Changes in soil microbial community structure and activity in a cedar plantation invaded by moso bamboo

Applied Soil Ecology 91 (2015) 1–7

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Changes in soil microbial community structure and activity in a cedar plantation invaded by moso bamboo Ed-Haun Chang a , Chih-Yu Chiu b, * a b

Mackay Junior College of Medicine, Nursing, and Management, Beitou, Taipei 112, Taiwan Biodiversity Research Center, Academia Sinica, Nankang, Taipei 11529, Taiwan

A R T I C L E I N F O

A B S T R A C T

Article history: Received 26 August 2014 Received in revised form 3 February 2015 Accepted 5 February 2015 Available online xxx

Moso bamboo is fast-growing and can invade a neighboring forest with its rhizome system. We investigated the effect of bamboo invasion on an adjacent Japanese cedar plantation in terms of soil microbial biomass, activities and community structure by analysis of phospholipid-derived fatty acid (PLFA) and denaturing gradient gel electrophoresis (DGGE) profiles. In the cedar-plantation soil invaded by bamboo, soil microbial biomass C (Cmic) decreased and biomass N (Nmic) increased, which reduced Cmic/Nmic. Similarly, soil cellulase and xylanase activities decreased with invasion, indicating that bamboo invasion into the cedar plantation facilitated changes in microbial biomass and activities by changing soil biochemical properties. The proportion of total PLFAs that was attributed to all bacteria, Gram-positive (G+) bacteria and Gram-negative (G
) bacteria, was reduced with invasion. The ratio of G +/G
bacteria, 16:v7t to 16:1v7c, and cyclopropyl fatty acids to their precursors (i.e., cy17:0/16:1v7 and cy19:0/18:1v7c) was highest in cedar-plantation soil, suggesting that environmental stress for soil bacterial communities is alleviated in bamboo invaded soils. Low ratio of G+/G
in the bambooplantation and transition-zone soil was associated with increased level of easily decomposable organic matter (Cmic/Corg and Nmic/Ntot) in bamboo-invaded soil. Principle component analysis of PLFA content separated the cedar-plantation soil from bamboo and transition-zone soil. DGGE analysis revealed that change in both bacterial and fungal community structure was associated with bamboo invasion. Bamboo invasion caused significant changes in soil microbial activities and community structure in the Japanese cedar plantation. ã 2015 Elsevier B.V. All rights reserved.

Keywords: Microbial community Bamboo Phospholipid-derived fatty acids Denaturing gradient gel electrophoresis

1. Introduction Soil microorganisms are essential for maintaining soil fertility and plant growth because they play an important role in nutrient cycling and availability (Fritze et al., 1994). Forest species substitution has resulted in forest ecosystem changes (Van Calster et al., 2007). Such changes directly affect microbial communities and may alter physico-chemical properties, such as soil C, N and other biological properties (Chen et al., 2004; Ushio et al., 2010; Lucas-Borja et al., 2012). This influence may be attributed to species differences in litter quality, root exudates, and nutrient uptake (Grayston and Prescott, 2005; Lucas-Borja et al., 2012). Bamboo is one of the most important economic plants in East Asia. Bamboo’s culms are used similar to wood as a construction material, and its shoots are widely consumed as a healthy and delicious food source. Bamboo is one of the fastest-growing plants

* Corresponding author. Tel.: +886 2 2787 1180; fax: +886 2 2789 9624. E-mail address: [email protected] (C.-Y. Chiu). http://dx.doi.org/10.1016/j.apsoil.2015.02.001 0929-1393/ ã 2015 Elsevier B.V. All rights reserved.

in the world (Cho et al., 2011) because of a unique rhizomedependent system that spreads laterally and generates sprouts at the soil surface. Consequently, bamboo spreads and easily invades natural and secondary forests. The invasion of bamboo into adjacent forests has become a critical problem in forest management. With an aggressive rhizome system, bamboo expands easily into adjacent forests. The fast-growing shoots persistently occupy the gaps of trees under the canopy, making the forest floor too dark to regenerate the seedlings of young trees. Finally, bamboo gradually takes over the territory of adjacent forest. Thus, the expansion of bamboo leads to reduced biodiversity (Okutomi et al., 1996). Meanwhile, bamboo leaves can release allelochemicals that reduce the growth of understory plants (Chou and Yang, 1982) and cause changes in plant community composition and species diversity (Larpkern et al., 2011). Plant species composition plays a major role in governing soil microbial community structure (Grayston et al., 2004). However, there are few reports on the impact of bamboo invasion on the structure and diversity of the microbial community of forest soils (Wang et al., 2009).

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Our previous bar-coded pyrosequencing study of 16S rRNA gene clone libraries (Lin et al., 2014) indicated that soil bacterial diversity increased and the bacterial community changed when bamboo invaded a cedar plantation. However, little is known about the effect of bamboo invasion on soil microbial activities, or on the microbial biomass of bacteria and fungi. Denaturing gradient gel electrophoresis (DGGE) (Lorenzo et al., 2010) and phospholipid-derived fatty acid (PLFA) analysis (Sun et al., 2013) have been used to determine changes in the soil microbial community structure associated with plant invasion in forest ecosystems. PLFA analysis can provide information on the viable biomass of the microbial community, such as Gram-positive bacteria, Gram-negative bacteria, actinomycetes and fungi (White et al., 1996), because PLFAs are quickly degraded after cell death (Tabuchi et al., 2008; Frostegård et al., 2011). PLFA analysis has been used to determine the changes in soil microbial community structure resulting from different forest management practices, including different coniferous species composition (Demoling et al., 2008), moisture stress (Wilkinson et al., 2002) and prescribed burning regimes (Sun et al., 2011). In addition, PLFAs have been used to assess the physiological status of microorganisms by measuring specific fatty acids indicative of stresses, such as nutrient limitation and severe pH conditions (Bååth and Anderson, 2003). Although versatile, the use of PLFA analysis to examine the microbial community at the species level is limited. DGGE is a simple, rapid and reproducible technique to interrogate diverse taxa, such as bacteria, fungi and target groups, using different primers to amplify from the same soil DNA extract (Nakatsu, 2007). Here, we examined the microbial biomass and community structure by measuring PLFAs and used DGGE profiling to clarify how the microbial activity, biomass and community structure changed in Japanese cedar-plantation soil with invasion by bamboo. 2. Materials and methods 2.1. Site description and soil sampling This study was conducted at Shanlinshi (23 400 N, 120 460 E), a subtropical montane area in Nantou County in central Taiwan. The elevation is approximately 1350 m a.s.l., the mean annual precipitation approximately 2600 mm and the mean annual temperature 17  C. Parts of this area were reforested with Japanese cedar (Cryptomeria japonica) about 40 years ago after large-scale cutting of the natural camphor forest. Almost at the same time, moso bamboo (Phyllostachys edulis), a temperate species of giant timber bamboo, was introduced and established adjacent to the cedar plantation. Currently, a transition zone, with both cedar and bamboo plants of 30–50 m wide, stretches in the boundary between the bamboo and cedar plantations. Farmers occasionally cut bamboo stems to induce the regeneration of bamboo, leaving many stumps in the bamboo plantation. In addition, frequent harvesting of bamboo shoots (e.g., monthly during harvesting season) also caused severe or moderate disturbance for the soils in bamboo plantation and transition zone. By contrast, the soil in cedar plantation is largely undisturbed and has lush understory plants. Four parallel transect lines separated by more than 50 m within each vegetation type (moso bamboo plantation, the transition zone and cedar plantation) were surveyed in February 2011. Four replicate plots (25  25 m) were selected for each vegetation zone, for 12 sampling plots in total. The soils were derived from sandstone, and classified as Dystrudept in Soil Taxonomy (Soil Survey Staff, 2010). The studied soils were clayey loam and usually moderately well drained. Two to 3 cm depth of litter covered on the

surface of soils. Soil samples were collected with use of a soil core 8 cm in diameter by 10 cm deep. Three cores collected from each plot composed a sample in each plot. Visible detrital material, such as roots and litter, were manually removed when soil was passed through a 2-mm sieve. The 10 cm depth soil samples from each sampling plot were all in A horizon. Samples were stored at 4  C in the dark. Biochemical analyses, including analysis of microbial biomass and enzymatic activities, were completed within 1 month after field collection. Portions of soil samples were freeze-dried at
20  C immediately after sampling for analyses of PLFAs and DNA extraction. 2.2. Analytical methods Organic C and total N contents in soil samples were calculated by use of an NSC elemental analyzer (NA1500 Series 2, Fisons, Italy). Soil subsamples were weighed and oven-dried for 72 h at 105  C to determine moisture content. Soil pH values in air-dried samples were measured by use of a combination of glass electrodes (soil: water ratio 1:2.5) (McLean, 1982). Soil microbial biomass was analyzed by the chloroform fumigation–extraction method (Vance et al., 1987). Total organic C in the extracted solution was measured by use of a total organic C analyzer (Model 1010 O.I. Analytical, Texas) and converted to microbial biomass C (Cmic) assuming a conversion factor of 2.22 (Wu et al.,1990). Microbial N (Nmic) was calculated from ninhydrin-reactive N released from the biomass and determined colorimetrically at 560 nm (Amato and Ladd, 1988). Cellulase activity (EC 3.2.1.4) was determined as described by Schinner and von Mersi (1990). Fresh soil samples (5 g) were incubated with water-soluble carboxymethylcellulose (1.4%) for 24 h at 50  C (pH 5.5). The low-molecular-weight products and sugars resulting from the enzymatic degradation of carboxymethylcellulose were measured at 690 nm absorbance. Xylanase activity (EC 3.2.1.8) was determined as described by Schinner and von Mersi (1990). Fresh soil samples (1 g) were incubated with 5 ml xylan (3.4%) and 5 ml acetate buffer (pH 5.5) at 50  C for 24 h. Absorbance was measured at 690 nm. Substrate concentrations were tested to maximize enzyme activities in soil samples. Phosphatase activity (EC 3.1.3.2) was analyzed colorimetrically by measuring the absorbance by p-nitrophenol at 400 nm after incubation of 1 g fresh soil with 4 ml modified universal buffer (pH 6.5) and 1 ml of 100 mM p-nitrophenyl phosphate at 37  C for 1 h, following Tabatabai and Bremner (1969). Urease activity (EC 3.5.1.5) was determined as described by Kandeler and Gerber (1988). A fresh soil sample (2.5 g) was mixed with 2.5 ml urea (80 mM) and 20 ml of 0.1 M borate buffer (pH 10.0). The mixture was allowed to react for 2 h at 37  C. After incubation, 30 ml of KCl (2 M) was added before shaking for 30 min. Ammonium content was determined through the indophenol reaction. The color intensity of the final solution was measured at 690 nm. Extraction and analysis of PLFAs were as described by Frostegård et al. (1993). Lipids were extracted in a single-phase mixture of chloroform–methanol–citrate (1:2:0.8). Phospholipids were split into neutral, glyco- and phospholipids by use of a solidphase extraction column and eluted with chloroform, acetone and methanol. Phospholipids then underwent methylation to form fatty acid methyl esters (FAMEs). FAME identification and quantification were analyzed by capillary gas chromatography (GC) with a flame ionization detector (Thermo Finnigan Trace chromatograph) as described by Chang et al. (2011). Fatty acid nomenclature was in accordance with Frostegård et al. (1993). The fatty acids i15:0, a15:0, 15:0, i16:0, 16:1v7c, 17:0, i17:0, cy17:0, 18:1v7c, and cy19:0 represent bacteria; 18:2v6 fungi; i15:0, a15:0, i16:0, and i17:0 Gram-positive (G+) bacteria; 16:1v7c, cy17:0, 18:1v7c, and cy19:0 Gram-negative (G
) bacteria; and 10Me18:0 actinomycetes (Zogg et al., 1997; Zelles, 1999).

E.-H. Chang, C.-Y. Chiu / Applied Soil Ecology 91 (2015) 1–7 Table 1 Soil chemical properties of the different vegetation types (mean  standard deviation). Vegetation

pH

Organic C (g kg
1)

Total N (g kg
1)

C/N ratio

Bamboo Transition Japanese cedar

4.13  0.13 a 4.37  0.66 a 3.35  0.12 b

80.0  10 b 68.0  34 b 214  35 a

6.66  0.80 b 6.11  2.70 b 13.3  1.59 a

12.0  0.80 b 10.9  1.50 b 16.1  0.94 a

Values in each column followed by different letters indicate differences between different vegetation types (Duncan’s multiple range test, p < 0.05).

Soil DNA was extracted by using the PowerSoil DNA isolation kit (MO BIO lab, Solana Beach, CA). For soil bacteria, PCR conditions for primers F968-GC and R1401 (Nübel et al., 1996) were as follows. The PCR mixtures (50 ml) contained 20 ml 2X Quick Taq HS DyeMix (Toyobo), 10 pmol each primer, 1 ml template DNA and deionized water to bring the final volume to 50 ml. The PCR program was as follows: 2 min 94  C, followed by 34 cycles of 94  C for 30 s, 61  C for 30 s and 68  C for 1 min. For soil fungi, PCR conditions for primer NS1 and GC fungi were as follows. The PCR mixtures (50 ml) contained 20 ml 2X Quick Taq HS DyeMix (Toyobo), 10 pmol each primer, and 1 ml template DNA and deionized water to bring the final volume to 50 ml. The PCR program was as follows: 2 min 94  C, followed by 32 cycles of 94  C for 30 s, 52  C for 30 s and 68  C for 1 min. The PCR products were analyzed on a DGGE gel as described (Suzuki et al., 2009). A DGGE gel comprised 6% polyacrylamide gel and 50–70% denaturants for bacterial analysis and 7% polyacrylamide and 20–45% denaturants for fungal analysis. The electrophoresis was performed for 18 h at 50 V in 1x TAE buffer at a constant temperature of 58  C for bacteria and 60  C for fungi with a Dcode Universal Detection system (Bio-Rad Laboratories, Hercules, CA, USA). The gel was stained with Gelstar (20 ml of Gelstar in 200 ml of 1x TAE buffer) for 30 min, then the DGGE band profiles were visualized under UV light. Digital image capturing involved use of a Gel Doc XR gel imaging system (Bio-Rad). The obtained DGGE fingerprints were analyzed by use of Quantity One (Bio-Rad USA). 2.3. Statistical analysis Data from biochemical and chemical analysis were converted on an oven-dried basis. One way analysis of variance with Duncan’s multiple range test were used to test significant differences in the soil properties, soil enzyme activities and microbial communities. Principal component analysis (PCA) was used to test relative concentrations (mol%) of individual fatty acids. Statistical analyses involved use of SPSS v12.0 (SPSS Inc., Chicago, USA). p < 0.05 was considered to be statistically significant. Hierarchical cluster analysis with an unweighted pair-group method with arithmetic mean (UPGMA) algorithm was used to process the DGGE banding pattern produced from the forest sites. 3. Results 3.1. Soil characteristics and microbial biomass pH, organic C content and total nitrogen content significantly differed between the Japanese cedar-plantation and bamboo-

3

associated (bamboo plantation and transition zone) soils (Table 1). The lowest soil pH and the highest values of soil organic C, total N and C/N ratio were found in the Japanese cedar plantation. There was no significant difference between the bamboo-plantation and transition-zone soils in these factors. The highest Cmic and lowest Nmic were in cedar-plantation soil, for the highest Cmic/Nmic in the soil (Table 2). Both Cmic/Corg and Nmic/Ntot were the lowest in the Japanese cedar-plantation soil. Cmic, Nmic, Cmic/Nmic, Cmic/Corg and Nmic/Ntot did not differ between the bamboo-plantation and transition-zone soil. 3.2. Soil enzyme activities Soil cellulase and xylanase activities were the highest in the cedar-plantation soil, with no significant difference between the bamboo-plantation and transition-zone soil (Table 3). Urease and phosphatase activities did not differ between the cedar and bamboo-plantation soil. 3.3. PLFA analyses Total PLFA concentration, as an indicator of active soil microbial biomass, was highest in the cedar plantation. Bacterial biomarkers, G+ and G
bacteria, and ratio of G+ to G
bacteria were also highest in the cedar plantation with no significant difference between the bamboo-plantation and transition-zone soil (Table 4). Soil fungi, arbuscular mycorrhizal fungi, actinomycetes, and ratio of fungal to bacterial biomass did not differ among the vegetation types. Ratios of 16:v7t (trans-unsaturated fatty acid) to 16:1v7c (cisunsaturated fatty acid) and cyclopropyl fatty acids to their precursors (i.e., cy17:0/16:1v7 and cy19:0/18:1v7c), indicators of physiological and nutritional stress within microbial communities (Morre-Kucera and Dick, 2008), were highest in the Japanese cedar-plantation soil, with no significant difference between bamboo-plantation and transition-zone soil (Table 5). Soil communities, analyzed by PCA of PLFA levels, significantly differed among different vegetation types. The PLFA levels in the soil could be divided into two large clusters, Japanese cedar (nonbamboo invaded) and bamboo-invaded soil. The first and second principal component (PC1, PC2) accounted for 87% of the variation in PLFA levels (Fig. 1). PC1 differentiated the Japanese cedar soil from other plantation soil, whereas PC2, the VAM biomarker (16:1v5), had positive loading and appeared to differentiate bamboo-plantation from transition-zone soil. High positive loadings by the G+ bacteria (i15:0, a15:0, i16:0, 10Me16:0 and 10Me17:0) and G
bacteria (cy17:0 and cy19:0) contributed to separating cedar soil from other plantation soils along the PC1 axis. 3.4. DGGE analysis Dendrograms of genetic similarity obtained by DGGE analysis of bacterial communities in the soil samples clustered into different plantation types (Fig. 2). One group consisted of microbial communities from bamboo-associated soils (bamboo plantation and transition zone), whereas the other group was from nonbamboo-associated soils (Japanese cedar).

Table 2 Soil microbial biomass characteristics of the different vegetation types (mean  standard deviation). Vegetation

Cmic (mg kg
1)

Nmic (mg kg
1)

Cmic/Nmic

Cmic/Corg (%)

Nmic/Ntot (%)

Bamboo Transition Japanese cedar

2160  960 b 1700  1200 b 3370  430 a

547  110 a 419  160 ab 366  56 b

3.96  1.66 b 3.97  1.96 b 9.31  1.44 a

2.67  1.11 a 2.44  1.04 a 1.58  0.21 b

8.21  1.25 a 7.04  0.91 a 2.78  0.52 b

Cmic: microbial biomass C; Nmic: microbial biomass N. Values in each column followed by different letters indicate differences between different vegetation types (Duncan’s multiple range test, p < 0.05).

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Table 3 Soil enzymatic activities of the different vegetation types (mean  standard deviation). Vegetation

Cellulase (mg glucose g
1 d
1)

Xylanase (mg glucose g
1d
1)

Urease (mmol NH4+-N g
1 h
1)

Phosphatase (mg nitrophenol g
1 h
1)

Bamboo Transition Japanese cedar

1860  680 b 1120  780 b 12000  2400 a

5460  2900 b 2900  1900 17600  4500 a

55.9  21 a 47.9  35 a 86.8  44 a

1190  300 a 646  230 b 1190  270 a

Values in each column followed by different letters indicate differences between different vegetation types (Duncan’s multiple range test, p < 0.05). Table 4 Content of phospholipid fatty acid biomarkers (nmol g
1 soil) and ratios of biomarkers in different vegetation types (mean  standard deviation). Vegetation

Total PLFA (nmol g
1)

Bacteriaa

G+e

G
f

G+/G


Fungib

AMF Fungic

Actinomycetesd

Fungi/bacteria

Bamboo Transition Japanese cedar

266  77 b 235  140 b 456  40 a

130  34 b 119  73 b 225  14 a

54.0  16 b 52.1  34 b 108  9.8 a

65.0  15 b 56.3  34 b 99.3  5.9 a

0.82  0.08 b 0.93  0.10 b 1.09  0.12 a

7.55  5.58 a 4.54  1.70 a 8.76  4.66 a

8.13  2.41 a 6.35  2.65 a 7.61  0.69 a

5.65  1.81 a 4.61  2.55 a 5.39  0.46 a

0.038  0.017 a 0.050  0.027 a 0.053  0.027 a

PLFA: phospholipid fatty acids. Values in each column followed by different letters indicate differences between different vegetation types (Duncan’s multiple range test, p < 0.05). a As indicated by the sum of fatty acid biomarkers i15:0, a15:0, 15:0, i16:0, 16:1v7c, 17:0, i17:0, cy17:0, 18:1v7c, and cy19:0. b As indicated by the biomarker 18:2v6,9c. c As indicated by the biomarker 16:1v5c. d As indicated by the biomarker 10Me18:0. e As indicated by the sum of fatty acid biomarkers i15:0, a15:0, i16:0, and i17:0. f As indicated by the sum of fatty acid biomarkers cy17:0, 16:1v7c;18:1v7c, and cy19:0.

4. Discussion We observed a significant decrease in soil organic matter (SOM) and increase in pH in moso bamboo-invaded soil compared with adjacent Japanese cedar-plantation soil. Litter decomposition is directly influenced by litter quality. The soil C cycling was found to be extremely slow in the cedar plantation due to the strong resistance of cedar litter to degradation (Nakane, 1995). The decay of plant residues might proceed more slowly in the cedarplantation than bamboo-invaded soil. In addition, frequent human disturbances, such as bamboo shoot harvesting in the bamboo plantation, effectively tilled the soil, which might accelerate the degradation of the organic matter in the bamboo soil. The increase in pH in bamboo-invaded soil may be due to changes in some chemical characteristics such as cation exchange capacity (Sasaki, 2012). Umemura and Takenaka (2014) also found that increase in soil pH was a distinct change in soil chemistry as a result of moso bamboo invading hinoki cypress forests. The soil microbial biomass Cmic, Nmic, organic C and total N of bambooinvaded and non-invaded sites were significantly different; therefore, the bamboo invasion may increase the fraction of labile SOM. The decrease in microbial biomass in the bamboo plantation and in transitional sites could be due to decreased SOM, caused by high mineralization of SOM and less litter input (Xu et al., 2008). The ratio of Cmic/Corg reflects the potential for SOM mineralization after fresh input of organic materials (Pascual et al., 1997). A high ratio of Cmic/Corg indicates high levels of labile organic C in the

Table 5 PLFA ratio indicators of metabolic stress of microbial communities in different vegetation types(mean  standard deviation). Vegetation

16:1v7t/16:1v7c

cy17:0/16:1v7c

cy19:0/18:1v7c

Bamboo Transition Japanese cedar

0.062  0.012 b 0.058  0.018 b 0.118  0.011 a

0.344  0.029 b 0.350  0.021 b 0.414  0.043 a

1.03  0.07 b 0.956  0.26 b 1.85  0.32 a

Values in each column followed by different letters indicate differences between different vegetation types (Duncan’s multiple range test, p < 0.05).

soil (Sparling, 1992). The easily decomposed herbaceous litter and extensive rhizome structure of moso bamboo could provide more labile organic C than Japanese cedar. Zhang et al. (2003) also found higher Cmic/Corg in a bamboo forest soil than in a Cryptomeria fortunei forest because the soil may have more labile organic substrates. In general, soil enzyme activities were closely associated with levels of organic matter (Grandy et al., 2009); high SOM sustains high microbial biomass and high enzyme activities (Chodak and  ska, 2010). Badiane et al. (2001) found that forest types have Niklin a direct impact on the quality of organic matter, resulting in changes in soil enzyme activity. Low cellulase and xylanase activities in bamboo-invaded plantations might be due to the low SOM. Despite the lack of any significant differences in urease and phosphatase activities among the different vegetation types, bacterial biomass based on PLFAs was significantly associated with urease and phosphatase activity. Such results agreed with Zheng et al. (2008), who found that soil phosphatase and urease activities in different plantations were positively correlated with the abundance of bacteria.

3 Bamboo Transition Japanese cedar

2

PC2 (13.2%)

Similarly, cluster analysis of fungal communities showed two main clusters for these vegetation types (Fig. 3). Soils of the bamboo plantation and transition zone clustered together and apart from the cedar-plantation soils.

1

0

-1

-2 -2.0

-1.5

-1.0

-0.5

0.0

0.5

1.0

1.5

2.0

PC1 (73.8%) Fig. 1. Plots of the two main principal components (PCs) from principal component analysis of the mol% of microbial phospholipid fatty acid content in soil samples from different vegetation types.

E.-H. Chang, C.-Y. Chiu / Applied Soil Ecology 91 (2015) 1–7

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Fig. 2. Cluster analysis of bacterial community structure from different vegetation types measured by unweighted pair group method with arithmetic mean (UPGMA).

A bamboo rhizome system reduces opportunities for the establishment and growth of other plants under a bamboo canopy (Okutomi et al., 1996). In addition, bamboo leaves release allelochemicals that can reduce seedling abundance and species richness under bamboo (Chou and Yang, 1982; Larpkern et al., 2011), which might cause changes in soil microbial communities when bamboo invaded into cedar plantation. Changes in the ratio of G+/G
bacteria are associated with changes in the quality of SOM; a low ratio of G+/G
may be due to induced growth of G
bacteria under substrate-rich conditions (Margesin et al., 2009; Djukic et al., 2010), which results in high levels of G
bacteria and a low G+/G
ratio in bamboo soil. Low ratios of G+/G
in the bamboo plantation and the transition zone agree with an increase in easily decomposable organic matter (Cmic/Corg and Nmic/Ntot) in bamboo-invaded soil (Table 2). Guckert et al. (1986) found increased ratios of 16:1v7t/16:1v7c isomers to be associated with stress and starvation conditions for bacteria. The ratio of 16:1v7t/16:1v7c in soil of Japanese cedar was higher than in other soil (Table 5), which suggests that sources of C and N are more labile in bamboo than in cedar-plantation soil.

Microbes growing under low available C and low pH environment showed increased ratios of cyclopropyl PLFAs to their monoenoic precursors (Ratledge and Wilkinson, 1988). High ratios of cy17:0/16:1v7c and cy19:0/18:1v7c are indicative of physiological and nutritive stress (Morre-Kucera and Dick, 2008). In this study, the ratios of cy17:0/16:1v7c and cy19:0/18:1v7c were highest in the Japanese cedar plantation (Table 5). The low ratios of cy17:0/16:1v7c and cy19:0/18:1v7c indicate that bamboo invasion creates a less stressful, more favorable environment for the soil microbial community. High values of these ratios (both 16:1v7t/ 16:1v7c and cy17:0/16:1v7c) are due to increased C limitations (Moore-Kucera and Dick, 2008; Sampedro et al., 2009). Additionally, the ratio of cy19:0/18:1w7c was found to be negatively associated with pH value (Chang et al., 2011). The decreased ratio of cy19:0/18:1w7c might be a response to the elevated pH caused by bamboo invasion. Dendrograms of soil bacteria and fungi based on PCR-DGGE showed that the greatest differences between microbial communities related to bamboo invasion (Figs. 2 and 3). Using bar-coded pyrosequencing techniques on 16S rRNA gene clone libraries

Fig. 3. Cluster analysis of fungal community structure from different vegetation types measured by UPGMA.

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constructed from this same study site, Lin et al. (2014) found that the soil bacterial communities of the bamboo and transition zone were more similar to each other than to the Japanese plantation communities. These findings support our results. Bamboo exhibits strong allelopathy and can reform soil microbial communities. In addition, bamboo itself also has antibacterial activity (Tanaka et al., 2013), which results in lower soil bacterial biomass and/or activities in bamboo-invaded soils compared to Japanese cedarplantation soil. In this study, the DGGE findings agreed with the PCA of PLFAs, showing the distinct composition of bacterial and fungal communities between Japanese cedar and bamboo-invaded soil. Collecting bamboo shoots disturbs the soil in a process similar to tilling. Tilling can reduce fungal biomass and proliferation (Kabir, 2005). Some studies (Oehl et al., 2003; Alguacil et al., 2008) indicated that intensified agricultural practices such as tilling have a negative impact on fungal biomass and diversity. Our PCR-DGGE findings revealed the existence of different soil fungal communities in different soil plantation types. However, PLFA analysis revealed no significant differences in fungal biomass between plantation soils. Rahman et al. (2008) found an inconsistent relationship between microbial abundance and DNA band count as measured by PLFA and DGGE, respectively. PLFA diversity is used as an indicator of microbial community structure because certain groups of microbes are associated with specific fatty acids. However, coexisting species with similar PLFA profiles may be underrepresented by PLFA measurements of microbial diversity (Hedrick et al., 2000); therefore, information from DGGE profiles supplements PLFA analysis and may be more accurate at the species level (Zaady et al., 2010). 5. Conclusions Bamboo invasion of a Japanese cedar plantation increased the fraction of the labile SOM, thereby leading to an increase in the ratio of Cmic/Corg in bamboo-invaded soil. Similarly, soil cellulase and xylanase activities decreased with invasion, so the bamboo invasion into the cedar plantation may have facilitated the change in microbial biomass and activities by changing soil biochemical properties. In addition to the input of relatively decomposable bamboo litter, frequent human disturbances, such as bamboo shoot harvesting in the bamboo plantation, might also accelerate degradation of SOM in bamboo soil. The proportion of total PLFAs and the fractions attributed to bacteria, G+ and G
bacteria all decreased with invasion. Low ratios of G+/G
in the bamboo soil coincide with the increase in easily decomposable organic matter (Cmic/Corg and Nmic/Ntot) in bamboo-invaded soil. DGGE cluster analysis revealed that changes in both the bacterial and fungal community structure were associated with bamboo invasion. Therefore, the bamboo invasion caused significant changes in soil microbial activities and community structure. Acknowledgment This work was supported by the Taiwan National Science Council, Taiwan (NSC 101-2621-B-001-002-MY3). References Alguacil, M.M., Lumini, E., Roldán, A., Salinas-García, J.R., Bonfante, P., Bianciotto, V., 2008. The Impact of tillage pratices on arbuscular mycorrhizal fungal diviersity in subtropical crpos. Ecol. Appl. 18, 527–536. Amato, M., Ladd, J.N., 1988. Assay for microbial biomass based on ninhydrin-reactive nitrogen in extracts of fumigated soils. Soil Biol. Biochem. 20, 107–114. Bååth, E., Anderson, T.H., 2003. Comparison of soil fungal/bacterial ratios in a pH gradient using physiological and PLFA-based techniques. Soil Biol. Biochem. 35, 955–963.

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