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.     

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.     

Relationships betweenDendroctonus micans Kug. (Coleoptera: Scolytidae) survival and development and biochemical changes in Norway Spruce,Picea abies (L.) Karst., phloem caused by mechanical wounding

The bark of Norway spruce trees,Picea abies (L.) Karst., was wounded to produce areas of bark with differing biochemical characteristics. Adults and larvae ofDendroctonus micans Kug. were inserted into these trees at points around the wounds. Larval survival was higher, larval dry weights were higher, and adults were more likely to oviposit at sites around the wound that had higher phloem moisture contents. Larvae showed higher survival and dry weights in phloem with low terpene contents. Adults oviposited in phloem with lower stilbene glucoside contents. Phloem moisture differed at sites around the wound compared with that at a control site away from the wound, and the nitrogen content of entire bark cores was higher above wounds than at control sites away from the wounds. Phloem moisture correlated with the nitrogen content of entire bark. Phloem terpene contents correlated with each other, as did the stilbene contents. Associations between the host exploitation pattern ofD. micans and the biochemistry of the host phloem are discussed in relation to preselection by adults of sites suitable for larval development.     

Degradation of Leaf Litter Phenolics by Aquatic and Terrestrial Isopods

To investigate species-specific decomposition rates of litter from native (Quercus faginea) and introduced (Eucalyptus globulus) tree species in Portugal, we monitored changes in the phenolic signature of leaf litter during decomposition as mediated by an aquatic, Proasellus coxalis (Isopoda: Asellota), and two terrestrial, Porcellio dispar and Eluma caelatum (Isopoda: Oniscidea), detritivores. Although the litter of Eucalyptus and Quercus did not differ in overall protein precipitation capacity, we detected differences in terms of contents of particular phenolic compounds and phenol oxidation products. Accordingly, we observed food-specific consumption rates in Proasellus, but not in the terrestrial isopods. Proasellus digested Eucalyptus at significantly higher rates than Quercus, whereas the opposite was the case for Eluma, and Porcellio digested both litter types equally well. Despite slight differences in detail, effects of Proasellus on changes in the signature of litter phenolics were similar for both litter types, whereas terrestrial isopods—Porcellio and Eluma, although they differed from each other—digestively degraded phenolic compounds in Eucalyptus and Quercus litter, respectively, in different ways. Overall, however, degradation of litter phenolics was similarly effective on both litter types. From these data, we conclude that decomposition of Eucalyptus litter does not proceed more slowly than of litter from native Portuguese trees.     

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.     

Phenolics in black oak bark and leaves

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

Drought Stress Alters the Concentration of Wood Terpenoids in Scots Pine and Norway Spruce Seedlings

Drought is known to have an impact on the resistance of conifers to various pests, for example, by affecting resin flow in trees. Little is known, however, about the quantitative and qualitative changes in resin when trees are growing in low moisture conditions. We exposed Scots pine (Pinus sylvestris L.) and Norway spruce (Picea abies (L.) Karst.) seedlings to medium and severe drought stress for two growing seasons and analyzed the monoterpenes and resin acids in the main stem wood after two years of treatment. In addition to secondary chemistry, we measured the level of nutrients in the needles and the growth response of seedlings. After the first year of treatment, drought stress did not affect the growth of seedlings, but in the second year, shoot growth was retarded, especially in Scots pine. In both conifer species, severe drought increased the concentrations of several individual monoterpenes and resin acids. Total monoterpenes and resin acids were 39 and 32% higher in severe drought-treated Scots pine seedlings than in the controls, and 35 and 45% higher in Norway spruce seedlings. In Scots pine needles, the concentrations of nitrogen and phosphorus increased, while magnesium and calcium decreased compared to controls. In Norway spruce needles, nutrient concentrations were not affected. The results suggest that drought stress substantially affects both the growth of conifers and the chemical quality of the wood. We discuss the potential trade-off in growth and defense of small conifer seedlings.     

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.     

Loss of Tannins and Other Phenolics from Willow Leaf Litter

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

Casuarina cunninghamiana Tissue Extracts Stimulate the Growth of Frankia and Differentially Alter the Growth of Other Soil Microorganisms

Aqueous extracts of host plant Casuarina cunninghamiana tissue altered the in vitro growth of its diazotrophic microsymbiont Frankia and a selection of other soil microorganisms. The growth of actinomycetous Frankia strains, 55005. AvcI1, CesI5, CjI82 001, and Cj was stimulated by aqueous extracts of C. cunninghamiana tissue. Green cladodes (photosynthetic branches), unsuberized roots, and suberized roots were more stimulatory than dry cladodes and seed tissue. Aqueous extracts of green cladodes of C. cunninghamiana most stimulated the growth of Casuarina-derived Frankia strains CjI82 001 and 55005. The growth of isolates of soil bacteria Bradyrhizobium japonicum, Arthrobacter globiformis and Bacillus subtillis and of the soil fungi Penicillium oxalicum and Arthroderma cookiellum was either inhibited or not affected by cladode extracts. Cladode extracts stimulated the growth of the actinomycete Streptomyces albus and the fungus Rhizopus homothallicus. The magnitude (as great as 100%) of the increase in growth caused by tissue extracts for the Casuarina-derived Frankia strains relative to other soil microbes suggests a host-specific enhancement of the microsymbiont.     

Folivory in Fruit Bats: Leaves Provide a Natural Source of Calicum

Leaves are an important dietary source of carbohydrates and protein, and an especially rich source of calcium for bats. Most studies of leaf eating by fruit bats have suggested that only male bats feed on leaves. In this study, 23 wild-caught Tongan fruit bats (Pteropus tonganus) were used in feeding trials conducted in an outdoor enclosure. The number of leaves and percentage of each leaf eaten were recorded for each bat on a daily basis, and these data were then multiplied by a calcium constant that was derived from a chemical analysis of leaves from Callophylum neo-ebudicum. Leaves of C. neo-ebudicum that were available in the enclosure were consumed by 82.7% of the bats. Overall, males consumed leaves in greater quantities and with higher frequency than females. Bats that consumed leaves on a regular basis consumed up to 46% more calcium to their diet compared with bats that did not regularly consume leaves. Leaves may represent a readily available, widely used, concentrated source of minerals for foraging bats, and have the potential to contribute significantly to the total amount of ingested calcium.     

Seasonal Changes in Foliar Terpenes Indicate Suitability Of Douglas-fir Buds for Western Spruce Budworm

The terpene composition of current-year buds of Douglas-fir, Pseudotsuga menziesii (Mirb.) Franco, was analyzed from before budburst to after buds were fully flushed. Terpene composition was measured at weekly intervals for several seasons at eight different locations in the southern interior of British Columbia, Canada. Discriminant functions relating terpene composition to suitability of buds for newly emerged western spruce budworm, Choristoneura occidentalis Freeman, were developed based on terpene profiles of the buds and bioassays measuring the suitability to budworms of a sister group of buds. Changes in percent composition of bud terpenes before and during budburst were closely associated with changes in the suitability of the buds to utilization by budworms at both the tree and site by date levels. Use of a degree-day scale for bud suitability removed much of the year-to-year variation, but remaining differences among sites suggest additional sources of variation influencing the insect-host plant relationship. The success of correctly classifying bud suitability using terpene profiles demonstrates the value of foliar terpenes as indicators of seasonal changes in suitability of Douglas-fir foliage during the critical spring emergence period of western spruce budworm. This indicator could be used to screen individual trees susceptible to budworm damage and identify sites at high risk of damaging defoliation.     

Effect of Phenolic Acids in Soil under and Between Rows of a Prior Sorghum (Sorghum bicolor) Crop on Germination, Emergence, and Seedling Growth of Peanut (Arachis hypogea)

Sorghum bicolor is an allelopathic crop that reduces the yield of succeeding crops. We have assessed its effect on the germination, emergence, and seedling growth of Arachis hypogea sown in soil that had had a prior sorghum cropping. A. hypogea was sown on rows and interrows of a previous sorghum crop in 1997 and 1998 in Senegal. Seedling establishment (germination rate and seedling weight) was better between rows than on rows of the previous crop. The highest concentrations of phenolic compounds occurred in the rows in 1998, while contents of row and interrow soils were similar in 1997. Vanillic acid was the main component of the six chemicals found in 1997 soils, whereas the 1998 soil samples contained mainly p-hydroxybenzoic acid, p-hydroxybenzaldehyde, vanillic, and p-coumaric acids (10 phenolics identified). The germination of peanut seeds in water (control), soil water extracts, and mixtures of pure phenolics (equivalent to those in 1997 and 1998 soil samples) was tested. All extracts inhibited germination compared to controls, but there was no significant difference among treatments, i.e., the inhibition was the same for seeds in soil solutions and those in the respective phenolic mixtures. Similarly, there were no significant differences among the germination rates in soil water extracts of rows and interrows or in the pure phenolic mixtures of rows and interrows. We propose a geometrical sowing pattern for peanuts between the rows of the previous sorghum crop to escape the latter's "allelopathic heritage."     

Potential Allelopathic Activity of Natural Plant Heliannanes: A Proposal of Absolute Configuration and Nomenclature

Proposals for the biogenesis and absolute configuration of 12 heliannanes (based on modified Mosher methodology) are presented. The proposal of biogenesis is discussed for both possible origins—terrestrial or marine. Due to the different skeleton types that have been found, a systematic nomenclature based on the different heterocycle ring size is proposed. Phytotoxicity of compounds 5–11 suggests that these new bioactive sesquiterpenes may be involved in sunflower defense against dicotyledon species, and they could be used as natural herbicide templates.     

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.     

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.     

<Emphasis Type="Italic">Arabidopsis thaliana:</Emphasis> A New Test Species for Phytotoxic Bioassays

Lettuce seeds (Lactuca sativa L.) and other crop species are often used in phytotoxic bioassays that are designed to detect allelochemicals. The seeds of these species are considered ideal because they are readily available, germinate rapidly and uniformly, and are routinely used in laboratories around the world. Despite their common use, however, the seeds of these species are often not as sensitive or responsive to some phytotoxic chemicals as are the seeds of other species. While searching for a more sensitive test species for phytotoxic bioassays, the Columbia ecotype of Arabidopsis thaliana exhibited greater sensitivity to seven potent allelochemicals than did lettuce seeds, which, in some cases, did not respond at all to those substances. Sensitivity satisfies one of the criteria for selecting a test species for bioassays. We now report on the results of our study and offer additional reasons for using A. thaliana seeds.     

Chemical response of Picea glehnii seed-epiphytic Penicillium species to Pythium vexans under in vitro competitive conditions for mycelial growth

The potential protection of Picea glehnii seedlings from damping-off by seed-epiphytic Penicillium species was investigated. We studied the chemical response of seed-epiphytic Penicillium species (Pen. cyaneum, Pen. damascenum, and Pen. implicatum) to Pythium vexans, a damping-off fungus, in vitro. Penicillium species were cultured singly or cocultured with Pyt. vexans for 14 or 18 d, and mycelial growth, pH of culture filtrate, antifungal activity of the culture filtrate against Pyt. vexans, and the amount of antifungal compound produced by each Penicillium species, were examined. The filtrate of both the single culture of Penicillium and the coculture of Penicillium and Pyt. vexans showed antifungal activity against Pyt. vexans. In a coculture with Pyt. vexans, Pen. cyaneum produced an antifungal compound (patulin) as in the single culture. Pen. damascenum cocultured with Pyt. vexans produced an antifungal compound (citrinin), as it did in the single culture and in larger amounts on day 10. Pen. implicatum produced two antifungal compounds, frequentin and palitantin, and the ratio of frequentin (with higher antifungal activity than palitantin) to palitantin was higher in the coculture with Pyt. vexans than in the single culture. Our results indicate that these Penicillium species have the ability to produce antifungal compounds and to keep antifungal activity under competitive condition with Pyt. vexans. The chemical response of these Penicillium species to Pyt. vexans may contribute to protect P. glehnii seedlings from damage by Pyt. vexans.     

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.