applied soil ecology 36 (2007) 32–40
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Decomposition dynamics of Myrtus communis and Quercus ilex leaf litter: Mass loss, microbial activity and quality change A. Fioretto *, S. Papa, A. Pellegrino, A. Fuggi Dipartimento di Scienze della Vita, Seconda Universita` di Napoli, Via Vivaldi no. 43, 81100 Caserta, Italy
article info
abstract
Article history:
The patterns of microbial respiration and enzyme activity (cellulase, xylanase, laccase and
Received 7 April 2006
peroxidase) in the leaf litter of two evergreen sclerophyll species, Myrtus communis and
Received in revised form
Quercus ilex, were compared during decay in a low shrubland and in a coppice wood in a
17 November 2006
Natural Reserve in the Mediterranean area.
Accepted 21 November 2006
The two litter types had similar initial amounts of lignin, cellulose and acid-detergentsoluble substances, but the litter from M. communis had a lower N content and higher C/N ratio. In spite of this, the decomposition rate of M. communis litter was higher than for Q. ilex.
Keywords:
Whilst no seasonal variation was observed in the rate of organic matter degradation,
Litter decomposition
respiration and extractable cellulase and xylanase activities showed seasonal variation in
Enzyme activity
both litter types with the highest values in winter–early spring and the lowest in summer,
Microbial respiration
and showed positive correlations with water content. Peroxidase activity exhibited a
Fungal biomass
seasonal pattern and was higher in Q. ilex than in M. communis, while laccase activity
Cellulose
was higher in M. communis and increased with fungal biomass as degradation progressed.
Lignin
Nevertheless, no relationship was found between laccase and peroxidase activity and lignin degradation. The results obtained indicate that the seasonal alternation of moist and dry periods is the major factor influencing soil biological activity in the Mediterranean area. The occurrence of pronounced seasonal variation in litter enzyme activity prevents the determination of possible correlations with litter mass loss. # 2006 Elsevier B.V. All rights reserved.
1.
Introduction
The decomposition of organic matter is mediated by extracellular enzymes released into the environment by microbial communities by secretion or cellular lysis. The decomposition process can be represented as a successional loop (Sinsabaugh et al., 2002) in which strong interactions are established among substrate, microdecomposers, and extracellular enzymes. Substrate quality affects the microbial community, inducing the release of specific extracellular enzymes that degrade and modify the substrate. In turn, substrate break down products drive microbial succession. In this view, three major types of relationships
can be recognized: enzyme activity and litter composition, enzyme activity and mass loss rate, and enzyme activity and microbial community composition. Differences in the initial litter composition, notably in the relative contents of lignin, cellulose, hemicellulose and pectin, lead to differences in enzyme activity through enzyme substrate-interactions (Sinsabaugh et al., 1991). Along with variations in enzyme production, differential enzyme activity leads to differences in the degradation rate. Climate and, in particular, microclimate also plays an important role in the decomposition process, generating patterns of temperature, moisture and nutrient availability that affect the decay of organic matter and in the end, through the release of
* Corresponding author. Tel.: +39 0823 274550; fax: +39 0823 274571. E-mail address:
[email protected] (A. Fioretto). 0929-1393/$ – see front matter # 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.apsoil.2006.11.006
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applied soil ecology 36 (2007) 32–40
nutrients, primary productivity. Productivity, in turn, affects litter quality and thus its decomposition rate. Litter quality changes during decomposition and as a consequence changes the degree of enzyme expression. Activities of enzymes involved in the soluble saccharide degradation peak early in the decomposition process, then decline markedly; the activities of the main cellulases generally peak about midway through decomposition. On the contrary, enzyme activity involved in the breakdown of more recalcitrant compounds tends to be higher in fast-decomposing litter (Linkins et al., 1990; Sinsabaugh et al., 1992, 2002). As concerns the link between community composition or biodiversity and extracellular enzyme activities, there are few, sometimes contradictory, reports (Raviraja et al., 1998; Zak et al., 1995; Maire et al., 1999; Maamri et al., 1999). Changes in enzyme activity as related to litter mass loss provide diagnostic information on the dynamics of decomposition. The activities of several enzymes, in particular those involved in the breakdown of major plant components (cellulose, hemicellulose and lignin) as well as in the cycling of nutrients such as nitrogen and phosphorus, have been studied and in some instances correlated with the rate of disappearance of specific litter constituents or with mass loss. In previous studies on the decomposition dynamics of leaf litter of Cistus incanus, a summer semi-deciduos species, and Myrtus communis, an evergreen, in a Mediterranean shrubland, it was shown that cellulase and xylanase activity patterns during early phases of decay were affected more by season and its influence on soil water availability, than by litter quality changes (Fioretto et al., 2000, 2001). Here, we compare the pattern of decomposition of leaf litter from Myrtus communis L. with that of leaf litter from Quercus ilex L. in the early and faster phases of decay. The decomposition dynamics of both evergreen sclerophyll species was followed through about 26 months in their respective stands, a shrubland for M. communis, and a pure wood for Q. ilex, placed within the same Natural Reserve but in different areas. Microbial enzyme activities mainly related to the degradation of cellulose and lignin (cellulase, xylanase, laccase and peroxidase) were measured at regular intervals and related to the rate of substrate disappearance and fungal biomass, as these enzyme are mainly of fungal origin. The aim of the present study was to establish if differences in microclimatic conditions and substrate characteristics of the two incubation sites, plus differences in composition of the two litter types, affect microbial succession and, therefore, the pattern of enzyme activity.
2.
Material and methods
2.1.
Site description
The study was carried out in the Natural Reserve of Castel Volturno (Campania, Southern Italy). The Reserve is a flat coastal area of about 268 ha, south to the Volturno estuary (408570 N; 138550 E), and has a typical Mediterranean vegetation that presents itself as a mosaic of different plant patches. The local climate is typically Mediterranean with mild and rainy winters and hot and dry summers.
Table 1 – Soil characteristics in low maquis under Myrtus communis shrubs, and in oak wood within the Natural Reserve of Castel Volturno Low maquis Sand (coarse + fine) Water holding capacity (H2O g/100 g d wt) pH Potential pH C org (%) N (%) C/N
Oak wood
99 57
99 62
8.2 7.4 2.4 0.21 11
8.0 6.8 13.8 0.51 27
Gravel was absent.
We selected two experimental plots of about 3000 m2. The first one was within a coppice wood of Quercus ilex. Despite the sporadic occurrence of Pinus pinea trees, the plot was chosen in an area covered only with holm-oak. The second plot was within a stand of low Mediterranean maquis that was burned in 1976. The shrub cover included Cistus salvifolius L., Cistus incanus L., Myrtus communis L., Rhamnus alaternus L., Asparagus acutifolius L., Phillyrea angustifolia L. and Pistacia lentiscus L. The local soil originated from loose siliceous–calcareous marine sand and pyroclastic products. The main physical characteristics of the soil in the two stands are reported in Table 1. Both soils were sandy, with a high percentage of coarse sand (about 55% in the maquis and 75% in the oak wood). The pH was sub-alkaline or alkaline in both soils although the potential pH was considerablyly lower in the oak wood. In contrast, the two soils showed very different organic carbon and nitrogen contents. Senescent leaves of myrtle had less nitrogen and a higher C/N ratio than those of holm-oak (Table 2). The amounts of lignin and cellulose as well as acid-detergent-soluble substances were similar in the 2 litters. In addition, the amount of nitrogen in the acid-detergent-soluble substances and bound to lignin was smaller in myrtle than in holm-oak litter
Table 2 – Qualitative characteristics of undecomposed leaf litter of M. communis and Q. ilex M. communis 1
N content (mg g d wt) C/N pH Acid-detergent-soluble substance (ADSS) (% IOM) Cellulose (% IOM) Lignin (% IOM) N of ADSS (% ILN) N of cellulose (%ILN) N of lignin (% ILN) Tannins (% d wt)
Q. ilex
7.8 62 5.4 45
11.1 46 5.8 47
37 18 22 22 54 5.5
39 15 35 23 37 1.9
Acid-detergent-soluble substances (ADSS), lignin and cellulose content are expressed as percentages of the leaf litter organic matter (IOM) before exposure. Similarly, nitrogen linked with cellulose, lignin and of ADSS is showed as percentage of the total nitrogen of undecomposed litter (ILN).
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applied soil ecology 36 (2007) 32–40
(Table 2). The myrtle litter had highest soluble polyphenol content.
2.2.
Sample preparation and processing
Freshly abscissed leaves of myrtle and holm-oak were collected when most of the litter fall occurs. After the removal of contaminating debris, each litter was mixed to provide an homogeneous sample, air dried and stored in polyethylene bags at room temperature (about 20 8C). Aliquots of 3–3.5 g of leaf material were enclosed in terylene net bags with a mesh of 1 mm2. Because of morphological differences in the two leaf types, the bags were of two different sizes (16 cm 10 cm for myrtle leaves; 26 cm 22 cm for holm-oak leaves). The litter bags containing the leaves of myrtle and holm-oak (240 for each litter type) were set out in January and December 1998, respectively. The myrtle litter bags were incubated under 10 shrubs of the same species chosen randomly in an area where the canopy cover by M. communis was about 40%. Similarly, the holm-oak litter bags were placed in 10 microsites randomly selected in the Q. ilex wood. The bags were fixed on the top of the standing litter layer by metal pegs. The bags were sampled in the first year every 2 months for myrtle litter and every 3 months for holm-oak. In the second and third years, the samplings of both litter types were carried out about every 3 months. At each sampling, two bags were collected from each of the nine microsites. Bags from the 10th microsite were sampled and processed only when the bags were destroyed by rodents. The litter bags were placed in individual plastic bags to minimise litter and water loss and taken to the laboratory. Here the litter samples from each bag were cleaned 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 determination. In order to provide enough material for all the analyses planned (enzyme activities, microbial respiration, fungal biomass, lignin and cellulose content, N content, ash content) and to permit statistical treatment of the data, the remainder of the litter in the bags from groups of three sites were pooled. The three composite samples were obtained from bags of the same group of microsites at each sampling. Each of these samples was divided into subsamples for the various analyses.
2.3.
Mass loss
Litter mass loss was measured for each litter bag as the difference in weight before (time 0) and after (time X) in situ incubation at each sampling time. Data were expressed as percentage of residual organic matter (% IOM) relative to the initial mass. Litter organic matter was calculated by subtracting its ash content from the dry mass of the litter.
2.4.
Enzyme extraction and assays
Litter samples were ground in the appropriate cold buffer (anhydrous sodium acetate M 0.05, pH 5.5) 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 for enzyme
activity assays. 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 (Fioretto et al., 2000). In particular, 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 solution 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, l ml of the diluted mixture, l ml reagent A (16.0 g anhydrous sodium carbonate and 0.9 g potassium cyanide dissolved in 1 l distilled water), l ml reagent B (0.5 g potassium ferric hexacyanide 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 far 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 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 extinction was measured within 30 min at 690 nm against the reagent blank. The extinction, after subtracting the control values, was used to determine the glucose equivalents on a calibration curve (conc. range 2.8–28 mg mll). Activities were expressed as mmol of glucose equivalents gl dry weight hl. 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 activity of laccase (EC 1.10.3.2) was estimated according to Leatham and Stahamann (1981), with minor modifications (Fioretto et al., 2000). 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 (3-30 dimethyl 4-40 diamino biphenyl). Peroxidase (EC 1.11.1.7) activity, determined in the same enzyme extract as for the assay of laccase, was measured in the same conditions and in the same reaction mixture with the addition of 0.1 ml of 4 mM H2O2 (Ander and Eriksson, 1976). Peroxidase activity was evaluated by subtracting the laccase activity from the overall assay activity. The activities were calculated as mmol of tolidine oxidised per minute using a molar extinction coefficient of 6340 (McClaugherty and Linkins, 1990). AlI enzyme assays were performed in triplicate for each litter sample.
2.5.
Respiration from litter
Respiration from litter was measured as CO2 evolution for samples kept at field moisture. About 1 g of leaf litter was incubated in airtight jars (500 ml) for 2 days at 25 8C in total darkness. The CO2 released was adsorbed in a 0.5 M NaOH solution and determined by two-phase titration with HCl (0.1 M) (Froment, 1972). The CO2 output from leaf litter was expressed in mmol g1 dry l day1. The measurement was performed in triplicate on each litter combined sample.
applied soil ecology 36 (2007) 32–40
2.6.
Fungal biomass
Total hyphal length was determined by the intersection method of Olson (1950). The dry litter (1 g) was milled and homogenized in water by a laboratory mixer for 5 min. Subsequently, an aliquot of the homogenate was diluted to obtain four samples with a final dilution of 1 mg litter ml1 of water. A membrane filter from each diluted sample was prepared and stained as described by Sundman and Sivela¨ (1978). The values are reported as mg dry fungal biomass per gram dry litter as described in Berg and So¨derstro¨m (1979), on the basis of an average 9.3 mm2 cross section of the hyphae, a density of 1.1 g ml1 and a dry mass of 15%.
2.7.
Chemical analyses
The chemical composition of the two kinds of leaf litter was determined on oven-dried subsamples powdered by a Fritsch Pulverisette (type 00.502, Oberstein, Germany) equipped with an agate pocket and ball mill. Carbon and nitrogen contents were determined by combustion in an Elemental Analyzer NA 1500 (Carlo Erba Strumentazione, Milan, Italy). Lignin and cellulose contents were determined according to Van Soest and Wine (1968) with modifications (Fioretto et al., 2005). Nitrogen aliquots linked to cellulose and lignin were also evaluated by combustion in an Elemental Analyzer of the acid-detergent-fibre (ADF), obtained by treatment of finely powered litter samples with sulphuric acid and cetyltrimethylammonium bromide and containing lignin, cellulose and ashes, and of the acid-detergent-fibre after oxidation (ADFAO), obtained by treatment of ADF with buffered permanganate solution at 20–25 8C for 90 min and containing cellulose and ash. The aliquot of nitrogen linked to lignin was calculated by subtracting the aliquot linked to cellulose (nitrogen content of ADFAO) from total (nitrogen content of ADF) and correcting for the ash content. Further details are given in Fioretto et al. (2005). The tannin content of leaves was measured according to Hagerman and Butler (1972). This method measures the amount of condensed or hydrolysable tannin precipitaded by a standard protein, bovine serum albumin (BSA). The precipitate was dissolved at high pH in the presence of a detergent, and the coloured iron-phenolate complex was determined spectrophotometrically. In particular, 100 mg of oven-dried litter was extracted three times by ultrasonication in 20 ml water/methanol solution (50%, vv) at 4 8C. The extract, after centrifugation (14,000 rpm for 5 min) was dried, liophylized and redissolved in a water/methanol solution (80/20%, v/ v). An aliquot corresponding to 3.5 mg of liophylized extract was added to 1 ml BSA and allowed to sit for 24 h at 4 8C. After centrifugation (15 min at 3000 g), the pellet was redissolved in 2 ml SDS/TEA (5%, v/v triethanolamine, 1%, v/v SDS–50 ml triethanolamine, 10 g sodium dodecyl sulphate up to 1 l of water) and 1 ml FeCl3. After 15 min the absorbance was read at 510 nm. A calibration straight line was built using different aliquots of tannic acid. All chemical analyses were performed in triplicate for each litter sample.
2.8.
35
Statistical analyses
In the figures, mass loss data are reported as means S.E. of 18 field measurements, while other data are means S.D. of three field replicates, each with three laboratory determinations. Correlations were determined using the simple Pearson correlation coefficient.
3.
Results
3.1.
Mass loss
The decomposition rates of the two litters (Fig. 1A) were similar in the first phase of degradation (0–250 days). Subsequently, decomposition of holm-oak litter slowed down. After 26 months, the remaining organic matter was about 40% and 10% of the initial mass for holm-oak and myrtle, respectively. The best fit for residual mass was a first-order exponential decay curve (Fig. 1B).
3.2.
Biological activity
The respiration rate and the moisture content of the litter (Fig. 2A and B) at each sampling time showed seasonal variations, with the highest values in winter–early spring and the lowest in summer. As the litter bags were set out in the experimental plots in December and January, the winter period includes the first 100 days of decomposition and from days 350 to 450 whereas summer included days 200 to 300. A
Fig. 1 – Organic matter decay of leaf litter of M. communis (- - -) and Q. ilex (—) during about 18 months of decomposition. The remaining organic matter (ROM) is expressed as percentage of initial organic matter (IOM) before litter exposure (A), and in semilogarithmic plot (B). Bar indicates standard error.
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applied soil ecology 36 (2007) 32–40
Fig. 2 – Respiration rate (A) and moisture content (B) of decomposing litters of M. communis (- - -) and Q. ilex (—) at sampling. Values are means W S.D. of three measurements with three replicates of each.
significant positive correlation was found between respiration and litter moisture content (Table 3). When comparing the respiration rates of the litter from the two species, the seasonal variations appeared less pronounced in oak litter than in myrtle.
Table 3 – Pearson’s correlation coefficient (r2) between some properties (M: moisture content of the litter; R: microbial respiration; F: fungal biomass; 1: cellulase activity; 2: xylanase activity; 3: peroxidase activity; 4: laccase activity) during the decomposition of M. communis (up) and Q. ilex (below) litters M
R
F
M. communis R F 1 2 3 4
0.78 ** 0.20 NS 0.77 ** 0.76 ** 0.83 *** 0.18 NS
0.014 NS 0.89 *** 0.78 ** 0.66 * 0.14 NS
0.18 NS 0.23 NS 0.12 NS 0.96 ***
Q. ilex R F 1 2 3 4
0.62 * 0.16 NS 0.70 * 0.26 NS 0.25 NS 0.14 NS
0.014 0.71 * 0.76 * 0.87 ** 0.03 NS
0.56 NS 0.27 NS 0.01 NS 0.86 **
Significance for *P < 0.05, **P < 0.01, ***P < 0.001. NS: not significant. N = 9 for M. communis and n = 6 for Q. ilex.
Fig. 3 – Cellulase (A) and xylanase (B) activities in litters of M. communis (- - -) and Q. ilex (—) during their decomposition. Values are means W S.D. of three measurements with three replicates of each.
Extractable cellulase activity showed a similar seasonal pattern and was often closely similar in the two litters (Fig. 3A). As observed for respiration, this enzyme activity had a maximum in winter–early spring (although with higher values in the late phase of decomposition) in both litters (Fig. 3A), and a minimum in summer. Similar patterns were detected for extractable xylanase activity (Fig. 3B), although the measured values were higher in the holm-oak litter. After 400 days of decomposition, cellulase and xylanase activities decreased. The decrease was quite sudden in holmoak and more gradual in myrtle. Both cellulase and xylanase activity patterns matched those of litter microbial respiration and a positive correlation between these activities and respiration was found (Table 3). In addition, these activities, as well as respiration, were correlated with litter water content in both litters (Table 3). The enzymes involved in lignin degradation, laccase and peroxidase, showed different behaviours. Laccase activity (Fig. 4A), was low in the first and late phase of decomposition, but high in the intermediate phase. Peroxidase activity (Fig. 4B), on the contrary, showed a seasonal pattern similar to that observed for cellulase and xylanase activity. In particular, laccase activity in the myrtle litter was already detectable at a low level in the undecomposed litter and remained low during the first 8 months. Subsequently, laccase activity increased rapidly as degradation progressed (Fig. 4A). Laccase activity was below the assay detection limit, in the undecomposed holm-oak litter, and rapidly increased as decomposition progressed, but remained always below the
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applied soil ecology 36 (2007) 32–40
Fig. 4 – Laccase (A) and peroxidase (B) activities in litters of M. communis (- - -) and Q. ilex (—) during their decomposition. Values are means W S.D. of three measurements with three replicates of each.
maximum values measured in myrtle litter (Fig. 4A). Peroxidase activity (Fig. 4B) was already present in the undecomposed myrtle litter; however, it remained low until 300 days. In the holm-oak litter peroxidase activity was non detectable in the unexposed material. After exposure it rapidly increased and was higher than in the mirtle litter throughout the study period (Fig. 4B). The maximum level of activity was detected after about 90 days (March) for the holm-oak litter and 1 year for the myrtle litter.
3.3.
Fungal biomass
The two undecomposed litters showed similar values for fungal biomass (Fig. 5). Fungal biomass showed little changes
Fig. 6 – Cellulose and lignin dynamics during decomposition of M. communis (- - -) and Q. ilex (—) litter. Values are means W S.D. of three measurements with three replicates of each.
during the early phase of decomposition but exhibited a 4–5fold increase after 250–300 days of decomposition, with values which were considerably higher in myrtle than in holm-oak litter.
3.4.
Quality changes
Cellulose and lignin did not change substantially in the first 8 months of exposure in myrtle litter (Fig. 6 A and B). During the following 2 months, lignin was reduced to half the initial content and afterwards showed no further decrease. Cellulose, in contrast, was degraded at a constant rate after the initial lag-phase. In holm-oak litter, cellulose degradation started 2 months after exposure and then continued at a constant rate, whereas there was no lignin degradation during the entire study period (Fig. 6A and B).
Table 4 – Residual nitrogen in the whole remaining litter (RL), cellulose (C), lignin (L) and acid detergent soluble substances (ADSS) of M. communis and Q. ilex after about 1 and 2 years of decomposition, calculated as percentage of initial content M. communis
Fig. 5 – Fungal biomass in litter of M. communis (- - -) and Q. ilex (—) during decomposition. Values are means W S.D. of three measurements with four replicates of each.
RL C L ADSS
Q. ilex
1 Year
2 Years
1 Year
92 98 25 257
58 53 3 212
100 70 35 170
2 Years 110 70 26 174
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Nitrogen was immobilized in the holm-oak litter during the entire study period, whereas in the myrtle litter a considerable loss of N was recorded, particularly during the second year of decomposition (Table 4). Nitrogen linked to cellulose in holmoak was lost during the first year and in myrtle only during the second year (Table 4). By contrast, nitrogen linked to lignin was lost from the start of decomposition in both litters but more quickly in myrtle. The amount of nitrogen in ADSS with time increased substantially.
4.
Discussion
The rate of litter decomposition is related to the initial concentration of N (Witkamp, 1966), which is recognized as the main factor limiting the growth of decomposer populations (Berg and Staaf, 1987). A higher concentration of lignin implies the presence of relatively more abundant recalcitrant material, but also determines a greater proportion of N in recalcitrant forms (Berg, 1986). In this connection, interest has focused on the lignin-to-N ratio (Harmon et al., 1990) and C-toN ratio (Bosatta and Staaf, 1982) that are assumed to control the rate of decomposition and to determine whether N will be immobilized or released during decomposition (Berg and Staaf, 1981; Enrı´quez et al., 1993). The critical C/N threshold may vary with climate and organic matter composition, but nitrogen immobilization has been reported in litters where the C/N ratio was above 25–34 (Blair, 1988). In the present study the myrtle litter decomposed faster than the holm-oak, despite a lower N content and higher C/N ratio (Table 2). As expected, N was released from myrtle litter during decay (Table 4). By contrast, in holm-oak litter, N was immobilized even though the C/N ratio exceeded the abovementioned threshold (Table 4). As suggested for other ecosystems (Melillo et al., 1982; Taylor et al., 1989; Berg et al., 1996), the lower decomposition rate of holm-oak litter could depend on the low initial lignin-to-N ratio in this species, and its further decrease during decomposition because of nitrogen immobilization. Tannins are defined as naturally occurring water-soluble polyphenols of varying molecular weight and inhibit the growth of a number of microorganisms. For this reason, tannins generally retard the rate of organic matter decomposition (Bhat et al., 1998). Surprisingly, the higher content of tannins in myrtle litter seems to have no effect on its decomposition rate. Respiration in both litter types was mainly influenced by moisture content. Holm-oak litter generally showed lower respiration rates than myrtle litter, particularly during the wet periods (Fig. 2). This is consistent with the presence of lower fungal biomass in the former (Fig. 5). The moisture contents of both myrtle and holm-oak litters were highest during the winter of the second year of decomposition, due to the increased water-holding capacity of the residual litter as mass loss progressed (Virzo De Santo et al., 1993). Cellulase, xylanase and peroxidase had seasonal patterns of activity. Similar findings have been reported for cellulase and chitinase activities in beech leaf litter of European forest (Andersson et al., 2004). The alternation of maximum and
minimum activity was strongly related to succession of cold and warm or moist and dry periods. The last condition is a characteristic of the Mediterranean climate that strongly affects soil biological activity. Rainfall affects litter moisture which in turn affects microbial biomass and activity. Donnely et al. (1990) and Berg et al. (1998) showed that moisture was more important than temperature as a factor influencing the growth of microbial biomass in litter and soil. In addition, Criquet et al. (2002) detected a direct correlation between the moisture of decomposing litter and the number of heterotrophic bacteria and the expression of extracellular enzymes such as cellulases and hemicellulases. The good correlations found in the present study between moisture and litter respiration, moisture and enzyme activity and litter respiration and enzyme activity as well as in other studies (Joshi et al., 1993; Fioretto et al., 2000; Papa et al., 2000; Criquet et al., 2002; Andersson et al., 2004) confirmed these hypotheses and showed that in the Mediterranean area environmental fluctuations that alter mainly moisture may obscure or overwhelm patterns linked with litter quality. The occurrence of detectable cellulase and xylanase activities at the start of decomposition suggests that xylan and cellulose are among the first compounds decomposed. In fact, cellulose degradation dynamics showed that the decay of this compound started immediately in holm-oak but after 8 months in myrtle (Fig. 6). Cellulase activity generally increases more slowly throughout decomposition and early peaks are typically found in litter types particularly rich in lignin (Linkins et al., 1990; Sinsabaugh et al., 1992). Nevertheless, high activity in the first stage of decomposition has been reported for decomposing leaf litter of the evergreen oak in forests of the French Mediterranean area (Criquet et al., 2002). The persistence of high cellulase and xylanase activities in myrtle and holm-oak litters after a year of exposure, when the residual contents of organic matter were about 60% and 75% of the initial value, respectively, is indicative of the persistence of substantially amounts of xylan and cellulose. Consistent with this, the measured amount of cellulose after 18 months of decomposition was about 30% and 25% of the initial organic matter in myrtle and holm-oak, respectively, that is 53% and 70% of the initial amount of cellulose (Table 4). The highest cellulase activities observed in the second year for both litters correspond with a mass loss of about 30% and 40% for holm-oak and myrtle, respectively. In this connection, it is reported that the maximum activity is generally reached when the litter mass loss is around 40–80% (Sinsabaugh et al., 2002). Peroxidase and laccase are involved in lignin degradation. The detection of peroxidase activity since the onset of decomposition, although with higher values in holm-oak than myrtle litter, suggests that the microorganisms responsible for this activity, were among the early colonisers and lignin degradation started immediately. The differences in the degree of expression of peroxidase activity between the two litter types could be explained assuming that a different number of microrganisms are involved in enzyme production. On the other hand, laccase activity, with no seasonal pattern, increased as decomposition progressed. The substantial increase in laccase activity after 3 months for holm-oak and
applied soil ecology 36 (2007) 32–40
8 months for myrtle coincided with an increase in fungal biomass, suggesting that this enzymatic activity was mainly associated with the fungal population. In myrtle, lignin degradation started, as for cellulose, after about 8 months of exposure, whereas in holm-oak litter lignin did not degrade at all during the entire decay period (Fig. 6). After about 3 years of decomposition lignin and cellulose were 3% and 8% of IOM in myrtle, while in holm-oak these components were around 30% IOM (Fioretto et al., 2005). No relationship was therefore found between laccase and peroxidase activity and lignin degradation. In fact, although from 8 to 10 months the lignin in decomposing myrtle litter was reduced to half the initial content, afterwards only a small further decrease was measured, but still laccase and peroxidase activities continued to rise. In holm-oak no increase was recorded in lignin degradation even after 3 months of decay, when laccase activity had increased considerablyly. Since the analytical methods employed to determine lignin content do not distinguish between true lignin and partially humified products, this discrepancy could be explained assuming that part of the lignin in the holm-oak litter was converted into humified products that underwent further degradation. Although most enzymes showed a clear seasonal pattern, no seasonal variation was observed in organic matter degradation. This discrepancy could be at least in part explained considering that enzyme activities were measured in vitro as potential activities in an adequate extraction medium and under standard conditions. In the field, the presence of inhibitors and/or activators as well as the presence of other enzymes operating simultaneously could attenuate the sharp variations measured under laboratory conditions. The turnover activities were calculated from models of mass loss as a function of cumulative enzyme activities (Sinsabaugh et al., 2002). Cumulative enzyme activity is expressed in units of activity-days, and calculated by integrating the area under a curve of enzyme activity versus time. A linear regression generates a first-order rate constant called apparent enzymatic efficiency. The seasonal variation in cellulase, xylanase and peroxidase enzyme activities as well as the drastic decrease in laccase activity after about 1.5 years in litter of myrtle and holm-oak in the Mediterranean area do not correlate with litter mass loss, and therefore do not permit calculation of the turnover activities. In conclusion, the two litter types, in spite of differences in the soil characteristics of the two incubation sites and especially differences in litter quality which led to different rate of decay, showed generally a similar pattern of biological activity suggesting a succession of functionally similar microbial communities,with changes more linked with climatic conditions than litter quality and decomposition rate.
Acknowledgements The research was supported by MURST (Ministero dell’Universita` e Ricerca Scientifica e Tecnologica, Italy) and ModMedIII project contract n. ENV4CT970680 of the European Community). We thank Dr. N. Costantino, for granting perission to work in the Natural Reserve of Castel Volturno, Warrant Officer, N. Ricciardi and the staff of the ‘‘Ispettorato
39
della Forestale di Castel Volturno’’ for their help. We also thank Dr. A. Izzo for providing the data on polyphenol contents in the litters.
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