Annual variations of phenoloxidase activities in an evergreen oak
litter: in¯uence of certain biotic and abiotic factors
S. Criquet*, A.M. Farnet, S. Tagger, J. Le Petit
Institut MeÂditerraneÂen d'Ecologie et de PaleÂoeÂcologie, UPRES A CNRS, Laboratoire de Microbiologie, Service 452, Faculte des Sciences et Techniques de Saint-JeÂroÃme, 13397 Marseille cedex 20, France
Abstract
This study concerns ligninolysis phenomena occurring over 13 months in forest litter. Evergreen oak (Quercus ilex L.) litter was taken as a model because Quercus ilex L. is the most abundant tree species in forests of the French Mediterranean area. Several biotic and abiotic factors potentially involved in transformations of polyphenolic compounds, were measured between October 1997 and October 1998. These factors were: global fungal micro¯ora, the fungi producing phenoloxidases (PO+), the activities of several phenoloxidases, hydrosoluble phenols, and temperature, humidity and pH of the litter. Results showed that the annual dynamics of fungi and phenoloxidase activities appear to be seasonal, i.e. that these biotic factors such as, were optimal in autumn. A multiple regression analysis showed that there was no correlation between biotic factors such as, fungal populations and phenoloxidase activity and abiotic factors such as, temperature, humidity and pH. Laccases were the preponderant phenoloxidase activities during the year, while those of Mn-peroxidases only appeared in the autumn of 1997. Other phenoloxidases, lignin-peroxidases and tyrosinases were never detected. Interactions between laccases and humic substances were also investigated. Adsorption of laccases on humic substances leads to a shift in the optimal temperature activity of these enzymes from 50 to 308C. Activities of laccases also shifted towards more acidic values when laccases were not adsorbed on humic substances. Nevertheless, the optimal pH was the same (5.7) whether laccases were adsorbed or not to humic substances. Electrophoresis analysis showed little variations in the number of phenoloxidase isoenzymes. Indeed, laccases showed three isoenzymes during the year (Rf 0.23, 0.34 and 0.43). Only one isoform of Mn-peroxidase, with an Rf 0.21, was detected in the litter.72000 Elsevier Science Ltd. All rights reserved.
Keywords:Litter; Fungi; Laccase; Peroxidase; Humic substances
1. Introduction
Litter decomposition on the forest ¯oor is an essen-tial process in nutrient cycles and soil formation. These processes are controlled by abiotic factors, such as climate and by biotic factors, such as, chemical composition and microbial communities of litter and soil (CouÃteaux et al., 1995). The continuous degra-dation of dead plant materials by saprophytic microor-ganisms, such as fungi, requires the release of various enzymes that are physically and chemically associated
with insoluble organic debris and inorganic particles (Stozky and Burns, 1982). In litter, lignin is particu-larly important because it represents the most abun-dant aromatic polymer. Of particular importance are enzymes excreted by microorganisms for the initial depolymerization of lignin (Burns, 1978). The extra-cellular activities of the enzymes derive from exoen-zymes produced by living cells or from endoenexoen-zymes released during disintegration of cells (Tabatabai and Fu, 1992). Various studies have shown the activities of numerous enzymes in soil and litter (Roberge, 1978; Sinsabaugh et al., 1991), but there is a lack of studies about the microorganisms implicated in the production of phenoloxidases in litter and the temporal
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bution of these enzymes. Indeed, polyphenoloxidases (i.e. Mn- and lignin-peroxidases, laccases and tyrosi-nases) oxidize phenol and polyphenol compounds to radical cations and are able to cleave lateral chain or aryl±alkyl bounds in a molecule of lignin. Criquet et al. (1999) have developed a methodology for the measurement of the activities of laccases in evergreen oak litter. This litter was taken as a model, because evergreen oak forms characteristic forest climax popu-lations that are widely distributed throughout the wes-tern Mediterranean area (Rapp, 1969). Leaves are the principal (50±60%) component of the litter followed by wood, ¯owers and fruits (Chabert et al., 1984). Cel-lulose (22.6%), hemicelCel-lulose (15.1%) and lignin (14.9%) are the main chemical components of these leaves (Gillon et al., 1994). Hydrolizable (0.54%) and condensed tannins (2.14%) (Racon et al., 1988) are also important components, since they are defensive chemicals inhibiting proliferation of insects and micro-organisms (KuÈhnelt, 1963; Feeny and Bostock, 1968; Poinsot-Balaguer et al., 1993). Based on these charac-teristics, this study focused on the development of phe-noloxidases in evergreen oak litter over one year. Interactions between the enzymes of the litter and humic substances were studied, using laccases as a model, because this type of enzyme is also important in the biosynthesis and the decomposition of humic compounds (Su¯ita and Bollag, 1981; Martin et al., 1982; Haider and Martin, 1988; Stevenson, 1994). Throughout the year, we investigated global fungal micro¯ora de®ned as fungi which is able to grow on a non-speci®c culture medium, fungi releasing phenoloxi-dases, water soluble phenols, pH, humidity and tem-perature of the litter. We compared these parameters with the activity of phenoloxidases over the same period.
This paper represents part of a larger study of the processes involved in litter degradation and, in particu-lar, interactions between microorganisms, enzymes and abiotic factors in Mediterranean evergreen oak litter.
2. Materials and methods
An evergreen oak litter (Quercus ilex L.) was sampled monthly for one year, between October 1997 and 1998, from a dense copse at ``La Gardiole de Rians'' (Var, France). Samples were collected from the Lv layer de®ned by Babel (1971): leaves still recogniz-able despite decay and often compressed in lumps. Five fresh samples (80 g each) were collected from the litter immediately before analysis. For the study of the micro¯ora and electrophoretic analysis, these samples were pooled before analysis.
According to Criquet et al. (1999), phenoloxidases were extracted by shaking, at room temperature for 1
h on a reciprocal shaker, 80 g of litter in 700 ml of a 0.1 M CaCl2solution added with 0.05% Tween 80 and
20 g polyvinylpolypyrrolidone. The supernatant of each extract was concentrated in a cellulose-dialysis tube (Polylabo, Strasbourg, France), with a 10 kDa molecular mass cut-o, covered with polyethylene gly-col, until a ®nal volume of 1/10±1/20 of the initial volume. The activities of laccases in the concentrated extracts were measured spectrophotometrically (Kon-tron, model Uvikon 860) using syringaldazine (Sigma) as the substrate (Harkin and Obst, 1973). For kinetic measurements, 0.5 ml of enzyme extract, 2.5 ml of 0.1 M phosphate buer (pH 5.7), and 10 ml of a solution of syringaldazine (5mM) were mixed in a 4 ml cuvette. The rate of oxidation of syringaldazine to quinone was measured for 5 min at 525 nm xM 65, 000 Mÿ1 cmÿ1). The results are expressed in units de®ned as mmol of quinone formed from syringaldazine per min-ute (U) and per gram of dry matter (U gÿ1DM).
To determine dry weight, the litter material was dried at 1008C for 24 h. The activity of Mn-peroxi-dases (MnP) was measured according to Pasczcynski et al. (1985) using vanillylacetone as the substrate (synthetized in our laboratory). For kinetic measure-ments, 0.5 ml of enzyme extract, 2.17 ml of 0.1 M lac-tate buer (pH 4.5), 0.2 ml of a 0.05 mM H2O2
solution, 0.1 ml of 0.1 mM MnSO4and 30 ml of a 0.4
mM vanillylacetone solution were mixed in a 4 ml cuv-ette. Controls did not contain H2O2, to subtract
lac-case activity on vanillylacetone. The results are expressed in unit (U) gÿ1 DM, where U is mmole of vanillylacetone disappearing per minute. The activities of lignin peroxidases (LiP) and tyrosinases were measured according to Tien and Kirk (1984) and Daw-ley and FlurkDaw-ley (1993), using 0.5 ml of litter extract. Litter extracts boiled for 15 min served as controls for the activities of all enzymes, respectively. No activities were detected in these boiled extracts.
The laccase fraction not adsorbed on humic sub-stances (free laccase fraction) was estimated in the lit-ter extract by precipitating humic substances (Mayaudon and Sarkar, 1974) with a solution of 8 g lÿ1 of protamine sulfate grade II (Sigma). The litter extract was then centrifuged for 20 min at 12,000 g and ®ltered through a 1.2 m GF/C ®lter. The residual laccase activity was measured in the ®ltrate as described above and expressed in percentage of the total activity of laccase.
mixed with gel loading buer (50 mM Tris±HCl pH 6.8, 0.1% bromophenol blue and 10% glycerol) and loaded on a non-denaturing (without sodium dodecyl sulfate or b-mercaptoethanol) polyacrylamide gel elec-trophoresis (PAGE) according to Laemmli (1970). For the analysis of phenoloxidase isoenzymes, polyacryl-amide gels (7.5%) layered with 4% staking gels and Tris-glycine buer were used. The mini-gels (Mini-Pro-tean II, Biorad) were run at 200 V for 45 min. The electrophoresis gel were stained using p -phenylenedia-mine (Sigma) 0.1% (W/V) in Na-phosphate buer (100 mM, pH 5.7) to reveal activity of the laccases. The ac-tivities of the Mn-peroxidase isoenzymes were visual-ized using lactate buer (100 mM, pH 5.7) supplemented with 0.1% 2, 7-diamino¯uorene (Sigma) and 3 mM MnSO4. H2O2 (20 mM) was added to the
lactate buer only after 30 min to eliminate laccase ac-tivity on 2,7-diamino¯uorene.
For counting fungi, the litter (2.5 g) was powdered (<0.5 mm) using a Moulinex mixer, and suspended in 250 ml of a 8.5% NaCl solution with 0.05% Tween 80. The mixture was agitated for 2 h on a reciprocal shaker (120 rpm) and sonicated in a sonic bath (Bio-Block, ultrasons 320 W, 35 kHz) for 1 min (Kilbertus et al., 1982). This treatment provided the best de-sorption of microorganisms from the litter. Dilutions of the mixture were plated on a medium containing 5 g malt, 15 g agar, 50 mg chloramphenicol per liter for global fungal micro¯ora. Fungi producing phenoloxi-dases (PO+) were counted on the same medium added with 0.5 ml guaiacol (Flegel et al., 1982). Results are expressed in unit forming colony per gram of dry mat-ter (UFC gÿ1 DM). Water soluble phenolic com-pounds were measured by incubating, without agitation, the powered litter (5 g) in 75 ml of double distilled water for 24 h. The water soluble fraction was centrifuged for 10 min at 12,000 rpm, ®ltered through a 1.2 mm Whatman ®lter, and a clear supernatant was obtained following this treatment. It was then analyzed by the method of Box (1983). A calibration curve was established with catechin, and results are expressed in microgram equivalent of catechin per-gram of dry matter (mg eq. catechin gÿ1DM).
The pH of the litter was measured (pH meter, Metrohm, Herisau, Switzerland) monthly by incubat-ing 5 g of powdered leaves in 100 ml of double dis-tilled water for 2 h. The temperature in the litter during the year was measured with a temperature data logger (Tinytalk II, Radiospares, Beauvais, France) and OTLM software (Orion Tiny Logger Manager, Gemini data Loggers, UK). The humidity of the litter was deducted from measure of the dry weight.
2.1. Statistical analysis
Correlation coecients of Pearson (r) were
calcu-lated between all the variables measured during the year, except for the electrophoresis analysis. Signi®cant correlations were retained for p R0.05 or p R0.01. The in¯uence of humidity, pH and temperature on fungal populations and phenoloxidase activities was evaluated by multiple regression. Multiple regression
models were analyzed by ANOVA. Levels of signi®-cance are indicated aspR0:05 or pR0:01: All the cal-culations were eected using Statview software version 1.03.
3. Results and discussion
3.1. Relationships between phenoloxidases, fungi and abiotic factors
The activities of laccases, measured at pH 5.7 and 308C, reached a ®rst peak in autumn of 1997 (Fig. 1). This peak was followed by a decrease in the activity until March 1998. During the spring of 1998, the ac-tivities increased again, albeit slightly, and then sharply decreased in July 1998 because of the typical dry Med-iterranean summer. Over the year, there was no signi®-cant correlation between humidity and temperature of the litter and the activity of laccases (Table 1). Once again, we observed a maximum activity of laccase in the autumn of 1998. This phenomenon may re¯ect a seasonal cycle of the expression of laccases in ever-green oak litter.
It was only in the autumn of 1997 that activity of Mn-peroxidases (Fig. 1) was observed in the litter. The best pH and temperature for the measurement of the activity of Mn-peroxidases were 4.5 and 308C, respect-ively (Fig. 2). The same pH was found by Galliano et al. (1991) using vanillylacetone as the substrate, and a MnP puri®ed from a culture of Rigidosporus lignosus. Bartha and Bordeleau (1969) extracted peroxidases from soil using a 50 mM phosphate buer (pH 6.0) and measured their activities in the same buer using o-dianisidine as the substrate. The peroxidase detected with this method had activities between 0.610ÿ2and 2.35 10ÿ2 U gÿ1 DM, which are comparable with the activities obtained in our study with the evergreen oak litter (Fig. 1). Bollag et al. (1987) found an
opti-mal pH value of 5.0 for a peroxidase extracted from soil. The Mn-peroxidases derived from the evergreen oak litter were totally inactive for pH values above 5.5. Consequently, distinction between the activities of peroxidases and laccases seems possible because the ac-tivities of both these enzymes are at 4.5 and 5.7,
re-Table 1
Pearson correlation coecients between microbial, enzymatic, chemical and environmental factorsa
Hr t8C pH HP Lac F Lac MnP GF PO+
Hr 1 ÿ0.43 ÿ0.05 0.08 0.48 ÿ0.43 0.38 0.47 0.38
t8C 1 ÿ0.12 0.34 ÿ0.46 0.82b ÿ0.31 ÿ0.22 ÿ0.22
pH 1 ÿ0.69b ÿ0.17 0.004 ÿ0.54c ÿ0.56c ÿ0.57c
HP 1 0.01 0.26 0.32 0.44 0.36
Lac 1 ÿ0.71b 0.64b 0.15 0.23
F. Lac 1 ÿ0.42 ÿ0.19 ÿ0.31
MnP 1 0.58c 0.66b
GF 1 0.94b
PO+ 1
aHr: water content of the litter;t8C: temperature; HP: hydrosoluble phenols; Lac: laccase activity; F. Lac: free laccase activity; MnP: Mn-per-oxidase activity; GF: global fungal micro¯ora; PO+: fungi producing phenoloxidases.
bP< 0.01. c
P< 0.05.
spectively. Furthermore, for pH 4.5, no phenoloxidase activity was detected without adding H2O2 to the
enzyme extract. No activity was detected for other phenoloxidases, (i.e. lignin peroxidase and tyrosinase). Laccases appeared to be the most important phenolox-idases in the evergreen oak litter because these were present throughout the year and could be excreted in signi®cant quantities, especially in autumn. These results agree with those of Ruggiero and Radogna (1984), who reported that a humus-phenoloxidase sys-tem extracted from soil showed only, or prevalently, laccase activity.
The global fungal micro¯ora and fungi producing polyphenoloxidases (PO+) were evaluated using the plate counts. Such a technique is known to be ambigu-ous because some colonies may arise from spores and hyphal fragments. Nevertheless, according to Witkamp (1966), this method can re¯ect signi®cantly the quanti-tative variations of the fungal micro¯ora in soil and litter, which is one of the purposes of this study.
The global fungal micro¯ora and the PO+ fungi were well correlated (r = 0.94, Table 1) and showed a signi®cant increase in number during the autumn of
1997 (Fig. 1). This sudden increase may be attributable to a typical and seasonal augmentation of the number of fungi, particularly basidiomycetes, in litter. Seasonal trends in fungal population dynamics are well known, and Berg et al. (1998) have recently described the dynamics and strati®cation of fungi in the organic layers of a Scots pine forest soil. The number of fungi PO+ only showed a new increase in the autumn of 1998. In the case of global fungal micro¯ora, other smaller variations were observed during the year and may be the result of meteorological conditions (Berg et al., 1998), especially rainfalls, which present quite vary-ing frequency and intensity under Mediterranean cli-mate. The maximal expression of the activities of laccases and Mn-peroxidases occurred during the period of proliferation of the fungi PO+(Fig. 1). Even if certain bacteria, such as Bacillus sphaericus (Claus and Filip, 1997) and Streptomyces strains (Crawford, 1978), are able to synthesize laccases, Rosenbrock et al. (1995) suggested that the laccase activity in litters was essentially attributable to lignolytic fungi.
The pH of the evergreen oak litter ranged from 5.5 to 7.0 during the year (Fig. 3). During autumn, the pH values were the lowest and favorable to the develop-ment of PO+ fungi r ÿ0:57, Table 1). These fungi synthesized laccases and Mn-peroxidases, whose activi-ties measured in vitro were well correlated r0:64, Table 1). However, Figs. 3 and 4 show that the pH values in autumn were favorable for the in situ activity of laccases, but not of peroxidases since the lowest pH observed in autumn (5.5±5.7) was still too high for Mn-peroxidase activity (pH optimum 4.5). Mn-peroxi-dase activity was negatively correlated with the pH r ÿ0:54, Table 1). Thus, it is dicult to hypothesize that peroxidases were really active in the evergreen oak litter during autumn, since the optimum pH for Mn-peroxidases and the pH measured in the litter extract were dierent.
As indicated above, signi®cant negative coecients were observed between pH and PO+ fungi, and between pH and Mn-peroxidase activities (Table 1). Nevertheless, multiple regression analysis (Tables 2 and 3) shows there was no relationship between biotic factors (fungal populations and phenoloxidase activity) and the abiotic ones (temperature, humidity, and pH). Thus, variations of these biological parameters over one year cannot be predicted by these classical abiotic factors.
3.2. Relationships between humic compounds and laccases
Among the water soluble components of the litter, water soluble phenols are chemical compounds usually integrated in humic substances, which interact with enzymes of litter. The origin of water soluble phenols
in the evergreen oak litter is not clear, because they may be due to degradation of lignin and polyphenolic compounds by phenoloxidases or due to leaching from the upper layer of the litter, which is rich in water sol-uble components (GourbieÁre, 1983; McClaugherty, 1983). Furthermore, water soluble phenols may have an important role in the acidi®cation observed in the Lv layer, which may also be due to the simultaneous release of various metabolic organic acids. The pH values were negatively correlated r ÿ0:69, Table 1) with the concentration of water soluble phenols (Fig. 3). The values of water soluble phenols were cor-related neither with the temporal dynamics of fungi nor with the activities of the phenoloxidases (Table 1). The presence of these water soluble phenols may be explained by disintegration over the year of plant cells releasing hydrolyzable and condensed tannins in ever-green oak litter (Racon et al., 1988). These tannins undergo leaching of the litter layers and, probably, water soluble phenols do not re¯ect the in situ cata-bolic activities of microorganisms on polyphenolic components of evergreen oak litter.
According to Burns (1982), high proportions of extracellular enzymes are associated with soil or litter colloids, such as humic compounds. According to Tabatabai and Fu (1992), these systems may allow great stability of laccases in soil and may aect the
properties of soil or litter enzymes. However, little work has been done to ®nd the degree of adsorption of extracellular enzymes on humic colloids over a period of a year (Burns, 1982). Laccases were selected for the present study, because these were the sole phe-noloxidase observed in evergreen oak litter throughout the year.
Criquet et al (1999) has mentioned the role of humic substances on the thermic optimum for the activity of laccases in litter extracts. Maximum temperature for activity of laccases of the litter extract shifted from 30
Table 2
Coecients of the regression equations calculated between activities of laccases and Mn-peroxidases, fungal numbers (global and PO+), moist-ure, temperature and pHa
Variable namea Constant Moisture (coecienta) Temperature (coecientb) pH (coecientc) r2
Laccases 4.073 0.017 ÿ0.064 ÿ0.423 0.326
Mnÿperoxidases 9.714b 0.011 ÿ0.046 ÿ1.472b 0.479
Global fungi 27.436b 0.054 ÿ0.063 ÿ4.341b 0.522
Fungi PO+ 16.676 0.024
ÿ0.048 ÿ2.618b 0.465
aVariable name = Constant + (aMoisture) + (bTemperature) + (cpH). bLevels of signi®cance of the coecients are indicated forP
R0.05.
Table 3
Analysis of variance of the multiple regression models established between activities of laccases and Mn-peroxidases, fungal numbers (global and PO+), moisture, temperature and pHa
Source SS df MS F-ratio Prob >F
Laccase model 4.016 3 1.339 1.450 0.293
Error 8.308 9 0.923
MnP model 4.721 3 1.574 2.756 0.104
Error 5.138 9 0.571
Global fungi model 45.443 3 15.148 3.270 0.073
Error 41.690 9 4.632
Fungi PO+model 14.091 3 4.697 2.608 0.116
Error 16.209 9 1.801
a
SS : Sum of squares; df : degrees of freedom; MS : Mean of Squares.
to 508C after total precipitation of humic substances using protamine sulfate (Fig. 4). However, Ruggiero and Radogna (1984) have shown that the thermic opti-mum for laccases adsorbed on humic substances shifted to higher temperatures. Furthermore, Nanni-pieri et al. (1982) have shown that immobilization of enzymes on humic substances usually increases their thermic stability. These contradictory results may be explained by the observations of Ladd and Butler (1975), who reported certain exceptions for thermal properties of soil enzymes.
There was also a shift in laccase activity towards acidic values of the pH, when humic substances were removed from the litter extracts with protamine sulfate (Fig. 4), although the optimum pH values were the same in both extracts, with or without humic sub-stances. A similar phenomenon has also been described for urease activity in soil (Nannipieri et al., 1978).
Fig. 5 shows that the optimal laccase activity was negatively correlated to the ``free laccase fraction'' r ÿ0:71, Table 1). Thus, it appears that the adsorp-tion of laccases on the humic substances is a favorable factor, which allows a higher activity of these enzymes
in the litter. These results could agree with the hypoth-esis of Gramss et al. (1999), who suggested an impli-cation of laccases and other phenoloxidases in the biosynthesis and decomposition of humic substances in the litter. Moreover, the high correlation coecient observed between the ``free laccase fraction'' and tem-perature r0:82, Table 1) may indicate that humi®-cation processes in the evergreen oak litter are probably slackened or stopped during summer dryness, which is typical of Mediterranean climate.
3.3. Electrophoretic analysis
The laccases detected over the year in the evergreen oak litter (Fig. 6) showed three isoenzymes with Rf
0.23, 0.34 and 0.43 (Fig. 7). These three isoenzymes were observed in the litter between October 1997 and January 1998 and in September and October 1998, when the activities of laccases generally peaked. For the rest of the year, the isoenzyme with Rf 0.23 was
not detected in the litter extracts (Fig. 7). In August 1998, only one isoenzyme (Rf 0.34) was present in the
litter (Fig. 7), which was probably due to the dryness of the Mediterranean summer (Daget, 1984). This dry-ness was probably unfavorable for the development of global fungal micro¯ora and fungi producing polyphe-noloxidases, thereby, for the expression of several lac-case isoenzymes in the litter.
Only one isoenzyme of Mn-peroxidases with an Rf
of 0.21 (Fig. 7) was observed in November and December 1997, in accordance with the peak activity of this enzyme detected during the same period.
4. Conclusion
This study has shown certain aspects of the mi-crobial degradation of polyphenolic compounds in evergreen oak litter in the Mediterranean area. The phenoloxidases which oxidize these compounds seem
Fig. 5. Variation in activities of ``total laccases'' from extracts con-taining humic substances and of ``free laccase fraction'' devoid of humic substances (HS) in the evergreen oak litter studied over 13 consecutive months. Values represent the averages of ®ve replicates.
to be limited to laccases and Mn-peroxidases, since lig-nin-peroxidases and tyrosinases were never detected under these experimental conditions. During the autumn, we observed the maximum activities of phe-noloxidases followed by an increase in the number of fungi producing polyphenoloxidases.
Laccases were the only phenoloxidases present in the litter throughout the year and were always rep-resented by one isoenzyme (Rf 0.34) with a high
ac-tivity staining. The two other isoenzymes were not detected in all the samples and their activity staining was always weaker than that observed with the perma-nent isoenzyme (Rf 0.34). The highest activity of
lac-cases was observed, when they were adsorbed to humic substances. This adsorption was weak during the summer (hot, as is typical of a Mediterranean cli-mate), since it is negatively correlated to temperature.
Since laccases are present in the litter throughout the year, polyphenolic compounds, such as lignin, are probably subjected to continuous transformations by these enzymes. Nevertheless, in the evergreen oak lit-ter, transformation of polyphenolic compounds at an optimum rate appeared to be a seasonal phenomenon, involving the activity of phenoloxidases such as lac-cases or Mn-peroxidases produced by ligninolytic fungi. The presence of water soluble phenols was apparently not correlated with the development of fungi and with activities of phenoloxidases. It may be correlated with other phenomena such as the enzy-matic disintegration of cells containing tannins in the evergreen oak leaf.
It will be interesting to study the evergreen oak litter at length in autumn, with samples very closely col-lected at very short intervals. The tannin concentration measured in the litter would allow us to study closely how these compounds are released over time. Further-more, under these conditions, correlation between
temperature, humidity, pH and other biological par-ameters would allow us to de®ne the seasonal cycle, which seems so important for fungi. In other words, we should investigate the direct in¯uence of abiotic factors on fungal enzymatic activities.
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