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Department of Biological Sciences, Sir Alexander Fleming Building, Imperial College London, London SW7 2AZ, UK
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ABSTRACT |
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The ability of two wood-decay basidiomycetes to produce extracellular mucilaginous material (ECMM) and its relationship with total biomass production has been investigated. Growth and ECMM production by the white-rot fungus Coriolus versicolor and the brown-rot fungus Gloeophyllum trabeum were assessed in liquid culture under different nutritional and environmental conditions. Nutritional, pH and temperature factors all influenced significantly the proportion of ECMM in the total biomass produced. When total biomass production was reduced due to unfavorable growth conditions (stress), the proportion of ECMM in the biomass was elevated. The results are discussed with regard to the possible role(s) of ECMM in the responses of these fungi to stress.
Key words: Coriolus versicolor, extracellular mucilaginous material (ECMM), Gloeophyllum trabeum, growth conditions
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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, Abu Ali 1998, Abu Ali et al 1999). Various roles for ECMM such as adhesion to substrata, protection against desiccation and nutrient sinks have been proposed ( Jones 1994, Bes et al 1987, Nicholson 1984).
Several studies have provided visual data on the production of ECMM by wood-decay basidiomycetes (Foisner et al 1985a, b; Green et al 1990, Ruel and Joseleau 1991, Abu Ali et al 1999) but relatively little quantitative data is available. Sarkar et al (1986) isolated and quantified the ECMM from the ascomycete Moniliella pollinis growing in the presence of increasing concentrations of carbon. In a later study Jellison et al (1997) attempted to quantify ECMM produced by the basidiomycetes Gloeophyllum trabeum and Postia placenta with optical microscopy and by expressing the area covered by ECMM in relation to the area covered by the hyphae. However both experimental methods had limitations and while the former was unable to link ECMM production to the production of mycelium, the latter study relied on the quantification of tri-dimensional structures by analyzing two-dimensional data.
ECMM is composed primarily of a polysaccharide matrix, in which proteins and lipids also may be included (Foisner et al 1985a, b; Doss 1999, Jones 1994, Cooper et al 2000). It is secreted at the tip of actively growing hyphae and accumulates in the region just behind the secretion zone (Evans et al 1981). However secretion is dependent on the age of the hyphae. Old and necrotic hyphae lose the ability to secrete ECMM (Evans et al 1981, Palmer et al 1983, Ruel and Joseleau 1991). Thus its production seems to be affected by the physiological status of the organism.
ECMM has been observed during the growth of basidiomycetes and other wood-inhabiting fungi on a variety of surfaces. Its production was stimulated particularly during growth on wood, suggesting a probable role during the decay process (Abu Ali 1998, Abu Ali et al 1999). Wood presents a nutritionally challenging substrate for fungal growth due to its low nitrogen content and the complexity of the bulk carbon present. Nitrogen sources known to occur in wood include glutamic acid, aspartic acid, valine, alanine, leucine and threonine (Laidlaw and Smith 1965). The presence of histidine, glycine and serine also has been detected in some wood species (Merrill and Cowling 1966a, b). Carbon sources in wood include glucose (the major source as both cellulose and hemicelluloses), mannose, galactose, rhamnose, xylose, fucose, inositol and sorbitol (Eaton and Hale 1993, Weißmann et al 1989). All occur in polymerised forms and are intermingled with lignin. Successful growth of wood inhabiting fungi in wood also is dependent on suitable moisture content, temperature, pH and aeration.
The aim of the present study was to obtain quantitative data on the production of ECMM by wood-rotting basidiomycetes when grown on various substrates likely to be encountered in wood. This was intended to permit identification of any particular nutritional stimuli that might be associated with the substantial ECMM production, which was noted in the qualitative work by Abu Ali et al (1999). In view of the anticipated variation in biomass production when grown on different nutrient sources and under different conditions, ECMM production was expressed with reference to total biomass (mycelium and ECMM) present.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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A defined liquid medium after Beever (1969), with a modified trace element solution, was used. The medium contained (per liter of deionized water): 1 g KH2PO4, 0.5 g MgSO40.7H2O, 0.3 g CaSO4, 22.5 g glucose, 2.14 g l-glutamic acid, 10 mL thiamine HCl solution (10 mg l–1) and 0.5 mL trace elements solution (1 L deionized water, 0.1 g ZnSO40.7H2O, 0.3 g MnCl.4H2O, 0.2 g CoCl.6H2O, 0.01 g CuCl20.2H2O, 0.02 g NiCl20.6H2O, 2.556 g FeSO4, 0.01 g CuCl2, 5 g NaMoO4, 0.3 g H3BO3 and sufficient HCl to dissolve salts). The initial pH of the medium was adjusted to pH 5.0 (±0.2) with HCl and NaOH and was measured again after incubation before harvesting the cultures.
A standard 15-mm inoculum plug taken from the growing margin of 1 wk old cultures on 2% MA was used to inoculate 90 x 20 mm Petri dishes (Greiner) containing 25 mL of the defined liquid medium. Cultures were incubated 1 wk at 22 C (±1 C), 70% RH (±5%), unless otherwise specified.
Investigations on growth under different conditions.— – Fungi were grown in the defined medium described above, which was used as a control. The medium was modified each time according to the nutritional and parameters being investigated.
We investigated several alternative carbon and nitrogen sources to those in the basic medium, which reflected those known to occur in wood. Mannose, cellobiose or sorbitol were added to the medium to provide equivalent carbon levels to glucose. The nitrogen sources investigated were L-arginine, L-isoleucine, NH4NO3, glycine or threonine. These were added to the medium to provide equivalent nitrogen levels to L-glutamic acid.
The effects of different initial pH and incubation temperature were investigated as "environmental" stress factors. Initial pH was modified to 3.0, 4.0, 5.0 and 6.0 (±0.2) using HCl and NaOH. The incubation temperatures used were 10, 15, 25 and 30, 35 C (also 40 C in the case of G. trabeum).
The effect of incubation time on biomass and ECMM production also was assessed. Cultures were grown on the basic defined medium, incubated up to 7 wk at 22 C and sampled at weekly intervals.
Extraction and preparation of ECMM.— –
Procedure for isolation of ECMM followed those of Sarkar et al (1985, 1986), Madi et al (1997) and Krcmar et al (1999) with modifications as reported below. Each "replicate" consisted of five liquid cultures, which were bulked before measurement. The bulked liquid cultures were filtered through a single layer of nylon mesh. Individual colonies were transferred into a 250 mL Erlenmeyer flask, washed with 20 mL deionized water (dH2O) at room temperature and refiltered on nylon mesh. This was repeated three times. The washed mycelium was dried to constant weight at 105 C and the mycelial biomass determined by weighing.
Preliminary studies compared the extraction efficacy of cold (room temperature) and hot (ca. 85 C). To reduce the possibility of unwanted extraction from the hyphal cell wall, the use of cold water was preferred throughout the study. Assessment of extraction efficacy of the washing procedure was by visual observation of washed and unwashed specimens by scanning electron microscopy (SEM) throughout the study. The samples were mounted on a copper stub using colloidal graphite. They were cryofixed by rapid quenching in liquid nitrogen and transferred under vacuum to the cold stage of a Philips SEM 501B microscope. Ice crystals were removed under observation by raising the temperature of the cryostage to allow for sublimation. The samples were coated with gold for 6 min and returned to the cryostage of the microscope for observation.
The supernatant obtained from the initial filtration of the liquid cultures and the liquid obtained from the washings contained the ECMM. They were combined and concentrated under rotary vacuum evaporation (40 C) to a volume of approximately 30 mL. After concentration three volumes of 95% ethanol were added with shaking at room temperature (20 C ± 2 C). The precipitated ECMM was centrifuged 10 min at 6000 rpm at 15 C, resuspended in deionized H2O and dialyzed in visking tubes (Medicell International) against dH2O for 14 h. After dialysis the ECMM was centrifuged at 10 min 16 000 rpm at 15 C. This led to the identification of two fractions. The pellet fraction, which could be resuspended but not dissolved in dH2O, was referred to as the "insoluble" ECMM. The supernatant, which also contained a form of ECMM, was referred to as the "soluble" ECMM. Both fractions were freeze-dried ca. 24 h, weighed and stored at 4 C in a desiccator. For the purpose of simplicity, the data for these two fractions have been combined into a single value for ECMM.
Quantification of insoluble ECMM was carried out in triplicate. Quantification of soluble ECMM also was carried out in triplicate for the control cultures. In all other cases individual factors for the insoluble:soluble ECMM ratio, derived from single soluble ECMM determinations, were used to calculate the amount of soluble ECMM.
The data for total ECMM dry weight were combined with the data for mycelial dry weight to give a value for total biomass produced. The proportion of ECMM was expressed as percentage of the total biomass to evaluate the ECMM production pattern.
The set of experiments was repeated and consistent effects obtained. The data presented are from a single experimental series and statistical significance was assessed by the analysis of deviance deletion method using the GLIM package (Royal Statistical Society).
ECMM compositional analysis.— –
Total carbohydrate content of the ECMM was determined with the method of Dubois et al (1956) against glucose. Total protein content was determined against bovine serum albumin by the method of Lowry et al (1951).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). It was estimated from several visual observations that more than 80% of the ECMM was removed by the procedure. Quantitative data were obtained on the extraction efficiency of dH2O water at the temperatures tested (data not shown), and little difference was observed between the temperatures tested.
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Nutritional factors.— –
C. versicolor produced its highest total biomass on glucose and mannose as carbon sources (FIG. 2). Biomass was reduced by half and two-thirds, respectively on cellobiose and sorbitol. On the glucose and on the mannose media, ECMM comprised approximately 15–20% of the total biomass. The ECMM proportion increased significantly to ca. 40% of the total biomass on the cellobiose and sorbitol media. The highest biomass in G. trabeum was produced on cellobiose as a carbon source (FIG. 3), with ECMM accounting for 20% of this biomass. On the other carbon sources biomass production was reduced to about two-thirds of that on cellobiose but ECMM proportion rose to approximately 35% of the total biomass. In both species, therefore, the proportion of ECMM in the total biomass varied significantly (P < 0.001) with carbon source and showed an opposite trend to biomass production (i.e. high total biomass = low ECMM proportion, low total biomass = high ECMM proportion).
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). Total biomass production was reduced by about half to three quarters on most other N-sources compared with L-glutamic acid. A significantly (P < 0.001) higher proportion of ECMM in the total biomass was observed on threonine (40% of total biomass) and on isoleucine (50%) compared with L-glutamic acid (15%) or the other N-sources (25%). With G. trabeum the highest biomass occurred on L-glutamic acid and glycine. Isoleucine was the poorest N-source giving ca half the biomass production. All other N-sources gave intermediate levels of biomass production. A significant (P < 0.001) difference also was found in the proportion of ECMM within the total biomass produced according to N-source. Low ECMM proportion in the total biomass (15%) was associated with glycine, which yielded the highest total biomass production, and the highest ECMM proportion (ca. 43%) was on isoleucine, which supported the lowest total biomass production. In general the pattern of low ECMM proportion under high total biomass conditions and high ECMM proportion under low biomass conditions seen with the carbon sources also occurred with these N-sources. The total amount of ECMM showed little variation throughout the range of nutritional conditions tested. Generally the total amount of ECMM was approximately 40 mg. The greatest variation (decrease) was observed when G. trabeum was grown in the presence of glycine and threonine (ca. 20 mg).
Temperature and pH.— –
The optimal temperature for growth of C. versicolor was 25 C (FIG. 4). G. trabeum had a higher optimal temperature, with the most biomass produced at 35 C. High proportions of ECMM were found in the biomass of both fungi at the lower incubation temperatures which, as expected, substantially reduced total biomass production. Low ECMM proportions also were associated in both fungi with high total biomass yields. An exception to this trend was observed with G. trabeum at its highest growth temperature (40 C), which, despite yielding quite high biomass production, also was associated with reasonably high ECMM proportion (about 45% of total biomass). At this temperature it is clear that the yield of ECMM, however, is about double that found under any other of the growth conditions studied. Once more the total amount of ECMM remained rather constant throughout the range of temperature tested, especially in the case of C. versicolor. In G. trabeum the lowest amount of total ECMM was observed during growth at 25 C and the highest during growth at 40 C.
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). For both species high total biomass production was associated with low proportions of ECMM and vice versa. However little variation was observed in the total amount of ECMM produced by the two fungi over the range of pH tested, with the greatest variation being observed at the lowest pH (also pH 4 in the case of C. versicolor).
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) and thereafter remained constant up to the end of the incubation period. Biomass production in G. trabeum peaked at 5 and 6 wk and showed a decline at 7 wk (FIG. 6). Both species showed their highest proportions of ECMM in the first week of growth, thereafter showing a decline for the majority of the incubation period at levels of about 7% of total biomass. In the case of G. trabeum a clear trend of progressively increasing total biomass was mirrored by a trend of progressively decreasing ECMM proportion, despite little variation in the total amount of ECMM. At the longest period of incubation, the proportion of ECMM showed a slight increase, especially in cultures of G. trabeum.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Removal of ECMM, which is known to surround the hyphae of fungi growing on wood (Abu Ali et al 1999) and in liquid cultures (as seen in the results reported here) required careful washing (Sarkar et al 1985, 1986; Madi et al 1997). Assessment of the washed mycelia by cryo-SEM revealed that the majority of the ECMM was removed by the washing procedure. The extent of the ECMM removal was estimated by visual cryo-SEM assessment throughout the study to be ca 80%. The amount of ECMM removed was observed to be consistent. We therefore assumed that the method was a reliable tool for the quantification of stress-induced changes in ECMM secretion.
Both fungi showed different preferences for carbon and nitrogen sources. However a consistent relationship between total biomass production and the proportion of ECMM within the biomass is observed in the data. When these two parameters are considered relative to each other, an inverse relationship is seen between total biomass production and the proportion of ECMM. Thus low biomass production (as a consequence of nutritional stress) was associated strongly with elevated proportions of ECMM and vice versa. This is well illustrated in the data for carbon sources with C. versicolor, where those that supported the highest levels of total biomass production (glucose and mannose) yielded the two lowest proportions of ECMM in the biomass (FIG. 2). Conversely the two poorest carbon sources also yielded the two highest proportions of ECMM.
Data presented by Sarkar et al (1986) with Moniliella pollinis have shown an increased total biomass production and ECMM production when carbon stress (due to limited dextrose supply) on the cultures was alleviated. We have recalculated their data according to the approach of the present study (i.e. considering ECMM as a proportion of the total biomass) and observed a high proportion of ECMM when M. pollinis is carbon limited and a reduction in this ECMM proportion when carbon stress is reduced (TABLE I). Our results also are consistent with the observations of Bes et al (1987) and with the trend reported by Jellison et al (1997), who noted that the production of ECMM in nitrogen-stressed cultures of wood-decay basidiomycetes was highest under conditions of moderate stress and was reduced under nitrogen-rich conditions. In our data it is clear that ECMM proportion is reduced in cultures growing under optimum nitrogen nutrition and elevated when nitrogen stress is induced by unfavorable nitrogen sources (e.g. l-glutamic acid and isoleucine respectively, see FIG. 3).
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The results for the experiment on time of incubation showed a similar inverse relationship between ECMM proportion and total biomass, however this trend cannot be linked to stress factors. Rather it emphasises how the secretion of ECMM is associated with young, actively growing hyphae. In both C. versicolor and G. trabeum ECMM proportion was high at the earlier incubation times, when total biomass was low and hyphae were young, and decreased as biomass increased with time. In cultures of G. trabeum, a steady decrease in the proportion of ECMM within the biomass can be observed in 2–5 wk. However it is of note that after 5 wk incubation, G. trabeum produced a low total amount of ECMM. This was unexpected because total ECMM production seemed to be constant throughout the duration of the study for both species. Such sudden decrease in total ECMM production might lead to a skewed interpretation of the results. This is supported by the fact that the proportion of ECMM increases to about 7% thereafter.
Taken overall our results indicate that the proportion of ECMM in the total biomass produced by these two fungi is strongly influenced by the degree of stress experienced by the cultures. This response was consistent for several stress factors. The factors that exhibited a wide range of stress for C. versicolor were carbon source, nitrogen source and temperature and for G. trabeum were temperature and pH. Under low-stress conditions (from carbon source, nitrogen source and temperature) C. versicolor developed total biomass levels of approximately 300 mg, with ECMM proportions of ca. 10–15%. Under high stress from these factors reductions in C. versicolor biomass to ca 60 mg biomass occurred, but the proportion of ECMM increased significantly to ca 50% of this biomass. G. trabeum exhibited similar effects with ca. 250 mg biomass and 18% ECMM proportion when under low stress (from temperature and pH) reducing to about 40–100 mg biomass with approx 50% ECMM proportion when stress was high. In general terms ECMM represented 10–20% of biomass under low stress conditions rising to 40–50% of the biomass under high stress conditions.
Throughout the range of growth conditions tested, little variation was observed in the total amount of ECMM produced, with a few exceptions. This constant pattern might be due to a variation in the area of ECMM secretion in the hyphae when the fungi are exposed to certain types of stress, which might be mediated by regulation of the hyphal morphology. Research in this direction is being undertaken.
From a compositional point of view the results presented here fit well with those described in the literature (Foisner et al 1985b, Jones 1994, Doss 1999, Krcmar et al 1999). They revealed, however, that, mineral precipitation can occur from the culture medium and indicate that preliminary tests to determine the stability of the medium itself should be considered for future studies.
The functional role of elevated proportions of ECMM in stressed cultures of these fungi is uncertain, but its development appears to be a general phenomenon irrespective of the nature of stress. It is possible that ECMM somehow affords a protective role to the mycelium by enabling continued growth or survival. Furthermore the general nature of the effect observed in these studies is suggestive of a common mechanism providing this response. Preliminary observations on the morphology of stressed cultures of these fungi indicates the development of a more highly branched form than under low-stress conditions, which suggests a possible role of increased branching and changes in hyphal morphology in delivery of elevated proportions of ECMM within the biomass.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Corresponding author. Present address: Ensis, Sala Street, Private Bag 3020, Rotorua, New Zealand. E-mail: damiano.vesentini{at}ensisjv.com. Tel. +64 (0)7 3435595. Fax +64 (0)7 3435507.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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