Effects of Microbial Utilization of Phenolic Acids and their Phenolic Acid Breakdown Products on Allelopathic Interactions

Reversible sorption of phenolic acids by soils may provide some protection to phenolic acids from microbial degradation. In the absence of microbes, reversible sorption 35 days after addition of 0.5–3 mol/g of ferulic acid or p-coumaric acid was 8–14% in Cecil A_p horizon and 31–38% in Cecil B_t, horizon soil materials. The reversibly sorbed/solution ratios (r/s) for ferulic acid or p-coumaric acid ranged from 0.12 to 0.25 in A_p and 0.65 to 0.85 in B_t horizon soil materials. When microbes were introduced, the r/s ratio for both the A_p and B_t horizon soil materials increased over time up to 5 and 2, respectively, thereby indicating a more rapid utilization of solution phenolic acids over reversibly sorbed phenolic acids. The increase in r/s ratio and the overall microbial utilization of ferulic acid and/or p-coumaric acid were much more rapid in A_p than in B_t horizon soil materials. Reversible sorption, however, provided protection of phenolic acids from microbial utilization for only very short periods of time. Differential soil fixation, microbial production of benzoic acids (e.g., vanillic acid and p-hydroxybenzoic acid) from cinnamic acids (e.g., ferulic acid and p-coumaric acid, respectively), and the subsequent differential utilization of cinnamic and benzoic acids by soil microbes indicated that these processes can substantially influence the magnitude and duration of the phytoxicity of individual phenolic acids.     

Phenolic acids affect photosynthesis and protein synthesis by isolated leaf cells of velvet-leaf

The effects ofp-coumaric, ferulic, chlorogenic, and vanillic acids on photosynthesis and protein synthesis by isolated leaf cells of velvetleaf (Abutilon theophrasti Medik) were investigated. Photosynthesis and protein synthesis were measured in cell suspensions by the incorporation of¹⁴CO₂ and [¹⁴C]leucine, respectively. None of the tested phenolic acids except vanillic reduced photosynthesis by more than 50% at the highest concentration and 30 min of incubation. At 100M concentrations and 60-min incubation periods,p-coumaric, ferulic, chlorogenic, and vanillic acids inhibited photosynthesis by 33, 37, 57, and 65%, respectively. Ferulic acid was the most inhibitory to protein synthesis and reduced the incorporation of [¹⁴C]leucine by 50% at about 1.0M after 60 min of incubation. At the highest concentrations tested in this study, vanillic and ferulic acids were inhibitory to photosynthesis and protein synthesis, respectively, whereas chlorogenic andp-coumaric acids did not inhibit either physiological process. The maximum inhibition of protein synthesis by chlorogenic acid was 19% and by vanillic acid was 28% at 100M concentrations. Chlorogenic, vanillic, andp-cou-maric acids at 0.1M caused increased protein synthesis over the untreated control. Overall, photosynthesis was more sensitive than protein synthesis to the four phenolic acids tested.Florida Agricultural Experiment Station Journal Series No. 9228.     

Effects of mixtures of four phenolic acids on leaf area expansion of cucumber seedlings grown in Portsmouth B1 soil materials

Cucumber seedlings growing in a 12 mixture of soil (Portsmouth B₁) and sand adjusted to pH 5.2 were treated every other day five times with 0, 0.0625, 0.125, 0.25, or 0.5 mol/g soil of ferulic, caffeic,p-coumaric,p-hydroxybenzoic, protocatechuic, sinapic, syringic, or vanillic acids. Treatments began when seedlings were 8 days old. The effects on mean absolute rates of leaf expansion were used to estimate the relative potencies of these phenolic acids to ferulic acid. Based on the results of this experiment, ferulic,p-coumaric,p-hydroxybenzoic, and vanillic acids were chosen for further study. Materials and procedures were identical in the second study, but treatments consisted of mixtures of the four phenolic acids at concentration combinations designed to achieve 40 % or 60 % inhibition of absolute rates of leaf expansion. Using joint action analysis, a model describing the action of the phenolic acid mixtures was developed. A model involving only two factor terms was sufficient to describe the observed responses of cucumber leaf area to the phenolic acid mixtures. The action ofp-hydroxybenzoic acid on absolute rates of leaf expansion was inhibited by the presence of the other three phenolic acids. No other antagonisms or synergisms existed among the four compounds.This research was partially supported by the North Carolina Agricultural Research Service, Raleigh, North Carolina 27695-7643 and by the US-Spain Joint Committee for Scientific and Technological Cooperation project CCA-8309/166.     

Effects of mixtures of phenolic acids on leaf area expansion of cucumber seedlings grown in different pH portsmouth A1 soil materials

Cucumber seedlings growing in A₁horizon Portsmouth soil material adjusted to pH 5.2, 6.0, or 6.9 were treated with 0, 0.25 or 0.5, mol/ g soil ferulic acid,p-coumaric acid, vanillic acid, or an equal mixture (0.5 mol/g total) of two acids every other day. A total of five treatments was given starting with day 7 from seeding. Absolute rates of leaf expansion were determined for seedlings. The experiment was terminated when seedlings were 17 days old. All three phenolic acids inhibited leaf expansion. The dose required for 50% inhibition of absolute rates of leaf expansion increased as pH of the soil systems increased. The order of toxicity based on 50% dose and relative potency were as follows: ferulic acid > vanillic acid =p-coumaric acid. Effects of mixtures of phenolic acids on absolute rates of leaf expansion, when compared to the effects of individual phenolic acids, were found to be antagonistic for the ferulic-vanillic acid mixture and the ferulic-p-coumaric acid mixture in the pH 5.2 soil systems. Several phenolic acid treatments were required before antagonistic effects of mixtures were evident. In all other instances, when treatment effects were significant, the effects of individual phenolic acids were additive.Paper No. 11875 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, North Carolina 27695-7601. The use of trade names in this publication does not imply endorsement by North Carolina Agricultural Research Service of the product named, nor criticism of similar products not mentioned. This research was partially supported by US-Spain Joint Committee for Scientific and Technological Corporation project CCA-8309/166.     

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.     

Allelopathy in agroecosystems: Wheat phytotoxicity and its possible roles in crop rotation

The germination rates of cotton and wheat seeds were significantly affected by various extracts of wheat mulch and soils collected from the wheat field. This toxicity was even more pronounced against seedling growth. Five allelochemics: ferulic,p-coumaric,p-OH benzoic, syringic, and vanillic acids, were identified from the wheat mulch and its associated soil. Quantitatively, ferulic acid was found at higher concentrations thanp-coumaric acid in the soil. Various concentrations of ferulic andp-coumaric acids were toxic to the growth of radish in a bioassay. The functional aspects of allelochemic transfer from decaying residue to soil and the subsequent microbial degradation within agroecosystems are discussed, particularly as they relate to wheat crop rotation, with wheat and cotton, in Pakistan.     

Influence of Phenolic acids on microbial populations in the rhizosphere of cucumber

Experiments were conducted to determine whether changes in soil microbial populations that occur in response to additions of certain allelopathic phenolic acids to bulk soil also occur in the rhizosphere. Cucumber seedlings were transplanted into cups containing a nutrient-enriched mixture of Portsmouth B₁, soil and sand and were watered five times (once every 48 hr) with aqueous solutions of ferulic,p-coumaric, or vanillic acid (each at 0, 0.25, or 0.50ol/g soil material). Nutrient solution was applied on alternate days. Leaf growth was suppressed by up to 42% by phenolic acids, but changes in root growth varied with the compound and concentration in solution. Significant increases (over 600% relative to controls) in populations of fast-growing bacteria in the rhizosphere were detected after two but not after five treatments, and increases (400% relative to controls) in numbers of fungal propagules were detected after five treatments. Such increases suggested that chronic exposure to a phenolic acid might resuit in high populations of rhizosphere microorganisms that could metabolize the compounds and thus alter observable responses by the plant. To test this, plants were watered repeatedly with a low-concentration solution of ferulic acid (chronic treatments; 0.0 or 0.1mol/g soil material in one experiment, 0.000 or 0.025imol/g soil material in a second) and then once with a highconcentration solution (acute treatment; 0.0, 0.5, or 1.0mol/g soil material in the first experiment; 0.000, 0.125, or 0.250mol/g soil material in the second).Paper No. 12385 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, North Carolina. The use of trade names in this publication does not imply endorsement by the North Carolina Agricultural Research Service or the United States Department of Agriculture of the products named, nor criticism of similar ones not mentioned. Acute treatments and some chronic treatments suppressed leaf growth, but results were inconsistent for root growth. Acute treatments increased numbers of several types of bacteria in the rhizosphere but had inconsistent effects on fungi. Chronic treatments had no effect on numbers of bacteria or fungal propagules in the rhizosphere. Furthermore, chronic treatments did not alter responses of plants or microbial populations to the subsequent acute treatment. Results demonstrated that phenolic acids in soil, which must pass through the rhizosphere before interaction with plant roots can occur, alter the microbial ecology of the rhizosphere. However, microbially mediated acclimation of plants to relatively high concentrations of ferulic acid was not observed.     

Phenolic acid content of soils from wheat-no till,wheat-conventional till,and fallow-conventional till soybean cropping systems

Soil core (0–2.5 and/or 0–10 cm) samples were taken from wheat no till, wheat-conventional till, and fallow-conventional till soybean cropping systems from July to October of 1989 and extracted with water in an autoclave. The soil extracts were analyzed for seven common phenolic acids (p-coumaric, vanillic,p-hydroxybenzoic, syringic, caffeic, ferulic, and sinapic; in order of importance) by high-performance liquid chromatography. The highest concentration observed was 4 g/g soil forp-coumaric acid. Folin & Ciocalteu's phenol reagent was used to determine total phenolic acid content. Total phenolic acid content of 0- to 2.5-cm core samples was approximately 34% higher than that of the 0- to 10-cm core samples. Phenolic acid content of 0- to 2.5-cm core samples from wheat-no till systems was significantly higher than those from all other cropping systems. Individual phenolic acids and total phenolic acid content of soils were highly correlated. The last two observations were confirmed by principal component analysis. The concentrations were confirmed by principal component analysis, tions of individual phenolic acids extracted from soil samples were related to soil pH, water content of soil samples, total soil carbon, and total soil nitrogen. Indirect evidence suggested that phenolic acids recovered by the water-autoclave procedure used came primarily from bound forms in the soil samples.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.     

Induction and/or Selection of Phenolic Acid-Utilizing Bulk-Soil and Rhizosphere Bacteria and Their Influence on Phenolic Acid Phytotoxicity

Bulk-soil and rhizosphere bacteria are thought to exert considerable influence over the types and concentrations of phytotoxins, including phenolic acids, that reach a root surface. Induction and/or selection of phenolic acid-utilizing (PAU) bacteria within the bulk-soil and rhizosphere have been observed when soils are enriched with individual phenolic acids at concentrations 0.25 mol/g soil. However, since field soils frequently contain individual phenolic acids at concentrations well below 0.1 mol/g soil, the actual importance of such induction and/or selection remains uncertain. Common bacteriological techniques (e.g., isolation on selective media, and plate dilution frequency technique) were used to demonstrate in Cecil A_p soil systems: (1) that PAU bacterial communities in the bulk soil and the rhizosphere of cucumber seedlings were induced and/or selected by mixtures composed of individual phenolic acids at concentrations well below 0.25 mol/g soil; (2) that readily available carbon sources other than phenolic acids, such as glucose, did not modify induction and/or selection of PAU bacteria; (3) that the resulting bacterial communities readily utilize mixtures of phenolic acids as a carbon source; and (4) that depending on conditions (e.g., initial PAU bacterial populations, and phenolic acid concentration) there were significant inverse relationships between PAU bacteria in the rhizosphere of cucumber seedlings and absolute rates of leaf expansion and/or shoot biomass. The decline in seedling growth could not be attributed to resource competition (e.g., nitrogen) between the seedlings and the PAU bacteria in these studies. The induced and/or selected rhizosphere PAU bacteria, however, reduced the magnitude of growth inhibition by phenolic acid mixtures. For a 0.6 mol/g soil equimolar phenolic acid mixture composed of p-coumaric acid, ferulic acid, p-hydroxybenzoic acid, and vanillic acid, modeling indicated that an increase of 500% in rhizosphere PAU bacteria would lead to an approximate 5% decrease (e.g., 20–25%) in inhibition of absolute rates of leaf expansion. As far as we know, this is the first time that such a relationship has been quantified.     

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.     

Use of water and EDTA extractions to estimate available (free and reversibly bound) phenolic acids in Cecil soils

Sterile and microbe reinfested Cecil A_p and B_t soil materials amended with 0 to 5 µmol/g of ferulic acid,p-coumaric acid,p-hydroxybenzoic acid, or vanillic acid were extracted after varying time intervals with water, EDTA, or NaOH to characterize sorption of cinnamic and benzoic acid derivatives and to determine the effectiveness of water and EDTA extractions in estimating concentrations of free and reversibly bound phenolic acids in soils. Basic EDTA (0.5 M, pH 8) extractions and water extractions provided good estimates of both free and reversibly bound cinnamic acid derivatives, but not of benzoic acid derivatives. Neutral EDTA (0.25 M, pH 7) and water extractions, however, were effective for both cinnamic and benzoic acid derivatives Rapid initial sorption of both cinnamic and benzoic acid derivatives was followed by slow long-term sorption of the cinnamic acid derivatives. Slow long-term sorption was not observed for the benzoic acid derivatives. The amount of sorption of phenolic acids in soil materials was directly related to the concentration of phenolic acids added to soil materials. The addition of a second phenolic acid to the soil materials did not substantially affect the sorption of each individual phenolic acid. Sodium hydroxide extractions, which were made only after phenolic acids in phenolic acid-amended and non-amended soil material were depleted by microbes, confirmed that neutral EDTA and water extractions of soils can be used to make accurate estimates of baseline (residual) levels of free and reversibly bound phenolic acids available to soil microbes and, thus, potentially to seeds and roots.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.     

Inducible Production of Phenolic Acids in Wheat and Antibiotic Resistance to Sitodiplosis mosellana

Larvae of the wheat midge, Sitodiplosis mosellana (Géhin) feed on the surface of wheat seeds for about 10 days beginning when pollination occurs. A few wheats have a high level of antibiotic resistance to the larvae, which suppresses their growth and development. Nearly all larvae develop successfully on susceptible wheats. Analysis by HPLC of seed extracts produced by alkaline hydrolysis revealed rapid changes in the levels of p-coumaric and ferulic acids during early seed development. Seed infested by wheat midge larvae showed induced changes in the dynamics of these phenolic acids. The most resistant wheats had a higher constitutive level and a more rapid induction of ferulic acid than susceptible wheats. Levels of ferulic acid exceeding 0.35 g/g fresh weight were associated with a high mortality of newly hatched larvae. In one wheat line, resistance also was associated with induced production of p-coumaric acid. The induction of ferulic acid was similar in wheat from the laboratory and field, except in one resistant wheat that produced higher levels in the field. In ripe seeds, resistant and susceptible wheats had similar levels of phenolic acids.     

Effects of soil nitrogen level on ferulic acid inhibition of cucumber leaf expansion

It has been suggested that the allelopathic activity of phenolic acids should be primarily important in soils of low fertility. If this is true, then plant growth inhibition by phenolic acids may be unimportant in managed agricultural soils. The objective of this study was to determine how soil nitrogen (N) level might modify phenolic acid inhibition of growth. Cucumber seedlings (Cucumis sativus cv Early Green Cluster) grown in containers in growth chambers under varying N levels (5, 10, 15, 20, and 25 g N/g soil) in Portsmouth B,-horizon soil material were treated with ferulic acid (0 or 10 g/g soil). Nitrogen and ferulic acid (FA) were applied every other day to the soil surface. The amount of FA in the soil solution declined with depth in the containers. A more rapid disappearance of FA from the soil solution was observed for the last FA treatment (0% recovered after 10 hr on day 23) than the first treatment (44% recovered after 10 hr on day 13). Both low N (5 g N/g soil) and FA treatments reduced shoot dry weight, the mean absolute (AGR) and the mean relative (RGR) rates of leaf expansion, and increased the root-shoot ratio. High N treatments reduced shoot dry weight and the AGR. Ferulic acid inhibited cucumber seedling growth over a range of N concentrations, suggesting that the allelopathic activity of phenolic acids may be important in both nutrient limiting and nonlimiting soils for some species.Paper No. 12219 of the journal series of the North Carolina Agricultural Research Service, Raleigh, North Carolina 27695-7601. The use of tradenames in this publication does not imply endorsement by the North Carolina Agricultural Research Service of products named, nor criticism of similar ones not mentioned.     

Effects of three phenolic acids on chlorophyll content and growth of soybean and grain sorghum seedlings

Experiments were designed to test the hypothesis that interference with chlorophyll metabolism may be one mechanism of inhibition of plant growth in allelopathic interactions. Effects of ferulic,p-coumaric, and vanillic acids on soybean and grain sorghum growth and chlorophyll content were quantified and compared after seedlings were treated with these compounds in a nutrient culture. Following a 6-day treatment cycle, dry weights of soybean seedlings were reduced by both 10^–3 M and 5 × 10^–4 M treatments of ferulic,p-coumaric and vanillic acids. Soybean weight reductions in each case were paralleled by a significant reduction in the concentration (g Chl/mg dry wt) of chlorophylls a and b and total chlorophyll in the unifoliate leaves. Sorghum seedling growth was also reduced by each of the compounds at the 5 × 10^–4 M level, but leaf chlorophyll concentration was not below that of control plants.     

Allelopathy of yellow fieldcress (Rorippa sylvestris): Identification and characterization of phytotoxic constituents

Both the neutral and acidic fractions of the acetone extract of yellow fieldcress (Kireha-inugarashi,Rorippa sylvestris Besser) inhibited lettuce seed germination. Salicylic,p-hydroxybenzoic, vanillic, and syringic acid were identified in the acidic fraction. In the neutral fraction, hirsutin (8-methylsulfinyloctyl isothiocyanate), 4-methoxyindole-3-acetonitrile, and pyrocatechol were identified. Bioassay using a root exudate recirculating system showedR. sylvestris during flowering inhibited the lettuce seedling growth. Hirsutin (13g/plant/day) and pyrocatechol (9.3g/plant/day) were the major compounds released into the rhizosphere. Several combinations of pyrocatechol,p-hydroxybenzoic acid, vanillic acid, and hirsutin reduced lettuce seedling growth. These compounds seemed to be allelochemicals.     

Autointoxication mechanism ofOryza sativa I. Phytotoxic effects of decomposing rice residues in soil

The aqueous extracts of decomposing rice residues in soil exhibited inhibition on the radicle growth of lettuce and rice seeds and the growth of rice seedlings. The phytotoxicity was found in extracts obtained from the early stage of decomposition (first month), and gradually declined thereafter. The inhibition was also found in extracts obtained from rice fields, and was persistent for 4 months. The root initiation of hypocotyl cuttings of mungbeans was suppressed by extracts of decaying rice residues and extracts obtained from paddy soil. Five phytotoxins,p-hydroxybenzoic,p-coumaric, vanillic, ferulic, ando-hydroxyphenylacetic acids, and several unknowns were found in the decomposing rice residues under waterlogged conditions. At 25 ppm,o-hydroxyphenylacetic acid revealed significant inhibition on the radicle growth of rice and lettuce seeds and suppressed root initiation of mungbean seedlings. It was concluded that the growth of rice seedlings was retarded by decaying rice residues in soil; thus, this appeared to be an autointoxication phenomenon.Paper No. 176 of the Scientific Journal Series, Institute of Botany, Academia Sinica. This study was financially supported by the National Science Council, the Republic of China.     

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."     

Effects of various mixtures of ferulic acid and some of its microbial metabolic products on cucumber leaf expansion and dry matter in nutrient culture

Cucumber seedlings (Cucumis sativus cv. Early Green Cluster) ranging from 6 to 16 days of age were treated with various concentrations (0– 1 mM) of caffeic, ferulic,p-coumaric,p-hydroxybenzoic, protocatechuic, sinapic, syringic, and vanillic acids and mixtures of ferulic acid and one or two of the other phenolic acids. Seedlings were grown in full-strength Hoagland's solution which was changed every other day. Phenolic acid treatments were given with each nutrient solution change starting at day 6 or given once when seedlings were 13 or 14 days old. Leaf area, mean relative rates of leaf expansion, transpiration rates, water utilization, and the concentrations of the phenolic acids in nutrient solution were determined at one- or two-day intervals. Seedling dry weight was determined at final harvest. Seedling leaf area and dry weight were linearly related. Since leaf areas can be easily obtained without destructive sampling and leaf area expansion responds rapidly to phenolic acid treatments, it was utilized as the primary indicator of plant response. The resulting data suggested that a number of ferulic acid microbial metabolic products, as well as two other phenolic acids observed in soils (p-coumaric and syringic acid), can reduce seedling dry weight, leaf expansion, and water utilization of cucumber seedlings in a similar manner. The magnitude of impact of each of the phenolic acids, however, varied with phenolic acid and concentration. It appears that the inhibitory activity of these phenolic acids involved water relations of cucumber seedlings, since the phenolic acid treatments resulted in closure of stomata which then remained closed for several days after treatment. The data also demonstrated that the effects of mixtures of phenolic acids on cucumber seedlings may be synergistic, additive, or antagonistic. The type of response observed appeared to be related to the factor measured, the compounds in the nmixture, and the magnitued of inhibition associated with each compounds. The data also indicated that the effects of the various phenolic acids were reversible, since seedling leaf area increased rapidly once phenolic acids were removed from the root environment. Mean relative rates of leaf expansion recovered even in the presence of the various phenolic acids.Paper No. 9396 of the Journal Series of the North Carolina Agricultural Research Service. Raleigh, North Carolina. Mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by the Agricultural Research Service and does not imply its approval to the exclusion of other products that may be suitable.     

Solution volume and seed number: Often overlooked factors in allelopathic bioassays

Cucumber seeds were germinated under various combinations of solution volume and seed number with a range of ferulic acid concentrations. At each concentration, radicle growth decreased as the relative amount of ferulic acid available per seed increased from (25 seeds/5 ml) to 5 (5 seeds/5 ml) to 19 (25 seeds/95 ml). With 2.0 mM ferulic acid in buffered solution, radicle lengths after 48 hr ranged from 71 to 47% of control. The amount of ferulic acid remaining in 2.0 mM solution after 48 hr was directly proportional to the amount initially available per seed, and ranged from 9 to 91%. Solution volume and seed number also significantly affected inhibition by vanillic acid, caffeic acid, and juglone. With 0.1 mM juglone, radicle lengths after 48 hr were 88% of control with 25 seeds/5 ml, 68% with 5 seeds/5 ml, and 56% with 25 seeds/90 ml. The data demonstrated that lower phytotoxin concentrations can produce equivalent or greater inhibitory effects than higher concentrations when the amount available per seed for uptake is greater. Equivalent inhibition of radicle growth was observed with 1.0 mM (5 seeds/5 ml) and 2.0 mM (25 seeds/5 ml) ferulic acid. Available literature on herbicides indicates that similar effects occur in greenhouse and field studies.     

Interactions betweenKalmia and black spruce: Isolation and identification of allelopathic compounds

Aqueous extracts of fresh leaves and organic soil of northern sheep laurel (Kalmia angustifolia var.angustifolia) were found to be inhibitory to the growth of black spruce (Picea mariana) germinants. Primary root growth of black spruce was more affected by the extracts than was shoot growth. The growth inhibition caused by the leaf extract was most pronounced under acidic conditions (pH 3–4). The aqueous extract ofKalmia leaves contained ferulic, vanillic, syringic, gentisic,m-coumaric,p-coumaric,o-hydroxyphenylacetic, andp-hydroxybenzoic acids as well as some other unknown compounds. These compounds were isolated from the aqueous extract ofKalmia leaves by ethyl acetate extraction and identified using thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC). Bioassay indicated that the overall toxicity of the phenolic compounds to black spruce appeared to increase in the order ofo-hydroxyphenylacetic,p-hydroxybenzoic, vanillic,p-coumaric, gentisic, syringic, ferulic, andm-coumaric acids.