Journal of Chemical Ecology, Vol. 30, No. 10, October 2004 ( C 2004) NITROGEN-INDUCED CHANGES IN PHENOLICS OF Vaccinium myrtillus—IMPLICATIONS FOR INTERACTION WITH A PARASITIC FUNGUS JOHANNA WITZELL 1,∗ and ANNA SHEVTSOVA 2 1 Department of Forest Genetics and Plant Physiology Swedish University of Agricultural Sciences SE-90183 Ume ˚ a, Sweden 2 Department of Forest Vegetation Ecology Swedish University of Agricultural Sciences SE-90183 Ume ˚ a, Sweden (Received November 19, 2003; accepted June 30, 2004) Abstract—The effects of nitrogen (N) fertilization on the phenolic status of Vaccinium myrtillus leaves were studied to assess whether N amendment affects the potentially defensive phenolic metabolites in a way that could have con- sequences for the interaction with a parasitic fungus (Valdensia heterodoxa). Healthy (symptomless) and V. heterodoxa-infected leaves were collected from plants grown in the understorey of a boreal coniferous forest, where they re- ceived no additional N or either a moderate or a high dose of N fertilizer. Leaf samples were taken during a single growth season and analyzed for individual phenolics using HPLC. The effect of a moderate N dose on the concentration and content of phenolics was in most cases nonsignificant. In contrast, the high N dose resulted in pronounced effects. In healthy leaves, N fertilization reduced concentration of three of five individual phenolics. Moreover, fertilization with high dose of N accompanied by infection by V. heterodoxa often increased the concentration and content of phenolics as compared to unfertilized plants. Addition of N had no significant effect on the growth of the analyzed V. myr- tillus leaves, and the N-induced variation in phenolic levels seemed to be due to changed rate of their production. The concentration and content of pheno- lic metabolites in healthy leaves collected from unfertilized plots fluctuated compound-specifically during the growth season, and the phenolic responses to N and infection showed temporal and compound-specific variations. Key Words—Boreal forest, bilberry, chemical defense, disease resistance, nitrogen fertilization, Valdensia heterodoxa. ∗ To whom correspondence should be addressed. E-mail: [email protected]1937 0098-0331/04/1000-1937/0 C 2004 Springer Science+Business Media, Inc.
NITROGEN-INDUCED CHANGES IN PHENOLICS OFVaccinium myrtillus—IMPLICATIONS FOR INTERACTION
WITH A PARASITIC FUNGUS
JOHANNA WITZELL1,∗ and ANNA SHEVTSOVA2
1Department of Forest Genetics and Plant PhysiologySwedish University of Agricultural Sciences
SE-90183 Umea, Sweden2Department of Forest Vegetation Ecology
Swedish University of Agricultural SciencesSE-90183 Umea, Sweden
(Received November 19, 2003; accepted June 30, 2004)
Abstract—The effects of nitrogen (N) fertilization on the phenolic status ofVaccinium myrtillus leaves were studied to assess whether N amendment affectsthe potentially defensive phenolic metabolites in a way that could have con-sequences for the interaction with a parasitic fungus (Valdensia heterodoxa).Healthy (symptomless) and V. heterodoxa-infected leaves were collected fromplants grown in the understorey of a boreal coniferous forest, where they re-ceived no additional N or either a moderate or a high dose of N fertilizer. Leafsamples were taken during a single growth season and analyzed for individualphenolics using HPLC. The effect of a moderate N dose on the concentrationand content of phenolics was in most cases nonsignificant. In contrast, the highN dose resulted in pronounced effects. In healthy leaves, N fertilization reducedconcentration of three of five individual phenolics. Moreover, fertilization withhigh dose of N accompanied by infection by V. heterodoxa often increasedthe concentration and content of phenolics as compared to unfertilized plants.Addition of N had no significant effect on the growth of the analyzed V. myr-tillus leaves, and the N-induced variation in phenolic levels seemed to be dueto changed rate of their production. The concentration and content of pheno-lic metabolites in healthy leaves collected from unfertilized plots fluctuatedcompound-specifically during the growth season, and the phenolic responses toN and infection showed temporal and compound-specific variations.
Nitrogen (N) is one of the limiting factors for primary production of plants, andits availability also affects the growth and reproduction of herbivores and mi-croorganisms. Anthropogenic activities (e.g., industry and agriculture) often leadto increased N deposition in the environment. This is likely to have a variety ofeffects, both positive and negative, on the different trophic levels of terrestrialecosystems (Aber et al., 1989; Vitousek and Howard, 1991; Lee, 1998). Ecolog-ical consequences of N deposition may be particularly profound for ecosystemsthat are inherently N poor and have historically low background levels of Ndeposition, such as dry heathlands and boreal forests (Hunter and Price, 1991;Nordin et al., 1998; Strengbom et al., 2002). In these ecosystems, addition ofN leads to changes in field layer vegetation, usually involving increased abun-dance of fast-growing graminoids (grasses) at the expense of slow-growing andnutrient conserving species, such as ericaceous dwarf shrubs (e.g., Calluna andVaccinium species) (Aerts and Berendse, 1988; Aerts et al., 1990; Strengbom et al.,2002, 2003). This transition may be mediated, at least in part, by N increasingthe susceptibility of the slow-growing species to damage by abiotic factors (e.g.,frosts or drought), herbivores, or parasites (Heil and Diemont, 1983; Strengbomet al., 2002). This, in turn, reduces competitive ability and causes perturbationof the coverage, which allows fast-growing species to establish and take over(Aerts et al., 1990; Lee, 1998, and references therein; Strengbom et al., 2002,2003).
In the understorey of a Norway spruce-dominated boreal forest, N additiondecreases the abundance of Vaccinium myrtillus L. (bilberry), while that of a grassspecies, Deschampsia flexuosa L., increases (Strengbom, 2002; Strengbom et al.,2002). Concurrently, an increase in the incidence of a fungal parasite, Valdensiaheterodoxa Peyr., on V. myrtillus leaves has been recorded on N-fertilized plots(Strengbom et al., 2002). The fungus causes brown spot disease, which may leadto premature shedding of infected leaves and patchiness of V. myrtillus cover.The fungus overwinters on fallen leaves as sclerotia, from which the fruit bodiesdevelop in the following spring. The initial infection of host plants takes place inthe early summer, and the infection develops from conidia, which are formed onnecrotic lesions (Norwell and Redhead, 1994; Vogelgsang and Shamoun, 2002).The phytochemical basis of the observed N-induced increase in the susceptibilityof V. myrtillus to V. heterodoxa is not yet clear. Increased N (amino acid) concen-trations of leaves in fertilized plants may at least partially explain this increase.Earlier studies have shown that concentration of glutamine increases in fertilizedV. myrtillus plants, and spraying of leaves with glutamine solution increases the in-cidence of V. heterodoxa (Nordin et al., 1998; Strengbom, 2002; Strengbom et al.,2002). However, increased N availability may affect the phytochemical qualityof plants in several ways, and we still know little about the possible N-induced
NITROGEN-INDUCED CHANGES IN PHENOLICS OF Vaccinium myrtillus 1939
changes in other phytochemicals, which may predispose V. myrtillus to infestationby parasitic fungi.
The vegetative tissues of V. myrtillus plants contain a variety of carbon-basedsecondary metabolites, such as phenolic acids and flavonoids (Gallet, 1994; Fraisseet al., 1996; Witzell et al., 2003). Some of these compounds possess antimicrobialactivity (Kokubun and Harborne, 1994; Ho et al., 2001; Puupponen-Pimia et al.,2001) and are, therefore, potentially important in V. myrtillus–V. heterodoxa asso-ciation (see also Nicholson and Hammerschmidt, 1992; Bennett and Wallsgrove,1994; Dixon and Paiva, 1995, for general aspects of phenolics in plant defense).Increased N availability in the environment may decrease the levels of phenolicmetabolites in plants (Haukioja et al., 1998; Koricheva et al., 1998). Althoughsimple and mechanistic explanations for environmentally induced changes in al-location of resources to carbon-based defenses have been searched for during thelast decades [e.g., studies testing the validity of the carbon–nutrient balance (CNB)hypothesis; Bryant et al., 1983], recent evaluations have emphasized that the pro-duction of phenolic metabolites responds to changes in nutrient availability in ahighly complex manner (Haukioja et al., 1998; Koricheva et al., 1998; Penuelasand Estiarte, 1998; Jones and Hartley, 1999; Hamilton et al., 2001; Koricheva,2002; Lerdau and Coley, 2002). For instance, the ontogenetic and phenologicalvariation in plant phenolic metabolism (Kause et al., 1999; Riipi et al., 2002;Kleiner et al., 2003) needs to be better considered in studies addressing phenolicresponses to abiotic and biotic factors. Our understanding of carbon allocationpatterns under different nutrient regimes may be further refined by methodolog-ical improvements. Traditionally, predictions about treatment-induced shifts inresource allocation to secondary metabolites have been based on concentrationdata. Variation in concentrations of secondary metabolites across treatments may,however, be due simply to differences in plant growth and accumulation of biomass(Koricheva, 1999). Thus, it has been suggested that the measurement of simulta-neous shifts in biomass production and absolute content of secondary metabolitesmay provide more accurate information about the mechanisms underlying changesin their concentrations (Koricheva, 1999; Koricheva and Shevtsova, 2002).
The aim of our study was to investigate the effects of N fertilization on thephenolic chemistry of healthy and V. heterodoxa-infected V. myrtillus leaves. Wehypothesized that N amendment decreases the constitutive levels of defensivephenolics in leaves, or depresses the parasite-induced changes in phenolics, whichcould explain their increased susceptibility to fungal parasites associated with in-creases in N supply (cf. Nordin et al., 1998; Strengbom, 2002; Strengbom et al.,2002, 2003). To assess plant quality for parasites (and, potentially other naturalenemies) at different levels of N fertilization, we analyzed changes in concentra-tions of phenolics. In addition, to distinguish changes in phenolic levels due totreatment-induced effects on biomass accumulation from changes in allocationpatterns, we also determined the total amount of compounds per individual leaf.
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The measurements were done on four occasions to study the temporal variationsin phenolics in relation to the progress of the infection. This study is a part ofa research project attempting to define the N effects on various phytochemicalcharacters that are critical for the parasite resistance of V. myrtillus.
METHODS AND MATERIALS
Study Area and Treatments. The experimental area, Svartberget ResearchStation, is situated in northern Sweden, 60 km NW from Umea (64◦14′N, 19◦46′E).The experimental forest is a late successional spruce (Picea abies L. Karst) for-est, with a field layer dominated by the ericaceous dwarf shrub V. myrtillus. Theexperiment was set up as a randomized complete block design in 1996. Withineach of five blocks (150 × 150 m), three 31.6 × 31.6 m plots were randomly as-signed to N fertilization treatments of 0 (N0-plots, unfertilized control), 12.5 (N1-plots, moderate N level), and 50 (N2-plots, high N level) kg ha−1 y−1. The ferti-lizer was given as NH4NO3 in the form of granules once a year (at the beginningof the growth season) for 5 years (1996–2000). In the study area, the backgroundlevel of N deposition is low (about 3.5 kg N ha−1 y−1; Strengbom, 2002). In gen-eral, the level of air-borne N deposition in Sweden has been reported to vary from2–3 to 20 kg ha−1 y−1 in northern and southern parts, respectively (Strengbomet al., 2003, and references therein), whereas in Central Europe higher amountshave been reported (e.g., in the Netherlands from 50 to 85 kg ha−1 y−1; Berendseet al., 1993, cited by Vitousek, 1994; Dise et al., 1998; Tietema et al., 1998). Thus,the N doses used in our study were ecologically realistic for Northern and CentralEuropean conditions.
Sample Collection. Samples were collected on four occasions during thegrowth season of 2000: in mid-June, mid-July, mid-August, and late August.These sampling occasions were considered to represent, respectively, the initialphase of V. heterodoxa infection by ascospores (mid-June), the expansive phaseby the conidial infection (mid-July and mid-August), and the late phase, when thefirst overwintering structures are initiated (late-August). Within each plot, severalramets of V. myrtillus were chosen randomly. From these ramets, we haphazardlyselected one ramet without visible symptoms of infection by V. heterodoxa (June–July) or with less than 10% of infection (August) and one ramet with visiblesymptoms of infection (>50% of infected leaves). The distance between thetwo ramets did not exceed 2 m to reduce possible chemical variation due topatchiness of soil and microclimate. A leaf without visible symptoms (healthy)was chosen from a healthy ramet, and a leaf bearing necrotic lesions symptomaticof V. heterodoxa infection (infected) was collected from the other ramets foranalysis. Care was taken to choose leaves of similar developmental stage (i.e.,leaves in a phase where they were no longer actively expanding, but not yetsenescent). The presence of parasites or damage from herbivores on other leaves
NITROGEN-INDUCED CHANGES IN PHENOLICS OF Vaccinium myrtillus 1941
during the study season or preceding growth seasons was regarded as backgroundvariation and not recorded in this study. The analysis of single leaves rather thanwhole shoots was preferred because of the difficulty of finding whole shoots freeof parasite infestations during the entire experimental period. During the firstsampling occasion, parasite frequency on the plots was still low, and in both Juneand July whole annual shoots in which none of the leaves had visible symptomscould be found. However, during August this was often impossible, because ofthe high incidence of parasites. The disease incidences for the N treatments weremeasured during 1996–2000 (Strengbom, 2002).
Collected leaves were air-dried in a well-ventilated room at ambient temper-ature. After this, the dry leaves were kept in a desiccator for 48 hr, and each wasweighed. A higher drying temperature (e.g., 105◦C) for dry weight determinationwas not used because of the risk of causing changes in phenolics (Waterman andMole, 1994). After weighing, the necrotic tissue was separated with a scalpelfrom the symptom-bearing leaves and discarded to ensure that the analyzed tissuerepresented living cells. Leaves were stored at −20◦C and equilibrated to roomtemperature in a desiccator for 24 hr before analysis.
Extraction and Analysis of Phenolics. Phenolics were analyzed using anHPLC system equipped with a photodiode array detector according to the methoddescribed by Witzell et al. (2003). The methanol extracts contained several pheno-lics, of which five compounds, which represented different types of phenolics, wereselected for a more detailed analysis. On the basis of retention times and specificUV-spectra (200-400 nm), which were compared to authentic standards, thesecompounds were identified as chlorogenic acid, catechin, arbutin, p-coumaricacid, and quercetin-3-glucoside.
Data Analyses. We estimated the effects of N fertilization, V. heterodoxainfection, and sampling time on phenolic compounds by using mixed analysis ofvariance with SAS statistical software (proc MIXED, SAS Release 8.2). Leaf dryweight and the concentrations and contents of the selected phenolic compoundswere the dependent variables. The N fertilization treatment (with three levels)served as a fixed factor and was further decomposed into orthogonal contraststesting for linear and quadratic trends. Since the area covered by the individ-ual clones of V. myrtillus can be extensive (up to 5–15 m diam; Ritchie, 1956;Flower-Ellis, 1971), the diseased and healthy leaves may have originated from thesame clone of V. myrtillus and could not be considered as independent samples.Therefore, infection was treated as a split-plot factor and the fertilization as awhole-plot factor. The random factors included block and block × fertilization.Samples that were successively harvested from the same microhabitats of thesame plots were also considered as dependent samples, and the sampling timewas treated as a repeated-measures factor. The covariance matrix of the modelwas chosen by fitting several different correlation models and comparing their re-spective Akaike’s Information criteria. A mixed ANOVA model was used with the
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ddfm=satterth option, invoking the Satterthwait approximation (Schabenbergerand Pierce, 2002). Mean comparisons between different levels of treatments anddifferent sampling dates were performed by Tukey–Kramer and Dunnett’s tests(LSMEANS/diff option in proc MIXED; SAS Institute, 1999). In addition, in thecase of significant interactions between main factors, the simple effects were stud-ied by using LSMEANS/slice option (proc MIXED; SAS Institute, 1999). Prior toanalysis, each data set was tested for normality (Shapiro–Wilk’s test) and homo-geneity of variances (Levene’s test) to verify that it conformed to the assumptionsof ANOVA, and the data were transformed when necessary. Untransformed valuesare presented in the figures.
Changes in leaf growth over the season, and in the concentration and contentof phenolics, were visualized and interpreted with the help of graphical vectoranalysis (GV analysis; Koricheva, 1999; Koricheva and Shevtsova, 2002; see alsoFigure 2f for interpretation of GV diagrams). For this, mean concentrations andcontents of individual phenolics for each treatment and sampling occasion wereexpressed as values relative to the corresponding values of the control (healthyleaves from unfertilized plots). The relative changes in concentrations and contentsof the phenolic were plotted against each other on a vector diagram. The mean leafweights represented the inverse of the slope factor. The effects of the treatmentswere expressed by vectors, connecting values for the control (x = y = z = 1) withthe values for the treatments. Seasonal variations for each individual phenoliccompound studied were analyzed in the healthy leaves from unfertilized plotsrelative to the levels observed at the first sampling occasion (mid-June) (for moredetails on vector diagrams construction and interpretation, see Koricheva, 1999).
RESULTS
Leaf Dry Weight. There were no significant effects of N, sampling time or in-fection on the dry weight of leaves, although the interactive effect of infection andsampling time on leaf weight was significant (Table 1). In the healthy leaves fromthe unfertilized plots, we found no changes in the dry weight of the samples col-lected between mid-June and late August [F (3, 36) = 0.63, P = 0.600]. Infectedleaves were marginally lighter than healthy leaves in June [F (1, 80.8) = 3.52,P = 0.064], but in mid-August infected leaves were heavier than the healthy ones[F (1, 81.5) = 6.05, P = 0.016] (Figure 1).
Seasonal Changes in Phenolics in Healthy Leaves from Unfertilized Plots.A significant sampling time effect was found on individual phenolic concentrationand on the absolute content of all phenolics, with the exception of quercetin-3-glucoside (Table 1). The GV analysis also indicated that there were seasonalvariations in individual phenolic compounds (Figure 2). For instance, the level ofarbutin was lowest in July, but increased close to the initial (June) level duringAugust (Figure 2c). Both the concentration and content of the other four phenolics
NITROGEN-INDUCED CHANGES IN PHENOLICS OF Vaccinium myrtillus 1943
TABLE 1. RESULTS OF REPEATED-MEASURES ANOVA FOR THE EFFECTS OF NITROGEN
FERTILIZATION (N), Valdensia heterodoxa INFECTION (INF) AND SAMPLING TIME (TIME)ON LEAF DRY WEIGHT (DW), AND CONCENTRATIONS AND LEAF CONTENTS OF
INDIVIDUAL PHENOLIC COMPOUNDS OF Vaccinium myrtillus
aTransformation of the data; bThe covariance matrix of the model (ar(1) – first-order autoregressive;Sp(exp) – exponential; vc – variance components).
were highest in mid-July (catechin and quercetin-3-glucoside; Figure 2b and d) ormid-August (chlorogenic acid and p-coumaric acid; Figure 2a and e). After thesedates, levels were reduced, and by late August they had returned close to theirinitial (June) levels (Figure 2). The magnitude of seasonal changes was highestfor catechin, with a twofold increase in concentration from mid-June to mid-July.
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FIG. 1. Dry weight (mg) of healthy and infected leaves of Vaccinium myrtillus collectedfrom plots with different levels of N fertilization (N0 – no additional N; N1 – addition of12.5 kg N ha−1 y−1; N2 – addition of 50 kg N ha−1 y−1) collected on four occasions duringthe growth season. Values are means ± SE.
Effects of N Fertilization on Phenolics of Healthy Leaves During the GrowthSeason. The concentrations of arbutin, chlorogenic acid, and p-coumaric acidwere reduced by N fertilization (Table 1, Figures 3–7). In healthy plants, N amend-ment reduced the concentration of arbutin in mid-June (Figure 3) and chlorogenicacid (Figure 4) and p-coumaric acid (Figure 5) in mid-July and mid-August(Table 1). Differences in phenolic concentration were detected most often be-tween control and N2-treated plants (Figures 3–7). However, the concentrationsof chlorogenic acid (Figure 4) and catechin (Figure 6) in healthy leaves werereduced by the moderate level of N fertilization in mid-July.
The absolute contents of any of the individual phenolics were generally notsignificantly affected by N additions (Table 1), although the changes generallyfollowed the same trend as that of their concentration (Figures 3–5). However,N significantly reduced the content of chlorogenic acid on N2-treated plots inmid-July (Figure 4).
FIG. 2. Seasonal changes in concentrations and contents of individual phenolic compoundsin healthy leaves of Vaccinium myrtillus on control (unfertilized) plots relative to thelevel of these compounds in mid-June: (a) chlorogenic acid; (b) catechin; (c) arbutin; (d)quercetin-3-glucoside; (e) p-coumaric acid; and (f) an interpretation key to GV analysis.For each individual phenolic compound, concentration and content are expressed as valuesrelative to the levels observed at the first (reference) sampling occasion (mid-June). SeeMethods and Materials for more details on vector diagram construction. The values forthe absolute concentrations (µmol/g DW ± SE) and leaf contents (µmol/leaf ± SE) of thephenolic compounds are indicated along the corresponding axes for the reference sampling(mid-June).
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FIG. 3. Vector diagrams of effects of N fertilization (N0 – control; N1 – addition of 12.5 kgN ha−1 y−1; N2 – addition of 50 kg N ha−1 y−1) on the concentration and content of arbutinin Vaccinium myrtillus measured in healthy leaves (−h) and leaves infected by Valdensiaheterodoxa (−i) on four occasions during the growth season. For each sampling occasion,effects of treatments are represented by vectors, connecting values of the control (N0-h),for which (x = y = 1), with the values of the treatments expressed as values relative tothe corresponding value of the control. See text and Figure 2f for more details on vectordiagram construction. The significance of differences between treatments and the controlis indicated by asterisks (for content/concentration, as marked), where ns, o, ∗, and ∗∗indicate P > 0.1, 0.05 < P < 0.1, P < 0.05, and P < 0.01, respectively (Dunnett’s test).
Effects of N Fertilization on Infection-Associated Changes During the GrowthSeason. Infection by V. heterodoxa significantly affected both the concentrationand absolute content of quercetin-3-glucoside and p-coumaric acid (Table 1).However, the magnitude and direction of phenolic changes in V. heterodoxa-infected leaves depended on the fertilization level (Figures 5 and 7), as indicated
NITROGEN-INDUCED CHANGES IN PHENOLICS OF Vaccinium myrtillus 1947
FIG. 4. Vector diagram of effects of N fertilization on the concentration and content ofchlorogenic acid in Vaccinium myrtillus measured in healthy leaves and leaves infected byValdensia heterodoxa on four occasions during the growth season. See Figure 3 for key tothe treatment codes.
by the significant or marginally significant interactions between the linear trendfor N and infection (Table 1).
In general, the concentration and absolute content of most phenolics tendedto increase in infected leaves of plants growing on N2-plots, but the timing ofincreases seemed to vary compound-specifically. For instance, the concentrationand content of p-coumaric acid and quercetin-3-glucoside were elevated in in-fected leaves collected from N2-plots in mid-July and August (Figures 5 and 7),and those of arbutin and chlorogenic acid in mid-June and July (Figures 3 and 4),respectively (Tukey–Kramer test, P < 0.05). The changes in concentration and
1948 WITZELL AND SHEVTSOVA
FIG. 5. Vector diagram of effects of N fertilization on the concentration and content ofp-coumaric acid in Vaccinium myrtillus measured in healthy leaves and leaves infected byValdensia heterodoxa on four occasions during the growth season. See Figure 3 for key tothe treatment codes.
content of catechin in infected leaves in mid-August, accompanied by increase inleaf biomass (Figure 6) suggest that excess catechin synthesis occurred in infectedleaves during this period. This trend was significant not only in N2-treated butalso in unfertilized plants. These results indicate that in most cases, significantchanges in the allocation of resources to phenolic acids (chlorogenic acid andp-coumaric acid) and polyphenols (quercetin-3-glucoside and catechin) in re-sponse to N treatments and infection occurred during July and mid-August(Figures 3–7). For arbutin, on the other hand, N-induced reduction in allocationhad already occurred in June (see healthy leaves of N2-treated plants in Figure 3).
NITROGEN-INDUCED CHANGES IN PHENOLICS OF Vaccinium myrtillus 1949
FIG. 6. Vector diagram of effects of N fertilization on the concentration and content ofcatechin in Vaccinium myrtillus measured in healthy leaves and leaves infected by Valdensiaheterodoxa on four occasions during the growth season. See Figure 3 for key to the treatmentcodes.
DISCUSSION
Fertilization with N usually accelerates the vegetative growth of plants, sothe developmental stage and size of plants under different N regimes may varyconsiderably (Gebauer et al., 1998). Phenolic levels also show developmental andtemporal variations (Kause et al., 1999; Riipi et al., 2002; Kleiner et al., 2003).Thus, careful selection of plant material is necessary to avoid comparisons ofplants or plant tissues at different developmental stages (Coleman et al., 1994).The fact that we found no significant effect of N treatment, sampling time, or
1950 WITZELL AND SHEVTSOVA
FIG. 7. Vector diagram of effects of N fertilization on the concentration and content ofquercetin-3-glucoside in Vaccinium myrtillus measured in healthy leaves and leaves infectedby Valdensia heterodoxa on four occasions during the growth season. See Figure 3 for keyto the treatment codes.
infection on leaf dry weight suggests that the leaves collected for analyses at dif-ferent times had reached a similar growth phase and size, and, thus, the chemicaldata obtained across the sampling period were comparable. Lack of N effect onleaf size is in agreement with results of other studies that have shown nonsignif-icant increase in growth (shoot biomass and length) of V. myrtillus in responseto N amendment (Richardson et al., 2002; Strengbom, 2002; Strengbom et al.,2002). The smaller size of infected leaves in June suggests that infection had anegative initial impact on leaf growth. This may reflect an increased investment ofresources to processes other than growth, for instance to respiration, which is often
NITROGEN-INDUCED CHANGES IN PHENOLICS OF Vaccinium myrtillus 1951
accelerated during parasite infection (Simons et al., 1999). The higher dry weightof infected leaves later in the growth season may reflect increased accumulation ofstructural components and strengthening of the cell walls as a protective responseagainst parasite invasion.
Our results indicate that marked within-seasonal variation occurred in the lev-els of individual phenolic compounds in healthy leaves of unfertilized V. myrtillus.Dry weights in mid-June and late August were similar, suggesting that the shifts inphenolic levels were not due to the distribution of a constant amount of phenolicsin a varying amount of biomass but rather affected by other processes. The sizeof the pool of any phenolic compound may be affected by production, catabolism,turnover, and transport (Reichardt et al., 1991). However, recent studies indicatethat some plant phenolics may be more stable than previously thought, and thatthey often accumulate in the cells in which they are synthesized (Ruuhola et al.,2001, and references within). Thus, we suggest that the observed within-seasonalincrease in concentration and content of phenolics may be mainly due to within-seasonal increase in their rate of production (cf. Koricheva, 1999; Koricheva andShevtsova, 2002), indicating that in V. myrtillus phenolic biosynthesis is activelyregulated in response to inherent or external cues. The fluctuation in chemicalquality of leaf tissues may act as a constitutive buffer against damage caused byherbivore and pathogen attacks, which peak at different times during the season(cf. Osier et al., 2000).
We found that N fertilization significantly reduced the concentrations ofchlorogenic acid, arbutin, and p-coumaric acid in V. myrtillus leaves, especially inhealthy leaves. Considering the nonsignificant N effect on leaf dry weight, theseresults may suggest that N-induced reduction in concentrations of these com-pounds were primarily caused by reductions in their production (cf. Koricheva,1999; Koricheva and Shevtsova, 2002). This is in agreement with earlier resultsconcerning N effect on phenolics in V. myrtillus leaves (Grellmann, 2001) and withthe general tendency of reduction in phenolic production in woody plants in re-sponse to fertilization (reviewed by Haukioja et al., 1998; Koricheva et al., 1998).However, levels of two of the other analyzed compounds, quercetin-3-glucosideand catechin, were not significantly affected by N, indicating that individual phe-nolics in V. myrtillus leaves show variations in response to N amendment. Observedvariation in response of individual phenolics to N fertilization may be explained bydifferences in their chemical structures and biosynthetic pathways (Haukioja et al.,1998; Koricheva et al., 1998). The lack of an unambiguous N effect on quercetin-3-glucoside and catechin may also indicate that their levels in V. myrtillus were morestrongly influenced by factors other than N availability. For example, flavonolglycosides are important in plant protection against UV light (Booij-James et al.,2000), and light conditions may be a regulator of their synthesis and accumulation.
Our results suggest that phenolic metabolism of V. myrtillus was not markedlyaltered by the moderate N dose applied in our 5-year study. Possibly, vegetative
1952 WITZELL AND SHEVTSOVA
reproduction provides V. myrtillus with buffering mechanisms that attenuate theeffects of moderate N addition on secondary metabolism. Sharing of photosyn-thates and nutrients among ramets is crucial for the success of clonal plants (Oriansand Jones, 2001). Nevertheless, with the higher N dose, more pronounced alter-ations in the secondary metabolism were detected, suggesting that any potentialbuffering capacity is exceeded at a level of 50 kg ha−1 y−1, which is high for bo-real forest ecosystems, but realistic for Central Europe. This finding accentuatesthe importance of cautiously interpreting results of studies (e.g., those testing thevalidity of the predictions of the CNB hypothesis), where the levels of nutritionmanipulations are outside the regime that the plants have evolved to “expect” intheir natural environments.
In vitro effects of these compounds on growth and development ofV. heterodoxa are the subjects of future studies. However, chlorogenic acid, cate-chins, quercetin, and arbutin (or hydroquinone released from it) have been pointedout as antimicrobial agents in earlier literature (Kokubun and Harborne, 1994;Ho et al., 2001; Puupponen-Pimia et al., 2001). We, thus, anticipated that ourcompounds were potential antifungal agents in the V. myrtillus–V. heterodoxa as-sociation. We expected that their constitutive or induced levels might be lowestin fertilized plants, which show the lowest resistance to V. heterodoxa parasite(Strengbom, 2002; Strengbom et al., 2002). Indeed, we found an N-induced re-duction in concentrations of individual phenolics in healthy leaves. If the analyzedphenolics have activity as constitutive antifungal agents in V. myrtillus leaves, suchreductions would partly explain the increased disease susceptibility reported inearlier studies in plants growing on the same N-fertilized plots (Strengbom et al.,2002).
Interestingly, we observed higher concentrations and contents of some indi-vidual phenolics in infected leaves, mainly at the N2 level. Parallel results havebeen reported by Strengbom et al. (2003), who found that the N2 treatment tendsto increase N concentration in V. myrtillus plants whereas C concentrations remainthe same as in unfertilized controls, or are elevated by N addition (Strengbom et al.,2003; J. Strengbom, unpublished results). A plausible biochemical mechanism be-hind the phenolic increases at high N treatment might be that the higher N dose re-sulted in enhanced C fixation or stimulated phenolic synthesis, possibly because ofincreased levels of photosynthetic enzymes and amino acid precursors for phenolicsynthesis (Gebauer et al., 1998; Haukioja et al., 1998; Jones and Hartley, 1999).
The compound-specific and within-seasonal variation in phenolic levels im-ply that an individual phenolic compound may be functional (i.e., have eithernegative or positive effects on the parasite) only at a certain phase of an infection.For instance, the phenolic glycoside arbutin responded most markedly during theinitial, ascospore infection, while the phenolic acids and the flavonoid quercetin-3-glucoside appeared to be involved in defensive or stress responses induced by
NITROGEN-INDUCED CHANGES IN PHENOLICS OF Vaccinium myrtillus 1953
the conidial stage of the fungus. The increased synthesis of catechin in infectedleaves in mid-August, on the other hand, implies that this compound (and theproanthocyanidins derived from it) may interact with the initiation of fungal over-wintering structures (sclerotia). Future studies with controlled inoculations and invitro tests assessing the direct effects of phenolics on the growth and developmentof V. heterodoxa should provide the necessary confirmation or challenge to thesehypotheses.
In our study, the lack of controlled inoculations precludes detection of anycausal relationships between fungal infection and phenolic induction. Thus, thepossible connections between the growth and development of V. heterodoxa andthe observed N-dependent increases in phenolics in infected V. myrtillus leaves arenot yet clear. Phenolic increases in infected plants may represent a defensive re-sponse (Nicholson and Hammerschmidt, 1992; Dixon and Paiva, 1995). However,since the highest incidence of V. heterodoxa has been reported for the N2 treatment(Strengbom, 2002; Strengbom et al., 2002), contribution of the above-mentionedphenolic increases under high N amendment to protection against V. heterodoxaappears limited. Rather, it seems likely that the observed phenolic increases were ageneral stress response to infection that was conditional on the high N amendmentin the environment. Since the fungal infections were not experimentally manipu-lated, and since the detected phenolic responses in infected leaves may representlocal, short-term responses to the parasitic fungus, assessment of their broaderecological importance for the herbaceous layer of the boreal forest ecosystem isnot straightforward. Nevertheless, because all shifts in plant chemistry may changethe quality of plants for consumers (cf. Ball et al., 2000; Hamback et al., 2002), it isconceivable that quantitative changes in phenolics of infected V. myrtillus leavescould have consequences for other pathogens or herbivores as well. Moreover,the changed phenolic content of prematurely fallen, diseased leaves may affectvarious soil-mediated ecological relationships and the microbial flora in the soil(Gallet, 1994; Nilsson et al., 1998; Schimel et al., 1998).
In summary, our results indicate that N-induced reductions in the constitutivelevels of phenolics may be linked to increased susceptibility of boreal V. myr-tillus to V. heterodoxa infection under N amendment (cf. Strengbom et al., 2002).They also confirm that the magnitude and direction of abiotic and biotic effectson phenolic metabolism of V. myrtillus leaves have a marked seasonal compo-nent. Moreover, the changes in levels of certain phenolics in V. myrtillus leavesoccur mainly in plants treated with an N loading that is over 10-fold comparedto the natural N level that the plants are adapted to “expect.” These results em-phasize the importance of sampling on multiple occasions, as well as recognizingthe natural N regimes of the studied plant species, in any study that addressesthe ecological consequences of environmentally induced alterations in plant phe-nolic metabolism.
1954 WITZELL AND SHEVTSOVA
Acknowledgments—We thank Julia Koricheva, Marie-Charlotte Nilsson, Annika Nordin, TorgnyNasholm, and Joachim Strengbom, and anonymous referees for critically reading the article and makingvaluable suggestions to improve it. We also thank John Blackwell for improving the English language.The study was financially supported by research grants from the Academy of Finland (Grant No.48102) and the Jenny and Antti Wihuri Foundation (JW) and by the Swedish Research Council forEnvironment, Agricultural Sciences and Spatial Planning (JW, AS).
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