REGULAR ARTICLE

Assessment of soil nitrogen and phosphorous availabilityunder elevated CO2 and N-fertilization in a short rotationpoplar plantation

A. Lagomarsino & M. C. Moscatelli &M. R. Hoosbeek & P. De Angelis & S. Grego

Received: 28 December 2007 /Accepted: 31 March 2008 / Published online: 30 April 2008# Springer Science + Business Media B.V. 2008

Abstract Photosynthetic stimulation by elevated[CO2] is largely regulated by nitrogen and phosphorusavailability in the soil. During a 6 year Free Air CO2

Enrichment (FACE) experiment with poplar trees intwo short rotations, inorganic forms of soil nitrogen,extractable phosphorus, microbial and total nitrogenwere assessed. Moreover, in situ and potentialnitrogen mineralization, as well as enzymatic activi-ties, were determined as measures of nutrient cycling.The aim of this study was to evaluate the effects ofelevated [CO2] and fertilization on: (1) N mineraliza-tion and immobilization processes; (2) soil nutrientavailability; and (3) soil enzyme activity, as an

indication of microbial and plant nutrient acquisitionactivity. Independent of any treatment, total soil Nincreased by 23% in the plantation after 6 years due toafforestation. Nitrification was the main processinfluencing inorganic N availability in soil, whileammonification being null or even negative. Ammo-nium was mostly affected by microbial immobiliza-tion and positively related to total N and microbialbiomass N. Elevated [CO2] negatively influencednitrification under unfertilised treatment by 44% andconsequently nitrate availability by 30% on average.Microbial N immobilization was stimulated by [CO2]enrichment and probably enhanced the transformationof large amounts of N into organic forms lessaccessible to plants. The significant enhancement ofenzyme activities under elevated [CO2] reflected anincrease in nutrient acquisition activity in the soil, aswell as an increase of fungal population. Nitrogenfertilization did not influence N availability andcycling, but acted as a negative feed-back onphosphorus availability under elevated CO2.

Keywords Enzymes .Microbial biomass . N cycling .

Soil nutrients

Introduction

Terrestrial net primary productivity (NPP) and soilorganic matter (SOM) dynamics are affected byinteractions between plants and soil microorganisms.

Plant Soil (2008) 308:131–147DOI 10.1007/s11104-008-9614-4

Responsible Editor: Barbara Wick.

A. Lagomarsino (*) :M. C. Moscatelli : S. GregoDepartment of Agrobiology and Agrochemistry,University of Tuscia,V. S. Camillo de Lellis s.n.c.,01100 Viterbo, Italye-mail: lagomarsino@unitus.it

A. Lagomarsino : P. De AngelisDeparment of Forest Environment and Resources,University of Tuscia,V. S. Camillo de Lellis s.n.c.,01100 Viterbo, Italy

M. R. HoosbeekDepartment of Environmental Sciences,Earth System Science group, Wageningen University,P.O. Box 47, 6700 AA Wageningen, The Netherlands

Microbial activity affects the availability of nutrients insoils, which in turn influences the extent of plantresponses to elevated [CO2] (Cardon 1996). Anassessment of future net carbon (C) sequestrationshould therefore consider the limitations imposed byavailable nutrients in the soil (Van Groenigen et al.2002).

Short term experiments indicate that mineralnitrogen (N) availability in the soil can decrease,increase or exhibit no significant change beneathtemperate trees grown under elevated atmospheric[CO2] (Zak et al. 2000). Explanations for lower soil Navailability under elevated [CO2] include decreasedleaf litter N concentrations (Cotrufo et al. 1998, 2005)and increased N immobilization due to increasedlabile C exudates causing increased microbial activity(Janssens et al. 1998). However, some studies suggestthat N mineralization could increase in response toenhanced labile C supply from roots (Zak et al. 1993).

At the POP-EuroFACE site elevated [CO2] en-hanced C inputs to soil through a larger fine rootbiomass (Lukac et al. 2003) and higher fine rootturnover rates (Godbold et al. 2006), thus increasingthe labile C fraction in soil (Hoosbeek et al. 2006). Atthe same time [CO2] enrichment induced a decreaseof N concentration in all plant tissues and in allspecies (Cotrufo et al. 2005). The consequent increaseof the C/N ratio of litter (Cotrufo et al. 2005) and oflabile SOM (Lagomarsino et al. 2006) favoured amicrobial immobilization process. N availabilitylimitation due to increased NPP under elevated[CO2] may be overcome by plants through somecombination of increased N uptake from the soil and/or more efficient use of the assimilated N (Finzi et al.2007). During the first cycle of the plantation,increased N use efficiency (NUE) sustained higherproductivity rates (Calfapietra et al. 2007) andHoosbeek et al. (2004) observed a priming-effect onsoil organic matter due to increased C inputs to thesoil. However, during the second cycle the priming-effect had ceased due to decreasing inorganic soil Navailability. As a result, the increase of soil C waslarger under elevated [CO2] than under ambientconditions (Hoosbeek et al. 2006). The same authorsdid not find changes in N mineralization rates in thefirst 2 years of the second cycle.

Next to N, available inorganic phosphorus couldbecome a limiting factor for photosynthetic activityand could provoke down-regulation responses under

elevated [CO2] (Harley et al. 1992; Demetz and Insam1999; Syvertsen and Graham 1999). The plant growthresponse under P limitation appears to be closelylinked to the mycorrhizal colonization rates. Increasedmycorrhizal colonization enhances P uptake at lowavailability, but also invokes belowground C cost formycorrhizal root growth and maintenance (Syvertsenand Graham 1999). Elevated [CO2] may reduce thisrelative cost of ectomycorrhizas, presumably byincreasing belowground carbohydrate supply fromincreased photosynthesis (Morgan et al. 1994; Lewisand Strain 1996) and mycorrhizal hyphal abundance(Treseder and Allen 2000). Differently, mycorrhizalabundance generally declines in response to N and Pfertilization across studies (Treseder 2004).

Elevated [CO2] and fertilization treatment can alsoalter the turnover and composition of the soilmicrobial community, its respiration rates and theextracellular enzyme activity, changing the utilizationof plant derived substrates (Phillips et al. 2002).Decomposition processes are the result of the overallactivity of microbial communities driven by theacquisition of nutrients released by extracellulardegradation of detritus (Sinsabaugh and Moorhead1994). The concentration of substrates available tosoil microorganisms affects the synthesis of extracel-lular enzymes. Assaying the degradation of severaldifferent substrates is therefore an approach tounderstand the turnover of organic matter and theavailability of inorganic nutrients. This can provideindications on the function and efficiency of anecosystem and on the interactions among subsystems(Dick and Tabatabai 1993).

The aim of this work is to evaluate the effects ofelevated [CO2] and fertilization on: (1) N mineraliza-tion and immobilization processes; (2) soil nutrientavailability; and (3) soil enzyme activity, as anindication of microbial and plant nutrient acquisitionactivity.

Materials and methods

Site description

The POP-EuroFACE experimental plantation andFACE facility are located in central Italy, Tuscania(VT; 42°22′ N, 11°48′ E, alt. 150 m) on about a 9 hafield. The soil has a loam to silt-loam texture, the A

132 Plant Soil (2008) 308:131–147

horizon (0–60 cm deep) was classified as a PachicXerumbrept (US taxonomy), total C range is 0.65–1.18%, total N range is 0.11–0.14%. For furtherinformation on soil physical and chemical propertiessee Hoosbeek et al. (2004). The mean values oftemperature and precipitation (calculated over a periodof 14 years, from meteorological data collected at arural station about 10 Km from the site) are 14.1°C and818 mm, respectively.

Six experimental plots were planted in 1999 withthree different poplar genotypes in order to highlight thegenotypic differences. The genotypes used were P. ×euramericana (P. deltoides Bart. ex Marsh. × P. nigra,Clone I-214), a local selection of P. alba L. (Clone2AS11) and a genotype of P. nigra L. (Clone JeanPourtet). Further information on genotypic properties isdetailed in Calfapietra et al. (2001). The selection ofthe three species was motivated by the needs to includea wide range of morphological and functional charac-teristics, which could interact with the elevatedatmospheric CO2 concentration, like: branchiness, leafmorphology, water relations, total leaf surface andmass, phenology. The clone I-214 was included as“standard”, because the wide diffusion in the world andin the scientific literature. The six plots were treatedeither with atmospheric (control) or enriched (550 μmolmol−1 CO2) CO2 concentration with FACE technology(Free-air CO2 enrichment; Miglietta et al. 2001). Eachplot is divided into six triangular sectors, with twosectors per poplar genotype: three species × twonitrogen levels. The trees were coppiced after the firstthree growing seasons (1999–2001) and the experi-ment continued with a second rotation (2002–2004).Nitrogen fertilization started in July 2002. Navarson 20(16 ammoniacal and 4 nitric)−6−6 N, P, K + micro-elements was supplied weekly during the growingseason for 16 weeks to a final total amount of 212 kgN ha−1. During the 2003 and 2004 growing seasons thefertilizer Ammonium Nitrate 34 (17 ammoniacal and17 nitric) was supplied weekly in amounts proportionalto the growth rate for 20 weeks and provided a totalamount of 290 kg N ha−1.

Soil sampling

Soil samples were collected, after removal of litterlayer, from the 0–20 cm layer inside each of the threesectors in each plot, fertilized (F) and unfertilized (UF)for a total of 36 soil cores (2 CO2×2 fertilization×3

species×3 replicates). In 2002, 2003 and 2004 soilsamples were collected in spring and autumn for atotal of six sampling dates. Ammonium and nitrateswere analysed also on samples collected in October2000 and in the four seasons of 2001; total N wasanalysed on samples collected in each autumn ofyears 1999, 2000, 2001, 2003 and 2004. Soil sampleswere immediately sieved (<2 mm) and the moisturecontent adjusted to 60% of their water holdingcapacity (WHC). The soil samples were then left toequilibrate at room temperature in the dark for 1 weekprior to biochemical analyses.

In June 2002 soil samples were collected also infertilized sub-plots although the addition of nitrogenstarted the following month; however, data related tothese samples are not considered in the calculation ofthe fertilization effect.

Chemical and biochemical analyses

Ammonium was extracted in 1 M KCl and wasdetermined following Anderson and Ingram (1993);nitrate was determined colorimetrically after extractionin 0.5 M K2SO4 (Cataldo et al. 1975). Extractablephosphorus was determined according to Olsen andSommers (1982) after acid extraction with 1 M NH4F.

Total nitrogen was determined by flash combustionin an elemental analyzer (EA 1108; Van Lagen 1996).The change of soil N was calculated as: (N%t1−N%1999)/N%t1 for UF soil and as (N%t1−N%2001)/N%t1 for F soil.

Net N mineralization and net nitrification weremeasured according to Eno (1960). Two adjacent soilcores (10 cm in diameter and 20 cm in depth) werecollected in June and October 2004 for each species ineach plot. The first core (initial content) was immedi-ately returned in laboratory, sieved and the nitrate andammonium content were measured following Cataldoet al. (1975) and Anderson and Ingram (1993), re-spectively. The second core (final content) was replacedin the field in a thin polyethylene plastic bag to avoidwater entry and incubated in situ for 1 month. After1 month it was removed from the field and treated inthe same way as the first one. Net mineralization, netammonification and net nitrification were calculated asfollows: Final N� NHþ

4 þ N� NO�3

� �� Initial N�ðNHþ

4 þ N� NO�3 Þ ¼ net Nmineralization; Final N�ð

NHþ4 Þ � Initial N� NHþ

4

� � ¼ net ammonification;Final N�NO�

3

� �� Initial N�NO�3

� �¼ net nitrification.

Plant Soil (2008) 308:131–147 133

Potential nitrification was measured, in soilssampled in June and October 2004, after inhibitionof N� NO�

2 oxidation with sodium chlorate (10 mM)according to the short nitrification assay (SNA)reported by Hopkins et al. (1988).

Microbial biomass nitrogen (MBN) was estimatedfollowing the Fumigation Extraction (FE) method(Vance et al. 1987). Two portions of moist soil (20 goven-dry soil) were weighed: the first one (nonfumigated) was extracted with 80 ml of 0.5 MK2SO4 for 30 min by oscillating shaking at 200 rpmand filtered (Whatman no. 42), the second one wasfumigated for 24 h at 25°C with ethanol-free CHCl3.Following fumigant removal the soil was extractedsimilarly for the non fumigated one. Ninhydrinreagent solution (Sigma) was used to measureninhydrin-reactive nitrogen in 0.5 M K2SO4 soilextracts (fumigated and non-fumigated) in order todetermine microbial biomass N (MBN; Joergensenand Brookes 1990).

The enzyme activities were detected colorimetrical-ly using a SHIMADZU UV-VIS 1240 spectrophotom-eter. Acid phosphatase activity was determined afterincubation at 37°C with 0.115 M p-nitrophenylphos-phate at pH 5.0 as substrate, the pNP (p-nitrophenol)released was determined at 410 nm (Garzillo et al.1996). Arylsulphatase activity was detected accordingto Elsgaard et al. (2002) using 25 mM p-nitrophenylsulphate at pH 5.8 as substrate and the pNP releasedwas determined at 400 nm. 5 mM p-nitrophenyl-N-acetyl-β-D-glucosaminide at pH 5.8 was used assubstrate to test chitinase (N-acetyl-β-D-glucosamini-

dase) activity following Parham and Deng (2000); thepNP released was determined at 400 nm.

Statistical analysis

Analysis of variance (ANOVA) was performed toevaluate the main effects of time, elevated [CO2],fertilization, species and their interaction on theparameters analysed. Data were tested for normalitywith the Shapiro–Wilk statistic. Data from in situnitrogen mineralization and nitrification were normal-ized using a logarithmic transformation. A rando-mised block design was applied using the generallinear model procedure with CO2, N and species asfactors of variation. The two replicates for each plot

-15

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Oct-99 Oct-00 Oct-01 Oct-02 Oct-03 Oct-04

ch

ang

e o

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(%

)

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Fig. 1 Total nitrogenexpressed as variation withrespect to the year 1999 forsoil under control and ele-vated [CO2] UF and to theyear 2001 for soils undercontrol and elevated [CO2]F. Values from the threeclones were pooled togethersince no significant differ-ences were found. Errorbars represent standarderrors, n=9

Table 1 Analysis of variance for temporal variation (time),elevated CO2, fertilization, species and their interactions on:total nitrogen, N–NH4 and N–NO3 content

Total nitrogen N–NH4 N–NO3

Time 0.000 0.000 0.000CO2 n.s. n.s. 0.000Fertilization n.s. n.s. 0.000Species n.s. 0.005 0.002CO2 × time n.s. n.s. 0.002CO2 × fertilization n.s. n.s. n.s.Time × fertilization n.s. 0.001 0.000Time × CO2 × fertilization n.s. n.s. 0.008

p values <0.05 were reported, n.s. not significant. The notsignificant interactions were removed from the analysis

134 Plant Soil (2008) 308:131–147

were averaged and plot (three control plots and threeFACE plots) was the unit of replication. Thesignificance of elevated [CO2] and fertilization treat-ments were determined in F and UF plots from 2002to 2004 (n=72). When there were no significantvariations due to the different poplar species, datafrom different poplar genotypes were pooled together.When interactions were not significant they wereexcluded from analysis. When significant interactionsbetween factors were found, a Fisher’s LSD post hoctest was performed. Differences were consideredstatistically significant when p<0.05. All statisticalanalyses were performed with the Systat 11.0 statis-tical software package (SPSS Inc.).

Results

Nitrogen

The total soil nitrogen content increased significantlybetween 1999 and 2004 irrespective of treatments,particularly after the year 2001 (Fig. 1). In theyear 2001 a temporary decrease of total N in soilunder elevated [CO2] was observed. After 2001, nosignificant effects due to elevated [CO2] treatment orfertilization were found (Table 1).

At the end of the first rotation cycle there was asignificant depletion of ammonium (Fig. 2). Thedecrease was similar for the three clones, although

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Fig. 2 N–NH4 content insoils under Control and ele-vated [CO2] F and UF for P.alba (a), P. nigra (b) andP. × euramericana (c). Er-ror bars represent standarderrors, n=3

Plant Soil (2008) 308:131–147 135

in P. × euramericana a stronger depletion wasdetected. In the second cycle ammonium showed anincrease in June 2004, which was followed by adecrease in October to reach the previous values(Fig. 2). No significant differences due to elevated[CO2] or N fertilization were found (Table 1).Interactions with [CO2] enrichment were not foundeither.

Nitrate concentrations were on average threefoldhigher than ammonium and peaked twice in October2001 and 2002 in control soil (Fig. 3). Under P. ×euramericana nitrate concentrations were relativelylow, never exceeding 20 μg N–NO3 g

−1 (Fig. 3c). The[CO2] enrichment induced a significant reduction of

N–NO3 content in soil, unrelated to the species, butdepending on time (Table 1): the largest decrease dueto the treatment was in the first and second year afterthe coppicing (p<0.01, post hoc test), while in 2004this effect vanished. The nitrogen fertilization induceda strong increase of nitrates with the highest effect atthe end of the first year of fertilization, October 2002(p<0.001, post hoc test). As result of the interactionamong treatments and time (Table 1), at October 2002the increase was higher in Control than in FACE plots(Fig. 3).

Both the ammonium and nitrate concentrations insoil were significantly affected by the species(Table 1), with mean values of inorganic N N�ð

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Fig. 3 N–NO3 content insoils under Control and ele-vated [CO2] F and UF for P.alba (a), P. nigra (b) andP. × euramericana (c). Er-ror bars represent standarderrors, n=3

136 Plant Soil (2008) 308:131–147

NHþ4 þ N� NO3Þ for the whole study period of 14.8

for P. alba, 13.4 for P. nigra and 12 μg N g−1 for P. ×euramericana.

The microbial biomass N (MBN) had the highestvalues in October 2004 (Fig. 4a). The [CO2]enrichment did not induce significant changes,whereas fertilization had a positive effect in June2003 (Table 2; p<0.01, post hoc test). Whenexpressed as fraction of total N, the microbialbiomass N (MBN/TN) was significantly increasedby elevated [CO2] all over the period of measure-

ment, both in F and UF soils (Fig. 4b and Table 2).The fertilization treatment caused a significantincrease of the MBN/TN ratio in June 2003 (Table 2;p<0.01, post hoc test).

In situ N mineralization revealed that nitrifica-tion was the main process determining N mineral-ization in the plantation. Ammonification wasnegligible or negative, and was not affected by thetreatments (Fig. 5a). Nitrification had a significantseasonal trend, with higher values in June, comparedto October (2004). The three clones had similar

Table 2 Analysis of variance for temporal variation (time), elevated CO2, fertilization, species and their interactions on: in situnitrification, in situ N mineralization, potential nitrification, the microbial biomass N (MBN), the ratio MBN/TN (total N) and theirinteractions

In situ nitrification In situ N mineralization Potential nitrification MBN MBN/TN

Time 0.003 n.s. n.s. 0.000 0.000CO2 0.044 n.s. 0.000 n.s. 0.046Fertilization n.s. n.s. n.s. n.s. n.s.Species n.s. n.s. n.s. n.s. n.s.Fertilization × CO2 n.s. n.s. 0.007 n.s. n.s.Fertilization × time n.s. n.s. n.s. 0.002 0.003

p values <0.05 were reported; n.s. not significant. The not significant interactions were removed from the analysis

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Fig. 4 a Microbial biomassN (MBN) in soils undercontrol and elevated [CO2]F and UF. b MBN/TN ratioin soils under control andelevated [CO2] F and UF.Values from the three cloneswere pooled together sinceno significant differenceswere found. Error bars rep-resent standard errors, n=9

Table 2 Analysis of variance for temporal variation (time),elevated CO2, fertilization, species and their interactions on: insitu nitrification, in situ N mineralization, potential nitrification,

the microbial biomass N (MBN), the ratio MBN/TN (total N)and their interactions

Plant Soil (2008) 308:131–147 137

values, and did not induce modifications in thenitrification activity (Fig. 5a and Table 2). Theelevated [CO2] treatment decreased significantlythe nitrification at the two sampling dates in UF soilof about 46% (p<0.05, post hoc test). In situmineralization was not significantly affected by elevat-ed [CO2], although the [CO2] effect was close to thesignificance level (p=0.06). N fertilization did notinduce any significant change neither on nitrificationnor on N mineralization.

The potential nitrification (SNA) decreased signifi-cantly under elevated [CO2] by 51% and 45% in Juneand October 2004 in UF plots (Fig. 5b; p<0.01, posthoc test). Nitrogen fertilization treatment did not causesignificant changes in potential nitrification activity.

From the regression analysis ammonium relatedpositively and significantly to the total soil N content(Fig. 6a) and MBN (Fig. 6b); whereas the amount ofnitrate in the soil correlated significantly with soilnitrification activity (Fig. 6c).

Phosphorus and enzymatic activities

A negative temporal trend on soil P content wasevident after June 2003 (Fig. 7). A negative effect of

CO2 enrichment was observed only in F soils, with amean reduction of about 27% (p<0.01, post hoc test,Table 3). N fertilization induced a significant increaseof the phosphorus amount in soil, but this increasewas dependent on time: the largest effect wasobserved in October 2002 in control plots and inJune 2003 in control and FACE plots, although inOctober of 2003 and 2004 only a slight increase wasobserved (Fig. 7). The poplar species did not affectthe P amount neither in the first nor in the secondcycle.

Acid phosphatase activity increased between June2003 and October 2004 (Fig. 8). The highest valueswere observed in P. nigra soil, while the lowestvalues were observed in P. × euramericana soil. The[CO2] enrichment induced a significant increaseunrelated to the N level in soil and to the species(Table 3). The N fertilization induced a consistentincrease at the end of the first year of the secondrotation (October 2002; p<0.001, post hoc test), whilethe effect was absent during following years (Fig. 8).Extractable phosphorus and acid-phosphatase weresignificantly inversely correlated during the wholeperiod of study (2000–2004) independently of thetreatments, as shown in Fig. 10a.

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Fig. 5 a In situ N mineral-ization (min), calculatedfrom nitrification (nitr) andammonification (amm) pro-cesses in soils under controland elevated [CO2] F andUF. b Potential nitrificationactivity in soils under con-trol and elevated [CO2] Fand UF sampled in June andOctober 2004. Values fromthe three clones were pooledtogether since no significantdifferences were found. Er-ror bars represent standarderrors, n=9

138 Plant Soil (2008) 308:131–147

Arylsulphatase showed a seasonal fluctuation withmaximum levels in June (particularly June 2004) andminimum in October (Fig. 9a). The poplar clones didnot influence significantly arylsulphatase, neither ininteraction with time nor with treatments (Table 3).

The elevated [CO2] treatment induced a significantincrease of the enzymatic activity all over the periodof study while no interactions with nitrogen werefound (Table 3). On the contrary, the N fertilizationdecreased the enzyme activity, in particular in the

y = 0.06x + 1.30R2 = 0.308, p<0.001

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Fig. 6 Linear regressionsbetween a soil total N andammonium content for theyears 2003–2004; b micro-bial biomass N and ammo-nium content for theyears 2002–2004; c andsoil nitrification activity forthe year 2004, as average ofJune and October

Plant Soil (2008) 308:131–147 139

period October 2003–June 2004 (p<0.001, post hoctest; Table 3; Fig. 9a).

The activity of chitinase (N-acetyl-glucosamini-dase) was similar to that of arylsulphatase with thehighest values in June and the lowest in October(Fig. 9b). The [CO2] enrichment induced a significantincrease in the enzymatic activity without interactionswith time, fertilization and poplar clones (Table 3).Neither the fertilization treatment, nor the speciesinfluenced significantly this enzyme. Arylsulphataseand chitinase activities were significantly correlated(Fig. 10b).

Discussion

Soil N availability and mineralization-immobilizationprocesses

A great depletion of soil ammonium, unrelated to theFACE treatment, was observed during the third

growing season of the first cycle, the most productiveone (Calfapietra et al. 2003), whereas nitrate declinedin FACE but not in control soils. We can advancesome hypotheses for ammonium depletion in theplantation soil. A first mechanism might be thepreferential uptake of ammonium by poplars. Thispossibility was also suggested by Rothstein et al.(2000) and Bassirirad et al. (1997) in studies onpoplars excised roots that reported more rapid kineticsof NHþ

4 uptake than NO�3 , as considerable more

energy is generally required by plants for uptake andassimilation of the latter. A second process was theconversion of ammonium-N produced by nitrogenmineralization to nitrate-N, which was extremelyrapid at POP-EuroFACE (Calfapietra et al. 2007):net nitrification and net N mineralization wereequivalent in the plantation, and nitrification was themain process influencing the inorganic N amount insoil. Finally we observed the immobilization ofammonium-N in microbial biomass, as showed bythe significant regression between the two.

Table 3 Analysis of variance for temporal variation (time), elevated CO2, fertilization, species and their interactions on: extractablephosphorus, acid phosphatase, arylsulphatase, chitinase and their interactions

Phosphorus Acid phosphatase Arylsulphatase Chitinase

Time 0.000 0.000 0.000 0.000CO2 0.035 0.030 0.017 0.016Fertilization 0.015 0.000 0.000 n.s.Species n.s. 0.035 n.s. n.s.Fertilization × CO2 0.034 n.s. n.s. n.s.Fertilization × time n.s. 0.000 0.000 n.s.

p values <0.05 were reported, n.s. not significant. The not significant interactions were removed from the analysis

0

5

10

15

20

25

30

35

Jun-02 Oct-02 Jun-03 Oct-0 3 Oct-04

µg P

g-1

Control UF Control F CO2 UF CO2 F

Fig. 7 Extractable phos-phorus in soils under Con-trol and elevated [CO2] Fand UF. Values from thethree clones were pooledtogether since no significantdifferences were found. Er-ror bars represent standarderrors, n=9

140 Plant Soil (2008) 308:131–147

Both poplar uptake and microbial nitrificationactivity could explain the significant decrease ofnitrates in FACE soils found all over the period ofstudy. Under elevated [CO2], availability of respira-tory carbon substrates can enhance root energy statusand could thus positively influence NO�

3 uptake rates(BassiriRad et al. 1997). The same authors report infact enhancement of nitrate uptake in seedlings ofPinus spp. under elevated [CO2].

In a synthesis based on four forest FACE experi-ments, Finzi et al. (2007) postulated that increasedforest growth under elevated atmospheric CO2 con-centrations can only be sustained if (1) more N is

taken up from the soil, or (2) the N present in thevegetation is used more efficiently, i.e. the NUEincreases. At the Duke Forest, Oak Ridge and AspenFACE experiments, increased N uptake was found tobe the mechanism in order to sustain higher produc-tivity under FACE.

On the contrary, at the POP-EuroFACE experi-ment, based on data from the first rotation, higherproduction was sustained through an increase of NUEunder elevated [CO2] (Calfapietra et al. 2007).Similarly, in the second rotation lower leaf Nconcentrations (Marinari et al. 2006) in combinationwith higher growth rates (Liberloo et al. 2006, 2007)

0

500

1000

1500

2000

2500

3000

3500

Jun-02 Oct-02 Jun-03 Oct-03 Jun-04 Oct-04

µg p

NP

g-1

Control UF Control F CO2 UF CO2 F

0

500

1000

1500

2000

2500

3000

3500

µg p

NP

g-1

0

500

1000

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2500

3000

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µg p

NP

g-1

a

b

c

0

500

1000

1500

2000

2500

3000

3500

Jun-02 Oct-02 Jun-03 Oct-03 Jun-04 Oct-04

µg p

NP

g-1

Control UF Control F CO2 F

0

500

1000

1500

2000

2500

3000

3500

µg p

NP

g-1

0

500

1000

1500

2000

2500

3000

3500

µg p

NP

g-1

a

b

c

Fig. 8 Acid phosphatase insoils under control and ele-vated [CO2] F and UF of P.alba (a), P. nigra (b) andP. × euramericana (c). Er-ror bars represent standarderrors, n=3

Plant Soil (2008) 308:131–147 141

were found under elevated [CO2]. From these remarkswe infer that not increased N uptake, but ratherincreased NUE was the mechanism by whichincreased growth was still sustained during the secondrotation under elevated [CO2]. Significant differencesof N concentrations in plant tissues and consequentlyon NUE among the three species were showed byCalfapietra et al. (2007), with the highest values in P.alba and the lowest in P. × euramericana. A similartrend was observed in soil, with the highest values ofinorganic N under P. alba and the lowest in P. xeuramericana. The possible explanation could berelated to the different productivity of the threespecies, with continues adjustment of NUE accordingto the increasing soil nitrogen limitation.

The lower values of soil nitrate concentrationsobserved under elevated [CO2] were related to thedecrease of nitrification rate observed by both in situand laboratory determination. Other studies reportedunaltered or even decreased net N mineralization rateafter carbohydrates amendments to temperate peren-nial grassland (Jonasson et al. 1996) as well asagricultural (Rutherford and Juma 1992) and silvicul-tural systems (Turner and Olson 1976). On the

contrary, Carnol et al. (2002) reported an increase ofnitrification under elevated [CO2], related to adecrease in microbial biomass and activity thatreduced competition for NHþ

4 . This is not the caseof POP-EuroFACE soils, where, under elevated [CO2]conditions, the size of microbial biomass increased(Moscatelli et al. 2005a) and the competition forammonium was supposed to be high in the plantation(Calfapietra et al. 2003). Hungate et al. (1999) foundthat elevated [CO2] depressed gross mineralizationand ammonium consumption, and caused an increasein the specific rate of ammonium immobilization bysoil microorganisms. In fact microorganisms appar-ently assimilates NHþ

4 considerably more rapidly thanNO�

3 , because less energy is required for theassimilation of the former (Haynes 1986). Mycorrhi-zal fungi in particular are known to prefer NHþ

4 toNO�

3 and, as Verstraete (1981) indicated, it seemspossible that mycorrhizae may act as agents ofbiological control of nitrification under vegetatedconditions.

Several theories asserted that the increase of easilydegradable C may stimulate microbial activity andnutrient cycling, but a decrease in litter quality (high

0

20

40

60

80

100

120

Jun-02 Oct-02 Jun-03 Oct-03 Jun-04 Oct-04

µg p

NP

g-1

h-1

Control UF Control F CO2 UF CO2 F

0

20

40

60

80

100

µg p

NP

g-1

h-1

a

0

20

40

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120

Jun-02 Oct-02 Jun-03 Oct-03 Jun-04 Oct-04

µg p

NP

g-1

h-1

Control UF Control F CO2 UF CO2 F

0

20

40

60

80

100

µg p

NP

g-1

h-1

b

Fig. 9 a Arylsulphatase ac-tivity in soils under controland elevated [CO2] F andUF; b Chitinase activity insoils under control and ele-vated [CO2] F and UF.Error bars represent stan-dard errors, n=9

142 Plant Soil (2008) 308:131–147

C/N ratio) may slow the rate of nutrient cycling andincrease N immobilization (Allen et al. 2000). and theresults of Magill and Aber (2000) indicated that in thepresence of a labile C source, available soil N wasrapidly immobilized, reducing soil N availability(Janssens et al. 1998). The labile C increasedsignificantly in the FACE plots (Hoosbeek et al.2006), and a higher C/N ratio of litter (Cotrufo et al.2005) and of labile SOM (Lagomarsino et al. 2006)was also observed. We can therefore hypothesize: (1)under elevated [CO2] microbial immobilization wasthe dominant process, as demonstrated by the increasein the ratio MBN/TN, (2) a preferential uptake ofammonium by microbial biomass and (3) a ratherrapid conversion of ammonium into organic forms, assuggested by the relationship between MBN andammonium and between this last and total N:microbial immobilization probably enhanced the

transformation of large amounts of N into organicforms less accessible to plants, balancing the reduc-tion in the N input via litter fall found by Cotrufo etal. (2005). Abiotic fixation of NHþ

4 in soils has alsobeen well documented (Schimel and Firestone 1989;Trehan 1996) and may be another important mecha-nism occurring in the plantation soil. Nevertheless,the increase of total N in 2004 followed a similartrend of ammonium and microbial biomass N in soil,confirming the role of microbes in N� NHþ

4 immo-bilization and suggesting a possible increase oforganic forms of N as litter accumulated in soilduring years.

The N addition did not influence neither the Ncontent in FACE soil, nor the described processes, butthe treatments interaction acted significantly in Octo-ber 2002, resulting in a strong increase of nitratesafter fertilization only in Control soil. Rothstein et al.

y = 0.59x + 7.46R2 = 0.483, p<0.001

0

20

40

60

80

100

120

0 20 40 60 80 100 120 140Chitinase ( µg pNP g -1h-1)

Ary

lsul

phat

ase

(µg

pNP

g-1

h-1)

y = -48.48x + 1698.5R2 = 0.358, p<0.001

0

500

1000

1500

2000

2500

0 10 20 30 40

acid

pho

spha

tase

(µg

pN

P g

-1 h

-1)

a

b

y = 7.46R2 = 0.483, p<0.001

0

20

40

60

80

100

120

0 20 40 60 80 100 120 140g pNP g -1h-1)

y = -48.48x + 1698.5R2 = 0.358, p<0.001

0

500

1000

1500

2000

2500

0 10 20 3

Phosphorus (µg P g-1)

Fig. 10 Linear regressionsbetween a extractable phos-phorus and acid phospha-tase and b arylsulphataseand chitinase activity, forthe period 2000–2004

Plant Soil (2008) 308:131–147 143

(2000) found for P. tremuloides that the fertilizationtreatment resulted in decreases in specific NHþ

4 andNO�

3 uptake rates of approximately 50% comparedwith the rates measured in the low-N treatment, andsuggested a down regulation of N uptake system inthe response to improved soil N status. We can arguethat in soil under elevated [CO2] this down regulationdid not occur and the poplar uptake remained high.This hypothesis is confirmed by the findings ofLiberloo et al. (2004) that in the same year 2002observed a positive effect of the interaction ofelevated [CO2] and N fertilization in plant production.

Phosphorus availability and nutrient acquisitionactivity

At our site soil phosphorus content was not limitingplant growth and was present in relatively highconcentrations. However, 4 years after the initiationof the experiment, P availability showed a negativetrend. In addition, we found an opposite effect ofelevated [CO2] on phosphorus availability betweenthe first and the second cycle. In the first cycleextractable phosphorus increased under elevated[CO2] (Moscatelli et al. 2005b), probably because oforganic P mineralization as a consequence of thepriming effect on soil organic matter reported byHoosbeek et al. (2004). After the coppicing a decreasedue to the FACE treatment was observed instead,being significant in F soils. It is known thatphosphorus availability is under biotic control ofenzymatic activity of plant and microorganisms andelevated [CO2] may increase the potential for plantnutrient acquisition by stimulating mycorrhizal colo-nization of roots and root exudation of phosphorus-mobilizing carbon compounds (De Lucia et al. 1997),as confirmed by the negative relationship between Pand acid phosphatase found in this study. Thesignificant enhancement of acid phosphatase underelevated [CO2] might therefore reflect a microbialand/or plant demand for this nutrient. The require-ment of P did not decrease with fertilization, and thepositive effect of elevated [CO2] was evident also inN-rich soils. For this purpose we assume that thefertilization treatment, reducing mycorrhizal coloni-zation, could even act as a negative feedback onphosphorus availability under elevated [CO2].

Chitinolitic and arylsulphatase enzymes are indi-cators of N and S demand (Parham and Deng 2000;

Tabatabai and Bremner 1970). Moreover, theseenzymatic activities are also considered as indirectindicators of the presence of fungi population becauseesther sulphates (substrates of arylsulphatase) are onlypresent in fungal cells (Bandick and Dick 1999) andchitin is the main constituent of fungal cell wall tissue(Miller et al. 1998). These last authors found a directrelationship of chitinase with two independent indica-tors of fungal biomass (phospholipid fatty acid andergosterol analyses) and concluded that measuringchitinase activity may provide a sensitive measure ofsoil fungal biomass. Both the enzymatic activities werealso significantly correlated between themselves andwith the C/N ratio of microbial biomass (Pearsoncorrelation, p<0.001, data not shown), which is anindicator of the fungi/bacteria ratio in soils (Joergensenet al. 1995). The positive effect of FACE treatmenton arylsulphatase and chitinase activity observedconstantly during the period of study couldtherefore indicate an increase of fungal populationunder higher [CO2], as well as an increase in the Sand N acquisition activity. These data were consistentwith Phillips et al. (2002) reporting an increase of 13C-N-acetylglucosaminidase metabolism under elevated[CO2]. Moreover mycorrhizas colonisation of Populusfine roots increased under FACE, which indicatesincreased fungi biomass due to elevated CO2 (Lukac etal. 2003).

Conclusion

Total N increased in the plantation due to land usechange; however a temporary decrease was observedat the end of the first cycle as a consequence of apriming-effect mechanism induced by elevated [CO2].Nitrification was the main process influencing inor-ganic N concentration in the soil and its decreaseunder elevated [CO2] was the main mechanismunderlying nitrate depletion in the plantation, underunfertilised treatment. Under elevated [CO2] ammo-nium was mostly affected by microbial immobiliza-tion, followed by its rather rapid conversion intoorganic forms. The significant enhancement of en-zyme activities under elevated [CO2] reflected anincrease in nutrient acquisition activity in the soil, aswell as an increase of fungal population. The Nfertilization treatment, reducing mycorrhizal coloni-zation, acted as a negative feedback on phosphorusavailability under elevated CO2.

144 Plant Soil (2008) 308:131–147

Soil inorganic N availability reflected speciesspecific differences in plant N pools and NUE,whereas the total N remains unaffected. Furthermore,specific differences on roots metabolism producedsignificant changes in the enzyme activity related tothe P uptake.

Acknowledgements The authors are grateful to Prof. GiuseppeScarascia Mugnozza coordinator of EU EUROFACE (EVR1-CT-2002-40027) and MIUR Centre of Excellence “Forests andclimate” projects for allowing the use of POP-EuroFACEexperimental station.

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