Linking bacterial endophytic communities to essential oils: clues from Lavandula angustifolia Mill.

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Research Article

Linking Bacterial Endophytic Communities to Essential Oils:

Clues from

Lavandula angustifolia Mill

Giovanni Emiliani,

1

Alessio Mengoni,

2

Isabel Maida,

2

Elena Perrin,

2

Carolina Chiellini,

2

Marco Fondi,

2

Eugenia Gallo,

3

Luigi Gori,

4

Valentina Maggini,

3

Alfredo Vannacci,

3

Sauro Biffi,

5

Fabio Firenzuoli,

4

and Renato Fani

2

1_{Trees and Timber Institute, National Research Council, Via Madonna del Piano, No. 10, Sesto Fiorentino, 50019 Florence, Italy}

2_{Laboratory of Microbial and Molecular Evolution, Department of Biology, University of Florence, Via Madonna del Piano 6,}

Sesto Fiorentino, 50019 Florence, Italy

3_{Department of Neuroscience, Psychology, Drug Research and Child Health, University of Florence,}

Viale Pieraccini 6, 50139 Florence, Italy

4_{Center for Integrative Medicine, Careggi University Hospital, University of Florence, Viale Pieraccini 6, 50139 Florence, Italy}

5_{Il giardino delle Erbe, via Del Corso 6, Casola Valsenio, 48010 Ravenna, Italy}

Correspondence should be addressed to Renato Fani; renato.fani@unifi.it Received 17 January 2014; Accepted 29 April 2014; Published 26 May 2014 Academic Editor: Gyorgyi Horvath

Copyright © 2014 Giovanni Emiliani et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Endophytic bacteria play a crucial role in plant life and are also drawing much attention for their capacity to produce bioactive compounds of relevant biotechnological interest. Here we present the characterisation of the cultivable endophytic bacteria of Lavandula angustifolia Mill.—a species used since antiquity for its therapeutic properties—since the production of bioactive metabolites from medical plants may reside also in the activity of bacterial endophytes through their direct production, PGPR activity on host, and/or elicitation of plant metabolism. Lavender tissues are inhabited by a tissue specific endophytic community dominated by Proteobacteria, highlighting also their difference from the rhizosphere environment where Actinobacteria and Firmicutes are also found. Leaves’ endophytic community resulted as the most diverse from the other ecological niches. Overall, the findings reported here suggest: (i) the existence of different entry points for the endophytic community, (ii) its differentiation on the basis of the ecological niche variability, and (iii) a two-step colonization process for roots endophytes. Lastly, many isolates showed a strong inhibition potential against human pathogens and the molecular characterization demonstrated also the presence of not previously described isolates that may constitute a reservoir of bioactive compounds relevant in the field of pathogen control, phytoremediation, and human health.

1. Introduction

A diverse range of bacteria, including pathogens, mutualists, and commensals is supported by plants. These bacteria grow in and around roots, in the vasculature, and on aerial tissues [1, 2]. In particular, endophytic bacteria can be defined as those bacteria that colonize the internal tissue of the plants with no external sign of infection or negative effect on their host [2, 3]. It is increasingly evident that bacte-rial endophytes influence plant physiology, facilitating the uptake of nutrients such as nitrogen, phosphorus, sulphur,

magnesium, and calcium [4] and showing plant growth-promoting activity (PGPR) related to the production of phytohormones involved in regulatory metabolism, such as ethylene [5], indole-3-acetic acid (IAA), and acetoin, 2,3-butanediol, [6–8]. Moreover, endophytic bacteria can also improve plant growth via nitrogen fixation [9]. Other relevant functions performed by endophytic bacteria are represented by the decrease or prevention of the pathogenic effects of certain parasitic microorganisms with the production of antimicrobial compounds or by increasing plant tolerance to pollution or stresses [3,10]. Bacterial endophytes are drawing Volume 2014, Article ID 650905, 16 pages

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increasing interest as together with fungal endophytes they are reported to produce a number of bioactive metabolites relevant to human health, such as antibiotics [11], antitumor compounds [12,13], and anti-inflammatory agents [14].

Endophytic bacteria can be further classified as “obli-gate” or “facultative” in accordance with their life strategies. Obligate endophytes are strictly dependent on the host plant for their growth and survival, besides, transmission to other plants could occur only by seeds or via vectors, while faculta-tive endophytes could grow outside host plants [15]. Several studies have shown that facultative endophytes constitute the largest fraction of the endophytic bacterial communities [5]; in fact large plant-by-plant (both at interspecific and intraspe-cific level) differences in the bacterial communities composi-tion have been found [16–20], supporting the idea that the ability to enter plant tissues is a widespread phenotype of soil and rhizosphere bacteria, and that plants exert only a limited selectivity on the colonizing bacterial communities [21], even if clues for a control over the bacterial colonizers operated by yet not completely clarified plant mechanism(s) are reported [5,22]. Actually, a recent bioinformatic study has shown that inside the class of Alphaproteobacteria (which includes well known bacterial endophytes, such as rhizobia and methy-lobacteria) few genomic signatures only could distinguish endophytes from nonendophytes [23]. Another common feature of endophytic bacterial community is their strong temporal and spatial variability [16,19,24]; the community composition not only varies in the soil compartment between the rhizosphere and the root internal tissues in response to complex and not yet fully clarified biotic (plant species and genotype dependent) and abiotic (soil characteristics) driven processes [16, 24], but also among different aerial tissues (stems, leaves, flowers) of the same plant [25,26]. Total endophytes are influenced not only by the location within the plant but also by the presence of the main components of the essential oil in the leaves of aromatic plants [27].

However, all the studies performed so far have anal-ysed mainly the bacterial communities in terms of species composition (or higher taxonomic ranks), especially using cultivation-independent techniques, as 16S rRNA gene libraries or metagenetic sequencing [20, 28, 29]. While cultivation-independent techniques allow a deep coverage of taxonomic diversity of bacterial communities, they provide only partial information on community structure and clearly hamper the possibility to test the actual presence of PGPR activities and bioactive molecule production in the bacterial community.

Among crops, medicinal plants are stirring much atten-tion due to the increasing demand for green chemistry approaches, sustainable practices, and especially in the quest for novel antibiotics able to tackle the increasing multidrug resistance of pathogenic bacteria. In spite of the high rele-vance of medicinal plants, to the best of our knowledge, very little, if nothing at all, is known about the endophytic bacterial communities isolated from medicinal plants. For this reason, in this work we isolated and preliminary characterized from a taxonomical viewpoint the aerobic heterotrophic cultivable endophytic bacterial community of lavender (Lavandula angustifolia Mill.syn. Lavandula officinalis Chaix, Lavandula

vera DC, Lavandula spica L. var angustifolia Auct.). Lavender has a long history of medicinal use and is purported to possess relaxant, neurological, and antibacterial effects [30].

The essential oil of Lavender has been used since antiq-uity for a variety of medical application [31]; in particular anxiolytic activity of Lavender oil was confirmed [32] and its antimicrobial activity was demonstrated against different pathogens [33]. Moon et al. [34] reported that low (≤1%) concentrations of L. angustifolia and L.× intermedia oil can completely eliminate Giardia duodenalis, Trichomonas vagi-nalis, and Hexamita inflata in vitro. Preliminary results also highlighted the possibility for lavender essential oil to be used as antibiotic resistance modifying agent in microorganisms [35].

It has also been demonstrated that the production of bioactive compounds is significantly impacted by the genetic milieu of the plant and by environmental factors [31]; in this framework, it is increasingly evident that the collection of microorganism living in association with complex organisms, generally defined as microbiota, plays a fundamental role in shaping their phenotypic features. Since the qualiquantitative production of bioactive metabolites in medical plants may reside also in the activity of bacterial endophytes through the direct production, PGPR activity, and/or elicitation of plant metabolism [36], the purpose of this research was therefore to perform a cultivation-dependent study of the mesophilic aerobic heterotrophic bacterial endophytic community of the relevant medical and balsamic species L. angustifolia in order to (i) identify its composition, (ii) its diversity between different plant compartments (roots, stems, and leaves), and (iii) its diversity in relation to the rhizosphere bacterial community, and (iv) build a collection of isolates to be screened for the production of bioactive compounds, and (v) test a panel of randomly selected endophytic bacteria versus opportunistic human pathogens belonging to the Burkholde-ria cepacia complex (Bcc). We have chosen these bacteBurkholde-ria since many strains of the complex are opportunistic human pathogens and represent a serious concern for cystic fibrosis (CF) patients and immunocompromised individuals [37], responsible for the “cepacia syndrome,” characterized by high fever, severe progressive respiratory failure, leukocytosis, and elevated erythrocyte sedimentation rate. Moreover, Bcc strains are (naturally) resistant to many antibiotics such as cephalosporin,𝛽-lactams, polymyxins, and aminoglycosides; therefore, Bcc infections are very problematic to eradicate [37,38]. In spite of this high degree of resistance to many antibiotics, it has been recently demonstrated that essential oils extracted from six medicinal plants are able to completely inhibit the growth of Bcc members, including those with a clinical origin and exhibiting resistance to many antibiotics [39].

2. Materials and Methods

2.1. Plant Sampling and Isolation of Mesophilic Cultivable Endophytic and Rhizosphere Bacteria. Five agamically prop-agated potted L. angustifolia plants were collected from the “Giardino delle Erbe,” located in Casola Valsenio, Italy,

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in June 2012. On the same day, plants were transported to the laboratory for processing. Pieces of 200 mg of fresh leaves, stems, and roots tissues were collected from each of the 5 plants and bulked (for a final sample of 1 g) to account for plants to plants variability. Roots and stems samples were divided into 1 cm long pieces and surface-sterilized for 40 s with 70% ethanol followed by 10 min with 2.5% sodium hypochlorite. Plant leaves were surface-sterilized for 20 s with 70% ethanol followed by 5 min with 2.5% sodium hypochlo-rite. To remove the disinfectant, sections were rinsed three times for 5 min in sterile distilled water. Samples were then dried with sterile filter paper and subsequently ground with 2 mL 0.9% sodium chloride with a sterile mortar. Aliquots (100𝜇L) of the last washing water were plated in triplicate as sterility controls. Samples (100𝜇L) of tissue extracts and their different dilutions were plated in triplicate. Aliquots of 200 mg of soil from each pot were collected and bulked for 1 g total. The soil samples were then resuspended in 5 mL of 10 mM Mg₂SO₄ and placed under stirring for 1 h at room temperature.

Endophytic and rhizospheric bacteria were grown in trip-licate on solid 5% tryptone soya broth (TSB) medium (Oxoid Ltd., Basingstoke, Hampshire, UK) at 30∘C for 72 h. The number of aerobic heterotrophic bacteria was determined as colony-forming units (CFUs). Each CFU determination was performed in triplicate and an average value of bacterial titre was determined. From each sample, colonies were randomly selected and singularly plated on 5% solid TSB Petri dishes. From each isolate, glycerol stock (25% final concentration) was prepared and stored at−80∘C.

2.2. PCR Amplification and Sequencing of 16S rRNA Coding Genes from Bacterial Endophytes. Cell lysates were pre-pared by dissolving a bacterial colony in 100𝜇L sterile distilled water and incubation at 99∘C for 10 min, followed by 5 min at 4∘C. An aliquot of 2𝜇L lysate was used for the amplification reaction by polymerase chain reaction (PCR). Amplification of 16S rRNA genes was performed in a total volume of 20𝜇L containing 2 𝜇L of 10X reaction buffer (Polymed, Firenze, Italy), 1.5 mM MgCl₂, 10 pmol of each primer [27f, 5󸀠-GAGAGTTTGATCCTGGCTCAG, and 1495r, 5󸀠-CTACGGCTACCTTGTTACGA], 0.25 mM of dNTP mix, 2 U of Taq DNA polymerase (Polymed). PCR reaction conditions were as described by Mengoni et al. [40]. For sequencing reaction, amplified 16S rDNA fragments were excised from 1% agarose gels and purified using the QIAquick Gel Extraction (Qiagen) according to the manu-facturer’s instructions. Direct sequencing of amplicons was performed at the Genechron laboratory (Ylichron Srl, Italy) with primer 27f on an ABI3730 DNA analyser (Applied Biosystems, Foster City, CA, USA) using the Big Dye Termi-nator Kit.

2.3. Sequence Analysis and Phylogenetic Tree Construction. The sequences presented in this work have been deposited in GeneBank database under the accession numbers KF202531-KF202915. Partial 16S rDNA sequences were matched against

nucleotide sequences available in GenBank database using the BLASTn program [41].

MUSCLE [42] (http://www.drive5.com/muscle) was used to align the 16S rDNA sequences obtained with the most similar orthologous sequences retrieved from the Ribosomal Database Project (RDP) database (http://rdp.cme.msu.edu/). Alignments were trimmed to eliminate poorly aligned region and used to build Bayesian, Maximum Parsimony (MP), and the Neighbor joining (NJ) dendrograms.

Bayesian dendrograms were obtained with MrBayes 3.2 [43] with GTR substitution model with gamma-distributed rate variation across sites with 1000000 generations. MP dendrograms were obtained with MEGA 5 [44] (http://www .megasoftware.net/) using the Tree-Bisection-Regrafting (TBR) algorithm with search level 3 in which the initial trees were obtained by the random addition of sequences (500 replicates); the robustness of the inferred trees was evaluated by 1000 bootstrap resamplings; consistency and retention indexes (CI and RI) were calculated with Mesquite software 2.75 (http://mesquiteproject.org). NJ dendrograms were obtained after calculation of a Kimura two-parameter distance matrix with the software Mega 5. The robustness of the inferred trees was evaluated by 1000 bootstrap resamplings.

2.4. Statistical Analysis. Pairwise sequence identity values (not taking deletion into account) were calculated using the stand-alone Clustal Omega [45]. Genetic distances among OTU were calculated using the Kimura 2 parameter model and 1000 bootstraps replications in the MEGA 5.2.1 software [44]. Diversity indexes were calculated with the PAST3 software [46]. Pairwise differentiation (Fst) values were cal-culated inside the GenoDive v 2.0b25 software (http://www .patrickmeirmans.com/software/GenoDive.html), using vec-tor of presence/absence of the bacterial genera in the different samples with 999 permutations.

2.5. Cross-Streaking Experiments. Antibacterial activity was determined by using the cross-streak method [47, 48]. Hereinafter, endophytic bacterial isolates to be tested for inhibitory activity will be termed “tester” strains, whereas Bcc strains used as a target will be called “target” strains. Cross-streaking experiments were carried out as previously described [47] by using Petri dishes. Tester strains were streaked across one-half of an agar plate with PCA medium and incubated at 30∘C. After 2 days of incubation, target strains were streaked on PCA medium perpendicular to the initial streak and plates were further incubated at 30∘C for 2 days. The antagonistic effect was indicated by the failure of the target strains to grow. The list of target Bcc strains used in this work is reported inTable 3.

3. Results

3.1. Composition of Endophytic Bacterial Communities Isolated from L. Angustifolia. Aerobic heterotrophic culturable bac-teria were isolated from leaves, stems, and roots of 5 plants of Lavandula angustifolia. Leaves samples had the highest

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number of CFU/g fresh weight (6.8 × 105), whereas roots had the lowest values (1.6× 104), the difference being anyway of one order of magnitude only (stem and rhizospheric soil showed titers of 6.5× 104 and 3.4× 105, resp.). Plates were visually inspected and no increase in colony number was observed with an extended incubation time of up to 7 days.

On the triplicate plates of the same plant portion, 100 colonies were randomly selected and isolated on TSA agar plates. A collection of 400 colonies was then prepared with isolates from the four samples types, namely, rhizospheric soil, roots, stems, and leaves.

In order to determine the taxonomic composition of the bacterial communities isolated from the different compart-ments of L. angustifolia plants and from rhizospheric soil, 16S rRNA genes were PCR-amplified from the isolates of the collection as described in Materials and Methods. An ampli-con of the expected size was obtained from each bacterial isolate (data not shown) and the nucleotide sequence of the amplicons was then determined. In this way we obtained 395 sequences, each of which was used as seed to probe databases. Table 1 reports the percentage distribution of the iden-tified bacterial phyla in the different plant compartments and in the rhizospheric soil, along with standard diver-sity indexes calculated on genera distribution; the results show a relative peak of diversity in the leaf and a min-imum in the stem (Shannon index 2.29 and 1.06, resp.). Figure 1 depicts the taxonomic composition of the total lavender aerobic heterotrophic cultivable endophytic com-munity made up by a total of 11 genera; the majority of the isolated strains (88%) belonged to proteobacteria, with a dominance of gammaproteobacteria (74%), Pseudomonas being the most abundant genus accounting for the 51% of total community, followed by Stenotrophomonas (13%) and Pantoea (9%). Rhizobium is also frequently found in the internal tissue of lavender, representing 14% of the total collection. Actinomycetales are also represented (8%) with the genus Microbacterium being the most abundant (6%). Other genera were found, namely, Bacillus, Plan-tibacter, Psychrobacter, Sanguibacter, Salinibacterium, and Jeotgalibacillus representing collectively 6% of the endophytic collection.

Bacteria isolated from the rhizospheric soil showed a quite different taxonomic composition (Figure 2(a)) when compared to the overall endophytic community; in the rhizo-sphere, Bacillus is the most abundant genus (44%), followed by Pseudomonas (30%), Microbacterium (10%), Rhizobium (7%), and Arthrobacter (6%), a genus that is not detected in the endophytic community.Figure 2shows also the com-position of the endophytic communities isolated from the different tissues (panels (b), (c), and (d), for roots, stem, and leaves, resp.); the three compartments are characterized by strikingly different communities, for the differential presence of genera (e.g., Bacillus is found in roots and stem, but absent in the leaves, Sanguibacter and Plantibacter are detected in the leaves only, Jeotgalibacillus is found only in the roots) and the overall diversity (8 genera are found in the leaves and only 5 genera are in the stem) and the different relative presence of the other most abundantgenera.

Table 1: Percentage distribution of bacterial phyla isolated from L. angustifolia roots, stem, leaves, and rhizospheric soil; standard diversity indexes built on genera distribution are also presented.

Soil Roots Stem Leaves

Proteobacteria 40 94 95 93 Firmicutes 44 6 3 — Actinobacteria 16 — 2 7 Richness 7 6 5 8 Evenness 0.73 0.72 0.45 0.76 Shannon index 2.06 1.88 1.06 2.29

Figure 3shows the distribution of main genera (occurring in>5% of total isolates) in the 3 lavender tissues and in its rhizosphere. Isolates belonging to the genus Bacillus decrease their abundance from the rhizosphere to the stem and disappear in the leaves, while Pantoea and Microbacterium show a similar distribution in stems and leaves, but are both absent in the roots. Isolates from the Pseudomonas genus dominate in the stem community and are similarly occurring (ca. 30% of total) in the other 3 samples. Rhizobium spp. was found in soil and represented 40% of roots isolates. Lastly, Stenotrophomonas spp. is abundant in the roots and leaves, but absent in the stem. To further analyse the diversity of the bacterial communities isolated, pairwise differentiation (Fst) values were calculated on vector of presence/absence of bacterial genera; the lowest differentiation was registered among roots and rhizospheric soil (Fst = 0.198) communities, while the highest among stems and leaves (Fst = 0.512). Overall, the stem endophytic community resulted as the most differentiated from the others (Fst = 0.428 and 0.394 versus roots and rhizospheric soil, resp.) while the leaves community showed similar differentiation values when compared to rhizospheric soil (Fst = 0.245) and roots (Fst = 0.239).

Considering the low number of bacterial phyla found in the endophytic community (>95% of isolates were assigned to only 6 genera), we analysed the intrageneric level of diversity by means of 16S rRNA gene sequence identity values. Data obtained are shown in Table 2. From the 16S rRNA gene sequence diversity indices reported that the isolates assigned to the genus Rhizobium possessed the lowest overall diversity, suggesting the presence of a low number of species belonging to the genus Rhizobium. On the other extreme, the Pantoea isolates showed a stronger differentiation supporting the idea of the presence of dif-ferent species (sequence identity< 97%). To further char-acterize the isolated endophytic and rhizosphere bacteria, Bayesian, Maximum Parsimony and Neighbor Joining trees based on 16S rRNA genes were constructed for the most abundant genera, namely, Stenotrophomonas (Figure 4 and Suppl. Figures 3 and 9 in Supplementary Material available online at http://dx.doi.org/10.1155/2014/650905), Rhizobium (Figure 5 and Suppl. Figures 4 and 10), Pantoea (Figure 6 and Suppl. Figures 5 and 11), Microbacterium (Figure 7and Suppl. Figures 6 and 12), Bacillus (Suppl. Figures 1, 7, and 13), and Pseudomonas (Suppl. Figures 2, 8, and 14). Overall, Bayesian and NJ dendrograms showed a stronger support

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Bacillus, 2.7 _{Jeotgalibacillus, 0.3} Microbacterium, 6.4 Pantoea, 9.2 Plantibacter, 0.3 Pseudomonas, 51.2 Psychrobacter, 0.7 Rhizobium, 13.9 Salinibacterium, 1.0 Sanguibacter, 1.0 Stenotrophomonas, 13.2

Figure 1: Composition of the aerobic heterotrophic endophytic (sensu stricto) bacterial community in L. angustifolia tissues. The composition is reported as percentages of the total number of characterized isolates (𝑛 = 395).

Acinetobacter 1% Stenotrophomonas 2% Arthrobacter 6% Rhizobium 7% Microbacterium 10% Pseudomonas 30% Bacillus 44% (a) Psychrobacter 1% Jeotgalibacillus 1% Bacillus 5% Stenotrophomonas 24% Pseudomonas 28% Rhizobium 41% (b) Pseudomonas 90% Pantoea 5% Bacillus 3% Microbacterium 1% _{Salinibacterium} 1% (c) Pseudomonas 36% Pantoea 23% Microbacterium 19% Stenotrophomonas 15% Sanguibacter 3% Salinibacterium 2% Plantibacter 1% Psychrobacter 1% (d)

Figure 2: Composition of the aerobic heterotrophic bacterial community of L. angustifolia rhizospheric soil (a), roots (b), stem (c), and leaves (d). The composition is reported as percentages of the total number of characterized isolates for each sample.

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100 90 80 70 60 50 40 30 20 10 0 P res ence (%) Soil Roots Stem Leaves B a cill u s M ic rob a cte rium Pa nt oe a P se udomona s R h iz obium Ste n otr ophomona s

Figure 3: Comparison of the relative abundance of bacterial genera in the different samples; only genera with percentages>5% in at least one of the samples are reported.

Table 2: Intragenus diversity analysis based on 16S rRNA gene sequence identity; mean identity of all versus all 16S rRNA gene sequences (for each genus); mean distance (Kimura 2 parameter); number of pairwise comparisons of 16S sequences with identity>97%, considered as threshold of species identity (in brackets the total number of comparisons and the percentage); number of OTU with at least 1 pairwise comparison with identity>97%.

Genus Number of isolates Mean identity among_{OTU (%)} Mean distance among OTU

Number of pairwise comparisons with

identity>97%

Number of OTU with identity>97% Pseudomonas 172 94.25 0.013 3840 (14878, 25.8%) 170 Bacillus 47 93.04 0.027 329 (1128, 29%) 46 Rhizobia 46 98.61 0.016 952 (1081, 88%) 46 Stenotrophomonas 37 95.34 0.023 415 (703, 59%) 37 Microbacterium 30 95.00 0.059 120 (465, 25%) 29 Pantoea 26 86.95 0.126 24 (351, 6.8%) 7

(and consistency of the results) than the MP ones (see also Suppl. Table 4). The analysis of the Bayesian phylogenetic trees revealed the following.

(i) Endophytic bacteria isolated from the leaves and roots of lavender and assigned to the genus

Stenotrophomonas (Figure 4) are clearly split in two

groups. One group contains isolates clustering with Stenotrophomonas chelatiphaga; interestingly this cluster comprises isolates from both roots and leaves (no Stenotrophomonas were isolated from the stem), suggesting that two compartments might share bacteria belonging to the same species, even though the polymorphism shown by the aligned partial 16S sequences might suggest a diversification at the strain level. The second cluster embeds roots isolates (with the exception of leaves isolate LL44), clustering with Stenotrophomonas rizophila.

(ii) Concerning rhizobia (Figure 5), a grouping in one main cluster of low differentiate sequences is present.

These groups might contain new clades of plant-associated rhizobia, which deserve more investigation in the future.

(iii) From the phylogenetic tree ofFigure 6, Pantoea iso-lated endophytes (from lavender stem and leaves) cluster with already described Pantoea spp.; another group of isolates (all from the leaves) are diverging and thus may represent none yet described strains are found to be associated with plants.

(iv)Figure 7 depicts the phylogenetic position of Microbacterium isolates (mainly from leaves and rhizosphere) in the context of the genus reference strains; noticeably many leaves isolates cluster with M. hydrocarbonoxydans and M. oleivorans, species whose members are able to perform crude oil degradation [49].

(v) Concerning Bacillus isolates (Suppl. Figure 1), a cluster of soil isolates including Bacillus mojavensis was disclosed. Another group, again comprising soil

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0.09 LR48 LR31 LR41 LL38 LL43 LL94 LT48 LR98 LR86 LL88 LR72 LL17 LR28 LR8 LRL93 LL39 LR61 LR76 LL37 LR7 LR29 LR34 LL77 LL76 LR79 LL96 LR95 LL44 LL45 LL13 LR26 LR99 LL75 LR80 LR32 LR60 LR75 0.66 0.91 1 1 0.89 0.91 0.91 0.87 0.93 0.57 0.78 0.7 0.91 S_{001020552 Escherichia coli} S001576206 S daejeonensis MJ03 S000559371 S ginsengisoli S_{000007629 P geniculata ATCC 193} S_{000386319 S koreensis TR6-01} S_{000841904 S terrae R-32768} S000003927 S nitritireducens L2 S_{000841903 S humi R-32729} S000824093 S maltophilia IAM 124 S000010261 P hibiscicola ATCC 19 S_{000129976 S rhizophila e-p10} S000130243 P pictorum LMG 981 S001611233 S pavanii ICB 89 S001097383 S chelatiphaga LPM-5 S000390459 S acidaminiphila AMX1 S000009493 P beteli ATCC 19861T6

Figure 4: Bayesian dendrogram showing the relationships among the 16S rDNA sequences of 37 isolates belonging to the genus Stenotrophomonas and those of reference type strains. Posterior probability values are indicated at the node. Nodes are collapsed at 70% probability. LL = bacteria isolated from the leaves, LR = bacteria isolated from the roots (seeSection 2for details).

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S000413524 A sp K-Ag-3

S000364392 R sullae DSM 14623

S000559201 R mesosinicum CCBAU 2 S000421679 R rubi ICMP 11833

S000388919 R yanglingense CCBAU S000967698 Mycoplana peli AN419

S000000452 R genosp R BDV5365 S000967537 R tibeticum CCBAU 850 S000926235 R multihospitium CCBA S000776010 R fabae CCBAU 33202 S000262741 R mongolense S110 S000775995 R leguminosarum bv vi S002911602 R grahamii CCGE 502 S000265215 R pisi DSM 30132 S000769770 R phaseoli ATCC 14482 S000437446 R etli CFN 42 USDA 90 S000261327 R etli S1 S000979412 R indigoferae CCBAU 7 S001187435 R leguminosarum bv tr S002222203 R leguminosarum bv ph S000635888 R mesosinicum CCBAU 4 S000410286 R etli S001168838 R oryzae Alt 505 S000979020 R yanglingense SH2262 S001014241 R alamii GBV016 S000367571 R etli PRF51 S002035399 A rubi F266 S001096426 R loessense CCBAU 251 S000967565 R sullae CCBAU 85011 S003746557 M ciceri CPN7 S000413521 R rhizogenes IFO 1325 S003284111 R vallis R3-65 S001294595 R phenanthrenilyticum S000769681 R borbori DN316 S000752081 R miluonense CCBAU 41 S000111802 R indigoferae CCBAU 7 S000438747 R mongolense USDA 184 S002290486 A radiobacter K84 ATC S000379907 R mesoamericanum tpud S000014582 M sp N36 S000979022 R loessense CCBAU 719 S000393807 R gallicum DASA12020 S002034822 R lusitanum CCBAU 150 S000055319 R sp WSM749 S000364339 R tropici LMG 9518 S000541167 R gallicum bv gallicu S000981736 R hainanense I66 S000387012 R gallicum CbS-1 S003284683 R endophyticum S6-260 S000093085 R leguminosarum WSM16 S002961235 R genosp TUXTLAS-27 4 S000364330 A rhizogenes LMG 152 S000769199 R sp BSV16 S000393812 R leguminosarum DASA2 S000262202 R mongolense S152 S000639628 S sp DAO10 S000903294 R alkalisoli CCBAU 01 S000721040 A tumefaciens MAFF 03 S000392228 S sp 9702-M4 S000261905 R galegae S163 S001328174 R cnuense KSW 4-12 S001745941 R vignae CCBAU 05176 S002411294 R undicola OURAN110 S000967567 R tubonense CCBAU 850 S000942308 R selenitireducens B1 S003301457 A tumefaciens MM10 S000262203 S fredii S150 S000413447 R galegae ATCC 43677 S000364391 R sp Esparseta 3 S000967566 R cellulosilyticus CC S002914131 Ensifer sp KJ018 S003717587 A tumefaciens KFB 233 S000431871 Candidatus R massilia S000456906 A vitis PHX1 S003753424 Candidatus R massilia S002221963 R pusense NRCPB10 S002958314 M sp CCNWGS0211 S001188792 endophytic bacterium S002233057 R taibaishanense CCNW S001170532 A tumefaciens CCNWGS0 S003289155 endophytic bacterium S000427818 R huautlense SO2 S001548991 R rosettiformans W3 S000393816 R galegae DASA12028 S000015022 R sp DUS470 S000444220 R vignae CCBAU 23084 S000389830 BradyR japonicum PRY6

S002233877 R helanshanense CCNWM S002958452 A tumefaciens B2 S000021216 R larrymoorei 3-10 S000824040 Amorphomonas oryzae B S003289156 endophytic bacterium S000964676 A fabrum str C58 S000444931 P viscosum CICC10215 S003717524 Shinella sp M80 S002229177 A tumefaciens RR5 S001588055 Beijerinckia fluminen S000017731 A vitis CFBP 2617 S000323103 R sp TCK S000437650 R vitis NCPPB3554 S000015668 A sp LMG 11915 S002232356 R sphaerophysae CCNWQ S000434676 R loessense CCBAU 719 S000140466 R daejeonense L61T KC S000421666 A larrymoorei ICMP 15 S000330242 S sp S1-T-10 S003262687 A tumefaciens NBRC 15 S001098292 R huautlense CCBAU 65 S000996028 R giardinii CCBAU 012 S002445816 R skierniewicense AL9 S000127045 Azospirillum sp Z2962 S002408292 A rubi LMG 159 S000721033 A vitis G-Ag-19 S001241092 Blastobacter aggregat S000021974 S sp S009 S003749510 A viscosum SDGD01 S000635884 A sp CCBAU 23089 S001589495 R pongamiae VKLR-01 S000438628 R giardinii H152 S000002681 A sp LMG 11936 S000391412 A albertimagni AOL15 S000721046 R radiobacter IAM 120 S002228822 A larrymoorei BAC1011 S000722695 R cellulosilyticum AL S000635883 B sp CCBAU 23024 S003262668 A vitis NBRC 15140 S000021625 R aggregatum IFAM 100 Caulobacter mirabilis S000003864 A sp MSMC211 S003287632 S sp MG−2011−4-AO S000429561 A sp PB S000806232 R soli DS-42 0.3 LR51 LR78 LR50 LT74 LR68 LR9 LR22 LR67 LR69 LR23 LR24 LR5 LR21 LT92 LR19 LR49 LR62 LR57 LR54 LT91 LR55 LR12 LR20 LR47 LR18 LR11 LR65 LR25 LR45 LR56 LT12 LR64 LR16 LR6 LR70 LR7 LR17 LT73 LR63 LR52 LR77 LR46 LT87 LR53 LR15 LR66 0.9489 0.9085 0.7606 0.9525 0.7316 0.7803 0.5579 1 0.685 0.6484 0.5141 0.8108 1

Figure 5: Bayesian dendrogram showing the relationships among the 16S rRNA gene sequences of 46 isolates belonging to the genus Rhizobium and those of reference type strains. Posterior probability values are indicated at each node. Nodes are collapsed at 70% probability. LT = bacteria isolated from the rhizosphere, LR = bacteria isolated from the roots (seeSection 2for details).

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0.05 LS99 LL78 LL46 LL70 LL54 LS9 LL56 LS11 LL40 LL48 LL41 LL53 LL89 LL9 LL90 LL95 LS100 LL69 LL47 LS93 LL92 LL86 LL61 LL55 LL62 0.95 0.9 0.74 0.55 0.99 0.5 1 0.81 0.88 0.78 1 0.84 1 0.71 1 0.83 1 0.91 1 0.99 S001187454 Pantoea anthophila LM S001610452 Pantoea calida S001187455 Pantoea deleyi LMG 24

S001610453 Pantoea gaviniae A18

S000691510 Pantoea dispersa LMG2 S001020552 Escherichia coli J016 S001187643 Pantoea eucrina LMG 2 S001187644 Pantoea conspicua LMG S001187456 Pantoea vagans LMG 24 S001187453 Pantoea eucalypti LMG S000412194 Pantoea allii BD 390 S000000125 Pantoea cypripedii DS S001187641 Pantoea septica LMG 5 S000016079 Pantoea agglomerans D

S000381168 Pantoea ananatis ATCC S001187642 Pantoea brenneri LMG

S000381179 Pantoea stewartii ATC

Figure 6: Bayesian dendrogram showing the relationships among the 16S rRNA gene sequences of 25 isolates belonging to the genus Pantoea and those of reference type strains. Posterior probability values are indicated at the node. Nodes are collapsed at 70% probability. LS = bacteria isolated from the stem, LL = bacteria isolated from the leaves (seeSection 2for details).

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0.5 LL6 LL67 LL81 LL5 LL8 LL29 LS87 LL100 LL82 LL85 LL83 LL7 LT3 LT5 LT67 LT56 LL32 LL84 LL57 LL59 LT7 LL30 LL2 LT85 LL31 LL1 LT4 LT6 LT8 LT2 0.8882 0.679 0.997 0.9977 0.5035 0.644 0.9964 0.9961 0.9758 0.7238 0.9846 0.5929 0.504 0.7041 0.7642 0.6421 1 0.6098 0.5001 0.5655 0.6027 S000964148 M flavum S000381731 M keratanolyticum S000003131 M imperiale S000967183 M insulae S000006251 M dextranolyticum S000650697 M thalassium S000000028 M flavescens S000440800 M maritypicum S000711001 M terricola S000014753 M foliorum S000871546 M soli S000541107 M koreense S000019591 M liquefaciens S000001603 M arabinogalactanolyt S000622938 M deminutum S000469185 M kitamiense S000892535 M profundi S000841857 M indicum S000012860 M phyllosphaerae S000965858 M binotii S001155658 M lindanitolerans S000381738 M chocolatum S000001703 M resistens S000121408 M paraoxydans S000381732 M luteolum S000440798 M xylanilyticum S000018525 M esteraromaticum S001577784 M mitrae S000964149 M lacus S001187351 M invictum S000381734 M terrae S000083932 M aerolatum S000650694 M hominis S001351455 M agarici S000009659 M schleiferi S000390332 M gubbeenense S000473677 M halotolerans S000622940 M aoyamense S000020165 M testaceum S000539538 M oleivorans S001417385 M pseudoresistens S000013457 M lacticum S000381735 M terregens S000010831 M laevaniformans S001577352 M radiodurans S000901905 M aquimaris S000727788 M ginsengisoli S000539539 M hydrocarbonoxydans S000381737 M ketosireducens S000427347 M halophilum S001020552 Escherichia coli S000006247 M aurum S000022611 M barkeri S000891240 M luticocti S000622939 M pumilum S001417386 M humi S000843265 M kribbense S000381733 M saperdae S000021147 M trichothecenolyticu S000964146 M awajiense S000711011 M pygmaeum S001014065 M hatanonis S000427348 M aurantiacum S000002734 M arborescens S000964147 M fluvii S000503539 M natoriense S000007793 M oxydans

Figure 7: Bayesian dendrogram showing the relationships among the 16S rRNA gene sequences of 30 isolates belonging to the genus Microbacterium and those of reference type strains. Posterior probability values are indicated at the node. Nodes are collapsed at 70% probability. LT = bacteria isolated from the rhizosphere, LS = bacteria isolated from the stem, LL = bacteria isolated from the leaves (see Section 2for details).

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Table 3: The 40 Burkholderia cepacia complex strains used as targets in the cross streak experiments.

Target strain

Strain Species Origin

FCF1 B. cepacia CF FCF3 LMG17588 B. multivorans ENV FCF16 B. cenocepacia (IIIA) CF J2315 FCF18 B. cenocepacia (IIIB) CF FCF20 FCF23 FCF24 FCF27 FCF29 FCF30 LMG16654 C5424 CEP511 MVPC1/16 ENV MVPC1/73 LMG19230

B. cenocepacia (IIIC) ENV

LMG19240 FCF38 _{B. cenocepacia (IIID)} _CF LMG21462 FCF41 B. stabilis CF FCF42 B. vietnamiensis CF TVV75 ENV LMG18941 B. dolosa CF LMG18942 LMG18943 MCI7 B. ambifaria ENV LMG19467 CF LMG19182 ENV LMG16670 B. anthina ENV FCF43 B. pyrrocina CF LSED4 B. lata CF LMG24064 B. latens CF LMG24065 B. diffusa CF LMG23361 B. contaminans AI LMG24067 B. seminals CF LMG24068 B. metallica CF LMG24066 B. arboris ENV LMG24263 B. ubonensis NI

Abbreviations: CF: strains isolated from cystic fibrosis patients; AI: strains isolated from animal infection; NI: strains isolated from nosocomial infec-tion; ENV: environmental strain.

isolates, showed similarities with the stress resistant B. safensis.

(vi) Lastly, more than the half of the bacterial isolates were affiliated to the genus Pseudomonas; the phylo-genetic tree reported in Supplemental Figure 2 is quite

complex. Regardless of the overall low support of the tree, (typical of Pseudomonas phylogenies), some observations may be drawn; the presence of large unresolved clusters shows clearly a low divergence of the isolated colonies, implying the existence of clonal populations; Pseudomonas spp. isolated from the different compartments are intermixed, suggest-ing the presence of a continuum rhizosphere-leaves (Pseudomonas is the only genus found in all the 4 sample analysed).

3.2. Inhibition of Burkholderia Cepacia Complex Strains Growth by L. angustifolia Endophytic Bacteria. In order to check the ability of L. angustifolia bacterial endophytes to antagonize the growth of (opportunistic) human pathogenic bacteria, cross-streak experiments were carried out as described in Section 2 using a panel of the 19 randomly chosen different endophytic strains isolated from soil, roots, or leafs and phylogenetically assigned to six different gen-era (Bacillus, Microbacterium, Pantoea, Plantibacter, Pseu-domonas, and Rhizobium), as testers versus 40 Bcc strains representative of seventeen species (out of eighteen) and with either environmental or clinical origin, with some of which being multidrug-resistant. Data obtained are shown in Table 4. The analysis of these data revealed that all the tester strains were able to completely inhibit the growth of Bcc strains, including those with a clinical source and that exhibited multidrug resistance; this finding suggested that lavender endophytic bacteria are able to synthesize (strong) antimicrobial compounds. However, tester strains showed a different pattern of Bcc growth inhibition, even those affiliated to the same genus. In addition to this, there is no apparent difference in the antimicrobial potential between strains belonging to different plant compartments. Concerning the sensitivity of Bcc strains to the antimicrobial activity of lavender endophytes, strains belonging to different species exhibited a different sensitivity spectrum. However, it is quite interesting that strains with clinical origin appeared to be more sensitive to the antimicrobials synthesized by endophytic bacteria than their environmental counterparts (see for instance strains belonging to the species Burkholderia

cenocepacia IIIB) (Supplementary Table3).

4. Discussion

Medicinal plants are stirring the attention of many researchers, due to the presence of compounds that constitute a large fraction of the current pharmacopoeias. Natural products have been the source of most of the active ingredients of medicines and more than 80% of drug substances are natural products or inspired by a natural compound, including most of the of anticancer and anti-infective agents [50]. Lavender plants are grown mainly for the production of essential oil, which has antiseptic and anti-inflammatory properties. In spite of the relative harsh environment for bacterial colonization, L. angustifolia tissues were found to be rich in bacterial diversity. However, the endophytic community isolated from different plant

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Ta b le 4 :G ro w th o f 4 0 B u rkho lder ia cepacia co m p lex st ra in s in th e p re se nce/a b se nce o f endo p h yt ic b ac te ri al st ra in s is o la te d fr o m la vender . Sp ecies St ra in O ri gin LL1 LL2 LL3 LL4 LL6 LL7 LL9 LL10 L S1 LS2 L S3 LS4 L S5 LS6 L R1 LR2 L R3 LR4 L R5 C-B. ce pac ia FCF 1 CF −− − − − − − − − − − − − − − − − − − + B. ce pac ia FCF 3 CF +/ −− − − − − − − − − − − − − − − − − − + B. m u lt iv or an s LM G 17 588 En v + −− ++ /− +/ −− +/ − + − ++ /− +/ −− + − + −− + B. ce nocepac ia (III A ) FCF 16 C F −− − − − − − − − − − − − − − − − − − + B. ce nocepac ia (III A) J2 31 5 C F + −− +/ − +/ −− − +/ −− − + −−−− − − − − + B. ce nocepac ia (IIIB ) FCF 18 C F + −− − − − − − − − − − − − +/ −− − − − + B. ce nocepac ia (IIIB) F CF 20 CF + + + + + + /− ++ /− +/ − +/ − +/ − ++ /− ++ /− ++ + + + B. ce nocepac ia (IIIB) F CF 23 CF + −− − − − − − +/ −− − ++ /−− − + −− − − + B. ce nocepac ia (IIIB) F CF 24 CF + −− − − − − − +/ −− +/ −− − − + −− − − + B. ce nocepac ia (IIIB) F CF 27 CF + −− − − − − − +/ −− − − − − − − − − − + B. ce nocepac ia (IIIB) F CF 29 CF + −− +/ − +/ −− − +/ −− − − − − − + − + −− + B. ce nocepac ia (IIIB) F CF 30 CF + −− +/ − +/ −− − +/ − +/ −− − − − − + − + −− + B. ce nocepac ia (IIIB) L M G 16 65 4 C F + +/ −− ++ /− + − ++ − ++ −− + − + −− + B. ce nocepac ia (IIIB) C 54 24 CF + + /−− +/ −− − − +/ −− − +/ −− − − +/ −− + −− + B. ce nocepac ia (IIIB) C EP5 11 CF + −− +/ −− − − +/ − +/ −− +/ − +/ −− − + − + −− + B. ce nocepac ia (IIIB) M VPC 1/1 6 E n v + + /−− +/ − ++ /−− +/ − +/ − +/ − +/ −− +/ −− +/ −− + − +/ − + B. ce nocepac ia (IIIB) M VPC 1/7 3 E n v + + + + /− ++ + /− + + ++++++ + + + + /− + B. ce nocepac ia (IIII C) LM G 19 23 0 En v + + + + + + + /− + + ++++++ + + + + + B. ce nocepac ia (III C) LM G 19 24 0 En v + + + + + /− + − ++ + /− +++ + /− ++ + + + /− + B. ce nocepac ia (IIID) F CF 38 CF + −− − − − − − − − − − − − − − − − − + B. ce nocepac ia (IIID) L M G 21 4 62 C F −− − − − − − − − − − − − − − − − − − + B. sta bili s FCF 4 1 C F + +/ − ++ + /− +/ −− ++ + − ++ /−− ++ + + /−− + B. vi etna m ie n si s FCF 4 2 C F −− − − − − − − − − − − − − − − − − − + B. vi etna m ie n si s TV V 75 E n v + + /−− +/ −− − − +/ −− − ++ /−− − +/ −− + −− + B. do los a LM G 18 941 CF + −− +/ − +/ − +/ −− +/ −− − ++ /−− − +/ − +/ − + −− + B. do los a LM G 18 94 2 CF + + −− +/ − +/ −− ++ + /− ++ /−− − ++ /− ++ /−− + B. do los a LM G 18 94 3 CF + + /−− ++ /− +/ −− +/ − +/ −− ++ /−− − ++ /− + −− + B. am bifa ri a MC I 7 E n v + + − +/ − +/ − +/ −− ++ /−− ++ /−− − + − + −− + B. am bifa ri a LM G 194 67 CF + + − +/ − +/ −− − ++ /−− ++ /−− − + − +/ −− − + B. am bifa ri a LM G 19 18 2 En v + + − +/ − +/ −− − + −− + −−− + − +/ −− − + B. an th ina LM G 16 6 70 En v + −− − − + − + −− ++ /−− − + − +/ −− − + B. py rr oc in ia FCF 43 C F + /−− − − − − − − − − +/ −− − − − − +/ −− − + B. la ta LS ED 4 C F + − + −− +/ −− + −− ++ /−− − +/ − +/ − +/ −− − + B. la te n s LM G 24 0 6 4 CF + − + +/ −− +/ −− ++ − ++ − ++ + + + − + B. diff u sa LM G 24 0 65 CF + + /− + +/ − +/ − + − ++ − ++ − +/ − ++ + + − + B .c on tamin an s LM G 23 36 1 AI + + /− +/ − +/ − +/ − + − ++ + + + + + /− ++ + + − + B. se m inali s LM G 24 0 67 CF + + /− ++ /− + + + + + + +++++ + + + − + B. metallica L M G 24068 C F + + + + /− + + + + + + +++++ + + + − + B. ar bor is LM G 24 0 6 6 En v + +/ − ++ /− ++ + /− ++ + + + + /− +/ − ++ + + /−− + B. u bone n si s LM G 24 26 3 NI + −− − − +/ −− +/ − + − ++ −− +/ −− +/ −− − + A b b re via tio n s: CF :s tra in s is o la ted fr o m cy stic fib ro sis p at ien ts; A I: stra in s is o la te d fr o m animal inf ec tio n ;NI: stra in s is o la te d fr o m n os o co m ia li n fe ct ion ;E N V :e n vi ron m en ta ls tr ai n ;L :s tr ai ns is o la te d fr om th e le af ; S: st rains is o la te d fr o m the stem; R :s tr ains is ol at ed fr o m th e ro o t. Sy m b o ls: +: gr o w th ;+ /− :r ed uced gr o w th; + /−− :v er y red uced gr o w th; − :n o gro w th ;C -: P et ri d ishe s co n tai n ing on ly th e targe t st rai ns .

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compartments showed different levels of diversity, with the leaf showing the highest level and the stem the lowest (Table 1). It is noteworthy that the same level of diversity (regardless of community composition) showed by the leaf and roots community is already reported in other species [26] even if there are few direct comparisons of roots and phyllosphere bacterial communities, especially comparisons using material from the same plants. The microbial community residing in the phyllosphere is faced with a nutrient poor and variable environment that is characterized by fluctuating temperature, humidity, and UV radiation and so it is predicted to be more diverse than that within the rhizosphere, a more homeostatic environment. These findings, supported also by the similar bacterial titre, are in agreement with the idea that the source of inoculum for the leaf community (except for the vertically transferred via seeds) may be exclusive (e.g., aerosol, even if the process is not yet clarified, Bulgarelli et al. 2013) and not related to transmission from the roots. Moreover, the low diversity of the community isolated in the stem, dominated by clonally (Table 2) populations of Pseudomonas, could be related to the highly specific main tissue (phloem) present there, which could provide an homeostatic environment, rich in nutrient (sucrose contained in the phloem tissues) but poorer (if compared to leaf and roots) in secondary metabolites that may promote ecological niche differentiation.

Concerning the taxonomic community composition, dif-ferent taxonomic profiles were found for endosphere and rhizosphere. More in detail the rhizosphere community showed quite similar diversity indexes if compared to the roots one, but with a different composition; as an example, members of the phylum Actinobacteria are completely absent from the root internal tissues that are largely dominated by Proteobacteria, while they are present in the soil. To explain this finding a two-step model has been proposed [16]: soil abiotic properties determine the structure of the initial bacterial communities that is then modified by rhi-zodeposits with plant roots cell wall features and released metabolites promoting the growth of organotrophic bacteria, thereby initiating a soil community shift. In the second step, convergent host genotype-dependent selection [51,52] fine-tunes community profiles thriving on the rhizoplane and within plant roots.

Moreover, endosphere communities were different between plant organs (roots, stems, and leaves), even if the internal tissues are dominated, as already reported [25,52– 54] by members of the phylum Proteobacteria; an example, while rhizobia were isolated from root internal tissues, they are completely absent from stems and leaves (Figures2and 3); isolates belonging to the genus Pantoea, on the contrary, were isolated from aerial parts but not from root tissues. Hence, it is possible that plant might “select” specific taxa for entering inside its compartment. Such hypothesis is supported also by the pairwise differentiation values; the communities isolated from the different tissues, even those in physical continuity, are in fact strongly differentiated with the stem, representing a low diversity compartment separating the two high diversity organs roots and leaves. In this view it is noteworthy that in lavender only Pseudomonas represents

a ubiquitous and abundant genus of both rhizosphere and endosphere (Figure 3). The analysis of intrageneric diversity pointed out also that Pseudomonas isolates belong to a low differentiated clonal population (Table 2 and Suppl. Figure 2), suggesting also that this component of the community either diffused inside the organs from a radical or foliar entry point or established during plants development, putatively from the seed inoculum. A similar consideration can be proposed for isolates of rhizobia but for the rhizosphere-root internal tissues continuum: a low taxonomically differentiated community from the soil entered (increasing its relative abundance) inside the plant organ. An entry point from the leaves is on the contrary consistent with the distribution of Pantoea isolates with their strong diversity (Table 2) suggesting a diversification in response to the high variable leaf environment. The isolates belonging to the genus Stenotrophomonas show an interesting distribution pattern (Figure 3); they are rare in the rhizosphere, abundant in the roots and in the leaves, and absent in the stem; this finding suggests a different entry point or a negative selection in the stem; the second explanation is supported by admixture of root and leaves isolates (Figure 4).

The detailed phylogenetic analyses of the isolates enabled us also to putatively ascribe some isolates to already described endophytic strains that can be targeted for functional charac-terisation; many Microbacterium leaves isolates cluster with M. hydrocarbonoxydans and M. oleivorans, two species with crude oil degrading activity [49] being this metabolic activity that is found frequently associated with a phyllosphere adapted lifestyle [55]. Several isolates cluster with bacteria with interesting biotechnological or ecological applications, such as S. chelatiphaga, a remarkable species with biotech-nological potential in phytoremediation [56] and S. rizophila a plant associated bacterium with antifungal activity [57] or Bacillus mojavensis a bacteriumclosely related to B. subtilis and already reported having an endophytic lifestyle and showing an antagonistic role against plant pathogenic fungi [58]. On the contrary, many lavender Pseudomonas isolates cluster with known plant pathogen strains highlighting the sometimes subtle difference among pathogenic and endo-phytic lifestyle and with Pantoea agglomerans (and also with lavender isolates clustering withinthe B. licheniformis and B. soronensis groups), a known plant associated bacterium, and an opportunistic pathogen in immune-compromised human individuals drawing attention to the possible pathogenic action of endophytic bacteria in humans [59,60].

The overall analysis of the dendrograms points out that the characterization of isolates from endophytic communities can lead to the identification (as an example for Pantoea and rhizobia) of not yet described strains that may represent untapped resources of bioactive compounds of agricultural or medical interest.

Even though it has not been still completely demon-strated, it cannot be excluded that the possibility that the qualiquantitative spectrum of bioactive metabolites in med-ical plants and their essential oils may be related also on the activity of bacterial endophytes as they may promote plant health and growth, elicit plant metabolism, or directly produce biotechnologically relevant compounds [36]. In this

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context, the characterization of cultivable bacterial commu-nities is a fundamental step to this purpose since it paves the way to the possibility to build collections of isolates that may be phenotypically typed for important traits. In our opinion, data obtained in this work (Table 4and Supplementary Tables 2 and 3) offers a preliminary but very promising example of the biotechnological potential of bacteria isolated from medicinal plants. In this pilot study in fact the majority of the tested endophytes showed to inhibit the growth of several (in some case all) human pathogenic strains belonging to the Bcc complexes that are known for their resistance to traditional antibiotics. These data are particularly interesting if correlated with the recent finding that essential oils from different medicinal plants are able to strongly (or completely) interfere with the growth of the same Bcc strains [61] and that bacterial endophytes isolated from plants of the same species exhibited the same bioactivity (Maida et al., in preparation). It is therefore possible that the therapeutic properties of the essential oil might reside also on bioactive compounds of microbial origin. We are completely aware that the determi-nation of the correlation (eventually) existing between the composition of bacterial endophytic communities and the bioactivity of essential oils extracted from a medicinal plant would require the parallel analysis of the bacterial community and the essential oil activity extracted from the same plant. However, data obtained in this work and those from Maida et al. [62] support this idea.

It is also particularly intriguing, the idea that the antimi-crobial activity of essential oils may reside on the action of multiple compounds, some of them produced or elicited by the microbiota, limiting the observed rapid evolution of human pathogenic bacteria resistant to single molecule antimicrobials.

The characterization of the heterotrophic aerobic endo-phytic bacterial community of L. angustifolia highlighted the existence of a diversified community between different organs/tissues that may be related to the coexistence of different sources of inoculum and/or a selection of the com-munities promoted by nutrient sand metabolites availability and antagonistic forces. Several not previously characterized strains have been isolated and molecularly typed, confirming that the analysis of the bacterial communities inhabiting extreme or unconventional environments may represent a proper strategy for the discovery of untapped sources of functional biodiversity and bioactive molecule production.

Conflict of Interests

The authors declare that there is no conflict of interests regarding the publication of this paper.

Acknowledgments

This work was supported by grants from the Ente Cassa di Risparmio di Firenze (Grant 2013.0657, Herbiome: new antimicrobial compounds from endophytic bacteria isolated from medicinal plants). Giovanni Emiliani is financially

supported by the Regione Toscana POR CRO FSE 2007-2013 Asse IV Grant Sysbiofor (CNR-9). Marco Fondi and Elena Perrin are financially supported by the FEMS Advanced Fellowship (FAF2012) and the Buzzati-Traverso Foundation, respectively.

References

[1] B. Reinhold-Hurek and T. Hurek, “Living inside plants: bacterial endophytes,” Current Opinion in Plant Biology, vol. 14, no. 4, pp. 435–443, 2011.

[2] M. Rosenblueth and E. Mart´ınez-Romero, “Bacterial endo-phytes and their interactions with hosts,” Molecular Plant-Microbe Interactions, vol. 19, no. 8, pp. 827–837, 2006.

[3] R. P. Ryan, K. Germaine, A. Franks, D. J. Ryan, and D. N. Dowling, “Bacterial endophytes: recent developments and applications,” FEMS Microbiology Letters, vol. 278, no. 1, pp. 1–9, 2008.

[4] R. Ç akmakçi, F. Dönmez, A. Aydın, and F. S¸ahin, “Growth promotion of plants by plant growth-promoting rhizobacteria under greenhouse and two different field soil conditions,” Soil Biology and Biochemistry, vol. 38, pp. 1482–1487, 2006. [5] P. R. Hardoim, L. S. van Overbeek, and J. D. V. Elsas,

“Prop-erties of bacterial endophytes and their proposed role in plant growth,” Trends in Microbiology, vol. 16, no. 10, pp. 463–471, 2008.

[6] S. Compant, C. Cl´ement, and A. Sessitsch, “Plant growth-promoting bacteria in the rhizo- and endosphere of plants: their role, colonization, mechanisms involved and prospects for utilization,” Soil Biology and Biochemistry, vol. 42, no. 5, pp. 669–678, 2010.

[7] B. R. Glick, “Bacterial ACC deaminase and the alleviation of plant stress,” Advances in Applied Microbiology, vol. 56, pp. 291– 312, 2004.

[8] B. R. Glick, B. Todorovic, J. Czarny, Z. Cheng, J. Duan, and B. McConkey, “Promotion of plant growth by bacterial ACC deaminase,” Critical Reviews in Plant Sciences, vol. 26, no. 5-6, pp. 227–242, 2007.

[9] S. L. Doty, B. Oakley, G. Xin et al., “Diazotrophic endophytes of native black cottonwood and willow,” Symbiosis, vol. 47, no. 1, pp. 23–33, 2009.

[10] B. Lugtenberg and F. Kamilova, “Plant-growth-promoting rhi-zobacteria,” Annual Review of Microbiology, vol. 63, pp. 541–556, 2009.

[11] U. F. Castillo, G. A. Strobel, E. J. Ford et al., “Munumbicins, wide-spectrum antibiotics produced by Streptomyces NRRL 30562, endophytic on Kennedia nigriscans,” Microbiology, vol. 148, no. 9, pp. 2675–2685, 2002.

[12] Y. Igarashi, M. E. Trujillo, E. Mart´ınez-Molina et al., “Anti-tumor anthraquinones from an endophytic actinomycete Micromonospora lupini sp. nov,” Bioorganic & Medicinal Chem-istry Letters, vol. 17, no. 13, pp. 3702–3705, 2007.

[13] S. Shweta, J. H. Bindu, J. Raghu et al., “Isolation of endophytic bacteria producing the anti-cancer alkaloid camptothecine from Miquelia dentata Bedd. (Icacinaceae),” Phytomedicine, vol. 20, pp. 913–917, 2013.

[14] T. Taechowisan, A. Wanbanjob, P. Tuntiwachwuttikul, and J. Liu, “Anti-inflammatory activity of lansais from endophytic Streptomyces sp. SUC1 in LPS-induced RAW 264.7 cells,” Food and Agricultural Immunology, vol. 20, no. 1, pp. 67–77, 2009.

(15)

[15] P. R. Hardoim, C. C. P. Hardoim, L. S. van Overbeek, and J. D. van Elsas, “Dynamics of seed-borne rice endophytes on early plant growth stages,” PLoS ONE, vol. 7, no. 2, Article ID e30438, 2012.

[16] D. Bulgarelli, K. Schlaeppi, S. Spaepen, E. V. L. van Themaat, and P. Schulze-Lefert, “Structure and functions of the bacterial microbiota of plants,” Annual Review of Plant Biology, vol. 64, pp. 807–838, 2013.

[17] A. Mengoni, S. Mocali, G. Surico, S. Tegli, and R. Fani, “Fluctu-ation of endophytic bacteria and phytoplasmosis in elm trees,” Microbiological Research, vol. 158, no. 4, pp. 363–369, 2003. [18] A. Mengoni, F. Pini, L.-N. Huang, W.-S. Shu, and M.

Bazzicalupo, “Plant-by-plant variations of bacterial commu-nities associated with leaves of the nickel hyperaccumulator Alyssum bertolonii desv,” Microbial Ecology, vol. 58, no. 3, pp. 660–667, 2009.

[19] S. Mocali, E. Bertelli, F. di Cello et al., “Fluctuation of bacteria isolated from elm tissues during different seasons and from different plant organs,” Research in Microbiology, vol. 154, no. 2, pp. 105–114, 2003.

[20] F. Pini, A. Frascella, L. Santopolo et al., “Exploring the plant-associated bacterial communities in Medicago sativa L,” BMC Microbiology, vol. 12, article 78, 2012.

[21] A. Mengoni, L. Cecchi, and C. Gonnelli, “Nickel hyperac-cumulating plants and Alyssum bertolonii: model systems for studying biogeochemical interactions in serpentine soils,” in Bio-Geo Interactions in Metal-Contaminated Soils, E. Kothe and A. Varma, Eds., pp. 279–296, Springer, Berlin, Germany, 2012. [22] S. Ikeda, T. Okubo, M. Anda et al., “Community- and

genome-based views of plant-associated bacteria: plant-bacterial inter-actions in soybean and rice,” Plant and Cell Physiology, vol. 51, no. 9, pp. 1398–1410, 2010.

[23] F. Pini, M. Galardini, M. Bazzicalupo, and A. Mengoni, “Plant-bacteria association and symbiosis: are there common genomic traits in alphaproteobacteria?” Genes, vol. 2, no. 4, pp. 1017–1032, 2011.

[24] K. Aleklett and M. Hart, “The root microbiota, a fingerprint in the soil?” Plant Soil, vol. 370, pp. 671–686, 2013.

[25] A. Beneduzi, F. Moreira, P. B. Costa et al., “Diversity and plant growth promoting evaluation abilities of bacteria isolated from sugarcane cultivated in the South of Brazil,” Applied Soil Ecology, vol. 63, pp. 94–104, 2013.

[26] N. Bodenhausen, M. W. Horton, and J. Bergelson, “Bacterial communities associated with the leaves and the roots of Ara-bidopsis thaliana,” PLoS ONE, vol. 8, no. 2, Article ID e56329, 2013.

[27] T. F. da Silva, R. E. Voll´u, D. Jurelevicius et al., “Does the essential oil of Lippia sidoides Cham. (pepper-rosmarin) affect its endophytic microbial community?” BMC Microbiology, vol. 13, no. 1, article 29, 2013.

[28] M. E. Lucero, A. Unc, P. Cooke, S. Dowd, and S. Sun, “Endophyte microbiome diversity in micropropagated Atriplex canescens and Atriplex torreyi var griffithsii,” PLoS ONE, vol. 6, no. 3, Article ID e17693, 2011.

[29] D. S. Lundberg, S. L. Lebeis, S. H. Paredes et al., “Defining the core Arabidopsis thaliana root microbiome,” Nature, vol. 487, no. 7409, pp. 86–90, 2012.

[30] H. M. Cavanagh and J. M. Wilkinson, “Biological activities of lavender essential oil,” Phytotherapy Research, vol. 16, no. 4, pp. 301–308, 2002.

[31] G. Woronuk, Z. Demissie, M. Rheault, and S. Mahmoud, “Biosynthesis and therapeutic properties of Lavandula essential oil constituents,” Planta Medica, vol. 77, no. 1, pp. 7–15, 2011. [32] R. Perry, R. Terry, L. K. Watson, and E. Ernst, “Is lavender an

anxiolytic drug? A systematic review of randomised clinical trials,” Phytomedicine, vol. 19, pp. 825–835, 2012.

[33] S. de Rapper, G. Kamatou, A. Viljoen, and S. van Vuuren, “The in vitro antimicrobial activity of Lavandula angustifolia essential oil in combination with other aroma-therapeutic oils,” Evidence-Based Complementary and Alternative Medicine, vol. 2013, Article ID 852049, 10 pages, 2013.

[34] T. Moon, J. M. Wilkinson, and H. M. Cavanagh, “Antiparasitic activity of two Lavandula essential oils against Giardia duode-nalis, Trichomonas vaginalis and Hexamita inflata,” Parasitology Research, vol. 99, no. 6, pp. 722–728, 2006.

[35] P. S. Yap, S. H. Lim, C. P. Hu, and B. C. Yiap, “Com-bination of essential oils and antibiotics reduce antibiotic resistance in plasmid-conferred multidrug resistant bacteria,” Phytomedicine, vol. 20, pp. 710–713, 2013.

[36] G. Brader, S. Compant, B. Mitter, F. Trognitz, and A. Sessitsch, “Metabolic potential of endophytic bacteria,” Current Opinion in Biotechnology, vol. 27, pp. 30–37, 2014.

[37] P. Drevinek and E. Mahenthiralingam, “Burkholderia ceno-cepacia in cystic fibrosis: epidemiology and molecular mecha-nisms of virulence,” Clinical Microbiology and Infection, vol. 16, no. 7, pp. 821–830, 2010.

[38] S. Bazzini, C. Udine, and G. Riccardi, “Molecular approaches to pathogenesis study of Burkholderia cenocepacia, an important cystic fibrosis opportunistic bacterium,” Applied Microbiology and Biotechnology, vol. 92, no. 5, pp. 887–895, 2011.

[39] I. Maida, A. lo Nostro, G. Pesavento et al., “Exploring the anti-Burkholderia cepacia complex activity of essential oils: a preliminary analysis,” Evidence-Based Complementary and Alternative Medicine, vol. 2014, Article ID 573518, 10 pages, 2014. [40] A. Mengoni, R. Barzanti, C. Gonnelli, R. Gabbrielli, and M. Bazzicalupo, “Characterization of nickel-resistant bacteria isolated from serpentine soil,” Environmental Microbiology, vol. 3, no. 11, pp. 691–698, 2001.

[41] S. F. Altschul, T. L. Madden, A. A. Schaffer et al., “Gapped BLAST and PSI-BLAST: a new generation of protein database search programs,” Nucleic Acids Research, vol. 25, no. 17, pp. 3389–3402, 1997.

[42] R. C. Edgar, “MUSCLE: multiple sequence alignment with high accuracy and high throughput,” Nucleic Acids Research, vol. 32, no. 5, pp. 1792–1797, 2004.

[43] F. Ronquist, M. Teslenko, P. van der Mark et al., “Mrbayes 3.2: efficient bayesian phylogenetic inference and model choice across a large model space,” Systematic Biology, vol. 61, no. 3, pp. 539–542, 2012.

[44] K. Tamura, D. Peterson, N. Peterson, G. Stecher, M. Nei, and S. Kumar, “MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and max-imum parsimony methods,” Molecular Biology and Evolution, vol. 28, no. 10, pp. 2731–2739, 2011.

[45] F. Sievers, A. Wilm, D. Dineen et al., “Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega,” Molecular Systems Biology, vol. 7, article 539, 2011.

[46] Ø. Hammer, D. A. T. Harper, and P. D. Ryan, “PAST: pale-ontological statistics software package for education and data analysis,” Palaeontologia Electronica, vol. 4, no. 1, pp. 1–9, 2001.

(16)

[47] M. C. Papaleo, M. Fondi, I. Maida et al., “Sponge-associated microbial Antarctic communities exhibiting antimicrobial activity against Burkholderia cepacia complex bacteria,” Biotech-nology Advances, vol. 30, no. 1, pp. 272–293, 2012.

[48] A. lo Giudice, V. Bruni, and L. Michaud, “Characterization of Antarctic psychrotrophic bacteria with antibacterial activities against terrestrial microorganisms,” Journal of Basic Microbiol-ogy, vol. 47, no. 6, pp. 496–505, 2007.

[49] A. Schippers, K. Bosecker, C. Spr¨oer, and P. Schumann, “Microbacterium oleivorans sp. nov. and Microbacterium hydrocarbonoxydans sp. nov., novel crude-oil-degrading Gram-positive bacteria,” International Journal of Systematic and Evo-lutionary Microbiology, vol. 55, no. 2, pp. 655–660, 2005. [50] J. D. McChesney, S. K. Venkataraman, and J. T. Henri, “Plant

natural products: back to the future or into extinction?” Phyto-chemistry, vol. 68, no. 14, pp. 2015–2022, 2007.

[51] D. K. Manter, J. A. Delgado, D. G. Holm, and R. A. Stong, “Pyrosequencing reveals a highly diverse and cultivar-specific bacterial endophyte community in potato roots,” Microbial Ecology, vol. 60, no. 1, pp. 157–166, 2010.

[52] K. Ulrich, A. Ulrich, and D. Ewald, “Diversity of endophytic bacterial communities in poplar grown under field conditions,” FEMS Microbiology Ecology, vol. 63, no. 2, pp. 169–180, 2008. [53] N. R. Gottel, H. F. Castro, M. Kerley et al., “Distinct microbial

communities within the endosphere and rhizosphere of Populus deltoides roots across contrasting soil types,” Applied and Envi-ronmental Microbiology, vol. 77, no. 17, pp. 5934–5944, 2011. [54] B. Ma, X. Lv, A. Warren, and J. Gong, “Shifts in diversity and

community structure of endophytic bacteria and archaea across root, stem and leaf tissues in the common reed, Phragmites australis, along a salinity gradient in a marine tidal wetland of northern China,” Antonie van Leeuwenhoek, vol. 104, no. 5, pp. 759–768, 2013.

[55] J. A. Vorholt, “Microbial life in the phyllosphere,” Nature Reviews Microbiology, vol. 10, no. 12, pp. 828–840, 2012. [56] R. P. Ryan, S. Monchy, M. Cardinale et al., “The versatility

and adaptation of bacteria from the genus Stenotrophomonas,” Nature Reviews Microbiology, vol. 7, no. 7, pp. 514–525, 2009. [57] A. Wolf, A. Fritze, M. Hagemann, and G. Berg,

“Stenotrophomo-nas rhizophila sp. nov., a novel plant-associated bacterium with antifungal properties,” International Journal of Systematic and Evolutionary Microbiology, vol. 52, no. 6, pp. 1937–1944, 2002. [58] C. W. Bacon and D. M. Hinton, “Endophytic and biological

control potential of Bacillus mojavensis and related species,” Biological Control, vol. 23, no. 3, pp. 274–284, 2002.

[59] G. Berg, L. Eberl, and A. Hartmann, “The rhizosphere as a reservoir for opportunistic human pathogenic bacteria,” Envi-ronmental Microbiology, vol. 7, no. 11, pp. 1673–1685, 2005. [60] H. L. Tyler and E. W. Triplett, “Plants as a habitat for

ben-eficial and/or human pathogenic bacteria,” Annual Review of Phytopathology, vol. 46, pp. 53–73, 2008.

[61] R. Perry, R. Terry, L. K. Watson, and E. Ernst, “Is lavender an anxiolytic drug? A systematic review of randomised clinical trials,” Phytomedicine, vol. 19, no. 8-9, pp. 825–835, 2012. [62] I. Maida, A. lo Nostro, G. Pesavento et al., “Exploring the

anti-Burkholderia cepacia complex activity of essential oils: a preliminary analysis,” Evidence-Based Complementary and Alternative Medicine, vol. 2014, Article ID 573518, 10 pages, 2014.

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