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Allelochemical momilactone B from rice plants

Hisashi Kato-Noguchi and Takeshi Ino

Department of Biochemistry and Food Science, Faculty of Agriculture, Kagawa University, Miki, Kagawa 761-0795, Japan.
Email hisashi@ag.kagawa-u.ac.jp

Abstract

The putative compound causing the inhibitory effect of rice (Oryza sativa L.) seedlings was isolated from their culture solution and purified with several chromatographies, and the chemical structure of the inhibitor was determined by spectral data as 3,20-epoxy-3α-hydroxy-9β-pimara-7,15-dien-19,6β-olide (momilactone B). Momilactone B inhibited the root and shoot growth of cress seedlings at concentrations greater than 3 nmol mL-1. The inhibition was increased with increasing concentrations of momilactone B. Momilactone B was released into the neighboring environment from rice roots throughout its life cycle. The rate of momilactone B release from rice roots increased until flowering initiation and then decreased. The release rate of momilactone B at the day of flowering started was 2.1 μg plant-1 day-1, which was ca. 44-fold greater than that of at day 30. On an average, a single rice plant released about 100 μg of momilactone B into the neighboring environment over its life cycle. The release level of momilactone B from rice plants and its effectiveness on the growth inhibition of species tested suggest that momilactone B may act as an allelochemical causing growth suppression of neighboring plants.

Media summary

The release level of momilactone B from rice plants and its effectiveness on the growth inhibition of species tested suggest that momilactone B may act as an allelochemical causing growth suppression of neighboring plants.

Key Words

Allelopathy, growth inhibitor, momilactone B, Oryza sativa, rice, root exudates.

Introduction

Rice (Oryza sativa L.) has been extensively studied with respect to its allelopathy as part of a strategy for sustainable weed management, such as breeding allelopathic rice strains. A large number of rice varieties were found to inhibit the growth of several plant species when these rice varieties were grown together with the tested species under field or/and laboratory conditions (Dilday et al. 1994; Kim et al. 1999; Olofsdotter et al. 1999; Azmi et al. 2000). These findings suggest that rice probably produces and releases allelochemical(s) into the environment. However, rice allelochemicals released from living rice plants have not yet been identified.

Aqueous extracts of rice plants inhibited the growth of several plant species (Kawaguchi et al. 1997 Ebana et al. 2001) and aqueous extracts of decomposing rice residues inhibited root growth of lettuce seedlings (Chou and Lin 1976). Several phenolic compounds, such as p-hydroxybenzoic acid, vanillic acid, p-coumaric acid and ferulic acid were found in aqueous extracts of rice residues and straw (Rimando et al. 2001; Chung et al. 2002). It is not clear, however, whether these compounds are released from living rice plants into the neighboring environment. In this paper, a growth inhibitor was isolated from rice root exudates and the biological activity and concentration of the allelochemical were determined.

Methods

Isolation and quantification of allelochemical

Seeds of rice (Oryza sativa cv. Koshihikari) were germinated and hydroponically grown for 14 days as described by Kato-Noguchi et al. (2002). Then, the culture solution was filtrated and separated by several chromatographic fractionations keeping track of the biological activity and finally putative compound causing the inhibitory effect of the rice seedlings was isolated. The active compound was characterized by high-resolution mass, 1H NMR and 13C NMR spectra.

Bioassay

Test samples were evaporated to dryness, dissolved in a small volume of methanol or water, added to a sheet of filter paper (No. 2) in a 2.8-cm Petri dish and dried. Then, the filter paper in the Petri dishes was moistened with 0.6 ml of a 0.05% (v/v) aqueous solution of Tween 20, and 10 cress seeds were arranged on the filter paper and grown in the dark at 25C. The length of the shoots and roots of their seedlings was measured after 36 h.

Quantification of momilactone B

Seeds of rice were germinated and uniform seedlings were hydroponically grown for 120 days in greenhouse under natural day light condition. The culture solution was renewed every two days and only the roots of the rice plants were immersed in the solution during the incubation. According to the methods described by Kato-Noguchi et al. (2003), release level of momilactone B from rice plants into the culture solution and concentrations of momilactone B in shoots and roots of the rice plants were determined.

Results

Isolation and identification of rice allelochemical

About 5,000 rice seedlings, cv. Koshihikari, were hydroponically grown for 14 days in order to find out a potent allelochemical in rice root exudates. Keeping track of the biological activity, their culture solution was purified by several chromatographic fractionations and finally 2.1 mg of putative compound causing the inhibitory effect of the rice seedlings was isolated (Kato-Noguchi et al. 2002). The chemical structure of the inhibitor was determined from its high-resolution MS, and 1H- and 13C-NMR spectral data as 3,20-epoxy-3α-hydroxy-9β- pimara-7,15-dien- 19,6β-olide (momilactone B; Figure 1).

Figure 1

Momilactone B was first isolated from rice husks with momilactone A (Kato et al. 1973; Takahashi et al. 1976) and later found in rice leaves and straw (Cartwright et al. 1977; Kodama et al. 1988). The function of momilactone A as a phytoalexin has been extensively studied and several lines of evidence indicate that momilactone A has an important role in rice defense system against pathogens (Takahashi et al. 1999; Agrawal et al. 2002). Although the growth inhibitory activity of momilactone B was much greater than that of momilactone A (Takahashi et al. 1976; Kato et al. 1977), the function of momilactone B is obscure.

Biological activity of momilactone B

Momilactone B inhibited the root and shoot growth of cress seedlings at concentrations greater than 3 nmol mL-1. The inhibition was increased with increasing concentrations of momilactone B. The concentrations required for 30 % and 50% inhibition in the assay were 12 and 16 nmol mL-1 , and 36 and 41 nmol mL-1 on cress roots and shoots, respectively, as interpolated from the concentration-response curves (Kato-Noguchi et al. 2002).

Concentrations of momilactone B in rice shoots and roots

Rice plants were hydroponically grown for 130 days and momilactone B concentrations in their shoots and roots were determined. Momilactone B was found in shoots and roots of rice plants throughout the experimental periods. The level of momilactone B in the shoots and roots increased over vegetative growth stage until flowering initiation and then decreased. However, the increase in the shoots was much greater than that in the roots. At day 80, the level of momilactone B in the shoots was 3.8-fold greater than that in the roots.

Rice plants release momilactome B into their neighboring environments

Rice plants were hydroponically grown for 130 days and the release level of momilactone B from their plants were determined. Rice plants released momilactone B throughout the experimental periods. The release rate of momilactone B increased rapidly from day 30 until day 80 when flowering started and then decreased sharply. The release rate of momilactone B at day 80 was 2.1 μg plant-1 day-1, which was ca. 44-fold greater than that at day 30 (Figure 2). In the present experiments, only rice roots were immersed in the culture solution, hence, the rice plants probably release momilactone B from their roots into the solution or their neighboring environments.

One rice plant released about 100 μg of momilactone B into the culture solution as estimated by integrating the release rate of momilactone B between day 0 - 130. Rice crop having a trait of very high density in the paddy field and the water in the field is not replaced usually during their growing season (Rao et al. 1989, Akita and Tanaka 1992). Thus, accumulation of momilactone B released from rice plants may occur in the field conditions sufficiently to inhibit germination and growth of neighboring plants.

Conclusion

Momilactone B was isolated from rice root exudates as a putative compound causing the inhibitory effect of rice plants. Momilactone B inhibited the germination and growth of other plant species, and the inhibitory activity was comparable to that of ABA (Takahashi et al. 1976; Kato et al. 1977). Rice plants released momilactone B into their neighboring environment. On an average, a single rice plant released about 100 μg of momilactone B into the neighboring environment over its life cycle. The release level of momilactone B from rice plants and its effectiveness on the growth inhibition of species tested suggest that momilactone B may act as an allelochemical causing growth suppression of neighboring plants.

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