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An allelopathic substance in red pine needles (Pinus densiflora)
ARTICLE IN PRESS Journal of Plant Physiology 166 (2009) 442—446
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SHORT COMMUNICATION
An allelopathic substance in red pine needles (Pinus densiflora) Hisashi Kato-Noguchia,, Yoshiko Fushimia, Hideyuki Shigemorib a
Department of Applied Biological Science, Faculty of Agriculture, Kagawa University, Miki, Kagawa 761-0795, Japan Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba 305-8572, Japan
b
Received 29 April 2008; received in revised form 12 June 2008; accepted 25 June 2008
KEYWORDS Allelopathy; Growth inhibitor; Phytoalexin; Pinaceae
Summary Aqueous methanol extracts of red pine (Pinus densiflora) needles inhibited the growth of roots and shoots of cress (Lepidium sativum), lettuce (Lactuca sativa), alfalfa (Medicago sativa), ryegrass (Lolium multiflorum), timothy (Pheleum pratense), Digitaria sanguinalis and Echinochloa crus-galli. Increasing the extract concentration increased inhibition, suggesting that the pine needles may have growth inhibitory substances and possess allelopathic potential. The aqueous methanol extract of the pine needles was purified, and a main inhibitory substance was isolated and determined by spectral data as 9a,13b-epidioxyabeit-8(14)en18-oic acid. This substance inhibited root and shoot growth of cress and Echinochloa crus-galli seedlings at concentrations greater than 0.1 mM. The endogenous concentration of the substance was 0.13 mmol/kg pine needle. These results suggest that 9a,13b-epidioxyabeit-8(14)en-18-oic acid may contribute to the growth inhibitory effect of the pine needles and may play an important role in the allelopathy of red pine. & 2008 Elsevier GmbH. All rights reserved.
Introduction The vegetation under red pine forests is sparse compared with other forests that have dense undergrowths of herbaceous plants, despite the fact that the sunlight intensity under red pine forests is sufficient for these herbaceous plants to grow (Rice, 1984). Lee and Monsi (1963) suspected Corresponding author. Tel./Fax: +81 87 891 3086.
E-mail address: [email protected] (H. Kato-Noguchi).
that failure of these herbaceous plants to grow in the red pine forest was due to an allelopathic effect of the pines. They investigated the allelopathic potential of the pine against the seed germination of 15 plant species and found that the needle extracts of the pine markedly inhibited the germination of three plant species. It was also reported that several other pine species showed strong allelopathic potential (Rice, 1984; Harris et al., 2003; Jia et al., 2003; Nektarios et al., 2005; Caboun, 2006; Fernandez et al., 2006, 2008).
0176-1617/$ - see front matter & 2008 Elsevier GmbH. All rights reserved. doi:10.1016/j.jplph.2008.06.012
ARTICLE IN PRESS Allelopathy of Pinus Much research has been conducted to identify the allelochemicals in these pines, and several phenolic acids and their related compounds were isolated from pine needles, bark and soils under the pine trees as putative allelopathic substances (Lee and Monsi, 1963; Chu-Chou 1978; Lodhi and Killingbeck, 1982; Kil and Yim, 1983; Son et al., 1996; Node et al., 2003). However, these compounds are universal in many plant species and growth inhibitory activities were not sufficient to cause growth inhibition of neighboring plants (Olofsdotter et al., 2002). Therefore, the level of allelopathic potential in pines cannot be distinguished from that of other plant species by these compounds. Other compounds may thus contribute to the allelopathy of pines. In this study, the allelopathic potential of red pine needles was determined and a growth inhibitor causing the allelopathic effect was isolated and characterized.
Materials and methods Plant material Needles of red pine (Pinus densiflora Sieb. et Zucc.) were randomly collected from 20 trees (young to old needles from young to old trees) in a 10 10 m2 area, in mountainous terrain in Takamatsu, Japan, in April 2007. Extraction and bioassay Needles of red pine (100 g fresh weight) were extracted with 200 mL of 80% aqueous methanol in darkness at 15–20 1C for 2 d. After filtration using filter paper (No. 2; Toyo Ltd., Tokyo), the residue was extracted again with 200 mL of methanol for 2 d and filtered, and the two filtrates were combined. An aliquot of the extract (final assay concentration was 10, 30 or 100 mg fresh weight pine needle equivalent extract/mL) was evaporated to dryness, dissolved in a small volume of methanol and added to a sheet of filter paper (No. 2) in a 3-cm Petri dish, and methanol was evaporated in the draft chamber. The filter paper in the Petri dishes was then moistened with 0.8 mL of a 0.05% (v/v) aqueous solution of Tween 20, which was used for surfactant and did not cause any toxic effects. After germination in darkness at 25 1C for 18–120 h, 10 seeds of cress (Lepidium sativum L.), lettuce (Lactuca sativa L.), alfalfa (Medicago sativa L.), ryegrass (Lolium multiflorum Lam), timothy (Pheleum pratense L.), Digitaria sanguinalis L. or Echinochloa crus-galli (L.) Beauv were sown on it. The lengths of their shoots and roots were measured after 48 h of incubation in darkness at 25 1C. Control seedlings were sown on the filter paper moistened with the aqueous solution of Tween 20. The bioassay was repeated three times using a randomized design with 10 plants for each determination. Significant
443 differences between treatment and control plants were examined by Welch’s t-test for each test plant species. Purification of active substance in ethyl acetate fraction Needles of red pine (100 g fresh weight) were extracted as described above and the extract was concentrated at 40 1C in vacuo to produce an aqueous residue. The aqueous residue was adjusted to pH 7.0 with 1 M phosphate buffer, partitioned three times against an equal volume of ethyl acetate. The ethyl acetate fraction was evaporated to dryness and chromatographed on a column of silica gel (60 g, silica gel 60, 70–230 mesh; Merck), eluted stepwise with n-hexane containing increasing amounts of ethyl acetate (10% per step, v/v; 100 mL per step). The biological activity of the fractions was determined using a cress bioassay as described above, and activity was found in fractions obtained by elution with 60–70% of ethyl acetate in n-hexane. After evaporation, the residue was purified by a column of Sephadex LH-20 (50 g, Amersham Pharmacia Biotech, Buckinghamshire, UK), and eluted with 20%, 40%, 60% and 80% (v/v) aqueous methanol (100 mL per step) and methanol (200 mL). The active fraction was eluted by 60% aqueous methanol and evaporated to dryness. The residue was dissolved with 20% (v/v) aqueous methanol (2 mL) and loaded onto reverse-phase C18 Sep-Pak cartridges (Waters). The cartridges were eluted with 20%, 40%, 60% or 80% (v/v) aqueous methanol and methanol (15 mL per step). The active fraction was eluted by 80% aqueous methanol and evaporated to dryness. The residue was finally purified by reverse-phase HPLC (10 mm i.d. 50 cm, ODS AQ-325; YMC Ltd., Kyoto, Japan) eluted at a flow rate of 2 mL/min with 75% aqueous methanol, detected at 220 nm. Inhibitory activity was found in a peak fraction eluted between 110 and 112 min, yielding an active substance (4.3 mg) as colorless oil. The active substance was characterized by 1 H- and 13C-NMR spectra.
Results and discussion Allelopathic potential of pine needle extract Aqueous methanol extract of pine needles inhibited root and shoot growth of all plant species including weed species (Digitaria sanguinalis and Echinochloa crus-galli), and increasing the extract concentration increased the inhibition of all plants (Figure 1). The extract obtained from 100 mg fresh weight pine needles inhibited the root growth of cress, lettuce, alfalfa, ryegrass, timothy, Digitaria sanguinalis and Echinochloa crus-galli to 9.2%, 7.1%, 14.5%, 13.9%, 5.8%, 18% and 7.5% of control root growth, respectively, and inhibited the shoot growth of cress, lettuce, alfalfa, ryegrass, timothy,
ARTICLE IN PRESS 444
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Figure 1. Effects of aqueous methanol extract of pine needles on root and shoot growth of cress, lettuce, alfalfa, ryegrass, timothy, D. sanguinalis and E. crus-galli. Concentrations of tested samples corresponded to the extract obtained from 10, 30 and 100 mg fresh weight pine needles. Means7SE from three independent experiments with 10 plants for each determination are shown. Asterisk indicates significant difference between control and treatment: *Po0.05, **Po0.01, ***Po0.001 (Welch’s t-test).
Digitaria sanguinalis and Echinochloa crus-galli to 14.8%, 20.2%, 16.5%, 26.2%, 22.1%, 52.4% and 65.7% of control shoot growth, respectively. The pine extract therefore had an inhibitory effect on a wide range of plant species, both of the monocotyledonous plants (alfalfa, ryegrass, timothy, Digitaria sanguinalis and Echinochloa crus-galli) and the dicotyledonous plants (cress, lettuce and alfalfa). These results suggest that the extract of pine needles may contain growth inhibitory substances and the pine needles may possess allelopathic potential.
Identification of allelochemical and its biological activity An active substance (4.3 mg) was isolated as colorless oil from the aqueous methanol extract of pine needles (100 g fresh weight). The 1H-NMR (400 MHz, CD3OD) spectrum of the compound showed d 0.97 (6H, d, J ¼ 6.9 Hz), 1.08 (3H, s), 1.28 (3H, s), 1.41 (1H, m), 1.46 (1H, m), 1.47 (1H, m), 1.55 (1H, m), 1.60 (1H, m), 1.62 (1H, m), 1.65 (1H, m), 1.67 (1H, m), 1.74 (1H, m), 1.90 (1H, m), 1.91 (1H, m), 1.97 (1H, m), 2.09 (1H, m), 2.45 (1H,
m), 2.52 (1H, m), 2.54 (1H, m) and 6.09 (1H, s). The 13 C-NMR (100 MHz, CD3OD, TMS as internal standard) spectrum of the compound showed d 17.1, 17.5, 17.7, 19.1, 19.8, 21.7, 24.2, 25.0, 30.8, 32.1, 36.8, 38.1, 39.0, 46.3, 79.1, 80.8, 127.1, 144.2 and 182.0. From the comparison of these data with those reported in the literature (Barrero et al., 1991), the compound was identified as an abietane diterpenoid, 9a,13b-epidioxyabeit-8(14)en-18-oic acid (Figure 2). This compound has been isolated from needles and bark of another Pinaceae species (Barrero et al., 1991; Tanaka et al., 1997; Kinouchi et al., 2000); however, it has not been reported that 9a,13b-epidioxyabeit-8(14)en-18-oic acid has plant growth inhibitory effect. This substance inhibited root and shoot growth of cress and Echinochloa crus-galli at concentrations greater than 0.1 mM (Figure 3). When the percentage length of test plants was plotted against a logarithm of the concentrations, the concentration–response curves were linear between 20% and 80% inhibition. The concentrations required for 50% inhibition in the roots and shoots of cress in the assay, determined by a logistic regression analysis, were 0.47 and 0.49 mM, respectively, and on those of Echinochloa crus-galli
ARTICLE IN PRESS Allelopathy of Pinus were 0.52 and 1.2 mM, respectively. Therefore, 9a,13b-epidioxyabeit-8(14)en-18-oic acid inhibited the root and shoot growth of cress and Echinochloa crus-galli seedlings, and inhibition was increased with increasing concentrations of 9a,13b-epidioxyabeit8(14)en-18-oic acid.
Conclusion An aqueous methanol extract of red pine needles was purified, and a main inhibitory substance was
Figure 2. Chemical structure of 9a,13b-epidioxyabeit8(14)en-18-oic acid.
445 isolated and determined by spectral data as 9a,13bepidioxyabeit-8(14)en-18-oic acid (Figure 2). This compound was active at concentrations greater than 0.1 mM (Figure 3). Chemicals with alleloapthic activity are present in many plants and in many plant organs. Under certain conditions, these compounds are released into the plant rhizosphere, either as exudates from living plants or by decomposition of plant residues in sufficient quantities to inhibit the growth of neighboring or successional plants (Rice, 1984; Putnam, 1988; Seigler, 1996; Einhellig, 1999). The endogenous concentration of 9a,13b-epidioxyabeit-8(14)en18-oic acid was at least 0.13 mmol/kg pine needles because 4.3 mg of the substance (MW 334) was isolated from 100 g pine needles. Considering the endogenous level and the inhibitory activity, the compound may provide a competitive advantage to pine as an allelopathic substance involved in the defense mechanism through the inhibition of the growth of neighboring plant species. This could provide a competitive advantage to pine in terms of space, water, minerals and nutrients. In addition, 9a,13b-epidioxyabeit8(14)en-18-oic acid has been found in other Pinaceae plant species including Abies marocana, Larix kaempferi and Picea glehni (Barrero et al., 1991; Tanaka et al., 1997; Kinouchi et al., 2000). The present results suggest that 9a,13b-epidioxyabeit-8(14)en-18-oic acid may contribute to the growth inhibitory effect of pine, and may play an important role in the allelopathy of Pinaceae plant species.
Figure 3. Effects of 9a,13b-epidioxyabeit-8(14)en-18-oic acid on the root and shoot growth of cress and E. crus-galli seedlings. Other details as for Figure 1.
ARTICLE IN PRESS 446
References Barrero AF, Sanchez JF, Alvarez-Manzaneda EJR, Mun ˜oz Dorado M. Phytochemistry 1991;30:593–7. Caboun V. Allelopathy J 2006;17:17–31. Chu-Chou M. Ann Appl Biol 1978;90:407–16. Einhellig FA. In: Inderjit, Dakshini KMM, Foy CL, editors. Principals and practices in plant ecology: allelochemical interactions. Boca Raton, FL: CRC Press; 1999. p. 479–94. Fernandez C, Lelong B, Vila B, Me´vy JP, Robles C, Greff S, et al. Chemoecology 2006;16:97–105. Fernandez C, Voiriota S, Me´vya JP, Vilaa B, Ormen ˜oa B, Dupouyeta S, et al. Forest Ecol Manage 2008;255: 2928–36. Harris MR, Lamb D, Erskine PD. Aust J Bot 2003;51: 93–102. Jia L, Zhai M, Feng C. J Beijing Forest Univ 2003;25:6–10. Kil BS, Yim YJ. J Chem Ecol 1983;9:1135–51.
H. Kato-Noguchi et al. Kinouchi Y, Ohtsu H, Tokuda H, Nishino H, Matsunaga S, Tanaka R. J Nat Prod 2000;63:817–20. Lee IK, Monsi M. Bot Mag 1963;76:400–13. Lodhi MAK, Killingbeck KT. J Chem Ecol 1982;8:275–83. Nektarios PA, Economou G, Avgoulas C. HortScience 2005; 40:246–50. Node M, Tomita-Yokotani K, Suzuki T, Kosemura S, Hirata H, Hirata K, et al. Weed Biol Manage 2003;3:111–6. Olofsdotter M, Rebulanan M, Madrid A, Dali W, Navarez D, Olk DC. J Chem Ecol 2002;28:229–42. Putnam AR. Weed Technol 1988;2:510–8. Rice EL. Allelopathy, 2nd ed. Orlando: Academic Press; 1984. Seigler DS. Agron J 1996;88:876–85. Son DS, Eon TJ, Seo JD, Lee SR. J Korean For Soc 1996;85: 372–80. Tanaka R, Ohtsu H, Matsunaga S. Phytochemistry 1997; 46:1051–7.
www.elsevier.de/jplph
SHORT COMMUNICATION
An allelopathic substance in red pine needles (Pinus densiflora) Hisashi Kato-Noguchia,, Yoshiko Fushimia, Hideyuki Shigemorib a
Department of Applied Biological Science, Faculty of Agriculture, Kagawa University, Miki, Kagawa 761-0795, Japan Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba 305-8572, Japan
b
Received 29 April 2008; received in revised form 12 June 2008; accepted 25 June 2008
KEYWORDS Allelopathy; Growth inhibitor; Phytoalexin; Pinaceae
Summary Aqueous methanol extracts of red pine (Pinus densiflora) needles inhibited the growth of roots and shoots of cress (Lepidium sativum), lettuce (Lactuca sativa), alfalfa (Medicago sativa), ryegrass (Lolium multiflorum), timothy (Pheleum pratense), Digitaria sanguinalis and Echinochloa crus-galli. Increasing the extract concentration increased inhibition, suggesting that the pine needles may have growth inhibitory substances and possess allelopathic potential. The aqueous methanol extract of the pine needles was purified, and a main inhibitory substance was isolated and determined by spectral data as 9a,13b-epidioxyabeit-8(14)en18-oic acid. This substance inhibited root and shoot growth of cress and Echinochloa crus-galli seedlings at concentrations greater than 0.1 mM. The endogenous concentration of the substance was 0.13 mmol/kg pine needle. These results suggest that 9a,13b-epidioxyabeit-8(14)en-18-oic acid may contribute to the growth inhibitory effect of the pine needles and may play an important role in the allelopathy of red pine. & 2008 Elsevier GmbH. All rights reserved.
Introduction The vegetation under red pine forests is sparse compared with other forests that have dense undergrowths of herbaceous plants, despite the fact that the sunlight intensity under red pine forests is sufficient for these herbaceous plants to grow (Rice, 1984). Lee and Monsi (1963) suspected Corresponding author. Tel./Fax: +81 87 891 3086.
E-mail address: [email protected] (H. Kato-Noguchi).
that failure of these herbaceous plants to grow in the red pine forest was due to an allelopathic effect of the pines. They investigated the allelopathic potential of the pine against the seed germination of 15 plant species and found that the needle extracts of the pine markedly inhibited the germination of three plant species. It was also reported that several other pine species showed strong allelopathic potential (Rice, 1984; Harris et al., 2003; Jia et al., 2003; Nektarios et al., 2005; Caboun, 2006; Fernandez et al., 2006, 2008).
0176-1617/$ - see front matter & 2008 Elsevier GmbH. All rights reserved. doi:10.1016/j.jplph.2008.06.012
ARTICLE IN PRESS Allelopathy of Pinus Much research has been conducted to identify the allelochemicals in these pines, and several phenolic acids and their related compounds were isolated from pine needles, bark and soils under the pine trees as putative allelopathic substances (Lee and Monsi, 1963; Chu-Chou 1978; Lodhi and Killingbeck, 1982; Kil and Yim, 1983; Son et al., 1996; Node et al., 2003). However, these compounds are universal in many plant species and growth inhibitory activities were not sufficient to cause growth inhibition of neighboring plants (Olofsdotter et al., 2002). Therefore, the level of allelopathic potential in pines cannot be distinguished from that of other plant species by these compounds. Other compounds may thus contribute to the allelopathy of pines. In this study, the allelopathic potential of red pine needles was determined and a growth inhibitor causing the allelopathic effect was isolated and characterized.
Materials and methods Plant material Needles of red pine (Pinus densiflora Sieb. et Zucc.) were randomly collected from 20 trees (young to old needles from young to old trees) in a 10 10 m2 area, in mountainous terrain in Takamatsu, Japan, in April 2007. Extraction and bioassay Needles of red pine (100 g fresh weight) were extracted with 200 mL of 80% aqueous methanol in darkness at 15–20 1C for 2 d. After filtration using filter paper (No. 2; Toyo Ltd., Tokyo), the residue was extracted again with 200 mL of methanol for 2 d and filtered, and the two filtrates were combined. An aliquot of the extract (final assay concentration was 10, 30 or 100 mg fresh weight pine needle equivalent extract/mL) was evaporated to dryness, dissolved in a small volume of methanol and added to a sheet of filter paper (No. 2) in a 3-cm Petri dish, and methanol was evaporated in the draft chamber. The filter paper in the Petri dishes was then moistened with 0.8 mL of a 0.05% (v/v) aqueous solution of Tween 20, which was used for surfactant and did not cause any toxic effects. After germination in darkness at 25 1C for 18–120 h, 10 seeds of cress (Lepidium sativum L.), lettuce (Lactuca sativa L.), alfalfa (Medicago sativa L.), ryegrass (Lolium multiflorum Lam), timothy (Pheleum pratense L.), Digitaria sanguinalis L. or Echinochloa crus-galli (L.) Beauv were sown on it. The lengths of their shoots and roots were measured after 48 h of incubation in darkness at 25 1C. Control seedlings were sown on the filter paper moistened with the aqueous solution of Tween 20. The bioassay was repeated three times using a randomized design with 10 plants for each determination. Significant
443 differences between treatment and control plants were examined by Welch’s t-test for each test plant species. Purification of active substance in ethyl acetate fraction Needles of red pine (100 g fresh weight) were extracted as described above and the extract was concentrated at 40 1C in vacuo to produce an aqueous residue. The aqueous residue was adjusted to pH 7.0 with 1 M phosphate buffer, partitioned three times against an equal volume of ethyl acetate. The ethyl acetate fraction was evaporated to dryness and chromatographed on a column of silica gel (60 g, silica gel 60, 70–230 mesh; Merck), eluted stepwise with n-hexane containing increasing amounts of ethyl acetate (10% per step, v/v; 100 mL per step). The biological activity of the fractions was determined using a cress bioassay as described above, and activity was found in fractions obtained by elution with 60–70% of ethyl acetate in n-hexane. After evaporation, the residue was purified by a column of Sephadex LH-20 (50 g, Amersham Pharmacia Biotech, Buckinghamshire, UK), and eluted with 20%, 40%, 60% and 80% (v/v) aqueous methanol (100 mL per step) and methanol (200 mL). The active fraction was eluted by 60% aqueous methanol and evaporated to dryness. The residue was dissolved with 20% (v/v) aqueous methanol (2 mL) and loaded onto reverse-phase C18 Sep-Pak cartridges (Waters). The cartridges were eluted with 20%, 40%, 60% or 80% (v/v) aqueous methanol and methanol (15 mL per step). The active fraction was eluted by 80% aqueous methanol and evaporated to dryness. The residue was finally purified by reverse-phase HPLC (10 mm i.d. 50 cm, ODS AQ-325; YMC Ltd., Kyoto, Japan) eluted at a flow rate of 2 mL/min with 75% aqueous methanol, detected at 220 nm. Inhibitory activity was found in a peak fraction eluted between 110 and 112 min, yielding an active substance (4.3 mg) as colorless oil. The active substance was characterized by 1 H- and 13C-NMR spectra.
Results and discussion Allelopathic potential of pine needle extract Aqueous methanol extract of pine needles inhibited root and shoot growth of all plant species including weed species (Digitaria sanguinalis and Echinochloa crus-galli), and increasing the extract concentration increased the inhibition of all plants (Figure 1). The extract obtained from 100 mg fresh weight pine needles inhibited the root growth of cress, lettuce, alfalfa, ryegrass, timothy, Digitaria sanguinalis and Echinochloa crus-galli to 9.2%, 7.1%, 14.5%, 13.9%, 5.8%, 18% and 7.5% of control root growth, respectively, and inhibited the shoot growth of cress, lettuce, alfalfa, ryegrass, timothy,
ARTICLE IN PRESS 444
H. Kato-Noguchi et al.
Figure 1. Effects of aqueous methanol extract of pine needles on root and shoot growth of cress, lettuce, alfalfa, ryegrass, timothy, D. sanguinalis and E. crus-galli. Concentrations of tested samples corresponded to the extract obtained from 10, 30 and 100 mg fresh weight pine needles. Means7SE from three independent experiments with 10 plants for each determination are shown. Asterisk indicates significant difference between control and treatment: *Po0.05, **Po0.01, ***Po0.001 (Welch’s t-test).
Digitaria sanguinalis and Echinochloa crus-galli to 14.8%, 20.2%, 16.5%, 26.2%, 22.1%, 52.4% and 65.7% of control shoot growth, respectively. The pine extract therefore had an inhibitory effect on a wide range of plant species, both of the monocotyledonous plants (alfalfa, ryegrass, timothy, Digitaria sanguinalis and Echinochloa crus-galli) and the dicotyledonous plants (cress, lettuce and alfalfa). These results suggest that the extract of pine needles may contain growth inhibitory substances and the pine needles may possess allelopathic potential.
Identification of allelochemical and its biological activity An active substance (4.3 mg) was isolated as colorless oil from the aqueous methanol extract of pine needles (100 g fresh weight). The 1H-NMR (400 MHz, CD3OD) spectrum of the compound showed d 0.97 (6H, d, J ¼ 6.9 Hz), 1.08 (3H, s), 1.28 (3H, s), 1.41 (1H, m), 1.46 (1H, m), 1.47 (1H, m), 1.55 (1H, m), 1.60 (1H, m), 1.62 (1H, m), 1.65 (1H, m), 1.67 (1H, m), 1.74 (1H, m), 1.90 (1H, m), 1.91 (1H, m), 1.97 (1H, m), 2.09 (1H, m), 2.45 (1H,
m), 2.52 (1H, m), 2.54 (1H, m) and 6.09 (1H, s). The 13 C-NMR (100 MHz, CD3OD, TMS as internal standard) spectrum of the compound showed d 17.1, 17.5, 17.7, 19.1, 19.8, 21.7, 24.2, 25.0, 30.8, 32.1, 36.8, 38.1, 39.0, 46.3, 79.1, 80.8, 127.1, 144.2 and 182.0. From the comparison of these data with those reported in the literature (Barrero et al., 1991), the compound was identified as an abietane diterpenoid, 9a,13b-epidioxyabeit-8(14)en-18-oic acid (Figure 2). This compound has been isolated from needles and bark of another Pinaceae species (Barrero et al., 1991; Tanaka et al., 1997; Kinouchi et al., 2000); however, it has not been reported that 9a,13b-epidioxyabeit-8(14)en-18-oic acid has plant growth inhibitory effect. This substance inhibited root and shoot growth of cress and Echinochloa crus-galli at concentrations greater than 0.1 mM (Figure 3). When the percentage length of test plants was plotted against a logarithm of the concentrations, the concentration–response curves were linear between 20% and 80% inhibition. The concentrations required for 50% inhibition in the roots and shoots of cress in the assay, determined by a logistic regression analysis, were 0.47 and 0.49 mM, respectively, and on those of Echinochloa crus-galli
ARTICLE IN PRESS Allelopathy of Pinus were 0.52 and 1.2 mM, respectively. Therefore, 9a,13b-epidioxyabeit-8(14)en-18-oic acid inhibited the root and shoot growth of cress and Echinochloa crus-galli seedlings, and inhibition was increased with increasing concentrations of 9a,13b-epidioxyabeit8(14)en-18-oic acid.
Conclusion An aqueous methanol extract of red pine needles was purified, and a main inhibitory substance was
Figure 2. Chemical structure of 9a,13b-epidioxyabeit8(14)en-18-oic acid.
445 isolated and determined by spectral data as 9a,13bepidioxyabeit-8(14)en-18-oic acid (Figure 2). This compound was active at concentrations greater than 0.1 mM (Figure 3). Chemicals with alleloapthic activity are present in many plants and in many plant organs. Under certain conditions, these compounds are released into the plant rhizosphere, either as exudates from living plants or by decomposition of plant residues in sufficient quantities to inhibit the growth of neighboring or successional plants (Rice, 1984; Putnam, 1988; Seigler, 1996; Einhellig, 1999). The endogenous concentration of 9a,13b-epidioxyabeit-8(14)en18-oic acid was at least 0.13 mmol/kg pine needles because 4.3 mg of the substance (MW 334) was isolated from 100 g pine needles. Considering the endogenous level and the inhibitory activity, the compound may provide a competitive advantage to pine as an allelopathic substance involved in the defense mechanism through the inhibition of the growth of neighboring plant species. This could provide a competitive advantage to pine in terms of space, water, minerals and nutrients. In addition, 9a,13b-epidioxyabeit8(14)en-18-oic acid has been found in other Pinaceae plant species including Abies marocana, Larix kaempferi and Picea glehni (Barrero et al., 1991; Tanaka et al., 1997; Kinouchi et al., 2000). The present results suggest that 9a,13b-epidioxyabeit-8(14)en-18-oic acid may contribute to the growth inhibitory effect of pine, and may play an important role in the allelopathy of Pinaceae plant species.
Figure 3. Effects of 9a,13b-epidioxyabeit-8(14)en-18-oic acid on the root and shoot growth of cress and E. crus-galli seedlings. Other details as for Figure 1.
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References Barrero AF, Sanchez JF, Alvarez-Manzaneda EJR, Mun ˜oz Dorado M. Phytochemistry 1991;30:593–7. Caboun V. Allelopathy J 2006;17:17–31. Chu-Chou M. Ann Appl Biol 1978;90:407–16. Einhellig FA. In: Inderjit, Dakshini KMM, Foy CL, editors. Principals and practices in plant ecology: allelochemical interactions. Boca Raton, FL: CRC Press; 1999. p. 479–94. Fernandez C, Lelong B, Vila B, Me´vy JP, Robles C, Greff S, et al. Chemoecology 2006;16:97–105. Fernandez C, Voiriota S, Me´vya JP, Vilaa B, Ormen ˜oa B, Dupouyeta S, et al. Forest Ecol Manage 2008;255: 2928–36. Harris MR, Lamb D, Erskine PD. Aust J Bot 2003;51: 93–102. Jia L, Zhai M, Feng C. J Beijing Forest Univ 2003;25:6–10. Kil BS, Yim YJ. J Chem Ecol 1983;9:1135–51.
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