Centro de Investigaciones Biológicas, CSIC, Velázquez 144, 28006-Madrid, Spain1
Instituto de Química Orgánica, Departamento de Química Orgánica Biológica, CSIC, Juan de la Cierva 3, 28006-Madrid, Spain2
Author for correspondence: Antonio Leal. Tel: +34 91 5611800 ext. 4437. Fax: +34 91 5627518. e-mail: aleal{at}cib.csic.es
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ABSTRACT |
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Keywords: complex galactans, Hypocreales, chemotaxonomy
Abbreviations: TMS, trimethylsilyl
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INTRODUCTION |
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The type species of the genus Nectria is Nectria cinnabarina (Tode: Fr.) Fr. The characteristics of species related to N. cinnabarina, included in the N. cinnabarina group, were discussed by Rossman (1989) . In a later work this author expressed the opinion that the genus Nectria should be restricted to the N. cinnabarina group (Rossman, 1993
). The large number of species included in the genus Nectria made it necessary to divide them into groups according to morphological features (Booth, 1959
). Other workers have added new species, elucidated additional characters and substantiated the groups (Samuels & Rossman, 1979
; Seifert, 1985
; Samuels & Seifert, 1987
; Rossman, 1989
, 1993
).
Various species of Sesquicillium have been misidentified because of the morphological similarities with other genera. As an example, some strains of S. candelabrum were named Gliocladium penicilloides and Verticillium candelabrum prior to their final inclusion in Sesquicillium (Domsch et al., 1980 ). Morphological characters are sometimes insufficient to delineate unequivocally fungal genera; therefore, chemical and biochemical characters and, most recently, rDNA sequencing are increasingly used in fungal systematics.
Monosaccharide composition of the cell wall has been used for yeast and fungal taxonomy, systematics and phylogeny (Weijman & Golubev, 1987 ; Prillinger et al., 1990
, 1991
, 1993
; Messner et al., 1994
). Bartnicki-García (1968)
proposed the use of dual combinations of major polysaccharides of the cell wall to classify fungal species and divided the whole spectrum of fungi into eight categories. As Pfyffer (1998)
stated: cell wall polysaccharides appear to be extremely conservative and therefore may be considered reliable markers for fungal taxonomy. The alkali-extractable and water-soluble polysaccharides from the cell walls have been proposed as taxonomic characters for various fungal genera (Leal & Bernabé, 1998
). Among the Hypocreales, some polysaccharides have been proposed as markers for Calonectria and its anamorph, Cylindrocladium (Ahrazem et al., 1997
), and for Gibberella and Fusarium (Ahrazem et al., 2000
). As stated above, the perfect state of Sesquicillium species belongs to Nectria. This genus is heterogeneous, since it comprises the teleomorphic states of 21 anamorphic genera (Samuels & Seifert, 1987
). The concept of Nectria has changed according to the weight given to the morphological and biological characteristics of both the teleomorph and the anamorph by different authors (Rossman, 1993
; Rossman et al., 1999
).
In this work we characterize the alkali-extractable, water-soluble cell-wall polysaccharides from isolates of N. cinnabarina, and of Sesquicillium and Nectria with Sesquicillium anamorphs, in an attempt to find out if the morphological differences which allowed the arrangement of Nectria with Sesquicillium anamorphs in a group are reflected in the composition of the cell-wall polysaccharides.
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METHODS |
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Isolation of polysaccharides from F1S.
A 100 mg aliquot of fraction F1S was dissolved in 1·5 ml distilled water and centrifuged at 13000 g for 15 min to eliminate insolubles. The supernatant was added to a column (90x2·6 cm) of Sepharose CL-6B and eluted with distilled water with a flow of 22 ml h-1. Fractions were collected and monitored for carbohydrate by the phenol-sulfuric acid method (Dubois et al., 1956 ). The fractions which gave a positive test for carbohydrates were combined in batches of six successive fractions, concentrated to a small volume and freeze-dried. The column was previously calibrated with a mixture of standards: T500, T70 and T10 dextrans (Pharmacia).
Chemical analysis.
Neutral sugars were analysed by methanolysis followed by hydrolysis with 3 M trifluoroacetic acid (1 h at 121 °C). Neutral sugars were converted into their corresponding alditol acetates (Laine et al., 1972 ) and identified and quantified by GLC as described previously (Gómez-Miranda et al., 1981
). Uronic acid content was measured by the carbazole reaction (Bitter & Muir, 1962
) and the acidic monosaccharides identified on the hydrolysates as their trimethylsilyl (TMS)-derivatives by GLC-MS. The carboxylic groups of the uronic acids in the polysaccharide were reduced with sodium borodeuteride (NaBD4) according to the method of Taylor & Conrad (1972)
. The complete reduction was checked by IR spectroscopy. IR spectra were obtained by the KBr technique (Price, 1972
) on a Perkin-Elmer 1420 infrared spectrophotometer. Aminosugar content was determined after hydrolysis of the polysaccharides (6 M HCl, 4 h, 100 °C) according to the method of Chen & Johnson (1983)
and glucosamine was identified by HPLC in an amino acid analyser Biochrom 20 (Pharmacia) using commercial standards.
Absolute configuration of the monosaccharides.
The monosaccharides released in the hydrolysates were derivatized according to the method of Gerwig et al. (1979) and their absolute configuration determined by GLC-MS of the tetra-O-TMS-(+)-2-butylglycosides obtained.
Linkage analyses.
The reduced polysaccharides (15 mg) were methylated according to the method of Ciucanu & Kerek (1984) . The polysaccharides that contained aminosugars were also methylated by the same protocol without reducing the carboxylic group. The methylated material was extracted with chloroform/methanol (1:1, v/v), dialysed sequentially against water and 50% ethanol, and evaporated. Methylated fractions, which showed negligible IR absorption for hydroxyl groups, were hydrolysed with 3 M trifluoroacetic acid (121 °C, 1 h), and the products were reduced with NaBD4, then acetylated and subjected to GLC-MS, using a SPB-1 column (30 mx0·22 mm i.d.x0·25 µm film thickness), a temperature programme of 160200 °C with 1 min initial hold and ramp rate 2 °C min-1, and a mass detector (Q-Mass; Perkin-Elmer). Quantification was performed according to peak area. Analyses by the reductive-cleavage method were performed in two steps (Lee & Gray, 1988
), with TMS triflate as catalyst, but the reactions were carried out under Ar and the time during the reductive cleavage step was shortened to 56 h, to minimize unwanted by-products. The partially methylated anhydroalditol acetates obtained were analysed by GLC-MS using a fused silica SPB-1 column and a temperature programme of 150200 °C with 3 min initial hold and ramp rate 3 °C min-1.
NMR analyses.
The 1-D 1H-NMR spectra were recorded for solutions of the polysaccharides previously stirred with Amberlite IR-120 to convert the sodium salt of uronic acids into the free carboxylic acids, deuterated with D2O and dissolved in 99·9% D2O. The 1-D 1H-NMR spectra were determined at 40 °C on a Varian INOVA-300 spectrometer (1H, 300 MHz). 2-D 1H- and 13C-NMR experiments were carried out at 40 °C on a Varian Unity 500 spectrometer. Proton chemical shifts refer to residual HDO at =4·61 p.p.m. Carbon chemical shifts refer to internal acetone at
=31·07 p.p.m.
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RESULTS |
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Linkage analyses
The results of methylation and reductive cleavage analyses are shown in Table 2. The polysaccharides from Sesquicillium spp., N. lasiacidis and N. impariphialis contained 5-O-substituted (
5)-Galf-(1
) and 2,6-di-O-substituted galactofuranose (
2,6)-Galf-(1
) and terminal residues of glucopyranose (Glcp-(1
). All these polysaccharides also contained a small proportion of terminal glucuronic acid. In addition to these residues, the polysaccharide from N. impariphialis contained 6-O-substituted glucopyranose (
6)-Glcp-(1
) and the polysaccharides from S. candelabrum and N. lasiacidis contained 5,6-di-O-substituted galactofuranose (
5,6)-Galf-(1
), and terminal N-acetylglucosamine (GlcNAc-(1
). This last residue was only detected in unreduced samples.
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1H-NMR analysis
The 1H-NMR spectra of F1S-II (Fig. 2) from S. buxi and S. pseudosetosum revealed a coincidence in the position of the signals, differing in their intensity, and showed five signals at
=5·03, 5·09, 5·12, 5·22 and 5·35 p.p.m. The three major peaks (
=5·03, 5·09 and 5·35 p.p.m.) appear in the 1H-NMR spectra of the polysaccharides of all the species analysed, except in that of N. cinnabarina.
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To find additional connections among residues, we conducted an HMBC experiment, which provides signals corresponding to long-range connections among protons and the carbons placed at two and three bonds from them. In this way, in addition to expected intra-ring connections, peaks corresponding to H-1A/C-5B, 1A/C-5E, H-1B/C-5C, H-1C/C-5E, H-1D/C-2A, H-1E/C-6A and H-1F/C-6C could be observed (Fig. 6).
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From the combined evidence, it is therefore proposed that the cell-wall polysaccharide isolated from S. candelabrum has an irregular structure (Fig. 7). The derivatives detected in the methylation analyses suggest that the chains are connected to some 4-O positions of a small (1
6)-mannan core.
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The 1H-NMR spectra of polysaccharides F1S-II from the three N. cinnabarina strains were different from those for the polysaccharides of the Sesquicillium species and the other species of Nectria. The signals detected in the anomeric region were at =5·03, 5·10 and 5·16 p.p.m.
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DISCUSSION |
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The results show that changes in morphological features of certain species of a genus are reflected in the structure of the alkali-extractable, water-soluble polysaccharides, and they confirm that these polysaccharides are reliable characteristics for fungal systematics at genus or subgenus level and for establishing teleomorphanamorph and phylogenetic relationships, since: the nature of the polysaccharides in any particular fungus is not capricious, but is related to its taxonomic position and thus reflects its evolutionary history (Bartnicki-García, 1987 ).
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ACKNOWLEDGEMENTS |
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Received 5 February 2001;
accepted 18 March 2001.
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