European Journal of Soil Biology 44

This article appeared in a journal published by Elsevier. The attachedcopy is furnished to the author for internal non-commercial researchand education use, including for instruction at the authors institution

and sharing with colleagues.

Other uses, including reproduction and distribution, or selling orlicensing copies, or posting to personal, institutional or third party

websites are prohibited.

In most cases authors are permitted to post their version of thearticle (e.g. in Word or Tex form) to their personal website orinstitutional repository. Authors requiring further information

regarding Elsevier’s archiving and manuscript policies areencouraged to visit:

http://www.elsevier.com/copyright

http://www.elsevier.com/copyright

Author's personal copy

Original article

Experimental drought reduced acid and alkaline phosphataseactivity and increased organic extractable P in soil in a Quercusilex Mediterranean forest

J. Sardans*, J. Penuelas, R. Ogaya

Unitat d’Ecofisiologia i Canvi Global CSIC-CEAB-CREAF, CREAF (Centre de Recerca Ecologica i d’Aplicacions Forestals),

Universitat Autonoma de Barcelona, E-08193 Bellaterra (Espanya), Spain

a r t i c l e i n f o

Article history:

Received 24 December 2007

Accepted 14 September 2008

Published online 12 October 2008

Keywords:

Acid phosphatase activity

Alkaline phosphatase activity

Drought

Leaf P concentration

Litter

Quercus ilex

Soil

Short-term available-P

Soil moisture

Soil organic matter

Total soil-P

a b s t r a c t

A six-year (1999–2005) experiment of drought manipulation was conducted in a Quercus ilex

Mediterranean forest (Southern Catalonia) to simulate predicted climatic conditions pro-

jected for the decades to come. The aim was to investigate the direct and indirect effects of

drought conditions on acid and alkaline phosphatase activity in soil and on P concentra-

tions in soil, leaves and litter throughout the year. Soil acid phosphatase activity was

higher than soil alkaline phosphatase activity. Drought reduced acid phosphatase activity

in soil in all seasons, including summer and winter, the seasons with less biological activity

due to water and cold stress. Reductions of soil water content between 13 and 29% reduced

soil acid phosphatase activity between 22 and 27% depending on the season. Drought

reduced alkaline phosphatase activity (by 28%) only in winter. Soil acid and alkaline

phosphatase activities were positively correlated with soil water content in all seasons. In

contrast short-term available-P which increased under drought in several seasons was

weakly correlated with soil phosphatase activities. As a result, immediately/short-term

available-P concentration ratios decreased in all the seasons (between 10 and 71%).

Drought increased foliar P concentration and reduced the C/P concentration ratio in litter

fall of the dominant tree Q. ilex. Drought also decreased the ratio between organic C and

short-term available-P in soil. The results show that soil phosphatase activity is more

directly dependent on changes in water availability than on changes in its substrate, short-

term available-P. These effects of drought have several implications: the accumulation in

the soil of labile P not directly available to plants, the increase in potential P losses from

leaching and erosion during the torrential rainfalls typical of the Mediterranean climate,

and changes in plant, litter and soil C:P stoichiometry that may lead to changes in soil

trophic chains.

ª 2008 Elsevier Masson SAS. All rights reserved.

* Corresponding author. Tel.: þ34 93 581 29 34.E-mail address: [email protected] (J. Sardans).

ava i lab le at www.sc ienced i rec t . com

journa l homepage : h t tp : / /www.e lsev ie r . com/ loca te /e jsob i

1164-5563/$ – see front matter ª 2008 Elsevier Masson SAS. All rights reserved.doi:10.1016/j.ejsobi.2008.09.011

e u r o p e a n j o u r n a l o f s o i l b i o l o g y 4 4 ( 2 0 0 8 ) 5 0 9 – 5 2 0


1. Introduction

Mediterranean ecosystems are frequently water and nutrient

limited [1,2]. The latter is an important factor in the growth,

structure and distribution of Mediterranean communities

[3–6]. Mediterranean ecosystems often suffer from nutrient

deficiencies [3,7] that may be accentuated by climate change.

Along with nitrogen [8], phosphorus is a frequent limiting

factor in Mediterranean ecosystems [2,5,9]. A reduction in P

availability can aggravate the direct effects of drought because

a high P availability has a positive effect on water use effi-

ciency (WUE) [6,10,11]. In Mediterranean ecosystems, the

effects of climate change on plant growth and nutrient cycles

could be linked to the availability of P [12,13]. Observational

and experimental evidence supports the assumption that

water stress affects the levels of nutrients in trees [14,15], and

P tissue concentrations play a very important role in plant

biology [16,17]. Soil enzymes are critical in maintaining

nutrient availability. Soil enzymes depend on soil microbe

activity which can be affected by drought. Because of this,

drought can affect nutrient availability and soil nutrient

contents.

Soil enzymes are frequently used as indicators in studies

on the influences of soil treatments on soil fertility [18]. They

integrate information about soil microbial status and soil

physio-chemical traits such as nutrient contents, litter

composition and soil pH. These enzymes may therefore

provide useful information on the effects of environmental

changes on soil fertility [19]. Nutrient availability may corre-

late well with their activity [20]. Bacteria and fungi synthesize

and secrete enzymes such as phosphatases extracellularly.

These enzymes are biologically synthesized but are separated

from the cells producing them and can be stabilized through

association with humic substances [21,22]. Therefore, factors

influencing soil microbial activity such as drought exert

control over soil enzyme production and consequently affect

nutrient availability and soil fertility [23].

The re-wetting effect [19] can also reduce the bacterial

population, which decreases soil enzyme activity and even-

tually leads to increasing nutrient leaching. Furthermore,

when the environment evolves towards drier conditions,

sclerophylly, a typical Mediterranean trait, can increase

[24–26], raising the proportion of structural compounds in

litter, and consequently decreasing decomposition rates,

slowing P release and increasing P in non-available forms.

In Mediterranean ecosystems, over the last century,

precipitation has either exhibited a long-term downward

trend, principally in the dry season [27,28] or no significant

change at all [29–31] although in all cases a rise in the

evapotranspiration potential has led to increased aridity

[29,30,32]. Global circulation models predict drier conditions

in these ecosystems in the next decades [33].

In previous studies in an evergreen holm-oak Mediterra-

nean forest located in South Catalonia at 80 km south-west of

Barcelona, we observed that drought decreased the activity of

some soil enzymes, including acid and alkaline phosphatases,

in some seasons and at particular soil depths, whereas there

were no effects in other seasons and soil depths [34]. We also

found that drought affected P in some soil fractions but not

the overall P soil stocks [35]. It is important now to gain

knowledge on the relation between the changes in the activity

of phosphatases and the changes in soil P stocks and avail-

ability, soil organic matter and soil moisture, since drought

can have a direct effect on soil phosphatase activity by

changing soil water availability or an indirect effect by

changing soil organic matter, short-term available-P, soil C/P

ratio or other soil traits. Our current aim is to investigate

whether the decreased phosphatase activity was a direct

effect of reduced soil water content or an indirect effect

through changes in soil organic matter content or in C/P ratios

in litter and soil or in soil pH. For example, under drought an

increase in litter production and/or soil organic matter can

stimulate soil enzyme activity [36,37], however, a decrease in

soil moisture can decrease soil enzyme activity. These

changes in soil phosphatase activity can affect the soil organic

matter and the stoichiometry between C and P in soil, which

in turn can have several other effects at the ecosystem level.

We also aimed to further investigate whether the effects of the

decrease in soil water content on soil phosphatase activity can

change throughout the year, in the different seasons, since

the effects of changes in water availability are likely to be

sensitive to changes in temperature and phenology

throughout the year.

We thus posed the following questions: (i) what are the

effects of the drought predicted for the next decades by

climatic and ecophysiological models [31,33,38] on the activity

of soil phosphatases and on soil P content? (ii) How do the

possible changes in soil phosphatase relate to the changes in

litter, soil organic matter and total soil-P and extractable P

fractions (immediately available-P and short-term available-P)?

and (iii) What is the seasonality of those effects and those

relationships? Hence, we conducted a field experiment of

drought manipulation in a Mediterranean holm oak forest

throughout the four seasons between July 15, 2004 and April

20, 2005.

2. Material and methods

2.1. Field site

The study was carried out in a natural Quercus ilex oak forest in

the Prades mountains at 80 km south-west of Barcelona, in

Southern Catalonia (NE Spain) (41�130 N, 0�550 E) on a south-

facing slope (25%) at 930 m above sea level. The soil is a stoney

Dystric Xerochrept [39] lying on a bedrock of metamorphic

sandstone. Its depth ranges between 35 and 100 cm, with the

depth of Horizon A ranging between 25 and 30 cm. Soil prop-

erties are shown in Table 1. The average annual temperature

is 12 �C, the mean of the minimum temperatures in the

coldest month (January) is �8.5 �C and the mean of the

maximum temperatures in the hottest month (July) is 31.8 �C.

The average rainfall is 658 mm, with maximum rainfall in the

period between September and November. Summer drought

is pronounced and usually lasts for 3 months, from July to

September. The vegetation consists of a dense forest domi-

nated by Q. ilex L. (holm oak) (20.8 m2 ha�1 trunk basal area at

50 cm height) accompanied by abundant Phillyrea latifolia



(mock privet) (7.7 m2 ha�1 trunk basal area at 50 cm height)

and Arbutus unedo L, (strawberry tree). In winter 1999, the

aboveground biomass of Q. ilex represented 77.1% of the total

aboveground biomass, while P. latifolia represented 12.6% and

A. unedo 7.8%. These three species thus represent 97.6% of the

whole ecosystem aboveground biomass. In winter 2005, the

figures for the same three species were 75.6% 13.3% and 8.7%,

respectively, representing in total 97.6 % of the total above-

ground biomass. This data is shown in Table 1.

2.2. Experimental design

Eight 15 m� 10 m plots were established at the same altitude

(930 m above sea level) on a slope. Four of the plots received

the drought treatment and four were left as control plots. All

the plots were established in an area with the same aspect and

altitude, with a minimum distance between plots of 15 m and

a maximum distance of 30 m. The treatments were randomly

assigned to different plots. The drought treatment consisted

of partial rainfall exclusion achieved by suspending trans-

parent PVC strips at a height of 0.5–0.8 m above the soil that

covered approximately 30% of the total soil surface. Four

plastic strips 14 m long and 1 m wide were placed along the

drought treatment plots from top to bottom and a 0.8–1 m

deep ditch was dug along the entire top edge of the upper part

of the treatment plots to intercept any runoff water. The water

intercepted by the strips and ditches was channeled to the

bottom edge of the plots. The litter fallen on PVC strips was

removed manually and periodically spread on the soil below

the strips. The drought treatment began on 1 March 1999 [40].

In the drought plots we distinguished two levels of drought

at soil level: that of the soil between the strips (D) (runoff

exclusion) and that under the strips (DD) (runoff exclusion

plus rainfall exclusion).

2.3. Soil moisture determination

Soil moisture content was measured every two weeks

throughout the experiment by time domain reflectometry

(Tektronix 1502 C, Beaverton, OR, USA), connecting the time

domain reflectometer to the ends of three stainless steel

cylindrical rods, 20 cm long, fully driven into the soil [40]. Four

sites per plot were randomly selected to install the steel

cylindrical rods for soil moisture measurements. Moreover,

soil moisture was measured gravimetrically in each soil

sample used in the analysis of soil enzyme activity, pH and P

and C content. An aliquot of each soil sample was weighed

immediately after arrival from the field (fresh weight) and

then was dried at 70 �C until constant weight (dry weight). The

soil gravimetrical analyses of water content were conducted

separately in 0–15 cm and 15–30 cm of soil depth layers.

2.4. Leaf and litter-fall sampling

After six years of drought treatment (January 2005), we con-

ducted litter-fall and leaf sampling. Eight samples of leaves

from the three dominant species (Q. ilex, P. latifolia and

A. unedo) were randomly sampled in each plot (four samples in

the sun and four samples in the shade). The leaves were

sampled between 1.5 and 6 m height where most foliar

Table 1 – Soil properties at 0–15 cm depth.

Soil variable January 2005

pH (in water extracts) 6.5� 0.5

pH (in KCl 0.1 M extracts) 6.2� 0.5

CEC (cation exchange capacity) (meq/100 g soil) 14.5� 0.9

Base exchangeable (meq/100 g soil) 10.0� 0.8

Percent base saturation (%) 70.0� 7.1

Total soil N (mg g�1) 2.5� 1.3

NH4þ in 0.1 M KCl extracts (mg kg�1) 2.7� 1.4

Total soil P (mg g�1) 0.60� 0.03

Immediately available-P (Olsen-Pi) 0.0055� 0.0006

Total soil K (mg g�1) 15.5� 0.3

Biomass (kg ha�1) Treatment March 1999 January 2005

Quercus ilex

Leaf C 5721� 1368 5763� 1496

D 6023� 1527 5890� 1656

Wood C 80349� 21083 84057� 23982

D 85779� 24826 87134� 27565

Phillyrea latifolia

Leaf C 863� 413 931� 450

D 774� 316 845� 349

Wood C 14404� 6925 15586þ 7557

D 12895� 5270 14122þ 5840

Arbutus unedo

Leaf C 920� 277 1081� 323

D 496� 161 663� 180

Wood C 14771� 5078 18149� 6333

D 7737� 2706 8418� 2508

Total leaf and wood biomass at the beginning of the experiment (March 1999) and 6 years later (January 2005).

e u r o p e a n j o u r n a l o f s o i l b i o l o g y 4 4 ( 2 0 0 8 ) 5 0 9 – 5 2 0 511


biomass was located. The sample collection was standardized

in order to avoid biased results due to different age of tissues

and position with respect to sunlight. The leaves sampled

were current year leaves in 2004 which represented the

majority of the leaves on the plants of these three species

from that year.

Litter-fall was collected in 20 circular baskets (27 cm

diameter with 1.5 mm mesh diameter) randomly distributed

on the ground of each one of the eight plots. The fallen litter

was collected every 2 months during 2004 (February 2, April

4, June 6, August 16, October 10 and December 13). Total

litter-fall was estimated from the proportion of the surface

area of the plots covered by the collecting baskets. The litter

fall on the plastic strips was spread on the soil under the

plastic strips every 1–2 months (February 2, April 4, June 6,

August 16, September 15(??), October 10, November 14(??)

and December 13).

2.5. Soil sampling

We sampled the soil in July 2004, October 2004, January 2005

and April 2005. In each sampling campaign we randomly

sampled five cores from the first 30 cm of soil profile (Horizon

A) in control plots and 10 in each drought plot at a minimum

distance of 1 m from the nearest tree or shrub. We sampled

five soil cores between strips and five soil cores under strips in

drought plots. All soil cores are 5 cm in diameter and 30 cm

deep. We collected and separately analyzed the 0–15 cm deep

soil and the 15–30 cm deep soil in each soil core. Thus in

horizon A we distinguished an A1 subhorizon (first 15 cm)

rich in organic matter (7.25% w/w) and an A2 subhorizon

(15–30 cm) with only moderate amounts of organic matter

(1.3% w/w).

2.6. Chemical analyses

All the samples were stored at 4 �C until analyzed. Soil

samples were first sieved (B< 2 mm) and afterwards divided

into five aliquots. One aliquot of each soil sample was dried at

105 �C for soil water content determination. A second soil

aliquot was stored at 4 �C for soil bicarbonate-extractable Pi

and Po determination and total soil organic C analyses. A third

soil sample aliquot was dried at 105 �C for total soil P and C

analyses. This soil aliquot was ground using a FRITSCH Pul-

verisette (Rudolstadt, Germany). A fourth soil sample aliquot

was used to determine soil pH, and finally, the rest of each soil

sample was kept without drying at 4 �C until soil enzyme

determination was conducted during the next six days. The

leaf and litter samples were dried in an oven at 60 �C until

reaching constant weight and then ground in a FRITSCH Pul-

verisette (Rudolstadt, Germany) to analyze their C and P

concentrations.

2.6.1. Determination of total P and C in leaf, litter and soilWe measured the concentration of P in plant and soil samples

by ICP-OES (Optic Emission Spectroscopy with Inductively

Coupled Plasma) [41] using a model JOBIN IBON JY 38 (Long-

jumeau, HORIBA Jobin Ibon S.A.S., France).

Before the biomass ICP-OES analyses, we carried out an

acid digestion of the leaves and litter-fall with an acid mixture

of HNO3 (60%) and HClO4 (60%) (2:1) in a microwave oven

(SAMSUNG, TDS, Seoul, South Korea). Two milliliters of the

mixed acid solution were added to 100 mg of dry biomass for

each sample. The digested solutions were made up to 10 ml

final volume. During the acid digestion process, two blank

solutions (2 ml of acid mixture without any sample biomass)

were also analyzed. In order to assess the precision of the

analytical procedures of biomasses, we used standard certi-

fied biomass (DC73351).

To determine total P in soil samples, digestion was carried

out in a microwave oven at 120 �C for 8 h with 0.25 g of ground

sample in 9 ml of HNO3 (65%) and 4 ml HF (40%) [42]. The

digested solutions were made up to 50 ml final volume with

HNO3 (3%). The digested solutions were filtered with a Millex

0.45 mm filter and stored at 4 �C awaiting analysis. Analytical

precision for soil and bedrock assays, as verified by parallel

analyses of international (GSR-6) standard, was better than 5%

for all trace elements analyzed. We determined total P in soil

using the soil samples from winter 2005.

To determine C concentration in biomasses, 1–2 mg of fine

sieved biomass sample plus 2 mg of V2O5 (as oxidant) were

used. C biomass concentrations were determined by organic

elemental analysis, by combustion coupled to gas chroma-

tography (Carlo Erba Method). We used a Thermo Electron Gas

Chromatograph model NA 2100 (C.E. instruments-Thermo

Electron, Milano, Italy). In order to assess the accuracy of the

biomass digestion and analytical procedures, we used stan-

dard certified biomass (DC73351). For soil analyses, the

analytical precision (as verified by parallel analyses of inter-

national (GSR-6) standard) was better than 5% in all samples.

To determine total organic C in soil samples we used the

Walkley–Black Method [43]. Briefly, 10 mL of 1 N potassium

dichromateþ 20 mL of concentrated H2SO4 solution were

added to 1 g of sieved and dried soil. They were mixed by

gentle rotation for 1 min, heated to 150 �C for 10 min and then

cooled to room temperature. They were then diluted to 200 mL

with deionized water and 10 mL of phosphoric acid, 0.2 g of

ammonium fluoride and 10 drops of diphenylamine indicator

were added. Finally, we conducted a titration with Morh salt

solution (0.5 N SO4FeNH4 and 0.1 N H2SO4).

2.6.2. Soil bicarbonate-extractable Pi and Po determinationThe P available to plants in the soil was determined by Olsen’s

method [44]. This method measures inorganic P extracted in

0.5 M NaHCO3 at pH 8.5 (Olsen Pi). Total P in the extract (Olsen

Pt) was determined in an aliquot of the extract after adding an

equal volume of 5 N H2SO4 containing 167 g KS2O8 L�1 and

digesting at 150 �C. Organic-P in the extract (Olsen Po) was

calculated by the difference between Olsen Pt and Olsen Pi.

With the Olsen-Po and total soil organic C we calculated the

ratio between these two soil variables in the different seasons

to evaluate the effects of drought on the ratio between the

mobile part of organic P with respect to soil organic C.

2.7. pH determination

We measured soil pH in a 1:2.5 soil solution (in both water and

0.01 M KCl) at room temperature using a glass electrode

(ORION 960 Autochemistry SYSTEM, Thermo Electron Corpo-

ration, MA, USA) for all the soil samples.



2.8. Acid and alkaline phosphatase activitymeasurements

Soil phosphatase activity was determined using para-nitro-

phenyl phosphate ( pNPP) as an orthophosphate monoester

analogue substrate [45,46]. Briefly, we put 1 g of each soil

sample (<2 mm) in a 50 ml Erlenmeyer flask, added 4 ml of

THAM solution (Tris-hydroxymethyl-aminomethane, with

citric, maleic and boric acids), buffer (pH 6.5 for acid phos-

phatase assay or pH 11 for alkaline phosphatase assay) and

1 ml of p-nitrophenyl phosphate solution prepared in the

same buffer, and swirled the flask for a few seconds to mix the

contents. After stoppering the flask, we placed it in an incu-

bator at 37 �C. After 1 h of incubation, we removed the

stopper, added 1 ml of 0.5 M CaCl2 and 4 ml of 0.5 M NaOH, and

swirled the flask for a few seconds to stop the reaction. Then,

we filtered (0.45 mm HA nitrocellulose, Millipore) the soil

suspension in order to prevent the interference of possible

precipitates. The fading of the intensity of yellow colour in the

calibration standards, samples and controls was measured

with a spectrophotometer Spectronic 20 Genesys (Spectronic

Instruments Inc, Rochester NY, USA) at 398 nm against the

reagent blank. We calculated the p-nitrophenol content by

referring to a calibration curve obtained with standards con-

taining 0, 10, 20, 30, 40 and 50 ppm of p-nitrophenol. Then, the

phosphomonoesterase activity was expressed as mg p-nitro-

phenol ( pNP) per gram dry matter and incubation time

(hours).

2.9. Statistical analyses

The effects of the drought treatment on each variable studied

were investigated by means of ANOVA using a post-hoc test

(Bonferroni/Dunn) to compare the results of the analyses of

soil variables from control plots with those from the two

levels of drought that were established in the drought plots

(runoff exclusion and runoff exclusion plus rainfall exclu-

sion). These analyses were conducted with the Statview 6.0

program (Abacus Concepts, SAS Institute Inc. Berkeley

Ca, USA).

We correlated the seasonal values of soil phosphatase

activity with the corresponding mean of soil moisture of each

control plot, D (runoff exclusion) and DD (runoff exclusion

plus rainfall exclusion) plot, i.e. 12 points (4 Cþ 4Dþ 4 DD) in

each correlation. Each value of soil phosphatase activity and

soil moisture were the arithmetic mean values of the soil acid

or alkaline phosphatase activity, and the soil moisture

obtained gravimetrically from the five soil samples of

each plot.

3. Results

3.1. Effects of drought on soil moisture

During the period 1999–2005 the soil in drought treatment

plots submitted to runoff exclusion (D) had an average soil

moisture of 17.5� 0.5% v/v (n¼ 100), which was 9% lower

than that of soil from control plots, which had an average

soil moisture of 19.2� 0.5% v/v (n¼ 100) (P¼ 0.026). The soil

submitted to both runoff and partial rainfall exclusion (DD)

had an average soil moisture of 15.4� 1.0% v/v (n¼ 100),

which was 20% lower than that of soil from control plots

(P¼ 0.0018). In the three previous months to sampling data,

the soils submitted to both runoff and partial rainfall exclu-

sion (DD) had lower soil moisture in the four seasons (25%,

29%, 13% and 24% in summer 2004, autumn 2004, winter

2004–2005 and spring 2005, respectively) than the soil of

control plots (Table 2). The soils submitted to runoff exclu-

sion (D) had lower soil moisture than control plots in spring

2005 (19%) (Table 2).

The soils sampled with runoff plus rainfall exclusion had

lower water content than control soils in autumn, winter and

spring, both at 0–15 cm (13%, 65% and 58%, respectively) and

15–30 cm of depth (38%, 64% and 48%, respectively). In

summer this effect was only observed at 0–15 cm of soil depth

(28%) (data not shown). The soils with runoff exclusion had

lower soil water content than controls in spring both at

0–15 cm and 15–30 cm of soil depth (58% and 18%) and in

autumn at 15–30 cm of soil depth (37%). This effect was also

observed in soil samples in summer of 15–30 cm of depth

(28%) (data not shown).

3.2. Effects of drought on soil acid and alkalinephosphatase activity

Drought tended to reduce acid phosphatase activity both in

the 0–15 cm and the 15–30 cm soil layers in the four seasons

which were analyzed (Fig. 1). This effect was significant at

0–15 cm depth under plastic strips, in autumn, winter and

spring (24%, 27% and 22%, respectively) in response to a soil

moisture reduction of 41%, 28% and 19%, respectively (Fig. 1).

At 15–30 cm depth, drought reduced acid phosphatase activity

by 27% in soils between the strips in summer 2004 (with 23%

soil moisture reduction) (Fig. 1)

DD reduced alkaline phosphatase activity by 37% at

0–15 cm and 63% at 15–30 cm depths in response to a 27% soil

moisture reduction in winter 2005 (Fig. 1). Drought had no

clear effects on alkaline phosphatase activity in summer,

autumn and spring.

3.3. Correlation between acid and alkaline phosphataseactivity and soil moisture

At 0–15 cm depth, soil phosphatase activity (both acid and

alkaline) and soil water content were positively correlated in

summer, autumn and winter (Figs. 2 and 3).

At 15–30 cm depth, acid soil phosphatase activity and soil

water content were also positively correlated in summer,

autumn and winter (Figs. 2 and 3). Alkaline soil phosphatase

activity and soil moisture were positively correlated in

summer and winter (Figs. 2 and 3).

3.4. Total soil-P, immediately available-P andshort-term available-P

The total soil P was not determined in 1999. Immediately

available-P was determined in 1999 prior to the beginning of

the treatment. No differences were observed between control

and drought plots, and the values were similar to those



obtained in 2005. Drought had no effect on total soil P, all the

soil samples having between 0.520 and 0.630 mg P g�1 soil

(data not shown).

At 0–15 cm depth, drought increased short-term avail-

able-P in summer, autumn and winter (Fig. 1). Immediately

available-P was not affected by drought (Fig. 1). DD treat-

ment decreased the ratio immediately/short-term avail-

able-P in the four seasons analyzed (34%, 47%, 56% and 54%

in summer, autumn, winter and spring, respectively)

(Fig. 4).

At 15–30 cm depth, drought increased short-term avail-

able-P in autumn, winter and spring (Fig. 1). In spring drought

decreased immediately available-P (Fig. 1). As a consequence

of these results a tendency for the ratio immediately/short-

term available-P to decrease was observed in all seasons. This

decrease was significant in the three rainy seasons (71%, 50%

and 10% in autumn, winter and spring, respectively) (Fig. 4).

Drought decreased by 27% immediate available-P/total soil P

in spring 2005 (data not shown).

3.5. Relation of soil phosphatase activities withimmediately available-P and with short-term available-P

The soil phosphatase activities were mostly not correlated

with immediately and short-term available soil fractions of P.

In 0–15 cm of soil depth, soil acid phosphatase activity was

correlated with short-term available-P in spring (P¼ 0.004,

R¼ 0.76), in 15–30 cm depth soil acid phosphatase activity was

correlated with immediately available-P in spring (P¼ 0.03,

R¼ 0.63), with short-term available-P in summer (P¼ 0.06,

R¼ 0.56), and with the immediately/short-term available-P

ratio in spring (P¼ 0.009, R¼ 0.71). Soil alkaline phosphatase

activity was correlated with short-term available-P in summer

(P¼ 0.05, R¼ 0.56) and winter (P¼ 0.04, R¼ 0.58), and with

Table 2 – Soil water content (%, v/v) in summer and autumn 2004 and in winter and spring 2005.

Season Treatment

Control Runoff exclusion Runoff exclusion plusrainfall exclusion

Soil water content (%, v/v in the first 30 cm) Summer 13.3� 0.9(a) 11.6� 0.8ab 10.0� 0.1(b)

Autumn 14.2� 0.9a 10.7� 0.6a 8.4� 1.0b

Winter 27.8� 1.1a 26.0� 0.7a 20.0� 2.1b

Spring 10.1� 0.1a 8.2� 0.5b 7.8� 0.5b

Different letters indicate statistically significant differences (P< 0.05). Different letters between brackets indicate marginally significant

differences between treatments (P< 0.1).

2

4

6

8

10

ControlRunoff exclussionRunoff + partial rainfall exclussion

*

*

*

0-15 cm soil depth

*

15-30 cm soil depth

2

4

6

8 *

*

0

20

40

60

80

100

Summer Autumn Winter Spring

Plan

t availab

le-P

(m

g kg

-1 so

il)

Short-term

Immediate

*

*

Summer Autumn Winter Spring

**

Immediate

Short term

Alkalin

e p

ho

sp

hatase

activity

Acid

p

ho

sp

hatase

activity

(m

icro

g

p

-N

P g

-1 so

il h

-1)

Fig. 1 – Acid and alkaline soil phosphatase activities (mg p-NP gL1 hL1), immediately available-P (mg kgL1) and short-term

available-P (mg kgL1) in summer and autumn 2004 and in winter and spring 2005, after 5–6 years of drought treatment. Soil

variables were calculated in the upper A1 0–15 cm soil layer and in the lower A2 15–30 cm soil layer.



immediately/short-term available-P ratio in summer (P¼ 0.04,

R¼ 0.59). In all these cases short-term available-P decreased

when soil phosphate activity increased, whereas immediately

available-P and the immediately/short-term available-P ratio

increased when soil phosphatase activity increased (data

not shown).

3.6. Soil carbon and organic matter content, C/P ratioand soil pH

Runoff exclusion plus partial rainfall exclusion increased soil

organic C at 15–30 cm soil depth by 71% in summer and 39% in

winter and also decreased soil organic C at 0–15 cm soil depth

by 58% in summer (Table 3). Drought had no effects on soil pH

either in water or in KCl extracts (data not shown). Runoff

exclusion plus partial rainfall exclusion increased the ratio of

soil organic C/total soil P by 36% at 0–15 cm and 22% at 15–

30 cm (Fig. 5). Drought decreased the ratio of soil organic C/

short-term available-P at 0–15 cm soil depth in autumn and

spring and at 15–30 cm soil depth in autumn and winter

(Fig. 5).

3.7. P and C concentrations and C/P concentration ratioin leaves and litter-fall

Drought increased P concentrations in leaves and litter-fall

of the dominant tree, Q. ilex (Fig. 6). As a result, drought

decreased the C/P ratio in the litter-fall of this species

(Fig. 5). Drought did not induce changes in C and P

concentrations nor the C/P concentration ratio of P. latifolia

(Fig. 6). Drought decreased litter-fall C concentrations (Fig. 6)

and the C/P concentration ratio in the litter-fall of A. unedo

(Fig. 6).

After six years of drought, during the period from July 2004

to July 2005 drought tended to increase the losses of P in leaf

litter fall, but the differences were not significant. During this

period the losses were 1594� 964 g ha�1 in control plots and

1970� 198 g ha�1 in drought-treated plots.

4

8

12Summer

y = 4.34 + 0.16x R = 0.47, P = 0.024

Summer

y = 1.07 + 0.35x R = 0.81, P = 0.0015

4

8

12Autumn

y = 4.23 + 0.29x R = 0.71, P = 0.0094

Autumn

y = 3.69 + 0.13x R = 0.59, P = 0.053

4

8

12Winter

y = -0.86 + 0.36x R = 0.80, P = 0.002

Winter

y = -1.61 + 0.25x R = 0.63, P = 0.028

0

4

8

12

Spring

10 15 20 25 30 35105 15 20 25 30 35

Spring

Soil moisture (%, w/w)

So

il acid

p

ho

sp

hatase activity (µg

p

-N

P g

-1

h

-1)

0-15 cm soil depth 15-30 cm soil depth

Fig. 2 – Relationships between soil acid phosphatase activity (mg p-NP gL1 hL1) and soil water content (average % w/w) at

different soil depths (0–15 and 15–30 cm) and in the four seasons analyzed.



4

8

12

y = 1.29+ 0.25x R = 0.52, P = 0.085

Summer

y = -0.076 + 0.20xR = 0.55, P = 0.063

Summer

4

8

12Autumn

y = 1.91+ 0.24x R = 0.56, P = 0.027

Autumn

4

8

12Winter

y = -1.54 + 0.28x R = 0.71, P = 0.027 y = -4.27 + 0.28x R = 0.89, P = 0.0093

Winter

0

4

8

12

5 10 15 20 25 30 35

Spring

10 15 20 25 30 35

Spring

0-15 cm soil depth 15-30 cm soil depth

So

il alkalin

e p

ho

sp

hatase activity (µg

p

-N

P g

-1 h

-1)

Soil moisture (%, w/w)

Fig. 3 – Relationships between soil alkaline phosphatase activity (mg p-NP gL1 hL1) and soil water content (average % w/w) at

different soil depths (0–15 and 15–30 cm) and in the four seasons analyzed.

0,2

0,4

0,6

0,8

aab

b

Summer 2004

a

b b

Autumn 2004

aab b

Winter 2004-2005 Spring 2005

a

bb

0

0,2

0,4

0,6

0,8

C DD

a

b ba

abb

a a

bIm

med

iately/sh

ort-term

availab

le-P

Treatment

0-15 cm

d

ep

th

15-30 cm

d

ep

th

D C DDD C DDD C DDD

Fig. 4 – Immediately/short-term available-P concentration ratio at different soil depths (0–15 and 15–30 cm) in the four

seasons analyzed. Different letters indicate significant differences between treatments at P < 0.05. C, control; D, runoff

exclusion, DD, runoff plus partial rainfall exclusion.



4. Discussion

The soil acid phosphatase activity was higher than the soil

alkaline phosphatase activity, which is consistent with the

natural pH of these soils (6.5� 0.2) being closer to the

optimum pH for activity of the former enzyme than that of

the latter. The activities of soil acid and alkaline phosphatases

were in the range observed in previous studies in this area [34]

and are in the highest range observed in other Mediterranean

soils [27,47,48]. The effects of drought were greater on acid

phosphatase than on alkaline phosphatase. These results

reinforced previous findings which reported a decrease in soil

acid and alkaline phosphatase activities after 4 years of

drought treatment. Previous studies [34] focused on the effects

of drought on phosphatase activities in the plant growth

seasons, spring and autumn, but not in summer and winter.

We have now observed that drought reduces soil phosphatase

activity in all seasons and not only in the most biologically

active seasons, spring and autumn.

The activity of acid phosphatase was reduced in all the

seasons analyzed whereas alkaline phosphatase activity was

only reduced in winter 2004–2005. This different response was

not produced by any change in soil pH since pH was not

affected by drought. The impact of drought on total soil

phosphatase activity was significant because acid phospha-

tase activity accounted for 60% of the total soil phosphatase

activity.

The results indicate that changes in phosphatase activity

in soil under drought were mainly the consequence of a direct

effect of soil moisture reduction and less a result of an indirect

effect through changes in soil P status. We observed that soil

phosphatase activities increased whereas short-term avail-

able-P decreased in spring. In general, drought treatment

presented lower soil phosphatase activity and higher short-

term available-P than control soils throughout the year. On

the other hand, the P losses from plant to soil through litter

fall tended to increase in these 6 years of drought, incorpo-

rating more organic-P to soil in drought plots. All these find-

ings reinforce the idea that soil phosphatase activities are not

stimulated by the availability of its substrate. Furthermore,

the observed effects of drought increasing short-term avail-

able-P and decreasing the ratio immediately/short-term

available-P are similar to those observed three years before in

the same experiment [35].

The results confirmed the limiting role of soil water

content on soil acid and alkaline phosphatase activity, and

that this effect lasts the whole year. Soil water content and

soil phosphatase activities were positively correlated also in

summer and winter and not only in spring and autumn as was

observed in previous reports [34]. Our results show that

a moderate drought of 20% reduction in soil water content as

predicted for the coming decades [31,33], is sufficient to

reduce soil phosphatase activity in a sub-humid Mediterra-

nean forest. Li and Sarah [27] in a study of diverse soil traits

with increasing aridity along a climate transect in Israel

observed a sharp change in soil phosphatase activity from

260 mm to 120 mm of annual rainfall.

The reduction in soil water content is the most probable

mechanism involved in the reduction of soil phosphatase

activity under prolonged drought conditions in this Mediter-

ranean forest. This reduction was most likely due to

a decrease in the soil microbial population resulting from

a reduction in the leaching of soluble organic carbon from

litter. Thus, if drought increases in the coming decades in

Mediterranean areas, the decrease in soil phosphatase activity

due to reduced soil water content may be significant and

independent from the seasons.

The effects of the increase in soil air content under drier

conditions lead to instability of macro- and micronutrient

aggregates and consequently to the release of organic P after

rewetting [49]. This release together with the decrease in soil

0

20

40

60

80

0-15 cm 15-30 cm

ControlRunoff exclusion

Runoff +rainfall exclussion

a

bab b b

a

2000

4000

6000

8000

10000Control

Runoff exclusion

Runoff +rainfall exclusion

Summer

1000

2000a

a

b b bb

Autumn

400

800

1200

1600

a

b b

Winter

0

300

600

900

1200

0-15 cm 15-30 cm

a a

b

Spring

So

il o

rg

an

ic C

/sh

ort-term

availab

le-P

So

il o

rg

an

ic C

/to

tal so

il P

Fig. 5 – Soil organic C/total soil P ratio in winter and soil

organic C/short-term available-P ratio in the four seasons

at different soil depths. Different letters indicate significant

differences between treatments at P < 0.05.



phosphatase activity explains the observed increase in short-

term available-P. This increase led to a reduction in the

immediately/short-term available-P ratio. In drought plots,

the decrease in soil phosphatase activities did not imply less

immediately available-P. The decreases in P liberated from

short-term available-P were counterbalanced by a lower plant

uptake capacity. This reduced capacity was due to the lower

soil water content, which decreases the soil nutrient diffusion

[50], and to a slowing in plant growth [51,52]. As a result, the

soil soluble P fraction increased under drought. Thus, the

results confirm the relation between the decreases in soil

water content, soil phosphatase activity and immediate/

short-term available-P in the different seasons in this Medi-

terranean forest. The increase in short-term available-P in

soil, which is the substrate of soil phosphatases, did not imply

enhanced phosphatase activity or increased microbial

synthesis of more soil phosphatases. Thus, the increase of

short-term available-P, litter P contents and total amount of

soil organic matter in some seasons under drought did not

counterbalance the direct effects of reduction in soil water

content which decreased soil phosphatase activity.

The increases in short-term available-P in soil heighten the

risk of potential losses of soil P by erosion and leaching during

rainfall. This, in fact, occurred during these six years of

drought as indicated by the absence of change in total soil P

and the significant loss of P in stand biomass in drought

treated plots [53]. The P which has not been captured by stand

biomass is not in the soil either, indicating a net ecosystem P

loss during the six years of drought treatment. The general

decrease in soil phosphatase activities together with the

greater amounts of soil organic-P (short-term available-P) in

the soil solution, which is easily leached in torrential rainfalls,

can account for the great losses of soil P in drought plots, in

spite of lower plant capture and greater litter production.

Drought-induced increases in leaf P concentrations have

also been observed in other experiments and have been linked

to a mechanism to improve WUE [10]. The increased leaf P

concentration in Q. ilex in drought plots can be explained by

a concentration effect due to reduction in growth and by an

increase in leaf P contents with respect to stem P contents. In

spite of these increased foliar P concentrations, in a recent

study in the same ecosystem, Sardans and Penuelas [53]

observed that the accumulated P in total aboveground

biomass tended to decrease in drought plots due to a reduc-

tion in growth. Thus, in drought plots the tendency to

Treatments

1

2a

b

Leaves

(b) (a)

Litterfall Leaves Litterfall Leaves Litterfall

20

40

60

80

a b

0200400600800

1000

1

2

20

40

60

80

0200400600800

1000

1

2

1.5

0.5

20

40

60

80

0200400600800

1000

Control Drought Control Drought Control Drought Control Drought Control Drought Control Drought

ab

(a)

(b)

Treatments Treatments

Quercus ilex Phillyrea latifolia Arbutus unedo

C/P

C co

ncen

tratio

n

(%

)

P co

ncen

tratio

n

(m

g g

-1)

Fig. 6 – P (mg gL1) and C (%, dw) concentrations and C/P concentration ratio in Quercus ilex, Phillyrea latifolia and Arbutus

unedo leaves and litter-fall in control and drought plots. Different letters indicate significant differences between treatments

at P < 0.05. Different letters between brackets indicate marginally significant differences between treatments (P < 0.1).

Table 3 – Soil organic-C concentrations (%, w/w).

Season Treatment 0–15 cm 15–30 cm

Summer 2004 C 4.3� 0.4a 1.4� 0.2b

D 3.3� 0.3a 1.8� 0.1b

DD 1.8� 0.4b 2.4� 0.2a

Autumn 2004 C 2.6� 0.2 1.9� 0.1

D 2.7� 0.3 1.8� 0.1

DD 2.6� 0.2 2.0� 0.1

Winter 2004–2005 C 1.9� 0.2 1.8� 0.1b

D 2.3� 0.3 1.6� 0.3b

DD 2.3� 0.2 2.5� 0.2a

Spring 2005 C 2.3� 0.4 1.5� 0.3

D 2.1� 0.3 1.4� 0.2

DD 2.0� 0.3 1.9� 0.2

Soil variables were calculated in the upper A1 0–15 cm soil layer and

in the lower A2 15–30 cm soil layer.

Different letters indicate statistically significant differences

(P< 0.05). Significant differences are highlighted in bold type.

C, soil of control plots; D, soil of drought plots situated between

plastic strips (runoff exclusion); DD, soil of drought plots situated

below plastic strips (runoff plus rainfall exclusion).



decrease immediately available-P in soil and the immediately/

short-term available-P, which means a reduction of soil-P

available for plants, was accompanied by a reduction in

aboveground plant content. On the other hand, drought

decreased the C/P concentration ratio in litter fall as a result of

a slow P liberation related to the decrease in soil phosphatase

activity and in the plant P uptake capacity.

These changes in the soil stoichiometry between C and P

can have also implications in the detritivore community

composition by affecting the nutritional quality of litter and

soil organic matter [54–56].

4.1. Final remarks

Here, we have studied the effects of drought on phosphatase

activity and on different soil P fractions in the same soil

samples, together with litter and leaves. Drought by reducing

soil water content decreases soil phosphate activity, plant

growth and P uptake, changing the ratios between soil

P fractions. Coinciding with a decrease in soil water content

throughout the year in drought soils, a constant decrease of

soil acid phosphatase activity was observed in all seasons.

Drought reduced soil phosphatase activity mainly as a direct

effect of reduced soil water content on the soil microbial

population; decreasing P mineralization, reducing the

immediately/short-term available-P concentration ratio,

accumulating greater amounts of short-term available-P and

increasing P content in the litter of the dominant tree

species. These effects imply that (i) soil phosphatase activity

is more dependent on the changes in soil water content than

on the changes in its substrate, short-term available-P, (ii)

the effects of drought decreasing soil phosphatase activity

may be significant in any season if there is a decrease in soil

water content, (iii) there is an accumulation of short-term

available-P in soil that increases potential P losses by

leaching and erosion during the torrential rainfalls typical of

the Mediterranean climate, (iv) there is a reduction in the

P available to plant capture, and (v) there is a change in soil

C:P stoichiometry with the corresponding implications for

soil trophic chains.

Acknowledgements

This research was supported by the Spanish Government

CGL2004-01402/BOS and CGL2006-04025/BOS grants, the EC

Integrated project FP6 ALARM (GOCE-CT-2003-506675), a Fun-

dacion BBVA 2004 and a Catalan government SGR2005-00312

grant. This experiment complied in all aspects with the laws

of the Spain and the European Union.

r e f e r e n c e s

[1] G.S. McMaster, W.M. Jow, J. Kummerow, Response ofAdenostoma fasciculatum and Ceanothus gregii chaparral tonutrient additions, J. Ecol. 70 (1982) 745–756.

[2] J. Sardans, F. Roda, J. Penuelas, Phosphorus limitation andcompetitive capacities of Pinus halepensis and Quercus ilex

subsp. rotundifolia on different soils, Plant Ecol. 174 (2004)305–317.

[3] R.L. Specht, The sclerophyllous heath vegetation ofAustralia: the eastern and central states, in: R.L. Specht (Ed.),Heathlands and Related Shrublands, Descriptive Studies,Elsevier, Amsterdam, 1979, pp. 125–210.

[4] J.A. Carreira, F. Sanchez-Vazquez, F.X. Niell, Short-term andsmall-scale patterns of post-fire regeneration in a semi-ariddolomitic basin of Southern Spain, Acta Oecol. 13 (1992)241–253.

[5] J. Sardans, F. Roda, J. Penuelas, Effects of water anda nutrient pulse supply on Rosmarinus officinalis growth,nutrient content and flowering in the field, Environ. Exp. Bot.53 (2005) 1–11.

[6] J. Sardans, J. Penuelas, F. Roda, Changes in nutrient status,retranslocation and use efficiency in young post-fireregeneration Pinus halepensis in response to sudden N and Pinput, irrigation and removal of competing vegetation, TreesStruct. Funct. 19 (2005) 233–250.

[7] J. Terradas, Ecologıa de la Vegetacion, De la Ecofisiologıa delas Plantas a la Dinamica de Comunidades y Paisajes, Omega,Barcelona, 2001.

[8] X. Mayor, F. Roda, Effects of irrigation and fertilization onstem diameter growth in a Mediterranean holm oak forest,For.t Ecol. Manage. 68 (1994) 119–126.

[9] M. Hanley, M. Fenner, Growth of Aleppo pine (Pinushalepensis) deprived of single mineral nutrients, J.Mediterranean Ecol. 2 (2001) 107–112.

[10] D.K. Sing, P.W.G. Sale, C.K. Pallaghy, B.M. McKenzie,Phosphorus concentrations in the leaves of defoliated whiteclover affect abcisic acid formation and transpiration indrying soil, New Phytol. 146 (2000) 249–259.

[11] J.M. Ruiz-Lozano, C. Collados, J.M. Barea, R. Azccon,Arbuscular mycorrhizal symbiosis can alleviate drought-induced nodule senescence in soybean plants, New Phytol.151 (2001) 493–502.

[12] H.J. Deacon, The comparative evolution of Mediterranean-type ecosystems: a southern perspective, in: F.J. Kruger, F.J.Mitchell, J.U.M. Jarvis (Eds.), Mediterranean TypeEcosystems: The Role of Nutrients, Springer-Verlag, Berlin,1983, pp. 3–40.

[13] S. Minerbi, What effect does climate have on the appearanceof the ‘‘new type’’ of forest damage? Dendronatura 8 (1987) 24.

[14] A. Escudero, J.M. Del Arco, I.C. Sanz, J. Ayala, Effects of leaflongevity and retranslocation on the efficiency on theretention time of the nutrients in the leaf biomass ofdifferent wood species, Oecologia 90 (1992) 80–87.

[15] E. Dıaz, A. Roldan, Effects of reforestation techniques on thenutrient content, photosynthetic rate and stomatalconductance of Pinus halepensis seedlings under semiaridconditions, Land Degrad. Dev. 11 (2000) 475–486.

[16] P.V. Sundareshwar, J.T. Morris, E.T. Koepfler, B. Fornwalt,Phosphorus limitation of coastal ecosystems processes,Science 299 (2003) 563–565.

[17] G.D. Paoli, L.M. Curran, D.R. Zak, Phosphorus efficiency ofBornean rain forest productivity. Evidence against the unimodel efficiency hypothesis, Ecology 86 (2005) 1548–1561.

[18] S.K. Chen, C.A. Edwards, S. Subler, The influence of twoagricultural biostimulants on nitrogen transformations,microbial activity, and plant growth in soil microcosms, SoilBiol. Biochem. 35 (2003) 9–19.

[19] C. Baum, P. Leinweber, A. Schlichting, Effets of chemicalconditions in re-wetted peats temporal variation in microbialbiomass and acid phosphatase activity within the growingseason, Appl. Soil Ecol. 22 (2003) 167–174.

[20] F. Asmar, F. Eiland, N.E. Nielsen, Effect of extracellular-enzyme activities on solubilization rate of soil organicnitrogen, Biol. Fertil. Soils 17 (1994) 32–38.



[21] R.L. Sinsabaugh, Enzymic analysis of microbial pattern andprocess, Biol. Fertil. Soils 17 (1994) 69–74.

[22] J. Skujins, Extracellular enzymes in soil, CRC Crit. Rev.Microbiol. 4 (1976) 383–421.

[23] R.L. Sinsabaugh, R. Antibus, A.E. Linkins, C.A. McClaugherty,Wood decomposition: nitrogen and phosphorus dynamics inrelation to extracellular enzyme activity, Ecology 74 (1993)1586–1593.

[24] W.K. Smith, P.S. Nobel, Influences of seasonal changes in leafmorphology on water-use efficiency for three desertbroadleaf shrubs, Ecology 58 (1977) 2227–2233.

[25] E.L. Dunn, F.M. Shropshire, L.C. Strong, H.A. Mooney, Thewater factor and convergent evolution in Mediterranean-type vegetation, in: O.L. Lange, L. Kappen, E. Schulze (Eds.),Water and Life. Problems and Modern Approaches, Springer-Verlag, Berlin, 1983, pp. 492–505.

[26] G. Oliveira, O. Correira, M.A. Martins-Loucao, F.M. Catarino,Phenological and growth patterns of the Mediterranean oakQuercus suber, Trees Struct. Funct. 9 (1994) 41–49.

[27] X.Z. Li, P. Sarah, Enzyme activities along a climate transect inthe Judean Desert, Catena 53 (2003) 349–363.

[28] M.J. Esteban-Parra, F.S. Rodrigo, Y. Castro-Diez, Spatial andtemporal patterns of precipitation in Spain for the period1880–1992, Int. J. Climatol. 18 (1998) 1557–1574.

[29] J. Pinol, J. Terradas, F. Lloret, Climate warming, wildfirehazard, and wildfire occurrence in coastal eastern Spain,Climatic Change 38 (1998) 347–357.

[30] J. Penuelas, I. Filella, P. Comas, Changed plant and animal lifecycles from 1952 to 2000 in Mediterranean Region, Glob.Chang. Biol. 8 (2002) 531–544.

[31] J. Penuelas, I. Filella, S. Sabate, C. Gracia, Natural systems:terrestrial ecosystems, in: J.E. Llebot (Ed.), Report on ClimateChange in Catalonia, Institut d’Estudis Catalans, Barcelona,2005, pp. 517–553.

[32] J. Penuelas, M. Boada, A global change-induced biome shift inthe Montseny mountains (NE Spain), Glob. Chang. Biol. 9(2003) 131–140.

[33] J.T. Hougton, Y. Dung, Climate change 2001: the scientific basis.Contribution of Working Group I, Third Assessement Report ofIntergovermental Panel on Climate Change, IPCC, 2001.

[34] J. Sardans, J. Penuelas, Drought decreases soil enzymeactivity in a Mediterranean Quercus ilex L. forest, Soil Biol.Biochem. 37 (2005) 455–461.

[35] J. Sardans, J. Penuelas, Increasing drought decreasesphosphorus availability in an evergreen Mediterraneanforest, Plant Soil 267 (2004) 367–377.

[36] M. Zaman, H.J. Di, K.C. Cameron, A field study of gross ratesof N mineralization and nitrification and their relationshipsto microbial biomass and enzyme activities in soil treatedwith dairy effluent and ammonium fertilizer, Soil UseManage. 15 (1999) 188–194.

[37] D.E. Dodor, M.A. Tabatabai, Effect of cropping systems onphosphatases in soils, J. Plant Nutr. Soil Sci. 166 (2003) 7–13.

[38] S. Sabate, C. Gracia, A. Sanchez, Likely effects of climatechange on growth of Quercus ilex, Pinus halepensis, Pinuspinaster, Pinus sylvestris and Fagus sylvatica forests in theMediterranean region, For. Ecol. Manage. 162 (2002) 23–37.

[39] Soil Survey Staff Soil Taxonomy: A Basis System of SoilClassification for Making and Interpreting Soil Surveys, , In:USDA Agricultural Handbook, vol. 436, US GovernmentPrinting Office, Washington, D.C., 1998.

[40] S.J. Zegelin, I. White, D.R. Jenkins, Improved field probes forsoil water content and electrical conductivity measurementsusing time domain reflectometry, Water Resour. Res. 25(1989) 2367–2376.

[41] M.A. Mateo, S. Sabate, Vegetal tissue wet digestion usinga domestic microwave, Anal. Chim. Acta 279 (1993) 273–279.

[42] R. Bargagli, D.F. Brown, L. Nelli, Metal biomonitoring withmosses: procedures for correcting for soil contamination,Environ. Pollut. 89 (1995) 169–175.

[43] A. Walkley, I.A. Black, An examination of Degtjareff methodfor determination soil organic matter and a proposedmodification of the chromic acid titration method, Soil Sci.37 (1934) 29–37.

[44] F.S.I. Watanabe, S.R. Olsen, Test of an ascorbic acid methodfor determining phosphorus in water and NaHCO3 extractsfrom soil, Soil Sci. Soc. Am. J. 29 (1965) 677–678.

[45] M.A. Tabatabai, Soil enzymes, in: R.W. Weaver, J.S. Angel, P.S. Bottomley (Eds.), Methods of Soil Analysis. Part 2:Microbiological and Biochemical Properties, Soil ScienceSociety of America, Madison, Wisconsin, 1982, pp. 775–833.

[46] F. Schinner, R. Ohlinger, E. Kandeler, R. Margesin, Methods inSoil Biology, Springer, Berlin, Heidelberg, New York, Tokyo, 1996.

[47] F.A. Rutigliano, R.D. Ascoli, A.V. De Santo, Soil microbialmetabolism and nutrient status in a Mediterranean area asaffected by plant cover, Soil Biol. Biochem. 36 (2004) 1719–1729.

[48] A. Piotrowska, G. Iamarino, M.A. Rao, L. Gianfreda, Short-term effects of olive mill waste water (OMW) on chemicaland biochemical properties of a semiarid Mediterranean soil,Soil Biol. Biochem. 38 (2006) 600–610.

[49] C.H. Dickinson, G.J. Pugh, Biology of Plant LitterDecomposition, Academic Press, London, 1974.

[50] A. Jungk, N. Classen, Ion diffusion in the soil-root system,Adv. Agron. (1997) 53–110.

[51] R. Ogaya, J. Penuelas, J. Martinez-Vilalta, M. Mangiron, Effectof drought on diameter increment of Quercus ilex, Phillyrealatifolia, and Arbutus unedo in a holm oakk forest of NE Spain,For. Ecol. Manage. 180 (2003) 175–184.

[52] R. Ogaya, J. Penuelas, Leaf mass per area ratio in Quercus ilexleaves under a wide range of climatic conditions. Theimportance of low temperatures, Acta Oecol. 31 (2007) 168–173.

[53] J. Sardans, J. Penuelas, Drought changes P and Kaccumulation patterns in an evergreen Mediterranean forest,Funct. Ecol. 21 (2007) 191–201.

[54] W. Makino, J.B. Cotner, R.W. Sterner, J.J. Elser, Are bacteriamore like plants or animals? Growth rate and resourcedependence of bacterial C:N:P stoichiometry, Funct. Ecol. 17(2003) 121–130.

[55] J.T. Ngai, R.J. Jefferies, Nutrient limitation of plant growthand forage quality in Artic coastal marshes, J. Ecol. 92 (2004)1001–1010.

[56] S. Diehl, S. Berger, R. Wohrl, Flexible nutrient stoichiometrymediates environmental influences, on phytoplakton and itsresources, Ecology 86 (2005) (2005) 2931–2945.


Documents

European Journal of Soil Biology 44