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Enzyme dynamics on decomposing leaf litter of Cistus incanus and Myrtus

communis in a Mediterranean ecosystem

A. Fioretto*, S. Papa, E. Curcio, G. Sorrentino, A. Fuggi

Dipartimento di Scienze della Vita, Seconda Universita` di Napoli. Via Vivaldi, 43. 81100 Caserta, Italy

Accepted 5 April 2000

Abstract

The decomposition of Cistus incanus leaf litter, a summer deciduous species, was compared to that of Myrtus communis, an evergreen species, during 15 months of incubation in a Mediterranean low shrubland. The litterbags were placed under randomly selected shrubs of Myrtus and Cistus, respectively. Owing to the different microclimatic conditions under deciduous and evergreen shrubs Cistus litter was also incubated under Myrtus shrubs. Microbial activity was evaluated by measuring litter respiration and enzyme activities (cellulase, xylanase,

a-amylase,b-amylase, laccase and peroxidase). During the first 8 months of incubation the decomposition rate of both litters was inde-pendent of litter quality and incubation conditions. The average decay constant (k) ranged between 0:29^0:03 and 0:33^0:03 yr21: Subsequently, it increased only for litters incubated under Myrtus shrubsk0:57^0:15 and 0:48^0:16 yr21for Cistus and Myrtus litters, respectively). The dry summer affected the decay rate of litters incubated under Myrtus but not under Cistus. Microbial respiration showed seasonal changes (from 25 to 150mmol CO2g21dry wt. 24 h21), with low levels in summer, mainly because of the low litter water content. After samples were placed in the field,a-amylase activity decreased rapidly, dropping to zero in Cistus litter, whereas it remained detectable in Myrtus litter (.0.02mmol glucose g21dry wt. h21). Theb-amylase activity was low over the entire period. The activities of cellulase and xylanase ranged from 1 to 30mmol glucose equivalents (reducing sugar) g21dry wt. h21. Both litters showed the lowest enzyme activities in summer, when litter respiration was also at the lowest level. Peroxidase activity was detected in the litter of Myrtus(from 0 to 50mmol o-tolidine oxidised g21dry wt. h21) and had a seasonal pattern similar to cellulase and xylanase. It was undetectable in Cistus. In both litters laccase increased significantly going from 10 to 140mmol o-tolidine oxidised g21_{dry wt. h}21_{between eight and nine months when a large} increase of fungal biomass occurred (from 0.5 to 2.5 mg g21dry wt.). The analyses of these enzymes have shown qualitative and quantitative differences depending on the litter type and the microclimatic conditions, suggesting changes in the microbial succession.q_{2000 Elsevier} Science Ltd. All rights reserved.

Keywords: Litter decomposition; Xylanase; Cellulase; Amylases; Laccase; Peroxidase; Cistus incanus, Myrtus communis

1. Introduction

Plant litter decomposition is a critical process in nutrient cycling within temperate forest ecosystems (Swift et al., 1979). Over the past 30 years, many studies have been carried out on litter decomposition and the dynamics of nutrient release to analyse the effect of climate and litter quality (Taylor et al., 1989; Arianoutsou, 1993; Virzo De Santo et al., 1993; Berg et al., 1995; Rutigliano et al., 1996; Fioretto et al., 1998) as well as of soil organisms (Anderson and Ineson, 1984; Moore and Walter, 1988; Persson, 1989; Ponge, 1991; Dilly and Irmler, 1998). In recent years, atten-tion has turned to the degrading capacity of microorganisms by evaluating their enzyme activities. Microbial species and

communities release enzymes into the environment in order to degrade macromolecular and insoluble organic matter prior to cell uptake and metabolism (Burns, 1982). This important property may allow decomposition rates to be related to the enzymes that directly mediate the degradation of the major structural components of plant material and can provide functional information on specific aspects of the microbial community and succession (Sinsabaugh et al., 1991). Moreover, both the enzymes involved in the degra-dation of main litter components, such as cellulose, hemi-cellulose and lignin, and those involved in the cycling of nitrogen, phosphorus and sulphur, are of primary interest in understanding the factors controlling plant litter decomposi-tion. Lignocellulolytic enzyme activities have been reported as good indicators of mass loss rates (Sinsabaugh et al., 1994).

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and is influenced by temperature, moisture, pH, and quality and quantity of available substrate (Linkins et al., 1984; Sinsabaugh and Linkins, 1987).

Studies on litter decomposition analysing enzyme activ-ities have been performed in aquatic and forest ecosystems (Sinsabaugh et al., 1991; 1992, 1993; Joshi et al., 1993; Rosenbrock et al., 1995; Dilly and Munch, 1996) but no data are available for Mediterranean ecosystems.

In this paper, we compare the pattern of microbial enzyme activities (cellulase, xylanase, a-amylase, b -amylase, laccase and peroxidase) and respiration during litter decomposition of two species in a Mediterranean low shrub land: Cistus incanus L., a summer deciduous species, and Myrtus communis L., an evergreen sclerophyll species. The aim of the work was to analyse the time of appearance and the activity of enzymes in qualitatively different litter in order to obtain functional information on microbial succession. The effect of microclimatic condi-tions on litter decomposition rate and enzyme activities was also evaluated.

2. Materials and methods

2.1. Site description

The study was carried out in a stand of Mediterranean low macchia within the Natural Reserve of Castel Volturno (Campania, Italy). The Reserve (268 ha) is located to the South of the Volturno estuary. The climate is typically Mediterranean with mild, wet winters (mean temperature of the coldest month 10.68C) and hot, dry summers (mean temperature of the hottest month: 288C). The annual rainfall is approximately 680 mm.

We selected an experimental plot (2500 m2, 6 m a.s.l.), burned in 1976, where the shrub canopy cover was about 70% and characterised by Cistus incanus, Cistus salvifolius,

Myrtus communis, Rhamnus alaternus, Asparagus acutifo-lius, Phillirea angustifolia and Pistacia lentiscus. Tree

canopy cover by Quercus ilex and Pinus halepensis was low (10%) while the herbaceous canopy cover was about 40%. Cistus and Myrtus species were the main components of the shrub canopy.

2.2. Sample preparation

Freshly abscised leaves of C. incanus and M. communis were collected by shaking shrubs over a large net in May– June, when most of the litterfall occurs. Contaminating debris (e.g. leaves of other species, small stems or branches, flowers) was removed carefully from each collection. The litter was mixed to provide an homogeneous sample, air-dried and stored in polyethylene bags at room temperature (about 208C) until sample preparation. About 3 g of leaf material were placed in each terylene net bag (16×10 cm2length×height) with a mesh size of 1 mm2.

2.3. Sample processing

The litter bags (240 for each litter type) were set out in the experimental plot on January 1998 in ten randomly selected sites under either C. incanus or M. communis shrubs. To evaluate the effect of microclimatic conditions on decom-position, bags enclosing Cistus litter were also incubated under Myrtus shrubs, because Cistus is a pioneer species after fire but is replaced by more competitive species, i.e.

Myrtus, during succession. All the bags were fixed on top of

the litter layer by metal pegs. The soil characteristics of the incubation sites are reported in Table 1.

The bags were collected every two months in the first year of decomposition and every three months thereafter. At the sampling date two bags were collected from each of the ten sites and placed in individual plastic bags to minimise litter loss and avoid dehydration. The bags were brought to the laboratory, where the litter of each bag was cleaned to remove soil using a small brush and weighed.

A subsample of litter from each bag (30–40% fresh weight) was oven dried at 75₈C to constant weight and used for dry matter and water content determinations.

To provide enough material to perform all the analyses and to allow statistical treatment of data, the remainder of the litter enclosed in the bags from groups of three sites was pooled. The samples of the tenth site were used when rodents destroyed bags of the other sites. At each sampling, the three composite samples were obtained by bags from the same group of incubation sites. Each of these samples, was divided into subsamples and used to determine litter respira-tion, enzyme activities, pH and fungal biomass.

2.4. Litter respiration

Litter respiration was measured as CO2 evolution from

litter at field moisture level. Approximately 1 g of leaf litter was incubated in airtight jars for 2 d at 25₈C in total Table 1

Physical and chemical properties of the top soil (0–5 cm) under C. incanus and M. communis

Cistus Myrtus

Sand (%) 98.8 99.9

Field capacity (H2O g 100 g21d.wt)

57 57

pHa 8.5 8.2

Potential pHb 7.9 7.4

C org (%) 1.32 2.40

N (%) 0.19 0.21

C to N ratio 6.9 11.43

CO2evolution rate (mmol CO2g21dry wt. 24 h21)

1.95 1.84

a _{The pH of the soil was determined as described for the litter but shaking} 10 g of dry soil in 25 ml of distilled water.

b_{The potential pH was determined shaking 10 g of dry soil in 25 ml 1 M} KCl.

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darkness. The CO2released was absorbed in NaOH solution

(0.5 M) and its amount was determined by two phase titra-tion with HCl (0.05 M) (Froment, 1972). The CO2output

from leaf litter was expressed inmmol g21dry litter d21. All measures were performed in triplicate on the three litter subsamples.

2.5. Enzyme extraction and assays

Litter samples to be assayed for enzyme activity were ground in the appropriate cold buffer using a Polytron homogeniser for 1 min. The homogenate was centrifuged at 10,000 g at 4₈C for 20 min. The supernatant was filtered through a Whatman No 1 filter paper and used as the enzyme extract. No significant enzyme activities were detected in the pellet.

Xylanase (EC 3.2.1.8) and CM-cellulase (EC 3.2.1.4) activities were determined according to Schinner and Von Mersi (1990) with minor modifications: 0.5 g of leaf litter were transferred to a test tube, suspended in 10 ml cold acetate buffer 0.2 M pH 5.5 (1.2 w/v). Xylanase activity was determined by shaking 0.4 ml of the enzyme extract, 1.3 ml 0.2 M acetate buffer and 1.5 ml xylan substrate solu-tion for 24 h at 50₈C. A xylan-free control was prepared. After incubation, 1.5 ml xylan substrate solution was added to the control. The mixtures were shaken, filtered and diluted 1:50 with distilled water. For photometric analysis, 1 ml of the diluted mixture, 1 ml reagent A (16.0 g anhy-drous sodium carbonate and 0.9 g potassium cyanide dissolved in 1 l distilled water), 1 ml reagent B (0.5 g potas-sium ferric hexa-cyanide dissolved in 1 l distilled water and stored in the dark) were mixed in a test-tube (pH.10.5) and boiled in a water bath at 1008C for 15 min. After cooling in a water bath at 208C for 5 min, 5 ml of reagent C (1.5 g ferric ammonium sulphate, 1.0 g sodium-dodecyl-sulfate, and 4.2 ml conc. sulphuric acid dissolved in 1 l distilled water at 508C) were added, mixed (pH,2.0) and allowed to stand for 60 min at 208C to develop colour. The extinc-tion was measured within 30 min at 690 nm against the reagent blank. The extinction, after subtracting the control values from the sample value, was used to determine the glucose equivalents on a calibration curve (conc. range 2.8– 28mg ml21). Activities were expressed asmmol of glucose equivalents g21dry weight h21. The CM-cellulase activity was assayed in the same way using CM-cellulose (0.7% w/v) as substrate. The filtrates were diluted 1:30 with distilled water for the photometric measurement of glucose.

The activities ofa-amylase (EC 3.2.1.1) andb-amylase (EC 3.2.1.2) were estimated according to Bernfeld (1955) with minor modifications: 0.5 g leaf litter were transferred into a test tube, suspended in 10 ml of buffer (0.02 M phos-phate pH 6.9 fora-amylase and 0.2 M acetate pH 4.8 forb -amylase) and kept at 4₈C. The mixture was homogenised, centrifuged and filtered as described above. The enzyme activities were determined by incubating at 37₈C for 2 h a

mixture containing: 2 ml enzyme extract, 1 ml a-amylase substrate (1 g soluble starch in 100 ml 0.02 M phosphate buffer, pH 6.9), or 1 ml b-amylase substrate (1 g soluble starch in 100 ml 0.2 M acetate buffer, pH 4.8). The enzyme reaction was stopped by adding 2 ml dinitrosalicylic acid solution (1 g dinitrosalicylic acid dissolved in 20 ml 2 M NaOH and 50 ml distilled water; 30 g potassium sodium tartrate added and made up to 100 ml with water) and diluted 1:20 with distilled water to measure the extinction at 575 nm. The reducing sugars formed were determined using a calibration curve (conc. range 2.8–28mg ml21). The enzyme activities are expressed as mmol of glucose equivalents g21dry weight h21.

The activity of laccase (EC 1.10.3.2) was estimated according to Leatham and Stahmann (1981), with minor modifications: 0.5 g leaf litter were transferred into a test tube, suspended in 10 ml of buffer (50 mM acetate pH 5.0) and kept at 4₈C. The mixture was homogenised, centrifuged and filtered as described above. Soluble laccase activity was measured by recording the increase of absorbance at 600 nm for 1 min at 30₈C in a mixture containing: 1 ml enzyme extract, 1 ml 50 mM pH 5.0 acetate buffer and 0.2 ml 25 mM o-tolidine (3-30dimethyl 4-40diamino biphenyl).

Peroxidase (EC 1.11.1.7) activity, determined in the same enzyme extract as used to assay laccase, was measured in the same conditions and in the same reaction mixture to which 0.1 ml of 4 mM H2O2was added (Ander and

Eriks-son, 1976). The peroxidase activity was evaluated by subtracting the laccase activity from the overall assay activ-ity. The activities were calculated as mmol of tolidine oxidised min21 using a molar extinction coefficient of 6340 (McClaugherty and Linkins, 1990).

All enzyme assays were performed in triplicate for each litter sample.

2.6. Chemical analyses

The chemical composition of the two kinds of leaf litter was determined as follows: the oven-dried litter subsamples were ground to fine powder by a Fritsch Pulverisette (type 00.502, Oberstein, Germany) equipped with an agate pocket and ball mill. The analyses of each sample were carried out in triplicate.

Carbon and nitrogen content were determined by combustion in an Elemental Analyzer NA 1500 (Carlo Erba Strumentazione, Milan, Italy).

Total P, K, Ca, Mg, Na, Mn contents were determined by a SpectrAA-20 atomic absorption spectrophotometer (Varian-Techtron, Mulgrave Victoria, Australia) after digestion of the samples in a mixture of nitric and hydro-fluoric acid (2:1 v:v) by a Digestore Milestone MLS 1200 (Microwave Laboratory System, Sorisole BG, Italy). The elemental composition is given in mg g21dry litter.

2.7. Fungal biomass

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intersection method. The dry litter (1 g) was milled and homogenised in water (100 ml) for 5 min. Subsequently, an aliquot of this homogenate was diluted to obtain four samples (1 mg litter per ml of water). Four membrane filters were prepared according to Sundman and Sivela¨ (1978). The values are reported as mg dry fungal biomass g21of dry litter on the basis of the average 9.3mm2cross section of the hyphae, a density of 1.1 g ml21and a dry mass of 15% (Berg and So¨derstro¨m, 1979). The measures were made on the three samples, each with three replicates.

2.8. pH and soluble substance measurements

The pH of the litter was determined by shaking litter in distilled water for 10 min (0.5 g dry litter in 10 ml water). The suspension was left to stand for 10 min and the super-natant used to measure the pH with an electronic pH meter (HI 8424, HANNA Instruments, Sarmeola di Rubano PD, Italy).

Water soluble substances were determined by soaking the undecomposed leaf litter in distilled water (1 g of dry litter in 70 ml of water) at 20–228C for 24 h (Berg and Wessen, 1984, modified). The samples underwent a two-fold sonica-tion for 2 h at the start and at the end of leaching period in a sonicator bath filled with water and ice. To prevent micro-bial growth, 2–3 drops of sodium hypochlorite were added. The dry weight of these substances was obtained by calcu-lating the difference between the dry weight of the litter before and after leaching. Measurements on each sample were made in triplicate.

2.9. Statistics

The mass loss over time was fitted to a simple exponential curve (Olson, 1963)

lnxt=x0 2kt

where x0is the original mass of leaf litter, xtthe amount of

litter remaining after time t, t is time (year) and k the decom-position rate (yr21). The half-life for decomposition (t1/2),

that is the time necessary to reach 50% mass loss, was calculatedt1=20:691=k:

The significance of differences among the litters was tested by two-way analysis of variance (ANOVA) followed by Tukey test. Correlations were determined using the simple Pearson correlation coefficient.

3. Results

3.1. Initial chemical composition

Among the macronutrients, the initial content of the C, N, P, K and Ca was higher in the Cistus than in the Myrtus litter (Table 2). In contrast, the initial content of Mg was higher in the Myrtus litter while that of Na was similar in both litters. Among the micronutrients, particular attention was given to

Table 2 Initial pH, nutrient and water solubl e substanc es contained in Cistu s an d M yrtus Litter pH Water sol uble substa nces (%) C (mg g 2 1dry wt.) N (mg g 2 1dry w t.) P (mg g 2 1dry wt.) K (mg g 2 1dry wt.) Mg (mg g 2 1dry wt.) Ca (mg g 2 1dr y w t.) Na (mg g 2 1dry wt.) Mn (mg g 2 1dry wt.) Cistus 5.3 10.8 414 10.8 0.68 5.84 1.22 11.7 1.22 0.09 Myrtus 5.4 16.8 375 9.8 0.55 3.86 2.62 10.7 1.35 0.07

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Mn because it is essential for lignin-degrading enzymes (Perez and Jeffrey, 1992; Archibald and Roy, 1992). Its initial content was higher in the Cistus than in the Myrtus litter (Table 2).

The water soluble substances were about 35% more abun-dant in the undecomposed leaf litter of Myrtus than of the

Cistus. The pH values were similar (Table 2).

3.2. Litter decomposition

Fig. 1 shows the decomposition dynamics over 15 months of Cistus leaf litter incubated under Cistus and under Myrtus (Myrtus replaced Cistus in the succession) as well as Myrtus litter incubated under Myrtus.

The average decomposition rates were similar for all the litter types during the first eight months of incubation. Subsequently, they were higher for litters under Myrtus than under Cistus shrubs (Fig. 1). After 15 months, litter under Cistus had lost 30 of the initial mass and those under Myrtus had lost 40%. Significant differences P_,

0:001were found between the mass loss patterns of Myrtus, as well as of the Cistus under Myrtus, and that of Cistus under Cistus.

The data conformed to a first order exponential decay

0:48 and 0.57 yr21 for Myrtus and Cistus, respectively. Over the experimental period the half-lives of Cistus and

Myrtus litters incubated under Myrtus were similar t1=2 1:5 yrand lower in Cistus under Cistust1=22:1 yr:

Both litters incubated under Myrtus showed reduced decomposition rate during the dry summer. In the same period the decomposition rate of Cistus litter incubated under Cistus, did not change.

3.3. Litter respiration

Fig. 2 shows respiration rates as well as water contents for litters at each sampling. The respiration rates showed seaso-nal variations: the highest value occurring in the wet seasons, and the lowest in summer. In addition, they were correlated with the water content of the litter that, however, had higher values in autumn than in spring. Only Cistus incubated under Myrtus showed a respiration pattern signif-icantly different from the othersP_,0:001:

3.4. Enzyme activities

During the decay process, extractable cellulase and Fig. 1. Residual mass (% of the initial) of decomposing leaf litter of Cistus

(B_—B_{) and Myrtus (}V_—V_{) incubated under the relative shrubs. The} residual mass of Cistus litter incubated under Myrtus (O_{– –-}O_{) is also} reported. The values are means^SE of 20 measurements.

Table 3

Decay constant k (yr21)^SE of leaf litter of M. communis and C. incanus incubated in situ calculated at different incubation times. The coefficient of determination (r2_{) of the exponential decay interpolation of weight loss is} also reported. Significance for * P,0:05;** P,0:01;*** P,0:001

0–242 days 242–460 days 0–460 days

Cistus 0:32^0:02 0:30^0:03 0:33^0:02 Under Cistus r20:98*** r20:94** r20:99***

Cistus 0:29^0:03 0:57^0:15 0:46^0:04 Under Myrtus r20:97*** r20:88** r20:95***

Myrtus 0:33^0:03 0:48^0:16 0:45^0:04 Under Myrtus r20:98*** r20:82* r20:96***

Fig. 2. Respiration rates of decomposing litters at the field moisture and water content of litter at sampling. Values are means ^SD of three measurements with three replications. Cistus (B_—B_{) and Myrtus (}V_— V_{) litter incubated under respective shrubs; (}O_{– –-}O₎_{Cistus litter} incu-bated under Myrtus shrubs.

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xylanase activities showed seasonal variations with a maxi-mum in autumn and a minimaxi-mum in late spring and summer (Fig. 3). These patterns were similar to those of microbial respiration (Fig. 2) and a correlation between these activities and respiration was found (Table 4). The highest activities occurred in Myrtus litterP_,0:001:No significant differ-ences were found in the Cistus in relation to the two incuba-tion sites.

Thea-amylase activity in both litters was high at the start of incubation and declined rapidly as decomposition progressed (Fig. 3). In the Cistus litters the activity fell to

almost zero after two months. Consequently, no seasonal variation was observed. Between the litters, Myrtus had the highest average a-amylase activity although the initial value was the lowest one. However, there was no significant difference between them.

Theb-amylase activity was similar in both kinds of litter but showed an irregular pattern (Fig. 3). The highest activ-ities ofa- andb-amylase were less than 5–10% of those of cellulase and xylanase.

Laccase activity remained at low initial levels during the first 8 months (Fig. 4), subsequently increasing rapidly as Fig. 3. Cellulase, xylanase,a- andb-amylase activities of litters of Cistus incubated under Cistus (B_—B_{) and of Myrtus (}V_—V_{) and Cistus incubated under} Myrtus (O_{– –-}O_{) during their decomposition. Values are means}_^_{SD of three measurements with three replicates of each.}

Table 4

Coefficient of determination (r2) between properties examined during litter decomposition of Cistus and Myrtus. Significance for * P,0:05;, ** P,0:01; *** P,0:001)

Cistus under Cistus Cistus under Myrtus Myrtus under Myrtus

Water content — respiration r20:56* r20:36 r20:79** Water content — cellulase

activity

r20:80** r20:80** r20:50*

Water content — xylanase activity

r20:70** r20:67* r20:59*

Water content — peroxidase activity

ND ND r20:60*

Respiration — cellulase activity r20:50* r20:69* r20:51* Respiration — xylanase activity r20:60* r20:73** r20:74** Fungal biomass — laccase

activity

r20:88*** r20:85** r20:94*** Fungal biomass — mass loss r20:50* r20:94*** r20:77*

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decomposition progressed. The highest activities occurred in Myrtus litter while the lowest were found in Cistus litter under Cistus. Differences, although more evident in the last phase of incubation, were significantP_,0:001between

Myrtus and Cistus, apart from the incubation site.

Peroxidase activity was found only in Myrtus litter (Fig. 4). It showed a seasonal pattern, with a maximum in autumn and a minimum in late spring and summer. In Cistus, peroxidase was below the detection limit of the assay for almost the entire incubation period. However, a low activity was measured after a year of decay. Significant differences were found between Myrtus and Cistus littersP,0:05: 3.5. Fungal biomass

During the first eight months of incubation, the fungal biomass of decomposing litter under Myrtus slowly increased in the wet period and decreased in the dry summer, while under Cistus it also increased in summer. Subsequently, biomass rapidly increased in autumn (Fig. 5), mainly in the litters under Myrtus. At the end of the incubation period, the fungal biomass was 4–5 fold higher than the initial value for all the litters.

No correlation was found between the overall fungal biomass of Cistus and Myrtus litters and the xylanase and cellulase enzyme complexes. Positive and significant

corre-lation were found between fungal biomass and laccase activity (Table 4) and between fungal biomass and mass loss (Table 4).

3.6. Changes in pH during decomposition

Although litters of Myrtus and Cistus had similar pH values at the start (Fig. 6), they showed significantly differ-ent changes during decompositionP_,0:001:The pH in

Myrtus litter stayed at 5.0 for about a year and then

increased to 6.5, whilst Cistus increased to 6–6.5 after just two months of incubation and stayed at this value for the remaining incubation period. The Cistus litter incubated under Cistus and Myrtus shrubs showed a similar pattern.

4. Discussion

Litter decomposition under Myrtus and under Cistus Fig. 4. Laccase and peroxidase activities of litters of Cistus incubated under

Cistus (B_—B_{), and of Myrtus (}V_—V_{) and Cistus (}O_{– –-}O_{) incubated} under Myrtus during their decomposition. Values are means^SD of three measurements with three replicates of each.

Fig. 5. Fungal biomass of decomposing litters of Cistus incubated under

Cistus (B_—B_{), and of Myrtus (}V_—V_{) and Cistus (}O_{– –-}O_{) incubated} under Myrtus. Values are means^SD of three measurements with three replicates of each.

Fig. 6. pH pattern of decomposing litters of Cistus incubated under Cistus (B_—B_{), and of Myrtus (}V_—V_{) and Cistus (}O_{– –-}O_{) incubated under} Myrtus. Values are means^SD of three measurements with three repli-cates of each.

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shrubs showed two main differences (Fig. 1) that can be attributed to the abiotic and biotic characteristics of the plot. The litter under Myrtus (but not under Cistus) was not decomposed in summer, in contrast to what occurs in some ecosystems (Dilly and Munch, 1996) and in particular in the mediterranean area (Fioretto et al., 1998). Cistus (summer deciduous) and Myrtus(evergreen) create different microcli-matic conditions on the top soil. In summer, the soil temperature in the morning at 5 cm was .28C higher under Cistus than under Myrtus. In winter there was no temperature difference.

At the time of sampling (about 3 h after dawn) there was no significant change in water availability even in summer; the litter water content showed similar values (Fig. 2B). However, some dew, formed by the high nocturnal humid-ity, moistened the litter under the bare Cistus but not under

Myrtus. The experimental plot was near the sea and the

relative humidity was often 100% in summer, especially the evening and early morning. Therefore, under Cistus there were drying and rewetting cycles that can favour mechanical fragmentation and decomposition as reported in leaf litters of sweet chestnut and oak (Anderson, 1973; Witkamp and Olson, 1963). Secondly, the increased decay rate in litters under Myrtus, but not under Cistus, in autumn, could be related to the increase in the fungal biomass in that period (Figs. 1 and 5).

The patterns of cellulase and xylanase matched microbial respiration. The positive and significant correlation of respiration with these enzyme activities (Table 4) suggests that enzyme activities are closely linked to microbial activ-ity (Linkins et al., 1990). As expected a good correlation also occurred between these enzyme activities and litter moisture (i.e. litter water content affected microbial activ-ity) (Table 4). Similar results have been reported in the decomposing leaf litter of Alnus nepalensis and Pinus kesiya (Joshi et al., 1993).

The increase in enzyme activities during the early stages of incubation supports the view that cellulose and xylan are among the first decomposed compounds. The soluble substances present in the litters from the beginning of the incubation (Table 2), together with the cellulase activity, suggests that the opportunistic early colonisers (which have no need to produce extracellular enzymes) may func-tion at the same time as the cellulolytic microorganisms. After a year of decomposition, when the mass loss was about 30–40%, enzyme activities remained high, probably because cellulose and xylan were not exhausted even though lignin degradation had started (Fig. 4). High cellulase activ-ity in the first decomposition period has been reported on decomposing leaf litter of flowering dogwood (Cornus

flor-ida L.), red maple (Acer rubrum L.) and chestnut (Quercus prinus L.) in microcosms (Linkins et al., 1990).

Thea- andb-amylase activities, high at the start, fell as decomposition progressed (Fig. 3), suggesting that remain-ing starch in the litter was rapidly degraded in the early stages of incubation.

The sudden increase of laccase activity after eight months of litter decay (Fig. 4) suggests that it was at this stage that the decomposition of lignin began to accelerate. This is supported by the significant increase of fungal biomass (Fig. 5) after the same time period (lignolytic microorgan-isms are largely fungi-reference). Peroxidase showed a seasonal pattern similar to cellulase and xylanase, but only in the Myrtus litter. No peroxidase activity was detected in

Cistus under our assay conditions.

Both litter types incubated under Myrtus showed the same rate of decomposition even though the in vitro enzyme activities of Myrtus were higher than those of Cistus (Figs. 1, 3 and 4). Such differences could be due to inhibitor compounds occurring in Myrtus litter and/or to enzyme isoforms produced by the colonising microorganisms. This is suggested by the shift in pH patterns during decomposi-tion (Fig. 6). The differences in composidecomposi-tion can determine the preference of the heterotrophs thriving on the litters because of their nutritional requirements (Sinsabaugh and Linkins, 1987).

In conclusion, the study in this mediterranean site with wet and mild winters and hot and dry summers has shown that: (a) cellulase and xylanase activities, from the start of incubation, were high during the wet season and low during the dry season, and correlated with microbial respiration; (b) a similar pattern existed for peroxidase activity in Myrtus litter; (c) laccase, involved in lignin degradation, increased at a later stage in the decomposition and showed high activ-ity in the wet season when fungal biomass significantly increased; (d) no seasonal relationship was observed for

a-amylase and high activity occurred only at the start of incubation.

Myrtus and Cistus litters incubated under myrtle showed

the same overall decomposition pattern: the decay rates were low in the dry and high in wet (and warm) season. Nevertheless, the two litters differed in their enzyme activ-ities (always higher in Myrtus than in Cistus) as well as in pH changes during decomposition, probably because of differences in their chemical properties.

The respiration rate and decay rate of Cistus litter incu-bated under Cistus was different to that of Cistus under

Myrtus. This was probably related to the microenvironment:

temperature and water availability at the top soil differed in summer because Myrtus is an evergreen while Cistus is a summer deciduous shrub. The effect of different rhizosphere microbial populations and of run-off from leaves could not be excluded.

Acknowledgements

The research was supported by MURST (Ministero dell’Universita` e Ricerca Scientifica of Italy) and by ModMedIII project contract n. ENV4CT970680 (European Community). We thank Dr N. Costantino, who permitted us

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to work in the Natural Reserve of Castel Volturno, to Warrant Officer, N. Ricciardi, and the staff of the “Ispettor-ato della Forestale di Castel Volturno” for their help. Thanks also to Dr G. Bartoli for help in nutrient analysis.

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pulver-ulentum. Archives of Microbiology 109, 1–8.

Anderson, J.M., 1973. The breakdown and decomposition of sweet chestnut (Castanea sativa Mill.) and beech (Fagus sylvatica L.) leaf litter in two deciduous woodland soils. Oecologia 12, 251–274.

Anderson, J.M., Ineson, P., 1984. Interactions between microorganisms and soil invertebrates in nutrient flux pathways of forest ecosystems. In: Anderson, J.M., Rayner, A.D.M., Walton, D.W.H. (Eds.). Inverte-brate-Microbial Interactions, Cambridge University Press, Cambridge, pp. 59–88.

Archibald, F., Roy, B., 1992. Production of manganic chelates by laccase from the lignin-degrading fungus Trametes (Coriolus) versicolor. Applied and Environmental Microbiology 58, 1496–1499.

Arianoutsou, M., 1993. Leaf litter decomposition and nutrient release in a maquis (evergreen sclerophyllous) ecosystem of North Eastern Greece. Pedobiologia 37, 65–71.

Berg, B., Calvo de Anta, R., Escudero, A., Ga¨rdena¨s, A., Johansson, M.B., Laskowski, R., Madeira, M., Ma¨lka¨nen, E., McClaugherty, C., Meent-meyer, V., Virzo De Santo, A., 1995. The chemical composition of newly shed needle litter of Scots pine and some other pine species in a climatic transect. Long term decomposition in a Scots pine forest. X. Canadian Journal of Botany 73, 1423–1435.

Berg, B., So¨derstro¨m, B., 1979. Fungal biomass and nitrogen in decompos-ing Scots pine needle litter. Soil Biology & Biochemistry 11, 339–341. Berg, B., Wessen, B., 1984. Changes in organic-chemical components and ingrowth of fungal mycelium in decomposing birch leaf litter as compared to pine needles. Pedobiologia 26, 285–298.

Bernfeld, P., 1955. Amylases a and b. In: Colowick, S.P., Kaplan, N.O. (Eds.). Methods in Enzymology, vol. 1. Academic Press, New York, pp. 149–158.

Burns, R.G., 1982. Enzyme activity in soil: location and a possible role in microbial ecology. Soil Biology & Biochemistry 14, 423–427. Dilly, O., Irmler, U., 1998. Succession in the food web during the

decom-position of leaf litter in a black alder (Alnus glutinosa (Gaertn.) L.) forest. Pedobiologia 42, 109–123.

Dilly, O., Munch, J.C., 1996. Microbial biomass content, basal respiration and enzyme activities during the course of decomposition of leaf litter in a black alder (Alnus glutinosa (L.) Gaertn.) forest. Soil Biology & Biochemistry 28, 1073–1081.

Fioretto, A., Musacchio, A., Andolfi, A., Virzo De Santo, A., 1998. Decom-position dynamics of litters of various pine species in a Corsican pine forest. Soil Biology & Biochemistry 30, 721–727.

Froment, A., 1972. Soil respiration in a mixed oak forest. Oikos 23, 273– 277.

Joshi, S.R., Sharma, G.D., Mishra, R.R., 1993. Microbial enzyme activities related to litter decomposition near a highway in a sub-tropical forest of North East India. Soil Biology & Biochemistry 25, 1763–1770. Leatham, G.F., Stahmann, M.A., 1981. Studies on the laccase of Lentinus

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Linkins, A.E., Sinsabaugh, R.L., McClaugherty, C.A., Melillo, J.M., 1990. Cellulase activity on decomposing leaf litter in microcosms. Plant and Soil 123, 17–25.

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2.9. Statistics

The mass loss over time was fitted to a simple exponential curve (Olson, 1963)

lnxt=x0 2kt

where x0is the original mass of leaf litter, xtthe amount of litter remaining after time t, t is time (year) and k the decom-position rate (yr21). The half-life for decomposition (t1/2),

that is the time necessary to reach 50% mass loss, was calculatedt1=20:691=k:

3. Results

3.1. Initial chemical composition

Table

Initial

pH,

nutrient

and

water

solubl

substanc

contained

Cistu

yrtus

Litter

Water

sol

uble

substa

nces

(%)

C (mg

1dry

wt.)

N (mg

1dry

t.)

P (mg

1dry

wt.)

K (mg

1dry

wt.)

Mg (mg

1dry

wt.)

Ca (mg

1dr

Cistus

5.3

10.8

414

10.8

0.68

5.84

1.22

11.7

Myrtus

5.4

16.8

375

9.8

0.55

3.86

2.62

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Mn because it is essential for lignin-degrading enzymes (Perez and Jeffrey, 1992; Archibald and Roy, 1992). Its initial content was higher in the Cistus than in the Myrtus litter (Table 2).

The water soluble substances were about 35% more abun-dant in the undecomposed leaf litter of Myrtus than of the

Cistus. The pH values were similar (Table 2).

3.2. Litter decomposition

0:001were found between the mass loss patterns of Myrtus, as well as of the Cistus under Myrtus, and that of Cistus under Cistus.

The data conformed to a first order exponential decay

curve. The decay constants, determined until 240 d when decomposition was independent from incubation microsites, ranged from 0.30 to 0.33 yr21(Table 3). Subsequently, they increased for litters under Myrtus reaching the value k 0:48 and 0.57 yr21 for Myrtus and Cistus, respectively. Over the experimental period the half-lives of Cistus and

Myrtus litters incubated under Myrtus were similar t1=2

1:5 yrand lower in Cistus under Cistust1=22:1 yr:

Both litters incubated under Myrtus showed reduced decomposition rate during the dry summer. In the same period the decomposition rate of Cistus litter incubated under Cistus, did not change.

3.3. Litter respiration

3.4. Enzyme activities

During the decay process, extractable cellulase and

Fig. 1. Residual mass (% of the initial) of decomposing leaf litter of Cistus (B_—B_{) and Myrtus (}V_—V_{) incubated under the relative shrubs. The} residual mass of Cistus litter incubated under Myrtus (O_{– –-}O_{) is also} reported. The values are means^SE of 20 measurements.

Table 3

0–242 days 242–460 days 0–460 days

Cistus 0:32^0:02 0:30^0:03 0:33^0:02 Under Cistus r20:98*** r20:94** r20:99*** Cistus 0:29^0:03 0:57^0:15 0:46^0:04 Under Myrtus r20:97*** r20:88** r20:95*** Myrtus 0:33^0:03 0:48^0:16 0:45^0:04 Under Myrtus r20:98*** r20:82* r20:96***

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Thea-amylase activity in both litters was high at the start of incubation and declined rapidly as decomposition progressed (Fig. 3). In the Cistus litters the activity fell to

Laccase activity remained at low initial levels during the first 8 months (Fig. 4), subsequently increasing rapidly as

Fig. 3. Cellulase, xylanase,a- andb-amylase activities of litters of Cistus incubated under Cistus (B_—B_{) and of Myrtus (}V_—V_{) and Cistus incubated under} Myrtus (O_{– –-}O_{) during their decomposition. Values are means}_^_{SD of three measurements with three replicates of each.}

Table 4

Coefficient of determination (r2) between properties examined during litter decomposition of Cistus and Myrtus. Significance for * P,0:05;, ** P,0:01; *** P,0:001)

Cistus under Cistus Cistus under Myrtus Myrtus under Myrtus

Water content — respiration r20:56* r20:36 r20:79**

Water content — cellulase activity

r20:80** r20:80** r20:50* Water content — xylanase

activity

r20:70** r20:67* r20:59* Water content — peroxidase

activity

ND ND r20:60*

Respiration — cellulase activity r20:50* r20:69* r20:51*

Respiration — xylanase activity r20:60* r20:73** r20:74**

Fungal biomass — laccase activity

r20:88*** r20:85** r20:94***

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Myrtus and Cistus, apart from the incubation site.

3.5. Fungal biomass

No correlation was found between the overall fungal biomass of Cistus and Myrtus litters and the xylanase and cellulase enzyme complexes. Positive and significant

corre-lation were found between fungal biomass and laccase activity (Table 4) and between fungal biomass and mass loss (Table 4).

3.6. Changes in pH during decomposition

Although litters of Myrtus and Cistus had similar pH values at the start (Fig. 6), they showed significantly differ-ent changes during decompositionP_,0:001:The pH in

Myrtus litter stayed at 5.0 for about a year and then

4. Discussion

Litter decomposition under Myrtus and under Cistus

Fig. 4. Laccase and peroxidase activities of litters of Cistus incubated under Cistus (B_—B_{), and of Myrtus (}V_—V_{) and Cistus (}O_{– –-}O_{) incubated} under Myrtus during their decomposition. Values are means^SD of three measurements with three replicates of each.

Fig. 5. Fungal biomass of decomposing litters of Cistus incubated under Cistus (B_—B_{), and of Myrtus (}V_—V_{) and Cistus (}O_{– –-}O_{) incubated} under Myrtus. Values are means^SD of three measurements with three replicates of each.

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the litter water content showed similar values (Fig. 2B). However, some dew, formed by the high nocturnal humid-ity, moistened the litter under the bare Cistus but not under

Myrtus. The experimental plot was near the sea and the

flor-ida L.), red maple (Acer rubrum L.) and chestnut (Quercus prinus L.) in microcosms (Linkins et al., 1990).

Thea- andb-amylase activities, high at the start, fell as decomposition progressed (Fig. 3), suggesting that remain-ing starch in the litter was rapidly degraded in the early stages of incubation.

compounds occurring in Myrtus litter and/or to enzyme isoforms produced by the colonising microorganisms. This is suggested by the shift in pH patterns during decomposi-tion (Fig. 6). The differences in composidecomposi-tion can determine the preference of the heterotrophs thriving on the litters because of their nutritional requirements (Sinsabaugh and Linkins, 1987).

Myrtus and Cistus litters incubated under myrtle showed

The respiration rate and decay rate of Cistus litter incu-bated under Cistus was different to that of Cistus under

Myrtus. This was probably related to the microenvironment:

Acknowledgements

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References

Ander, P., Eriksson, K.E., 1976. The importance of phenol oxidase activity in lignin degradation by the white-rot fungus Sproventriculum pulver-ulentum. Archives of Microbiology 109, 1–8.