Allelopathic effect of phenolic acids from humic solutions on two spruce mycorrhizal fungi:Cenococcum graniforme andLaccaria laccata

The aim of this investigation was to determine the impact ofp-hydroxyacetophenone,p-hydroxybenzoic acid, catechol, and protocatechuic acid on respiration of two spruce mycorrhizal fungi:Laccaria laccata andCenococcum graniforme. These phenols are produced byVaccinium myrtillus,Athyrium filixfemina, andPicea abies, predominant species of spruce forests in the Alps, and they are also present in humic solutions at 10^–10 M or 10^–5 M. Respiration of the two fungi was inhibited by the four phenolic acids, even at concentrations ranging from 10^–5 M to 10^–7 M. These data show phenolic acids from humic solutions have biological activity at extremely low concentrations, suggesting a contribution ofV. myrtillus, A. filixfemina, andP. abies to allelopathic inhibition of mycorrhizal fungi.     

Allelopathic potential in bilberry-spruce forests: Influence of phenolic compounds on spruce seedlings

Regeneration failure ofPicea abies in a subalpine bilberry-spruce forest was studied in relation to phenolic compounds, their occurrence and toxicity. Germination bioassays with natural leachates of bilberry (Vaccinium myrtillus) and spruce showed negative effects on root elongation of spruce seedlings. Growth bioassays on litter and humus demonstrated inhibitory effects of these organic layers.p-Hydroxyacetophenone, a spruce-specific metabolite, was isolated in spruce throughfall (10^–6 M), in water extracts of litter (between 1 and 8 µg/g dry wt) and organic layer (less than 1 µg/g dry wt) in addition to tannins and several common phenolic acids. Potential relationships between vegetation cover and phenolic pattern of the soil are discussed, since organic layers under bilberry heath exhibited higher amounts of phenolic acids and tannins than those under spruce.p-Hydroxyacetophenone and caffeic acid reduced, even at 5 × 10^–5 M, spruce seedling growth, especially root development, with additive effects for these two monomers. Autotoxicity involving spruce trees and allelopathy of understory species, mediated byp-hydroxy-acetophenone and other phenolic compounds, including tannins, deserves further attention in regeneration studies.     

Effects of Vaccinium myrtillus on Spruce Regeneration: Testing the Notion of Coevolutionary Significance of Allelopathy

Failure of natural regeneration of conifers, such as subalpine spruce (Picea abies) and black spruce (Picea mariana), has been reported in the presence of dominant ericaceous understory plants of boreal forests of North America, Fino-Scandinavia, and northern Europe. Among other factors such as competition for light and nutrients, conifer regeneration failure has been attributed to allelopathic effects of the understory ericaceous plants. Rabotnov theorized that (the manifestation of) allelopathy is a result of long-term coevolution within established plant communities and that it may have maximum inhibitory effects on introduced species. Our objectives were to determine what components of the understory ericaceous plant, Vaccinium myrtillus, affect spruce regeneration and to test Rabotnov's hypothesis. Field experiments were complemented with laboratory studies in which seed germination and primary growth of the two spruces were used as response variables. We found that P. mariana was generally more affected than P. abies by V. myrtillus allelochemicals, both in field and in vitro experiments. Field germination of P. abies was only 2% and 3% in the undisturbed sowed plots and in Vaccinium-removed sowed plots, respectively, but P. marana did not germinate at all in these treatments. In humus-removed sowed plots, P. abies had 27% germination, while P. marian had only 15%. In a controlled experiment, P. mariana had the highest decrease in dry weight of primary root in the fresh leaf treatment of V. myrtillus (77%), followed by its leaf leachate (71%), humus (29%), and humus leachate (13%). The decreases in root dry weights of P. abies due to these treatments were 67, 47, 30, and 10%, respectively. Our results provide support for Rabotnov's hypothesis. It is possible that both V. myrtillus and Kalmia angustifolia, involved in the growth inhibition process of P. abies and P. mariana, respectively adopted similar "strategies" of allelopathic inhibition of conifers, by allocating a large part of their carbon pool to the production of secondary metabolites.     

Allelopathic effects of phenolic mixtures on respiration of two spruce mycorrhizal fungi

Investigations were conducted to determine whether the allelopathic effect (inhibition of oxygen consumption) of mull- and mor-type humic solutions on ectomycorrhizal fungi was due to the solutions' phenolic contents. In the first experiment, two concentrations (10^–3 M and 10^–7 M) of binary equimolar phenolic mixtures were tested on the oxygen consumption ofLaccaria laccata andCenococcum graniforme. The high concentrations of most of the mixtures induced an increase of fungal respiration, whereas the 10^–7 M treatments all reduced the rate of respiration. In the second experiment, the effects on respiration of the phenolic mixture reproducing mull- and mortype humic solutions were compared with the effects of natural humic solutions. The resulting data suggest that allelopathic effect of humic solutions can at least in part be attributed to their phenolic contents.     

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.     

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.     

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.     

Relationships Between Phenolics and Soil Microorganisms in Spruce Forests: Significance for Natural Regeneration

Plant-produced phenolic compounds inhibit soil microbial activity and are suspected to be involved in the failure of natural regeneration of French subalpine Norway spruce (Picea abies) forests. This work evaluated relationships between phenolic compounds and soil microorganisms in humus collected in spruce forest during winter and summer seasons. After having identified and quantified different phenolics from humus samples, we incubated another set of humus samples with 10 times the concentration of naturally occurring phenolics, and effects on the density of microorganisms were compared to humus with no addition of phenolic compounds. Furthermore, in order to follow the degradation of phenolic compounds by microbes, the concentration of phenolics in the incubated humus samples was determined by HPLC after three and six days of incubation. The results indicate that humus microorganisms use phenolics as a carbon source, since almost all phenolic compounds had disappeared after six days of incubation. Addition of phenolic compounds to the humus samples also affected the soil microbial populations so that bacteria were inhibited in the humus collected both in the winter and in the summer. However, there were differences between the experiments. Fungi and cellulose hydrolyzers were stimulated in the winter humus experiment, while fungi were unaffected and cellulose hydrolyzers were inhibited in the summer experiment. The addition of phenolic compounds stimulated ammonifiers in the summer experiment. We anticipate that the role of soil microorganisms in the problem of natural regeneration failure is probably more important than previously thought.     

Selected Ectomycorrhizal Fungi of Black Spruce (Picea mariana) can Detoxify Phenolic Compounds of Kalmia angustifolia

Allelopathy has been implicated as a factor contributing toward failure of black spruce (Picea mariana) regeneration in Kalmia angustifolia-dominated sites in eastern Canada. Several phenolic acids of Kalmia origin inhibit primary root growth of black spruce. We tested the hypothesis that some well-adapted conifer ectomycorrhizae can degrade and detoxify water-soluble phenolic compounds produced by Kalmia and use the degraded products as a carbon source to stimulate growth. We found that hyphal growth of Paxillus involutus, a common ectomycorrhizal fungus of black spruce, was stimulated by water leachates of Kalmia leaf and litter. An equimolar mixture of three phenolic acids (ferulic, o-coumaric, and o-hydroxyphenylacetic acid), commonly found in Kalmia, had no negative effects on fungal growth at 1 mM concentration. The o-hydroxyphenylacetic (o-HPA) acid, which is known to be toxic to black spruce, was found to stimulate the growth of Laccaria laccata, L. bicolor, and P. involutus (isolates 211804 and 196554) by 38.4, 29.3, 25.0, and 18.9%, respectively, at 1 mM. Pure ferulic, o-coumaric, and o-HPA acids were degraded by 100, 98, and 79.5%, respectively, within 10 d in the presence of P. involutus 211804. However, L. laccata could not tolerate high concentrations of the Kalmia leachates. P. involutus and L. bicolor used o-HPA acid as a carbon source when cultured in noncarbon nutrient medium. The 0.5 and 0.2 mM o-HPA acid inhibited the root growth of black spruce. However, after solutions had been exposed to a culture of P. involutus, they had no significant effect on seedling growth of black spruce. We concluded that some ectomycorrhizal fungi, such as P. involutus and L. bicolor, are able to degrade Kalmia phenolics. Our findings point to a mechanism by which ectomycorrhizal species can control species interactions in higher plants by changing the rhizosphere chemistry.     

Mycorrhizal Colonization and Plant Growth Affected by Aqueous Extract of Artemisia princeps var. orientalis and Two Phenolic Compounds

The effects of an aqueous extract of Artemisia princeps var. orientalis and two phenolic compounds on mycorrhizal colonization and plant growth have been investigated. Greenhouse studies showed that the inhibitory effect of the extract on mycorrhizal colonization and plant growth increased in proportion to the concentration of the extract. When the mycorrhizal test plants were treated with an increasing concentration of phenolic compounds, the mycorrhizal colonization in roots of the test plant and the plant growth were decreased. There were strong indications that mycorrhizal fungi mitigated the inhibitory influence of shoot extract of A. princeps var. orientalis and phenolic compounds.     

Juglone reduces growth,nitrogenase activity,and root respiration of actinorhizal black alder seedlings

European black alder trees [Alnus glutinosa (L.) Gaertn.] fix nitrogen with nodular symbionts and are interplanted with valuable black walnut trees (Juglans nigra L.) to increase soil nitrogen fertility. However, on some soils interplanted alder can be killed by black walnut's allelochemical juglone. In order to better understand the effects of juglone directly on the growth, nitrogen fixation, and root respiration of black alder, we grew nodulated alders hydroponically in a nitrogen-free nutrient solution at juglone levels of 2 × 10^–6, 2 × 10^–5, and 0 molar (M). Results indicate that nitrogenase activity (acetylene reduction) of alders growing in 2 × 10^–3 M juglone was reduced relative to alders without added juglone after one day, and in 2 × 10^–6 M juglone after five days. Root respiration (CO₂ evolution) and the relative increase of plant fresh weight were reduced in the 2 × 10^–5 M juglone treatment. In a related experiment, black alder germinants were grown in Flanagan silt loam soil dosed with 10^–3, 10^–4, and 0 M juglone. The inhibitory effects of 10^–3 M juglone on radicle elongation ceased 22–37 days after juglone treatments were started, suggesting that this soil can readily detoxify juglone.     

Antifungal Diketopiperazines from Symbiotic Fungus of Fungus-Growing Ant Cyphomyrmex minutus

The attine fungus Tyridiomyces formicarum, the symbiont of the fungus-growing ant Cyphomyrmex minutus, produces several antifungal diketopiperazines. This represents the first identification of antifungal compounds from an attine symbiont and contradicts previous suggestions that attine fungi do not produce metabolites with antifungal activity. T. formicarum probably produces antifungal compounds in defense (1) against other fungi that invade the gardens and escape the weeding activity of the ants, or (2) against ant-pathogenic fungi that could harm the host ants. Fungi cultivated by fungus-growing ants may represent a rich source of additional bioactive metabolites.     

Variation of enantiomeric composition of α-pinene in norway spruce,Picea abies,and its influence on production of verbenol isomers byIps typographus in the field

The enantiomeric composition of -pinene in individual Norway spruce trees [Picea abies (L.) Karst.] was determined on a chiral GC column after stereoselective hydroboration-oxidation followed by a reaction with isopropyl isocyanate to form the carbamate derivative. The enantiomeric composition varied considerably between trees of different genetic origin. There was a strong correlation between the chirality of -pinene in host spruce trees and thecis/trans ratio of verbenols found in the hindguts of the bark beetleIps typographus (L.) infesting the trees.Coleoptera; Scolytidae.Parts of this work have been presented at the 14th International Symposium on the Chemistry of Natural Products in Poznan, Poland, July 9–14, 1984.     

Occurrence and Correlations of Monoterpene Hydrocarbon Enantiomers in Pinus sylvestris and Picea abies

The relative amounts and enantiomeric compositions of monoterpene hydrocarbons in branch and trunk xylem, in needles, and in resin from apical buds in 18 Pinus sylvestris trees have been determined and compared with the terpene content in branch xylem and needles of Picea abies. Besides the high amount of (+)-3-carene, an excess of (+)--pinene has been found in P. sylvestris, whereas in P. abies (–)--pinene dominates over (+)--pinene. In P. sylvestris, clear positive correlations were found between (+)--pinene and (+)-camphene in the four tissues analyzed. Good positive correlations were also observed between (–)--pinene and (–)-camphene in the two types of xylem, between (+)--pinene and (+)--pinene in the resin, and between tricyclene and (–)-camphene in resin and needles. In P. abies, positive correlations were found between (+)--pinene and (+)-camphene in the branch xylem and between tricyclene and (–)-camphene as well as between (–)--pinene and (–)-camphene in the needles. Complex relationships between (–)--pinene and (–)--pinene were found both in the P. abies and in the P. sylvestris tissues. The importance of the enantiomeric composition of -pinene for the host selection of Ips typographus, Tomicus piniperda, and Hylobius abietis is discussed.     

Plant Phenolics as Dietary Antioxidants for Herbivorous Insects: A Test with Genetically Modified Tobacco

High foliar phenolics are generally assumed to increase resistance to insect herbivores, but recent studies show that tobacco lines modified to over– and underexpress phenolics do not exhibit higher constitutive resistance to caterpillars. This is contrary to the expectation that ingestion of tobacco phenolics, particularly chlorogenic acid, should cause oxidative stress in herbivores. We investigated free radical production and antioxidant capacity of fresh crushed leaves of tobacco lines exhibiting over a sixfold difference in chlorogenic acid content to test whether high phenolic concentrations are associated with increased production of reactive oxygen species (ROS). The effects of in planta phenolic levels on feeding behavior, growth, biochemical markers of oxidative stress, and the antioxidant capacity of midgut fluid and hemolymph were assessed in tobacco budworm, Heliothis virescens. The experiments showed that high phenolic foliage was more prooxidant than low phenolic foliage, but the net balance in crushed tissue was antioxidant in comparison to buffer and the commercial antioxidant standard, Trolox. In H. virescens, the antioxidant capacity of midgut fluid was also powerful, and caterpillars fed high phenolic foliage did not exhibit the expected markers of oxidative stress in midgut tissues (altered ascorbate ratios, disulfides, or total hydroperoxides). Instead, hemolymph of larvae fed high phenolic foliage exhibited improved total Trolox equivalent antioxidant capacity (TEAC). These results suggest that the elevated foliar phenolics in some plants may have beneficial antioxidant properties for herbivorous insects, much as dietary phenolics do in mammals.     

Disruption of Secondary Attraction of the Spruce Beetle, Dendroctonus rufipennis, by Pheromones of Two Sympatric Species

Capture of spruce beetles, Dendroctonus rufipennis, in multiple-funnel traps baited with frontalin and -pinene was reduced by up to 42% in the presence of synthetic (+)-exo- and (+)-endo-brevicomin, aggregation pheromones of the sympatric species Dryocoeles affaber. (±)-endo-Brevicomin was inhibitory to spruce beetles in two experiments and (±)-exo-brevicomin was inhibitory in one experiment, reducing spruce beetle trap catches by up to 87% and 75%, respectively. Spruce beetle trap catches were also reduced by 85% by (±)- or (+)-ipsdienol, but not by (–)-ipsdienol. Ips tridens, a second sympatric species, produces both enantiomers of ipsdienol in its pheromone blend. Responses by D. affaber to its own pheromone were significantly enhanced by addition of the spruce beetle lure. Enantiospecific pheromones of secondary competing species, or less costly racemic substitutes, may be useful for managing spruce beetles using competitive displacement or exclusion. Baiting susceptible hosts with pheromones of secondary species may enhance attack by secondary species, while partially repelling spruce beetles.     

Sex Pheromone of Nuttall's Sheep Moth, Hemileuca nuttalli, from the Eastern Sierra Nevada Mountains of California

The sex attractant pheromone blend of Hemileuca nuttalli (Lepidoptera: Saturniidae) from the eastern slope of the Sierra Nevada Mountains in California was determined to be a combination of E10,Z12-hexadeca-10,12-dien-1-yl acetate (E10,Z12–16:Ac) and E10,E12-hexadeca-10,12-dien-1-yl acetate (E10,E12–16:Ac). Ratios of the compounds in extracts of female pheromone glands varied around a mean of 100:48, although the ratio was not critical in field trials. Blends of synthetic E10,Z12–16:Ac and E10,E12–16:Ac in 100:50, 50:100, and 100:100 ratios attracted equal numbers of male moths. Field trials also indicated that E10,Z12–hexadeca-10,12-dien-1-ol (E10,Z12–16:OH) and E10,Z12-hexadeca-10,12-dienal (E10,Z12–16:Ald) were antagonistic at rates of 3.3% and 10%, respectively, of the E10,Z12–16:Ac, despite being found in female moth extracts. E10,E12–16:Ac, E10,Z12–16:OH, and E10,Z12–16:Ald all appear to have roles in maintenance of reproductive isolation between H. nuttalli and H. eglanterina.     

Geographic Variation in Sex Pheromone Blend of Hemileuca electra from Southern California

Differences were found in the pheromonally mediated mate location systems of two subspecies of Hemileuca electra, H. electra electra (Hee) and H. e. mojavensis (Hem), from southern California. Hem female pheromone gland extracts contained eight times as much (10E,12Z)-hexadeca-10,12-dienal (E10,Z12–16:Ald) and half as much hexadecyl acetate (16:Ac) as Hee extracts. Relative amounts of the other major component of the pheromone blends, (10E,12Z)-hexadeca-10,12-dien-1-ol (E10,Z12–16:OH) did not differ between the two subspecies. In coupled gas chromatography–electroantennogram studies, responses of male antennae to 1:1:1 mixtures of the three principal components (E10,Z12–16:Ac, E10,Z12–16:OH, E10,Z12–16:Ald) also differed, with Hem antennae producing significantly larger responses to E10,Z12–16:Ald and significantly smaller responses to 10E,12Z–16:Ac than Hee. In field trials, male Hem were attracted to Hem females in preference to Hee females. Males of a second species, H. burnsi, which is sympatric with Hem but not Hee, also were attracted to females of Hee transported to their range. Field tests of blends of synthesized pheromone components confirmed that male Hem preferred E10,Z12–16:Ald ratios of 10–100% of the major component, E10,Z12–16:Ac, whereas males of Hee and H. burnsi responded optimally to ratios of 0.3–1% E10,Z12–16:Ald to E10,Z12–16:Ac. 16:Ac added to lures increased attraction of Hee but not Hem males. The data presented are consistent with reproductive character displacement, whereby the Hem subspecies has modified its pheromone-based mating system to reduce interference from sympatric H. burnsi.     

Pyrrolizidine alkaloids from Senecio jacobaea affect fungal growth

We investigated the growth-reducing effects of pyrrolizidine alkaloids (PAs) from Senecio jacobaea on nine plant-associated fungi (five strains of Fusarium oxysporum, two of F. sambucinum, and two of Trichoderma sp). Fungal growth was monitored on water agar media containing different concentrations of monocrotaline, retrorsine, or a purified extract of PAs from S. jacobaea. The growth rate of six strains was inhibited by PAs at the highest test concentration (3.33 mM), with the magnitude of the inhibition (7–35%) being dependent upon the specific fungus-PA interaction. In general, the PA extract caused the largest inhibition. However, the fungi isolated from S. jacobaea were positively affected by the PA extract (7–9%). Retrorsine N oxide was as effective as retrorsine in its inhibition of mycelium growth.     

Identification of sex pheromone component of spruce budmothZeiraphera canadensis

The analyses of virgin female sex pheromone gland extracts by gas chromatography (GC), GC-electroantennographic detection (GC-EAD) and GC-mass spectrometry (GC-MS) followed by field-trapping experiments, have identified (E)-9-tetradecenyl acetate (E9–14:Ac) as the primary sex pheromone component of the spruce budmoth,Zeiraphera canadensis. Dosages of 1.0–100.0 g ofE9–14:Ac impregnated in rubber septa provide effective trap baits.Lepidoptera: Tortricidae: Eucosminae.