Applied Soil Ecology 86 (2014) 82–90

Douglas-fir reforestation in North Apennine (Italy): Performance onsoil carbon sequestration, nutrients stock and microbial activity

Livia Vittori Antisari a, Gloria Falsone a, Serena Carbone a, Sara Marinari b,*,Gilmo Vianello a

aDipartimento di Scienze Agrarie, Alma Mater Studiorum, Università di Bologna, Via Fanin, 40, 40127 Bologna, ItalybDipartimento per la Innovazione dei Sistemi Biologici. Agroalimentari e Forestali, Università degli Studi della Tuscia, Via S. Camillo de Lellis, Viterbo 01100,Italy

A R T I C L E I N F O

Article history:Received 18 April 2014Received in revised form 12 September 2014Accepted 15 September 2014Available online xxx

Keywords:Douglas-firNatural beech forestSoil qualityNutrientStocksPedotransfer

A B S T R A C T

The aim of the study was to determine the effect of mature Douglas fir reforestation (DR) on nutrients andC stocks in mineral soils compared with beech forests (BF) at two altitudes in North Apennine, Italy, ataround (1) 1028 and (2) 1281 m a.s.l. To explore the effects of Douglas fir reforestation with respect tonatural beech forest, the microbial biomass and its activity were also taken in account as a driving force oforganic C mineralization. For each fixed-depth mineral layer (0–5, 5–10 and 10–30 cm) the bulk densityvalues were determined, also estimated using common pedotransfer functions (PTF). Total organic C(TOC) stock in the 0–30 cm layer was determined using both the measured and the PTF-estimated bulkdensity. The nutrients stocks (C pools, N, P, S, Ca) as well as potential microbial respiration and enzymeactivities per centimeter were also calculated for the whole depth. TOC and C pools were positivelyaffected by Douglas fir at 1028 m a.s.l. with respect to the other altitude and trees cover. In DR1 withrespect to DR2, BF1 and BF2, double contents of TOC (344 �13 vs. 157 � 1, 122 � 6, 162 � 1 g m�2 cm�1)and residual C pool (220 � 8 vs. 65 �1, 63 � 3, 116 � 1 g m�2 cm�1) were observed. Conversely, P stockswere significantly higher under beech at 1300 m a.s.l. because highest P litter content were found in BF2.Moreover, the activity of enzyme involved in C cycle (SEIc) suggest that to establish the effects on soilorganic matter biochemical degradation an interaction between plant cover and altitudes may occur. Thefast biochemical degradation of organic matter produced by beech forest and Douglas fir plantationoccurred at 1281 and 1028 m a.s.l., respectively (BF1 vs. BF2 = 1.40 vs. 4.35 DF1 vs. DF2 = 2.54 vs. 2.14 mmolMUF m�2 h�1 cm�1). In conclusion, in the North Apennine (Italy) Douglas fir old reforestation withrespect to natural beech forest caused substantial improvement on soil C sequestration, N stock andmicrobial activity at lower altitudes (1028 m vs.1281 m a.s.l.). Moreover, to assess soil nutrient stocks, theapplicability of PTFs might be improved by the use of soil depth in BD estimation.

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1. Introduction

Forest ecosystems are an invaluable resource for biodiversityand environmental sustainability (Anderson, 2003; Lejon et al.,2005). In Europe, in the second half of 1900, the production ofhigh-quality wood has led to a major forests exploitation in whichthe native deciduous species were replaced with monospecificconifer plantations, often of exotic origin, characterized by highproductivity (Lejon et al., 2005). The fear, that exotic species couldchange the ecosystems, has stimulated the study in the short andlong-term of their impact on soil fertility, biological diversity

* Corresponding author: Tel.: +39 761 357288.E-mail address: marinari@unitus.it (S. Marinari).

http://dx.doi.org/10.1016/j.apsoil.2014.09.0090929-1393/ã 2014 Elsevier B.V. All rights reserved.

(Herbauts and de Buyl, 1981; Ranger et al., 1990), giving rise toquestions about the forest ecosystems functioning (Noirfalise andVanesse, 1975; Hobbie, 1992 Hobbie, 1992). At the end of 2000,increased concerns were observed for the ecological risks posed bymonoculture conifers (Augusto et al., 2002; Rothe et al., 2002) andthe studies were addressed to applied to the sustainable forestmanagement maintaining ecosystem functioning (Zerbe, 2002;Janowiak and Webster, 2010).

Douglas-fir played an important role in Italian plantationforestry because, within its optimal vegetation zone ranged from600 to 1000 m a.s.l., no indigenous conifer has similar character-istics of productivity and timber quality (Corona et al., 1998). Theforest management in many areas of the Apennines is failed withthe consequent abandonment, no-renewal and degradation offorests. The tendency in Europe is to replace monocultures of

L.V. Antisari et al. / Applied Soil Ecology 86 (2014) 82–90 83

conifers with native broadleaves, anyhow, a detailed knowledge ofthe effect of tree species on soil processes is necessary, in order topreserve the ecosystem processes (Malchair and Carnol, 2009).

The forest floor, seldom confused with humus, is very importantin that it provides a source of food and habitat for myriad microflora and fauna, whose activity is essential for the maintenance ofthe nutrient cycles of nitrogen, phosphorous and sulfur.

Generally, coniferous species have often acidified soils, reducedthe cycling of nutrients, depressing microbial activity (VittoriAntisari et al., 2010) and these effects are probably partly due torecalcitrant compounds in coniferous needles (Berger et al., 2012),while little information was available for Douglas-fir ecosystems.However, Douglas-fir compared with domestic coniferous species,showed lower acidifying effects on upper soil layers andcontributes to better humus forms, recycling nutrients moreeffectively and producing litter which could be easily decomposed(Kupka et al., 2013). Beech forest (Fagus sylvatica, L.) is common inphytoclimatic zone of Fagetum from 900 to 1600 m a.s.l. in NorthApennine (Italy) (De Philippis, 1951) as well as Douglas-fir(Pseudotsuga menziesii var. menziesii) reforestation stands but ina narrow range of elevation, from 600 to 1000 m a.s.l. (Corona et al.,1998). Douglas-fir plantations were performed as recovering ofdegraded agricultural and forest land, because of fast growth ofDouglas-fir and consequent increase of soil organic matter alongsoil profile. However, the optimum Douglas-fir vegetation zone toobtain high productivity and soil quality improvement has neverbeen tested in the North Apennine (Italy). Moreover, the fear thatexotic species could change the ecosystems has stimulated thestudy in the short and long-term of their impact on soil fertility andnutrient stocks, no expectation was made with respect to soilorganic carbon sequestration. In fact, little is known about theeffect of this exotic species on nutrients biogeochemical cyclealong soil profiles (Welke and Hope, 2005). For this reason, the aimof the study was to determine the effect of Douglas-fir old

Fig. 1. Localization of the survey a

reforestation across two elevations (1028 and 1281 m a.s.l.) in theNorth Apennine (Italy), on nutrients and organic C stocks inmineral soils. To explore the effects of Douglas-fir reforestationwith respect to natural beech forest, the microbial biomass and itsactivity were also taken in account as a driving force of organic Cmineralization.

2. Materials and methods

2.1. Site description and field survey

The study area is included between 1000–1300 m a.s.l., and it islocated in the Corno alle Scale park (Northern Emilia-TuscanyAppenines, Italy). Natural beech forests (F. sylvatica, L.) andDouglas-fir (P. menziesii Franco) reforestations dominate theFagetum warm phytoclimatic subzone (below 1300 m a.s.l.according to the Pavari’s phytoclimatic classification; De Philippis,1951). The mean air temperature is 9 �C and the annualprecipitation ranges from 1270 mm (weather station in Sestola,1020 m a.s.l.) to 2100 mm (weather station in Passo Porretta,1313 m a.s.l.). Soil develop on Cervarola massive sandstone, withparticle size from coarse to fine, consisting in a prevailing silica–clastic components such as quartz, feldspar, mica, biotite andchlorite. In the District “Monte Cavallo Corno alle Scale”, beechforest (F. sylvatica L.) is prevailing and the stands are monospecific.The beech forest is natural pure and the majestic century-oldspecimens of tall trees are rare, replaced by plants of smaller sizefor the prevailing coppice management. The minimum cuttingcycle is 30 years. However, in this last 50 years the beech forest isabandoned and for this reason the plants have reached heightsbetween 18 and 22 meters and diameters of logs between 160 and180 cm. Some tree specimens have bigger size, up to 30–35 m talland trunk diameter of 2 m. The Douglas-fir reforestation is 70 years

rea and soil sampling scheme.

Table 1Main descriptive elements of investigated soil profiles and soil classification.

Profile Horizons Color Munsell Structure Texture Consistence Roots Rock fragments Soil classification

Master Depth (cm) Boundary Dry Moist

D T G S T D M S P Q S S V% R

BF1 Oi 0.5–0 A SA1 0–3 A S 10YR 4/3 7.5YR 3/3A2 03-07-2009 C W 7.5YR 4/2 7.5YR 3/2 1 vf gr sl S VFR (w) ss (w) po 3 vf FGR 4 1 Epileptic CambisolA3 7/9–11/13 G W 7.5YR 4/2 7.5YR 3/2 1 f sbk sl S L (w) so (w) po 3 fm FGR 5 1 (Eutric)Bw 11/13–28 G W 7.5YR 5/4 7.5YR 3/4 0 f sg ls L L (w) ss (w) po 1 m FGR 13 3C 28–33 G I 7.5YR 5/4 7.5YR 4/4 0 m sg sl L L (w) so (w) po 1 f MGR 21 3C/R 33+ U Moderately weathered sandstone (Cervarola formation)

DR1 Oi/Oe 5/8–3/6 A S 10YR 4/4 7.5YR 3/2Oa 3/6–0 C W 7.5YR 2./2 7.5YR 2/1 1 f gr sl S FR (w) so (w) po 1 f FGR 11 1 Epileptic CambisolA 0–5/21 G I 7.5YR 4/3 7.5YR 3/2 1 f sbk sl S VFR (w) ss (w) ps 1 fm MGR 12 1 (Humic, Dystric)Bw 5/21–22/32 D I 7.5YR 5/6 7.5YR 4/4 2 f sbk sil S FR (w)s (w) p 1 m FGR 14 3C 22/32–37 D I 7.5YR 5/8 7.5YR 5/4 0 m sg sl L L (w) ss (w) ps 1 m MGR 22 3C/R 37+ U Moderately weathered sandstone (Cervarola formation)

DR2 Oi 8.5–8 A SOe 8–5 C W 10YR 3/3 10YR 3/2Oa 5–0 G W 10YR 3/3 10YR 3/3 1 vf gr sl S L (w) ss (w)po 2 fm FGR 12 2 Epileptic CambisolA 5–13 C W 10YR 4/3 10YR 3/3 1 f gr sl L L (w) so (w) po 3 mco MGR 13 1 (Humic, Dystric)Bw1 13–26 G W 10YR 5/6 10YR 4/4 1 m abk ls S VFR (w) ss (w) po 1 m MGR 15 2Bw2 26–40 D I 10YR 5/4 10YR 4/3 0 m sg ls S L (w) ss (w) po 0 MGR 23 2C/R 40+ U Moderately weathered sandstone (Cervarola formation)

BF2 Oi 2–0 A SA 0–3 C W 10YR 3/3 10YR 3/2 1 vf gr s L L (w) so (w)po 2 vf FGR 4 3 Epileptic CambisolAB 3–12/18 G I 10YR 5/4 10YR 4/4 1 f abk ls S VFR (w) ss (w)po 1 m MGR 12 2 (Humic, Dystric)Bw 12/18–38/45 D I 5YR 4/6 5YR 3/4 1 f abk sl L L (w) so (w) po 1 m CGR 24 1C/R 38/45+ U Moderately weathered sandstone (Cervarola formation)

Codes according to Schoeneberger et al. (2002)Horizon boundary: (D) Distinctness: A = abrupt, C = clear, G = gradual, D = diffuse (T) Topography: S = smooth, W = wavy, I = irregular, U = unknown.Structure: (G) Grade: 0 = structureless, 1 = weak–2 = moderate (S) Size: vf = very fine, f = fine, m = medium–co = coarse T) Type: gr = granular, abk = angular blocky,sbk = subangular blocky, sg = single grain.Texture: field estimation: s = sand, ls = loamy sand, l = loam, sil = silt loam, sl = sandy loam.Consistence: Rupture resistence: (D) Dry: L =loose, S = soft, SH = slightly hard- (M) Moist: L = loose, VFR = very friable. (S) Stickiness: (w) so = non-sticky, (w) ss = slightly sticky,(w) s = moderately sticky, (P) Plasticity: (w) po = non-plastic, (w) ps = slightly plastic, (w) p = moderately plastic.Roots: (Q) Quantity: 0 = very few, 1 = few, 2= common, 3 = many. (S) Size: vf = very fine, f = fine, m = medium, co = coarse.Rock fragments: (S) Size: FGR = fine gravely, MGR = medium gravely; CGR = coarse gravely; VGR = very gravely; XGR = extremely gravely. (V%) Fragment content % by volume–(R) Roundness: 1 = angular, 2 = subangular, 3 = subrounded.

84 L.V. Antisari et al. / Applied Soil Ecology 86 (2014) 82–90

old and the tree height is variable from 60 to 75 m and the diameterof the trunk is around 1–1.5 m. Also this stand is now abandoned.

Four sites in similar and comparable topographic position andrepresentative of vegetation cover were selected: two sites inbeech forest (BF1 and BF2) and two sites in Douglas-firreforestation (DR1 and DR2) were chosen from 1028 to 1281 m(Fig. 1). The soil profiles investigated in this paper were chosenafter the study of pre-existing pedological survey due to soils mapsmade by Emilia Romagna Region (RER, 1985a; Ungaro et al., 2005).The soil profiles called DR1, BF1, DR2 and BF2 fall in soil unitsP5 and P6, respectively, of mountain band (800–1200 m a.s.l.). Thephytoclimatic zone is characterized by beech forest; the parentmaterial is flysch rock and the morphology is relatively steep (RER,1985b,c,c). In particular, the P5 soil unit is between 900–1000 m a.s.l. and P6 soil unit from 1000–1200 m a.s.l.; all physicochemicalcharacters of our soil profiles correspond with those determined bysoil units survey in which one soil profile was open each 150 acresand one control each 50 acres (scale 1:50,000) according to Goosen(1967).

The spatial variability of soil properties was determined withsoil units survey and in the soil profiles studied in this paper,skeleton, pH, total carbonate content and texture were stackablewith those obtained by the RER survey, as well as the slope,erosion, rockiness, stoniness.

All the soil profiles investigated fall in beech phytoclimaticzone, but the DR1 and DR2 profiles were chosen under Douglas-fir

reforestation. After the second world war (1947/48) many areas ofApennine have been converted and evergreen species (Picea abiesL., Picea alba Mill., Pseudotsuga menziesii Franco, Pinus mugo Turraand Pinus nigra Arnold) have been planted in place of the beechforest. In these areas, Douglas-fir was used for valuable timber(Corona et al., 1998). Additional sources of insight into the studyarea are derived by forest and vegetation map (RER, 2002) andgeolithologic map (ISPRA, 2011).

In the study area, the moderate elevation marks the pedocli-matic mesic soil temperature regime and udic soil moistureregime.

At each site, a representative soil profile was open anddescribed (Table 1) according to Schoeneberger et al., 2002. Rockfragments content was estimated visually in the soil profileaccording to FAO (2006). All soils were classified as EpilepticCambisols (IUSS Working Group WRB, 2007), and the thickness oftheir solum varied from 28 to 45 cm. For soil characterization, soilsamples of each genetic horizon were collected. In order todetermine the bulk density values undisturbed samples werecollected from two pits opened at each site using the core methodfrom fixed-depth mineral layer (0–5, 5–10 and 10–30 cm).

2.2. Soil physico-chemical properties and litter nutrient content

All the samples from the genetic horizons were air-dried andsieved (<2 mm), and the soil analyses were carried out on the

Table 2P, S and Ca content in leaves and needles litter collected in BF (BF1 and BF2) and DR(DR1 and DR2) sites, respectively. Mean with standard deviation values arereported. Different letters indicate significant difference (p < 0.05) between sites.

Site P S Ca

g kg�1 g kg�1 g kg�1

BF 1.06 � 0.20a 1.38 � 0.28 16.76 � 2.21a

DR 0.72 � 0.07b 1.66 � 0.26 10.34 � 0.77b

L.V. Antisari et al. / Applied Soil Ecology 86 (2014) 82–90 85

obtained fine earth fraction. The pH was determined potentio-metrically in a 1:2.5 soil: deionised water suspension. The texturewas obtained by the pipette method after dispersion of the samplewith a sodium hexametaphosphate solution (Gee and Bauder,1986). Total organic C (TOC) was determined by wet oxidation at160 �C with K2Cr2O7 1/3 M, according to the method of Springerand Klee (1954). The total N content was determined following theKjeldahl method (Bremner, 1965). The exchangeable cations (Ca,Mg, K, Na) were determined after exchange with NH4

+-acetate atpH 7 (Summer and Miller, 1996). The exchangeable elementconcentrations were measured by inductively coupled plasmaoptical emission spectroscopy (ICP-OES, Ametek, Spectro) and theeffective cation exchangeable capacity (ECEC) was calculated bythe sum of the exchangeable cations and the exchange aciditydetermined in KCl 1 M. The total element concentrations (P, S andCa) were measured by X-ray fluorescence spectroscopy.

Litter samples were also collected from each site in order toanalyze nutrient content of litterfall. The litters were air-dried andground in a mill to a mesh fraction less than 50 mm. The totalconcentration of P, S and Ca was determined by ICP-OES afterdigestion by HNO3 (Bergmann et al., 1994). Nutrient contents oflitter are showed in Table 2.

2.3. Soil carbon pools

Different soil C pools characterized by increasing persistence insoil were separated: K2SO4-extractable C from CHCl3 non-fumigated soil samples is used to estimate the labile C pool(Badalucco et al., 1992), as well as the microbial biomass C (Cmic)that is considered to be a major part of the active or labile C pools(von Lützow et al., 2007); humic C (C of fulvic acids – CFA and C ofhumic acids – CHA) is stabilized by humification processes and isconsidered to be refractory thus belonging to the passive soilorganic matter pools (von Lützow et al., 2007); residual C pool isstabilized by interactions with mineral surfaces and complexationthus belonging to the passive pool of soil organic matter (vonLützow et al., 2008).

For each genetic horizon Cmic was measured using thefumigation–extraction method of Vance et al. (1987), carried outon soil samples previously conditioned by incubation for one weekat 60% of their water holding capacity in a thermostat at 20 �C. Theorganic C extracted by 0.5 M K2SO4 from CHCl3-fumigated and non-fumigated samples were measured by a TOC-TN analyzer(Shimadzu, TOC-V/CPN).

Organic C was (TEC) extracted with a solution of 0.1 M NaOHand 0.1 M Na4P2O7 at 65 �C for 24 h (Vittori Antisari et al., 2010).The humic acids were separated from TEC by acidification (pH < 2)and centrifugation, while fulvic acids were separated from thenon-humified organic material by solid chromatography withpolyvinyl pyrrolidone resin, according to the method proposed byCiavatta et al. (1990). The organic C content in the humic and fulvicacid fractions (CHA and CFA) were determined by wet oxidation at160 �C with K2Cr2O7 1/3 M, according to the method of Springerand Klee (1954).

The residual C was calculated by the difference between thetotal organic C (TOC) and the separated active (K2SO4-extractable C

and Cmic) and passive (CFA and CHA) pools of C, thus it representedthe C fraction insoluble in alkali (i.e., humin C fraction).

2.4. Microbial respiration and enzyme activities

The respiratory activity of the soil biomass was determinedusing a slightly modified version of the Isermayer methodIsermeyer, 1952. The soil samples (10 g) were conditioned at60% of the WHC and pre-incubated for 3 days at 4 �C; then eachsample was placed in a hermetically sealed container (500 ml),which was fitted with a beaker containing 4 ml of 0.5 M NaOH totrap the evolved CO2; the basal respiration (BR) of each soilsamples (mg C–CO2 kg�1 soil) was computed as the average of thevalues measured during the 28-day period of analysis, at 1–3–7–10–14–21–28 days.

The metabolic (qCO2, Dilly and Munch 1998) and mineralization(qM, Pinzari et al., 1999) quotients were calculated according to thefollowing equations:

qCO2= BR/Cmic (mg C–CO2 basal h�1 g Cmic�1)

qM = 28 days cumulative respiration/TOC (mg C–CO2 g TOC�1)

Soil enzyme activities were measured according to the methodsof Marx et al. (2001) and Vepsäläinen et al. (2001), based on the useof fluorogenic methylumbelliferyl (MUF)-substrates. Soils wereanalyzed for b-cellobiohydrolase (EC 3.2.1.91), N-acetyl-&beta;-glucosaminidase (EC 3.2.1.30), b-glucosidase (EC 3.2.1.21), a-glu-cosidase (EC 3.2.1.20), acid phosphatase (EC 3.1.3.2), arylsulfatase(EC 3.1.6.1) and xylosidase (EC 3.2.2.27) using 4-MUF-b-D-cellobioside, 4-MUF- N-acetyl-b-glucosaminide, 4- MUF-b-D-glucoside, 4- MUF - a-D-glucoside, 4- MUF-phosphate, 4-MUF-sulphate and 4-MUF-7-b-D-xyloside as substrates, respectively.The four enzyme activities involved in carbon cycle were expressedby a synthetic enzyme index (SEIc= b-cellobiohydrolase + b-glucosidase + a-glucosidase + xylosidase). A moist sample (equiv-alent weight to 2 g oven-dry material) was weighed into a sterilejar and 50 ml of Na-acetate buffer pH 5.5 were added. Ahomogenous suspension was obtained by homogenising withUltraTurrax at 9600 rev min�1 for 3 min. Aliquots of 100 ml werewithdrawn and dispensed into a 96 well microplate (threeanalytical replicates sample�1 substrate�1). Finally, 100 ml of1 mM substrate solution were added giving a final substrateconcentration of 500 mM. Fluorescence was measured after 0, 30,60, 120, 180 min of incubation at 30 �C. Fluorescence (excitation360 nm; emission 450 nm) was measured with an automatedfluorimetric plate-reader (Fluoroskan Ascent).

2.5. Measurement and estimation of bulk density

From the undisturbed soil core the bulk density (BD) of the fineearth (<2 mm) of each fixed-depth mineral layer (0–5, 5–10 and10–30 cm) was quantified from the mass of the oven-dry soil(105� C) divided by the volume of the soil cores (Blake and Hartge,1986). For each fixed-depth mineral layer the bulk density valueswere also estimated using common pedotransfer functions (PTF)for BD. The well-known PTF of Adams (1973) was used to calculateBD. This approach is solely based on organic matter content andwas applied in several SOC studies (De Vos et al., 2005). The PTFs ofHarrison and Bocock (1981) were used to estimate BD as functionsof OM content and soil depth, whereas the PTFs of Leonavi9ciutè(2000) were used as function of OM content, texture and soilhorizons. Values of measured and estimated BD are reported inTable 3. The low variability of the measured BD further confirmedthe homogeneity of the studied soil based on the soil unitsidentified by RER survey.

Table 3Measured and estimated bulk density values. Mean with standard deviation values are reported for measured BD.

Soil profiles Horizons Fixed-depth mineral layers Measured BD Estimated BD by PTF (1) Estimated BD by PTF (2) Estimated BD by PTF (3)

cm g cm�3 g cm�3 g cm�3 g cm�3

BF1 A1, A2 0–5 0.999 � 0.02 1.19 0.980 1.24A2, A3 5–10 0.955 � 0.03 1.26 1.090 1.35A3, Bw, C 10–30 0.986 � 0.06 1.38 1.383 1.49

BF2 A, AB 0–5 0.805 � 0.01 1.23 1.037 1.31AB 5–10 0.894 � 0.02 1.30 1.169 1.40AB, Bw 10–30 0.926 � 0.09 1.37 1.330 1.54

DR1 A 0–5 0.970 � 0.03 0.97 0.727 0.73A 5–10 0.822 � 0.04 0.97 0.727 0.73A, Bw, C 10–30 0.882 � 0.04 1.26 1.087 1.44

DR2 A 0–5 0.905 � 0.05 1.19 0.973 1.24A, Bw1 5–10 0.931 � 0.05 1.23 1.037 1.30Bw1, Bw2 10–30 0.883 � 0.07 1.34 1.256 1.49

PTF (1) by Adams (1973); PTF (2) by Harrison and Bocock (1981); PTF (3) by Leonavi9ciutè (2000).

86 L.V. Antisari et al. / Applied Soil Ecology 86 (2014) 82–90

2.6. Nutrient concentration and microbial activity on soil volume basis

The stocks of total organic C, C pools (K2SO4-extractable C, Cmic,CFA, CHA and residual C), total N, total P, total S and total Ca werecalculated on a volume basis. For each mineral layer (0–5, 5–10,10–30 cm) the concentration of OC and C pools, N, P, S and Ca wascalculated by weighed mean of their concentration in thecorresponding horizons according to the depth. The stocks werethus calculated by taking into account the calculated concentra-tion, the bulk density of fine earth (<2 mm) of each pit, thethickness of layer and the coarse fragments (>2 mm) volume in thelayer. In the same way, potential microbial respiration and enzymeactivities were also calculated on a soil volume basis for eachmineral layer.

The total organic C stock of the 0–30 cm layer (i.e., the sum ofthe OC stocks of all fixed-depth mineral layers) was calculatedusing both the measured and the PTF-estimated BD values.

The mean value of organic C pools and nutrient stocks percentimeter in the 0–30 cm layer was used as synthetic index inorder to compare our data. For each nutrient, the stock percentimetre (expressed as g nutrient m�2 cm�1) was calculated asthe sum of the nutrient stock in the 0–5, 5–10 and 10–30 cm layersdivided by 30 cm (i.e., S nutrient stock of each layer/30 cm).Similarly, potential microbial respiration and enzyme activities percentimetre in the 0–30 cm layer were also calculated (expressed as

Table 4Main properties averaged for fixed-depth mineral layers of soils under Beech forest (B

Soil profiles Horizons Fixed-depth mineral layers pH

cm

BF1 A1, A2 0–5 6.2

A2, A3 5–10 6.3

A3, Bw, C 10–30 6.4

BF2 A, AB 0–5 5.1

AB 5–10 5.2

AB, Bw 10–30 5.2

DR1 A 0–5 6.3

A 5–10 6.3

A, Bw, C 10–30 5.4

DR2 A 0–5 4.9

A, Bw1 5–10 5.1

Bw1, Bw2 10–30 5.3

SOM: soil organic matter determined by multiply the content of organic C by 1.72; EC

mg C–CO2 m�2 h�1 cm�1 and mmol MUF m�2 h�1 cm�1, respec-tively).

2.7. Statistical analysis

The data on soil volume basis calculated for the two pits eachsites were averaged and standard deviation values of each sitewere determined. The correlation between the averaged organic Cpools stock and soil properties was evaluated using the Pearsoncoefficient. A two-way analysis of variance was carried out on thedata from each pit considering both site factors (i.e., vegetation andelevation). The normal distribution of data was checked using theShapiro–Wilk test. The threshold used for significance in allstatistical tests was set at 0.05. All data treatments were carried outusing SPSS software package (SPSS Inc. Chicago, IL).

3. Results

3.1. Soil properties averaged for fixed-depth mineral layers

In order to characterize the selected fixed-depth mineral layersof soil profiles in the different forest covers, soil properties of eachgenetic horizon were averaged on the basis of horizon thickness(Table 4). Soil pH values ranged from 4.9 to 6.4. For beech forest soiltexture was quite comparable along all fixed-depth mineral layers

F) and Douglas-fir reforestation (DR).

Sand Silt Clay SOM C/N ECEC

g kg�1 g kg�1 g kg�1 g kg�1 cmol+ kg�1

790 195 15 62.8 9.0 24.4773 210 17 44.2 8.8 14.8760 218 23 17.4 8.6 6.5

791 189 20 52.3 8.6 8.2669 285 46 34.4 13.3 5.3787 183 30 20.6 6.4 5.0

755 214 31 139.8 16.6 22.5755 214 31 139.8 16.6 22.5493 393 114 44.7 14.8 7.8

840 153 7 64.2 15.5 5.1769 205 26 52.3 15.5 4.4664 278 58 26.1 13.6 2.7

EC: effective cation exchange capacity.

Table 5Total organic C (TOC) stocks for 0–30 cm mineral layers of beech forest (BF) andDouglas-fir reforestation (DR) calculated using measured bulk density values (BD)and estimated using pedotransfer funcionts (PTF) for BD. Mean with standarddeviation values are reported for TOC stocks calculated by measured BD

Soil profile TOC stocks by measured BD TOC stocks calculated by PTF for BD

PTF (1) PTF (2) PTF (3)

kg m�2 kg m�2 kg m�2 kg m�2

BF1 4.71 � 0.04 6.13 5.54 6.51BF2 3.67 � 0.18 5.46 5.01 5.98DR1 10.31 � 0.38 12.49 10.01 11.62DR2 4.87 � 0.04 6.85 6.04 7.40

PTF (1) Adams (1973); PTF (2) Harrison and Bocock (1981); PTF (3) Leonavi9ciutè(2000).

Table 7Correlation matrix of organic C pools stock and soil properties.

TOC K2SO4-extractable C Cmic CFA CHA Residual C

pH 0.323 �0.325 0.333 0.361 0.664* 0.110Sand �0.543 �0.434 �0.111 �0.142 -0.095 �0.662*

Silt 0.504 0.384 0.068 0.090 0.082 0.622*

Clay 0.615* 0.534 0.203 0.256 0.120 0.733**

SOM 0.521 �0.419 0.186 0.147 0.646* 0.408ECEC 0.395 �0.417 0.393 0.231 0.863* 0.136

SOM: soil organic matter; ECEC: effective cation exchange capacity; TOC: totalorganic C; Cmic: microbial biomass C; CFA and CHA: C of fulvic and humic acids,respectively.

* p < 0.05.** p < 0.01.

L.V. Antisari et al. / Applied Soil Ecology 86 (2014) 82–90 87

while Douglas-fir sites showed higher variability of texture alongdepth, sand contents decreasing slightly with soil depth, whereasclay contents increasing accordingly. In soils, the organic mattercontent varied from 17.4 to 139.8 g kg�1, showing as expected adecreasing trend with depth. The C-to-N ratio ranged from 6.4 to13.3 for beech forest, whereas higher values of 13.6–16.6 weredetected for Douglas-fir reforestation. The ECEC values varied from2.7 to 24.4 cmol+ kg�1 overlapping the trend of organic matter.

3.2. Calculated and estimated stocks and storage of functional organiccarbon pools

The total organic C (TOC) stocks down to 30 cm of soil depth fordifferent forest type are shown in Table 5. The amount of TOCstocks, calculated using measured bulk density (BD) values,varied from 3.67 to 10.31 kg m�2. The site DR1 had the highestTOC stocks that decreased, increasing altitude. TOC stocks werealso calculated using different PTFs for BD and underestimationand overestimation were observed (Table 5). The application ofPTFs by Adams (1973) and Leonavi9ciutè (2000) led to aconsiderable overestimation of TOC stocks of 21–49% and 13–63%, respectively; whereas by Harrison and Bocock (1981) theTOC stocks were overestimated of 18–36% and underestimated of3% in the case of DR1.

Data of total organic C stock per centimetre and its distributionin the active and passive C pools are reported in Table 6. The TOCstock per centimetre varied from 122 to 344 g m�2 cm�1. Amongthe passive OC pools stored, the residual C represented the largestpart and varied from 41 to 72% of the TOC stored, whereas CFA andCHA represented the 8–13% and 17–44% respectively. Generally, theamount of passive OC pools stored decreased with site elevationand between forest cover Douglas-fir reforestation had higheramount of passive C stored. Data thus confirmed that the DR1 sitewas the most OC-rich soil, having higher passive OC stocks than theother sites. These features suggested that vegetation type was notthe only significant site factor on passive OC stocks, as interactionbetween vegetation and elevation was significant (p always <0.05).

Table 6Mean of organic C pools and nutrient stocks per centimetre (g m�2 cm�1) calculated for 0at different elevation (mean with standard deviation values are reported) and results o

Site Vegetation Elevation TOC K2SO4-extractable C Cmic

BF1 BF 1010 157 � 1 1.0 � <0.1 1.4 � 0BF2 BF 1284 122 � 6 1.4 � 0.1 1.8 � 0DR1 DR 1028 344 � 13 1.4 � 0.1 2.0 �

DR2 DR 1281 162 � 1 1.9 � <0.1 0.5 �

Vegetationa ** **

Elevationa ** ** *

Vegetation � Elevationa ** ** *

a Significance level for the effect.

In all sites, the percentage of the active C pool, both the labileK2SO4-extractable C and Cmic, represented less than 1–2% of thetotal storage of organic C. Also active OC pool was susceptible tosite factors, and whereas microbial biomass clearly had mostfavourable condition in DR1 site (Douglas-fir at lower elevation;interaction effect significant at p level <0.01), the K2SO4-extractable C was preferentially stored under Douglas-fir (p < 0.01)and at higher site (p < 0.01).

A correlation matrix was calculated to assess the relationshipsbetween soil properties and the soil carbon accumulation (Table 7).Total organic C showed significant (p < 0.05) correlation with claycontent and, as expected, also residual C was highly correlated(p < 0.01) with clay fraction. The CHA was significant correlatedwith pH, organic matter content and effective cation exchangecapacity.

3.3. N, P, S and Ca stocks

The total N stored ranged from 11 to 22 g m�2 cm�1 (Table 6). Nodifferences in N amount were detected between vegetation cover.However, DR1 showed the highest storage as result of interactionbetween elevation and vegetation site factors (p < 0.01) followingthe observed trend of passive OC pools. The P stored varied from3.3 to 9.0 g m�2 cm�1 and a higher stock was present under beechat higher elevation (p < 0.01). The amount of S stored in the soilsranged between 0.7 and 2.1 g m�2 cm�1, and similarly to P storagehigher stock was present under beech at higher elevation(p < 0.01). The stocks of Ca varied from 12 to 58 g m�2 cm�1 andthe site at lower elevation (DR1 and specially BF1) had the highestCa stored.

3.4. Microbial activity on soil volume base

The potential microbial activity was measured in terms of bothrespiration and enzyme activities. The basal respiration (BR) variedfrom 2.03 to 9.27 mg C–CO2 m�2 h�1 cm�1. The two sites at lowerelevation (BF1 and DR1) had four-fold microbial respiration thansites at higher elevation (BF2 and DR2) (Table 8). In addition, the

–30 cm layer of soils under both Beech forest (BF) and Douglas-fir reforestation (DR)f the two-way ANOVA.

CFA CHA Residual C N P S Ca

.5 21 � <1 69 � <1 65 � 1 18 � <1 6.4 � 0.1 0.7 � 0.1 58 � 1

.1 17 � 1 40 � 2 63 � 3 16 � 1 9.0 � 0.6 2.1 � 0.2 17 � 10.1 26 � 1 94 � 3 220 � 8 22 � 1 4.4 � 0.1 1.6 � 0.1 37 � 1<0.1 18 � <1 27 � <1 116 � 1 11 � <1 3.3 � 0.1 1.6 � 0.1 12 � <1

** * ** ** * **** ** ** ** * ** **** ** ** ** ** **

Table 8Mean of potential microbial respiration and enzyme activities per centimetre (mg C–CO2 m�2 h�1 cm�1 and mmol MUF m�2 h�1 cm�1, respectively) in the 0–30 cm layer ofsoils for under beech forest (BF) and Douglas-fir reforestation (DR) at different elevation (mean with standard deviation values are reported) and results of the two-wayANOVA.

Site Vegetation Elevation BR SEIc Chitinase activity Acid phosphatase activity Arylsulfatase activity

BF1 BF 1010 8.75 � 0.07 1.40 � 0.01 1.40 � 0.01 5.97 � 0.02 1.38 � 0.02BF2 BF 1284 2.03 � 0.10 4.35 � 0.09 2.23 � 0.06 15.01 � 0.33 6.59 � 0.18DR1 DR 1028 9.27 � 0.34 2.54 � 0.05 2.32 � 0.04 4.93 � 0.09 1.52 � 0.03DR2 DR 1281 2.71 � 0.01 2.14 � 0.01 1.09 � 0.01 7.63 � 0.01 3.48 � 0.02

Vegetationa ** ** * ** **

Elevationa ** ** ** ** **

Vegetation � elevationa ** ** ** **

BR: basal respiration; SEIc: synthetic enzyme index of C cycle.a Significance level for the effect.* p < 0.05.** p < 0.01.

88 L.V. Antisari et al. / Applied Soil Ecology 86 (2014) 82–90

mineralization quotient (qM,) had higher value in BF1, while themetabolic quotient (qCO2) was significantly lower at higheraltitude under both tree covers (Fig. 2). The activity of theenzymes involved in C cycle (SEIc) ranged from 1.40 to 4.35 mmolMUF m�2 h�1 cm�1 (Table 8), the highest value was observed underbeech at higher elevation (BF2) and under Douglas-fir at lowerelevation (DR1). The chitinase activity varied from 1.1 to 2.3 mmolMUF m�2 h�1 cm�1 with considerable variation among sites. Theacid phosphatase and arylsulfatase activities ranged from 4.90 to15.01 and from 1.38 to 6.59 mmol MUF m�2 h�1 cm�1, respectively.These two enzyme showed the highest activities under beech at1281 m a.s.l similarly to SEIc.

4. Discussions

Land use change, such as the replacement of native forest ofbeech by Douglas-fir plantation, affects various processes involvedin the soil organic matter turnover. In fact, the Douglas-fir

Fig. 2. Mean values of mineralization quotient (qM) and metabolic quotient (qCO2) in thand results of the two-way ANOVA. Bars are standard deviations.

plantations offer a rapid increase of soil organic matter but alsodifferences in SOM composition and quality (Jaffrain et al., 2007).In this case of study, the organic carbon stock, as well as the labile(K2SO4-extractable C) and passive C pools, were significantlyhigher under Douglas-fir reforestation; while the passive C storeddecreased with site elevation, the labile C pool increased. In theCzech Republic conditions, Douglas-fir was recognized as one ofthe most productive species (Kantor and Mareš, 2009; Podrázskýet al., 2009; Remeš et al., 2010). At lower altitudes the averageannual increment of the standing volume was three times higherfor Douglas-fir than for hardwood (Kupka et al., 2013). The highproductivity of Douglas-fir may also cause a relatively favorableeffects on the forest soil (Menšík et al., 2009; Podrázský and Kupka,2011). In fact, higher accumulation of the surface humus in thestands of Douglas-fir was found with respect to broadleavesspecies (Kupka et al., 2013). In agreement with previous results,our C stocks suggest that enhancement of C storage was occurredunder Douglas-fir at 1028 m a.s.l. probably due to an increase of

e 0–30 cm layers of soils under beech forest (BF) and Douglas fir reforestation (DR)

L.V. Antisari et al. / Applied Soil Ecology 86 (2014) 82–90 89

above and underground organic matter input since all C poolsincreased. Moreover, in our case the basal respiration was higher insoil under Douglas-fir with respect to beech forest, especially atlow elevation, but the C mineralization slowed down, as indicatedby the low value of qM. Therefore, our data confirmed a sloworganic C turnover under Douglas-fir plantation than natural beechforest. It is known that, the soil organic matter quality of epipendonunder coniferous resulted in a more recalcitrant and hydrophobiccomposition than that of epipedon under beech. For this reason alow mineralization quotient with consequent C accumulation issupposed to occur. However, under different climatic conditionscontrasting results have been obtained. In particular, undercontinental climate, with oceanic influence at 638 m a.s.l,Douglas-fir plantation led to decrease inputs of carbon to the soil,mineralization rates and size of the labile C pool (Gauthier et al.,2010). The growth of Douglas-fir is limited by the availability ofwater which is a function of soil physiography, and evaporativedemand (Brix, 1972; Spittlehouse, 1985; Carter and Klinka, 1990).Beedlow et al. (2013) found that Douglas-fir growth rate increasedwith temperature according to elevation, where water was not alimiting factor. Suboptimal temperatures, as well as reduced solarradiation and high atmospheric humidity deficits interact withbiological constraints to further limit productivity (Littell andPeterson, 2005). Moreover, in the north Apennines climatic factorsrelated to water balance have a greater influence on theproductivity of Douglas-fir plantations than topographic and soilfactors (Corona et al., 1998). Thus, given the complexity ofinteracting factors, a relatively small change in climate is likelyto alter Douglas-fir growth and forest composition. For this reason,to extend the soil quality assessment to a wide area, climaticfactors have to be very similar; even though the microenvironmentconditions cannot be excluded. In our study, the beneficial effectsof Douglas-fir on soil quality improvement was reflected by highorganic C stocks at 1028 m a.s.l., probably because at this elevationin North Apennines the exotic species found more favourableclimate conditions. At 1028 m a.s.l., soil under Douglas-fir hadhigher residual C stocks than under beech, therefore the vegetationshowed a pronounced effect on C pool storage, due to both the highplant productivity and the interactions of organic matter withminerals. In fact, at 1028 m a.s.l., soil showed a high content of claywhich may promote the preservation carbon passive pool. A moreintense mineral weathering may occur under Douglas-fir thanunder beech (Mareschal et al., 2013), as consequence clay fractionincreased in Bw horizon. As regards as the other nutrient stocks isconcerned, the total nitrogen stock was positively affected byDouglas-fir at 1028 m a.s.l. similarly to OC. Conversely, since P and Sstocks were significantly higher under beech at 1300 m a.s.l., theircontents were measured in leaves and needles litterfall in order toassess differences in terms of plants litter composition in BF and DRsites. A higher P content was observed in beech than in needleslitterfall (1.06 � 0.2 and 0.72 � 0.1 g kg�1 in litterfall from BF and DRsites, respectively). Thus, high P stocks under beech trees was inagreement with the high P content in litterfall. Conversely, Scontent in litterfall was similar among plant species (1.38 � 0.3 and1.66 � 0.3 g kg�1, respectively) and difference in S stock seemed tobe less affected by litter input and its quality. The highest soil P andS stocks under BF2 may also affect enzyme activities involved intheir biogeochemical cycles. In fact, acid phosphatase andarylsulphatase reached the highest values in BF2. Moreover, thehighest values of SEIc suggest an interaction between plant coverand altitudes to establish the effects on soil organic matterbiochemical degradation. The fast biochemical degradation oforganic matter produced by beech forest and Douglas-fir planta-tion occurred at 1281 and 1028 m a.s.l., respectively. Finally,according to previous investigations a decrease of basic nutrientsuch as Ca, was observed in the organic-mineral horizon of

Douglas-fir with respect to broadleaves (Martiník 2003; Kupkaet al., 2013). This result could be due to the different litterfallelement composition consisting in lower Ca content in Douglas-firwith respect to beech (10 � 1 vs. 17 � 2 g Ca kg�1). According to theclimate change point of view, the temperature increase withdecreasing summer precipitation (at least in central and southernEurope) and more intense events (Cristensen et al., 2007), may leadto increased dry periods. In this context, the Douglas-fir could finda progressive adaptation at altitudes above 1300 m asl. On the basisof a silvicultural management program, instead of a mixed forest, amosaic of pure species (Kolström et al., 2011; Keenan, 2012) mightbe suggested. Douglas-fir, due to its rapid growth, can promote Csequestration and organic components distribution along theprofile of soils with limited natural fertility.

As regards to the use of PTFs for estimation of C stocks, forAdams's PTF, our data was in agreement to Wiesmeier et al. (2012),who found considerable overestimation of SOC stocks in forestsoils and concluded that Adams's PTF is only applicable for roughestimation of SOC under cropland and grassland sites. Similarly, inour study the SOC stocks were overestimated according toLeonavi9ciutè's PTF, probably due to the overweighed effect oftexture variation; since Leonavi9ciutè's PTF was performed for soilswhich have an active redistribution of colloidal phase (i.e., eluvialand illuvial horizons). In our case the use of Harrison and Bocock'sPTF seemed to improve the SOC stocks estimation since the soilsunder study are Cambisols. Finally, the results suggest that theapplicability of PTFs in few developed forest soils could probablybe improved when OM content and soil depth is taken into accountin BD estimation.

5. Conclusion

The reforestation with exotic coniferous, such as Douglas-fircompared with indigenous beech forest in the North Apennine(Italy), caused substantial improvement on soil C sequestration, Nstock and microbial activity at lower with respect to the higheraltitudes (1028 m vs. 1281 m a.s.l.). However, the Douglas-fir couldfind a progressive adaptation at altitudes above 1300 m asl in theperspective of climatic change. Moreover, in this study it has beendemonstrated that the applicability of PTFs might be improved bythe use of soil depth in BD estimation.

Acknowledgements

The results reported here come from the labors of two SoilScience laboratories at Bologna and Tuscia Universities. Theauthors thank the “Centro Sperimentale per lo Studio e l'Analisidel Suolo” of Alma Mater Studiorum, Dr. Andrea Simoni, Dr. RositaMarabottini for technical support in soil analyses, two anonymousreviewers whose thoughtful comments have greatly improved thismanuscript.

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