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.     

Chemosensitivity of lobster,Homarus americanus,to secondary plant compounds: Unused receptor capabilities

Chemosensitivity to secondary plant substances was examined electrophysiologically and behaviorally for the lobsterHomarus americanus. Neurophysiological experiments show that some chemoreceptor cells in the antennules (representing the sense of smell) and walking legs (representing the sense of taste) were excited by secondary compounds from plants of marine and terrestrial origin. These compounds include amygdalin, atropine sulfate, bromoform, caffeine,p-coumaric acid, diiodomethane, ferulic acid, heliotropin, phloroglucinol, quinine sulfate, salicin, sinigrin, tannic acid, and tomatine. The possible behavioral function of three of these compounds was tested. Phloroglucinol and ferulic acid had no observable effect on any aspect of feeding behavior at any concentration tested. Tannic acid, which is related to polyphenols found in marine algae, had no observable effect at any concentration tested on orientation to and grasping of food (activities controlled primarily by antennular and leg chemoreceptors, respectively) but did have an inhibitory effect on food ingestion (an activity controlled primarily by mouthpart chemoreceptors). These electrophysiological and behavioral results suggest that potential chemoreceptive information derived from many secondary plant compounds may not be used in feeding behavior. The receptors sensitive to these compounds may represent a common chemical sense as suggested by Dethier (1980). However, at least one compound, tannic acid, that is smelled and tasted by lobsters can function as a feeding inhibitor at the level of the mouthparts.     

Differences in Effects of Pyrrolizidine Alkaloids on Five Generalist Insect Herbivore Species

The evolution of the diversity in plant secondary compounds is often thought to be driven by insect herbivores, although there is little empirical evidence for this assumption. To investigate whether generalist insect herbivores could play a role in the evolution of the diversity of related compounds, we examined if (1) related compounds differ in their effects on generalists, (2) there is a synergistic effect among compounds, and (3) effects of related compounds differed among insect species. The effects of pyrrolizidine alkaloids (PAs) were tested on five generalist insect herbivore species of several genera using artificial diets or neutral substrates to which PAs were added. We found evidence that structurally related PAs differed in their effects to the thrips Frankliniella occidentalis, the aphid Myzus persicae, and the locust Locusta migratoria. The individual PAs had no effect on Spodoptera exigua and Mamestra brassicae caterpillars. For S. exigua, we found indications for synergistic deterrent effects of PAs in PA mixtures. The relative effects of PAs differed between insect species. The PA senkirkine had the strongest effect on the thrips, but had no effect at all on the aphids. Our results show that generalist herbivores could potentially play a role in the evolution and maintenance of the diversity of PAs.     

Pellioditis pellio: Substituted phenolic compounds as chemoattractants

Vanillin, vanillic acid, and other related compounds are chemoattractants either for nematodes or arthropods. We tested vanillin, vanillic acid, and 2,4-dihydroxybenzaldehyde in an in vitro bioassay system for their ability to attractPellioditis pellio males and females. Females were not attracted to any of the three compounds tested. Males responded to vanillin and vanillic acid, but not to 2,4-dihydroxybenzaldehyde. Males responded to vanillin and vanillic acid when these compounds were present at 10^–5 M concentration. Vanillin at 10^–3 and 10^–7 M concentrations was not attractive.     

Role of Detoxification of Plant Secondary Compounds on Diet Breadth in a Mammalian Herbivore, Trichosurus vulpecula

Theory predicts that mammalian herbivores detoxify different classes of plant secondary compounds via separate metabolic pathways and that generalist herbivores maintain broad diet breadth to avoid overloading individual detoxification pathways. We tested the hypothesis that a generalist marsupial herbivore, the common brushtail possum, Trichosurus vulpecula, can maintain a higher intake of food when allowed to select from two diets containing different profiles of secondary compounds (phenolics and terpenes) than when given access to the diets individually. Diets consisted of a fruit and vegetable mash to which was added ground leaves of either Eucalyptus melliodora or E. radiata. E. melliodora and E. radiata differ in their concentrations and types of secondary compounds. Brushtail possums include these eucalypt species as part of their natural diet. We measured food consumption and detoxification metabolites of possums on these diets. Consistent with the hypothesis, animals presented with a choice of both diets consumed more food than animals given diets singly. One of the two indicators of detoxification, acid load in urine, differed significantly between diets while the other, glucuronic acid, did not. These results provide partial support for the hypothesis that diet breadth is governed by detoxification abilities.     

Forest Successional Stage Affects the Cortical Secondary Chemistry of Three Old Forest Lichens

Three epiphytic old forest lichens (Usnea longissima, Pseudocyphellaria crocata, and Lobaria pulmonaria) were transplanted along a natural shade–sun gradient comprising three successional stages in boreal spruce forests (dense young forest, open old forest, and clear-cut) for one summer. After harvest, extractable secondary compounds were analyzed by high-performance liquid chromatography, and the brown pigmentation in melanic species was quantified by reflectance measurements. Cortical compounds in all species increased from shady young forests to exposed clear-cuts. Usnic acid, the major cortical, secondary compound in U. longissima, showed consistently higher concentration in the clear-cut than in the two forested stands. Pseudocyphellaria crocata and L. pulmonaria, lacking extractable secondary compounds in the cortex, significantly increased their amounts of cortical melanins in well-lit stands. The medullary compounds showed more complex responses. Many were not influenced by environmental conditions during the transplantation, whereas the majority of those that responded showed the lowest concentration in clear-cut transplants. Only a few medullary compounds showed the highest concentration in the clear-cut, and at a low level of significance. The synthesis of UV-B-absorbing usnic acid and melanins seems to be part of an acclimation to increased light exposure. The medullary compounds in studied species barely function as solar screens despite their strong UV-B absorbance.     

Chemical Constituents of the Aquatic Plant Schoenoplectus lacustris: Evaluation of Phytotoxic Effects on the Green Alga Selenastrum capricornutum

Forty-nine secondary metabolites were isolated from aqueous and alcoholic extracts of the aquatic plant Shoenoplectus lacustris. All compounds were characterized based on spectroscopic data. Eleven free and glycosylated low-molecular polyphenols, 17 cinnamic acid and dihydrocinnamic acid derivatives, 11 flavonoids, and 10 C₁₃ nor-isoprenoids were identified. The structure of the new compound, 1-benzoyl-glycerol-2-α-l-arabinopyranoside, was elucidated by 2D NMR experiments (COSY, HSQC, HMBC, NOESY). To evaluate potential phytotoxic effects, all compounds were tested on the green alga Selenastrum capricornutum, a unicellular organism commonly used in tests of toxicity as a bioindicator of eutrophic sites. The most active compound was (−)-catechin, showing an inhibition similar to that of the algaecide CuSO₄.     

Differences in host use efficiency of larvae of a generalist moth, Operophtera brumata on three chemically divergent Salix species

The food selection, growth, and fecundity of insect herbivores are largely dictated by the chemical composition and nutritive values of plant foliage. We studied the host-use efficiency of larvae of the generalist moth, Operophtera brumata (Lepidoptera: Geometridae) on three chemically divergent but nutritively similar willows (Salix spp.). The 4th instars were able to use the salicylate-free leaves of S. phylicifolia efficiently. Growth was slightly reduced on S. pentandra, which contained a moderate level of acetylated salicylates. The high concentration of salicylates found in the leaves of S. myrsinifolia seemed to provide efficient protection against non-specialized O. brumata. We also studied assimilation of nutrients and degradation of salicylates and other secondary compounds in the digestive tract of O. brumata larvae. Neither the assimilation of nitrogen nor of carbon were affected by secondary chemicals of ingested food. Salicylates were shown to be degraded to salicin and catechol, while further degradation of salicin to saligenin was rather slow. In an artificial diet experiment, we showed that two degradation products of salicylates, catechol and saligenin markedly reduced the growth of the larvae. Neither salicin nor chlorogenic acid affected larval growth. We conclude that salicylates reduced the growth of the generalist winter moth mainly by feeding deterrence caused by 6-hydroxy-2-cyclohexenone and catechol. Compared to the deleterious effects of salicylates the effects of other secondary compounds were minor.     

Physiological and behavioral effects of coniferyl benzoate on avian reproduction

Various plant secondary metabolites related to cinnamic acid are of interest because of their repellency to birds and their occurrence in ecologically important food items. Coniferyl benzoate (CB), a phenylpropanoid ester that occurs in quaking aspen (Populus tremuloides) is of particular ecological interest because of its effect on ruffed grouse (Bonasa umbellus) feeding behavior and its possible influence on the population dynamics of this bird. During detoxification processes, CB and other analogous compounds are metabolized into by-products, such as ferulic acid (FA), that can cause anti-reproductive effects. We tested whether consumption of CB produces antire-productive effects similar to FA using male and female Japanese quail (Coturnix coturnix) as avian models for ruffed grouse. The parameters we investigated included: the production, morphology, and development of eggs; reproductive characteristics influenced by estrogen; serum prolactin levels; and male reproductive behavior. Dietary CB did not produce antireproductive effects similar to FA at intake levels that Japanese quail and ruffed grouse would freely consume. Consumption of CB by Japanese quail significantly reduced egg production and body mass but did not affect male reproductive performance. Coniferyl benzoate's effect on egg production may be explained by lower energy acquisition and retention rather than endocrine changes per se. Contrary to previous reports, it is unlikely that FA, or similar compounds act directly as estrogen mimics or antagonists. Although, CB did reduce egg production in quail, it is unlikely that it would affect egg production in wild ruffed grouse. Detoxification costs and the effects of CB on nutrient utilization may explain why ruffed grouse avoid high dietary levels of CB.     

Change in acceptability of barley plants to aphids after exposure to allelochemicals from couch-grass (Elytrigia repens)

The response of the bird cherry-oat aphid, Rhopalosiphum padi, to barley plants was investigated following exposure of the plants to root allelochemicals from the aggressive weed couch-grass, Elytrigia (Agropyron) repens. Plants were treated either with root exudates from living couch-grass plants or with previously identified couch-grass root compounds [5-hydroxyindole-3-acetic acid, DL-5-hydroxytryptophan, L-5-hydroxytryptophan hydrate, and 6-hydroxy-1,2,3,4-tetrahydro--carboline-3-carboxylic acid (carboline)] either separately or in mixtures. In choice and no-choice settling tests, aphid acceptance of barley plants was significantly reduced following treatment with root exudates, and the carboline when tested alone or in combination with the other compounds. In contrast, the other compounds without the carboline were less active in reducing aphid acceptance. In a probing bioassay, individual substances were either neutral or stimulatory to aphids, indicating that the reduced settling was probably not due to direct effects on aphids, but rather due to effects on the plant. This was confirmed in olfactometer assays, in which aphids were repelled by odors from barley plants following treatment with a mixture containing all four chemicals.     

Isolation of the diterpenoids, ent-kauran-16alpha-ol and ent-atisan-16alpha-ol, from sunflowers, as oviposition stimulants for the banded sunflower moth, Cochylis hospes

Two diterpenoid alcohols, ent-kauran-16-ol (1) and ent-atisan-16-ol (2), were isolated from pre bloom (R3-R4 stage) sunflower heads as oviposition stimulants for the banded sunflower moth, Cochylis hospes. Fractionation of a sunflower head extract, by normal-phase flash column chromatography, resulted in an early eluting fraction exhibiting significant activity in an egg-laying bioassay. Compounds 1 and 2, along with ent-trachyloban- 19-oic acid (3) and ent-kaur-16-en-19-oic acid (4), were isolated as the major components of this fraction and identified by their NMR and mass spectra. The purified compounds were individually tested for ovipositional activity in dose-response bioassays. In these bioassays, compounds 1 and 2 gave linear dose responses, with increasing numbers of eggs laid as the dosage of either increased. Compounds 3 and 4 failed to stimulate significant egg-laying at any of the dosages tested. A factorial design bioassay, using compounds 1 and 2, showed that 1 was relatively more stimulatory than 2, and that there was no synergistic effect on oviposition when the two compounds were combined.     

Taste Sensitivity of Insect Herbivores to Deterrents is Greater in Specialists Than in Generalists: A Behavioral Test of the Hypothesis with Two Closely Related Caterpillars

Sensitivity of caterpillars of Heliothis virescens, a generalist, and H. subflexa, a specialist, to eight different plant secondary compounds was examined behaviorally. The compounds were nicotine hydrogen tartrate, hordenine, caffeine, sinigrin, linamarin, arbutin, chlorogenic acid, and salicin. All compounds deterred feeding, at least at the higher concentrations, but the generalist was less affected than the specialist. Thus the hypothesis that specialists have greater sensitivity to deterrents than generalists was supported. In most cases deterrence occurred on first encounter, indicating that the response was sensory; in some cases short-term postingestive effects also appeared to play a role. The larger quantities of deterrent-containing food ingested by H. virescens sometimes resulted in measurable postingestive effects during the second control test. This did not occur in H. subflexa, which more commonly rejected the deterrent-containing food on first contact. The contrast between the species is discussed in relation to tradeoffs involved in different diet breadths.     

The Male Abdominal Glands of <Emphasis Type="Italic">Leucophaea maderae</Emphasis>: Chemical Identification of the Volatile Secretion and Sex Pheromone Function

In Leucophaea maderae, male calling behavior involves the release of a sex pheromone from the abdominal sternal glands. An extract of sternal glands attracted conspecific females over a distance. The compounds present were identified as hydroxy-3-butan-2-one, (2R, 3R)-butanediol, senecioic acid, and (E)-2-octenoic acid. The same components are also present in male tergal glands. The identified compounds were tested on their own and in mixtures. Their biological function is discussed.     

Induction of Phenolic Compounds in Two Cultivars of Cucumber by Treatment of Healthy and Powdery Mildew-infected Plants with Extracts of Reynoutria sachalinensis

Accumulation of phenolic compounds (p-coumaric, caffeic, and ferulic acids and p-coumaric acid methyl ester) was followed in susceptible (Mustang) and tolerant (Flamingo) cucumber (Cucumis sativus) cultivars. The objective was to determine whether these compounds played a role in resistance against powdery mildew following a prophylactic treatment with Milsana (leaf extracts from the giant knot weed Reynoutria sachalinensis, polygonaceae). This treatment significantly reduced the incidence of powdery mildew in both cultivars. Phenolic compounds were extracted from leaves. In the hydrolyzed fraction containing phenolic aglycones, levels of p-coumaric, caffeic, and ferulic acids and of p-coumaric acid methyl ester increased in all treatments (with leaf extracts of R. Sachalinensis, powdery mildew, or both) except the control, one or two days after treatment. In the fraction containing free phenolics, from the tested compounds, only ferulic acid showed an increase in cv. Flamingo (tolerant), and was particularly evident following treatments. On the other hand, the amounts of hydroxycinnamic acids increased rapidly in the two cultivars following Milsana treatment, suggesting their role in disease reduction. All compounds showed antifungal activity when tetsed against common pathogens of cucumber (Botrytis cinerea, Pythium ultimum, and P. aphanidermatum), but in general methyl esters were more fungitoxic than their corresponding free acids. This study suggests that cucumber is able to release antifungal compounds that are instrumental in repressing powdery mildew infection. This response is seemingly independent from the level of genetic resistance associated with each cultivar.     

Inhibition of growth ofPhytophthora parasitica var.nicotianae by aromatic acids and coumarins in a laboratory bioassay

Hydroxy-, dihydroxy-, trihydroxy-, methoxy-, dimethoxy-, hydroxy-+methoxy-, amino-, chloro-, and nitro-substituted benzoic, phenylacetic, phenylpropanoic, and phenylpropenoic (cinnamic) acids were evaluated for activity against the growth ofPhytophthora parasitica var.Nicotianae, Races 0 and 1, in a laboratory bioassay. Several substituted coumarins were also tested. In general, for Race 0, the phenylpropenoic acids were more active (on a millimolar basis), than the corresponding benzoic, phenylacetic, or phenylpropionic acids (9 of 14 series). Among the most active acids wereo-hydroxycinnamic and the chloro- and methoxycinnamic acids. The activities of unsubstituted benzoic and phenylpropionic acids were comparable to the most active compounds tested. Monohydroxyaromatic acids were more active than most dihydroxy acids of the same chain length. Dihydro-3,4-dihydroxycinnamic acid was slightly more active than the corresponding cinnamic acid, while the reverse was true for the mono-p-hydroxycinnamic acid versusp-hydroxyphenylpropionic acid. Coumarin was more active than its hydroxy, methyl, hydroxymethyl, or methoxy derivatives. In general, Race 1 was even more significantly affected by the aromatic acids. Glycosylated coumarins were inactive in the bioassay, compared to their aglycones.     

Lichen Compounds Restrain Lichen Feeding by Bank Voles <Emphasis Type="Italic">(Myodes glareolus</Emphasis>)

Some lichen compounds are known to deter feeding by invertebrate herbivores. We attempted to quantify the deterring efficiency of lichen compounds against a generalist vertebrate, the bank vole (Myodes glareolus). In two separate experiments, caged bank voles had the choice to feed on lichens with natural or reduced concentrations of secondary compounds. We rinsed air-dry intact lichens in 100% acetone to remove extracellular compounds non-destructively. In the first experiment, pairs of control and rinsed lichen thalli were hydrated and offered to the bank voles. Because the lichens desiccated fast, we ran a second experiment with pairs of ground control and compound-deficient thalli, each mixed with water to porridge. Eight and six lichen species were tested in the first and second experiment, respectively. In the first, bank voles preferred compound-deficient thalli of Cladonia stellaris and Lobaria pulmonaria, but did not discriminate between the other thallus pairs. This was likely a result of deterring levels of usnic and stictic acid in the control thalli. When lichens were served as porridge, significant preference was found for acetone-rinsed pieces of Cladonia arbuscula, C. rangiferina, Platismatia glauca, and Evernia prunastri. The increased preference was caused mainly by lower consumption of control thalli. Grinding and mixing of thallus structures prevented bank voles from selecting thallus parts with lower concentration of secondary compounds and/or strengthened their deterring capacity. We conclude that some lichen secondary compounds deter feeding by bank voles.     

Food experience on the predatory behavior of the ant Myrmica rubra towards a specialist moth,Acrolepiopsis assectella

Entomophagous insects are often repelled by the secondary compounds of the plants eaten by their prey. These compounds, therefore, take on a defensive role for the phytophagous species that sequester them. Given that numerous entomophagous species are capable of learning, the effects on the foraging behavior of a repeated experience were investigated in the predatory ant Myrmica rubra. The sulfur amino acids methyl-cysteine sulfoxide (MCSO) and propyl-cysteine sulfoxide (PCSO) produced by Allium plants were identified in caterpillars of the leek moth Acrolepiopsis assectella. Three behavioral studies were carried out, with or without prior familiarization with caterpillars reared either on leek or on an artificial diet containing no Allium compounds. In choice tests with the two types of caterpillars, unfamiliarized ants displayed a preference for caterpillars reared on the artificial diet, but this preference disappeared or was reversed in both young and old ants after familiarization.     

The Effects of Nectar–Nicotine on Colony Fitness of Caged Honeybees

Nectar of many bee flowers contains secondary compounds, which are considered toxic for honeybees on repeated exposure. Although many anecdotal reports indicate the toxicity of secondary compounds to bees, only a few studies have tested the extent of toxicity at different honeybee ages, especially at the larval stages. Honeybees encounter nicotine at trace concentrations (between 0.1 and 5 ppm) in floral nectar of a few Nicotiana spp. and in Tilia cordata. Adult honeybee workers tolerate these nicotine concentrations. In controlled nonchoice feeding experiments with caged bees, we investigated the effect of nicotine on hatching success and larval and forager survival. Naturally occurring concentrations of nectar–nicotine did not affect hatching success of larvae or their survival, but the latter was negatively affected by higher concentrations of nicotine (50 ppm). Concentrations of nicotine in fresh honey samples from the hives were 90% lower than the concentrations in the offered experimental sucrose solutions. Our results indicate that honeybees can cope with naturally occurring concentrations of nicotine, without notable mortality, even when consumed in large quantities for more than 3 weeks.     

Variation in Birch (<Emphasis Type="Italic">Betula pendula</Emphasis>) Shoot Secondary Chemistry due to Genotype,Environment, and Ontogeny

Plant secondary chemistry is determined by both genetic and environmental factors, and large intraspecific variation in secondary chemistry has frequently been reported. The heritability of specific tree secondary metabolites is, however, mostly unknown. We tested the effect of plant genotype, environment, and ontogeny on the variation in shoot secondary chemistry of juvenile and mature European white birches (Betula pendula). Phenolic compounds and triterpenoids were analyzed in 30 naturally regenerated 20-year-old parental trees and micropropagated plantlets that originated from 14 of those same parental trees, planted at four growing sites. Most of the variation for phenolic compounds was explained by differences between parental trees, whereas triterpenoids had a high variation both between parental trees and within the canopy of individual tree. The effect of ontogeny was strong for some individual compounds. In mature trees, the amount of triterpenoids was less than 1 mg/g (DW), whereas the concentration in juvenile plantlets was up to 64 mg/g (DW). Clonal plantlets and parental trees were generally quite similar in their phenolic contents, but there were significant differences for all analyzed compounds among clones. Environment had no significant effect on the accumulation of some compounds, whereas for others, a significant environmental effect and/or significant genotype by environment interaction was found. These results suggest that birch shoot secondary chemistry is under strong genetic control and that the environmental effects depend on the studied chemical trait.     

Responses of braconid parasitoidsMicroplitis croceipes (Cresson) andM. demolitor Wilkinson to stereoisomers of kairomone 13-methylhentriacontane

The responses ofMicroplitis croceipes (Cresson) andMicroplitis demolitor Wilkinson to theR andS stereoisomers of the kairomone 13-meth-ylhentriacontane were evaluated. Singly and in combination at 5, 50-, and 500-ng dosage levels each were tested. There was a linear dosage response by both species to the three dosages. Furthermore, there was no difference between responses to the two stereoisomers or their combination by either species at any dosage level. The effects of the two stereoisomers were fully interchangeable and additive. This is the first report of parasitoid responses to stereoisomers of a kairomone.