1 Department of Biology, Faculty of Science, Silpakorn University, Nakorn Pathom 73000, Thailand
2 School of Life Sciences, Xiamen University, Xiamen 361005, China
3 Kunming Institute of Botany (KIB), The Chinese Academy of Sciences, Kunming 650204, China
4 Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai 50200, Thailand
Correspondence
Thongchai Taechowisan
tewson84{at}hotmail.com
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ABSTRACT |
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INTRODUCTION |
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METHODS |
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In vitro assay for antagonism.
An in vitro plate-assay technique was developed to test the inhibitory effects of CMUAc130 on the phytopathogenic fungi. Tests for inhibitory activity were made on ISP-2 in Petri dishes. A 0·8 cm diameter ISP-2 agar plug, covered fully with a lawn of CMUAc130, was placed 1·5 cm from the edge of the Petri dish and incubated at 30 °C for 5 days. This was done to allow the culture to be established on the agar surface and to sporulate prior to inoculation of the plates with fungal strains. For each test fungus, a 0·8 cm diameter PDA plug covered with actively growing mycelium was placed about 6 cm from CMUAc130. The inoculated plates were placed in an incubator at 30 °C for 4 days. The inhibition zone was determined by measuring the distance between the fungi and CMUAc130 in dual cultures. Morphology of fungal mycelium along the edges of the inhibited colonies facing CMUAc130 was examined under a dissecting microscope at 400x magnification.
Preparation of inoculum and fermentation.
A spore suspension of CMUAc130 was prepared in distilled water from cultures grown on ISP-4 medium at 30 °C for 10 days. The suspension was added to ISP-2 broth in each 500 ml Erlenmeyer flask at a rate of 108 spores in 100 ml liquid medium. Cultures were kept on a shaker at 120 r.p.m. at 30 °C for 48 h and used as seed stocks. For large production of culture filtrates, CMUAc130 was grown in a modified 3000 ml glass container containing 1500 ml ISP-2 broth, and then with orbital shaking for 5 days under the same conditions. The 5-day-old cultures were filtrated by Whatman paper no. 1 under vacuum. The mycelial mats were washed with distilled water and separated by centrifugation at 5000 r.p.m. for 20 min. The culture filtrate and mycelial mats of CMUAc130 were extracted with ethyl acetate. Both ethyl acetate extracts of liquid filtrate and mycelial mats showed antifungal activity; hence, they were combined.
Fractionation and purification of the compounds.
The residue (320 mg) was dissolved in 10 ml methanol and fractionated on a reverse-phase column (Li Chroprep RP-18; Merck) with increasing concentrations of methanol as eluent (50, 70 and 100 %). Fractions were combined on the basis of TLC results and concentrated under vacuum to produce a black gum (120 mg). The black gum was mixed with 2·0 g silica gel (Walk gel). This mixture was then subjected to bioassay-guided separation through a chromatography column (50x7·5 cm) over silica gel (50 g) and eluted with chloroformmethanol (100 : 0, 95 : 5, 90 : 10, 85 : 15, 80 : 20, 70 : 30, 60 : 40, 50 : 50 and 0 : 100). About 50 ml per fraction was collected. Purifications were performed by column chromatography over silica gel (finer than 200-mesh). The fractions were combined and made into four pooled fractions on the basis of their TLC results. The pooled fractions, A-1 (eluted with chloroformmethanol from 95 : 5 to 90 : 10), A-2 (eluted with chloroformmethanol from 90 : 10 to 80 : 20), A-3 (eluted with chloroformmethanol from 80 : 20 to 60 : 40) and A-4 (eluted with chloroformmethanol from 60 : 40 to 30 : 70), were screened again for their antifungal activity against C. musae and F. oxysporum by using the paper-disc method (Rothrock & Gottlieb, 1984). Fractions A-2 and A-3 were most active against these fungi and they showed one major spot with the same Rf value (0·63, chloroformmethanol 20 : 1) in TLC. As they were identical, we combined these fractions and purified them by repeated chromatography over a silica-gel column, followed by recrystallization in a mixture of hexane and chloroform (9 : 1). This yielded active compound (i). Purification of A-1 resulted respectively in compounds (ii), (iii) and (iv). The structures of the active compounds have been identified by using NMR and mass-spectral data.
Structure elucidation of the compounds.
The melting point of the compounds was determined on a Buchi-540 melting-point apparatus. Optical rotations were measured on a Perkin-Elmer 241 polarimeter, IR spectra on a Perkin-Elmer 1 spectrometer, 1H- and 13C-NMR spectra on a Bruker DRX 500 spectrometer and electron impact-MS and fast atom bombardment-MS, respectively, on a Hewlett Packard 5989 B and a Finnigan/Thermo Quest Mat 95 XL mass spectrometer.
Antifungal-activity assay.
Supernatants of CMUAc130 from ISP-2 broth, the extracted fractions or purified compounds were tested for antibiosis against C. musae and F. oxysporum by using the paper-disc method. Two 8 mm discs of sterile paper (Advantec; Toyo Roshi Kaisha) were soaked respectively in culture filtrate (50 µl), crude extract (50 µl) and each of four purified compounds (0·25 mg in 50 µl). The air-dried discs were placed on a PDA plate. Each plate was then inoculated with an agar block (8 mm diameter) containing mycelial mats of the fungi in the centre of the plate. The paper discs were 2·2 cm from the phytopathogen. Percentage inhibition was obtained 4 days after treatment at 30 °C from the following equation:
Inhibition (%)=[(growth diameter in untreated controlgrowth diameter in treatment) x100]/growth diameter in untreated control
Each treatment consisted of three replicates. The experiment was repeated twice.
MICs.
Purified compounds (i) and (ii) were assayed on PDA in Petri dishes to determine the MICs of these compounds against C. musae (Elson et al., 1994). The purified compound (2 mg) was dissolved in DMSO (200 µl), diluted serially in the same solvent and added to PDA at 48 °C. The medium (5 ml) was added to a 5 cm diameter Petri dish. The final concentrations were 0, 1, 5, 10, 30, 60, 90, 120, 150 and 180 µg ml1. A 5 mm diameter plug of the fungi, removed from the margin of a 4-day-old colony on PDA, was placed 1·5 cm from the edge of the plate. Linear growth of the fungi at 30 °C was recorded 2 days after treatment. Each treatment consisted of three replicates. Percentage inhibition was obtained from the equation described above. The experiment was repeated twice.
Data analysis.
Data from the antifungal-activity assay and MICs of CMUAc130 were analysed by SPSS for Windows 11.01. Means of treatments for each experiment were compared by using Duncan's multiple-range test (P0·05).
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RESULTS |
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Structure elucidation of compound (i)
5,7-Dimethoxy-4-p-methoxylphenylcoumarin (C18H16O5; Fig. 2) was a white, amorphous powder: melting point, 151152 °C (EtOH); UV
max (MeOH) nm (log
): 250 (4·07), 325 (4·29); 1H NMR (CDCl3):
7·20 (2H, d, J=8·5 Hz, H-2', H-6'), 6·87 (2H, d, J=8·5 Hz, H-3', H-5'), 6·50 (1H, d, J=2·5 Hz, H-8), 6·22 (1H, d, J=2·5 Hz, H-6), 5·96 (1H, s, H-3), 3·83 (6H, s, OMe-7, OMe-4'), 3·46 (3H, s, OMe-5); IR vmax (CHCl3) cm1: 1710, 1610, 1595, 1510, 1158, 1111, 1052, 952, 872, 860, 830; MS m/z (relative intensities): 312 [M]+ (80), 284 [MCO]+ (100), 269 [MMeCO]+ (37), 241 [M43CO]+ (2).
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Compounds (iii) (14 mg) and (iv) (11 mg), obtained from A-1, were identified as vanillin and 3-methoxy-4-hydroxytoluene (Fig. 2) by comparison with their authentic samples.
The antifungal activity of different fractions from culture broth of S. aureofaciens CMUAc130 was evaluated by using C. musae and F. oxysporum. The paper-disc assay method indicated that compound (i) was most effective at inhibiting the growth of C. musae and F. oxysporum (Table 1). The same percentage of inhibition was detected for compound (ii). Both the culture filtrate of S. aureofaciens CMUAc130 and its crude extract as the control also showed efficacy at suppressing C. musae and F. oxysporum. However, vanillin and 3-methoxy-4-hydroxytoluene extracted from culture broth of S. aureofaciens CMUAc130 showed weak inhibition of fungal growth.
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DISCUSSION |
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Previous reports indicated that 5,7-dimethoxy-4-p-methoxylphenylcoumarin was produced by numerous species of plants, including Hintonia latiflora (Mata et al., 1990), Exostema caribaeum (Mata et al., 1988
) and Coutarea hexandra (Delle Monache et al., 1983
), but nothing is known about its activity. Our study is the first in which 5,7-dimethoxy-4-p-methoxylphenylcoumarin from culture filtrates of an endophytic Streptomyces species was isolated from the root tissue of Z. officinale. It further proved that 5,7-dimethoxy-4-p-methoxylphenylcoumarin or 5,7-dimethoxy-4-phenylcoumarin was one of the antifungal components, as evidenced by the fact that the culture filtrate, crude extract, pure 5,7-dimethoxy-4-p-methoxylphenylcoumarin and 5,7-dimethoxy-4-phenylcoumarin from S. aureofaciens CMUAc130 were all inhibitory to C. musae and F. oxysporum. In our study, hyphae of C. musae treated with culture filtrates of S. aureofaciens CMUAc130, pure 5,7-dimethoxy-4-p-methoxylphenylcoumarin and 5,7-dimethoxy-4-phenylcoumarin showed signs of necrosis and fractures when examined under a scanning electron microscope (data not shown). This suggests that 4-arylcoumarins from S. aureofaciens CMUAc130 may be related to hyphal collapse of C. musae. The results of this study conclude that 5,7-dimethoxy-4-p-methoxylphenylcoumarin is a major ingredient in the culture filtrate of S. aureofaciens CMUAc130 and it may play an important role in the inhibition of the damping-off pathogen C. musae and F. oxysporum.
As stated in several reports, Streptomyces activity in plants not only protects against pathogens, but the metabolic products of Streptomyces also influence plant growth and physiology (Katznelson & Cole, 1965; Mishra et al., 1987
). As 5,7-dimethoxy-4-p-methoxylphenylcoumarin has been isolated from numerous plants, but not from members of the Zingiberaceae, further investigations are necessary to determine the relationship between 5,7-dimethoxy-4-p-methoxylphenylcoumarin and this plant family. Also, its cytotoxic activity against human cell lines should be studied.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 9 November 2004;
revised 28 January 2005;
accepted 11 February 2005.
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