Agronomy Journal 93:16-20 (2001)
© 2001 American Society of Agronomy
ALLELOPATHY SYMPOSIUM
Searching for Rice Allelochemicals
An Example of Bioassay-Guided Isolation
Agnes M. Rimandoa,
Maria Olofsdotterb,
Franck E. Dayana and
Stephen O. Dukea
a USDA-ARS-NPURU, National Center for Natural Products Research, P.O. Box 8048, University, MS 38677
b Weed Science, IRRI-KVL, Thorvaldsens vej 40, 1871 Fredericksberg C, Copenhagen, Denmark
Corresponding author (arimando{at}ars.usda.gov)
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ABSTRACT
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A bioactivity-guided isolation method was developed with the objective of isolating the allelochemicals in rice (Oryza sativa L.). Roots of the allelopathic rice cultivar Taichung Native 1, grown hydroponically, were extracted and fractionated, with the activity of the fractions followed using a 24-well culture plate microbioassay. Some of the fractions obtained consisted of pure compounds, but none inhibited the growth of barnyardgrass [Echinochloa crusgalli (L.) Beauv.] at the lower concentration at which they were tested. Identified compounds were azelaic acid; 
-coumaric acid; 1H-indole-3-carboxaldehyde; 1H-indole-3-carboxylic acid; 1H-indole-5-carboxylic acid; and 1,2-benzenedicarboxylic acid bis(2-ethylhexyl)ester. -Coumaric acid, a known allelochemical, inhibited the germination of lettuce (Lactuca sativa L.) seedlings at 1 mM. However, -coumaric acid was active against barnyardgrass only at concentrations higher than 3 mM. The two most active fractions obtained from the bioassay-guided isolation were still a mixture of compounds as analyzed by gas chromatography–mass spectrometry (GC-MS). Further fractionation is being done to isolate and identify the allelochemical(s) in these active fractions. This work has demonstrated the use of bioassay-guided isolation in identifying allelochemicals in rice and has correlated observed field activity with laboratory experiments.
Abbreviations: GLM, general linear model • GC-MS, gas chromatography–mass spectrometry • NMR, nuclear magnetic resonance
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INTRODUCTION
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OBSERVATION of apparent allelopathy in rice (Oryza sativa L.) has recently drawn great attention (Olofsdotter, 1998), and there is much interest in identification of the allelochemical(s). Identification of the phytotoxic compound(s) responsible for allelopathy will allow efficient generation of more allelopathic cultivars through traditional breeding or biotechnology-based genetic alterations. Such cultivars could become important tools in the development of advanced integrated weed management strategies for rice, which would be less dependent on synthetic herbicides.
The search for allelochemicals in rice necessitates evaluating the activity in a laboratory set-up to distinguish between competition and allelopathy, which cannot be distinctly separated in field studies (Olofsdotter et al., 1997). Depending on one's objectives, different methods could be followed in searching for active constituents from plants. These include bioassay-guided isolation, fractionation-driven bioassay, isolate and assay, and biochemical combinatorial chemistry approaches. The advantages and disadvantages of each of these methods are discussed in more detail by Duke et al. (2000a). We chose bioassay-guided isolation as the best way to proceed because the active component is not known.
Bioassay-guided isolation integrates the processes of separation of compounds in a mixture, using various analytical methods, with results obtained from biological testing. The process begins with testing an extract to confirm its activity, followed by crude separation of the compounds in the matrix and testing the crude fractions (Fig. 1)
. Further fractionation is carried out on the fractions that are determined to be active, at a certain concentration threshold, whereas the inactive fractions are set aside or discarded. The process of fractionation and biological testing is repeated until pure compound(s) are obtained. Structural identification of the pure compound then follows. This methodology precludes overlooking novel compounds that are often missed in studies that only identify those compounds with which the investigator is familiar. Moreover, the possibility of discovering an unknown molecular site of action is maximized (Duke et al., 2000b).
In carrying out bioassay-guided isolations of allelochemicals from rice, there are several important factors that must be considered. First, the rice cultivar to be extracted has to be chosen with care to make sure that allelopathy is a likely explanation of the effects on weeds seen in the field and laboratory. Second, an appropriate bioassay needs to be chosen that eliminates nonactive fractions without giving too many false positive results. And third, the target weed to test in the bioassay is an important factor to make sure that laboratory screening actually targets weeds of interest from a field perspective. The use of miniaturized whole organism bioassay is most desirable in a bioassay-guided isolation from an economic standpoint.
Although there have been attempts to identify allelochemicals from rice (Kim and Shin, 1998; Mattice et al., 1998) to date, no laboratory has attempted to conduct bioassay-directed isolation of rice-generated allelochemicals. This paper describes the general strategy and methodology used in the bioassay-guided isolation of allelochemicals as applied in the isolation of phytotoxic compounds from the rice cultivar Taichung Native 1 (TN1), an accession reported to be allelopathic (Dilday et al., 1998).
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Materials and methods
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Plant Material
The rice cultivar TN1 was selected as the plant material to be extracted after several experiments, both in the laboratory and the field, indicated that this cultivar was allelopathic against several weed species in the rice ecosystem (Dilday et al., 1998; Olofsdotter and Navarez, 1996). Roots were chosen as the most likely plant source of allelochemicals in the field situation.
Cultivar TN1 was grown hydroponically. The seeds were surface-sterilized with NaOCl for 30 min, soaked in distilled water, and germinated in 9-cm petri dishes (20 seeds dish-1) under room temperature. Twelve days after soaking, uniform seedlings were placed in holes in a styrofoam float that was placed in a 24-L pail. The float allowed the roots to be submerged in hydroponic solution. There were five seedlings and 20 L of hydroponic solution per pail. The five rice seedlings were planted in holes, 10 cm apart, in a Styrofoam float. The pail was wrapped with aluminum foil to inhibit algae growth and placed in a cooling bench (25°C). The hydroponic culture solution (Yoshida et al., 1976, p. 61–67) was changed every 2.5 d and pH was maintained at 5.5 throughout the experiment.
After 1 mo of growth, the roots were separated from the shoots, and roots were dried. The dried roots were powdered and extracted with MeOH/H2O (50:50), and the extract dried under vacuum. The dried extract was subjected to column chromatographic separations as outlined in Fig. 2
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Phytotoxicity Assay
The activity of TN1 root extract and its column fractions were monitored using a 24-well plate assay previously reported (Rimando et al., 1998). Briefly described, barnyardgrass [Echinochloa crusgalli (L.) Beauv.] seeds were placed on filter paper set at the bottom of the well (5 seeds well-1). Samples were dissolved in acetone and tested at determined concentrations (1 g L-1 for extracts and less pure fractions, 0.5 g L-1, for purer factions; 3.0 mM for pure compounds) in a final volume of 200 µL (10% acetone in H2O) in the wells. Sample was added in duplicate wells. Each plate had duplicate H2O only and 10% acetone in H2O control wells. The plate was sealed with parafilm and placed in a growth chamber (25°C with a 16-h photoperiod at 400 µmol m-2 s-1 photosynthetically active radiation) for 4 d, after which barnyardgrass shoot and root lengths were measured. Barnyardgrass was used as the test species, as this is an important weed in the rice ecosystem. As a parallel check, phytotoxicity of the extracts and fractions against lettuce (Lactuca sativa L.) seedlings was also monitored. Effects on growth of lettuce seedlings were rated visually on a scale of 0 (no effect) to 5 (complete inhibition of growth). Phytotoxicity of -coumaric acid against barnyardgrass and lettuce was tested a concentrations as shown in Table 5. Analysis of variance using the general linear model (GLM) procedure (SAS Inst., 1998) was carried out on the data (barnyardgrass root and shoot length), and the means were separate by LSD at the 
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Chemical Identification
Thin layer chromatography was done on Silica gel plates (Macherey-Nagel, Alugram Sil G/UV254, 0.25-mm layer) using a combination of CH2Cl2 and MeOH as developing solvent. Column chromatography was performed on Silica gel (CH2Cl2 to MeOH gradient elution) and C18 sorbents (H2O to MeOH gradient elution). All organic solvents used were purchased from Fisher Scientific (Norcross, GA). Identification of pure compounds was carried out using nuclear magnetic resonance (NMR) (Bruker Avance DPX 300) and mass spectroscopy (Hewlett Packard 5989A). Fractions were also analyzed by gas chromography–mass spectrometry (GC-MS) (Hewlett Packard 5890 Series II gas chromatograph coupled to Hewlett Packard 5989A MS engine). Gas chromography–mass spectrometry conditions are as follows: DB-5MS 30-m length by 0.25 mm i.d. by 0.25-µm film capillary column (J&W Scientific, Folsom, CA); injector temp. 250°C, oven temperature programmed at 120°C initial temperature held for 1 min, then increased 12°C min-1 to 320°C and held at this temperature for 5 min. The carrier gas was helium at a flow rate of 1.9 mL min-1. Mass spectrometry zone temperature was: transfer line at 280°C, source at 250°C, quadruple at 100°C. Ionization voltage was at 70 eV.
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Results and discussion
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Several rice cultivars have demonstrated allelopathic property against barnyardgrass or other weeds. Among the rice cultivars evaluated from prior screening procedures to distinguish between competition and allelopathy, field experiments, and greenhouse studies, TN1 had shown allelopathic effect against barnyardgrass, horse-purslane (Trianthema portulacastrum L.), ducksalad (Heteranthera limosa), and Ammannia sp. (Dilday et al., 1998; Olofsdotter et al., 1997). Cultivar TN1 was chosen for extraction and isolation of allelochemicals, not only for being the "parent" allelopathic cultivar but also because of results obtained from preliminary studies. In a blinded study, root extracts from three rice samples (V69, V216, and VO1) were tested for activity against lettuce and barnyardgrass using 24-well culture plate assay. Sample V01, which was an extract of TN1, inhibited the growth of barnyardgrass significantly (Table 1). The TN1 cultivar also carries the gene for semidwarfism, which is present in modern rice varieties, and which may explain why many modern rice varieties have allelopathic potential (Olofsdotter et al., 1997).
Production of plant material was done in hydroponics, which makes it very easy to separate plant parts from each other. Furthermore, the plant material is clean and without pollutants from soil or other solid growth medium. The rice was grown for 1 mo, which is a short production time for plant material.
Because no studies have ever been done as far as isolation of the allelochemicals in TN1 is concerned, it was decided to do a bioassay-directed isolation in order not to miss any of the active compounds/allelochemicals. The simplicity, economy (small amount of sample required for testing), and the fast turn-around time at which assay results are obtained (4 d) made the 24-well plate assay an ideal and convenient assay for the purposes of this study. Furthermore, this assay allowed the observation of gross physiological effects in the whole plant, and most importantly, results from this assay correlated with the activity of a known allelopathic rice cultivar TN1 (Table 1).
The dried, powdered roots of TN1 were initially extracted with MeOH/H2O (50:50) and tested for phytotoxic activity. The extract was active, and it was then partitioned between EtOAc and H2O. The EtOAc fraction was active, whereas the H2O fraction was not. Following a bioassay-guided fractionation of the organic portion, seven fractions (G-M, Fig. 2) were obtained, which had activity against barnyardgrass at assay concentration of 1 g L-1. Preparative layer chromatographic work on these fractions yielded further fractions and also some pure compounds. These fractions were analyzed by GC-MS to determine the identity of the pure compounds and in the case of a mixture, to identify the components of the mixture. Fractions comprised of only one peak were analyzed further by 1H-NMR for identity verification. Pure compounds were tested at 3 mM, while fractions were tested at a lower concentration (0.5 g L-1) than was used for routine isolation work, to determine where the activity resides among these samples. Results showed fractions M-1 and L-2 to have the highest activity, inhibiting growth of barnyardgrass roots (Table 2) and shoots (Table 3) significantly.
The GC-MS analysis of M-1 and L-2 showed that these two most active fractions were still a mixture of compounds (Fig. 3)
. Two peaks appear to be in common between these two fractions, i.e., retention times 4.9 and 6.2 min. Conclusions cannot be made at this point as to whether either one or both of these compounds is/are the allelochemical(s). Further bioassay-directed fractionation is being carried out to isolate the allelochemical(s) from these fractions.
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Fig. 3 Chromatogram of TN1 active fractions (A) L-2 and (B) M-1. Peaks at 4.9 and 6.2 min appear to be common between these two fractions
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None of the pure compounds isolated (G-1, G-2, H-1, H-3, I-2, I-3, J-1, J-2, K-1, K-4) strongly inhibited barnyardgrass. These compounds were identified, where possible, by matching GC-MS data with mass spectral library and by 1H-NMR (Table 4). One of the compounds was identified as p-coumaric acid (fraction G-2). -Coumaric acid has been reported to be weakly phytotoxic (Lydon and Duke, 1989; Reynolds, 1978) and an allelochemical (Chou, 1992; Einhellig, 1987; Koch and Wilson, 1977). It was also identified by GC-MS as one of the compounds present in rice samples in a study on the effect of allelopathic rice varieties on ducksalad (Mattice et al., 1998). In our studies, however, -coumaric acid inhibited the growth of barnyardgrass roots only at higher concentrations (10 and 5 mM), but was inactive at 3 mM (Table 5). At 5 mM, it did not inhibit growth of the shoots. -Coumaric acid was phytotoxic to lettuce at 10, 5, and 3 mM.
A bioassay-guided isolation procedure to identify the allelochemicals in rice was shown to be advantageous in this study. It enabled the correlation of field activity of allelopathic rice on barnyardgrass with laboratory experiments. Results were obtained which showed -coumaric acid not likely to be the allelochemical in TN1, but rather other fractions that do not contain this reportedly phytotoxic compound showing better activity. Within a relatively short period of time, the groundwork for more detailed isolation and identification of the allelochemicals in rice was established. Although phytotoxicity was used as the indication of biological activity, final isolation of the phytotoxic compound(s) will lead to further work in demonstrating its activity in the field to prove allelopathy. Future work also includes the identification of the phytotoxin(s) isolated from the roots in the hydroponic culture solution.
Identification of the allelochemical(s) will also allow the chemical fingerprinting of other rice cultivars. Isolation and identification of the allelochemical provides a basis for studies to determine its biosynthesis, identification of the enzymes and the genes encoding the enzymes, and applying genetic engineering techniques to enhance the production of the allelochemical. The ultimate goal of these studies is to produce highly allelopathic rice variety in order for the crop to have its own defense against associated weeds.
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Summary
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This study has shown the utility of bioassay-guided search for the allelochemicals in Taichung Native 1 rice. A known phytotoxin and an allelochemical, -coumaric acid was shown not to inhibit the growth of barnyardgrass following activity-guided isolation. None of the other identified compounds isolated showed activity against barnyardgrass when tested using a 24-well plate microbioassay. The bioassay-guided isolation work directed the activity to two fractions that have two peaks in common as determined from a GC-MS analysis. Work is continuing on the isolation and identification of the allelochemicals.
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ACKNOWLEDGMENTS
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We thank Stacy Allen, Amber Hale, and Artemio Madrid for their invaluable technical assistance.
Received for publication November 30, 1999.
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REFERENCES
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TOP
ABSTRACT
INTRODUCTION
