil, nutrient deficiency, acid rain, impact of the relatively high ozone concentration level in rural areas, impact of airborne NaCl from the North Sea (280 kg ha-’ year-‘), and relatively low water potential. However, large individual variation was observed within the genetic pool which suggests the presence of adaptive properties to the acidic and nutrient-poor soil in the Klosterhede Plantation. The results are consistent with the results obtained from a field study of 29-yearold P. abies conducted in the Gribskov Forest, Northern Zealand, where the trees are growing under similar stress conditions (Jensen and Lokke, 1990). In that study the mean concentration of picein was 1.24 mg g-’ (range 0.62-2.05 mg g-‘) and of piceol 0.95 mg g-l (range 0.47-l .74 mg g-l). CONCLUSIONS On the basis of the present knowledge, the concentrations of picein and of piceol may be used as indicators for stress in P. abies. However, changes in the concentra-
308
HANS
LBKKE
TABLE 4 MEANVALUES ANDRANGE(INBRACKETS)OF CONCENTRATIONS OFPICEINANDPICEOLIN THENEEDLES OF P.abies FROMA NATURALPOPULATION, KLOSTERHEDEPLANTATION, WITHTHE SOIL COVEREDWITHAROOF
Treatment
No. of trees
Control (no roof)
7
Irrigation, rain water
8
Irrigation, neutral Fertilized Note. The concentrations (P > 0.05). * Expressed as piceol.
10 9
Picein 2.60” (0.1 l-7.29) 3.43”
Piceol 2.49’ ( 13y
(1.36-6.07)
(“.0lO;%493)
3.87” (0.62-10.8) 4.64” (0.9 1-9.28)
(0.050-2.53) 1.14’ (0.010-3.16)
are given in mg g-l. Means
Sumof piceol and picein* 3.62d (1.80-5.06) 3.27d (2.22-5.97) 2.96d (1.04-7.20) 3.14d (1.53-4.49)
with the same letter are not significantly
different
tions of picein and of piceol are not causedby all stressfactors and negligible hydroly-
sis of picein to piceol may be observed in trees showing visible damage. The concentrations of picein and piceol in P. abies are highly dependent on the provenance and the ozone level. Apparently, the following single-impact variables are of minor importance: -herbicide treatment with atrazine, glyphosate, or hexazinone -drought -mechanical damage, e.g., cutting of top shoots -soil acidification -acid rain (direct effect). Probably the temperature, the light quality, and the dose are of importance for the concentration level of picein and piceol. These variables should be included in future studies. Nutrient deficiency, infection with fungi and insects, atmospheric deposition of NaCl, and genetic variations with respect to adaptive properties should be investigated further as well as the influence of the age of the trees and the annual endogene variations. ACKNOWLEDGMENTS It is acknowledged that the Danish Natural Science Research Council has funded the HPLC equipment, and thanks are due to Mr. Jakob Maag for valuable technical assistance, to MSChE Jan Steen Jensen, Institute of Organic Chemistry, Technical University of Denmark, for synthesis of picein, and to Dr. Hans Rovlund, The Arboretum of The Danish Royal Veterinary and Agricultural University, for supplying the cloned plant material.
REFERENCES DITTRICH, P., AND K~~NDLER, pem der Fichte (Picea dies
0. (197 1). Einfluss der Jahreszeit auf Bildung [L.] Karst.). Ber. Dfsch. Bot. Ges. 84,465-472.
und Umzats
von PhenolkBr-
PICEIN AND PICEOL IN NORWAY
SPRUCE
309
ESTERBAUER, H., GRILL, D., AND BECK, G. (1974). Spectrophotometric determination of picein and phydroxyacetophenone in needles of Picea abies with 2,4-dinitrophenylhydrazine. Anal. Chem. 46,789791. ESTERBAUER, H., GRILL, D., AND BECK, G. (1975). Untersuchungen iiber Phenole in Nadeln von Picea abies. Phyton (Horn, Austria) 17,87-99. HERBERT, R. B. (I 98 1). The Biosynthesis of Secondary Metabolites. Chapman and Hall, London. HOMANS, D. L., AND FUCHS, S. (1970). Direct bioautrography on thin layer chromatograms as a method for detecting fungitoxic substances. J. Chromatogr. S&327-329. HOQUE, E. (1984a). Norway spruce die-back: Isolation, biological activity, measurement of concentration ofphydroxy acetophenone and its O-ghtcoside (Picein) by gas chromatography. Eur. J. For. Pathol. 14, 377-382.
HOQUE, E. (1984b). Spruce die-back: Isolation of phydroxyacetophenone from diseased shoots of Picea abies. Phytochemistry 23,923-925. HOQUE, E. (1985). Norway spruce die-back: Occurrence, isolation, biological activity of phydroxy acetophenone and phydroxy acetophenone-O-glucoside and their possible roles during stress phenomena. Eur. J. For. Pathol. 15, 129-145. HOQUE, E. (1986). High-performance liquid chromatographic analysis of phydroxyacetophenone and phydroxyacetophenone-P-D-glucopyranoside, two major phenolic compounds in Norway spruce. J. Chromatogr. 360,452-458. HOQUE, E. (1989). Effects of phydroxyacetophenone and its ghrcoside on the enzymatic oxidation of indole-3-acetic acid. Agric. Biol. Chem. 53,239-240. JENSEN,J. S., AND LBKKE, H. ( 1990). 4-Hydroxyacetophenone and its ghtcoside picein as chemical indicators for stressin Pieea abies. Z. Pflanzenkrankh. Pflanzenschutz. 97(3), in press. J~~TTNER,F. (1987). Quantitative Analyse der Terpene und anderer VOC in Fichtennadels von natiirlichen Standorten und aus unterschiedhch begasten (03/S02) Open-Top-Kammer Bestlnden. 3. Statuskolloquium des PEF vom 10. bis 12. MHrz 1987 in Karlsruhe (F. Horsch, W. G. Filby, N. Fund, S. Gross, B. Hanisch, E. Kilz, and A. Seidel, Eds.), pp. 149-I 57. Projekt Europlisches Forschungszentrum fur Massnahmen zur Luftreinhaltung (PEF). Kernforschungszentrum Karlsruhe, GFR. MISH, F. C., Ed. ( 1984). Webster’s Ninth New Collegiate Dictionary. Merriam-Webster, Springfield, MA. OSSWALD, W. F., HEINISCH, H., AND ELSTNER, E. F. (1986). Einfluss von Mineralstoffernahrung, Ozon und saurem Nebel auf den Gehalt der fungitoxischen Substanz p-Hydroxyacetophenon in Fichtennadeln (Picea abies (L.) Karst.). Forstwiss. Centralbl. 105,26 1-264. SCHINDLBECK, E. (1978). Identifizierung eines Wachstuminhibitors (4-Hydroxy-Acetophenonrest) aus mlnnlichen Fichtenbhiten (Picea abies (L.) Karst.). Silvae Genet. 27(l), 42-47. SCHLIEMANN, W. (1986). p-Hydroxyacetophenone and its fl-D-Glucopyranoside from shoots of Picea abies. Pharmazie41. 138-l 39.