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Basic and Applied Ecology 6 (2005) 367—383

KEYWORDPrimary suGlacier foAlpine ecoPhospholipacids;Bacteria;Fungi;Enzymes;CSR mode

1439-1791/$ - sdoi:10.1016/j.

�CorrespondE-mail addr

www.elsevier.de/baae

Plant succession and rhizosphere microbialcommunities in a recently deglaciated alpineterrain

Dagmar Tscherkoa,�, Ute Hammesfahra, Georg Zeltnerb,Ellen Kandelera, Reinhard Bockerb

aInstitute of Soil Science and Land Evaluation, University of Hohenheim, Emil-Wolff-Straße 27,70599 Stuttgart, GermanybInstitute of Landscape and Plant Ecology, University of Hohenheim, August-von-Hartmann-Straße 3,70599 Stuttgart, Germany

Received 2 February 2004; accepted 14 February 2005

Sccession;reland;system;id fatty

l

ee front matter & 200baae.2005.02.004

ing author. Tel.: +49 7ess: tscherko@uni-hoh

SummaryThis study describes how early and late successional plant species affect soilmicroorganisms in alpine ecosystems. We quantify the relative importance of plantspecies and soil properties as determinants of belowground microbial communities.Sixteen plant species were selected from six successional stages (4–14–20–43–75–135years) within the foreland of the Rotmoosferner glacier, Austria, and at one(reference) site outside the foreland. The size, composition and function of thecommunities of microorganism in the bulk soil and the rhizosphere were characterizedby ninhydrin-reactive nitrogen, phospholipid fatty acids and enzyme activities(b-glucosidase, b-xylosidase, N-acetyl-b-glucosaminidase, leucine aminopeptidase,acid phosphatase, sulphatase). The results show that the microbial data could begrouped according to early (up to 43 years) and late-colonizing plant species (75 ormore years). In early succession, no plant species or soil age effect was detected onthe microbial biomass, phospholipid fatty acids, or enzyme activity. The rhizospheremicrobial community was similar to that in the bulk soil, which in turn was determinedby the abiotic environmental conditions. In late succession, improved soil conditionsprobably mediated plant species effects on the belowground microbial community.The most pronounced rhizosphere effects were attributed to plant species of the 75-and 135-year-old sites. The microbial colonization (size, composition, activity) of thebulk soil predominantly followed changes in vegetation cover, plant life forms and soil

5 Gesellschaft fur Okologie. Published by Elsevier GmbH. All rights reserved.

144 858999; fax: +49 711 459 3117.enheim.de (D. Tscherko).

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D. Tscherko et al.368

organic matter. In summary, the observed successional pattern of the above- andbelowground communities provides an example of the facilitation models of primarysuccession.& 2005 Gesellschaft fur Okologie. Published by Elsevier GmbH. All rights reserved.

ZusammenfassungZiel dieser Arbeit ist die Charakterisierung der Rhizospharenmikroorganismen vonPionier- und Folgearten entlang des Rotmoosferner Gletschervorfeldes im Otztal,Osterreich. Sechs Standorte innerhalb (Alter: 4–14–20–43–75–135 Jahre) und einStandort ausserhalb des Gletschervorfeldes (Referenz) und 16 Pflanzenarten wurdenunter dem Aspekt der fortschreitenden Sukzession ausgewahlt, und Rhizospharenbo-den der fur das jeweilige Sukzessionsstadium representativen Pflanzenarten, sowieder Gesamtboden beprobt. Die mikrobielle Gemeinschaftsstruktur wurde anhand vonPhospholipidfettsauremustern, mikrobielle Umsetzungsraten anhand von Enzymmes-sungen (b-Glucosidase, b-Xylosidase, N-Acetyl-b-Glucosaminidase, Leucin-Aminopep-tidase, saure Phosphatase, Sulfatase) mittels fluorogener Substrate charakterisiert.Innerhalb der ersten 43 Sukzessionsjahre konnte kein artspezifischer Effekt auf diemikrobielle Rhizospharengemeinschaft nachgewiesen werden. Die Aktivitat undZusammensetzung wurde uberwiegend von den extremen abiotischen Standortsbe-dingungen gepragt. Ab 75 Sukzessionsjahren konnte aufgrund verbesserter Boden-und Nahrstoffbedingungen ein pflanzenspezifischer Einfluss auf die mikrobielleGemeinschaft nachgewiesen werden. Der starkste Rhizosphareneffekt wurde aufden 75- und 135 Jahre alten Standorten beobachtet. Die mikrobielle Kolonisierung desBodens wurde durch die Vegetationsbedeckung, die Lebensformen und vomHumusgehalt bestimmt.& 2005 Gesellschaft fur Okologie. Published by Elsevier GmbH. All rights reserved.

Introduction

In the Alps, primary succession on glacier fore-lands has been frequently studied since the 19thcentury, and vegetation development along thechronosequences has been described. The pro-cesses of primary succession; however, remainincompletely understood. Grime (2001) identifiedthree primary plant strategies: competitors (C),stress-tolerators (S) and ruderals (R) and suggestedaccording to his CSR model that shifting equilibriabetween competition, mineral nutrient stress, anddisturbance are determinants of vegetation com-position in primary succession. On a more plant-specific level, Callaway, Brooker, Choler, Kikvidze,Lortie, et al. (2002) proposed a model in whichfacilitative interactions among alpine plant com-munities predominate when abiotic conditions areharsh, but that competitive interactions predomi-nate when abiotic conditions are less stressful.Both concepts provide an example of the facilita-tion model of vegetation succession (Connell &Slatyer, 1977). How this shift in plant speciescomposition and interaction in primary successionaffects the plant–microbial interface has receivedlittle attention, although soil colonization and theestablishment of plants may depend on this process(Allen, Allen, Zink, Harney, Yoshida, et al., 1999).The species of plant is known to affect the

composition of the rhizosphere microbial commu-nity (Fang, Radosevich, & Fuhrmann, 2001; Marsch-ner, Yang, Lieberei, & Crowley, 2001; Kuske,Ticknor, Miller, Dunbar, Davis, et al., 2002; Knauff,Schulz, & Scherer, 2003). The composition andactivity of the soil microbiota along primarysuccessional gradients of glacier forelands havealso been characterized (Ohtonen, Fritze, Penna-nen, Jumpponen, & Trappe, 1999; Schipper, De-gens, Sparling, & Duncan, 2001; Sigler, Crivii, &Zeyer, 2002; Tscherko, Rustemeier, Richter, Wanek,& Kandeler, 2003). Much less is known about themicrobial colonization of rhizospheres in primarysuccession. Only two studies have investigated theimpact of colonizing plants on microbial communitygrowth and composition on recently deglaciatedterrain (Bardgett & Walker, 2004; Tscherko, Ham-mesfahr, Marx, & Kandeler, 2004). To date, how-ever, no attempt has been made to study microbialpopulations in the rhizosphere of plants exposed toenvironmental gradients such as increasing speciesrichness and soil productivity in alpine landscapes.

The aim of this study is therefore to comparethe rhizosphere microorganisms of successionalplant species along a vegetational gradient ofrecently deglaciated terrain. Based on the modelsproposed by Grime (2001) and Callaway et al.(2002), we specifically investigated (i) the impactof successional plant species on the soil microbial

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Plant succession and rhizosphere microorganisms in alpine terrain 369

community and (ii) this community’s interactionwith soil properties in primary succession. Toexamine these issues, we investigated the commu-nity structure and function of the microbiota in therhizospheres of a sequence of successional plantspecies across the Rotmoosferner glacier foreland,Austria, using phospholipid fatty acids and enzymeactivity measurements. This study is intended tofoster our understanding of how plants and soildetermine belowground microbial populations in adeveloping alpine ecosystem.

Material and methods

Rotmoosferner glacier foreland

The study sites are located on the foreland of theRotmoosferner glacier (461500N, 111030E) in the Otzvalley (Austria) at an altitude of 2280–2450mabove sea level. The annual mean temperature is�1.3 1C (1997–1998) and mean annual precipitationis 820mm (1970–1996) (Kaufmann, 2001). Theglacier foreland starts with the youngest sites(4 yr), about 10m away from the glacier terminus,and ends at the terminal moraine, dated 1850,about 1500m north of the glacier snout. The area infront of the glacier foreland has been ice free forabout 9500 yr (Patzelt & Bortenschlager, 1979) andserved as an undisturbed reference site. The aspectof the entire foreland is slightly north with anaverage slope of 8%.

Vegetation survey

The vegetation was mapped on 4-, 14-, 20-, 30-,43-, 48-, 75- and 135-year-old successional sitesand on the reference plot. The size of theinvestigation plots was 25m2. The abundance ofeach plant species was estimated according toBraun-Blanquet (1964), but with a more detailedscale according to Wilmanns (1993). The nomen-clature of the taxa follows Wisskirchen and Haeu-pler (1998). The plant species were classified intothe following life forms (Raunkiaer, 1934): chamae-phytes (herbaceous stems, buds on or just abovesoil surface but never more than 0.5m aboveground); woody chamaephytes (chamaephytes withwoody, persistent stems), hemicryptophytes (her-baceous stems, often dying back after the growingseason, buds on or below soil level surviving);therophytes (annuals, surviving unfavourable sea-sons in the form of seeds, completing life-cycleduring the favourable season), geophytes (hemi-cryptophytes, surviving unfavourable seasons in the

form of rhizome, bulb, tuber or root bud, budsbelow soil level), and nanophanerophytes (woodystems, buds above soil surface but normally lessthan 3m above ground). The percentage of surfacecover by each life form was calculated. Forsimplicity, the species names as listed in Table 1were replaced, where appropriate, by genus namesin the following sections.

Soils

The parent material of the soil is mainlyneoglacial moraine till (mica slate and granite withlocal grains of carbonate minerals). Soils (loamysand) are classified as Leptic and Eutric Regosols.The chemical properties were determined for thebulk soil and are listed in Table 2. In brief, soil pHwas potentiometrically measured in 0.01M CaCl2,and the total carbon (Ct) and total nitrogen (Nt)contents were determined by dry combustion at1200 1C using an elemental analyser (LECO CNS2000) with infra-red and thermal conductivitydetector, respectively. The carbonate content wasestimated by treating the sample with 85% phos-phoric acid and subsequently quantifying theevolved CO2 by electrical conductivity. Carbonatecarbon (Ccarb) was then subtracted from Ct toobtain the organic carbon (Corg) concentration.Nitrate (NO3

�–N) and ammonium (NH4+–N) were

measured in 0.01M CaCl2 extracts, and availablephosphorus (P) and potassium (K) were photome-trically determined after 0.05M acetate–lactateextraction (Schlichting, Blume, & Stahr, 1995).

Sampling

Seven sampling sites were selected between themoraines according to the year of deglaciation(Table 2). All sampling sites were similar inaltitude, aspect and slope. The most representa-tive plant species of selected successional stageswere collected, yielding 16 rhizosphere soils(Saxifraga aizoides (14 years), Linaria alpina,Trifolium pallescens, Poa alpina (20 years), T.pallescens, Campanula cochleoriifolia, Elyna myo-suroides, Gentiana bavaria (43 years), T. badium,Leontodon helveticus, Poa alpina (75 years),Potentilla aurea (135 years), Poa alpina, Carexsempervirens, Crepis aurea, Nardus stricta (refer-ence)). Samples were taken in September 2001.Rhizosphere soil was collected by pulling the plantout of the ground and mechanically separating theadhering soil from the roots. The soil from eachplant was pooled. Three individuals of each plantspecies were taken from an area of 25m2, yielding

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Table 1. Abundance (coverage) of plant species according to successional stage

Plant species Successional age (years)

4 14 20 30 43 48 75 135 Reference

Arabis caerulea rKoeleria hirsuta rArabis hirsuta r rArtemisia genipi r 1 + 2m +Saxifraga oppositifolia r + 1 + + rSaxifraga aizoides + + + + + +Hutchinsia alpina rLinaria alpina + rSilene exscapa + 1 + + 3 + +Leontodon helveticus r r + 1 2b 1Trifolium pallescens r + + 1 1 1Poa alpina r 2m 1 2a 2b 2b 2bPolytrichum alpinum rSenecio incanus subsp. Carniolicus rSedum atratum rSaxifraga exarata agg. r rCerastium uniflorum r +Cerastium alpinum r +Saxifraga paniculata r 1Sempervivum montanum r +Campanula cochleariifolia r + +Luzula spicata r + rRacomitriuim canescens r 1Gnaphalium supinum r + r +Geum reptans + r r +Oxyria digyna + rAchillea moschata + + + +Saxifraga bryoides 1 r + + +Minuartia verna r + r + +Campanula scheuchzeri r + 1 1 1Agrostis rupestris r + 1 1 1Alchemilla vulgaris r r 1 + 1Euphrasia rostkoviana r r 2m 2m 2b +Festuca pumila r 2a 1 1 2aSibbaldia procumbens r r 1 2b 1Salix retusa retusa 1Taraxacum officinale rAntennaria carpatica rSalix reticulata rGentiana bavarica rErigeron uniflorus + +Parnassia palustris + +Taraxacum alpinum r +Salix helvetica r +Botrychium lunaria r r rDeschampsia cespitosa r +Cirsium spinosissimum r r + rSalix herbacea + r +Veronica alpina + + + +Leontodon hispidus + + 1 +Elyna myosuroides + 1 +Polygonum viviparum 1 2a 1Epilobium fleischeri 1Moehringia ciliata +Myosotis alpestris +

D. Tscherko et al.370

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Table 1 (continued )

Plant species Successional age (years)

4 14 20 30 43 48 75 135 Reference

Galium helveticum +Gentiana austriaea +Anthyllis vulneraria subsp. alpestris rHieracium intybaceum rAndrosace obtusifolia + rArtemisia mutellina + +Astragalus alpinus r rTrifolium badium + 2b +Anthoxanthum alpinum rThymus pulegioides rCardamine alpina rVaccinium uliginosum rGentianella tenella rCardamine resedifolia rVeronicum doronicum rHomogyne alpina +Antennaria dioica +Gentiana verna + +Primula hirsuta r rTrifolium pratense subsp. Nivale r +Veronica bellidioides r +Phleum alpinum agg. + rCrysanthemum alpinum + 1 +Potentilla aurea + 2b 2aBartsia alpina 1 +Hieracium piloselloides rHieracium alpinum rCarex fusca + +Crepis aurea 1Ligusticum mutellina 2aSoldanella pusilla +Luzula campestris +Geum montanum +Nardus stricta 2aFestuca violacea +Festuca rubra +Ranunculus montanus +Euphrasia minima +Carex sempervirens 1Trifolium alpinum rHelictotrichon versicolor +Anthoxanthum odoratum +Pinguicula vulgaris rGentiana nivale rViola palustris +Phyteuma hemisphaericum +

r: 1–3 individuals with less than o1% coverage; +:o1%; 1: 1–5%; 2m450 individuals, but less than o5%; 2a: 5–15%; 2b: 16–25%; 3:26–50%.

Plant succession and rhizosphere microorganisms in alpine terrain 371

three replicate samples. Four replicates of non-rhizosphere soil (bulk soil) were collected fromthe surface (0–10 cm) of the same area andserved as a control. Bulk soil and rhizosphere soil

were stored in plastic bags at �20 1C. Prior toanalyses of enzyme activities, the samples wereallowed to thaw and were passed through a 2mmsieve.

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Table

2.

Selected

soilproperties

ofthesuccession

alsitesac

ross

theRo

tmoo

sferne

rglac

ierforeland

Succession

alag

e(yea

rs)

Corg(m

gg�

1)

Nt(m

gg�

1)

C=N

ratio

Cca

rb(m

gg�

1)

pH

NH4+–N(mgg�

1)

NO3�

–N(mgg�

1)

P(mgg�

1)

K(mgg�

1)

410

.32b

c0.28

a39

.1a

11.40a

7.5a

0.54

a—

0.07

12.08a

147.80

b0.85

a20

.9abc

8.40

a7.5a

0.54

a0.07

0.06

10.56a

b

207.00

b0.28

a26

.0abc

10.11a

7.5a

0.21

a—

—7.37

ab

302.93

a0.33

a8.8c

3.87

b7.3a

0.12

a—

0.07

4.46

cd

4312

.20c

d0.39

a31

.5ab

7.56

a7.3a

0.94

a—

0.05

4.05

cd

486.70

b1.08

ab

11.2

c1.65

c7.1a

0.90

a—

0.05

a3.60

c

7516

.01c

d1.35

b11

.9abc

0.02

6.0b

7.92

b0.11

0.17

b10

.50a

b

135

18.71d

1.79

b10

.3bc

—4.5c

9.91

b—

0.47

c6.56

bc

Referenc

e48

.87e

3.91

c12

.4abc

—4.2d

18.25b

—1.50

d9.30

ab

Resultsaregive

nas

mea

ns.Fo

rtheeffectsof

succession

alag

e,va

lues

withthesamesuffixletter

areno

tsign

ifica

ntly

different

atthePp

0.05

leve

l.Mea

nswitho

utsuffixletters:

insufficien

tnu

mber

ofreplica

tesdue

toze

rova

lues.

D. Tscherko et al.372

Microbial biomass and phospholipid fatty acidanalysis

Ninhydrin-reactive nitrogen (NHR-N), as an in-dicator of microbial biomass, was extracted afterfumigation of the soils with chloroform, and thenitrogen compounds in the 2M KCl extracts werecolourimetrically determined at 570 nm (Amato &Ladd, 1988).

Phospholipids were extracted from 2 g soil,fractionated and quantified using the proceduredescribed by Frostegard, Baath, and Tunlid (1993)and Bardgett, Hobbs, and Frostegard (1996).Separated fatty acid methyl-esters were identifiedusing gas chromatography and a flame ionizationdetector. Fatty acid nomenclature was usedas described by Frostegard et al. (1993). The fattyacids i15:0, a15:0, 15:0, i16:0, 16:1 o7c, 17:0,i17:0, cy17:0, 18:1 o7c and cy19:0 were chosen torepresent bacterial biomass (bacterial PLFA), and18:2 o6,9c (fungal PLFA) was taken to indicatefungal biomass (Federle, 1986; Zelles, 1999). Theratio of bacterial PLFA to fungal PLFA (bacterialPLFA/fungal PLFA) was calculated to indicate shiftsin the ratio between bacterial and fungal biomass.The Gram-positive specific fatty acids i15:0, a15:0,i16:0 and i17:0 and the Gram-negative specificfatty acids cy17:0, 18:1 o7c and cy19:0 were takenas a measure of the ratio between Gram-positiveand Gram-negative bacterial biomass (Gram-positives/Gram-negatives ratio). The fatty acid20:5 o3c was used as an indicator for soil algae(Fleurence, Gutbier, Maubeau, & Leray, 1994;Khotimchenko, Vaskovsky, & Titlyanova, 2002).Results are given in [nmol g�1].

Enzyme activity

A range of hydrolytic enzymes involved in C, N, Pand S processes were investigated using a fluori-metric microplate assay, described by Marx, Woodand Jarvis (2001). The activities of b-glucosidase(E.C. 3.2.1.21), N-acetyl-b-glucosaminidase (E.C.3.2.1.30), leucine aminopeptidase (E.C. 3.4.11.1),b-xylosidase (E.C. 3.2.2.37), acid phosphatase(E.C. 3.1.3.2) and sulphatase (E.C. 3.1.6.1) weremeasured using 4-methylumbelliferyl (MUB)-b-1,4-glucoside, MUB-N-acetyl-b-1,4-glucosaminide, L-leucine-7-amido-4-methyl-coumarin, MUB-b-xylo-pyranoside, MUB-phosphate and MUB-sulphate assubstrates, respectively. Enzyme activities weredetermined as the rate of release of MUB and 7-amino-methylcoumarin (AMC) from the MUB- orAMC-labelled substrates, respectively. The soil-substrate solutions were incubated at 30 1C and

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Plant succession and rhizosphere microorganisms in alpine terrain 373

the release of MUB and AMC was measuredcontinuously for 74min, using an automated lumi-nescence spectrophotometer (emission 446 nm,excitation 377 nm). Activities are reported in [nmolMUB/AMCg�1min�1] units.

Data handling and statistical analysis

Microbial biomass (NHR-N), enzyme activity, andphospholipid fatty acids were calculated on anoven-dry weight (105 1C) basis. Soil chemical andmicrobiological data were log-transformed to ob-tain normal distribution. Differences in chemicalproperties between soils were assessed by univari-ate analysis of variance, followed by the multiplerange test (Student Newman Keuls test). Coeffi-cients of variance (CV) of enzyme activities andfatty acids were calculated for each species. Thearithmetic means of the CV are listed in Tables 3and 4. The plants T. pallescens, T. badium,Leontodon helveticus and Potentilla aurea werenot included in the calculation due to the insuffi-cient number of replicates (k ¼ 2). A principalcomponent analysis (PCA) was performed on thePLFA and enzyme activity data to ordinate the bulksoils and rhizospheres according to the successionalstage, and to identify the importance of eachmicrobial property. The unrotated factor scores ofthe PLFA and enzyme activity data were displayedfor each sample in two individual PCA plots. Thefactor scores (PCA 1, PCA 2) were tested forsignificant differences between soils as describedabove (sample size n ¼ 66). The relationship (r)between environmental properties (number ofplant species, coverage, life forms, Corg, Nt, C=Nratio) and bulk soil microbial properties was testedby bivariate correlation analysis. Due to the highautocorrelation of Corg and Nt with other soilproperties, partial correlation analysis was appliedto eliminate the influence of Corg and Nt on thevariables of interest (pH, NO3

�–N, NH4+–N, P and K)

(n ¼ 25 for microbial data, n ¼ 7 for vegetationproperties). Significance was accepted at thePp0:05 level of probability.

Results

Vegetation development

The gradual retreat of the Rotmoosferner glacierover the last 150 years has resulted in a series ofplant communities whose successional age dependson the time since glacial retreat. Although thedelineation of vegetation units across the glacier

moraine field was difficult because of the transi-tional changes of vegetation between the succes-sional stages, a clear trend from a short-livedpioneer vegetation to an alpine grassland wasobserved (Table 1). In detail, the 4-year-old sitewas devoid of vegetation due to the short time ofecosystem development. The 14-year-old siteharboured exclusively early pioneer species,namely Saxifraga aizoides and Saxifraga oppositi-folia; less dominant species were Arabis caeruleaand Arabis hirsuta as well as Koeleria hirsuta andArtemisia genipi. The height of these plants neverexceeded 5 cm. Cover and abundance was only 3%.In the subsequent stage (20 years) an increasingnumber of species became established. The highestcoverage was observed for Artemisia genipi.Further species were Linaria alpina and thepioneers mentioned above. T. pallescens and SiIeneexscapa also appeared, but were more typical forlater stages. Poa alpina, the most common plantspecies within the deglaciated area, occurred therefor the first time. Poa alpina and Leontodonhelveticus represented intrinsic species of thealpine grassland outside the glacier foreland.Vegetation cover reached 5%, maximum plantheight was 10 cm, and the number of species wasten. Within 30 years of succession the number ofspecies increased up to 30. Total plant coveragereached 25%, and the vegetation was about 15 cmhigh. The dominant species were Saxifragabryoides, Saxifraga oppositifolia, Saxifraga ai-zoides, Silene exscapa and Poa alpina. Accompany-ing species were Achillea moschata, Campanulacochlearifolia, Campanula scheuchzeri and Sem-pervivum montanum. The perennials Luzula spicataand Festuca pumilla occurred, and both were alsotypical species on the reference site. The 43-year-old plot was dominated by Artemisia genipi,Polygonum viviparum and Poa alpina. Elyna myo-suroides, Agrostis rupestris and Deschampsia ce-spitosa represented further grasses. For the firsttime, dwarf shrubs of the genus Salix appeared,with Salix herbacea, Salix reticulata, Salix retusasubsp. retusa and Salix helvetica as most importantrepresentatives. Pioneer plants were still present,but low in abundance (except for Artemisia genipi).Total coverage remained at 25%, with 34 plantspecies present. The herbaceous layer and shrubsreached 15 and 20 cm height, respectively. After 48years of succession, only few annual plants werepresent and these had a low coverage, whereas theabundance of the grasses Poa alpina and Festucapumilla increased. At the same time the impor-tance of species belonging to the legume family(T. pallescens, T. badium) grew. The vegetationheight did not change compared to earlier

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Table 3. Abundance of ninhydrin-reactive nitrogen (NHR-N) and fatty acids in the rhizosphere of early and late colonizer plants. Age: successional age; REF:reference; CV: mean coefficients of variance

Age(years)

Plant species NHR-N(mgN g�1)

14:0(nmol g�1)

i15:0(nmol g�1)

a15:0(nmol g�1)

15:1(nmol g�1)

15:0(nmol g�1)

i16:0(nmol g�1)

16:1 o7c(nmol g�1)

16:0(nmol g�1)

i17:0(nmol g�1)

17:1(nmol g�1)

4 Soil 1.01 0.10 0.15 1.02 0.20 0.66 — — 0.42 — —

14 Soil 0.04 — — 1.60 0.20 — — 0.24 0.51 — —Saxifraga 6.09 — 0.06 1.11 0.20 — — 0.82 3.46 — —

20 Soil 0.24 — — 0.29 0.07 — 0.89 0.04 0.24 — —Linaria 2.27 — — 1.04 0.02 — — 0.07 0.19 — —Poa 11.29 — 0.24 0.16 0.08 — 0.35 2.40 6.39 0.18 —Trifolium pallescens 0.00 0.50 0.49 1.35 0.21 — — 3.38 9.64 — —

43 Soil 2.85 0.32 1.24 0.02 — 0.09 1.52 2.64 — —Trifolium pallescens 10.53 2.56 6.30 8.18 0.61 1.68 3.27 18.64 39.25 1.08 —Campanula 20.11 0.47 2.58 2.00 — 0.55 1.01 10.25 20.64 0.78 —Elyna 9.20 0.12 0.94 1.31 0.05 0.18 0.37 2.88 4.64 0.21 —Gentiana 17.06 0.24 1.82 1.02 — 0.30 0.79 6.41 11.03 0.58 —

75 Soil 14.67 0.17 1.81 1.26 0.05 0.12 1.10 5.81 12.00 0.71 0.44Poa 91.86 4.75 22.25 23.55 0.37 4.27 20.98 69.88 114.40 7.87 7.89Trifolium badium 207.50 25.01 94.83 80.95 1.75 12.70 56.74 142.81 297.77 18.99 —Leontodon 87.60 12.52 58.11 46.04 1.37 7.82 28.08 114.03 176.70 12.09 —

135 Soil 12.95 1.05 6.90 4.42 0.37 0.85 3.73 8.63 18.78 2.62 —Potentilla 210.16 18.41 76.95 75.95 3.06 12.86 42.35 127.46 248.16 15.27 —

REF Soil 14.18 0.73 5.62 3.67 1.04 1.43 5.97 7.06 27.66 4.16 0.21Poa 107.38 5.90 37.67 27.12 2.21 7.21 31.80 49.53 127.66 13.79 2.34Crepis 63.01 3.68 23.21 21.95 1.08 4.56 10.20 36.63 92.05 7.08 —Nardus 80.55 4.63 35.35 21.42 0.76 5.18 15.35 38.45 86.77 8.47 —Carex 81.62 8.72 45.19 33.46 1.73 6.99 24.21 60.33 138.59 10.59 —

CV 0.75 0.80 0.69 0.66 0.89 0.80 0.63 0.57 0.48 0.44 1.56

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PRES

S

Age(years)

Plant species cy17:0(nmol g�1)

17:0(nmol g�1)

18:3 o3c(nmol g�1)

18:1 o9c(nmol g�1)

18:1 o7c(nmol g�1)

18:0(nmol g�1)

cy19:0(nmol g�1)

20:2(nmol g�1)

20:0(nmol g�1)

22:0(nmol g�1)

24:0(nmol g�1)

20:5 o3c(nmol g�1)

4 Soil — 0.17 0.14 1.00 — 0.40 — 0.20 — 0.12 0.25 1.56

14 Soil — 0.07 0.07 0.49 0.28 0.04 — — — — — 1.35Saxifraga — 0.07 0.73 7.61 0.16 0.29 — — — — — 0.73

20 Soil — 0.06 — 0.01 — 0.02 — — — — — 0.55Linaria — — 0.13 0.71 — 0.01 — — — — 0.12 1.24Poa 0.14 0.33 — 3.15 4.23 1.11 — — — 0.07 0.26 0.87Trifolium pallescens 0.00 0.45 0.66 13.10 1.62 1.66 0.31 — — 0.41 0.66 0.76

43 Soil — — — 1.48 1.69 0.34 — — — — — 0.52Trifolium pallescens 2.35 1.20 0.91 13.13 18.64 5.07 0.51 — 1.03 1.26 1.57 1.90Campanula 1.13 0.83 0.13 9.61 11.88 2.57 0.25 0.37 0.83 1.20 1.69 2.26Elyna 0.33 0.14 0.05 3.18 5.23 0.53 0.19 — 0.05 0.36 0.45 1.86Gentiana 0.71 0.48 0.08 5.80 8.71 1.40 0.17 0.24 0.37 0.80 0.91 2.24

75 Soil 1.31 0.41 0.15 5.68 8.54 2.44 0.69 — 0.44 0.74 0.99 0.35Poa 11.42 4.57 5.65 60.16 89.46 18.76 3.24 0.85 5.00 6.15 8.38 6.01Trifolium badium 26.86 11.60 4.95 140.54 206.46 42.48 6.34 0.45 12.61 12.44 12.13 2.96Leontodon 20.06 6.52 8.06 103.86 150.25 12.69 9.73 2.54 5.88 9.28 10.79 5.38

135 Soil 3.06 1.11 0.30 10.21 10.93 3.14 2.11 — 1.22 1.73 1.33 0.70Potentilla 26.93 12.04 6.48 124.11 150.17 41.61 11.19 8.25 11.38 12.40 9.99 6.02

REF Soil 4.55 2.32 - 27.25 19.43 9.77 2.64 — 3.05 3.08 2.25 0.22Poa 20.54 7.39 0.87 128.09 99.83 34.53 11.06 1.31 9.21 11.16 7.33 2.53Crepis 15.01 3.84 0.93 57.99 65.22 11.56 34.42 2.53 5.08 6.06 4.27 0.90Nardus 16.65 4.19 - 68.05 59.51 15.13 31.49 1.35 7.10 7.06 4.99 0.54Carex 17.58 5.34 0.93 86.81 93.47 21.07 29.92 0.92 7.22 9.94 6.83 1.60

CV 0.38 0.64 0.86 0.54 0.45 0.84 0.63 0.84 0.46 0.50 0.56 0.82

Table 3 (continued)

Plant

successionand

rhizosphere

microorganism

sin

alpine

terrain375

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Table 4. Enzyme activity in the rhizosphere of the early and late colonizer plants

Age(years)

Plant species Leucineaminopeptidase(nmolAMCg�1 h�1)

NAG b-Glucosidase(nmol MUBg�1 h�1)

b-Xylosidase(nmol MUBg�1 h�1)

Phosphatase Sulfatase

4 Soil 1.14 0.43 0.00 — 3.47 0.00

14 Soil 1.78 0.44 0.07 0.02 2.50 —Saxifraga 2.38 0.24 0.47 — 3.06 —

20 Soil 1.69 0.23 — 0.04 2.69 —Linaria 0.39 0.32 0.67 0.06 4.43 0.01Poa 3.69 2.20 10.36 0.18 15.03 0.73Trifoliumpallescens

4.63 1.79 18.31 0.41 36.66 —

43 Soil 2.50 1.09 3.02 0.08 3.38 0.02Trifoliumpallescens

8.80 11.93 41.33 3.27 80.24 0.99

Campanula 14.43 8.38 29.51 1.59 32.72 1.13Elyna 7.63 13.05 17.31 2.25 27.39 1.00Gentiana 10.01 3.10 14.38 0.96 13.35 0.93

75 Soil 9.19 12.17 43.64 5.30 179.40 3.35Poa 55.73 54.21 240.50 25.68 405.06 15.57Trifoliumbadium

80.11 59.76 407.64 35.69 580.48 35.56

Leontodon 38.16 34.16 125.99 15.22 383.68 15.56

135 Soil 6.50 7.55 18.99 4.36 149.45 3.55Potentilla 103.88 61.21 435.85 63.00 642.61 24.20

REF Soil 7.51 12.34 21.72 16.80 317.80 13.33Poa 22.91 375.74 38.01 43.60 21.30 569.31Crepis 2.72 39.81 54.95 60.55 45.13 1067.05Nardus 22.28 47.24 46.20 49.19 764.60 30.52Carex 21.18 68.02 125.54 59.73 1094.54 19.53

CV 0.35 0.49 0.59 0.78 0.42 0.69

Age: successional age; NAG: N-acetyl-glucosaminidase; REF: reference; CV: mean coefficient of variance.

D. Tscherko et al.376

stages, whereas the total vegetation cover reached35%.

A big change in vegetation occurred between the48-year- and the 75-year-old stage. Vegetationcovered 70% of the surface in the latter stage,and the number of species increased to 46. Themost dominant plant species was Silene exscapa,followed by Poa alpina, Festuca pumilla, T.pallescens and T. badium, which formed an initialgrassland vegetation. Further accompanying spe-cies were Euphrasia rostkoviana, Sibbaldia pro-cumbens and Polygonum viviparum. Many of thepioneer plants were replaced, except for someSaxifraga species. After 135 years of succession,the vegetation still covered 70%, but the numberof species declined to 31. The vegetation was

dominated by Poaceae and Fabaceae, along withPotentilla aurea, SibbaIdia procumbens, Campanu-la scheuchzeri, Euphrasia rostkoviana and Leonto-don helveticus. Pioneers became extremely rare.Although this vegetation was similar to an alpinegrassland community, many of the colourful alpinemeadow species were still lacking. The vegetationof the reference site, about 9500 years old,represented a typical Poa–Carex–Nardus alpinegrassland. The vegetation covered 80%, contained50 different species, and reached about 20 cm inheight. Many species not found within the glacierforeland were present, including Crepis aurea,Ligusticum mutellina, Nardus stricta and Carexsempervirens. Further dominant species were Poaalpina, Potentilla aurea, Polygonum viviparum,

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Plant succession and rhizosphere microorganisms in alpine terrain 377

Festuca pumilla, Agrostis rupestris, Campanulascheuchzeri and Sibbaldia procumbens. The le-gumes T. pallescens and T. badium were stillpresent, completed by T. pratense subsp. nivaleand T. alpinum. The pioneers were no longerpresent.

Fig. 1 shows the change in vegetation cover andplant life forms according to the process of soildevelopment. The vegetation cover slowly in-creased to nearly 80%, which indicates that this

0

5

10

15

20 Geophytes

0

5

10

15

20 Therophytes

0

25

50

75

100 Chamaephytes

0

25

50

75

100 Hemicryptophytes

0

25

50

75

100 Vegetation cover

0 20 40 60 80 100 120 140 REF

Surface age [years]

Figure 1. Surface cover [%] of the vegetation and ofdifferent life forms according to the successional stage;REF ¼ reference.

process still had not been completed even aftermore than 135 years. There were still gaps and baresoil within the site. The different plant life formshad invaded at different stages. The hemicrypto-phytes and herbal chamaephytes were the mostabundant plant life forms along the glacier fore-land. The coverage of herbal chamaephytes in thevegetation steadily decreased during succession,while the cover of hemicryptophytes increased.Therophytes (annuals) represented the typicalplant life form of the youngest sites (up to 20years) and almost completely disappeared in theolder ones. The geophytes showed no clear trendacross the chronosequence. This plant life formreached its highest abundance in the early (20years) and late successional stages (75 years andolder), while the 30–48-year-old sites were low ingeophytes. The woody chamaephytes were absenton the sites deglaciated for 30 years, but primarilyoccurred and peaked on the 43-year-old stage, witha constant decline from 9% to 2% as successionprogressed. The nanophanerophytes were patchilydistributed across the chronosequence, with sur-face coverage below 3%. This plant life form wasthe least abundant.

Environmental interrelations

Most of the microbial changes in the bulk soilwere related to the vegetation cover, plant lifeform and soil organic matter. The rise in vegetationcover and the increase in abundance of hemicryp-tophytes were positively correlated with microbialbiomass (r ¼ 0:82–0.99), fungal PLFA (r ¼ 0:85–

0.98), bacterial fatty acids (except for a15:0)(r ¼ 0:78–0.97), the fatty acids listed in Table 3(r ¼ 0:57–0.93), and the enzyme activities (r ¼0:78–0.95). The proportion of therophytes in thevegetation was related to the Gram-positives/Gram-negatives ratio (r ¼ 0:93) and soil algae(20:5 o3c) (r ¼ 0:89). The other plant life forms(chamaephytes, geophytes, nanophanerophytes)were weakly correlated with the properties of thebelowground microbial community. Plant speciesrichness was correlated with microbial biomass(r ¼ 0:87), a few fatty acids (fungal PLFA, 16:1 o7c,16:0, 18:1 o7c, 20:5 o3c) (r ¼ 0:79–0.92) and theactivities of N-acetyl-glucosaminidase (r ¼ 0:87),b-glucosidase (r ¼ 0:79), phosphatase (r ¼ 0:82)and leucine aminopeptidase (r ¼ 0:87). A strongpositive relationship between soil organic matter(Corg, Nt) and microbial biomass (r ¼ 0:82), fattyacids (r ¼ 0:54–0.84) and enzyme activity (r ¼0:59–0.94) could be established. Solely the fattyacids 15:0, 17:1, 18:3 o3c, cy19:0 and 20:5 o3c

ARTICLE IN PRESS

0

20

40

60

80

nmol

g-1

nmol

g-1

200400600800

Soi

lS

oil

Sax

ifrag

aS

oil

Lina

riaP

oaT

. pal

l.S

oil

T. p

all.

Cam

panu

laE

lyna

Gen

tiana Soi

lP

oaT

. bad

ium

Leon

todo

nS

oil

Pot

entil

laS

oil

Poa

Cre

pis

Nar

dus

Car

ex

fungal PLFAbacterial PLFA

0

5

10

15

20

25

100

200

300

il il a il ia a ll. il ll. a a a il a n il la il a is s x

Gram-positivesGram-negatives

4 14 20 43 75 135 referencePlant species / surfaceage [years]

A

B

D. Tscherko et al.378

(soil algae) were not correlated with Corg orNt. The decrease in C=N ratio was related toan increase in microbial biomass (r ¼ �0:79), in thebacterial fatty acid i15:0 (r ¼ �0:80) and in theactivities of xylosidase (r ¼ �0:87) and sulphatase(r ¼ �0:90), while other bacterial fatty acids(i16:0, 17:0, bacterial PLFA, Gram-positives) wereonly weakly correlated with the C=N ratio(r ¼ �0:54–�0.60). Soil nutrients were mainlyrelated with the activity of enzymes, whereas nosignificant relationship could be established withthe microbial biomass and fatty acids. In particular,ammonium was positively correlated with enzymeactivity (r ¼ 0:54–0.99), whereas phosphorus wasnegatively correlated with sulphatase (r ¼ �0:99),b-xylosidase (r ¼ �0:99) and leucine aminopepti-dase (r ¼ �0:62). Nitrate and potassium showed nocorrelation with growth and activity of the below-ground microbial community. Soil pH was nega-tively related to fungal PLFA (r ¼ �0:51), thebacterial fatty acids i15:0, i16:0, 16:1 o7c, i17:0,cy17:0, 18:1 o7c, cy19:0 (r ¼ �0:55–�0.95), Gram-positives (r ¼ �0:78), Gram-negatives (r ¼ �0:76),bacterial PLFA/fungal PLFA ratio (r ¼ �0:66) and tophosphatase activity (r ¼ �0:46).

So

So

Sax

ifrag S

oLi

nar

Po

T. p

a So

T. p

aC

ampa

nul

Ely

nG

entia

n So

Po

T. b

adiu

mLe

onto

do So

Pot

entil S

oP

oC

rep

Nar

duC

are

0

4

8

12

16

Soi

lS

oil

Sax

ifrag

aS

oil

Lina

riaP

oaT

. pal

l.S

oil

T. p

all.

Cam

panu

laE

lyna

Gen

tiana Soi

lP

oaT

. bad

ium

Leon

todo

nS

oil

Pot

entil

laS

oil

Poa

Cre

pis

Nar

dus

Car

ex

Gram-positives/Gram-negativesbacterial PLFA/fungal PLFA

4 14 20 43 75 135 reference

4 14 20 43 75 135 reference

Plant species / surface age [years]

Plant species / surface age [years]

C

Figure 2. Successional changes of the phospholipid fattyacid pattern in the rhizosphere of different plant speciesand in the bulk soil: bacterial PLFA, fungal PLFA, Gram-positives, Gram-negatives, bacterial PLFA/fungal PLFAratio, Gram-positives/Gram-negatives ratio. Bars aremeans, whiskers indicate standard deviation (meandeviation is given for Trifolium pallescens, Trifoliumbadium, Leontodon, Potentilla). Note break in y-axisscale (T. pall: Trifolium pallescens).

Plant species effect

Microbial biomass and phospholipid fatty acidpattern

The microbial biomass (NHR-N) in the rhizo-sphere of the early colonizer plants was very lowwithin the first 43 years of succession and similar tothe bulk soil concentrations (Table 3). In subse-quent successional stages, the rhizosphere micro-bial biomass grew one order of magnitude(Leontodon, Poa, Carex, Crepis, Nardus) andreached its maximum in the root zone of T. badiumand Potentilla in the 75- and 135-year-old stages,respectively. Rhizosphere fatty acid concentrationswere similar between plant species of the earlystages (up to 43 years), whereas the differencesbecame more distinct as the ecosystem developed.Bacterial PLFA, fungal PLFA, Gram-positives, Gram-negatives and 20:5 o3c (soil algae) increased 20-fold under plant species of the 75- and 135-yearsite (Fig. 2A and B, Table 3). In particular, T. badiumand Potentilla frequently contained highest con-centrations of fatty acids in their rhizosphere. Thebacterial PLFA/fungal PLFA ratio peaked in therhizosphere of Poa, Carex, Nardus and Crepis atthe reference site (Fig. 2C). The Gram-positives/Gram-negatives ratio tended to be higher for latesuccessional plant species (75 years and older) thanfor early colonizers (except for Saxifraga). How-

ever, no species effect was detected within thesetwo groups. No ratio could be calculated for Linaria

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0

1

2

3

4

5

6

PC

A 2

(8

%)

T. badium

LeontodonLeontodon

Poa

Poa PoaPotentilla

PotentillaCarex

NardusPoa

Phospholipid fatty acids

4 - 43 years

reference135 years75 years

SaxifragaLinariaPoa (20 years)T. pallescensCampanulaElynaGentiana

bulk soils

A

Plant succession and rhizosphere microorganisms in alpine terrain 379

due to the lack of Gram-negatives (Fig. 2C). Theother fatty acids, listed in Table 3, showed a similarpattern: within the first 43 years of succession, allplant species were similar in their fatty acidabundance and composition (except for T. palles-cens at the 43-year site), whereas the amount offatty acids in the rhizosphere of the late succes-sional plant species was 5–10-fold higher comparedto early successional species.

-1

0

1

2

3

4

5

PC

A 2

(26

%)

T. badiumT. badium

LeontodonLeontodon

Poa

PoaPoa Potentilla

Potentilla

Carex Carex

Carex

Crepis

Crepis

Crepis

NardusNardus

Nardus

PoaPoa

Poa

4 - 43 years

Enzyme activity

-3

-2

-1

-1 0 1 2 3 4PCA 1 (78 %)

T. badiumCarex

Carex

Crepis

Crepis

CrepisNardus

Nardus

PoaPoa

reference135 years75 years

SaxifragaLinariaPoa (20 years)T. pallescensCampanulaElynaGentiana

bulk soils

B

Enzyme activity

Rhizosphere enzyme activity was not affected byplant species or site age during the first 43 years ofsuccession and was similar to the activity levels ofthe bulk soil. As succession progressed, differencesbetween plant species and between rhizosphereand bulk soil became significant (Table 4). Plantspecies of the 75- and 135-year-old site (T. badium,Potentilla) revealed highest activities of leucineaminopeptidase and b-glucosidase, while plantspecies of the reference site were highest inN-acetyl-b-glucosaminidase (Poa), phosphatase(Carex, Nardus) and sulphatase (Poa, Crepis). Plantspecies of the 135-year-old site (Potentilla) and thereference were highest in b-xylosidase.

-2-2 -1 0 1 2 3 4

PCA 1 (52 %)

Figure 3. Principal component plot of phospholipid fattyacid pattern and enzyme activity. Distinction of samplesby plant species and successional stage (symbols indicateyears since deglaciation).

PCA ordination

Two principal components were extracted fromthe fatty acid data (Fig. 3A). PC 1 separated therhizospheres of the younger sites (up to 43 years)from those of the later stages. The bulk soilsclustered with the early colonizing plant speciesregardless of the successional stage. The latesuccessional plant species tended to group accord-ing to surface age, although there was highheterogeneity. T. badium and Potentilla wereseparated from Leontodon, Poa (reference), Carex,Crepis and Nardus along PC 1. PC 2 differentiatedPoa (75 years) from T. badium, Nardus, Carex,Crepis and Poa (reference). Nineteen out of 24fatty acids were highly correlated with PC 1, whichaccounted for 78% of the total variance among thePLFA data. PC 2 explained 8% of the total varianceand was dominated by the fatty acids 17:1, 18:3o3c, cy19:0 (Gram-negatives) and 20:5 o3c (soilalgae).

The enzyme activity data revealed a similarordination pattern. Along PC 1 the bulk soils andthe plant species of the early stages of succession(up to 43 years) formed a central core, with nodifferences between plant species, bulk soils orsuccessional stage (Fig. 3B). The late successionalplant species (75 years or older) were distant from

the central core. PC 2 allowed the differentiationof the late colonizers Crepis and Poa (reference)from T. badium, Leontodon, Potentilla, Nardus andCarex. PC 1 accounted for 52% of the variation inthe enzyme activity data and was dominated by theenzymes b-glucosidase, b-xylosidase, leucine ami-nopeptidase and acid phosphatase. N-acetyl-b-glucosaminidase and sulphatase were highly corre-lated with PC 2, explaining 26% of the variability ofthe data.

Discussion

Vegetation development

The vegetation survey showed that a 4-yearperiod of ecosystem development was too shortto enable plant species to colonize the bare soil. Atypical pioneer vegetation appeared within 14–20

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D. Tscherko et al.380

years. Those pioneer plants that were able to settleprobably exhibit a specific ecological adaptation tothis harsh environment and, in the long term,possibly facilitated germination and growth ofsubsequent colonizer plant species. N-fixing le-gumes (T. pallescens, T. badium) occurred rela-tively late in the succession. Legumes, andparticularly nodulation, are known to require highphosphorus supply (Marschner, 1995). Probably, thelow phosphorus availability on the up to 48-year-oldsites constrained the growth of legumes, whereashighest densities of Trifolium were found on the 75-year-old site. Woody plants did not permanentlycolonize the deglaciated moraine field. For exam-ple, Salix shrubs appeared on the 43–48-year-oldstages, but were replaced by more competitivegrasses (e.g., Poaceae) as succession progressed.This finding is in accordance with the hypothesisthat shrubs dominate the intermediate phase ofvegetation succession because they are less com-petitive due to their low growth rates compared toperennial herbs (Grime, 2001).

Based on the distribution of plant life formsacross the moraine field, the data suggest that theabundance of annuals (therophytes) decreased andthe perennials increased during succession. More-over, the increase in vegetation cover and plantdiversity was related to an increase in plantbiomass, which most likely resulted in enhancedcompetition for available nutrient resources in thesoil (Tilman, 1999; Heer & Korner, 2002). On theother hand, the increase in plant surface coverindicates a decreased disturbance with ecosystemdevelopment. Grime (2001) proposed the CSRmodel, which suggests the importance of distur-bance, competition and mineral nutrient stress asdeterminants of the vegetation in primary succes-sion. Three types of plant strategies can bedistinguished: the competitors which exploit condi-tions of low stress and low disturbance, the stress-tolerators (high stress, low disturbance), and theruderals (low stress, high disturbance). If we applythis model to the Rotmoosferner glacier foreland,the result suggests that the plant community shiftedfrom a more ruderal-dominated plant community inthe early stages (up to 48 years) to a morecompetitive vegetation in the late successionalstages. The latter community represents the bal-ance between nutrient availability and competitionand is characterized by high productivity.

Environmental interrelations

The vegetation cover, hemicryptophytes and soilorganic matter proved to be the most important

drivers of microbial changes during succession. Theplant cover regulates the temperature regime atthe soil surface (Roxburgh, Wilson, & Mark, 1988;Korner, 2003) because barren soil surfaces exposedto full sunlight can heat up to temperatures of50–80 1C (Reisigel & Keller, 1994). Heat stress andsoil drying also adversely affect soil nutrientavailability (Sah & Mikkelsen, 1986), root growthand exudation, which in turn affect microbialgrowth and activity (Drew, 1990; Paul & Clark,1996). Our data suggest that the increase invegetation cover decreased heat stress and drynessat the soil surface, favouring microbial growth andactivity. With the increase in vegetation cover, theherbaceous hemicryptophytes became more abun-dant; this in turn promoted aboveground biomassproduction, which successively allowed soil organicmatter to accumulate. In a subsequent step, soilorganic matter favoured microbial growth andactivity (Wardle, 2002). The enhanced input offresh litter with increasing plant cover probablyfurther improved soil organic matter quality, whichfinally could have favoured faster-growing bacteriaversus fungi. In accordance with previous studies(Yao, He, Wilson, & Campbell, 2000; Tscherko etal., 2003), these close relationships confirm thedependence of heterotrophic microorganisms onthe quantity and quality of organic matter in thesoil. The weak correlation between plant speciesrichness and the properties of the belowgroundmicrobial community suggests that short-termeffects on microbial populations caused by specificplant species are less important than long-term soilprocesses, such as the build-up of soil organicmatter (Wardle, Bonner, Barker, Yeates, Nicholson,et al., 1999; Spehn, Joshi, Schmid, Alphei, &Korner, 2000).

Successional age

In our study, microbial abundance and activity inthe rhizospheres increased during succession.Based on the CSR framework (Grime, 2001), thecompetitive plant communities of the later stagesare characterized by high productivity, which inturn favoured the rhizosphere microbiota. Differ-ences to colonizers of the younger stages might beinterpreted as a sign of better soil conditions (e.g.,more humus and mineral nutrients). Additionally,the increase in rhizosphere microbial biomass, fattyacids and activity during succession might berelated to the shift from a vegetation dominatedby annuals in early succession to a vegetationdominated by perennials in late succession. Annualsgenerally release less C than perennials, partly due

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Plant succession and rhizosphere microorganisms in alpine terrain 381

to perennials having to invest more of theirassimilates to survive year round (Harris, Santanto-nia, & McGinty, 1980). Higher rates of carbonrelease by perennials might have favoured growthand activity of rhizosphere microorganisms (Grays-ton, Vaughan, & Jones, 1996; Bardgett, Mawdsley,Edwards, Hobbs, Rodwell, et al., 1999).

Plant species effect

Most pronounced plant species effects on thesize, composition and activity of the rhizospheremicroorganisms were observed in the late succes-sional stages (75 years and later), while no effectswere detected within the first 43 years of succes-sion. Based on the concept proposed by Callaway etal. (2002), the distinct clustering of the earlycolonizers (within 43 years) and the differencesbetween late successional plant species might beexplained by a shift from a non-plant-specificmicrobial community in early succession to plant-specific microbial communities in subsequentstages. During early succession, plant specieseffects on rhizosphere microbiota were probablyrestricted by abiotic constraints. Those plantspecies capable of colonizing under such difficultconditions seem to harbour no specific microorgan-isms in their rhizospheres. These rhizosphere andindigenous soil microorganisms might be deter-mined by physical and chemical limitations (e.g.excess or shortage of water and heat). This is inclear contrast to the findings reported by Bardgettand Walker (2004), who observed a significanteffect of Rhacomitrium, Alnus and Equisetum onthe rhizosphere fatty acid composition within thefirst 15 years of succession at Glacier Bay, Alaska.They argued that moisture retention, and build-upof humus and nitrogen by nitrogen fixing Alnus,were probably responsible for promoting microbialgrowth. These contrasting results might be ex-plained by the much faster vegetation succession atGlacier Bay. Whereas woody plants occurred in thechronosequence of Glacier Bay within 15 years,they appeared in the Rotmoosferner glacier fore-land only after 40 years, indicating less favourableconditions for plant and microbial growth in theharsh alpine environment.

The better coverage of different microhabitatsand a more competitive interaction caused by anincreasing range of plant species and functionalgroups (the niche differentiation effect) in latesuccession could have resulted in more diversesubstrate supplies to microorganisms, which in turnfavoured the development of specific rhizospheremicrobial communities. Among the late colonizers,

T. badium, Leontodon and Potentilla of the 75- and135-year-old stage harboured the highest amountof microorganisms in their rhizospheres. Spehn etal. (2000) reported that the presence of legumesfavoured microbial biomass and decompositionrates, probably due to nitrogen fixation andsubsequently higher nitrogen availability, in grass-land soil. The higher density of legumes and thehigher nitrate concentration compared to the othersuccessional sites probably help to explain theenhanced amount and activity of the rhizospheremicroorganisms at the 75-year-old stage.

This study demonstrates that changes in thebelowground microbial community are related tovegetation changes and soil productivity. Highestmicrobial growth and activity were found at siteswhere sufficient nutrient supply favoured therhizosphere microbial community. We concludethat a shift from more abiotically dominatedinteractions to more competitive interactionsbetween plants, microorganisms and soil resourcesin primary succession affects the rhizospheremicrobial community and its function.

Acknowledgements

We would like to acknowledge Rudiger Kaufmannand Mainhardt Strobl, who assisted in the field,Karin Breuer, Sabine Rudolph and Cornelia Ruf fortechnical assistance with the laboratory analysis,Dr. Breuer (Federal Institute for Agricultural Chem-istry Hohenheim) for the determination of soilchemical properties, and Dr. Michael Stachowitschfor improving the language. We are also indebted toMarie-Claude Marx for her help with the enzymemeasurements and to the University of Reading,UK, for technical support. Logistic support wasprovided by the Alpine Research station of theUniversity of Innsbruck, Austria.

References

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Amato, M., & Ladd, J. N. (1988). Assay for microbialbiomass based on ninhydrin-reactive nitrogen inextracts of fumigated soils. Soil Biology and Biochem-istry, 20, 107–114.

Bardgett, R. D., Hobbs, P. J., & Frostegard, A. (1996).Changes in soil fungal:bacterial biomass ratios following

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