Interactions oftrans-cinnamic acid,its related phenolic allelochemicals,and abscisic acid in seedling growth and seed germination of lettuce

Phenolic compounds have been identified as the most common allelochemicals produced by higher plants. Inhibitions of cinnamic acid, its related phenolic derivatives, and abscisic acid (ABA) on seedling growth and seed germination of lettuce were studied.trans-Cinnamic acid, ando-,m-, andp-coumaric acids inhibited the growth of etiolated seedlings of lettuce at concentrations higher than 10^–4 M and seed germination above 10^–3 M. Coumarin inhibited seedling growth and seed germination at 10^–5 M or above. Chlorogenic acid inhibited seedling growth above 10^–4 M, but did not inhibit seed germination at 10^–5–5×10^–3 M. Low concentrations (below 10^–3 M) of caffeic and ferulic acids promoted the elongation of hypocotyls, but higher concentrations (over 10^–3 M) inhibited seedling growth and seed germination. These phenolic compounds and abscisic acid had additive inhibitory effects both on seedling growth and seed germination. The inhibition on lettuce was reversed by caffeic and ferulic acids at concentrations lower than 10^–3 M except for the inhibition of germination by coumarin. These results suggest that in naturetrans-cinnamic acid,o-, m-, p-coumaric acids, coumarin, and chlorogenic acid inhibit plant growth regardless of their concentration. However, caffeic and ferulic acids can either promote or inhibit plant growth according to their concentration.     

Allelopathy ofSasa cernua

Sasa (Sasa cernua Makino) is a very serious weed pest. Its allelopathy was studied using lettuce, wheat, timothy, and green amaranth as testing species. Cultured in the rhizosphere soil ofSasa cernua, the seedlings were inhibited by 42–80% compared with the controls cultured in normal soil and vermiculite. The phenolic fraction extracted with 1 M NaOH from the rhizosphere soil ofS. cernua caused significant inhibitions on the seed germination and seedling growth of lettuce, timothy, green amaranth, and barnyard grass.p-Coumaric, ferulic, vanillic, andp-hydroxybenzoic acids andp-hydroxybenzaldehyde were identified as the main allelochemicals in sasa soil by HPLC and [¹H]NMR. Their contents in the rhizosphere soil were 5640, 1060, 860, 810 and 630 μg/100 g soil. The neutral fraction inhibited the seed germination and seedling growth of lettuce in the TLC direct bioassay. Volatile compounds released from sasa leaves also inhibited the growth of lettuce, wheat, timothy, and green amaranth grown under light, and the growth of etiolated seedlings of barley and wheat. These results confirm thatS. cernua produces typical allelopathy through its rhizosphere soil and air space.     

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.     

Phytotoxicity of <Emphasis Type="Italic">Phytolacca americana</Emphasis> Leaf Extracts on the Growth,and Physiological Response of <Emphasis Type="Italic">Cassia mimosoides</Emphasis>

We examined the allelochemical effects of control soil, native soil (treated soil), and leaf extracts of Phytolacca americana (pokeweed) on the germination rate and seedling growth of Cassia mimosoides var. nomame. We also studied the resulting changes in root-tip ultrastructure and peroxidase isozyme biochemistry. P. americana leaf extract inhibited seed germination, seedling growth, and biomass when compared to control and treated soil. Root and shoot growth in treated soil was stimulated relative to control soil, but root growth was inhibited by 50% in the leaf extract treatment. Biomass of C. mimosoides seedlings grown on leaf extract was reduced sevenfold when compared to the control seedlings. The amounts of total phenolic compounds in the leaf extract, treated soil, and control soil were 0.77, 0.14, and 0.03 mg l⁻¹, respectively. The root tips of C. mimsoides treated with leaf extracts of P. americana showed amyloplasts and large central vacuoles with electron-dense deposits inside them when compared to control root tips. The activity of guaiacol peroxidase (GuPOX) in whole plant, roots, and shoots of C. mimosoides increased as leaf extract increased; maximum activity was observed in extract concentrations of 75% and higher. Root GuPOX activity was three times higher than in shoots. Therefore, we conclude that inhibition of C. mimosoides growth is related to the phenolic compounds in the P. americana leaf extract and the ultrastructure changes in root-tip cells and increased GuPOX activity is a response to these allelochemicals.     

Effects of ferulic acid and some of its microbial metabolic products on radicle growth of cucumber

An initial survey of the effects of aqueous solutions of ferulic acid and three of its microbial metabolic products at pH 4.5, 6.0, and 7.5 was determined on radicle growth of 11 crop species in Petri dishes. These bioassays indicated that cucumber, ladino clover, lettuce, mung bean, and wheat were inhibited by ferulic, caffeic, protocatechuic, and/or vanillic acids and that the magnitude of inhibition varied with concentration (0–2 mM), phenolic acid, and pH of the initial solution. The pH values of the initial solutions changed considerably when added to the Petri dishes containing filter paper and seeds. The final pH values after 48 hr were 6.6, 6.8, and 7.1, respectively, for the initial 4.5, 6.0, and 7.5 pH solutions. The amounts of the phenolic acids in the Petri dishes declined rapidly over the 48 hr of the bioassay, and the rate of phenolic acid decline was species specific. Cucumber was subsequently chosen as the bioassay species for further study. MES buffer was used to stabilize the pH of the phenolic acid solutions which ranged between 5.5 and 5.8 for all subsequent studies. Inhibition of radicle growth declined in a curvilinear manner over the 0–2 mM concentration range. At 0.125 and 0.25 mM concentrations of ferulic acid, radicle growth of cucumber was inhibited 7 and 14%, respectively. A variety of microbial metabolic products of ferulic acid was identified in the Petri dishes and tested for toxicity. Only vanillic acid was as inhibitory as ferulic acid. The remaining phenolic acids were less inhibitory to noninhibitory. When mixtures of phenolic acids were tested, individual components were antagonistic to each other in the inhibition of cucumber radicle growth. Depending on the initial total concentration of the mixture, effects ranged from 5 to 35% lower than the sum of the inhibition of each phenolic acid tested separately. Implications of these findings to germination bioassays are discussed.Paper No. 9000 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, North Carolina. Mention of trade or company names does not constitute a guarantee or warranty of the product by the North Carolina Agricultural Research Service and does not imply its approval to the exclusion of other products that may be suitable.     

Possible allelopathic constituents ofCoffea arabica

The aqueous extracts of leaves, stems, and roots ofCoffea arabica significantly inhibited the seed germination and radicle growth of rye grass, lettuce, and fescue. When the extracts were diluted to 1% solution, significant suppression of lettuce growth was still found and was particularly pronounced in the extract of young seedlings. The paper chromato-gram of the ether fraction of an aqueous extract of coffee leaves was bioassayed with lettuce seeds and revealed a remarkable inhibition throughout the chromatogram except for the segment ofR _f 0.00–0.12. Paper without spotting extract was used as a standard. The phytotoxins present in coffee tissue were identified by paper and thin-layer chro-matography and mass spectrometry. The compounds include caffeine, theobromine, theophylline, paraxanthine, scopoletin, and chlorogenic, ferulic,p-coumaric,p-hydroxybenzoic, caffeic, and vanillic acids. All compounds except caffeic acid exhibited significant phytotoxicity to lettuce growth at a concentration of 100 ppm.Paper no. 222 of the Scientific Journal Series, Institute of Botany, Academia Sinica, Taiwan, and Journal Article 3582 of the Agricultural Experiment Station, Oklahoma State University, Stillwater, Oklahoma. The abstract was published in the Proceedings of 63rd Annual Meeting of American Societies for Experimental Biology, held on April 1–10, 1979, Dallas, Texas.     

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."     

Synergistic inhibitory effects ofp-coumaric and ferulic acids on germination and growth of grain sorghum

The data support the hypothesis that there is a synergistic phytotoxic effect whenp-coumaric and ferulic acids are found together. Equimolar mixtures of both acids showed greater reduction in sorghum seed germination, shoot elongation, and total seedling growth than either phytotoxin caused when alone. Repeated experiments showed mixtures containing 5×10^–3 Mp-coumaric and 5×10^–3 M ferulic acids reduced germination to 34% of controls after 24 hr and 59% by 48 hr. The same concentration of either phenol-treated seeds alone showed 69 and 92% germination at comparable times. The phytotoxic action of the combination approximated the inhibitory effect on germination of 10^–2 M ferulic acid and was a greater reduction than caused by 10^–2 Mp-coumaric treatments. Sorghum seedling growth was more sensitive than germination, with an equimolar mixture of 2.5×10^–4 Mp-coumaric and 2.5×10^–4 M ferulic acids reducing seedling dry weight significantly below weights of seedlings treated separately with 2.5×10^–4 Mp-coumaric or ferulic acids. Further dilutions showed a 1.25×10^–4 M concentration of either phenol was stimulatory to seedling growth, whereas a mixture of these two produced inhibition.     

Synergistic inhibitory effects of vanillic andp-hydroxybenzoic acids on radish and grain sorghum

Radish and grain sorghum germination and sorghum growth were inhibited in a synergistic manner by combinations of vanillic andp-hydroxybenzoic acids. At threshold inhibition levels, 2.5 × 10^–3 M vanillic acid-treated radish seeds had 71 % of control germination after 24 hr and 2.5 × 10^–3 Mp-hydroxybenzoic acid-treated radish yielded 95% germination. A mixture of 2.5 × 10^–3 M of each of these two phytotoxins showed 52% germination after 24 hr. Equimolar mixtures of 5 × 10^–3 M vanillic andp-hydroxybenzoic acids allowed sorghum germination of 60% of untreated seeds after 24 hr, whereas separate treatments of individual phenols had 93% and 96% of control seed germination. Sorghum root and shoot elongation and total seedling growth were more sensitive than germination to vanillic andp-hydroxybenzoic acid treatments, and synergistic effects also were apparent. A combination of 5 × 10^–3 M vanillic with 5 × 10^–3 Mp-hydroxybenzoic reduced root length more than either did individually, and a mixture of 5 × 10^–4 M vanillic with 5 × 10^–4 Mp-hydroxybenzoic acid reduced sorghum seedling growth to approximately that resulting from a 10^–3 M concentration of either phenol alone. Phytotoxin levels inhibitory to sorghum growth caused small increases in lower leaf surface diffusive resistance, but did not close stomates, and this effect was not judged to be the cause of reduced sorghum growth.     

HPLC identification of allelopathic compounds fromLantana camara

Aqueous extracts ofLantana camara L. leaves inhibited ryegrass (Lolium multiflorum Lam.) germination and seedling growth. Phytotoxic compounds were fractionated from crude aqueous extracts and fractions were evaluated for their phytotoxicity. Inhibition was most pronounced with the alkaline and acid hydrolysates. Plant inhibition by the crude extract reflected a complex interaction of numerous individual components of diverse chemical compositions and potencies. Presumptive identification of the individual components was accomplished with high-performance liquid chromatography (HPLC). Thirteen phenolic compounds were identified, and most of these compounds were phytotoxic to ryegrass seedlings. Radicle elongation was more sensitive to the toxins than shoot elongation.Florida Agricultural Experiment Station Journal Series No. 7906.     

Biological activity and tentative identification of flavonoid components in velvetleaf (Abutilon theophrasti Medik.) seed coats

The biological activity of aqueous extracts of velvetleaf (Abutilon theophrasti Medik.) seed coats and their flavonoid components against three plant species and five soil fungi was investigated. Aqueous extracts slightly inhibited germination and significantly inhibited radicle growth of all plant species tested. Fungal growth was inhibited or not affected, depending on species, by aqueous extracts. The aqueous extracts were extracted into methanol and separated using paper chromatography for identification of compounds responsible for inhibition of seedling and fungal growth. Six flavonoid compounds were isolated and tentatively identified as delphinidin, cyanidin, quercetin, myricetin, (+)-catechin, and (–)-epicatechin. Bioassays revealed that the flavonoid compounds significantly inhibited germination and radicle growth of all test species at a concentration of 1.0 mM. Individual flavonoids had variable effects on fungi but appeared to inhibit growth and sporulation of potential seed-decomposing fungi rather than beneficial fungi. These results indicate that the flavonoid complement of velvetleaf seed coats may function in a dual defensive role against competing seedlings and seed-attacking fungi.Journal article No. 9878 of the Missouri Agricultural Experiment Station.     

Allelopathic activity in wheat-conventional and wheat-no-till soils: Development of soil extract bioassays

The primary objective of this research was to determine if soil extracts could be used directly in bioassays for the detection of allelopathic activity. Here we describe: (1) a way to estimate levels of allelopathic compounds in soil; (2) how pH, solute potential, and/or ion content of extracts may modify the action of allelopathic compounds on germination and radicle and hypocotyl length of crimson clover (Trifolium incarnatum L.) and ivyleaved morning glory (Ipomoea hederacea L. Jacquin.); and (3) how biological activity of soil extracts may be determined. A water-autoclave extraction procedure was chosen over the immediate-water and 5-hr EDTA extraction procedures, because the autoclave procedure was effective in extracting solution and reversibly bound ferulic acid as well as phenolic acids from wheat debris. The resulting soil extracts were used directly in germination bioassays. A mixture of phenolic acids similar to that obtained from wheat-no-till soils did not affect germination of clover or morning glory and radicle and hypocotyl length of morning glory. The mixture did, however, reduce radicle and hypocotyl length of clover. Individual phenolic acids also did not inhibit germination, but did reduce radicle and hypocotyl length of both species. 6-MBOA (6-methoxy-2,3-benzoxazolinone), a conversion product of 2-o-glucosyl-7-methoxy-1,4-benzoxazin-3-one, a hydroxamic acid in living wheat plants, inhibited germination and radicle and hypocotyl length of clover and morning glory. 6-MBOA, however, was not detected in wheat debris, stubble, or soil extracts. Total phenolic acids (FC) in extracts were determined with Folin and Ciocalteu's phenol reagent. Levels of FC in wheat-conventionaltill soil extracts were not related to germination or radicle and hypocotyl length of either species. Levels of FC in wheat-no-till soil extracts were also not related to germination of clover or morning glory, but were inversely related to radicle and hypocotyl length of clover and morning glory. FC values, solute potential, and acidity of wheat-no-till soil extracts appeared to be independent (additive) in action on clover radicle and hypocotyl length. Radicle and hypocotyl length of clover was inversely related to increasing FC and solute potential and directly related to decreasing acidity. Biological activity of extracts was determined best from slopes of radicle and hypocotyl length obtained from bioassays of extract dilutions. Thus, data derived from the water-autoclave extraction procedure, FC analysis, and slope analysis for extract activity in conjunction with data on extract pH and solute potential can be used to estimate allelopathic activity of wheat-no-till soilsThe use of trade names in this publication does not imply endorsement by the North Carolina Agricultural Research Service of products named, or criticism of similar ones not mentioned.     

Allelochemicals fromPolygonum sachalinense Fr. Schm. (Polygonaceae)

The root exudates fromPolygonum sachalinense in a recirculating system significantly inhibited lettuce seedling growth. The rhizomes and roots ofP. sachalinense were extracted with 80% acetone. Bioassay of the neutral-acidic fraction on the TLC agar plate showed the inhibitory activity corresponded to the two yellow pigment bands. Two orange needles were isolated and identified as anthraquinone compounds: emodin and physcion. Both compounds exhibited inhibitory activities against the seedling growth of several testing plant species. Glucosides were isolated fromP. sachalinense and were identified as emodin-1-O-β-D-glucoside and physcion-1-O-β-D-glucoside, respectively. On plant growth bioassay, these glucosides showed no phytotoxic activity against lettuce seedlings. The concentrations of emodin, physcion, and their glucosides from rhizome with roots, aerial parts, fallen leaves, and soil were determined. The rhizome with roots and fallen leaves contained emodin and physcion at relatively high concentrations. Emodin also occurs in the soil of this plant community with effective concentrations in the fall. The results indicate that these anthraquinones are responsible for the observed interference and are potent allelopathic substances.     

Use of Mixtures of Allelochemicals to Compare Bioassays Using Red Maple, Pin Cherry, and American Elm

Toxicity threshold levels for five different bioassays (germination in combination with radicle length, seedling growth, callus growth, plating, and cell suspension growth) were determined on three invasive arborescent species [American elm (Ulmus americana), red maple (Acer rubrum), and pin cherry (Prunus pensylvanica)] using two different mixtures of allelochemicals. Thresholds for the duckweed (Lemna minor) bioassay and lettuce (Lactuca sativa) seed germination bioassay were also determined for comparison with the same mixtures. All five bioassays showed a sensitivity similar to those of duckweed and lettuce bioassays. The lowest sensitivity was in mixtures containing about 5000 μg/liter of each chemical, but the species tested and the mixture applied influenced the outcome. Some bioassays could not be used with red maple (plating and cell suspension) and pin cherry (germination and seedling growth). Cell suspension growth was the most sensitive in in vitro culture, whereas callus culture was the least sensitive. Cell suspension growth in multiwell culture plates should be favored in allelopathy studies because of its sensitivity and the need for relatively small volumes of sample. Detection of phytotoxicity using whole plant systems (germination and seedling growth) and tissue culture appeared not to be directly correlated. We conclude that using many bioassays produces the most conclusive evidence of allelopathic activity.     

Phytotoxic properties of nordihydroguaiaretic acid,a lignan fromLarrea tridentata (Creosote bush)

The phytotoxic properties of nordihydroguaiaretic acid (NDGA) isolated from creosote bush,Larrea tridentata (Zygophyllaceae), were examined. NDGA dramatically reduces the seedling root growth of barnyard grass, green foxtail, perennial ryegrass, annual ryegrass, red millet, lambsquarter, lettuce, and alfalfa, and reduces the hypocotyl growth of lettuce and green foxtail. It has no effect on the germination of lettuce seeds. NDGA almost certainly contributes to the observed allelopathic nature of creosote bush.Work performed during sabbatical year leave at Western Regional Research Center.     

Chemical inhibition of fire-prone grasses by fire-sensitive shrub,Conradina canescens

In an investigation of potential chemical activity of fire-sensitive shrubs in Florida's sand pine scrub community, bioassays of foliar washes ofConradina canescens showed significant inhibitory activity on three native grasses that are known to fuel frequent surface fires; inhibition was concentrated seasonally in spring and summer. Application of runoff fromConradina leaves to one of the grasses caused a 50% reduction in growth over a 20-week period. Isolation of the biologically active fractions from the fresh leaves ofC. canescens yielded numerous monoterpenes, a number of which were identified from a GC-MS reference library and/or MS comparison to authentic compounds: 11 from the diethyl ether extract, 11 from steam distillation, and four from the foliar leaf wash. Numerous other monoterpenes present in the extractions were unknown. The terpenoid fraction completely inhibited seed germination of one of the native grasses and of lettuce. Saturated aqueous solutions of nine of the monoterpenes inhibited germination and radicle growth of two native grasses. SEM views of the leaf surfaces ofConradina reveal secretory trichomes that appear to be the source of the monoterpenes as well as the triterpene, ursolic acid. The biological activity ofC. canescens as a fire-sensitive component of the scrub community is reviewed in light of the chemical evidence.     

Allelopathic effects ofPolygonum aviculare L. II. Isolation,characterization, and biological activities of phytotoxins

In earlier work, we found thatPolygonum aviculare had pronounced allelopathic effects against several test species. Four inhibitors were isolated from livingPolygonum plants, three of which were glucosides. Four different inhibitors were isolated fromPolygonum residues and soil underPolygonum stands, and none of these occurred in soil fromCynodon dactylon (L.) Pers. stands. Three of these were glycosides containing both fructose and cellobiose as the sugars. Color reactions of all the inhibitors indicated that they are phenolic in nature. All the inhibitors reduced seed germination and/or seedling growth ofChenopodium album L. Moreover some of them inhibited growth of different strains ofRhizobium andAzotobacter.     

Role of benzoxazinones in allelopathy by rye (Secale cereale L.)

Two phytotoxic compounds [2,4-dihydroxy-1,4(2H)-benzoxazin-3-one (DIBOA) and 2(3H)-benzoxazolinone (BOA)] were previously isolated and identified in 35-day-old greenhouse-grown rye shoot tissue. Both compounds were also detected by TLC in greenhouse-grown root and fieldgrown shoot tissue. The concentration of DIBOA varied in the tissues, with the greatest quantity detected in greenhouse-grown shoots. DIBOA and BOA were compared with -phenyllactic acid (PLA) and -hydroxybutyric acid (HBA) for activity on seed germination and seedling growth and were consistently more toxic than either compound. Dicot species tested, including lettuce (Lactuca sativa L.), tomato (Lycopersicon esculentum Mill.), and redroot pigweed (Amaranthus retroflexus L.), were 30% more sensitive than the monocots tested. Of the two benzoxazinone compounds, DIBOA was most toxic to seedling growth. DIBOA and BOA reduced chlorophyll production inChlamydomonas rheinhardtii Dangeard, by 50% at 7.5 × 10^–5 M and 1.0 × 10^–3 M, respectively. Both DIBOA and BOA inhibited emergence of barnyardgrass (Echinochloa crusgalli L. Beauv.), cress (Lepidium sativum L.), and lettuce when applied to soil, indicating their potential for allelopathic activity.Journal Article No. 11945 of the Michigan Agricultural Experiment Station.     

Allelopathy of oats. I. Assessment of allelopathic potential of extract of oat shoots and identification of an allelochemical

The allelopathic potential of oat (Avena sativa L.) extracts was investigated under laboratory conditions. The ethyl ether-, acetone-, and water-soluble fractions obtained from the extract of oat shoots inhibited the germination and growth of roots and hypocotyls of lettuce (Lactuca sativa L.). The inhibitory activity of the water-soluble fraction was maximum, followed by that of ethyl ether-soluble and acetone-soluble fraction. An active principle of the water-soluble fraction was isolated and its structure was determined by spectral data asl-tryptophan.l-Tryptophan inhibited the growth of hypocotyls and roots of lettuce seedlings at concentrations greater than 0.03 and 0.1 mM, respectively. These results suggested thatl-tryptophan may be an allelochemical which affects the growth or germination of different plant species.     

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.