Department of Plant Sciences, Montana State University, Bozeman, MT 59717, USA1
Department of Applied Biological Science, Science University of Tokyo, Noda, Chiba 278-8510, Japan2
The Institute of Physical and Chemical Research, Wako, Saitama 351- 0198, Tokyo, Japan3
Department of Chemistry, University of Utah, Salt Lake City, UT 84112, USA4
Department of Botany and Range Science, Brigham Young University, Provo, UT 84602, USA5
Author for correspondence: Gary Strobel. Tel: +1 406 994 5148. Fax: +1 406 994 7600. e-mail: uplgs{at}montana.edu
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ABSTRACT |
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Keywords: antimycotic, phycomycetes, aquatic ecology , epiphyte
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INTRODUCTION |
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The oomycetes can be devastating to terrestrial plants; however, the appropriate moisture requirements must be met (Buczacki, 1983 ). Yet, conceivably these organisms could also attack and infect plants normally existing in strictly aquatic environments but nevertheless, aquatic plants seem to thrive and are relatively disease-free in their respective ecosystems. This biological conundrum may be related to either intrinsic or extrinsic factors controlling plant disease resistance, a phenomenon little understood or studied in aquatic plants. With regard to the possibility of extrinsic factors controlling disease, certain epiphytic or endophytic microbes may associate with aquatic plants and produce anti-oomycetous compounds. This may contribute to the defence of the plant by killing, inhibiting or warding off invading oomycetes. This rationale served as a basis for a search for microbes participating in associations with aquatic plants in which antimycotics may be produced by epiphytes or endophytes. If such compounds exist, they may prove agriculturally applicable to plant disease control situations in which one or more phytopathogenic oomycetes are a potential problem.
Rivers arising and flowing in the Venezuelan-Guyana of South America make up the regions primary bodies of fresh water. Rhyncholacis pedicillata is a small highly specialized aquatic plant of the family Podostemaceae that grows in colonies and thrives in some of the brown-black rivers of the Venezuelan-Guyana (Steyermark et al., 1995 ). These plant colonies are comprised of hundreds of individuals that grow firmly attached to rock surfaces and prefer swift water currents. R. pedicillata is 0·10·5 m in size and it has a slightly enlarged bulbous-like base and an extensive root system that anchors the plant to rock. Its stems are multi-branched, are lace-like and covered with numerous small leaves. Close examination of individual plants in the Carrao river revealed animal or environmentally inflicted wounds on the stems. Normally, it would seem that such wounds would serve as entry points for one or more pathogenic oomycetes. However, little or no disease symptoms on the plants were observed.
Small stem pieces were examined for their associated microbes and each of those recovered was assayed against Pythium ultimum. The most commonly observed microbe was a strongly red-pigmented bacterium that was identified as Serratia marcescens. It colonized the surface of the stems of R. pedicillata, rather than internally. In culture, this bacterium produced a novel macrocyclic chlorinated lactone termed oocydin A that demonstrated selective toxicity (antimycotic) towards the oomycetes with extremely low MICs (e.g. 0·03 µg ml-1 for various Phytophthora spp.). This report deals primarily with the isolation of S. marcescens and the chemical characterization of oocydin A. Some specific details are also given on the biological activity of the compound. Finally, its relationship to the biology of the plantmicrobe interaction is discussed, along with its potential usefulness to agriculture.
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METHODS |
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Fungal strains.
All strains of Phytophthora used in bioassays were the generous gift of Dr John Menge, University of California, Riverside, CA, USA. P. ultimum and all other plant-pathogenic fungi used in bioassays were a generous gift of Dr Don Mathre, Montana State University. Standard isolates of S. marcescens, used for comparative purposes, were obtained from the Montana State University bacterial culture collection.
Bioassays.
A simple plate bioassay was used to detect bioactivity of various fractions during the purification of oocydin A. Aliquots (1020 µl) of sample were placed on a PDA plate and dried. The plates were then inoculated with four 7x7 mm plugs of agar infested with P. ultimum, one in each quadrant, and incubated for 3648 h at 23 °C. Antimycotic activity was apparent as a zone of growth inhibition. The antimycotic activity of pure oocydin A was also tested against a series of plant pathogens by dissolving 50 µg of the compound in 100 µl methanol, spotting 10 µl portions onto PDA plates and allowing the droplets to dry. Each plate was overlaid or sprayed with an aqueous suspension of the test fungus in water (containing mycelial fragments and/or spores), sealed with a piece of Parafilm and then incubated at 23 °C for 45 d. In each case, a positive control test was also conducted (the residue obtained from the methylene- chloride-extracted medium without the bacterium). MICs of oocydin A against various phycomycetes were determined by preparing a stock solution of the compound (1 mg ml-1 in methanol). This was dispensed, by serial twofold dilution, into a 24-well plate. Each well of the plate contained 500 µl potato dextrose (PD) broth. A small plug of the test fungus was placed into each well and the plate incubated for 4872 h at 23 °C. The MIC was taken as the concentration of oocydin A in the well where no growth was detected visually after either 48 or 72 h.
MIC tests were also conducted for several fungal pathogens of humans using the microbroth dilution assay as recommended by the subcommittee on antifungal susceptibility testing of the US National Committee for Clinical Laboratory Standards (NCCLS). The effects of the well- established antimycotics metalaxyl, pseudomycin B and amphotericin B were determined concomitantly.
Oocydin A was tested against human cancer cell lines BT-20, MCF 7 (both from ATCC, Manassas, VA, USA) and a normal human mammary cell line, cc2551 HMEC (from Clonetics, San Diego, CA, USA). These cells were exposed to serial dilutions of oocydin A. After 3 d, they were stained with neutral red and the absorbance measured at 540 nm. For non-adherent cells, a modification of the staining protocol was used (Berent et al., 1986 ). The results were recorded as median inhibitory concentrations (IC50 values).
Oocydin A isolation procedure.
Cells from a single colony of S. marcescens MSU-97 were used to inoculate 15 ml PD broth and the culture was grown overnight with shaking at 23 °C. This culture was then used to inoculate two 2·0 litre flasks, each containing 1 l of medium consisting of 24 g PD; 6 g soytone; 4 g yeast extract and 100 mg NaCl in H2O. The cultures were incubated at 23 °C for 15 d. The micro-organism was removed by centrifugation and the culture fluid extracted twice with two equal volumes of methylene chloride. This solvent was taken to dryness by flash evaporation at 4550 °C under vacuum. Approximately 2·1 g of residue was present after evaporation of the solvent. This material was dissolved in chloroform (5 ml) and placed on a 3·0x20·0 cm column of silica gel. The column was eluted with a chloroform/methanol (10:1, v/v) solution. The first 100 ml to elute from the column was discarded and the next 50100 ml contained the antimycotic activity. This material was placed onto another silica gel column (the same size as initially used) and eluted with methylene chloride/methanol (10:1, v/v) solution. In this case, the first 50 ml of eluate was discarded and the next 80 ml contained the bioactivity (approx. 5060 mg). The mixture of compounds was then subjected to a series of preparative TLC steps on 20x20 cm plates to yield a pure product. The solvent systems used in succession were as follows: solvent A, chloroform/methanol/acetic acid (12:1: 0·1, by vol.); solvent B, chloroform/methanol/ammonium hydroxide (6:2:0·1, by vol.); and solvent C, methylene chloride/methanol (6:2, v/v). After each successive TLC step, each band on the plate was eluted with methanol and the material possessing bioactivity (see bioassay procedure above) was reapplied to the next plate prior to separation. Once obtained and authenticated, a small amount of oocydin A was applied at the margin to the TLC plate as an appropriate reference for larger scale preparations. It appeared as a brownish spot with the vanillin-sulfuric reagent after gentle heating (Cardellina, 1991 ). Approximately 1518 mg oocydin A was recovered per litre of culture fluid. It was obtained as a whitish powder by dissolving it in a methanolic-aqueous solution, which was then frozen and subjected to lyophilization. This was the preparation that was subjected to physical, chemical and biological characterization, once the purity of the sample was firmly established.
HPLC.
The putative TLC-purified sample of oocydin A (20 µg) was subjected to HPLC using an Altima C-18 column (7·0x250 mm) (Alltech) and eluting with a linear gradient starting with a solution of 0·1% TFA/30% acetonitrile in water and finishing with a solution of 0·1% TFA/50% acetonitrile in water, over the course of 30 min. Detection was performed with a Waters variable wavelength detector at 208 nm.
Spectroscopic analyses.
After the purity of oocydin A had been established it was subjected to a series of analyses using standard spectroscopic techniques. Electrospray mass spectroscopy was performed by dissolving the sample in methanol/water/acetic acid (50:50:1, by vol.). The sample was then injected into Montana State Universitys custom-built mass spectrometer with a spray flow of 2 µl min -1 and a spray voltage of 2·2 kV via the loop injection method. Oocydin A was also subjected to laser desorption mass spectroscopy on a Perspectives Biosystems instrument. The sample was incorporated into a matrix of 3,5-dihydroxybenzoic acid and scanned accordingly. The following parameters were used: accelerating voltage 30000 V, grid voltage 70%, mirror ratio 1·06, laser 260 and cytochrome c to standardize the instrument. Negative high resolution FAB mass spectroscopy was done to acquire empirical formula data. Elemental analysis was done by degradative techniques at Atlantic Microlab, Norcross, GA, USA. Infrared spectroscopy was done on a Perkin Elmer instrument with oocydin A embedded in a matrix of anhydrous KBr and pressed into a pellet. An average of 16 scans was taken on the sample. The UV absorption spectrum of oocydin A was determined in 1 ml 100% methanol (1·0 cm light path, Beckman DU 50 spectrophotometer). Its optical rotation was determined in a sample dissolved in 100% methanol and analysed in a JASCO P1010 instrument.
Oocydin A was subjected to NMR techniques after being dissolved in 100% deuterated methanol. Initially, the 1H spectrum was obtained on a Bruker DRX 500 instrument with 64 scans and a delay cycle of 2 s, and collected as 32k real-time domain points using a transmitter frequency of 500·13 MHz. The 1H spectrum was referenced to the MeOD (deuterated methanol) signal at 3·3 p.p.m. For acquisition of the 13C spectrum, 4096 scans were made with a recycle delay of 10 s and collected as 32k real-time domain points using a transmitter frequency of 125·77 MHz. The spectrum was referenced to the residue MeOD signal at 49·0 p.p.m.
Oocydin A was also analysed by 2D INADEQUATE analysis on a 500 MHz Varian Inova spectrometer operating at 125·892 MHz and 26 °C. Oocydin A (58 mg) was dissolved in 200 µl CDCl3 and the analysis performed using a Varian 5 mm probe and a Shigemi microtube whose susceptibility was matched to the solvent used. Analysis parameters included a 10 µs 13C 90° degree pulse and a pulse sequence delay optimized for detection of 55 Hz carboncarbon scalar coupling constants. A total of 64 evolution increments of 800 transients each were used for an analysis time of 4·7d. Digital resolution of the acquisition and evolution dimensions were 0·2 and 176·1 Hz per point, respectively. The gross structure of oocydin A was deduced from the various NMR data, particularly gradient DQF-COSY, gradient HMQC, gradient HMBC and difference 1D-NOE spectra in a JEOL JNM-alpha 600 spectrometer. Stereochemistry of oocydin A was primarily determined by 1D-NOE and selective NOE experiments. INADEQUATE spectral processing and signal assignments were done in a near automated fashion using software described by Dunkel et al. (1990 , 1992
) and Harper et al. (1996)
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Scanning electron microscopy.
Materials to be examined were placed in 2% glutaraldehyde in 0·1 M sodium cacodylate buffer (pH 7·27·4) (Upadhyay et al., 1991 ). The samples were critical-point-dried, gold-coated with a sputter coater, and observed and photographed with a JEOL 6100 scanning electron microscope. Bacterial preparations were supported on dried
-irradiated carnation leaves.
Materials.
All solvents used for HPLC and TLC were HPLC grade. Solvents used for extraction of oocydin A were ACS grade. All TLC was conducted on EM- Merck precoated glass silica gel plates at a thickness of 0·25 mm.
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RESULTS AND DISCUSSION |
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Electrospray mass spectroscopic analysis of oocydin A produced a major peak at 493·9 m/z. This is consistent with a single charged species of (M + Na)+ (Fig. 3). The laser desorption spectrum also produced a major peak at 493·3 consistent with (M+Na) +. However, there was also a significant M+2 peak at 495·3, which could be accounted for by oocydin A possessing a chlorine atom. This may be true because of a high abundance of the isotope 37Cl. Negative HRFAB data yielded (M -H)+ of 469·1640, which accounted for the empirical formula C23H30O8Cl1 . The difference between the negative HRFAB data [469=(M -H)+] and the electrospray laser desorption data [M =(470+Na)+] is equivalent to 23 m/z, which is the atomic mass of sodium. Therefore, in both the electrospray and laser desorption mass spectral analysis, oocydin A sequestered a sodium ion, which accounted for its molecular mass plus 23 m/z . In addition, the molecule assumed a net positive charge by picking up H+. Elemental analysis of oocydin A for its halogen content revealed that it was=7·1% (the expected Cl content is 7·4%). In the elemental analysis, the halogen was not specifically identified as chlorine. Although the negative HRFAB and the laser desorption mass spectral data gave a strong indication for the presence of chlorine in oocydin A, these analyses were not totally definitive. Therefore, further supportive evidence was obtained with an Oxford Instrument energy-dispersive X-ray microanalysis system on a JEOL scanning electron microscope. The test was conducted on about 1520 µg oocydin A supported on an Al stub. A peak distinctive for chlorine and no other halogen appeared in the spectrum. Thus, the empirical formula for oocydin A is C23H31 O8Cl1.
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The 1H-NMR spectrum was consistent with a compound having methyl, methylene and hydrogen bonded on carbons bearing oxygen (Fig. 4). In the 13C-NMR spectrum, it was possible to account for 23 carbon atoms (Fig. 5
). This NMR analysis also showed double- bonded carbons, carbons bearing carbonyl groups and methyl carbons.
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Other biological considerations
Oocydin A is a novel bioactive compound possessing some unique biological and chemical properties. Overall, it appears that it has selective lethal activity against oomycetes at MICs lower or similar to those noted for metalaxyl (Table 1). Metalaxyl is well established as the fungicide to which the oomyctes are the most sensitive (Lyr, 1995
). However, in the past 10 years, increasing resistance to metalaxyl is developing in certain oomycete populations and this fungicide is being rendered useless or less effective (Lyr, 1995
). Thus, a tremendous need exists for new compounds to combat these plant pathogens. These observations suggest that oocydin A may have potential as a novel antimycotic against some of the most important plant pathogens the oomycetes. The cursory examination of the biological selectivity of oocydin A described here suggests that it targets one or more unique structural sites in the oomycetes. However, no mode of action studies on the compound have yet been done to confirm this suggestion. Oocydin A appears to have selective toxicity against various human cell lines. In preliminary studies, IC50 values of 0·2 µg ml-1 against BT-20 (breast cancer cell line), 0·42 µg ml-1 against MCF-7 (breast cancer cell line) and 0·6 µg ml -1 against a normal mammary cell line were noted. In future it may be possible to chemically modify oocydin A so that the compound is more selectively toxic to cancer cells. This would increase the likelihood that an oocydin A derivative could be more seriously considered as an anticancer drug candidate. However, a more immediate goal should be the exploration of oocydin A as a candidate compound for agricultural applications. This would necessitate the acquisition of large-scale amounts of the compound for plant testing and perhaps chemical modification to reduce any toxicity problems that may arise if the compound is to be used in controlling diseases of crops caused by oomycetes.
In natural settings, S. marcescens may play a role in the R. pedicillata relationship by first establishing itself on the plant surface and then producing oocydin A. This may eventually diffuse into the plant and/or, due to its solubility and charge characteristics, it may adhere to the plant surface. The presence of oocydin A in the area of the bacterial colony may prevent oomyceteous pathogens from causing rot or decay of the plant by virtue of the selective toxicity of this novel compound.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 20 April 1999;
revised 4 August 1999;
accepted 27 August 1999.