Plant phenolics as chemical defenses: Effects of natural phenolics on survival and growth of prairie voles (Microtus ochrogaster)

Very few studies have shown experimentally that plant chemical defenses actually reduce the performance of individual mammalian herbivores, much less the density of mammalian populations. We investigated the effects of representatives of three classes of plant phenoiics on the survival and growth of prairie voles by incorporating the compounds into artificial diets and feeding them to weanlings for three weeks. At low levels of protein, both quercetin (a flavonoid) and tannic acid (a hydrolyzable tannin) caused reduced growth rates; no effect occurred at high levels of protein. Quebracho (a condensed tannin) inhibited feeding and thus was lethal at all levels of protein. These results indicate that plant phenolics are likely to influence the performance and dynamics of natural populations of microtine rodents by reducing the quality of available forage. The hypothesis that the primary mode of action of the phenoiics is the reduction of digestibility of protein was not supported. The reduced growth caused by both quercetin and tannic acid could be attributed primarily to their toxicity. The effect of quebracho resulted from reduced intake (unpalatability).     

Role of phenolics of coniferous trees as deterrents against debarking behavior of meadow voles (Microtus pennsylvanicus)

Preferences of meadow voles (Microtus pennsylvanicus) toward various coniferous species were tested under winter conditions. Cafeteria tests showed preferences for Norway spruce (Picea abies) and Norway pine (Pinus resinosa) and rejection of white pine (Pinus strobus) and white spruce (Picea glauca). When phenolic extracts of these species were prepared and added to a low 8% protein diet, food intake of voles and preferences by rank followed the same patterns. Determination of the concentration of phenolics in branch samples using the Singleton and Rossi procedure showed that Norway pines contained the smallest amount (2.15%/g dry matter) while spruces yielded the most (2.76%/g dry matter). These experiments lead us to expect high losses of Norway pine and Norway spruce in habitats harboring high vole densities.     

Preference of meadow voles (Microtus pennsylvanicus) for conifer seedlings: Chemical components and nutritional quality of bark of damaged and undamaged trees

Meadow voles (Microtus pennsylvanicus Ord.) of the 1987–1988 population density peak left several seedling plantations of Norway spruce (Picea abies) and Norway pine (Pinus resinosa) severely damaged in southern Quebec, Canada, while white spruce (Picea glauca) and white pine (Pinus strobus) suffered no damage. We compared levels of crude protein, total nonstructural carbohydrates (TNC), total phenols, and monoterpenes in the bark to detect differences between damaged and undamaged seedlings within and between plantations. There were no significant differences in levels of nutritional components between both types of seedlings within the same plantation. However, differences were noted among tree plantations for protein, TNC, and phenolics content but these differences could not be assigned to the voles' selection for particular species. Quantitative and qualitative differences also were registered in monoterpene levels between species. -Myrcene and bornyl acetate were found only in seedlings of undamaged species. Moreover, the latter contained higher levels of limonene. We conclude that the presence of specific monoterpenes is more important in defense mechanisms of conifer seedlings than relative levels of nutrients (protein or TNC) or total phenolics.     

Effect of birch (Betula pendula) bark and food protein level on root voles (Microtus oeconomus): I. food consumption,growth, and mortality

The effect of protein and birch bark powder (BBP) content of forage on food consumption and growth of root voles (Microtus oeconomus) was studied by feeding young voles with eight different diets for two weeks. Voles ate more when the protein content of forage was moderate (6%) or low (3%), and when BBP was added to the diets, as compared to a high (12%) protein diet without BBP addition. On high and moderate protein diets voles gained weight, whereas on low protein diets they lost weight. Addition of BBP caused reduced growth or weight loss. Consuming a diet with moderate (6%) protein and high BBP (20%) resulted in high mortality (9 of 10 died). These results show that root voles need at least 6% protein for growth and that the effect of birch bark depends on the protein content of the forage.     

Effects of resource manipulation on the correlation between total phenolics and astringency in Douglas-fir

The correlation between total phenolics and tannin content within a species is often considered to be suitably strong to allow researchers to assume, with some degree of confidence, that levels of one will approximately parallel the other. However, the manipulation of resource availability could lead to disproportionate changes in total phenolics and tannins and/or in the specific monomers of which these fractions are composed, thus altering the correlation between these components. In order to test this hypothesis, we examined the correlation between foliar levels of total phenolics (as measured by the ferric chloride assay) and tannins (as measured by an astringency assay) in Douglas-fir (Pseudotsuga menziesii Mirb. Franco) before and after the manipulation of nitrogen and water availability. Prior to manipulation of resources, the correlation between total phenolics and tannins was strong and highly significant (r²=0.869;P < 0.001). This correlation was considerably weaker and not statistically significant following resource manipulation (r² = 0.392; 0.20 <P < 0.50). These results demonstrate that manipulation of resource availability can alter the correlation between total phenolics and tannins in intraspecific comparisons. The causes underlying the observed degradation in the correlation between these measures (whether qualitative, quantitative, or both) are unknown and require further investigation.     

Effects of dietary protein and lupine alkaloids on growth and survivorship ofSpodoptera eridania

Toxic chemicals and nutrients are often positively correlated within and among plants. We studied how such correlations affect the suitability of plants as food for herbivores by assessing the growth and survivorship ofSpodoptera eridania (army worm) on artificial diets containing lupine alkaloids and casein. We found that (1) the effects of casein were determined by other dietary components: increased dietary casein led to increased larval growth only when the diet was also high in wheat germ. (2) Dietary alkaloids were effective at very low concentrations, reducing both growth and survivorship. The alkaloids lupanine and sparteine were not synergistic in their effects, and the interaction between alkaloids and casein was significant only in the low-wheat-germ diets. (3) The effects of casein and alkaloids were generally apparent only in the first instar, and the growth of fifth-instar larvae was unaffected by diet. (4) Using these artificial diet experiments, we can make simple predictions about the food quality of plants grown under various nutrient regimes. These predictions are consistant with recent ideas about optimal plant defenses.     

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.     

The effects of chronic tannic acid intake on prairie vole (Microtus ochrogaster) reproduction

The hypothesis was tested that the reproductive performance of voles would be reduced when fed diets containing tannins either because of increased metabolic rates, decreased intake, or decreased digestive efficiency. We fed a ration containing 4% tannic acid (TA) (dry mass basis) to 24 pair of prairie voles (Microtus ochrogaster) and compared reproductive performance (litter size, birth weights, body mass of the young until weaning, and mass changes in the dams) to that of 24 pair of prairie vole fed a control ration. We also compared the intake rates, digestive efficiency, and metabolic rates [as measured by Vo₂ consumption (cm³/hr)] of dams and young fed both rations. Voles consuming 4% TA diets produced litters of similar size and mass as did voles fed the control ration. Furthermore, the mass of the young of dams fed the tannic acid ration were similar to the young of dams consuming the control ration through day 19 postpartum. However, dams consuming the tannic acid ration lost mass throughout lactation while the control dams maintained mass. Because the Vo₂ rates of both treated and control dams and litters were similar, we conclude increased metabolic costs were not the reason for the observed mass loss but, rather, reduced digestive efficiency, reduced intake of digestible dry mass, and apparent digestible nitrogen.     

Effects of birch phenolics on a grazing and a browsing mammal: A comparison of hares

Mountain hares (Lepus timidus L.) commonly have high proportions of birch (Betula spp.) in their winter diets, whereas European hares (Lepus europaeus) do not. The effects of a birch extract added to laboratory diets offered to mountain hares and European hares on the digestibility and sodium balance were measured. The extract added contained total phenolics equivalent in amount to that occurring in diets containing 0, 40, 60 and 80% dry matter of birch twigs. Increasing dietary phenolic concentration led to reduced voluntary food intake and apparent protein digestibility in both hare species. No effects on apparent dry matter digestibility were observed. The highest concentration of birch extract caused severe sodium losses via the urine by European hares but not by mountain hares. The effects of the birch extract within the digestive system appear to be similar in the two hare species, whereas the mountain hare appears to be better adapted to the toxic effects that disrupted sodium balance in the European hare. These preliminary results suggest that detoxification rather than digestive abilityper se may contribute to different mammalian herbivores' feeding strategies.     

Allelopathic Effects of Humus Phenolics on Growth and Respiration of Mycorrhizal Fungi

Allelopathy due to humus phenolics is a cause of natural regeneration deficiency in subalpine Norway spruce (Picea abies) forests. If inhibition of spruce germination and seedling growth due to allelochemicals is generally accepted, in contrast there is a lack of knowledge about phenolic effects on mycorrhizal fungi. Thus, this work tested effects of a humic solution and its naturally occurring phenolics on the growth and respiration of two mycorrhizal fungi: Hymenoscyphus ericae (symbiont of Vaccinium myrtillus, the main allelochemical-producing plant) and Hebeloma crustuliniforme (symbiont of P. abies, the target plant). Growth and respiration of H. crustuliniforme were inhibited by growth medium with the original humic solution (–6% and –30%), respectively, whereas the same humic solution did not affect growth but decreased respiration of H. ericae (–55%). When naturally occurring phenolics (same chemicals and concentrations in the original humic solution) were added to the growth medium, growth of H. crustuliniforme was not affected, whereas that of H. ericae significantly increased (+10%). We conclude that H. ericae is better adapted to the allelopathic constraints of this forest soil than H. crustuliniforme and that the dominance of V. myrtillus among understory species could be explained in this way.     

Effect of birch (Betula pendula) bark and food protein level on root voles (Microtus oeconomus): II. detoxification capacity

The effect of protein and birch bark powder (BBP) content of forage on detoxification capacity of root voles (Microtus oeconomus) was studied. Young voles were fed with eight different diets for two weeks. Individuals on low (3%) protein diets had significantly lighter livers and kidneys than those on moderate (6%) or high (12%) protein diets. Birch bark powder addition did not have significant effect on organ weight. Detoxification was significantly induced, apparently due to secondary compounds in BBP. The activity of ethoxyresorufin-O-dealkylase (EROD) was high when protein content or BBP concentration in forage was high as compared to low protein diets or diets containing no BBP. Glucuronidation, on the other hand, was not induced by BBP. High BBP content caused serious physiological stress to the voles. The only individuals surviving were those capable of sufficiently allocating energy and protein to detoxification.     

Effects of phenolic acids on germination and early growth of herbaceous woodland plants

Four herbaceous plant species from woodland (clearings),Deschampsia flexuosa, Scrophularia nodosa, Senecio sylvaticus, andChamaenerion angustifolium, were tested for their sensitivity to phenolic acids. Seven commonly occurring phenolic compounds were used in a germination experiment in concentrations ranging from 0.01 to 10 mM, i.e., salicylic,p-hydroxybenzoic, syringic, caffeic, vanillic,p-coumaric, and ferulic acids. Germination was delayed rather than inhibited. Radicle elongation was strongly affected; at lower concentrations stimulatory effects were observed, whereas at high concentrations radicle elongation was severely reduced. Salicylic acid was the most effective phenolic compound, whereas caffeic acid caused no effects. Early growth was studied in more detail in a second experiment withDeschampsia flexuosa andSenecio sylvaticus and the phenolic acids, ferulic and p-coumaric acid. Primary root length, number and length of secondary roots, and dry weight were stimulated at 0.01 mM but were inhibited at 10 mM of both compounds. The results are discussed in view of the allelopathic relations between trees and herbaceous understory vegetation.     

Effects of biotic and abiotic stress on induced accumulation of terpenes and phenolics in red pines inoculated with bark beetle-vectored fungus

This study characterized the chemical response of healthy red pine to artificial inoculation with the bark beetle-vectored fungusLeptographium terebrantis. In addition, we sought to determine whether stress altered this induced response and to understand the implications of these interactions to the study of decline diseases. Twenty-five-year-old trees responded to mechanical wounding or inoculation withL. terebrantis by producing resinous reaction lesions in the phloem. Aseptically wounded and wound-inoculated phloem contained higher concentrations of phenolics than did constitutive tissue. Trees inoculated withL. terebrantis also contained higher concentrations of six monoterpenes,-pinene,-pinene, 3-carene, limonene, camphene, and myrcene, and higher total monoterpenes than did trees that were mechanically wounded or left unwounded. Concentrations of these monoterpenes increased with time after inoculation. Total phenolic concentrations in unwounded stem tissue did not differ between healthy and root-diseased trees. Likewise, constitutive monoterpene concentrations in stem phloem were similar between healthy and root-diseased trees. However, when stem phloem tissue was challenged with fungal inoculations, reaction tissue from root-diseased trees contained lower concentrations of-pinene, the predominant monoterpene in red pine, than did reaction tissue from healthy trees. Seedlings stressed by exposure to low light levels exhibited less extensive induced chemical changes when challenge inoculated withL. terebrantis than did seedlings growing under higher light. Stem phloem tissue in these seedlings contained lower concentrations of-pinene than did nonstressed seedlings also challenge inoculated withL. terebrantis. It is hypothesized that monoterpenes and phenolics play a role in the defensive response of red pine against insect-fungal attack, that stress may predispose red pine to attack by insect-fungal complexes, and that such interactions are involved in red pine decline disease. Implications to plant defense theory and interactions among multiple stress agents in forest decline are discussed.To whom correspondence should be addressed at USDA Forest Service, 2500 Shreveport Hwy., Pineville, Louisiana 71360.     

Effects of basic modifiers on sfe efficiencies of ephedrine derivatives from plant matrix

The effect of basified modifiers in supercritical CO₂ (SCF-CO₂) such as diethylamine has been investigated on the SFE efficiencies of ephedrine derivatives (e.g. methylephedrine, norephedrine, ephedrine, and pseudoephedrine) from aerial parts ofEphedra sinica. The addition of basified modifiers in SCF-CO₂ showed a greater enhancement of SFE efficiency of ephedrine derivatives than pure modifiers. These results might be due to the fact that the salts ofEphedra alkaloids (insoluble in CO₂) in plant tissues would be changed to their free bases (freely soluble in CO₂) by basified modifiers such as diethylamine in methanol. In addition to enhancing the solubilities, it could increase the degree of desorption of the compounds from plant matrix. Also, the SCF-CO₂ modified with methanol basified with diethylamine showed a difference of extractability of ephedrine and its diastereomer, pseudoephedrine. This paper was presented at The 5th International Symposium on Separation Technology-Korea and Japan held at Seoul between August 19 and 21, 1999.     

A comparison of the effectiveness of predator odor and plant antifeedant in deterring small mammal feeding damage on lodgepole pine seedlings

The influence of mustelid (weasel) anal-gland compounds (semiochemicals) and a plant antifeedant (pinosylvin) on snowshoe hare (Lepus americanus) and meadow vole (Microtus pennsylvanicus) feeding damage was investigated. This study has shown conclusively that when applied to spring-planted lodgepole pine seedlings, pinosylvin is effective in significantly reducing feeding damage. Seedlings treated with pinosylvin suffered damage levels of 2.3% and 21.2% compared to the controls with damage levels of 24.5% and 41.0%. Weasel scent was found to reduce damage levels, but the reduction was not statistically significant in all blocks.     

Effects of nitrogen fertilization on secondary chemistry and ectomycorrhizal state of Scots pine seedlings and on growth of grey pine aphid

Effects of nitrogen availability on secondary compounds, mycorrhizal infection, and aphid growth of 1-year-old Scots pine (Pinus sylvestris L.) seedlings were studied during one growing season. Seedlings were fertilized with nutrient solutions containing low, optimum, and two elevated (2 × and 4 × optimum) levels of NH₄NO₃. At the end of growing season foliar nitrogen concentration, needle biomass, needle length, water contents of needles, root collar diameter, and number of buds increased with enhanced nitrogen availability. Addition of nitrogen did not have effect on concentrations of monoterpenes in growing needles, but in mature needles significantly decreased concentrations of some individual and total monoterpenes were detected. In growing needles the concentrations of some individual resin acids decreased, and in mature needles concentrations of some individual and total resin acids increased with increased nitrogen fertilization. Higher numbers of resin ducts were found in mature needles with nitrogen fertilization. Nitrogen fertilization decreased total phenolic concentrations in growing and mature needles of the current year, but in needles of the previous year no significant differences occurred. Mycorrhizal infection was highest at medium (optimum and 2 × optimum) nitrogen fertilization levels. The relative growth rate (RGR) of grey pine aphid [Schizolachnus pineti (F.)] responded positively to the increase in foliar nitrogen content. However, the increase in aphid performance between optimum and the highest fertilization level was slight. This may indicate a deterring effect of resin acids on aphids. The results indicate that carbon/nutrient balance hypothesis fails to predict directly the effects of nitrogen availability on concentrations of carbon-based defensive compounds in mature foliage. Altered nitrogen supply affects allocation to secondary metabolites differently, depending on the developmental state of the plant and the biosynthesis pathway, cost of synthesis, and storage of compounds.     

Feeding responses ofChilo partellus (Swinhoe) (Lepidoptera: Pyralidae) larvae to sorghum plant phenolics and their analogs

Phagostimulatory responses of third-instar larvae ofChilo Partellus to phenolic components identified in an ethyl acetate extract of the leaf whorls of 3-week-old plants ofSorghum bicolor cultivar IS 18363 were studied in no-choice bioassays. The major components in the extract were identified as 4-hydroxybenzaldehyde and 4-hydroxybenzoic acid, with 4-hydroxy-3-methoxycinnamic acid, 3,4-dihydroxycinnamic acid, and 4-hydroxycinnamic acid present in minor amounts. All but 4-hydroxycinnamic acid were stimulatory at the doses tested. 4-Hydroxybenzaldehyde was more stimulatory than other potential biogenetic analogs. Hydroxybenzoic acids generally elicited greater feeding response than cinnamic acids, and the pattern of oxygen substitution in the benzene ring was related to bioactivity.     

Stability of phenolic and protein measures in excised oak foliage

The stability of protein and phenolic measures in excised foliage from two oak species was measured under conditions that simulated the handling and treatment of foliage during insect rearing trials. Excised foliage kept hydrated under refrigeration or insect-rearing conditions maintained stable levels of protein content, proanthocyanidins, gallotannins, total phenolics, and protein-binding capacity for up to 48 hr following field sampling. Measures of protein content, total phenolics, protein-binding capacity, and proanthocyanidins were significantly greater 48–72 hr after field sampling, followed by declines to near field levels within 120 hr.     

Effects of mineral nitrogen andBradyrhizobium inoculation on growth and iron nutrition of groundnut

Experiments were conducted in a glasshouse to determine the effects of the mineral N supplied as ammonium nitrate andBradyrhizobium inoculation on the growth and iron nutrition of nodulating and non-nodulating groundnut (Arachis hypogaea L.) lines. In a sterilized sand-vermiculite medium supplied with N-free nutrient solution (pH 7.0), inoculation of nodulating groundnut withBradyrhizobium strain NC 43.3 enhanced dry matter production and O-phenanthroline extractable iron and N contents of the plants. The supply of mineral N at a rate of 100 mg N L^–1 (as NH₄NO₃) through deionized water (pH 8.5) induced iron chlorosis symptoms in the nodulating groundnuts grown in Vertisols, but these symptoms were not observed at higher N levels (200–400 mg N L^–1). The induced chlorosis was only partially corrected by inoculation withBradyrhizobium strains NC 92 and NC 43.3. The iron deficiency chlorosis was, however, corrected by application of higher rates of ammonium nitrate.Submitted as JA No. 942 of the International Crops Research Institute for the Semi-Arid Tropics (ICRISAT)