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 10.6
8C and hot, dry summers mean temperature of the hottest month: 28
8C. The annual rainfall is approximately 680 mm.
We selected an experimental plot 2500 m
2
, 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 20
8C until sample preparation. About 3 g of leaf material
were placed
in each
terylene net
bag 16 × 10 cm
2
length × height with a mesh size of 1 mm
2
. 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 75
8C 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 CO
2
evolution from litter at field moisture level. Approximately 1 g of leaf litter
was incubated in airtight jars for 2 d at 25 8C in total
A. Fioretto et al. Soil Biology Biochemistry 32 2000 1847–1855 1848
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 H
2
O g 100 g
21
d.wt 57
57 pH
a
8.5 8.2
Potential pH
b
7.9 7.4
C org 1.32
2.40 N
0.19 0.21
C to N ratio 6.9
11.43 CO
2
evolution rate mmol CO
2
g
21
dry wt. 24 h
21
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.
darkness. The CO
2
released 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 CO
2
output from leaf litter was expressed in mmol g
21
dry litter d
21
. 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 4
8C 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 wv. 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 50
8C. 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 100
8C for 15 min. After cooling in a water bath at 20
8C 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 50
8C were added, mixed pH , 2.0 and allowed to stand for 60 min at 20
8C 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– 28 mg ml
21
. Activities were expressed as mmol of glucose equivalents g
21
dry weight h
21
. The CM-cellulase activity was assayed in the same way using CM-cellulose 0.7
wv as substrate. The filtrates were diluted 1:30 with distilled water for the photometric measurement of
glucose.
The activities of a-amylase EC 3.2.1.1 and b-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 for a-amylase and 0.2 M acetate pH 4.8 for b- amylase and kept at 4
8C. The mixture was homogenised, centrifuged and filtered as described above. The enzyme
activities were determined by incubating at 37 8C 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 2.8–28 mg ml
21
. The enzyme activities are expressed as mmol of glucose
equivalents g
21
dry weight h
21
. 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 4
8C. 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 30
8C 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 3-3 dimethyl 4-4
diamino 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 H
2
O
2
was 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 min
21
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 g
21
dry litter. 2.7. Fungal biomass
Total hyphal length was determined by the Olson 1950
A. Fioretto et al. Soil Biology Biochemistry 32 2000 1847–1855 1849
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 g
21
of dry litter on the basis of the average 9.3 mm
2
cross section of the hyphae, a density of 1.1 g ml
21
and 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 20–22 8C 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 ln
x
t
= x
2kt
where x is the original mass of leaf litter, x
t
the amount of litter remaining after time t, t is time year and k the decom-
position rate yr
21
. The half-life for decomposition t
12
, that is the time necessary to reach 50 mass loss, was
calculated
t
1=2
0:691=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
