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Article

Allelopathy and Allelochemicals in Chamaecyparis obtusa Leaves for the Development of Sustainable Agriculture

by
Hisashi Kato-Noguchi
1,*,
Kumpei Mori
1,
Arihiro Iwasaki
2 and
Kiyotake Suenaga
2
1
Department of Applied Biological Science, Faculty of Agriculture, Kagawa University, Miki 761-0795, Kagawa, Japan
2
Department of Chemistry, Faculty of Science and Technology, Keio University, 3-14-1 Hiyoshi, Kohoku, Yokohama 223-8522, Kanagawa, Japan
*
Author to whom correspondence should be addressed.
Agronomy 2024, 14(7), 1557; https://doi.org/10.3390/agronomy14071557
Submission received: 5 June 2024 / Revised: 3 July 2024 / Accepted: 3 July 2024 / Published: 17 July 2024
(This article belongs to the Special Issue Extraction and Analysis of Bioactive Compounds in Crops—2nd Edition)
Browse Figures
Versions Notes

Abstract

:
Chamaecyparis obtusa (Siebold et Zucc.) Endl. is cultivated in the temperate region of East Asia for its high-quality and profitable timber. The timber-processing industry produces a large amount of waste, such as tree bark, branches, and leaves, and finding ways to minimize such waste is economically and environmentally efficient. In the present study, the allelopathic activity and allelochemicals of the C. obtusa leaves were investigated to develop potential application methods. C. obtusa leaves were phytotoxic and associated leaf extracts significantly suppressed the growth of the weeds; Echinochloa crus-galli, Phleum pratense and Lolium multiflorum under laboratory conditions. The leaf powder applied onto the soil surface also suppressed the germination of E. crus-galli and P. pratense under greenhouse conditions. Hinokiic acid and (+)-dihydrosesamin were isolated from the leaves and structurally identified. Both compounds suppressed the growth of cress and P. pratense in a concentration dependent manner. When the leaves of C. obtua were applied as a soil additive, hinokiic acid and (+)-dihydrosesamin in the leaves potentially cause the growth inhibitory activity by leaching into the soil. These experiments showed that the leaves and the leaf extracts of C. obtusa are phytotoxic. These experiments also demonstrated that the leaves and the leaf extracts of C. obtusa have growth inhibitory potential against several plant species, and the leaves produce allelochemicals. Therefore, the leaves and leaf extracts of C. obtusa may be useful for weed suppression purposes. The leaf biomass of C. obtusa is frequently large and the timber processing industry produces significant leaf waste. The development of weed control products using waste leaves may be a solution to minimize the timber processing waste to reduce environmental impact and provide economic value. However, leaf material should be evaluated for weed suppression and herbicidal activity as a soil additive and also possible as a foliar spray under field conditions.

1. Introduction

The gymnospermous species, Chamaecyparis obtusa (Siebold et Zucc.) Endl., belonging to the Cupressaceae family, grows 20–30 m in height and is up to 1 m in diameter. It has red-brown bark. The alternate leaves are scale-like, 2–4 mm in length, and obtusa (Figure 1). This species is native to Taiwan and Japan and is widely cultivated in the temperature regions in East Asia for its high-quality and profitable timber [1,2,3,4,5]. However, the timber-processing industry produces a large amount of waste, such as tree bark, branches, and leaves, and minimizing waste would reduce environmental impacts and economic concerns [6,7]. It is, therefore, valuable to develop possible uses for the waste from timber processing to reduce the negative impacts of timber waste from C. obtusa.
One variety of C. Obtusa, C. Obtusa var. formosana, occurs in the Yuanyang Lake Nature Preserve area in Taiwan as a dominant species in the forest. When individuals of the species fall due to aging, typhoons, and diseases in the forest, the gaps created by the fallen trees have been observed to be replaced by the seedlings of C. obtusa, and other plant seedlings did not grow well [8]. Salicylic acid was isolated from the fallen tree bark and was found to inhibit the seedling growth of several other plant species [8]. Therefore, salicylic acid in the bark may be involved in the persistence of the species in the forest as a dominant species. The C. obtusa bark from timber-processing waste was allelopathic, and benzoic acid and gallic acid were identified in the bark as allelochemicals [9]. Allelopathy is the phenomenon in which certain plants suppress the germination, growth, and regeneration process of neighboring plants by releasing allelochemicals [10,11,12]. Therefore, plant parts containing allelochemicals can be applied to weed management to reduce the application of commercial herbicides. In addition, the essential oil extracted from the bark, wood, and fruits of C. obtusa and the sawdust from its timber-processing industry showed antioxidant activity, anti-bacterial activity, and anti-fungal activity, and several related compounds for the activity have been identified in the bark, branches, fruits, and sawdust [13,14,15,16]. The plant parts contain bioactive substances that may be utilized as useful resources, contributing to the effective and sustainable management of the timber-processing waste from forestry biomass [12,17].
The essential oil obtained from the leaves of C. obtusa also exhibited anti-bacterial and anti-fungal activity, and α-terpinyl acatate, limonene, hibaene sabinene, and cis-thujopsene were isolated as major components of its essential oil [18,19,20]. It was reported that the leaves of several Gymnospermous species, such as Pinus species and Ginko biloba L., are allelopathic and contain several allelochemicals [21,22,23]. These allelochemicals in the leaves were carried onto the soil under the trees by deforestation and liberated into the soil due to the decomposition process of the leaves. The liberated allelochemicals can suppress the germination, growth, and regeneration process of the neighboring plant species and contribute to the domination of the host plants in the natural ecosystem [24,25,26,27]. Although volatile substances from the essential oil extracted from the C. obtusa leaves showed allelopathic activity [28], there has been no information available on the allelochemicals in the leaves. The objective of the present investigation was to develop the possible application of the C. obtusa leaves for timber-processing waste to reduce the ecological impact because the leaf biomass of the species is substantial [29,30,31,32]. Therefore, we determined the allelopathic activity of the C. obtusa leaves on the weed species Echinochloa crus-galli, Phleum pratense, and Lolium multiflorum and characterized the allelochemicals from the leaves. We also determined the allelopathic activity of the leaves applied onto the soil surface under greenhouse conditions.

2. Materials and Methods

2.1. Plant Materials

The leaves of C. obtusa were sampled on the campus of Faculty of Agriculture, Kagawa University (Ikenobe 2393, Miki, Kagawa, Japan) on April 2022 (voucher number CB-L-2022-4-1). Weed species: Echinochloa crus-galli (L.) P. Beauv., Phleum pratense L., and Lolium multiflorum Lam. were selected for the evaluation of the allelopathic activity of tested samples. E. crus-galli, P. pretense, and L. multiflorum seeds were germinated on moist filter paper (No 2; Advantec-Toyo Ltd., Tokyo, Japan) in darkness at 25 °C for 48 h, as described by Hossen et al. [31]. Cress (Lepidum sativum L.) was selected as a bioassay species for the separation process of allelochemicals because of its easy operation and stable germination.

2.2. Determination of Allelopathic Activity of C. obtusa Leaves under a Laboratory Condition

Extract of the leaves of C. obtusa (100 g fresh weight) was obtained by soaking in 1500 mL of aqueous methanol (80%, v/v) for 48 h. After filtration using filter paper (No. 2), the residue was re-extracted by soaking it in 1500 mL of methanol for 24 h and filtered. Two filtrates were mixed and evaporated in vacuo at 40 °C.
The concentrated extract was dissolved in 100 mL of methanol, and 0.001, 0.03, 0.01, 0.3, or 0.1 mL of the extract solution was added onto filter paper (No. 2) in a Petri dish (3 cm, i.d.). After complete evaporation of the methanol in the Petri dishes in a fume hood, Tween 20 solution (1 mL, 0.05% w/v, Nacalai-Tesque, Kyoto, Japan) was added onto the filter paper. Therefore, the Petri dishes contained the substances in the extract obtained from 1, 3, 10, 30, or 100 mg of fresh weight of C. obtusa leaves per mL.
Ten germinated seeds of each weed species were transferred and arranged on the filter paper in the Petri dishes. The length of the roots and coleoptiles of these weed species was measured after 48 h incubation in darkness at 25 °C. The root and coleoptile length of these weed species were compared with respective control root and coleoptile length, and the percentage length against controls was calculated. Control plants were incubated in exactly same treatment but without adding the leaf extract. The experiment was performed four times with 10 germinated seeds for each determination.
The allelopathic activity of the C. obtusa leaf extracts was also evaluated using a cress seed bioassay. The bioassay was carried out with same treatment as the weed species except for the germination process. Cress seeds without the germination process were directly incubated with the leaf extracts of C. obtusa on the filter paper in the Petri dishes.

2.3. Allelopathic Activity of C. obtusa Leaves under Greenhouse Conditions

Sandy loam soil (pH of 6.5, dried, 8 kg) was filled into plastic pots (30 cm i.d. × 20 cm high, surface area; 0.07 m2). The leaves of C. obtusa were dried and ground into powder using a mechanical blender. The powder was spread onto the soil surface in the pots at 0 (control), 0.21, 0.7, 2.1, 7, and 21 g of powder per pot, and these pots were kept in a greenhouse for 9 days after watering. Then, 100 seeds of E. crus-galli and P. pratense were separately sown on the soil in the pots and kept in a greenhouse under natural daylight (average daylight of 12.5 h) and temperature (average 14.7 °C) conditions in April. Water (500 mL) was supplied when the soil surface was dry. The germination rate of E. crus-galli was determined on day 14 after sowing, comparing that of control pots. The experiment was performed four times with 100 seeds for each determination. E. crus-galli and P. pratense were germinated as described in Section 2.1, and 100 germinated seeds of E. crus-galli and P. pratense were separately transplanted into the soil in the pots and kept in a greenhouse under the same conditions as described above. After 20 days, the length of above-ground parts of both species was determined.

2.4. Isolation of Allelochemicals from C. obtusa Leaves

The C. obtusa leaves (100 g fresh weight) were extracted as described above and evaporated to produce an aqueous residue. The aqueous residue was adjusted to pH of 7.0 with phosphate buffer (1 M) and partitioned three times against ethyl acetate for the separation of the ethyl acetate and aqueous fraction. The biological activity of the aqueous and ethyl acetate fractions was determined using the cress bioassay as described in Section 2.2. The ethyl acetate fraction showed higher inhibitory activity than that of the aqueous fraction.
The ethyl acetate fraction after evaporation was subjected to a silica gel column (silica gel 60, 40 g, Merck, Darmstadt, Germany) and separated into 8 fractions using the stepwise solvent system with n-hexane and ethyl acetate mixtures. The solvent composition was fraction 1 (n-hexane:ethyl acetate = 80:20, v/v, total volume 100 mL), fraction 2 (70:30; other conditions were the same as fraction 1), fraction 3 (60:40), fraction 4 (50:50), fraction 5 (40:60), fraction 6 (30:70), fraction 7 (20:80), and fraction 8 (0:100). The biological activity of all separated fractions was determined using the cress bioassay, and inhibitory activity was found in fractions 2 and 6.

2.5. Isolation of an Allelochemical from Fraction 2 Obtained in Section 2.4

The active fraction 2 was evaporated to dryness and separated into 9 fractions using a Sephadex LH-20 column (40 g, Sigma-Aldrich, Burlington, VT, USA) according to the stepwise solvent system with water and methanol mixtures. The solvent composition was fraction 1 (water:methanol = 80:20; v/v, total volume of 100 mL), fraction 2 (70:30), fraction 3 (60:40), fraction 4 (50:50), fraction 5 (40:60), fraction 6 (30:70), fraction 7 (20:80), fraction 8 (10:90), and fraction 9 (0:100). Inhibitory activity was found in fraction 6. After evaporation of fraction 6, the residue was separated using a reverse-phase ODS cartridge (Dispo-SPE; YMC Ltd., Kyoto, Japan). The ODS cartridge was eluted with following solvent composition, fraction 1 (water:methanol = 80:20, v/v, total volume 30 mL), fraction 2 (70:30), fraction 3 (60:40), fraction 4 (50:50), fraction 5 (40:60), fraction 6 (30:70), fraction 7 (20:80), fraction 8 (10:90), and fraction 9 (0:100). Fraction 6 showed inhibitory activity, and the residue of the fraction 6 was separated using a reverse-phase HPLC (column; ODS-AQ, 10 mm i.d. × 500 mm, YMC Ltd.; detection 220 nm) eluted at a flow rate of 1.5 mL/min with 75% (v/v) aqueous methanol. An active compound (1) was isolated in a fraction eluted between 111 and 122 min, and the chemical structure was determined using the spectrum data of HRESI-MS and 1H-NMR (400 MHz, CDCl3) and optical rotation.

2.6. Isolation of an Allelochemical from Fraction 6 Obtained in Section 2.4

Active fraction 6 was evaporated to dryness and separated using a Sephadex LH-20 column as described above, and inhibitory activity was detected in fraction 6. Fraction 6 after evaporation was separated using a reverse-phase ODS cartridge, and inhibitory activity was found in fraction 5. Fraction 5 was separated using a reverse-phase HPLC (column; ODS-AQ, 10 mm i.d. × 500 mm; detection 220 nm) eluted at a flow rate of 1.5 mL/min with 85% (v/v) aqueous methanol. An active compound (2) was isolated in a fraction eluted between 85 and 93 min and characterized by the spectrum analyses of HRESI-MS, 1H-NMR (400 MHz, CDCl3), 13C-NMR (100 MHz, CDCl3), and optical rotation.

2.7. Spectrum Data for Isolated Compounds

Compound 1: [α]D28 -25 (c 0.073, MeOH); ESIMS m/z 257.1508 [M+Na]+ (calcd for C15H22O2Na 257.1512); 1H NMR (400 MHz, CDCl3) δ 6.68 (brs, 1H), 2.08 (brs, 1H), 1.93 (brd, J = 18.1 Hz, 1 H), 1.80–1.69 (m, 2 H), 1.50–1.39 (m, 2 H), 1.33–1.14 (m, 3 H), 1.18 (s, 3 H), 1.12 (s, 3 H), 0.80 (dd, J = 9.1, 4.5 Hz, 1 H), 0.70 (t, J = 4.1 Hz, 1 H), 0.67 (s, 3 H); 13C NMR (100 MHz, CDCl3) δ 41.7, 40.2, 37.2, 34.8, 34.1, 31.6, 29.2, 28.5, 26.9, 19.6, 17.1, 11.6.
Compound 2: [α]D26 = +5.7 (c 0.18, pyridine); ESIMS m/z 357.1341 [M+H]+ (calcd for C20H21O6, 357.1338); 1H NMR (400 MHz, CDCl3) δH 6.83 (s, 1 H, Ar-H), 6.75–6.77 (m, 2 H, Ar-H), 6.73 (d, J = 7.7 Hz, 1 H, Ar-H), 6.68 (d, J = 1.8 Hz, 1 H, Ar-H), 6.64 (dd, J = 7.7, 1.8 Hz, 1 H, Ar-H), 5.94 (s, 2 H, OCH2O), 5.93 (s, 2 H, OCH2O), 4.79 (d, J = 5.9 Hz, 1 H, H7), 4.05 (dd, J = 8.6, 6.3 Hz, 1 H, H18a), 3.90 (dd, J = 10.4, 5.2 Hz, 1 H, H9a), 3.76 (dd, J = 10.4, 5.4 Hz, 1 H, H9b), 3.72 (dd, J = 8.6, 6.3 Hz, 1 H, H18b), 2.87 (dd, J = 13.6, 6.8 Hz, 1 H, H16a), 2.70 (m, 1 H, H17), 2.54 (dd, J = 13.6, 10.4 Hz, 1 H, H16b), 2.35 (m, 1 H, H8).

2.8. Allelopathic Activity of the Isolated Compounds

The allelopathic activity of hinokiic acid and (+)-dihydrosesamin isolated from C. obtusa leaves was determined using cress and P. pratense bioassay. The compounds were dissolved in methanol and added onto filter paper (No. 2) in a 3 cm Petri dish. After complete evaporation of the methanol in the Petri dishes in a fume hood, Tween 20 solution was added onto the filter paper as per the procedures described in Section 2.2. The concentrations of hinokiic acid and (+)-dihydrosesamin in the bioassay solution was 0.03, 0.1, 0.3, 1, and 3 mM. The experiment was performed four times with 10 cress seeds and 10 P. pratense germinated seeds for each determination.
Benzoxazolin-2(3H)-one (BOA; Wako chemicals, Kyoto, Japan) were also dissolved in methanol and added onto filter paper (No. 2) in a 3 cm Petri dish. After complete evaporation of the methanol in the Petri dishes and adding Tween 20, cress and P. pratense bioassays were performed as described above.

2.9. Statistics

The biological activity of all tested samples was determined with four replications using a randomized design. Significant differences between treatment and control were examined using Welch’s t-test. Significant differences in the inhibitory activity of isolated compounds were examined using an ANOVA one-way analysis (SPSS, version 16.0) and post hoc analysis with Tukey’s HSD test at p < 0.05 level. IC50 value was obtained using GraphPad Prism 6.0 based on the bioassay data.

3. Results

3.1. Allelopathic Activity of C. obtusa Leaves

The extracts of C. obtusa leaves significantly suppressed the root and coleoptile growth of E. crus-galli, P. pratense, and L. multiflorum at concentrations ≥ 10 mg leaf equivalent extract per mL (Figure 2). The concentration of the extracts causing 50% growth inhibition (IC50 value) for the root growth was 20.6 mg, 11.5 mg, and 53.4 mg of leaf equivalent extract per mL for E. crus-galli, P. pratense, and L. multiflorum, respectively, and the IC50 value for the coleoptile growth was 24.5 mg, 12.1 mg, and 81.2 mg of leaf equivalent extract per mL, respectively. The extracts also suppressed the root and hypocotyl growth of cress at concentrations ≥ 10 mg leaf equivalent extract per mL (Figure 3). The IC50 values of the extracts for the cress root and hypocotyl growth were 15.0 mg and 10.9 mg of leaf equivalent extract per mL, respectively.
The leaf powder of C. obtusa significantly inhibited the germination of E. crus-galli and P. pratense under greenhouse conditions at dosages ≥ 0.7 g of powder per pot (Figure 4). The inhibition was increased with the increasing powder dosage. The IC50 value of the powder dosage against the germination of E. crus-galli and P. pratense was 3.7 g and 5.2 g of powder per pot, respectively. Control germination rates for E. crus-galli and P. pratense were 68.3% and 73.8% 14 days after seed sowing, respectively.
The leaf powder also inhibited the growth of the above-ground parts of E. crus-galli and P. pratense under greenhouse conditions at dosages ≥ 2.1 g of powder per pot (Figure 5). The inhibition was increased with the increasing powder dosage. The IC50 value of the powder dosage against the growth of E. crus-galli and P. pratense was 4.6 g and 6.7 g of powder per pot, respectively.

3.2. Isolation and Identification of Allelochemicals in C. obtusa Leaves

The leaf extract of C. obtusa was purified through the bioassay-guided separation process. The ethyl acetate fraction obtained by the partition process of the extract was separated into eight fractions using silica gel chromatography. The biological activity of all separated fractions was determined using a cress bioassay, and allelopathic activity was detected in fractions 2 and 6.
Fraction 2 was further separated using Sephadex LH-20 and ODS cartridges. Finally, compound 1, showing allelopathic activity, was isolated using HPLC. The molecular formula of compound 1 was determined as C15H22O2 using HRESI-MS analysis. The 1H-NMR and 13C-NMR spectrum and the optical rotation suggest that compound 1 is (-)-hinokiic acid (Figure 6) based on the spectrum data in the literature [33].
Fraction 6 was also separated using Sephadex LH-20 and ODS cartridge, and compound 2, showing allelopathic activity, was finally isolated using HPLC. The molecular formula of compound 2 was determined to be C20H20O6 using HRESI-MS analysis. The 1H-NMR spectrum and the optical rotation suggest that compound 2 is (+)-dihydrosesamin (Figure 6) based on the spectrum data in the literature [34,35].

3.3. Allelopathic Activity of the Isolated Compounds

(-)-Hinokiic acid showed significant growth inhibition on the cress roots and hypocotyls at concentrations ≥ 0.3 mM and on the P. pratense roots and coleoptiles at concentrations ≥ 0.3 mM and 1 mM, respectively (Figure 7). The IC50 value of (-)-hinokiic for the cress root and hypocotyl growth was 0.4 mM, and that for the P. pratense root and coleoptile growth was 0.6 mM and 1.6 mM, respectively.
(+)-Dihydrosesamin also showed significant growth inhibition on the cress roots and hypocotyls, as well as the P. pratense roots and coleoptiles at concentrations ≥ 0.3 mM (Figure 8). The IC50 value of (+)-dihydrosesamin for the cress root and hypocotyl growth was 0.5 mM and 0.6 mM, respectively, and that for the P. pratense root and coleoptile growth was 1.3 mM and 1.5 mM, respectively. The IC50 value of BOA for the cress root and hypocotyl growth was 1.7 mM and 2.9 mM, respectively, and that for the P. pratense root and coleoptile growth was 3.7. mM and 4.5 mM, respectively.

4. Discussion

4.1. Allelopathic Activity of C. obtusa Leaves

C. obtusa is widely cultivated in the temperature region in East Asia for its high-quality and profitable timber [1,2,3]. The timber industry produces a large amount of waste, including tree leaves, and developing possible applications for wastes from the leaves would reduce environmental concerns and improve economic efficiency [6,7]. We have examined the allelopathic activity of C. obtusa leaves under laboratory and greenhouse conditions. The leaf extracts showed significant inhibition of the growth of E. crus-galli, P. pratense, and L. multiflorum under laboratory conditions (Figure 2). However, the sensitivity of these weed species to the extracts differed among the species. Comparing their IC50 values, the sensitivity of the P. pratense roots to the extracts was the highest, and that of the L. multiflorum coleoptiles was the lowest among the roots and coleoptiles of these weed species (Table 1). These results suggest that the C. obtusa leaves may contain certain allelochemicals that are extractable with a mixture of methanol and water.
The leaf powder of C. obtusa applied to the soil surface significantly suppressed the germination and growth of E. crus-galli and P. pratense under greenhouse conditions (Figure 4 and Figure 5). The dosages of 0.21, 0.7, 2.1, 7, and 21 g of leaf powder per pot (surface area; 0.07 m2) correspond to approximately 0.03, 0.1, 0.3, 1, and 3 tons of leaf powder per hectare (10,000 m2), respectively. This result suggests that allelochemicals in the C. obtusa leaves may be leached into soils and cause the germination inhibition of E. crus-galli. E. crus-galli is one of the most noxious weeds in agricultural fields [36]. In addition, the application of the powder on the soil surface may be much easier than the incorporation of the powder into the soil.
Allelochemicals are synthesized and stored in plant tissues such as leaves, stems, and roots. According to need, the allelochemicals are released into the neighboring environments from the donor plants, including the rhizosphere, through root exudation and volatilization. Some allelochemicals are also released during the decomposition processes of the fallen leaves and residues of the donor plants and accumulate in the soil [10,37,38]. The allelochemicals provide advantages to the donor plants in resource competition for water, nutrients, and light with neighboring plant species by inhibiting their germination and growth [10,11,12]. It was reported that when fresh and/or dry leaves of some allelopathic plant species were incorporated into the soil, the germination and growth of several weed species were significantly suppressed in greenhouse and field conditions. It was also reported that when the extracts and soaking water of some allelopathic plants were applied to several weed species as irrigation water and foliar spray, the growth of these weed species was significantly inhibited [37,38,39,40,41,42,43,44,45]. In addition, the extracts and residues of several allelopathic plants suppressed the symbiosis of some weeds with arbuscular mycorrhizal fungi and rhizobia [39,40,41]. Rhizobium colonization increases the growth of legumes through the supply of nitrogen [46,47]. The colonization of arbuscular mycorrhizal fungi elevates the growth and fitness of most territorial plants by increasing nutrient absorption, photosynthesis, and tolerance to stress conditions [48,49,50]. The inhibition of symbiosis probably reduces the growth and fitness of these weed species. Therefore, the allelochemicals and plant tissues of allelopathic plants, such as leaves and bark, can be applied as weed-management options in some agriculture practices, reducing man-made herbicide dependency and contributing to developing sustainable agriculture [37,51].
C. obtusa leaves may contain extractable allelochemicals (Figure 2), and some of the allelochemicals in the leaves may be leached into soils and cause germination and growth inhibition (Figure 4). Therefore, C. obtusa leaves may be potentially useful as soil additive materials to control weeds in a variety of agricultural settings. The leaf extracts may also be applied as a foliar spray to control weeds. However, the weed-control activity of the leaves should first be evaluated as a soil additive material, and the weed-control activity of the leaf extract should first be evaluated as a foliar spray in the field condition.

4.2. Allelochemicals in C. obtusa Leaves

In our study, extracts of C. obtusa leaves were separated through the bioassay-guided separation process using the stepwise solvent system and bioassay using cress seeds. The germination of weed species, E. crus-galli, P. pratense, and L. multiflorum, was unstable, and the germination rate was 20–40% at 48 h after sowing. On the other hand, the germination of cress was high (>90%) and stable, as described by Kato-Noguchi et al. [52]. In addition, the extract of C. obtusa leaves also inhibited the cress roots and hypocotyls in a concentration-dependent manner (Figure 3). The sensitivity of the cress roots and hypocotyl to the C. obtusa leaf extract was relatively high in comparison with the IC50 values of these weed species exposed to the extracts (Figure 2 and Figure 3). The biological activity of all separated fractions after every separation step needed to be determined for the bioassay-guided separation process, and the most active fraction in each separation step was subjected to the next separation step. Therefore, cress was selected as a bioassay plant species because of its stable germination rate and easy handling.
The leaf extract of C. obtusa was separated using silica gel chromatography, Sephadex LH-20 chromatography, ODS cartridge, and reverse-phase HPLC, and two active compounds, 1 and 2, were isolated. Those compounds were characterized by the spectrum data as hinokiic acid and (+)-dihydrosesamin, respectively (Figure 6). Hinokiic acid and (+)-dihydrosesamin suppressed the growth of cress and P. pratense in a concentration-dependent manner (Figure 7 and Figure 8). Compared to the IC50 value, the inhibitory activity of hinokiic acid and (+)-dihydrosesamin is very similar (Table 1).
Hinokiic acid (sesquiterpene) has been isolated and identified in the leaves and heartwood of C. obtusa, Sabina gaussenni, and Juniperus species [33,35,53,54]. However, biological activity, including the growth inhibitory activity and allelopathic activity of the compound, has not yet been reported. (+)-Dihydrosesamin (furano lignan) has been isolated in the leaves of Juniperus thurifera [55] and stereoselectively synthesized from (3R)-3-(3,4-methylenedioxyphenyl)-4-butanolide [34]. (-)-Dihydrosesamin showed inhibitory activity in human cancer cell lines [56]. However, the biological activity and allelopathic activity of (+)-dihydrosesamin has not been documented. Hinokiic and (+)-dihydrosesamin in the leaves of C. obtusa may also contribute to the allelopathic activity of the leaf extracts and leaf powder (Figure 2, Figure 3 and Figure 4) and support the findings described in Section 4.1. However, the weed-control activity of hinokiic and (+)-dihydrosesamin should be evaluated in natural conditions in the future. In addition, the growth-inhibitory activity of (+)-dihydrosesamin and hinokiic acid was greater than the well-known phytotoxic substance BOA when considering their IC50 values.
C. obtusa is an evergreen species with scale-like leaves (Figure 1). However, its old leaves are dropped and replaced by young leaves in several years, and the fallen leaves accumulate in the soil under the tree as litter (Figure 9). The fallen leaves of the species reportedly amount to 2.9 t–6.4 t ha−1 per year [57,58,59]. The litter of scale-like leaves is easily broken into small pieces [60], is decomposed by 16% and 45% after 3 and 15 months, respectively, and forms a topsoil layer on the forest floor [57,61]. Hinokiic acid and (+)-dihydrosesamin in the leaves may be released into the soil of the forest floor during the decomposition process of the fallen leaves of C. obtusa. It has been reported that several allelochemicals, including the well-known allelopathic substance juglone, are contained in the leaves of the respective species and liberated into the soil during the decomposition process of the leaves [10]. Two potent allelochemicals, 7-oxodehydroabietic acid and 15-hydroxy-7-oxodehydroabietate, were found in the soil under Japanese red pine trees (Pinus densiflora Sieb. et Zucc.). The pine is also an evergreen species and is part of the Gymnosperm. These compounds originate from the fallen leaves of the pine and are generated during the decomposition process of the leaves [23]. Therefore, hinokiic and (+)-dihydrosesamin in the leaves may work as allelopathic agents of C. obtusa and contribute to its dominance in the natural ecosystem [8].

5. Conclusions

C. obtusa leaves were found to be phytotoxic, and the leaf extracts significantly suppressed the growth of the weeds E. crus-galli, P. pratense, and L. multiflorum in laboratory conditions. The leaf powder applied onto the soil surface also suppressed the germination of E. crus-galli and P. pratense under greenhouse conditions. Hinokiic acid and (+)-dihydrosesamin were isolated from the leaves and identified. Both compounds suppressed the growth of cress and P. pratense in a concentration-dependent manner. When the leaves of C. obtusa are applied as a soil additive, hinokiic acid and (+)-dihydrosesamin in the leaves may leach into the soil and potentially cause growth-inhibitory activity. These experiments showed that the leaves and the leaf extracts of C. obtusa have growth inhibition. These experiments showed that the leaves and the leaf extracts of C. obtusa have growth-inhibitory activity against several plant species, and the leaves potentially contain allelochemicals. Therefore, the leaves and leaf extracts of C. obtusa may be useful for weed-suppression purposes. The leaf biomass of C. obtusa is quite substantial, and the timber-processing industry produces significant leaf waste. The development of weed -control products using waste leaves may be helpful in minimizing the timber processing waste to reduce environmental impacts and provide economic value. However, first, the leaf material should be evaluated for weed-suppression and herbicidal activity as a soil additive and a foliar spray under field conditions.

Author Contributions

Conceptualization, H.K.-N.; methodology, K.M., A.I., K.S. and H.K.-N.; software K.M.; validation, K.S. and H.K.-N.; formal analysis, K.M.; investigation, K.M. and A.I.; data curation, K.S. and H.K.-N.; writing H.K.-N.; visualization, H.K.-N.; supervision, H.K.-N. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Data Availability Statement

Data are contained within the article.

Acknowledgments

The author acknowledges David R. Clements, Trinity Western University, BC, Canada, for editing the English of the manuscript.

Conflicts of Interest

The authors declare no conflicts of interest.

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Agronomy 14 01557 g001
Figure 1. C. obtusa and its leaves.
Figure 1. C. obtusa and its leaves.
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Figure 2. Effects of the methanolic extract of C. obtusa leaves on the root and coleoptile growth of E. crus-galli, P. pratense, and L. multiflorum under laboratory conditions. The concentrations indicate the extracts obtained from 1, 3, 10, 30, or 100 mg fresh weight of C. obtusa leaves per mL. Means ± SE were calculated from 4 independent experiments with 10 seedlings for each determination. Asterisks show significant differences between treatment and control: *; p < 0.05, **; p < 0.01, ***; p < 0.001.
Figure 2. Effects of the methanolic extract of C. obtusa leaves on the root and coleoptile growth of E. crus-galli, P. pratense, and L. multiflorum under laboratory conditions. The concentrations indicate the extracts obtained from 1, 3, 10, 30, or 100 mg fresh weight of C. obtusa leaves per mL. Means ± SE were calculated from 4 independent experiments with 10 seedlings for each determination. Asterisks show significant differences between treatment and control: *; p < 0.05, **; p < 0.01, ***; p < 0.001.
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Figure 3. Effects of the methanolic extract of C. obtusa leaves on the growth of the roots and coleoptiles of cress. The concentrations indicate the extracts obtained from 1, 3, 10, 30, or 100 mg fresh weight of C. obtusa leaves per mL. Means ± SE were calculated from 4 independent experiments with 10 seedlings for each determination. Asterisks show significant differences between treatment and control: *; p < 0.05, **; p < 0.01, ***; p < 0.001.
Figure 3. Effects of the methanolic extract of C. obtusa leaves on the growth of the roots and coleoptiles of cress. The concentrations indicate the extracts obtained from 1, 3, 10, 30, or 100 mg fresh weight of C. obtusa leaves per mL. Means ± SE were calculated from 4 independent experiments with 10 seedlings for each determination. Asterisks show significant differences between treatment and control: *; p < 0.05, **; p < 0.01, ***; p < 0.001.
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Figure 4. Effects of the leaf powder of C. obtusa on the germination of E. crus-galli and P. pratense under greenhouse conditions. Leaf power (0.21, 0.7, 2.1, 7, and 21 g powder per pot) was spread over soil surface of the pots. Means ± SE were calculated from 4 independent experiments with 100 seeds for each determination. Asterisks show significant differences between treatment and control: *; p < 0.05, **; p < 0.01, ***; p < 0.001.
Figure 4. Effects of the leaf powder of C. obtusa on the germination of E. crus-galli and P. pratense under greenhouse conditions. Leaf power (0.21, 0.7, 2.1, 7, and 21 g powder per pot) was spread over soil surface of the pots. Means ± SE were calculated from 4 independent experiments with 100 seeds for each determination. Asterisks show significant differences between treatment and control: *; p < 0.05, **; p < 0.01, ***; p < 0.001.
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Figure 5. Effects of the leaf powder of C. obtusa on the growth of above-ground parts of E. crus-galli and P. pratense under greenhouse conditions. Leaf power (0.21, 0.7, 2.1, 7, and 21 g powder per pot) was spread over soil surface of the pots. Means ± SE were calculated from 4 independent experiments with 100 plants for each determination. Asterisks show significant differences between treatment and control: *; p < 0.05, **; p < 0.01, ***; p < 0.001.
Figure 5. Effects of the leaf powder of C. obtusa on the growth of above-ground parts of E. crus-galli and P. pratense under greenhouse conditions. Leaf power (0.21, 0.7, 2.1, 7, and 21 g powder per pot) was spread over soil surface of the pots. Means ± SE were calculated from 4 independent experiments with 100 plants for each determination. Asterisks show significant differences between treatment and control: *; p < 0.05, **; p < 0.01, ***; p < 0.001.
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Figure 6. Chemical structures of hinokiic acid and (+)-dihydrosesamin.
Figure 6. Chemical structures of hinokiic acid and (+)-dihydrosesamin.
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Figure 7. Effects of hinokiic acid on the root and hypocotyl/coleoptile growth of cress and P. pratense. Means ± SE were calculated from 4 independent experiments with 10 seedlings for each determination. Different letters on the bars in the same panels indicate significant differences at the p < 0.05 level.
Figure 7. Effects of hinokiic acid on the root and hypocotyl/coleoptile growth of cress and P. pratense. Means ± SE were calculated from 4 independent experiments with 10 seedlings for each determination. Different letters on the bars in the same panels indicate significant differences at the p < 0.05 level.
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Figure 8. Effects of (+)-dihydrosesamin on the root and hypocotyl/coleoptile growth of cress and P. pratense. Means ± SE were calculated from 4 independent experiments with 10 seedlings for each determination. Different letters on the bars in the same panels indicate significant differences at the p < 0.05 level.
Figure 8. Effects of (+)-dihydrosesamin on the root and hypocotyl/coleoptile growth of cress and P. pratense. Means ± SE were calculated from 4 independent experiments with 10 seedlings for each determination. Different letters on the bars in the same panels indicate significant differences at the p < 0.05 level.
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Figure 9. The accumulation of fallen C. obtusa leaves on the soil under the tree.
Figure 9. The accumulation of fallen C. obtusa leaves on the soil under the tree.
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Table 1. IC50 values of the extracts, leaf powder of C. obtusa, and isolated compounds from C. obtusa on the growth or germination of bioassay test plant species. The values were determined by the regression equation of the concentration response, as described in the text.
Table 1. IC50 values of the extracts, leaf powder of C. obtusa, and isolated compounds from C. obtusa on the growth or germination of bioassay test plant species. The values were determined by the regression equation of the concentration response, as described in the text.
Extract (mg of leaf equivalent extract per mL)
Test plantRoot growthColeoptile/hypocotyl growth
E. crus-galli20.624.5
P. pratense11.512.1
L. multiflorum53.481.2
Cress1510.9
Leaf powder (g powder per pot)
Test plantGermination
E. crus-galli4.6
P. pratense6.7
(-)-Hinokiic (mM)
Test plantRoot growthColeoptile/hypocotyl growth
P. pratense0.61.6
Cress0.40.4
(+)-Dihydrosesamin (mM)
Test plantRoot growthColeoptile/hypocotyl growth
P. pratense1.31.5
Cress 0.50.6
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Kato-Noguchi, H.; Mori, K.; Iwasaki, A.; Suenaga, K. Allelopathy and Allelochemicals in Chamaecyparis obtusa Leaves for the Development of Sustainable Agriculture. Agronomy 2024, 14, 1557. https://doi.org/10.3390/agronomy14071557

AMA Style

Kato-Noguchi H, Mori K, Iwasaki A, Suenaga K. Allelopathy and Allelochemicals in Chamaecyparis obtusa Leaves for the Development of Sustainable Agriculture. Agronomy. 2024; 14(7):1557. https://doi.org/10.3390/agronomy14071557

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Kato-Noguchi, Hisashi, Kumpei Mori, Arihiro Iwasaki, and Kiyotake Suenaga. 2024. "Allelopathy and Allelochemicals in Chamaecyparis obtusa Leaves for the Development of Sustainable Agriculture" Agronomy 14, no. 7: 1557. https://doi.org/10.3390/agronomy14071557

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