Clonal Saplings of Trembling Aspen Do Not Coordinate Defense Induction

Induction of plant chemical defenses in response to insect feeding may be localized to the site of damage or expressed systemically, mediated by signal transduction throughout the plant. Such systemic induction processes have been widely investigated in plants with single stems, but rarely in clonal plants comprised of multiple ramets with vascular connections. For a clonal tree species such as trembling aspen (Populus tremuloides Michx), integration of induced defense within clones could be adaptive, as clones are spatially extensive and susceptible to outbreak herbivores. We used pairs of aspen saplings with shared roots, replicated from three genotypes, to determine whether defense-induction signals are communicated within clones. One ramet in each pair was subjected to a damage treatment (feeding by Lymantria dispar, followed by mechanical damage), and subsequent changes in leaf defensive chemistry were measured in both ramets. Responses to damage varied by defense type: condensed tannins (CTs) increased in damaged ramets but not in connected undamaged ramets, whereas salicinoid phenolic glycosides (SPGs) were not induced in any ramets. Genotypes varied in their levels of CTs, but not in their levels of SPGs, and responded similarly to damage treatment. These results suggest that, even with both vascular and volatile information available, young aspen ramets do not induce defenses based on signals or metabolites from other ramets. Thus, unlike other clonal plant species, aspen do not appear to coordinate defense induction within clones. Lack of coordinated early induction in aspen may be related to the function of CTs in tolerance, rather than resistance.     

Age-Related Shifts in Leaf Chemistry of Clonal Aspen (Populus tremuloides)

Developmental changes in plant structure and function can influence both mammalian and arthropod feeding preferences for many woody plant species. This study documents age-related changes that occur in the leaf chemistry of trembling aspen (Populus tremuloides Michx., Salicaceae) and discusses implications for the herbivore community and ecosystem processes. We collected leaves from replicate ramets from six age classes (1–25+ yr) in each of seven aspen clones growing in south central Wisconsin, USA. Chemical analyses were conducted to determine concentrations of condensed tannins, phenolic glycosides (salicortin and tremulacin), nitrogen, starch, and soluble sugars. Each variable differed significantly among clones and among age classes. On average, condensed tannin concentrations doubled in the first five years and then remained fairly constant among older age classes. Combined phenolic glycoside (salicortin + tremulacin) concentrations were high in the youngest ramets (ca. 19%) and decreased sharply with age. Developmental changes in tannin, salicortin, and tremulacin concentrations exceeded those of nitrogen and carbohydrates. Developmental shifts of this magnitude, and the age-related tradeoff that occurs between condensed tannins and phenolic glycosides, are likely to have significant influence on the herbivore community of aspen and may influence leaf litter decomposition and nutrient cycling.     

Induction of Phenolic Glycosides by Quaking Aspen (<Emphasis Type="Italic">Populus tremuloides)</Emphasis> Leaves in Relation to Extrafloral Nectaries and Epidermal Leaf Mining

We studied the effect of epidermal leaf mining on the leaf chemistry of quaking aspen, Populus tremuloides, during an outbreak of the aspen leaf miner, Phyllocnistis populiella, in the boreal forest of interior Alaska. Phyllocnistis populiella feeds on the epidermal cells of P. tremuloides leaves. Eleven days after the onset of leaf mining, concentrations of the phenolic glycosides tremulacin and salicortin were significantly higher in aspen leaves that had received natural levels of leaf mining than in leaves sprayed with insecticide to reduce mining damage. In a second experiment, we examined the time course of induction in more detail. The levels of foliar phenolic glycosides in naturally mined ramets increased relative to the levels in insecticide-treated ramets on the ninth day following the onset of leaf mining. Induction occurred while some leaf miner larvae were still feeding and when leaves had sustained mining over 5% of the leaf surface. Leaves with extrafloral nectaries (EFNs) had significantly higher constitutive and induced levels of phenolic glycosides than leaves lacking EFNs, but there was no difference in the ability of leaves with and without EFNs to induce phenolic glycosides in response to mining. Previous work showed that the extent of leaf mining damage was negatively related to the total foliar phenolic glycoside concentration, suggesting that phenolic glycosides deter or reduce mining damage. The results presented here demonstrate that induction of phenolic glycosides can be triggered by relatively small amounts of mining damage confined to the epidermal tissue, and that these changes in leaf chemistry occur while a subset of leaf miners are still feeding within the leaf.     

Browse Quality in Quaking Aspen (Populus tremuloides): Effects of Genotype, Nutrients, Defoliation, and Coppicing

The consequences of interactions among genetic, ontogenetic, and environmental factors for the quality of winter-dormant tissues as food for browsing herbivores is poorly understood. We conducted two sequential common garden studies to assess the impacts of intraspecific genetic variation, nutrient availability, prior defoliation, and ontogenetic stage on the chemical quality of winter-dormant tissue in quaking aspen (Populus tremuloides Michx.). In the first study, saplings of 12 aspen genotypes were grown under low and high soil nutrient conditions, with or without two successive seasons of defoliation. Quantity and quality of current year’s twig growth were assessed. Twig production varied among genotypes and declined under low nutrient availability, but showed little response to prior defoliation. Chemical quality of sapling twigs varied substantially among genotypes, and in response to nutrient availability and prior defoliation. Overall, browse quality improved (nitrogen levels increased while phenolic glycoside and condensed tannin levels decreased) after defoliation. Growth and chemical variables exhibited low to moderate clonal repeatability (broad sense heritability) values. Our second study employed the same 12 genotypes, grown under high-nutrient conditions and with or without two seasons of defoliation. The trees were coppiced to produce root sprouts, which were chemically assessed 1 yr later. Rejuvenation via coppicing led to increased levels of nitrogen, phenolic glycosides (salicortin), and tannins in root sprouts, and the magnitude of change varied among aspen genotypes. Signatures of defoliation nearly 2 yr earlier persisted in terms of elevated levels of phenolic glycosides in root sprouts of previously defoliated trees. Aspen forests likely present browsing herbivores with chemically heterogeneous environments because of the interactions of genetic, ontogenetic, and environmental factors that vary over space and time.     

Effects of genotype, nutrient availability, and defoliation on aspen phytochemistry and insect performance

Genetic and environmental variability, and their interactions, influence phytochemical composition and, in turn, herbivore performance. We evaluated the independent and interactive effects of plant genotype, nutrient availability, and defoliation on the foliar chemistry of quaking aspen (Populus tremuloides) and consequences for performance of gypsy moths (Lymantria dispar). Saplings of four genotypes were grown under two conditions of nutrient availability and subjected to three levels of artificial defoliation. Concentrations of all secondary and primary metabolites evaluated responded to at least one or more of the experimental treatments. Of the secondary metabolites, phenolic glycosides were affected strongly by genotype, less so by nutrient availability, and not induced by defoliation. Condensed tannins were strongly dependent upon genotype, soil nutrient availability, and their interaction, and, in contrast to phenolic glycosides, were induced by artificial defoliation. Of the primary metabolites, foliar nitrogen was affected by genotype and soil nutrient availability. Starch concentrations were affected by genotype, nutrient availability, defoliation and interactions among these factors. Foliar water content responded to genotype, nutrient availability, and defoliation, and the effect of nutrient availability depended on genotype. Herbivore performance on these plants was strongly influenced by plant genotype and soil nutrient availability, but much less so by defoliation. Although several of the compound types (condensed tannins, starch, and water) responded to defoliation, quantitative variation in these compounds did not contribute to substantive changes in herbivore performance. Rather, the primary source of variation in insect performance was due to plant genotype (phenolic glycoside levels), while nutrient availability (foliar nitrogen levels) was of secondary importance. These results suggest that genetic variation in aspen plays a major role in determining patterns of insect performance, whereas environmental variation, such as was tested, here is of negligible importance.     

Root Chemistry in Populus tremuloides: Effects of Soil Nutrients,Defoliation, and Genotype

Although genetic, environmental, and G x E effects on aboveground phytochemistry have been well documented in trembling aspen (Populus tremuloides), little work has focused on the same factors affecting tissues underground. Belowground plant defenses are likely important mediators of root-feeding herbivores that can strongly influence plant fitness. We used a common garden of potted aspen trees to explore the individual and interactive effects of soil nutrient availability, foliar damage, genotype, and their interactions, on concentrations of phytochemicals in aspen roots. Our common garden experiment employed 12 aspen genotypes that were planted into either low- or high-nutrient soil environments. Half of the trees were subjected to defoliation for two successive years, while the others were protected from damage. At the end of the growing season after the second defoliation, we harvested the trees to obtain root samples for which we assessed levels of phenolic glycosides, condensed tannins, nitrogen, and starch. Phenolic glycosides were most affected by genotype, while the other root phytochemicals were most responsive to soil nutrient conditions. The effects of defoliation were observed in interaction with soil nutrient environment and/or genotype. Interestingly, the effect of defoliation on phenolic glycosides was mediated by soil nutrients, whereas the effect of defoliation on condensed tannins was observed in concert with effects of both soil nutrients and genotype. Comparison of data from this study with an earlier, related study revealed that concentrations of phenolic glycosides and condensed tannins are lower in roots than leaves, and less responsive to defoliation. That soil nutrient environment affects root phytochemical concentrations is not unexpected given the intimate association of roots and soil, but the complex interactions between soil nutrients, aboveground damage, and genotype, and their effects on root phytochemistry, are intriguing. Variation in root chemistry could have wide-reaching effects on soil microbial communities, nutrient cycling, and herbivores. Additionally, the response of phytochemicals to damage across organs can link different, spatially separated herbivores as they use different parts of the same plant resource.     

Reserves Accumulated in Non-Photosynthetic Organs during the Previous Growing Season Drive Plant Defenses and Growth in Aspen in the Subsequent Growing Season

Plants store non-structural carbohydrates (NSC), nitrogen (N), as well as other macro and micronutrients, in their stems and roots; the role of these stored reserves in plant growth and defense under herbivory pressure is poorly understood, particularly in trees. Trembling aspen (Populus tremuloides) seedlings with different NSC and N reserves accumulated during the previous growing season were generated in the greenhouse. Based on NSC and N contents, seedlings were assigned to one of three reserve statuses: Low N–Low NSC, High N–Medium NSC, or High N–High NSC. In the subsequent growing season, half of the seedlings in each reserve status was subjected to defoliation by forest tent caterpillar (Malacosoma disstria) while the other half was left untreated. Following defoliation, the effect of reserves was measured on foliar chemistry (N, NSC) and caterpillar performance (larval development). Due to their importance in herbivore feeding, we also quantified concentrations of phenolic glycoside compounds in foliage. Seedlings in Low N-Low NSC reserve status contained higher amounts of induced phenolic glycosides, grew little, and supported fewer caterpillars. In contrast, aspen seedlings in High N-Medium or High NSC reserve statuses contained lower amounts of induced phenolic glycosides, grew faster, and some of the caterpillars which fed on these seedlings developed up to their fourth instar. Furthermore, multiple regression analysis indicated that foliar phenolic glycoside concentration was related to reserve chemistry (NSC, N). Overall, these results demonstrate that reserves accumulated during the previous growing season can influence tree defense and growth in the subsequent growing season. Additionally, our study concluded that the NSC/N ratio of reserves in the previous growing season represents a better measure of resources available for use in defense and growth than the foliar NSC/N ratios.     

Performance and Secondary Chemistry of Two Hybrid Aspen (<Emphasis Type="Italic">Populus tremula</Emphasis> L. x <Emphasis Type="Italic">Populus tremuloides</Emphasis> Michx.) Clones in Long-Term Elevated Ozone Exposure

The effects of moderately elevated ozone (ca. 35 ppb) on the growth and secondary chemistry of the leaves of two soil-grown Finnish hybrid aspen (Populus tremula L. x Populus tremuloides Michx.) clones with different ozone sensitivities were studied at an open-air exposure field in Kuopio, Finland. Stomatal conductance, photosynthetic rate, and chlorophyll fluorescence were measured during the third growing season. Foliar phenolic concentrations, ergosterol concentration of fine roots, and final dry mass of the trees were determined at the end of the third growing season. Elevated ozone increased the ectomycorrhizal status of the fine roots but had no effect on gas exchange or on the final biomass of either of the clones, indicating equal sensitivity to ozone and no effect of elevated ozone on the intraspecific competitive ability of the clones after three growing seasons. However, in agreement with the data from potted plants of the same clones after two growing seasons, significant differences between the clones were found in all parameters measured. A negative correlation between growth and high concentrations of foliar phenolics indicated that allocation to secondary chemistry also was costly in terms of growth under high resource availability.     

Consequences of Enriched Atmospheric CO2 and Defoliation for Foliar Chemistry and Gypsy Moth Performance

Elevated concentrations of atmospheric CO₂ are likely to interact with other factors affecting plant physiology to alter plant chemical profiles and plant–herbivore interactions. We evaluated the independent and interactive effects of enriched CO₂ and artificial defoliation on foliar chemistry of quaking aspen (Populus tremuloides) and sugar maple (Acer saccharum), and the consequences of such changes for short-term performance of the gypsy moth (Lymantria dispar). We grew aspen and maple seedlings in ambient (~360 ppm) and enriched (650 ppm) CO₂ environments at the University of Wisconsin Biotron. Seven weeks after budbreak, trees in half of the rooms were subjected to 50% defoliation. Afterwards, foliage was collected for chemical analyses, and feeding trials were conducted with fourth-stadium gypsy moths. Enriched CO₂ altered foliar levels of water, nitrogen, carbohydrates, and phenolics, and responses generally differed between the two tree species. Defoliation induced chemical changes only in aspen. We found no significant interactions between CO₂ and defoliation for levels of carbon-based defenses (phenolic glycosides and tannins). CO₂ treatment altered the performance of larvae fed aspen, but not maple, whereas defoliation had little effect on performance of insects. In general, results from this experimental system do not support the hypothesis that induction of carbon-based chemical defenses, and attendant effects on insects, will be stronger in a CO₂-enriched world.     

Phytochemical Variation in Quaking Aspen: Effects on Gypsy Moth Susceptibility to Nuclear Polyhedrosis Virus

The performance of gypsy moths (Lymantria dispar) feeding on quaking aspen (Populus tremuloides) is strongly influenced by host foliar chemistry and susceptibility to a nuclear polyhedrosis virus (LdNPV), but the relationship of susceptibility to chemistry is poorly understood. We investigated the effects of genetic and resource-mediated variation in phytochemistry on viral pathogenicity. Trees were grown in pots in a common garden. Disks were punched from aspen leaves, inoculated with LdNPV and fed to third instars. Additional leaves were analyzed for levels of nitrogen, starch, phenolic glycosides, and condensed tannins. Despite marked variation among trees in levels of phenolic glycosides and tannins, we observed minimal variation in larval susceptibility to LdNPV. Viral pathogenicity was only weakly (inversely) correlated with tannin concentrations in one of two experiments. These results suggest that differential defoliation of aspen by gypsy moths in the field is due to the direct effects of host chemistry on larval performance rather than to the indirect effects of host chemistry on efficacy of this natural enemy.     

Genotypic Differences and Prior Defoliation Affect Re-Growth and Phytochemistry after Coppicing in <Emphasis Type="Italic">Populus tremuloides</Emphasis>

Although considerable research has explored how tree growth and defense can be influenced by genotype, the biotic environment, and their interaction, little is known about how genotypic differences, prior defoliation, and their interactive effects persist in trees that re-grow after damage that severs their primary stem. To address these issues, we established a common garden consisting of twelve genotypes of potted aspen (Populus tremuloides) trees, and subjected half of the trees to defoliation in two successive years. At the beginning of the third year, all trees were severed at the soil surface (coppiced) and allowed to regenerate for five months. Afterwards, we counted the number of root and stump sprouts produced and measured the basal diameter (d) and height (h) of the tallest ramet in each pot. We collected leaves one and two years after the second defoliation and assessed levels of phenolic glycosides, condensed tannins, and nitrogen. In terms of re-growth, we found that the total number of sprouts produced varied by 3.6-fold among genotypes, and that prior defoliation decreased total sprout production by 24%. The size (d²h) of ramets, however, did not differ significantly among genotypes or defoliation classes. In terms of phytochemistry, we observed genotypic differences in concentrations of all phytochemicals assessed both one and two years after the second defoliation. Two years after defoliation, we observed effects of prior defoliation in a genotype-by-defoliation interaction for condensed tannins. Results from this study demonstrate that genotypic differences and impacts of prior defoliation persist to influence growth and defense traits in trees even after complete removal of above-ground stems, and thus likely influence productivity and plant-herbivore interactions in forests affected by natural disturbances or actively managed through coppicing.     

Effects of Light and Nutrient Availability on Aspen: Growth, Phytochemistry, and Insect Performance

This study explored the effect of resource availability on plant phytochemical composition within the framework of carbon–nutrient balance (CNB) theory. We grew quaking aspen (Populus tremuloides) under two levels of light and three levels of nutrient availability and measured photosynthesis, productivity, and foliar chemistry [water, total nonstructural carbohydrates (TNC), condensed tannins, and phenolic glycosides]. Gypsy moths (Lymantria dispar) and forest tent caterpillars (Malacosoma disstria) were reared on foliage from each of the treatments to determine effects on insect performance. Photosynthetic rates increased under high light, but were not influenced by nutrient availability. Tree growth increased in response to both the direct and interactive effects of light and nutrient availability. Increasing light reduced foliar nitrogen, while increasing nutrient availability increased foliar nitrogen. TNC levels were elevated under high light conditions, but were not influenced by nutrient availability. Starch and condensed tannins responded to changes in resource availability in a manner consistent with CNB theory; levels were highest under conditions where tree growth was limited more than photosynthesis (i.e., high light–low nutrient availability). Concentrations of phenolic glycosides, however, were only moderately influenced by resource availability. In general, insect performance varied relatively little among treatments. Both species performed most poorly on the high light–low nutrient availability treatment. Because phenolic glycosides are the primary factor determining aspen quality for these insects, and because levels of these compounds were minimally affected by the treatments, the limited response of the insects was not surprising. Thus, the ability of CNB theory to accurately predict allocation to defense compounds depends on the response of specific allelochemicals to changes in resource availability. Moreover, whether allelochemicals serve to defend the plant depends on the response of insects to specific allelochemicals. Finally, in contrast to predictions of CNB theory, we found substantial allocation to storage and defense compounds under conditions in which growth was carbon-limited (e.g., low light), suggesting a cost to defense in terms of reduced growth.     

Preservation of salicaceae leaves for phytochemical analyses: Further assessment

The chemistry of the plant family Salicaceae has been of interest to researchers as diverse as chemical ecologists, chemosystematists, and paper chemists. Continuing the debate on proper methods for preservation of plant material prior to analysis, vacuum-drying was recently advocated, because freeze-drying may cause degradation of phenolic glycosides. This study was conducted to clarify the consequences of freeze-drying for foliar secondary chemicals and to evaluate the consequences of vacuum-drying for primary compounds (protein and carbohydrates). Leaves of quaking aspen (Populus tremuloides) were flash-frozen in liquid nitrogen and freeze-dried or vacuum-dried at room temperature. We then analyzed samples for levels of salicortin and tremulacin (phenolic glycosides), condensed tannins, nitrogen, soluble protein, sugars, and starch. Freeze-drying did not alter the concentrations of phenolic glycosides or tannins, relative to vacuum-drying. Freeze-drying did cause a small and inexplicable decline in nitrogen and soluble protein. Vacuum-drying, however, reduced starch concentrations by 38%. We suggest that the vacuum-drying method be used in studies in which carbohydrates are of no interest. For studies measuring carbohydrates, however, freeze-drying is a better alternative, and should effect no changes in levels of secondary compounds if samples are not allowed to thaw during the drying process.     

Interactions between Bacteria And Aspen Defense Chemicals at the Phyllosphere – Herbivore Interface

Plant- and insect-associated microorganisms encounter a diversity of allelochemicals, and require mechanisms for contending with these often deleterious and broadly-acting compounds. Trembling aspen, Populus tremuloides, contains two principal groups of defenses, phenolic glycosides (salicinoids) and condensed tannins, which differentially affect the folivorous gypsy moth, Lymantria dispar, and its gut symbionts. The bacteria genus Acinetobacter is frequently associated with both aspen foliage and gypsy moth consuming that tissue, and one isolate, Acinetobacter sp. R7-1, previously has been shown to metabolize phenolic glycosides. In this study, we aimed to characterize further interactions between this Acinetobacter isolate and aspen secondary metabolites. We assessed bacterial carbon utilization and growth in response to different concentrations of phenolic glycosides and condensed tannins. We also tested if enzyme inhibitors reduce bacterial growth and catabolism of phenolic glycosides. Acinetobacter sp. R7-1 utilized condensed tannins but not phenolic glycosides or glucose as carbon sources. Growth in nutrient-rich medium was increased by condensed tannins, but reduced by phenolic glycosides. Addition of the P450 enzyme inhibitor piperonyl butoxide increased the effects of phenolic glycosides on Acinetobacter sp. R7-1. In contrast, the esterase inhibitor S,S,S,-tributyl-phosphorotrithioate did not affect phenolic glycoside inhibition of bacterial growth. Degradation of phenolic glycosides by Acinetobacter sp. R7-1 appears to alleviate the cytotoxicity of these compounds, rather than provide an energy source. Our results further suggest this bacterium utilizes additional, complementary mechanisms to degrade antimicrobial phytochemicals. Collectively, these results provide insight into mechanisms by which microorganisms contend with their environment within the context of plant-herbivore interactions.     

Gypsy Moth Caterpillar Feeding has Only a Marginal Impact on Phenolic Compounds in Old-Growth Black Poplar

Species of the Salicaceae produce phenolic compounds that may function as anti-herbivore defenses. Levels of these compounds have been reported to increase upon herbivory, but only rarely have these changes in phenolics been studied under natural conditions. We profiled the phenolics of old-growth black poplar (Populus nigra L.) and studied the response to gypsy moth (Lymantria dispar L.) herbivory in two separate field experiments. In a first experiment, foliar phenolics of 20 trees were monitored over 4 weeks after caterpillar infestation, and in a second experiment the bark and foliar phenolics of a single tree were measured over a week. Of the major groups of phenolics, salicinoids (phenolic glycosides) showed no short term response to caterpillar feeding, but after 4 weeks they declined up to 40 % in herbivore damaged and adjacent undamaged leaves on the same branch when compared to leaves of control branches. Flavonol glycosides, low molecular weight flavan-3-ols, and condensed tannins were not affected by herbivory in the first experiment. However, in the single-tree experiment, foliar condensed tannins increased by 10–20 % after herbivory, and low molecular weight flavan-3-ols decreased by 10 % in the leaves but increased by 10 % in the bark. Despite 15 % experimental leaf area loss followed by a 5-fold increase in foliar jasmonate defense hormones, we found no evidence for substantial induction of phenolic defense compounds in old growth black poplar trees growing in a native stand. Thus, if phenolics in these trees function as defenses against herbivory, our results suggest that they act mainly as constitutive defenses.     

Both Gas Chromatography and an Electronic Nose Reflect Chemical Polymorphism of Juniper Shrubs Browsed or Avoided by Sheep

Chemical polymorphism may contribute to variation in browsing damage by mammalian herbivores. Earlier, we demonstrated that essential oil concentration in juniper, Juniperus communis, was negatively associated with herbivore browsing. The aim of the present study was to characterize the volatile chemical composition of browsed and non-browsed J. communis. By using either gas chromatography with flame ionization detection (GC-FID) or an electronic nose device, we could separate sheep-browsed or non-browsed juniper shrubs by their essential oil pattern and complex odor matrix. The main components of the essential oil from J. communis were monoterpenes. We distinguished three chemotypes, dominated either by α-pinene, sabinene, or δ-3-carene. Shrubs belonging to the α-pinene- or sabinene-dominated groups were browsed, whereas all individuals with the δ-3-carene chemotype were unused by the local herbivores. The electronic nose also separated the browsed and non-browsed shrubs indicating that their odor matrix could guide sheep browsing. Responses of sheep could integrate the post-ingestive effects of plant secondary metabolites with sensory experience that stems from odor–phytotoxin interactions. Chemotype diversity could increase the survival rate in the present population of J. communis as certain shrubs could benefit from relatively better chemical protection against the herbivores.     

Inter- and Intra-Specific Variation in Stem Phloem Phenolics of Paper Birch (<Emphasis Type="Italic">Betula papyrifera</Emphasis>) and European White Birch (<Emphasis Type="Italic">Betula pendula</Emphasis>)

Outbreaks of bronze birch borer (BBB) (Agrilus anxius), a wood-boring beetle endemic to North America, have been associated with widespread mortality of birch (Betula spp.). There is substantial inter- and intra-specific variation in birch resistance to BBB. Species endemic to North America, such as paper birch (B. papyrifera), have coevolved with BBB and are more resistant than European and Asian birch species, such as European white birch (B. pendula), which lack an evolutionary history with BBB. Borer larvae feed on stem phloem tissue. Therefore, in search of potential resistance mechanisms against BBB, we compared the constitutive phenolic profile of stem phloem tissue of paper birch with that of European white birch. We also analyzed intraspecific variation in phenolic composition among clones and/or half-siblings of both species. Three phenolics (coumaroylquinic acid, betuloside pentoside A, and a diarylheptanoid hexoside) were detected only in paper birch, and concentrations of six other phenolics were significantly higher in paper birch. These differences may contribute to the high resistance of paper birch to BBB relative to European white birch. There was significant intraspecific variation in four of 17 phenolics found in paper birch and in five of 14 found in European white birch, but clones and half-siblings within each species could not be distinguished by phenolic composition using multivariate analysis.     

Plant Community Chemical Composition Influences Trembling Aspen (<Emphasis Type="Italic">Populus tremuloides</Emphasis>) Intake by Sheep

Nutrients and plant secondary compounds in aspen (Populus tremuloides) may interact with nutrients in the surrounding vegetation to influence aspen use by herbivores. Thus, this study aimed to determine aspen intake and preference by sheep in response to supplementary nutrients or plant secondary compounds (PSC) present in aspen trees. Thirty-two lambs were randomly assigned to one of four molasses-based supplementary feeds to a basal diet of tall fescue hay (N = 8) during three experiments. The supplements were as follows: (1) high-protein (60% canola meal), (2) a PSC (6% quebracho tannins), (3) 25% aspen bark, and (4) control (100% molasses). Supplements were fed from 0700 to 0900, then lambs were fed fresh aspen leaves collected from stands containing high (Experiment 1, 2) or low (Experiment 3) concentrations of phenolic glycosides (PG). In Experiment 2, lambs were simultaneously offered aspen, a forb (Lathyrus pauciflorus), and a grass (Bromus inermis) collected from the aspen understory. Animals supplemented with high protein or tannins showed greater intake of aspen leaves than animals supplemented with bark or the control diet (P < 0.05), likely because some condensed tannins have a positive effect on protein nutrition and protein aids in PSC detoxification. Overall, animals supplemented with bark showed the lowest aspen intake, suggesting PSC in bark and aspen leaves had additive inhibitory effects on intake. In summary, these results suggest that not only the concentration but also the types and proportions of nutrients and chemical defenses available in the plant community influence aspen use by herbivores.     

Leaf Ontogeny Influences Leaf Phenolics and the Efficacy of Genetically Expressed Bacillus thuringiensis cry1A(a) d-Endotoxin in Hybrid Poplar Against Gypsy Moth

We tested the hypothesis that ontogenetic variation in leaf chemistry could affect the efficacy of genetically expressed Bacillus thuringiensis cry1A(a) d-endotoxin, and thus provide spatial variation in (1) foliage protection and (2) selective pressures that could delay the resistance of folivores. Our model consisted of clonal hybrid Populus plants (NC5339). Consumption of foliage and relative growth rates of gypsy moth, Lymantria dispar (L.) increased, and phenolic glycoside concentrations decreased, as leaves from transformed plants containing the cry1A(a) d-endotoxin and nontransformed plants matured from leaf plastochron index (LPI) 1–6. Feeding and growth rates were negatively correlated with phenolic glycosides in both transformed and nontransformed foliage. The presence of the B. thuringiensis d-endotoxin was at most, additive to the effect of the phenolic glycosides. Feeding and growth rates were positively correlated with condensed tannins in transformed foliage, but there was no relationship with condensed tannins in nontransformed foliage. The results indicate that the presence of foliar allelochemicals of poplar can enhance the effectiveness of genetically expressed B. thuringiensis d-endotoxin against gypsy moth larvae. However, the spatial variation in gypsy moth performance in response to the combination of foliar allelochemicals and d-endotoxin was not greater than the effect of ontogenetic variation in foliar allelochemicals alone. These results suggest that for this important pest, foliage protection may be obtained without genetically engineered defenses, and instead, by relying on ontogenetic and clonal variation in allelochemicals. The benefits of combining novel resistance mechanisms with natural ones will depend upon the specific folivore's adaptation to natural resistance mechanisms, such as allelochemicals. Moreover, some of the greatest benefits from transgenic resistance may arise from the need to protect trees from multiple pests, some of which may not be deterred by, or may even prefer, allelochemicals that confer protection from a few species.     

Variation among and within mountain birch trees in foliage phenols,carbohydrates, and amino acids,and in growth ofEpirrita autumnata larvae

Leaf quality of the mountain birch (Betula pubescens ssp.tortuosa) for herbivores was studied at several hierarchical levels: among trees, among ramets within trees, among branches within ramets, and among short shoots within branches. The experimental units at each level were chosen randomly. The indices of leaf quality were the growth rate of the larvae of a geometrid,Epirrita autumnata, and certain biochemical traits of the leaves (total phenolics and individual phenolic compounds, total carbohydrates and individual sugars, free and protein-bound amino acids). We also discuss relationships between larval growth rate and biochemical foliage traits. Larval growth rates during two successive years correlated positively at the level of tree, the ramet, and the branch, indicating that the relationships in leaf quality remained constant between seasons both among and within trees. The distribution of variation at different hierarchical levels depended on the trait in question. In the case of larval growth rate, ramets and short shoots accounted for most of the explained variation. In the case of biochemical compounds, trees accounted for most of the variance in the content of total phenolics and individual low-molecular-weight phenolics. In the content of carbohydrates (total carbohydrates, starch, fructose, glucose, and sucrose) and amino acids, variation among branches was generally larger than variation among trees. Variation among ramets was low for most compounds. No single leaf trait played a paramount role in larval growth. Secondary compounds, represented by phenolic compounds, or primary metabolites, particularly sugars, may both be important in determining the suitability of birch leaves for larvae. If phenols are causally more important, genet-specific analyses of foliage chemistry are needed. If sugars are of primary importance, within-genet sampling and analysis of foliage chemistry are necessary.