Effects of large mammalian herbivores and ant symbionts on condensed tannins of Acacia drepanolobium in Kenya

Condensed tannins have been considered to be important inducible defenses against mammalian herbivory. We tested for differences in condensed tannin defenses in Acacia drepanolobium in Kenya over two years among different large mammalian herbivore treatments [total exclusion, antelope only, and megaherbivore (elephants and giraffes) + antelope] and with four different ant symbiont species on the trees. We predicted that (1) condensed tannin concentrations would be lowest in the mammal treatment with the lowest level of herbivory (total exclusion), (2) trees occupied by mutualist ants that protect the trees most aggressively would have lower levels of tannins, and (3) if chemical defense production is costly, there would be a trade-off between tannin concentrations, growth, and mechanical defenses. Mean tannin concentrations increased from total exclusion treatments to wildlife-only treatments to megaherbivore + antelope treatments. In 1997, condensed tannin concentrations were significantly lower in trees occupied by the ant Crematogaster nigriceps, the only ant species that actively removed axillary buds. Contrary to our prediction, trees occupied by ant species that protect the trees more aggressively against mammalian herbivores did not have lower overall levels of condensed tannins. There was no consistent evidence of a trade-off between tannin concentrations and growth rate, but there was a positive correlation between mean thorn length and mean tannin concentrations across species of ant inhabitants and across herbivore treatments in 1997. Contrary to our expectation, trees had higher tannin concentrations in the upper parts of the canopy where there is little herbivory by mammals.     

Secondary chemistry of hybrid and parental willows: Phenolic glycosides and condensed tannins inSalix sericea,S. eriocephala,and their hybrids

Salix sericea andS. eriocephala differ markedly in secondary chemistry.S. sericea produces phenolic glycosides, salicortin and 2-cinnamoylsalicortin, and low concentrations of condensed tannin. In contrast,S. eriocephala produces no phenolic glycosides, but high concentrations of condensed tannins. Hybrid chemistry is intermediate for both types of chemicals, suggesting predominantly additive inheritance of these two defensive chemical systems from the parental species. However, there is extensive variation among hybrids. This variation may be due to genetic variation among parental genotypes, which genes were passed on, or to subsequent back-crossing. The differences in chemistry are likely to exert a strong effect on the relative susceptibility of hybrid and parental willows to herbivores.     

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.     

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.     

Chemical and Biological Assays to Evaluate Bacterial Inhibition by Tannins

The interactions among purified tannins from three different plants with five strains of ruminal bacteria were investigated. Tannins were purified from crude quebracho (Schinopsis balansae spp.), desmodium (Desmodium ovalifolium), and myrtle (Mirtus communis). The ruminal bacteria studied were Streptococcus bovis JB1, Ruminococcus albus 8, Fibrobacter succinogenes S85, Prevotella ruminicola H₁4, and a recently isolated tannin-tolerant bacterium. Bacterial binding to tannins, rate of tannin binding, and the inhibitory concentrations for each bacterium to each tannin were evaluated. The effects of tannins on glucose utilization and fermentation product formation also were studied. Tannin binding was rapid with little additional binding occurring 10 min after tannin addition. There was variation among bacteria in the amount of tannins bound and in the amount of tannin required to inhibit growth. Condensed tannins from myrtle and desmodium had the highest binding capacity and were most inhibitory to microbes. Absorbance readings from the total phenolics assay were highest for myrtle and lowest for quebracho. Similarly, readings for condensed tannins were highest for desmodium, which also had the largest polymer size. Myrtle and quebracho gave the largest precipitate rings in the radial diffusion assay, a biological assay that measures precipitation of bovine serum albumin by tannins. The relative absorbance values obtained from the total phenolics assay were the most useful predictor of tannin inhibition of microbial growth. The acid–butanol assay and the radial diffusion assay using bovine serum albumin as the precipitable protein were much less useful predictors of tannin biological activity against the bacteria studied.     

The chemistry and development of tannin-based adhesives for exterior plywood

Condensed tannins are phenolic in nature and undergo reaction with formaldehyde to form resins. Tannin/formaldehyde condensates tend to have a low degree of condensation resulting in bonds lacking strength and water resistance. Small amounts of short-length phenol/resorcinol/formaldehyde, phenol/formaldehyde, and urea/formaldehyde polymers are used to increase the degree of polymerization of tannin-formaldehyde resins, decreasing brittleness and increasing water resistance. Furfural is used to plasticize tannin/formaldehyde resins to improve the distribution of stress forces on the glueline, overcoming weakness due to stress concentration. Exterior plywood panels were prepared using these modified tannin/formaldehyde resins as adhesives. The modified tannin/formaldehyde adhesives perform well, have similar characteristics to and are as easy to use as phenol/formaldehyde adhesives, and also have a few advantages, especially tolerance to faster pressing times.     

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.     

Synthesis,characterization, and thermal behaviors of tannin stearates prepared from quebracho and pine bark extracts

Reported is the preparation of various condensed tannin stearates and the associated chemical characterization and thermal properties of these products. Stearate esters of condensed tannins from both quebracho and pine bark extracts were prepared in generally high, isolated yields from reaction with stearic acid chloride. Tannin esterification was confirmed by both Fourier transform infrared and nuclear magnetic resonance (NMR), and the average degree of stearate substitution was calculated from ¹H‐NMR analysis. Product degree of substitution (DS) was observed to proportionately increase with higher stearic acid chloride ratio with maximum DS values of 4.0 and 5.1 achieved for quebracho and pine tannins, respectively. Thermal analysis revealed that tannin stearate products have increased thermal stability with a degradation onset at significantly higher temperature for those samples possessing relatively greater DS. Analysis using differential scanning calorimetry revealed isolated products to consist of multiple components which exhibit interesting melt behaviors, a likely result from their work up on isolation. However, multiple melt features of the individual components in products were lost on heating to give single, broad melt endotherms due to coalescence of sample components. Furthermore, tannin stearate samples with high DS show relatively greater endothermic melting at higher temperature than those samples with lower DS. © 2010 Wiley Periodicals, Inc. J Appl Polym Sci, 2010     

Ecological implications of condensed tannin structure: A case study

Condensed tannins were isolated from bitterbnish (Purshia tridentata) and blackbrush (Coleogyne ramosissima). Structural analyses showed that both tannins were procyanidins of similar polymer length. The overall stereochemistries at C-3 and C-4, however, differed between the two tannins. These changes in stereochemistry resulted in blackbrush tannins being less preferred than bitterbrush tannins when offered to snowshoe hares (Lepus americanus). It is unlikely that differences in protein-precipitating abilities are the cause for the preference of the bitterbrush over the blackbrush tannins. Instead, we hypothesize that condensed tannins may be depolymerized and absorbed following ingestion. Differences in tannin structure can lead to different depolymerized products and rates of depolymerization, both of which may affect herbivore preferences.     

Interactions amongHeliothis virescens larvae,cotton condensed tannin and the CryIA(c) δ-endotoxin ofBacillus thuringiensis

The potential interactions among a plant-produced allelochemical, a phytophagous insect, and an endotoxin produced byBacillus thuringiensis were investigated using purified cotton condensed tannins, the CryIA(c)-endotoxin fromB. thuringiensis subsp.kurstaki strain HD-73, and larvae ofHeliothis virescens. Purified condensed tannin from cotton fed to neonateH. virescens reduced feeding and mortality caused by insecticidal crystals ofB. thuringiensis. In fifth instars, tannin reduced relative growth rate (RGR), relative consumption rate (RCR), but antagonized the effects of the crystal-endotoxin. Tannin did not deter feeding of fifth instars in choice tests with cellulose-ester disks. Masking tannin from interacting with the dietary ingredients of artificial diets and crystal protein by encapsulation in alginate gel suggested that tannin adversely affected feeding after ingestion.These results suggest that insect control tactics that employ-endotoxins in microbial insecticides and transgenic cotton plants may not be compatible when used in conjunction with plants containing high tannin concentrations.     

Differential effects of condensed and hydrolyzable tannin on polyphenol oxidase activity of attine symbiotic fungus

The leaf-cutting antAtta cephalotes is a generalist herbivore of the neotropics and collects leaf material to cultivate a fungus. It appears that this fungus, a Basidiomycete, is responsible for the ability of the ants to utilize most of the available woody plant species. Tannins and other phenolics are ubiquitous secondary chemicals in woody plants, and Basidiomycete fungi produce enzymes, such as polyphenol oxidase, that are capable of polymerizing and inactivating the phenolics. This study evaluates the effects of a condensed and a hydrolyzable tannin on the activity of polyphenoi oxidase and the growth of the fungus. I hypothesized that low concentrations of tannin would not inhibit polyphenol oxidase activity but high concentrations would inhibit the enzyme. Consequently, I predicted that only high concentrations of tannin would inhibit fungal growth. Laboratory assays with the fungus indicated that hydrolyzable tannin (tannic acid) and condensed tannin (quebracho tannin) differ in the mechanism of inhibition. Tannic acid does not inhibit polyphenol oxidase activity but does inhibit fungal growth. Quebracho tannin, however, inhibits both polyphenol oxidase activity and fungal growth. As predicted, both tannic acid and quebracho tannin primarily inhibit the fungus at high concentrations.     

Effects of Acacia condensed tannins on urinary parameters,body mass,and diet choice of an Acacia specialist rodent,Thallomys nigricauda

The aim of this study was to investigate the dietary and physiological effects of condensed tannin ingestion on foregut fermenters, using Thallomys nigricauda, a folivorous rodent, as a model. We initially investigated the variability in physiological parameters, such as daily body mass (DM_b), daily feed intake, daily fecal energy loss (FE), daily energy intake (DEI), daily urine pH, and daily urinary ammonia and urea concentrations, in response to different diets with low condensed tannin levels. This experiment was conducted to identify which physiological variables showed the least variation in the absence of tannin. In a second experiment, we investigated the response of the same dietary and physiological parameters to the effects of high dietary condensed tannin ingestion in T. nigricauda. We hypothesized that DM_b, daily feed intake, FE, and DEI of T. nigricauda would be adversely affected by high dietary tannin content. We predicted that detoxification activity by T. nigricauda would increase at higher tannin levels. Ingestion of tannins affected the nutritional status of T. nigricauda, as shown by a decrease in body mass at high tannin levels. We also found that fewer ammonium ions were excreted in the urine by T. nigricauda, as would be expected if this were a means of regulating metabolic acidosis. The urine produced was more alkaline. This result indicates that T. nigricauda is not metabolizing these allelochemicals. Urea production was initially reduced, indicating conservation of bicarbonate ions that will neutralize blood acidity if there is detoxification. A diet choice experiment showed that tree rats avoid high tannin diets, even to the extent that they lose body mass on an alternative diet. This last-mentioned result is noteworthy because previous studies of the effects of tannins on herbivorous mammals have shown that there is physiological control rather than behavioral avoidance of the negative effects of tannin ingestion.     

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.     

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.     

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.     

Response of total tannins and phenolics in loblolly pine foliage exposed to ozone and acid rain

Tannin and total phenolic levels in the foliage of loblolly pine (Pinus taeda L.) were examined in order to evaluate the effect of atmospheric pollution on secondary plant metabolism. The trees were exposed to four ozone concentrations and three levels of simulated acid rain. Tannin concentration (quantity per gram) and content (quantity per fascicle) were increased in foliage exposed to high concentrations of ozone in both ozone-sensitive and ozone-tolerant families. No effect of acid rain on tannins was observed. Neither total phenolic concentration nor content was significantly affected by any treatment, indicating that the ozone-related increase in foliar tannins was due to changes in allocation within the phenolic group rather than to increases in total phenolics. The change in allocation of resources in the production of secondary metabolites may have implications in herbivore defense, as well as for the overall energy balance of the plant.AAES Journal no. 9-902690P.     

Evaluation of Methods for Measuring Polyphenols in Conifer Foliage

There is a resurgence of interest in the quantification of polyphenols in plant tissues because of their presumed ecological importance in plant–litter–soil and plant–animal interactions. The influence of sample preparation, extracting solvent, foliage quality, and assay method was investigated for the quantification of total phenols and condensed tannins in conifer foliage. Our results suggest that it is not possible to recommend a single optimal protocol for quantification of total phenol and condensed tannin fractions from plant materials. In general, the use of aqueous acetone (50–70% v/v) with freeze-dried materials gave the highest recovery. The Folin-Ciocalteau method for total phenols and the butanol–HCl hydrolysis method for condensed tannins appear superior to other common assays tested. There were large differences (1.4–2.2 times) in the reactivity of purified condensed tannins among species, indicating the importance of an appropriate standard for polyphenol quantification. A solid-state ¹³C NMR method with an improved "interrupted decoupling" pulse sequence yielded the highest concentrations for condensed tannins. Assuming that ¹³C NMR provides an accurate measure of total condensed tannin, the other extraction/assay methods used in this study recovered 50–86% of the condensed tannin fraction. The recovery rate is correlated with the nitrogen content of the foliage, which suggests that the formation of protein–tannin complexes may limit the extractability of condensed tannins. While ¹³C NMR condensed tannin values may give the best value for total condensed tannin concentrations, the water-soluble fraction may have the greatest physiological and/or ecological significance.     

Antinutritional effects and ecological significance of dietary condensed tannins may not be due to binding and inhibiting digestive enzymes

The digestive enzymes alkaline phosphatase and 5-nucleotide phosphodiesterase, solubilized from bovine intestinal mucosa and purified to homogeneity, were found to be strongly inhibited in vitro by condensed tannins (proanthocyanidins) purified from sorghum seeds and from quebracho. Tannin inhibition was prevented and reversed by the detergent Triton X-100 (protein-binding agent), by soluble polyvinylpyrrolidone (tannin-binding agent), or by phosphatidylcholine (membrane component). When tested as a crude particulate membrane fraction more characteristic of their in vivo condition, both enzymes were inhibited much less than either purified enzyme at the same tannin concentration. Because the enzymes appear to be relatively insensitive to inhibition by tannin in conditions which mimic in vivo conditions, and because the proportion of the dietary tannin which is available to interact with these enzymes in the digestive tract is likely to be rather small, we suggest that the antinutritional effects and ecological significance of dietary tannins are not due to tannin inhibition of these or other digestive enzymes by direct binding to them.     

Rapid Herbivore-Induced Changes in Mountain Birch Phenolics and Nutritive Compounds and Their Effects on Performance of the Major Defoliator, Epirrita autumnata

Insect damage changes plant physiology and chemistry, and such changes may influence the performance of herbivores. We introduced larvae of the autumnal moth (Epirrita autumnataBorkh.) on individual branches of its main host plant, mountain birch (Betula pubescens ssp. czerepanovii (Orlova) Hämet-Ahti) to examine rapid-induced plant responses, which may affect subsequent larval development. We measured systemic responses to herbivory by analyzing chemistry, photosynthesis, and leaf growth, as well as effects on larval growth and feeding, in undamaged branches of damaged and control trees. Larvae reared on leaves from intact branches of the herbivore-damaged trees grew faster than those reared on leaves of control trees, indicating systemic-induced susceptibility. Herbivore damage did not lead to systemic changes in levels of primary nutrients or phenolic compounds. The analyses of photosynthetic activity and individual hydrolyzable tannins revealed a reversal of leaf physiology-herbivore defense patterns. On control trees, consumption by E. autumnata larvae was positively correlated with photosynthetic activity; on damaged trees, this correlation was reversed, with consumption being negatively correlated with photosynthetic activity. A similar pattern was found in the relationship between monogalloylglucose, the most abundant hydrolyzable tannin of mountain birch, and leaf consumption. Among the control trees, consumption was positively correlated with concentrations of monogalloylglucose, whereas among herbivore-damaged trees, this correlation was reversed and became negative. Our results suggest that herbivore performance is related to both concentrations of phenolic compounds and photosynthetic activity in leaves. This linkage between herbivore performance, leaf chemistry, and physiology was sensitive to induced plant responses caused by slight herbivore damage.     

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.