ABSTRACT It was hypothesized that seasonality and resource availability altered through tree girdling were major determinants of the phylogenetic composition of the archaeal and bacterial

community in a temperate beech forest soil. During a 2-year field experiment, involving girdling of beech trees to intercept the transfer of easily available carbon (C) from the canopy to

roots, members of the dominant phylogenetic microbial phyla residing in top soils under girdled versus untreated control trees were monitored at bimonthly intervals through 16S rRNA

gene-based terminal restriction fragment length polymorphism profiling and quantitative PCR analysis. Effects on nitrifying and denitrifying groups were assessed by measuring the abundances

soil nutritional and soil climatic changes associated with seasonality, indicating their high metabolic versatility and capability to adapt to environmental changes. For these phyla,

significant interrelations with soil chemical and microbial process data were found suggesting their potential, but poorly described contribution to nitrification or denitrification in

temperate forest soils. In conclusion, our extensive approach allowed us to get novel insights into effects of seasonality and resource availability on the microbial community, in particular

on hitherto poorly studied bacterial phyla and functional groups. SIMILAR CONTENT BEING VIEWED BY OTHERS STRUCTURAL AND FUNCTIONAL SHIFTS OF SOIL PROKARYOTIC COMMUNITY DUE TO _EUCALYPTUS_

PLANTATION AND ROTATION PHASE Article Open access 03 June 2020 RELATIONSHIPS BETWEEN NITROGEN CYCLING MICROBIAL COMMUNITY ABUNDANCE AND COMPOSITION REVEAL THE INDIRECT EFFECT OF SOIL PH ON

OAK DECLINE Article Open access 16 October 2020 PLANT-ASSOCIATED FUNGI SUPPORT BACTERIAL RESILIENCE FOLLOWING WATER LIMITATION Article Open access 09 September 2022 INTRODUCTION Trees

release large proportions of their accumulated carbon (C) and nitrogen (N) in the form of tree residues (for example, litter, dead roots) and root exudates to the soil organic matter

(Yarwood et al., 2009; Fontaine et al., 2004). The soil organic matter pool provides an important energy source for soil microorganisms, which are the major performing agents in

decomposition and soil organic matter transformation, the key processes in terrestrial C and N cycling (Buckley and Schmidt, 2002). Quantity and quality of available C and N control soil

microbial population dynamics and microbial processes including nitrification or denitrification (Schimel and Weintraub, 2003; Magill and Aber, 2000). In particular, it was shown that

microbial community structures were shaped by N cycle dynamics in forest soils (Högberg et al., 2007; Lejon et al., 2005; Grayston and Prescott, 2005). Belowground C and N transfer, shaped

by trees and through microbial processes, is influenced by external factors such as seasonality. Seasonally alternating climatic conditions take a decisive control on tree physiology,

photosynthesis and discharge of C and N into soil (Cannell and Dewar, 1994; Waring and Running, 1998). It can be concluded that cyclic changes in tree physiology have a significant influence

on soil microbial communities. In addition, temporal variability in the soil microbial community composition was shown in response to seasonal variation in temperature, moisture and plant

activity (Koch et al., 2007; Waldrop and Firestone, 2006; Horz et al., 2004; Buckley and Schmidt, 2002). Seasonal and other temporal alternations in climatic conditions are determinants of

soil N cycle dynamics (Cookson et al., 2006; Wolsing and Priemé, 2004; Horz et al., 2004). Soil N cycling includes both reductive and oxidative processes, in which soil microbes have a

predominant role (Cabello et al., 2004). Key microbial processes within the soil N cycle are catalyzed by key enzymes, including _amoA_ gene encoding a subunit of ammonia monooxygenase in

nitrification, as well as _nirS_ and _nirK_ gene (nitrite reductases) and _nosZ_ gene (nitrous oxide (N2O) reductase) involved in denitrification. The diversity and abundance of

microorganisms carrying these genes and the actual link to N2O emission have been extensively studied in diverse soil ecosystems (for example, Henderson et al., 2010, Leininger et al., 2006;

Philippot et al., 2006). Tree girdling is a procedure to remove the bark and phloem from a tree down to the youngest xylem effectively excluding rhizodeposition into soil and thus

restricting resource availability without disturbing the soil-root-microbe ecosystem (Högberg et al., 2001). It was shown that manipulation of C and N availability by tree girdling leads to

significant modifications in soil nutrient stoichiometry. Weintraub et al. (2007) measured lower dissolved organic C and N as well as an increase over time in nitrate and ammonium in girdled

plots of a subalpine forest. Högberg et al. (2007) observed a tendency towards higher inorganic N levels in girdled plots of a boreal forest, whereas Ekberg et al. (2007) detected a

decrease in total organic C in girdled plots of temperate spruce stand. It is thus likely that girdling-related changes in soil chemistry, in particular labile C and N pools have

considerable effects on the soil microbial community structure (Dannenmann et al., 2009; Weintraub et al., 2007). Although these examples show that soil microbial communities are influenced

by C and N availability as well as by seasonality, the effects on different bacterial phyla or functional groups in temperate forest soils are still poorly understood. The objective of this

study therefore was to assess in depth the microbial community response to C and N availability and to seasonal changes. A 2-year field experiment was carried out in a temperate beech forest

(Klausenleopoldsdorf, Lower Austria). The major hypothesis was that seasonality along with altered C and N availability achieved through tree girdling control the community structure of the

affected bacterial and archaeal population. Abundance and community structure of the total soil bacterial and archaeal population and specific phyla, that is, acidobacteria, alpha- and

beta-proteobacteria and verrucomicrobia, as well as nitrifying and denitrifying microbial communities were investigated. Detected community changes were related to seasonality and tree

girdling induced alterations in labile C and N pools as well as to soil moisture and soil temperature variations. MATERIALS AND METHODS EXPERIMENTAL SITE, SAMPLINGS AND GEOCHEMICAL DATA The

experimental study site was situated in a 65-year old beech forest (forest community _Hordelymo-Fagetum_ with main tree species _Fagus sylvatica_ L.) in Klausenleopoldsdorf (geographical

location: 48°07′N, 16°03′E, 510 m above see level), Lower Austria, approximately 40 km southwest of Vienna. At the study site, representing an extensively managed forest-monitoring site

according to the International Co-operative Programme on Assessment and Monitoring of Air Pollution Effects on Forests, a mean annual temperature of 7.6 °C and a mean annual precipitation of

768 mm were determined. The soil defined as a dystric cambisol had developed from the Laab formation (Eocene) and major geochemical properties were determined previously (pH value: 4.0;

total organic C: 4.36%; total N: 0.33%, C-to-N ratio: 13.1) (Hackl et al., 2004). The field experiment was started with girdling of trees in May 2006. Tree bark was removed at 10 cm length

around the trunk at about 1.5 m above ground. Three girdling plots of 20 × 20 m were installed, of which only the inner 10 × 10 m were used for soil samplings. Six replicate control plots

without tree girdling measuring 5 × 5 m were installed. Understory vegetation was removed from all plots and was repeated in the second spring season wherever necessary. During the whole

experimental phase, soil temperature and soil moisture were measured continuously (Kaiser et al., 2010). Further details about tree vitality, leaf litter amount and quality as well as fine

root biomass in girdled and control plots have been reported by Kaiser et al. (2010). Sampling was performed bimonthly from May 2006 until May 2008. Two replicate samples were taken from

each plot (six replicates in total), with four subsamples (soil cores of 10 × 10 × 5–10 cm, depending on the depth of A horizon) collected in each subplot. Generally, sampling was based on a

predetermined sampling scheme to avoid sampling of already disturbed soil and to warrant independent soil samples throughout the experimental period. The four sub-samples of each plot were

pooled, sieved through 2 mm mesh (5 mm mesh in case of wet soils) and stored at −20 °C. C and N pool data used for statistical purposes in this study were taken from Kaiser et al. (2010) and

Kitzler _et al._ (unpublished data). MICROBIAL COMMUNITY STRUCTURE (TERMINAL RESTRICTION FRAGMENT LENGTH POLYMORPHISM (T-RFLP) ANALYSIS) Bulk soil DNA was isolated (FastDNA Spin for Soil

Kit, MP Biomedicals, Solon, OH, USA) and extracts were quantified photometrically (Nanodrop ND-1000, Nanodrop Technologies, Wilmington, DE, USA). Bacterial and archaeal 16S rRNA genes were

PCR-amplified using primers sets targeting total bacteria and archaea as well as four selected bacterial phyla. Based on a soil 16S rRNA gene library of one hundred clones generated before

start of the field experiment, acidobacteria (28%), alpha- and beta-proteobacteria (18% and 14%, respectively), as well as verrucomicrobia (16%) have been selected as the four most dominant

bacterial community members in the soils of the studied experimental site (Supplementary Table S1; NCBI accession numbers HM364804 to HM364903). For T-RFLP analysis, all forward primers were

labeled with 6-carboxyfluorescein at the 5′ ends. From each DNA sample, two replicate PCRs were done. Composition of PCR cocktails and amplification details are provided in Table 1.

Amplicons (5 μl) were checked on ethidium bromide-stained 1% (w/v) agarose gels. Replicate amplicons were pooled, purified (Sephadex G-50, GE Healthcare Biosciences, Waukesha, WI, USA), and

approximately 200 ng of each amplicon were digested with 5 U _Alu_I (Invitrogen, Carlsbad, CA, USA) at 37 °C for 4 hrs. Before the T-RFLP analysis, digests were purified (Sephadex G-50) and

an aliquot of 5 μl was mixed with 15 μl HiDi formamide (Applied Biosystems, Foster City, CA, USA) and 0.3 μl internal size standard (500 ROX Size Standard, Applied Biosystems). Labeled

terminal-restriction fragments were denatured at 92 °C for 2 min, chilled on ice and detected on an ABI 3100 automatic DNA sequencer (Applied Biosystems) in the GeneScan mode. Gelquest

software package (version 2.2.1, SequentiX, Klein Raden, Germany) was used to compare relative lengths of terminal-restriction fragments with the 500 ROX size standard and to compile

electropherograms into a numeric data set, in which fragment length and peak height >50 fluorescence units were used for profile comparison. T-RFLP profiles used for statistical analyses

were normalized according to Dunbar et al. (2000). MICROBIAL ABUNDANCE (QUANTITATIVE PCR (QPCR) ANALYSIS) For standard preparation, amplicons from each investigated taxonomic group and

functional genes (Table 2) were purified (Invisorb Spin PCRapid kit, Invitek, Berlin, Germany), ligated into the StrataClone PCR cloning vector pSC-A (Stratagene, La Jolla, CA, USA), and

ligation products were transformed with StrataClone SoloPack competent cells (Stratagene). Specificity of clones used as qPCR standards were checked with Basic Local Alignment Search Tool.

Plasmid DNA was isolated (Plasmid Miniprep Kit, Bio-Rad Laboratories, Hercules, CA, USA) and quantified as described above. For qPCR, each 25 μl PCR cocktail contained 12.5 μl iQ SybrGreen

Supermix (Bio-Rad Laboratories), 0.4 μM of each oligonucleotide (Table 2), 1.0 mg ml−1 bovine serum albumin and 10 and 50 ng template DNA for taxonomic groups and functional genes,

respectively. Apart from the bacterial _amoA_ gene assay, all functional gene qPCRs were supplemented with 0.625 μl dimethyl sulfoxide. PCR reactions were run on an iCycler iQ Multicolor

Real-time PCR Detection System (Bio-Rad Laboratories) and were started with 3 min at 95 °C, followed by amplification cycles specific for each phylum and functional gene (Table 2). Melting

curve analysis of amplicons was conducted to confirm that fluorescence signals originated from specific amplicons and not from primer-dimers or other artifacts. Each DNA sample was processed

in triplicate reactions, whereas standard curves were generated using duplicate 10-fold dilutions of isolated plasmid DNA. Automated analysis of PCR amplicon quality (for example, PCR

baseline subtraction, Ct-threshold setting to the linear amplification phase) and quantity was performed with iCycler Optical System Software Version 3.1 (Bio-Rad Laboratories). STATISTICAL

ANALYSES Analysis of variance combined with _post hoc_ Tukey-B tests (SPSS for Windows, version 11.7, SPSS Inc., Chicago, IL, USA) was performed according to Rasche et al. (2006) to

determine significant treatment effects (tree girdling, seasonality) on abundance and community structure of the investigated microbial groups and functional genes. Pearson's linear

correlation coefficients were calculated for assessing significant relations between microbial abundance and geochemical parameters (SPSS for Windows). Effect of tree girdling on T-RFLP data

sets obtained from each target group was further assayed based on Bray–Curtis similarity coefficients (Legendre and Legendre, 1998). Therefore, a similarity matrix was generated for all

possible pairs of samples of each target group. This similarity matrix was used for analysis of similarity (ANOSIM) statistics (Clarke and Green, 1988) to test the hypothesis that bacterial,

archaeal and taxonomic communities were altered by tree girdling over the investigation period. ANOSIM generates a test statistics, _R_. The magnitude of _R_ indicates the degree of

separation between two communities, with a score of 1 indicating complete separation and 0 indicating no separation. Calculation of similarity coefficients and ANOSIM were carried out using

Primer six for Windows (version 6.1.5, Primer-E Ltd., Plymouth, UK). To test the influence of environmental variables on the microbial community structure, canonical correspondence analyses

were carried out in Canoco (version 4.5 for Windows, PRI Wageningen, the Netherlands) (Lepš and Šmilauer, 2003). Presence or absence as well as relative height of terminal-restriction

fragments were used as ‘species’ data whereas geochemical data were included in the analysis as ‘environmental’ variables. Resulting ordination biplots approximated the weighted differences

between the individual communities (T-RFLP patterns) with respect to each of the geochemical factors, which were represented as arrows. The length of the corresponding arrows indicated the

relative importance of the geochemical factor in explaining variation in the six microbial T-RFLP profiles, whereas the angle between arrows indicated the degree to which they were

correlated. A Monte Carlo permutation test based on 1000 random permutations was used to calculate the impact of geochemical variables on community patterns. RESULTS MICROBIAL COMMUNITY

STRUCTURE (T-RFLP ANALYSIS) Compared with tree girdling, seasonality had the greatest, significant influence on the total bacterial and archaeal community structure as well as on the four

selected phyla (_P_<0.001) (Table 3). The community structure of the total archaea was changed by tree girdling (_P_<0.001), whereas total bacterial was not (_P_>0.05).

Alpha-proteobacteria and acidobacteria showed a detectable community change due to tree girdling (_P_<0.01), whereas beta-proteobacteria and verrucomicrobia appeared not altered

(_P_>0.05). A clear interaction between the two factors ‘seasonality’ and ‘tree girdling’ was determined indicating an interrelated influence of both factors on the community dynamics of

the bacterial and archaeal community (_P_<0.05) (Table 3). ANOSIM detected a distinct tree girdling-induced community change among archaea, which was confirmed by several significant _R_

values ranging between 0.124 and 0.913 indicating distinct structural differences between two individual archaeal communities (control versus tree girdling) (_P_<0.05) (Table 4). Based on

_R_ values, greatest community differentiations became measurable during early fall and winter months. A comparable trend was determined for total bacteria, although the differences were

less pronounced as compared with the total archaea explained by a smaller number of significant _R_ values. For alpha-proteobacteria and acidobacteria, ANOSIM calculated several significant

_R_ values, whereas for beta-proteobacteria and verrucomicrobia only at three sampling dates significant tree girdling effects (_P_<0.05) were found. Canonical correspondence analysis was

used to test the significant dependence of the community differentiations on tree girdling and seasonality-related changes in geochemical parameters (Table 5, Figure 1). Multivariate

testing, based on 1000 Monte Carlo permutations, confirmed the significance of the two canonical axes (_P_<0.05). The total percentage variance of the microbiota-environment relation

ranged between 47.6% (verrucomicrobia) and 70.8% (total bacteria) (Table 5). The first canonical axis attributed the greatest influence explaining the microbiota-environment relation. The

strong relation between the T-RFLP data sets and geochemical data was substantiated by high correlation coefficients of at least 0.390 (Table 5). Figure 1 illustrates the relationship

between the community changes and geochemical data and shows further the clear seasonality and tree girdling related community differentiations overall the 2-year experimental period.

Generally, higher soil moisture was determined in the second experimental year, whereas higher soil temperatures were measured in the first year of the field experiment (Kaiser et al.,

2010). These soil climatic differentiations were clearly reflected in all six T-RFLP community patterns, in which distinct community shifts were observed between the first and the second

year (Figure 1). The first year samples tended to cluster if higher soil temperatures and lower soil moisture were observed, whereas the opposite effect was determined for the second year

samples. When explaining the effect of determined chemical parameters on assayed microbial communities, nitrate contents were in average highest in the first year of investigation and peaked

during summer months (Kaiser et al., 2010). Dissolved organic nitrogen (DON) showed a clear decrease in the second year as compared to the first year, whereas no clear trend was observable

for dissolved organic carbon (DOC) and ammonia (Kaiser et al., 2010). These changes in chemical parameters were clearly reflected in alterations within the soil microbial communities (Figure

1). In particular, the first year samples tended to cluster with high nitrate values, whereas the second year samples indicated a clear correlation with high DON values. In general,

seasonality effect was overwhelming the effect of tree girdling. MICROBIAL ABUNDANCE (QPCR ANALYSIS) Analysis of variance determined significant effects of seasonality on bacterial and

archaeal abundance as measured by qPCR of 16S rRNA genes and functional genes (_P_<0.001) (Table 3, Figure 2). Seasonality effect was most pronounced for beta-proteobacteria, which showed

abundance shifts of 236 and 242% for the control (average 1.14 × 1010 16S rRNA gene copies) and girdled (average 1.18 × 1010) plots, respectively, over the whole experimental period. The

smallest seasonality effect was determined for total archaea (average 6.13 × 107) with 63% variation in the control plots over the whole experiment. However, abundance of total archaea

measured in the girdled plots (average 1.09 × 108) revealed a seasonality related fluctuation of 112%. The other microbial groups assayed in the control and girdled plots took intermediate

positions with seasonal changes ranging between 65% (verrucomicrobia in control plots) and 97% (alpha-proteobacteria in girdled plots). In contrast to seasonality, tree girdling had a minor

effect and was only significant for total archaea, alpha-proteobacteria and acidobacteria (_P_<0.05). For these three groups, significantly higher 16S RNA gene copies were determined in

the girdled plots as compared with the control plots (44%, total archaea; 18%, alpha-proteobacteria and acidobacteria). No significant differences were found for beta-proteobacteria (3%

higher in girdled plots) and verrucomicrobia (2% higher in girdled plots). Contrastingly, 16S rRNA gene copies tended to be 7% greater in control plots in comparison with girdled plots when

analyzing total bacterial abundance (_P_>0.05). Except for beta-proteobacteria, a clear interaction between both factors could be calculated indicating an interrelated influence of

‘seasonality’ and ‘tree girdling’ on the abundance of bacterial and archaeal communities (_P_<0.05) (Table 3). A highly significant effect of seasonality was detected for archaeal and

bacterial _amoA_ gene, _nirS_ gene and _nosZ_ gene abundance (_P_<0.001) (Table 3, Figure 3). All assayed functional genes revealed higher gene copies in the second experimental year

(June 2007 to May 2008) as compared with the first investigation period (May 2006 to May 2007). Abundance of bacterial and archaeal _amoA_ genes showed greater seasonal fluctuations as

compared with _nirS_ and _nosZ_ genes. Over the whole project period, greatest seasonal changes on archaeal _amoA_ gene abundance were found in control plots (average 1.41 × 107, 187%

variation), whereas the effect was smallest for archaeal _amoA_ gene copies in girdled plots (average 3.14 × 107, 145%). Bacterial _amoA_ gene copies took an intermediate position and their

seasonal responses were similar to those of archaeal _amoA_ genes. Although seasonality related shifts of the quantities of _nirS_ and _nosZ_ genes were highly significant (_P_<0.001),

their overall seasonal alternation was less intense as compared with the assayed _amoA_ genes. Greatest seasonal differentiations were determined for _nosZ_ gene in the control plots (107%),

whereas smallest differences were determined for _nirS_ gene copies in the control plots (91%). Copy numbers of the _nirS_ gene behaved similarly to those of the _nosZ_ gene, but with

slightly greater seasonal changes. Distinct differences between copy numbers of assayed functional genes were determined between control and tree girdling plots. Archaeal _amoA_ gene

revealed 55% higher numbers in girdled plots (6.83 × 107) as compared with control plots (1.41 × 107) (_P_<0.01), whereas 71% higher copies of bacterial _amoA_ gene were measured in

girdled plots (average 6.83 × 107) as compared with the corresponding controls (average 1.99 × 107) (_P_<0.001). Effect of tree girdling was not significant for _nirS_ and _nosZ_ genes.

Copy numbers of _nosZ_ gene showed 14% lower values in girdled plots as compared with controls (2.04 × 108 versus 2.33 × 108), whereas _nirS_ gene abundance was not significantly changed by

tree girdling (2.43 × 108 (controls) versus 2.35 × 108 (tree girdling)) (_P_>0.05). Significant interactions between seasonality and tree girdling were determined for both _amoA_ genes

(_P_<0.05). Generally, it needs to be pointed out that very small abundance differences may have occurred due to variations in the used qPCR assays. In the latter discussion only those

abundance differences were considered for which statistical significances were obtained. LINEAR CORRELATION COEFFICIENTS BETWEEN MICROBIAL ABUNDANCE AND GEOCHEMICAL DATA Positive

correlations, except for total archaea, verrucomicrobia and functional genes, were calculated for DOC (range from _r_=0.164 (_P_<0.05, acidobacteria) to _r_=0.239 (_P_<0.01,

alpha-proteobacteria)), showing increased gene abundance when high DOC contents were determined. DON revealed a positive correlation with total archaea (_r_=0.299, _P_<0.01),

acidobacteria (_r_=0.166, _P_<0.05) both _amoA_ genes (bacterial _amoA_ gene, _r_=0.228; archaeal _amoA_ gene, _r_=0.226) and the _nosZ_ gene (_r_=0.352) (_P_<0.01) (Table 6). For soil

nitrate, positive correlations were determined between total bacteria (_r_=0.254, _P_<0.01), and soil ammonia was positively correlated with beta-proteobacteria (_r_=0.187, _P_<0.05)

and acidobacteria (_r_=0.301, _P_<0.01). Negative correlations were calculated between soil ammonia and total archaea (_r_= −0.278, _P_<0.01) as well as verrucomicrobia (_r_= −0.269,

_P_<0.01) reflecting decreased abundance with increasing soil ammonia levels. Although soil nitrate was only correlated with _nosZ_ gene (_r_= −0.256), soil ammonia was negatively

correlated with all functional genes (at least _r_= −0.235) (_P_<0.01). Total N2O emission was positively correlated with total archaeal abundance (_r_=0.476), acidobacteria (_r_=0.527),

verrucomicrobia (_r_=0.393), archaeal _amoA_ (_r_=0.283), _nirS_ (_r_=0.445) and _nosZ_ (_r_=0.424) (_P_<0.01) genes. Soil temperature was negatively correlated with all assayed groups

(range from _r_= −0.163 (_P_<0.05, acidobacteria) to _r_= −0.421 (_P_<0.01, verrucomicrobia)), except with beta-proteobacteria (_r_= 0.475, _P_<0.01), indicating an abundance

decrease with higher soil temperatures. Similar trends were observed for soil moisture (range from _r_= −0.233 (_P_<0.01, total bacteria) to _r_= −0.398 (_P_<0.01,

beta-proteobacteria)); however, for total archaea and acidobacteria an increase in abundance was determined when higher soil moisture occurred (_r_=0.327 (_P_<0.01, total archaea) and

_r_=0.289 (_P_<0.01, acidobacteria)). Functional genes were negatively correlated with soil temperature (range from _r_= −0.259 (bacterial _amoA_ gene) to _r_= −0.456 (_nosZ_ gene)),

whereas positive correlations were determined between functional genes and soil moisture (range from _r_=0.247 (archaeal _amoA_ gene) to _r_=0.418 (_nosZ_ gene)) (_P_<0.01). DISCUSSION

Previous studies on the effects of seasonality and resource availability on dynamics of soil microbial communities under field conditions have been restricted in resolution by the use of

wide sampling intervals and short investigation periods, and have focused on broad microbial domains rather than specific phyla. To overcome these limitations, we used a bi-monthly sampling

scheme during a 2-year tree girdling field experiment period to study in detail the effect of seasonality and resource availability on the soil microbial community. Structure (T-RFLP

analysis) and abundance (qPCR) of microbial communities were analyzed at different taxonomic scales including bacterial and archaeal domains and specific bacterial phyla, which occur

prominently in the assayed soil, that is, acidobacteria, alpha- and beta-proteobacteria and verrucomicrobia, as well as nitrifying and denitrifying bacteria and archaea. Our results showed

that resource availability due to seasonal variation, but also due to tree girdling resulted in specific short- and medium-term changes in community structure and abundance of archaea and

bacteria as well as representatives of selected phyla. Generally, microbial communities were altered by seasonal effects to a larger extent than by altered root exudation. These community

alterations were partly ascribed to the influence of seasonality and tree girdling on physicochemical parameters such as DOC) and DON, nitrate, ammonia, as well as soil temperature and soil

moisture. Seasonality in temperate forest soils is reflected by alterations in soil moisture and soil temperature, being acknowledged control factors of soil microbial communities (Stres et

al., 2008; Tabuchi et al., 2008; Cleveland et al., 2007). Both parameters were responsible for compositional shifts in soil bacterial communities determined in this study. Similarly, changes

in soil temperature and moisture appeared to be determinants of the archaeal community in the present field study. Also Shen et al. (2008) and Tourna et al. (2008) have found temporal

shifts in archaeal abundance, and Stres et al. (2008) and Tourna et al. (2008) evidenced responsiveness of soil archaea to variations in soil temperature and soil moisture. Seasonal changes

in soil climate were further closely linked to short- and medium-term variations in resource availability, which further correlated with the quantity and quality of organic matter entering

the soil, as it was also previously suggested (Bell et al., 2009; Cookson et al., 2006; Krave et al., 2002). Consequently, total bacterial communities and individual phyla studied were

clearly shaped by supply of DOC, DON and mineral N (that is, ammonia, nitrate), which is in agreement with previously published data (Drenovsky et al., 2004; Zak et al., 2003; Alden et al.,

2001). Abundance of acidobacteria was positively correlated with soil DOC and mineral N contents, which is in agreement with previous reports (Tabuchi et al., 2008; Hayatsu et al., 2008;

Ruppel et al., 2007). Several members of this phylum have been evidenced to be facultative or obligatory anaerobic organotrophs (Jones et al., 2009; Fierer et al., 2007). Hence, their

abundance may be particularly favored by high soil moisture together with effects on community structure, as we found in the present field study and was proven by other reports (for example,

Janssen, 2006). Throughout the field experiment, acidobacterial communities were more abundant and underwent significant structural changes in girdled, C-limited plots. This may signify

their high metabolic versatility to be well-adapted to resource limitation and their ability to decompose complex C substrates deriving from the rather recalcitrant soil organic matter pool

(Ward et al., 2009; Hansel et al., 2008; Eichorst et al., 2007; Fierer et al., 2007). Girdling prevents the uptake of available nutrients such as ammonia and nitrate by trees (Högberg et

al., 2001), and therefore resulted in relatively higher mineral N concentrations in soils of girdled plots. We found significantly higher bacterial _amoA_ gene copies in girdled plots than

in controls substantiating our assumption that N availability is a crucial controlling factor for ammonia oxidizing bacteria (Fierer et al., 2009). No correlation was seen between ammonia

oxidizing bacteria and soil nitrate content. However, net changes in the soil nitrate pool do not reflect nitrifying activity, as apart from microbes plants also utilize nitrate as N source

(Adair and Schwartz, 2008). Copies of bacterial _amoA_ genes were further positively correlated with DON. DON is the precursor of ammonia (mineralization) and thus is essential for the

constant replenishment of the ammonia pool as substrate for nitrification, thus indicating that DON is essential for maintaining ammonia oxidizing bacteria metabolism (You et al., 2009;

Brierley et al., 2001). Moist soil conditions pronouncing the diffusion of substrates (for example, nitrate and ammonia) to microbes offered obviously a favourable environment for sustaining

and increasing the abundance of ammonia oxidizing bacteria, which is in agreement with previously published information (Fierer et al., 2009; Adair and Schwartz, 2008). We found that

seasonality and varying resource availability changed the abundance of ammonia oxidizing archaea (AOA). Decreasing soil temperature was correlated with increasing AOA abundance, which is in

contrast to the results of a soil microcosm study by Tourna et al. (2008). However, Urakawa et al. (2008) and Caffrey et al. (2007) investigated marine ecosystems in which decreased

phylogenetic diversity and abundance of AOA were found with increasing temperature, respectively. Based on these contradictory results, we suggest further experiments under field conditions

to substantiate that decreasing soil temperature promotes AOA abundance. Our results suggested a potential dependence of AOA on ammonia availability, which was supported by recent studies

(He et al., 2007; Santoro et al., 2008). Because of the negative correlation between AOA and ammonia concentrations, we conclude that the ammonia decrease may have been the consequence of

pronounced ammonia oxidation activity in the assayed temperate soils, whereas Valentine, (2007) proposed that AOA seem to be better adapted to low ammonia concentrations in soil. However, it

remains poorly investigated to which extent soil AOA react to different concentrations of ammonium in soils (Jia and Conrad, 2009; Chen et al., 2008). N2O emissions were positively

correlated with the abundance of total archaea and AOA indicating that archaea may be directly involved in denitrification processes. Although denitrification is often considered a bacterial

process, the measured high abundance of archaea suggested that denitrification was probably also widespread among the archaea studied in this field experiment. But further research is

required to substantiate this assumption as only limited information is available for archaeal denitrification in soil ecosystems so far (for example, Bartossek et al., 2010; Hayatsu et al.,

2008). However, it has been confirmed that several archaeal members perform both assimilatory and dissimilatory reduction processes to produce for example, N2O (Hayatsu et al., 2008;

Cabello et al., 2004; Zehr and Ward, 2002), and their actual contribution to denitrification was proven by the presence of denitrification genes (for example, _nir_ and _nos_ genes) in the

genomes of several archaeal species (Bartossek et al., 2010; Cabello et al., 2004). In conclusion, our field study in a temperate ecosystem including tree girdling to induce soil C

limitation is the first field survey that has been performed for two consecutive years with a bi-monthly sampling scheme. This approach allowed us to get a detailed insight into short- and

medium-term effects of seasonality and resource availability on the soil microbial community, which has been explored at domain level as well as at a smaller taxonomic scale using selected

bacterial phyla and functional groups. We showed that community structure and abundance of archaea and acidobacteria appeared to be particularly altered by these two factors, reflecting

their potentially high metabolic versatility in the assayed soils. Further, our extensive field survey revealed that belowground C allocation along with seasonal climatic influences changed

the abundance of nitrifying and denitrifying bacteria and archaea and showed a sound correlation with treatment-related dynamics of physicochemical parameters in the investigated soils.

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ACKNOWLEDGEMENTS This study was financed by the Austrian Science Fund (FWF, Project number: P18495-B03). We thank Dr. Evelyn Hackl (AIT) for her valuable comments and suggestions on the

manuscript. AUTHOR INFORMATION Author notes * Frank Rasche Present address: Present address: University of Hohenheim, Department of Plant Production and Agroecology in the Tropics and

Subtropics, D-70593 Stuttgart, Germany, AUTHORS AND AFFILIATIONS * AIT Austrian Institute of Technology GmbH, Bioresources Unit, Seibersdorf, Austria Frank Rasche, Daniela Knapp & Angela

Sessitsch * Department of Chemical Ecology and Ecosystem Research, Christina Kaiser, Marianne Koranda & Andreas Richter * Faculty of Life Sciences, University of Vienna, Vienna, Austria

Christina Kaiser, Marianne Koranda & Andreas Richter * Department of Forest Ecology, Federal Research and Training Centre for Forests, Natural Hazards and Landscape, Vienna, Austria

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_et al._ Seasonality and resource availability control bacterial and archaeal communities in soils of a temperate beech forest. _ISME J_ 5, 389–402 (2011).

https://doi.org/10.1038/ismej.2010.138 Download citation * Received: 01 March 2010 * Revised: 02 June 2010 * Accepted: 11 July 2010 * Published: 30 September 2010 * Issue Date: March 2011 *

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currently available for this article. Copy to clipboard Provided by the Springer Nature SharedIt content-sharing initiative KEYWORDS * tree girdling * abundance and community structure of

archaea and bacteria * nutrient cycling * resource use * soil moisture and soil temperature