Abstract The dehydrogenase activity (DHA) in the up-per 10 cm of forest soils was measured in three experi-mental plots (1 ha) in Los Alcornocales Natural Park(southern Spain). In each plot, a sylviculture treatment ofthinning and shrub-clearing had been previously carriedout in one half, while the other half was left as a forestcontrol. Soil samples were taken during the dry season(July 2000) and after the first autumn rains (October2000). The DHA of forest soil in autumn [527165 nmolp-iodonitrotetrazolium formazan (INTF) g1 h1] was almost double that in summer (28995 nmol INTF g1h1), for one of the studied plots. During the dry season,DHA of forest control soils (32485 nmol INTF g1 h1)was higher than in the thinned and shrub-cleared forest(25393 nmol INTF g1 h1). During the autumn (wetseason), however, the effects of the sylvicultural practic-es on the soil dehydrogenase were negligible. Significantdifferences in DHA were found between the three sites.Multiple regression analysis identified pH as the bestpredictor of DHA of these soils. Other soil properties(pH, K, Ca, Mg, and soil moisture) also showed signifi-cant correlations with DHA. In addition, clay content ap-peared to enhance the enzyme activity. Our results sug-gest that thinning and shrub-clearing in Mediterraneanforests seem to affect negatively the soil DHA, and theirimpact is more marked during the dry season. However,season and site effects are better determinants of DHAthan management practices.

Keywords Soil enzyme activity Dehydrogenase activity Shrub-clearing practices Mediterranean forest Quercus suber

Introduction

Soil enzyme activities are very sensitive to both naturaland anthropogenic disturbances, and show a quick re-sponse to the induced changes (Dick 1997). Therefore,enzyme activities can be considered effective indicatorsof soil quality changes resulting from environmentalstress or management practices. Enzyme activities havebeen found to be very responsive to different agriculturalsoil conservation practices such as non-tillage (Dick1992; Bergstrom et al. 1998), organic amendments (Dick1992; Perucci 1992; Miller and Dick 1995; Banerjee et al. 1997), crop rotation (Dick 1992; Miller and Dick1995), and organic cultivation (Beyer et al. 1992). Like-wise, Garcia et al. (1998) found that restoration practicesof degraded arid soils in marginal areas strongly influ-enced soil enzyme activities. On the other hand, little in-formation exists about enzyme activity in Mediterraneanforest soils, with most studies being related to nutrientcycling and soil fertility (Schneider 1998).

To explore the potentiality of biochemical propertiesin the assessment of soil quality and sustainability, it isdesirable to have a database of these properties in nativesoils with late-successional vegetation from differentecosystems around the world. Native soils supportingmature vegetation are used as the high-quality referencesoils, since a balance exists between their physical,chemical, biological and biochemical properties that en-sures the ecosystem sustainability (Dick 1997; Pankhurstet al. 1997). Trasar-Cepeda et al. (1999) and Leirs et al.(2000) thoroughly evaluated several biological and bio-chemical properties of an Atlantic forest ecosystem ofQuercus robur in north-west Spain. However, basic in-formation about biochemical properties of Mediterra-nean forest soils is still needed. Moreover, the effects offorest management on soil biology and biochemistry inMediterranean ecosystems are not clearly understood.

The activity of dehydrogenase is considered an indi-cator of the oxidative metabolism in soils and thus of themicrobiological activity (Skujins 1973), because, beingexclusively intracellular, it is linked to viable cells. How-

C. Quilchano () T. MaranInstituto de Recursos Naturales y Agrobiologa de Sevilla, Consejo Superior de Investigaciones Cientficas, Avda. Reina Mercedes 10, Apartado 1052, 41080 Sevilla, Spaine-mail: cquilchano@irnase.csic.esTel.: +34-954-624711, Fax: +34-954-624002

Biol Fertil Soils (2002) 35:102107DOI 10.1007/s00374-002-0446-8

O R I G I N A L PA P E R

Consuelo Quilchano Teodoro MaranDehydrogenase activity in Mediterranean forest soils

Received: 30 July 2001 / Published online: 1 March 2002 Springer-Verlag 2002

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ever, the relationship between an individual biochemicalproperty and the total microbial activity is not alwaysobvious, especially in the case of complex systems likesoils, where the microorganisms and processes involvedin the degradation of the organic compounds are highlydiverse (Nannipieri et al. 1990). Nevertheless, dehydro-genase activity (DHA) has been used as an indicator ofthe microbiological activity in Mediterranean arid soils(Garcia et al. 1994), and in agricultural soils of more humid regions of north-west Germany (Beyer et al.1992).

Mediterranean forests, in particular Quercus suber L.(cork oak) forests, are usually subjected to shrub-clear-ing practices (Montoya 1988). This sylvicultural practiceconsists of the removal by slashing of the woody under-storey vegetation with two main objectives: (1) to im-prove cork productivity by decreasing the competitionby shrubs for soil nutrients, and (2) to reduce the risk ofsummer fires by avoiding the accumulation of fuel bio-mass. However, these sylvicultural practices may havedetrimental side-effects on the forest ecology, by simpli-fying the ecosystem structure, decreasing the biodiver-sity, favouring opportunistic plant species, and limitingnatural regeneration. Removal of the shrubs above-ground biomass may also affect the forest soil properties,especially the biological and biochemical ones, sinceshrub-clearing modifies the microclimatic conditions atground level, and the amount and quality of the potentialorganic inputs to soil.

The main objectives in this study were: (1) to mea-sure DHA in Mediterranean forests soils, during condi-tions of two contrasting seasons (dry summer and wetautumn); (2) to study the relations between DHA andvarious soil properties; and (3) to evaluate the potentialimpact of the shrub-clearing management practice onDHA in forest soils.

Materials and methods

Study area

The study was carried out in Los Alcornocales Natural Park (provinces of Cdiz and Mlaga, in southern Spain), where threeexperimental forest plots were established: Panera (363154N,53429W), Buenas Noches (362256N, 53458W), and Tira-dero (36946N, 53539W). The dominant tree species are Q.suber (cork oak) and Quercus canariensis Willd. (semi-deciduous

oak). These forests have a dense and diverse woody understorey,e.g. 22 different shrub species were found in a 0.1-ha plot of corkoak forest (Ojeda et al. 2000). Dominant arborescent shrubs areArbutus unedo and Phillyrea latifolia; heaths (Erica spp.) and leguminous shrubs (Genista spp.) are also abundant.

The soils are developed over Oligo-Miocene sandstone of theEl Aljibe geological unit. Consequently, most of them show asandy texture with good drainage, although soils with a consider-able amount of clay can also be found scattered in the studied area. In general, soil samples had low pH values and high organicmatter content (Table 1).

The climate is defined as mild Mediterranean type withwarm, dry summers, and humid, cool winters. The influence of both the Atlantic Ocean and Mediterranean Sea acts as a tem-perature buffer, preserving the ecosystem from extreme tempera-ture values and providing some humidity during the summerdrought.

Soil sampling and soil analyses

Each forest plot (1 ha) was divided into two parts: one was sub-jected to the shrub-clearing treatment (in March 2000) whereas theother one was left untreated (control). Soil sampling was carriedout in two different seasons: firstly during the dry season (end ofJuly 2000; only in one plot), and secondly in autumn, just after thefirst rains (October 2000; in all three plots). Soil samples weretaken from the upper 10-cm layer (once the litter had been re-moved), following a regular pattern. Each soil sample was com-posed by pooling three subsamples (3 cm in diameter10 cm indepth) collected in a 11-m area. Soil samples were collectedalong each transect (established for vegetation studies, T. Maranet al., unpublished study) at regular intervals of 6 m. A total of 24 samples (12 in treated and 12 in control zones) were collectedfrom each forest plot. Samples were placed in plastic bags andkept in ice-boxes. Once in the laboratory, soil samples were sieved(

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Statistical analysis

Students t-test was used to compare the treated half plot and thecorresponding control plot to explore the possible impact of theshrub-clearing practice on soil DHA. However, in the Buenas Noches plot, where DHA values did not fit a normal distribution, aMann-Whitney U-test was used instead. To compare soil DHA be-tween the three sites, a two-way ANOVA was performed (DHAdata was previously log-transformed). After a significant ANOVA,the post-hoc multiple comparisons were made with a Scheff test.Relationships between soil variables and DHA were studied bymeans of correlation tests (Pearsons correlation coefficient). Amultivariate linear regression model (forward stepwise) was fittedto test which soil properties could be used as predictors of DHA(data of some variables were log-transformed to meet the multiplelinear regression assumptions). Statistical analyses were per-formed with Statistica 5.1 for Windows (Statsoft 1997).

Results and discussion

In general, the mean DHA measured (in autumn) in theforest soils of Los Alcornocales Natural Park was393155 nmol INTF g1 h1 in the upper 10-cm soil layer(range 192.6976.4 nmol INTF g1 h1). The literaturecontains only a few reports (Camia et al. 1998; Leirs etal. 2000) of DHA in forest soils measured by using INTas electron acceptor. The values of DHA obtained are almost double those reported by Leirs et al. (2000) in anAtlantic forest of Q. robur, exposed to a temperate climate in north-west Spain, in soils containing a higheramount of soil organic matter than those of the Los Alcornocales forest. A reasonable hypothesis could bethat the lower soil pH values, together with a higher C:Nratio, in northern forests leads to a slow decompositionrate and hence less DHA than in the southern soils studied.

Seasonal effects

DHA measured in autumn samples (527165 nmol INTFg1 h1) was almost double that measured in summersamples (28995 nmol INTF g1 h1) at the same loca-tion (Panera site; Fig. 1); accordingly, significant differ-ences were detected between the DHA values in the twoseasons (t=6.52, P=0.000001, n=24).

The increase in soil water content in autumn wouldfavour the increase in microbiological activity (andhence in DHA), especially considering the low water po-tential values measured in summer (12.6 MPa and10.1 MPa in the control and shrub-cleared zones, re-spectively). Garcia et al. (1994) also found that the rainyseason enhanced the enzyme activity (DHA) of soils inthe south-east arid region of Spain. Other authors also attributed the increase in microbial activity in forest(Grres et al. 1998) and in grassland soils (Banerjee etal. 2000) to higher soil moisture contents.

The higher inputs of organic residues during autumn(leaf-shedding season) in this forest ecosystem couldalso contribute to the observed seasonal increase inDHA. The litterfall inputs favour the overall soil oxida-

tive activity; as the litterfall undergoes decomposition,smaller and simpler organic molecules are leached down from the litter layer to the surface soil horizon, as water-soluble organic matter, thus providing a labile or-ganic substrate for soil microorganisms (McBrayer andCromack 1980; Grres et al. 1998).

Forestry management effects

In summer, the DHA activity was higher in the controlzone (32485.1 nmol INTF g1 h1) than in the managedhalf-plot (25393.2 nmol INTF g1 h1), although thisdifference was only marginally significant (t=1.955,P=0.06). Spatial variability in soil properties is high inthe studied experimental plots (T. Maran et al., unpub-lished data) and this soil heterogeneity could mask thepossible management effects on soil microbiological activity. On the other hand, when we analysed the effectof the shrub-clearing management practice on DHA,considering the sampling transect as a nested factor, we did find a significant management effect (F=9.2,P=0.008, df=1, 16). These results showed that shrub-clearing negatively affected the soil DHA during the dryseason.

However, no significant effect of the managementpractice on soil dehydrogenase was found in autumn inany of the three sites studied. Only in one site (Tiradero)was a marginally significant trend (P=0.066) observed,with higher DHA values in the control zone than in theshrub-cleared zone (Fig. 2), as found in summer for drysoils. A possible interpretation of the different quantita-tive effects of the forest management practices on theDHA activity is that the negative effect of shrub-clearingon soil microbiological activity is produced underdrought conditions, but is almost negligible when soilsare moist. The microclimatic changes induced by thepractice of shrub-clearing, such as higher radiation and amore dramatic oscillation in soil water content and soiltemperature, would be more influential on soil processesduring extreme conditions of drought. Besides, it may bealso possible that in the shrub-cleared zones the practiceof shrub-clearing provokes a shift in the composition of

Fig. 1 Dehydrogenase activity (DHA) in forest soil (Panera site)measured in summer and autumn. Control and shrub-cleared zonesare compared (n=12). Vertical bars represent SE. INTF p-Iodo-nitrotetrazolium formazan

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soil microorganisms with populations more sensitive todrought. The non-significant effects of shrub-clearing onsoil DHA measured in autumn could also be due to therecovery of enzyme activity to background levels. Theextent of soil recovery will depend on the degree of thedisturbance caused by the management practice, thesoils resilience, and the time elapsed from the last timethe practice was applied. Perucci (1992) found that theeffect of compost addition on soil DHA was only shown1 month after the compost was applied, and thereafter,no effect of this practice was observed. In this case, toassess better the impact of shrub-clearing on soil DHA,more frequent monitoring is needed.

Site effects

Significant differences were found between the soilDHA measured in the three forest plots in autumn, (Table 2). The Panera site showed the highest DHA values (Fig. 2).

The three experimental plots, located in the same for-ested area, were dominated by cork oak trees; butshowed differences in plant species composition and soilproperties (Table 1), and these differences could be re-sponsible for the variations in DHA. Firstly, the speciescomposition of the forest canopy plays an important rolein determining the type of soil organic matter and its associated decomposition processes. Thus, the semi-deciduous oak Q. canariensis has a higher percentagecover than Q. suber in the Panera plot (the ratio of Q. canariensis to Q. suber is 1.29) whereas the oppositeoccurs in the Tiradero site (the ratio of Q. canariensisto Q. suber is 0.53), and in the Buenas Noches site Q. canariensis is totally absent (M. Diaz-Villa, personalcommunication). This varying proportion of semi-decid-uous versus evergreen oaks in the forest canopy will bereflected in soil biological and biochemical properties.The leaf-shedding period of the semi-deciduous oak ismainly in autumn, while the evergreen cork oak shedsleaves throughout the year. Moreover, the decompositionrate of semi-deciduous oak litter is higher than that of

cork oak, as shown by Gallardo and Merino (1993) in alitter bag experiment near the Panera site, because of thelower cutin content and the lower toughness of the semi-deciduous leaves. Indeed, in this particular Mediterra-nean ecosystem, the rate of litter decomposition is moreaffected by the leaf cutin content and leaf toughness than by other chemical constituents or physical features(Gallardo and Merino 1993).

Secondly, differences in soil DHA between sitescould also be due to the different soil texture (Table 1).The Panera site showed both the highest clay content andhighest DHA. Soil texture has been reported as a key de-terminant of microbial ecology (Stotzky 1986). Ladd et al. (1996) found a positive relationship between claycontent and the soil microbial biomass content, andBeyer et al. (1992) found a positive correlation betweenclay content and enzyme activity (DHA). Soil texture affects other soil properties, such as water availability,nutrient supply (especially cations) and, to some extent,pH values, which in turn determine microbial growthand activity (Stotzky 1986; Ladd et al. 1996; Grres et al. 1998; Leirs et al. 2000; Zeller et al. 2001). In general, fine-textured soils have more micropores thansandy soils. Soil micropores protect mineralising micro-organisms against grazers (Killham 1994) and this canbe one of the reasons for the higher microbial biomassand enzyme activity of fine-textured soils.

Relationships with soil variables

Soil DHA was positively and significantly correlatedwith soil pH, Ca, Mg, K and water content (Table 3). Onthe other hand, no correlation was found between dehy-drogenase enzyme activity and total soil C and N. Theseresults agree with those reported by Beyer et al. (1992).However, Leirs et al. (2000) reported a clear positiverelationship between soil DHA and soil C. Probably inthese forest ecosystems soil microorganisms are nutrientrather than C limited, since DHA did not respond to thevariation of C contents or to the C:N ratio. The observedincrease in DHA from dry (summer) to wet (autumn)season, shows a positive relationship between DHA andwater content. The positive effect of increasing watercontent and nutrient addition on soil DHA has been already reported (Nannipieri et al. 1990).

Fig. 2 DHA in forest soil measured in the three selected plots dur-ing autumn. Control and shrub-cleared zones are compared(n=12). Vertical bars represent SE. B Noches Buenas Noches;for other abbreviations, see Fig. 1

Table 2 Two-way ANOVA for DHA of forest soils, measured inautumn. F-values for main effects (management and site) and two-way interactions are presented

df F PMain effectsManagement 1 0.51 0.47 n.s.Site 2 21.19 1.00107***Two-way interactionsManagementsite 2 1.27 0.28 n.s.

***P

Acknowledgements This study was supported by the CICYT-FEDER, 1FD97-0743-C03-03, project. We thank Luis V. Garcia,Eduardo Gutierrez and Malole Diaz-Villa for their field assistance,and Juan Arroyo for advice on the plot design. We also thank Felipe Oliveros (director) and the staff of Los Alcornocales Natu-ral Park for their support and cooperation in setting up the experi-mental plots. Personnel of TRAGSA conducted the sylviculturalpractices. Rafael Lpez and the Soil Laboratory staff at the IRNAS made chemical analyses of soil samples.

References

Allen SE (1989) Chemical analysis of ecological materials. Black-well, Oxford

Banerjee MR, Burton DL, Depoe S (1997) Impact of sewagesludge application on soil biological characteristics. AgricEcosyst Environ 66:241249

Banerjee MR, Burton DL, McCaughery WPP, Grant CA (2000)Influence of pasture management on soil biological quality. J Range Manage 53:127133

Benefield CB, Howard PJA, Howard DM (1977) The estimationof dehydrogenase activity in soil. Soil Biol Biochem 9:6770

Bergstrom DW, Monreal CM, King DJ (1998) Sensitivity of soilenzyme activity to conservation practices. Soil Sci Soc Am J62:12861295

Beyer L, Wachendorf C, Balzer FM, Balzer-Graf UR (1992) Theeffect of soil texture and soil management on microbial bio-mass and soil enzyme activities in arable soils of NorthwestGermany. Agrobiol Res 45:276283

Camia F, Trasar-Cepeda C, Gil-Sotres F, Leirs C (1998) Measurement of dehydrogenase activity in acid soils rich inorganic matter. Soil Biol Biochem 30:10051011

Deka RN, Wairiu M, Mtakwa PW, Mullins CE, Veenendaal EM,Townsend J (1995) Use and accuracy of the filter-paper tech-nique for measurement of soil matric potential. Eur J Soil Sci46:233238

Dick RP (1992) A review: long-term effects of agricultural sys-tems on soil biochemical and microbial parameters. AgricEcosyst Environ 40:2536

Dick RP (1997) Soil enzyme activities as integrative indicators ofsoil health. In: Pankhurst CE, Doube BM, Gupta VVSR (eds)Biological indicators of soil health. CAB International, NewYork, pp 121156

Gallardo A, Merino J (1993) Leaf decomposition in two Mediter-ranean ecosystems of southwest Spain: influence of substratequality. Ecology 74:152161

Garcia C, Hernandez T, Costa F (1994) Microbial activity in soilsunder Mediterranean environmental conditions. Soil Biol Bio-chem 26:11851191

Garcia C, Hernandez T, Albaladejo J, Castillo V, Roldn A (1998)Revegetation in semiarid zones: influence of terracing and organic refuse on microbial activity. Soil Sci Soc Am J62:670676

Gee GW, Bauder JW (1986) Particle-size analysis. In: Klute A(ed) Methods of soil analysis. Part 1. Physical and mineralogi-cal methods. American Society of Agronomy, Madison, Wis.pp 383411

Grres JH, Dichiaro MJ, Lyons JB, Amador JA (1998) Spatial andtemporal patterns of soil biological activity in a forest and anold field. Soil Biol Biochem 30:219230

Killham K (1994) Soil ecology. Cambridge University Press,Cambridge

Ladd JN, Foster RC, Nannipieri P, Oades MJ (1996) Soil structureand biological activity. In: Bollag JM, Stotzky G (eds) Soilbiochemistry, vol 9. Dekker, New York, pp 2377

Leirs MC, Trasar-Cepeda C, Seoane S, Gil-Sotres F (2000) Biochemical properties of acid soils under climax vegetation(Atlantic oakwood) in an area of the European temperate-humid zone (Galicia, NW Spain): general parameters. SoilBiol Biochem 32:733745

106

Multiple linear regression parameters for dehydroge-nase are shown in Table 4. Several soil properties (pH,water content, C, N, C/N, Ca, Mg, K and P) measured inautumn samples (n=72) were considered to fit the multi-variate linear regression model. The forward stepwise re-gression analysis included in the model the followingvariables: pH, K, and N. However, only pH and K vari-ables were significant regression components, while N(although included in the regression model) made no sig-nificant contribution to R2 (Table 4). According to theseresults, soil pH is the best predictor of DHA, and ac-counts for the highest proportion of the overall varianceexplained.

In conclusion, the DHA in forest soils of Los Alcornocales Natural Park (S. Spain) is high, comparedto the few reported data. In general, DHA was higher inautumn (with a higher soil moisture content) than duringthe dry summer. The shrub-clearing management prac-tice induced a decrease in the soil DHA during summer(dry season), but this effect was not apparent during the autumn (wet season). However, site factors (forestcanopy species composition, soil texture, pH, and avail-able nutrients) and seasonal sampling date were greaterdeterminants of the variation in DHA than managementfactors. Nutrient supply and soil pH were better predic-tors of DHA than the amount and quality (based on itsC:N ratio) of the soil organic matter.

Table 3 Pearson correlation coefficients between some selectedsoil properties and DHA

DHA pH K N

pH 0.55** 1.00 0.35** 0.04Water content 0.35** 0.58** 0.14 0.18Total C 0.12 0.26* 0.33** 0.68**Total N 0.04 0.04 0.53** 1.00C/N ratio 0.08 0.36** 0.03 0.02Available Ca 0.32** 0.41** 0.58** 0.46**Available Mg 0.32** 0.40** 0.65** 0.55**Available K 0.32** 0.35** 1.00 0.53**Available P 0.06 0.17 0.05 0.39**

*P

Schneider K (1998) P availability in forest soils and its role for thenutrition of Quercus pyrenaica Willd. in the Sierra de Gata,western Spain. Thesis. Universitaet Hohenheim of Stuttgart,Stuttgart

Skujins J (1973) Dehydrogenase: an indicator of biological activi-ties in arid soils. Bull Ecol Res Commun [Stockh] 17:235241

StatSoft (1997) Statistica for Windows, version 5.1. Statsoft, Tulsa, Okla.

Stotzky G (1986) Influence of soil mineral colloids on metabolicprocesses, growth, adhesion and ecology of microbes and viruses. In: Huang PA, Schnitzer M (eds) Interactions of soilminerals with natural organics and microbes. Soil Science Society of America, Madison, Wis. pp 305428

Trasar-Cepeda C, Leirs C, Gil-Sotres F (1999) Biochemical proper-ties of acid soils under climax vegetation (Atlantic oak-wood) inan area of the European temperate-humid zone (Galicia, NWSpain): specific parameters. Soil Biol Biochem 32:747755

Zeller V, Bardgett RD, Tappeiner U (2001) Site and managementeffects on soil microbial properties of subalpine meadows: a study of land abandonment along a north-south gradient inthe European Alps. Soil Biol Biochem 33:639649

107

McBrayer JF, Cromack K Jr (1980) Effect of snow-pack on oak-litter breakdown and nutrient release in a Minnesota forest. Pedobiologia 20:4754

Miller M, Dick RP (1995) Thermal stability and activities of soilenzymes as influenced by crop rotations. Soil Biol Biochem27:11611166

Montoya JM (1988) Los alcornocales. Ministry of Agriculture,Fisheries and Food, Madrid

Nannipieri P, Grego S, Ceccanti B (1990) Ecological significanceof biological activity. In: Bollag J-M, Stotzky G (eds) Soil bio-chemistry, vol 6. Dekker, New York, pp 293355

Ojeda F, Maran T, Arroyo J (2000) Plant diversity patterns inthe Aljibe Mountains (S. Spain): a comprehensive account.Biodiv Conserv 9:13231343

Pankhurst CE, Doube BM, Gupta VVSR (1997) Biological indica-tors of soil health: synthesis. In: Pankhurst CE, Doube BM,Gupta VVSR (eds) Biological indicators of soil health. CABInternational, New York, pp 419435

Perucci P (1992) Enzyme activity and microbial biomass in a field soil amended with municipal refuse. Biol Fertil Soils14:5460