Journal of Chemical Ecology, Vol. 22, No. 4, 1996
EFFECTS OF NITROGEN FERTILIZATION ON SECONDARY CHEMISTRY A N D ECTOMYCORRHIZAL STATE OF SCOTS PINE SEEDLINGS A N D ON GROWTH OF GREY PINE APHID
PIRJO KAINULAINEN,*
JARMO
HOLOPAINEN,
VIRPI PALOM,~KI,
and T O I N I H O L O P A I N E N
Department of Ecology and Environmental Science, University of Kuopio, P.O. Box 162 7, FTN-70211 Kuopio, Finland (Received April 10, 1995; accepted November 20, 1995)
Abstract--Effects of nitrogen availability on secondary compounds, mycorrhizal infection, and aphid growth of l-year-old Scots pine (Pinus sylvestris L.) seedlings were studied during one growing season. Seedlings were fertilized with nutrient solutions containing low, optimum, and two elevated (2 x and 4 x optimum) levels of NH4NO~. At the end of growing season foliar nitrogen concentration, needle biomass, needle length, water contents of needles, root collar diameter, and number of buds increased with enhanced nitrogen availability. Addition of nitrogen did not have effect on concentrations of monoterpenes in growing needles~ but in mature needles significantly decreased concentrations of some individual and total monoterpenes were detected. In growing needles the concentrations of some individual resin acids decreased, and in mature needles concentrations of some individual and total resin acids increased with increased nitrogen fertilization. Higher numbers of resin ducts were found in mature needles with nitrogen fertilization. Nitrogen fertilization decreased total phenolic concentrations in growing and mature needles of the current year, but in needles of the previous year no significant differences occurred. Mycorrbizal infection was highest at medium (optimum and 2 × optimum) nitrogen fertilization levels. The relative growth rate (RGR) of grey pine aphid [Schizolachnus pineti (F.)] responded positively to the increase in foliar nitrogen content. However, the increase in aphid performance between optimum and the highest fertilization level was slight. This may indicate a deterring effect of resin acids on aphids, The results indicate that carbon/nutrient balance hypothesis fails to predict directly the effects of nitrogen availability on concentrations of carbon-based defensive compounds in mature foliage. Altered nitrogen supply affects allocation to secondary
*To whom correspondence should be addressed. 617 0098-0331/96/0,~0-0617$09.5010 @ 1996 Plenum Publishing Corporation
618
KAINULAINEN, HOLOPAINEN, PALOM,~KI, AND HOLOPAINEN metabolites differently, depending on the developmental state of the plant and the biosynthesis pathway, cost of synthesis, and storage of compounds. Key Words--Nitrogen, Pinus sylvestris, monoterpenes, resin acids, total phenolics, resin canals, plant-insect interactions, Schizolachnus pineti, mycorrhiza,
INTRODUCTION
Scots pine (Pinus sylvestris L.) is normally adapted to grow on nutrient-poor sites. An excess of nitrogen increases the growth and changes the phenology of pine seedlings (Will, 1971; L/ims/i et al., 1990; Rikala and Huurinainen, 1990; Dewald et al., 1992). Pines produce significant amounts of secondary compounds: phenolic compounds and oleoresin, which mainly contains monoterpenes and resin acids. These substances may have an important role in defense responses against herbivory (Alfaro et al., 1980; Larsson et al., 1986; Schopf, 1986; Elliott and Loudon, 1987; Ross and Berisford, 1990) and pathogen attacks (Flodin and Fries, 1978; Haars et al., 1981; Stenlind and Johansson, 1987; Lieutier et al., 1991; Himejima et al., 1992). Investigations of the interactions between leaf nitrogen and carbon-based chemical defense suggest that both nitrogen and allelochemicals are important factors in determining the nutritional quality of plant tissue for herbivores (Watt et al., 1990), but the response of sucking insects to host plant quality on conifers is poorly known (Waring and Cobb, 1992). Variations in environmental conditions can modify the quantities of secondary compounds found in plants (Gref and Tenow, 1987; Bjrrkrnan et al., 1991; Kainulainen et al., 1992; Muzika, 1993). Limited availability of resources, particularly nitrogen, may determine the plant investment in secondary compounds (Bryant et al., 1983; Coley et al., 1985). Bryant et al. (1983) have proposed that the carbon/nutrient balance of individual plants strongly affects their allocation of resources to primary and secondary metabolism. They predicted that increase in availability of nitrogen to plants would increase allocation of carbon to growth, resulting in a corresponding reduction of carbon-based secondary compounds, e.g., terpenes and phenolics. However, changes in terpenoid levels have not conformed to this hypothesis as well as do changes in the levels of other carbon-based compounds, such as phenolic compounds (Bryant et al., 1987; Muzika and Pregitzer, 1992; Muzika, 1993). For example, in pine needles, concentrations of terpenoid compounds increased after nitrogen fertilization (Bjrrkman et al., 1991; McCullough and Kulman, 1991). Terpenoid compounds are synthesized and stored in resin canals, which are specialized anatomical structures. Bjrrkman et al. (1991) suggested that the formation of resin ducts is limited by the low availability of nitrogen.
FERTILIZATION, SCOTS PINE, AND GREY PINE APHID
619
In mycorrhizal root systems the carbon allocation and nitrogen uptake are closely related (Scheromm et al., 1990; Plassard et al., 1991). Both shortage and surplus of nitrogen are reported to affect adversely ectomycorrhiza development (Marx et al., 1977; Alexander and Fairley, 1983; V/ire, 1990; Hotopainen and Heinonen-Tanski, 1993). Plants with well-developed ectomycorrhiza were able to allocate more carbon to shoot growth and antiherbivore defense due to better water and nutrient uptake (Gehring and Whitham, 1994). However, carbon/nutrient balance hypothesis (Bryant et al., 1983) does not take into account the changes in nutrient uptake by mycorrhiza and costs of mycorrhiza formation. The present study measures the responses of secondary compounds, mycorrhiza, and parasite interactions of Scots pine seedlings to different nitrogen availability regimes (low, optimal, and elevated levels). The primary objective was to find out if there is variation in concentrations of secondary compounds during shoot and needle development and if secondary chemical compounds originating from separate metabolic pathways have similar responses to nitrogen availability. A further objective of the study was to detect the possible role of increased nitrogen availability to mycorrhizal symbiosis and phloem-feeding herbivores, because these biotic interactions are often disturbed when the host plant is subjected to increased nitrogen deposition, and they may affect resource allocation patterns in host plant.
METHODS AND MATERIALS
Plant Material and Fertilizer Treatment. Scots pine (Pinus sylvestris L.) seedlings (seed origin: Kaskim/iki, Toivakka, M29-90-0(K~, 1 Mk PS-508) were obtained from the forest nursery of Suonenjoki (62°38'N, 27°04'E), central Finland. Seeds were sown in spring in paper pots (PS-508:124 cm 3) filled with Sphagnum peat (Vapo Tree nursery peat; mixture fertilizer 11-11-18 (N-P-K) 1 kg/m 3, dolomite lime 3 kg/m 3, total nitrogen 8 g/m2). The plants were overwintered in an open field at the Botanical Garden of the University of Kuopio. In the beginning of January, the plants were moved into a greenhouse and kept at a temperature of 5°C for a few days. The seedlings with their peat pot were planted in sand (grain size 0.5-1.2 mm) in 0.5-liter plastic pots (diameter 10.5 cm) with bottom perforations to prevent flooding and kept in a greenhouse at 14°C for one week. After that the plants were grown in a controlled growth chamber simulating the weather conditions of central Finland in June: day/night temperature was 19/12°C, the light/dark cycle 22/2 hr and the maximum light irradiance about 300/zmol photons/m 2 sec. At the beginning of the period of new shoot growth, the seedlings were arranged into four blocks. One block of seedlings was fertilized with a solution
620
KAINULAINEN, HOLOPAINEN, PALOM,~KI,~AND HOLOPAINEN
(50 mg N/liter) that contained the nitrogen and cation nutrition requirement for optimal growth of Scots pine (Ingestad, 1962). Two other blocks were fertilized with nitrogen levels two (100 mg N/liter) and four times (200 mg N/liter) higher than the optimum level. Seedlings were fertilized three times a week and the fourth block of seedlings received the equivalent volume of distilled water (100 ml) at each fertilization time. Sampling. Eight seedlings of each fertilization level were sampled when the seedlings had received nitrogen equivalent to 0, 9.8, 19.6, and 39.3 g/m 2 (0, 85, 170, and 340 mg N per pot) and needles were still growing. The second sampling was made when the growth of new shoots and needles had ceased with nitrogen concentrations equivalent to 0, 16.7, 33.5, and 67.0 g/m 2 (0, 145, 290, and 580 mg N per pot). Needles and roots of seedlings were collected for secondary chemistry, light microscopy, and mycorrhizal analysis in both sampies. Secondary chemistry and light microscopy samples were collected from needles of the current year. Total phenolics were also analyzed from needles of the previous year. Roots were carefully washed and divided into two lots, which were used for mycorrhizal and total phenolic analysis, respectively. In the latter sample, total nitrogen was analyzed from current-year needles of separate seedlings, which had been used in the measurement of the aphid growth rate. Morphological and Biomass Measurements. At both sampling dates, the height of the seedlings and the water content of the needles of the current and previous years were measured. The fresh weights of current-year shoots were measured from actively growing seedlings in the first sampling. In the latter sampling root collar diameter, needle length, number of buds, and biomass of foliage were measured. Needles and stems for biomass and water content measurements were oven-dried at 60°C for two days and weighed. Chemical Analysis. Monoterpenes were extracted from fresh needles with n-hexane (Kainulainen et al., 1992). Resin acids were extracted from freezedried needles following the procedures of Gref and Ericsson (1985). Only abietane and pimarane diterpene resin acids were investigated. Extracts were analyzed using gas chromatography-mass spectrometry (Hewlett Packard GC type 5890, MSD type 5970) using a 25-m-long HP-5 (0.2 mm ID, 0.11 p.m film thickness, Hewlett Packard) capillary column as described earlier by Kainulainen et al. (1994). Total phenolic analysis of current-year needles were made from the same needle powder as resin acids. For total phenolic analysis previous-year needles and roots were oven-dried at 45°C for two days and milled. The samples were stored in a desiccator at 4°C. Needle powder was extracted with 80% (v/v) aqueous acetone (Kainulainen et al., 1993), and total phenolics were analyzed by the Folin-Ciocalteu technique as described by Julkunen-Tiitto (1985). Tannic acid was used as a standard and results expressed as tannic acid equivalents.
FERTILIZATION. SCOTS PINE, AND GREY PINE APHID
621
Needles for total nitrogen analysis were oven-dried at 60°C for 48 hr and milled. Total nitrogen was analyzed by the standard Kjeldahl technique (Allen, 1989). Sample Preparation for Light Microscopy. Needle samples were immediately placed in 2% glutaraldehyde in 0.05 M phosphate buffer (pH 7.0). Pieces (about 1 mm) cut from a region 1/3 behind the tip of each needle were prefixed for 20 hr in glutaraldehyde fixative and postfixed in 1% OsO4 solution for 6 hr at 6°C. After fixation, samples were dehydrated in a graded ethanol series and embedded in Epon LX-112. Blocks were polymerized for one day at 37°C and three days at 60°C. Sections for light microscopy (five replicates) were stained with toluidine blue and studied at magnifications of 100 x or 400 ×. The number and diameter of resin ducts and the cross-sectional area of needles were measured. Mycorrhizal Infection. The washed root systems of the seedlings that were used for secondary chemistry analysis were stored at - 2 4 ° C until analyzed. Each root system was positioned over mesh and a total of 1 m of young long roots were randomly sampled. The mycorrhizal root tips were identified under a dissecting microscope after 15 min staining by Ponceau S (Daughtridge et al., 1986). The root systems were highly dominated by one mycorrhizal type--thinsheathed brown mycorrhizae. Therefore, only the total number of mycorrhizae was counted, and the percentage of rootlets infected by mycorrhizal fungi was used for treatment comparisons. Aphid Growth. Bioassay with aphids was accomplished on plants that were later used for total nitrogen analysis. Third-instar nymphs of the grey pine aphid [Schizolachnus pineti (F.)] reared on pine seedlings receiving optimum fertilization were placed on needles of the current year when the plants had received nitrogen at 0, 16.7, 33.5, and 67.0 g/m 2. The relative growth rate (RGR) was determined by weighing the nymphs before transfer and after three days of feeding on experimental seedlings. The nymphs were weighed individually on a Sartorius Microbalance MP3 (resolution 1 #g). RGR for nymphs was calculated with the formula: (lnW2 - lnWt)/t, where W2 is final weight, WI is initial weight, and t is time in days between the two measurements. Statistical Analysis. Data were analyzed with one-way analysis of variance and differences between optimal and other nitrogen treatments were compared with orthogonal contrasts (Mize and Schultz, 1985).
RESULTS
Total Nitrogen and Growth. The total nitrogen concentration of needles was significantly increased with higher nitrogen fertilization (Table 1). No sig-
15.0 (17) 3. I (17) 2.9 (17) 3.4 (17) 0.65 (9) 56.2 (17) 49.1 (16) 3.8 (8)
16.5 (18) 4.4 (18) 9.3 (18) 4.8 (18) 2.85 (10) 68.3 (18) 56.0 (17) I 1.5 (8)
16.7
16.3 (17) 4,9 (17) 11.0 (17) 5.5 (17) 3.31 (9) 69.4 (17) 55.4 (17) 15.5 (81
33,5 16.6 (16) 5. I (16) 11.0 (16) 5.7 (16) 3.82 (8) 70.7 (16) 55.8 (16) 18.9 (8)
67.0
626
KAINULAINEN, HOLOPAINEN, PALOM,~KI, AND HOLOPAINEN
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{rag/I) FIG. 1. Number of resin ducts at four different nitrogen fertilization levels in growing (11) and mature (A) needles. Each value is the mean (+SE) of five samples. Mean values within a same line with the same letter are not significantly different at 0.05 level (Tukey's multiple range test).
Mycorrhizal Infection. Mycorrhizal infection responded similady to fertilization at both sampling times (Figure 2). Both the lowest and highest nitrogen availabilities resulted in lower mycorrhizal infection levels compared to the medium fertilization levels. At the first sampling, the maximal infection was achieved by higher nitrogen fertilization than in the second sampling. Total phenolic concentration in roots did not correlate with mycorrhizal infection. Aphid Growth. RGR of S. pineti nymphs was significantly (F3.21 = 3.93, P = 0.023) affected by nitrogen fertilization, being significantly lower (P = 0.013) at nitrogen fertilization of 0 g/m 2 than at the optimum level (16.7 g/m2). RGR of nymphs was positively and nonlinearly correlated with foliar nitrogen concentration of pine seedlings (Figure 3). Secondary metabolites were not analyzed from the same seedlings that were used for bioassay. When the ratio of total nitrogen to total resin acids was calculated using treatment means, a
627
FERTILIZATION, SCOTS PINE, AND GREY PINE APHID
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(mg/I) FIG. 2. Mycorrhizal infection of roots at four different nitrogen fertilization levels, in the first sampling when needles were growing (11) and the second sampling when needles were mature (&). Each value is the mean (+SE) of eight plants. Mean values of the same line with the same letter are not significantly different at 0.05 level (Tukey's multiple range test).
significant linear positive correlation with the mean RGR of S. pineti nymphs per treatment was found (Figure 4). Concentrations of total nitrogen, total phenolics, total monoterpenes, and total resin acids and the nitrogen/phenolics and nitrogen/total monoterpenes ratios did not correlate with aphid RGR when treatment means were used in analyses.
DISCUSSION
Plant Growth Responses. The most distinctive growth responses of pine seedlings to increasing nitrogen availability were increases in needle length and
628
KAINULAINEN, HOLOPAINEN, PALOM,/~KI, AND HOLOPAINEN
0.30 r2 = 0 . 2 9 0 , p = 0 . 0 0 4
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FIG. 3. Relationship between the relative growth rate of nymphs of the grey pine aphid and foliar nitrogen concentration of pine seedlings grown at four different nitrogen fertilization levels: • = 0 g / m 2, • = 16.7g/m z, • = 33,5g/m 2,and • = 67.0g/m 2.
foliar biomass. The lack of a height growth response to nitrogen availability is explained by the fixed height growth, which is determined in Scots pine in the previous growing season during bud formation (Lanner, 1976; Kozlowski et al., 1991; Rikala and Huudnainen, 1990; Rikala, 1992). Growth allocation to photosynthesizing needle biomass reflects greater productivity and enhanced competitive ability of seedlings in response to light in a resource-rich environment (e.g., Herms and Mattson, 1992; Van der W e f t et al., 1993). Secondary Chemistry. In the present study concentrations of resin acids in mature needles increased with elevated nitrogen availability, contrary to the carbon/nutrient balance hypothesis (Bryant et al., 1983). By contrast, concentrations of foliar monoterpenes and total phenolics decreased with elevated nitro-
629
FERTILIZATION. SCOTS PINE, AND GREY PINE APHID
0.30 r2 = 0.905, p = 0.049
0.20 °
m
EFFECTS OF NITROGEN FERTILIZATION ON SECONDARY CHEMISTRY A N D ECTOMYCORRHIZAL STATE OF SCOTS PINE SEEDLINGS A N D ON GROWTH OF GREY PINE APHID
PIRJO KAINULAINEN,*
JARMO
HOLOPAINEN,
VIRPI PALOM,~KI,
and T O I N I H O L O P A I N E N
Department of Ecology and Environmental Science, University of Kuopio, P.O. Box 162 7, FTN-70211 Kuopio, Finland (Received April 10, 1995; accepted November 20, 1995)
Abstract--Effects of nitrogen availability on secondary compounds, mycorrhizal infection, and aphid growth of l-year-old Scots pine (Pinus sylvestris L.) seedlings were studied during one growing season. Seedlings were fertilized with nutrient solutions containing low, optimum, and two elevated (2 x and 4 x optimum) levels of NH4NO~. At the end of growing season foliar nitrogen concentration, needle biomass, needle length, water contents of needles, root collar diameter, and number of buds increased with enhanced nitrogen availability. Addition of nitrogen did not have effect on concentrations of monoterpenes in growing needles~ but in mature needles significantly decreased concentrations of some individual and total monoterpenes were detected. In growing needles the concentrations of some individual resin acids decreased, and in mature needles concentrations of some individual and total resin acids increased with increased nitrogen fertilization. Higher numbers of resin ducts were found in mature needles with nitrogen fertilization. Nitrogen fertilization decreased total phenolic concentrations in growing and mature needles of the current year, but in needles of the previous year no significant differences occurred. Mycorrbizal infection was highest at medium (optimum and 2 × optimum) nitrogen fertilization levels. The relative growth rate (RGR) of grey pine aphid [Schizolachnus pineti (F.)] responded positively to the increase in foliar nitrogen content. However, the increase in aphid performance between optimum and the highest fertilization level was slight. This may indicate a deterring effect of resin acids on aphids, The results indicate that carbon/nutrient balance hypothesis fails to predict directly the effects of nitrogen availability on concentrations of carbon-based defensive compounds in mature foliage. Altered nitrogen supply affects allocation to secondary
*To whom correspondence should be addressed. 617 0098-0331/96/0,~0-0617$09.5010 @ 1996 Plenum Publishing Corporation
618
KAINULAINEN, HOLOPAINEN, PALOM,~KI, AND HOLOPAINEN metabolites differently, depending on the developmental state of the plant and the biosynthesis pathway, cost of synthesis, and storage of compounds. Key Words--Nitrogen, Pinus sylvestris, monoterpenes, resin acids, total phenolics, resin canals, plant-insect interactions, Schizolachnus pineti, mycorrhiza,
INTRODUCTION
Scots pine (Pinus sylvestris L.) is normally adapted to grow on nutrient-poor sites. An excess of nitrogen increases the growth and changes the phenology of pine seedlings (Will, 1971; L/ims/i et al., 1990; Rikala and Huurinainen, 1990; Dewald et al., 1992). Pines produce significant amounts of secondary compounds: phenolic compounds and oleoresin, which mainly contains monoterpenes and resin acids. These substances may have an important role in defense responses against herbivory (Alfaro et al., 1980; Larsson et al., 1986; Schopf, 1986; Elliott and Loudon, 1987; Ross and Berisford, 1990) and pathogen attacks (Flodin and Fries, 1978; Haars et al., 1981; Stenlind and Johansson, 1987; Lieutier et al., 1991; Himejima et al., 1992). Investigations of the interactions between leaf nitrogen and carbon-based chemical defense suggest that both nitrogen and allelochemicals are important factors in determining the nutritional quality of plant tissue for herbivores (Watt et al., 1990), but the response of sucking insects to host plant quality on conifers is poorly known (Waring and Cobb, 1992). Variations in environmental conditions can modify the quantities of secondary compounds found in plants (Gref and Tenow, 1987; Bjrrkrnan et al., 1991; Kainulainen et al., 1992; Muzika, 1993). Limited availability of resources, particularly nitrogen, may determine the plant investment in secondary compounds (Bryant et al., 1983; Coley et al., 1985). Bryant et al. (1983) have proposed that the carbon/nutrient balance of individual plants strongly affects their allocation of resources to primary and secondary metabolism. They predicted that increase in availability of nitrogen to plants would increase allocation of carbon to growth, resulting in a corresponding reduction of carbon-based secondary compounds, e.g., terpenes and phenolics. However, changes in terpenoid levels have not conformed to this hypothesis as well as do changes in the levels of other carbon-based compounds, such as phenolic compounds (Bryant et al., 1987; Muzika and Pregitzer, 1992; Muzika, 1993). For example, in pine needles, concentrations of terpenoid compounds increased after nitrogen fertilization (Bjrrkman et al., 1991; McCullough and Kulman, 1991). Terpenoid compounds are synthesized and stored in resin canals, which are specialized anatomical structures. Bjrrkman et al. (1991) suggested that the formation of resin ducts is limited by the low availability of nitrogen.
FERTILIZATION, SCOTS PINE, AND GREY PINE APHID
619
In mycorrhizal root systems the carbon allocation and nitrogen uptake are closely related (Scheromm et al., 1990; Plassard et al., 1991). Both shortage and surplus of nitrogen are reported to affect adversely ectomycorrhiza development (Marx et al., 1977; Alexander and Fairley, 1983; V/ire, 1990; Hotopainen and Heinonen-Tanski, 1993). Plants with well-developed ectomycorrhiza were able to allocate more carbon to shoot growth and antiherbivore defense due to better water and nutrient uptake (Gehring and Whitham, 1994). However, carbon/nutrient balance hypothesis (Bryant et al., 1983) does not take into account the changes in nutrient uptake by mycorrhiza and costs of mycorrhiza formation. The present study measures the responses of secondary compounds, mycorrhiza, and parasite interactions of Scots pine seedlings to different nitrogen availability regimes (low, optimal, and elevated levels). The primary objective was to find out if there is variation in concentrations of secondary compounds during shoot and needle development and if secondary chemical compounds originating from separate metabolic pathways have similar responses to nitrogen availability. A further objective of the study was to detect the possible role of increased nitrogen availability to mycorrhizal symbiosis and phloem-feeding herbivores, because these biotic interactions are often disturbed when the host plant is subjected to increased nitrogen deposition, and they may affect resource allocation patterns in host plant.
METHODS AND MATERIALS
Plant Material and Fertilizer Treatment. Scots pine (Pinus sylvestris L.) seedlings (seed origin: Kaskim/iki, Toivakka, M29-90-0(K~, 1 Mk PS-508) were obtained from the forest nursery of Suonenjoki (62°38'N, 27°04'E), central Finland. Seeds were sown in spring in paper pots (PS-508:124 cm 3) filled with Sphagnum peat (Vapo Tree nursery peat; mixture fertilizer 11-11-18 (N-P-K) 1 kg/m 3, dolomite lime 3 kg/m 3, total nitrogen 8 g/m2). The plants were overwintered in an open field at the Botanical Garden of the University of Kuopio. In the beginning of January, the plants were moved into a greenhouse and kept at a temperature of 5°C for a few days. The seedlings with their peat pot were planted in sand (grain size 0.5-1.2 mm) in 0.5-liter plastic pots (diameter 10.5 cm) with bottom perforations to prevent flooding and kept in a greenhouse at 14°C for one week. After that the plants were grown in a controlled growth chamber simulating the weather conditions of central Finland in June: day/night temperature was 19/12°C, the light/dark cycle 22/2 hr and the maximum light irradiance about 300/zmol photons/m 2 sec. At the beginning of the period of new shoot growth, the seedlings were arranged into four blocks. One block of seedlings was fertilized with a solution
620
KAINULAINEN, HOLOPAINEN, PALOM,~KI,~AND HOLOPAINEN
(50 mg N/liter) that contained the nitrogen and cation nutrition requirement for optimal growth of Scots pine (Ingestad, 1962). Two other blocks were fertilized with nitrogen levels two (100 mg N/liter) and four times (200 mg N/liter) higher than the optimum level. Seedlings were fertilized three times a week and the fourth block of seedlings received the equivalent volume of distilled water (100 ml) at each fertilization time. Sampling. Eight seedlings of each fertilization level were sampled when the seedlings had received nitrogen equivalent to 0, 9.8, 19.6, and 39.3 g/m 2 (0, 85, 170, and 340 mg N per pot) and needles were still growing. The second sampling was made when the growth of new shoots and needles had ceased with nitrogen concentrations equivalent to 0, 16.7, 33.5, and 67.0 g/m 2 (0, 145, 290, and 580 mg N per pot). Needles and roots of seedlings were collected for secondary chemistry, light microscopy, and mycorrhizal analysis in both sampies. Secondary chemistry and light microscopy samples were collected from needles of the current year. Total phenolics were also analyzed from needles of the previous year. Roots were carefully washed and divided into two lots, which were used for mycorrhizal and total phenolic analysis, respectively. In the latter sample, total nitrogen was analyzed from current-year needles of separate seedlings, which had been used in the measurement of the aphid growth rate. Morphological and Biomass Measurements. At both sampling dates, the height of the seedlings and the water content of the needles of the current and previous years were measured. The fresh weights of current-year shoots were measured from actively growing seedlings in the first sampling. In the latter sampling root collar diameter, needle length, number of buds, and biomass of foliage were measured. Needles and stems for biomass and water content measurements were oven-dried at 60°C for two days and weighed. Chemical Analysis. Monoterpenes were extracted from fresh needles with n-hexane (Kainulainen et al., 1992). Resin acids were extracted from freezedried needles following the procedures of Gref and Ericsson (1985). Only abietane and pimarane diterpene resin acids were investigated. Extracts were analyzed using gas chromatography-mass spectrometry (Hewlett Packard GC type 5890, MSD type 5970) using a 25-m-long HP-5 (0.2 mm ID, 0.11 p.m film thickness, Hewlett Packard) capillary column as described earlier by Kainulainen et al. (1994). Total phenolic analysis of current-year needles were made from the same needle powder as resin acids. For total phenolic analysis previous-year needles and roots were oven-dried at 45°C for two days and milled. The samples were stored in a desiccator at 4°C. Needle powder was extracted with 80% (v/v) aqueous acetone (Kainulainen et al., 1993), and total phenolics were analyzed by the Folin-Ciocalteu technique as described by Julkunen-Tiitto (1985). Tannic acid was used as a standard and results expressed as tannic acid equivalents.
FERTILIZATION. SCOTS PINE, AND GREY PINE APHID
621
Needles for total nitrogen analysis were oven-dried at 60°C for 48 hr and milled. Total nitrogen was analyzed by the standard Kjeldahl technique (Allen, 1989). Sample Preparation for Light Microscopy. Needle samples were immediately placed in 2% glutaraldehyde in 0.05 M phosphate buffer (pH 7.0). Pieces (about 1 mm) cut from a region 1/3 behind the tip of each needle were prefixed for 20 hr in glutaraldehyde fixative and postfixed in 1% OsO4 solution for 6 hr at 6°C. After fixation, samples were dehydrated in a graded ethanol series and embedded in Epon LX-112. Blocks were polymerized for one day at 37°C and three days at 60°C. Sections for light microscopy (five replicates) were stained with toluidine blue and studied at magnifications of 100 x or 400 ×. The number and diameter of resin ducts and the cross-sectional area of needles were measured. Mycorrhizal Infection. The washed root systems of the seedlings that were used for secondary chemistry analysis were stored at - 2 4 ° C until analyzed. Each root system was positioned over mesh and a total of 1 m of young long roots were randomly sampled. The mycorrhizal root tips were identified under a dissecting microscope after 15 min staining by Ponceau S (Daughtridge et al., 1986). The root systems were highly dominated by one mycorrhizal type--thinsheathed brown mycorrhizae. Therefore, only the total number of mycorrhizae was counted, and the percentage of rootlets infected by mycorrhizal fungi was used for treatment comparisons. Aphid Growth. Bioassay with aphids was accomplished on plants that were later used for total nitrogen analysis. Third-instar nymphs of the grey pine aphid [Schizolachnus pineti (F.)] reared on pine seedlings receiving optimum fertilization were placed on needles of the current year when the plants had received nitrogen at 0, 16.7, 33.5, and 67.0 g/m 2. The relative growth rate (RGR) was determined by weighing the nymphs before transfer and after three days of feeding on experimental seedlings. The nymphs were weighed individually on a Sartorius Microbalance MP3 (resolution 1 #g). RGR for nymphs was calculated with the formula: (lnW2 - lnWt)/t, where W2 is final weight, WI is initial weight, and t is time in days between the two measurements. Statistical Analysis. Data were analyzed with one-way analysis of variance and differences between optimal and other nitrogen treatments were compared with orthogonal contrasts (Mize and Schultz, 1985).
RESULTS
Total Nitrogen and Growth. The total nitrogen concentration of needles was significantly increased with higher nitrogen fertilization (Table 1). No sig-
15.0 (17) 3. I (17) 2.9 (17) 3.4 (17) 0.65 (9) 56.2 (17) 49.1 (16) 3.8 (8)
16.5 (18) 4.4 (18) 9.3 (18) 4.8 (18) 2.85 (10) 68.3 (18) 56.0 (17) I 1.5 (8)
16.7
16.3 (17) 4,9 (17) 11.0 (17) 5.5 (17) 3.31 (9) 69.4 (17) 55.4 (17) 15.5 (81
33,5 16.6 (16) 5. I (16) 11.0 (16) 5.7 (16) 3.82 (8) 70.7 (16) 55.8 (16) 18.9 (8)
67.0
626
KAINULAINEN, HOLOPAINEN, PALOM,~KI, AND HOLOPAINEN
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Nitrogen in fertilization solution
{rag/I) FIG. 1. Number of resin ducts at four different nitrogen fertilization levels in growing (11) and mature (A) needles. Each value is the mean (+SE) of five samples. Mean values within a same line with the same letter are not significantly different at 0.05 level (Tukey's multiple range test).
Mycorrhizal Infection. Mycorrhizal infection responded similady to fertilization at both sampling times (Figure 2). Both the lowest and highest nitrogen availabilities resulted in lower mycorrhizal infection levels compared to the medium fertilization levels. At the first sampling, the maximal infection was achieved by higher nitrogen fertilization than in the second sampling. Total phenolic concentration in roots did not correlate with mycorrhizal infection. Aphid Growth. RGR of S. pineti nymphs was significantly (F3.21 = 3.93, P = 0.023) affected by nitrogen fertilization, being significantly lower (P = 0.013) at nitrogen fertilization of 0 g/m 2 than at the optimum level (16.7 g/m2). RGR of nymphs was positively and nonlinearly correlated with foliar nitrogen concentration of pine seedlings (Figure 3). Secondary metabolites were not analyzed from the same seedlings that were used for bioassay. When the ratio of total nitrogen to total resin acids was calculated using treatment means, a
627
FERTILIZATION, SCOTS PINE, AND GREY PINE APHID
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200
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(mg/I) FIG. 2. Mycorrhizal infection of roots at four different nitrogen fertilization levels, in the first sampling when needles were growing (11) and the second sampling when needles were mature (&). Each value is the mean (+SE) of eight plants. Mean values of the same line with the same letter are not significantly different at 0.05 level (Tukey's multiple range test).
significant linear positive correlation with the mean RGR of S. pineti nymphs per treatment was found (Figure 4). Concentrations of total nitrogen, total phenolics, total monoterpenes, and total resin acids and the nitrogen/phenolics and nitrogen/total monoterpenes ratios did not correlate with aphid RGR when treatment means were used in analyses.
DISCUSSION
Plant Growth Responses. The most distinctive growth responses of pine seedlings to increasing nitrogen availability were increases in needle length and
628
KAINULAINEN, HOLOPAINEN, PALOM,/~KI, AND HOLOPAINEN
0.30 r2 = 0 . 2 9 0 , p = 0 . 0 0 4
0.20 °~
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Foliar nitrogen mg.g
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-1
FIG. 3. Relationship between the relative growth rate of nymphs of the grey pine aphid and foliar nitrogen concentration of pine seedlings grown at four different nitrogen fertilization levels: • = 0 g / m 2, • = 16.7g/m z, • = 33,5g/m 2,and • = 67.0g/m 2.
foliar biomass. The lack of a height growth response to nitrogen availability is explained by the fixed height growth, which is determined in Scots pine in the previous growing season during bud formation (Lanner, 1976; Kozlowski et al., 1991; Rikala and Huudnainen, 1990; Rikala, 1992). Growth allocation to photosynthesizing needle biomass reflects greater productivity and enhanced competitive ability of seedlings in response to light in a resource-rich environment (e.g., Herms and Mattson, 1992; Van der W e f t et al., 1993). Secondary Chemistry. In the present study concentrations of resin acids in mature needles increased with elevated nitrogen availability, contrary to the carbon/nutrient balance hypothesis (Bryant et al., 1983). By contrast, concentrations of foliar monoterpenes and total phenolics decreased with elevated nitro-
629
FERTILIZATION. SCOTS PINE, AND GREY PINE APHID
0.30 r2 = 0.905, p = 0.049
0.20 °
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