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Native bacteria promote plant growth under drought stress condition without impacting the rhizomicrobiome

Native bacteria promote plant growth under drought stress condition without impacting the... ABSTRACT Inoculation of plants with beneficial plant growth-promoting bacteria (PGPB) emerges a valuable strategy for ecosystem recovery. However, drought conditions might compromise plant-microbe interactions especially in semiarid regions. This study highlights the effect of native PGPB after 1 year inoculation on autochthonous shrubs growth and rhizosphere microbial community composition and activity under drought stress conditions. We inoculated three plant species of semiarid Mediterranean zones, Thymus vulgaris, Santolina chamaecyparissus and Lavandula dentata with a Bacillus thuringiensis strain IAM 12077 and evaluated the impact on plant biomass, plant nutrient contents, arbuscular mycorrhiza fungi (AMF) colonization, soil rhizosphere microbial activity and both the bacterial and fungal communities. Inoculation with strain IAM 12077 improved the ability of all three plants species to uptake nutrients from the soil, promoted L. dentata shoot growth (>65.8%), and doubled the AMF root colonization of S. chamaecyparissus. Inoculation did not change the rhizosphere microbial community. Moreover, changes in rhizosphere microbial activity were mainly plant species-specific and strongly associated with plant nutrients. In conclusion, the strain IAM 12077 induced positive effects on plant growth and nutrient acquisition with no impact on the rhizosphere microbiome, indicating a rhizosphere microbial community resilient to native bacteria inoculation. Bacillus thuringiensis, plant growth-promoting bacteria, microbial activity, degraded soil, 16S rRNA gene, 18S rRNA gene, arbuscular mycorrhiza fungi INTRODUCTION Knowledge of plant-microbe interaction led to the application of microbes as plant growth promoters due to their capability to improve plant development, commonly used to improve crop yields, in ecosystem recovering strategies (Bashan et al. 2012). Plant growth-promoting bacteria (PGPB) may play a decisive role in facilitating plant growth in soil and might be especially relevant to vegetation recovery strategies. However, modification in plant microbial community structure caused by inoculation of specific microbes might be buffered by ecosystem resilience (Shade et al. 2012), which is driven by the level of diversity of soil biota (Kennedy 1999; Nannipieri et al. 2003) and therefore, may compromise the efficiency of applied PGPB. On the other hand, the inoculated microbes may change the microbial community, thus impacting the soil ecosystem functioning and the environment. Hence, it is crucial to determine if effects due to inoculation with PGPB are long-lasting concerning the beneficial impacts on plant growth and effects on soil biota. The effect of PGPB in plants is mainly studied in crops (Lottmann et al. 2000; Scherwinski, Grosch and Berg 2008; Chowdhury et al. 2013), however studies on the application of PGPB as a strategy for ecosystem recovery (Bashan et al. 2008; Bashan et al. 2012) lack an understanding on the impact of the applied microbes in soil microflora on the long term. Plant-microbe interactions might differ in degraded soil or soil under stress conditions such as drought (Jackson et al. 2003; Aboim et al. 2008; Peixoto et al. 2010). Alternatively, changes in soil microbial structure may result in undesirable effects if native species critical to plant growth are lost or subsequently have reduced roles. This is particularly relevant as plants interact with multiple soil microorganisms in addition to the inoculated bacteria. For example, native arbuscular mycorrhizal fungi (AMF) perform critical ecological ecosystem functions, especially in semiarid Mediterranean regions (Requena, Jeffries and Barea 1996). AMF affect plant diversity and productivity and contribute to determining the stability and sustainability of the ecosystem (van der Heijden et al. 1998; van der Heijden et al. 2006). Therefore, the impact of PGPB inoculation on other native soil microorganisms may be even more important when we consider a degraded environment with reduced biodiversity, what might be more vulnerable to changes in soil microbial composition. In the best-case scenario, re-vegetation programs should combine the beneficial effects of PGPB with AMF and other microorganisms associated with plant roots as microbial sources for growth and nutrient uptake (Armada, Roldán and Azcón 2014; Mengual et al. 2014; Armada et al. 2015a, 2016). The selection of autochthonous plant species capable of hosting a high diversity of AMF in combination with PGPB in their rhizospheres may increase plant establishment in stressful environments such as degraded areas in the semiarid Mediterranean. To our knowledge, there is still a lack of understanding how the inoculation of native microbes affects rhizosphere soil microbes, especially mycorrhiza. Several studies have evaluated the impact of inoculated PGPB, however, they usually apply non-native microbes (Schreiter et al. 2014) in short-term experiments (Scherwinski, Grosch and Berg 2008) and in agricultural fields aiming to increase crop productivity (Lottmann et al. 2000). Research should focus on impacts of native PGPB in facilitating plant growth under stress conditions of both soil degradation and drought. Such an evaluation would best combine molecular biological (i.e. next-generation sequencing) and biochemical (assessment of biomarkers and fatty acids) approaches, since an understanding that embraces soil community structure and activity is required. Next generation sequencing of phylogenetic markers such as ribosomal RNA genes provides information on the overall community structure, whereas the analysis of phospholipid fatty acids (PLFA) and enzyme activities provide information on microbial activities. The sequence of ribosomal RNA gene provides the identification of microbial groups that comprise the microbial community through environmental DNA present in the sample, which result in information more closely related to the microbial seedbank concept (Philippot et al. 2013). Accordingly, a more comprehensive characterization of microbial activity could also rely on the identification of biomarkers based on the composition of PLFA, on dehydrogenase activity (estimator of basal respiration), urease activity (microbial hydrolyzation of urea) (Lloyd and Sheaffe 1973), and phosphatase activity (microbial mediated phosphorus availability). Altogether, the combined analysis provides information about community structure and potentially viable microbes and their functional activity and helps to highlight the potential impacts of bacterial inoculation in plant rhizosphere. The aim of this study was to examine the long-lasting impact of a beneficial native bacterial strain IAM 12077 of B. thuringiensis inoculated in three plant species (Thymus vulgaris, Santolina chamaecyparissus and Lavandula dentata), cultured under drought stress conditions on (1) plant growth and nutrition and (2) the root AMF colonization, the rhizosphere bacterial and fungal community structure (assessed by next-generation sequencing) and the rhizosphere microbial enzyme activities. We hypothesize that after 1 year, the inoculation of PGPB strain IAM 12077 on plants affects the rhizosphere microbial community assembly and mycorrhiza colonization. MATERIALS AND METHODS Soil bacteria isolation and molecular identification The bacterial strain used in this study was isolated from the same natural soil used in the microcosm experiment (see description below). The bacterium was isolated from a mixture of rhizosphere soils from several autochthonous shrub species. A homogenate of 1 g soil in 9 mL sterile water was diluted (10−2–10−4), plated on three different media [Yeast Mannitol Agar, Potato Dextrose Agar and Luria-Bertani Agar (Bertani 1951)] and incubated at 28°C for 48 h to isolate bacteria from different taxonomic groups. Molecular characterization of the selected bacterium was by sequencing the 16S rRNA gene. Bacterial cells were collected, diluted, lysed, and the DNA extracted, which was used as PCR template. The amplification reactions were performed in 25 µL volume containing 10× PCR buffer, 50 mM MgCl2, 10 µL of each primer [27F (AGAGTTTGATCCTGGCTCAG) and 1492R (GGTTACCTTGTTACGACTT)] and 1 µL of 5 U/µl Taq polymerase (Platinum, Invitrogen, Waltham, USA). PCR was carried out in a thermal cycler using following conditions: 5 min at 95°C, followed by 30 cycles of 45 s at 95°C, 45 s at 44°C and 2 min at 72°C and finally one cycle of 10 min at 72°C. PCR products were analyzed by 1% agarose gel electrophoresis and purified with the QIAquick Gel extraction kit (Qiagen) for subsequent sequencings using an automated DNA sequencer (Perkin–Elmer ABI Prism 373, USA). Sequence data were compared to databases (EMBL and GenBank) using BLAST program. Similarity searches at NCBI using BLAST program unambiguously identified the bacterial species similar to B. thuringiensis strains. The genetic similarity was also confirmed by phylogenetic analysis of the 16S rRNA gene sequence (Fig. S1, Supporting Information). The strain is B. thuringiensis IAM 12077 (Accession NR 043403.1 or accession D16281). In previous studies (Armada et al. 2015b, 2016), the same strain was characterized for growth under non-stress and osmotic stress conditions, ability to synthesize proline or poly-β-hydroxybutyrate, lipid peroxidation and PGPB characteristics that included α-ketobutyrate [ACC (1-aminocyclopropane-1-carboxylate) deaminase] and indole acetic acid (IAA) production and phosphate solubilization. Microcosm experimental design and soil characteristics The microcosm experimental design was based on two factors: (1) three different autochthonous shrub species: T. vulgaris (T), S. chamaecyparissus (S) and L. dentata (L) and (2) inoculation or not of the autochthonous species with the selected B. thuringiensis strain IAM 12077 (non-inoculated, -; inoculated with strain IAM 12077, Bt). Each of six treatments included five biological replicates resulting in 30 pots. The soil used in this experiment is natural soil from the 'Vicente Blanes' natural park in Molina de Segura, Murcia, Spain, (coordinates: 38° 12′ N and 1′ 13′ W, 393 m altitude). The soil is a Typic Torriorthent (SSS 2006) containing: organic C 0.94%, total N 0.22%, P 1.36 · 10−3 g kg−1 (Olsen test), pH 8.9 and an electric conductivity of 1.55 dS·m−1. The substrate used in this assay consisted of a 5 cm layer of the natural park soil mixed with sterile sand [5/2 (v/v)]. The substrate was added to 0.5 kg capacity pots. One milliliter of pure strain IAM 12077 culture (108 CFU·mL−1), grown in LB medium broth (Bertani 1951) for 48 h at 28°C was applied to the potted seeds at sowing time. The bacterial inoculum (108 CFU·mL−1) was again applied to the seedlings 15 days later. The control consisted of inoculations with sterilized LB medium. The three plant species were grown for 1 year in pots under greenhouse conditions (temperature 19°C–25°C, 16 h/8 h light/dark photoperiod). The photosynthetic photon flux density measured with a light-meter (LICOR, model LI-188B) was 400–700 µmol m−2 s−1. During the experiment, plants were subject to drought conditions by limiting the soil water-holding capacity to 50% each day after water application and allowing the water-holding capacity to decrease to ∼30% before the next water application. Plant biomass and nutrient analysis Plants were harvested 1 year after planting (five replicates per treatment, n=5), shoots were excised from the roots, and fresh weights of both shoots and roots determined. Subsequently, samples were dried for 48 h at 75°C and dry masses determined. Shoot P, K, Ca and Mg (mg plant−1), as well as Zn, Fe, Mn and Cu (µg plant−1) contents, were determined by inductively coupled plasma optical emission spectrometry at the Analytical Service of the Centro de Edafología y Biología Aplicada del Segura, CSIC, Murcia, Spain. AMF root colonization, spores isolation and identification The roots of the three plant species were carefully washed and stained (Phillips and Hayman 1970). The percentage of mycorrhizal root length was determined by microscopic examination of stained root samples (Phillips and Hayman 1970) using the gridline intersect method of Giovannetti and Mosse (1980). The AMF spores were isolated from the soil samples by a wet sieving process (Sieverding 1991). The morphological spore characteristics and their subcellular structures were described from a specimen mounted in: polyvinyl alcohol-lactic acid-glycerine (PVLG) (Koske and Tessier 1983), a mixture of PVLG and Melzer’s reagent (Brundrett 1994), a mixture of lactic acid to water at 1:1, Melzer’s reagent, and water (Spain 1990). For identification of the AMF species, the spores were examined by microscopy at up to 400-fold magnification as described for glomeromycotean classification (Oehl et al. 2011). Rhizospheric soil enzymatic activity Dehydrogenase activity was determined following Skujins’ method (1976) as modified by García, Hernández and Costa (1997). One gram of rhizosphere soil was exposed to 0.2 mL of 0.4% 2-p-iodophenyl-3-p-nitrophenyl-5-phenyl tetrazolium chloride in distilled water for 20 h at 22°C in the dark. The formed iodo-nitrotetrazolium formazan (INTF) was extracted with 10 mL of methanol by shaking vigorously for 1 min and filtering through a Whatman no. 5 filter paper. INTF was measured spectrophotometrically at 490 nm. β-glucosidase was determined using 0.05 M p-nitrophenyl-β-D-glucopyranoside, (Masciandaro, Ceccanti and García 1994) as substrate. This assay is also based on the release and detection of p-nitrophenol (PNP). Two milliliters of 0.1 M maleate buffer (pH 6.5) and 0.5 mL of the substrate were added to 0.5 g of sample and incubated at 37°C for 90 min. The reaction was stopped by addition of tris-hydroxymethyl aminomethane according to Tabatabai (1982). The amount of generated PNP was determined by absorbance at 398 nm (Tabatabai and Bremner 1969). Urease activity was determined by the method of Nannipieri et al. (1980) and expressed as µmol N-NH3·g−1 soil · h−1. Alkaline phosphatase activity was determined using 0.115 M p-nitrophenyl phosphate disodium as substrate. Two milliliters of 0.5 M sodium acetate buffer adjusted to pH 5.5 using acetic acid (Naseby and Lynch 1997) and 0.5 mL of substrate was added to 0.5 g of soil and incubated at 37°C for 90 min. The reaction was stopped by cooling to 2°C for 15 min. Then, 0.5 mL of 0.5 M CaCl2 and 2 mL of 0.5 M NaOH were added, and the mixture was centrifuged at 1649 xg for 5 min. The PNP formed was determined by absorbance at 398 nm (Tabatabai and Bremner 1969). For controls, the substrate was added before the additions of CaCl2 and NaOH. Rhizospheric soil microbial lipid extraction and PLFA analysis Lipid extraction, fractionation, mild alkaline methanolysis and GC analysis were according to Frostegard, Bååth and Tunlio (1993a,b). PLFA analysis was carried out in freeze-dried frozen samples (−80°C). Lipids were extracted from 3 g lyophilized soil samples using a one-phase mixture (1:2:0.8 v/v/v) of chloroform/methanol/0.15 M, pH 4.0 citrate buffer. After extraction, the lipids were separated into neutral lipids, glycolipids and polar lipids (phospholipids) on silicic acid columns (Merck Kieselgel 60 63–200 µm) followed by a mild alkaline methanolysis to form the corresponding fatty acid methyl esters for GC analysis. The fatty acids were identified by their retention times in relation to that of the internal standard (fatty acid methyl esters 19:0 and 12:0). The following fatty acids were assessed as biomarkers for bacterial biomass: i14:0, i15:0, a15:0, i16:0, 16:1w7t, 17:1w7, a17:1w7, i17:0, cy17:0, 18:1w7c and cy19:0 (Mauclaire et al. 2003). PLFA 16:1w5 was used as an indicator of AMF (Olsson et al. 1995; Drigo et al. 2010). C18:2w6.9 was used as a measure of fungal biomass (Bååth 2003). Methylated fatty acid (10Me16:0) was used as a specific biomarker for Actinomycetes (Frostegard, Bååth and Tunlio 1993a,b; Welc et al. 2010). The ratios of Gram-positive (G+) to Gram-negative (G−) bacteria were calculated by taking the sum of the PLFAs i-C14:0, i-C15:0, a-C15:0, i-C16:0, i-C17:0 and a-C17:0 reflected the amount of G+ bacteria, whereas C16:1w7, C17:0 cy and C18:1w7 reflected the amount of G− bacteria (Frostegård and Bååth 1996; Zelles 1997). Rhizospheric soil DNA extraction, PCR conditions for fungal and bacterial tag-encoded amplification and sequencing DNA was extracted from 0.5 g of soil using the fast DNA Spin Kit for soil (MO BIO Laboratories Inc., Carlsbad CA, USA) and quantified by 260 nm absorbance (Nanodrop Technology, Wilmington, DE, USA). The integrity of the DNA was verified by 1% agarose gel electrophoresis using TBE buffer (45 mM Tris-borate, 1 mM EDTA, pH 8.0). For fungal 18S rRNA partial gene amplification, primers described by Verbruggen et al. (2012) were used. The 5′ terminus of primers contained an adaptor sequence and a multiplex identifier tag (MID, 12 different 10-bp-long tags), which resulted in the following primer constructs (adaptor in boldface): Forward (FF390.1), 5′-CTATGCGCCTTGCCAGCCCGCTCAG-(MID)-CGWTAACGAACGAGACCT-3′, Reverse (FR1), 5′-CGTATCGCCTCCCTCGCGCCATCAG-(MID)-AICCATTCAATCGGTAIT-3′. PCR reactions contained 2.0 µL of 10 µM each forward and reverse primer, 5.0 µL 10× PCR-buffer, 5.0 µL of 2 mM dNTP’s, 0.5 µL BSA, 33.10 µL Milli-Q water and 0.40 µL FastStar Expand TAQ Taq DNA polymerase (5 U/µL). The PCR conditions were 95°C for 5 min followed by 25 cycles at 95°C for 30 s, 57°C for 1 min and 72°C for 1 min, a final elongation step at 72°C for 10 min. Products were purified using QIAquick PCR Purification Kit (Qiagen). For bacteria, the V4 region of the 16S rRNA gene was amplified by PCR using 515F (5’-GTGCCAGCMGCCGCGGTAA-3’) and 806R (5’-GGACTACHVGGGTWTCTAAT -3’) primers. The 515F primer included the Roche 454-B pyrosequencing adapter (a 10-bp barcode) unique to each sample, and a GT linker, while 806R included the Roche 454-A sequencing adapter (a 10-bp barcode), unique to each sample, and a GG linker. PCRs contained 1.0 µL of 5 µM each forward and reverse primers, 2.5 µL 10× PCR-buffer, 2.5 µL of 2 mM dNTPs, 16.80 µL Milli-Q water and 0.20 µL of 5 U/µL FastStar Expand TAQ Taq DNA polymerase under the following conditions: 95°C for 5 min followed by 30 cycles of 95°C for 30 s, 53°C for 1 min and 72°C for 1 min, a final elongation step at 72°C for 10 min. Products were purified using QIAquick PCR Purification Kit (Qiagen). Amplicons were quantified and equimolar pooled. The samples were sequenced (Macrogen Company Inc., South Korea) on a Roche 454 automated sequencer and GS FLX system using titanium chemistry (454 Life Sciences, Branford, CT, USA). Statistical analysis Basic and univariate statistics Kolmogorov–Smirnov and Lilliefors tests were used for assessing the normality of the data. Most variables of plant biomass were normally distributed or could be normalized by data transformation. Homogeneity of variance was checked by using the Brown–Forsythe test for unequal replication numbers. Statistical analysis consisted of two-way ANOVA followed by Tukey-Kramer’s test. Correlation and regression analyzes were also performed to identify univariate interactions among variables. For the regression analysis, two values of shoot biomass were considered outliers and therefore, excluded from this analysis. To reduce both family-wise error and the false discovery rate, all multiple comparisons passed to a step-down resampling algorithm (Westfall and Young 1993) while the correlation analysis had their P-values corrected by the Benjamini–Hochberg false discovery rate adjustment (Benjamini and Hochberg 1995). Multivariate statistics and generalized linear models The sequenced datasets (bacterial and fungal) were analyzed using the 'phyloseq' package in R (McMurdie and Holmes 2013). The bacterial and fungal operational taxonomic unit (OTU) abundances were summarized at the class and genus taxonomical levels, respectively, and the Hellinger transformation was adopted prior to ordination methods (Legendre and Gallagher 2001) in order to stabilize the mean-variance relationship and to avoid confounding location and dispersion effects (Warton, Wright and Wang 2012). The 'ade4' R package (Dray and Dufour 2007) compared the effect of treatment on the various datasets (plant biomass and nutrients, microbial enzymatic activity and soil microbial community). Between-class analysis (BCA) measured the amount of variance restricted to the grouping factor as a percentage of the total inertia (Dray and Jombart 2011; Thioulouse, Prin and Duponnois 2012). BCA is an alternative method to linear discriminant analysis for which the number of samples is smaller than the number of variables (Thioulouse, Prin and Duponnois 2012). Principal component analysis was applied to each community data set prior to BCA. Monte–Carlo tests of the treatment groups were performed with 999 permutations. We also performed Co-inertia analysis to investigate the degree of data co-structure (Dray, Chessel and Thioulouse 2003) by evaluating the covariance between our four different datasets: (i) plant biomass and nutrients, (ii) microbial activity, (iii) bacterial and (iv) fungal community structure. Because both microbial enzymatic activity and soil microbial community (fungi and bacteria) datasets presented an overdispersal variance, we applied a generalized linear model (GLM) based on tweedie and negative binomial distribution to investigate the effects of both plant and bacterial inoculum on microbial activity and structure, respectively. To avoid sequencing bias common in next-generation platforms (Lee et al. 2012; McMurdie and Holmes 2014), we decided to use the total number of reads per sample as a covariance effect in our GLMs. The effect of each treatment in the microbial community was evaluated by the Wald’s test (W) and Wilks’s lambda test for the enzymatic activity. The R environment (R Development Core Team 2007) and 'ade4' (Chessel, Dufour and Thioulouse 2004), 'mvabund' (Wang et al. 2012) and 'multcomp' (Hothorn, Bretz and Westfall 2008) packages were used. RESULTS Plant growth, nutrition and symbiotic parameters Inoculation with strain IAM 12077 increased the shoot biomass of all three plant species compared to the control: 65.8% for L. dentata, 31.8% for S. chamaecyparissus and 17% for T. vulgaris (Fig. 1A). However, inoculation weakly influenced root biomass (P < 0.06) (Fig. 1B). Figure 1. View largeDownload slide Influence of a native B. thuringiensis strain IAM 12077 on both shoot and root biomass of three autochthonous plant species native to natural arid Mediterranean soil under drought stress conditions. Means + CI95%, data followed by the same capital letter do not differ statistically among plants, while small-case letters refer to differences between inoculated and control plants. Tukey’s test at 5% probability. Figure 1. View largeDownload slide Influence of a native B. thuringiensis strain IAM 12077 on both shoot and root biomass of three autochthonous plant species native to natural arid Mediterranean soil under drought stress conditions. Means + CI95%, data followed by the same capital letter do not differ statistically among plants, while small-case letters refer to differences between inoculated and control plants. Tukey’s test at 5% probability. Inoculation with strain IAM 12077 increased both macro and micronutrients in shoots, but all three plant species responded differently: P and K increased by 51% and 47%, respectively, for T. vulgaris , while K, Ca and Mg increased by 63%, 27% and 36% for L. dentata compared to non-inoculated controls (Table 1). Inoculation with strain IAM 12077 also increased the shoot content of the micronutrients Zn, Fe and Cu for T. vulgaris and Zn, Mn and Cu for S. chamaecyparissus compared to non-inoculated controls (Table 1). In general, the shoot nutrient content of S. chamaecyparissus contained the largest amount of P, while L. dentata accumulated more K, Ca and Mg than the other two species in this study (Table 1). Table 1. Total content of macro- (P, K, Ca and Mg) and micronutrients (Zn, Fe, Mn and Cu) in the shoot of three autochthonous plants species Thymus vulgaris (T), Santolina chamaecyparissus (S) andLavandula dentata (L) grown in natural arid Mediterranean soil under drought stress conditions as affected by the inoculation of the autochthonous bacteria strain Bacillus thuringiensis (Bt). P (mg plant−1) K (mg plant−1) Ca (mg plant−1) Mg (mg plant−1) Zn (µg plant−1) Fe (µg plant−1) Mn (µg plant−1) Cu (µg plant−1) T(-) 0.57 ± 0.11Ab 7.07 ± 0.25Bb 6.01 ± 1.85Bb 1.70 ± 0.62Ab 33.28 ± 1.69Ba 42.79 ± 3.67ABb 40.35 ± 14.51Ba 4.21 ± 0.27Bb TBt 0.86 ± 0.09Aa 10.42 ± 0.07Ba 7.94 ± 0.28Ba 2.21 ± 0.24Aa 54.06 ± 5.90Bb 115.22 ± 17.83ABa 46.49 ± 0.74Ba 6.38 ± 1.04Ba S(-) 0.87 ± 0.07Ab 10.10 ± 0.45Ba 8.45 ± 1.00Bb 1.01 ± 0.06Bb 60.93 ± 3.78Ab 87.16 ± 20.55Ba 100.19 ± 3.02Ab 11.05 ± 0.59Ab SBt 1.01 ± 0.08Aa 12.93 ± 0.70Bb 11.95 ± 0.67Bb 1.43 ± 0.10Ba 89.72 ± 7.46Aa 78.43 ± 19.76Ba 121.93 ± 2.57Aa 13.17 ± 0.27Aa L(-) 0.62 ± 0.09Ba 13.51 ± 0.45Ab 13.30 ± 1.68Ab 2.14 ± 0.13Ab 38.50 ± 4.69Ab 104.25 ± 16.89Aa 13.41 ± 1.32Cb 5.19 ± 0.48Bb LBt 0.64 ± 0.11Ba 21.97 ± 1.48Aa 16.83 ± 0.51Aa 2.91 ± 0.04Aa 47.39 ± 2.42Aa 100.21 ± 15.00Aa 20.60 ± 0.69Ca 7.23 ± 0.69Ba P (mg plant−1) K (mg plant−1) Ca (mg plant−1) Mg (mg plant−1) Zn (µg plant−1) Fe (µg plant−1) Mn (µg plant−1) Cu (µg plant−1) T(-) 0.57 ± 0.11Ab 7.07 ± 0.25Bb 6.01 ± 1.85Bb 1.70 ± 0.62Ab 33.28 ± 1.69Ba 42.79 ± 3.67ABb 40.35 ± 14.51Ba 4.21 ± 0.27Bb TBt 0.86 ± 0.09Aa 10.42 ± 0.07Ba 7.94 ± 0.28Ba 2.21 ± 0.24Aa 54.06 ± 5.90Bb 115.22 ± 17.83ABa 46.49 ± 0.74Ba 6.38 ± 1.04Ba S(-) 0.87 ± 0.07Ab 10.10 ± 0.45Ba 8.45 ± 1.00Bb 1.01 ± 0.06Bb 60.93 ± 3.78Ab 87.16 ± 20.55Ba 100.19 ± 3.02Ab 11.05 ± 0.59Ab SBt 1.01 ± 0.08Aa 12.93 ± 0.70Bb 11.95 ± 0.67Bb 1.43 ± 0.10Ba 89.72 ± 7.46Aa 78.43 ± 19.76Ba 121.93 ± 2.57Aa 13.17 ± 0.27Aa L(-) 0.62 ± 0.09Ba 13.51 ± 0.45Ab 13.30 ± 1.68Ab 2.14 ± 0.13Ab 38.50 ± 4.69Ab 104.25 ± 16.89Aa 13.41 ± 1.32Cb 5.19 ± 0.48Bb LBt 0.64 ± 0.11Ba 21.97 ± 1.48Aa 16.83 ± 0.51Aa 2.91 ± 0.04Aa 47.39 ± 2.42Aa 100.21 ± 15.00Aa 20.60 ± 0.69Ca 7.23 ± 0.69Ba Standard Errors are given. Within each parameter, values followed by the same capital letter did not differ between plants while the values followed by the same small letter did not differed between inoculation within plant species (P ≤ 0.05) as determined by Tukey’s test (n= 3). View Large Table 1. Total content of macro- (P, K, Ca and Mg) and micronutrients (Zn, Fe, Mn and Cu) in the shoot of three autochthonous plants species Thymus vulgaris (T), Santolina chamaecyparissus (S) andLavandula dentata (L) grown in natural arid Mediterranean soil under drought stress conditions as affected by the inoculation of the autochthonous bacteria strain Bacillus thuringiensis (Bt). P (mg plant−1) K (mg plant−1) Ca (mg plant−1) Mg (mg plant−1) Zn (µg plant−1) Fe (µg plant−1) Mn (µg plant−1) Cu (µg plant−1) T(-) 0.57 ± 0.11Ab 7.07 ± 0.25Bb 6.01 ± 1.85Bb 1.70 ± 0.62Ab 33.28 ± 1.69Ba 42.79 ± 3.67ABb 40.35 ± 14.51Ba 4.21 ± 0.27Bb TBt 0.86 ± 0.09Aa 10.42 ± 0.07Ba 7.94 ± 0.28Ba 2.21 ± 0.24Aa 54.06 ± 5.90Bb 115.22 ± 17.83ABa 46.49 ± 0.74Ba 6.38 ± 1.04Ba S(-) 0.87 ± 0.07Ab 10.10 ± 0.45Ba 8.45 ± 1.00Bb 1.01 ± 0.06Bb 60.93 ± 3.78Ab 87.16 ± 20.55Ba 100.19 ± 3.02Ab 11.05 ± 0.59Ab SBt 1.01 ± 0.08Aa 12.93 ± 0.70Bb 11.95 ± 0.67Bb 1.43 ± 0.10Ba 89.72 ± 7.46Aa 78.43 ± 19.76Ba 121.93 ± 2.57Aa 13.17 ± 0.27Aa L(-) 0.62 ± 0.09Ba 13.51 ± 0.45Ab 13.30 ± 1.68Ab 2.14 ± 0.13Ab 38.50 ± 4.69Ab 104.25 ± 16.89Aa 13.41 ± 1.32Cb 5.19 ± 0.48Bb LBt 0.64 ± 0.11Ba 21.97 ± 1.48Aa 16.83 ± 0.51Aa 2.91 ± 0.04Aa 47.39 ± 2.42Aa 100.21 ± 15.00Aa 20.60 ± 0.69Ca 7.23 ± 0.69Ba P (mg plant−1) K (mg plant−1) Ca (mg plant−1) Mg (mg plant−1) Zn (µg plant−1) Fe (µg plant−1) Mn (µg plant−1) Cu (µg plant−1) T(-) 0.57 ± 0.11Ab 7.07 ± 0.25Bb 6.01 ± 1.85Bb 1.70 ± 0.62Ab 33.28 ± 1.69Ba 42.79 ± 3.67ABb 40.35 ± 14.51Ba 4.21 ± 0.27Bb TBt 0.86 ± 0.09Aa 10.42 ± 0.07Ba 7.94 ± 0.28Ba 2.21 ± 0.24Aa 54.06 ± 5.90Bb 115.22 ± 17.83ABa 46.49 ± 0.74Ba 6.38 ± 1.04Ba S(-) 0.87 ± 0.07Ab 10.10 ± 0.45Ba 8.45 ± 1.00Bb 1.01 ± 0.06Bb 60.93 ± 3.78Ab 87.16 ± 20.55Ba 100.19 ± 3.02Ab 11.05 ± 0.59Ab SBt 1.01 ± 0.08Aa 12.93 ± 0.70Bb 11.95 ± 0.67Bb 1.43 ± 0.10Ba 89.72 ± 7.46Aa 78.43 ± 19.76Ba 121.93 ± 2.57Aa 13.17 ± 0.27Aa L(-) 0.62 ± 0.09Ba 13.51 ± 0.45Ab 13.30 ± 1.68Ab 2.14 ± 0.13Ab 38.50 ± 4.69Ab 104.25 ± 16.89Aa 13.41 ± 1.32Cb 5.19 ± 0.48Bb LBt 0.64 ± 0.11Ba 21.97 ± 1.48Aa 16.83 ± 0.51Aa 2.91 ± 0.04Aa 47.39 ± 2.42Aa 100.21 ± 15.00Aa 20.60 ± 0.69Ca 7.23 ± 0.69Ba Standard Errors are given. Within each parameter, values followed by the same capital letter did not differ between plants while the values followed by the same small letter did not differed between inoculation within plant species (P ≤ 0.05) as determined by Tukey’s test (n= 3). View Large Remarkably, the plant P content was not significantly correlated with the percentage of AMF colonization (P > 0.20). The predominant AMF species identified in the native consortium in soil of this study were as follows: Septoglomus constrictum, Diversispora aunantia, Archaespora trappei, Glomus versiforme and Paraglomus ocultum, which were cataloged and included in the collection of EEZ (codes EEZ 198 to EEZ 202). The analysis of AMF root colonization revealed a distinct pattern. Among all non-inoculated species, T. vulgaris had the highest percentage of AMF root colonization. Bacteria inoculation on S. chamaecyparissus significantly increased the percentage AMF root colonization by 92% and total AMF colonization by 145% with respect to the non-inoculated controls (Table 2). However, we did not detect any statistical differences in AMF root colonization for L. dentata with or without inoculation. We also report a significant relationship between the percentage of AMF root colonization and shoot dry mass (r²=0.48, P<0.01) (Figure S1, Supporting Information). Table 2. Soil enzymatic activities and AMF root colonization in the rhizosphere of three autochthonous plants Thymus vulgaris (T), Santolina chamaecyparissus (S) andLavandula dentata (L) grown in natural arid Mediterranean soil under drought stress conditions as affected by the inoculation of the autochthonous bacteria strain Bacillus thuringiensis (Bt). Dehydrogenase (µg INTF g−1) β-glucosidase (µmol PNF g−1 soil h−1) Urease (µmol N-NH3 g−1 h−1) Alkaline Phosphatase (µmol PNF g soil−1 h−1) AMF (%) Total AMF colonization T(-) 71.9 ± 0.49Ba 137.8 ± 0.00Ba 606.5 ± 19.96Aa 181.2 ± 26.90Ba 54 ± 3.0Aa 231 ± 37.6Aa TBt 66.5 ± 3.19Ba 177.8 ± 0.02Ba 577.3 ± 21.56Aa 141.7 ± 28.96Ba 48 ± 3.5Aa 162 ± 24.2Aa S(-) 88.6 ± 3.36Aa 201.5 ± 0.02Ba 526.5 ± 16.95Ab 302.4 ± 17.71Aa 24 ± 4.1Bb 64 ± 22.4Bb SBt 72.9 ± 5.11Aa 186.9 ± 0.04Ba 687.2 ± 71.87Aa 215.1 ± 5.00Aa 46 ± 4.6Aa 157 ± 25.7Aa L(-) 87.3 ± 6.65Aa 367.6 ± 56.37Aa 622.1 ± 35.97Ab 221.4 ± 36.02Aa 27 ± 2.1Ba 97 ± 16.2Ba LBt 80.6 ± 4.08Aa 346.2 ± 37.05Aa 679.6 ± 12.09Aa 224.5 ± 12.58Aa 30 ± 1.7Aa 153 ± 1.8Aa Dehydrogenase (µg INTF g−1) β-glucosidase (µmol PNF g−1 soil h−1) Urease (µmol N-NH3 g−1 h−1) Alkaline Phosphatase (µmol PNF g soil−1 h−1) AMF (%) Total AMF colonization T(-) 71.9 ± 0.49Ba 137.8 ± 0.00Ba 606.5 ± 19.96Aa 181.2 ± 26.90Ba 54 ± 3.0Aa 231 ± 37.6Aa TBt 66.5 ± 3.19Ba 177.8 ± 0.02Ba 577.3 ± 21.56Aa 141.7 ± 28.96Ba 48 ± 3.5Aa 162 ± 24.2Aa S(-) 88.6 ± 3.36Aa 201.5 ± 0.02Ba 526.5 ± 16.95Ab 302.4 ± 17.71Aa 24 ± 4.1Bb 64 ± 22.4Bb SBt 72.9 ± 5.11Aa 186.9 ± 0.04Ba 687.2 ± 71.87Aa 215.1 ± 5.00Aa 46 ± 4.6Aa 157 ± 25.7Aa L(-) 87.3 ± 6.65Aa 367.6 ± 56.37Aa 622.1 ± 35.97Ab 221.4 ± 36.02Aa 27 ± 2.1Ba 97 ± 16.2Ba LBt 80.6 ± 4.08Aa 346.2 ± 37.05Aa 679.6 ± 12.09Aa 224.5 ± 12.58Aa 30 ± 1.7Aa 153 ± 1.8Aa Standard Errors are given. Within each parameter, values followed by the same capital letter did not differ between plants while the values followed by the same small letter did not differed between inoculations within plant species (P ≤ 0.05) as determined by Tukey’s test. View Large Table 2. Soil enzymatic activities and AMF root colonization in the rhizosphere of three autochthonous plants Thymus vulgaris (T), Santolina chamaecyparissus (S) andLavandula dentata (L) grown in natural arid Mediterranean soil under drought stress conditions as affected by the inoculation of the autochthonous bacteria strain Bacillus thuringiensis (Bt). Dehydrogenase (µg INTF g−1) β-glucosidase (µmol PNF g−1 soil h−1) Urease (µmol N-NH3 g−1 h−1) Alkaline Phosphatase (µmol PNF g soil−1 h−1) AMF (%) Total AMF colonization T(-) 71.9 ± 0.49Ba 137.8 ± 0.00Ba 606.5 ± 19.96Aa 181.2 ± 26.90Ba 54 ± 3.0Aa 231 ± 37.6Aa TBt 66.5 ± 3.19Ba 177.8 ± 0.02Ba 577.3 ± 21.56Aa 141.7 ± 28.96Ba 48 ± 3.5Aa 162 ± 24.2Aa S(-) 88.6 ± 3.36Aa 201.5 ± 0.02Ba 526.5 ± 16.95Ab 302.4 ± 17.71Aa 24 ± 4.1Bb 64 ± 22.4Bb SBt 72.9 ± 5.11Aa 186.9 ± 0.04Ba 687.2 ± 71.87Aa 215.1 ± 5.00Aa 46 ± 4.6Aa 157 ± 25.7Aa L(-) 87.3 ± 6.65Aa 367.6 ± 56.37Aa 622.1 ± 35.97Ab 221.4 ± 36.02Aa 27 ± 2.1Ba 97 ± 16.2Ba LBt 80.6 ± 4.08Aa 346.2 ± 37.05Aa 679.6 ± 12.09Aa 224.5 ± 12.58Aa 30 ± 1.7Aa 153 ± 1.8Aa Dehydrogenase (µg INTF g−1) β-glucosidase (µmol PNF g−1 soil h−1) Urease (µmol N-NH3 g−1 h−1) Alkaline Phosphatase (µmol PNF g soil−1 h−1) AMF (%) Total AMF colonization T(-) 71.9 ± 0.49Ba 137.8 ± 0.00Ba 606.5 ± 19.96Aa 181.2 ± 26.90Ba 54 ± 3.0Aa 231 ± 37.6Aa TBt 66.5 ± 3.19Ba 177.8 ± 0.02Ba 577.3 ± 21.56Aa 141.7 ± 28.96Ba 48 ± 3.5Aa 162 ± 24.2Aa S(-) 88.6 ± 3.36Aa 201.5 ± 0.02Ba 526.5 ± 16.95Ab 302.4 ± 17.71Aa 24 ± 4.1Bb 64 ± 22.4Bb SBt 72.9 ± 5.11Aa 186.9 ± 0.04Ba 687.2 ± 71.87Aa 215.1 ± 5.00Aa 46 ± 4.6Aa 157 ± 25.7Aa L(-) 87.3 ± 6.65Aa 367.6 ± 56.37Aa 622.1 ± 35.97Ab 221.4 ± 36.02Aa 27 ± 2.1Ba 97 ± 16.2Ba LBt 80.6 ± 4.08Aa 346.2 ± 37.05Aa 679.6 ± 12.09Aa 224.5 ± 12.58Aa 30 ± 1.7Aa 153 ± 1.8Aa Standard Errors are given. Within each parameter, values followed by the same capital letter did not differ between plants while the values followed by the same small letter did not differed between inoculations within plant species (P ≤ 0.05) as determined by Tukey’s test. View Large Microbial enzymatic activity and PFLA composition in the rhizosphere of the three plant species β-glucosidase activity was highest in L. dentata rhizospheres, alkaline phosphatase activity was highest in S. chamaecyparissus rhizospheres, and dehydrogenase activity was highest in both plant species. Inoculation with strain IAM 12077 did not affect any of the enzymatic activities measured for all three plant species, except that the urease activity of S. chamaecyparissus increased by 30.5% compared with the non-inoculated control (Table 2). The lipid abundance of the microbial community including bacteria, fungi, Actinomycetes, AMF, G+ and G− bacteria, total PLFA and total neutral lipids fatty acids (NLFA) was lower in L. dentata rhizosphere. Bacteria, fungi, AMF and total PLFA significantly increased in S. chamaecyparissus, while Actinomycetes, G+ and G− bacteria increased for both T. vulgaris and S. chamaecyparissus. ANOVA analysis revealed significant differences (P ≤0.05) for the microbial biomarkers of bacteria, fungi, Actinomycetes, G+ and G− bacteria and total PLFA of the rhizosphere of all three autochthonous plants species. Inoculation with strain IAM 12077 for each plant species did not significantly affect the lipid abundance of the rhizosphere microbial community, although inoculated L. dentata had a low content of acidic phospholipid biomarkers, but the content of NLFA was significantly increased compared to the non-inoculated control (L(-)) (Table 3). Table 3. Content of phospholipid acid (µg PLFA g−1 sed) and neutral lipids acid biomarkers (µg NLFA gr−1 sed) in the rhizosphere of the three autochthonous plants species [Thymus vulgaris (T),Santolina chamaecyparissus (S) andLavandula dentata (L)] inoculated (Bt) or not (-) with bacteria strain IAM 12077 Bacillus thuringiensis grown in natural arid Mediterranean soil under drought stress conditions. Bacteria Fungi Actinomycetes Mycorrhiza Gram+ Gram− Total PLFA Total NLFA T(-) 0.307 ± 0.11ABa 0.143 ± 0.052ABa 0.059 ± 0.018Aa 0.042 ± 0.012Aa 0.015 ± 0.005Aa 0.020 ± 0.006Aa 0.552 ± 0.19ABa 14.37 ± 2.7Ab TBt 0.601 ± 0.48ABa 0.237 ± 0.187ABa 0.207 ± 0.111Aa 0.098 ± 0.078Aa 0.046 ± 0.020Aa 0.041 ± 0.008Aa 1.144 ± 0.84ABa 18.48 ± 0.8Aa S(-) 0.581 ± 0.25Aa 0.266 ± 0.101Aa 0.126 ± 0.054Aa 0.069 ± 0.025Aa 0.028 ± 0.010ABa 0.036 ± 0.013Aa 1.042 ± 0.43Aa 9.91 ± 5.2Ab SBt 0.754 ± 0.10Aa 0.323 ± 0.054Aa 0.165 ± 0.021Aa 0.110 ± 0.019Aa 0.026 ± 0.013ABa 0.039 ± 0.018Aa 1.353 ± 0.18Aa 19.42 ± 4.7Aa L(-) 0.015 ± 0.004Ba 0.012 ± 0.007Ba 0.001 ± 0.000Ba 0.003 ± 0.000Aa 0.001 ± 0.000Ba 0.002 ± 0.000Ba 0.032 ± 0.01Ba 10.34 ± 2.7Ab LBt 0.093 ± 0.003Ba 0.046 ± 0.017Ba 0.020 ± 0.000Ba 0.022 ± 0.000Aa 0.006 ± 0.000Ba 0.012 ± 0.000Ba 0.181 ± 0.02Ba 22.77 ± 2.5Aa Bacteria Fungi Actinomycetes Mycorrhiza Gram+ Gram− Total PLFA Total NLFA T(-) 0.307 ± 0.11ABa 0.143 ± 0.052ABa 0.059 ± 0.018Aa 0.042 ± 0.012Aa 0.015 ± 0.005Aa 0.020 ± 0.006Aa 0.552 ± 0.19ABa 14.37 ± 2.7Ab TBt 0.601 ± 0.48ABa 0.237 ± 0.187ABa 0.207 ± 0.111Aa 0.098 ± 0.078Aa 0.046 ± 0.020Aa 0.041 ± 0.008Aa 1.144 ± 0.84ABa 18.48 ± 0.8Aa S(-) 0.581 ± 0.25Aa 0.266 ± 0.101Aa 0.126 ± 0.054Aa 0.069 ± 0.025Aa 0.028 ± 0.010ABa 0.036 ± 0.013Aa 1.042 ± 0.43Aa 9.91 ± 5.2Ab SBt 0.754 ± 0.10Aa 0.323 ± 0.054Aa 0.165 ± 0.021Aa 0.110 ± 0.019Aa 0.026 ± 0.013ABa 0.039 ± 0.018Aa 1.353 ± 0.18Aa 19.42 ± 4.7Aa L(-) 0.015 ± 0.004Ba 0.012 ± 0.007Ba 0.001 ± 0.000Ba 0.003 ± 0.000Aa 0.001 ± 0.000Ba 0.002 ± 0.000Ba 0.032 ± 0.01Ba 10.34 ± 2.7Ab LBt 0.093 ± 0.003Ba 0.046 ± 0.017Ba 0.020 ± 0.000Ba 0.022 ± 0.000Aa 0.006 ± 0.000Ba 0.012 ± 0.000Ba 0.181 ± 0.02Ba 22.77 ± 2.5Aa Standard Errors are given. Within each parameter, values followed by the same capital letter did not differ between plants while the values followed by the same small letter did not differed between inoculations within plant species (P ≤ 0.05) as determined by Tukey’s test (n= 3). View Large Table 3. Content of phospholipid acid (µg PLFA g−1 sed) and neutral lipids acid biomarkers (µg NLFA gr−1 sed) in the rhizosphere of the three autochthonous plants species [Thymus vulgaris (T),Santolina chamaecyparissus (S) andLavandula dentata (L)] inoculated (Bt) or not (-) with bacteria strain IAM 12077 Bacillus thuringiensis grown in natural arid Mediterranean soil under drought stress conditions. Bacteria Fungi Actinomycetes Mycorrhiza Gram+ Gram− Total PLFA Total NLFA T(-) 0.307 ± 0.11ABa 0.143 ± 0.052ABa 0.059 ± 0.018Aa 0.042 ± 0.012Aa 0.015 ± 0.005Aa 0.020 ± 0.006Aa 0.552 ± 0.19ABa 14.37 ± 2.7Ab TBt 0.601 ± 0.48ABa 0.237 ± 0.187ABa 0.207 ± 0.111Aa 0.098 ± 0.078Aa 0.046 ± 0.020Aa 0.041 ± 0.008Aa 1.144 ± 0.84ABa 18.48 ± 0.8Aa S(-) 0.581 ± 0.25Aa 0.266 ± 0.101Aa 0.126 ± 0.054Aa 0.069 ± 0.025Aa 0.028 ± 0.010ABa 0.036 ± 0.013Aa 1.042 ± 0.43Aa 9.91 ± 5.2Ab SBt 0.754 ± 0.10Aa 0.323 ± 0.054Aa 0.165 ± 0.021Aa 0.110 ± 0.019Aa 0.026 ± 0.013ABa 0.039 ± 0.018Aa 1.353 ± 0.18Aa 19.42 ± 4.7Aa L(-) 0.015 ± 0.004Ba 0.012 ± 0.007Ba 0.001 ± 0.000Ba 0.003 ± 0.000Aa 0.001 ± 0.000Ba 0.002 ± 0.000Ba 0.032 ± 0.01Ba 10.34 ± 2.7Ab LBt 0.093 ± 0.003Ba 0.046 ± 0.017Ba 0.020 ± 0.000Ba 0.022 ± 0.000Aa 0.006 ± 0.000Ba 0.012 ± 0.000Ba 0.181 ± 0.02Ba 22.77 ± 2.5Aa Bacteria Fungi Actinomycetes Mycorrhiza Gram+ Gram− Total PLFA Total NLFA T(-) 0.307 ± 0.11ABa 0.143 ± 0.052ABa 0.059 ± 0.018Aa 0.042 ± 0.012Aa 0.015 ± 0.005Aa 0.020 ± 0.006Aa 0.552 ± 0.19ABa 14.37 ± 2.7Ab TBt 0.601 ± 0.48ABa 0.237 ± 0.187ABa 0.207 ± 0.111Aa 0.098 ± 0.078Aa 0.046 ± 0.020Aa 0.041 ± 0.008Aa 1.144 ± 0.84ABa 18.48 ± 0.8Aa S(-) 0.581 ± 0.25Aa 0.266 ± 0.101Aa 0.126 ± 0.054Aa 0.069 ± 0.025Aa 0.028 ± 0.010ABa 0.036 ± 0.013Aa 1.042 ± 0.43Aa 9.91 ± 5.2Ab SBt 0.754 ± 0.10Aa 0.323 ± 0.054Aa 0.165 ± 0.021Aa 0.110 ± 0.019Aa 0.026 ± 0.013ABa 0.039 ± 0.018Aa 1.353 ± 0.18Aa 19.42 ± 4.7Aa L(-) 0.015 ± 0.004Ba 0.012 ± 0.007Ba 0.001 ± 0.000Ba 0.003 ± 0.000Aa 0.001 ± 0.000Ba 0.002 ± 0.000Ba 0.032 ± 0.01Ba 10.34 ± 2.7Ab LBt 0.093 ± 0.003Ba 0.046 ± 0.017Ba 0.020 ± 0.000Ba 0.022 ± 0.000Aa 0.006 ± 0.000Ba 0.012 ± 0.000Ba 0.181 ± 0.02Ba 22.77 ± 2.5Aa Standard Errors are given. Within each parameter, values followed by the same capital letter did not differ between plants while the values followed by the same small letter did not differed between inoculations within plant species (P ≤ 0.05) as determined by Tukey’s test (n= 3). View Large There was a significant effect for all three plant species with respect to the profile of microbial PLFA in the rhizospheres. Significant differences were found for the bacterial biomarkers (C17:1w8c and C18:1w9t) and fungal biomarkers (C18:1w9c and C18:2w6c) (Table S1, Supporting Information). Inoculation did not significantly influence the profiles of fatty acids (Wilk’s = 0.39, P > 0.05). BCA analysis also pointed to statistically relevant differences for non-inoculated plants and to a non-significant difference for bacterial inoculation. The results showed that 98.5% of the total variance in this dataset relates to the differences between plant species (Fig. 2) where we observed that the microbial activity (enzymatic activity) and PFLA composition of L. dentata presented a distinct profile compared to those of T. vulgaris and S. chamaecyparissus. Figure 2. View largeDownload slide Effect of three autochthonous plant species of natural arid Mediterranean under drought stress condition on rhizosphere microbial activity and abundance (enzymatic activity and PFLA composition). The interclass inertia was 98.5%, and the Monte Carlo permutation level of significance was P = 0.001 after 999 permutations. Figure 2. View largeDownload slide Effect of three autochthonous plant species of natural arid Mediterranean under drought stress condition on rhizosphere microbial activity and abundance (enzymatic activity and PFLA composition). The interclass inertia was 98.5%, and the Monte Carlo permutation level of significance was P = 0.001 after 999 permutations. Bacterial and fungal community structure The numbers of total sequences were 107,667 of fungi and 81,135 of bacteria. The sequence reads resulted in 900 OTUs for fungi and 3,756 OTUs for bacteria. Table S2 (Supporting Information) presents the total abundance medians and interquartile range for both bacterial and fungal communities according to treatments. Analysis of absolute abundance of both bacterial (W = 119.1, P = 0.82) and fungal (W = 64.7, P = 0.65) OTUs at the family level for the various treatments did not reveal any significant effects after 1 year of strain IAM 12077 inoculation. Most bacterial abundance (47%) was related to five major taxa: Rubrobacter (13.3%), unclassified Solirubrobacterales (10.8%), unclassified Actinobacteria (9.2%), Solirubrobacter (8.2%) and unclassified Geodermatophilaceae (5.3%). Contrastingly, fungal taxa accounted for 55% of total abundance reported for the experiment, and the fungi belonged to taxa that included Hypocreales (22%), Hypocreomicetidea unclassified (9.4%), Dothideomycetesincertaesedis (8.6%), Agaricales (7.8%) and Glomus (7.6%). The population abundance of Bacillales order that comprises our strain IAM 12077 inoculum also did not show any difference among treatments, after 1 year of inoculation (W = 5.47, P> 0,05). In summary, our analysis of changes in the microbial communities due to inoculation showed no significant effects. We also observed only a small impact due to the total number of reads in our analysis (W = 1.72×10−12, P > 0.05). Interactions of microbial community structure and activity, and shoot nutrient acquisition The co-inertia analysis showed a significant covariance between bacterial and fungal soil rhizosphere communities, and between microbial activity and abundance (enzymatic activity and PFLA composition) and plant biomass and nutrients (Fig. 3). We found that 55% of total data variance for plant nutrients and biomass correlated with microbial activity and abundance, while 72% of fungal community variability strongly associated with bacterial community variance. Figure 3. View largeDownload slide Co-inertia analysis results from plant biomass and nutrients (pln), microbial activity and abundance (enzymatic activity and PFLA composition) (act), and bacterial (bac) and fungi (fun) community datasets. Figure 3. View largeDownload slide Co-inertia analysis results from plant biomass and nutrients (pln), microbial activity and abundance (enzymatic activity and PFLA composition) (act), and bacterial (bac) and fungi (fun) community datasets. Based on the co-inertia analysis results, we investigated how plant biomass and nutrients interacted with microbial activity and abundance. We found 20 positive interactions and 8 negative interactions (Fig. 4). Total fungi activity and abundance correlates negatively with root dry weight (r = −0.51, P< 0.05) and is negatively associated with plant Mg-content (r = −0.55, P < 0.05). β-glucosidase relates positively with plant K (r = 0.75), Ca (r = 0.67) and Fe contents (r = 0.51) and was negatively associated with the percentage of AMF (r = 0.53) and Mn (r = 0.60) content. We also report a negative relationship between dehydrogenase and the percentage of AMF (r = −0.67, P < 0.05) and the total AMF colonization. The same was true for alkaline phosphatase activity, which correlated negatively with the percentage of AMF (r = −0.63, P< 0.05) and total AMF colonization (r = −0.55, P < 0.05). On the other hand, enzymatic activity, lipid acid abundance, fatty acid composition of total bacteria, total fungi, Actinomycetes and mycorrhiza likely contributed to the accumulation of P (rbac = 0.58, rfun = 0.55, ract = 0.58 and rmyc = 0.57), Zn (rbac = 0.54, rfun = 0.50, ract = 0.54 and rmyc = 0.54), Mn (rbac = 0.72, rfun = 0.73, ract = 0.67 and rmyc = 0.71) and Cu (rbac = 0.52, rfun = 0.52 and rmyc = 0.52). We also report a strong relationship between the presence of G− bacteria and plant Mn content (r = 0.57, P< 0.05). Figure 4. View largeDownload slide Diagram of correlations between microbial activity, acidic lipid abundance, fatty acid composition and plant biomass and nutrients. Blue squares represent positive correlations, and red squares represent negative correlations. Darker/lighter colors indicate stronger/weaker correlations. White squares are non-significant relationships. Figure 4. View largeDownload slide Diagram of correlations between microbial activity, acidic lipid abundance, fatty acid composition and plant biomass and nutrients. Blue squares represent positive correlations, and red squares represent negative correlations. Darker/lighter colors indicate stronger/weaker correlations. White squares are non-significant relationships. We also evaluated the co-occurrence between bacterial and fungal communities in the rhizosphere of all the three plant species (Fig. 5). We found 122 positive associations and 61 negative associations between the rhizosphere bacterial and fungal communities. Presence of the genus Glomus correlated negatively with bacteria from the groups Sphingosinicella and Acidobacteria Gp6, while the presence of Glomus correlated positively with Acidobacteria Gp16. We observed two positive interactions between the genus Paraglomus with Pseudomonadaceae (unclassified) and Microvirga. In summary, our analysis showed the interactions between microbial activity and abundance (enzyme activity and PFLA composition) and plant biomass, and between bacterial and fungal communities, under drought conditions are majorly positive, while interactions of two genera of mycorrhiza (Glomus and Paraglomus) associated both positively and negatively with bacterial groups. Figure 5. View largeDownload slide Diagram of correlations between bacterial and fungal community inhabiting the rhizosphere of the three plant species. The shades of blue squares represent positive correlations, and the shades of red squares represent negative correlations, white squares are non-significant relationships. Figure 5. View largeDownload slide Diagram of correlations between bacterial and fungal community inhabiting the rhizosphere of the three plant species. The shades of blue squares represent positive correlations, and the shades of red squares represent negative correlations, white squares are non-significant relationships. DISCUSSION Inoculation with strain IAM 12077 on seed and seedlings of three plant species resulted in plant growth and nutrient content acquisition, after 1 year under drought stress conditions. The inoculation increased shoot growth of all three autochthonous plant species studied. Strain IAM 12077 may enhance plant growth by various mechanisms such as optimizing the supply of nutrients and the solubilization of inorganic phosphorus (Glick 1995; He et al. 1997; Leggett, Gleddie and Holloway 2001). Hence, the application of phosphate solubilizing bacteria (PSB) could be a reasonable substitute for chemical phosphate fertilizers (Khan and Zaidi 2006). Previously, Armada et al. (2015b) showed that inoculation with strain IAM 12077 enhanced maize nutrient uptake of P by 37% and Fe, Zn and Cu, indicating the capacity of this strain on solubilization of non-available nutrients and the production of siderophores. Thus, this strain alone strongly impacted the plant nutrient uptake. The enhanced access to soil nutrients likely explains the increase of plant biomass, but strain IAM 12077 also seems to possess mechanism to improve plant tolerance under adverse conditions (Armada et al. 2016), and other growth promoting compounds such as indole-3-acetic acid (IAA). Altogether, the bacteria alone promoted plant growth without resulting in long-term changes on AMF colonization and the rhizosphere microbial community. Our analysis also showed no significant effect of strain IAM 12077 in both percentages of AMF and total AMF colonization. In general, AMF present host preference or host specificity (Vandenkoornhuyse et al. 2003; Öpik et al. 2006; Alguacil Roldán and Torres 2009), which might explain the lack of inoculated strain influence on this plant-fungi relationship. Nevertheless, the impact of bacteria in increasing drought tolerance processes seems to be more associated with the proportion of intraradical structures such as arbuscules than to the percentage of root colonized as previously reported (Marulanda, Azcón and Ruíz-Lozano 2003; Vivas, Barea and Azcón 2005; Armada et al. 2016). This specificity has ecological importance for revegetation programs in ecosystems that include autochthonous shrubs (Armada, Roldán and Azcón 2014; Mengual et al. 2014; Armada et al. 2015a,b, 2016), and our analysis suggests that inoculation with strain IAM 12077 may contribute to promoting P uptake without compromising plant-AMF relationships. However, we found that microbial activity of β-glucosidase, dehydrogenase and alkaline phosphatase were negatively correlated with the percentage of AMF colonization in roots. When measuring soil enzyme activities, it should be considered that potential activities are determined (Schloter, Dilly and Munch 2003). Notwithstanding, evaluating soil enzymatic activity remains useful as an indicator of biochemical potential, possible resilience and a sensor of changes in soil key functions (Taylor et al. 2002). Therefore, soil microbial activity, especially that of alkaline phosphatase points to a role for non-AMF microbial activity in solubilizing P for uptake in plants (Nakas, Gould and Klein 1987), which may contribute to the lack of significance between plant P-content and percentage of AMF root colonization. We report a positive correlation between bacterial and fungal activities. Therefore, strain IAM 12077 induced plant P uptake and plants interacted with soil-active microbes for continuous acquisition of soil nutrients, without the dependence on AMF for nutrient uptake. The strain IAM 12077 in the present study presents a great potential for improving nutrient acquisition, especially compared to organic fertilizer sources (Güneş et al. 2014). This might explain the increase of phosphorus content in T. vulgaris due to strain inoculation (+51%). Inoculation with strain IAM 12077 increased shoot K content for the three plant species studied and had the greatest influence on L. dentata (+63%). Potassium is one the most important soluble inorganic nutrients and regulates water uptake capacity by the roots (Wang et al. 2013), likely an essential process during plant growth under water stress conditions. According to Armada et al. (2016), the strain IAM 12077 modulates the plant antioxidant responses by decreasing oxidative stress, which contributes to improve the nutrient uptake and plant growth performance under stress conditions. The lack of significant changes in rhizosphere microbial community after 1 year of inoculation together with the increased nutrient concentration in plant tissue may suggest the nutrient acquisition resulted from a long-lasting effect of strain IAM 12077. We also found that strain IAM 12077-inoculated seeds and seedlings exhibited increase in both Ca and Mg contents compared to non-inoculated plants. Calcium acts in membrane protection, and magnesium contributes to modulation of ionic currents across chloroplasts and vacuole membranes (thus, regulating the stomatal opening and ion balance in cells), both of which are phenomena of particular relevance under drought conditions (Parida and Jha 2013). The enhancement of Mg content for inoculated plants suggests a reduced impact of drought on the functioning of the photosynthetic apparatus in these three plant species when colonized by strain IAM 12077. The plants used in our study belong to two different families (Lamiaceae and Asteraceae) but are all autochthonous drought-tolerant shrub species with deep roots that help to cope with nutrient stress in eroded soils (Francis and Thornes 1990). They belong to the natural succession of the shrubland community of semiarid Mediterranean ecosystems in the southeast of Spain (Alguacil et al. 2011). T. vulgaris is heliophylous plant that grows well in drained and calcareous soils, usually reaching 15–30 cm height (Dorling 2008). S. chamaecyparissus grows well on rocky soils with button-like flower-heads in summer; consists of an aromatic shrub that grows up to 75 cm (Dorling 2008). Finally, L. dentata prefers well-drained alkaline soils and sunny conditions (González 2007) and grows to 60 cm with linear or lance-shaped leaves and the whole plant is also strongly aromatic with a widely known fragrance (Bayer 2006). Considering that three plants in this study prefer well drained soil and they are naturally tolerant to drought conditions, the 1 year of controlled water-holding capacity simulated the aspects of stressful water conditions likely inducing the plants to intensify their interactions with soil microbiome, as indicated by the significant covariance between the soil microbial activity and plant nutrients and biomass. The microbial abundances, bacterial (C17:1w8c, C18:1w9t) and fungal biomarkers (C18:1w9c, C18:2w6c) and enzymatic activities differed significantly in the rhizosphere of the three plant species studied. However, strain IAM 12077 inoculation did not significantly influence the profile of fatty acids in the rhizosphere of these species. Therefore, after 1 year of inoculation, there was nearly no effect of the inoculated bacteria with respect to determining rhizosphere microbial activity and abundance (enzyme activity and PLFA composition), despite measurable contributions to plant growth and nutrient uptake. These results suggest a potential application of strain IAM 12077 as part of a revegetation strategy for enhancing plant growth and uptake of nutrients with a minimized impact on rhizosphere microbial activity and abundance. The PLFA technique provides a rapid and inexpensive method to access microbial biomass and composition (Frostegård, Tunlid and Bååth 2011), which may be even more sensitive in detecting shifts than methods based on DNA or RNA (Ramsey et al. 2006). However, PLFA lacks specificity since many different (and unknown) groups of organisms may present the same biomarker. Thus, molecular methods allow obtaining more accurate information on the soil microbial community by detecting not only the active microbes, but also the whole soil microbiota including the seedbank (Philippot et al. 2013). Based on that, we confirm that strain IAM 12077 did not induce a significant shift in the rhizozpshere microbial community after 1 year of inoculation. Moreover, the co-inertia results point to significant interactions between rhizosphere microbial activity and plant nutrition, and we later identified part of this covariance as single variable correlations. The β-glucosidase activity that was highest in L. dentata suggests carbohydrate transformation, which is important as an alternative energy source for microorganisms. Indeed, β-glucosidase activity is positively associated with plant K, Ca and Fe contents, which suggest that metabolically active microbes may directly contribute to plant nutrient uptake. S. chamaecyparissus had the greatest phosphorus content in shoot biomass and the increase of alkaline phosphatase activity. The cycling of N, C and P are controlled by hydrolase enzymes such as urease (N), β-glucosidase (C) and phosphatases (P), which are mainly synthesized by soil microorganisms (Ros et al. 2006). These hydrolases are involved in the mineralization of compounds that provide nutrients that include N, P and C. Therefore, rhizosphere active microbes contribute to plant nutrient uptake, while the strain IAM 12077 improved nutrient uptake. As our reported differences in microbial activities are plant-specific, we infer that plants might shift their microbial rhizosphere composition by activating microbes able to mediate soil nutrient uptake in plants. The Rv coefficient suggested that part of plant nutrient content results from microbial activity, which according to the BCA analysis is a plant-specific selection. Furthermore, ANOVA results also highlighted the role of strain IAM 12077 inoculation in plant nutrients. Therefore, beyond the role of strain IAM 12077 in the rhizosphere, the plants likely drive the microbial activity towards their nutrient necessities. Plant nutrient uptake probably occurred as a combination of plant selection of active microbes together with the beneficial roles of strain IAM 12077. The strongest link found in our study relates the rhizosphere bacterial and fungal community covariance for plants under water stress. Here we found major positive associations that were nearly double the number of negative microbial interactions. According to the stress-gradient hypothesis, species interactions increase their importance due to shifts from competition to facilitation with respect to stress (He and Bertness 2014). Our results provide support for this hypothesis, the strong covariance between bacterial and fungal communities indicates that the variance of some bacterial groups links with fungal community changes. In fact, fungal groups were positively associated with many of the evaluated bacterial groups (e.g. the genus Paraglomus was positively associated with Pseudomonadaceae and Microvirga). Furthermore, the bacterial community likely employs several physiological modifications in response to changing soil moisture, such as the production of exopolysaccharides (Kohler, Caravaca and Roldán 2009), sporulation (Landesman and Dighton 2010) and adjustment of internal water potential to match that of the external environment. Effects of microbial inoculum on the rhizosphere microbial community structure have often been reported for only short-term experiments ranging from 30 to 90 days (Cipriano et al. 2016) and usually for short-cycle crops (Schreiter et al. 2014). Since we aimed to describe the longer-term impact of strain IAM 12077 in rhizosphere community by evaluating their effects after 1 year, this may explain why we found weak changes induced by inoculation with respect to the total rhizosphere microbial community (results of DNA analysis). In addition, it is likely that the strain IAM 12077 produced a primer effect on plants (Cipriano et al. 2016) that resulted in positive effect on plant growth after 1 year of inoculation. Previous studies have shown the potential of PGPB as a strategy for successful ecosystem recovery strategies (Bashan et al. 2008,2012) and our study goes beyond by showing the dismal impact of strain IAM 12077 in rhizosphere microbial community. Thus, strategies for land recovery might be more effective by the use and application of existent PGPB present in soil. In conclusion, in greenhouse conditions, inoculation using native bacterial strain with PGPB properties enhanced the growth of three autochthonous shrubs species and nutrient uptake without changing the rhizosphere microbial diversity and did not affect AMF groups after 1 year of inoculation under water stress conditions. Plants possess ecological advantages by fostering soil microbial seedbank and assembly their rhizosphere microbial populations (Mendes et al. 2014; Barbosa Lima et al. 2015; Cipriano et al. 2016; Schlemper et al. 2017a,b). The inoculation of autochthonous shrubs species with strain IAM 12077 may be a sustainable option for recovering degraded soils without harming and impacting the rhizosphere microbial community structure and activity over the long-term. However, future studies in field conditions are needed to evaluate the responses of plants inoculated with this strain. SUPPLEMENTARY DATA Supplementary data are available at FEMSEC online. ACKNOWLEDGEMENTS We thank Domingo Álvarez for the morphological identification of autochthonous mycorrhizal fungus. Publication number 6531 of the NIOO-KNAW, Netherlands Institute of Ecology. FUNDING E. Armada was financed by the Ministry of Science and Innovation, Spain. This work was carried out in the framework of the project reference AGL2009-12530-C02-02 with a grant of short stay (ref. BES-2010-042736) at NIOO-KNAW in Wageningen, and the grant The Netherlands Organization for Scientific Research (NWO, 729.004.016), M.F.A. Leite was financed by CAPES A116-2013 program. Conflict of interest. None declared. REFERENCES Aboim MCR , Coutinho HLC , Peixoto RS et al. Soil bacterial community structure and soil quality in a slash-and-burn cultivation system in Southeastern Brazil . Appl Soil Ecol . 2008 ; 38 : 100 – 8 . Google Scholar CrossRef Search ADS Alguacil MM , Roldán A , Torres MP . Complexity of semiarid gypsophilous shrub communities mediates the AMF biodiversity at the plant species level . Microb Ecol . 2009 ; 57 : 718 – 27 . Google Scholar CrossRef Search ADS PubMed Alguacil MM , Torres MP , Torrecillas E et al. Plant type differently promote the arbuscular mycorrhizal fungi biodiversity in the rhizosphere after revegetation of a degraded semiarid land . Soil Biol Biochem . 2011 ; 43 : 167 – 73 . Google Scholar CrossRef Search ADS Armada E , Azcón R , López-Castillo OM et al. Autochthonous arbuscular mycorrhizal fungi and Bacillus thuringiensis from a degraded Mediterranean area can be used to improve physiological traits and performance of a plant of agronomic interest under drought conditions . Plant Physiol Biochem . 2015a ; 90 : 64 – 74 . Google Scholar CrossRef Search ADS Armada E , Barea JM , Castillo P et al. Characterization and management of autochthonous bacterial strains from semiarid soils of Spain and their interactions with fermented agrowastes to improve drought tolerance in native shrub species . Appl Soil Ecol . 2015b ; 96 : 306 – 18 . Google Scholar CrossRef Search ADS Armada E , Probanza A , Roldán A et al. Native plant growth promoting bacteria Bacillus thuringensis and mixed or individual mycorrhizal species improved drought tolerance and oxidative metabolism in Lavandula dentata plants . J Plant Physiol . 2016 ; 192 : 1 – 12 . Google Scholar CrossRef Search ADS PubMed Armada E , Roldán A , Azcón R . Differential activity of autochthonous bacteria in controlling drought stress in native Lavandula and Salvia plants species under drought conditions in natural arid soil . Microb Ecol . 2014 ; 67 : 410 – 20 . Google Scholar CrossRef Search ADS PubMed Bååth E . The use of neutral lipid fatty acids to indicate the physiological conditions of soil fungi . Microb Ecol . 2003 ; 45 : 373 – 83 . Google Scholar CrossRef Search ADS PubMed Barbosa Lima A , Souza Cannavan FS , Navarrete AA et al. Amazonian Dark Earth and plant species from the Amazon region contribute to shape rhizosphere bacterial communities . Microb Ecol . 2015 ; 69 : 855 – 66 . Google Scholar CrossRef Search ADS PubMed Bashan Y , Puente ME , de-Bashan LE et al. Environmental uses of plant growth-promoting bacteria , In Plant-Microbe interactions 2008. , E . Ait Barka , Clément C (Ed). in Chapter 4 , ( Trivandrun, Kerala, India : Research Signpost ), 69 – 93 . Bashan Y , Salazar BG , Moreno M et al. Restoration of eroded soil in the Sonoran Desert with native leguminous trees using plant growth-promoting microorganisms and limited amounts of compost and water . J Environ Manage . 2012 ; 102 : 26 – 36 . Google Scholar CrossRef Search ADS PubMed Bayer E . Plantas del Mediterráneo : Blume , 2006 . Benjamini Y , Hochberg Y . Controlling the false discovery rate:a practical and powerful approach to multiple testing . J R Statistl Soc Series B (Methodological) . 1995 ; 57 : 289 – 300 . Bertani G . Studies on lysogenes I. The mode of phage liberation by lysogenic Escherichia coli . J Bacteriol . 1951 ; 62 : 293 – 300 . Google Scholar PubMed Brundrett M Practical Methods in Mycorrhizal Research , Mycologue Publications 1994 . Chessel D , Dufour AB , Thioulouse J . The ade4 package-I- One-table methods . R News . 2004 ; 4 : 5 – 10 . Chowdhury SP , Dietel K , Rändler M et al. Effects of Bacillus amyloliquefaciens FZB42 on lettuce growth and health under pathogen pressure and its impact on the rhizosphere bacterial community . PLoS ONE . 2013 ; 8 : e68818 . Google Scholar CrossRef Search ADS PubMed Cipriano MAP , Lupatini M , Lopes-Santos L et al. Lettuce and rhizosphere microbiome responses to growth promoting Pseudomonas species under field conditions . FEMS Microbiol Ecol . 2016 ; 92 : 1 – 12 . Google Scholar CrossRef Search ADS Dorling K . RHS AZ Encyclopedia of Garden Plants , Publisher DK, 3 edition , 2008 . Dray S , Chessel D , Thioulouse J . Co-inertia analysis and the linking of ecological data tables . Ecology . 2003 ; 84 : 3078 – 89 . Google Scholar CrossRef Search ADS Dray S , Dufour AB . The ade4 package:implementing the duality diagram for ecologists . J Statist Softw . 2007 ; 22 : 1 – 20 . Google Scholar CrossRef Search ADS Dray S , Jombart T . Revisiting Guerry’s data:introducing spatial constraints in multivariate analysis . Ann Appl Stat . 2011 ; 5 : 2278 – 99 . Google Scholar CrossRef Search ADS Drigo B , Pijl AS , Duyts H et al. Shifting carbon flow from roots into associated microbial communities in response to elevated atmospheric CO2 . P Nat Acad Sci USA . 2010 ; 107 : 10938 – 42 . Google Scholar CrossRef Search ADS Francis DF , Thornes JB . Matorral:erosion and reclamation . In: Albaladejo J , Stocking MA , Díaz , E (eds). Soil Degradation and Rehabilitation in Mediterranean Environmental Conditions . Murcia, Spain : Consejo Superior de Investigaciones Científicas , 1990 , 87 – 115 . Frostegård Å , Bååth E , Tunlio A . Shifts in the structure of soil microbial communities in limed forests as revealed by phospholipid fatty acid analysis . Soil Biol Biochem . 1993a ; 25 : 723 – 30 . Google Scholar CrossRef Search ADS Frostegård A , Bååth E . The use of phospholipid fatty acid analysis to estimate bacterial and fungal biomass in soil . Biol Fert Soils . 1996 ; 22 : 59 – 65 . Google Scholar CrossRef Search ADS Frostegard A , Tunlid A , Baath E . Phospholipid fatty acid composition, biomass, and activity of microbial communities from two soil types experimentally exposed to different heavy metals . Appl Environ Microbiol . 1993b ; 59 : 3605 – 17 . Frostegård Å , Tunlid A , Bååth E . Use and misuse of PLFA measurements in soils . Soil Biol Biochem . 2011 ; 43 : 1621 – 5 . Google Scholar CrossRef Search ADS García C , Hernández MT , Costa F . Potential use of dehydrogenase activity as an index of microbial activity in degraded soils . Commun Soil Sci Plant Nut . 1997 ; 28 : 123 – 34 . Google Scholar CrossRef Search ADS Giovannetti M , Mosse B . Evaluation of techniques for measuring vesicular arbuscular mycorrhizal infection in roots . New Phytol . 1980 ; 84 : 489 – 500 . Google Scholar CrossRef Search ADS Glick BR . The enhancement of plant growth by free-living bacteria . Can J Microbiol . 1995 ; 41 : 109 – 17 . Google Scholar CrossRef Search ADS González GAL Guía de los árboles y arbustos de la Península Ibérica y Baleares:(especies silvestres y las cultivadas más comunes): Editorial S. A. Mundi-prensa livros, 3rd edition , 2007 . Güneş A , Turan M , Güllüce M et al. Nutritional content analysis of plant growth-promoting rhizobacteria species . Eur J Soil Biol . 2014 ; 60 : 88 – 97 . Google Scholar CrossRef Search ADS He Q , Bertness MD . Extreme stresses, niches, and positive species interactions along stress gradients . Ecology . 2014 ; 95 : 1437 – 43 . Google Scholar CrossRef Search ADS PubMed He ZL , Baligar VC , Martens DC et al. Effect of phosphate rock, lime and cellulose on soil microbial biomass in acidic forest soil and its significance in carbon cycling . Biol Fert Soils . 1997 ; 24 : 329 – 34 . Google Scholar CrossRef Search ADS van der Heijden MGA , Klironomos JN , Ursic M et al. Mycorrhizal fungal diversity determines plant biodiversity, ecosystem variability and productivity . Nature . 1998 ; 396 : 69 – 72 . Google Scholar CrossRef Search ADS van der Heijden MGA , Streitwolf-Engel R , Riedl R et al. The mycorrhizal contribution to plant productivity, plant nutrition and soil structure in experimental grassland . New Phytol . 2006 ; 172 : 739 – 52 . Google Scholar CrossRef Search ADS PubMed Hothorn T , Bretz F , Westfall P . Simultaneous inference in general parametric models . Biometrical J . 2008 ; 50 : 346 – 63 . Google Scholar CrossRef Search ADS Jackson LE , Calderon FJ , Steenwerth KL et al. Responses of soil microbial processes and community structure to tillage events and implications for soil quality . Geoderma . 2003 ; 114 : 305 – 17 . Google Scholar CrossRef Search ADS Kennedy AC . Bacterial diversity in agroecosystems . Agric Ecosyst Environ . 1999 ; 74 : 65 – 76 . Google Scholar CrossRef Search ADS Khan MS , Zaidi A . Influence of composite inoculations of phosphate solubilizing organisms and an arbuscular mycorrhizal fungus on yield, grain protein and phosphorus and nitrogen uptake by greengram . Arch Agron Soil Sci . 2006 ; 52 : 579 – 90 . Google Scholar CrossRef Search ADS Kohler J , Caravaca F , Roldán A . Effect of drought on the stability of rhizosphere soil aggregates of Lactuca sativa grown in a degraded soil inoculated with PGPR and AM fungi . Appl Soil Ecol . 2009 ; 42 : 160 – 5 . Google Scholar CrossRef Search ADS Koske RE , Tessier BA . A convenient permanent slide mounting medium . Mycol Soc Amer Newslet . 1983 ; 34 : 59 . Landesman WJ , Dighton Response of soil microbial communities and the production of plant-available nitrogen to a two-year rainfall manipulation in the New Jersey Pinelands . Soil Biol Biochem . 2010 ; 42 : 1751 – 8 . Google Scholar CrossRef Search ADS Lee CK , Herbold CW , Polson SW et al. Groundtruthing next-gen sequencing for microbial ecology–biases and errors in community structure estimates from PCR amplicon pyrosequencing . PLoS ONE . 2012 ; 7 : e44224 . Google Scholar CrossRef Search ADS PubMed Legendre P , Gallagher E . Ecologically meaningful transformations for ordination of species data . Oecologia . 2001 ; 129 : 271 – 80 . Google Scholar CrossRef Search ADS PubMed Leggett M , Gleddie S , Holloway G . Phosphate-solubilizing microorganisms and their use . In: Ae N , Arihara J , Okada K , Srinivasan A (eds). Plant Nutrient Acquisition . Japan : Springer , 2001 , 299 – 318 . Google Scholar CrossRef Search ADS Lloyd A , Sheaffe MJ . Urease activity in soils . Plant Soil . 1973 ; 39 : 71 – 80 . Google Scholar CrossRef Search ADS Lottmann J , Heuer H , de Vries J et al. Establishment of introduced antagonistic bacteria in the rhizosphere of transgenic potatoes and their effect on the bacterial community . FEMS Microbiol Ecol . 2000 ; 33 : 41 – 9 . Google Scholar CrossRef Search ADS PubMed Marulanda A , Azcón R , Ruíz-Lozano J . Contribution of six arbuscular mycorrhizal fungal isolates to water uptake by Lactuca sativa plants under drought stress . Physiol Plant . 2003 ; 119 : 526 – 33 . Google Scholar CrossRef Search ADS Masciandaro G , Ceccanti B , García C . Anaerobic digestion of straw and piggery wastewater: II. Optimization of the process . Agrochimica . 1994 ; 3 : 195 – 203 . Mauclaire L , Pelz O et al. Assimilation of toluene carbon along a bacteria-protist food chain determined by 13C-enrichment of biomarker fatty acids . J Microbiol Methods . 2003 ; 55 : 635 – 49 . Google Scholar CrossRef Search ADS PubMed McMurdie PJ , Holmes S . Phyloseq:an R Package for reproducible interactive analysis and graphics of microbiome census data . PLoS ONE . 2013 ; 8 : e61217 . Google Scholar CrossRef Search ADS PubMed McMurdie PJ , Holmes S . Waste not, want not:Why rarefying microbiome data is inadmissible . PLoS Comput Biol . 2014 ; 10 : e1003531 . Google Scholar CrossRef Search ADS PubMed Mendes LW , Kuramae EE , Navarrete AA et al. Taxonomical and functional microbial community selection in soybean rhizosphere . ISME J . 2014 ; 8 : 1577 – 87 . Google Scholar CrossRef Search ADS PubMed Mengual C , Schoebitz M , Azcón R et al. Microbial inoculants and organic amendment improves plant establishment and soil rehabilitation under semiarid conditions . J Environ Manage . 2014 ; 134 : 1 – 7 . Google Scholar CrossRef Search ADS PubMed Nakas JP , Gould WD , Klein DA . Origin and expression of phosphatase activity in a semi-arid grassland soil . Soil Biol Biochem . 1987 ; 19 : 13 – 8 . Google Scholar CrossRef Search ADS Nannipieri P , Ascher J , Ceccherini MT et al. Microbial diversity and soil functions . Eur J Soil Sci . 2003 ; 54 : 655 – 70 . Google Scholar CrossRef Search ADS Nannipieri P , Ceccanti B , Cervelli S et al. Extraction of phosphatase, urease, proteases, organic-carbon, and nitrogen from soil . Soil Sci Soc Amer J . 1980 ; 44 : 1011 – 6 . Google Scholar CrossRef Search ADS Naseby DC , Lynch JM . Rhizosphere soil enzymes as indicators of perturbations caused by enzyme substrate addition and inoculation of a genetically modified strain of Pseudomonas fluorescens on wheat seed . Soil Biol Biochem . 1997 ; 29 : 1353 – 62 . Google Scholar CrossRef Search ADS Oehl F . Advances in glomeromycota taxonomy and classification . IMA fungus . 2011 ; 2 : 191 – 9 . Google Scholar CrossRef Search ADS PubMed Olsson PA , Baath E , Jakobsen I et al. The use of phospholipid and neutral lipid fatty acids to estimate biomass of arbuscular mycorrhizal fungi in soil . Mycolog Res . 1995 ; 99 : 623 – 9 . Google Scholar CrossRef Search ADS Öpik M , Moora M , Liira J et al. Composition of root-colonizing arbuscular mycorrhizal fungal communities in different ecosystems around the globe . J Ecol . 2006 ; 94 : 778 – 90 . Google Scholar CrossRef Search ADS Parida AK , Jha B . Physiological and biochemical responses reveal the drought tolerance efficacy of the halophyte Salicornia brachiata . J Plant Growth Regul . 2013 ; 32 : 342 – 52 . Google Scholar CrossRef Search ADS Peixoto RS , Chaer GM , Franco N et al. A decade of land use contributes to changes in the chemistry, biochemistry and bacterial community structures of soils in the Cerrado . Antonie Van Leeuwenhoek . 2010 ; 98 : 403 – 13 . Google Scholar CrossRef Search ADS PubMed Philippot L , Raaijmakers JM , Lemanceau P et al. Going back to the roots:the microbial ecology of the rhizosphere . Nat Rev Microbiol . 2013 ; 11 : 789 – 99 . Google Scholar CrossRef Search ADS PubMed Phillips JM , Hayman DS . Improved procedure of clearing roots and staining parasitic and vesicular-arbuscular mycorrhizal fungi for rapid assessment of infection . T Brit Mycol Soc . 1970 ; 55 : 159 – 61 . Google Scholar CrossRef Search ADS R Development Core Team R:A Language and Environment for Statistical Computing . Vienna, Austria : R Foundation for Statistical Computing , 2007 . Ramsey PW , Rillig MC , Feris KP et al. Choice of methods for soil microbial community analysis:PLFA maximizes power compared to CLPP and PCR-based approaches . Pedobiologia . 2006 ; 50 : 275 – 80 . Google Scholar CrossRef Search ADS Requena N , Jeffries P , Barea JM . Assessment of natural mycorrhizal potential in a desertified semiarid ecosystem . Appl Environ Microbiol . 1996 ; 62 : 842 – 7 . Google Scholar PubMed Ros M , Pascual JA , Garcia C et al. Hydrolase activities, microbial biomass and bacterial community in a soil after long-term amendment with different composts . Soil Biol Biochem . 2006 ; 38 : 3443 – 52 . Google Scholar CrossRef Search ADS Scherwinski K , Grosch R , Berg G . Effect of bacterial antagonists on lettuce:active biocontrol of Rhizoctonia solani and negligible, short-term effects on nontarget microorganisms . FEMS Microbiol Ecol . 2008 ; 64 : 106 – 16 . Google Scholar CrossRef Search ADS PubMed Schlemper TR , Leite MFA , Reis Lucheta A et al. Rhizobacterial community structure differences among sorghum cultivars in different growth stages and soils . FEMS Microbiol Ecol . 2017a ; 93 , Doi.org/10.1093/femsec/fix096 the pages are not yet available in the FEMS Mircobiology Journal . Schlemper TR , van Veen JA , Kuramae EE . Co-variation of bacterial and fungal communities in different sorghum cultivars and growth stages is soil dependent , Microb Ecol . 2017b , https://doi.org/Doi: 10.1007/s00248-017-1108-6. Schloter M , Dilly O , Munch JC . Indicators for evaluating soil quality . Agricult Ecosys Environ . 2003 ; 98 : 255 – 62 . Google Scholar CrossRef Search ADS Schreiter S , Ding G-C , Grosch R et al. Soil type-dependent effects of a potential biocontrol inoculant on indigenous bacterial communities in the rhizosphere of field-grown lettuce . FEMS Microbiol Ecol . 2014 ; 90 : 718 – 30 . Google Scholar CrossRef Search ADS PubMed Shade A , Peter H , Allison SD et al. Fundamentals of microbial community resistance and resilience . Front Microbiol . 2012 ; 3 : 417 . Google Scholar CrossRef Search ADS PubMed Sieverding E , Mulhern K. Vesicular‐Arbuscular Mycorrhiza Management in Tropical Agrosystems , Eschborn, Germany: Deutsche Gesellschaft fur Technische Zusammenarbeit (GTZ) GmbH 1991 . Skujins J . Extracellular enzymes in soil . CRC Crit Rev Microbiol . 1976 ; 4 : 383 – 421 . Google Scholar CrossRef Search ADS PubMed Spain JL . Arguments for diagnoses based on unaltered wall structures . Mycotaxon . 1990 ; 38 : 71 – 6 . SSS Soil Survey Staff (SSS) . “Keys to Soil Taxonomy” 10th ed. USDA. Natural Resources, Conservation Service, Washington DC . 2006 . Tabatabai MA , Bremner JM . Use of p-nitrophenyl phosphate for assay of soil phosphatase activity . Soil Biol Biochem . 1969 ; 1 : 301 – 7 . Google Scholar CrossRef Search ADS Tabatabai MA . Soil enzymes . In: Page AL , Miller EM , Keeney DR (eds). Methods of Soil Analysis. Part 2 2nd ed. Agron Monogr 9 . Madison, Wisconsin : ASA and SSSA , 1982 , 501 – 38 . Taylor JP , Wilson B , Mills MS et al. Comparison of microbial numbers and enzymatic activities in surface soils and subsoils using various techniques . Soil Biol Biochem . 2002 ; 34 : 387 – 401 . Google Scholar CrossRef Search ADS Thioulouse J , Prin Y , Duponnois R . Multivariate analyses in soil microbial ecology:a new paradigm . Environ Ecol Statist . 2012 ; 19 : 499 – 520 . Google Scholar CrossRef Search ADS Vandenkoornhuyse P , Ridgway KP , Watson IJ et al. Co-existing grass species have distinctive arbuscular mycorrhizal communities . Mol Ecol . 2003 ; 12 : 3085 – 95 . Google Scholar CrossRef Search ADS PubMed Verbruggen E , Kuramae EE , Hillekens R et al. Testing potential effects of maize expressing the Bacillus thuringiensis Cry1Ab endotoxin (Bt Maize) on mycorrhizal fungal communities via DNA- and RNA-based pyrosequencing and molecular fingerprinting . Appl Environ Microbiol . 2012 ; 78 : 7384 – 92 . Google Scholar CrossRef Search ADS PubMed Vivas A , Barea JM , Azcón R . Brevibacillus brevis isolated from cadmium-or zinc-contamined soils improves in vitro spore germination and growth of Glomus mosseae under high Cd or Zn concentrations . Microb Ecol . 2005 ; 49 : 416 – 24 . Google Scholar CrossRef Search ADS PubMed Wang M , Zheng Q , Shen Q et al. The critical role of potassium in plant stress response . Int J Mol Sci . 2013 ; 14 : 7370 . Google Scholar CrossRef Search ADS PubMed Wang Y , Naumann U , Wright ST et al. mvabund– an R package for model-based analysis of multivariate abundance data . Methods Ecol Evol . 2012 ; 3 : 471 – 4 . Google Scholar CrossRef Search ADS Warton DI , Wright ST , Wang Y . Distance-based multivariate analyses confound location and dispersion effects . Methods Ecol Evol . 2012 ; 3 : 89 – 101 . Google Scholar CrossRef Search ADS Welc M , Ravnskov S , Kieliszewska-Rokicka B et al. Suppression of other soil microorganisms by mycelium of arbuscular mycorrhizal fungi in root-free soil . Soil Biol Biochem . 2010 ; 42 : 1534 – 40 . Google Scholar CrossRef Search ADS Westfall PH , Young SS Resampling-Based Multiple Testing . New York, NY : John Wiley & Sons , 1993 . Zelles L . Phospholipid fatty acid profiles in selected members of soil microbial communities . Chemosphere . 1997 ; 35 : 275 – 94 . Google Scholar CrossRef Search ADS PubMed © FEMS 2018. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png FEMS Microbiology Ecology Oxford University Press

Native bacteria promote plant growth under drought stress condition without impacting the rhizomicrobiome

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Oxford University Press
Copyright
© FEMS 2018.
ISSN
0168-6496
eISSN
1574-6941
DOI
10.1093/femsec/fiy092
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Abstract

ABSTRACT Inoculation of plants with beneficial plant growth-promoting bacteria (PGPB) emerges a valuable strategy for ecosystem recovery. However, drought conditions might compromise plant-microbe interactions especially in semiarid regions. This study highlights the effect of native PGPB after 1 year inoculation on autochthonous shrubs growth and rhizosphere microbial community composition and activity under drought stress conditions. We inoculated three plant species of semiarid Mediterranean zones, Thymus vulgaris, Santolina chamaecyparissus and Lavandula dentata with a Bacillus thuringiensis strain IAM 12077 and evaluated the impact on plant biomass, plant nutrient contents, arbuscular mycorrhiza fungi (AMF) colonization, soil rhizosphere microbial activity and both the bacterial and fungal communities. Inoculation with strain IAM 12077 improved the ability of all three plants species to uptake nutrients from the soil, promoted L. dentata shoot growth (>65.8%), and doubled the AMF root colonization of S. chamaecyparissus. Inoculation did not change the rhizosphere microbial community. Moreover, changes in rhizosphere microbial activity were mainly plant species-specific and strongly associated with plant nutrients. In conclusion, the strain IAM 12077 induced positive effects on plant growth and nutrient acquisition with no impact on the rhizosphere microbiome, indicating a rhizosphere microbial community resilient to native bacteria inoculation. Bacillus thuringiensis, plant growth-promoting bacteria, microbial activity, degraded soil, 16S rRNA gene, 18S rRNA gene, arbuscular mycorrhiza fungi INTRODUCTION Knowledge of plant-microbe interaction led to the application of microbes as plant growth promoters due to their capability to improve plant development, commonly used to improve crop yields, in ecosystem recovering strategies (Bashan et al. 2012). Plant growth-promoting bacteria (PGPB) may play a decisive role in facilitating plant growth in soil and might be especially relevant to vegetation recovery strategies. However, modification in plant microbial community structure caused by inoculation of specific microbes might be buffered by ecosystem resilience (Shade et al. 2012), which is driven by the level of diversity of soil biota (Kennedy 1999; Nannipieri et al. 2003) and therefore, may compromise the efficiency of applied PGPB. On the other hand, the inoculated microbes may change the microbial community, thus impacting the soil ecosystem functioning and the environment. Hence, it is crucial to determine if effects due to inoculation with PGPB are long-lasting concerning the beneficial impacts on plant growth and effects on soil biota. The effect of PGPB in plants is mainly studied in crops (Lottmann et al. 2000; Scherwinski, Grosch and Berg 2008; Chowdhury et al. 2013), however studies on the application of PGPB as a strategy for ecosystem recovery (Bashan et al. 2008; Bashan et al. 2012) lack an understanding on the impact of the applied microbes in soil microflora on the long term. Plant-microbe interactions might differ in degraded soil or soil under stress conditions such as drought (Jackson et al. 2003; Aboim et al. 2008; Peixoto et al. 2010). Alternatively, changes in soil microbial structure may result in undesirable effects if native species critical to plant growth are lost or subsequently have reduced roles. This is particularly relevant as plants interact with multiple soil microorganisms in addition to the inoculated bacteria. For example, native arbuscular mycorrhizal fungi (AMF) perform critical ecological ecosystem functions, especially in semiarid Mediterranean regions (Requena, Jeffries and Barea 1996). AMF affect plant diversity and productivity and contribute to determining the stability and sustainability of the ecosystem (van der Heijden et al. 1998; van der Heijden et al. 2006). Therefore, the impact of PGPB inoculation on other native soil microorganisms may be even more important when we consider a degraded environment with reduced biodiversity, what might be more vulnerable to changes in soil microbial composition. In the best-case scenario, re-vegetation programs should combine the beneficial effects of PGPB with AMF and other microorganisms associated with plant roots as microbial sources for growth and nutrient uptake (Armada, Roldán and Azcón 2014; Mengual et al. 2014; Armada et al. 2015a, 2016). The selection of autochthonous plant species capable of hosting a high diversity of AMF in combination with PGPB in their rhizospheres may increase plant establishment in stressful environments such as degraded areas in the semiarid Mediterranean. To our knowledge, there is still a lack of understanding how the inoculation of native microbes affects rhizosphere soil microbes, especially mycorrhiza. Several studies have evaluated the impact of inoculated PGPB, however, they usually apply non-native microbes (Schreiter et al. 2014) in short-term experiments (Scherwinski, Grosch and Berg 2008) and in agricultural fields aiming to increase crop productivity (Lottmann et al. 2000). Research should focus on impacts of native PGPB in facilitating plant growth under stress conditions of both soil degradation and drought. Such an evaluation would best combine molecular biological (i.e. next-generation sequencing) and biochemical (assessment of biomarkers and fatty acids) approaches, since an understanding that embraces soil community structure and activity is required. Next generation sequencing of phylogenetic markers such as ribosomal RNA genes provides information on the overall community structure, whereas the analysis of phospholipid fatty acids (PLFA) and enzyme activities provide information on microbial activities. The sequence of ribosomal RNA gene provides the identification of microbial groups that comprise the microbial community through environmental DNA present in the sample, which result in information more closely related to the microbial seedbank concept (Philippot et al. 2013). Accordingly, a more comprehensive characterization of microbial activity could also rely on the identification of biomarkers based on the composition of PLFA, on dehydrogenase activity (estimator of basal respiration), urease activity (microbial hydrolyzation of urea) (Lloyd and Sheaffe 1973), and phosphatase activity (microbial mediated phosphorus availability). Altogether, the combined analysis provides information about community structure and potentially viable microbes and their functional activity and helps to highlight the potential impacts of bacterial inoculation in plant rhizosphere. The aim of this study was to examine the long-lasting impact of a beneficial native bacterial strain IAM 12077 of B. thuringiensis inoculated in three plant species (Thymus vulgaris, Santolina chamaecyparissus and Lavandula dentata), cultured under drought stress conditions on (1) plant growth and nutrition and (2) the root AMF colonization, the rhizosphere bacterial and fungal community structure (assessed by next-generation sequencing) and the rhizosphere microbial enzyme activities. We hypothesize that after 1 year, the inoculation of PGPB strain IAM 12077 on plants affects the rhizosphere microbial community assembly and mycorrhiza colonization. MATERIALS AND METHODS Soil bacteria isolation and molecular identification The bacterial strain used in this study was isolated from the same natural soil used in the microcosm experiment (see description below). The bacterium was isolated from a mixture of rhizosphere soils from several autochthonous shrub species. A homogenate of 1 g soil in 9 mL sterile water was diluted (10−2–10−4), plated on three different media [Yeast Mannitol Agar, Potato Dextrose Agar and Luria-Bertani Agar (Bertani 1951)] and incubated at 28°C for 48 h to isolate bacteria from different taxonomic groups. Molecular characterization of the selected bacterium was by sequencing the 16S rRNA gene. Bacterial cells were collected, diluted, lysed, and the DNA extracted, which was used as PCR template. The amplification reactions were performed in 25 µL volume containing 10× PCR buffer, 50 mM MgCl2, 10 µL of each primer [27F (AGAGTTTGATCCTGGCTCAG) and 1492R (GGTTACCTTGTTACGACTT)] and 1 µL of 5 U/µl Taq polymerase (Platinum, Invitrogen, Waltham, USA). PCR was carried out in a thermal cycler using following conditions: 5 min at 95°C, followed by 30 cycles of 45 s at 95°C, 45 s at 44°C and 2 min at 72°C and finally one cycle of 10 min at 72°C. PCR products were analyzed by 1% agarose gel electrophoresis and purified with the QIAquick Gel extraction kit (Qiagen) for subsequent sequencings using an automated DNA sequencer (Perkin–Elmer ABI Prism 373, USA). Sequence data were compared to databases (EMBL and GenBank) using BLAST program. Similarity searches at NCBI using BLAST program unambiguously identified the bacterial species similar to B. thuringiensis strains. The genetic similarity was also confirmed by phylogenetic analysis of the 16S rRNA gene sequence (Fig. S1, Supporting Information). The strain is B. thuringiensis IAM 12077 (Accession NR 043403.1 or accession D16281). In previous studies (Armada et al. 2015b, 2016), the same strain was characterized for growth under non-stress and osmotic stress conditions, ability to synthesize proline or poly-β-hydroxybutyrate, lipid peroxidation and PGPB characteristics that included α-ketobutyrate [ACC (1-aminocyclopropane-1-carboxylate) deaminase] and indole acetic acid (IAA) production and phosphate solubilization. Microcosm experimental design and soil characteristics The microcosm experimental design was based on two factors: (1) three different autochthonous shrub species: T. vulgaris (T), S. chamaecyparissus (S) and L. dentata (L) and (2) inoculation or not of the autochthonous species with the selected B. thuringiensis strain IAM 12077 (non-inoculated, -; inoculated with strain IAM 12077, Bt). Each of six treatments included five biological replicates resulting in 30 pots. The soil used in this experiment is natural soil from the 'Vicente Blanes' natural park in Molina de Segura, Murcia, Spain, (coordinates: 38° 12′ N and 1′ 13′ W, 393 m altitude). The soil is a Typic Torriorthent (SSS 2006) containing: organic C 0.94%, total N 0.22%, P 1.36 · 10−3 g kg−1 (Olsen test), pH 8.9 and an electric conductivity of 1.55 dS·m−1. The substrate used in this assay consisted of a 5 cm layer of the natural park soil mixed with sterile sand [5/2 (v/v)]. The substrate was added to 0.5 kg capacity pots. One milliliter of pure strain IAM 12077 culture (108 CFU·mL−1), grown in LB medium broth (Bertani 1951) for 48 h at 28°C was applied to the potted seeds at sowing time. The bacterial inoculum (108 CFU·mL−1) was again applied to the seedlings 15 days later. The control consisted of inoculations with sterilized LB medium. The three plant species were grown for 1 year in pots under greenhouse conditions (temperature 19°C–25°C, 16 h/8 h light/dark photoperiod). The photosynthetic photon flux density measured with a light-meter (LICOR, model LI-188B) was 400–700 µmol m−2 s−1. During the experiment, plants were subject to drought conditions by limiting the soil water-holding capacity to 50% each day after water application and allowing the water-holding capacity to decrease to ∼30% before the next water application. Plant biomass and nutrient analysis Plants were harvested 1 year after planting (five replicates per treatment, n=5), shoots were excised from the roots, and fresh weights of both shoots and roots determined. Subsequently, samples were dried for 48 h at 75°C and dry masses determined. Shoot P, K, Ca and Mg (mg plant−1), as well as Zn, Fe, Mn and Cu (µg plant−1) contents, were determined by inductively coupled plasma optical emission spectrometry at the Analytical Service of the Centro de Edafología y Biología Aplicada del Segura, CSIC, Murcia, Spain. AMF root colonization, spores isolation and identification The roots of the three plant species were carefully washed and stained (Phillips and Hayman 1970). The percentage of mycorrhizal root length was determined by microscopic examination of stained root samples (Phillips and Hayman 1970) using the gridline intersect method of Giovannetti and Mosse (1980). The AMF spores were isolated from the soil samples by a wet sieving process (Sieverding 1991). The morphological spore characteristics and their subcellular structures were described from a specimen mounted in: polyvinyl alcohol-lactic acid-glycerine (PVLG) (Koske and Tessier 1983), a mixture of PVLG and Melzer’s reagent (Brundrett 1994), a mixture of lactic acid to water at 1:1, Melzer’s reagent, and water (Spain 1990). For identification of the AMF species, the spores were examined by microscopy at up to 400-fold magnification as described for glomeromycotean classification (Oehl et al. 2011). Rhizospheric soil enzymatic activity Dehydrogenase activity was determined following Skujins’ method (1976) as modified by García, Hernández and Costa (1997). One gram of rhizosphere soil was exposed to 0.2 mL of 0.4% 2-p-iodophenyl-3-p-nitrophenyl-5-phenyl tetrazolium chloride in distilled water for 20 h at 22°C in the dark. The formed iodo-nitrotetrazolium formazan (INTF) was extracted with 10 mL of methanol by shaking vigorously for 1 min and filtering through a Whatman no. 5 filter paper. INTF was measured spectrophotometrically at 490 nm. β-glucosidase was determined using 0.05 M p-nitrophenyl-β-D-glucopyranoside, (Masciandaro, Ceccanti and García 1994) as substrate. This assay is also based on the release and detection of p-nitrophenol (PNP). Two milliliters of 0.1 M maleate buffer (pH 6.5) and 0.5 mL of the substrate were added to 0.5 g of sample and incubated at 37°C for 90 min. The reaction was stopped by addition of tris-hydroxymethyl aminomethane according to Tabatabai (1982). The amount of generated PNP was determined by absorbance at 398 nm (Tabatabai and Bremner 1969). Urease activity was determined by the method of Nannipieri et al. (1980) and expressed as µmol N-NH3·g−1 soil · h−1. Alkaline phosphatase activity was determined using 0.115 M p-nitrophenyl phosphate disodium as substrate. Two milliliters of 0.5 M sodium acetate buffer adjusted to pH 5.5 using acetic acid (Naseby and Lynch 1997) and 0.5 mL of substrate was added to 0.5 g of soil and incubated at 37°C for 90 min. The reaction was stopped by cooling to 2°C for 15 min. Then, 0.5 mL of 0.5 M CaCl2 and 2 mL of 0.5 M NaOH were added, and the mixture was centrifuged at 1649 xg for 5 min. The PNP formed was determined by absorbance at 398 nm (Tabatabai and Bremner 1969). For controls, the substrate was added before the additions of CaCl2 and NaOH. Rhizospheric soil microbial lipid extraction and PLFA analysis Lipid extraction, fractionation, mild alkaline methanolysis and GC analysis were according to Frostegard, Bååth and Tunlio (1993a,b). PLFA analysis was carried out in freeze-dried frozen samples (−80°C). Lipids were extracted from 3 g lyophilized soil samples using a one-phase mixture (1:2:0.8 v/v/v) of chloroform/methanol/0.15 M, pH 4.0 citrate buffer. After extraction, the lipids were separated into neutral lipids, glycolipids and polar lipids (phospholipids) on silicic acid columns (Merck Kieselgel 60 63–200 µm) followed by a mild alkaline methanolysis to form the corresponding fatty acid methyl esters for GC analysis. The fatty acids were identified by their retention times in relation to that of the internal standard (fatty acid methyl esters 19:0 and 12:0). The following fatty acids were assessed as biomarkers for bacterial biomass: i14:0, i15:0, a15:0, i16:0, 16:1w7t, 17:1w7, a17:1w7, i17:0, cy17:0, 18:1w7c and cy19:0 (Mauclaire et al. 2003). PLFA 16:1w5 was used as an indicator of AMF (Olsson et al. 1995; Drigo et al. 2010). C18:2w6.9 was used as a measure of fungal biomass (Bååth 2003). Methylated fatty acid (10Me16:0) was used as a specific biomarker for Actinomycetes (Frostegard, Bååth and Tunlio 1993a,b; Welc et al. 2010). The ratios of Gram-positive (G+) to Gram-negative (G−) bacteria were calculated by taking the sum of the PLFAs i-C14:0, i-C15:0, a-C15:0, i-C16:0, i-C17:0 and a-C17:0 reflected the amount of G+ bacteria, whereas C16:1w7, C17:0 cy and C18:1w7 reflected the amount of G− bacteria (Frostegård and Bååth 1996; Zelles 1997). Rhizospheric soil DNA extraction, PCR conditions for fungal and bacterial tag-encoded amplification and sequencing DNA was extracted from 0.5 g of soil using the fast DNA Spin Kit for soil (MO BIO Laboratories Inc., Carlsbad CA, USA) and quantified by 260 nm absorbance (Nanodrop Technology, Wilmington, DE, USA). The integrity of the DNA was verified by 1% agarose gel electrophoresis using TBE buffer (45 mM Tris-borate, 1 mM EDTA, pH 8.0). For fungal 18S rRNA partial gene amplification, primers described by Verbruggen et al. (2012) were used. The 5′ terminus of primers contained an adaptor sequence and a multiplex identifier tag (MID, 12 different 10-bp-long tags), which resulted in the following primer constructs (adaptor in boldface): Forward (FF390.1), 5′-CTATGCGCCTTGCCAGCCCGCTCAG-(MID)-CGWTAACGAACGAGACCT-3′, Reverse (FR1), 5′-CGTATCGCCTCCCTCGCGCCATCAG-(MID)-AICCATTCAATCGGTAIT-3′. PCR reactions contained 2.0 µL of 10 µM each forward and reverse primer, 5.0 µL 10× PCR-buffer, 5.0 µL of 2 mM dNTP’s, 0.5 µL BSA, 33.10 µL Milli-Q water and 0.40 µL FastStar Expand TAQ Taq DNA polymerase (5 U/µL). The PCR conditions were 95°C for 5 min followed by 25 cycles at 95°C for 30 s, 57°C for 1 min and 72°C for 1 min, a final elongation step at 72°C for 10 min. Products were purified using QIAquick PCR Purification Kit (Qiagen). For bacteria, the V4 region of the 16S rRNA gene was amplified by PCR using 515F (5’-GTGCCAGCMGCCGCGGTAA-3’) and 806R (5’-GGACTACHVGGGTWTCTAAT -3’) primers. The 515F primer included the Roche 454-B pyrosequencing adapter (a 10-bp barcode) unique to each sample, and a GT linker, while 806R included the Roche 454-A sequencing adapter (a 10-bp barcode), unique to each sample, and a GG linker. PCRs contained 1.0 µL of 5 µM each forward and reverse primers, 2.5 µL 10× PCR-buffer, 2.5 µL of 2 mM dNTPs, 16.80 µL Milli-Q water and 0.20 µL of 5 U/µL FastStar Expand TAQ Taq DNA polymerase under the following conditions: 95°C for 5 min followed by 30 cycles of 95°C for 30 s, 53°C for 1 min and 72°C for 1 min, a final elongation step at 72°C for 10 min. Products were purified using QIAquick PCR Purification Kit (Qiagen). Amplicons were quantified and equimolar pooled. The samples were sequenced (Macrogen Company Inc., South Korea) on a Roche 454 automated sequencer and GS FLX system using titanium chemistry (454 Life Sciences, Branford, CT, USA). Statistical analysis Basic and univariate statistics Kolmogorov–Smirnov and Lilliefors tests were used for assessing the normality of the data. Most variables of plant biomass were normally distributed or could be normalized by data transformation. Homogeneity of variance was checked by using the Brown–Forsythe test for unequal replication numbers. Statistical analysis consisted of two-way ANOVA followed by Tukey-Kramer’s test. Correlation and regression analyzes were also performed to identify univariate interactions among variables. For the regression analysis, two values of shoot biomass were considered outliers and therefore, excluded from this analysis. To reduce both family-wise error and the false discovery rate, all multiple comparisons passed to a step-down resampling algorithm (Westfall and Young 1993) while the correlation analysis had their P-values corrected by the Benjamini–Hochberg false discovery rate adjustment (Benjamini and Hochberg 1995). Multivariate statistics and generalized linear models The sequenced datasets (bacterial and fungal) were analyzed using the 'phyloseq' package in R (McMurdie and Holmes 2013). The bacterial and fungal operational taxonomic unit (OTU) abundances were summarized at the class and genus taxonomical levels, respectively, and the Hellinger transformation was adopted prior to ordination methods (Legendre and Gallagher 2001) in order to stabilize the mean-variance relationship and to avoid confounding location and dispersion effects (Warton, Wright and Wang 2012). The 'ade4' R package (Dray and Dufour 2007) compared the effect of treatment on the various datasets (plant biomass and nutrients, microbial enzymatic activity and soil microbial community). Between-class analysis (BCA) measured the amount of variance restricted to the grouping factor as a percentage of the total inertia (Dray and Jombart 2011; Thioulouse, Prin and Duponnois 2012). BCA is an alternative method to linear discriminant analysis for which the number of samples is smaller than the number of variables (Thioulouse, Prin and Duponnois 2012). Principal component analysis was applied to each community data set prior to BCA. Monte–Carlo tests of the treatment groups were performed with 999 permutations. We also performed Co-inertia analysis to investigate the degree of data co-structure (Dray, Chessel and Thioulouse 2003) by evaluating the covariance between our four different datasets: (i) plant biomass and nutrients, (ii) microbial activity, (iii) bacterial and (iv) fungal community structure. Because both microbial enzymatic activity and soil microbial community (fungi and bacteria) datasets presented an overdispersal variance, we applied a generalized linear model (GLM) based on tweedie and negative binomial distribution to investigate the effects of both plant and bacterial inoculum on microbial activity and structure, respectively. To avoid sequencing bias common in next-generation platforms (Lee et al. 2012; McMurdie and Holmes 2014), we decided to use the total number of reads per sample as a covariance effect in our GLMs. The effect of each treatment in the microbial community was evaluated by the Wald’s test (W) and Wilks’s lambda test for the enzymatic activity. The R environment (R Development Core Team 2007) and 'ade4' (Chessel, Dufour and Thioulouse 2004), 'mvabund' (Wang et al. 2012) and 'multcomp' (Hothorn, Bretz and Westfall 2008) packages were used. RESULTS Plant growth, nutrition and symbiotic parameters Inoculation with strain IAM 12077 increased the shoot biomass of all three plant species compared to the control: 65.8% for L. dentata, 31.8% for S. chamaecyparissus and 17% for T. vulgaris (Fig. 1A). However, inoculation weakly influenced root biomass (P < 0.06) (Fig. 1B). Figure 1. View largeDownload slide Influence of a native B. thuringiensis strain IAM 12077 on both shoot and root biomass of three autochthonous plant species native to natural arid Mediterranean soil under drought stress conditions. Means + CI95%, data followed by the same capital letter do not differ statistically among plants, while small-case letters refer to differences between inoculated and control plants. Tukey’s test at 5% probability. Figure 1. View largeDownload slide Influence of a native B. thuringiensis strain IAM 12077 on both shoot and root biomass of three autochthonous plant species native to natural arid Mediterranean soil under drought stress conditions. Means + CI95%, data followed by the same capital letter do not differ statistically among plants, while small-case letters refer to differences between inoculated and control plants. Tukey’s test at 5% probability. Inoculation with strain IAM 12077 increased both macro and micronutrients in shoots, but all three plant species responded differently: P and K increased by 51% and 47%, respectively, for T. vulgaris , while K, Ca and Mg increased by 63%, 27% and 36% for L. dentata compared to non-inoculated controls (Table 1). Inoculation with strain IAM 12077 also increased the shoot content of the micronutrients Zn, Fe and Cu for T. vulgaris and Zn, Mn and Cu for S. chamaecyparissus compared to non-inoculated controls (Table 1). In general, the shoot nutrient content of S. chamaecyparissus contained the largest amount of P, while L. dentata accumulated more K, Ca and Mg than the other two species in this study (Table 1). Table 1. Total content of macro- (P, K, Ca and Mg) and micronutrients (Zn, Fe, Mn and Cu) in the shoot of three autochthonous plants species Thymus vulgaris (T), Santolina chamaecyparissus (S) andLavandula dentata (L) grown in natural arid Mediterranean soil under drought stress conditions as affected by the inoculation of the autochthonous bacteria strain Bacillus thuringiensis (Bt). P (mg plant−1) K (mg plant−1) Ca (mg plant−1) Mg (mg plant−1) Zn (µg plant−1) Fe (µg plant−1) Mn (µg plant−1) Cu (µg plant−1) T(-) 0.57 ± 0.11Ab 7.07 ± 0.25Bb 6.01 ± 1.85Bb 1.70 ± 0.62Ab 33.28 ± 1.69Ba 42.79 ± 3.67ABb 40.35 ± 14.51Ba 4.21 ± 0.27Bb TBt 0.86 ± 0.09Aa 10.42 ± 0.07Ba 7.94 ± 0.28Ba 2.21 ± 0.24Aa 54.06 ± 5.90Bb 115.22 ± 17.83ABa 46.49 ± 0.74Ba 6.38 ± 1.04Ba S(-) 0.87 ± 0.07Ab 10.10 ± 0.45Ba 8.45 ± 1.00Bb 1.01 ± 0.06Bb 60.93 ± 3.78Ab 87.16 ± 20.55Ba 100.19 ± 3.02Ab 11.05 ± 0.59Ab SBt 1.01 ± 0.08Aa 12.93 ± 0.70Bb 11.95 ± 0.67Bb 1.43 ± 0.10Ba 89.72 ± 7.46Aa 78.43 ± 19.76Ba 121.93 ± 2.57Aa 13.17 ± 0.27Aa L(-) 0.62 ± 0.09Ba 13.51 ± 0.45Ab 13.30 ± 1.68Ab 2.14 ± 0.13Ab 38.50 ± 4.69Ab 104.25 ± 16.89Aa 13.41 ± 1.32Cb 5.19 ± 0.48Bb LBt 0.64 ± 0.11Ba 21.97 ± 1.48Aa 16.83 ± 0.51Aa 2.91 ± 0.04Aa 47.39 ± 2.42Aa 100.21 ± 15.00Aa 20.60 ± 0.69Ca 7.23 ± 0.69Ba P (mg plant−1) K (mg plant−1) Ca (mg plant−1) Mg (mg plant−1) Zn (µg plant−1) Fe (µg plant−1) Mn (µg plant−1) Cu (µg plant−1) T(-) 0.57 ± 0.11Ab 7.07 ± 0.25Bb 6.01 ± 1.85Bb 1.70 ± 0.62Ab 33.28 ± 1.69Ba 42.79 ± 3.67ABb 40.35 ± 14.51Ba 4.21 ± 0.27Bb TBt 0.86 ± 0.09Aa 10.42 ± 0.07Ba 7.94 ± 0.28Ba 2.21 ± 0.24Aa 54.06 ± 5.90Bb 115.22 ± 17.83ABa 46.49 ± 0.74Ba 6.38 ± 1.04Ba S(-) 0.87 ± 0.07Ab 10.10 ± 0.45Ba 8.45 ± 1.00Bb 1.01 ± 0.06Bb 60.93 ± 3.78Ab 87.16 ± 20.55Ba 100.19 ± 3.02Ab 11.05 ± 0.59Ab SBt 1.01 ± 0.08Aa 12.93 ± 0.70Bb 11.95 ± 0.67Bb 1.43 ± 0.10Ba 89.72 ± 7.46Aa 78.43 ± 19.76Ba 121.93 ± 2.57Aa 13.17 ± 0.27Aa L(-) 0.62 ± 0.09Ba 13.51 ± 0.45Ab 13.30 ± 1.68Ab 2.14 ± 0.13Ab 38.50 ± 4.69Ab 104.25 ± 16.89Aa 13.41 ± 1.32Cb 5.19 ± 0.48Bb LBt 0.64 ± 0.11Ba 21.97 ± 1.48Aa 16.83 ± 0.51Aa 2.91 ± 0.04Aa 47.39 ± 2.42Aa 100.21 ± 15.00Aa 20.60 ± 0.69Ca 7.23 ± 0.69Ba Standard Errors are given. Within each parameter, values followed by the same capital letter did not differ between plants while the values followed by the same small letter did not differed between inoculation within plant species (P ≤ 0.05) as determined by Tukey’s test (n= 3). View Large Table 1. Total content of macro- (P, K, Ca and Mg) and micronutrients (Zn, Fe, Mn and Cu) in the shoot of three autochthonous plants species Thymus vulgaris (T), Santolina chamaecyparissus (S) andLavandula dentata (L) grown in natural arid Mediterranean soil under drought stress conditions as affected by the inoculation of the autochthonous bacteria strain Bacillus thuringiensis (Bt). P (mg plant−1) K (mg plant−1) Ca (mg plant−1) Mg (mg plant−1) Zn (µg plant−1) Fe (µg plant−1) Mn (µg plant−1) Cu (µg plant−1) T(-) 0.57 ± 0.11Ab 7.07 ± 0.25Bb 6.01 ± 1.85Bb 1.70 ± 0.62Ab 33.28 ± 1.69Ba 42.79 ± 3.67ABb 40.35 ± 14.51Ba 4.21 ± 0.27Bb TBt 0.86 ± 0.09Aa 10.42 ± 0.07Ba 7.94 ± 0.28Ba 2.21 ± 0.24Aa 54.06 ± 5.90Bb 115.22 ± 17.83ABa 46.49 ± 0.74Ba 6.38 ± 1.04Ba S(-) 0.87 ± 0.07Ab 10.10 ± 0.45Ba 8.45 ± 1.00Bb 1.01 ± 0.06Bb 60.93 ± 3.78Ab 87.16 ± 20.55Ba 100.19 ± 3.02Ab 11.05 ± 0.59Ab SBt 1.01 ± 0.08Aa 12.93 ± 0.70Bb 11.95 ± 0.67Bb 1.43 ± 0.10Ba 89.72 ± 7.46Aa 78.43 ± 19.76Ba 121.93 ± 2.57Aa 13.17 ± 0.27Aa L(-) 0.62 ± 0.09Ba 13.51 ± 0.45Ab 13.30 ± 1.68Ab 2.14 ± 0.13Ab 38.50 ± 4.69Ab 104.25 ± 16.89Aa 13.41 ± 1.32Cb 5.19 ± 0.48Bb LBt 0.64 ± 0.11Ba 21.97 ± 1.48Aa 16.83 ± 0.51Aa 2.91 ± 0.04Aa 47.39 ± 2.42Aa 100.21 ± 15.00Aa 20.60 ± 0.69Ca 7.23 ± 0.69Ba P (mg plant−1) K (mg plant−1) Ca (mg plant−1) Mg (mg plant−1) Zn (µg plant−1) Fe (µg plant−1) Mn (µg plant−1) Cu (µg plant−1) T(-) 0.57 ± 0.11Ab 7.07 ± 0.25Bb 6.01 ± 1.85Bb 1.70 ± 0.62Ab 33.28 ± 1.69Ba 42.79 ± 3.67ABb 40.35 ± 14.51Ba 4.21 ± 0.27Bb TBt 0.86 ± 0.09Aa 10.42 ± 0.07Ba 7.94 ± 0.28Ba 2.21 ± 0.24Aa 54.06 ± 5.90Bb 115.22 ± 17.83ABa 46.49 ± 0.74Ba 6.38 ± 1.04Ba S(-) 0.87 ± 0.07Ab 10.10 ± 0.45Ba 8.45 ± 1.00Bb 1.01 ± 0.06Bb 60.93 ± 3.78Ab 87.16 ± 20.55Ba 100.19 ± 3.02Ab 11.05 ± 0.59Ab SBt 1.01 ± 0.08Aa 12.93 ± 0.70Bb 11.95 ± 0.67Bb 1.43 ± 0.10Ba 89.72 ± 7.46Aa 78.43 ± 19.76Ba 121.93 ± 2.57Aa 13.17 ± 0.27Aa L(-) 0.62 ± 0.09Ba 13.51 ± 0.45Ab 13.30 ± 1.68Ab 2.14 ± 0.13Ab 38.50 ± 4.69Ab 104.25 ± 16.89Aa 13.41 ± 1.32Cb 5.19 ± 0.48Bb LBt 0.64 ± 0.11Ba 21.97 ± 1.48Aa 16.83 ± 0.51Aa 2.91 ± 0.04Aa 47.39 ± 2.42Aa 100.21 ± 15.00Aa 20.60 ± 0.69Ca 7.23 ± 0.69Ba Standard Errors are given. Within each parameter, values followed by the same capital letter did not differ between plants while the values followed by the same small letter did not differed between inoculation within plant species (P ≤ 0.05) as determined by Tukey’s test (n= 3). View Large Remarkably, the plant P content was not significantly correlated with the percentage of AMF colonization (P > 0.20). The predominant AMF species identified in the native consortium in soil of this study were as follows: Septoglomus constrictum, Diversispora aunantia, Archaespora trappei, Glomus versiforme and Paraglomus ocultum, which were cataloged and included in the collection of EEZ (codes EEZ 198 to EEZ 202). The analysis of AMF root colonization revealed a distinct pattern. Among all non-inoculated species, T. vulgaris had the highest percentage of AMF root colonization. Bacteria inoculation on S. chamaecyparissus significantly increased the percentage AMF root colonization by 92% and total AMF colonization by 145% with respect to the non-inoculated controls (Table 2). However, we did not detect any statistical differences in AMF root colonization for L. dentata with or without inoculation. We also report a significant relationship between the percentage of AMF root colonization and shoot dry mass (r²=0.48, P<0.01) (Figure S1, Supporting Information). Table 2. Soil enzymatic activities and AMF root colonization in the rhizosphere of three autochthonous plants Thymus vulgaris (T), Santolina chamaecyparissus (S) andLavandula dentata (L) grown in natural arid Mediterranean soil under drought stress conditions as affected by the inoculation of the autochthonous bacteria strain Bacillus thuringiensis (Bt). Dehydrogenase (µg INTF g−1) β-glucosidase (µmol PNF g−1 soil h−1) Urease (µmol N-NH3 g−1 h−1) Alkaline Phosphatase (µmol PNF g soil−1 h−1) AMF (%) Total AMF colonization T(-) 71.9 ± 0.49Ba 137.8 ± 0.00Ba 606.5 ± 19.96Aa 181.2 ± 26.90Ba 54 ± 3.0Aa 231 ± 37.6Aa TBt 66.5 ± 3.19Ba 177.8 ± 0.02Ba 577.3 ± 21.56Aa 141.7 ± 28.96Ba 48 ± 3.5Aa 162 ± 24.2Aa S(-) 88.6 ± 3.36Aa 201.5 ± 0.02Ba 526.5 ± 16.95Ab 302.4 ± 17.71Aa 24 ± 4.1Bb 64 ± 22.4Bb SBt 72.9 ± 5.11Aa 186.9 ± 0.04Ba 687.2 ± 71.87Aa 215.1 ± 5.00Aa 46 ± 4.6Aa 157 ± 25.7Aa L(-) 87.3 ± 6.65Aa 367.6 ± 56.37Aa 622.1 ± 35.97Ab 221.4 ± 36.02Aa 27 ± 2.1Ba 97 ± 16.2Ba LBt 80.6 ± 4.08Aa 346.2 ± 37.05Aa 679.6 ± 12.09Aa 224.5 ± 12.58Aa 30 ± 1.7Aa 153 ± 1.8Aa Dehydrogenase (µg INTF g−1) β-glucosidase (µmol PNF g−1 soil h−1) Urease (µmol N-NH3 g−1 h−1) Alkaline Phosphatase (µmol PNF g soil−1 h−1) AMF (%) Total AMF colonization T(-) 71.9 ± 0.49Ba 137.8 ± 0.00Ba 606.5 ± 19.96Aa 181.2 ± 26.90Ba 54 ± 3.0Aa 231 ± 37.6Aa TBt 66.5 ± 3.19Ba 177.8 ± 0.02Ba 577.3 ± 21.56Aa 141.7 ± 28.96Ba 48 ± 3.5Aa 162 ± 24.2Aa S(-) 88.6 ± 3.36Aa 201.5 ± 0.02Ba 526.5 ± 16.95Ab 302.4 ± 17.71Aa 24 ± 4.1Bb 64 ± 22.4Bb SBt 72.9 ± 5.11Aa 186.9 ± 0.04Ba 687.2 ± 71.87Aa 215.1 ± 5.00Aa 46 ± 4.6Aa 157 ± 25.7Aa L(-) 87.3 ± 6.65Aa 367.6 ± 56.37Aa 622.1 ± 35.97Ab 221.4 ± 36.02Aa 27 ± 2.1Ba 97 ± 16.2Ba LBt 80.6 ± 4.08Aa 346.2 ± 37.05Aa 679.6 ± 12.09Aa 224.5 ± 12.58Aa 30 ± 1.7Aa 153 ± 1.8Aa Standard Errors are given. Within each parameter, values followed by the same capital letter did not differ between plants while the values followed by the same small letter did not differed between inoculations within plant species (P ≤ 0.05) as determined by Tukey’s test. View Large Table 2. Soil enzymatic activities and AMF root colonization in the rhizosphere of three autochthonous plants Thymus vulgaris (T), Santolina chamaecyparissus (S) andLavandula dentata (L) grown in natural arid Mediterranean soil under drought stress conditions as affected by the inoculation of the autochthonous bacteria strain Bacillus thuringiensis (Bt). Dehydrogenase (µg INTF g−1) β-glucosidase (µmol PNF g−1 soil h−1) Urease (µmol N-NH3 g−1 h−1) Alkaline Phosphatase (µmol PNF g soil−1 h−1) AMF (%) Total AMF colonization T(-) 71.9 ± 0.49Ba 137.8 ± 0.00Ba 606.5 ± 19.96Aa 181.2 ± 26.90Ba 54 ± 3.0Aa 231 ± 37.6Aa TBt 66.5 ± 3.19Ba 177.8 ± 0.02Ba 577.3 ± 21.56Aa 141.7 ± 28.96Ba 48 ± 3.5Aa 162 ± 24.2Aa S(-) 88.6 ± 3.36Aa 201.5 ± 0.02Ba 526.5 ± 16.95Ab 302.4 ± 17.71Aa 24 ± 4.1Bb 64 ± 22.4Bb SBt 72.9 ± 5.11Aa 186.9 ± 0.04Ba 687.2 ± 71.87Aa 215.1 ± 5.00Aa 46 ± 4.6Aa 157 ± 25.7Aa L(-) 87.3 ± 6.65Aa 367.6 ± 56.37Aa 622.1 ± 35.97Ab 221.4 ± 36.02Aa 27 ± 2.1Ba 97 ± 16.2Ba LBt 80.6 ± 4.08Aa 346.2 ± 37.05Aa 679.6 ± 12.09Aa 224.5 ± 12.58Aa 30 ± 1.7Aa 153 ± 1.8Aa Dehydrogenase (µg INTF g−1) β-glucosidase (µmol PNF g−1 soil h−1) Urease (µmol N-NH3 g−1 h−1) Alkaline Phosphatase (µmol PNF g soil−1 h−1) AMF (%) Total AMF colonization T(-) 71.9 ± 0.49Ba 137.8 ± 0.00Ba 606.5 ± 19.96Aa 181.2 ± 26.90Ba 54 ± 3.0Aa 231 ± 37.6Aa TBt 66.5 ± 3.19Ba 177.8 ± 0.02Ba 577.3 ± 21.56Aa 141.7 ± 28.96Ba 48 ± 3.5Aa 162 ± 24.2Aa S(-) 88.6 ± 3.36Aa 201.5 ± 0.02Ba 526.5 ± 16.95Ab 302.4 ± 17.71Aa 24 ± 4.1Bb 64 ± 22.4Bb SBt 72.9 ± 5.11Aa 186.9 ± 0.04Ba 687.2 ± 71.87Aa 215.1 ± 5.00Aa 46 ± 4.6Aa 157 ± 25.7Aa L(-) 87.3 ± 6.65Aa 367.6 ± 56.37Aa 622.1 ± 35.97Ab 221.4 ± 36.02Aa 27 ± 2.1Ba 97 ± 16.2Ba LBt 80.6 ± 4.08Aa 346.2 ± 37.05Aa 679.6 ± 12.09Aa 224.5 ± 12.58Aa 30 ± 1.7Aa 153 ± 1.8Aa Standard Errors are given. Within each parameter, values followed by the same capital letter did not differ between plants while the values followed by the same small letter did not differed between inoculations within plant species (P ≤ 0.05) as determined by Tukey’s test. View Large Microbial enzymatic activity and PFLA composition in the rhizosphere of the three plant species β-glucosidase activity was highest in L. dentata rhizospheres, alkaline phosphatase activity was highest in S. chamaecyparissus rhizospheres, and dehydrogenase activity was highest in both plant species. Inoculation with strain IAM 12077 did not affect any of the enzymatic activities measured for all three plant species, except that the urease activity of S. chamaecyparissus increased by 30.5% compared with the non-inoculated control (Table 2). The lipid abundance of the microbial community including bacteria, fungi, Actinomycetes, AMF, G+ and G− bacteria, total PLFA and total neutral lipids fatty acids (NLFA) was lower in L. dentata rhizosphere. Bacteria, fungi, AMF and total PLFA significantly increased in S. chamaecyparissus, while Actinomycetes, G+ and G− bacteria increased for both T. vulgaris and S. chamaecyparissus. ANOVA analysis revealed significant differences (P ≤0.05) for the microbial biomarkers of bacteria, fungi, Actinomycetes, G+ and G− bacteria and total PLFA of the rhizosphere of all three autochthonous plants species. Inoculation with strain IAM 12077 for each plant species did not significantly affect the lipid abundance of the rhizosphere microbial community, although inoculated L. dentata had a low content of acidic phospholipid biomarkers, but the content of NLFA was significantly increased compared to the non-inoculated control (L(-)) (Table 3). Table 3. Content of phospholipid acid (µg PLFA g−1 sed) and neutral lipids acid biomarkers (µg NLFA gr−1 sed) in the rhizosphere of the three autochthonous plants species [Thymus vulgaris (T),Santolina chamaecyparissus (S) andLavandula dentata (L)] inoculated (Bt) or not (-) with bacteria strain IAM 12077 Bacillus thuringiensis grown in natural arid Mediterranean soil under drought stress conditions. Bacteria Fungi Actinomycetes Mycorrhiza Gram+ Gram− Total PLFA Total NLFA T(-) 0.307 ± 0.11ABa 0.143 ± 0.052ABa 0.059 ± 0.018Aa 0.042 ± 0.012Aa 0.015 ± 0.005Aa 0.020 ± 0.006Aa 0.552 ± 0.19ABa 14.37 ± 2.7Ab TBt 0.601 ± 0.48ABa 0.237 ± 0.187ABa 0.207 ± 0.111Aa 0.098 ± 0.078Aa 0.046 ± 0.020Aa 0.041 ± 0.008Aa 1.144 ± 0.84ABa 18.48 ± 0.8Aa S(-) 0.581 ± 0.25Aa 0.266 ± 0.101Aa 0.126 ± 0.054Aa 0.069 ± 0.025Aa 0.028 ± 0.010ABa 0.036 ± 0.013Aa 1.042 ± 0.43Aa 9.91 ± 5.2Ab SBt 0.754 ± 0.10Aa 0.323 ± 0.054Aa 0.165 ± 0.021Aa 0.110 ± 0.019Aa 0.026 ± 0.013ABa 0.039 ± 0.018Aa 1.353 ± 0.18Aa 19.42 ± 4.7Aa L(-) 0.015 ± 0.004Ba 0.012 ± 0.007Ba 0.001 ± 0.000Ba 0.003 ± 0.000Aa 0.001 ± 0.000Ba 0.002 ± 0.000Ba 0.032 ± 0.01Ba 10.34 ± 2.7Ab LBt 0.093 ± 0.003Ba 0.046 ± 0.017Ba 0.020 ± 0.000Ba 0.022 ± 0.000Aa 0.006 ± 0.000Ba 0.012 ± 0.000Ba 0.181 ± 0.02Ba 22.77 ± 2.5Aa Bacteria Fungi Actinomycetes Mycorrhiza Gram+ Gram− Total PLFA Total NLFA T(-) 0.307 ± 0.11ABa 0.143 ± 0.052ABa 0.059 ± 0.018Aa 0.042 ± 0.012Aa 0.015 ± 0.005Aa 0.020 ± 0.006Aa 0.552 ± 0.19ABa 14.37 ± 2.7Ab TBt 0.601 ± 0.48ABa 0.237 ± 0.187ABa 0.207 ± 0.111Aa 0.098 ± 0.078Aa 0.046 ± 0.020Aa 0.041 ± 0.008Aa 1.144 ± 0.84ABa 18.48 ± 0.8Aa S(-) 0.581 ± 0.25Aa 0.266 ± 0.101Aa 0.126 ± 0.054Aa 0.069 ± 0.025Aa 0.028 ± 0.010ABa 0.036 ± 0.013Aa 1.042 ± 0.43Aa 9.91 ± 5.2Ab SBt 0.754 ± 0.10Aa 0.323 ± 0.054Aa 0.165 ± 0.021Aa 0.110 ± 0.019Aa 0.026 ± 0.013ABa 0.039 ± 0.018Aa 1.353 ± 0.18Aa 19.42 ± 4.7Aa L(-) 0.015 ± 0.004Ba 0.012 ± 0.007Ba 0.001 ± 0.000Ba 0.003 ± 0.000Aa 0.001 ± 0.000Ba 0.002 ± 0.000Ba 0.032 ± 0.01Ba 10.34 ± 2.7Ab LBt 0.093 ± 0.003Ba 0.046 ± 0.017Ba 0.020 ± 0.000Ba 0.022 ± 0.000Aa 0.006 ± 0.000Ba 0.012 ± 0.000Ba 0.181 ± 0.02Ba 22.77 ± 2.5Aa Standard Errors are given. Within each parameter, values followed by the same capital letter did not differ between plants while the values followed by the same small letter did not differed between inoculations within plant species (P ≤ 0.05) as determined by Tukey’s test (n= 3). View Large Table 3. Content of phospholipid acid (µg PLFA g−1 sed) and neutral lipids acid biomarkers (µg NLFA gr−1 sed) in the rhizosphere of the three autochthonous plants species [Thymus vulgaris (T),Santolina chamaecyparissus (S) andLavandula dentata (L)] inoculated (Bt) or not (-) with bacteria strain IAM 12077 Bacillus thuringiensis grown in natural arid Mediterranean soil under drought stress conditions. Bacteria Fungi Actinomycetes Mycorrhiza Gram+ Gram− Total PLFA Total NLFA T(-) 0.307 ± 0.11ABa 0.143 ± 0.052ABa 0.059 ± 0.018Aa 0.042 ± 0.012Aa 0.015 ± 0.005Aa 0.020 ± 0.006Aa 0.552 ± 0.19ABa 14.37 ± 2.7Ab TBt 0.601 ± 0.48ABa 0.237 ± 0.187ABa 0.207 ± 0.111Aa 0.098 ± 0.078Aa 0.046 ± 0.020Aa 0.041 ± 0.008Aa 1.144 ± 0.84ABa 18.48 ± 0.8Aa S(-) 0.581 ± 0.25Aa 0.266 ± 0.101Aa 0.126 ± 0.054Aa 0.069 ± 0.025Aa 0.028 ± 0.010ABa 0.036 ± 0.013Aa 1.042 ± 0.43Aa 9.91 ± 5.2Ab SBt 0.754 ± 0.10Aa 0.323 ± 0.054Aa 0.165 ± 0.021Aa 0.110 ± 0.019Aa 0.026 ± 0.013ABa 0.039 ± 0.018Aa 1.353 ± 0.18Aa 19.42 ± 4.7Aa L(-) 0.015 ± 0.004Ba 0.012 ± 0.007Ba 0.001 ± 0.000Ba 0.003 ± 0.000Aa 0.001 ± 0.000Ba 0.002 ± 0.000Ba 0.032 ± 0.01Ba 10.34 ± 2.7Ab LBt 0.093 ± 0.003Ba 0.046 ± 0.017Ba 0.020 ± 0.000Ba 0.022 ± 0.000Aa 0.006 ± 0.000Ba 0.012 ± 0.000Ba 0.181 ± 0.02Ba 22.77 ± 2.5Aa Bacteria Fungi Actinomycetes Mycorrhiza Gram+ Gram− Total PLFA Total NLFA T(-) 0.307 ± 0.11ABa 0.143 ± 0.052ABa 0.059 ± 0.018Aa 0.042 ± 0.012Aa 0.015 ± 0.005Aa 0.020 ± 0.006Aa 0.552 ± 0.19ABa 14.37 ± 2.7Ab TBt 0.601 ± 0.48ABa 0.237 ± 0.187ABa 0.207 ± 0.111Aa 0.098 ± 0.078Aa 0.046 ± 0.020Aa 0.041 ± 0.008Aa 1.144 ± 0.84ABa 18.48 ± 0.8Aa S(-) 0.581 ± 0.25Aa 0.266 ± 0.101Aa 0.126 ± 0.054Aa 0.069 ± 0.025Aa 0.028 ± 0.010ABa 0.036 ± 0.013Aa 1.042 ± 0.43Aa 9.91 ± 5.2Ab SBt 0.754 ± 0.10Aa 0.323 ± 0.054Aa 0.165 ± 0.021Aa 0.110 ± 0.019Aa 0.026 ± 0.013ABa 0.039 ± 0.018Aa 1.353 ± 0.18Aa 19.42 ± 4.7Aa L(-) 0.015 ± 0.004Ba 0.012 ± 0.007Ba 0.001 ± 0.000Ba 0.003 ± 0.000Aa 0.001 ± 0.000Ba 0.002 ± 0.000Ba 0.032 ± 0.01Ba 10.34 ± 2.7Ab LBt 0.093 ± 0.003Ba 0.046 ± 0.017Ba 0.020 ± 0.000Ba 0.022 ± 0.000Aa 0.006 ± 0.000Ba 0.012 ± 0.000Ba 0.181 ± 0.02Ba 22.77 ± 2.5Aa Standard Errors are given. Within each parameter, values followed by the same capital letter did not differ between plants while the values followed by the same small letter did not differed between inoculations within plant species (P ≤ 0.05) as determined by Tukey’s test (n= 3). View Large There was a significant effect for all three plant species with respect to the profile of microbial PLFA in the rhizospheres. Significant differences were found for the bacterial biomarkers (C17:1w8c and C18:1w9t) and fungal biomarkers (C18:1w9c and C18:2w6c) (Table S1, Supporting Information). Inoculation did not significantly influence the profiles of fatty acids (Wilk’s = 0.39, P > 0.05). BCA analysis also pointed to statistically relevant differences for non-inoculated plants and to a non-significant difference for bacterial inoculation. The results showed that 98.5% of the total variance in this dataset relates to the differences between plant species (Fig. 2) where we observed that the microbial activity (enzymatic activity) and PFLA composition of L. dentata presented a distinct profile compared to those of T. vulgaris and S. chamaecyparissus. Figure 2. View largeDownload slide Effect of three autochthonous plant species of natural arid Mediterranean under drought stress condition on rhizosphere microbial activity and abundance (enzymatic activity and PFLA composition). The interclass inertia was 98.5%, and the Monte Carlo permutation level of significance was P = 0.001 after 999 permutations. Figure 2. View largeDownload slide Effect of three autochthonous plant species of natural arid Mediterranean under drought stress condition on rhizosphere microbial activity and abundance (enzymatic activity and PFLA composition). The interclass inertia was 98.5%, and the Monte Carlo permutation level of significance was P = 0.001 after 999 permutations. Bacterial and fungal community structure The numbers of total sequences were 107,667 of fungi and 81,135 of bacteria. The sequence reads resulted in 900 OTUs for fungi and 3,756 OTUs for bacteria. Table S2 (Supporting Information) presents the total abundance medians and interquartile range for both bacterial and fungal communities according to treatments. Analysis of absolute abundance of both bacterial (W = 119.1, P = 0.82) and fungal (W = 64.7, P = 0.65) OTUs at the family level for the various treatments did not reveal any significant effects after 1 year of strain IAM 12077 inoculation. Most bacterial abundance (47%) was related to five major taxa: Rubrobacter (13.3%), unclassified Solirubrobacterales (10.8%), unclassified Actinobacteria (9.2%), Solirubrobacter (8.2%) and unclassified Geodermatophilaceae (5.3%). Contrastingly, fungal taxa accounted for 55% of total abundance reported for the experiment, and the fungi belonged to taxa that included Hypocreales (22%), Hypocreomicetidea unclassified (9.4%), Dothideomycetesincertaesedis (8.6%), Agaricales (7.8%) and Glomus (7.6%). The population abundance of Bacillales order that comprises our strain IAM 12077 inoculum also did not show any difference among treatments, after 1 year of inoculation (W = 5.47, P> 0,05). In summary, our analysis of changes in the microbial communities due to inoculation showed no significant effects. We also observed only a small impact due to the total number of reads in our analysis (W = 1.72×10−12, P > 0.05). Interactions of microbial community structure and activity, and shoot nutrient acquisition The co-inertia analysis showed a significant covariance between bacterial and fungal soil rhizosphere communities, and between microbial activity and abundance (enzymatic activity and PFLA composition) and plant biomass and nutrients (Fig. 3). We found that 55% of total data variance for plant nutrients and biomass correlated with microbial activity and abundance, while 72% of fungal community variability strongly associated with bacterial community variance. Figure 3. View largeDownload slide Co-inertia analysis results from plant biomass and nutrients (pln), microbial activity and abundance (enzymatic activity and PFLA composition) (act), and bacterial (bac) and fungi (fun) community datasets. Figure 3. View largeDownload slide Co-inertia analysis results from plant biomass and nutrients (pln), microbial activity and abundance (enzymatic activity and PFLA composition) (act), and bacterial (bac) and fungi (fun) community datasets. Based on the co-inertia analysis results, we investigated how plant biomass and nutrients interacted with microbial activity and abundance. We found 20 positive interactions and 8 negative interactions (Fig. 4). Total fungi activity and abundance correlates negatively with root dry weight (r = −0.51, P< 0.05) and is negatively associated with plant Mg-content (r = −0.55, P < 0.05). β-glucosidase relates positively with plant K (r = 0.75), Ca (r = 0.67) and Fe contents (r = 0.51) and was negatively associated with the percentage of AMF (r = 0.53) and Mn (r = 0.60) content. We also report a negative relationship between dehydrogenase and the percentage of AMF (r = −0.67, P < 0.05) and the total AMF colonization. The same was true for alkaline phosphatase activity, which correlated negatively with the percentage of AMF (r = −0.63, P< 0.05) and total AMF colonization (r = −0.55, P < 0.05). On the other hand, enzymatic activity, lipid acid abundance, fatty acid composition of total bacteria, total fungi, Actinomycetes and mycorrhiza likely contributed to the accumulation of P (rbac = 0.58, rfun = 0.55, ract = 0.58 and rmyc = 0.57), Zn (rbac = 0.54, rfun = 0.50, ract = 0.54 and rmyc = 0.54), Mn (rbac = 0.72, rfun = 0.73, ract = 0.67 and rmyc = 0.71) and Cu (rbac = 0.52, rfun = 0.52 and rmyc = 0.52). We also report a strong relationship between the presence of G− bacteria and plant Mn content (r = 0.57, P< 0.05). Figure 4. View largeDownload slide Diagram of correlations between microbial activity, acidic lipid abundance, fatty acid composition and plant biomass and nutrients. Blue squares represent positive correlations, and red squares represent negative correlations. Darker/lighter colors indicate stronger/weaker correlations. White squares are non-significant relationships. Figure 4. View largeDownload slide Diagram of correlations between microbial activity, acidic lipid abundance, fatty acid composition and plant biomass and nutrients. Blue squares represent positive correlations, and red squares represent negative correlations. Darker/lighter colors indicate stronger/weaker correlations. White squares are non-significant relationships. We also evaluated the co-occurrence between bacterial and fungal communities in the rhizosphere of all the three plant species (Fig. 5). We found 122 positive associations and 61 negative associations between the rhizosphere bacterial and fungal communities. Presence of the genus Glomus correlated negatively with bacteria from the groups Sphingosinicella and Acidobacteria Gp6, while the presence of Glomus correlated positively with Acidobacteria Gp16. We observed two positive interactions between the genus Paraglomus with Pseudomonadaceae (unclassified) and Microvirga. In summary, our analysis showed the interactions between microbial activity and abundance (enzyme activity and PFLA composition) and plant biomass, and between bacterial and fungal communities, under drought conditions are majorly positive, while interactions of two genera of mycorrhiza (Glomus and Paraglomus) associated both positively and negatively with bacterial groups. Figure 5. View largeDownload slide Diagram of correlations between bacterial and fungal community inhabiting the rhizosphere of the three plant species. The shades of blue squares represent positive correlations, and the shades of red squares represent negative correlations, white squares are non-significant relationships. Figure 5. View largeDownload slide Diagram of correlations between bacterial and fungal community inhabiting the rhizosphere of the three plant species. The shades of blue squares represent positive correlations, and the shades of red squares represent negative correlations, white squares are non-significant relationships. DISCUSSION Inoculation with strain IAM 12077 on seed and seedlings of three plant species resulted in plant growth and nutrient content acquisition, after 1 year under drought stress conditions. The inoculation increased shoot growth of all three autochthonous plant species studied. Strain IAM 12077 may enhance plant growth by various mechanisms such as optimizing the supply of nutrients and the solubilization of inorganic phosphorus (Glick 1995; He et al. 1997; Leggett, Gleddie and Holloway 2001). Hence, the application of phosphate solubilizing bacteria (PSB) could be a reasonable substitute for chemical phosphate fertilizers (Khan and Zaidi 2006). Previously, Armada et al. (2015b) showed that inoculation with strain IAM 12077 enhanced maize nutrient uptake of P by 37% and Fe, Zn and Cu, indicating the capacity of this strain on solubilization of non-available nutrients and the production of siderophores. Thus, this strain alone strongly impacted the plant nutrient uptake. The enhanced access to soil nutrients likely explains the increase of plant biomass, but strain IAM 12077 also seems to possess mechanism to improve plant tolerance under adverse conditions (Armada et al. 2016), and other growth promoting compounds such as indole-3-acetic acid (IAA). Altogether, the bacteria alone promoted plant growth without resulting in long-term changes on AMF colonization and the rhizosphere microbial community. Our analysis also showed no significant effect of strain IAM 12077 in both percentages of AMF and total AMF colonization. In general, AMF present host preference or host specificity (Vandenkoornhuyse et al. 2003; Öpik et al. 2006; Alguacil Roldán and Torres 2009), which might explain the lack of inoculated strain influence on this plant-fungi relationship. Nevertheless, the impact of bacteria in increasing drought tolerance processes seems to be more associated with the proportion of intraradical structures such as arbuscules than to the percentage of root colonized as previously reported (Marulanda, Azcón and Ruíz-Lozano 2003; Vivas, Barea and Azcón 2005; Armada et al. 2016). This specificity has ecological importance for revegetation programs in ecosystems that include autochthonous shrubs (Armada, Roldán and Azcón 2014; Mengual et al. 2014; Armada et al. 2015a,b, 2016), and our analysis suggests that inoculation with strain IAM 12077 may contribute to promoting P uptake without compromising plant-AMF relationships. However, we found that microbial activity of β-glucosidase, dehydrogenase and alkaline phosphatase were negatively correlated with the percentage of AMF colonization in roots. When measuring soil enzyme activities, it should be considered that potential activities are determined (Schloter, Dilly and Munch 2003). Notwithstanding, evaluating soil enzymatic activity remains useful as an indicator of biochemical potential, possible resilience and a sensor of changes in soil key functions (Taylor et al. 2002). Therefore, soil microbial activity, especially that of alkaline phosphatase points to a role for non-AMF microbial activity in solubilizing P for uptake in plants (Nakas, Gould and Klein 1987), which may contribute to the lack of significance between plant P-content and percentage of AMF root colonization. We report a positive correlation between bacterial and fungal activities. Therefore, strain IAM 12077 induced plant P uptake and plants interacted with soil-active microbes for continuous acquisition of soil nutrients, without the dependence on AMF for nutrient uptake. The strain IAM 12077 in the present study presents a great potential for improving nutrient acquisition, especially compared to organic fertilizer sources (Güneş et al. 2014). This might explain the increase of phosphorus content in T. vulgaris due to strain inoculation (+51%). Inoculation with strain IAM 12077 increased shoot K content for the three plant species studied and had the greatest influence on L. dentata (+63%). Potassium is one the most important soluble inorganic nutrients and regulates water uptake capacity by the roots (Wang et al. 2013), likely an essential process during plant growth under water stress conditions. According to Armada et al. (2016), the strain IAM 12077 modulates the plant antioxidant responses by decreasing oxidative stress, which contributes to improve the nutrient uptake and plant growth performance under stress conditions. The lack of significant changes in rhizosphere microbial community after 1 year of inoculation together with the increased nutrient concentration in plant tissue may suggest the nutrient acquisition resulted from a long-lasting effect of strain IAM 12077. We also found that strain IAM 12077-inoculated seeds and seedlings exhibited increase in both Ca and Mg contents compared to non-inoculated plants. Calcium acts in membrane protection, and magnesium contributes to modulation of ionic currents across chloroplasts and vacuole membranes (thus, regulating the stomatal opening and ion balance in cells), both of which are phenomena of particular relevance under drought conditions (Parida and Jha 2013). The enhancement of Mg content for inoculated plants suggests a reduced impact of drought on the functioning of the photosynthetic apparatus in these three plant species when colonized by strain IAM 12077. The plants used in our study belong to two different families (Lamiaceae and Asteraceae) but are all autochthonous drought-tolerant shrub species with deep roots that help to cope with nutrient stress in eroded soils (Francis and Thornes 1990). They belong to the natural succession of the shrubland community of semiarid Mediterranean ecosystems in the southeast of Spain (Alguacil et al. 2011). T. vulgaris is heliophylous plant that grows well in drained and calcareous soils, usually reaching 15–30 cm height (Dorling 2008). S. chamaecyparissus grows well on rocky soils with button-like flower-heads in summer; consists of an aromatic shrub that grows up to 75 cm (Dorling 2008). Finally, L. dentata prefers well-drained alkaline soils and sunny conditions (González 2007) and grows to 60 cm with linear or lance-shaped leaves and the whole plant is also strongly aromatic with a widely known fragrance (Bayer 2006). Considering that three plants in this study prefer well drained soil and they are naturally tolerant to drought conditions, the 1 year of controlled water-holding capacity simulated the aspects of stressful water conditions likely inducing the plants to intensify their interactions with soil microbiome, as indicated by the significant covariance between the soil microbial activity and plant nutrients and biomass. The microbial abundances, bacterial (C17:1w8c, C18:1w9t) and fungal biomarkers (C18:1w9c, C18:2w6c) and enzymatic activities differed significantly in the rhizosphere of the three plant species studied. However, strain IAM 12077 inoculation did not significantly influence the profile of fatty acids in the rhizosphere of these species. Therefore, after 1 year of inoculation, there was nearly no effect of the inoculated bacteria with respect to determining rhizosphere microbial activity and abundance (enzyme activity and PLFA composition), despite measurable contributions to plant growth and nutrient uptake. These results suggest a potential application of strain IAM 12077 as part of a revegetation strategy for enhancing plant growth and uptake of nutrients with a minimized impact on rhizosphere microbial activity and abundance. The PLFA technique provides a rapid and inexpensive method to access microbial biomass and composition (Frostegård, Tunlid and Bååth 2011), which may be even more sensitive in detecting shifts than methods based on DNA or RNA (Ramsey et al. 2006). However, PLFA lacks specificity since many different (and unknown) groups of organisms may present the same biomarker. Thus, molecular methods allow obtaining more accurate information on the soil microbial community by detecting not only the active microbes, but also the whole soil microbiota including the seedbank (Philippot et al. 2013). Based on that, we confirm that strain IAM 12077 did not induce a significant shift in the rhizozpshere microbial community after 1 year of inoculation. Moreover, the co-inertia results point to significant interactions between rhizosphere microbial activity and plant nutrition, and we later identified part of this covariance as single variable correlations. The β-glucosidase activity that was highest in L. dentata suggests carbohydrate transformation, which is important as an alternative energy source for microorganisms. Indeed, β-glucosidase activity is positively associated with plant K, Ca and Fe contents, which suggest that metabolically active microbes may directly contribute to plant nutrient uptake. S. chamaecyparissus had the greatest phosphorus content in shoot biomass and the increase of alkaline phosphatase activity. The cycling of N, C and P are controlled by hydrolase enzymes such as urease (N), β-glucosidase (C) and phosphatases (P), which are mainly synthesized by soil microorganisms (Ros et al. 2006). These hydrolases are involved in the mineralization of compounds that provide nutrients that include N, P and C. Therefore, rhizosphere active microbes contribute to plant nutrient uptake, while the strain IAM 12077 improved nutrient uptake. As our reported differences in microbial activities are plant-specific, we infer that plants might shift their microbial rhizosphere composition by activating microbes able to mediate soil nutrient uptake in plants. The Rv coefficient suggested that part of plant nutrient content results from microbial activity, which according to the BCA analysis is a plant-specific selection. Furthermore, ANOVA results also highlighted the role of strain IAM 12077 inoculation in plant nutrients. Therefore, beyond the role of strain IAM 12077 in the rhizosphere, the plants likely drive the microbial activity towards their nutrient necessities. Plant nutrient uptake probably occurred as a combination of plant selection of active microbes together with the beneficial roles of strain IAM 12077. The strongest link found in our study relates the rhizosphere bacterial and fungal community covariance for plants under water stress. Here we found major positive associations that were nearly double the number of negative microbial interactions. According to the stress-gradient hypothesis, species interactions increase their importance due to shifts from competition to facilitation with respect to stress (He and Bertness 2014). Our results provide support for this hypothesis, the strong covariance between bacterial and fungal communities indicates that the variance of some bacterial groups links with fungal community changes. In fact, fungal groups were positively associated with many of the evaluated bacterial groups (e.g. the genus Paraglomus was positively associated with Pseudomonadaceae and Microvirga). Furthermore, the bacterial community likely employs several physiological modifications in response to changing soil moisture, such as the production of exopolysaccharides (Kohler, Caravaca and Roldán 2009), sporulation (Landesman and Dighton 2010) and adjustment of internal water potential to match that of the external environment. Effects of microbial inoculum on the rhizosphere microbial community structure have often been reported for only short-term experiments ranging from 30 to 90 days (Cipriano et al. 2016) and usually for short-cycle crops (Schreiter et al. 2014). Since we aimed to describe the longer-term impact of strain IAM 12077 in rhizosphere community by evaluating their effects after 1 year, this may explain why we found weak changes induced by inoculation with respect to the total rhizosphere microbial community (results of DNA analysis). In addition, it is likely that the strain IAM 12077 produced a primer effect on plants (Cipriano et al. 2016) that resulted in positive effect on plant growth after 1 year of inoculation. Previous studies have shown the potential of PGPB as a strategy for successful ecosystem recovery strategies (Bashan et al. 2008,2012) and our study goes beyond by showing the dismal impact of strain IAM 12077 in rhizosphere microbial community. Thus, strategies for land recovery might be more effective by the use and application of existent PGPB present in soil. In conclusion, in greenhouse conditions, inoculation using native bacterial strain with PGPB properties enhanced the growth of three autochthonous shrubs species and nutrient uptake without changing the rhizosphere microbial diversity and did not affect AMF groups after 1 year of inoculation under water stress conditions. Plants possess ecological advantages by fostering soil microbial seedbank and assembly their rhizosphere microbial populations (Mendes et al. 2014; Barbosa Lima et al. 2015; Cipriano et al. 2016; Schlemper et al. 2017a,b). The inoculation of autochthonous shrubs species with strain IAM 12077 may be a sustainable option for recovering degraded soils without harming and impacting the rhizosphere microbial community structure and activity over the long-term. However, future studies in field conditions are needed to evaluate the responses of plants inoculated with this strain. SUPPLEMENTARY DATA Supplementary data are available at FEMSEC online. ACKNOWLEDGEMENTS We thank Domingo Álvarez for the morphological identification of autochthonous mycorrhizal fungus. Publication number 6531 of the NIOO-KNAW, Netherlands Institute of Ecology. FUNDING E. Armada was financed by the Ministry of Science and Innovation, Spain. This work was carried out in the framework of the project reference AGL2009-12530-C02-02 with a grant of short stay (ref. BES-2010-042736) at NIOO-KNAW in Wageningen, and the grant The Netherlands Organization for Scientific Research (NWO, 729.004.016), M.F.A. Leite was financed by CAPES A116-2013 program. Conflict of interest. None declared. REFERENCES Aboim MCR , Coutinho HLC , Peixoto RS et al. Soil bacterial community structure and soil quality in a slash-and-burn cultivation system in Southeastern Brazil . Appl Soil Ecol . 2008 ; 38 : 100 – 8 . Google Scholar CrossRef Search ADS Alguacil MM , Roldán A , Torres MP . Complexity of semiarid gypsophilous shrub communities mediates the AMF biodiversity at the plant species level . Microb Ecol . 2009 ; 57 : 718 – 27 . Google Scholar CrossRef Search ADS PubMed Alguacil MM , Torres MP , Torrecillas E et al. Plant type differently promote the arbuscular mycorrhizal fungi biodiversity in the rhizosphere after revegetation of a degraded semiarid land . Soil Biol Biochem . 2011 ; 43 : 167 – 73 . Google Scholar CrossRef Search ADS Armada E , Azcón R , López-Castillo OM et al. Autochthonous arbuscular mycorrhizal fungi and Bacillus thuringiensis from a degraded Mediterranean area can be used to improve physiological traits and performance of a plant of agronomic interest under drought conditions . Plant Physiol Biochem . 2015a ; 90 : 64 – 74 . Google Scholar CrossRef Search ADS Armada E , Barea JM , Castillo P et al. Characterization and management of autochthonous bacterial strains from semiarid soils of Spain and their interactions with fermented agrowastes to improve drought tolerance in native shrub species . Appl Soil Ecol . 2015b ; 96 : 306 – 18 . Google Scholar CrossRef Search ADS Armada E , Probanza A , Roldán A et al. Native plant growth promoting bacteria Bacillus thuringensis and mixed or individual mycorrhizal species improved drought tolerance and oxidative metabolism in Lavandula dentata plants . J Plant Physiol . 2016 ; 192 : 1 – 12 . Google Scholar CrossRef Search ADS PubMed Armada E , Roldán A , Azcón R . Differential activity of autochthonous bacteria in controlling drought stress in native Lavandula and Salvia plants species under drought conditions in natural arid soil . Microb Ecol . 2014 ; 67 : 410 – 20 . Google Scholar CrossRef Search ADS PubMed Bååth E . The use of neutral lipid fatty acids to indicate the physiological conditions of soil fungi . Microb Ecol . 2003 ; 45 : 373 – 83 . Google Scholar CrossRef Search ADS PubMed Barbosa Lima A , Souza Cannavan FS , Navarrete AA et al. Amazonian Dark Earth and plant species from the Amazon region contribute to shape rhizosphere bacterial communities . Microb Ecol . 2015 ; 69 : 855 – 66 . Google Scholar CrossRef Search ADS PubMed Bashan Y , Puente ME , de-Bashan LE et al. Environmental uses of plant growth-promoting bacteria , In Plant-Microbe interactions 2008. , E . Ait Barka , Clément C (Ed). in Chapter 4 , ( Trivandrun, Kerala, India : Research Signpost ), 69 – 93 . Bashan Y , Salazar BG , Moreno M et al. Restoration of eroded soil in the Sonoran Desert with native leguminous trees using plant growth-promoting microorganisms and limited amounts of compost and water . J Environ Manage . 2012 ; 102 : 26 – 36 . Google Scholar CrossRef Search ADS PubMed Bayer E . Plantas del Mediterráneo : Blume , 2006 . Benjamini Y , Hochberg Y . Controlling the false discovery rate:a practical and powerful approach to multiple testing . J R Statistl Soc Series B (Methodological) . 1995 ; 57 : 289 – 300 . Bertani G . Studies on lysogenes I. The mode of phage liberation by lysogenic Escherichia coli . J Bacteriol . 1951 ; 62 : 293 – 300 . Google Scholar PubMed Brundrett M Practical Methods in Mycorrhizal Research , Mycologue Publications 1994 . Chessel D , Dufour AB , Thioulouse J . The ade4 package-I- One-table methods . R News . 2004 ; 4 : 5 – 10 . Chowdhury SP , Dietel K , Rändler M et al. Effects of Bacillus amyloliquefaciens FZB42 on lettuce growth and health under pathogen pressure and its impact on the rhizosphere bacterial community . PLoS ONE . 2013 ; 8 : e68818 . Google Scholar CrossRef Search ADS PubMed Cipriano MAP , Lupatini M , Lopes-Santos L et al. Lettuce and rhizosphere microbiome responses to growth promoting Pseudomonas species under field conditions . FEMS Microbiol Ecol . 2016 ; 92 : 1 – 12 . Google Scholar CrossRef Search ADS Dorling K . RHS AZ Encyclopedia of Garden Plants , Publisher DK, 3 edition , 2008 . Dray S , Chessel D , Thioulouse J . Co-inertia analysis and the linking of ecological data tables . Ecology . 2003 ; 84 : 3078 – 89 . Google Scholar CrossRef Search ADS Dray S , Dufour AB . The ade4 package:implementing the duality diagram for ecologists . J Statist Softw . 2007 ; 22 : 1 – 20 . Google Scholar CrossRef Search ADS Dray S , Jombart T . Revisiting Guerry’s data:introducing spatial constraints in multivariate analysis . Ann Appl Stat . 2011 ; 5 : 2278 – 99 . Google Scholar CrossRef Search ADS Drigo B , Pijl AS , Duyts H et al. Shifting carbon flow from roots into associated microbial communities in response to elevated atmospheric CO2 . P Nat Acad Sci USA . 2010 ; 107 : 10938 – 42 . Google Scholar CrossRef Search ADS Francis DF , Thornes JB . Matorral:erosion and reclamation . In: Albaladejo J , Stocking MA , Díaz , E (eds). Soil Degradation and Rehabilitation in Mediterranean Environmental Conditions . Murcia, Spain : Consejo Superior de Investigaciones Científicas , 1990 , 87 – 115 . Frostegård Å , Bååth E , Tunlio A . Shifts in the structure of soil microbial communities in limed forests as revealed by phospholipid fatty acid analysis . Soil Biol Biochem . 1993a ; 25 : 723 – 30 . Google Scholar CrossRef Search ADS Frostegård A , Bååth E . The use of phospholipid fatty acid analysis to estimate bacterial and fungal biomass in soil . Biol Fert Soils . 1996 ; 22 : 59 – 65 . Google Scholar CrossRef Search ADS Frostegard A , Tunlid A , Baath E . Phospholipid fatty acid composition, biomass, and activity of microbial communities from two soil types experimentally exposed to different heavy metals . Appl Environ Microbiol . 1993b ; 59 : 3605 – 17 . Frostegård Å , Tunlid A , Bååth E . Use and misuse of PLFA measurements in soils . Soil Biol Biochem . 2011 ; 43 : 1621 – 5 . Google Scholar CrossRef Search ADS García C , Hernández MT , Costa F . Potential use of dehydrogenase activity as an index of microbial activity in degraded soils . Commun Soil Sci Plant Nut . 1997 ; 28 : 123 – 34 . Google Scholar CrossRef Search ADS Giovannetti M , Mosse B . Evaluation of techniques for measuring vesicular arbuscular mycorrhizal infection in roots . New Phytol . 1980 ; 84 : 489 – 500 . Google Scholar CrossRef Search ADS Glick BR . The enhancement of plant growth by free-living bacteria . Can J Microbiol . 1995 ; 41 : 109 – 17 . Google Scholar CrossRef Search ADS González GAL Guía de los árboles y arbustos de la Península Ibérica y Baleares:(especies silvestres y las cultivadas más comunes): Editorial S. A. Mundi-prensa livros, 3rd edition , 2007 . Güneş A , Turan M , Güllüce M et al. Nutritional content analysis of plant growth-promoting rhizobacteria species . Eur J Soil Biol . 2014 ; 60 : 88 – 97 . Google Scholar CrossRef Search ADS He Q , Bertness MD . Extreme stresses, niches, and positive species interactions along stress gradients . Ecology . 2014 ; 95 : 1437 – 43 . Google Scholar CrossRef Search ADS PubMed He ZL , Baligar VC , Martens DC et al. Effect of phosphate rock, lime and cellulose on soil microbial biomass in acidic forest soil and its significance in carbon cycling . Biol Fert Soils . 1997 ; 24 : 329 – 34 . Google Scholar CrossRef Search ADS van der Heijden MGA , Klironomos JN , Ursic M et al. Mycorrhizal fungal diversity determines plant biodiversity, ecosystem variability and productivity . Nature . 1998 ; 396 : 69 – 72 . Google Scholar CrossRef Search ADS van der Heijden MGA , Streitwolf-Engel R , Riedl R et al. The mycorrhizal contribution to plant productivity, plant nutrition and soil structure in experimental grassland . New Phytol . 2006 ; 172 : 739 – 52 . Google Scholar CrossRef Search ADS PubMed Hothorn T , Bretz F , Westfall P . Simultaneous inference in general parametric models . Biometrical J . 2008 ; 50 : 346 – 63 . Google Scholar CrossRef Search ADS Jackson LE , Calderon FJ , Steenwerth KL et al. Responses of soil microbial processes and community structure to tillage events and implications for soil quality . Geoderma . 2003 ; 114 : 305 – 17 . Google Scholar CrossRef Search ADS Kennedy AC . Bacterial diversity in agroecosystems . Agric Ecosyst Environ . 1999 ; 74 : 65 – 76 . Google Scholar CrossRef Search ADS Khan MS , Zaidi A . Influence of composite inoculations of phosphate solubilizing organisms and an arbuscular mycorrhizal fungus on yield, grain protein and phosphorus and nitrogen uptake by greengram . Arch Agron Soil Sci . 2006 ; 52 : 579 – 90 . Google Scholar CrossRef Search ADS Kohler J , Caravaca F , Roldán A . Effect of drought on the stability of rhizosphere soil aggregates of Lactuca sativa grown in a degraded soil inoculated with PGPR and AM fungi . Appl Soil Ecol . 2009 ; 42 : 160 – 5 . Google Scholar CrossRef Search ADS Koske RE , Tessier BA . A convenient permanent slide mounting medium . Mycol Soc Amer Newslet . 1983 ; 34 : 59 . Landesman WJ , Dighton Response of soil microbial communities and the production of plant-available nitrogen to a two-year rainfall manipulation in the New Jersey Pinelands . Soil Biol Biochem . 2010 ; 42 : 1751 – 8 . Google Scholar CrossRef Search ADS Lee CK , Herbold CW , Polson SW et al. Groundtruthing next-gen sequencing for microbial ecology–biases and errors in community structure estimates from PCR amplicon pyrosequencing . PLoS ONE . 2012 ; 7 : e44224 . Google Scholar CrossRef Search ADS PubMed Legendre P , Gallagher E . Ecologically meaningful transformations for ordination of species data . Oecologia . 2001 ; 129 : 271 – 80 . Google Scholar CrossRef Search ADS PubMed Leggett M , Gleddie S , Holloway G . Phosphate-solubilizing microorganisms and their use . In: Ae N , Arihara J , Okada K , Srinivasan A (eds). Plant Nutrient Acquisition . Japan : Springer , 2001 , 299 – 318 . Google Scholar CrossRef Search ADS Lloyd A , Sheaffe MJ . Urease activity in soils . Plant Soil . 1973 ; 39 : 71 – 80 . Google Scholar CrossRef Search ADS Lottmann J , Heuer H , de Vries J et al. Establishment of introduced antagonistic bacteria in the rhizosphere of transgenic potatoes and their effect on the bacterial community . FEMS Microbiol Ecol . 2000 ; 33 : 41 – 9 . Google Scholar CrossRef Search ADS PubMed Marulanda A , Azcón R , Ruíz-Lozano J . Contribution of six arbuscular mycorrhizal fungal isolates to water uptake by Lactuca sativa plants under drought stress . Physiol Plant . 2003 ; 119 : 526 – 33 . Google Scholar CrossRef Search ADS Masciandaro G , Ceccanti B , García C . Anaerobic digestion of straw and piggery wastewater: II. Optimization of the process . Agrochimica . 1994 ; 3 : 195 – 203 . Mauclaire L , Pelz O et al. Assimilation of toluene carbon along a bacteria-protist food chain determined by 13C-enrichment of biomarker fatty acids . J Microbiol Methods . 2003 ; 55 : 635 – 49 . Google Scholar CrossRef Search ADS PubMed McMurdie PJ , Holmes S . Phyloseq:an R Package for reproducible interactive analysis and graphics of microbiome census data . PLoS ONE . 2013 ; 8 : e61217 . Google Scholar CrossRef Search ADS PubMed McMurdie PJ , Holmes S . Waste not, want not:Why rarefying microbiome data is inadmissible . PLoS Comput Biol . 2014 ; 10 : e1003531 . Google Scholar CrossRef Search ADS PubMed Mendes LW , Kuramae EE , Navarrete AA et al. Taxonomical and functional microbial community selection in soybean rhizosphere . ISME J . 2014 ; 8 : 1577 – 87 . Google Scholar CrossRef Search ADS PubMed Mengual C , Schoebitz M , Azcón R et al. Microbial inoculants and organic amendment improves plant establishment and soil rehabilitation under semiarid conditions . J Environ Manage . 2014 ; 134 : 1 – 7 . Google Scholar CrossRef Search ADS PubMed Nakas JP , Gould WD , Klein DA . Origin and expression of phosphatase activity in a semi-arid grassland soil . Soil Biol Biochem . 1987 ; 19 : 13 – 8 . Google Scholar CrossRef Search ADS Nannipieri P , Ascher J , Ceccherini MT et al. Microbial diversity and soil functions . Eur J Soil Sci . 2003 ; 54 : 655 – 70 . Google Scholar CrossRef Search ADS Nannipieri P , Ceccanti B , Cervelli S et al. Extraction of phosphatase, urease, proteases, organic-carbon, and nitrogen from soil . Soil Sci Soc Amer J . 1980 ; 44 : 1011 – 6 . Google Scholar CrossRef Search ADS Naseby DC , Lynch JM . Rhizosphere soil enzymes as indicators of perturbations caused by enzyme substrate addition and inoculation of a genetically modified strain of Pseudomonas fluorescens on wheat seed . Soil Biol Biochem . 1997 ; 29 : 1353 – 62 . Google Scholar CrossRef Search ADS Oehl F . Advances in glomeromycota taxonomy and classification . IMA fungus . 2011 ; 2 : 191 – 9 . Google Scholar CrossRef Search ADS PubMed Olsson PA , Baath E , Jakobsen I et al. The use of phospholipid and neutral lipid fatty acids to estimate biomass of arbuscular mycorrhizal fungi in soil . Mycolog Res . 1995 ; 99 : 623 – 9 . Google Scholar CrossRef Search ADS Öpik M , Moora M , Liira J et al. Composition of root-colonizing arbuscular mycorrhizal fungal communities in different ecosystems around the globe . J Ecol . 2006 ; 94 : 778 – 90 . Google Scholar CrossRef Search ADS Parida AK , Jha B . Physiological and biochemical responses reveal the drought tolerance efficacy of the halophyte Salicornia brachiata . J Plant Growth Regul . 2013 ; 32 : 342 – 52 . Google Scholar CrossRef Search ADS Peixoto RS , Chaer GM , Franco N et al. A decade of land use contributes to changes in the chemistry, biochemistry and bacterial community structures of soils in the Cerrado . Antonie Van Leeuwenhoek . 2010 ; 98 : 403 – 13 . Google Scholar CrossRef Search ADS PubMed Philippot L , Raaijmakers JM , Lemanceau P et al. Going back to the roots:the microbial ecology of the rhizosphere . Nat Rev Microbiol . 2013 ; 11 : 789 – 99 . Google Scholar CrossRef Search ADS PubMed Phillips JM , Hayman DS . Improved procedure of clearing roots and staining parasitic and vesicular-arbuscular mycorrhizal fungi for rapid assessment of infection . T Brit Mycol Soc . 1970 ; 55 : 159 – 61 . Google Scholar CrossRef Search ADS R Development Core Team R:A Language and Environment for Statistical Computing . Vienna, Austria : R Foundation for Statistical Computing , 2007 . Ramsey PW , Rillig MC , Feris KP et al. Choice of methods for soil microbial community analysis:PLFA maximizes power compared to CLPP and PCR-based approaches . Pedobiologia . 2006 ; 50 : 275 – 80 . Google Scholar CrossRef Search ADS Requena N , Jeffries P , Barea JM . Assessment of natural mycorrhizal potential in a desertified semiarid ecosystem . Appl Environ Microbiol . 1996 ; 62 : 842 – 7 . Google Scholar PubMed Ros M , Pascual JA , Garcia C et al. Hydrolase activities, microbial biomass and bacterial community in a soil after long-term amendment with different composts . Soil Biol Biochem . 2006 ; 38 : 3443 – 52 . Google Scholar CrossRef Search ADS Scherwinski K , Grosch R , Berg G . Effect of bacterial antagonists on lettuce:active biocontrol of Rhizoctonia solani and negligible, short-term effects on nontarget microorganisms . FEMS Microbiol Ecol . 2008 ; 64 : 106 – 16 . Google Scholar CrossRef Search ADS PubMed Schlemper TR , Leite MFA , Reis Lucheta A et al. Rhizobacterial community structure differences among sorghum cultivars in different growth stages and soils . FEMS Microbiol Ecol . 2017a ; 93 , Doi.org/10.1093/femsec/fix096 the pages are not yet available in the FEMS Mircobiology Journal . Schlemper TR , van Veen JA , Kuramae EE . Co-variation of bacterial and fungal communities in different sorghum cultivars and growth stages is soil dependent , Microb Ecol . 2017b , https://doi.org/Doi: 10.1007/s00248-017-1108-6. Schloter M , Dilly O , Munch JC . Indicators for evaluating soil quality . Agricult Ecosys Environ . 2003 ; 98 : 255 – 62 . Google Scholar CrossRef Search ADS Schreiter S , Ding G-C , Grosch R et al. Soil type-dependent effects of a potential biocontrol inoculant on indigenous bacterial communities in the rhizosphere of field-grown lettuce . FEMS Microbiol Ecol . 2014 ; 90 : 718 – 30 . Google Scholar CrossRef Search ADS PubMed Shade A , Peter H , Allison SD et al. Fundamentals of microbial community resistance and resilience . Front Microbiol . 2012 ; 3 : 417 . Google Scholar CrossRef Search ADS PubMed Sieverding E , Mulhern K. Vesicular‐Arbuscular Mycorrhiza Management in Tropical Agrosystems , Eschborn, Germany: Deutsche Gesellschaft fur Technische Zusammenarbeit (GTZ) GmbH 1991 . Skujins J . Extracellular enzymes in soil . CRC Crit Rev Microbiol . 1976 ; 4 : 383 – 421 . Google Scholar CrossRef Search ADS PubMed Spain JL . Arguments for diagnoses based on unaltered wall structures . Mycotaxon . 1990 ; 38 : 71 – 6 . SSS Soil Survey Staff (SSS) . “Keys to Soil Taxonomy” 10th ed. USDA. Natural Resources, Conservation Service, Washington DC . 2006 . Tabatabai MA , Bremner JM . Use of p-nitrophenyl phosphate for assay of soil phosphatase activity . Soil Biol Biochem . 1969 ; 1 : 301 – 7 . Google Scholar CrossRef Search ADS Tabatabai MA . Soil enzymes . In: Page AL , Miller EM , Keeney DR (eds). Methods of Soil Analysis. Part 2 2nd ed. Agron Monogr 9 . Madison, Wisconsin : ASA and SSSA , 1982 , 501 – 38 . Taylor JP , Wilson B , Mills MS et al. Comparison of microbial numbers and enzymatic activities in surface soils and subsoils using various techniques . Soil Biol Biochem . 2002 ; 34 : 387 – 401 . Google Scholar CrossRef Search ADS Thioulouse J , Prin Y , Duponnois R . Multivariate analyses in soil microbial ecology:a new paradigm . Environ Ecol Statist . 2012 ; 19 : 499 – 520 . Google Scholar CrossRef Search ADS Vandenkoornhuyse P , Ridgway KP , Watson IJ et al. Co-existing grass species have distinctive arbuscular mycorrhizal communities . Mol Ecol . 2003 ; 12 : 3085 – 95 . Google Scholar CrossRef Search ADS PubMed Verbruggen E , Kuramae EE , Hillekens R et al. Testing potential effects of maize expressing the Bacillus thuringiensis Cry1Ab endotoxin (Bt Maize) on mycorrhizal fungal communities via DNA- and RNA-based pyrosequencing and molecular fingerprinting . Appl Environ Microbiol . 2012 ; 78 : 7384 – 92 . Google Scholar CrossRef Search ADS PubMed Vivas A , Barea JM , Azcón R . Brevibacillus brevis isolated from cadmium-or zinc-contamined soils improves in vitro spore germination and growth of Glomus mosseae under high Cd or Zn concentrations . Microb Ecol . 2005 ; 49 : 416 – 24 . Google Scholar CrossRef Search ADS PubMed Wang M , Zheng Q , Shen Q et al. The critical role of potassium in plant stress response . Int J Mol Sci . 2013 ; 14 : 7370 . Google Scholar CrossRef Search ADS PubMed Wang Y , Naumann U , Wright ST et al. mvabund– an R package for model-based analysis of multivariate abundance data . Methods Ecol Evol . 2012 ; 3 : 471 – 4 . Google Scholar CrossRef Search ADS Warton DI , Wright ST , Wang Y . Distance-based multivariate analyses confound location and dispersion effects . Methods Ecol Evol . 2012 ; 3 : 89 – 101 . Google Scholar CrossRef Search ADS Welc M , Ravnskov S , Kieliszewska-Rokicka B et al. Suppression of other soil microorganisms by mycelium of arbuscular mycorrhizal fungi in root-free soil . Soil Biol Biochem . 2010 ; 42 : 1534 – 40 . Google Scholar CrossRef Search ADS Westfall PH , Young SS Resampling-Based Multiple Testing . New York, NY : John Wiley & Sons , 1993 . Zelles L . Phospholipid fatty acid profiles in selected members of soil microbial communities . Chemosphere . 1997 ; 35 : 275 – 94 . Google Scholar CrossRef Search ADS PubMed © FEMS 2018. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices)

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Published: May 16, 2018

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