Comparison of cell-wall polysaccharides from Nectria cinnabarina with those from the group of Nectria with Sesquicillium anamorphs

Oussama Ahrazem1, Alicia Prieto1, Begoña Gómez-Miranda1, Manuel Bernabé2 and J. Antonio Leal1

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


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Alkali-extractable and water-soluble polysaccharides were purified from cell walls of five species of Sesquicillium or its teleomorphs, Nectria lasiacidis and Nectria impariphialis, and from Nectria cinnabarina, the type species of Nectria, a heterogeneous genus that belongs to the Hypocreales. Methylation and NMR analyses for determination of linkage types and structure were performed and indicated differences between the polysaccharides purified during the present study and those isolated from other nectrioid fungi, namely the presence of 5-O-substituted galactofuranose (->5)-Galf-(1->) in the main chain together with 2,6-di-O-substituted galactofuranose (->2,6)-Galf-(1->) residues in Sesquicillium buxi and Sesquicillium pseudosetosum. The polysaccharide from N. impariphialis was similar to those obtained from the above species, although an additional residue of 6-O-substituted glucopyranose (->6)-Glcp-(1->), was detected in some side chains. In N. lasiacidis and Sesquicillium candelabrum the polysaccharide contained an additional branching point of 5,6-di-O-substituted galactofuranose (->5,6)-Galf-(1->) linked to terminal N-acetylglucosamine GlcNAc-(1->). These chains were linked to a small mannan core. All these polysaccharides showed major differences to the polysaccharide of N. cinnabarina, which was formed by a main chain of (1->6)-ß-linked galactofuranose units almost fully branched at positions 2-O by either single residues of glucopyranose or acidic chains containing glucuronic acid and mannose.

Keywords: complex galactans, Hypocreales, chemotaxonomy

Abbreviations: TMS, trimethylsilyl


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The genus Sesquicillium Gams was created by the transfer (Gams, 1968 ) of Fusidium buxi Schmidt in Link and Verticillium candelabrum Bonorden. It is characterized by the presence of verticillate conidiophores, whose ultimate branches consist of terminal tapering phialides and subterminal cells with a lateral sporiferous neck. Five new species of Nectria with Sesquicillium anamorphs were described by Samuels (1989) . He found that the anamorphs of Nectria sesquicilli and Nectria lasiacidis were indistinguishable from Sesquicillium buxi and he erected three species of Sesquicillium with the anamorphs of the other species of Nectria: Sesquicillium asymmetricum (Nectria sesquiphialis), Sesquicillium impariphiale (Nectria impariphialis) and Sesquicillium pseudosetosum (Nectria parviphialis). These species of Nectria form a homogeneous group characterized by superficial, non-stromatic, smooth globose, non-papillate, orange perithecia. The known species of Sesquicillium and their Nectria teleomorphs were keyed by Samuels (1989) . Recently, Rossman et al. (1999) erected a new genus, Bionectria, in which the species of Nectria with anamorphs in Sesquicillium are included.

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.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Micro-organisms and culture media.
The strains of Nectria and Sesquicillium listed in Table 1 were maintained on slants of Bacto potato dextrose agar supplemented with 1 g l-1 yeast extract (Difco). N. lasiacidis and N. impariphialis are teleomorphs of S. buxi (Gams, 1968 ) and S. impariphiale (Samuels, 1989 ), respectively. The culture medium and growth conditions were as previously described (Gómez-Miranda et al., 1988 ).


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Table 1. Sugar composition (%) of fraction F1S-II of Sesquicillium species, N. impariphialis, N. cinnabarina and N. lasiacidis

 
Wall material preparation and fractionation.
Wall material was obtained as reported elsewhere (Prieto et al., 1988 ). Cell-wall material (8 g) was repeatedly extracted with 1 M NaOH (300 ml) at 20 °C. After centrifugation, the supernatants were combined and absolute ethanol (v/v) was added. The precipitate was collected by centrifugation, dialysed against running tap water and then freeze-dried (F1). This polysaccharidic material (500 mg) was suspended in distilled water (10 ml), stirred at room temperature for 2 h and centrifuged. The precipitate was extracted again with distilled water and the supernatant solutions were combined and freeze-dried (F1S).

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 (1–5 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 160–200 °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 5–6 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 150–200 °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 {delta}=4·61 p.p.m. Carbon chemical shifts refer to internal acetone at {delta}=31·07 p.p.m.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Fractionation of polysaccharides from F1S
The alkali-extractable, water-soluble material from all the species was filtered through a column of Sepharose CL-6B (Fig. 1). Two peaks were detected in the three strains of S. candelabrum and in S. pseudosetosum. The first one (F1S-I) was a minor excluded fraction (<=10%) which contained a glucan of high molecular mass, which was eliminated, and the second one (F1S-II) amounted to approximately 90% and eluted in a wide peak. A single peak (F1S-II) was obtained in S. buxi, N. lasiacidis the three N. cinnabarina isolates and N. impariphialis. The 1H-NMR spectra of the polysaccharidic material of different batches from a peak were similar, which indicated that F1S-II was a unique polysaccharide.



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Fig. 1. Column chromatography of F1S from Sesquicillium species, N. lasiacidis, N. cinnabarina CBS 278.48 and N. impariphialis on Sepharose CL-6B. The arrows indicate the elution of the different dextrans (500, 70 and 10 kDa) used as standards for calibration.

 
Chemical analysis
The polysaccharide of fraction F1S-II from the species analysed gave glucose, mannose, glucuronic acid and higher percentages of galactose (Table 1). Glucuronic acid was detected in all the polysaccharides by GLC-MS analysis of their TMS derivatives. Colorimetric determination of aminosugars gave around 7% in all strains of S. candelabrum and in N. lasiacidis, and negative results for the polysaccharides F1S-II from the remaining species. This aminosugar was identified as glucosamine by HPLC in comparable amounts (8%) to those detected by the colorimetric method. All the monosaccharides had the D configuration.

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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Table 2. Methylation and reductive cleavage analyses (%) from F1S-II of several species of Sesquicillium, N. impariphialis, N. cinnabarina and N. lasiacidis

 
The results of the methylation and reductive cleavage (Table 2) indicated that the polysaccharides isolated from strains of N. cinnabarina contained 2,6-di-O-substituted galactofuranose (->2,6)-Galf-(1->), terminal residues of glucopyranose (Glcp-(1->) and mannopyranose (Manp-(1->), 6-O-substituted mannopyranose (->6)-Manp-(1->), and 4-O-substituted glucuronic acid (->4)-GlcpA-(1->).

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 {delta}=5·03, 5·09, 5·12, 5·22 and 5·35 p.p.m. The three major peaks ({delta}=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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Fig. 2. Anomeric region of the 1H-NMR spectra of fraction F1S-II from N. cinnabarina, N. lasiacidis, N. impariphialis and species of Sesquicillium.

 
As the polysaccharide from S. candelabrum contained signals observed in most of the spectra of the other fungi, it was chosen for further studies. The 1H-NMR spectrum of S. candelabrum (CBS 190.38) contained six signals in the anomeric region (Fig. 3a). The DQCOSY spectrum displayed H-1/H-2 crosspeaks for six distinct residues, in the proportion 3:1:1:3:3:1, which were labelled A–F, from low to high field, according to their anomeric protons. A 2-D TOCSY (HOHAHA) experiment (mixing time=85 ms) allowed the establishment of clear connectivities to the rest of the protons for residues A, C, D and F (Fig. 4a). The signals corresponding to unit D readily identified it as {alpha}-Glcp, as deduced from the coupling constants (J1,2=3·8, J2,3=9·8, J3,4=9·7 Hz). The low position of proton H-2 (3·94 p.p.m.) and the values of the coupling constants from unit F (J1,2=3·6, J2,3>9, J3,4=9·3 Hz), in addition to the presence of a singlet at 2·05 p.p.m., probably due to an acetyl group, suggested it was a GlcpNAc moiety. The chemical shifts of protons H-2, H-3 and H-4 corresponding to residues A, B, C and E were very similar ({Delta}{delta}<0·2 p.p.m.) and appeared at low field, indicating the presence of Galf units. The 13C-NMR spectrum (Fig. 3b) showed a C=O carbon (175·3 p.p.m.), six anomeric singlets, four of them in the range 106–109 p.p.m., and a methyl singlet at 23·0 p.p.m.



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Fig. 3. 1H-NMR spectrum (D2O, 40 °C, 300 MHz) (a), and 13C-NMR spectrum (D2O, 40 °C, 125 MHz) (b) of the F1S-II polysaccharide from S. candelabrum CBS 190.38. The C=O and CH3 singlets are not shown in the carbon spectrum.

 


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Fig. 4. 2-D NMR spectra (40 °C, 500 MHz) for selected regions of the F1S-II polysaccharide from S. candelabrum: (a) TOCSY (HOHAHA, mixing time=85 ms); (b) HMQCl; and (c) NOESY (mixing time=300 ms) subspectra. The anomeric protons and relevant crosspeaks have been labelled.

 
To confirm the assignments and to find the glycosylation sites, we used a HMQC experiment (Fig. 4b), which maps the connectivities between carbon atoms and their directly bonded protons. The crosspeaks in the anomeric region corroborated the presence of six residues. For non-anomeric atoms, the chemical shift of C-2 from unit F (54·6 p.p.m.) demonstrated that it was a GlcpNAc unit. The crowding of non-anomeric atoms, for both protons and carbons (Fig. 4b), did not allow unambiguous assignment of all signals; therefore it seemed advisable to run a HMQC-TOCSY experiment which, starting from each HMQC crosspeak of the 2-D spectrum, provides additional signals in the same row, caused by TOCSY transfer, thus allowing the identification of carbon chemical shifts pertaining to each residue (Fig. 5). In this way, most of the remaining carbon and protons were assigned to their corresponding units (Table 3). Comparison of the observed values with those reported in the literature (Bock & Pedersen, 1983 ; Parra et al., 1994 ; Leal et al., 1996 ) led to the identification of residue A as being 2,6-di-O-substituted Galf, B and E as 5-O-substituted Galf moieties, and C as 5,6-di-O-substituted Galf. Concerning the anomeric configuration of these residues, the vicinal J1,2 coupling constants of all Galf moieties (J<2 Hz) suggested that all have ß-configuration, which is supported by the chemical shifts of their anomeric carbons ({delta} >106 p.p.m.).



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Fig. 5. 2-D HMQC-TOCSY spectrum for relevant region of the F1S-II polysaccharide from S. candelabrum. The crosspeaks corresponding to the different relevant rows are labelled.

 

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Table 3. 1H- and 13C-NMR chemical shifts ({delta}) for the alkali-extractable, water-soluble cell-wall polysaccharide isolated from S. candelabrum

 
A 2-D NOESY spectrum (mixing time=300 ms, Fig. 4c) allowed the observation of connections H-1A/H-5E, H-1D/H-2A, H-1E/H-6a+6bA and H-1F/H-6a+6bC, indicating connections E->6A2<-D, A->5E and F->6C.

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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Fig. 6. Selected region of the HMBC spectrum for the F1S-II polysaccharide from S. candelabrum. Relevant crosspeaks are labelled.

 
Minor signals, corresponding to mannose residues, as deduced from the methylation analyses, are also observed in the proton and carbon 1-D spectra. Due to their low proportion (<6·5%), no additional information on these residues could be obtained, although they are most probably forming part of a small mannan core, which has been found in the polysaccharides isolated from other fungi (Prieto et al., 1997 ; Ahrazem et al., 1999 ).

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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Fig. 7. Repeating units of polysaccharides from several species of Sesquicillium, N. lasiacidis, N. cinnabarina and N. impariphialis, deduced from structural and chemical analyses.

 
The 1H-NMR spectra of the other two strains of S. candelabrum, and also that of N. lasiacidis, are very similar; therefore, the structure of the polysaccharides of these species are closely related, the only expected differences being small variations in the proportions of the different residues. Following analogous methodology, the structure of the polysaccharides of S. buxi, S. pseudosetosum and N. impariphialis were deduced as being those depicted in Fig. 7.

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 {delta}=5·03, 5·10 and 5·16 p.p.m.


   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The polysaccharides from species of Sesquicillium and related species of Nectria had in common the most abundant residues: terminal glucopyranose (Glcp-(1->), 5-O-substituted galactofuranose (->5)-Galf-(1->), and 2,6-di-O-substituted galactofuranose (->2,6)-Galf-(1->), which revealed a fairly similar basic structure and the relatedness of these fungi. The 1H-NMR spectra of these polysaccharides also revealed a common pattern, with three major signals at {delta}=5·03, 5·09 and 5·35 p.p.m. (Fig. 2). Additional residues might permit the identification of certain species. The structures depicted in Fig. 7 are proposed from the results of the chemical analyses of the polysaccharides, the full structural analysis of S. candelabrum presented here and the coincidence of chemical shifts of the signals of 1H-NMR spectra with those reported for similar residues (Table 4). The polysaccharides of S. candelabrum and N. lasiacidis had around 10% of the 5-O-substituted galactofuranose, (->5)-Galf-(1->), substituted also at O-6. These species also contain about 10% of N-acetylglucosamine. These results indicated the connection of our isolates of N. lasiacidis with S. candelabrum. Since it has been reported that the anamorph of N. lasiacidis is S. buxi (Samuels, 1989 ), it is possible that this strain of N. lasiacidis is misplaced. It is interesting to note that differences in conidium morphology between S. buxi and S. candelabrum have been described (Bisset, 1983 ). The polysaccharide from N. impariphialis contained 6-O-substituted glucopyranose (->6)-Glcp-(1->), in addition to the three main residues. This species was distinguished from other species of this group by the absence of the ring in the ascal apex (Samuels, 1989 ).


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Table 4. Assignments of the major signals of the anomeric region of the 1H-NMR spectra of polysaccharides from Sesquicillium spp., N. cinnabarina, N. lasiacidis and N. impariphialis

 
The polysaccharide obtained from the three isolates of N. cinnabarina (structure 1, Fig. 7) was similar to the polysaccharides found in species of Gibberella and Fusarium (Jikibara et al., 1992 ; Ahrazem et al., 2000 ) and in Penicillium vermoesenii (=Gliocladium vermoesenii) (Ahrazem et al., 1999 ) since they have similar residues, mainly 2,6-di-O-substituted galactofuranose (->2,6)-Galf-(1->), and terminal glucopyranose (Glcp-(1->), and almost identical 1H-NMR spectra. The differences found in the polysaccharide from N. cinnabarina and those species of Sesquicillium and Nectria with Sesquicillium anamorphs were in agreement with the separation of these species according to their morphological characters (Samuels, 1989 ) and support the creation of the genus Bionectria (Rossman et al., 1999 ).

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 teleomorph–anamorph 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 ).


   ACKNOWLEDGEMENTS
 
This work was supported by Grants PB 95/0078 and PB 93/0127 from Dirección General de Investigación Científica y Técnica. We thank Mr J. López for technical assistance.


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Ahrazem, O., Prieto, A., Leal, J. A., Gómez-Miranda, B., Domenech, J., Jiménez-Barbero, J. & Bernabé, M. (1997). Structural elucidation of acidic fungal polysaccharides isolated from the cell-wall of genera Cylindrocladium and Calonectria. Carbohydr Res 303, 67-72.[Medline]

Ahrazem, O., Gómez-Miranda, B., Prieto, A., Barasoaín, I., Bernabé, M. & Leal, J. A. (1999). Structural characterisation of a cell wall polysaccharide from Penicillium vermoesenii: chemotaxonomic application. Can J Bot 77, 961-968.

Ahrazem, O., Gómez-Miranda, B., Prieto, A., Barasoaín, I., Bernabé, M. & Leal, J. A. (2000). A characteristic water-soluble cell wall polysaccharide for Fusarium and Gibberella species. Mycol Res 104, 603-610.

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Ciucanu, I. & Kerek, F. (1984). A simple and rapid method for the permethylation of carbohydrates. Carbohydr Res 131, 209-217.

Domsch, K. H., Gams, W. & Anderson, T. H. (1980). Compendium of Soil Fungi, vol. 1. London: Academic Press.

Dubois, M., Giller, K. A., Rebers, P. A. & Smith, F. A. (1956). Colorimetric method for determination of sugars and related substances. Anal Chem 28, 350-356.

Gams, K. W. (1968). Die systematische Stellung der Schimmelpilze Fusidium buxi und Verticillium candelabrum. Acta Bot Neerl 17, 455-460.

Gerwig, G. J., Kamerling, J. P. & Vliegenthart, J. F. G. (1979). Determination of the absolute configuration of monosaccharides in complex carbohydrates by capillary G.L.C. Carbohydr Res 77, 10-17.[Medline]

Gómez-Miranda, B., Rupérez, P. & Leal, J. A. (1981). Changes in chemical composition during germination of Botrytis cinerea sclerotia. Curr Microbiol 6, 243-246.

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Received 5 February 2001; accepted 18 March 2001.



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