Differential impact on soil microbes of allelopathic compounds released by the invasive Acacia dealbata Link

Soil Biology & Biochemistry 57 (2013) 156e163

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Differential impact on soil microbes of allelopathic compounds released by the invasive Acacia dealbata Link Paula Lorenzo a, *, Carla Sofia Pereira b, Susana Rodríguez-Echeverría a a b

Centre for Functional Ecology, Department of Life Sciences, University of Coimbra, Calçada Martim de Freitas, 3000-455 Coimbra, Portugal IMAR e CMA, Department of Life Sciences, University of Coimbra, 3001-401 Coimbra, Portugal

a r t i c l e i n f o

a b s t r a c t

Article history: Received 28 February 2012 Received in revised form 19 July 2012 Accepted 22 August 2012 Available online 6 September 2012

Acacia dealbata Link, an Australian tree legume, is one of the most invasive species in south-eastern Europe. The invasive success of A. dealbata is partially attributed to its ability to release allelopathic compounds that affect native plant species, but the allelopathic effect on soil microbes has been little explored. Here, we used natural leachates to assess the bioactivity of these allelochemicals on soil microorganisms in native Mediterranean pine and mixed forests. Soil samples were treated either with acacia canopy leachate or the corresponding canopy leachate. Soil microbial communities were analyzed using Biolog EcoplatesÔ and PCReDGGE. Allelochemicals naturally released by A. dealbata clearly modified soil bacterial functional diversity in the pine forest where acacia leachate significantly increased the consumption of carbohydrates and amino acids and reduced the utilization of carboxylic acids. Acacia leachates also lead to a significant reduction in bacterial richness and diversity in the pine forest soil. However, the soil microorganisms of mixed oak forest were insensitive to allelochemical activity. Our results show that the allelopathic effects of A. dealbata on soil microbes depend on ecosystem type and that soil bacteria are more sensitive than soil fungi to the allelochemicals released by A. dealbata. We conclude that the higher sensitivity of pine forest soil microbiota to allelochemicals introduced by A. dealbata can contribute to the process of invasion. Ó 2012 Elsevier Ltd. All rights reserved.

Keywords: Allelopathy Bacteria Fungi Natural leachates Plant invasion Richness and diversity species Soil community structure Soil functional diversity

1. Introduction Exotic plants might release allelochemical compounds that are novel to native species in the new range contributing to its invasive success (Callaway and Ridenour, 2004). This theory, named “novel weapons hypothesis”, was originally proposed for plant species (Callaway and Aschehoug, 2000) but can be also applied to microbial communities (Inderjit and van der Putten, 2010). This theory, based on Rabotnov’s hypothesis (1982), states that allelopathic interactions are involved in the functioning of ecosystems and that there is an adaptation of co-existing plants to the chemical released by other species. Following this hypothesis, exotic plant species can introduce novel chemicals into the non-native range that would have negative impacts on native communities as the exotic and native species have not developed a mutual tolerance in the course of a joint evolutionary process (Rabotnov, 1982; Callaway and Aschehoug, 2000). Although allelopathy has been

* Corresponding author. Tel.: þ351 239 855 238; fax: þ351 239 855 211. E-mail addresses: [email protected] (P. Lorenzo), carlasofiaropereira@ gmail.com (C.S. Pereira), [email protected] (S. Rodríguez-Echeverría). 0038-0717/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.soilbio.2012.08.018

little tested under field conditions (Gómez-Aparicio and Canham, 2008; Peguero et al., in press), there is considerable evidence for allelopathic effects of invaders on native plant communities. For example, chemicals released by some invasive Centaurea species were found to suppress native species and to play a role in structuring native plant communities (Bais et al., 2003; Hierro and Callaway, 2003; Callaway and Ridenour, 2004). The “novel weapons hypothesis” is also strongly supported by comparisons of the allelopathy associated to invasive species in native and introduced ranges (e.g. Thorpe et al., 2009; Kim and Lee, 2011; Inderjit et al., 2011b). Recent reviews also suggest that the progression of invasion is mediated by the interactions between exotic plants and soil communities including the effect and fate of allelochemicals produced by the invader (Inderjit and van der Putten, 2010; Inderjit et al., 2011a). Soil microbiota is an important factor in determining the structure and dynamics of plant communities and can be affected by plant invasions (e.g. Mangla et al., 2008; Lorenzo et al., 2010c; Rodríguez-Echeverría, 2010; Sanon et al., 2009; Elgersma and Ehrenfeld, 2011), but the interaction between new allelochemicals and local soil microbes has started to be explored only recently. Some components of root exudates identified in the

P. Lorenzo et al. / Soil Biology & Biochemistry 57 (2013) 156e163

rhizosphere of Pinus radiata change bacterial communities of pasture soils (Shi et al., 2011). Also, chemical compounds secreted by the invasive Alliaria petiolata inhibit ecto- and arbuscular mycorrhizal fungi in invaded fields (Wolfe et al., 2008; Cantor et al., 2011). The root exudates of yet another invader, Chromolaena odorata, promote the accumulation of local fungal pathogens in its rhizosphere, creating negative feedbacks for native plant species (Mangla et al., 2008). Soil microorganisms can also play a role in the fate of allelochemicals released into the soil (Inderjit, 2005; Inderjit and van der Putten, 2010). In fact, Lankau (2010) and Zhu et al. (2011) have found allelopathic effects in sterilized soils compared to live soils, which could be explained by microbial transformations of the new chemicals into less toxic forms (Inderjit, 2005; Inderjit and van der Putten, 2010). However, it is also possible that native soil microorganisms cannot degrade the novel allelochemicals, which can lead to the accumulation of these compounds up to toxic levels (Inderjit and van der Putten, 2010). Following the Rabotnov’s hypothesis (1982), a recent study has also shown that a soil community can develop resistance over time to glucosinolates released during A. petiolata invasion (Lankau, 2011). Acacia dealbata Link is an aggressive Australian invader in southeast Europe, southern Africa and South America (Lorenzo et al., 2010a; Richardson and Rejmánek, 2011). This legume benefits from disturbance events (Le Maitre et al., 2011), but it is also able to invade relatively intact forest understories, abandoned arable lands and watercourses (Lorenzo, 2010; Lorenzo et al., 2010a). A. dealbata invasive success seems to be related to its phenotypic plasticity, high capacity of resprouting, rapid response to anthropogenic disturbance (Fuentes-Ramírez et al., 2011) and the production of allelopathic compounds that are detrimental for native understory species (Lorenzo et al., 2008, 2010b; 2011; Lorenzo, 2010; Fuentes-Ramírez et al., 2010). In addition, A. dealbata has been shown to alter nutrient pools, soil microbial community structure and the diversity and richness of soil microorganisms (Lorenzo et al., 2010c). Several studies have shown the effect of natural washes of A. dealbata on germination, seedling growth, net photosynthetic rate and respiration of agricultural or native species (Lorenzo et al., 2008, 2010b; 2011) but whether allelochemicals can modify soil microbial communities in the invaded areas remains unknown. The current study focuses on this putative effect of novel allelopathic compounds on soil microbial communities. Our hypothesis was that A. dealbata releases allelochemicals that lead to changes in soil microbial community function and structure and to decreased biodiversity of soil bacteria and fungi. We assessed the allelopathic effect of natural leachates, which is a more realistic approach than extracting secondary compounds from plant material in the laboratory (Lorenzo et al., 2008, 2010b; 2011). These leachates represent the natural concentrations of chemical compounds, a necessary condition to validate allelopathy under field conditions (Cantor et al., 2011). A. dealbata natural leachates were applied to soil collected in two native forests, an open maritime pine forest and a mixed oak forest and the functional and genetic diversity of soil microbial communities were subsequently analyzed using Biolog EcoplatesÔ and PCReDGGE. 2. Material and methods

area: a mixed typical deciduous Mediterranean forest dominated by Quercus suber L. and Quercus canariensis Willd with a rich understorey of cistaceae and leguminous shrubs and annual forbs, grasses and legumes; an open forest of Pinus pinaster Aiton with an understorey dominated by grasses and some Ulex and Cytisus shrubs, and an area covered by A. dealbata without understory vegetation. 2.2. Collection of natural leachates Five plastic trays (43  31  9 cm) covered with a 1-cm mesh plastic net were placed underneath the canopy of dominant species in each vegetation patch. To mimic natural conditions, litter removed from an area equivalent to the tray surface was placed on top of the mesh. Leachate collection was done every two days during the rainy season in February 2011. Leachates from each vegetation type were transported to the laboratory, passed through filter paper and kept frozen in 300 mL aliquots at 20  C pending use (Lorenzo et al., 2010b, 2011). The pH was 6.25, 5.95 and 5.90 for acacia, pine and mixed forest leachates respectively. Collection date was chosen to coincide with the flowering stage of A. dealbata, since this is the peak of production and release of allelochemicals for this species (Lorenzo et al., 2010b, 2011). 2.3. Soil sampling and analysis of soil chemical properties Soil collection was conducted in the pine and mixed oak forests in March 2011. Six samples of the top soil layer (0.25 m2 and 10 cm deep) were randomly collected in each studied forest and transported immediately to the laboratory. Soil samples from the same origin were pooled and little stones and roots were manually removed. One part of the fresh soil was immediately used for the soil experimental set-up and other part was air-dried at room temperature for chemical analysis. Soil analysis for pH, organic matter, total nitrogen, available phosphorus and potassium were performed following standard protocols after soils were sieved through a 2-mm sieve as described by Rodríguez-Echeverría et al. (2009). The mixed-forest soil was characterized by higher amounts of organic matter, total nitrogen and available phosphorous than the pine-forest soil, which presented higher values of pH and available potassium (Table 1). 2.4. Experimental set-up Twelve pots were filled with 100 mL of each fresh soil type and placed in a growth chamber at 16 h light/8 h night and average temperature of 29  C. Both soil types, from pine forest and mixed forest, were watered with 10 mL of either acacia leachate or with each own leachate twice a week. When necessary, additional 10 mL of distilled water were added to prevent desiccation. After 45 days, all pots were removed from the growth chamber, half of each pot was stored at 4  C for two days before conducting the functional diversity analysis and the other half was frozen at 20  C to be used for DNA extraction and subsequent genetic analysis.

Table 1 Chemical characteristics of the two soils used in this study.

2.1. Study area Soil sampling and collection of natural canopy leachates were conducted in a seminatural area in Coimbra, Portugal (40120 3200 N 8 240 0400 W) characterized by a Mediterranean climate and Lithosoils. There are three well-defined patches of vegetation in this

157

pH O.M. (%) N total (%) P2O5 (mg/kg) K2O (mg/kg)

Pine soil

Mixed soil

5.9 6.73 0.199 4 137

5.1 8.04 0.243 12 105

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2.5. Soil microbial functional diversity Biolog EcoplatesÔ (Biolog inc., Hayward, CA, USA) were used to study the metabolic profiles of the microbial communities (Garland and Mills, 1991). The six original samples were grouped in pairs to have a final number of three replicates per soil and treatment. Briefly, 2 g of fresh soil was added to 20 mL of sterile 0.2% sodium pyrophosphate solution into a test tube and shaken for 10 s to achieve a 101 dilution. A 102 dilution was then prepared adding more sodium pyrophosphate solution (18 mL). The tube was mixed and 140 mL aliquots of this solution were placed in wells of the Biolog EcoplateÔ. The inoculated plates (one for each subsample) were incubated at 22  C in the dark, for 15 days and the absorbance of plates was measured twice a day with a microplate reader (Tecan, Sunrise remote, Austria) at a wavelength of 590 nm to evaluate the color development over the incubation period. Each plate contains 96 wells with 31 different carbon sources and water as control (each substrate in three wells; Table S1). Metabolism of the substrate of each well by soil microorganisms resulted in the reduction of tetrazolium, which changed the color of the substrate from colorless to purple. Optical density readings were blanked against the initial color intensity (ODb) and then corrected against the control well (ODct) on each plate (ODb  ODct) before data analysis. Negative values were set to zero. The average well color development (AWCD) was calculated for each plate in each reading time. These AWCD were used to calculate kinetic parameters for each substrate by the non-linear regression model: AWCD ¼ k/ (1 þ exp(r*(Time-s))), where k is the threshold or asymptote (the maximum absorbance in a well), r is the maximum rate of color development (reflected by the slope of the curve), and s is the lag time (time needed to the beginning of color development) (Stefanowicz, 2006). U ManneWhitney tests (using SPSS v.19.0 for Windows) were used to determine differences between kinetic parameters of the soils treated with the two leachate types within each vegetation type. g-Hydroxybutyric acid and Glucose-1phosphate substrates were excluded from all analyzes because curves did not reach the asymptote phase. To analyze the functional profile of soils from test treatments a principal components analysis (PCA, using Canoco for Windows 4.5) was performed using the AWCDt50 (a mid to late reading point of the exponential phase of color development) previously normalized (dividing each well color development with the AWCD of the respective reading time) to reduce the impact of the differences in inoculum densities of the Biolog plates (Garland, 1996).

were carried out in a final volume of 25 mL containing 2.5 mL of buffer (160 mM (NH4)2SO4, 670 mM TrisHCL pH8.8, 0.1% Tween20, 25 mM MgCl2) (BIORON, Germany), 400 nM of each primer, 200 mM dNTPs, 0.5 U of DFS-Taq polymerase (BIORON, Germany), and 1 mL of template DNA. The polymerase chain reaction (PCR) conditions for bacteria were: an initial denaturing step at 94  C for 5 min followed by 30 cycles of 30 s at 94  C, 30 s at 55  C and 30 s at 72  C, followed by a final extension step at 72  C for 30 min. The PCR conditions for fungi were: an initial denaturing step at 94  C for 3 min followed by 35 cycles consisting of 1 min at 94  C, 1 min at 50  C and 1 min at 72  C, followed by a final extension step at 72  C for 30 min. Aliquots (5 mL) of each PCR mixture were examined by electrophoresis in an agarose gel (1% w/v) stained with GelRedÔ to check fragment size and integrity. All PCRs were performed using a GeneAmp 9700 (Applied Biosystems, Perkin Elmer, CA, USA). Denaturant gradient gel electrophoresis (DGGE) was performed with a DGGE-2001 system from CBS Scientific (CA, USA). A 10 mL aliquot of each bacteria PCR product and 15 mL aliquot of each fungi PCR product was used for DGGE analysis. Gels contained 6% (w/v) acrylamide for bacteria PCR products and 8% (w/v) acrylamide for fungi PCR products. The linear gradient used was from 40 to 75% denaturant for bacteria and from 20 to 50% for fungi, while 100% denaturing acrylamide was defined as containing 7 M urea and 40% (v/v) formamide. Gels (22  17 cm) were run in 21 L 1 TAE buffer at 20 V for 15 min, followed by 16 h at 65 V and maintained at a constant temperature of 60  C. Gels were stained for 20 min in 1.0 GelStarÒ and destained for 30 min in distilled water prior to visualization. All gels were digitalized using BioRad ChemiDoc XRS. Digital images of the gels were analyzed using GelCompar II (Applied Maths, Belgium). Detrended Correspondence analysis (DCA) was performed to analyze the obtained DGGE fingerprintings using CANOCO version 4.5 (Microcomputer Power, Ithaca, NY, USA). Richness, defined as number of species, was calculated as the total number of bands per sample. To calculate diversity, defined as number of different species and their relative frequency, gel bands were classified according to their intensity in four categories. Diversity was calculated using a modification of the Shannon index P [(ni/N)Ln(ni/N)] where ni (Shannon and Weaver, 1949), H0 ¼  had one of four possible values (1e4) depending on band intensity. U ManneWhitney analyses (SPSS v.19.0 for Windows) were used to check the differences in bacterial and fungal richness and diversity between acacia and pine or acacia and mixed leachates within each forest. The level of significance for all statistical analyses was fixed at P  0.05.

2.6. Genetic diversity of microbial communities 3. Results Soil DNA extractions were performed from 0.25 g of soil using a PowerSoilÔ DNA Isolation Kit (MO BIO Laboratories, Inc., CA). DNA extracted from soil samples was amplified using eubacteriaspecific primers for the 16S rRNA gene (Muyzer et al., 1993) and fungal ITS1F-GC and ITS4 primers targeted at the fungal 18S rRNA gene (White et al., 1990; Gardes and Bruns, 1993). All reactions

3.1. Soil microbial functional diversity Leachate type had a significant effect on the kinetic parameters of consumption of carbon substrates analyzed either as carbon source type or separately in both studied soils.

Table 2 Values of kinetic parameters from AWDC curves for functional group of carbon sources in the pine soil. Kinetic parameter

Leachate type

Carbohydrates

k

Pine Acacia Pine Acacia Pine Acacia

1.64 1.92 0.04 0.07 186 142

r s

     

0.03 0.19* 0.002 0.03 7.11 16.70*

Carboxylic acids 1.60 1.36 0.05 0.07 172 141

     

0.13 0.05* 0.002 0.003* 2.70 4.54*

Amino acids 1.66 1.92 0.04 0.06 183 162

     

0.08 0.05* 0.005 0.003 2.56 17.30

Miscellaneous 1.08 0.99 0.05 0.19 138 107

     

0.02 0.06 0.01 0.10 29.05 28.65

Polymers 1.64 1.97 0.04 0.04 178 192

     

0.07 0.20 0.002 0.01 1.10 9.43

Amines 0.81 1.17 0.02 0.11 221 152

     

0.08 0.36 0.004 0.02* 5.61 30.07*

The means values and SE are shown, n ¼ 3. Asterisks indicate statistical significance between leachate types in each kinetic parameter (U ManneWhitney test, P ¼ 0.05).

P. Lorenzo et al. / Soil Biology & Biochemistry 57 (2013) 156e163

159

Table 3 Values of kinetic parameters from AWDC curves for functional group of carbon sources in the mixed soil. Kinetic parameter

Leachate type

Carbohydrates

k

Mixed Acacia Mixed Acacia Mixed Acacia

1.94 1.80 0.05 0.06 171 164

r s

     

0.18 0.07 0.01 0.002 8.30 5.73

Carboxylic acids 1.44 1.28 0.06 0.07 144 146

     

Amino acids

0.10 0.11 0.003 0.005* 7.97 5.17

     

1.75 1.63 0.05 0.06 159 164

0.05 0.15 0.01 0.01 7.34 10.46

Miscellaneous 0.97 0.97 0.11 0.09 65 80

     

Polymers

0.11 0.11 0.03 0.02 1.13 7.94

2.19 1.56 0.04 0.04 180 185

     

0.17 0.08* 0.003 0.002 31.48 4.90

Amines 0.75 1.08 0.04 0.06 144 170

     

0.13 0.09 0.01 0.02 21.00 11.59

The means values and SE are shown. n ¼ 3. Asterisks indicate statistical significance between leachate types in each kinetic parameter (U ManneWhitney test, P ¼ 0.05).

1.5

(Itaconic acid, 0.850) and one carbohydrate (a-D-Lactose, 0.876). Meanwhile, two amino acids (L-Arginine, 0.850; LAsparagine, 0.963), one polymer (Glycogen, 0.972) and one carboxylic acid (a-Ketobutyric acid, 0.852) had the greatest

Pine forest

A

PC2 (23.7%)

AL 6-4

AL 2-5

PL 1-4 PL 3-5 PL 2-6

-1.0

AL 1-3

1.0

-1.0

PC1 (54.0%)

1.5

Mixed forest

B

ML 4-2

PC2 (33.6%)

AL 2-3

AL 6-5

AL 1-4

-0.8

The pine forest soil watered with acacia leachate showed a significantly higher consumption (k) of carbohydrates and amino acids and a significantly lower consumption of carboxylic acids (Table 2). Acacia leachate also significantly increased the rate of consumption (r) of carboxylic acids and amines (Table 2) and reduced lag time (s) for the consumption of carbohydrates, carboxylic acids and amines in pine forest soil (Table 2). Analyzing each carbon source individually, there was a significant lower consumption (k) of D-Galacturonic acid, L-Phenylalanine and D,L-aGlycerol phosphate in the pine forest soil watered with acacia leachate (Fig. S1A). Acacia leachate also led to changes in the rate of consumption (r) of D-Xylose, D-Cellobiose, Pyruvic acid methyl ester, Tween 80 and Putrescine (Fig. S1B). A shorter lag phase (s) in the consumption of D-Xylose, 4-Hydroxy benzoic acid, L-Arginine, LAsparagine, L-Serine, and Putrescine substrates was observed in the pine forest soil watered with acacia leachate, while this treatment lead to a significantly longer lag phase for a-Ketobutyric acid and Tween 40 carbon sources (Fig. S1C). The soil of the studied mixed oak forest watered with acacia leachate showed a significantly higher consumption of polymers and a lower consumption rate of carboxylic acids than mixed-forest soils watered with their own leachate (Table 3). The kinetic parameters of the other functional groups of carbon sources were not affected by acacia leachate. Acacia leachate led to a significant decrease in the consumption (k) of 4-Hydroxy benzoic acid, LArginine, L-Asparagine, L-Threonine and Glycogen (Fig. S2A). The consumption rate (r) was significantly increased by acacia leachate for b-Methyl-D-glucoside, D-Galacturonic acid, 2-Hydroxy benzoic acid and Glycyl-L-glutamic acid but was reduced for Tween 40 (Fig. S2B). The lag phase was significantly increased by acacia leachate only for N-Aceryl-D-glucosamine and Tween 40 (Fig. S2C). AWCD data at t50 for the two soils types were used for PCA analysis and the resulting plots are shown in Fig. 1. The first and second axes explained 77.7% of the variation of the utilization of sole carbon sources data for the pine forest soil microbial communities (PC1 and PC2). The main factors contributing to the PC1 axis were three carboxylic acids (a-Ketobutyric acid, 0.942; D-Glucosaminic acid, 0.914; Itaconic acid, 0.886), two polymers (Tween 80, 0.882; Glycogen, 0.890), one amino acid (L-Phenylalanine, 0.897), one miscellaneous (Pyruvic acid methyl ester, 0.939) and one amine (Putrescine, 0.942). The PC2 axis was mainly determined by one carbohydrate (N-Aceryl-D-glucosamine, 0.852), one amino acid (L-Asparagine, 0.872) and one amine (Phenylethyl-amine, 0.958) (Fig. 1A). Pine soil samples treated with acacia leachate or with pine leachate were clearly separated along the first axis of the PCA (Fig. 1A). The microbial functional diversity of soil samples watered with its own leachate was more homogenous and mainly related to the consumption of a-Ketobutyric acid, L-Threonine, Glycogen, D-Glucosaminic acid, i-Erythriol substrates. Acacia leachate led to a more heterogenous utilization of carbon sources (Fig. 1A). The two first axes of the PCA for the mixed-forest soil explained 68.9% of the variance. The PC1 axis was strongly related to the utilization of one polymer (Tween 40, 0.924), one carboxylic acid

ML 6-1 ML 3-5

-1.0

PC1 (35.3%)

1.5

Fig. 1. Principal component analysis of carbon substrate utilization patterns obtained with Biolog EcoPlates for pine (A) and mixed forest soils (B). All values are based on AWCD data at t50 incubation (n ¼ 3) PL: pine leachate; ML: mixed leachate; AL: acacia leachate. Numbers indicates coupled replicates. Abbreviations of carbon sources are listed in Table S1.

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influence on the PC2 axis. The PCA plot of mixed-forest soil did not find a clear difference between soil samples treated with acacia or mixed leachates (Fig. 1B). Two soil samples watered with its own leachate clustered together and apart from the remaining samples and were related to the consumption of i-Erythriol, 2-Hydroxy benzoic acid and Phenylethyl-amine. The remaining soil samples were scattered along the PCA plot (Fig. 1B). 3.2. Soil microbial genetic diversity Watering with acacia or native leachates did not have a clear effect on the community structure of soil bacterial or fungal communities from pine and mixed forests (Figs. 2 and 3). Within each ecosystem, the community structure of soil bacteria and fungi were very heterogenous and independent of leachate type. However, significant differences were observed in species richness and diversity of bacteria in the pine forest soil watered with its own leachate or with acacia leachate (P  0.05) (Fig. 4A). Species diversity and number of bacterial species in the soil watered with acacia leachate were 1.1 and 1.2 times lower respectively than in control soil (Fig. 4A). These parameters were not affected by leachate type in the soil from mixed forest (P > 0.05) (Fig. 4B). No significant differences in fungal diversity and species richness for pine or mixed-forest soils were found (P > 0.05) (Fig. 5A,B). 4. Discussion The results obtained partially support our original hypothesis that the allelopathic compounds of A. dealbata were responsible for changes in functional and genetic diversity of soil microbial communities. We have found different patterns for both sole and groups of carbon source utilization in pine and mixed forest soils. More effects of acacia leachates were observed in the microbial communities of the pine forest than in those of the mixed forest. Watering with acacia leachate leads to a different functional microbial composition in the pine forest soil, with an increase of bacterial groups consuming carbohydrates, amino acids and amines and a reduction of bacteria using carboxylic acids. Accordingly, the PCA analysis showed a clear separation between pine-forest soils watered with pine or acacia leachate. Significant differences in the mixed forest soil were observed only in the consumption of polymers. Since pH and ionic concentration were not toxic for either

acacia or native leachates (Abrol et al., 1988; Carballeira and Reigosa, 1999; Lorenzo et al., 2008, 2010b, 2011) the observed effects are attributed to the release of chemical compounds by A. dealbata. The observed changes on the physiological profile are caused by culturable bacteria able to develop rapidly in high-nutrient conditions in the Biolog EcoplatesÔ (Stefanowicz, 2006). Therefore, the decrease in the consumption of some carbon sources indicates a decline in bacterial species that are using these substrates and that may be naïve to the new allelochemicals (Inderjit and van der Putten, 2010; Wolfe and Klironomos, 2005). DGGE analyses confirmed these results showing that allelochemicals released by A. dealbata reduced the number and diversity of bacterial species in the pine forest soil. Following the novel weapon hypothesis (Callaway and Aschehoug, 2000), acacia compounds could have a phytotoxic effect on soil microbes in the invaded range because they have not experienced these chemicals in a common coevolutionary process (Rabotnov, 1982; Inderjit and van der Putten, 2010). Nevertheless, acacia leachate also lead to a significant reduction of the lag phase for the consumption of several carbon sources in the pine forest soil, which indicates that acacia leachate might be favoring some groups of fast-growing bacteria. Marchante et al. (2008) have shown that the catabolic diversity of soil microbial communities is different in native soils and soils invaded for a long time by Acacia longifolia. A shift in soil functional microbe diversity or community composition may influence nutrient cycling and ecosystem processes and be related with the invasion by exotic plant species (Wolfe and Klironomos, 2005; Rout and Callaway, 2009). Plant invasions usually lead to changes in nutrient pools in the invaded soils (Liao et al., 2008), which can influence the soil microbial communities (Liao et al., 2008; Rout and Callaway, 2009). This study shows that invasive plants could alter the soil microbial community through allelopathic mechanisms and these changes might also drive changes in soil nutrient fluxes. This suggests that soil microbes might not be just passengers in the processes of nutrient changes associated with the invasion (Rout and Callaway, 2009), but instead might be an active part of the modifications that allow the colonization by exotic species. On the other hand, in spite of finding differences in the functional profile of soil bacteria from pine forests due to acacia leachate, the DGGE analysis did not detect changes in the community structure of soil bacteria. This suggests that a change in

Fig. 2. DCA plots based on the DGGE data of the bacterial (A) and fungal (B) communities of pine forest soil, watered with its own canopy leachate (PL) or with acacia canopy leachate (AL). Axis 1 and 2 explain 60.1% and 42.1% of the variance for bacteria and fungi respectively.

P. Lorenzo et al. / Soil Biology & Biochemistry 57 (2013) 156e163

161

Fig. 3. DCA plots based on the DGGE data of the bacterial (A) and fungal (B) communities of mixed forest soil, watered with its own canopy leachate (ML) or with acacia canopy leachate (AL). Axis 1 and 2 explain 51.9% and 67.9% of the variance for bacteria and fungi respectively.

functional diversity does not necessarily imply that the microbial community structure has changed (Orwin et al., 2006), or that more time would be necessary to see these changes. This result might also be attributed to the limitations of the DGGE technique (van Elsas and Boersma, 2011). Although DGGE is a widely accepted methodology to study and compare microbial communities (Wallis et al., 2010; Ben-David et al., 2011), different sequences might comigrate to the same position in the gel thus limiting the conclusions that can be drawn from this analysis (Jackson et al., 2000). Samples with dissimilar DGGE banding patterns can be considered different but this technique might fail in detecting variation between closely related communities. Also, co-migration of bands

Pine forest

A

with different sequence might lead to an underestimation of the community richness and diversity. Although a previous study has shown that the invasion by A. dealbata can change the soil fungal communities of shrublands and grasslands (Lorenzo et al., 2010c), we could not detect changes in the community of soil fungi in any of the two studied ecosystems. Soil fungal communities in forests might have a higher stability than soil bacterial communities (Agnelli et al., 2004). However, fungi and other slowly growing organisms are difficult to detect using the Biolog EcoplatesÔ technique as discussed by Zhang et al. (2009). This result might also be due to the short time used for this experiment, which might be not enough to affect soil

B

Mixed forest

a

Species diversity of bacteria (H’)

3.5

a 3.4

b

3.3 3.2 3.1 3.0

35

b

30

Species richnes of bacteria

a

a

25 20 15 10 5 0

Pine leachate

Acacia leachate

Mixed leachate

Acacia leachate

Fig. 4. Species richness and diversity of soil bacteria for pine (A) and mixed forests (B). Bars are mean  SE, n ¼ 3. Different letters indicate significant differences between leachate types after U ManneWhitney test (P  0.05).

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P. Lorenzo et al. / Soil Biology & Biochemistry 57 (2013) 156e163

A

Pine forest

Species diversity of fungi (H’)

3.5

B

Mixed forest

a

3.0

a

2.5 2.0 1.5 1.0 0.5 0.0

Species richnes of fungi

20

a a

15 10 5 0

Pine leachate

Acacia leachate

Mixed leachate

Acacia leachate

Fig. 5. Species richness and diversity of soil fungi for pine (A) and mixed forests (B). Bars are means  SE, n ¼ 3. Significant differences (P  0.05) between leachate types using U ManneWhitney test were not found.

fungi that are slower growers than soil bacteria and metabolize more recalcitrant substrates (Paterson et al., 2008). However, soil fungi communities of forests An alternative explanation is that soil fungi in the studied ecosystems are less affected by allelochemicals than by other mechanisms such as changes in litter deposition or micro-environmental conditions introduced by A. dealbata. Although mycorrhizal fungi can be affected by flavonoid and glucosinolates compounds released by the aggressive invader A. petiolata in North America (Callaway et al., 2008; Wolfe and Klironomos, 2005), the general allelopathic effects of chemical compounds on other soil fungal groups, such as the studied basidiomycota and ascomycota, are poorly understood. The fate and effectiveness of allelopathic compounds in soil are highly dependent on biotic and abiotic environmental conditions and evolutionary history as discussed by Inderjit et al. (2011a). Lankau (2010) and Zhu et al. (2011) showed that soil biota might be involved in the deactivation of allelochemicals released by the invasive A. petiolata and Eupatorium adenophorum respectively. Another factor indirectly influencing allelochemicals fate in soil can be related to the quality and quantity of soil organic matter. The studied mixed oak forest had higher understory plant diversity and soil nutrient content than the pine forest. The high diversity of understory plants is associated with annual inputs of different litter types that could support a higher diversity of soil microorganisms, thereby increasing the type of interactions with allelochemicals and increasing the probability of being degraded or transformed into not toxic forms. This might be a plausible explanation for the lack of effect of acacia leachates in the soil microbiota of native mixed forests, and could be part of the observed biotic resistance of well-preserved mixed oak forest to the invasion by A. dealbata (Lorenzo, 2010). 5. Conclusions The use of natural leachates allows us to suggest that chemicals released in field conditions by A. dealbata can affect the soil

microbiota of native ecosystems. Our results show that soil bacteria are more sensitive than soil fungi to these allelochemicals, and that the effect depends on the ecosystems studied. Allelopathy was more important for the bacterial community in the pine forest where soil functional diversity and bacteria richness and diversity were affected. On the contrary, soil microorganisms of the mixed Mediterranean oak forest were quite insensitive to the released chemicals. We conclude that the soil microbiota of pine forests can be affected by allelochemicals introduced by A. dealbata contributing to the process of invasion. Future work is needed to relate these changes in microbial functional profiles to ecosystem processes. Acknowledgments We would like to thank to Dr. José Paulo Sousa for the use of his laboratory equipment and Dr. Tiago Natal da Luz for his valuable help in the statistical procedures. Paula Lorenzo was supported by a posdoctoral fellowship from Fundación Juana de Vega. The work was supported by the project MUTUALNET (PTDC/BIA-BEC/103507/ 2008), funded by the Portuguese Foundation for Science and Technology. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.soilbio.2012.08. 018. References Abrol, I.P., Yadav, J.S.P., Massoud, F.I., 1988. Salt-affected Soils and Their Management. In: FAO Soils Bulletin, vol. 39. Food and Agriculture Organization of the United Nations, Rome. Agnelli, A., Ascher, J., Corti, G., Ceccherini, M.T., Nannipieri, P., Pietramellara, G., 2004. Distribution of microbial communities in a forest soil profile investigated by microbial biomass, soil respiration and DGGE of total and extracellular DNA. Soil Biology & Biochemistry 36, 859e868.

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