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Appl Environ Microbiol, February 1998, p. 613-617, Vol. 64, No. 2
Department of General Microbiology,
University of Copenhagen, DK-1307 Copenhagen K,
Denmark,1
GSF-Forschungszentrum
für Umwelt- und Gesundheit GmbH, Institut für
Bodenökologie, D-85764 Oberschleißheim,
Germany,2 and
Institute of Microbiology,
University of Innsbruck, A-6020 Innsbruck, Austria3
Received 9 June 1997/Accepted 24 November 1997
Our objective was to determine if 4-methylumbelliferyl-labelled
enzyme substrates could be used to detect and quantify specific components of chitinase and cellulase activities as specific indicators of the presence and activity of fungal biomass. The fluorogenic substrates 4-methylumbelliferyl (MUF)
N-acetyl- The determination of enzyme
activities is a simple approach to the study of microbially mediated
processes within the soil environment. Thus, soil enzyme activities
have been interpreted as indirect measures of microbial biomass,
rhizosphere effects, soil productivity, and mineralization potential of
naturally occurring substrates or xenobiotics (4). However,
few studies have attempted to correlate soil enzyme activities with the
presence and activities of specific components of the microbial
community. The ability of soil-inhabiting fungi to produce a range of
enzymes capable of degrading complex litter substances could make the
use of an enzymatic approach to study soil fungal populations possible. These enzymes must be specific for fungal presence and activity. In one
study of chitinase in soil (24), chitinase activity and the
number of fungal propagules in chitin-amended soils were strongly correlated. The same correlation was not found for actinomycetes or
bacteria. Thus, chitinase activity appears to be a suitable indicator
of actively growing fungi in the soil. The hydrolysis of cellulose
requires the interaction of a number of hydrolases produced by
cellulolytic microorganisms. A major role is played by the cellulase
system, which consists of several distinct enzymes that are produced by
a large number of microorganisms, including fungi, actinomycetes, and
bacteria. However, fungi have been suggested as the predominant source
of Fluorogenic 4-methylumbelliferyl (MUF)-labelled enzyme substrates have
been introduced for process-oriented studies in aquatic systems
(3, 18) and, more recently, in peatlands (11). MUF substrates have been used to assay cell-bound activities in pure
cultures of fungi, as the soluble substrate can enter the cell wall,
making periplasmic enzyme activity detectable (15). These
substrates have been used to detect fungal chitinolytic activities
(17a) and cellulases (6) in vitro. The substrates may be added to environmental samples and, when hydrolyzed, release 4-methyl-umbelliferone (4-MU), which fluoresces and can be quantified in nanomolar concentrations (3).
A variety of methods to quantify fungi in soil have been described. The
techniques include direct microscopic observation and extraction of
fungus-specific indicator molecules such as glucosamine or ergosterol
(9). More recently, the phospholipid fatty acid (PLFA)
18:2 Preparation, incubation, and sampling.
Soil samples were
collected from Danish and Italian sites in September 1994 (Table
1). The soils were sent to the
GSF-Forschungszentrum für Umwelt- und Gesundheit GmbH, Institute
für Bodenökologie, Neuherberg, Germany, where the soils
were incubated. Soils were incubated in containers 7 cm high and 10 cm
in diameter. To each container was added 700 g of rewetted
equilibrated soil with maize litter either incorporated or left on top.
Samples were taken after 14, 30, 60, 120, 240, and 360 days of
incubation. Ergosterol and enzymes were measured for each sample. PLFA
analysis was performed on the 14-, 30-, 120-, and 360-day-old samples.
Enzyme assays were performed at the University of Copenhagen,
Copenhagen, Denmark, ergosterol determinations were performed at the
University of Innsbruck, Innsbruck, Austria, and PLFA analyses were
performed at the GSF Institute für Bodenökologie.
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
The Use of Fluorogenic Substrates To Measure Fungal
Presence and Activity in Soil
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
-D-glucosaminide and MUF
-D-lactoside were used for the detection and
quantification of -N-acetylglucosaminidase (EC 3.2.1.30)
(NAGase) and endo 1,4--glucanase (EC 3.2.1.4)/cellobiohydrolase (EC
3.2.1.91) (CELase), respectively. Culture screenings on solid media
showed a widespread ability to produce NAGase among a taxonomically
diverse selection of fungi on media with and without added chitin.
NAGase activity was expressed only in a limited number of bacteria and on media supplemented with chitin. The CELase activity was observed only in a limited number of fungi and bacteria. Bacterial CELase activity was expressed on agar media containing a cellulose-derived substrate. In soil samples, NAGase activity was significantly correlated with estimates of fungal biomass, based on the content of
two fungus-specific indicator molecules, 18:2
6 phospholipid fatty
acid (PLFA) and ergosterol. CELase activity was significantly correlated with the PLFA-based estimate of fungal biomass in the soil,
but no correlation was found with ergosterol-based estimates of fungal
biomass.
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
-D-glucosidase (EC 3.2.1.21) (16, 17) and
endo 1,4--glucanase (EC 3.2.1.4) (23) activity in soils.
6 has been proposed as an indicator of fungal biomass (7,
12). Our objectives in the present study were to determine if (i)
components of chitinase and cellulase activities could be used as
indicators of the presence and activity of fungal biomass and (ii)
enzyme activities detected with specific MUF substrates in soil samples
were correlated with the content of the fungus-specific indicator
molecules 18:26 PLFA and ergosterol.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
TABLE 1.
Main chemical and physical parameters of the
experimental soils
Enzyme activity screening on solid media.
4-Methylumbelliferyl (MUF)
N-acetyl--D-glucosaminide and MUF
-D-lactoside (Sigma Chemical Co.) were used for the
detection of -N-acetylglucosaminidase (EC 3.2.1.30)
(NAGase) (19) and endo 1,4--glucanase (EC
3.2.1.4)/cellobiohydrolase (CELase) (EC 3.2.1.91) (6),
respectively. A diverse selection of fungi and bacteria were screened
on soil extract agar (SEA) containing either MUF
N-acetyl--D-glucosaminide or MUF
-D-lactoside, chitin agar containing MUF
N-acetyl--D-glucosaminide, and
carboxymethylcellulose (CMC) (Sigma Chemical Co.) agar containing
MUF--D-lactoside.
1 distilled water) soil
extract, 400 ml; glucose, 1; peptone, 1; yeast extract, 1;
K2HPO4, 1; and agar, 15. The CMC agar contained
(g liter1 distilled water) CMC, 10; asparagine, 0.5;
yeast extract, 0.5; Mg2SO4 · 7H2O; (NH4)2SO4, 0.5;
KH2PO4, 1; KCl, 0.5; CaCl2, 1; agar, 15. The chitin agar contained (g liter1 distilled
water) hydrolyzed chitin precipitate, 10;
K2HPO4, 1; MgSO4 · 7H2O, 0.5; NaCl, 0.5; CaCl2, 0.1;
Fe(NH4)2(SO4)2 · 6H2O, 0.05; NH4Cl, 0.1; agar, 15.
Hydrolyzed chitin precipitate was prepared by adding 10 g of
chitin (Fluka BioChemika) to 200 ml of distilled water and was kept
overnight at 4°C. Four hundred milliliters of 75%
H2SO4 was added, and the resulting solution was
left at 4°C for 24 h. The chitin was precipitated by mixing the
solution with 9 liters of 4°C distilled water. After 48 h, the
supernatant was decanted and the precipitate was filtered to remove
large pieces of undissolved chitin. The precipitate was centrifuged and
washed with 0.2% K2HPO4, and the supernatant
was removed in 5 to 6 repeated cycles. The resulting hydrolyzed chitin
precipitate was used in the preparation of the chitin agar.
After autoclaving, 2 ml of (200 µM) filter-sterilized (0.2-µm pore
size) MUF--D-lactoside or
MUF--D-glucosaminide was added to the agar media. Media
were dispensed into microtiter plates (24-well plate; Greiner
Labortechnik), and each well was inoculated with a pure fungal or
bacterial culture. The microtiter plates were incubated at room
temperature (21 to 23°C) and were visually examined daily under UV
light (366 nm) for 8 days. Activity against specific MUF substrates was
indicated by fluorescence. All screenings were done in triplicate.
Enzyme activity on soil samples.
NAGase and CELase
activities in soil samples were determined as follows. Bulk soil was
weighed into 10-ml plastic tubes. One hundred milligrams of soil in
four replicates was found to be representative of the soils in this
experiment (coefficient of variation < 15%); however, the amount
of soil and the number of replications necessary for the enzymatic
assay to be representative of a given soil may vary with soil type.
Soil samples were not physically treated prior to weighing. All assays
were conducted at 25°C in 2 ml of 50 mM Tris-maleate buffer (pH 5).
MUF N-acetyl--D-glucosaminide or
MUF--D-lactoside was added to a final concentration of
20 µM. Reactions were incubated for 30 min (NAGase) and 180 min
(CELase). Optimum pH was determined for each enzyme. Turnover rates
were constant during the assay time (unpublished data). Controls for extract and substrate fluorescence were processed in parallel. An
additional control was processed to correct for quenching agents in the
soil sample (11); however, quenching was not detected in
these experiments. The assay was terminated by adding 2 ml of ice-cold
96% ethanol. After centrifugation, 2.7 ml of supernatant was
transferred to plastic cuvettes (10 by 10 by 48 mm; Sarstedt) containing 300 µl of 2.5 M Tris buffer at pH 10. MUF substrate turnover was calculated by reference to a standard curve. Fluorescence derived from liberated 4-MU was determined with an LS50 luminescence spectrometer (Perkin-Elmer, Buckinghamshire, United Kingdom) at 446-nm
emission and 377-nm excitation. Enzyme activities in soil samples were
expressed as nanomoles of 4-MU per hour per gram (dru weight) of soil.
Extraction and quantification of ergosterol and PLFA.
Ergosterol analysis was performed as described by Roessner
(25) with minor modifications: 2 g of moist soil was
placed in 100-ml Schott flasks and treated with 20 ml of methanol, 5 ml of ethanol and 2 g of KOH and saponified for 40 min at 70°C.
After cooling, 5 ml of distilled water was added. The suspension was filtered through a paper filter (AGF 606, 125 mm; Frisenette) into a
separatory funnel. Soil residues were washed twice with 20 ml of
methanol. n-Hexane was added (30 ml), and the suspension was shaken for
2 min. The n-hexane phase containing the ergosterol fraction was
evaporated to dryness at 40°C with a rotary evaporator. The dried
extract was resuspended in 2 ml of methanol and filtered (0.02-µm
pore size, Anotrop; Whatman Ltd). This final extract (20 µl) was
analyzed by HPLC with an RFC-18 column (Merck no. 50829, LiChrospher
60, RP-select B, 5 µm diameter). The mobile phase was water-methanol
in a ratio of 5:95 with a flow rate of 1.5 ml min1.
Column eluant was monitored for absorbance at 282 nm. The retention time of ergosterol under these conditions was approximately 3 min.
6 was determined by the method of Frostegård
et al. (13) with minor modifications. Lipids were extracted from 2 g (wet weight) of soil with a one-phase mixture of
chloroform, methanol, and citrate buffer (0.15 M, pH 4) at a 1:2:0.8
ratio. Methyl nonadeconoate was added to the phospholipid fractions as an internal standard. The fatty acid methyl esters were separated and
quantified on a gas chromatograph (Varian) equipped with a flame
ionization detector and a 50-m nonpolar phenylmethyl silicone capillary
column (Hewlett-Packard, Palo Alto, Calif.). Identification of the
fatty acid methyl ester was done by mass spectrometry (Hewlett-Packard gas chromatography-mass spectometry system). Linear regression analysis
and analysis of variance with STATISTICA software (Statsoft, 1994) were
used to investigate the relationship between the variables examined.
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RESULTS |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Solid media screenings.
Eighty-six percent of the fungi tested
had NAGase activity, but only 14% had CELase activity when they were
inoculated onto SEA agar (Table 2). The
fungi were inoculated on chitin agar containing MUF
N-acetyl--D-glucosaminide and CMC agar
containing MUF--D-lactoside to determine if NAGase and
CELase activity were inducible. CELase activity could be induced in
three fungi, and NAGase activity could be induced in two of three
previously negative fungi (Table 2). When grown on SEA, all bacteria
tested negative with regard to NAGase or CELase activity (Table 2);
however, seven bacterial isolates produced NAGase or CELase when they
were cultured on chitin and CMC agar.
|
Correlation with fungal biomass.
We evaluated the utility of
MUF N-acetyl--D-glucosaminide and MUF
-D-lactoside substrates by correlating substrate
turnover rates with the amount of 18:26 PLFA and ergosterol. The
enzyme activity determinations were reproducible (coefficient of
variation < 15%). One-way analysis of variance identified
differences between individual sampling times with respect to enzyme
determinations (P < 0.001) and ergosterol
(P < 0.01) and PLFA (P < 0.001).
NAGase activity and PLFA were clearly correlated, and a weak but
significant relationship between NAGase activity and ergosterol was
detected (Fig. 1). CELase activity was
correlated with PLFA but not with ergosterol (Fig.
2). Finally, there was a significant
correlation between 18:26 PLFA and ergosterol
(r2 = 0.38, P < 0.01) and a
weak but significant correlation between NAGase and CELase activities
(r2 = 0.22, P < 0.05) (data not
shown).
|
) and Danish soil incubations (
).
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DISCUSSION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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We found that NAGase activity was expressed by a diverse group of fungi. Furthermore, we detected NAGase activity in cultures growing on SEA agar containing glucose and peptone but no chitin, suggesting that NAGase is constitutive in these fungi. These results are consistent with those of other studies in which constitutive expression of NAGase activity in fungi was reported (5, 22). In contrast, CELase activity was restricted to a limited number of fungal species and the activity was predominantly expressed on CMC agar.
We used ergosterol and 18:26 PLFA as indicators of fungal biomass in
order to correlate NAGase and CELase activities with soil fungal
biomass. The extraction of ergosterol is widely used for quantification
of fungal biomass (9, 20). Potential for error exists in
this method, which entails problems such as variability of specific
ergosterol content in fungal tissue and in the relationship to living
and nonliving fungal biomass (2, 27).
PLFA analysis has been used to describe soil microbial community
structure and to detect changes in response to various disturbances (1, 7, 28). More recently, the 18:26 PLFA was found to be
highly correlated with ergosterol content in 15 types of soil (r2 = 0.85, P < 0.001)
(12), consistent with the significant but weaker correlation
we found in this study.
The correlations between NAGase activity and the soil content of
ergosterol and 18:26 PLFA, in combination with the constitutive expression of NAGase by a diverse group of fungi, support the use of
this activity as an indicator of fungal biomass in soil samples. The
fact that CELase activity was expressed in a limited number of fungal
species excludes the use of this activity as a measure of fungal
biomass. However, the correlation between 18:26 PLFA and CELase
activity is consistent with a predominant fungal origin
(23).
In bacteria, NAGase and CELase activities were restricted to a limited number of species and expressed only on media containing chitin or CMC, suggesting a nutritional role for these enzymes. As fungi are considered to be the major important contributors to chitin turnover in soils (14), chitinolytic bacteria or actinomycetes are not likely to have a significant impact on the interpretation of NAGase activity as reflecting actively growing fungi in a soil sample. This conclusion is supported by our data and is in agreement with the significant, positive correlation found between chitinase activities, but not between bacteria or actinomycetes, and fungal propagules in chitin-amended soils (24).
The role of NAGase in fungal physiology is complex and has been linked
to morphogenetics (15), N acquisition (14), and mycoparasitism (8). In an environmental sample, NAGase
enzymes located in the periplasmic space will also contribute to a MUF substrate-derived signal, since MUF substrates have been used to
measure periplasmic activities of fresh intact cells (15). Consequently, NAGase activity as determined by the enzymatic cleavage of MUF N-acetyl--D-glucosaminide in soil
samples may reflect a nutritional as well as a possible morphogenetic
aspect of fungal growth. Furthermore, NAGase activity in a soil sample
may comprise enzyme activities of a recent biological origin as well as
activities derived from enzymes that have retained their activity in
nonliving soil compartments, e.g., dead cells, cell debris, and enzymes immobilized in the soil matrix (26). Further study has been initiated to investigate the relationship between NAGase activity and
fungal growth and to determine the extent to which NAGase activity
reflects enzyme activities of a recent biological origin, in order to
evaluate the relationship to living and nonliving fungal biomass.
No definitive method to quantify fungal biomass in soil exists. Direct microscopic techniques are laborious and tend to underestimate the amount of fungi (10, 21), and calculations of fungal biomass with pure culture-derived conversion factors and the soil ergosterol content tend to overestimate fungal biomass in relation to total microbial biomass (12). The determination of NAGase activity is simple, i.e., no laborious physical or chemical treatment of the sample is necessary and the assay is rapid and reproducible.
In conclusion, we have shown that (i) NAGase activity is present in a
diverse group of fungi, (ii) the activity appears to be constitutive,
(iii) the activity is correlated with two independent indicators of
fungal biomass, and (iv) the activity can be quantified in soil samples
with a fluorogenic substrate. The data in the present study suggest
that NAGase activity as determined by the turnover of
MUF-N-acetyl--D-glucosaminide may provide a
simple, sensitive, and rapid measure of soil fungal biomass.
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ACKNOWLEDGMENTS |
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This project was supported by the EU Environment and Climate Program project no. EV5V-CT940-034.
We thank Kirsten Jensen for technical assistance and Bo Jensen and Jacob Møller for useful discussions about the manuscript.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of General Microbiology, University of Copenhagen, Sølvgade 83 H, DK-1307 Copenhagen K, Denmark. Phone: 45 35 32 20 54. Fax: 45 35 32 20 40. E-mail: mmiller{at}mermaid.molbio.ku.dk.
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Abstract
Introduction
Materials & Methods
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Discussion
References
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Appl Environ Microbiol, February 1998, p. 613-617, Vol. 64, No. 2
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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