Patterns and sources of leaf tannin variation in yellow birch (Betula allegheniensis) and sugar maple (Acer saccharum)

Leaves from forest-grown sugar maple (Acer saccharum Marsh) and yellow birch (Betula allegheniensis Britt.) trees were analyzed for four tannin measures (hydrolyzable and condensed tannins, total phenolics, and protein binding) at three times during the growing season. Fifteen-year-old half-sib sugar maples from four provenances, representing the geographical extremes of the sugar maple range and growing in a common garden, were examined for the same traits. We found no significant geographic or seed source component to variation in three of the four tannin measures. We found significant seasonal changes in both birch and maple leaf tannins. Withincanopy leaf tannin variation tended to obscure differences between trees in maple, but in birches between-tree differences in leaf tannin content were more readily found. We also found a significant negative correlation between leaf protein binding capacity and leaf wet weight.     

Phenolics in black oak bark and leaves

Catechin, quercitrin, robinin, quercetin 3-methyl ether, scopoletin, cholorogenic acid, several leucoanthocyanins, and condensed and hydrolyzable tannins were identified in bark and leaves ofQuercus velutina Lamarck. The concentrations of most phenolics in leaves increased as the growing season progressed, whereas those of most phenolics in bark remained essentially unchanged. Qualitative differences in bark and leaf phenolics among different trees were negligible.     

Loss of Tannins and Other Phenolics from Willow Leaf Litter

A litter bag experiment was conducted to determine the fate of condensed tannins during the initial phase of willow leaf decomposition. Chemical methods and HPLC were used to identify and quantify the phenolics in aqueous acetone extracts of the leaves during the first eight weeks after leaf fall. Extractable phenolics and tannins were lost rapidly from the leaves and had half lives of about 2.4 weeks. Lower molecular weight tannins were lost more rapidly than higher molecular weight tannins, suggesting that the primary route for loss of tannins is leaching. There was no evidence for decomposition of the tannin during the leaf decay process. After leaching, the tannin apparently sorbs tightly to the soil.     

Methods and pitfalls of extracting condensed tannins and other phenolics from plants: Insights from investigations onEucalyptus leaves

Optimal conditions for extraction of tannins and other phenolics from tree foliage and their subsequent storage rarely have been investigated. We investigated methods of drying leaves, optimal solvents, and the effects of light and temperature on the extractability and stability of condensed tannins (proanthocyanidins) and total phenolics from leaves ofEucalyptus trees. Aqueous acetone was a better solvent than aqueous methanol for condensed tannins and total phenolics, but condensed tannins were less stable in aqueous acetone than aqueous methanol. Stability of condensed tannins also was decreased substantially by room temperature versus 4°C and by exposure to indirect sunlight, although the assay for total phenolics was unaffected. For quantitative estimation of condensed tannins, extraction with 50% acetone was better than methods of direct analysis of leaf tissue. The highest estimates of total condensed tannins were obtained by exhaustive extraction with 50% acetone followed by direct analysis of the residue. Lyophilization of fresh leaf increased yield of condensed tannin (although usually by less than 10%). Lyophilization and subsequent storage of extracts had little effect on assays for condensed tannins or total phenolics.     

Effects of Elevated CO<Subscript>2</Subscript> and Herbivore Damage on Litter Quality in a Scrub Oak Ecosystem

Atmospheric CO₂ concentrations have increased dramatically over the last century and continuing increases are expected to have significant, though currently unpredictable, effects on ecosystems. One important process that may be affected by elevated CO₂ is leaf litter decomposition. We investigated the interactions among atmospheric CO₂, herbivory, and litter quality within a scrub oak community at the Kennedy Space Center, Florida. Leaf litter chemistry in 16 plots of open-top chambers was followed for 3 years; eight were exposed to ambient levels of CO₂, and eight were exposed to elevated levels of CO₂ (ambient + 350 ppmV). We focused on three dominant oak species, Quercus geminata, Quercus myrtifolia, and Quercus chapmanii. Condensed tannin concentrations in oak leaf litter were higher under elevated CO₂. Litter chemistry differed among all plant species except for condensed tannins. Phenolic concentrations were lower, whereas lignin concentrations and lignin/nitrogen ratios were higher in herbivore-damaged litter independent of CO₂ concentration. However, changes in litter chemistry from year to year were far larger than effects of CO₂ or insect damage, suggesting that these may have only minor effects on litter decomposition.     

Inheritance patterns of phenolics in F1, F2, and back-cross hybrids of willows: implications for herbivore responses to hybrid plants

The aim of this study was to determine the inheritance pattern of phenolic secondary compounds in pure and hybrid willows and its consequences for plant resistance to leaf-feeding insects. F1, F2, and back-cross hybrids along with pure species were produced by hand pollination of pure, naturally-growing Salix caprea (L., Salicaceae) and S. repens (L.) plants. Leaf concentrations of condensed tannins and seven different phenolic glucosides were determined by using butanol-HCl and HPLC analyses. Insect herbivore leaf damage was measured on the same leaves as used for chemical analyses. We found hybrids to be approximately intermediate between the parental species: S. caprea with high levels of condensed tannins and no phenolic glucosides, and S. repens with low levels of condensed tannins and high levels of phenolic glucosides. We also found a negative correlation between concentrations of condensed tannins and phenolic glucosides, suggesting a trade-off in production of these two substances. F2 hybrids and the hybrid back-crossed to S. caprea were significantly more damaged by insect herbivores than the parental species and the F1 hybrid, indicating reduced resistance and possibly a selective disadvantage for these hybrid categories.     

Condensed tannins,attine ants,and the performance of a symbiotic fungus

Field experiments indicate that the foliar concentration of condensed tannin affects the selection of leaf material ofInga oerstediana Benth., a tropical legume tree, by leaf cutter ants. In one study an increase in tannin concentration was correlated with a decrease in the acceptability of leaves to leaf-cutter ants, except at low tannin concentrations. Protein concentration was not correlated with acceptability nor was the ratio of protein to tannin. Results from a second study suggest that when the concentration of tannin was low the ants appear to select leaves on the basis of nutrient availability. Laboratory assays with the ants indicated that quebracho tannin, a commercially available condensed tannin, inhibits foraging ants. Again, at lower concentrations, quebracho tannin appeared to have little affect on the ants. The fungus the ants cultivate is a wood-rotting Basidiomycete that produces enzymes, such as polyphenol oxidase (PPO), that are capable of inactivating tannins. The activity of these PPOs may explain why leaf-cutter ants are undeterred by low concentrations of condensed tannins. I hypothesized that PPO activity would be absent from fungal cultures without tannin and that only high concentrations of tannin would inhibit the fungus. Cultures with and without tannin showed similar PPO activity. Thus PPO activity is constitutive. In fact, as fungal biomass increased, so did PPO activity. As hypothesized, only high concentrations of quebracho tannin inhibited PPO activity and fungal growth. However, it is not clear whether the ants can discriminate between concentrations that do and do not inhibit the fungus.     

Do defoliation and subsequent phytochemical responses reduce future herbivory on oak trees?

Perennial plants are thought to respond to partial or complete defoliation by producing new foliage that is less susceptible to herbivores because of induction of allelochemicals. Here, I tested this hypothesis by manually removing primary foliage from branches ofQuercus emoryi (Fagaceae) at two different times in the season and monitoring changes in protein and tannin levels and the amount of herbivory relative to control branches. New, secondary leaves had 2.5 × greater hydrolyzable tannin content than mature foliage of control branches. Condensed tannins, which constitute a relatively low fraction of leaf mass, were lower, while protein content was temporarily greater, in new secondary leaves relative to mature leaves. Despite large increases in hydrolyzable tannins, herbivory levels were greater on refoliated branches than on control branches. New foliage is susceptible to herbivory regardless of when it is produced in the season, possibly because lower toughness and higher water content override any induced or developmentally related changes in allelochemistry. My results do not support the hypothesis that postherbivore changes in phytochemistry protect perennial plants from future herbivory, at least within a growing season.     

Tannin Composition Affects the Oxidative Activities of Tree Leaves

We examined whether tannin composition plays an important role in explaining the oxidative activities of tree leaves of Acer saccharum (sugar maple) and Quercus rubra (red oak). Sugar maple leaves contained substantial amounts of ellagitannins, condensed tannins, and galloyl glucoses, whereas red oak leaves contained almost exclusively condensed tannins. Oxidative activities of the crude phenolics from both species, and the phenolic fractions from sugar maple, were measured with electron paramagnetic resonance (EPR) spectrometry and UV-visible spectrophotometry. The two assays produced similar results: (1) sugar maple phenolics produced larger semiquinone radical concentrations,and higher semiquinone decay rates and browning rates than did red oak phenolics;(2) ellagitannin levels were positively associated with the three measures of oxidative activity; and (3) condensed tannin and galloyl glucose levels were negatively associated with these measures. The negative relationship between condensed tannin levels and oxidative activity resulted from the antioxidant effects of condensed tannins on hydrolyzable tannins; several purified condensed tannins significantly decreased the concentrations of semiquinone radicals and browning rates of pedunculagin (an ellagitannin) and pentagalloyl glucose. As expected, whole-leaf extracts from sugar maple produced elevated levels of semiquinone radicals, but none were observed in red oak extracts when the two species were compared with an EPR time-course assay. We conclude that the oxidative activities of tree leaves may be affected by tannin composition, and that the prooxidant activity of ellagitannins may be decreased by co-occurring condensed tannins.     

Light-induced variation in phenolic levels in foliage of rain-forest plants

Levels of phenolic secondary metabolites in the leaves of four west African rain-forest plants,Acacia pennata, Cynometra leonensis, Diopyros thomasii, andTrema guineensis, were correlated with incident light intensity at both the inter- and intraindividual level. Enhanced phenolic levels under high light intensity appeared to be due to production of both polyphenolics (condensed and hydrolyzable tannins) and simple phenolics. InTrema guineensis, where it is possible to separate leaves in terms of both their age and the light incident upon them, condensed tannin production progressed differently during the development of sun and shade leaves, suggesting continuing production of new oligomers in the former but not in the latter. The results of this study suggest that the production of phenolics in relation to variation in incident light is a finely tuned process, which must be explained in terms of plant physiology and intermediate metabolism rather than in terms of resource allocation or a direct response to herbivory.     

Variation of total phenolic content and individual low-molecular-weight phenolics in foliage of mountain birch trees (Betula pubescens ssp.tortuosa)

We studied seasonal and between-tree variation in the composition and content of total and individual low-molecular-weight phenolics (LMWP) in leaves of mountain birch trees (Betula pubescens ssp.tortuosa). The major phenolic compounds were chlorogenic acid, quercetin-3-O--D-glucuronopyranoside, myricetin-3-O-(5-acetyl)-L-rhamnopyranoside, and 1-O-galloyl--D-(2-O-acetyl)-glucopyranose. The content of total phenolics, as well as the sum of individual LMWP, varied only slightly among trees while variation in contents of individual LMWP was large. Concentrations of almost all phenolics decreased during the growing season but pairwise correlations between individual phenolics remained similar over the whole season indicating tree-specific LMWP profiles over the season. Among flavonoids, the between-tree component of variation was 2.6 times as large as the seasonal component, while for variation of nonflavonoids the between-tree component was larger than the seasonal one. To explain the significant correlations within both flavonoid and nonflavonoid compounds, we discuss the biogenesis of LMWP in birch leaves, as well as their ecological role.     

Factors affecting levels of some phenolic compounds,digestibility, and nitrogen content of the mature leaves ofBarteria fistulosa (Passifloraceae)

Levels of total phenolics, condensed tannins, acid detergent fiber, pepsin/cellulase digestibility, and nitrogen in mature leaves of 26 individuals of the ant-plant,Barteria fistulosa, have been determined. Analysis of the results in terms of the presence or absence of ants and the position of the branch from which the leaves were collected showed no relationship with concentrations of phenolics or fiber and only a weak relationship with digestibility and nitrogen. By contrast, light intensity strongly influenced levels of phenolics, notably condensed tannins, so that mature leaves of individuals growing in direct sunlight were less digestible and appeared to be of lower quality as food for herbivores than did mature leaves of individuals in shaded positions. Possible reasons for the variation in condensed tannin levels are discussed.     

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.     

Effects of bilberry (Vaccinium myrtillus L.) litter on seed germination and early seedling growth of four boreal tree species

Laboratory and greenhouse bioassays were used to test for inhibitory effects of senescent and decomposed leaves and aqueous extract from bilberry (Vaccinium myrtillus L.) against seed germination and seedling growth of aspen (Populus tremula L.), birch (Betula pendula Roth.), Scots pine (Pinus sylvestris L.), and Norway spruce [Picea abies (L.) Karst.]. Aqueous extracts from bilberry leaves were inhibitory to aspen seed germination and seedling growth and also induced root damage and growth abnormalities. Addition of activated carbon removed the inhibitory effects of extracts. Senescent leaves reduced pine and spruce seed germination, but rinsing of seeds reversed this inhibition. Senescent leaves were more inhibitory than decomposed leaf litter, suggesting that the inhibitory compounds in bilberry leaves are relatively soluble and released at early stages during decomposition. Spruce was generally less negatively affected by litter and aqueous extracts than the other tested species. This study indicates that chemical effects of bilberry litter have the potential to inhibit tree seedling recruitment, but these effects were not consistently strong. Phytotoxicity is unlikely to be of critical importance in determining success for spruce seedling establishment.     

Alkaloidal responses to damage inNicotiana native to North America

We performed field tests of alkaloid induction inNicotiana attenuata plants growing in southwestern Utah with mimicry of the two major types of damage inflicted by invertebrate and vertebrate herbivores: leaf damage and stalk removal, respectively. In undamaged plants, seasonal increases in leaf nicotine content occurred at a rate of 0.046% leaf dry mass/day. Leaf damage doubled the accumulation rate to 0.086–0.138% leaf dry mass/day, while stalk removal resulted in a quadrupling of the accumulation rate to 0.206% leaf dry mass/day. These damage-induced increases in nicotine accumulation are significantly larger than between-plant and phenological variations. Leaf damage to the nornicotine-(N. repanda andN. trigonophylla) and anabasine-accumulating (N. glauca)Nicotiana species native to North America resulted in 1.5- to 5-fold increases in their principal leaf alkaloid pools. We conclude that alkaloid induction is not limited to nicotine-accumulatingNicotiana species and that herbivores feeding on previously damaged plants are likely to encounter tissues with alkaloid titers significantly higher than those of undamaged plants.     

Preserving leaves for tannin and phenolic glycoside analyses: A comparison of methods using three willow taxa

Members of the Salicaceae often produce phenolic glycosides and condensed tannins. There is much debate on the best method for the preservation of leaf material prior to chemical analysis. Published results indicate freeze-drying, a method commonly used for tannin analysis, may be inappropriate for phenolic glycosides, unless done in a manner to prevent thawing during the drying process. Another commonly employed method, air-drying, is appropriate for phenolic glycosides but inappropriate for condensed tannins. I present evidence using willow leaves that demonstrates that: (1) leaves freeze-dried in external flasks without temperature control contain lower concentrations of phenolic glycosides (salicortin and 2-cinnamoylsalicortin), (2) air-dried leaves have reduced concentrations of condensed tannins, while (3) vacuum-dried fresh leaves have high concentrations of both phenolic glycosides and condensed tannins. Freeze-drying caused salicortin and 2-cinnamoyl salicortin concentrations to drop by 20 mg/g and 4 mg/g, respectively. Salicin, a product of salicortin and 2-cinnamoyl salicortin degradation, is absent in vacuum-dried leaves, present in air-dried leaves and very high in freeze-dried leaves. Thus, the presence of salicin in this system is an artifact of the preservation technique. Condensed tannin concentrations dropped nearly 20 mg/g when leaves were air-dried. Thus, vacuum-drying fresh leaves allows researchers to quantify phenolic glycosides and condensed tannins from the same leaf material.     

Comparative study of allelopathy as exhibited byProsopis Juliflora swartz andProsopis cineraria (L) druce

The allelopathic effects ofProsopis juliflora were studied both in the laboratory and in nature and compared with that ofProsopis cineraria to understand the chemical nature of allelochemics. Both species occupy the same habitats butP. cineraria does not appear to have any toxic effect on other plants under its canopy.P. juliflora is highly allelopathic and does not allow the growth of any other species. Leaf extracts and leaf leachates ofP. juliflora were inhibitory. Decaying leaves were also inhibitory at early stages of decomposition. Live roots were not found to be inhibitory in cogermination and interplanting of seeds. Chemical investigation of the extracts showed the allelopathic compounds to be phenolic in nature in both the species. Slow decomposition and heavy accumulation of leaf litter belowP. juliflora may possibly result in accumulation of toxic substances in soil layers, inhibiting growth of other species.     

Comparison of Chemical Compositions and Antioxidant Activities of Condensed Tannins From Different Parts of Calliandra haematocephala

In this study, the condensed tannins in 70% acetone extracts of leaf, twig, and stem bark of Calliandra haematocephala were purified by a Sephadex LH-20 column, and their structures were identified using MALDI-TOF-MS and thiolysis-HPLC-ESI-MS analyses. The results showed that the condensed tannin structures varied significantly among C. haematocephala organs. The condensed tannins from leaf were essentially of prodelphinidins, consisting mainly of (epi)gallocatechin and (epi)gallocatechin-3-O-gallate units; the condensed tannins from twig and stem bark were predominately of procyanidins, with catechin, epicatechin, and (epi)catechin-3-O-gallate as the main constitutive units for the former and epicatechin as the main constitutive unit for the latter, respectively. Furthermore, in vitro antioxidant activities of these condensed tannins were evaluated using the ABTS^·+ and FRAP methods. All of the condensed tannins exhibited stronger antioxidant activities compared to those of synthetic antioxidant BHA. For all organs, the two tested activities were found to be the highest in leaf condensed tannins, followed by that in twig condensed tannins and stem-bark condensed tannins. The results suggested that the number of hydroxyl groups on the B-ring and the level of 3-O-galloylation in condensed tannins were closely related with their antioxidant activities, and that the condensed tannins from different parts of C. haematocephala could be used as a natural additive in the food and cosmetic industries.     

Use of chemical variation and predation as plant defenses byEncelia farinosa against a specialist herbivore

Larvae of the monophagous herbivore,Trirhabda geminata, selectively eat particular plants and plant parts of its natural host,Encelia farinosa. Measurements of leaf damage and larval positions on branches through time support this observation. Time-lapse movie photography revealed that larvae are sufficiently mobile to search most of a plant in a 48-hr period and that aggregations were the result of larval activity and not directly the result of oviposition. Experiments withT. geminata larvae on artificial diets containing a range of natural concentrations of chemical extracts fromE. farinosa leaves showed that the larvae grew significantly slower and had a lower overall survivorship at the high concentration. Combining the results of all choice tests, larvae appeared unable to distinguish between high- and low-concentration agar diets. Considered individually, larval preferences for natural production concentrations changed as the season progressed. Early-season larvae preferred low-concentration leaves, while late-season larvae preferred high-concentrations. Measurements of chemical and nitrogen content of leaves selected by larvae in the field confirmed this pattern. Percent parasitism in field-collected larvae increased with season as the larval population decreased. This combination of slowed growth and increasing parasitism and predation is a putative defense strategy ofEncelia farinosa to prevent adaptation by a specialist herbivore to the total range of compounds elaborated.