Tree girdling affects the soil microbial community by modifying resource availability in two subtropical plantations

Applied Soil Ecology 53 (2012) 108–115

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Tree girdling affects the soil microbial community by modifying resource availability in two subtropical plantations Dima Chen a , Lixia Zhou a , Jianping Wu b , Joanna Hsu c , Yongbiao Lin a , Shenglei Fu a,∗ a Key Laboratory of Vegetation Restoration and Management of Degraded Ecosystems, South China Botanical Garden, Chinese Academy of Sciences, Xingke Road 723, Tianhe District, Guangzhou 510650, China b Institute of Ecology and Environmental Sciences, Nanchang Institute of Technology, Nanchang 330099, China c Department of Wildland Resources and the Ecology Center, Utah State University, Logan, UT 84322, USA

a r t i c l e

i n f o

Article history: Received 16 April 2011 Received in revised form 11 October 2011 Accepted 26 October 2011 Keywords: Tree girdling Rhizodeposition Microbial community composition Phospholipid fatty acid (PLFA) Acacia crassicarpa Eucalyptus urophylla

a b s t r a c t We used tree girdling and phospholipid fatty acid (PLFA) analysis to evaluate the effect of nutrient availability and rhizodeposition on soil microbial community composition in two plantations (Acacia crassicarpa and Eucalyptus urophylla) in subtropical China. The magnitude of the girdling effect was also evaluated as a function of tree species and time after girdling (2 months vs. 9 months). In both plantations, tree girdling reduced the concentration of fungal PLFAs and increased the concentration of bacterial PLFAs with a consequent decrease in the fungi/bacteria ratio, but did not affect the concentration of total PLFAs. Tree girdling affected the concentration of gram-negative PLFAs and the ratio of gram-positive bacteria to gram-negative bacteria at 9 months but not at 2 months after girdling. The ratio of cy17:0 to 16:1␻7c was increased by girdling of A. crassicarpa, indicating a stressful and nutrient-deficient habitat for soil microorganisms, but was inconsistent for girdling of E. urophylla. In the A. crassicarpa plantation, responses to girdling for most microbial groups were associated with changes in dissolved organic carbon (DOC), dissolved organic nitrogen (DON), the ratio of carbon to nitrogen, and soil pH. In the E. urophylla plantation, responses to girdling were associated with changes in DON, DOC, and NO3 − -N. These results confirm that (i) recent photosynthates allocated belowground affect soil C and N availability and therefore greatly affect microbial community composition in subtropical plantations; (ii) the magnitude of the tree girdling effect increases with time after girdling and differs between plant species; and (iii) soil microbial communities are closely linked to vegetation types and plant C allocation. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Soil microbial communities greatly affect soil organic matter decomposition and soil structure formation, which in turn control many belowground processes critical to ecosystem function (Allison et al., 2007). Among the many edaphic, climatic, and environmental factors that influence microbial community composition, soil pH, and carbon (C) and nitrogen (N) availability are especially important (Bossio and Scow, 1998; Drenovsky et al., 2004; Fierer and Jackson, 2006). For example, both the relative concentration and diversity of bacteria are positively related to pH (Fierer and Jackson, 2006; Rousk et al., 2010), and fungi use substrates with larger C/N ratios than bacteria (Sterner and Elser, 2002). Identifying the factors that control microbial community composition and dynamics may improve our understanding of biogeochemical processes (Keith-Roach et al., 2002), food web dynamics (Laakso et al., 2000; Schmidt et al., 2000), and overall

∗ Corresponding author. Tel.: +86 20 37252722; fax: +86 20 37252831. E-mail address: [email protected] (S. Fu). 0929-1393/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.apsoil.2011.10.014

soil quality (Calderon et al., 2001; Yao et al., 2000). However, the mechanisms responsible for changes in microbial community composition have been difficult to untangle because soil variables are highly correlated. Photosynthates recently shaped by plants and released by their roots in the form of rhizodeposition are a major source of C for soil microbes and shape the composition of the microbial community in the rhizosphere (Fu and Cheng, 2004; Priha et al., 2001; Rajaniemi and Allison, 2009; Yarwood et al., 2009). Microbial decomposition processes in soil are highly sensitive to the availability of labile C and N (Rasche et al., 2010). The physical, chemical, and biological properties of root-associated soil result in greater microbial activity in the rhizosphere than in the bulk soil (Bardgett et al., 1998; Wardle et al., 2004). Root exudates and decaying plant material provide C that is used by heterotrophic soil organisms as a source of energy and structural material (Barea et al., 2005; Kuzyakov and Cheng, 2001). In turn, microbial activity in the rhizosphere affects root branching patterns and controls the supply of nutrients available to plants, thereby modifying the quality and quantity of root exudates (Bowen and Rovira, 1999).

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Tree girdling is a tool used to stop rhizodeposition and to thereby measure the effect of tree rhizodeposition on the microbial community (Scott-Denton et al., 2006). Tree girdling is also used to control non-commercial trees in tropical forests (Ohlson-Kiehn et al., 2006) and to clear savannas of invasive brush and trees (Czarapata, 2005). Tree girdling, in which the bark and phloem are removed down to the youngest xylem, is useful as a research tool because it stops rhizodeposition without mechanically disturbing the soil–root–microbe system (Bhupinderpal-Singh et al., 2003; Chen et al., 2009, 2010, 2011; Dannenmann et al., 2009; Göttlicher et al., 2006; Högberg et al., 2001; Kaiser et al., 2010; Rasche et al., 2010; Scott-Denton et al., 2006; Subke et al., 2004; Weintraub et al., 2007; Yarwood et al., 2009). In a large-scale study in a boreal forest, tree girdling reduced the number and biomass of fruiting bodies of ectomycorrhizal (ECM) fungi in soil to almost zero after 2 months but did not affect the number or biomass of saprotrophic fungi (Högberg et al., 2001, 2007). In a 4-year tree-girdling experiment in a boreal Scots pine forest, tree girdling significantly altered fungal and bacterial communities (Yarwood et al., 2009). It is thus likely that girdling-related changes in soil chemistry, and especially in labile C and N pools, have considerable effects on the soil microbial community structure (Dannenmann et al., 2009; Högberg et al., 2007; Kaiser et al., 2010; Rasche et al., 2010; Weintraub et al., 2007). Weintraub et al. (2007) measured reduced quantities of 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 increased 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 a temperate spruce stand. One possible indicator of change in substrate availability is the ratio of cyclopropyl to cyclopropyl precursor (e.g., cy17:0/16:1␻7c) in microbial cell membranes. An increase in this ratio has been used as an indicator of microbial stress caused by insufficient substrate availability (Bossio and Scow, 1998; Feng and Simpson, 2009), anaerobic conditions (Kieft et al., 1997), and water limitations (Moore-Kucera and Dick, 2008). A lower cy17:0/16:1␻7c ratio has been related to higher substrate availability, an increase in bacterial growth rates, and a decrease in carbon limitation (Bååth et al., 1995; Bossio and Scow, 1998). As indicated above, links between responses of microbial stress indicators and the availability of substrates supporting microbial growth have been examined in only a few studies (Feng and Simpson, 2009). Although previous studies have used tree girdling to determine how soil microbial communities are influenced by C and N availability resulting from rhizodeposition (Kaiser et al., 2010; Rasche et al., 2010; Yarwood et al., 2009), to the best of our knowledge none of these considered the effects of girdling different tree species or were conducted in subtropical plantations. In the current study, we assessed how tree girdling alters microbial community composition in two plantations in southern China. One plantation was planted with Acacia crassicarpa and the other with Eucalyptus urophylla. Both are fast-growing and major subtropical tree species that are commonly used for pulpwood production because of their high productivity and short rotations. These two tree species respond differently to tree girdling; most importantly for our purposes, the biomass of living fine roots was reduced by 94% for A. crassicarpa but by only 18% for E. urophylla 6 months after tree girdling (Chen et al., 2010, 2011). This suggests that for at least 6 months after girdling, rhizodeposition is substantial for E. urophylla but not for A. crassicarpa. Thus, we hypothesized that the magnitude of the tree girdling effect on microbial community composition would depend on both the tree species and soil properties. The two main objectives of this study were: (i) to quantify the effects of tree girdling on C and N availability and soil microbial community composition for the two plantations at different times

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after girdling; and (ii) to determine whether changes in soil microbial community composition are correlated with changes in C and N availability and other soil properties induced by tree girdling. 2. Materials and methods 2.1. Site description The study was conducted in A. crassicarpa and E. urophylla monospecific plantations at the Heshan Hilly Land Interdisciplinary Experimental Station (112◦ 50 E, 22◦ 34 N), Chinese Academy of Sciences (CAS). The field station is located in Heshan County, Guangdong Province, which is a hilly region with laterite soils and a subtropical monsoon climate. The mean annual precipitation between 1984 and 2006 was 1295 mm. About 80% of the precipitation falls during the wet season, between March and September. The mean annual temperature at the site is 21.7 ◦ C. The tree plantations used in this study were established in 2005, with A. crassicarpa and E. urophylla saplings planted at 3 m × 2 m spacing. Each plantation occupies an area of 50 ha. In January 2008, the average height and diameter at breast height were about 6.3 m and 6.4 cm for A. crassicarpa and about 11.9 m and 9.1 cm for E. urophylla. 2.2. Tree girdling treatment The girdling treatments are described in detail in Chen et al. (2010), who conducted a study with the same plots. Briefly, in each A. crassicarpa and E. urophylla plantation, six plots (10 m × 10 m each) were established, and understory vegetation was mowed in January 2007. Three of the six plots were used as girdling plots and the other three were used as controls in a completely randomized design. The girdling treatment was applied to all trees for each girdling plot. Each plot contained about 20 trees. All plots were trenched to 50 cm before girdling to avoid root encroachment from outside. For girdling, 10 cm of bark and cambium were removed over the entire circumference of the stem at about 1 m height on 11 February 2007. The two plant species respond differently to tree girdling: A. crassicarpa, which belongs to the Leguminosae, resprouts (the ability of growing new branches under the girdled wound) only weakly after girdling, while E. urophylla resprouts vigorously after girdling (Fig. 1). Leaves of A. crassicarpa started to fall 3 months after girdling, and no leaves remained on the plants 5 months after girdling; leaf litter was removed periodically so that it did not accumulate on the soil surface. In contrast, E. urophylla leaves did not fall until 7 months after girdling, and 70% of the trees were still alive 1 year after girdling. 2.3. Soil sampling and analysis Soils were sampled on 19 April and 28 November 2007. Four soil cores (3 cm in diameter and 20 cm depth) were taken at an even distance between two neighboring girdled trees, and the soil cores were mixed to obtain a composite soil sample. For each plot, five composite soil samples were collected. We determined soil microbial biomass C (Cmic ) and soil microbial biomass N (Nmic ) using chloroform fumigation–extraction (Brookes et al., 1985; Vance et al., 1987). Briefly, a 20-g subsample of field-moist soil was used for the fumigation and non-fumigation treatments. The filtered soil K2 SO4 -extracts of both fumigated and non-fumigated samples were analyzed for dissolved organic carbon (DOC) and dissolved organic nitrogen (DON) using a total organic carbon analyzer (Shimadzu TOC-VCPH). Soil microbial biomass C (Cmic ) and soil microbial biomass N (Nmic ) were calculated as follows: Cmic (or Nmic ) = 2.22 × E (Wu et al., 1990), where E is the quantity of carbon (or nitrogen) extracted from fumigated soil minus the quantity of carbon (or nitrogen) extracted from non-fumigated soil. The

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fatty acid. The quantities (ng g−1 dry soil) of specific fatty acids in a given sample were determined using an internal standard (methyl ester C19:0, 30 ng ␮l−1 , Sigma–Aldrich) and the formula described by Abaye et al. (2004) Fatty acid(ng g−1 dry soil) =

PFAME × ng Std PISTD × dilution × W

where PFAME and PISTD are the peak areas of each fatty acid methyl ester and the internal standard, respectively; ng Std is the concentration of the internal standard (ng ␮l−1 solvent); and W is the oven-dry soil weight. 2.5. Statistical analysis

Fig. 1. Resprouting ability of A. crassicarpa (a, b) and E. urophylla (c, d) after tree girdling and as indicated by the production of new phloem 1.5 months after girdling. Note that new phloem is absent on the xylem of A. crassicarpa (b) but is present on the xylem of E. urophylla (d).

Principle component analysis (PCA) was used to separate and group the samples based on their soil microbial groups as represented by specific PLFA biomarkers. To avoid double counting, bacterial biomarkers and fatty acid ratios were not included in the PCA. Multivariate data from the analyses of specific soil microbial groups were subjected to PCA with correlation matrices to extract the information into a few principal components that could be tested for effects of tree girdling as described above. For PCA analysis, scores of individual soil microbial groups were expressed as a percentage of the total PLFAs in the sample. We used t-tests to evaluate treatment effects on soil variables and soil microbial groups. Student’s t-test was also used to investigate the differences between the two sample periods based on scores of PC 1 and PC 2. We used stepwise regression to identify the soil characteristics that best explained the PLFA concentrations of each soil microbial group. Significance was set at P ≤ 0.05. The PCA analysis, Student’s t-test, and stepwise regression analyses were performed using SPSS 15.0 software (SPSS, Chicago, IL, USA). 3. Results

non-fumigated C and N content will be hereafter referred to as K2 SO4 -extractable carbon and nitrogen, which are proxies for DOC (Scott-Denton et al., 2006) and DON (Calderon et al., 2000). A 20-g subsample of soil was oven-dried at 105 ◦ C for 24 h to determine soil moisture. For determination of NH4 + -N and NO3 − -N content, another 10-g subsample of field-moist soil was extracted with 50 ml of 2 mol/L KCl, and the extract was subjected to colorimetric determination on a Lachat FIA (Lachat QuickChem FIA + 8000 Series, Zellweger Analytics, Milwaukee, WI). Soil pH was measured in a 1:2.5 (soil:water) suspension. Soil organic carbon (SOC) was determined using the dichromate oxidation method. Total soil nitrogen concentration (TN) was determined by the micro-Kjeldahl digestion, followed by colorimetric determination on a Lachat FIA. All results are expressed on an oven-dry soil basis. 2.4. Microbial community composition Microbial community composition in soil samples was assessed using phospholipid fatty acids (PLFAs) as described by Bossio and Scow (1998). The resultant fatty acid methyl esters were separated, quantified, and identified using capillary gas chromatography (GC). Qualitative and quantitative fatty acid analyses were performed using an Agilent 6890 gas chromatograph (Agilent Technologies, Palo Alto, CA, USA) and the MIDI Sherlock Microbial Identification System (MIDI Inc., Newark, DE, USA). Fatty acids were quantified by calibration against standard solutions of FAME 19:0 (Matreya Inc., State College, PA, USA), which was added as an internal standard at a concentration of 50 ng ml−1 . The fatty acids used as biomarkers for specific groups of soil organisms (Kong et al., 2008; Zhang et al., 2010) are listed in Table 1. The prefixes a and i indicate anteisoand iso-branching, respectively, and cy indicates a cyclopropane

3.1. Changes in soil properties Tree girdling altered the soil properties in both the A. crassicarpa and the E. urophylla plantation. For A. crassicarpa, data collected in April 2007 indicated that tree girdling had increased SOC and C/N relative to control plots (Table 2), but decreased soil moisture, TN, Cmic , Nmic , DOC, and DON. In November 2007, tree girdling had increased SOC, C/N, and NO3 − -N, but decreased Cmic , Nmic , DOC, and DON. Soil pH and NH4 + -N were not significantly affected by tree girdling in April or November 2007. For E. urophylla, data collected in April 2007 indicated that tree girdling had deceased DON and marginally decreased Cmic and Nmic (Table 3). In November 2007, tree girdling had decreased Cmic , Nmic , DOC, and DON, but increased NO3 − -N. Soil pH, soil moisture, SOC, TN, and NH4 − -N were not significantly affected by tree girdling in April or November 2007. 3.2. Changes in soil microbial groups Tree girdling changed the PLFA concentrations of soil microbial groups, and the magnitude of the effect increased with time after girdling and differed between the two tree species. Compared to control plots, however, tree girdling did not change the total PLFAs in either plantation in April or November 2007 (Fig. 2a). Generally, tree girdling increased the concentrations of bacterial PLFAs (total bacteria and general bacteria PLFAs) (Fig. 2b and f) and decreased fungal PLFAs (fungi and vesicular arbuscular mycorrhizal fungi PLFAs) (Fig. 2g and i), with a consequent decrease in Fu/Ba for both species (Fig. 2h). Tree girdling affected fungal populations more than bacterial populations, as demonstrated by PLFA data for

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Table 1 Fatty acids used in the analysis of microbial community composition in A. crassicarpa and E. urophylla plantations. Soil microbial groups and ratios

Diagnostic fatty acids

Gram-positive bacteria (G+)

i14:0 + i15:0 + a15:0 + i16:0 + i17:0 + a17:0 (Kong et al., 2008; Sampedro et al., 2006; Waldrop and Firestone, 2004; Zak et al., 1996; Zelles et al., 1997; Zhang et al., 2010) 16:1␻7t + 17:1␻8c + 18:1␻7c + cy17:0 + cy19:0 (Kong et al., 2008; Sampedro et al., 2006; Zak et al., 1996; Zhang et al., 2010) 15:0 + 17:0 (Kong et al., 2008; Zak et al., 1996; Zelles et al., 1997; Zhang et al., 2010) i14:0 + i15:0 + a15:0 + i16:0 + 16:1␻7t + i17:0 + a17:0 + 18:1␻7c + cy19:0 (Frostegård et al., 1993; Kong et al., 2008; Sampedro et al., 2006; Zhang et al., 2010) 18:1␻9 + 18:2␻6,9 + 18:3␻6c (Bossio and Scow, 1998; Kong et al., 2008; Sampedro et al., 2006; Zhang et al., 2010) 16:1␻5c (Kong et al., 2008; Olsson and Alstrom, 2000; Zhang et al., 2010) cy17:0/16:1␻7c (Dickens and Anderson, 1999; Kong et al., 2008; Zelles et al., 1997; Zhang et al., 2010) (i14:0 + i15:0 + a15:0 + i16:0 + i17:0 + a17:0)/(16:1␻7t + 17:1␻8c + 18:1␻7c + cy17:0 + cy19:0) (18:1␻9 + 18:2␻6,9 + 18:3␻6c)/(i14:0 + i15:0 + a15:0 + i16:0 + 16:1␻7t + i17:0 + a17:0 + 18:1␻7c + cy19:0)

Gram-negative bacteria (G−) General bacteria (GBa) Bacteria (Ba) Fungi (Fu) Vesicular-arbuscular mycorrhizal fungi (VAM) cy17:0/16:1␻7c (C/P) Ratio of G+ to G− (G+/G−) Ratio of Fu to Ba (Fu/Ba) The abbreviations of PLFA biomarkers are in brackets.

Table 2 Effect of tree girdling on soil properties in the A. crassicarpa plantation. Soil properties

a

pH Soil moisture (%)a SOC (g kg−1 ) TN (mg kg−1 ) SOC/TN (C/N) Cmic (mg kg−1 ) Nmic (mg kg−1 ) DOC (mg kg−1 ) DON (mg kg−1 ) NO3 − -N (mg kg−1 ) NH4 + -N (mg kg−1 )

April 2007

November 2007

Control plots

Girdled plots

Control plots

Girdled plots

4.06(0.02) 25.07(0.84) 8.86(1.29) 0.97(0.07) 9.13(0.62) 337.04(1.20) 86.96(12.96) 632.82(65.69) 401.12(39.15) 3.33(0.37) 92.25(1.69)

4.08(0.01) 19.76(0.20)* 11.50(1.24)* 0.76(0.06)* 15.13(0.49)** 137.25(10.72)** 42.96(7.74)** 432.62(32.77)** 301.47(20.21)* 3.11(0.59) 88.31(1.61)

4.30(0.08) 14.08(0.02) 7.94(0.38) 0.58(0.07) 13.69(0.26) 247.52(22.42) 75.91(8.35) 433.77(38.69) 738.60(27.94) 1.70(0.14) 102.80(7.94)

4.13(0.03) 15.00(0.60) 10.99(0.46)** 0.50(0.05) 21.98(0.68)** 128.30(16.56)** 43.31(12.11)* 228.66(37.84)** 459.81(24.43)** 3.61(0.60)** 94.73(7.10)

Trees were girdled on 11 February 2007. a Data from Chen et al. (2010). Values are means (standard errors) of three replicate plots. Asterisks indicate a significant difference between the tree girdling treatment and the control (*P < 0.05 and **P < 0.01). Abbreviations of soil properties: SOC, soil organic carbon; TN, total soil nitrogen; SOC/TN, ratio of SOC to TN; Cmic , soil microbial biomass carbon; Nmic , soil microbial biomass nitrogen; DOC, dissolved organic carbon; DON, dissolved organic nitrogen; NO3 − -N, soil nitrate; and NH4 + -N, soil ammonia.

bacteria and fungi (Fig. 2b and g) and by data for specific groups (Fig. 2c, d, i and k). Girdling of A. crassicarpa increased the cy17:0 to 16:1␻7c (C/P) ratio in April and November, but girdling of E. urophylla decreased the C/P ratio in April and increased it in November (Fig. 2j). For both tree species, girdling increased the concentration of gram-negative bacteria PLFAs (G−) and decreased the G+ to G− (G+/G−) ratio only in November, but did not significantly affect G− or G+/G− in April (Fig. 2d and e).

The PCA analysis based on soil microbial groups as indicated by PLFA biomarkers clearly separated the control and girdled plots (Fig. 3). PC 1 and 2 explained 65% and 32%, respectively, of the variance in microbial community composition for A. crassicarpa, and 52% and 27%, respectively, for E. urophylla (Fig. 3a and b). For A. crassicarpa in November 2007 but not in April 2007, soil microbial groups significantly differed between the control and girdled plots based on the score of PC 1 and PC 2. For E. urophylla, a significant difference was detected only based on the score of PC 1.

Table 3 Effect of tree girdling on soil properties in the E. urophylla plantation. Soil properties

pHa Soil moisture (%)a SOC (g kg−1 ) TN (mg kg−1 ) SOC/TN (C/N) Cmic (mg kg−1 ) Nmic (mg kg−1 ) DOC (mg kg−1 ) DON (mg kg−1 ) NO3 − -N (mg kg−1 ) NH4 + -N (mg kg−1 )

April 2007

November 2007

Control plots

Girdled plots

Control plots

Girdled plots

4.24(0.05) 11.00(1.40) 7.24(1.29) 0.53(0.09) 13.63(1.06) 267.41(40.04) 71.11(14.26) 318.35(83.70) 367.76(26.61) 2.23(0.10) 94.20(2.56)

4.10(0.02) 14.67(2.24) 7.69(2.13) 0.68(0.07) 11.11(0.36) 200.30(25.62) 50.32(8.01) 305.09(48.40) 269.62(21.13)* 2.66(0.35) 90.81(1.51)

4.33(0.02) 7.21(1.13) 7.04(0.52) 0.58(0.08) 15.05(0.10) 223.58(22.12) 54.73(5.12) 355.93(31.28) 608.11(26.99) 1.12(0.12) 89.73(6.14)

4.27(0.03) 7.01(1.10) 7.73(0.99) 0.64(0.05) 13.44(0.33) 121.56(18.74)* 36.32(4.22)* 239.36(29.01)* 411.71(39.66)* 2.26(0.16)* 96.33(7.11)

Trees were girdled on 11 February 2007. a Data from Chen et al. (2010). Values are means (standard errors) of three replicate plots. Asterisks indicate a significant difference between the tree girdling treatment and the control (*P < 0.05 and **P < 0.01). See Table 2 for soil property abbreviations.

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Fig. 2. Concentrations or ratios of soil microbial PLFAs (based on % of total PLFA biomarkers) in girdled and control plots in A. crassicarpa and E. urophylla plantations. a: Total PLFAs (␮g g−1 dry soil). b: Bacteria PLFAs (%). c: G+ bacteria PLFAs (%). d: G− bacteria PLFAs (%). e: G+/G−. f: GBa bacteria PLFAs (%). g: Fu PLFAs (%). h: Fu/Ba. i: VAM PLFAs (%). j: C/P. k: 18:2␻6,9 PLFAs (%). Values are means + standard error of three replicate plots. Within each sample date, asterisks indicate significant differences between treatments: *P < 0.05; **P < 0.01 (Student’s t-test). Trees were girdled on 11 February 2007, and samples were collected on 19 April and 28 November 2007.

3.3. Influence of soil properties on the PLFA concentrations of soil microbial groups Soil properties explained 15–49% of the variance in the PLFA concentrations of soil microbial groups in both plantations. Stepwise regression indicated that, in the A. crassicarpa plantation, DOC, DON, C/N, and pH were related to the PLFA concentrations of most soil microbial groups (Table 4). In the E. urophylla plantation, DON, DOC, and NO3 − -N were related to the PLFA concentrations of most soil microbial groups. In the E. urophylla plantation, Fu PLFAs were not correlated with any of the measured soil properties. 4. Discussion 4.1. Changes in C and N availability Tree girdling changed C availability in both plantations, but the magnitude of the effect differed for the two tree species. The increase of SOC and decrease in Cmic in girdled plots of both plantations was probably due to the dead roots and associated materials that had not completely decomposed; the C in these undecomposed roots was evidently unavailable to the microorganisms, and

this contributed to a decrease in soil microbial biomass. Previous research has indicated that a major source of labile carbon input to soil is in the form of root exudates (Jones et al., 2004). Because of the presumed absence of root exudates from girdled trees (girdling blocked the flow of newly formed photosynthates to the roots and to mycorrhizal fungi), DOC content was lower in girdled plots than in control plots. The decrease in DOC would affect the abundance, composition, and activity of rhizosphere microbes (Högberg et al., 2007). These effects of girdling on labile C were similar to those reported in other studies (Göttlicher et al., 2006; Högberg et al., 2001; Subke et al., 2004; Weintraub et al., 2007; Zeller et al., 2008). That girdling effects on C availability were less in the E. urophylla plantation than in the A. crassicarpa plantation can be explained by the vigorous resprouting of E. urophylla. Our group previously reported that CO2 efflux from soil was substantially reduced by girdling in the A. crassicarpa plantation but only slightly reduced by girdling in the E. urophylla plantation (Chen et al., 2010), which is consistent with the inference that the magnitude of the tree girdling effect on labile C in soil depends on the tree species. N availability was also strongly affected by tree girdling. DON and Nmic were decreased and NO3 − -N concentrations were increased in the girdled plots. The difference in N availability

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Table 4 Relationships (determined by stepwise regression) between concentrations or ratios of soil microbial PLFAs (based on % of total PLFA biomarkers) and soil properties in girdled and control plots in the A. crassicarpa and E. urophylla plantations. Soil microbial group

A. crassicarpa

E. urophylla

Soil property TP G+ G− GBa Ba Fu C/P Fu/Ba G+/G− VAM

P value

+DOC +DOC, −C/N +DON +pH, +DON +DON, +C/N −DOC, −DON, −pH +DOC, +DON −DON, −C/N +DOC, −C/N −DOC, −DON

0.03 <0.01 <0.01 <0.01 0.01 <0.01 <0.01 <0.01 <0.01 <0.01

r2

Soil property

P value

r2

0.15 0.35 0.32 0.40 0.29 0.49 0.45 0.32 0.37 0.36

−NH4 -N +DON +DON +DON, −NO3 − -N +DON −DOC, −DON +DON −DOC, −DON +DOC, −NO3 − -N −DON, +NO3 − -N

0.02 0.02 0.02 <0.01 0.02 0.03 0.01 0.01 0.01 0.02

0.20 0.19 0.20 0.42 0.23 0.21 0.21 0.22 0.29 0.28

+

PLFA biomarker abbreviations: TP, total PLFAs; G+, gram-positive bacteria; G−, gram-negative bacteria; G+/G−, ratio of gram-positive bacteria to gram-negative bacteria; GBa, general bacteria; Ba, bacteria; Fu, fungi; Fu/Ba, ratio of fungi to bacteria; C/P, cy17:0/16:1␻7c; and VAM, vesicular arbuscular mycorrhizal fungi. See Table 2 for soil property abbreviations. “+” and “−” indicate soil properties positively and negatively correlated with concentrations or ratios of soil microbial PLFAs.

between treatments may be ascribed to differences in direct root N inputs to the soil. Previous studies have also found that root exudation is associated with increased N availability in the rhizosphere (Weintraub et al., 2007). We suspect that tree girdling, by reducing the supply of available C to microorganisms, 1.0

A. crassicarpa

a

VAM Fu

PC 2 (32%)

.5

Apr vs C

4.2. Changes in the concentration of bacterial PLFAs

Nov vs C G+

0.0

TP

Apr vs G

-.5 GBa

Nov vs G

G-

-1.0 -1.0

-.5

0.0

.5

1.0

PC 1 (65%) 1.0

E. urophylla

b

Fu VAM

PC 2 (27%)

.5

Nov vs C G+ TP

0.0

Nov vs G Apr vs C

-.5

GGBa

Apr vs G

-1.0 -1.0

-.5

stimulated the microbial N mineralization. The difference in N availability between treatments could also result from differences in plant N uptake caused by reductions in mycorrhizal hyphae and fine roots (Jordan et al., 1998). Increased NO3 − -N concentration in response to girdling has also been found in other studies (Dannenmann et al., 2009; Johnson and Edwards, 1979; Weintraub et al., 2007; Zeller et al., 2008) and may affect microbial community composition.

0.0

.5

1.0

PC 1 (52%) Fig. 3. Principal component (PC) analysis of PLFA data from soil of A. crassicarpa plots (a), E. urophylla plots (b), and plots of both tree species (c). Whiskers show standard error of the mean of PCA weighted loading values; solid squares show the PCA weighted loading values of microorganisms. PLFA biomarker abbreviations: TP, total PLFAs; G+, gram-positive bacteria; G−, gram-negative bacteria; GBa, general bacteria; Fu, fungi; and VAM, vesicular arbuscular mycorrhizal fungi.

Tree girdling increased the concentration of bacterial PLFAs in both plantations. Increases in GBa and Ba PLFAs after tree girdling were in agreement with previous reports (Brant et al., 2006; Högberg et al., 2007) in which the quantity of bacterial PLFAs but not total PLFAs was affected by manipulation of C inputs. Ba and GBa have different distributions, the former belong to phylogenetic bacteria and the latter belong to broad phylogenetic distribution bacteria. For example, Högberg et al. (2007) found that bacterial abundance increased considerably after tree girdling. We found that the differences in availability of C and N (DON, DOC, C/N) among treatments were associated with differences in the PLFA concentrations of bacterial groups (Ba, GBa, and G−) (Table 4). This is consistent with the finding that belowground C inputs affect the soil microbial community (Brant et al., 2006). The decrease in DOC and DON in girdled plots was probably caused by the decrease in root exudation because a substantial portion of DOC and DON originates from photoassimilates (Giesler et al., 2007; Weintraub et al., 2007). The concentrations of PLFAs for bacterial groups were also related to soil pH (Table 4). Soil pH is generally thought to greatly affect the composition of the soil microbial community, e.g., bacteria are generally favored by high pH (Fierer and Jackson, 2006; Rousk et al., 2010). Fierer and Jackson (2006) found that both the relative abundance and diversity of bacteria were positively related to pH. Soil pH influences the solubility of organic soil solution constituents (e.g., DOC and DON) and other factors that in turn affect the soil microbial community. The shift from a G+ dominated community to a G− dominated community may indicate a change from oligotrophic to copiotrophic conditions caused by tree girdling (Frostegård et al., 1993). The increase in G− bacteria was unexpected because G− bacteria are generally favored by the labile carbon substrates released by rhizodeposition (Treonis et al., 2004). Perhaps tree girdling increased G− bacteria because the decomposing dead roots provided labile carbon and other nutrients. Similarly, Waldrop and

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Firestone (2004) found an increase in G− PLFAs after the addition of carbon substrates to an oak woodland soil. 4.3. Changes in the concentration of fungal PLFAs An increase in bacterial PLFAs and a decrease in fungal PLFAs in plots subjected to tree girdling led to a decrease in Fu/Ba in both plantations. In our study, one ECM fungal biomarker (18:2␻6,9) decreased more rapidly in the E. urophylla than in the A. crassicarpa plantation (Fig. 2k). The same pattern was previously reported (Högberg et al., 2001, 2006, 2007; Högberg and Högberg, 2002; Kaiser et al., 2010). For example, Högberg et al. (2007) found that Fu and Fu/Ba were significantly lower in girdled plots than in control plots 4 years after tree girdling. The current study documented a significant shift in the composition of fungal communities (i.e., significant reductions in ECM fungi) in response to girdling, a shift that was associated with the availability of C and N (DON and DOC) for both plantations (Table 4). Eliminating belowground C allocation by tree girdling significantly affected C and N availability for microbes and led to decreased DOC and DON contents in the soil (Tables 2 and 3), which is consistent with results reported by Weintraub et al. (2007) and Yarwood et al. (2009). Our results confirmed that the dramatic decrease in fungi after tree girdling was associated with a decrease in C supply and that fungi depend more than other soil microorganisms on belowground C allocation. In our study, microbial community composition and the availability of C and N were governed by C allocation to roots, and the effect of tree girdling differed in E. urophylla and A. crassicarpa plantations. For example, the decreases caused by tree girdling in VAM fungal and 18:2w6c PFLA biomarkers were more pronounced for E. urophylla than for A. crassicarpa, but the opposite was true for decreases in fungal PLFAs and Fu/Ba ratios. This difference in the tree girdling effect between the tree species may be ascribed to differences in tree responses to girdling, and in particular to the ability to resprout and retain fine roots after girdling. Even in the absence of girdling, the effects of rhizodeposition on the soil fungal community are qualitatively and quantitatively different for different plant species (Broeckling et al., 2008). Although our data indicate that the two tree species have different affects on the microbial community and that this probably reflects differences in rhizodeposition, we did not determine the composition of the root exudates. Further research is needed to determine the influences of root exudates of diverse plant species on microbial community structure. 4.4. Changes in the cy17:0/16:1␻7c (C/P) ratio The increase in the PLFA ratio of two gram-negative bacteria (cy17:0/16:1␻7c or C/P) in plots with girdled trees indicated that girdling created a stressful, nutrient-deficient habitat for the soil microbial community. While C/P decreased in the E. urophylla plantation 2 months after girdling, C/P was higher in girdled plots than in control plots of both plantations by the end of the experiment. One possible reason for the higher C/P in girdled plots could be the increased soil water content and therefore reduced soil O2 . The reduced water uptake by the trees in girdled plots should increase soil water content, which would have a major effect on microbial community composition (Chen et al., 2010; Kieft et al., 1997; Wilkinson et al., 2002). Increases in microbial community stress as indicated by increases in the C/P ratio can also result from C limitation (lack of labile C inputs) (Bossio and Scow, 1998; Fierer et al., 2003; Kieft et al., 1997). 4.5. The effect of tree girdling increased with time Tree girdling changed the C and N availability and soil microbial composition in both plantations, and the effects of tree girdling

were much greater 9 months than 2 months after girdling. The increase in the magnitude of the tree girdling effect over time may be explained as follows: (1) for some limited time after girdling, roots may remain alive by utilizing starch reserves and may continue to release exudates; and (2) soil temperature and soil moisture changed over time (Chen et al., 2011). Differences in soil moisture and temperature between control and girdled plots increased with time because light interception on the soil surface increased over time as leaves fell from girdled trees. Leaves of A. crassicarpa began falling 3 months after girdling, and no leaves remained on the plants 5 months after girdling. For E. urophylla, leaves did not fall until 7 months after girdling, and 70% of the trees were still alive 1 year after girdling. Our findings are consistent with previous reports that the effects of girdling persist for long periods (Kaiser et al., 2010; Rasche et al., 2010; Yarwood et al., 2009). 5. Conclusion Overall, our findings indicate that through modification of soil resource availability and especially through belowground C allocation, trees alter the microbial community composition in soil. Our results show that the effect of tree girdling on microbial community composition depends on the tree species. We consider that the difference in the magnitude of the girdling effects results from differences in the maintenance of living roots after girdling. Acknowledgements We thank two anonymous reviewers for their comments and Bruce Jaffee for polishing the language on the manuscript. This work was supported by the National Science Fund for Distinguished Young Scholars, the Knowledge Innovation Program of the Chinese Academy of Science, and a supporting program of the National Ministry of Science and Technology. References Abaye, D.A., Lawlor, K., Hirsch, P.R., Brookes, P.C., 2004. Changes in the microbial community of an arable soil caused by long-term metal contamination. European Journal of Soil Science 48, 1–10. Allison, V.J., Yermakov, Z., Miller, R.M., Jastrow, J.D., Matamala, R., 2007. Using landscape and depth gradients to decouple the impact of correlated environmental variables on soil microbial community composition. Soil Biology and Biochemistry 39, 505–516. Bååth, E., Frostegård, A., Pennanen, T., Fritze, H., 1995. Microbial community structure and pH response in relation to soil organic matter quality in wood-ash fertilized, clear-cut or burned coniferous forest soils. Soil Biology and Biochemistry 2, 229–240. Bardgett, R.D., Keiller, S., Cook, R., Gilburn, A.S., 1998. Dynamic interactions between soil animals and microorganisms in upland grassland soils amended with sheep dung: a microcosm experiment. Soil Biology and Biochemistry 30, 531–539. Barea, J.M., Pozo, M.J., Azcon, R., Azcon-Aguilar, C., 2005. Microbial co-operation in the rhizosphere. Journal of Experimental Botany 56, 1761–1778. Bhupinderpal-Singh, Nordgren, A., Lofvenius, M.O., Högberg, M.N., Mellander, P.E., Högberg, P., 2003. Tree root and soil heterotrophic respiration as revealed by girdling of boreal Scots pine forest: extending observations beyond the first year. Plant, Cell and Environment 26, 1287–1296. Bossio, D.A., Scow, K.M., 1998. Impacts of carbon and flooding on soil microbial communities: phospholipid fatty acid profiles and substrate utilization patterns. Microbial Ecology 35, 265–278. Bowen, G.D., Rovira, A.D., 1999. The rhizosphere and its management to improve plant growth. Advances in Agronomy 66, 1–102. Brant, J.B., Myrold, D.D., Sulzman, E.W., 2006. Root controls on soil microbial community composition in forest soils. Oecologia 148, 650–659. Broeckling, C.D., Broz, A.K., Bergelson, J., Manter, D.K., Vivanco, J.M., 2008. Root exudates regulate soil fungal community composition and diversity. Applied and Environmental Microbiology 74, 738–744. Brookes, P.C., Landman, A., Pruden, G., Jenkinson, D.S., 1985. Chloroform fumigation and the release of soil nitrogen: a rapid direct extraction method to measure microbial biomass nitrogen in soil. Soil Biology and Biochemistry 17, 837–842. Calderon, F.J., Jackson, L.E., Scow, K.M., Rolston, D.E., 2000. Microbial responses to simulated tillage in cultivated and uncultivated soils. Soil Biology and Biochemistry 32, 1547–1559.

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