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.     

Inheritance patterns of phenolics in F1, F2, and back-cross hybrids of willows: implications for herbivore responses to hybrid plants

The aim of this study was to determine the inheritance pattern of phenolic secondary compounds in pure and hybrid willows and its consequences for plant resistance to leaf-feeding insects. F1, F2, and back-cross hybrids along with pure species were produced by hand pollination of pure, naturally-growing Salix caprea (L., Salicaceae) and S. repens (L.) plants. Leaf concentrations of condensed tannins and seven different phenolic glucosides were determined by using butanol-HCl and HPLC analyses. Insect herbivore leaf damage was measured on the same leaves as used for chemical analyses. We found hybrids to be approximately intermediate between the parental species: S. caprea with high levels of condensed tannins and no phenolic glucosides, and S. repens with low levels of condensed tannins and high levels of phenolic glucosides. We also found a negative correlation between concentrations of condensed tannins and phenolic glucosides, suggesting a trade-off in production of these two substances. F2 hybrids and the hybrid back-crossed to S. caprea were significantly more damaged by insect herbivores than the parental species and the F1 hybrid, indicating reduced resistance and possibly a selective disadvantage for these hybrid categories.     

Effects of mixtures of phenolic acids on phosphorus uptake by cucumber seedlings

To determine how individual phenolic acids in a mixture might affect phosphorus (P) uptake, 15-day-old cucumber seedlings grown in solution culture were treated with ferulic, vanillic,p-coumaric, or equimolar mixtures of these phenolic acids. Phenolic acid and P uptake were determined by solution depletion. The joint action of the mixtures of these phenolic acids on P uptake was primarily additive. Thus, as the number of phenolic acids increased in the mixture, the concentrations of the individual phenolic acids in the mixture required to bring about a given response declined. Seedling uptake of individual phenolic acids from solution mixtures of phenolic acids was reduced when compared to the uptake of phenolic acids from single phenolic acid solutions. The magnitude of the reduction varied with phenolic acid and concentration. The dose required for 50% inhibition of P uptake was approximately two to three times higher for vanillic acid (6.73 mM) than for ferulic (2.27 mM) andp-coumaric acids (3.00 mM) when dose was based on the initial treatment concentrations. The dose required for 50% inhibition of P uptake was not significantly different for the three phenolic acids (42 ± 5 mol/g root fresh weight) when dose was based on phenolic acid uptake. Potential reasons for these differences are discussed.Paper No. 12527 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, North Carolina 27695-7643. The use of trade names in this publication does not imply endorsement by the North Carolina Agricultural Research Service of products named, nor criticism of similar ones not mentioned. This research was partially supported by the US-Spain Joint Committee for Scientific and Technological Cooperation project CCA-8309/166.     

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.     

Benefits of Citrate Over EDTA for Extracting Phenolic Acids from Soils and Plant Debris

The effectiveness of various citrate extractions to recover ferulic acid, a phenolic acid, from Cecil A_p and B_t soil and plant surface debris (crimson clover, rye, subterranean clover, and wheat) was compared with that of EDTA extractions. Citrate extractions were equivalent to or better than EDTA in recovering phenolic acids from soil and plant debris. Citrate, unlike EDTA, did not interfere with the Folin & Ciocalteu's phenol reagent when determining total phenolic acid content in soil and plant debris extracts. Care, however, must be taken when using Folin & Ciocalteu's phenol reagent to estimate total phenolic acid content, particularly in the presence of soil organic matter. Citrate was also more effective in extracting phenolic acids from A-horizon soils. Thus, citrate extractions overcome some of the major limitations observed for EDTA extractions.     

Environmental and biotic factors affecting the phenolic content of different cultivars ofSorghum bicolor

Levels of phenolic acids in healthy plants ofSorghum bicolor differ considerably with cultivar and always decrease as the plant matures. Laboratory- and field-grown plants show significant differences in phenolics. Environmental factors, particularly light intensity, influence the concentration of phenolics in sorghum. Attack by insects and pathogenic fungi also increase the phenolic content of the plants to varying degrees dependent on the cultivar and the stage of growth of the plant.     

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.     

Phenolic Compounds in Red Oak and Sugar Maple Leaves Have Prooxidant Activities in the Midgut Fluids of <Emphasis Type="Italic">Malacosoma disstria</Emphasis> and <Emphasis Type="Italic">Orgyia leucostigma</Emphasis> Caterpillars

Phenolic compounds are generally believed to be key components of the oxidative defenses of plants against pathogens and herbivores. However, phenolic oxidation in the gut fluids of insect herbivores has rarely been demonstrated, and some phenolics could act as antioxidants rather than prooxidants. We compared the overall activities of the phenolic compounds in red oak (Quercus rubra) and sugar maple (Acer saccharum) leaves in the midgut fluids of two caterpillar species, Malacosoma disstria (phenolic-sensitive) and Orgyia leucostigma (phenolic-tolerant). Three hypotheses were examined: (1) ingested sugar maple leaves produce higher levels of semiquinone radicals (from phenolic oxidation) in caterpillar midgut fluids than do red oak leaves; (2) O. leucostigma maintains lower levels of phenolic oxidation in its midgut fluids than does M. disstria; and (3) phenolic compounds in tree leaves have overall prooxidant activities in the midgut fluids of caterpillars. Sugar maple leaves had significantly lower ascorbate:phenolic ratios than did red oak leaves, suggesting that phenolics in maple would oxidize more readily than those in oak. As expected, semiquinone radicals were at higher steady-state levels in the midgut fluids of both caterpillar species when they fed on sugar maple than on red oak, consistent with the first hypothesis. Higher semiquinone radical levels were also found in M. disstria than in O. leucostigma, consistent with the second hypothesis. Finally, semiquinone radical formation was positively associated with two markers of oxidation (protein carbonyls and total peroxides). These results suggest that the complex mixtures of phenolics in red oak and sugar maple leaves have overall prooxidant activities in the midgut fluids of M. disstria and O. leucostigma caterpillars. We conclude that the oxidative defenses of trees vary substantially between species, with those in sugar maple leaves being especially active, even in phenolic-tolerant herbivore species.     

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.     

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.     

Toxicity of Phenolic Compounds Extracted from Bark Residues of Different Ages

In Quebec, Canada, industrial bark wastelands cover several hundred hectares of land. Bark residue that has piled up for decades tends to remain free of vegetation for years. To assess the revegetation potential of such sites, we sought to determine those factors responsible for poor plant growth. Phenolic compounds from fresh to 20-year-old bark residues were extracted with four solvents and quantified by high-performance liquid chromatography (HPLC). We simulated solutions (mixtures of standard phenolic compounds) to evaluate the potential toxic effects of phenolic compounds on the rhizobial growth, germination index, plant growth, nodule number, and nitrogen fixation activity of two legume species under laboratory conditions. The concentration of individual phenolic compounds varied from none detected to 350 μg/g bark residue. The extracted phenolic compounds differed among solvents and bark residues. The highest concentration of total phenolic compounds was from fresh bark; most of these were soluble in water or 0.1 M NaOH. For older bark residues, the total phenolic content depended on solvent strength, generally in the order of 2.0 M NaOH > 0.1 M NaOH = hot water > cold water. The biological activity of the simulated bark extracts was not established with the rhizobial growth inhibition test but with the germination index and rhizobium–legume symbiosis tests. With these, the toxicity of the simulated phenolic extracts decreased from fresh to the older bark residues. Plant dry weight, nodule number, and nitrogen fixation activity of vetch (Vicia sativa L.) were less negatively affected by high concentrations of phenolics than birdsfoot trefoil (Lotus corniculatus L.), although birdsfoot trefoil grew at lower concentrations. The rhizobium–legume symbiosis has potential for revegetating bark wastelands with less than 1 year old and older bark residues.     

Induction of a p-Coumaroyl Trihydroxy Triterpene Acid in Psylla-Infested and Mechanically Damaged Pear Trees

The pattern of induction and the chemical structure of phenolic compounds in pear trees (Pyrus communis, cv. Conference) that were either infested by pear leaf suckers Psylla pyricola and P. pyri or mechanically damaged, or both, were studied. Chromatographic (HPLC) and mass spectral analysis performed on extracts of leaf samples collected at various time intervals from trees subjected to three treatments demonstrated the induction (and/or amplification) of a phenolic compound, identified as 3-O-trans-p-coumaroyltormentic acid (I). New mass spectrometric data on this phenolic compound are presented. HPLC revealed different peak patterns in the course of the period of Psylla infestation and the lapse of time since mechanical damage was inflicted, compared to a control tree. The new phenolic compound became apparent after 12 hr and reached the highest level 30 days after damage by pear leaf suckers. It was also observed after 24 hr at lower intensity in samples from a mechanically damaged tree and exclusively on day 30 at very low intensity in the leaf extracts from the uninfested control trees. We conclude that damage by pear leaf suckers, and to a lesser extent also mechanical damage, induce the synthesis of the new, late-eluting phenolic compound. We propose that this compound is involved in plant defense against pear leaf suckers.     

Leaf Ontogeny Influences Leaf Phenolics and the Efficacy of Genetically Expressed Bacillus thuringiensis cry1A(a) d-Endotoxin in Hybrid Poplar Against Gypsy Moth

We tested the hypothesis that ontogenetic variation in leaf chemistry could affect the efficacy of genetically expressed Bacillus thuringiensis cry1A(a) d-endotoxin, and thus provide spatial variation in (1) foliage protection and (2) selective pressures that could delay the resistance of folivores. Our model consisted of clonal hybrid Populus plants (NC5339). Consumption of foliage and relative growth rates of gypsy moth, Lymantria dispar (L.) increased, and phenolic glycoside concentrations decreased, as leaves from transformed plants containing the cry1A(a) d-endotoxin and nontransformed plants matured from leaf plastochron index (LPI) 1–6. Feeding and growth rates were negatively correlated with phenolic glycosides in both transformed and nontransformed foliage. The presence of the B. thuringiensis d-endotoxin was at most, additive to the effect of the phenolic glycosides. Feeding and growth rates were positively correlated with condensed tannins in transformed foliage, but there was no relationship with condensed tannins in nontransformed foliage. The results indicate that the presence of foliar allelochemicals of poplar can enhance the effectiveness of genetically expressed B. thuringiensis d-endotoxin against gypsy moth larvae. However, the spatial variation in gypsy moth performance in response to the combination of foliar allelochemicals and d-endotoxin was not greater than the effect of ontogenetic variation in foliar allelochemicals alone. These results suggest that for this important pest, foliage protection may be obtained without genetically engineered defenses, and instead, by relying on ontogenetic and clonal variation in allelochemicals. The benefits of combining novel resistance mechanisms with natural ones will depend upon the specific folivore's adaptation to natural resistance mechanisms, such as allelochemicals. Moreover, some of the greatest benefits from transgenic resistance may arise from the need to protect trees from multiple pests, some of which may not be deterred by, or may even prefer, allelochemicals that confer protection from a few species.     

Importance of phenolic glucosides in host selection of shoot galling sawfly,Euura amerinae,onSalix pentandra

The effects of phenolic glucosides on the oviposition behavior ofEuura amerinae L. (Hymenoptera: Tenthredinidae) were tested in multiple oviposition experiments using different shoot length categories ofSalix pentandra L. (with different amounts of phenolic glucosides) and in experiments with pure phenolic glucosides (salidroside, arbutin, salicin, 90% salicortin, 90% 2-O-acetylsalicortin) or composite total fractions of phenolic glucosides from three willow species (S. pentandra, S. myrsinifolia Salisb.,S. triandra L.). This was the first time that the effects of pure phenolic glucosides on the oviposition behavior of sawfly species were tested. Total fraction of phenolic glucosides fromS. pentandra and its main individual glucoside, 2-O-acetyl-salicortin, stimulated the strongest ovipositional behavior inE. amerinae. The results show clearly that females ofE. amerinae can recognize and choose their host willow,S. pentandra, on the basis of phenolic glucosides. Moreover, they are probably able to use phenolic glucosides as a cue in shoot selection within host-plant individuals.     

A simple model for regeneration of a polymeric sorbent saturated with phenol

Regeneration of spent resin particles is a crucial step in a cyclic adsorption process for removal of phenolic species from wastewater. A simple: model is developed for simulating the regeneration behavior in finite batch adsorbers. It is then applied to the regeneration by sodium hydroxide solutions, of a polymeric sorbent, SP206, saturated with phenol. Analytic solutions of the model are obtained based on both the reaction front assumption and the quasi-steady state approximation in the resin particle, considering the fact that sodium hydroxide reacts with phenol very quickly.     

Phenolic biosynthesis,leaf damage,and insect herbivory in birch (Betula pendula)

The effect of both caterpillar herbivory and artificial damage on phenylalanine ammonia lysase (PAL) activity of birch foliage was measured, using an intact cell assay. After artificial damage there was a small increase in PAL activity in damaged leaves but no change in adjacent undamaged ones. Insect grazing produced a larger increase in PAL activity, and the enzyme activity was also increased in adjacent undamaged leaves. Artificial damage increased the phenolic levels of the damaged leaves. Insect grazing caused a larger, longer-lasting increase in phenolic levels and also elevated phenolic levels in undamaged leaves. The possible role of these wound-induced biochemical changes in birch is discussed.     

Role of phenolic compounds in the antialgal activity of barley straw

Barley straw decomposing in well-aerated water releases a substance(s) that inhibits algal growth. Phenolic compounds are toxic to algae but are unlikely to be present in sufficient quantities to account for the extended antialgal action of straw. However, straw is antialgal under conditions that may promote oxidation of phenolic hydroxyl groups to quinones; tannins are antialgal under similar conditions. The toxicity of authentic quinones towardsMicrocystis is confirmed; the quinones are some 10³ times more antialgal than phenolic acids. The possibility that oxidized lignin derivatives may be involved in straw toxicity towards algae is discussed.     

Microbial decomposition of ferulic acid in soil

The suppression of plant growth by different phenolic acids is well known. This work was designed to determine if ferulic acid, a known phenolic inhibitor of plant growth, accumulates in the soil and if soil microorganisms could be isolated that metabolize it. Over 99% of the extractable ferulic acid was lost from decaying hackberry leaves in 300 days. During this time the amount in the top 15 cm of soil remained fairly constant at about 30 ppm, except for the March sample which was significantly higher than the rest. Addition of ferulic acid to soil caused an increase in CO₂ evolution and in numbers of a select group of microorganisms.Rhodotorula rubra andCepnalosporium curtipes, which actively metabolize ferulic acid, were isolated, but the metabolic pathways employed appear to be different from the reported one. The reported pathway for ferulic acid breakdown is ferulic acid to vanillic acid to protocatechuic acid to -keto-adipic acid.Rhodotorula Rubra was found to convert ferulic acid to vanillic acid, but no evidence was found for utilization of the rest of the pathway.Cephalosporium curtipes appears to use a different pathway or to metabolize intermediate compounds rapidly without accumulating them, because no phenolic compounds were found during the breakdown of ferulic acid. The presence in the soil of microorganisms that metabolize ferulic acid and other phenolic acids is ecologically significant because such organisms prevent long-term accumulations of these substances, which are toxic to many other microorganisms and higher plants.     

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.     

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."