Acta Ecologica Sinica 35 (2015) 122–129

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Effect of seasonal soil freeze–thaw cycles on bacterial diversity in theprocess of fine root decomposition at different critical periods

Yuanyun Wei a,b, Zhichao Wu a,c, Wanqin Yang a,⁎, Fuzhong Wu a

a State Key Laboratory of Ecological Forestry Engineering, Institute of Ecology & Forestry, Sichuan Agricultural University, Chengdu 611130, Chinab Institute of Wetland Research, Chinese Academy of Forestry, Beijing 100091, Chinac Beijing Products Quality Supervision and Inspection Institute, Beijing 101300, China

⁎ Corresponding author.E-mail address: scyangwq@163.com (W. Yang).

http://dx.doi.org/10.1016/j.chnaes.2015.07.0021872-2032/© 2015 Elsevier B.V. All rights reserved.

a b s t r a c t

a r t i c l e i n f o

Article history:Received 2 September 2013Received in revised form 23 May 2014Accepted 14 July 2014

Keywords:Bacterial diversityFine root decompositionSeasonal soil freeze–thawSubalpine/alpine forest

Soil microbial community plays a crucial role in fine roots decomposition in the forest ecosystem, and under-standing the dynamics of soil microbial diversity in the fine roots will be helpful to reveal the mechanism offine root decomposition at different critical periods. As yet, little is known about the dynamics of soil microbialdiversity in the process of fine roots decomposition during the soil freeze–thaw season in the high-frigid forestecosystem. In order to understand the effects of seasonal freeze–thaw cycles on bacterial diversity in the processof fine roots decomposition at different critical periods in cold winter, litterbags with spruce (Picea asperata), fir(Abies faxoniana) and birch (Betula albosinensis) fine roots were buried in the forest floor at altitudes of 3582 m,3298m, and 3023m in different sites in the eastern Tibet Plateau on October 26, 2009. The litterbagswere pickedup and stored in ice box at the periods of onset of freezing (OF), deep freezing (DF), early thawing stage (ETS),middle thawing stage (MTS), late thawing stage (LTS), and early growing season (EGS) from freeze–thaw seasonto early growing season. DNA of bacterial in fine roots taken back was extracted immediately. Based on the PCR-DGGE technique, diverse bacterial communities in the studied fine roots were found in the whole process of fineroots decomposition. Bacterial diversity in fine roots were significantly higher at the periods of onset of freezing,middle thawing and late thawing, but significantly lower at the periods of in deep freezing and early thawing.Bacterial diversity in decomposing fine roots varied greatly with seasonal soil freeze–thaw process and tree spe-cies, but varied slightly with the studied altitudes. Regardless of tree species, higher similarity of bacterial com-munity in fine roots was observed among different altitudes at the same period, while relatively lowersimilarity of bacterial community in fine roots was observed at different critical periods at the same altitude.However, the similarity of bacterial community in fine roots was always higher than 48% during the freeze–thaw season. Biodiversity indices of richness, Shannon–Wiener index, Simpson index and evenness of bacterialcommunity were correlated significantly with average, maximum and minimum temperatures in soils duringthe freeze–thaw season, only except for the correlation between soil average temperature and Simpson index.The nitrogen concentration in fine roots was correlated significantly with Simpson index and evenness of bacte-rial community, but thephosphorus concentrationwasnot significantly correlatedwith these bacterial indices. Inconclusion, continuous soil freezing reduced the level of bacterial community diversity in fine roots, and the bac-terial community diversity in fine roots at soil thawing period would recover the level at onset freezing period,regardless of the studied altitudes. The dynamics of bacterial community infine roots conformed to the dynamicsof root decomposition. Meanwhile, fine roots quality could also influence the level of bacterial communitydiversity.

© 2015 Elsevier B.V. All rights reserved.

1. Introduction

The soil microbial community participates in litter decomposition asindispensable decomposer [1,2] and contributes to the formation of soilorganicmatter [3]. In cold biomes, the soilmicrobial community evolvesunique adaption and plentiful biodiversity in response to the harshwin-ter environment, and is extremely sensitive to change in habitats [4,5].

High altitude and latitude regions are subject to the most profound cli-matic changes on the planet [6,7]. So, in such regions, the response ofthe soil microbial community to climate change would exert crucial ef-fects on litter decomposition [8,9], and in turn influence the terrestrialcarbon cycle [10]. Despite the complex structure of the soil microbialcommunity to be studied, traditional microbial cultivation technologycan only figure out limited species [11,12]. In the recent decade, the de-velopment of denaturing gradient gel electrophoresis (DGGE) technolo-gy strengthens the knowledge concerning the microbial communityduring the progress of litter decomposition [13]. Fine roots account for

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more than 40% of litter in normal forest ecosystem [14,15] and supplyimportant carbon source for the soil microbial community. As mostfine roots are growing in subsurface soil, the soil microbial communityhas already planted on fine roots when they alive, and contributes fineroots decomposition notmirror foliage litter decomposition [16]. Unfor-tunately, for cold biomes most researchers focus on the diversity of thesoil microbial community itself and its seasonal change [17,18], whilelittle is known about the soil microbial diversity during fine roots de-composition. So promoting relative study is considerable important.

Western Sichuan alpine/subalpine forests are typical low-latitudeboreal forest ecosystems which are located at the east edge of Qinghai–Tibet Plateau. Snow pack covers the ground for several months annuallywith frequent soil freeze–thaw cycles, and the soil microbial communitystructure exhibits significant difference between freeze–thaw seasonand growing season [19,20]. A recent research reported a complicatedprocess of fine roots decomposition during freeze–thaw season whichis characterized by a three-time fluctuation in decomposition rate [21].Whether this phenomenon is related with the microbial communityshift remains uncertain. Another research found that microbial biomassduring fine roots decomposition had been influenced by soil tempera-ture and fine roots quality [22]. Bacterial diversity in the process of fineroot decomposition following similar regulation has not been wellproved. Consequently, we conducted a litterbag experiment whichselected the fine roots of three dominant species in Western Sichuanalpine/subalpine forests, namely spruce (Picea asperata), fir (Abiesfaxoniana) and birch (Betula albosinensis), as research objects to investi-gate the dynamics of bacterial diversity during fine roots decompositionin freeze–thaw season and early growing season. Our experimentbelonged to a program which intended to enhance the knowledgeconcerning litter decomposing naturally in a boreal forest under climatechange.

2. Material and methods

2.1. Research area

The research area was located in the Bipenggou Nature Reserve(E102°53′–102°57′, N31°14′–31°19′, 2458–4619 m a.s.l.), Li County,Sichuan Province, China. The annual precipitation was about 850 mm,themean annual temperaturewas 2–4 °C, andmaximumandminimumtemperatures were 23 °C and −18 °C respectively. The dominantspecies of trees were fir, spruce and birch; common species of shrub in-cluded dwarf bamboo (Fargesia spathacea), azalea (Rhododendrondelavayi), sallowthorn (Hippophae rhamnoides), Sorbus rufopilosa,Berberis sargentiana and Rosa sweginzowii; common species of grass in-cluded Cystopteris montana, Cacalia auriculata, sedge (Carex spp.) andCyperus spp. [19].

2.2. Study sites

Three sites along altitude gradient with growing typical forest com-munities in the research area were selected as study sites. The A1 site islocated at 3582 mwith a 34° slope toward NE45°, and dominated by firand larch (Larix mastersiana) in canopy, and azalea, sedge andC. montana in understory; the A2 site is located at 3298 m with a 31°slope toward NE42°, and dominated by fir and birch in canopy, anddwarf bamboo, willow (Salix cupularis) and S. rufopilosa in shrublayer; the A3 site is located at 3023 m with a 24° slope toward NE38°,and dominated by spruce and fir in canopy, and R. sweginzowii,B. sargentiana and dwarf bamboo in shrub layer. Soil physical and chem-ical characteristics had been described by Liu et al. [23].

2.3. Sample collection and pretreatment

In October 2009, fine roots (≤2 mm) of spruce, fir and birch weretrimmed from mature standard forests in the research area. The fine

roots were transported to the State Key Laboratory of Ecological Forest-ry Engineering in Sichuan Agriculture University, where they werecleaned by washing with tap water, spread on trays, and air-dried atroom temperature to constant mass. About 10 g of air-dried fine rootswere weighed and placed in a 20 cm × 20 cm nylon litterbag withmesh size of 0.5 mm. Litterbags were buried at a depth of 10 cm below-ground on 10-Nov-2009, and 45 litterbags containing fine roots of onespecies were buried at A1, A2 and A3 sites. Meanwhile, the soil temper-ature was detected hourly by iButton DS1923-F5 recorder (Maxim Co.USA). According to previous data, these litterbags were harvested at13-Dec-2009 (onset of freezing, OF), 21-Jan-2010 (deep freezing, DF),11-Mar-2010 (early thawing stage, ETS), 03-Apr-2010 (middle thawingstage, MTS), 28-Apr-2010 (later thawing stage, LTS) and 23-May-2010(early growing season, EGS) respectively, which represented six differ-ent critical periods. For one time sampling, litterbags were sampledwithfive replicates of one species at every site. After sampling, litterbagswere placed into polyethylene bags and kept in a cooler carefully. Sincesampleswere brought back to the laboratory, total communityDNAwasimmediately extracted from fine roots, while parts of fine roots werekept remained for chemical analyses after being oven dried.

2.4. Extraction of total DNA and bacteria 16S rDNA V3 amplification

Total community DNAwas extracted using the Soil DNA isolation kit(Tianze Inc., Beijing, China), and DNA concentrations were determinedby 0.7% agarose gel electrophoresis. Purified DNA extracts were ampli-fied by touchdown PCR amplification to obtain increased specificityand sensitivity bacteria 16S rDNA V3. PCR amplification of bacterial16S rDNA V3 were cultured isolates using the primers 341F with a40 bp GC clamp to 5′ and 534R, and the products were about 200 bp.The PCR amplification reaction was performed in Bio-Rad iCycle PCR.Thermocycle conditions were as follows: initial denaturation of 5 minat 94 °C; followed by 20 cycles of 94 °C for 1 min, 65–55 °C for 1 min,and 72 °C for 1 min which decreased 0.5 °C after every cycle; and thenfollowed by 10 cycles of 94 °C for 1 min, 55 °C for 1 min, and 72 °C for1 min with a final extension of 72 °C for 5 min [24]. After each PCR,the size of the amplification products was verified on 2% agarose gel.

2.5. Analysis of DGGE pattern

DGGEwas performedusing theDcode system(Bio-Rad Laboratories,Hercules, CA, USA). The PCR products were loaded onto polyacrylamidegels which were made with denaturing gradient ranging from 30% to70%. The electrophoresis was run for 16 h at 60 °C and 100 V. After theelectrophoresis, the gels were stained by silver nitrate staining andphotographed on GS-800 (Bio-Rad Laboratories, Hercules, CA, USA).

Comparisons of DGGE patterns were made with Bio-Rad QuantityOne 4.41 and dendrogramswere constructed using the UPGMAmethodfor grouping and the Jaccard coefficient of similarity. The intensity of thebands was reflected as peak heights in the densitometric curve. The in-dexes of bacteria diversity including richness (S), Shannon–Wienerindex (H), Simpson index (D) and evenness (EH) were calculatedaccording to formulae as follows [20]:

H ¼ −XS

i¼1

Pi lnPi

D ¼Xn

i¼1

Pi2

EH ¼ H=Hmax ¼ H= lnS

where n is the number of bands in each lane; S is the sum of all peakheights in DGGE pattern (S ≥ nmax); and Pi represent the percentage ofpeak heights of each band to a lane.

Fig. 1. DGGE patterns of bacterial 16S rDNA V3 during the fine root decomposition of spruce, fir and birch. Note: OF—onset of freezing, DF—deep freezing, ETS—early thawing stage,MTS—middle thawing stage, LTS—later thawing stage, EGS—early growing season, the same below.

124 Y. Wei et al. / Acta Ecologica Sinica 35 (2015) 122–129

Table 1The three-way ANOVA of bacteria diversity during fine root decomposition.

Source F df P

Richness T 12.60 5.20 b0.0001⁎

A 1.14 2.20 0.3289S 3.09 2.20 0.0556

Shannon–Wiener index T 15.36 5.20 b0.0001⁎

A 0.86 2.20 0.4301S 14.87 2.20 b0.0001⁎

Simpson index T 8.85 5.20 b0.0001⁎

A 0.40 2.20 0.6719S 27.46 2.20 b0.0001⁎

Evenness T 18.75 5.20 b0.0001⁎

A 0.87 2.20 0.4252S 20.76 2.20 b0.0001⁎

Note: T means differences between periods, A means differences between sites, S meansdifferences between species.⁎ P b 0.05.

125Y. Wei et al. / Acta Ecologica Sinica 35 (2015) 122–129

2.6. Fine roots chemical analyses

The fine roots samples used for chemical analyses were ground andthen passed through a 0.5 mm stainless steel sieve and digested byH2SO4–H2O2 solution heating. The nitrogen concentration was deter-mined by Kjeldahl method, and the phosphorus concentration was de-termined by molybdenum-blue colorimetry.

2.7. Statistical analyses

The significance of bacteria diversity difference in different periodswas examined by LSD (P b 0.05). Difference of bacteria diversity in pe-riods, sites and specieswas analyzedwith three-wayANOVA. The corre-lations of bacteria diversity indexes and factors were examined bySpearman's coefficient. All of the statistical analyses were done in SAS9.1.3 (SAS Institute Inc. 2007).

3. Results

3.1. DGGE analysis

The DGGE patterns showed that the number of bands at differentlanes, which ranged from 25 to 58, was obviously discrepant (Fig. 1).The richness of bacteria community was only significantly different inperiods but was insignificantly different in sites and species (Table 1).The richness of bacteria community was comparably higher in OF, dra-matically declined in DF, and then soared in MTS, finally decreasedagain in LTS and EGS. The maximum of richness appeared in MTS, andthe minimum of richness occurred in ETS (Fig. 2).

Fig. 2. Richness, Shannon–Wiener index, Simpson index and evenness of bacteria commu-nity during fine root decomposition. Note: The different small letters mean significantdifference at the 0.05 level.

3.2. Bacteria diversity indexes

Shannon–Wiener index of bacteria community during fine root de-composition was significantly higher in OF, MTS and LTS periods,while was significantly lower in DF, ETS and EGS. Simpson index of bac-teria community during fine root decomposition exhibited a conversetrend to Shannon–Wiener indexwith a smaller fluctuation. The dynam-ics of evenness was similar with Shannon–Wiener index (Fig. 2). All ofthe bacteria diversity indexes were relatively close in OF period, bythen, bacteria diversity indexes diverged gradually accompanied withthe process of fine root decomposition. The results of three-wayANOVA indicated significant differences of Shannon–Wiener index,Simpson index and evenness existed in periods and species, but didnot appear in sites (Table 1).

3.3. Similarity of bacteria community

Cluster analysis indicated that the bacteria planted on one species offine roots in a particular periodwas comparably similar at different sites(Fig. 3). The bacteria community structure in one period also assimilat-ed with bacteria in adjoining periods, but was relatively different fromother periods. To be specific, bacteria community during the decompo-sition of spruce fine roots could be classified as two major groups, one

126 Y. Wei et al. / Acta Ecologica Sinica 35 (2015) 122–129

group included bacteria in OF, DF and ETS periods, another group in-cluded bacteria in MTS and LTS periods, and the similarity of thesetwo groupswas less than54%. A great change of the bacteria communitystructure happened on soil thawing time,marked by only 49% similaritybetween the bacteria community in EGS and other periods. Besides, thebacteria community at A1 site reached 81% similarity in DF and ETS pe-riods, which was the most similar bacteria community. The change ofthe bacteria community structure during decomposition of fir finerootswas comparably little for the similarity of thewhole bacteria com-munity and was above 65% except the bacteria in ETS period at A3 site.As the similarity of the bacteria community in EGS period and in LTS

Fig. 3. Cluster analysis based on DGGE bands of bacterial 16

period reached 70%, the change of the bacteria community structureon fir fine roots wasmuch smaller than on spruce fine roots at that cru-cial time. The most similar bacteria community appeared in OF periodwhen bacteria community at A1 and A2 sites exhibited 84% similarity.Like the bacteria planted on spruce fine roots, the bacteria communityduring birch fine root decomposition could also be classified as twomajor groups, and thefirst group included bacteria inOF, DF and ETSpe-riods, while the second group included bacteria inMTS and LTS periods.In spite of the bacteria community obviously changed in LTS to EGS pe-riod at A2 and A3 sites, the bacteria community at A1 site was similar as85% level in the same period.

S rDNA V3 region during the fine root decomposition.

Fig. 5. Dynamics of fine root nitrogen and phosphorus concentration.

Fig. 4. Soil temperature dynamics of the three sites from 10-Nov-2009 to 23-May-2010.

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3.4. Influence of soil temperature, fine root nitrogen and phosphorusconcentrations on bacteria diversity

Fig. 4 exhibited the hourly soil temperature dynamics from 10-Nov-2009 to 23-May-2010, and Table 2 demonstrated average soil tempera-ture, maximum soil temperature and minimum soil temperature ofeach period. The results indicated that among the three sites, soil tem-perature was lowest and most stable at A1 site which is located at thehighest elevation, and the frequency and extent of soil temperaturechange were also smallest at A1 site.

Initial concentrations of nitrogen and phosphorus were both thehighest in birch fine roots of the three species. During the process offine root decomposition, nitrogen concentrations of birch almostremained at the same level, while nitrogen concentrations of spruceand fir continually increased in freeze–thaw season with the peaksappearing inMTS or LTS period, and followed by a decline in EGS period.Nitrogen concentrations of spruce fine roots at A1 andA2 sites had risenmost dramatically. Phosphorus concentrations of the three species' fineroots all decreased in freeze–thaw season, and then were unchanged orslightly raised in EGS period (Fig. 5).

In the freeze–thaw season, richness, evenness and Shannon–Wienerindex of the bacteria community positively correlated with average soiltemperature, maximum soil temperature and minimum soil tempera-ture, while Simpson index of bacteria community negatively correlatedwithmaximum soil temperature andminimum soil temperature. In ad-dition, bacteria diversity exhibited themost significant correlation withminimum soil temperature. However, by analyzing soil temperaturefrom OF to EGS period, neither bacteria richness, evenness, Shannon–Wiener index, nor Simpson index appeared significantly correlatedwith soil temperature; only evenness positively correlated with mini-mum soil temperature. In the process of fine root decomposition,Simpson index of the bacteria community positively correlated with ni-trogen concentration, and bacteria evenness negatively correlated withit. Neither bacteria richness, evenness, Shannon–Wiener index, norSimpson index appeared significantly correlated with phosphorus con-centration (Table 3).

Table 2Average soil temperature, maximum soil temperature and minimum soil temperature of each

Period Average temperature (°C) Maximum tem

A1 A2 A3 A1

OF 0.883 1.652 2.377 4.503DF −1.315 −0.901 −0.563 −0.218ETS −0.756 −0.599 −0.450 −0.155MTS 0.005 0.883 1.730 1.166LTS 1.096 3.474 4.959 4.437EGS 5.675 7.053 7.968 8.520

4. Conclusion and discussion

Seasonal soil freeze–thaw cycles exerted profound effects on bacte-ria diversity in the process of fine root decomposition. Relatively higherbacteria diversity which appeared in OF, MTS and LTS periods should beclosely associatedwith the process of soil freeze–thaw cycles.When soilfreezes or thaws, rapid soil temperature changes and frequent soilfreeze–thaw cycles had constructed intermediate disturbances, leadingto a number of adapted bacteria species flourishing in the short term,and forming the high level of bacteria diversity. By contrast, soil contin-ually remained deeply frozen in DF and ETS periods, and the peaks ofminimum soil temperature all appeared in these two periods. Suchsoil circumstance profited a specific bacteria species to be predominantin interspecies competition, thereby depressing bacteria diversity in DFand ETS periods. In the research byHaei et al., majority of bacteria grow-ing in winter favored relative stable soil temperature which fluctuatedaround 0 °C, and inhibited by soil continual frozen or frequent freeze–thaw cycles, especially, the influence of soil frozen duration was moreimportant than the frequency of soil freeze–thaw cycles [25]. Theirfinding was in accordance with the bacteria diversity dynamics in ourresearch. Among all soil temperature indexes, minimum soil tempera-ture existedmost significant correlationwith bacteria diversity indexes,also indicated soil continual frozen exerted more impact on bacteria

period.

perature (°C) Minimum temperature (°C)

A2 A3 A1 A2 A3

4.891 5.111 −0.533 −0.051 1.0240.935 1.087 −2.925 −2.489 −2.5121.759 5.578 −1.792 −2.907 −3.8284.640 6.267 −0.161 −0.283 −0.3155.671 7.090 −0.092 0.704 0.5389.394 9.916 2.236 4.082 4.513

Table 3The correlation coefficients of soil temperature, fine root nitrogen and phosphorus concentrations with bacteria diversity.

Richness Shannon–Wiener index

Simpson index Evenness

Average temperaturea Correlation coefficient 0.4043 0.4035 −0.2896 0.4371P 0.0079⁎ 0.0060⁎ 0.0536 0.0027⁎

Maximun temperaturea Correlation coefficient 0.4233 0.4280 −0.3496 −0.4496P 0.0038⁎ 0.0034⁎ 0.0186⁎ 0.0019⁎

Minimun temperaturea Correlation coefficient 0.6325 0.6159 −0.4446 0.6404P b0.0001⁎ b0.0001⁎ 0.0022⁎ b0.0001⁎

Average temperatureb Correlation coefficient 0.0154 0.0657 −0.0167 0.1363P 0.9118 0.6369 0.9043 0.3255

Maximun temperatureb Correlation coefficient 0.0996 0.1493 −0.1092 0.2111P 0.4735 0.2814 0.4319 0.1254

Minimun temperatureb Correlation coefficient 0.1722 0.2140 −0.1258 0.2806P 0.2129 0.1202 0.3647 0.0399⁎

Nitrogen concentration Correlation coefficient 0.1331 −0.1793 0.3453 −0.2805P 0.3371 0.1946 0.0105⁎ 0.0399⁎

Phosphorus concentration Correlation coefficient 0.1847 −0.0573 0.1715 −0.2231P 0.1811 0.6806 0.2150 0.1048

a Soil temperature data only including freeze–thaw season.b Soil temperature data including both freeze–thaw season and early growing season.⁎ P b 0.05.

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community. However, bacteria diversity dynamics in early growing sea-son diverged from the trend of bacteria diversity in freeze–thaw season,so there could be other reasons that dominated bacteria diversity inearly growing season. On the other hand, bacteria diversity dynamicsin freeze–thaw season preformed as raise after fall rather than a simplelinear process, which was coincident with fine root decomposition rate[21], meant fine root decomposition and bacteria diversity in this pro-cess should exist mutual relationship, and both of them had influencedby seasonal soil freeze–thaw cycles. Besides, the difference of fine rootdecomposition rate was significant in sites while difference of bacteriadiversity was significant in species, thereby different bacteria speciesmight own similar capacity to contribute on fine root decomposition.

Along the altitude gradient, bacteria diversity did not appear signif-icantly different, in spite of soil freeze–thaw regimes being discrepant,which implied that for the bacteria community, the habitat changingacross different periods was overwhelming the difference of soilfreeze–thaw cycles at different sites. A similar finding was reported byLauber et al. which compared soil microbial diversity under differentland use; they believed diverse soil microbial species coexisted inevery site suggesting the difference of soil microbial diversity amongdifferent sites to be insignificant [26]. An earlier study in our researcharea found that the soil bacteria community at higher site A1 was obvi-ously different from the soil bacteria community at lower site A3 at theend of freeze–thaw season; the similarity between them was less than60% [20]. This research further confirmed that a bacteria communityat a higher site in LTS period was more similar to a bacteria communityat a lower site in MTS period rather than in the same period. Such phe-nomenon reflected that the shift of bacteria community structure waslater at higher site than at lower site as soil thawing was later too.

Besides soil temperature, substance quality changewas another cru-cial factor for the bacteria community in the process of fine root decom-position. Nitrogen was one of the indispensable elements for the soilmicrobial community, and a change in soil nitrogen concentrationcould induce a shift in the soil microbial community structure [27].Zhang et al. found that comparedwith litter of camphor and beech, litterof oak could improve microbial community structure during litter de-composition with higher nitrogen concentration [28]. While in borealforest ecosystems, phosphorus might restrict soil microbial activityand reproduction for it was comparable more limited in soil [29]. Inour research, spruce and fir were close in nitrogen and phosphorusconcentrations of fine roots, and bacteria on spruce and fir's fine rootsappeared similar in diversity. During the process of fine root decompo-sition, phosphorus concentration had only decreased slightly, and nitro-gen concentration had even increased, which supplied sufficient

nutrition for the recovery of bacteria community in MTS period, thusexcluding the possibility that nitrogen or phosphorus restricted the bac-teria community and avoided undermining the influence of soil temper-ature on bacteria diversity. However, as the nitrogen concentration offine roots was only significantly correlated with parts of bacteria diver-sity indexes and phosphorus concentration of fine roots was insignifi-cantly correlated with all of bacteria diversity indexes, the differencesin fine root nitrogen and phosphorus concentrations could notcompletely explain the formation of bacteria diversity in the processof fine root decomposition. According to the research by Aneja et al., lit-ter of beech and spruce could exert selective effect on the soil microbialcommunity when they planted on litter in the early decompositionstage [30]. So, the difference of bacteria diversity in the process of fineroot decomposition should be related to fine root physiological con-struction, which was supposed to promote or impede bacteria plantingon fine roots.

In summary, bacterial diversity in the process of fine root decompo-sition at different critical periodswas significantly different as it was in-fluenced by seasonal soil freeze–thaw cycles. Not only soil thawing butalso soil deep freezing could exert profound effects on bacterial diversityin the process of fine root decomposition. Bacterial diversity dynamicswere generally similar along altitude gradients while the occurrencesof bacteria community structure shift when soil thawing was earlier atlower sites. On the other hand, by the time seasonal soil freeze–thaw cy-cles leading bacteria community change influenced on fine root decom-position, quality change during the process of fine root decompositionalso could affect bacteria diversity.

Acknowledgments

The Project was financially supported by National Natural ScienceFoundation of China (31170423, 31270498); National “Twelfth Five-Year” Plan for Science& Technology Support (2011BAC09B05); Programof Sichuan Youth Sci-Tech Foundation (2012JQ0008, 2012JQ0059); andChina Postdoctoral Science Foundation (No. 2012T50782).

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