Striking Alterations in the Soil Bacterial Community Structure And

Soil Biology & Biochemistry 109 (2017) 176e187

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Soil Biology & Biochemistry

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Striking alterations in the soil bacterial community structure and functioning of the biological N cycle induced by Pennisetum setaceum invasion in a semiarid environment

* G. Rodríguez-Caballero a, F. Caravaca a, , M.M. Alguacil a, M. Fernandez-L opez b, A.J. Fernandez-Gonz alez b,A.Roldan a a CSIC-Centro de Edafología y Biología Aplicada del Segura, Department of Soil and Water Conservation, P.O. Box 164, Campus de Espinardo, 30100 Murcia, Spain b CSIC- Estacion Experimental del Zaidín, Soil Microbiology and Symbiotic Systems Department, Profesor Albareda, 1, 18008 Granada, Spain article info abstract

Article history: The objective of this study was to determine whether native and invasive plants harbor different bac- Received 24 May 2016 terial communities in their rhizospheres and whether there are bacterial indicator species associated Received in revised form with the invasive rhizosphere. Additionally, physico-chemical, biochemical, and biological properties 24 December 2016 have been determined in the native and invasive rhizospheres in order to ascertain the relationships Accepted 19 February 2017 between these soil properties and the rhizosphere bacterial communities. We carried out a study in five independent locations under Mediterranean semiarid conditions, where the native Hyparrhenia hirta is being displaced by Pennisetum setaceum. Partial 16S rRNA genes of the rhizosphere bacterial commu- Keywords: fi Invasive plant nities were ampli ed and 454-pyrosequenced. Principal coordinate analysis revealed differences in the Microbial activity composition and structure of the rhizosphere bacterial communities between native and invasive plants, Pyrosequencing the values of the richness index being higher in the invasive microbial community. Rhizosphere mi- Rhizosphere bacterial community crobial community structure was also influenced by invaded location. The indicator species analysis Semiarid environment showed a higher number of indicators for the invasive community at all the taxonomic levels studied, the genus Ohtaekwangia being the most abundant indicator. As shown by canonical correspondence analysis, the protease and dehydrogenase activities and soil respiration were related to the rhizosphere bacterial community of invasive plant. However, only protease activity was significantly affected by the plant type, being higher in the invasive plant rhizosphere. Our results show that P. setaceum invasion has produced an intense interaction with the soil bacterial community, shifting its structure, composition, and protease activity related to N cycling, which may be altering the function of the invaded ecosystem. © 2017 Elsevier Ltd. All rights reserved.

1. Introduction the oceans. The ecosystem is altered not only by direct competition of exotic with indigenous species but also by modification of the Invasive alien species are non-native organisms which are structure and functioning through different processes such as introduced by human action outside their natural range and are productivity, decomposition, hydrology, geomorphology, fire re- able to establish and spread, causing ecological and economic gimes, and nutrient cycling (Vitousek et al., 1997; Kourtev et al., damage (Sundseth, 2014). They have been included among the five 2002; Levine et al., 2003; Vila and Ibanez, 2011). This leads to main causes of biodiversity loss by the Secretariat of the biodiversity losses, which have a negative repercussion on the Convention on Biological Diversity (2014), along with direct ecosystem services, economy, human health and well-being (Diaz habitat destruction and fragmentation; overexploitation of bio- et al., 2006). logical resources; pollution; and climate change and acidification of Plant invasions have been a target of research for decades and have been addressed by different approaches, including evolutionary, biogeographical, and ecophysiological analysis, among others (Gordon, 1998). In order to identify plant characteristics * Corresponding author. which serve as predictors for invasiveness and to find and optimize E-mail address: [email protected] (F. Caravaca). http://dx.doi.org/10.1016/j.soilbio.2017.02.012 0038-0717/© 2017 Elsevier Ltd. All rights reserved. G. Rodríguez-Caballero et al. / Soil Biology & Biochemistry 109 (2017) 176e187 177 solutions, various hypotheses have been postulated to explain the Gomez et al., 2003; Devesa Alcaraz and Arnelas, 2006) where it success of invasion (Hierro et al., 2005), and several functional traits has escaped into the wild and is threatening the native flora. Pen- of successful alien plants have been described (van Kleunen et al., nisetum setaceum exhibits a certain preference for anthropogenic 2015). On the other hand, it has been demonstrated that invasive and disturbed environments like roadsides. No previous studies on plants can exert profound impacts on invaded ecosystems altering the P. setaceum rhizosphere microbiota have been performed to soil physical-chemical (Kourtev et al., 1998), chemical (Dassonville date in spite of the relevance of this information to a better un- et al., 2008; Chacon et al., 2009), physical (Eviner and Chapin, 2002) derstanding of the ecology of this plant and its colonization and biological properties (Souza-Alonso et al., 2014). Moreover, mechanisms. Likewise, P. setaceum rhizosphere metagenomics several studies have been addressed to know how the rhizosphere could be employed to identify the most susceptible habitats and to microbiota is related to plant invasiveness since that the plant- design effective control strategies which take into account all the microbe interactions can be decisive for the successful establish- factors involved in its invasive behavior. ment of a plant community in a given ecosystem (Kourtev et al., We proceeded from the hypothesis that the invasive plant may 2003; Batten et al., 2007; Souza-Alonso et al., 2015; Medina-Villar selectively alter soil biota, independently of the characteristics of et al., 2016). The interactions established between the invasive invaded site, harboring in its rhizosphere a microbial community plants and soil native biota can lead to positive feedback processes endowed with an advantageous functional capacity with regard to that reinforce the invasion and limit the resistance and resilience to nutrients cycling in comparison to that of native rhizosphere. the invasion of the affected ecosystems. In relation to this, van der Therefore, this study aimed to assess the differences between the Putten et al. (2007) reviewed the ecological interactions between rhizosphere bacterial communities of the exotic P. setaceum and the invasive plants and pathogens, symbionts, or saprophytic soil mi- native H. hirta, formerly predominant but now displaced by the crobes, pointing out the importance of these relationships in invader, in five independent locations exposed to Mediterranean environmental function and sustainability. semiarid conditions. Additionally, measurements of soil chemical It has been documented that one of the potential strategies that and physico-chemical properties and soil enzymatic activities have invasive plants employ to ensure success is the alteration of C and N been included in this research, to analyze the possible changes cycles in different ways, such as modification of the soil microbial driven by the invasive plant and to relate them to the composition community structure (Ehrenfeld, 2003; Liao et al., 2008; and structure of the rhizosphere bacterial communities. Dassonville et al., 2011) or increase in the abundance of bacterial populations responsible for N cycling (Rodrigues et al., 2015; 2. Materials and methods McLeod et al., 2016). The effect of plant invasion on soil microbial community functions has been previously evaluated measuring 2.1. Plant species, study sites and sampling microbial enzymatic activities responsible for soil organic matter decomposition and nutrient cycling (Allison et al., 2006; Souza- Pennisetum setaceum is a perennial bunchgrass with C4 meta- Alonso et al., 2014). The assessment of the microbial function of bolism that can perpetuate itself through apomictic reproduction. invaded ecosystems can contribute to a greater understanding of Its seeds, which are produced in abundance, are wind dispersed the mechanisms of invasion as well as to a better estimation of its (Rahlao et al., 2014). It is drought tolerant and exhibits a wide impact on environmental function. phenotypic and reproductive plasticity (Williams et al., 1995). In Previously, the effects of exotic invasive plants on the soil mi- Southeast Spain, it is usually used as an ornamental plant in public crobial community structure have been assessed by measuring gardens and roundabouts, so it can easily naturalize in the sur- phospholipid fatty acids (Kourtev et al., 2003; Batten et al., 2007). In rounding ruderal habitats - where it competes with the native the last few years, the development of high throughput sequencing population of plants. One of these co-occurring natives is Hypar- methods for metagenomics has provided the tools suitable for the rhenia hirta, a C4 perennial bunchgrass very resistant to drought study of soil microbial communities (Coats and Rumpho, 2014). periods and native to the Mediterranean region (Clayton, 1969). It Some of these molecular methodologies have implemented to forms dense stands in disturbed areas such as uncultivated lands identify how exotic invasive plants modify soil microbiota and how and roadsides, protecting the soil and stabilizing eroded places. The these microorganisms enhance the invader's superiority (Coats reproduction strategies for this plant are very similar to that of et al., 2014; Slabbert et al., 2014). However, to the best of our P. setaceum. knowledge, the use of these approaches to evaluate invaded We selected five different locations highly invaded by the Mediterranean semiarid ecosystems has only been reported in a invasive Pennisetum setaceum (about 70%) for less than 10 years, study simulating the process of exotic plant invasion in an experi- where the dominant native species was Hyparrhenia hirta and now mental semiarid grassland (Carey et al., 2015). The climate in these forming mixed stands with both plant species (Table 1). All the areas is characterized by low rainfall rates with frequent drought locations were subjected to a Mediterranean semiarid climate periods, which lead to severe erosive processes and nutrient scar- (mean annual temperature around 18 C and average annual rain- city. Such restrictive circumstances limit the formation of a vege- fall around 265 mm) but with different edaphic characteristics. The tation cover (Caravaca et al., 2003) and result in a fragile ecosystem soils from the locations “La Alcayna” (A), “El Tiro” (T) and “Espi- which becomes especially exposed to the inherent risks of global nardo” (E) are shallow anthropogenic soils taken from roadsides. change. However, this harsh environment is suitable for the natu- These soils are difficult to classify because they were highly ralization of Pennisetum setaceum (Forssk.) Chiov, a C4 perennial disturbed during road construction as well as they occasionally species of the Poaceae native to Northeast Africa and with rapid receive sediments dragged by the rains. Soil from the watercourse growth and abundant seed production (Milton, 2004; Rahlao et al., “Rambla de Corvera” (C) is a Calcaric Regosol (IUSS Working Group 2014). Owing to its ornamental potential, it has been introduced WRB, 2015) developed on unconsolidated materials with a weakly and become invasive in USA, Australia, South Africa, and Italy developed mineral A horizon, whereas the soil from Santa Pola (S) (Milton, 2004; European and Mediterranean Plant Protection taken in a coastal plain is a Gleyic Solonchak (IUSS Working Group Organization (EPPO), 2009). In Spain, it is especially aggressive in WRB, 2015) predominantly sandy. In these habitats, the vegetation the Canary Islands, where it is present in protected areas and is is mainly formed by ruderal herbaceous plants. At each location, replacing endemic species (Gonzalez-Rodriguez et al., 2010), but it three (2 m by 2 m) sampling plots where both species grow and can also be found in the South of the Iberian Peninsula (Sanchez separated by 10 m were established. Within each plot, we collected 178 G. Rodríguez-Caballero et al. / Soil Biology & Biochemistry 109 (2017) 176e187 one soil sample from the rhizosphere of either P. setaceum or employing the U341F and U519R primers (Baker et al., 2003). These H. hirta plants separated from each other by about 1 m to avoid the primers also contain the pyrosequencing A/B adapters and the 8 bp influence among both rhizospheres. Thus, three replicates per plant multiplex identifiers (barcodes) (Binladen et al., 2007). species were established in each location (30 samples in total). Each PCR mixture consisted of a total volume of 25 ml, including Sampling was performed during the last week of July 2014. The 25 pmol of each primer, 1.8 mM MgCl2, 0.2 mM dNTPs, 1 X the plants, including the root system, were harvested together with the corresponding Taq buffer, 1 U of TaqMaster (5 Prime Inc., Gai- soil that adhered firmly to the roots (rhizospheric soil). One portion thersburg, MD, USA), and 10 ng of the DNA template. The PCR of the rhizospheric soil was stored in polyethylene bags at 20 C, program consisted of an initial denaturation step at 94 C for 4 min, for DNA sequencing, and the remaining soil was divided into two 25 cycles of denaturation at 94 C for 15 s, primer annealing at 55 C subsamples. One soil subsample was sieved at 2 mm and stored at 4 for 45 s, and extension at 72 C for 1 min, followed by a final stage of C for biochemical and biological analyses and another subsample heating at 72 C for 10 min. The PCR products were purified with was allowed to dry at room temperature and sieved at 2 mm for ultracentrifugal filter units (Ultracel-100 K membranes; Amicon, physico-chemical and chemical analyses. Cork, Ireland). To measure the DNA concentration, a Quant-iT ® PicoGreen ds DNA Assay Kit (Life Technologies, Carlsbad, CA, 2.2. Plant analyses USA) was used. For each sequencing run, ten samples identified each with a barcode labelling were pooled in equimolar amounts Shoot tissues were dried (60 C, 48 h) and ground before and submitted to pyrosequencing with the 454 FLX Junior system chemical analysis. The foliar concentrations of total C and N were (Life Sciences, Brandford, CR, USA), at the “Zaidín Experimental determined by dry combustion, using a TruSpec CN Analyzer (LECO, Station” (Spanish Council for Scientific Research) in Granada, Spain. St. Joseph, MI, USA). Three sequencing runs were required for sequencing all samples (30 samples in total). 2.3. Soil physico-chemical, biochemical, and biological analyses 2.5. Pyrosequencing data processing Soil pH was measured in a 1:5 (w/v) aqueous extract. Water soluble organic carbon (WSOC) was determined in this aqueous The sequences obtained were analyzed using the mothur soft- extract using a Shimadzu TOC-5050A automatic carbon analyzer for ware package (Schloss et al., 2009) and the authors’ standard liquid samples (Shimadzu Corporation, Kyoto, Japan). The total operating procedure for 454 data (Schloss et al., 2011). The organic carbon (TC) and nitrogen (TN) concentrations were PyroNoise algorithm (Quince et al., 2011), implemented as a measured using a Euro EA elemental analyzer (EuroVector, Milan, mothur component, was used to reduce sequencing error through Italy), after destruction of carbonates by treating the samples with the flowgrams information. Readings with more than two mis- hydrochloric acid. matches to the barcodes and three mismatches to the primers were Dehydrogenase activity was determined by colorimetric esti- discarded, as well as those containing homopolymers greater than mation of the reaction product INTF (iodonitrotetrazolium for- 8 bp. The primers and barcodes were trimmed too and a minimum mazan) formed by reduction of the substrate INT (2-p-iodophenyl- sequence length of 390 bp was established. The alignment of the 3-p-nitrophenyl-5-phenyltetrazolium chloride), following the remaining unique sequences was carried out against the SILVA- procedure described by Garcia et al. (1997). Estimation of the ure- compatible alignment database (Schloss, 2009; Quast et al., 2013). ase and protease activities was based on the determination of the Afterwards, the sequences were screened and filtered to remove þ NH4 released during the incubation of soil with the substrates urea those which did not fit within the selected alignment range. They or N-a-benzoyl-L-arginine amide (BAA), respectively (Nannipieri were also subjected to the UCHIME algorithm (Edgar et al., 2011)to et al., 1980). Alkaline phosphomonoesterase and b-glucosidase ac- identify and eliminate chimeras. Chloroplastic and mitochondrial tivities were determined by measuring the formation of p-nitro- sequences were discarded too. The resulting filtered and trimmed phenol (PNP) by spectrophotometry, as described by Tabatabai and sequences were clustered into operational taxonomic units (OTUs), Bremner (1969). The substrates used were p-nitrophenyl-b-D-glu- applying the average neighbor algorithm with a similarity cutoff of copyranoside (PNG) (Tabatabai, 1982) and p-nitrophenyl phosphate 97%. The centroid sequence of each cluster was selected as the most disodium (PNPP), respectively (Naseby and Lynch, 1997). representative of the OTU and was taxonomically classified at the Soil microbial biomass carbon (SMBC) was estimated using the genus level according to the Ribosomal Database Project (RDP) Substrate Induced Respiration method (Anderson and Domsch, trainset v.10, with a confidence level of 80%. To avoid bias in some of 1978). Soil respiration (SR) was measured by incubating soil the further analytical comparisons, the number of sequences of moistened to 60% of its water holding capacity (Ross et al., 1982). each sample was rarefied, if necessary, to the lowest one (2217 The amount of CO2 emitted during both measurements was sequences of the CH1 sample). determined with a m-Trac 4200 automatic analyzer (SY-LAB The sequence files were submitted to the NCBI Sequence Read Microbiology, Vienna, Austria). Archive (www.ncbi.nlm.nih.gov/sra) and are accessible in the Bio- Project PRJNA312563. 2.4. DNA extraction, PCR amplification, and pyrosequencing 2.6. Statistical analyses The total genomic DNA was extracted from 0.25 g of each indi- vidual rhizosphere soil sample, employing the PowerSoil™ DNA After rarefying the number of sequences per sample, the extraction kit (MoBio Laboratories Inc., Carlsbad, CA, USA) and resultant OTUs were used by mothur to calculate a-diversity esti- following the manufacturer's protocol. Electrophoresis in a 1.5% (w/ mators such as Chao1, the Shannon-Weaver index (H’), and the v) agarose gel, stained with Gel Red™ (Biotium, Hayward, CA, USA), Pielou evenness index. The sampling effort and OTU diversity was performed in order to check the yield and quality of the ex- across soil samples were represented by normalized rarefaction tractions. The DNA extracted was used as a template for the PCR curves (1000 iterations, 2217 randomly chosen sequences per amplification of the 16S rRNA hypervariable V3-V5 region, sample). The main effects of invasiveness, sampling location, and G. Rodríguez-Caballero et al. / Soil Biology & Biochemistry 109 (2017) 176e187 179 their interaction on the diversity indices and the number of OTUs 3. Results observed at the same sampling effort (2217 sequences) were tested using a two-way ANOVA. 3.1. Diversity of rhizosphere bacterial communities The assessment of the bacterial community composition and structure, in the rhizospheric soil samples, was also addressed The pyrosequencing and subsequent cleaning and filtering of through a two-dimensional principal coordinate analysis (PCoA) at the reads produced 211,343 useful sequences across all the samples, the OTU level. With this ordination technique we obtained the with an average length of 403 nt. The number of sequences per spatial distribution of the communities according to their mem- sample varied from 2217 to 13038 and about 92% of them were able bership and structure. It was conducted with all the OTUs obtained, to be classified at the phylum level while about 82% and 59% were from rarefied data, by employing the mothur PHYLIP package after classified at the order and genus level, respectively. As a result of the construction of a lower triangular formatted distance matrix applying the clustering algorithm, 7810 OTUs were generated. The (“Yue & Clayton theta” similarity estimator (Yue and Clayton, normalization of the number of sequences per sample resulted in 2005)). Then, the resultant PCoA axes scores were used to values from 507 to 831 OTUs and the coverage was greater than 79% compare the communities from native and invasive plants, in every case for the normalized data and greater than 89% for the applying a two-way ANOVA. Likewise, the REGR factor score values trimmed data (Table 2). The Chao-1 richness estimations varied obtained for each community in the PCoA were subjected to a from 1606 ± 28 OTUs (recorded in P. setaceum rhizosphere samples Student t-test for paired samples (native versus invasive within from location E) to 851 ± 69 OTUs (H. hirta rhizosphere in location locations) to ascertain significant differences between native and C) and the Shannon-Weaver diversity index (H’) ranged from invasive bacterial communities across locations. 5.84 ± 0.14 (P. setaceum rhizosphere in location S) to 4.99 ± 0.17 The number of sequences per OTU, at different taxonomic levels, (H. hirta rhizosphere in location S). The communities with higher was used as a measure of relative abundance and it was organized values of the diversity indices also exhibited higher Pielou evenness with STAMP (Parks et al., 2014). We used the relative abundances to values. In addition, the ANOVA (Table 2) showed that all indices perform an Indicator Species Analysis (ISA). This analysis allows the were influenced by the sampling location and, in the case of the identification of species which are associated with groups of sam- Shannon and Pielou indices, it also indicated significance for the ples by calculating an Indicator Value (IndVal) (Dufrene^ and interaction between the two factors. Regarding rarefaction ana- Legendre, 1997). The ISA was conducted with the “indicspecies” lyses, the graphical representation of the normalized number of package (v. 1.7.5 (De Caceres and Legendre, 2009)), implemented in OTUs (Fig. 1) revealed slightly different rarefaction curves R(R Development Core Team, 2015). Since our interest was mainly depending on the invasive character of the plant. The ANOVA focused on finding indicator species for the rhizosphere commu- confirmed that the average richness of the invasive rhizosphere nities of invasive or native plants, the invasiveness status of the samples (727 ± 14 OTUs) was higher than that of the native ones plant was used as the only classifier factor. The analysis was run by (654 ± 24 OTUs), when both were considered at the sampling effort applying the original IndVal method, which avoids the consider- of 2217 sequences (P ¼ 0.001). ation of group combinations, and was performed at the phylum, fi order, and genus levels, including all of the OTUs identi ed. 3.2. Composition and structure of the rhizosphere bacterial Canonical correspondence analyses (CCAs) were generated with communities CANOCO for Windows v.4.5 (Ter Braak and Smilauer, 2002). This constrained ordination process was applied to assess the effect of A total of 24 phyla were identified across all rhizosphere soil the soil properties measured on the community composition. For samples from both types of plant, native and invasive. Seven of this purpose, communities from invasive plants were analyzed them presented relative abundances higher than 1%, Proteobacteria independently of those from native plants, and only abundant or- being the most abundant phylum with an average representation of > ders (total relative abundance 0.15%) were considered in the 38% of all the reads. It was followed by Actinobacteria (25%), Bac- analysis. The CCAs were carried out by biplot scaling (inter-orders teroidetes (12%), Acidobacteria (8%), Verrucomicrobia (1.5%), and distances) and Monte Carlo permutation tests with 999 random Planctomycetes (1%). The remaining phyla identified comprised 3.8% permutations. CanoDraw was used to visualize the CCA diagram of all the sequences. obtained. At the order level, 65 taxonomic groups were identified, of The normality of the soil data was tested with the Kolmogorov- which Actynomicetales (Actinobacteria) was the dominant order < Smirnov normality test at P 0.05 and the homogeneity of vari- with a representation of 20% of the total number of sequences. It ances was proved with Levene's test. A two-way ANOVA was con- was followed in abundance by Rhizobiales (10.2%) and Rhodospir- ducted to assess the effect of the two factors (location and illales (7%), both of the Alphaproteobacteria class. Other abundant invasiveness) and their interaction on soil physico-chemical, orders of Proteobacteria were Myxococcales (5.2%), Burkholderiales ’ biochemical, and biological parameters. Additionally, the Tukey s (3.8%), Xanthomonadales (2.4%), and Sphingomonadales (2.2%). The fi HSD (Honestly Signi cant Difference) test was applied for the post- Bacteroidetes were mainly represented by Cytophagales (5.9%) and hoc analysis. The statistical procedures were performed with the Sphingobacteriales (4.8%). Of the phylum Acidobacteria,8%was software IBM SPSS Statistics 22 for Windows. distributed in ten different orders - Gp6, Gp4, and Gp10 being the most abundant orders, comprising 5.6% of the total number of

Table 1 Sampling points location, geographical coordinates and climatic parameters.

Location Abbreviation (invasive, native) Geographical coordinates Mean annual rainfall (mm)a Mean annual temperature (C)a

La Alcayna (A) AP, AH 38 40 6.2400 N/1 90 10.4200 W 231.6 17.2 El Tiro (T) TP, TH 38 10 9.1100 N/1 90 38.6300 W 287.4 18.7 Rambla de Corvera (C) CP, CH 37 490 31.7800 N/1 90 44.9600 W 253.3 17.4 Santa Pola (S) SP, SH 38 120 55.5200 N/0 300 29.2400 W 264.4 18.4 Espinardo (E) EP, EH 38 10 14.6000 N/1 90 42.6600 W 287.4 18.7

a Mean annual rainfall and mean annual temperature have been calculated considering values from 1985 to 2014. 180 G. Rodríguez-Caballero et al. / Soil Biology & Biochemistry 109 (2017) 176e187

Table 2 Normalized (at the lowest number of sequences per sample) values of bacterial diversity estimators of the rhizospheres of invasive (P¼ P. setaceum) and native (H¼ H. hirta) plants grown in five different locations. Mean ± standard error, n ¼ 3. Significance of effects of sampling location, invasive character and their interaction on the measured variables is also shown (F-values (P-values)). When the interaction between factors was significant, values in columns sharing the same letter do not differ significantly (P < 0.05) as determined by the Tukey’s HSD test. N ¼ normalized; T ¼ trimmed.

0 Location Sample OTUs Coverage N (%) Coverage T (%) Chao1 H Pielou AAP737 ± 36 79 ± 193± 0 bc 1585 ± 114 5.67 ± 0.11 b 0.86 ± 0.01 b AH 734 ± 18 80 ± 092± 1 abc 1516 ± 41 5.76 ± 0.08 b 0.87 ± 0.01 b TTP717 ± 11 80 ± 094± 0 c 1552 ± 33 5.70 ± 0.01 b 0.87 ± 0.00 b TH 627 ± 18 83 ± 092± 1 abc 1292 ± 23 5.45 ± 0.06 ab 0.85 ± 0.01 b CCP678 ± 19 84 ± 190± 0 ab 1152 ± 48 5.68 ± 0.05 b 0.87 ± 0.01 b CH 510 ± 37 88 ± 191± 1 ab 851 ± 69 4.99 ± 0.17 a 0.80 ± 0.02 a SSP728 ± 49 82 ± 189± 0 a 1297 ± 86 5.84 ± 0.14 b 0.89 ± 0.01 b SH 659 ± 19 84 ± 191± 0 ab 1168 ± 66 5.63 ± 0.06 b 0.87 ± 0.01 b EEP762 ± 12 79 ± 092± 1 abc 1606 ± 28 5.80 ± 0.04 b 0.87 ± 0.03 b EH 744 ± 47 80 ± 293± 1 bc 1538 ± 111 5.83 ± 0.10 b 0.88 ± 0.01 b ANOVA (F-values (P values)) Location 8.81 (<0.001) 14.61 (<0.001) 10.43 (<0.001) 23.81 (<0.001) 8.16 (<0.001) 7.22 (0.001) Invasiveness 13.63 (0.001) 14.20 (0.001) 0.03 (0.853) 14.12 (0.001) 12.37 (0.002) 9.78 (0.005) L x I 2.45 (0.08) 1.40 (0.271) 3.48 (0.026) 1.23 (0.330) 5.31 (0.004) 6.49 (0.002)

sequences. with the rhizosphere of invasive and native plants (Table 3). Thir- The number of genera identified comprised 294 taxa. Skerma- teen bacterial genera were proposed as indicators for the nella was the most abundant genus with 5.2% of the total relative P. setaceum rhizosphere. The highest IndVal (0.817) was obtained by abundance. Other abundant genera were Microvirga (3.5%), Niastella and the most abundant indicator was the genus Ohtaek- Ohtaekwangia (2.6%), Nocardioides (2.1%), Acidobacteria Gp6 (2.1%), wangia, one of the most abundant genera identified. The ISA and Massilia (2.1%). revealed seven indicator genera for H. hirta rhizosphere bacterial The principal coordinate analysis (PCoA) was performed at the communities. Armatimonas/Armatimonadetes Gp1 was the genus OTU level in order to ascertain changes in the structure of the with the highest IndVal (0.829) and Massilia was the most abun- microbial communities across experimental factors (Fig. 2). The dant indicator for this group. At the order level, seven and two taxa first principal coordinate explained 30.5% of the microbial com- were revealed as indicators for invasive and native rhizosphere munity variability and the second one explained an additional communities, respectively. The ISA at the phylum level yielded 11.6%. In general, the rhizosphere communities seemed to cluster three taxa for the communities harbored in the P. setacetum according to the sampling location but also according to the inva- rhizosphere, whereas no indicator phylum was found for the native siveness of the plant. The significant influence of these two factors plant. on the spatial ordination of the communities was determined by running a two-way ANOVA on the scores of the PCO1 and PCO2 3.3. Plant shoot C/N ratio axes. This statistical test confirmed that both location and inva- fi siveness signi cantly affected the rhizosphere bacterial commu- The shoot C/N ratio was calculated as an indicator of above- fi nities. Moreover, the interaction of the factors only had a signi cant ground litter quality. Low values for this ratio indicate high litter effect on the rhizosphere bacterial communities along PCO1. Like- quality in terms of nutrient availability through the microbial wise, the Student t-test for paired samples calculated with the decomposition processes (Manzoni et al., 2008). The ANOVA fi regression factor scores provided by the PCA also con rmed that revealed that invasiveness and sampling location had a significant the community composition and structure was significantly different between native and invasive bacterial communities across locations for both axes PCO1 (P ¼ 0.050) and PCO2 (P ¼ 0.037). 0.4 The Indicator Species Analysis (ISA) was used to identify taxa that could serve as indicators of bacterial communities associated AP2

SH1 0.2 EH3 AP ) SH2 SH3

800 AH %

6 EH1 TP . TH2 TH 1 TP1 1 AH2 (

s EP SP2 AH1

2 AP3 U 600 EH TH1 T O 0.0 AP1 AH3 CP CH2 O CH C SP3

f TP3 P CH1 SP1

o EP3 SP EP1 r SH TH3 e 400 EP2 b TP2 m CH3 u CP1 EH2 N -0.2 CP2 200 CP3

0 -0.6 -0.4 -0.2 0.0 0.2 0.4 0 500 1000 1500 2000 PCO1 (30.5%) Number of sequences Fig. 2. Principal coordinate analysis (PCoA) based on a lower-triangular distance ma- Fig. 1. Normalized rarefaction curves (up to minimum of 2217 sequences) along the trix (“Yue & Clayton theta” similarity estimator) for bacterial communities at OTU level number of sequences obtained from of the rhizospheres of invasive (P¼ P. setaceum) of the rhizospheres of P. setaceum (symbols in black) and H. hirta (symbols in white) and native (H¼ H. hirta) plants grown in ﬁve different locations. plants grown in ﬁve different locations. G. Rodríguez-Caballero et al. / Soil Biology & Biochemistry 109 (2017) 176e187 181

Table 3

Indicator species analysis (IndVal index) and relative abundances of bacterial genera in the rhizosphere of Pennisetum setaceum (RAI) and Hyparrhenia hirta (RAN).

Genus IndVal A B p-value RAI (%) RAN (%) P. setaceum Niastella 0.817 0.67 1.00 0.029 0.19 0.10 Arenimonas 0.815 0.71 0.93 0.020 0.10 0.04 Planctomyces 0.796 0.63 1.00 0.014 0.15 0.09 Pirellula 0.792 0.63 1.00 0.029 0.17 0.10 Luteolibacter 0.796 0.86 0.73 0.011 0.05 0.01 Hyphomicrobium 0.791 0.63 1.00 0.026 0.21 0.13 Opitutus 0.786 0.62 1.00 0.035 0.79 0.49 Ohtaekwangia 0.772 0.60 1.00 0.032 2.92 1.99 Litorilinea 0.764 0.58 1.00 0.028 0.32 0.23 Azoarcus 0.658 0.93 0.50 0.028 0.03 0.00 Actinocorallia 0.656 0.81 0.53 0.034 0.01 0.00 Candidatus_Koribacter 0.632 1.00 0.40 0.018 0.01 0.00 Enterococcus 0.602 0.90 0.40 0.038 0.02 0.00 H. hirta Armatimonas/Armatimonadetes Gp1 0.829 0.69 1.00 0.033 0.10 0.22 Hymenobacter 0.814 0.66 1.00 0.044 0.22 0.44 Massilia 0.798 0.64 1.00 0.021 1.58 2.76 Kribbella 0.786 0.62 1.00 0.028 0.08 0.12 Adhaeribacter 0.785 0.62 1.00 0.011 1.17 1.88 Leifsonia 0.777 0.60 1.00 0.404 0.14 0.22 Pigmentiphaga 0.598 0.89 0.40 0.044 0.00 0.01

80 3 A P-values B P-values Location (L) <0.001 Location (L) <0.001 d Invasiveness (I) 0.019 ) Invasiveness (I) NS -1 60 L x I 0.012 cd L x I NS kg

c -1 2 g N /

C b t 40 o b b

o b h ab S a 1 20 a Total nitrogen ( nitrogen Total

0 0 ATCSE ATCSE

C P-values 14 D P-values 80 Location (L) <0.001 Location (L) <0.001 ) -1 Invasiveness (I) NS 12 Invasiveness (I) 0.035

kg L x I NS L x I NS -1

g 60 10 ( n

o 8 O, 1:5) O, b 2 r

a 40 c 6 pH (H 4

organic 20 l a t 2 o T 0 0 ATCSE ATCSE

Invasive plant Native plant

Fig. 3. Shoot C/N ratio and chemical and physico-chemical properties of the rhizospheres of invasive (P¼ P. setaceum) and native (H¼ H. hirta) plants grown in five different locations. Mean ± standard error, n ¼ 3. Significance of effects of sampling location, invasive character and their interaction on the measured variables is also shown (P- values). When the interaction between factors was significant, bars sharing the same letter do not differ significantly (P < 0.05) as determined by the Tukey’s HSD test. effect on the shoot C/N ratio (Fig. 3). The native and invasive plants ratio were recorded in the shoots of native plants, particularly in differed in their litter quality. In general, the highest values for this locations A and S. In contrast, the highest litter quality 182 G. Rodríguez-Caballero et al. / Soil Biology & Biochemistry 109 (2017) 176e187 corresponded to the invasive plant. communities were influenced by the invasiveness of the plant, two different CCAs were performed on the data of the most abundant orders, considering the P. setaceum and H. hirta datasets indepen- 3.4. Soil physico-chemical, biochemical, and biological properties dently, in order to assess the effect of the measured soil properties on the bacterial community composition of their rhizospheres. The results of the two-way ANOVA show that the sampling The CCA of the invasive plant rhizosphere data (Fig. 6) revealed location mediated a significant effect on all the soil physico- that protease, dehydrogenase, and SR had a significant effect on the chemical, biochemical, and biological parameters (Figs. 3e5). In structure of these bacterial communities. Its first two dimensions the case of SR, TN, TC, and alkaline phosphomonoesterase activity, explained 33.9% and 18.6%, respectively, of the relationship be- this factor was the only one determining the differences found tween the bacterial orders and the soil properties. Regarding the between samples. Excepting dehydrogenase activity and soil pH, CCA performed on the native plants rhizosphere dataset (Fig. 7), the highest values of the soil properties were observed in the WSOC, SR, and alkaline phosphomonoesterase were the properties samples from location E, whereas the lowest values of the prop- that significantly influenced the structure of the H. hirta rhizo- erties measured were often found in samples collected from loca- sphere bacterial communities. For this ordination plot, the first axis tion S. explained 42.1% of the orders-soil properties relationship, whereas The protease activity and pH exhibited significant differences the second axis explained 25.0%. based on the invasiveness of the plant. The values for protease and pH were significantly higher in invasive plant rhizosphere samples. Besides, the two-way ANOVA showed a significant interaction be- 4. Discussion tween sampling location and invasiveness regarding the WSOC, SMBC, and dehydrogenase, urease, and b-glucosidase activities. 4.1. Effect of plant invasiveness on soil microbial community structure and composition 3.5. Relationship between soil properties and rhizosphere bacterial communities The metagenomic results of this study clearly showed that the composition and structure of the rhizosphere bacterial community In accordance with the results of the previously mentioned harbored by the invasive P. setaceum and the native H. hirta were analyses, which indicated that the rhizosphere bacterial distinct. It is worth noting that the bacterial community associated

5000

500 )

) c

A P-values -1 B P-values -1

g Location (L) 0.037

Location (L) <0.001 kg k

g Invasiveness (I) NS 400 Invasiveness (I) 0.019 4000 m L x I 0.040 ( L x I 0.012

C c c i b 3000 bc bc bc n 300 bc ab

a b

g ab

r ab o ab a e 2000 l ab 200 a b a

u a

l a a a o s

r 100 1000 e t a W Soil microbial biomassSoil C (mg 0 0 ATCSE ATCSE

25 ) C P-values -1 Location (L) <0.001 kg

-1 20 Invasiveness (I) NS h

2 L x I NS O C -

C 15 g m (

n 10 o i t a r i p

s 5 e r l i o S 0 ATCSE

Invasive plant Native plant

Fig. 4. Water soluble organic carbon, microbial biomass carbon and basal respiration of the rhizospheres of invasive (P¼ P. setaceum) and native (H¼ H. hirta) plants grown in five different locations. Mean ± standard error, n ¼ 3. Significance of effects of sampling location, invasive character and their interaction on the measured variables is also shown (P- values). When the interaction between factors was significant, bars sharing the same letter do not differ significantly (P < 0.05) as determined by the Tukey’s HSD test. G. Rodríguez-Caballero et al. / Soil Biology & Biochemistry 109 (2017) 176e187 183

Fig. 5. Biochemical properties of the rhizospheres of invasive (P¼ P. setaceum) and native (H¼ H. hirta) plants grown in five different locations. Mean ± standard error, n ¼ 3. Significance of effects of sampling location, invasive character and their interaction on the measured variables is also shown (P- values). When the interaction between factors was significant, bars sharing the same letter do not differ significantly (P < 0.05) as determined by the Tukey’s HSD test. with the invasive rhizosphere had greater richness but was not fynbos ecosystems. more diverse. These results disagree with those of Rodrigues et al. Accordingly, the indicator species analysis (ISA) revealed several (2015), who found higher alpha-diversity indices in the root-zone indicator bacteria for the invasive and native plant rhizosphere bacterial communities from the invasive Microstegium vimineum, communities. In fact, the number of indicators in the P. setaceum a C4 member of the Poaceae, in comparison with native, co- rhizosphere was higher at all taxonomic levels, even to the extent occurring species. However, Carey et al. (2015) did not observe that there were no indicator phyla in the communities harbored by significant effects on richness and alpha-diversity indices after the the native plant. Therefore, the invasive plant seems to be pro- controlled introduction of invasive plants in semiarid grasslands. moting or reducing the presence of some species of bacteria. Meanwhile, Slabbert et al. (2014) found a decreased Shannon di- The ISA highlighted the genus Ohtaekwangia as an indicator in versity index when invasive Acacia species were present in riparian the invasive plant rhizosphere. This genus is the most abundant 184 G. Rodríguez-Caballero et al. / Soil Biology & Biochemistry 109 (2017) 176e187

1 Actinomycetales 2 Rhizobiales 3 Rhodospirillales 4 Myxococcales 5 Sphingobacteriales 0.8 6 Burkholderiales 7 Cytophagales 8 Xanthomonadales 9 Sphingomonadales 36 29 10 Acidobacteria Gp6 24 11 Acidobacteria Gp4 0.4 28 12 12 Acidobacteria Gp10 TN 13 Solirubrobacterales ALP 14 Rhodobacterales 23 PRT 15 Acidobacteria Gp3 22 BGL 33 16 Acidimicrobiales 4 17 Planctomycetales 8 27 21 13WSOC 18 Caulobacterales 0.0 25 126 10 17 URE 19 Acidobacteria Gp7 35 18 3 34SR 7 32 16 31 11 20 Bacillales 20 2 5 SMBC 6 15 19 21 Gemmatimonadales pH 9 22 Flavobacteriales 23 Opitutales TC 24 Anaerolineales -0.4 14 25 Sphaerobacterales 26 Caldilineales 30 27 Acidobacteria Gp16 28 Rhodocyclales DH 29 Nitrospirales 30 Gaiellales -0.8 31 Rubrobacterales -0.8 -0.4 0.0 0.4 0.8 32 Alphaproteobacteria inc. sed. 33 Chloroflexales 34 Armatimonadales 35 Pseudomonadales 36 Nitrosomonadales

Fig. 6. Canonical correspondence analysis (CCA) on P. setaceum dataset. Abundant orders (relative abundance > 0.15%) and soil physical-chemical, biochemical and biological properties (PRT ¼ protease, BGL ¼ b-glucosidase, ALP ¼ alkaline phosphomonoesterase, URE ¼ urease, DH ¼ dehydrogenase, SMBC ¼ soil microbial biomass carbon, SR ¼ soil respiration, WSOC ¼ water soluble organic carbon, TN ¼ total nitrogen, TC ¼ total organic carbon) are represented. The eigenvalues of the ﬁrst and second axes are 0.04 and 0.02 respectively.

1 Actinomycetales 2 Rhizobiales 3 Rhodospirillales 4 Myxococcales 5 Sphingobacteriales 1.2 6 Burkholderiales 7 Cytophagales 22 8 Xanthomonadales SR 9 Sphingomonadales 10 Acidobacteria Gp6 0.8 ALP WSOCPRT 11 Acidobacteria Gp4 URETN 12 Acidobacteria Gp10 23 SMBC 13 Solirubrobacterales BGL 14 Rhodobacterales 0.4 28 35 15 Acidobacteria Gp3 16 Acidimicrobiales DH8 18 12 5 25 17 Planctomycetales 7 4 18 Caulobacterales 26 33 TC 15 9 6 19 Acidobacteria Gp7 0.0 17 2 20 Bacillales 32 24 21 14 20 21 Gemmatimonadales 1 22 Flavobacteriales 163 36 23 Opitutales 13 2730 -0.4 101911 24 Anaerolineales 29 25 Sphaerobacterales 31 26 Caldilineales 34 pH 27 Acidobacteria Gp16 28 Rhodocyclales -0.8 29 Nitrospirales 30 Gaiellales 31 Rubrobacterales -0.8 -0.4 0.0 0.4 0.8 1.2 32 Alphaproteobacteria inc. sed. 33 Chloroflexales 34 Armatimonadales 35 Pseudomonadales 36 Nitrosomonadales

Fig. 7. Canonical correspondence analysis (CCA) on H. hirta dataset. Abundant orders (relative abundance > 0.15%) and soil physical-chemical, biochemical and biological properties (PRT ¼ protease, BGL ¼ b-glucosidase, ALP ¼ alkaline phosphomonoesterase, URE ¼ urease, DH ¼ dehydrogenase, SMBC ¼ soil microbial biomass carbon, SR ¼ soil respiration, WSOC ¼ water soluble organic carbon, TN ¼ total nitrogen, TC ¼ total organic carbon) are represented. The eigenvalues of the first and second axes are 0.08 and 0.05 respectively. indicator and the third in abundance of all the genera identified. It (Yoon et al., 2011). In our study b-glucosidase activity in the inva- is positive for b-glucosidase activity but does not hydrolyze urea sive plant rhizosphere was influenced by the characteristics of G. Rodríguez-Caballero et al. / Soil Biology & Biochemistry 109 (2017) 176e187 185 invaded location, reaching the highest rates in the location E alkaline phosphomonoesterase activities (Zhang et al., 2009a). possibly due to the highest contents of total and labile organic Hymenobacter spp. produce alkaline phosphomonoesterase but do carbon. Ohtaekwangia belongs to the family Cytophagaceae, which not show urease activity (Zhang et al., 2009b). is known to be a proteolytic family as it contains apr genes (Bach et al., 2001). What is more interesting is that it has been 4.2. Effect of plant invasiveness on soil properties described previously as a nitrifier (Tabassum et al., 2015). An additional advantage conferred by this genus might be the pro- Multiple reports have focused on modifications of nitrogen and duction of marinoquinolines - chemical compounds with antibiotic, carbon cycling mediated by plant invasiveness (Ehrenfeld, 2003; antifungal, and insecticidal properties - which could protect the Liao et al., 2008). Invasive plants may directly affect soil C and N rhizosphere from pathogens and predators (Okanya et al., 2011). pools by their litter (quantity and quality) and root exudates. The second most abundant indicator genus for P. setaceum was However, in the current research the contents of soil total organic C Opitutus, a member of the order Opitutales, which has been and N only varied depending on the sampling location but not on described as an obligate anaerobe typical of rice paddy soils (Chin invasiveness. Probably, such soil properties require longer duration et al., 2001). In spite of this characteristic, Weber et al. (2014) also of invasion by P. setaceum to be altered. Our observations are in reported its presence when they studied changes in soil microbial contrast to several other studies that showed increases in soil nu- communities exposed to different degrees of burn severity. A trients - such as C, N, or P - associated with Acacia dealbata Link possible explanation for this is that, even in aerated soils, anoxic invasion in a forest and shrubland ecosystem (Souza-Alonso et al., microzones are present since the oxygen distribution is heteroge- 2014, 2015) or with Mikania micrantha H.B.K. invasion in a native neous (Picek et al., 2000; Or et al., 2007). The ability of Opitutus to forest community (Li et al., 2006). Similarly, soil respiration and reduce nitrate to nitrite was reported by Chin et al. (2001) in their alkaline phosphomonoesterase activity were only affected by description of the genus. sampling location, with the highest values in the location E coin- Hyphomicrobium, from the order Rhizobiales, was another indi- ciding with the highest levels of total organic C and N. In general, cator genus for the invasive plant rhizosphere: it is related to the N the effect of invasiveness on soil parameters related to function of cycle through N2 fixation and denitrification processes. The nifH the microbial community depended on invaded location. It is worth gene for nitrogenase was found to be present in several Hyphomi- noting that the invasion of P. setaceum had an effect more relevant crobium isolates, together with other genes encoding denitrifying on microbial community structure and composition than on mi- enzymes (Kloos et al., 1995). Recently, Rodrigues et al. (2015) also crobial community function, which could be related to the fact that reported an increase in the abundance of the Hyphomicrobiaceae different bacterial species can carry out the same function, a phe- family in the rhizosphere of the invasive grass M. vimineum. nomenon defined as the bacterial redundancy (Kohler et al., 2010). Two members of the family Planctomycetaceae (Pirellula and Soil protease activity, involved in the nitrogen cycle through the Planctomyces) were found to be indicators for the P. setaceum decomposition of simple peptide substrates, was affected by the rhizosphere. They belong to the order Planctomycetales and to the invasive character of the plant tested, regardless of invaded loca- phylum Planctomycetes, which are also indicators for the invaded tion. The occurrence of the highest rates of protease activity in the sites at their respective taxonomic levels. Planctomyces is a genus P. setaceum rhizosphere may be the result of a greater availability of which is more resistant to drought and rewetting stress than other organic N compounds, released by a better quality leaf litter (lower Gram-negative bacteria and is thought to be well adapted to shoot C/N ratio). The proteolytic processes produce ammonium as environmental changes because it contains a relatively large well, but afterwards it can be oxidized to nitrate by nitrifier or- genome (Ward et al., 2006). ganisms, such as Ohtaekwangia. These findings may be supported Niastella was the indicator genus with the highest IndVal of the by the fact that the most abundant indicator for the invasive plant rhizosphere bacterial communities harbored by P. setaceum.Itis coincides with this nitrifier genus, highlighting the importance of one of the members of the Chitinophagaceae that can hydrolyze this bacterial indicator in soil N processes. It is, therefore, probable chitin and cellulose (Weon et al., 2006) and it shows b-glucosidase that higher nitrate availability triggered an increase in nitrate up- activity, encoding several proteins predicted to perform this func- take by the plants. This could be the reason for the higher pH values tion (Bailey et al., 2013). in the invader's rhizosphere (Kourtev et al., 1998) and is in agree- Regarding the indicators for the H. hirta rhizosphere commu- ment with the hypothesis that invasion leads to changes in soil pH nities, Massilia was the most abundant genus proposed. It belongs (Fan et al., 2010; Kuebbing et al., 2014). The alteration of soil N- to the family Oxalobacteraceae and to the order Burkholderiales. cycling processes in response to invasion by exotic species has been Some species from this genus are positive for urease activity, nitrate previously reported in forests, grasslands, and wetlands (Liao et al., reduction, and ammonia production (Zhang et al., 2006). Massilia 2008; McLeod et al., 2016), but this is the first evidence from a may be important after ecosystem disturbances (Weber et al., 2014) Mediterranean semiarid environment invaded by a perennial grass. and it rapidly colonizes roots and fungal hyphae, including those from arbuscular mycorrhizal fungi (Scheublin et al., 2010; Ofek 4.3. Relationships between soil microbial communities and et al., 2012). properties In the native plant rhizosphere, the genus with the highest IndVal was Armatimonas/Armatimonadetes Gp1. This aerobic bac- Many studies have demonstrated that abiotic and biotic prop- terium has been described recently by Tamaki et al. (2011). The type erties shape the microbial community structure (Lauber et al., species Armatimonas rosea is positive for b-glucosidase and alkaline 2008). In the current study, only soil properties related to soil mi- phosphomonoesterase (Tamaki et al., 2011). The order Armatimo- crobial activity played a role in driving the bacterial communities in nadales was an indicator of this group as well. invasive and native rhizospheres. In contrast to our results, Li et al. Other indicator genera for the H. hirta rhizosphere were (2006) showed that soil physico-chemical properties, such as soil Adhaeribacter and Hymenobacter, which are both members of the pH, were a significant factor in structuring bacterial communities Cytophagaceae family. Many members of this family are able to measured using PLFA analysis. Protease activity accounted for most degrade complex molecules like polysaccharides or proteins of the explained variation in the structure of bacterial communities (McBride et al., 2014; Calleja-Cervantes et al., 2015). Adhaeribacter associated with the P. setaceum rhizosphere. This finding, together spp. generate extracellular fibrillar material and show urease and with the fact that the protease activity was greatest in the invasive 186 G. Rodríguez-Caballero et al. / Soil Biology & Biochemistry 109 (2017) 176e187 rhizospheres, highlights the ability of invasive plant to promote Caravaca, F., Barea, J.M., Palenzuela, J., Figueroa, D., Alguacil, M.M., Roldan, A., 2003. rhizosphere bacterial communities preferably related to N cycling. Establishment of shrub species in a degraded semiarid site after inoculation with native or allochthonous arbuscular mycorrhizal fungi. Applied Soil Ecology Likewise, dehydrogenase activity and SR, both general indicators of 22, 103e111. soil microbial activity, were also drivers of the bacterial commu- Caravaca, F., Masciandaro, G., Ceccanti, B., 2002. Land use in relation to soil chemical nities in invasive rhizospheres. and biochemical properties in a semiarid Mediterranean environment. Soil and Tillage Research 68, 23e30. The rhizosphere bacterial community of H. hirta was also asso- Carey, C.J., Beman, J.M., Eviner, V.T., Malmstrom, C.M., Hart, S.C., 2015. Soil microbial ciated with indicators of microbial activity such as soil respiration community structure is unaltered by plant invasion, vegetation clipping, and and the labile fraction of soil organic matter (WSOC), which is nitrogen fertilization in experimental semi-arid grasslands. Frontiers in fl Microbiology 6, 466. considered a carbon and energy source for the soil micro ora Clayton, W.D., 1969. A revision of the genus Hyparrhenia (London, UK). In: Statio- (Caravaca et al., 2002). Meanwhile, phosphomonoesterase activity, nery Office, H.M. (Ed.), Kew Bulletin Additional Series 2. which is involved in P-cycling and is strongly related to soil organic Coats, V.C., Pelletreau, K.N., Rumpho, M.E., 2014. Amplicon pyrosequencing reveals the soil microbial diversity associated with invasive Japanese barberry (Berberis matter (Nannipieri et al., 2011; Adrover et al., 2012), was a relevant thunbergii DC.). Molecular Ecology 23, 1318e1332. driver of the native plant bacterial community. However, phos- Coats, V.C., Rumpho, M.E., 2014. The rhizosphere microbiota of plant invaders: an phomonoesterase activity was similar for the rhizosphere bacterial overview of recent advances in the microbiomics of invasive plants. Frontiers in communities of native and exotic plants. The close relationship Microbiology 5, 368. Chacon, N., Herrera, I., Flores, S., Gonzalez, J.A., Nassar, J.M., 2009. Chemical, phys- between alkaline phosphomonoesterase activity and the bacterial ical, and biochemical soil properties and plant roots as affected by native and community structure confirms that this enzyme is mainly derived exotic plants in Neotropical arid zones. 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