Research ArticleLinking Bacterial Endophytic Communities to Essential OilsClues from Lavandula angustifolia Mill

Giovanni Emiliani1 Alessio Mengoni2 Isabel Maida2 Elena Perrin2

Carolina Chiellini2 Marco Fondi2 Eugenia Gallo3 Luigi Gori4 Valentina Maggini3

Alfredo Vannacci3 Sauro Biffi5 Fabio Firenzuoli4 and Renato Fani2

1 Trees and Timber Institute National Research Council Via Madonna del Piano No 10 Sesto Fiorentino 50019 Florence Italy2 Laboratory of Microbial and Molecular Evolution Department of Biology University of Florence Via Madonna del Piano 6Sesto Fiorentino 50019 Florence Italy

3 Department of Neuroscience Psychology Drug Research and Child Health University of FlorenceViale Pieraccini 6 50139 Florence Italy

4Center for Integrative Medicine Careggi University Hospital University of Florence Viale Pieraccini 6 50139 Florence Italy5 Il giardino delle Erbe via Del Corso 6 Casola Valsenio 48010 Ravenna Italy

Correspondence should be addressed to Renato Fani renatofaniunifiit

Received 17 January 2014 Accepted 29 April 2014 Published 26 May 2014

Academic Editor Gyorgyi Horvath

Copyright copy 2014 Giovanni Emiliani et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Endophytic bacteria play a crucial role in plant life and are also drawing much attention for their capacity to produce bioactivecompounds of relevant biotechnological interest Here we present the characterisation of the cultivable endophytic bacteria ofLavandula angustifolia Millmdasha species used since antiquity for its therapeutic propertiesmdashsince the production of bioactivemetabolites from medical plants may reside also in the activity of bacterial endophytes through their direct production PGPRactivity on host andor elicitation of plant metabolism Lavender tissues are inhabited by a tissue specific endophytic communitydominated by Proteobacteria highlighting also their difference from the rhizosphere environment where Actinobacteria andFirmicutes are also found Leavesrsquo endophytic community resulted as the most diverse from the other ecological niches Overallthe findings reported here suggest (i) the existence of different entry points for the endophytic community (ii) its differentiationon the basis of the ecological niche variability and (iii) a two-step colonization process for roots endophytes Lastly many isolatesshowed a strong inhibition potential against human pathogens and the molecular characterization demonstrated also the presenceof not previously described isolates that may constitute a reservoir of bioactive compounds relevant in the field of pathogen controlphytoremediation and human health

1 Introduction

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

magnesium and calcium [4] and showing plant growth-promoting activity (PGPR) related to the production ofphytohormones involved in regulatory metabolism such asethylene [5] indole-3-acetic acid (IAA) and acetoin 23-butanediol [6ndash8] Moreover endophytic bacteria can alsoimprove plant growth via nitrogen fixation [9]Other relevantfunctions performed by endophytic bacteria are representedby the decrease or prevention of the pathogenic effects ofcertain parasitic microorganisms with the production ofantimicrobial compounds or by increasing plant tolerance topollution or stresses [3 10] Bacterial endophytes are drawing

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2014 Article ID 650905 16 pageshttpdxdoiorg1011552014650905

2 Evidence-Based Complementary and Alternative Medicine

increasing interest as together with fungal endophytes theyare reported to produce a number of bioactive metabolitesrelevant to human health such as antibiotics [11] antitumorcompounds [12 13] and anti-inflammatory agents [14]

Endophytic bacteria can be further classified as ldquoobli-gaterdquo or ldquofacultativerdquo in accordance with their life strategiesObligate endophytes are strictly dependent on the host plantfor their growth and survival besides transmission to otherplants could occur only by seeds or via vectors while faculta-tive endophytes could grow outside host plants [15] Severalstudies have shown that facultative endophytes constitute thelargest 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 [16ndash20] supporting the idea that theability to enter plant tissues is a widespread phenotype of soiland rhizosphere bacteria and that plants exert only a limitedselectivity on the colonizing bacterial communities [21] evenif clues for a control over the bacterial colonizers operated byyet not completely clarified plant mechanism(s) are reported[5 22] Actually a recent bioinformatic study has shown thatinside the class of Alphaproteobacteria (which includes wellknown bacterial endophytes such as rhizobia and methy-lobacteria) few genomic signatures only could distinguishendophytes from nonendophytes [23] Another commonfeature of endophytic bacterial community is their strongtemporal and spatial variability [16 19 24] the communitycomposition not only varies in the soil compartment betweenthe rhizosphere and the root internal tissues in responseto complex and not yet fully clarified biotic (plant speciesand genotype dependent) and abiotic (soil characteristics)driven processes [16 24] but also among different aerialtissues (stems leaves flowers) of the same plant [25 26] Totalendophytes are influenced not only by the location within theplant but also by the presence of the main components of theessential 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 speciescomposition (or higher taxonomic ranks) especially usingcultivation-independent techniques as 16S rRNA genelibraries or metagenetic sequencing [20 28 29] Whilecultivation-independent techniques allow a deep coverage oftaxonomic diversity of bacterial communities they provideonly partial information on community structure and clearlyhamper the possibility to test the actual presence of PGPRactivities and bioactive molecule production in the bacterialcommunity

Among crops medicinal plants are stirring much atten-tion due to the increasing demand for green chemistryapproaches sustainable practices and especially in the questfor novel antibiotics able to tackle the increasing multidrugresistance of pathogenic bacteria In spite of the high rele-vance of medicinal plants to the best of our knowledge verylittle if nothing at all is known about the endophytic bacterialcommunities isolated frommedicinal plants For this reasonin this work we isolated and preliminary characterized froma taxonomical viewpoint the aerobic heterotrophic cultivableendophytic bacterial community of lavender (Lavandulaangustifolia Millsyn Lavandula officinalis Chaix Lavandula

veraDC Lavandula spica L var angustifoliaAuct) Lavenderhas a long history ofmedicinal use and is purported to possessrelaxant neurological and antibacterial effects [30]

The essential oil of Lavender has been used since antiq-uity for a variety of medical application [31] in particularanxiolytic activity of Lavender oil was confirmed [32] andits antimicrobial activity was demonstrated against differentpathogens [33] Moon et al [34] reported that low (le1)concentrations of L angustifolia and L times intermedia oil cancompletely eliminate Giardia duodenalis Trichomonas vagi-nalis and Hexamita inflata in vitro Preliminary results alsohighlighted the possibility for lavender essential oil to be usedas antibiotic resistance modifying agent in microorganisms[35]

It has also been demonstrated that the production ofbioactive compounds is significantly impacted by the geneticmilieu of the plant and by environmental factors [31] in thisframework it is increasingly evident that the collection ofmicroorganism living in associationwith complex organismsgenerally defined as microbiota plays a fundamental role inshaping their phenotypic features Since the qualiquantitativeproduction of bioactive metabolites in medical plants mayreside also in the activity of bacterial endophytes through thedirect production PGPR activity andor elicitation of plantmetabolism [36] the purpose of this research was thereforeto perform a cultivation-dependent study of the mesophilicaerobic heterotrophic bacterial endophytic community ofthe relevant medical and balsamic species L angustifolia inorder to (i) identify its composition (ii) its diversity betweendifferent plant compartments (roots stems and leaves) and(iii) its diversity in relation to the rhizosphere bacterialcommunity and (iv) build a collection of isolates to bescreened for the production of bioactive compounds and (v)test a panel of randomly selected endophytic bacteria versusopportunistic human pathogens belonging to the Burkholde-ria cepacia complex (Bcc) We have chosen these bacteriasince many strains of the complex are opportunistic humanpathogens and represent a serious concern for cystic fibrosis(CF) patients and immunocompromised individuals [37]responsible for the ldquocepacia syndromerdquo characterized by highfever severe progressive respiratory failure leukocytosisand elevated erythrocyte sedimentation rate Moreover Bccstrains are (naturally) resistant to many antibiotics such ascephalosporin120573-lactams polymyxins and aminoglycosidestherefore Bcc infections are very problematic to eradicate[37 38] In spite of this high degree of resistance to manyantibiotics it has been recently demonstrated that essentialoils extracted from sixmedicinal plants are able to completelyinhibit the growth of Bcc members including those with aclinical origin and exhibiting resistance to many antibiotics[39]

2 Materials and Methods

21 Plant Sampling and Isolation of Mesophilic CultivableEndophytic and Rhizosphere Bacteria Five agamically prop-agated potted L angustifolia plants were collected fromthe ldquoGiardino delle Erberdquo located in Casola Valsenio Italy

Evidence-Based Complementary and Alternative Medicine 3

in June 2012 On the same day plants were transported to thelaboratory for processing Pieces of 200mg of fresh leavesstems and roots tissues were collected from each of the 5plants and bulked (for a final sample of 1 g) to account forplants to plants variability Roots and stems samples weredivided into 1 cm long pieces and surface-sterilized for 40 swith 70 ethanol followed by 10min with 25 sodiumhypochlorite Plant leaves were surface-sterilized for 20 s with70 ethanol followed by 5min with 25 sodium hypochlo-rite To remove the disinfectant sections were rinsed threetimes for 5 min in sterile distilled water Samples were thendried with sterile filter paper and subsequently ground with2mL 09 sodium chloride with a sterile mortar Aliquots(100 120583L) of the last washing water were plated in triplicateas sterility controls Samples (100120583L) of tissue extracts andtheir different dilutions were plated in triplicate Aliquots of200mg of soil from each pot were collected and bulked for1 g total The soil samples were then resuspended in 5mL of10mM Mg

2SO4and placed under stirring for 1 h at room

temperatureEndophytic and rhizospheric bacteria were grown in trip-

licate on solid 5 tryptone soya broth (TSB) medium (OxoidLtd Basingstoke Hampshire UK) at 30∘C for 72 h Thenumber of aerobic heterotrophic bacteria was determined ascolony-forming units (CFUs) Each CFU determination wasperformed in triplicate and an average value of bacterial titrewas determined From each sample colonies were randomlyselected and singularly plated on 5 solid TSB Petri dishesFrom each isolate glycerol stock (25 final concentration)was prepared and stored at minus80∘C

22 PCR Amplification and Sequencing of 16S rRNA CodingGenes from Bacterial Endophytes Cell lysates were pre-pared by dissolving a bacterial colony in 100120583L steriledistilled water and incubation at 99∘C for 10min followedby 5min at 4∘C An aliquot of 2 120583L lysate was used forthe amplification reaction by polymerase chain reaction(PCR) Amplification of 16S rRNA genes was performedin a total volume of 20120583L containing 2 120583L of 10X reactionbuffer (Polymed Firenze Italy) 15mM MgCl

2 10 pmol

of each primer [27f 51015840-GAGAGTTTGATCCTGGCTCAGand 1495r 51015840-CTACGGCTACCTTGTTACGA] 025mM ofdNTP mix 2U of Taq DNA polymerase (Polymed) PCRreaction conditions were as described by Mengoni et al [40]

For sequencing reaction amplified 16S rDNA fragmentswere excised from 1 agarose gels and purified using theQIAquick Gel Extraction (Qiagen) according to the manu-facturerrsquos instructions Direct sequencing of amplicons wasperformed at the Genechron laboratory (Ylichron Srl Italy)with primer 27f on an ABI3730 DNA analyser (AppliedBiosystems Foster City CA USA) using the Big Dye Termi-nator Kit

23 Sequence Analysis and Phylogenetic Tree ConstructionThe sequences presented in this work have been deposited inGeneBank database under the accession numbers KF202531-KF202915 Partial 16S rDNA sequences were matched against

nucleotide sequences available in GenBank database usingthe BLASTn program [41]

MUSCLE [42] (httpwwwdrive5commuscle)was usedto align the 16S rDNA sequences obtained with the mostsimilar orthologous sequences retrieved from the RibosomalDatabase Project (RDP) database (httprdpcmemsuedu)Alignments were trimmed to eliminate poorly aligned regionand used to build Bayesian Maximum Parsimony (MP) andthe Neighbor joining (NJ) dendrograms

Bayesian dendrograms were obtained with MrBayes 32[43] with GTR substitution model with gamma-distributedrate variation across sites with 1000000 generations MPdendrograms were obtained with MEGA 5 [44] (httpwwwmegasoftwarenet) using the Tree-Bisection-Regrafting(TBR) algorithm with search level 3 in which the initialtrees were obtained by the random addition of sequences(500 replicates) the robustness of the inferred trees wasevaluated by 1000 bootstrap resamplings consistency andretention indexes (CI and RI) were calculated with Mesquitesoftware 275 (httpmesquiteprojectorg) NJ dendrogramswere obtained after calculation of a Kimura two-parameterdistance matrix with the software Mega 5 The robustnessof the inferred trees was evaluated by 1000 bootstrapresamplings

24 Statistical Analysis Pairwise sequence identity values(not taking deletion into account) were calculated using thestand-alone Clustal Omega [45] Genetic distances amongOTU were calculated using the Kimura 2 parameter modeland 1000 bootstraps replications in the MEGA 521 software[44] Diversity indexes were calculated with the PAST3software [46] Pairwise differentiation (Fst) values were cal-culated inside the GenoDive v 20b25 software (httpwwwpatrickmeirmanscomsoftwareGenoDivehtml) using vec-tor of presenceabsence of the bacterial genera in the differentsamples with 999 permutations

25 Cross-Streaking Experiments Antibacterial activity wasdetermined by using the cross-streak method [47 48]Hereinafter endophytic bacterial isolates to be tested forinhibitory activity will be termed ldquotesterrdquo strains whereasBcc strains used as a target will be called ldquotargetrdquo strainsCross-streaking experiments were carried out as previouslydescribed [47] by using Petri dishes Tester strains werestreaked across one-half of an agar plate with PCA mediumand incubated at 30∘C After 2 days of incubation targetstrains were streaked on PCA medium perpendicular to theinitial streak and plates were further incubated at 30∘C for 2days The antagonistic effect was indicated by the failure ofthe target strains to growThe list of target Bcc strains used inthis work is reported in Table 3

3 Results

31 Composition of Endophytic Bacterial Communities Isolatedfrom L Angustifolia Aerobic heterotrophic culturable bac-teria were isolated from leaves stems and roots of 5 plantsof Lavandula angustifolia Leaves samples had the highest

4 Evidence-Based Complementary and Alternative Medicine

number of CFUg fresh weight (68 times 105) whereas rootshad the lowest values (16 times 104) the difference being anywayof one order of magnitude only (stem and rhizospheric soilshowed titers of 65 times 104 and 34 times 105 resp) Plates werevisually inspected and no increase in colony number wasobserved with an extended incubation time of up to 7 days

On the triplicate plates of the same plant portion 100colonies were randomly selected and isolated on TSA agarplates A collection of 400 colonies was then prepared withisolates from the four samples types namely rhizosphericsoil roots stems and leaves

In order to determine the taxonomic composition of thebacterial communities isolated from the different compart-ments of L angustifolia plants and from rhizospheric soil16S rRNA genes were PCR-amplified from the isolates of thecollection as described in Materials and Methods An ampli-con of the expected size was obtained from each bacterialisolate (data not shown) and the nucleotide sequence of theamplicons was then determined In this way we obtained 395sequences each of whichwas used as seed to probe databases

Table 1 reports the percentage distribution of the iden-tified bacterial phyla in the different plant compartmentsand in the rhizospheric soil along with standard diver-sity indexes calculated on genera distribution the resultsshow a relative peak of diversity in the leaf and a min-imum in the stem (Shannon index 229 and 106 resp)Figure 1 depicts the taxonomic composition of the totallavender aerobic heterotrophic cultivable endophytic com-munity made up by a total of 11 genera the majority ofthe isolated strains (88) belonged to proteobacteria witha dominance of gammaproteobacteria (74) Pseudomonasbeing the most abundant genus accounting for the 51of total community followed by Stenotrophomonas (13)and Pantoea (9) Rhizobium is also frequently found inthe internal tissue of lavender representing 14 of thetotal 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 andJeotgalibacillus representing collectively 6 of the endophyticcollection

Bacteria isolated from the rhizospheric soil showed aquite different taxonomic composition (Figure 2(a)) whencompared to the overall endophytic community in the rhizo-sphere Bacillus is the most abundant genus (44) followedby Pseudomonas (30) Microbacterium (10) Rhizobium(7) and Arthrobacter (6) a genus that is not detectedin the endophytic community Figure 2 shows also the com-position of the endophytic communities isolated from thedifferent tissues (panels (b) (c) and (d) for roots stem andleaves resp) the three compartments are characterized bystrikingly different communities for the differential presenceof genera (eg Bacillus is found in roots and stem but absentin the leaves Sanguibacter and Plantibacter are detected inthe leaves only Jeotgalibacillus is found only in the roots) andthe overall diversity (8 genera are found in the leaves and only5 genera are in the stem) and the different relative presence ofthe other most abundantgenera

Table 1 Percentage distribution of bacterial phyla isolated fromL angustifolia roots stem leaves and rhizospheric soil standarddiversity indexes built on genera distribution are also presented

Soil Roots Stem LeavesProteobacteria 40 94 95 93Firmicutes 44 6 3 mdashActinobacteria 16 mdash 2 7Richness 7 6 5 8Evenness 073 072 045 076Shannon index 206 188 106 229

Figure 3 shows the distribution ofmain genera (occurringin gt5 of total isolates) in the 3 lavender tissues and in itsrhizosphere Isolates belonging to the genus Bacillus decreasetheir abundance from the rhizosphere to the stem anddisappear in the leaves while Pantoea and Microbacteriumshow a similar distribution in stems and leaves but are bothabsent in the roots Isolates from the Pseudomonas genusdominate in the stem community and are similarly occurring(ca 30 of total) in the other 3 samples Rhizobium spp wasfound in soil and represented 40 of roots isolates LastlyStenotrophomonas spp is abundant in the roots and leavesbut absent in the stem To further analyse the diversity ofthe bacterial communities isolated pairwise differentiation(Fst) values were calculated on vector of presenceabsenceof bacterial genera the lowest differentiation was registeredamong roots and rhizospheric soil (Fst = 0198) communitieswhile the highest among stems and leaves (Fst = 0512)Overall the stem endophytic community resulted as themostdifferentiated from the others (Fst = 0428 and 0394 versusroots and rhizospheric soil resp) while the leaves communityshowed similar differentiation values when compared torhizospheric soil (Fst = 0245) and roots (Fst = 0239)

Considering the low number of bacterial phyla found inthe endophytic community (gt95 of isolates were assignedto only 6 genera) we analysed the intrageneric level ofdiversity by means of 16S rRNA gene sequence identityvalues Data obtained are shown in Table 2 From the 16SrRNA gene sequence diversity indices reported that theisolates assigned to the genus Rhizobium possessed thelowest overall diversity suggesting the presence of a lownumber of species belonging to the genus Rhizobium Onthe other extreme the Pantoea isolates showed a strongerdifferentiation supporting the idea of the presence of dif-ferent species (sequence identity lt 97) To further char-acterize the isolated endophytic and rhizosphere bacteriaBayesian Maximum Parsimony and Neighbor Joining treesbased on 16S rRNA genes were constructed for the mostabundant genera namely Stenotrophomonas (Figure 4 andSuppl Figures 3 and 9 in Supplementary Material availableonline at httpdxdoiorg1011552014650905) Rhizobium(Figure 5 and Suppl Figures 4 and 10) Pantoea (Figure 6and Suppl Figures 5 and 11) Microbacterium (Figure 7 andSuppl Figures 6 and 12) Bacillus (Suppl Figures 1 7 and13) and Pseudomonas (Suppl Figures 2 8 and 14) OverallBayesian and NJ dendrograms showed a stronger support

Evidence-Based Complementary and Alternative Medicine 5

Bacillus 27 Jeotgalibacillus 03

Microbacterium 64

Pantoea 92 Plantibacter 03

Pseudomonas 512 Psychrobacter 07

Rhizobium 139

Salinibacterium 10

Sanguibacter 10

Stenotrophomonas 132

Figure 1 Composition of the aerobic heterotrophic endophytic (sensu stricto) bacterial community in L angustifolia tissuesThe compositionis reported as percentages of the total number of characterized isolates (119899 = 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

6 Evidence-Based Complementary and Alternative Medicine

100

90

80

70

60

50

40

30

20

10

0Pr

esen

ce (

)

SoilRoots

StemLeaves

Bacil

lus

Micr

obac

teriu

m

Pant

oea

Pseu

dom

onas

Rhiz

obiu

m

Sten

otro

phom

onas

Figure 3 Comparison of the relative abundance of bacterial genera in the different samples only genera with percentages gt5 in at least oneof 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 gt97 consideredas threshold of species identity (in brackets the total number of comparisons and the percentage) number of OTU with at least 1 pairwisecomparison with identity gt97

Genus Number of isolates Mean identity amongOTU () Mean distance among OTU

Number of pairwisecomparisons withidentity gt97

Number of OTU withidentity gt97

Pseudomonas 172 9425 0013 3840 (14878 258) 170Bacillus 47 9304 0027 329 (1128 29) 46Rhizobia 46 9861 0016 952 (1081 88) 46Stenotrophomonas 37 9534 0023 415 (703 59) 37Microbacterium 30 9500 0059 120 (465 25) 29Pantoea 26 8695 0126 24 (351 68) 7

(and consistency of the results) than the MP ones (see alsoSuppl Table 4) The analysis of the Bayesian phylogenetictrees revealed the following

(i) Endophytic bacteria isolated from the leavesand roots of lavender and assigned to the genusStenotrophomonas (Figure 4) are clearly split in twogroups One group contains isolates clustering withStenotrophomonas chelatiphaga interestingly thiscluster comprises isolates from both roots and leaves(no Stenotrophomonas were isolated from the stem)suggesting that two compartments might sharebacteria belonging to the same species even thoughthe polymorphism shown by the aligned partial 16Ssequences might suggest a diversification at the strainlevel The second cluster embeds roots isolates (withthe exception of leaves isolate LL44) clustering withStenotrophomonas rizophila

(ii) Concerning rhizobia (Figure 5) a grouping in onemain cluster of low differentiate sequences is present

These groups might contain new clades of plant-associated rhizobia which deservemore investigationin the future

(iii) From the phylogenetic tree of Figure 6 Pantoea iso-lated endophytes (from lavender stem and leaves)cluster with already described Pantoea spp anothergroup of isolates (all from the leaves) are divergingand thus may represent none yet described strains arefound to be associated with plants

(iv) Figure 7 depicts the phylogenetic position ofMicrobacterium isolates (mainly from leaves andrhizosphere) in the context of the genus referencestrains noticeably many leaves isolates cluster withM hydrocarbonoxydans and M oleivorans specieswhose members are able to perform crude oildegradation [49]

(v) Concerning Bacillus isolates (Suppl Figure 1) acluster of soil isolates including Bacillus mojavensiswas disclosed Another group again comprising soil

Evidence-Based Complementary and Alternative Medicine 7

009

LR48

LR31

LR41

LL38

LL43

LL94

LT48

LR98

LR86

LL88

LR72

LL17

LR28LR8

LRL93

LL39

LR61

LR76

LL37

LR7

LR29

LR34

LL77LL76

LR79

LL96

LR95LL44

LL45

LL13

LR26

LR99

LL75

LR80

LR32

LR60

LR75

066

091

1

1

089

091

091

087

093

057

078

07

091

S001020552 Escherichia coli

S001576206 S daejeonensis MJ03

S000559371 S ginsengisoli

S000007629 P geniculata ATCC 193

S000386319 S koreensis TR6-01

S000841904 S terrae R-32768

S000003927 S nitritireducens L2

S000841903 S humi R-32729

S000824093 S maltophilia IAM 124

S000010261 P hibiscicola ATCC 19

S000129976 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 genusStenotrophomonas and those of reference type strains Posterior probability values are indicated at the node Nodes are collapsed at 70probability LL = bacteria isolated from the leaves LR = bacteria isolated from the roots (see Section 2 for details)

8 Evidence-Based Complementary and Alternative Medicine

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 trS002222203 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 NRCPB10S002958314 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 MGminus2011minus4-AO

S000429561 A sp PB

S000806232 R soli DS-42

03

LR51

LR78

LR50

LT74

LR68

LR9

LR22

LR67

LR69

LR23LR24

LR5

LR21

LT92

LR19

LR49

LR62

LR57

LR54

LT91

LR55

LR12

LR20

LR47

LR18

LR11

LR65

LR25LR45

LR56

LT12

LR64

LR16

LR6

LR70

LR7

LR17

LT73

LR63

LR52

LR77

LR46

LT87

LR53

LR15

LR66094890908507606

09525

07316

07803

05579

1

0685

06484

05141

08108

1

Figure 5 Bayesian dendrogram showing the relationships among the 16S rRNA gene sequences of 46 isolates belonging to the genusRhizobium and those of reference type strains Posterior probability values are indicated at each node Nodes are collapsed at 70 probabilityLT = bacteria isolated from the rhizosphere LR = bacteria isolated from the roots (see Section 2 for details)

Evidence-Based Complementary and Alternative Medicine 9

005

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

095

09074

055

099

05

1

081

088

078

1

084

1

071

1

083

1

091

1

099

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 Pantoeaand those of reference type strains Posterior probability values are indicated at the node Nodes are collapsed at 70 probability LS = bacteriaisolated from the stem LL = bacteria isolated from the leaves (see Section 2 for details)

10 Evidence-Based Complementary and Alternative Medicine

05

LL6

LL67

LL81

LL5

LL8

LL29

LS87

LL100

LL82

LL85

LL83

LL7

LT3

LT5

LT67LT56

LL32

LL84

LL57

LL59

LT7

LL30

LL2

LT85

LL31LL1

LT4

LT6

LT8

LT2

08882

0679

0997

09977

05035

0644

0996409961

09758

07238

09846

05929

0504

0704107642

06421

1

06098

05001

05655

06027

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 paraoxydansS000381732 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 genusMicrobacterium and those of reference type strains Posterior probability values are indicated at the node Nodes are collapsed at 70probability LT = bacteria isolated from the rhizosphere LS = bacteria isolated from the stem LL = bacteria isolated from the leaves (seeSection 2 for details)

Evidence-Based Complementary and Alternative Medicine 11

Table 3The 40Burkholderia cepacia complex strains used as targetsin the cross streak experiments

Target strainStrain Species OriginFCF1 B cepacia CFFCF3LMG17588 B multivorans ENVFCF16 B cenocepacia (IIIA) CFJ2315FCF18

B cenocepacia (IIIB) CF

FCF20FCF23FCF24FCF27FCF29FCF30LMG16654C5424CEP511MVPC116 ENVMVPC173LMG19230 B cenocepacia (IIIC) ENVLMG19240FCF38 B cenocepacia (IIID) CFLMG21462FCF41 B stabilis CFFCF42 B vietnamiensis CFTVV75 ENVLMG18941

B dolosa CFLMG18942LMG18943MCI7

B ambifariaENV

LMG19467 CFLMG19182 ENVLMG16670 B anthina ENVFCF43 B pyrrocina CFLSED4 B lata CFLMG24064 B latens CFLMG24065 B diffusa CFLMG23361 B contaminans AILMG24067 B seminals CFLMG24068 B metallica CFLMG24066 B arboris ENVLMG24263 B ubonensis NIAbbreviations CF strains isolated from cystic fibrosis patients AI strainsisolated from animal infection NI strains isolated from nosocomial infec-tion ENV environmental strain

isolates showed similarities with the stress resistantB safensis

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

complex Regardless of the overall low support ofthe tree (typical of Pseudomonas phylogenies) someobservations may be drawn the presence of largeunresolved clusters shows clearly a low divergenceof the isolated colonies implying the existence ofclonal populations Pseudomonas spp isolated fromthe different compartments are intermixed suggest-ing the presence of a continuum rhizosphere-leaves(Pseudomonas is the only genus found in all the 4sample analysed)

32 Inhibition of Burkholderia Cepacia Complex StrainsGrowth by L angustifolia Endophytic Bacteria In order tocheck the ability of L angustifolia bacterial endophytes toantagonize the growth of (opportunistic) human pathogenicbacteria cross-streak experiments were carried out asdescribed in Section 2 using a panel of the 19 randomlychosen different endophytic strains isolated from soil rootsor leafs and phylogenetically assigned to six different gen-era (Bacillus Microbacterium Pantoea Plantibacter Pseu-domonas and Rhizobium) as testers versus 40 Bcc strainsrepresentative of seventeen species (out of eighteen) andwith either environmental or clinical origin with some ofwhich being multidrug-resistant Data obtained are shownin Table 4 The analysis of these data revealed that all thetester strains were able to completely inhibit the growthof Bcc strains including those with a clinical source andthat exhibited multidrug resistance this finding suggestedthat lavender endophytic bacteria are able to synthesize(strong) antimicrobial compounds However tester strainsshowed a different pattern of Bcc growth inhibition eventhose affiliated to the same genus In addition to this thereis no apparent difference in the antimicrobial potentialbetween strains belonging to different plant compartmentsConcerning the sensitivity of Bcc strains to the antimicrobialactivity of lavender endophytes strains belonging to differentspecies exhibited a different sensitivity spectrum However itis quite interesting that strains with clinical origin appearedto be more sensitive to the antimicrobials synthesized byendophytic bacteria than their environmental counterparts(see for instance strains belonging to the species Burkholderiacenocepacia IIIB) (Supplementary Table 3)

4 Discussion

Medicinal plants are stirring the attention of manyresearchers due to the presence of compounds thatconstitute a large fraction of the current pharmacopoeiasNatural products have been the source of most of theactive ingredients of medicines and more than 80 of drugsubstances are natural products or inspired by a naturalcompound including most of the of anticancer and anti-infective agents [50] Lavender plants are grown mainlyfor the production of essential oil which has antisepticand anti-inflammatory properties In spite of the relativeharsh environment for bacterial colonization L angustifoliatissues were found to be rich in bacterial diversity Howeverthe endophytic community isolated from different plant

12 Evidence-Based Complementary and Alternative Medicine

Table4Growth

of40

Burkholderiacepacia

complex

strains

inthep

resenceabsenceo

fend

ophytic

bacterialstrains

isolated

from

lavend

er

Species

Strain

Orig

inLL

1LL

2LL

3LL

4LL

6LL

7LL

9LL

10LS

1LS

2LS

3LS

4LS

5LS

6LR

1LR

2LR

3LR

4LR

5C-

Bcepacia

FCF1

CFminusminusminusminusminusminusminusminus

minusminusminusminusminusminusminusminusminusminusminus

+B

cepacia

FCF3

CF+minusminusminusminusminusminusminusminus

minusminusminusminusminusminusminusminusminusminusminus

+B

multivoran

sLM

G17588

Env

+minusminus

++minus

+minusminus

+minus

+minus

++minus

+minusminus

+minus

+minusminus

+B

cenocepacia

(IIIA

)FC

F16

CFminusminusminusminusminusminusminusminus

minusminusminusminusminusminusminusminusminusminusminus

+B

cenocepacia

(IIIA

)J2315

CF+minusminus

+minus

+minusminusminus

+minusminusminus

+minusminusminusminusminusminusminusminus

+B

cenocepacia

(IIIB)

FCF18

CF+minusminusminusminusminusminusminus

minusminusminusminusminusminus

+minusminusminusminusminus

+B

cenocepacia

(IIIB)

FCF20

CF+

++

++

+minus

++minus

+minus

+minus

+minus

++minus

++minus

++

++

+B

cenocepacia

(IIIB)

FCF23

CF+minusminusminusminusminusminusminus

+minusminusminus

++minusminusminus

+minusminusminusminus

+B

cenocepacia

(IIIB)

FCF24

CF+minusminusminusminusminusminusminus

+minusminus

+minusminusminusminus

+minusminusminusminus

+B

cenocepacia

(IIIB)

FCF27

CF+minusminusminusminusminusminusminus

+minusminusminusminusminusminusminusminusminusminusminus

+B

cenocepacia

(IIIB)

FCF29

CF+minusminus

+minus

+minusminusminus

+minusminusminusminusminusminusminus

+minus

+minusminus

+B

cenocepacia

(IIIB)

FCF30

CF+minusminus

+minus

+minusminusminus

+minus

+minusminusminusminusminusminus

+minus

+minusminus

+B

cenocepacia

(IIIB)

LMG16654

CF+

+minusminus

++minus

+minus

++minus

++minusminus

+minus

+minusminus

+B

cenocepacia

(IIIB)

C5424

CF+

+minusminus

+minusminusminusminus

+minusminusminus

+minusminusminusminus

+minusminus

+minusminus

+B

cenocepacia

(IIIB)

CEP5

11CF

+minusminus

+minusminusminusminus

+minus

+minusminus

+minus

+minusminusminus

+minus

+minusminus

+B

cenocepacia

(IIIB)

MVPC

116

Env

++minusminus

+minus

++minusminus

+minus

+minus

+minus

+minusminus

+minusminus

+minusminus

+minus

+minus

+B

cenocepacia

(IIIB)

MVPC

173

Env

++

++minus

++

+minus

++

++

++

++

++

++minus

+B

cenocepacia

(IIIIC)

LMG19230

Env

++

++

++

+minus

++

++

++

++

++

++

+B

cenocepacia

(IIIC

)LM

G19240

Env

++

++

+minus

+minus

++

+minus

++

++minus

++

++

+minus

+B

cenocepacia

(IIID)

FCF38

CF+minusminusminusminusminusminusminus

minusminusminusminusminusminusminusminusminusminusminus

+B

cenocepacia

(IIID)

LMG21462

CFminusminusminusminusminusminusminusminus

minusminusminusminusminusminusminusminusminusminusminus

+B

stabilis

FCF41

CF+

+minus

++

+minus

+minusminus

++

+minus

++minusminus

++

++minusminus

+B

vietnamien

sisFC

F42

CFminusminusminusminusminusminusminusminus

minusminusminusminusminusminusminusminusminusminusminus

+B

vietnamien

sisTV

V75

Env

++minusminus

+minusminusminusminus

+minusminusminus

++minusminusminus

+minusminus

+minusminus

+B

dolosa

LMG18941

CF+minusminus

+minus

+minus

+minusminus

+minusminusminus

++minusminusminus

+minus

+minus

+minusminus

+B

dolosa

LMG18942

CF+

+minusminus

+minus

+minusminus

++

+minus

++minusminusminus

++minus

++minusminus

+B

dolosa

LMG18943

CF+

+minusminus

++minus

+minusminus

+minus

+minusminus

++minusminusminus

++minus

+minusminus

+B

ambifaria

MCI

7En

v+

+minus

+minus

+minus

+minusminus

++minusminus

++minusminusminus

+minus

+minusminus

+B

ambifaria

LMG1946

7CF

++minus

+minus

+minusminusminus

++minusminus

++minusminusminus

+minus

+minusminusminus

+B

ambifaria

LMG19182

Env

++minus

+minus

+minusminusminus

+minusminus

+minusminusminus

+minus

+minusminusminus

+B

anthina

LMG16670

Env

+minusminusminusminus

+minus

+minusminus

++minusminusminus

+minus

+minusminusminus

+B

pyrrocinia

FCF43

CF+minusminusminusminusminusminusminusminus

minusminus

+minusminusminusminusminusminus

+minusminusminus

+B

lata

LSED

4CF

+minus

+minusminus

+minusminus

+minusminus

++minusminusminus

+minus

+minus

+minusminusminus

+B

latens

LMG2406

4CF

+minus

++minusminus

+minusminus

++minus

++minus

++

++

+minus

+B

diffu

saLM

G24065

CF+

+minus

++minus

+minus

+minus

++minus

++minus

+minus

++

++minus

+B

contam

inan

sLM

G23361

AI

++minus

+minus

+minus

+minus

+minus

++

++

++

+minus

++

++minus

+B

seminalis

LMG24067

CF+

+minus

++minus

++

++

++

++

++

++

++minus

+B

metallica

LMG2406

8CF

++

++minus

++

++

++

++

++

++

++minus

+B

arboris

LMG2406

6En

v+

+minus

++minus

++

+minus

++

++

++minus

+minus

++

++minusminus

+B

ubonensis

LMG24263

NI

+minusminusminusminus

+minusminus

+minus

+minus

++minusminus

+minusminus

+minusminusminus

+Ab

breviatio

nsC

Fstr

ains

isolatedfro

mcysticfi

brosispatie

ntsAIstr

ains

isolated

from

anim

alinfectionNIstr

ains

isolated

from

nosocomialinfectio

nEN

Venvironm

entalstrainLstr

ains

isolatedfro

mtheleaf

Sstr

ains

isolatedfro

mthes

temR

strains

isolatedfro

mther

oot

Symbo

ls+

grow

th+

minusreduced

grow

th+

minusminusveryredu

cedgrow

thminus

nogrow

thC

-Petrid

ishes

containing

onlythetargetstrains

Evidence-Based Complementary and Alternative Medicine 13

compartments showed different levels of diversity withthe 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 theleaf and roots community is already reported in otherspecies [26] even if there are few direct comparisons ofroots and phyllosphere bacterial communities especiallycomparisons using material from the same plants Themicrobial community residing in the phyllosphere is facedwith a nutrient poor and variable environment that ischaracterized by fluctuating temperature humidity and UVradiation and so it is predicted to be more diverse than thatwithin the rhizosphere a more homeostatic environmentThese findings supported also by the similar bacterial titreare in agreement with the idea that the source of inoculumfor the leaf community (except for the vertically transferredvia seeds) may be exclusive (eg aerosol even if the processis not yet clarified Bulgarelli et al 2013) and not related totransmission from the roots Moreover the low diversity ofthe community isolated in the stem dominated by clonally(Table 2) populations of Pseudomonas could be related tothe highly specific main tissue (phloem) present there whichcould provide an homeostatic environment rich in nutrient(sucrose contained in the phloem tissues) but poorer (ifcompared to leaf and roots) in secondary metabolites thatmay promote ecological niche differentiation

Concerning the taxonomic community composition dif-ferent taxonomic profiles were found for endosphere andrhizosphere More in detail the rhizosphere communityshowed quite similar diversity indexes if compared to theroots one but with a different composition as an examplemembers of the phylumActinobacteria are completely absentfrom the root internal tissues that are largely dominatedby Proteobacteria while they are present in the soil Toexplain this finding a two-step model has been proposed[16] soil abiotic properties determine the structure of theinitial bacterial communities that is then modified by rhi-zodeposits with plant roots cell wall features and releasedmetabolites promoting the growth of organotrophic bacteriathereby initiating a soil community shift In the second stepconvergent host genotype-dependent selection [51 52] fine-tunes community profiles thriving on the rhizoplane andwithin plant roots

Moreover endosphere communities were differentbetween plant organs (roots stems and leaves) even if theinternal tissues are dominated as already reported [25 52ndash54] by members of the phylum Proteobacteria an examplewhile rhizobia were isolated from root internal tissues theyare completely absent from stems and leaves (Figures 2 and3) isolates belonging to the genus Pantoea on the contrarywere isolated from aerial parts but not from root tissuesHence it is possible that plant might ldquoselectrdquo specific taxafor entering inside its compartment Such hypothesis issupported also by the pairwise differentiation values thecommunities isolated from the different tissues even those inphysical continuity are in fact strongly differentiated with thestem representing a low diversity compartment separatingthe two high diversity organs roots and leaves In this view itis noteworthy that in lavender only Pseudomonas represents

a ubiquitous and abundant genus of both rhizosphere andendosphere (Figure 3) The analysis of intrageneric diversitypointed out also that Pseudomonas isolates belong to a lowdifferentiated clonal population (Table 2 and Suppl Figure2) suggesting also that this component of the communityeither diffused inside the organs from a radical or foliar entrypoint or established during plants development putativelyfrom the seed inoculum A similar consideration can beproposed for isolates of rhizobia but for the rhizosphere-root internal tissues continuum a low taxonomicallydifferentiated community from the soil entered (increasingits relative abundance) inside the plant organ An entrypoint from the leaves is on the contrary consistent with thedistribution of Pantoea isolates with their strong diversity(Table 2) suggesting a diversification in response to the highvariable leaf environmentThe isolates belonging to the genusStenotrophomonas show an interesting distribution pattern(Figure 3) they are rare in the rhizosphere abundant in theroots and in the leaves and absent in the stem this findingsuggests a different entry point or a negative selection in thestem the second explanation is supported by admixture ofroot and leaves isolates (Figure 4)

The detailed phylogenetic analyses of the isolates enabledus also to putatively ascribe some isolates to already describedendophytic strains that can be targeted for functional charac-terisation many Microbacterium leaves isolates cluster withM hydrocarbonoxydans and M oleivorans two species withcrude oil degrading activity [49] being this metabolic activitythat is found frequently associated with a phyllosphereadapted lifestyle [55] Several isolates cluster with bacteriawith interesting biotechnological or ecological applicationssuch as S chelatiphaga a remarkable species with biotech-nological potential in phytoremediation [56] and S rizophilaa plant associated bacterium with antifungal activity [57] orBacillus mojavensis a bacteriumclosely related to B subtilisand already reported having an endophytic lifestyle andshowing an antagonistic role against plant pathogenic fungi[58] On the contrary many lavender Pseudomonas isolatescluster with known plant pathogen strains highlighting thesometimes subtle difference among pathogenic and endo-phytic lifestyle and with Pantoea agglomerans (and also withlavender isolates clustering withinthe B licheniformis and Bsoronensis groups) a known plant associated bacterium andan opportunistic pathogen in immune-compromised humanindividuals drawing attention to the possible pathogenicaction of endophytic bacteria in humans [59 60]

The overall analysis of the dendrograms points out thatthe characterization of isolates from endophytic communitiescan lead to the identification (as an example for Pantoeaand rhizobia) of not yet described strains that may representuntapped resources of bioactive compounds of agricultural ormedical interest

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

14 Evidence-Based Complementary and Alternative Medicine

context the characterization of cultivable bacterial commu-nities is a fundamental step to this purpose since it paves theway to the possibility to build collections of isolates that maybe phenotypically typed for important traits In our opiniondata obtained in this work (Table 4 and Supplementary Tables2 and 3) offers a preliminary but very promising exampleof the biotechnological potential of bacteria isolated frommedicinal plants In this pilot study in fact the majority ofthe tested endophytes showed to inhibit the growth of several(in some case all) human pathogenic strains belonging tothe Bcc complexes that are known for their resistance totraditional antibiotics These data are particularly interestingif correlated with the recent finding that essential oils fromdifferent medicinal plants are able to strongly (or completely)interfere with the growth of the same Bcc strains [61] and thatbacterial endophytes isolated from plants of the same speciesexhibited the same bioactivity (Maida et al in preparation)It is therefore possible that the therapeutic properties of theessential oil might reside also on bioactive compounds ofmicrobial origin We are completely aware that the determi-nation of the correlation (eventually) existing between thecomposition of bacterial endophytic communities and thebioactivity of essential oils extracted from a medicinal plantwould require the parallel analysis of the bacterial communityand the essential oil activity extracted from the same plantHowever data obtained in this work and those from Maidaet al [62] support this idea

It is also particularly intriguing the idea that the antimi-crobial activity of essential oils may reside on the actionof multiple compounds some of them produced or elicitedby the microbiota limiting the observed rapid evolutionof human pathogenic bacteria resistant to single moleculeantimicrobials

The characterization of the heterotrophic aerobic endo-phytic bacterial community of L angustifolia highlightedthe existence of a diversified community between differentorganstissues that may be related to the coexistence ofdifferent sources of inoculum andor a selection of the com-munities promoted by nutrient sand metabolites availabilityand antagonistic forces Several not previously characterizedstrains have been isolated and molecularly typed confirmingthat the analysis of the bacterial communities inhabitingextreme or unconventional environments may represent aproper strategy for the discovery of untapped sources offunctional biodiversity and bioactive molecule production

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This work was supported by grants from the Ente Cassadi Risparmio di Firenze (Grant 20130657 Herbiome newantimicrobial compounds from endophytic bacteria isolatedfrom medicinal plants) Giovanni Emiliani is financially

supported by the Regione Toscana POR CRO FSE 2007-2013 Asse IV Grant Sysbiofor (CNR-9) Marco Fondi andElena Perrin are financially supported by the FEMSAdvancedFellowship (FAF2012) and the Buzzati-Traverso Foundationrespectively

References

[1] B Reinhold-Hurek andTHurek ldquoLiving inside plants bacterialendophytesrdquo Current Opinion in Plant Biology vol 14 no 4 pp435ndash443 2011

[2] M Rosenblueth and E Martınez-Romero ldquoBacterial endo-phytes and their interactions with hostsrdquo Molecular Plant-Microbe Interactions vol 19 no 8 pp 827ndash837 2006

[3] R P Ryan K Germaine A Franks D J Ryan and DN Dowling ldquoBacterial endophytes recent developments andapplicationsrdquo FEMSMicrobiology Letters vol 278 no 1 pp 1ndash92008

[4] R Cakmakci F Donmez A Aydın and F Sahin ldquoGrowthpromotion of plants by plant growth-promoting rhizobacteriaunder greenhouse and two different field soil conditionsrdquo SoilBiology and Biochemistry vol 38 pp 1482ndash1487 2006

[5] P R Hardoim L S van Overbeek and J D V Elsas ldquoProp-erties of bacterial endophytes and their proposed role in plantgrowthrdquo Trends in Microbiology vol 16 no 10 pp 463ndash4712008

[6] S Compant C Clement and A Sessitsch ldquoPlant growth-promoting bacteria in the rhizo- and endosphere of plantstheir role colonization mechanisms involved and prospects forutilizationrdquo Soil Biology and Biochemistry vol 42 no 5 pp669ndash678 2010

[7] B R Glick ldquoBacterial ACC deaminase and the alleviation ofplant stressrdquo Advances in Applied Microbiology vol 56 pp 291ndash312 2004

[8] B R Glick B Todorovic J Czarny Z Cheng J Duan andB McConkey ldquoPromotion of plant growth by bacterial ACCdeaminaserdquo Critical Reviews in Plant Sciences vol 26 no 5-6pp 227ndash242 2007

[9] S L Doty B Oakley G Xin et al ldquoDiazotrophic endophytesof native black cottonwood and willowrdquo Symbiosis vol 47 no 1pp 23ndash33 2009

[10] B Lugtenberg and F Kamilova ldquoPlant-growth-promoting rhi-zobacteriardquoAnnual Review ofMicrobiology vol 63 pp 541ndash5562009

[11] U F Castillo G A Strobel E J Ford et al ldquoMunumbicinswide-spectrum antibiotics produced by Streptomyces NRRL30562 endophytic on Kennedia nigriscansrdquo Microbiology vol148 no 9 pp 2675ndash2685 2002

[12] Y Igarashi M E Trujillo E Martınez-Molina et al ldquoAnti-tumor anthraquinones from an endophytic actinomyceteMicromonospora lupini sp novrdquo Bioorganic amp Medicinal Chem-istry Letters vol 17 no 13 pp 3702ndash3705 2007

[13] S Shweta J H Bindu J Raghu et al ldquoIsolation of endophyticbacteria producing the anti-cancer alkaloid camptothecinefromMiquelia dentata Bedd (Icacinaceae)rdquo Phytomedicine vol20 pp 913ndash917 2013

[14] T Taechowisan A Wanbanjob P Tuntiwachwuttikul and JLiu ldquoAnti-inflammatory activity of lansais from endophyticStreptomyces sp SUC1 in LPS-induced RAW 2647 cellsrdquo Foodand Agricultural Immunology vol 20 no 1 pp 67ndash77 2009

Evidence-Based Complementary and Alternative Medicine 15

[15] P R Hardoim C C P Hardoim L S van Overbeek and J Dvan Elsas ldquoDynamics of seed-borne rice endophytes on earlyplant growth stagesrdquo PLoS ONE vol 7 no 2 Article ID e304382012

[16] D Bulgarelli K Schlaeppi S Spaepen E V L van Themaatand P Schulze-Lefert ldquoStructure and functions of the bacterialmicrobiota of plantsrdquo Annual Review of Plant Biology vol 64pp 807ndash838 2013

[17] A Mengoni S Mocali G Surico S Tegli and R Fani ldquoFluctu-ation of endophytic bacteria and phytoplasmosis in elm treesrdquoMicrobiological Research vol 158 no 4 pp 363ndash369 2003

[18] A Mengoni F Pini L-N Huang W-S Shu and MBazzicalupo ldquoPlant-by-plant variations of bacterial commu-nities associated with leaves of the nickel hyperaccumulatorAlyssum bertolonii desvrdquo Microbial Ecology vol 58 no 3 pp660ndash667 2009

[19] S Mocali E Bertelli F di Cello et al ldquoFluctuation of bacteriaisolated from elm tissues during different seasons and fromdifferent plant organsrdquo Research in Microbiology vol 154 no2 pp 105ndash114 2003

[20] F Pini A Frascella L Santopolo et al ldquoExploring the plant-associated bacterial communities in Medicago sativa Lrdquo BMCMicrobiology vol 12 article 78 2012

[21] A Mengoni L Cecchi and C Gonnelli ldquoNickel hyperac-cumulating plants and Alyssum bertolonii model systems forstudying biogeochemical interactions in serpentine soilsrdquo inBio-Geo Interactions in Metal-Contaminated Soils E Kothe andA Varma Eds pp 279ndash296 Springer Berlin Germany 2012

[22] S Ikeda T Okubo M Anda et al ldquoCommunity- and genome-based views of plant-associated bacteria plant-bacterial inter-actions in soybean and ricerdquo Plant and Cell Physiology vol 51no 9 pp 1398ndash1410 2010

[23] F Pini M Galardini M Bazzicalupo and A Mengoni ldquoPlant-bacteria association and symbiosis are there common genomictraits in alphaproteobacteriardquoGenes vol 2 no 4 pp 1017ndash10322011

[24] K Aleklett and M Hart ldquoThe root microbiota a fingerprint inthe soilrdquo Plant Soil vol 370 pp 671ndash686 2013

[25] A Beneduzi F Moreira P B Costa et al ldquoDiversity and plantgrowth promoting evaluation abilities of bacteria isolated fromsugarcane cultivated in the South of BrazilrdquoApplied Soil Ecologyvol 63 pp 94ndash104 2013

[26] N Bodenhausen M W Horton and J Bergelson ldquoBacterialcommunities associated with the leaves and the roots of Ara-bidopsis thalianardquo PLoS ONE vol 8 no 2 Article ID e563292013

[27] T F da Silva R E Vollu D Jurelevicius et al ldquoDoes theessential oil of Lippia sidoides Cham (pepper-rosmarin) affectits endophytic microbial communityrdquo BMC Microbiology vol13 no 1 article 29 2013

[28] M E Lucero A Unc P Cooke S Dowd and S SunldquoEndophyte microbiome diversity in micropropagated Atriplexcanescens and Atriplex torreyi var griffithsiirdquo PLoS ONE vol 6no 3 Article ID e17693 2011

[29] D S Lundberg S L Lebeis S H Paredes et al ldquoDefining thecoreArabidopsis thaliana rootmicrobiomerdquoNature vol 487 no7409 pp 86ndash90 2012

[30] H M Cavanagh and J M Wilkinson ldquoBiological activities oflavender essential oilrdquo Phytotherapy Research vol 16 no 4 pp301ndash308 2002

[31] G Woronuk Z Demissie M Rheault and S MahmoudldquoBiosynthesis and therapeutic properties of Lavandula essentialoil constituentsrdquo Planta Medica vol 77 no 1 pp 7ndash15 2011

[32] R Perry R Terry L K Watson and E Ernst ldquoIs lavender ananxiolytic drug A systematic review of randomised clinicaltrialsrdquo Phytomedicine vol 19 pp 825ndash835 2012

[33] S de Rapper G Kamatou A Viljoen and S van VuurenldquoThe in vitro antimicrobial activity of Lavandula angustifoliaessential oil in combination with other aroma-therapeutic oilsrdquoEvidence-Based Complementary and Alternative Medicine vol2013 Article ID 852049 10 pages 2013

[34] T Moon J M Wilkinson and H M Cavanagh ldquoAntiparasiticactivity of two Lavandula essential oils against Giardia duode-nalis Trichomonas vaginalis andHexamita inflatardquo ParasitologyResearch vol 99 no 6 pp 722ndash728 2006

[35] P S Yap S H Lim C P Hu and B C Yiap ldquoCom-bination of essential oils and antibiotics reduce antibioticresistance in plasmid-conferred multidrug resistant bacteriardquoPhytomedicine vol 20 pp 710ndash713 2013

[36] G Brader S Compant B Mitter F Trognitz and A SessitschldquoMetabolic potential of endophytic bacteriardquo Current Opinionin Biotechnology vol 27 pp 30ndash37 2014

[37] P Drevinek and E Mahenthiralingam ldquoBurkholderia ceno-cepacia in cystic fibrosis epidemiology and molecular mecha-nisms of virulencerdquo Clinical Microbiology and Infection vol 16no 7 pp 821ndash830 2010

[38] S Bazzini C Udine and G Riccardi ldquoMolecular approaches topathogenesis study of Burkholderia cenocepacia an importantcystic fibrosis opportunistic bacteriumrdquo Applied Microbiologyand Biotechnology vol 92 no 5 pp 887ndash895 2011

[39] I Maida A lo Nostro G Pesavento et al ldquoExploring theanti-Burkholderia cepacia complex activity of essential oilsa preliminary analysisrdquo Evidence-Based Complementary andAlternativeMedicine vol 2014 Article ID 573518 10 pages 2014

[40] A Mengoni R Barzanti C Gonnelli R Gabbrielli andM Bazzicalupo ldquoCharacterization of nickel-resistant bacteriaisolated from serpentine soilrdquo Environmental Microbiology vol3 no 11 pp 691ndash698 2001

[41] S F Altschul T L Madden A A Schaffer et al ldquoGappedBLAST and PSI-BLAST a new generation of protein databasesearch programsrdquo Nucleic Acids Research vol 25 no 17 pp3389ndash3402 1997

[42] R C Edgar ldquoMUSCLE multiple sequence alignment with highaccuracy and high throughputrdquo Nucleic Acids Research vol 32no 5 pp 1792ndash1797 2004

[43] F Ronquist M Teslenko P van der Mark et al ldquoMrbayes32 efficient bayesian phylogenetic inference and model choiceacross a large model spacerdquo Systematic Biology vol 61 no 3 pp539ndash542 2012

[44] K Tamura D Peterson N Peterson G Stecher M Nei andS Kumar ldquoMEGA5 molecular evolutionary genetics analysisusing maximum likelihood evolutionary distance and max-imum parsimony methodsrdquo Molecular Biology and Evolutionvol 28 no 10 pp 2731ndash2739 2011

[45] F Sievers A Wilm D Dineen et al ldquoFast scalable generationof high-quality protein multiple sequence alignments usingClustal Omegardquo Molecular Systems Biology vol 7 article 5392011

[46] Oslash Hammer D A T Harper and P D Ryan ldquoPAST pale-ontological statistics software package for education and dataanalysisrdquo Palaeontologia Electronica vol 4 no 1 pp 1ndash9 2001

16 Evidence-Based Complementary and Alternative Medicine

[47] M C Papaleo M Fondi I Maida et al ldquoSponge-associatedmicrobial Antarctic communities exhibiting antimicrobialactivity againstBurkholderia cepacia complex bacteriardquoBiotech-nology Advances vol 30 no 1 pp 272ndash293 2012

[48] A lo Giudice V Bruni and L Michaud ldquoCharacterization ofAntarctic psychrotrophic bacteria with antibacterial activitiesagainst terrestrial microorganismsrdquo Journal of Basic Microbiol-ogy vol 47 no 6 pp 496ndash505 2007

[49] A Schippers K Bosecker C Sproer and P SchumannldquoMicrobacterium oleivorans sp nov and Microbacteriumhydrocarbonoxydans sp nov novel crude-oil-degrading Gram-positive bacteriardquo International Journal of Systematic and Evo-lutionary Microbiology vol 55 no 2 pp 655ndash660 2005

[50] J D McChesney S K Venkataraman and J T Henri ldquoPlantnatural products back to the future or into extinctionrdquo Phyto-chemistry vol 68 no 14 pp 2015ndash2022 2007

[51] D K Manter J A Delgado D G Holm and R A StongldquoPyrosequencing reveals a highly diverse and cultivar-specificbacterial endophyte community in potato rootsrdquo MicrobialEcology vol 60 no 1 pp 157ndash166 2010

[52] K Ulrich A Ulrich and D Ewald ldquoDiversity of endophyticbacterial communities in poplar grown under field conditionsrdquoFEMS Microbiology Ecology vol 63 no 2 pp 169ndash180 2008

[53] N R Gottel H F Castro M Kerley et al ldquoDistinct microbialcommunities within the endosphere and rhizosphere ofPopulusdeltoides roots across contrasting soil typesrdquo Applied and Envi-ronmental Microbiology vol 77 no 17 pp 5934ndash5944 2011

[54] B Ma X Lv A Warren and J Gong ldquoShifts in diversity andcommunity structure of endophytic bacteria and archaea acrossroot stem and leaf tissues in the common reed Phragmitesaustralis along a salinity gradient in a marine tidal wetland ofnorthern Chinardquo Antonie van Leeuwenhoek vol 104 no 5 pp759ndash768 2013

[55] J A Vorholt ldquoMicrobial life in the phyllosphererdquo NatureReviews Microbiology vol 10 no 12 pp 828ndash840 2012

[56] R P Ryan S Monchy M Cardinale et al ldquoThe versatilityand adaptation of bacteria from the genus StenotrophomonasrdquoNature Reviews Microbiology vol 7 no 7 pp 514ndash525 2009

[57] AWolf A FritzeMHagemann andG Berg ldquoStenotrophomo-nas rhizophila sp nov a novel plant-associated bacterium withantifungal propertiesrdquo International Journal of Systematic andEvolutionary Microbiology vol 52 no 6 pp 1937ndash1944 2002

[58] C W Bacon and D M Hinton ldquoEndophytic and biologicalcontrol potential of Bacillus mojavensis and related speciesrdquoBiological Control vol 23 no 3 pp 274ndash284 2002

[59] G Berg L Eberl and A Hartmann ldquoThe rhizosphere as areservoir for opportunistic human pathogenic bacteriardquo Envi-ronmental Microbiology vol 7 no 11 pp 1673ndash1685 2005

[60] H L Tyler and E W Triplett ldquoPlants as a habitat for ben-eficial andor human pathogenic bacteriardquo Annual Review ofPhytopathology vol 46 pp 53ndash73 2008

[61] R Perry R Terry L K Watson and E Ernst ldquoIs lavender ananxiolytic drug A systematic review of randomised clinicaltrialsrdquo Phytomedicine vol 19 no 8-9 pp 825ndash835 2012

[62] I Maida A lo Nostro G Pesavento et al ldquoExploring theanti-Burkholderia cepacia complex activity of essential oilsa preliminary analysisrdquo Evidence-Based Complementary andAlternativeMedicine vol 2014 Article ID 573518 10 pages 2014

4 Evidence-Based Complementary and Alternative Medicine

number of CFUg fresh weight (68 times 105) whereas rootshad the lowest values (16 times 104) the difference being anywayof one order of magnitude only (stem and rhizospheric soilshowed titers of 65 times 104 and 34 times 105 resp) Plates werevisually inspected and no increase in colony number wasobserved with an extended incubation time of up to 7 days

On the triplicate plates of the same plant portion 100colonies were randomly selected and isolated on TSA agarplates A collection of 400 colonies was then prepared withisolates from the four samples types namely rhizosphericsoil roots stems and leaves

In order to determine the taxonomic composition of thebacterial communities isolated from the different compart-ments of L angustifolia plants and from rhizospheric soil16S rRNA genes were PCR-amplified from the isolates of thecollection as described in Materials and Methods An ampli-con of the expected size was obtained from each bacterialisolate (data not shown) and the nucleotide sequence of theamplicons was then determined In this way we obtained 395sequences each of whichwas used as seed to probe databases

Table 1 reports the percentage distribution of the iden-tified bacterial phyla in the different plant compartmentsand in the rhizospheric soil along with standard diver-sity indexes calculated on genera distribution the resultsshow a relative peak of diversity in the leaf and a min-imum in the stem (Shannon index 229 and 106 resp)Figure 1 depicts the taxonomic composition of the totallavender aerobic heterotrophic cultivable endophytic com-munity made up by a total of 11 genera the majority ofthe isolated strains (88) belonged to proteobacteria witha dominance of gammaproteobacteria (74) Pseudomonasbeing the most abundant genus accounting for the 51of total community followed by Stenotrophomonas (13)and Pantoea (9) Rhizobium is also frequently found inthe internal tissue of lavender representing 14 of thetotal 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 andJeotgalibacillus representing collectively 6 of the endophyticcollection

Bacteria isolated from the rhizospheric soil showed aquite different taxonomic composition (Figure 2(a)) whencompared to the overall endophytic community in the rhizo-sphere Bacillus is the most abundant genus (44) followedby Pseudomonas (30) Microbacterium (10) Rhizobium(7) and Arthrobacter (6) a genus that is not detectedin the endophytic community Figure 2 shows also the com-position of the endophytic communities isolated from thedifferent tissues (panels (b) (c) and (d) for roots stem andleaves resp) the three compartments are characterized bystrikingly different communities for the differential presenceof genera (eg Bacillus is found in roots and stem but absentin the leaves Sanguibacter and Plantibacter are detected inthe leaves only Jeotgalibacillus is found only in the roots) andthe overall diversity (8 genera are found in the leaves and only5 genera are in the stem) and the different relative presence ofthe other most abundantgenera

Table 1 Percentage distribution of bacterial phyla isolated fromL angustifolia roots stem leaves and rhizospheric soil standarddiversity indexes built on genera distribution are also presented

Soil Roots Stem LeavesProteobacteria 40 94 95 93Firmicutes 44 6 3 mdashActinobacteria 16 mdash 2 7Richness 7 6 5 8Evenness 073 072 045 076Shannon index 206 188 106 229

Figure 3 shows the distribution ofmain genera (occurringin gt5 of total isolates) in the 3 lavender tissues and in itsrhizosphere Isolates belonging to the genus Bacillus decreasetheir abundance from the rhizosphere to the stem anddisappear in the leaves while Pantoea and Microbacteriumshow a similar distribution in stems and leaves but are bothabsent in the roots Isolates from the Pseudomonas genusdominate in the stem community and are similarly occurring(ca 30 of total) in the other 3 samples Rhizobium spp wasfound in soil and represented 40 of roots isolates LastlyStenotrophomonas spp is abundant in the roots and leavesbut absent in the stem To further analyse the diversity ofthe bacterial communities isolated pairwise differentiation(Fst) values were calculated on vector of presenceabsenceof bacterial genera the lowest differentiation was registeredamong roots and rhizospheric soil (Fst = 0198) communitieswhile the highest among stems and leaves (Fst = 0512)Overall the stem endophytic community resulted as themostdifferentiated from the others (Fst = 0428 and 0394 versusroots and rhizospheric soil resp) while the leaves communityshowed similar differentiation values when compared torhizospheric soil (Fst = 0245) and roots (Fst = 0239)

Considering the low number of bacterial phyla found inthe endophytic community (gt95 of isolates were assignedto only 6 genera) we analysed the intrageneric level ofdiversity by means of 16S rRNA gene sequence identityvalues Data obtained are shown in Table 2 From the 16SrRNA gene sequence diversity indices reported that theisolates assigned to the genus Rhizobium possessed thelowest overall diversity suggesting the presence of a lownumber of species belonging to the genus Rhizobium Onthe other extreme the Pantoea isolates showed a strongerdifferentiation supporting the idea of the presence of dif-ferent species (sequence identity lt 97) To further char-acterize the isolated endophytic and rhizosphere bacteriaBayesian Maximum Parsimony and Neighbor Joining treesbased on 16S rRNA genes were constructed for the mostabundant genera namely Stenotrophomonas (Figure 4 andSuppl Figures 3 and 9 in Supplementary Material availableonline at httpdxdoiorg1011552014650905) Rhizobium(Figure 5 and Suppl Figures 4 and 10) Pantoea (Figure 6and Suppl Figures 5 and 11) Microbacterium (Figure 7 andSuppl Figures 6 and 12) Bacillus (Suppl Figures 1 7 and13) and Pseudomonas (Suppl Figures 2 8 and 14) OverallBayesian and NJ dendrograms showed a stronger support

Evidence-Based Complementary and Alternative Medicine 5

Bacillus 27 Jeotgalibacillus 03

Microbacterium 64

Pantoea 92 Plantibacter 03

Pseudomonas 512 Psychrobacter 07

Rhizobium 139

Salinibacterium 10

Sanguibacter 10

Stenotrophomonas 132

Figure 1 Composition of the aerobic heterotrophic endophytic (sensu stricto) bacterial community in L angustifolia tissuesThe compositionis reported as percentages of the total number of characterized isolates (119899 = 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

6 Evidence-Based Complementary and Alternative Medicine

100

90

80

70

60

50

40

30

20

10

0Pr

esen

ce (

)

SoilRoots

StemLeaves

Bacil

lus

Micr

obac

teriu

m

Pant

oea

Pseu

dom

onas

Rhiz

obiu

m

Sten

otro

phom

onas

Figure 3 Comparison of the relative abundance of bacterial genera in the different samples only genera with percentages gt5 in at least oneof 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 gt97 consideredas threshold of species identity (in brackets the total number of comparisons and the percentage) number of OTU with at least 1 pairwisecomparison with identity gt97

Genus Number of isolates Mean identity amongOTU () Mean distance among OTU

Number of pairwisecomparisons withidentity gt97

Number of OTU withidentity gt97

Pseudomonas 172 9425 0013 3840 (14878 258) 170Bacillus 47 9304 0027 329 (1128 29) 46Rhizobia 46 9861 0016 952 (1081 88) 46Stenotrophomonas 37 9534 0023 415 (703 59) 37Microbacterium 30 9500 0059 120 (465 25) 29Pantoea 26 8695 0126 24 (351 68) 7

(and consistency of the results) than the MP ones (see alsoSuppl Table 4) The analysis of the Bayesian phylogenetictrees revealed the following

(i) Endophytic bacteria isolated from the leavesand roots of lavender and assigned to the genusStenotrophomonas (Figure 4) are clearly split in twogroups One group contains isolates clustering withStenotrophomonas chelatiphaga interestingly thiscluster comprises isolates from both roots and leaves(no Stenotrophomonas were isolated from the stem)suggesting that two compartments might sharebacteria belonging to the same species even thoughthe polymorphism shown by the aligned partial 16Ssequences might suggest a diversification at the strainlevel The second cluster embeds roots isolates (withthe exception of leaves isolate LL44) clustering withStenotrophomonas rizophila

(ii) Concerning rhizobia (Figure 5) a grouping in onemain cluster of low differentiate sequences is present

These groups might contain new clades of plant-associated rhizobia which deservemore investigationin the future

(iii) From the phylogenetic tree of Figure 6 Pantoea iso-lated endophytes (from lavender stem and leaves)cluster with already described Pantoea spp anothergroup of isolates (all from the leaves) are divergingand thus may represent none yet described strains arefound to be associated with plants

(iv) Figure 7 depicts the phylogenetic position ofMicrobacterium isolates (mainly from leaves andrhizosphere) in the context of the genus referencestrains noticeably many leaves isolates cluster withM hydrocarbonoxydans and M oleivorans specieswhose members are able to perform crude oildegradation [49]

(v) Concerning Bacillus isolates (Suppl Figure 1) acluster of soil isolates including Bacillus mojavensiswas disclosed Another group again comprising soil

Evidence-Based Complementary and Alternative Medicine 7

009

LR48

LR31

LR41

LL38

LL43

LL94

LT48

LR98

LR86

LL88

LR72

LL17

LR28LR8

LRL93

LL39

LR61

LR76

LL37

LR7

LR29

LR34

LL77LL76

LR79

LL96

LR95LL44

LL45

LL13

LR26

LR99

LL75

LR80

LR32

LR60

LR75

066

091

1

1

089

091

091

087

093

057

078

07

091

S001020552 Escherichia coli

S001576206 S daejeonensis MJ03

S000559371 S ginsengisoli

S000007629 P geniculata ATCC 193

S000386319 S koreensis TR6-01

S000841904 S terrae R-32768

S000003927 S nitritireducens L2

S000841903 S humi R-32729

S000824093 S maltophilia IAM 124

S000010261 P hibiscicola ATCC 19

S000129976 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 genusStenotrophomonas and those of reference type strains Posterior probability values are indicated at the node Nodes are collapsed at 70probability LL = bacteria isolated from the leaves LR = bacteria isolated from the roots (see Section 2 for details)

8 Evidence-Based Complementary and Alternative Medicine

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 trS002222203 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 NRCPB10S002958314 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 MGminus2011minus4-AO

S000429561 A sp PB

S000806232 R soli DS-42

03

LR51

LR78

LR50

LT74

LR68

LR9

LR22

LR67

LR69

LR23LR24

LR5

LR21

LT92

LR19

LR49

LR62

LR57

LR54

LT91

LR55

LR12

LR20

LR47

LR18

LR11

LR65

LR25LR45

LR56

LT12

LR64

LR16

LR6

LR70

LR7

LR17

LT73

LR63

LR52

LR77

LR46

LT87

LR53

LR15

LR66094890908507606

09525

07316

07803

05579

1

0685

06484

05141

08108

1

Figure 5 Bayesian dendrogram showing the relationships among the 16S rRNA gene sequences of 46 isolates belonging to the genusRhizobium and those of reference type strains Posterior probability values are indicated at each node Nodes are collapsed at 70 probabilityLT = bacteria isolated from the rhizosphere LR = bacteria isolated from the roots (see Section 2 for details)

Evidence-Based Complementary and Alternative Medicine 9

005

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

095

09074

055

099

05

1

081

088

078

1

084

1

071

1

083

1

091

1

099

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 Pantoeaand those of reference type strains Posterior probability values are indicated at the node Nodes are collapsed at 70 probability LS = bacteriaisolated from the stem LL = bacteria isolated from the leaves (see Section 2 for details)

10 Evidence-Based Complementary and Alternative Medicine

05

LL6

LL67

LL81

LL5

LL8

LL29

LS87

LL100

LL82

LL85

LL83

LL7

LT3

LT5

LT67LT56

LL32

LL84

LL57

LL59

LT7

LL30

LL2

LT85

LL31LL1

LT4

LT6

LT8

LT2

08882

0679

0997

09977

05035

0644

0996409961

09758

07238

09846

05929

0504

0704107642

06421

1

06098

05001

05655

06027

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 paraoxydansS000381732 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 genusMicrobacterium and those of reference type strains Posterior probability values are indicated at the node Nodes are collapsed at 70probability LT = bacteria isolated from the rhizosphere LS = bacteria isolated from the stem LL = bacteria isolated from the leaves (seeSection 2 for details)

Evidence-Based Complementary and Alternative Medicine 11

Table 3The 40Burkholderia cepacia complex strains used as targetsin the cross streak experiments

Target strainStrain Species OriginFCF1 B cepacia CFFCF3LMG17588 B multivorans ENVFCF16 B cenocepacia (IIIA) CFJ2315FCF18

B cenocepacia (IIIB) CF

FCF20FCF23FCF24FCF27FCF29FCF30LMG16654C5424CEP511MVPC116 ENVMVPC173LMG19230 B cenocepacia (IIIC) ENVLMG19240FCF38 B cenocepacia (IIID) CFLMG21462FCF41 B stabilis CFFCF42 B vietnamiensis CFTVV75 ENVLMG18941

B dolosa CFLMG18942LMG18943MCI7

B ambifariaENV

LMG19467 CFLMG19182 ENVLMG16670 B anthina ENVFCF43 B pyrrocina CFLSED4 B lata CFLMG24064 B latens CFLMG24065 B diffusa CFLMG23361 B contaminans AILMG24067 B seminals CFLMG24068 B metallica CFLMG24066 B arboris ENVLMG24263 B ubonensis NIAbbreviations CF strains isolated from cystic fibrosis patients AI strainsisolated from animal infection NI strains isolated from nosocomial infec-tion ENV environmental strain

isolates showed similarities with the stress resistantB safensis

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

complex Regardless of the overall low support ofthe tree (typical of Pseudomonas phylogenies) someobservations may be drawn the presence of largeunresolved clusters shows clearly a low divergenceof the isolated colonies implying the existence ofclonal populations Pseudomonas spp isolated fromthe different compartments are intermixed suggest-ing the presence of a continuum rhizosphere-leaves(Pseudomonas is the only genus found in all the 4sample analysed)

32 Inhibition of Burkholderia Cepacia Complex StrainsGrowth by L angustifolia Endophytic Bacteria In order tocheck the ability of L angustifolia bacterial endophytes toantagonize the growth of (opportunistic) human pathogenicbacteria cross-streak experiments were carried out asdescribed in Section 2 using a panel of the 19 randomlychosen different endophytic strains isolated from soil rootsor leafs and phylogenetically assigned to six different gen-era (Bacillus Microbacterium Pantoea Plantibacter Pseu-domonas and Rhizobium) as testers versus 40 Bcc strainsrepresentative of seventeen species (out of eighteen) andwith either environmental or clinical origin with some ofwhich being multidrug-resistant Data obtained are shownin Table 4 The analysis of these data revealed that all thetester strains were able to completely inhibit the growthof Bcc strains including those with a clinical source andthat exhibited multidrug resistance this finding suggestedthat lavender endophytic bacteria are able to synthesize(strong) antimicrobial compounds However tester strainsshowed a different pattern of Bcc growth inhibition eventhose affiliated to the same genus In addition to this thereis no apparent difference in the antimicrobial potentialbetween strains belonging to different plant compartmentsConcerning the sensitivity of Bcc strains to the antimicrobialactivity of lavender endophytes strains belonging to differentspecies exhibited a different sensitivity spectrum However itis quite interesting that strains with clinical origin appearedto be more sensitive to the antimicrobials synthesized byendophytic bacteria than their environmental counterparts(see for instance strains belonging to the species Burkholderiacenocepacia IIIB) (Supplementary Table 3)

4 Discussion

Medicinal plants are stirring the attention of manyresearchers due to the presence of compounds thatconstitute a large fraction of the current pharmacopoeiasNatural products have been the source of most of theactive ingredients of medicines and more than 80 of drugsubstances are natural products or inspired by a naturalcompound including most of the of anticancer and anti-infective agents [50] Lavender plants are grown mainlyfor the production of essential oil which has antisepticand anti-inflammatory properties In spite of the relativeharsh environment for bacterial colonization L angustifoliatissues were found to be rich in bacterial diversity Howeverthe endophytic community isolated from different plant

12 Evidence-Based Complementary and Alternative Medicine

Table4Growth

of40

Burkholderiacepacia

complex

strains

inthep

resenceabsenceo

fend

ophytic

bacterialstrains

isolated

from

lavend

er

Species

Strain

Orig

inLL

1LL

2LL

3LL

4LL

6LL

7LL

9LL

10LS

1LS

2LS

3LS

4LS

5LS

6LR

1LR

2LR

3LR

4LR

5C-

Bcepacia

FCF1

CFminusminusminusminusminusminusminusminus

minusminusminusminusminusminusminusminusminusminusminus

+B

cepacia

FCF3

CF+minusminusminusminusminusminusminusminus

minusminusminusminusminusminusminusminusminusminusminus

+B

multivoran

sLM

G17588

Env

+minusminus

++minus

+minusminus

+minus

+minus

++minus

+minusminus

+minus

+minusminus

+B

cenocepacia

(IIIA

)FC

F16

CFminusminusminusminusminusminusminusminus

minusminusminusminusminusminusminusminusminusminusminus

+B

cenocepacia

(IIIA

)J2315

CF+minusminus

+minus

+minusminusminus

+minusminusminus

+minusminusminusminusminusminusminusminus

+B

cenocepacia

(IIIB)

FCF18

CF+minusminusminusminusminusminusminus

minusminusminusminusminusminus

+minusminusminusminusminus

+B

cenocepacia

(IIIB)

FCF20

CF+

++

++

+minus

++minus

+minus

+minus

+minus

++minus

++minus

++

++

+B

cenocepacia

(IIIB)

FCF23

CF+minusminusminusminusminusminusminus

+minusminusminus

++minusminusminus

+minusminusminusminus

+B

cenocepacia

(IIIB)

FCF24

CF+minusminusminusminusminusminusminus

+minusminus

+minusminusminusminus

+minusminusminusminus

+B

cenocepacia

(IIIB)

FCF27

CF+minusminusminusminusminusminusminus

+minusminusminusminusminusminusminusminusminusminusminus

+B

cenocepacia

(IIIB)

FCF29

CF+minusminus

+minus

+minusminusminus

+minusminusminusminusminusminusminus

+minus

+minusminus

+B

cenocepacia

(IIIB)

FCF30

CF+minusminus

+minus

+minusminusminus

+minus

+minusminusminusminusminusminus

+minus

+minusminus

+B

cenocepacia

(IIIB)

LMG16654

CF+

+minusminus

++minus

+minus

++minus

++minusminus

+minus

+minusminus

+B

cenocepacia

(IIIB)

C5424

CF+

+minusminus

+minusminusminusminus

+minusminusminus

+minusminusminusminus

+minusminus

+minusminus

+B

cenocepacia

(IIIB)

CEP5

11CF

+minusminus

+minusminusminusminus

+minus

+minusminus

+minus

+minusminusminus

+minus

+minusminus

+B

cenocepacia

(IIIB)

MVPC

116

Env

++minusminus

+minus

++minusminus

+minus

+minus

+minus

+minusminus

+minusminus

+minusminus

+minus

+minus

+B

cenocepacia

(IIIB)

MVPC

173

Env

++

++minus

++

+minus

++

++

++

++

++

++minus

+B

cenocepacia

(IIIIC)

LMG19230

Env

++

++

++

+minus

++

++

++

++

++

++

+B

cenocepacia

(IIIC

)LM

G19240

Env

++

++

+minus

+minus

++

+minus

++

++minus

++

++

+minus

+B

cenocepacia

(IIID)

FCF38

CF+minusminusminusminusminusminusminus

minusminusminusminusminusminusminusminusminusminusminus

+B

cenocepacia

(IIID)

LMG21462

CFminusminusminusminusminusminusminusminus

minusminusminusminusminusminusminusminusminusminusminus

+B

stabilis

FCF41

CF+

+minus

++

+minus

+minusminus

++

+minus

++minusminus

++

++minusminus

+B

vietnamien

sisFC

F42

CFminusminusminusminusminusminusminusminus

minusminusminusminusminusminusminusminusminusminusminus

+B

vietnamien

sisTV

V75

Env

++minusminus

+minusminusminusminus

+minusminusminus

++minusminusminus

+minusminus

+minusminus

+B

dolosa

LMG18941

CF+minusminus

+minus

+minus

+minusminus

+minusminusminus

++minusminusminus

+minus

+minus

+minusminus

+B

dolosa

LMG18942

CF+

+minusminus

+minus

+minusminus

++

+minus

++minusminusminus

++minus

++minusminus

+B

dolosa

LMG18943

CF+

+minusminus

++minus

+minusminus

+minus

+minusminus

++minusminusminus

++minus

+minusminus

+B

ambifaria

MCI

7En

v+

+minus

+minus

+minus

+minusminus

++minusminus

++minusminusminus

+minus

+minusminus

+B

ambifaria

LMG1946

7CF

++minus

+minus

+minusminusminus

++minusminus

++minusminusminus

+minus

+minusminusminus

+B

ambifaria

LMG19182

Env

++minus

+minus

+minusminusminus

+minusminus

+minusminusminus

+minus

+minusminusminus

+B

anthina

LMG16670

Env

+minusminusminusminus

+minus

+minusminus

++minusminusminus

+minus

+minusminusminus

+B

pyrrocinia

FCF43

CF+minusminusminusminusminusminusminusminus

minusminus

+minusminusminusminusminusminus

+minusminusminus

+B

lata

LSED

4CF

+minus

+minusminus

+minusminus

+minusminus

++minusminusminus

+minus

+minus

+minusminusminus

+B

latens

LMG2406

4CF

+minus

++minusminus

+minusminus

++minus

++minus

++

++

+minus

+B

diffu

saLM

G24065

CF+

+minus

++minus

+minus

+minus

++minus

++minus

+minus

++

++minus

+B

contam

inan

sLM

G23361

AI

++minus

+minus

+minus

+minus

+minus

++

++

++

+minus

++

++minus

+B

seminalis

LMG24067

CF+

+minus

++minus

++

++

++

++

++

++

++minus

+B

metallica

LMG2406

8CF

++

++minus

++

++

++

++

++

++

++minus

+B

arboris

LMG2406

6En

v+

+minus

++minus

++

+minus

++

++

++minus

+minus

++

++minusminus

+B

ubonensis

LMG24263

NI

+minusminusminusminus

+minusminus

+minus

+minus

++minusminus

+minusminus

+minusminusminus

+Ab

breviatio

nsC

Fstr

ains

isolatedfro

mcysticfi

brosispatie

ntsAIstr

ains

isolated

from

anim

alinfectionNIstr

ains

isolated

from

nosocomialinfectio

nEN

Venvironm

entalstrainLstr

ains

isolatedfro

mtheleaf

Sstr

ains

isolatedfro

mthes

temR

strains

isolatedfro

mther

oot

Symbo

ls+

grow

th+

minusreduced

grow

th+

minusminusveryredu

cedgrow

thminus

nogrow

thC

-Petrid

ishes

containing

onlythetargetstrains

Evidence-Based Complementary and Alternative Medicine 13

compartments showed different levels of diversity withthe 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 theleaf and roots community is already reported in otherspecies [26] even if there are few direct comparisons ofroots and phyllosphere bacterial communities especiallycomparisons using material from the same plants Themicrobial community residing in the phyllosphere is facedwith a nutrient poor and variable environment that ischaracterized by fluctuating temperature humidity and UVradiation and so it is predicted to be more diverse than thatwithin the rhizosphere a more homeostatic environmentThese findings supported also by the similar bacterial titreare in agreement with the idea that the source of inoculumfor the leaf community (except for the vertically transferredvia seeds) may be exclusive (eg aerosol even if the processis not yet clarified Bulgarelli et al 2013) and not related totransmission from the roots Moreover the low diversity ofthe community isolated in the stem dominated by clonally(Table 2) populations of Pseudomonas could be related tothe highly specific main tissue (phloem) present there whichcould provide an homeostatic environment rich in nutrient(sucrose contained in the phloem tissues) but poorer (ifcompared to leaf and roots) in secondary metabolites thatmay promote ecological niche differentiation

Concerning the taxonomic community composition dif-ferent taxonomic profiles were found for endosphere andrhizosphere More in detail the rhizosphere communityshowed quite similar diversity indexes if compared to theroots one but with a different composition as an examplemembers of the phylumActinobacteria are completely absentfrom the root internal tissues that are largely dominatedby Proteobacteria while they are present in the soil Toexplain this finding a two-step model has been proposed[16] soil abiotic properties determine the structure of theinitial bacterial communities that is then modified by rhi-zodeposits with plant roots cell wall features and releasedmetabolites promoting the growth of organotrophic bacteriathereby initiating a soil community shift In the second stepconvergent host genotype-dependent selection [51 52] fine-tunes community profiles thriving on the rhizoplane andwithin plant roots

Moreover endosphere communities were differentbetween plant organs (roots stems and leaves) even if theinternal tissues are dominated as already reported [25 52ndash54] by members of the phylum Proteobacteria an examplewhile rhizobia were isolated from root internal tissues theyare completely absent from stems and leaves (Figures 2 and3) isolates belonging to the genus Pantoea on the contrarywere isolated from aerial parts but not from root tissuesHence it is possible that plant might ldquoselectrdquo specific taxafor entering inside its compartment Such hypothesis issupported also by the pairwise differentiation values thecommunities isolated from the different tissues even those inphysical continuity are in fact strongly differentiated with thestem representing a low diversity compartment separatingthe two high diversity organs roots and leaves In this view itis noteworthy that in lavender only Pseudomonas represents

a ubiquitous and abundant genus of both rhizosphere andendosphere (Figure 3) The analysis of intrageneric diversitypointed out also that Pseudomonas isolates belong to a lowdifferentiated clonal population (Table 2 and Suppl Figure2) suggesting also that this component of the communityeither diffused inside the organs from a radical or foliar entrypoint or established during plants development putativelyfrom the seed inoculum A similar consideration can beproposed for isolates of rhizobia but for the rhizosphere-root internal tissues continuum a low taxonomicallydifferentiated community from the soil entered (increasingits relative abundance) inside the plant organ An entrypoint from the leaves is on the contrary consistent with thedistribution of Pantoea isolates with their strong diversity(Table 2) suggesting a diversification in response to the highvariable leaf environmentThe isolates belonging to the genusStenotrophomonas show an interesting distribution pattern(Figure 3) they are rare in the rhizosphere abundant in theroots and in the leaves and absent in the stem this findingsuggests a different entry point or a negative selection in thestem the second explanation is supported by admixture ofroot and leaves isolates (Figure 4)

The detailed phylogenetic analyses of the isolates enabledus also to putatively ascribe some isolates to already describedendophytic strains that can be targeted for functional charac-terisation many Microbacterium leaves isolates cluster withM hydrocarbonoxydans and M oleivorans two species withcrude oil degrading activity [49] being this metabolic activitythat is found frequently associated with a phyllosphereadapted lifestyle [55] Several isolates cluster with bacteriawith interesting biotechnological or ecological applicationssuch as S chelatiphaga a remarkable species with biotech-nological potential in phytoremediation [56] and S rizophilaa plant associated bacterium with antifungal activity [57] orBacillus mojavensis a bacteriumclosely related to B subtilisand already reported having an endophytic lifestyle andshowing an antagonistic role against plant pathogenic fungi[58] On the contrary many lavender Pseudomonas isolatescluster with known plant pathogen strains highlighting thesometimes subtle difference among pathogenic and endo-phytic lifestyle and with Pantoea agglomerans (and also withlavender isolates clustering withinthe B licheniformis and Bsoronensis groups) a known plant associated bacterium andan opportunistic pathogen in immune-compromised humanindividuals drawing attention to the possible pathogenicaction of endophytic bacteria in humans [59 60]

The overall analysis of the dendrograms points out thatthe characterization of isolates from endophytic communitiescan lead to the identification (as an example for Pantoeaand rhizobia) of not yet described strains that may representuntapped resources of bioactive compounds of agricultural ormedical interest

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

14 Evidence-Based Complementary and Alternative Medicine

context the characterization of cultivable bacterial commu-nities is a fundamental step to this purpose since it paves theway to the possibility to build collections of isolates that maybe phenotypically typed for important traits In our opiniondata obtained in this work (Table 4 and Supplementary Tables2 and 3) offers a preliminary but very promising exampleof the biotechnological potential of bacteria isolated frommedicinal plants In this pilot study in fact the majority ofthe tested endophytes showed to inhibit the growth of several(in some case all) human pathogenic strains belonging tothe Bcc complexes that are known for their resistance totraditional antibiotics These data are particularly interestingif correlated with the recent finding that essential oils fromdifferent medicinal plants are able to strongly (or completely)interfere with the growth of the same Bcc strains [61] and thatbacterial endophytes isolated from plants of the same speciesexhibited the same bioactivity (Maida et al in preparation)It is therefore possible that the therapeutic properties of theessential oil might reside also on bioactive compounds ofmicrobial origin We are completely aware that the determi-nation of the correlation (eventually) existing between thecomposition of bacterial endophytic communities and thebioactivity of essential oils extracted from a medicinal plantwould require the parallel analysis of the bacterial communityand the essential oil activity extracted from the same plantHowever data obtained in this work and those from Maidaet al [62] support this idea

It is also particularly intriguing the idea that the antimi-crobial activity of essential oils may reside on the actionof multiple compounds some of them produced or elicitedby the microbiota limiting the observed rapid evolutionof human pathogenic bacteria resistant to single moleculeantimicrobials

The characterization of the heterotrophic aerobic endo-phytic bacterial community of L angustifolia highlightedthe existence of a diversified community between differentorganstissues that may be related to the coexistence ofdifferent sources of inoculum andor a selection of the com-munities promoted by nutrient sand metabolites availabilityand antagonistic forces Several not previously characterizedstrains have been isolated and molecularly typed confirmingthat the analysis of the bacterial communities inhabitingextreme or unconventional environments may represent aproper strategy for the discovery of untapped sources offunctional biodiversity and bioactive molecule production

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This work was supported by grants from the Ente Cassadi Risparmio di Firenze (Grant 20130657 Herbiome newantimicrobial compounds from endophytic bacteria isolatedfrom medicinal plants) Giovanni Emiliani is financially

supported by the Regione Toscana POR CRO FSE 2007-2013 Asse IV Grant Sysbiofor (CNR-9) Marco Fondi andElena Perrin are financially supported by the FEMSAdvancedFellowship (FAF2012) and the Buzzati-Traverso Foundationrespectively

References

[1] B Reinhold-Hurek andTHurek ldquoLiving inside plants bacterialendophytesrdquo Current Opinion in Plant Biology vol 14 no 4 pp435ndash443 2011

[2] M Rosenblueth and E Martınez-Romero ldquoBacterial endo-phytes and their interactions with hostsrdquo Molecular Plant-Microbe Interactions vol 19 no 8 pp 827ndash837 2006

[3] R P Ryan K Germaine A Franks D J Ryan and DN Dowling ldquoBacterial endophytes recent developments andapplicationsrdquo FEMSMicrobiology Letters vol 278 no 1 pp 1ndash92008

[4] R Cakmakci F Donmez A Aydın and F Sahin ldquoGrowthpromotion of plants by plant growth-promoting rhizobacteriaunder greenhouse and two different field soil conditionsrdquo SoilBiology and Biochemistry vol 38 pp 1482ndash1487 2006

[5] P R Hardoim L S van Overbeek and J D V Elsas ldquoProp-erties of bacterial endophytes and their proposed role in plantgrowthrdquo Trends in Microbiology vol 16 no 10 pp 463ndash4712008

[6] S Compant C Clement and A Sessitsch ldquoPlant growth-promoting bacteria in the rhizo- and endosphere of plantstheir role colonization mechanisms involved and prospects forutilizationrdquo Soil Biology and Biochemistry vol 42 no 5 pp669ndash678 2010

[7] B R Glick ldquoBacterial ACC deaminase and the alleviation ofplant stressrdquo Advances in Applied Microbiology vol 56 pp 291ndash312 2004

[8] B R Glick B Todorovic J Czarny Z Cheng J Duan andB McConkey ldquoPromotion of plant growth by bacterial ACCdeaminaserdquo Critical Reviews in Plant Sciences vol 26 no 5-6pp 227ndash242 2007

[9] S L Doty B Oakley G Xin et al ldquoDiazotrophic endophytesof native black cottonwood and willowrdquo Symbiosis vol 47 no 1pp 23ndash33 2009

[10] B Lugtenberg and F Kamilova ldquoPlant-growth-promoting rhi-zobacteriardquoAnnual Review ofMicrobiology vol 63 pp 541ndash5562009

[11] U F Castillo G A Strobel E J Ford et al ldquoMunumbicinswide-spectrum antibiotics produced by Streptomyces NRRL30562 endophytic on Kennedia nigriscansrdquo Microbiology vol148 no 9 pp 2675ndash2685 2002

[12] Y Igarashi M E Trujillo E Martınez-Molina et al ldquoAnti-tumor anthraquinones from an endophytic actinomyceteMicromonospora lupini sp novrdquo Bioorganic amp Medicinal Chem-istry Letters vol 17 no 13 pp 3702ndash3705 2007

[13] S Shweta J H Bindu J Raghu et al ldquoIsolation of endophyticbacteria producing the anti-cancer alkaloid camptothecinefromMiquelia dentata Bedd (Icacinaceae)rdquo Phytomedicine vol20 pp 913ndash917 2013

[14] T Taechowisan A Wanbanjob P Tuntiwachwuttikul and JLiu ldquoAnti-inflammatory activity of lansais from endophyticStreptomyces sp SUC1 in LPS-induced RAW 2647 cellsrdquo Foodand Agricultural Immunology vol 20 no 1 pp 67ndash77 2009

Evidence-Based Complementary and Alternative Medicine 15

[15] P R Hardoim C C P Hardoim L S van Overbeek and J Dvan Elsas ldquoDynamics of seed-borne rice endophytes on earlyplant growth stagesrdquo PLoS ONE vol 7 no 2 Article ID e304382012

[16] D Bulgarelli K Schlaeppi S Spaepen E V L van Themaatand P Schulze-Lefert ldquoStructure and functions of the bacterialmicrobiota of plantsrdquo Annual Review of Plant Biology vol 64pp 807ndash838 2013

[17] A Mengoni S Mocali G Surico S Tegli and R Fani ldquoFluctu-ation of endophytic bacteria and phytoplasmosis in elm treesrdquoMicrobiological Research vol 158 no 4 pp 363ndash369 2003

[18] A Mengoni F Pini L-N Huang W-S Shu and MBazzicalupo ldquoPlant-by-plant variations of bacterial commu-nities associated with leaves of the nickel hyperaccumulatorAlyssum bertolonii desvrdquo Microbial Ecology vol 58 no 3 pp660ndash667 2009

[19] S Mocali E Bertelli F di Cello et al ldquoFluctuation of bacteriaisolated from elm tissues during different seasons and fromdifferent plant organsrdquo Research in Microbiology vol 154 no2 pp 105ndash114 2003

[20] F Pini A Frascella L Santopolo et al ldquoExploring the plant-associated bacterial communities in Medicago sativa Lrdquo BMCMicrobiology vol 12 article 78 2012

[21] A Mengoni L Cecchi and C Gonnelli ldquoNickel hyperac-cumulating plants and Alyssum bertolonii model systems forstudying biogeochemical interactions in serpentine soilsrdquo inBio-Geo Interactions in Metal-Contaminated Soils E Kothe andA Varma Eds pp 279ndash296 Springer Berlin Germany 2012

[22] S Ikeda T Okubo M Anda et al ldquoCommunity- and genome-based views of plant-associated bacteria plant-bacterial inter-actions in soybean and ricerdquo Plant and Cell Physiology vol 51no 9 pp 1398ndash1410 2010

[23] F Pini M Galardini M Bazzicalupo and A Mengoni ldquoPlant-bacteria association and symbiosis are there common genomictraits in alphaproteobacteriardquoGenes vol 2 no 4 pp 1017ndash10322011

[24] K Aleklett and M Hart ldquoThe root microbiota a fingerprint inthe soilrdquo Plant Soil vol 370 pp 671ndash686 2013

[25] A Beneduzi F Moreira P B Costa et al ldquoDiversity and plantgrowth promoting evaluation abilities of bacteria isolated fromsugarcane cultivated in the South of BrazilrdquoApplied Soil Ecologyvol 63 pp 94ndash104 2013

[26] N Bodenhausen M W Horton and J Bergelson ldquoBacterialcommunities associated with the leaves and the roots of Ara-bidopsis thalianardquo PLoS ONE vol 8 no 2 Article ID e563292013

[27] T F da Silva R E Vollu D Jurelevicius et al ldquoDoes theessential oil of Lippia sidoides Cham (pepper-rosmarin) affectits endophytic microbial communityrdquo BMC Microbiology vol13 no 1 article 29 2013

[28] M E Lucero A Unc P Cooke S Dowd and S SunldquoEndophyte microbiome diversity in micropropagated Atriplexcanescens and Atriplex torreyi var griffithsiirdquo PLoS ONE vol 6no 3 Article ID e17693 2011

[29] D S Lundberg S L Lebeis S H Paredes et al ldquoDefining thecoreArabidopsis thaliana rootmicrobiomerdquoNature vol 487 no7409 pp 86ndash90 2012

[30] H M Cavanagh and J M Wilkinson ldquoBiological activities oflavender essential oilrdquo Phytotherapy Research vol 16 no 4 pp301ndash308 2002

[31] G Woronuk Z Demissie M Rheault and S MahmoudldquoBiosynthesis and therapeutic properties of Lavandula essentialoil constituentsrdquo Planta Medica vol 77 no 1 pp 7ndash15 2011

[32] R Perry R Terry L K Watson and E Ernst ldquoIs lavender ananxiolytic drug A systematic review of randomised clinicaltrialsrdquo Phytomedicine vol 19 pp 825ndash835 2012

[33] S de Rapper G Kamatou A Viljoen and S van VuurenldquoThe in vitro antimicrobial activity of Lavandula angustifoliaessential oil in combination with other aroma-therapeutic oilsrdquoEvidence-Based Complementary and Alternative Medicine vol2013 Article ID 852049 10 pages 2013

[34] T Moon J M Wilkinson and H M Cavanagh ldquoAntiparasiticactivity of two Lavandula essential oils against Giardia duode-nalis Trichomonas vaginalis andHexamita inflatardquo ParasitologyResearch vol 99 no 6 pp 722ndash728 2006

[35] P S Yap S H Lim C P Hu and B C Yiap ldquoCom-bination of essential oils and antibiotics reduce antibioticresistance in plasmid-conferred multidrug resistant bacteriardquoPhytomedicine vol 20 pp 710ndash713 2013

[36] G Brader S Compant B Mitter F Trognitz and A SessitschldquoMetabolic potential of endophytic bacteriardquo Current Opinionin Biotechnology vol 27 pp 30ndash37 2014

[37] P Drevinek and E Mahenthiralingam ldquoBurkholderia ceno-cepacia in cystic fibrosis epidemiology and molecular mecha-nisms of virulencerdquo Clinical Microbiology and Infection vol 16no 7 pp 821ndash830 2010

[38] S Bazzini C Udine and G Riccardi ldquoMolecular approaches topathogenesis study of Burkholderia cenocepacia an importantcystic fibrosis opportunistic bacteriumrdquo Applied Microbiologyand Biotechnology vol 92 no 5 pp 887ndash895 2011

[39] I Maida A lo Nostro G Pesavento et al ldquoExploring theanti-Burkholderia cepacia complex activity of essential oilsa preliminary analysisrdquo Evidence-Based Complementary andAlternativeMedicine vol 2014 Article ID 573518 10 pages 2014

[40] A Mengoni R Barzanti C Gonnelli R Gabbrielli andM Bazzicalupo ldquoCharacterization of nickel-resistant bacteriaisolated from serpentine soilrdquo Environmental Microbiology vol3 no 11 pp 691ndash698 2001

[41] S F Altschul T L Madden A A Schaffer et al ldquoGappedBLAST and PSI-BLAST a new generation of protein databasesearch programsrdquo Nucleic Acids Research vol 25 no 17 pp3389ndash3402 1997

[42] R C Edgar ldquoMUSCLE multiple sequence alignment with highaccuracy and high throughputrdquo Nucleic Acids Research vol 32no 5 pp 1792ndash1797 2004

[43] F Ronquist M Teslenko P van der Mark et al ldquoMrbayes32 efficient bayesian phylogenetic inference and model choiceacross a large model spacerdquo Systematic Biology vol 61 no 3 pp539ndash542 2012

[44] K Tamura D Peterson N Peterson G Stecher M Nei andS Kumar ldquoMEGA5 molecular evolutionary genetics analysisusing maximum likelihood evolutionary distance and max-imum parsimony methodsrdquo Molecular Biology and Evolutionvol 28 no 10 pp 2731ndash2739 2011

[45] F Sievers A Wilm D Dineen et al ldquoFast scalable generationof high-quality protein multiple sequence alignments usingClustal Omegardquo Molecular Systems Biology vol 7 article 5392011

[46] Oslash Hammer D A T Harper and P D Ryan ldquoPAST pale-ontological statistics software package for education and dataanalysisrdquo Palaeontologia Electronica vol 4 no 1 pp 1ndash9 2001

16 Evidence-Based Complementary and Alternative Medicine

[47] M C Papaleo M Fondi I Maida et al ldquoSponge-associatedmicrobial Antarctic communities exhibiting antimicrobialactivity againstBurkholderia cepacia complex bacteriardquoBiotech-nology Advances vol 30 no 1 pp 272ndash293 2012

[48] A lo Giudice V Bruni and L Michaud ldquoCharacterization ofAntarctic psychrotrophic bacteria with antibacterial activitiesagainst terrestrial microorganismsrdquo Journal of Basic Microbiol-ogy vol 47 no 6 pp 496ndash505 2007

[49] A Schippers K Bosecker C Sproer and P SchumannldquoMicrobacterium oleivorans sp nov and Microbacteriumhydrocarbonoxydans sp nov novel crude-oil-degrading Gram-positive bacteriardquo International Journal of Systematic and Evo-lutionary Microbiology vol 55 no 2 pp 655ndash660 2005

[50] J D McChesney S K Venkataraman and J T Henri ldquoPlantnatural products back to the future or into extinctionrdquo Phyto-chemistry vol 68 no 14 pp 2015ndash2022 2007

[51] D K Manter J A Delgado D G Holm and R A StongldquoPyrosequencing reveals a highly diverse and cultivar-specificbacterial endophyte community in potato rootsrdquo MicrobialEcology vol 60 no 1 pp 157ndash166 2010

[52] K Ulrich A Ulrich and D Ewald ldquoDiversity of endophyticbacterial communities in poplar grown under field conditionsrdquoFEMS Microbiology Ecology vol 63 no 2 pp 169ndash180 2008

[53] N R Gottel H F Castro M Kerley et al ldquoDistinct microbialcommunities within the endosphere and rhizosphere ofPopulusdeltoides roots across contrasting soil typesrdquo Applied and Envi-ronmental Microbiology vol 77 no 17 pp 5934ndash5944 2011

[54] B Ma X Lv A Warren and J Gong ldquoShifts in diversity andcommunity structure of endophytic bacteria and archaea acrossroot stem and leaf tissues in the common reed Phragmitesaustralis along a salinity gradient in a marine tidal wetland ofnorthern Chinardquo Antonie van Leeuwenhoek vol 104 no 5 pp759ndash768 2013

[55] J A Vorholt ldquoMicrobial life in the phyllosphererdquo NatureReviews Microbiology vol 10 no 12 pp 828ndash840 2012

[56] R P Ryan S Monchy M Cardinale et al ldquoThe versatilityand adaptation of bacteria from the genus StenotrophomonasrdquoNature Reviews Microbiology vol 7 no 7 pp 514ndash525 2009

[57] AWolf A FritzeMHagemann andG Berg ldquoStenotrophomo-nas rhizophila sp nov a novel plant-associated bacterium withantifungal propertiesrdquo International Journal of Systematic andEvolutionary Microbiology vol 52 no 6 pp 1937ndash1944 2002

[58] C W Bacon and D M Hinton ldquoEndophytic and biologicalcontrol potential of Bacillus mojavensis and related speciesrdquoBiological Control vol 23 no 3 pp 274ndash284 2002

[59] G Berg L Eberl and A Hartmann ldquoThe rhizosphere as areservoir for opportunistic human pathogenic bacteriardquo Envi-ronmental Microbiology vol 7 no 11 pp 1673ndash1685 2005

[60] H L Tyler and E W Triplett ldquoPlants as a habitat for ben-eficial andor human pathogenic bacteriardquo Annual Review ofPhytopathology vol 46 pp 53ndash73 2008

[61] R Perry R Terry L K Watson and E Ernst ldquoIs lavender ananxiolytic drug A systematic review of randomised clinicaltrialsrdquo Phytomedicine vol 19 no 8-9 pp 825ndash835 2012

[62] I Maida A lo Nostro G Pesavento et al ldquoExploring theanti-Burkholderia cepacia complex activity of essential oilsa preliminary analysisrdquo Evidence-Based Complementary andAlternativeMedicine vol 2014 Article ID 573518 10 pages 2014

Evidence-Based Complementary and Alternative Medicine 5

Bacillus 27 Jeotgalibacillus 03

Microbacterium 64

Pantoea 92 Plantibacter 03

Pseudomonas 512 Psychrobacter 07

Rhizobium 139

Salinibacterium 10

Sanguibacter 10

Stenotrophomonas 132

Figure 1 Composition of the aerobic heterotrophic endophytic (sensu stricto) bacterial community in L angustifolia tissuesThe compositionis reported as percentages of the total number of characterized isolates (119899 = 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

6 Evidence-Based Complementary and Alternative Medicine

100

90

80

70

60

50

40

30

20

10

0Pr

esen

ce (

)

SoilRoots

StemLeaves

Bacil

lus

Micr

obac

teriu

m

Pant

oea

Pseu

dom

onas

Rhiz

obiu

m

Sten

otro

phom

onas

Figure 3 Comparison of the relative abundance of bacterial genera in the different samples only genera with percentages gt5 in at least oneof 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 gt97 consideredas threshold of species identity (in brackets the total number of comparisons and the percentage) number of OTU with at least 1 pairwisecomparison with identity gt97

Genus Number of isolates Mean identity amongOTU () Mean distance among OTU

Number of pairwisecomparisons withidentity gt97

Number of OTU withidentity gt97

Pseudomonas 172 9425 0013 3840 (14878 258) 170Bacillus 47 9304 0027 329 (1128 29) 46Rhizobia 46 9861 0016 952 (1081 88) 46Stenotrophomonas 37 9534 0023 415 (703 59) 37Microbacterium 30 9500 0059 120 (465 25) 29Pantoea 26 8695 0126 24 (351 68) 7

(and consistency of the results) than the MP ones (see alsoSuppl Table 4) The analysis of the Bayesian phylogenetictrees revealed the following

(i) Endophytic bacteria isolated from the leavesand roots of lavender and assigned to the genusStenotrophomonas (Figure 4) are clearly split in twogroups One group contains isolates clustering withStenotrophomonas chelatiphaga interestingly thiscluster comprises isolates from both roots and leaves(no Stenotrophomonas were isolated from the stem)suggesting that two compartments might sharebacteria belonging to the same species even thoughthe polymorphism shown by the aligned partial 16Ssequences might suggest a diversification at the strainlevel The second cluster embeds roots isolates (withthe exception of leaves isolate LL44) clustering withStenotrophomonas rizophila

(ii) Concerning rhizobia (Figure 5) a grouping in onemain cluster of low differentiate sequences is present

These groups might contain new clades of plant-associated rhizobia which deservemore investigationin the future

(iii) From the phylogenetic tree of Figure 6 Pantoea iso-lated endophytes (from lavender stem and leaves)cluster with already described Pantoea spp anothergroup of isolates (all from the leaves) are divergingand thus may represent none yet described strains arefound to be associated with plants

(iv) Figure 7 depicts the phylogenetic position ofMicrobacterium isolates (mainly from leaves andrhizosphere) in the context of the genus referencestrains noticeably many leaves isolates cluster withM hydrocarbonoxydans and M oleivorans specieswhose members are able to perform crude oildegradation [49]

(v) Concerning Bacillus isolates (Suppl Figure 1) acluster of soil isolates including Bacillus mojavensiswas disclosed Another group again comprising soil

Evidence-Based Complementary and Alternative Medicine 7

009

LR48

LR31

LR41

LL38

LL43

LL94

LT48

LR98

LR86

LL88

LR72

LL17

LR28LR8

LRL93

LL39

LR61

LR76

LL37

LR7

LR29

LR34

LL77LL76

LR79

LL96

LR95LL44

LL45

LL13

LR26

LR99

LL75

LR80

LR32

LR60

LR75

066

091

1

1

089

091

091

087

093

057

078

07

091

S001020552 Escherichia coli

S001576206 S daejeonensis MJ03

S000559371 S ginsengisoli

S000007629 P geniculata ATCC 193

S000386319 S koreensis TR6-01

S000841904 S terrae R-32768

S000003927 S nitritireducens L2

S000841903 S humi R-32729

S000824093 S maltophilia IAM 124

S000010261 P hibiscicola ATCC 19

S000129976 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 genusStenotrophomonas and those of reference type strains Posterior probability values are indicated at the node Nodes are collapsed at 70probability LL = bacteria isolated from the leaves LR = bacteria isolated from the roots (see Section 2 for details)

8 Evidence-Based Complementary and Alternative Medicine

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 trS002222203 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 NRCPB10S002958314 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 MGminus2011minus4-AO

S000429561 A sp PB

S000806232 R soli DS-42

03

LR51

LR78

LR50

LT74

LR68

LR9

LR22

LR67

LR69

LR23LR24

LR5

LR21

LT92

LR19

LR49

LR62

LR57

LR54

LT91

LR55

LR12

LR20

LR47

LR18

LR11

LR65

LR25LR45

LR56

LT12

LR64

LR16

LR6

LR70

LR7

LR17

LT73

LR63

LR52

LR77

LR46

LT87

LR53

LR15

LR66094890908507606

09525

07316

07803

05579

1

0685

06484

05141

08108

1

Figure 5 Bayesian dendrogram showing the relationships among the 16S rRNA gene sequences of 46 isolates belonging to the genusRhizobium and those of reference type strains Posterior probability values are indicated at each node Nodes are collapsed at 70 probabilityLT = bacteria isolated from the rhizosphere LR = bacteria isolated from the roots (see Section 2 for details)

Evidence-Based Complementary and Alternative Medicine 9

005

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

095

09074

055

099

05

1

081

088

078

1

084

1

071

1

083

1

091

1

099

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 Pantoeaand those of reference type strains Posterior probability values are indicated at the node Nodes are collapsed at 70 probability LS = bacteriaisolated from the stem LL = bacteria isolated from the leaves (see Section 2 for details)

10 Evidence-Based Complementary and Alternative Medicine

05

LL6

LL67

LL81

LL5

LL8

LL29

LS87

LL100

LL82

LL85

LL83

LL7

LT3

LT5

LT67LT56

LL32

LL84

LL57

LL59

LT7

LL30

LL2

LT85

LL31LL1

LT4

LT6

LT8

LT2

08882

0679

0997

09977

05035

0644

0996409961

09758

07238

09846

05929

0504

0704107642

06421

1

06098

05001

05655

06027

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 paraoxydansS000381732 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 genusMicrobacterium and those of reference type strains Posterior probability values are indicated at the node Nodes are collapsed at 70probability LT = bacteria isolated from the rhizosphere LS = bacteria isolated from the stem LL = bacteria isolated from the leaves (seeSection 2 for details)

Evidence-Based Complementary and Alternative Medicine 11

Table 3The 40Burkholderia cepacia complex strains used as targetsin the cross streak experiments

Target strainStrain Species OriginFCF1 B cepacia CFFCF3LMG17588 B multivorans ENVFCF16 B cenocepacia (IIIA) CFJ2315FCF18

B cenocepacia (IIIB) CF

FCF20FCF23FCF24FCF27FCF29FCF30LMG16654C5424CEP511MVPC116 ENVMVPC173LMG19230 B cenocepacia (IIIC) ENVLMG19240FCF38 B cenocepacia (IIID) CFLMG21462FCF41 B stabilis CFFCF42 B vietnamiensis CFTVV75 ENVLMG18941

B dolosa CFLMG18942LMG18943MCI7

B ambifariaENV

LMG19467 CFLMG19182 ENVLMG16670 B anthina ENVFCF43 B pyrrocina CFLSED4 B lata CFLMG24064 B latens CFLMG24065 B diffusa CFLMG23361 B contaminans AILMG24067 B seminals CFLMG24068 B metallica CFLMG24066 B arboris ENVLMG24263 B ubonensis NIAbbreviations CF strains isolated from cystic fibrosis patients AI strainsisolated from animal infection NI strains isolated from nosocomial infec-tion ENV environmental strain

isolates showed similarities with the stress resistantB safensis

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

complex Regardless of the overall low support ofthe tree (typical of Pseudomonas phylogenies) someobservations may be drawn the presence of largeunresolved clusters shows clearly a low divergenceof the isolated colonies implying the existence ofclonal populations Pseudomonas spp isolated fromthe different compartments are intermixed suggest-ing the presence of a continuum rhizosphere-leaves(Pseudomonas is the only genus found in all the 4sample analysed)

32 Inhibition of Burkholderia Cepacia Complex StrainsGrowth by L angustifolia Endophytic Bacteria In order tocheck the ability of L angustifolia bacterial endophytes toantagonize the growth of (opportunistic) human pathogenicbacteria cross-streak experiments were carried out asdescribed in Section 2 using a panel of the 19 randomlychosen different endophytic strains isolated from soil rootsor leafs and phylogenetically assigned to six different gen-era (Bacillus Microbacterium Pantoea Plantibacter Pseu-domonas and Rhizobium) as testers versus 40 Bcc strainsrepresentative of seventeen species (out of eighteen) andwith either environmental or clinical origin with some ofwhich being multidrug-resistant Data obtained are shownin Table 4 The analysis of these data revealed that all thetester strains were able to completely inhibit the growthof Bcc strains including those with a clinical source andthat exhibited multidrug resistance this finding suggestedthat lavender endophytic bacteria are able to synthesize(strong) antimicrobial compounds However tester strainsshowed a different pattern of Bcc growth inhibition eventhose affiliated to the same genus In addition to this thereis no apparent difference in the antimicrobial potentialbetween strains belonging to different plant compartmentsConcerning the sensitivity of Bcc strains to the antimicrobialactivity of lavender endophytes strains belonging to differentspecies exhibited a different sensitivity spectrum However itis quite interesting that strains with clinical origin appearedto be more sensitive to the antimicrobials synthesized byendophytic bacteria than their environmental counterparts(see for instance strains belonging to the species Burkholderiacenocepacia IIIB) (Supplementary Table 3)

4 Discussion

Medicinal plants are stirring the attention of manyresearchers due to the presence of compounds thatconstitute a large fraction of the current pharmacopoeiasNatural products have been the source of most of theactive ingredients of medicines and more than 80 of drugsubstances are natural products or inspired by a naturalcompound including most of the of anticancer and anti-infective agents [50] Lavender plants are grown mainlyfor the production of essential oil which has antisepticand anti-inflammatory properties In spite of the relativeharsh environment for bacterial colonization L angustifoliatissues were found to be rich in bacterial diversity Howeverthe endophytic community isolated from different plant

12 Evidence-Based Complementary and Alternative Medicine

Table4Growth

of40

Burkholderiacepacia

complex

strains

inthep

resenceabsenceo

fend

ophytic

bacterialstrains

isolated

from

lavend

er

Species

Strain

Orig

inLL

1LL

2LL

3LL

4LL

6LL

7LL

9LL

10LS

1LS

2LS

3LS

4LS

5LS

6LR

1LR

2LR

3LR

4LR

5C-

Bcepacia

FCF1

CFminusminusminusminusminusminusminusminus

minusminusminusminusminusminusminusminusminusminusminus

+B

cepacia

FCF3

CF+minusminusminusminusminusminusminusminus

minusminusminusminusminusminusminusminusminusminusminus

+B

multivoran

sLM

G17588

Env

+minusminus

++minus

+minusminus

+minus

+minus

++minus

+minusminus

+minus

+minusminus

+B

cenocepacia

(IIIA

)FC

F16

CFminusminusminusminusminusminusminusminus

minusminusminusminusminusminusminusminusminusminusminus

+B

cenocepacia

(IIIA

)J2315

CF+minusminus

+minus

+minusminusminus

+minusminusminus

+minusminusminusminusminusminusminusminus

+B

cenocepacia

(IIIB)

FCF18

CF+minusminusminusminusminusminusminus

minusminusminusminusminusminus

+minusminusminusminusminus

+B

cenocepacia

(IIIB)

FCF20

CF+

++

++

+minus

++minus

+minus

+minus

+minus

++minus

++minus

++

++

+B

cenocepacia

(IIIB)

FCF23

CF+minusminusminusminusminusminusminus

+minusminusminus

++minusminusminus

+minusminusminusminus

+B

cenocepacia

(IIIB)

FCF24

CF+minusminusminusminusminusminusminus

+minusminus

+minusminusminusminus

+minusminusminusminus

+B

cenocepacia

(IIIB)

FCF27

CF+minusminusminusminusminusminusminus

+minusminusminusminusminusminusminusminusminusminusminus

+B

cenocepacia

(IIIB)

FCF29

CF+minusminus

+minus

+minusminusminus

+minusminusminusminusminusminusminus

+minus

+minusminus

+B

cenocepacia

(IIIB)

FCF30

CF+minusminus

+minus

+minusminusminus

+minus

+minusminusminusminusminusminus

+minus

+minusminus

+B

cenocepacia

(IIIB)

LMG16654

CF+

+minusminus

++minus

+minus

++minus

++minusminus

+minus

+minusminus

+B

cenocepacia

(IIIB)

C5424

CF+

+minusminus

+minusminusminusminus

+minusminusminus

+minusminusminusminus

+minusminus

+minusminus

+B

cenocepacia

(IIIB)

CEP5

11CF

+minusminus

+minusminusminusminus

+minus

+minusminus

+minus

+minusminusminus

+minus

+minusminus

+B

cenocepacia

(IIIB)

MVPC

116

Env

++minusminus

+minus

++minusminus

+minus

+minus

+minus

+minusminus

+minusminus

+minusminus

+minus

+minus

+B

cenocepacia

(IIIB)

MVPC

173

Env

++

++minus

++

+minus

++

++

++

++

++

++minus

+B

cenocepacia

(IIIIC)

LMG19230

Env

++

++

++

+minus

++

++

++

++

++

++

+B

cenocepacia

(IIIC

)LM

G19240

Env

++

++

+minus

+minus

++

+minus

++

++minus

++

++

+minus

+B

cenocepacia

(IIID)

FCF38

CF+minusminusminusminusminusminusminus

minusminusminusminusminusminusminusminusminusminusminus

+B

cenocepacia

(IIID)

LMG21462

CFminusminusminusminusminusminusminusminus

minusminusminusminusminusminusminusminusminusminusminus

+B

stabilis

FCF41

CF+

+minus

++

+minus

+minusminus

++

+minus

++minusminus

++

++minusminus

+B

vietnamien

sisFC

F42

CFminusminusminusminusminusminusminusminus

minusminusminusminusminusminusminusminusminusminusminus

+B

vietnamien

sisTV

V75

Env

++minusminus

+minusminusminusminus

+minusminusminus

++minusminusminus

+minusminus

+minusminus

+B

dolosa

LMG18941

CF+minusminus

+minus

+minus

+minusminus

+minusminusminus

++minusminusminus

+minus

+minus

+minusminus

+B

dolosa

LMG18942

CF+

+minusminus

+minus

+minusminus

++

+minus

++minusminusminus

++minus

++minusminus

+B

dolosa

LMG18943

CF+

+minusminus

++minus

+minusminus

+minus

+minusminus

++minusminusminus

++minus

+minusminus

+B

ambifaria

MCI

7En

v+

+minus

+minus

+minus

+minusminus

++minusminus

++minusminusminus

+minus

+minusminus

+B

ambifaria

LMG1946

7CF

++minus

+minus

+minusminusminus

++minusminus

++minusminusminus

+minus

+minusminusminus

+B

ambifaria

LMG19182

Env

++minus

+minus

+minusminusminus

+minusminus

+minusminusminus

+minus

+minusminusminus

+B

anthina

LMG16670

Env

+minusminusminusminus

+minus

+minusminus

++minusminusminus

+minus

+minusminusminus

+B

pyrrocinia

FCF43

CF+minusminusminusminusminusminusminusminus

minusminus

+minusminusminusminusminusminus

+minusminusminus

+B

lata

LSED

4CF

+minus

+minusminus

+minusminus

+minusminus

++minusminusminus

+minus

+minus

+minusminusminus

+B

latens

LMG2406

4CF

+minus

++minusminus

+minusminus

++minus

++minus

++

++

+minus

+B

diffu

saLM

G24065

CF+

+minus

++minus

+minus

+minus

++minus

++minus

+minus

++

++minus

+B

contam

inan

sLM

G23361

AI

++minus

+minus

+minus

+minus

+minus

++

++

++

+minus

++

++minus

+B

seminalis

LMG24067

CF+

+minus

++minus

++

++

++

++

++

++

++minus

+B

metallica

LMG2406

8CF

++

++minus

++

++

++

++

++

++

++minus

+B

arboris

LMG2406

6En

v+

+minus

++minus

++

+minus

++

++

++minus

+minus

++

++minusminus

+B

ubonensis

LMG24263

NI

+minusminusminusminus

+minusminus

+minus

+minus

++minusminus

+minusminus

+minusminusminus

+Ab

breviatio

nsC

Fstr

ains

isolatedfro

mcysticfi

brosispatie

ntsAIstr

ains

isolated

from

anim

alinfectionNIstr

ains

isolated

from

nosocomialinfectio

nEN

Venvironm

entalstrainLstr

ains

isolatedfro

mtheleaf

Sstr

ains

isolatedfro

mthes

temR

strains

isolatedfro

mther

oot

Symbo

ls+

grow

th+

minusreduced

grow

th+

minusminusveryredu

cedgrow

thminus

nogrow

thC

-Petrid

ishes

containing

onlythetargetstrains

Evidence-Based Complementary and Alternative Medicine 13

compartments showed different levels of diversity withthe 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 theleaf and roots community is already reported in otherspecies [26] even if there are few direct comparisons ofroots and phyllosphere bacterial communities especiallycomparisons using material from the same plants Themicrobial community residing in the phyllosphere is facedwith a nutrient poor and variable environment that ischaracterized by fluctuating temperature humidity and UVradiation and so it is predicted to be more diverse than thatwithin the rhizosphere a more homeostatic environmentThese findings supported also by the similar bacterial titreare in agreement with the idea that the source of inoculumfor the leaf community (except for the vertically transferredvia seeds) may be exclusive (eg aerosol even if the processis not yet clarified Bulgarelli et al 2013) and not related totransmission from the roots Moreover the low diversity ofthe community isolated in the stem dominated by clonally(Table 2) populations of Pseudomonas could be related tothe highly specific main tissue (phloem) present there whichcould provide an homeostatic environment rich in nutrient(sucrose contained in the phloem tissues) but poorer (ifcompared to leaf and roots) in secondary metabolites thatmay promote ecological niche differentiation

Concerning the taxonomic community composition dif-ferent taxonomic profiles were found for endosphere andrhizosphere More in detail the rhizosphere communityshowed quite similar diversity indexes if compared to theroots one but with a different composition as an examplemembers of the phylumActinobacteria are completely absentfrom the root internal tissues that are largely dominatedby Proteobacteria while they are present in the soil Toexplain this finding a two-step model has been proposed[16] soil abiotic properties determine the structure of theinitial bacterial communities that is then modified by rhi-zodeposits with plant roots cell wall features and releasedmetabolites promoting the growth of organotrophic bacteriathereby initiating a soil community shift In the second stepconvergent host genotype-dependent selection [51 52] fine-tunes community profiles thriving on the rhizoplane andwithin plant roots

Moreover endosphere communities were differentbetween plant organs (roots stems and leaves) even if theinternal tissues are dominated as already reported [25 52ndash54] by members of the phylum Proteobacteria an examplewhile rhizobia were isolated from root internal tissues theyare completely absent from stems and leaves (Figures 2 and3) isolates belonging to the genus Pantoea on the contrarywere isolated from aerial parts but not from root tissuesHence it is possible that plant might ldquoselectrdquo specific taxafor entering inside its compartment Such hypothesis issupported also by the pairwise differentiation values thecommunities isolated from the different tissues even those inphysical continuity are in fact strongly differentiated with thestem representing a low diversity compartment separatingthe two high diversity organs roots and leaves In this view itis noteworthy that in lavender only Pseudomonas represents

a ubiquitous and abundant genus of both rhizosphere andendosphere (Figure 3) The analysis of intrageneric diversitypointed out also that Pseudomonas isolates belong to a lowdifferentiated clonal population (Table 2 and Suppl Figure2) suggesting also that this component of the communityeither diffused inside the organs from a radical or foliar entrypoint or established during plants development putativelyfrom the seed inoculum A similar consideration can beproposed for isolates of rhizobia but for the rhizosphere-root internal tissues continuum a low taxonomicallydifferentiated community from the soil entered (increasingits relative abundance) inside the plant organ An entrypoint from the leaves is on the contrary consistent with thedistribution of Pantoea isolates with their strong diversity(Table 2) suggesting a diversification in response to the highvariable leaf environmentThe isolates belonging to the genusStenotrophomonas show an interesting distribution pattern(Figure 3) they are rare in the rhizosphere abundant in theroots and in the leaves and absent in the stem this findingsuggests a different entry point or a negative selection in thestem the second explanation is supported by admixture ofroot and leaves isolates (Figure 4)

The detailed phylogenetic analyses of the isolates enabledus also to putatively ascribe some isolates to already describedendophytic strains that can be targeted for functional charac-terisation many Microbacterium leaves isolates cluster withM hydrocarbonoxydans and M oleivorans two species withcrude oil degrading activity [49] being this metabolic activitythat is found frequently associated with a phyllosphereadapted lifestyle [55] Several isolates cluster with bacteriawith interesting biotechnological or ecological applicationssuch as S chelatiphaga a remarkable species with biotech-nological potential in phytoremediation [56] and S rizophilaa plant associated bacterium with antifungal activity [57] orBacillus mojavensis a bacteriumclosely related to B subtilisand already reported having an endophytic lifestyle andshowing an antagonistic role against plant pathogenic fungi[58] On the contrary many lavender Pseudomonas isolatescluster with known plant pathogen strains highlighting thesometimes subtle difference among pathogenic and endo-phytic lifestyle and with Pantoea agglomerans (and also withlavender isolates clustering withinthe B licheniformis and Bsoronensis groups) a known plant associated bacterium andan opportunistic pathogen in immune-compromised humanindividuals drawing attention to the possible pathogenicaction of endophytic bacteria in humans [59 60]

The overall analysis of the dendrograms points out thatthe characterization of isolates from endophytic communitiescan lead to the identification (as an example for Pantoeaand rhizobia) of not yet described strains that may representuntapped resources of bioactive compounds of agricultural ormedical interest

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

14 Evidence-Based Complementary and Alternative Medicine

context the characterization of cultivable bacterial commu-nities is a fundamental step to this purpose since it paves theway to the possibility to build collections of isolates that maybe phenotypically typed for important traits In our opiniondata obtained in this work (Table 4 and Supplementary Tables2 and 3) offers a preliminary but very promising exampleof the biotechnological potential of bacteria isolated frommedicinal plants In this pilot study in fact the majority ofthe tested endophytes showed to inhibit the growth of several(in some case all) human pathogenic strains belonging tothe Bcc complexes that are known for their resistance totraditional antibiotics These data are particularly interestingif correlated with the recent finding that essential oils fromdifferent medicinal plants are able to strongly (or completely)interfere with the growth of the same Bcc strains [61] and thatbacterial endophytes isolated from plants of the same speciesexhibited the same bioactivity (Maida et al in preparation)It is therefore possible that the therapeutic properties of theessential oil might reside also on bioactive compounds ofmicrobial origin We are completely aware that the determi-nation of the correlation (eventually) existing between thecomposition of bacterial endophytic communities and thebioactivity of essential oils extracted from a medicinal plantwould require the parallel analysis of the bacterial communityand the essential oil activity extracted from the same plantHowever data obtained in this work and those from Maidaet al [62] support this idea

It is also particularly intriguing the idea that the antimi-crobial activity of essential oils may reside on the actionof multiple compounds some of them produced or elicitedby the microbiota limiting the observed rapid evolutionof human pathogenic bacteria resistant to single moleculeantimicrobials

The characterization of the heterotrophic aerobic endo-phytic bacterial community of L angustifolia highlightedthe existence of a diversified community between differentorganstissues that may be related to the coexistence ofdifferent sources of inoculum andor a selection of the com-munities promoted by nutrient sand metabolites availabilityand antagonistic forces Several not previously characterizedstrains have been isolated and molecularly typed confirmingthat the analysis of the bacterial communities inhabitingextreme or unconventional environments may represent aproper strategy for the discovery of untapped sources offunctional biodiversity and bioactive molecule production

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This work was supported by grants from the Ente Cassadi Risparmio di Firenze (Grant 20130657 Herbiome newantimicrobial compounds from endophytic bacteria isolatedfrom medicinal plants) Giovanni Emiliani is financially

supported by the Regione Toscana POR CRO FSE 2007-2013 Asse IV Grant Sysbiofor (CNR-9) Marco Fondi andElena Perrin are financially supported by the FEMSAdvancedFellowship (FAF2012) and the Buzzati-Traverso Foundationrespectively

References

[1] B Reinhold-Hurek andTHurek ldquoLiving inside plants bacterialendophytesrdquo Current Opinion in Plant Biology vol 14 no 4 pp435ndash443 2011

[2] M Rosenblueth and E Martınez-Romero ldquoBacterial endo-phytes and their interactions with hostsrdquo Molecular Plant-Microbe Interactions vol 19 no 8 pp 827ndash837 2006

[3] R P Ryan K Germaine A Franks D J Ryan and DN Dowling ldquoBacterial endophytes recent developments andapplicationsrdquo FEMSMicrobiology Letters vol 278 no 1 pp 1ndash92008

[4] R Cakmakci F Donmez A Aydın and F Sahin ldquoGrowthpromotion of plants by plant growth-promoting rhizobacteriaunder greenhouse and two different field soil conditionsrdquo SoilBiology and Biochemistry vol 38 pp 1482ndash1487 2006

[5] P R Hardoim L S van Overbeek and J D V Elsas ldquoProp-erties of bacterial endophytes and their proposed role in plantgrowthrdquo Trends in Microbiology vol 16 no 10 pp 463ndash4712008

[6] S Compant C Clement and A Sessitsch ldquoPlant growth-promoting bacteria in the rhizo- and endosphere of plantstheir role colonization mechanisms involved and prospects forutilizationrdquo Soil Biology and Biochemistry vol 42 no 5 pp669ndash678 2010

[7] B R Glick ldquoBacterial ACC deaminase and the alleviation ofplant stressrdquo Advances in Applied Microbiology vol 56 pp 291ndash312 2004

[8] B R Glick B Todorovic J Czarny Z Cheng J Duan andB McConkey ldquoPromotion of plant growth by bacterial ACCdeaminaserdquo Critical Reviews in Plant Sciences vol 26 no 5-6pp 227ndash242 2007

[9] S L Doty B Oakley G Xin et al ldquoDiazotrophic endophytesof native black cottonwood and willowrdquo Symbiosis vol 47 no 1pp 23ndash33 2009

[10] B Lugtenberg and F Kamilova ldquoPlant-growth-promoting rhi-zobacteriardquoAnnual Review ofMicrobiology vol 63 pp 541ndash5562009

[11] U F Castillo G A Strobel E J Ford et al ldquoMunumbicinswide-spectrum antibiotics produced by Streptomyces NRRL30562 endophytic on Kennedia nigriscansrdquo Microbiology vol148 no 9 pp 2675ndash2685 2002

[12] Y Igarashi M E Trujillo E Martınez-Molina et al ldquoAnti-tumor anthraquinones from an endophytic actinomyceteMicromonospora lupini sp novrdquo Bioorganic amp Medicinal Chem-istry Letters vol 17 no 13 pp 3702ndash3705 2007

[13] S Shweta J H Bindu J Raghu et al ldquoIsolation of endophyticbacteria producing the anti-cancer alkaloid camptothecinefromMiquelia dentata Bedd (Icacinaceae)rdquo Phytomedicine vol20 pp 913ndash917 2013

[14] T Taechowisan A Wanbanjob P Tuntiwachwuttikul and JLiu ldquoAnti-inflammatory activity of lansais from endophyticStreptomyces sp SUC1 in LPS-induced RAW 2647 cellsrdquo Foodand Agricultural Immunology vol 20 no 1 pp 67ndash77 2009

Evidence-Based Complementary and Alternative Medicine 15

[15] P R Hardoim C C P Hardoim L S van Overbeek and J Dvan Elsas ldquoDynamics of seed-borne rice endophytes on earlyplant growth stagesrdquo PLoS ONE vol 7 no 2 Article ID e304382012

[16] D Bulgarelli K Schlaeppi S Spaepen E V L van Themaatand P Schulze-Lefert ldquoStructure and functions of the bacterialmicrobiota of plantsrdquo Annual Review of Plant Biology vol 64pp 807ndash838 2013

[17] A Mengoni S Mocali G Surico S Tegli and R Fani ldquoFluctu-ation of endophytic bacteria and phytoplasmosis in elm treesrdquoMicrobiological Research vol 158 no 4 pp 363ndash369 2003

[18] A Mengoni F Pini L-N Huang W-S Shu and MBazzicalupo ldquoPlant-by-plant variations of bacterial commu-nities associated with leaves of the nickel hyperaccumulatorAlyssum bertolonii desvrdquo Microbial Ecology vol 58 no 3 pp660ndash667 2009

[19] S Mocali E Bertelli F di Cello et al ldquoFluctuation of bacteriaisolated from elm tissues during different seasons and fromdifferent plant organsrdquo Research in Microbiology vol 154 no2 pp 105ndash114 2003

[20] F Pini A Frascella L Santopolo et al ldquoExploring the plant-associated bacterial communities in Medicago sativa Lrdquo BMCMicrobiology vol 12 article 78 2012

[21] A Mengoni L Cecchi and C Gonnelli ldquoNickel hyperac-cumulating plants and Alyssum bertolonii model systems forstudying biogeochemical interactions in serpentine soilsrdquo inBio-Geo Interactions in Metal-Contaminated Soils E Kothe andA Varma Eds pp 279ndash296 Springer Berlin Germany 2012

[22] S Ikeda T Okubo M Anda et al ldquoCommunity- and genome-based views of plant-associated bacteria plant-bacterial inter-actions in soybean and ricerdquo Plant and Cell Physiology vol 51no 9 pp 1398ndash1410 2010

[23] F Pini M Galardini M Bazzicalupo and A Mengoni ldquoPlant-bacteria association and symbiosis are there common genomictraits in alphaproteobacteriardquoGenes vol 2 no 4 pp 1017ndash10322011

[24] K Aleklett and M Hart ldquoThe root microbiota a fingerprint inthe soilrdquo Plant Soil vol 370 pp 671ndash686 2013

[25] A Beneduzi F Moreira P B Costa et al ldquoDiversity and plantgrowth promoting evaluation abilities of bacteria isolated fromsugarcane cultivated in the South of BrazilrdquoApplied Soil Ecologyvol 63 pp 94ndash104 2013

[26] N Bodenhausen M W Horton and J Bergelson ldquoBacterialcommunities associated with the leaves and the roots of Ara-bidopsis thalianardquo PLoS ONE vol 8 no 2 Article ID e563292013

[27] T F da Silva R E Vollu D Jurelevicius et al ldquoDoes theessential oil of Lippia sidoides Cham (pepper-rosmarin) affectits endophytic microbial communityrdquo BMC Microbiology vol13 no 1 article 29 2013

[28] M E Lucero A Unc P Cooke S Dowd and S SunldquoEndophyte microbiome diversity in micropropagated Atriplexcanescens and Atriplex torreyi var griffithsiirdquo PLoS ONE vol 6no 3 Article ID e17693 2011

[29] D S Lundberg S L Lebeis S H Paredes et al ldquoDefining thecoreArabidopsis thaliana rootmicrobiomerdquoNature vol 487 no7409 pp 86ndash90 2012

[30] H M Cavanagh and J M Wilkinson ldquoBiological activities oflavender essential oilrdquo Phytotherapy Research vol 16 no 4 pp301ndash308 2002

[31] G Woronuk Z Demissie M Rheault and S MahmoudldquoBiosynthesis and therapeutic properties of Lavandula essentialoil constituentsrdquo Planta Medica vol 77 no 1 pp 7ndash15 2011

[32] R Perry R Terry L K Watson and E Ernst ldquoIs lavender ananxiolytic drug A systematic review of randomised clinicaltrialsrdquo Phytomedicine vol 19 pp 825ndash835 2012

[33] S de Rapper G Kamatou A Viljoen and S van VuurenldquoThe in vitro antimicrobial activity of Lavandula angustifoliaessential oil in combination with other aroma-therapeutic oilsrdquoEvidence-Based Complementary and Alternative Medicine vol2013 Article ID 852049 10 pages 2013

[34] T Moon J M Wilkinson and H M Cavanagh ldquoAntiparasiticactivity of two Lavandula essential oils against Giardia duode-nalis Trichomonas vaginalis andHexamita inflatardquo ParasitologyResearch vol 99 no 6 pp 722ndash728 2006

[35] P S Yap S H Lim C P Hu and B C Yiap ldquoCom-bination of essential oils and antibiotics reduce antibioticresistance in plasmid-conferred multidrug resistant bacteriardquoPhytomedicine vol 20 pp 710ndash713 2013

[36] G Brader S Compant B Mitter F Trognitz and A SessitschldquoMetabolic potential of endophytic bacteriardquo Current Opinionin Biotechnology vol 27 pp 30ndash37 2014

[37] P Drevinek and E Mahenthiralingam ldquoBurkholderia ceno-cepacia in cystic fibrosis epidemiology and molecular mecha-nisms of virulencerdquo Clinical Microbiology and Infection vol 16no 7 pp 821ndash830 2010

[38] S Bazzini C Udine and G Riccardi ldquoMolecular approaches topathogenesis study of Burkholderia cenocepacia an importantcystic fibrosis opportunistic bacteriumrdquo Applied Microbiologyand Biotechnology vol 92 no 5 pp 887ndash895 2011

[39] I Maida A lo Nostro G Pesavento et al ldquoExploring theanti-Burkholderia cepacia complex activity of essential oilsa preliminary analysisrdquo Evidence-Based Complementary andAlternativeMedicine vol 2014 Article ID 573518 10 pages 2014

[40] A Mengoni R Barzanti C Gonnelli R Gabbrielli andM Bazzicalupo ldquoCharacterization of nickel-resistant bacteriaisolated from serpentine soilrdquo Environmental Microbiology vol3 no 11 pp 691ndash698 2001

[41] S F Altschul T L Madden A A Schaffer et al ldquoGappedBLAST and PSI-BLAST a new generation of protein databasesearch programsrdquo Nucleic Acids Research vol 25 no 17 pp3389ndash3402 1997

[42] R C Edgar ldquoMUSCLE multiple sequence alignment with highaccuracy and high throughputrdquo Nucleic Acids Research vol 32no 5 pp 1792ndash1797 2004

[43] F Ronquist M Teslenko P van der Mark et al ldquoMrbayes32 efficient bayesian phylogenetic inference and model choiceacross a large model spacerdquo Systematic Biology vol 61 no 3 pp539ndash542 2012

[44] K Tamura D Peterson N Peterson G Stecher M Nei andS Kumar ldquoMEGA5 molecular evolutionary genetics analysisusing maximum likelihood evolutionary distance and max-imum parsimony methodsrdquo Molecular Biology and Evolutionvol 28 no 10 pp 2731ndash2739 2011

[45] F Sievers A Wilm D Dineen et al ldquoFast scalable generationof high-quality protein multiple sequence alignments usingClustal Omegardquo Molecular Systems Biology vol 7 article 5392011

[46] Oslash Hammer D A T Harper and P D Ryan ldquoPAST pale-ontological statistics software package for education and dataanalysisrdquo Palaeontologia Electronica vol 4 no 1 pp 1ndash9 2001

16 Evidence-Based Complementary and Alternative Medicine

[47] M C Papaleo M Fondi I Maida et al ldquoSponge-associatedmicrobial Antarctic communities exhibiting antimicrobialactivity againstBurkholderia cepacia complex bacteriardquoBiotech-nology Advances vol 30 no 1 pp 272ndash293 2012

[48] A lo Giudice V Bruni and L Michaud ldquoCharacterization ofAntarctic psychrotrophic bacteria with antibacterial activitiesagainst terrestrial microorganismsrdquo Journal of Basic Microbiol-ogy vol 47 no 6 pp 496ndash505 2007

[49] A Schippers K Bosecker C Sproer and P SchumannldquoMicrobacterium oleivorans sp nov and Microbacteriumhydrocarbonoxydans sp nov novel crude-oil-degrading Gram-positive bacteriardquo International Journal of Systematic and Evo-lutionary Microbiology vol 55 no 2 pp 655ndash660 2005

[50] J D McChesney S K Venkataraman and J T Henri ldquoPlantnatural products back to the future or into extinctionrdquo Phyto-chemistry vol 68 no 14 pp 2015ndash2022 2007

[51] D K Manter J A Delgado D G Holm and R A StongldquoPyrosequencing reveals a highly diverse and cultivar-specificbacterial endophyte community in potato rootsrdquo MicrobialEcology vol 60 no 1 pp 157ndash166 2010

[52] K Ulrich A Ulrich and D Ewald ldquoDiversity of endophyticbacterial communities in poplar grown under field conditionsrdquoFEMS Microbiology Ecology vol 63 no 2 pp 169ndash180 2008

[53] N R Gottel H F Castro M Kerley et al ldquoDistinct microbialcommunities within the endosphere and rhizosphere ofPopulusdeltoides roots across contrasting soil typesrdquo Applied and Envi-ronmental Microbiology vol 77 no 17 pp 5934ndash5944 2011

[54] B Ma X Lv A Warren and J Gong ldquoShifts in diversity andcommunity structure of endophytic bacteria and archaea acrossroot stem and leaf tissues in the common reed Phragmitesaustralis along a salinity gradient in a marine tidal wetland ofnorthern Chinardquo Antonie van Leeuwenhoek vol 104 no 5 pp759ndash768 2013

[55] J A Vorholt ldquoMicrobial life in the phyllosphererdquo NatureReviews Microbiology vol 10 no 12 pp 828ndash840 2012

[56] R P Ryan S Monchy M Cardinale et al ldquoThe versatilityand adaptation of bacteria from the genus StenotrophomonasrdquoNature Reviews Microbiology vol 7 no 7 pp 514ndash525 2009

[57] AWolf A FritzeMHagemann andG Berg ldquoStenotrophomo-nas rhizophila sp nov a novel plant-associated bacterium withantifungal propertiesrdquo International Journal of Systematic andEvolutionary Microbiology vol 52 no 6 pp 1937ndash1944 2002

[58] C W Bacon and D M Hinton ldquoEndophytic and biologicalcontrol potential of Bacillus mojavensis and related speciesrdquoBiological Control vol 23 no 3 pp 274ndash284 2002

[59] G Berg L Eberl and A Hartmann ldquoThe rhizosphere as areservoir for opportunistic human pathogenic bacteriardquo Envi-ronmental Microbiology vol 7 no 11 pp 1673ndash1685 2005

[60] H L Tyler and E W Triplett ldquoPlants as a habitat for ben-eficial andor human pathogenic bacteriardquo Annual Review ofPhytopathology vol 46 pp 53ndash73 2008

[61] R Perry R Terry L K Watson and E Ernst ldquoIs lavender ananxiolytic drug A systematic review of randomised clinicaltrialsrdquo Phytomedicine vol 19 no 8-9 pp 825ndash835 2012

[62] I Maida A lo Nostro G Pesavento et al ldquoExploring theanti-Burkholderia cepacia complex activity of essential oilsa preliminary analysisrdquo Evidence-Based Complementary andAlternativeMedicine vol 2014 Article ID 573518 10 pages 2014

6 Evidence-Based Complementary and Alternative Medicine

100

90

80

70

60

50

40

30

20

10

0Pr

esen

ce (

)

SoilRoots

StemLeaves

Bacil

lus

Micr

obac

teriu

m

Pant

oea

Pseu

dom

onas

Rhiz

obiu

m

Sten

otro

phom

onas

Figure 3 Comparison of the relative abundance of bacterial genera in the different samples only genera with percentages gt5 in at least oneof 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 gt97 consideredas threshold of species identity (in brackets the total number of comparisons and the percentage) number of OTU with at least 1 pairwisecomparison with identity gt97

Genus Number of isolates Mean identity amongOTU () Mean distance among OTU

Number of pairwisecomparisons withidentity gt97

Number of OTU withidentity gt97

Pseudomonas 172 9425 0013 3840 (14878 258) 170Bacillus 47 9304 0027 329 (1128 29) 46Rhizobia 46 9861 0016 952 (1081 88) 46Stenotrophomonas 37 9534 0023 415 (703 59) 37Microbacterium 30 9500 0059 120 (465 25) 29Pantoea 26 8695 0126 24 (351 68) 7

(and consistency of the results) than the MP ones (see alsoSuppl Table 4) The analysis of the Bayesian phylogenetictrees revealed the following

(i) Endophytic bacteria isolated from the leavesand roots of lavender and assigned to the genusStenotrophomonas (Figure 4) are clearly split in twogroups One group contains isolates clustering withStenotrophomonas chelatiphaga interestingly thiscluster comprises isolates from both roots and leaves(no Stenotrophomonas were isolated from the stem)suggesting that two compartments might sharebacteria belonging to the same species even thoughthe polymorphism shown by the aligned partial 16Ssequences might suggest a diversification at the strainlevel The second cluster embeds roots isolates (withthe exception of leaves isolate LL44) clustering withStenotrophomonas rizophila

(ii) Concerning rhizobia (Figure 5) a grouping in onemain cluster of low differentiate sequences is present

These groups might contain new clades of plant-associated rhizobia which deservemore investigationin the future

(iii) From the phylogenetic tree of Figure 6 Pantoea iso-lated endophytes (from lavender stem and leaves)cluster with already described Pantoea spp anothergroup of isolates (all from the leaves) are divergingand thus may represent none yet described strains arefound to be associated with plants

(iv) Figure 7 depicts the phylogenetic position ofMicrobacterium isolates (mainly from leaves andrhizosphere) in the context of the genus referencestrains noticeably many leaves isolates cluster withM hydrocarbonoxydans and M oleivorans specieswhose members are able to perform crude oildegradation [49]

(v) Concerning Bacillus isolates (Suppl Figure 1) acluster of soil isolates including Bacillus mojavensiswas disclosed Another group again comprising soil

Evidence-Based Complementary and Alternative Medicine 7

009

LR48

LR31

LR41

LL38

LL43

LL94

LT48

LR98

LR86

LL88

LR72

LL17

LR28LR8

LRL93

LL39

LR61

LR76

LL37

LR7

LR29

LR34

LL77LL76

LR79

LL96

LR95LL44

LL45

LL13

LR26

LR99

LL75

LR80

LR32

LR60

LR75

066

091

1

1

089

091

091

087

093

057

078

07

091

S001020552 Escherichia coli

S001576206 S daejeonensis MJ03

S000559371 S ginsengisoli

S000007629 P geniculata ATCC 193

S000386319 S koreensis TR6-01

S000841904 S terrae R-32768

S000003927 S nitritireducens L2

S000841903 S humi R-32729

S000824093 S maltophilia IAM 124

S000010261 P hibiscicola ATCC 19

S000129976 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 genusStenotrophomonas and those of reference type strains Posterior probability values are indicated at the node Nodes are collapsed at 70probability LL = bacteria isolated from the leaves LR = bacteria isolated from the roots (see Section 2 for details)

8 Evidence-Based Complementary and Alternative Medicine

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 trS002222203 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 NRCPB10S002958314 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 MGminus2011minus4-AO

S000429561 A sp PB

S000806232 R soli DS-42

03

LR51

LR78

LR50

LT74

LR68

LR9

LR22

LR67

LR69

LR23LR24

LR5

LR21

LT92

LR19

LR49

LR62

LR57

LR54

LT91

LR55

LR12

LR20

LR47

LR18

LR11

LR65

LR25LR45

LR56

LT12

LR64

LR16

LR6

LR70

LR7

LR17

LT73

LR63

LR52

LR77

LR46

LT87

LR53

LR15

LR66094890908507606

09525

07316

07803

05579

1

0685

06484

05141

08108

1

Figure 5 Bayesian dendrogram showing the relationships among the 16S rRNA gene sequences of 46 isolates belonging to the genusRhizobium and those of reference type strains Posterior probability values are indicated at each node Nodes are collapsed at 70 probabilityLT = bacteria isolated from the rhizosphere LR = bacteria isolated from the roots (see Section 2 for details)

Evidence-Based Complementary and Alternative Medicine 9

005

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

095

09074

055

099

05

1

081

088

078

1

084

1

071

1

083

1

091

1

099

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 Pantoeaand those of reference type strains Posterior probability values are indicated at the node Nodes are collapsed at 70 probability LS = bacteriaisolated from the stem LL = bacteria isolated from the leaves (see Section 2 for details)

10 Evidence-Based Complementary and Alternative Medicine

05

LL6

LL67

LL81

LL5

LL8

LL29

LS87

LL100

LL82

LL85

LL83

LL7

LT3

LT5

LT67LT56

LL32

LL84

LL57

LL59

LT7

LL30

LL2

LT85

LL31LL1

LT4

LT6

LT8

LT2

08882

0679

0997

09977

05035

0644

0996409961

09758

07238

09846

05929

0504

0704107642

06421

1

06098

05001

05655

06027

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 paraoxydansS000381732 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 genusMicrobacterium and those of reference type strains Posterior probability values are indicated at the node Nodes are collapsed at 70probability LT = bacteria isolated from the rhizosphere LS = bacteria isolated from the stem LL = bacteria isolated from the leaves (seeSection 2 for details)

Evidence-Based Complementary and Alternative Medicine 11

Table 3The 40Burkholderia cepacia complex strains used as targetsin the cross streak experiments

Target strainStrain Species OriginFCF1 B cepacia CFFCF3LMG17588 B multivorans ENVFCF16 B cenocepacia (IIIA) CFJ2315FCF18

B cenocepacia (IIIB) CF

FCF20FCF23FCF24FCF27FCF29FCF30LMG16654C5424CEP511MVPC116 ENVMVPC173LMG19230 B cenocepacia (IIIC) ENVLMG19240FCF38 B cenocepacia (IIID) CFLMG21462FCF41 B stabilis CFFCF42 B vietnamiensis CFTVV75 ENVLMG18941

B dolosa CFLMG18942LMG18943MCI7

B ambifariaENV

LMG19467 CFLMG19182 ENVLMG16670 B anthina ENVFCF43 B pyrrocina CFLSED4 B lata CFLMG24064 B latens CFLMG24065 B diffusa CFLMG23361 B contaminans AILMG24067 B seminals CFLMG24068 B metallica CFLMG24066 B arboris ENVLMG24263 B ubonensis NIAbbreviations CF strains isolated from cystic fibrosis patients AI strainsisolated from animal infection NI strains isolated from nosocomial infec-tion ENV environmental strain

isolates showed similarities with the stress resistantB safensis

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

complex Regardless of the overall low support ofthe tree (typical of Pseudomonas phylogenies) someobservations may be drawn the presence of largeunresolved clusters shows clearly a low divergenceof the isolated colonies implying the existence ofclonal populations Pseudomonas spp isolated fromthe different compartments are intermixed suggest-ing the presence of a continuum rhizosphere-leaves(Pseudomonas is the only genus found in all the 4sample analysed)

32 Inhibition of Burkholderia Cepacia Complex StrainsGrowth by L angustifolia Endophytic Bacteria In order tocheck the ability of L angustifolia bacterial endophytes toantagonize the growth of (opportunistic) human pathogenicbacteria cross-streak experiments were carried out asdescribed in Section 2 using a panel of the 19 randomlychosen different endophytic strains isolated from soil rootsor leafs and phylogenetically assigned to six different gen-era (Bacillus Microbacterium Pantoea Plantibacter Pseu-domonas and Rhizobium) as testers versus 40 Bcc strainsrepresentative of seventeen species (out of eighteen) andwith either environmental or clinical origin with some ofwhich being multidrug-resistant Data obtained are shownin Table 4 The analysis of these data revealed that all thetester strains were able to completely inhibit the growthof Bcc strains including those with a clinical source andthat exhibited multidrug resistance this finding suggestedthat lavender endophytic bacteria are able to synthesize(strong) antimicrobial compounds However tester strainsshowed a different pattern of Bcc growth inhibition eventhose affiliated to the same genus In addition to this thereis no apparent difference in the antimicrobial potentialbetween strains belonging to different plant compartmentsConcerning the sensitivity of Bcc strains to the antimicrobialactivity of lavender endophytes strains belonging to differentspecies exhibited a different sensitivity spectrum However itis quite interesting that strains with clinical origin appearedto be more sensitive to the antimicrobials synthesized byendophytic bacteria than their environmental counterparts(see for instance strains belonging to the species Burkholderiacenocepacia IIIB) (Supplementary Table 3)

4 Discussion

Medicinal plants are stirring the attention of manyresearchers due to the presence of compounds thatconstitute a large fraction of the current pharmacopoeiasNatural products have been the source of most of theactive ingredients of medicines and more than 80 of drugsubstances are natural products or inspired by a naturalcompound including most of the of anticancer and anti-infective agents [50] Lavender plants are grown mainlyfor the production of essential oil which has antisepticand anti-inflammatory properties In spite of the relativeharsh environment for bacterial colonization L angustifoliatissues were found to be rich in bacterial diversity Howeverthe endophytic community isolated from different plant

12 Evidence-Based Complementary and Alternative Medicine

Table4Growth

of40

Burkholderiacepacia

complex

strains

inthep

resenceabsenceo

fend

ophytic

bacterialstrains

isolated

from

lavend

er

Species

Strain

Orig

inLL

1LL

2LL

3LL

4LL

6LL

7LL

9LL

10LS

1LS

2LS

3LS

4LS

5LS

6LR

1LR

2LR

3LR

4LR

5C-

Bcepacia

FCF1

CFminusminusminusminusminusminusminusminus

minusminusminusminusminusminusminusminusminusminusminus

+B

cepacia

FCF3

CF+minusminusminusminusminusminusminusminus

minusminusminusminusminusminusminusminusminusminusminus

+B

multivoran

sLM

G17588

Env

+minusminus

++minus

+minusminus

+minus

+minus

++minus

+minusminus

+minus

+minusminus

+B

cenocepacia

(IIIA

)FC

F16

CFminusminusminusminusminusminusminusminus

minusminusminusminusminusminusminusminusminusminusminus

+B

cenocepacia

(IIIA

)J2315

CF+minusminus

+minus

+minusminusminus

+minusminusminus

+minusminusminusminusminusminusminusminus

+B

cenocepacia

(IIIB)

FCF18

CF+minusminusminusminusminusminusminus

minusminusminusminusminusminus

+minusminusminusminusminus

+B

cenocepacia

(IIIB)

FCF20

CF+

++

++

+minus

++minus

+minus

+minus

+minus

++minus

++minus

++

++

+B

cenocepacia

(IIIB)

FCF23

CF+minusminusminusminusminusminusminus

+minusminusminus

++minusminusminus

+minusminusminusminus

+B

cenocepacia

(IIIB)

FCF24

CF+minusminusminusminusminusminusminus

+minusminus

+minusminusminusminus

+minusminusminusminus

+B

cenocepacia

(IIIB)

FCF27

CF+minusminusminusminusminusminusminus

+minusminusminusminusminusminusminusminusminusminusminus

+B

cenocepacia

(IIIB)

FCF29

CF+minusminus

+minus

+minusminusminus

+minusminusminusminusminusminusminus

+minus

+minusminus

+B

cenocepacia

(IIIB)

FCF30

CF+minusminus

+minus

+minusminusminus

+minus

+minusminusminusminusminusminus

+minus

+minusminus

+B

cenocepacia

(IIIB)

LMG16654

CF+

+minusminus

++minus

+minus

++minus

++minusminus

+minus

+minusminus

+B

cenocepacia

(IIIB)

C5424

CF+

+minusminus

+minusminusminusminus

+minusminusminus

+minusminusminusminus

+minusminus

+minusminus

+B

cenocepacia

(IIIB)

CEP5

11CF

+minusminus

+minusminusminusminus

+minus

+minusminus

+minus

+minusminusminus

+minus

+minusminus

+B

cenocepacia

(IIIB)

MVPC

116

Env

++minusminus

+minus

++minusminus

+minus

+minus

+minus

+minusminus

+minusminus

+minusminus

+minus

+minus

+B

cenocepacia

(IIIB)

MVPC

173

Env

++

++minus

++

+minus

++

++

++

++

++

++minus

+B

cenocepacia

(IIIIC)

LMG19230

Env

++

++

++

+minus

++

++

++

++

++

++

+B

cenocepacia

(IIIC

)LM

G19240

Env

++

++

+minus

+minus

++

+minus

++

++minus

++

++

+minus

+B

cenocepacia

(IIID)

FCF38

CF+minusminusminusminusminusminusminus

minusminusminusminusminusminusminusminusminusminusminus

+B

cenocepacia

(IIID)

LMG21462

CFminusminusminusminusminusminusminusminus

minusminusminusminusminusminusminusminusminusminusminus

+B

stabilis

FCF41

CF+

+minus

++

+minus

+minusminus

++

+minus

++minusminus

++

++minusminus

+B

vietnamien

sisFC

F42

CFminusminusminusminusminusminusminusminus

minusminusminusminusminusminusminusminusminusminusminus

+B

vietnamien

sisTV

V75

Env

++minusminus

+minusminusminusminus

+minusminusminus

++minusminusminus

+minusminus

+minusminus

+B

dolosa

LMG18941

CF+minusminus

+minus

+minus

+minusminus

+minusminusminus

++minusminusminus

+minus

+minus

+minusminus

+B

dolosa

LMG18942

CF+

+minusminus

+minus

+minusminus

++

+minus

++minusminusminus

++minus

++minusminus

+B

dolosa

LMG18943

CF+

+minusminus

++minus

+minusminus

+minus

+minusminus

++minusminusminus

++minus

+minusminus

+B

ambifaria

MCI

7En

v+

+minus

+minus

+minus

+minusminus

++minusminus

++minusminusminus

+minus

+minusminus

+B

ambifaria

LMG1946

7CF

++minus

+minus

+minusminusminus

++minusminus

++minusminusminus

+minus

+minusminusminus

+B

ambifaria

LMG19182

Env

++minus

+minus

+minusminusminus

+minusminus

+minusminusminus

+minus

+minusminusminus

+B

anthina

LMG16670

Env

+minusminusminusminus

+minus

+minusminus

++minusminusminus

+minus

+minusminusminus

+B

pyrrocinia

FCF43

CF+minusminusminusminusminusminusminusminus

minusminus

+minusminusminusminusminusminus

+minusminusminus

+B

lata

LSED

4CF

+minus

+minusminus

+minusminus

+minusminus

++minusminusminus

+minus

+minus

+minusminusminus

+B

latens

LMG2406

4CF

+minus

++minusminus

+minusminus

++minus

++minus

++

++

+minus

+B

diffu

saLM

G24065

CF+

+minus

++minus

+minus

+minus

++minus

++minus

+minus

++

++minus

+B

contam

inan

sLM

G23361

AI

++minus

+minus

+minus

+minus

+minus

++

++

++

+minus

++

++minus

+B

seminalis

LMG24067

CF+

+minus

++minus

++

++

++

++

++

++

++minus

+B

metallica

LMG2406

8CF

++

++minus

++

++

++

++

++

++

++minus

+B

arboris

LMG2406

6En

v+

+minus

++minus

++

+minus

++

++

++minus

+minus

++

++minusminus

+B

ubonensis

LMG24263

NI

+minusminusminusminus

+minusminus

+minus

+minus

++minusminus

+minusminus

+minusminusminus

+Ab

breviatio

nsC

Fstr

ains

isolatedfro

mcysticfi

brosispatie

ntsAIstr

ains

isolated

from

anim

alinfectionNIstr

ains

isolated

from

nosocomialinfectio

nEN

Venvironm

entalstrainLstr

ains

isolatedfro

mtheleaf

Sstr

ains

isolatedfro

mthes

temR

strains

isolatedfro

mther

oot

Symbo

ls+

grow

th+

minusreduced

grow

th+

minusminusveryredu

cedgrow

thminus

nogrow

thC

-Petrid

ishes

containing

onlythetargetstrains

Evidence-Based Complementary and Alternative Medicine 13

compartments showed different levels of diversity withthe 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 theleaf and roots community is already reported in otherspecies [26] even if there are few direct comparisons ofroots and phyllosphere bacterial communities especiallycomparisons using material from the same plants Themicrobial community residing in the phyllosphere is facedwith a nutrient poor and variable environment that ischaracterized by fluctuating temperature humidity and UVradiation and so it is predicted to be more diverse than thatwithin the rhizosphere a more homeostatic environmentThese findings supported also by the similar bacterial titreare in agreement with the idea that the source of inoculumfor the leaf community (except for the vertically transferredvia seeds) may be exclusive (eg aerosol even if the processis not yet clarified Bulgarelli et al 2013) and not related totransmission from the roots Moreover the low diversity ofthe community isolated in the stem dominated by clonally(Table 2) populations of Pseudomonas could be related tothe highly specific main tissue (phloem) present there whichcould provide an homeostatic environment rich in nutrient(sucrose contained in the phloem tissues) but poorer (ifcompared to leaf and roots) in secondary metabolites thatmay promote ecological niche differentiation

Concerning the taxonomic community composition dif-ferent taxonomic profiles were found for endosphere andrhizosphere More in detail the rhizosphere communityshowed quite similar diversity indexes if compared to theroots one but with a different composition as an examplemembers of the phylumActinobacteria are completely absentfrom the root internal tissues that are largely dominatedby Proteobacteria while they are present in the soil Toexplain this finding a two-step model has been proposed[16] soil abiotic properties determine the structure of theinitial bacterial communities that is then modified by rhi-zodeposits with plant roots cell wall features and releasedmetabolites promoting the growth of organotrophic bacteriathereby initiating a soil community shift In the second stepconvergent host genotype-dependent selection [51 52] fine-tunes community profiles thriving on the rhizoplane andwithin plant roots

Moreover endosphere communities were differentbetween plant organs (roots stems and leaves) even if theinternal tissues are dominated as already reported [25 52ndash54] by members of the phylum Proteobacteria an examplewhile rhizobia were isolated from root internal tissues theyare completely absent from stems and leaves (Figures 2 and3) isolates belonging to the genus Pantoea on the contrarywere isolated from aerial parts but not from root tissuesHence it is possible that plant might ldquoselectrdquo specific taxafor entering inside its compartment Such hypothesis issupported also by the pairwise differentiation values thecommunities isolated from the different tissues even those inphysical continuity are in fact strongly differentiated with thestem representing a low diversity compartment separatingthe two high diversity organs roots and leaves In this view itis noteworthy that in lavender only Pseudomonas represents

a ubiquitous and abundant genus of both rhizosphere andendosphere (Figure 3) The analysis of intrageneric diversitypointed out also that Pseudomonas isolates belong to a lowdifferentiated clonal population (Table 2 and Suppl Figure2) suggesting also that this component of the communityeither diffused inside the organs from a radical or foliar entrypoint or established during plants development putativelyfrom the seed inoculum A similar consideration can beproposed for isolates of rhizobia but for the rhizosphere-root internal tissues continuum a low taxonomicallydifferentiated community from the soil entered (increasingits relative abundance) inside the plant organ An entrypoint from the leaves is on the contrary consistent with thedistribution of Pantoea isolates with their strong diversity(Table 2) suggesting a diversification in response to the highvariable leaf environmentThe isolates belonging to the genusStenotrophomonas show an interesting distribution pattern(Figure 3) they are rare in the rhizosphere abundant in theroots and in the leaves and absent in the stem this findingsuggests a different entry point or a negative selection in thestem the second explanation is supported by admixture ofroot and leaves isolates (Figure 4)

The detailed phylogenetic analyses of the isolates enabledus also to putatively ascribe some isolates to already describedendophytic strains that can be targeted for functional charac-terisation many Microbacterium leaves isolates cluster withM hydrocarbonoxydans and M oleivorans two species withcrude oil degrading activity [49] being this metabolic activitythat is found frequently associated with a phyllosphereadapted lifestyle [55] Several isolates cluster with bacteriawith interesting biotechnological or ecological applicationssuch as S chelatiphaga a remarkable species with biotech-nological potential in phytoremediation [56] and S rizophilaa plant associated bacterium with antifungal activity [57] orBacillus mojavensis a bacteriumclosely related to B subtilisand already reported having an endophytic lifestyle andshowing an antagonistic role against plant pathogenic fungi[58] On the contrary many lavender Pseudomonas isolatescluster with known plant pathogen strains highlighting thesometimes subtle difference among pathogenic and endo-phytic lifestyle and with Pantoea agglomerans (and also withlavender isolates clustering withinthe B licheniformis and Bsoronensis groups) a known plant associated bacterium andan opportunistic pathogen in immune-compromised humanindividuals drawing attention to the possible pathogenicaction of endophytic bacteria in humans [59 60]

The overall analysis of the dendrograms points out thatthe characterization of isolates from endophytic communitiescan lead to the identification (as an example for Pantoeaand rhizobia) of not yet described strains that may representuntapped resources of bioactive compounds of agricultural ormedical interest

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

14 Evidence-Based Complementary and Alternative Medicine

context the characterization of cultivable bacterial commu-nities is a fundamental step to this purpose since it paves theway to the possibility to build collections of isolates that maybe phenotypically typed for important traits In our opiniondata obtained in this work (Table 4 and Supplementary Tables2 and 3) offers a preliminary but very promising exampleof the biotechnological potential of bacteria isolated frommedicinal plants In this pilot study in fact the majority ofthe tested endophytes showed to inhibit the growth of several(in some case all) human pathogenic strains belonging tothe Bcc complexes that are known for their resistance totraditional antibiotics These data are particularly interestingif correlated with the recent finding that essential oils fromdifferent medicinal plants are able to strongly (or completely)interfere with the growth of the same Bcc strains [61] and thatbacterial endophytes isolated from plants of the same speciesexhibited the same bioactivity (Maida et al in preparation)It is therefore possible that the therapeutic properties of theessential oil might reside also on bioactive compounds ofmicrobial origin We are completely aware that the determi-nation of the correlation (eventually) existing between thecomposition of bacterial endophytic communities and thebioactivity of essential oils extracted from a medicinal plantwould require the parallel analysis of the bacterial communityand the essential oil activity extracted from the same plantHowever data obtained in this work and those from Maidaet al [62] support this idea

It is also particularly intriguing the idea that the antimi-crobial activity of essential oils may reside on the actionof multiple compounds some of them produced or elicitedby the microbiota limiting the observed rapid evolutionof human pathogenic bacteria resistant to single moleculeantimicrobials

The characterization of the heterotrophic aerobic endo-phytic bacterial community of L angustifolia highlightedthe existence of a diversified community between differentorganstissues that may be related to the coexistence ofdifferent sources of inoculum andor a selection of the com-munities promoted by nutrient sand metabolites availabilityand antagonistic forces Several not previously characterizedstrains have been isolated and molecularly typed confirmingthat the analysis of the bacterial communities inhabitingextreme or unconventional environments may represent aproper strategy for the discovery of untapped sources offunctional biodiversity and bioactive molecule production

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This work was supported by grants from the Ente Cassadi Risparmio di Firenze (Grant 20130657 Herbiome newantimicrobial compounds from endophytic bacteria isolatedfrom medicinal plants) Giovanni Emiliani is financially

supported by the Regione Toscana POR CRO FSE 2007-2013 Asse IV Grant Sysbiofor (CNR-9) Marco Fondi andElena Perrin are financially supported by the FEMSAdvancedFellowship (FAF2012) and the Buzzati-Traverso Foundationrespectively

References

[1] B Reinhold-Hurek andTHurek ldquoLiving inside plants bacterialendophytesrdquo Current Opinion in Plant Biology vol 14 no 4 pp435ndash443 2011

[2] M Rosenblueth and E Martınez-Romero ldquoBacterial endo-phytes and their interactions with hostsrdquo Molecular Plant-Microbe Interactions vol 19 no 8 pp 827ndash837 2006

[3] R P Ryan K Germaine A Franks D J Ryan and DN Dowling ldquoBacterial endophytes recent developments andapplicationsrdquo FEMSMicrobiology Letters vol 278 no 1 pp 1ndash92008

[4] R Cakmakci F Donmez A Aydın and F Sahin ldquoGrowthpromotion of plants by plant growth-promoting rhizobacteriaunder greenhouse and two different field soil conditionsrdquo SoilBiology and Biochemistry vol 38 pp 1482ndash1487 2006

[5] P R Hardoim L S van Overbeek and J D V Elsas ldquoProp-erties of bacterial endophytes and their proposed role in plantgrowthrdquo Trends in Microbiology vol 16 no 10 pp 463ndash4712008

[6] S Compant C Clement and A Sessitsch ldquoPlant growth-promoting bacteria in the rhizo- and endosphere of plantstheir role colonization mechanisms involved and prospects forutilizationrdquo Soil Biology and Biochemistry vol 42 no 5 pp669ndash678 2010

[7] B R Glick ldquoBacterial ACC deaminase and the alleviation ofplant stressrdquo Advances in Applied Microbiology vol 56 pp 291ndash312 2004

[8] B R Glick B Todorovic J Czarny Z Cheng J Duan andB McConkey ldquoPromotion of plant growth by bacterial ACCdeaminaserdquo Critical Reviews in Plant Sciences vol 26 no 5-6pp 227ndash242 2007

[9] S L Doty B Oakley G Xin et al ldquoDiazotrophic endophytesof native black cottonwood and willowrdquo Symbiosis vol 47 no 1pp 23ndash33 2009

[10] B Lugtenberg and F Kamilova ldquoPlant-growth-promoting rhi-zobacteriardquoAnnual Review ofMicrobiology vol 63 pp 541ndash5562009

[11] U F Castillo G A Strobel E J Ford et al ldquoMunumbicinswide-spectrum antibiotics produced by Streptomyces NRRL30562 endophytic on Kennedia nigriscansrdquo Microbiology vol148 no 9 pp 2675ndash2685 2002

[12] Y Igarashi M E Trujillo E Martınez-Molina et al ldquoAnti-tumor anthraquinones from an endophytic actinomyceteMicromonospora lupini sp novrdquo Bioorganic amp Medicinal Chem-istry Letters vol 17 no 13 pp 3702ndash3705 2007

[13] S Shweta J H Bindu J Raghu et al ldquoIsolation of endophyticbacteria producing the anti-cancer alkaloid camptothecinefromMiquelia dentata Bedd (Icacinaceae)rdquo Phytomedicine vol20 pp 913ndash917 2013

[14] T Taechowisan A Wanbanjob P Tuntiwachwuttikul and JLiu ldquoAnti-inflammatory activity of lansais from endophyticStreptomyces sp SUC1 in LPS-induced RAW 2647 cellsrdquo Foodand Agricultural Immunology vol 20 no 1 pp 67ndash77 2009

Evidence-Based Complementary and Alternative Medicine 15

[15] P R Hardoim C C P Hardoim L S van Overbeek and J Dvan Elsas ldquoDynamics of seed-borne rice endophytes on earlyplant growth stagesrdquo PLoS ONE vol 7 no 2 Article ID e304382012

[16] D Bulgarelli K Schlaeppi S Spaepen E V L van Themaatand P Schulze-Lefert ldquoStructure and functions of the bacterialmicrobiota of plantsrdquo Annual Review of Plant Biology vol 64pp 807ndash838 2013

[17] A Mengoni S Mocali G Surico S Tegli and R Fani ldquoFluctu-ation of endophytic bacteria and phytoplasmosis in elm treesrdquoMicrobiological Research vol 158 no 4 pp 363ndash369 2003

[18] A Mengoni F Pini L-N Huang W-S Shu and MBazzicalupo ldquoPlant-by-plant variations of bacterial commu-nities associated with leaves of the nickel hyperaccumulatorAlyssum bertolonii desvrdquo Microbial Ecology vol 58 no 3 pp660ndash667 2009

[19] S Mocali E Bertelli F di Cello et al ldquoFluctuation of bacteriaisolated from elm tissues during different seasons and fromdifferent plant organsrdquo Research in Microbiology vol 154 no2 pp 105ndash114 2003

[20] F Pini A Frascella L Santopolo et al ldquoExploring the plant-associated bacterial communities in Medicago sativa Lrdquo BMCMicrobiology vol 12 article 78 2012

[21] A Mengoni L Cecchi and C Gonnelli ldquoNickel hyperac-cumulating plants and Alyssum bertolonii model systems forstudying biogeochemical interactions in serpentine soilsrdquo inBio-Geo Interactions in Metal-Contaminated Soils E Kothe andA Varma Eds pp 279ndash296 Springer Berlin Germany 2012

[22] S Ikeda T Okubo M Anda et al ldquoCommunity- and genome-based views of plant-associated bacteria plant-bacterial inter-actions in soybean and ricerdquo Plant and Cell Physiology vol 51no 9 pp 1398ndash1410 2010

[23] F Pini M Galardini M Bazzicalupo and A Mengoni ldquoPlant-bacteria association and symbiosis are there common genomictraits in alphaproteobacteriardquoGenes vol 2 no 4 pp 1017ndash10322011

[24] K Aleklett and M Hart ldquoThe root microbiota a fingerprint inthe soilrdquo Plant Soil vol 370 pp 671ndash686 2013

[25] A Beneduzi F Moreira P B Costa et al ldquoDiversity and plantgrowth promoting evaluation abilities of bacteria isolated fromsugarcane cultivated in the South of BrazilrdquoApplied Soil Ecologyvol 63 pp 94ndash104 2013

[26] N Bodenhausen M W Horton and J Bergelson ldquoBacterialcommunities associated with the leaves and the roots of Ara-bidopsis thalianardquo PLoS ONE vol 8 no 2 Article ID e563292013

[27] T F da Silva R E Vollu D Jurelevicius et al ldquoDoes theessential oil of Lippia sidoides Cham (pepper-rosmarin) affectits endophytic microbial communityrdquo BMC Microbiology vol13 no 1 article 29 2013

[28] M E Lucero A Unc P Cooke S Dowd and S SunldquoEndophyte microbiome diversity in micropropagated Atriplexcanescens and Atriplex torreyi var griffithsiirdquo PLoS ONE vol 6no 3 Article ID e17693 2011

[29] D S Lundberg S L Lebeis S H Paredes et al ldquoDefining thecoreArabidopsis thaliana rootmicrobiomerdquoNature vol 487 no7409 pp 86ndash90 2012

[30] H M Cavanagh and J M Wilkinson ldquoBiological activities oflavender essential oilrdquo Phytotherapy Research vol 16 no 4 pp301ndash308 2002

[31] G Woronuk Z Demissie M Rheault and S MahmoudldquoBiosynthesis and therapeutic properties of Lavandula essentialoil constituentsrdquo Planta Medica vol 77 no 1 pp 7ndash15 2011

[32] R Perry R Terry L K Watson and E Ernst ldquoIs lavender ananxiolytic drug A systematic review of randomised clinicaltrialsrdquo Phytomedicine vol 19 pp 825ndash835 2012

[33] S de Rapper G Kamatou A Viljoen and S van VuurenldquoThe in vitro antimicrobial activity of Lavandula angustifoliaessential oil in combination with other aroma-therapeutic oilsrdquoEvidence-Based Complementary and Alternative Medicine vol2013 Article ID 852049 10 pages 2013

[34] T Moon J M Wilkinson and H M Cavanagh ldquoAntiparasiticactivity of two Lavandula essential oils against Giardia duode-nalis Trichomonas vaginalis andHexamita inflatardquo ParasitologyResearch vol 99 no 6 pp 722ndash728 2006

[35] P S Yap S H Lim C P Hu and B C Yiap ldquoCom-bination of essential oils and antibiotics reduce antibioticresistance in plasmid-conferred multidrug resistant bacteriardquoPhytomedicine vol 20 pp 710ndash713 2013

[36] G Brader S Compant B Mitter F Trognitz and A SessitschldquoMetabolic potential of endophytic bacteriardquo Current Opinionin Biotechnology vol 27 pp 30ndash37 2014

[37] P Drevinek and E Mahenthiralingam ldquoBurkholderia ceno-cepacia in cystic fibrosis epidemiology and molecular mecha-nisms of virulencerdquo Clinical Microbiology and Infection vol 16no 7 pp 821ndash830 2010

[38] S Bazzini C Udine and G Riccardi ldquoMolecular approaches topathogenesis study of Burkholderia cenocepacia an importantcystic fibrosis opportunistic bacteriumrdquo Applied Microbiologyand Biotechnology vol 92 no 5 pp 887ndash895 2011

[39] I Maida A lo Nostro G Pesavento et al ldquoExploring theanti-Burkholderia cepacia complex activity of essential oilsa preliminary analysisrdquo Evidence-Based Complementary andAlternativeMedicine vol 2014 Article ID 573518 10 pages 2014

[40] A Mengoni R Barzanti C Gonnelli R Gabbrielli andM Bazzicalupo ldquoCharacterization of nickel-resistant bacteriaisolated from serpentine soilrdquo Environmental Microbiology vol3 no 11 pp 691ndash698 2001

[41] S F Altschul T L Madden A A Schaffer et al ldquoGappedBLAST and PSI-BLAST a new generation of protein databasesearch programsrdquo Nucleic Acids Research vol 25 no 17 pp3389ndash3402 1997

[42] R C Edgar ldquoMUSCLE multiple sequence alignment with highaccuracy and high throughputrdquo Nucleic Acids Research vol 32no 5 pp 1792ndash1797 2004

[43] F Ronquist M Teslenko P van der Mark et al ldquoMrbayes32 efficient bayesian phylogenetic inference and model choiceacross a large model spacerdquo Systematic Biology vol 61 no 3 pp539ndash542 2012

[44] K Tamura D Peterson N Peterson G Stecher M Nei andS Kumar ldquoMEGA5 molecular evolutionary genetics analysisusing maximum likelihood evolutionary distance and max-imum parsimony methodsrdquo Molecular Biology and Evolutionvol 28 no 10 pp 2731ndash2739 2011

[45] F Sievers A Wilm D Dineen et al ldquoFast scalable generationof high-quality protein multiple sequence alignments usingClustal Omegardquo Molecular Systems Biology vol 7 article 5392011

[46] Oslash Hammer D A T Harper and P D Ryan ldquoPAST pale-ontological statistics software package for education and dataanalysisrdquo Palaeontologia Electronica vol 4 no 1 pp 1ndash9 2001

16 Evidence-Based Complementary and Alternative Medicine

[47] M C Papaleo M Fondi I Maida et al ldquoSponge-associatedmicrobial Antarctic communities exhibiting antimicrobialactivity againstBurkholderia cepacia complex bacteriardquoBiotech-nology Advances vol 30 no 1 pp 272ndash293 2012

[48] A lo Giudice V Bruni and L Michaud ldquoCharacterization ofAntarctic psychrotrophic bacteria with antibacterial activitiesagainst terrestrial microorganismsrdquo Journal of Basic Microbiol-ogy vol 47 no 6 pp 496ndash505 2007

[49] A Schippers K Bosecker C Sproer and P SchumannldquoMicrobacterium oleivorans sp nov and Microbacteriumhydrocarbonoxydans sp nov novel crude-oil-degrading Gram-positive bacteriardquo International Journal of Systematic and Evo-lutionary Microbiology vol 55 no 2 pp 655ndash660 2005

[50] J D McChesney S K Venkataraman and J T Henri ldquoPlantnatural products back to the future or into extinctionrdquo Phyto-chemistry vol 68 no 14 pp 2015ndash2022 2007

[51] D K Manter J A Delgado D G Holm and R A StongldquoPyrosequencing reveals a highly diverse and cultivar-specificbacterial endophyte community in potato rootsrdquo MicrobialEcology vol 60 no 1 pp 157ndash166 2010

[52] K Ulrich A Ulrich and D Ewald ldquoDiversity of endophyticbacterial communities in poplar grown under field conditionsrdquoFEMS Microbiology Ecology vol 63 no 2 pp 169ndash180 2008

[53] N R Gottel H F Castro M Kerley et al ldquoDistinct microbialcommunities within the endosphere and rhizosphere ofPopulusdeltoides roots across contrasting soil typesrdquo Applied and Envi-ronmental Microbiology vol 77 no 17 pp 5934ndash5944 2011

[54] B Ma X Lv A Warren and J Gong ldquoShifts in diversity andcommunity structure of endophytic bacteria and archaea acrossroot stem and leaf tissues in the common reed Phragmitesaustralis along a salinity gradient in a marine tidal wetland ofnorthern Chinardquo Antonie van Leeuwenhoek vol 104 no 5 pp759ndash768 2013

[55] J A Vorholt ldquoMicrobial life in the phyllosphererdquo NatureReviews Microbiology vol 10 no 12 pp 828ndash840 2012

[56] R P Ryan S Monchy M Cardinale et al ldquoThe versatilityand adaptation of bacteria from the genus StenotrophomonasrdquoNature Reviews Microbiology vol 7 no 7 pp 514ndash525 2009

[57] AWolf A FritzeMHagemann andG Berg ldquoStenotrophomo-nas rhizophila sp nov a novel plant-associated bacterium withantifungal propertiesrdquo International Journal of Systematic andEvolutionary Microbiology vol 52 no 6 pp 1937ndash1944 2002

[58] C W Bacon and D M Hinton ldquoEndophytic and biologicalcontrol potential of Bacillus mojavensis and related speciesrdquoBiological Control vol 23 no 3 pp 274ndash284 2002

[59] G Berg L Eberl and A Hartmann ldquoThe rhizosphere as areservoir for opportunistic human pathogenic bacteriardquo Envi-ronmental Microbiology vol 7 no 11 pp 1673ndash1685 2005

[60] H L Tyler and E W Triplett ldquoPlants as a habitat for ben-eficial andor human pathogenic bacteriardquo Annual Review ofPhytopathology vol 46 pp 53ndash73 2008

[61] R Perry R Terry L K Watson and E Ernst ldquoIs lavender ananxiolytic drug A systematic review of randomised clinicaltrialsrdquo Phytomedicine vol 19 no 8-9 pp 825ndash835 2012

[62] I Maida A lo Nostro G Pesavento et al ldquoExploring theanti-Burkholderia cepacia complex activity of essential oilsa preliminary analysisrdquo Evidence-Based Complementary andAlternativeMedicine vol 2014 Article ID 573518 10 pages 2014

Evidence-Based Complementary and Alternative Medicine 7

009

LR48

LR31

LR41

LL38

LL43

LL94

LT48

LR98

LR86

LL88

LR72

LL17

LR28LR8

LRL93

LL39

LR61

LR76

LL37

LR7

LR29

LR34

LL77LL76

LR79

LL96

LR95LL44

LL45

LL13

LR26

LR99

LL75

LR80

LR32

LR60

LR75

066

091

1

1

089

091

091

087

093

057

078

07

091

S001020552 Escherichia coli

S001576206 S daejeonensis MJ03

S000559371 S ginsengisoli

S000007629 P geniculata ATCC 193

S000386319 S koreensis TR6-01

S000841904 S terrae R-32768

S000003927 S nitritireducens L2

S000841903 S humi R-32729

S000824093 S maltophilia IAM 124

S000010261 P hibiscicola ATCC 19

S000129976 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 genusStenotrophomonas and those of reference type strains Posterior probability values are indicated at the node Nodes are collapsed at 70probability LL = bacteria isolated from the leaves LR = bacteria isolated from the roots (see Section 2 for details)

8 Evidence-Based Complementary and Alternative Medicine

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 trS002222203 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 NRCPB10S002958314 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 MGminus2011minus4-AO

S000429561 A sp PB

S000806232 R soli DS-42

03

LR51

LR78

LR50

LT74

LR68

LR9

LR22

LR67

LR69

LR23LR24

LR5

LR21

LT92

LR19

LR49

LR62

LR57

LR54

LT91

LR55

LR12

LR20

LR47

LR18

LR11

LR65

LR25LR45

LR56

LT12

LR64

LR16

LR6

LR70

LR7

LR17

LT73

LR63

LR52

LR77

LR46

LT87

LR53

LR15

LR66094890908507606

09525

07316

07803

05579

1

0685

06484

05141

08108

1

Figure 5 Bayesian dendrogram showing the relationships among the 16S rRNA gene sequences of 46 isolates belonging to the genusRhizobium and those of reference type strains Posterior probability values are indicated at each node Nodes are collapsed at 70 probabilityLT = bacteria isolated from the rhizosphere LR = bacteria isolated from the roots (see Section 2 for details)

Evidence-Based Complementary and Alternative Medicine 9

005

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

095

09074

055

099

05

1

081

088

078

1

084

1

071

1

083

1

091

1

099

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 Pantoeaand those of reference type strains Posterior probability values are indicated at the node Nodes are collapsed at 70 probability LS = bacteriaisolated from the stem LL = bacteria isolated from the leaves (see Section 2 for details)

10 Evidence-Based Complementary and Alternative Medicine

05

LL6

LL67

LL81

LL5

LL8

LL29

LS87

LL100

LL82

LL85

LL83

LL7

LT3

LT5

LT67LT56

LL32

LL84

LL57

LL59

LT7

LL30

LL2

LT85

LL31LL1

LT4

LT6

LT8

LT2

08882

0679

0997

09977

05035

0644

0996409961

09758

07238

09846

05929

0504

0704107642

06421

1

06098

05001

05655

06027

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 paraoxydansS000381732 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 genusMicrobacterium and those of reference type strains Posterior probability values are indicated at the node Nodes are collapsed at 70probability LT = bacteria isolated from the rhizosphere LS = bacteria isolated from the stem LL = bacteria isolated from the leaves (seeSection 2 for details)

Evidence-Based Complementary and Alternative Medicine 11

Table 3The 40Burkholderia cepacia complex strains used as targetsin the cross streak experiments

Target strainStrain Species OriginFCF1 B cepacia CFFCF3LMG17588 B multivorans ENVFCF16 B cenocepacia (IIIA) CFJ2315FCF18

B cenocepacia (IIIB) CF

FCF20FCF23FCF24FCF27FCF29FCF30LMG16654C5424CEP511MVPC116 ENVMVPC173LMG19230 B cenocepacia (IIIC) ENVLMG19240FCF38 B cenocepacia (IIID) CFLMG21462FCF41 B stabilis CFFCF42 B vietnamiensis CFTVV75 ENVLMG18941

B dolosa CFLMG18942LMG18943MCI7

B ambifariaENV

LMG19467 CFLMG19182 ENVLMG16670 B anthina ENVFCF43 B pyrrocina CFLSED4 B lata CFLMG24064 B latens CFLMG24065 B diffusa CFLMG23361 B contaminans AILMG24067 B seminals CFLMG24068 B metallica CFLMG24066 B arboris ENVLMG24263 B ubonensis NIAbbreviations CF strains isolated from cystic fibrosis patients AI strainsisolated from animal infection NI strains isolated from nosocomial infec-tion ENV environmental strain

isolates showed similarities with the stress resistantB safensis

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

complex Regardless of the overall low support ofthe tree (typical of Pseudomonas phylogenies) someobservations may be drawn the presence of largeunresolved clusters shows clearly a low divergenceof the isolated colonies implying the existence ofclonal populations Pseudomonas spp isolated fromthe different compartments are intermixed suggest-ing the presence of a continuum rhizosphere-leaves(Pseudomonas is the only genus found in all the 4sample analysed)

32 Inhibition of Burkholderia Cepacia Complex StrainsGrowth by L angustifolia Endophytic Bacteria In order tocheck the ability of L angustifolia bacterial endophytes toantagonize the growth of (opportunistic) human pathogenicbacteria cross-streak experiments were carried out asdescribed in Section 2 using a panel of the 19 randomlychosen different endophytic strains isolated from soil rootsor leafs and phylogenetically assigned to six different gen-era (Bacillus Microbacterium Pantoea Plantibacter Pseu-domonas and Rhizobium) as testers versus 40 Bcc strainsrepresentative of seventeen species (out of eighteen) andwith either environmental or clinical origin with some ofwhich being multidrug-resistant Data obtained are shownin Table 4 The analysis of these data revealed that all thetester strains were able to completely inhibit the growthof Bcc strains including those with a clinical source andthat exhibited multidrug resistance this finding suggestedthat lavender endophytic bacteria are able to synthesize(strong) antimicrobial compounds However tester strainsshowed a different pattern of Bcc growth inhibition eventhose affiliated to the same genus In addition to this thereis no apparent difference in the antimicrobial potentialbetween strains belonging to different plant compartmentsConcerning the sensitivity of Bcc strains to the antimicrobialactivity of lavender endophytes strains belonging to differentspecies exhibited a different sensitivity spectrum However itis quite interesting that strains with clinical origin appearedto be more sensitive to the antimicrobials synthesized byendophytic bacteria than their environmental counterparts(see for instance strains belonging to the species Burkholderiacenocepacia IIIB) (Supplementary Table 3)

4 Discussion

Medicinal plants are stirring the attention of manyresearchers due to the presence of compounds thatconstitute a large fraction of the current pharmacopoeiasNatural products have been the source of most of theactive ingredients of medicines and more than 80 of drugsubstances are natural products or inspired by a naturalcompound including most of the of anticancer and anti-infective agents [50] Lavender plants are grown mainlyfor the production of essential oil which has antisepticand anti-inflammatory properties In spite of the relativeharsh environment for bacterial colonization L angustifoliatissues were found to be rich in bacterial diversity Howeverthe endophytic community isolated from different plant

12 Evidence-Based Complementary and Alternative Medicine

Table4Growth

of40

Burkholderiacepacia

complex

strains

inthep

resenceabsenceo

fend

ophytic

bacterialstrains

isolated

from

lavend

er

Species

Strain

Orig

inLL

1LL

2LL

3LL

4LL

6LL

7LL

9LL

10LS

1LS

2LS

3LS

4LS

5LS

6LR

1LR

2LR

3LR

4LR

5C-

Bcepacia

FCF1

CFminusminusminusminusminusminusminusminus

minusminusminusminusminusminusminusminusminusminusminus

+B

cepacia

FCF3

CF+minusminusminusminusminusminusminusminus

minusminusminusminusminusminusminusminusminusminusminus

+B

multivoran

sLM

G17588

Env

+minusminus

++minus

+minusminus

+minus

+minus

++minus

+minusminus

+minus

+minusminus

+B

cenocepacia

(IIIA

)FC

F16

CFminusminusminusminusminusminusminusminus

minusminusminusminusminusminusminusminusminusminusminus

+B

cenocepacia

(IIIA

)J2315

CF+minusminus

+minus

+minusminusminus

+minusminusminus

+minusminusminusminusminusminusminusminus

+B

cenocepacia

(IIIB)

FCF18

CF+minusminusminusminusminusminusminus

minusminusminusminusminusminus

+minusminusminusminusminus

+B

cenocepacia

(IIIB)

FCF20

CF+

++

++

+minus

++minus

+minus

+minus

+minus

++minus

++minus

++

++

+B

cenocepacia

(IIIB)

FCF23

CF+minusminusminusminusminusminusminus

+minusminusminus

++minusminusminus

+minusminusminusminus

+B

cenocepacia

(IIIB)

FCF24

CF+minusminusminusminusminusminusminus

+minusminus

+minusminusminusminus

+minusminusminusminus

+B

cenocepacia

(IIIB)

FCF27

CF+minusminusminusminusminusminusminus

+minusminusminusminusminusminusminusminusminusminusminus

+B

cenocepacia

(IIIB)

FCF29

CF+minusminus

+minus

+minusminusminus

+minusminusminusminusminusminusminus

+minus

+minusminus

+B

cenocepacia

(IIIB)

FCF30

CF+minusminus

+minus

+minusminusminus

+minus

+minusminusminusminusminusminus

+minus

+minusminus

+B

cenocepacia

(IIIB)

LMG16654

CF+

+minusminus

++minus

+minus

++minus

++minusminus

+minus

+minusminus

+B

cenocepacia

(IIIB)

C5424

CF+

+minusminus

+minusminusminusminus

+minusminusminus

+minusminusminusminus

+minusminus

+minusminus

+B

cenocepacia

(IIIB)

CEP5

11CF

+minusminus

+minusminusminusminus

+minus

+minusminus

+minus

+minusminusminus

+minus

+minusminus

+B

cenocepacia

(IIIB)

MVPC

116

Env

++minusminus

+minus

++minusminus

+minus

+minus

+minus

+minusminus

+minusminus

+minusminus

+minus

+minus

+B

cenocepacia

(IIIB)

MVPC

173

Env

++

++minus

++

+minus

++

++

++

++

++

++minus

+B

cenocepacia

(IIIIC)

LMG19230

Env

++

++

++

+minus

++

++

++

++

++

++

+B

cenocepacia

(IIIC

)LM

G19240

Env

++

++

+minus

+minus

++

+minus

++

++minus

++

++

+minus

+B

cenocepacia

(IIID)

FCF38

CF+minusminusminusminusminusminusminus

minusminusminusminusminusminusminusminusminusminusminus

+B

cenocepacia

(IIID)

LMG21462

CFminusminusminusminusminusminusminusminus

minusminusminusminusminusminusminusminusminusminusminus

+B

stabilis

FCF41

CF+

+minus

++

+minus

+minusminus

++

+minus

++minusminus

++

++minusminus

+B

vietnamien

sisFC

F42

CFminusminusminusminusminusminusminusminus

minusminusminusminusminusminusminusminusminusminusminus

+B

vietnamien

sisTV

V75

Env

++minusminus

+minusminusminusminus

+minusminusminus

++minusminusminus

+minusminus

+minusminus

+B

dolosa

LMG18941

CF+minusminus

+minus

+minus

+minusminus

+minusminusminus

++minusminusminus

+minus

+minus

+minusminus

+B

dolosa

LMG18942

CF+

+minusminus

+minus

+minusminus

++

+minus

++minusminusminus

++minus

++minusminus

+B

dolosa

LMG18943

CF+

+minusminus

++minus

+minusminus

+minus

+minusminus

++minusminusminus

++minus

+minusminus

+B

ambifaria

MCI

7En

v+

+minus

+minus

+minus

+minusminus

++minusminus

++minusminusminus

+minus

+minusminus

+B

ambifaria

LMG1946

7CF

++minus

+minus

+minusminusminus

++minusminus

++minusminusminus

+minus

+minusminusminus

+B

ambifaria

LMG19182

Env

++minus

+minus

+minusminusminus

+minusminus

+minusminusminus

+minus

+minusminusminus

+B

anthina

LMG16670

Env

+minusminusminusminus

+minus

+minusminus

++minusminusminus

+minus

+minusminusminus

+B

pyrrocinia

FCF43

CF+minusminusminusminusminusminusminusminus

minusminus

+minusminusminusminusminusminus

+minusminusminus

+B

lata

LSED

4CF

+minus

+minusminus

+minusminus

+minusminus

++minusminusminus

+minus

+minus

+minusminusminus

+B

latens

LMG2406

4CF

+minus

++minusminus

+minusminus

++minus

++minus

++

++

+minus

+B

diffu

saLM

G24065

CF+

+minus

++minus

+minus

+minus

++minus

++minus

+minus

++

++minus

+B

contam

inan

sLM

G23361

AI

++minus

+minus

+minus

+minus

+minus

++

++

++

+minus

++

++minus

+B

seminalis

LMG24067

CF+

+minus

++minus

++

++

++

++

++

++

++minus

+B

metallica

LMG2406

8CF

++

++minus

++

++

++

++

++

++

++minus

+B

arboris

LMG2406

6En

v+

+minus

++minus

++

+minus

++

++

++minus

+minus

++

++minusminus

+B

ubonensis

LMG24263

NI

+minusminusminusminus

+minusminus

+minus

+minus

++minusminus

+minusminus

+minusminusminus

+Ab

breviatio

nsC

Fstr

ains

isolatedfro

mcysticfi

brosispatie

ntsAIstr

ains

isolated

from

anim

alinfectionNIstr

ains

isolated

from

nosocomialinfectio

nEN

Venvironm

entalstrainLstr

ains

isolatedfro

mtheleaf

Sstr

ains

isolatedfro

mthes

temR

strains

isolatedfro

mther

oot

Symbo

ls+

grow

th+

minusreduced

grow

th+

minusminusveryredu

cedgrow

thminus

nogrow

thC

-Petrid

ishes

containing

onlythetargetstrains

Evidence-Based Complementary and Alternative Medicine 13

compartments showed different levels of diversity withthe 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 theleaf and roots community is already reported in otherspecies [26] even if there are few direct comparisons ofroots and phyllosphere bacterial communities especiallycomparisons using material from the same plants Themicrobial community residing in the phyllosphere is facedwith a nutrient poor and variable environment that ischaracterized by fluctuating temperature humidity and UVradiation and so it is predicted to be more diverse than thatwithin the rhizosphere a more homeostatic environmentThese findings supported also by the similar bacterial titreare in agreement with the idea that the source of inoculumfor the leaf community (except for the vertically transferredvia seeds) may be exclusive (eg aerosol even if the processis not yet clarified Bulgarelli et al 2013) and not related totransmission from the roots Moreover the low diversity ofthe community isolated in the stem dominated by clonally(Table 2) populations of Pseudomonas could be related tothe highly specific main tissue (phloem) present there whichcould provide an homeostatic environment rich in nutrient(sucrose contained in the phloem tissues) but poorer (ifcompared to leaf and roots) in secondary metabolites thatmay promote ecological niche differentiation

Concerning the taxonomic community composition dif-ferent taxonomic profiles were found for endosphere andrhizosphere More in detail the rhizosphere communityshowed quite similar diversity indexes if compared to theroots one but with a different composition as an examplemembers of the phylumActinobacteria are completely absentfrom the root internal tissues that are largely dominatedby Proteobacteria while they are present in the soil Toexplain this finding a two-step model has been proposed[16] soil abiotic properties determine the structure of theinitial bacterial communities that is then modified by rhi-zodeposits with plant roots cell wall features and releasedmetabolites promoting the growth of organotrophic bacteriathereby initiating a soil community shift In the second stepconvergent host genotype-dependent selection [51 52] fine-tunes community profiles thriving on the rhizoplane andwithin plant roots

Moreover endosphere communities were differentbetween plant organs (roots stems and leaves) even if theinternal tissues are dominated as already reported [25 52ndash54] by members of the phylum Proteobacteria an examplewhile rhizobia were isolated from root internal tissues theyare completely absent from stems and leaves (Figures 2 and3) isolates belonging to the genus Pantoea on the contrarywere isolated from aerial parts but not from root tissuesHence it is possible that plant might ldquoselectrdquo specific taxafor entering inside its compartment Such hypothesis issupported also by the pairwise differentiation values thecommunities isolated from the different tissues even those inphysical continuity are in fact strongly differentiated with thestem representing a low diversity compartment separatingthe two high diversity organs roots and leaves In this view itis noteworthy that in lavender only Pseudomonas represents

a ubiquitous and abundant genus of both rhizosphere andendosphere (Figure 3) The analysis of intrageneric diversitypointed out also that Pseudomonas isolates belong to a lowdifferentiated clonal population (Table 2 and Suppl Figure2) suggesting also that this component of the communityeither diffused inside the organs from a radical or foliar entrypoint or established during plants development putativelyfrom the seed inoculum A similar consideration can beproposed for isolates of rhizobia but for the rhizosphere-root internal tissues continuum a low taxonomicallydifferentiated community from the soil entered (increasingits relative abundance) inside the plant organ An entrypoint from the leaves is on the contrary consistent with thedistribution of Pantoea isolates with their strong diversity(Table 2) suggesting a diversification in response to the highvariable leaf environmentThe isolates belonging to the genusStenotrophomonas show an interesting distribution pattern(Figure 3) they are rare in the rhizosphere abundant in theroots and in the leaves and absent in the stem this findingsuggests a different entry point or a negative selection in thestem the second explanation is supported by admixture ofroot and leaves isolates (Figure 4)

The detailed phylogenetic analyses of the isolates enabledus also to putatively ascribe some isolates to already describedendophytic strains that can be targeted for functional charac-terisation many Microbacterium leaves isolates cluster withM hydrocarbonoxydans and M oleivorans two species withcrude oil degrading activity [49] being this metabolic activitythat is found frequently associated with a phyllosphereadapted lifestyle [55] Several isolates cluster with bacteriawith interesting biotechnological or ecological applicationssuch as S chelatiphaga a remarkable species with biotech-nological potential in phytoremediation [56] and S rizophilaa plant associated bacterium with antifungal activity [57] orBacillus mojavensis a bacteriumclosely related to B subtilisand already reported having an endophytic lifestyle andshowing an antagonistic role against plant pathogenic fungi[58] On the contrary many lavender Pseudomonas isolatescluster with known plant pathogen strains highlighting thesometimes subtle difference among pathogenic and endo-phytic lifestyle and with Pantoea agglomerans (and also withlavender isolates clustering withinthe B licheniformis and Bsoronensis groups) a known plant associated bacterium andan opportunistic pathogen in immune-compromised humanindividuals drawing attention to the possible pathogenicaction of endophytic bacteria in humans [59 60]

The overall analysis of the dendrograms points out thatthe characterization of isolates from endophytic communitiescan lead to the identification (as an example for Pantoeaand rhizobia) of not yet described strains that may representuntapped resources of bioactive compounds of agricultural ormedical interest

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

14 Evidence-Based Complementary and Alternative Medicine

context the characterization of cultivable bacterial commu-nities is a fundamental step to this purpose since it paves theway to the possibility to build collections of isolates that maybe phenotypically typed for important traits In our opiniondata obtained in this work (Table 4 and Supplementary Tables2 and 3) offers a preliminary but very promising exampleof the biotechnological potential of bacteria isolated frommedicinal plants In this pilot study in fact the majority ofthe tested endophytes showed to inhibit the growth of several(in some case all) human pathogenic strains belonging tothe Bcc complexes that are known for their resistance totraditional antibiotics These data are particularly interestingif correlated with the recent finding that essential oils fromdifferent medicinal plants are able to strongly (or completely)interfere with the growth of the same Bcc strains [61] and thatbacterial endophytes isolated from plants of the same speciesexhibited the same bioactivity (Maida et al in preparation)It is therefore possible that the therapeutic properties of theessential oil might reside also on bioactive compounds ofmicrobial origin We are completely aware that the determi-nation of the correlation (eventually) existing between thecomposition of bacterial endophytic communities and thebioactivity of essential oils extracted from a medicinal plantwould require the parallel analysis of the bacterial communityand the essential oil activity extracted from the same plantHowever data obtained in this work and those from Maidaet al [62] support this idea

It is also particularly intriguing the idea that the antimi-crobial activity of essential oils may reside on the actionof multiple compounds some of them produced or elicitedby the microbiota limiting the observed rapid evolutionof human pathogenic bacteria resistant to single moleculeantimicrobials

The characterization of the heterotrophic aerobic endo-phytic bacterial community of L angustifolia highlightedthe existence of a diversified community between differentorganstissues that may be related to the coexistence ofdifferent sources of inoculum andor a selection of the com-munities promoted by nutrient sand metabolites availabilityand antagonistic forces Several not previously characterizedstrains have been isolated and molecularly typed confirmingthat the analysis of the bacterial communities inhabitingextreme or unconventional environments may represent aproper strategy for the discovery of untapped sources offunctional biodiversity and bioactive molecule production

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This work was supported by grants from the Ente Cassadi Risparmio di Firenze (Grant 20130657 Herbiome newantimicrobial compounds from endophytic bacteria isolatedfrom medicinal plants) Giovanni Emiliani is financially

supported by the Regione Toscana POR CRO FSE 2007-2013 Asse IV Grant Sysbiofor (CNR-9) Marco Fondi andElena Perrin are financially supported by the FEMSAdvancedFellowship (FAF2012) and the Buzzati-Traverso Foundationrespectively

References

[1] B Reinhold-Hurek andTHurek ldquoLiving inside plants bacterialendophytesrdquo Current Opinion in Plant Biology vol 14 no 4 pp435ndash443 2011

[2] M Rosenblueth and E Martınez-Romero ldquoBacterial endo-phytes and their interactions with hostsrdquo Molecular Plant-Microbe Interactions vol 19 no 8 pp 827ndash837 2006

[3] R P Ryan K Germaine A Franks D J Ryan and DN Dowling ldquoBacterial endophytes recent developments andapplicationsrdquo FEMSMicrobiology Letters vol 278 no 1 pp 1ndash92008

[4] R Cakmakci F Donmez A Aydın and F Sahin ldquoGrowthpromotion of plants by plant growth-promoting rhizobacteriaunder greenhouse and two different field soil conditionsrdquo SoilBiology and Biochemistry vol 38 pp 1482ndash1487 2006

[5] P R Hardoim L S van Overbeek and J D V Elsas ldquoProp-erties of bacterial endophytes and their proposed role in plantgrowthrdquo Trends in Microbiology vol 16 no 10 pp 463ndash4712008

[6] S Compant C Clement and A Sessitsch ldquoPlant growth-promoting bacteria in the rhizo- and endosphere of plantstheir role colonization mechanisms involved and prospects forutilizationrdquo Soil Biology and Biochemistry vol 42 no 5 pp669ndash678 2010

[7] B R Glick ldquoBacterial ACC deaminase and the alleviation ofplant stressrdquo Advances in Applied Microbiology vol 56 pp 291ndash312 2004

[8] B R Glick B Todorovic J Czarny Z Cheng J Duan andB McConkey ldquoPromotion of plant growth by bacterial ACCdeaminaserdquo Critical Reviews in Plant Sciences vol 26 no 5-6pp 227ndash242 2007

[9] S L Doty B Oakley G Xin et al ldquoDiazotrophic endophytesof native black cottonwood and willowrdquo Symbiosis vol 47 no 1pp 23ndash33 2009

[10] B Lugtenberg and F Kamilova ldquoPlant-growth-promoting rhi-zobacteriardquoAnnual Review ofMicrobiology vol 63 pp 541ndash5562009

[11] U F Castillo G A Strobel E J Ford et al ldquoMunumbicinswide-spectrum antibiotics produced by Streptomyces NRRL30562 endophytic on Kennedia nigriscansrdquo Microbiology vol148 no 9 pp 2675ndash2685 2002

[12] Y Igarashi M E Trujillo E Martınez-Molina et al ldquoAnti-tumor anthraquinones from an endophytic actinomyceteMicromonospora lupini sp novrdquo Bioorganic amp Medicinal Chem-istry Letters vol 17 no 13 pp 3702ndash3705 2007

[13] S Shweta J H Bindu J Raghu et al ldquoIsolation of endophyticbacteria producing the anti-cancer alkaloid camptothecinefromMiquelia dentata Bedd (Icacinaceae)rdquo Phytomedicine vol20 pp 913ndash917 2013

[14] T Taechowisan A Wanbanjob P Tuntiwachwuttikul and JLiu ldquoAnti-inflammatory activity of lansais from endophyticStreptomyces sp SUC1 in LPS-induced RAW 2647 cellsrdquo Foodand Agricultural Immunology vol 20 no 1 pp 67ndash77 2009

Evidence-Based Complementary and Alternative Medicine 15

[15] P R Hardoim C C P Hardoim L S van Overbeek and J Dvan Elsas ldquoDynamics of seed-borne rice endophytes on earlyplant growth stagesrdquo PLoS ONE vol 7 no 2 Article ID e304382012

[16] D Bulgarelli K Schlaeppi S Spaepen E V L van Themaatand P Schulze-Lefert ldquoStructure and functions of the bacterialmicrobiota of plantsrdquo Annual Review of Plant Biology vol 64pp 807ndash838 2013

[17] A Mengoni S Mocali G Surico S Tegli and R Fani ldquoFluctu-ation of endophytic bacteria and phytoplasmosis in elm treesrdquoMicrobiological Research vol 158 no 4 pp 363ndash369 2003

[18] A Mengoni F Pini L-N Huang W-S Shu and MBazzicalupo ldquoPlant-by-plant variations of bacterial commu-nities associated with leaves of the nickel hyperaccumulatorAlyssum bertolonii desvrdquo Microbial Ecology vol 58 no 3 pp660ndash667 2009

[19] S Mocali E Bertelli F di Cello et al ldquoFluctuation of bacteriaisolated from elm tissues during different seasons and fromdifferent plant organsrdquo Research in Microbiology vol 154 no2 pp 105ndash114 2003

[20] F Pini A Frascella L Santopolo et al ldquoExploring the plant-associated bacterial communities in Medicago sativa Lrdquo BMCMicrobiology vol 12 article 78 2012

[21] A Mengoni L Cecchi and C Gonnelli ldquoNickel hyperac-cumulating plants and Alyssum bertolonii model systems forstudying biogeochemical interactions in serpentine soilsrdquo inBio-Geo Interactions in Metal-Contaminated Soils E Kothe andA Varma Eds pp 279ndash296 Springer Berlin Germany 2012

[22] S Ikeda T Okubo M Anda et al ldquoCommunity- and genome-based views of plant-associated bacteria plant-bacterial inter-actions in soybean and ricerdquo Plant and Cell Physiology vol 51no 9 pp 1398ndash1410 2010

[23] F Pini M Galardini M Bazzicalupo and A Mengoni ldquoPlant-bacteria association and symbiosis are there common genomictraits in alphaproteobacteriardquoGenes vol 2 no 4 pp 1017ndash10322011

[24] K Aleklett and M Hart ldquoThe root microbiota a fingerprint inthe soilrdquo Plant Soil vol 370 pp 671ndash686 2013

[25] A Beneduzi F Moreira P B Costa et al ldquoDiversity and plantgrowth promoting evaluation abilities of bacteria isolated fromsugarcane cultivated in the South of BrazilrdquoApplied Soil Ecologyvol 63 pp 94ndash104 2013

[26] N Bodenhausen M W Horton and J Bergelson ldquoBacterialcommunities associated with the leaves and the roots of Ara-bidopsis thalianardquo PLoS ONE vol 8 no 2 Article ID e563292013

[27] T F da Silva R E Vollu D Jurelevicius et al ldquoDoes theessential oil of Lippia sidoides Cham (pepper-rosmarin) affectits endophytic microbial communityrdquo BMC Microbiology vol13 no 1 article 29 2013

[28] M E Lucero A Unc P Cooke S Dowd and S SunldquoEndophyte microbiome diversity in micropropagated Atriplexcanescens and Atriplex torreyi var griffithsiirdquo PLoS ONE vol 6no 3 Article ID e17693 2011

[29] D S Lundberg S L Lebeis S H Paredes et al ldquoDefining thecoreArabidopsis thaliana rootmicrobiomerdquoNature vol 487 no7409 pp 86ndash90 2012

[30] H M Cavanagh and J M Wilkinson ldquoBiological activities oflavender essential oilrdquo Phytotherapy Research vol 16 no 4 pp301ndash308 2002

[31] G Woronuk Z Demissie M Rheault and S MahmoudldquoBiosynthesis and therapeutic properties of Lavandula essentialoil constituentsrdquo Planta Medica vol 77 no 1 pp 7ndash15 2011

[32] R Perry R Terry L K Watson and E Ernst ldquoIs lavender ananxiolytic drug A systematic review of randomised clinicaltrialsrdquo Phytomedicine vol 19 pp 825ndash835 2012

[33] S de Rapper G Kamatou A Viljoen and S van VuurenldquoThe in vitro antimicrobial activity of Lavandula angustifoliaessential oil in combination with other aroma-therapeutic oilsrdquoEvidence-Based Complementary and Alternative Medicine vol2013 Article ID 852049 10 pages 2013

[34] T Moon J M Wilkinson and H M Cavanagh ldquoAntiparasiticactivity of two Lavandula essential oils against Giardia duode-nalis Trichomonas vaginalis andHexamita inflatardquo ParasitologyResearch vol 99 no 6 pp 722ndash728 2006

[35] P S Yap S H Lim C P Hu and B C Yiap ldquoCom-bination of essential oils and antibiotics reduce antibioticresistance in plasmid-conferred multidrug resistant bacteriardquoPhytomedicine vol 20 pp 710ndash713 2013

[36] G Brader S Compant B Mitter F Trognitz and A SessitschldquoMetabolic potential of endophytic bacteriardquo Current Opinionin Biotechnology vol 27 pp 30ndash37 2014

[37] P Drevinek and E Mahenthiralingam ldquoBurkholderia ceno-cepacia in cystic fibrosis epidemiology and molecular mecha-nisms of virulencerdquo Clinical Microbiology and Infection vol 16no 7 pp 821ndash830 2010

[38] S Bazzini C Udine and G Riccardi ldquoMolecular approaches topathogenesis study of Burkholderia cenocepacia an importantcystic fibrosis opportunistic bacteriumrdquo Applied Microbiologyand Biotechnology vol 92 no 5 pp 887ndash895 2011

[39] I Maida A lo Nostro G Pesavento et al ldquoExploring theanti-Burkholderia cepacia complex activity of essential oilsa preliminary analysisrdquo Evidence-Based Complementary andAlternativeMedicine vol 2014 Article ID 573518 10 pages 2014

[40] A Mengoni R Barzanti C Gonnelli R Gabbrielli andM Bazzicalupo ldquoCharacterization of nickel-resistant bacteriaisolated from serpentine soilrdquo Environmental Microbiology vol3 no 11 pp 691ndash698 2001

[41] S F Altschul T L Madden A A Schaffer et al ldquoGappedBLAST and PSI-BLAST a new generation of protein databasesearch programsrdquo Nucleic Acids Research vol 25 no 17 pp3389ndash3402 1997

[42] R C Edgar ldquoMUSCLE multiple sequence alignment with highaccuracy and high throughputrdquo Nucleic Acids Research vol 32no 5 pp 1792ndash1797 2004

[43] F Ronquist M Teslenko P van der Mark et al ldquoMrbayes32 efficient bayesian phylogenetic inference and model choiceacross a large model spacerdquo Systematic Biology vol 61 no 3 pp539ndash542 2012

[44] K Tamura D Peterson N Peterson G Stecher M Nei andS Kumar ldquoMEGA5 molecular evolutionary genetics analysisusing maximum likelihood evolutionary distance and max-imum parsimony methodsrdquo Molecular Biology and Evolutionvol 28 no 10 pp 2731ndash2739 2011

[45] F Sievers A Wilm D Dineen et al ldquoFast scalable generationof high-quality protein multiple sequence alignments usingClustal Omegardquo Molecular Systems Biology vol 7 article 5392011

[46] Oslash Hammer D A T Harper and P D Ryan ldquoPAST pale-ontological statistics software package for education and dataanalysisrdquo Palaeontologia Electronica vol 4 no 1 pp 1ndash9 2001

16 Evidence-Based Complementary and Alternative Medicine

[47] M C Papaleo M Fondi I Maida et al ldquoSponge-associatedmicrobial Antarctic communities exhibiting antimicrobialactivity againstBurkholderia cepacia complex bacteriardquoBiotech-nology Advances vol 30 no 1 pp 272ndash293 2012

[48] A lo Giudice V Bruni and L Michaud ldquoCharacterization ofAntarctic psychrotrophic bacteria with antibacterial activitiesagainst terrestrial microorganismsrdquo Journal of Basic Microbiol-ogy vol 47 no 6 pp 496ndash505 2007

[49] A Schippers K Bosecker C Sproer and P SchumannldquoMicrobacterium oleivorans sp nov and Microbacteriumhydrocarbonoxydans sp nov novel crude-oil-degrading Gram-positive bacteriardquo International Journal of Systematic and Evo-lutionary Microbiology vol 55 no 2 pp 655ndash660 2005

[50] J D McChesney S K Venkataraman and J T Henri ldquoPlantnatural products back to the future or into extinctionrdquo Phyto-chemistry vol 68 no 14 pp 2015ndash2022 2007

[51] D K Manter J A Delgado D G Holm and R A StongldquoPyrosequencing reveals a highly diverse and cultivar-specificbacterial endophyte community in potato rootsrdquo MicrobialEcology vol 60 no 1 pp 157ndash166 2010

[52] K Ulrich A Ulrich and D Ewald ldquoDiversity of endophyticbacterial communities in poplar grown under field conditionsrdquoFEMS Microbiology Ecology vol 63 no 2 pp 169ndash180 2008

[53] N R Gottel H F Castro M Kerley et al ldquoDistinct microbialcommunities within the endosphere and rhizosphere ofPopulusdeltoides roots across contrasting soil typesrdquo Applied and Envi-ronmental Microbiology vol 77 no 17 pp 5934ndash5944 2011

[54] B Ma X Lv A Warren and J Gong ldquoShifts in diversity andcommunity structure of endophytic bacteria and archaea acrossroot stem and leaf tissues in the common reed Phragmitesaustralis along a salinity gradient in a marine tidal wetland ofnorthern Chinardquo Antonie van Leeuwenhoek vol 104 no 5 pp759ndash768 2013

[55] J A Vorholt ldquoMicrobial life in the phyllosphererdquo NatureReviews Microbiology vol 10 no 12 pp 828ndash840 2012

[56] R P Ryan S Monchy M Cardinale et al ldquoThe versatilityand adaptation of bacteria from the genus StenotrophomonasrdquoNature Reviews Microbiology vol 7 no 7 pp 514ndash525 2009

[57] AWolf A FritzeMHagemann andG Berg ldquoStenotrophomo-nas rhizophila sp nov a novel plant-associated bacterium withantifungal propertiesrdquo International Journal of Systematic andEvolutionary Microbiology vol 52 no 6 pp 1937ndash1944 2002

[58] C W Bacon and D M Hinton ldquoEndophytic and biologicalcontrol potential of Bacillus mojavensis and related speciesrdquoBiological Control vol 23 no 3 pp 274ndash284 2002

[59] G Berg L Eberl and A Hartmann ldquoThe rhizosphere as areservoir for opportunistic human pathogenic bacteriardquo Envi-ronmental Microbiology vol 7 no 11 pp 1673ndash1685 2005

[60] H L Tyler and E W Triplett ldquoPlants as a habitat for ben-eficial andor human pathogenic bacteriardquo Annual Review ofPhytopathology vol 46 pp 53ndash73 2008

[61] R Perry R Terry L K Watson and E Ernst ldquoIs lavender ananxiolytic drug A systematic review of randomised clinicaltrialsrdquo Phytomedicine vol 19 no 8-9 pp 825ndash835 2012

[62] I Maida A lo Nostro G Pesavento et al ldquoExploring theanti-Burkholderia cepacia complex activity of essential oilsa preliminary analysisrdquo Evidence-Based Complementary andAlternativeMedicine vol 2014 Article ID 573518 10 pages 2014

8 Evidence-Based Complementary and Alternative Medicine

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 trS002222203 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 NRCPB10S002958314 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 MGminus2011minus4-AO

S000429561 A sp PB

S000806232 R soli DS-42

03

LR51

LR78

LR50

LT74

LR68

LR9

LR22

LR67

LR69

LR23LR24

LR5

LR21

LT92

LR19

LR49

LR62

LR57

LR54

LT91

LR55

LR12

LR20

LR47

LR18

LR11

LR65

LR25LR45

LR56

LT12

LR64

LR16

LR6

LR70

LR7

LR17

LT73

LR63

LR52

LR77

LR46

LT87

LR53

LR15

LR66094890908507606

09525

07316

07803

05579

1

0685

06484

05141

08108

1

Figure 5 Bayesian dendrogram showing the relationships among the 16S rRNA gene sequences of 46 isolates belonging to the genusRhizobium and those of reference type strains Posterior probability values are indicated at each node Nodes are collapsed at 70 probabilityLT = bacteria isolated from the rhizosphere LR = bacteria isolated from the roots (see Section 2 for details)

Evidence-Based Complementary and Alternative Medicine 9

005

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

095

09074

055

099

05

1

081

088

078

1

084

1

071

1

083

1

091

1

099

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 Pantoeaand those of reference type strains Posterior probability values are indicated at the node Nodes are collapsed at 70 probability LS = bacteriaisolated from the stem LL = bacteria isolated from the leaves (see Section 2 for details)

10 Evidence-Based Complementary and Alternative Medicine

05

LL6

LL67

LL81

LL5

LL8

LL29

LS87

LL100

LL82

LL85

LL83

LL7

LT3

LT5

LT67LT56

LL32

LL84

LL57

LL59

LT7

LL30

LL2

LT85

LL31LL1

LT4

LT6

LT8

LT2

08882

0679

0997

09977

05035

0644

0996409961

09758

07238

09846

05929

0504

0704107642

06421

1

06098

05001

05655

06027

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 paraoxydansS000381732 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 genusMicrobacterium and those of reference type strains Posterior probability values are indicated at the node Nodes are collapsed at 70probability LT = bacteria isolated from the rhizosphere LS = bacteria isolated from the stem LL = bacteria isolated from the leaves (seeSection 2 for details)

Evidence-Based Complementary and Alternative Medicine 11

Table 3The 40Burkholderia cepacia complex strains used as targetsin the cross streak experiments

Target strainStrain Species OriginFCF1 B cepacia CFFCF3LMG17588 B multivorans ENVFCF16 B cenocepacia (IIIA) CFJ2315FCF18

B cenocepacia (IIIB) CF

FCF20FCF23FCF24FCF27FCF29FCF30LMG16654C5424CEP511MVPC116 ENVMVPC173LMG19230 B cenocepacia (IIIC) ENVLMG19240FCF38 B cenocepacia (IIID) CFLMG21462FCF41 B stabilis CFFCF42 B vietnamiensis CFTVV75 ENVLMG18941

B dolosa CFLMG18942LMG18943MCI7

B ambifariaENV

LMG19467 CFLMG19182 ENVLMG16670 B anthina ENVFCF43 B pyrrocina CFLSED4 B lata CFLMG24064 B latens CFLMG24065 B diffusa CFLMG23361 B contaminans AILMG24067 B seminals CFLMG24068 B metallica CFLMG24066 B arboris ENVLMG24263 B ubonensis NIAbbreviations CF strains isolated from cystic fibrosis patients AI strainsisolated from animal infection NI strains isolated from nosocomial infec-tion ENV environmental strain

isolates showed similarities with the stress resistantB safensis

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

complex Regardless of the overall low support ofthe tree (typical of Pseudomonas phylogenies) someobservations may be drawn the presence of largeunresolved clusters shows clearly a low divergenceof the isolated colonies implying the existence ofclonal populations Pseudomonas spp isolated fromthe different compartments are intermixed suggest-ing the presence of a continuum rhizosphere-leaves(Pseudomonas is the only genus found in all the 4sample analysed)

32 Inhibition of Burkholderia Cepacia Complex StrainsGrowth by L angustifolia Endophytic Bacteria In order tocheck the ability of L angustifolia bacterial endophytes toantagonize the growth of (opportunistic) human pathogenicbacteria cross-streak experiments were carried out asdescribed in Section 2 using a panel of the 19 randomlychosen different endophytic strains isolated from soil rootsor leafs and phylogenetically assigned to six different gen-era (Bacillus Microbacterium Pantoea Plantibacter Pseu-domonas and Rhizobium) as testers versus 40 Bcc strainsrepresentative of seventeen species (out of eighteen) andwith either environmental or clinical origin with some ofwhich being multidrug-resistant Data obtained are shownin Table 4 The analysis of these data revealed that all thetester strains were able to completely inhibit the growthof Bcc strains including those with a clinical source andthat exhibited multidrug resistance this finding suggestedthat lavender endophytic bacteria are able to synthesize(strong) antimicrobial compounds However tester strainsshowed a different pattern of Bcc growth inhibition eventhose affiliated to the same genus In addition to this thereis no apparent difference in the antimicrobial potentialbetween strains belonging to different plant compartmentsConcerning the sensitivity of Bcc strains to the antimicrobialactivity of lavender endophytes strains belonging to differentspecies exhibited a different sensitivity spectrum However itis quite interesting that strains with clinical origin appearedto be more sensitive to the antimicrobials synthesized byendophytic bacteria than their environmental counterparts(see for instance strains belonging to the species Burkholderiacenocepacia IIIB) (Supplementary Table 3)

4 Discussion

Medicinal plants are stirring the attention of manyresearchers due to the presence of compounds thatconstitute a large fraction of the current pharmacopoeiasNatural products have been the source of most of theactive ingredients of medicines and more than 80 of drugsubstances are natural products or inspired by a naturalcompound including most of the of anticancer and anti-infective agents [50] Lavender plants are grown mainlyfor the production of essential oil which has antisepticand anti-inflammatory properties In spite of the relativeharsh environment for bacterial colonization L angustifoliatissues were found to be rich in bacterial diversity Howeverthe endophytic community isolated from different plant

12 Evidence-Based Complementary and Alternative Medicine

Table4Growth

of40

Burkholderiacepacia

complex

strains

inthep

resenceabsenceo

fend

ophytic

bacterialstrains

isolated

from

lavend

er

Species

Strain

Orig

inLL

1LL

2LL

3LL

4LL

6LL

7LL

9LL

10LS

1LS

2LS

3LS

4LS

5LS

6LR

1LR

2LR

3LR

4LR

5C-

Bcepacia

FCF1

CFminusminusminusminusminusminusminusminus

minusminusminusminusminusminusminusminusminusminusminus

+B

cepacia

FCF3

CF+minusminusminusminusminusminusminusminus

minusminusminusminusminusminusminusminusminusminusminus

+B

multivoran

sLM

G17588

Env

+minusminus

++minus

+minusminus

+minus

+minus

++minus

+minusminus

+minus

+minusminus

+B

cenocepacia

(IIIA

)FC

F16

CFminusminusminusminusminusminusminusminus

minusminusminusminusminusminusminusminusminusminusminus

+B

cenocepacia

(IIIA

)J2315

CF+minusminus

+minus

+minusminusminus

+minusminusminus

+minusminusminusminusminusminusminusminus

+B

cenocepacia

(IIIB)

FCF18

CF+minusminusminusminusminusminusminus

minusminusminusminusminusminus

+minusminusminusminusminus

+B

cenocepacia

(IIIB)

FCF20

CF+

++

++

+minus

++minus

+minus

+minus

+minus

++minus

++minus

++

++

+B

cenocepacia

(IIIB)

FCF23

CF+minusminusminusminusminusminusminus

+minusminusminus

++minusminusminus

+minusminusminusminus

+B

cenocepacia

(IIIB)

FCF24

CF+minusminusminusminusminusminusminus

+minusminus

+minusminusminusminus

+minusminusminusminus

+B

cenocepacia

(IIIB)

FCF27

CF+minusminusminusminusminusminusminus

+minusminusminusminusminusminusminusminusminusminusminus

+B

cenocepacia

(IIIB)

FCF29

CF+minusminus

+minus

+minusminusminus

+minusminusminusminusminusminusminus

+minus

+minusminus

+B

cenocepacia

(IIIB)

FCF30

CF+minusminus

+minus

+minusminusminus

+minus

+minusminusminusminusminusminus

+minus

+minusminus

+B

cenocepacia

(IIIB)

LMG16654

CF+

+minusminus

++minus

+minus

++minus

++minusminus

+minus

+minusminus

+B

cenocepacia

(IIIB)

C5424

CF+

+minusminus

+minusminusminusminus

+minusminusminus

+minusminusminusminus

+minusminus

+minusminus

+B

cenocepacia

(IIIB)

CEP5

11CF

+minusminus

+minusminusminusminus

+minus

+minusminus

+minus

+minusminusminus

+minus

+minusminus

+B

cenocepacia

(IIIB)

MVPC

116

Env

++minusminus

+minus

++minusminus

+minus

+minus

+minus

+minusminus

+minusminus

+minusminus

+minus

+minus

+B

cenocepacia

(IIIB)

MVPC

173

Env

++

++minus

++

+minus

++

++

++

++

++

++minus

+B

cenocepacia

(IIIIC)

LMG19230

Env

++

++

++

+minus

++

++

++

++

++

++

+B

cenocepacia

(IIIC

)LM

G19240

Env

++

++

+minus

+minus

++

+minus

++

++minus

++

++

+minus

+B

cenocepacia

(IIID)

FCF38

CF+minusminusminusminusminusminusminus

minusminusminusminusminusminusminusminusminusminusminus

+B

cenocepacia

(IIID)

LMG21462

CFminusminusminusminusminusminusminusminus

minusminusminusminusminusminusminusminusminusminusminus

+B

stabilis

FCF41

CF+

+minus

++

+minus

+minusminus

++

+minus

++minusminus

++

++minusminus

+B

vietnamien

sisFC

F42

CFminusminusminusminusminusminusminusminus

minusminusminusminusminusminusminusminusminusminusminus

+B

vietnamien

sisTV

V75

Env

++minusminus

+minusminusminusminus

+minusminusminus

++minusminusminus

+minusminus

+minusminus

+B

dolosa

LMG18941

CF+minusminus

+minus

+minus

+minusminus

+minusminusminus

++minusminusminus

+minus

+minus

+minusminus

+B

dolosa

LMG18942

CF+

+minusminus

+minus

+minusminus

++

+minus

++minusminusminus

++minus

++minusminus

+B

dolosa

LMG18943

CF+

+minusminus

++minus

+minusminus

+minus

+minusminus

++minusminusminus

++minus

+minusminus

+B

ambifaria

MCI

7En

v+

+minus

+minus

+minus

+minusminus

++minusminus

++minusminusminus

+minus

+minusminus

+B

ambifaria

LMG1946

7CF

++minus

+minus

+minusminusminus

++minusminus

++minusminusminus

+minus

+minusminusminus

+B

ambifaria

LMG19182

Env

++minus

+minus

+minusminusminus

+minusminus

+minusminusminus

+minus

+minusminusminus

+B

anthina

LMG16670

Env

+minusminusminusminus

+minus

+minusminus

++minusminusminus

+minus

+minusminusminus

+B

pyrrocinia

FCF43

CF+minusminusminusminusminusminusminusminus

minusminus

+minusminusminusminusminusminus

+minusminusminus

+B

lata

LSED

4CF

+minus

+minusminus

+minusminus

+minusminus

++minusminusminus

+minus

+minus

+minusminusminus

+B

latens

LMG2406

4CF

+minus

++minusminus

+minusminus

++minus

++minus

++

++

+minus

+B

diffu

saLM

G24065

CF+

+minus

++minus

+minus

+minus

++minus

++minus

+minus

++

++minus

+B

contam

inan

sLM

G23361

AI

++minus

+minus

+minus

+minus

+minus

++

++

++

+minus

++

++minus

+B

seminalis

LMG24067

CF+

+minus

++minus

++

++

++

++

++

++

++minus

+B

metallica

LMG2406

8CF

++

++minus

++

++

++

++

++

++

++minus

+B

arboris

LMG2406

6En

v+

+minus

++minus

++

+minus

++

++

++minus

+minus

++

++minusminus

+B

ubonensis

LMG24263

NI

+minusminusminusminus

+minusminus

+minus

+minus

++minusminus

+minusminus

+minusminusminus

+Ab

breviatio

nsC

Fstr

ains

isolatedfro

mcysticfi

brosispatie

ntsAIstr

ains

isolated

from

anim

alinfectionNIstr

ains

isolated

from

nosocomialinfectio

nEN

Venvironm

entalstrainLstr

ains

isolatedfro

mtheleaf

Sstr

ains

isolatedfro

mthes

temR

strains

isolatedfro

mther

oot

Symbo

ls+

grow

th+

minusreduced

grow

th+

minusminusveryredu

cedgrow

thminus

nogrow

thC

-Petrid

ishes

containing

onlythetargetstrains

Evidence-Based Complementary and Alternative Medicine 13

compartments showed different levels of diversity withthe 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 theleaf and roots community is already reported in otherspecies [26] even if there are few direct comparisons ofroots and phyllosphere bacterial communities especiallycomparisons using material from the same plants Themicrobial community residing in the phyllosphere is facedwith a nutrient poor and variable environment that ischaracterized by fluctuating temperature humidity and UVradiation and so it is predicted to be more diverse than thatwithin the rhizosphere a more homeostatic environmentThese findings supported also by the similar bacterial titreare in agreement with the idea that the source of inoculumfor the leaf community (except for the vertically transferredvia seeds) may be exclusive (eg aerosol even if the processis not yet clarified Bulgarelli et al 2013) and not related totransmission from the roots Moreover the low diversity ofthe community isolated in the stem dominated by clonally(Table 2) populations of Pseudomonas could be related tothe highly specific main tissue (phloem) present there whichcould provide an homeostatic environment rich in nutrient(sucrose contained in the phloem tissues) but poorer (ifcompared to leaf and roots) in secondary metabolites thatmay promote ecological niche differentiation

Concerning the taxonomic community composition dif-ferent taxonomic profiles were found for endosphere andrhizosphere More in detail the rhizosphere communityshowed quite similar diversity indexes if compared to theroots one but with a different composition as an examplemembers of the phylumActinobacteria are completely absentfrom the root internal tissues that are largely dominatedby Proteobacteria while they are present in the soil Toexplain this finding a two-step model has been proposed[16] soil abiotic properties determine the structure of theinitial bacterial communities that is then modified by rhi-zodeposits with plant roots cell wall features and releasedmetabolites promoting the growth of organotrophic bacteriathereby initiating a soil community shift In the second stepconvergent host genotype-dependent selection [51 52] fine-tunes community profiles thriving on the rhizoplane andwithin plant roots

Moreover endosphere communities were differentbetween plant organs (roots stems and leaves) even if theinternal tissues are dominated as already reported [25 52ndash54] by members of the phylum Proteobacteria an examplewhile rhizobia were isolated from root internal tissues theyare completely absent from stems and leaves (Figures 2 and3) isolates belonging to the genus Pantoea on the contrarywere isolated from aerial parts but not from root tissuesHence it is possible that plant might ldquoselectrdquo specific taxafor entering inside its compartment Such hypothesis issupported also by the pairwise differentiation values thecommunities isolated from the different tissues even those inphysical continuity are in fact strongly differentiated with thestem representing a low diversity compartment separatingthe two high diversity organs roots and leaves In this view itis noteworthy that in lavender only Pseudomonas represents

a ubiquitous and abundant genus of both rhizosphere andendosphere (Figure 3) The analysis of intrageneric diversitypointed out also that Pseudomonas isolates belong to a lowdifferentiated clonal population (Table 2 and Suppl Figure2) suggesting also that this component of the communityeither diffused inside the organs from a radical or foliar entrypoint or established during plants development putativelyfrom the seed inoculum A similar consideration can beproposed for isolates of rhizobia but for the rhizosphere-root internal tissues continuum a low taxonomicallydifferentiated community from the soil entered (increasingits relative abundance) inside the plant organ An entrypoint from the leaves is on the contrary consistent with thedistribution of Pantoea isolates with their strong diversity(Table 2) suggesting a diversification in response to the highvariable leaf environmentThe isolates belonging to the genusStenotrophomonas show an interesting distribution pattern(Figure 3) they are rare in the rhizosphere abundant in theroots and in the leaves and absent in the stem this findingsuggests a different entry point or a negative selection in thestem the second explanation is supported by admixture ofroot and leaves isolates (Figure 4)

The detailed phylogenetic analyses of the isolates enabledus also to putatively ascribe some isolates to already describedendophytic strains that can be targeted for functional charac-terisation many Microbacterium leaves isolates cluster withM hydrocarbonoxydans and M oleivorans two species withcrude oil degrading activity [49] being this metabolic activitythat is found frequently associated with a phyllosphereadapted lifestyle [55] Several isolates cluster with bacteriawith interesting biotechnological or ecological applicationssuch as S chelatiphaga a remarkable species with biotech-nological potential in phytoremediation [56] and S rizophilaa plant associated bacterium with antifungal activity [57] orBacillus mojavensis a bacteriumclosely related to B subtilisand already reported having an endophytic lifestyle andshowing an antagonistic role against plant pathogenic fungi[58] On the contrary many lavender Pseudomonas isolatescluster with known plant pathogen strains highlighting thesometimes subtle difference among pathogenic and endo-phytic lifestyle and with Pantoea agglomerans (and also withlavender isolates clustering withinthe B licheniformis and Bsoronensis groups) a known plant associated bacterium andan opportunistic pathogen in immune-compromised humanindividuals drawing attention to the possible pathogenicaction of endophytic bacteria in humans [59 60]

The overall analysis of the dendrograms points out thatthe characterization of isolates from endophytic communitiescan lead to the identification (as an example for Pantoeaand rhizobia) of not yet described strains that may representuntapped resources of bioactive compounds of agricultural ormedical interest

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

14 Evidence-Based Complementary and Alternative Medicine

context the characterization of cultivable bacterial commu-nities is a fundamental step to this purpose since it paves theway to the possibility to build collections of isolates that maybe phenotypically typed for important traits In our opiniondata obtained in this work (Table 4 and Supplementary Tables2 and 3) offers a preliminary but very promising exampleof the biotechnological potential of bacteria isolated frommedicinal plants In this pilot study in fact the majority ofthe tested endophytes showed to inhibit the growth of several(in some case all) human pathogenic strains belonging tothe Bcc complexes that are known for their resistance totraditional antibiotics These data are particularly interestingif correlated with the recent finding that essential oils fromdifferent medicinal plants are able to strongly (or completely)interfere with the growth of the same Bcc strains [61] and thatbacterial endophytes isolated from plants of the same speciesexhibited the same bioactivity (Maida et al in preparation)It is therefore possible that the therapeutic properties of theessential oil might reside also on bioactive compounds ofmicrobial origin We are completely aware that the determi-nation of the correlation (eventually) existing between thecomposition of bacterial endophytic communities and thebioactivity of essential oils extracted from a medicinal plantwould require the parallel analysis of the bacterial communityand the essential oil activity extracted from the same plantHowever data obtained in this work and those from Maidaet al [62] support this idea

It is also particularly intriguing the idea that the antimi-crobial activity of essential oils may reside on the actionof multiple compounds some of them produced or elicitedby the microbiota limiting the observed rapid evolutionof human pathogenic bacteria resistant to single moleculeantimicrobials

The characterization of the heterotrophic aerobic endo-phytic bacterial community of L angustifolia highlightedthe existence of a diversified community between differentorganstissues that may be related to the coexistence ofdifferent sources of inoculum andor a selection of the com-munities promoted by nutrient sand metabolites availabilityand antagonistic forces Several not previously characterizedstrains have been isolated and molecularly typed confirmingthat the analysis of the bacterial communities inhabitingextreme or unconventional environments may represent aproper strategy for the discovery of untapped sources offunctional biodiversity and bioactive molecule production

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This work was supported by grants from the Ente Cassadi Risparmio di Firenze (Grant 20130657 Herbiome newantimicrobial compounds from endophytic bacteria isolatedfrom medicinal plants) Giovanni Emiliani is financially

supported by the Regione Toscana POR CRO FSE 2007-2013 Asse IV Grant Sysbiofor (CNR-9) Marco Fondi andElena Perrin are financially supported by the FEMSAdvancedFellowship (FAF2012) and the Buzzati-Traverso Foundationrespectively

References

[1] B Reinhold-Hurek andTHurek ldquoLiving inside plants bacterialendophytesrdquo Current Opinion in Plant Biology vol 14 no 4 pp435ndash443 2011

[2] M Rosenblueth and E Martınez-Romero ldquoBacterial endo-phytes and their interactions with hostsrdquo Molecular Plant-Microbe Interactions vol 19 no 8 pp 827ndash837 2006

[3] R P Ryan K Germaine A Franks D J Ryan and DN Dowling ldquoBacterial endophytes recent developments andapplicationsrdquo FEMSMicrobiology Letters vol 278 no 1 pp 1ndash92008

[4] R Cakmakci F Donmez A Aydın and F Sahin ldquoGrowthpromotion of plants by plant growth-promoting rhizobacteriaunder greenhouse and two different field soil conditionsrdquo SoilBiology and Biochemistry vol 38 pp 1482ndash1487 2006

[5] P R Hardoim L S van Overbeek and J D V Elsas ldquoProp-erties of bacterial endophytes and their proposed role in plantgrowthrdquo Trends in Microbiology vol 16 no 10 pp 463ndash4712008

[6] S Compant C Clement and A Sessitsch ldquoPlant growth-promoting bacteria in the rhizo- and endosphere of plantstheir role colonization mechanisms involved and prospects forutilizationrdquo Soil Biology and Biochemistry vol 42 no 5 pp669ndash678 2010

[7] B R Glick ldquoBacterial ACC deaminase and the alleviation ofplant stressrdquo Advances in Applied Microbiology vol 56 pp 291ndash312 2004

[8] B R Glick B Todorovic J Czarny Z Cheng J Duan andB McConkey ldquoPromotion of plant growth by bacterial ACCdeaminaserdquo Critical Reviews in Plant Sciences vol 26 no 5-6pp 227ndash242 2007

[9] S L Doty B Oakley G Xin et al ldquoDiazotrophic endophytesof native black cottonwood and willowrdquo Symbiosis vol 47 no 1pp 23ndash33 2009

[10] B Lugtenberg and F Kamilova ldquoPlant-growth-promoting rhi-zobacteriardquoAnnual Review ofMicrobiology vol 63 pp 541ndash5562009

[11] U F Castillo G A Strobel E J Ford et al ldquoMunumbicinswide-spectrum antibiotics produced by Streptomyces NRRL30562 endophytic on Kennedia nigriscansrdquo Microbiology vol148 no 9 pp 2675ndash2685 2002

[12] Y Igarashi M E Trujillo E Martınez-Molina et al ldquoAnti-tumor anthraquinones from an endophytic actinomyceteMicromonospora lupini sp novrdquo Bioorganic amp Medicinal Chem-istry Letters vol 17 no 13 pp 3702ndash3705 2007

[13] S Shweta J H Bindu J Raghu et al ldquoIsolation of endophyticbacteria producing the anti-cancer alkaloid camptothecinefromMiquelia dentata Bedd (Icacinaceae)rdquo Phytomedicine vol20 pp 913ndash917 2013

[14] T Taechowisan A Wanbanjob P Tuntiwachwuttikul and JLiu ldquoAnti-inflammatory activity of lansais from endophyticStreptomyces sp SUC1 in LPS-induced RAW 2647 cellsrdquo Foodand Agricultural Immunology vol 20 no 1 pp 67ndash77 2009

Evidence-Based Complementary and Alternative Medicine 15

[15] P R Hardoim C C P Hardoim L S van Overbeek and J Dvan Elsas ldquoDynamics of seed-borne rice endophytes on earlyplant growth stagesrdquo PLoS ONE vol 7 no 2 Article ID e304382012

[16] D Bulgarelli K Schlaeppi S Spaepen E V L van Themaatand P Schulze-Lefert ldquoStructure and functions of the bacterialmicrobiota of plantsrdquo Annual Review of Plant Biology vol 64pp 807ndash838 2013

[17] A Mengoni S Mocali G Surico S Tegli and R Fani ldquoFluctu-ation of endophytic bacteria and phytoplasmosis in elm treesrdquoMicrobiological Research vol 158 no 4 pp 363ndash369 2003

[18] A Mengoni F Pini L-N Huang W-S Shu and MBazzicalupo ldquoPlant-by-plant variations of bacterial commu-nities associated with leaves of the nickel hyperaccumulatorAlyssum bertolonii desvrdquo Microbial Ecology vol 58 no 3 pp660ndash667 2009

[19] S Mocali E Bertelli F di Cello et al ldquoFluctuation of bacteriaisolated from elm tissues during different seasons and fromdifferent plant organsrdquo Research in Microbiology vol 154 no2 pp 105ndash114 2003

[20] F Pini A Frascella L Santopolo et al ldquoExploring the plant-associated bacterial communities in Medicago sativa Lrdquo BMCMicrobiology vol 12 article 78 2012

[21] A Mengoni L Cecchi and C Gonnelli ldquoNickel hyperac-cumulating plants and Alyssum bertolonii model systems forstudying biogeochemical interactions in serpentine soilsrdquo inBio-Geo Interactions in Metal-Contaminated Soils E Kothe andA Varma Eds pp 279ndash296 Springer Berlin Germany 2012

[22] S Ikeda T Okubo M Anda et al ldquoCommunity- and genome-based views of plant-associated bacteria plant-bacterial inter-actions in soybean and ricerdquo Plant and Cell Physiology vol 51no 9 pp 1398ndash1410 2010

[23] F Pini M Galardini M Bazzicalupo and A Mengoni ldquoPlant-bacteria association and symbiosis are there common genomictraits in alphaproteobacteriardquoGenes vol 2 no 4 pp 1017ndash10322011

[24] K Aleklett and M Hart ldquoThe root microbiota a fingerprint inthe soilrdquo Plant Soil vol 370 pp 671ndash686 2013

[25] A Beneduzi F Moreira P B Costa et al ldquoDiversity and plantgrowth promoting evaluation abilities of bacteria isolated fromsugarcane cultivated in the South of BrazilrdquoApplied Soil Ecologyvol 63 pp 94ndash104 2013

[26] N Bodenhausen M W Horton and J Bergelson ldquoBacterialcommunities associated with the leaves and the roots of Ara-bidopsis thalianardquo PLoS ONE vol 8 no 2 Article ID e563292013

[27] T F da Silva R E Vollu D Jurelevicius et al ldquoDoes theessential oil of Lippia sidoides Cham (pepper-rosmarin) affectits endophytic microbial communityrdquo BMC Microbiology vol13 no 1 article 29 2013

[28] M E Lucero A Unc P Cooke S Dowd and S SunldquoEndophyte microbiome diversity in micropropagated Atriplexcanescens and Atriplex torreyi var griffithsiirdquo PLoS ONE vol 6no 3 Article ID e17693 2011

[29] D S Lundberg S L Lebeis S H Paredes et al ldquoDefining thecoreArabidopsis thaliana rootmicrobiomerdquoNature vol 487 no7409 pp 86ndash90 2012

[30] H M Cavanagh and J M Wilkinson ldquoBiological activities oflavender essential oilrdquo Phytotherapy Research vol 16 no 4 pp301ndash308 2002

[31] G Woronuk Z Demissie M Rheault and S MahmoudldquoBiosynthesis and therapeutic properties of Lavandula essentialoil constituentsrdquo Planta Medica vol 77 no 1 pp 7ndash15 2011

[32] R Perry R Terry L K Watson and E Ernst ldquoIs lavender ananxiolytic drug A systematic review of randomised clinicaltrialsrdquo Phytomedicine vol 19 pp 825ndash835 2012

[33] S de Rapper G Kamatou A Viljoen and S van VuurenldquoThe in vitro antimicrobial activity of Lavandula angustifoliaessential oil in combination with other aroma-therapeutic oilsrdquoEvidence-Based Complementary and Alternative Medicine vol2013 Article ID 852049 10 pages 2013

[34] T Moon J M Wilkinson and H M Cavanagh ldquoAntiparasiticactivity of two Lavandula essential oils against Giardia duode-nalis Trichomonas vaginalis andHexamita inflatardquo ParasitologyResearch vol 99 no 6 pp 722ndash728 2006

[35] P S Yap S H Lim C P Hu and B C Yiap ldquoCom-bination of essential oils and antibiotics reduce antibioticresistance in plasmid-conferred multidrug resistant bacteriardquoPhytomedicine vol 20 pp 710ndash713 2013

[36] G Brader S Compant B Mitter F Trognitz and A SessitschldquoMetabolic potential of endophytic bacteriardquo Current Opinionin Biotechnology vol 27 pp 30ndash37 2014

[37] P Drevinek and E Mahenthiralingam ldquoBurkholderia ceno-cepacia in cystic fibrosis epidemiology and molecular mecha-nisms of virulencerdquo Clinical Microbiology and Infection vol 16no 7 pp 821ndash830 2010

[38] S Bazzini C Udine and G Riccardi ldquoMolecular approaches topathogenesis study of Burkholderia cenocepacia an importantcystic fibrosis opportunistic bacteriumrdquo Applied Microbiologyand Biotechnology vol 92 no 5 pp 887ndash895 2011

[39] I Maida A lo Nostro G Pesavento et al ldquoExploring theanti-Burkholderia cepacia complex activity of essential oilsa preliminary analysisrdquo Evidence-Based Complementary andAlternativeMedicine vol 2014 Article ID 573518 10 pages 2014

[40] A Mengoni R Barzanti C Gonnelli R Gabbrielli andM Bazzicalupo ldquoCharacterization of nickel-resistant bacteriaisolated from serpentine soilrdquo Environmental Microbiology vol3 no 11 pp 691ndash698 2001

[41] S F Altschul T L Madden A A Schaffer et al ldquoGappedBLAST and PSI-BLAST a new generation of protein databasesearch programsrdquo Nucleic Acids Research vol 25 no 17 pp3389ndash3402 1997

[42] R C Edgar ldquoMUSCLE multiple sequence alignment with highaccuracy and high throughputrdquo Nucleic Acids Research vol 32no 5 pp 1792ndash1797 2004

[43] F Ronquist M Teslenko P van der Mark et al ldquoMrbayes32 efficient bayesian phylogenetic inference and model choiceacross a large model spacerdquo Systematic Biology vol 61 no 3 pp539ndash542 2012

[44] K Tamura D Peterson N Peterson G Stecher M Nei andS Kumar ldquoMEGA5 molecular evolutionary genetics analysisusing maximum likelihood evolutionary distance and max-imum parsimony methodsrdquo Molecular Biology and Evolutionvol 28 no 10 pp 2731ndash2739 2011

[45] F Sievers A Wilm D Dineen et al ldquoFast scalable generationof high-quality protein multiple sequence alignments usingClustal Omegardquo Molecular Systems Biology vol 7 article 5392011

[46] Oslash Hammer D A T Harper and P D Ryan ldquoPAST pale-ontological statistics software package for education and dataanalysisrdquo Palaeontologia Electronica vol 4 no 1 pp 1ndash9 2001

16 Evidence-Based Complementary and Alternative Medicine

[47] M C Papaleo M Fondi I Maida et al ldquoSponge-associatedmicrobial Antarctic communities exhibiting antimicrobialactivity againstBurkholderia cepacia complex bacteriardquoBiotech-nology Advances vol 30 no 1 pp 272ndash293 2012

[48] A lo Giudice V Bruni and L Michaud ldquoCharacterization ofAntarctic psychrotrophic bacteria with antibacterial activitiesagainst terrestrial microorganismsrdquo Journal of Basic Microbiol-ogy vol 47 no 6 pp 496ndash505 2007

[49] A Schippers K Bosecker C Sproer and P SchumannldquoMicrobacterium oleivorans sp nov and Microbacteriumhydrocarbonoxydans sp nov novel crude-oil-degrading Gram-positive bacteriardquo International Journal of Systematic and Evo-lutionary Microbiology vol 55 no 2 pp 655ndash660 2005

[50] J D McChesney S K Venkataraman and J T Henri ldquoPlantnatural products back to the future or into extinctionrdquo Phyto-chemistry vol 68 no 14 pp 2015ndash2022 2007

[51] D K Manter J A Delgado D G Holm and R A StongldquoPyrosequencing reveals a highly diverse and cultivar-specificbacterial endophyte community in potato rootsrdquo MicrobialEcology vol 60 no 1 pp 157ndash166 2010

[52] K Ulrich A Ulrich and D Ewald ldquoDiversity of endophyticbacterial communities in poplar grown under field conditionsrdquoFEMS Microbiology Ecology vol 63 no 2 pp 169ndash180 2008

[53] N R Gottel H F Castro M Kerley et al ldquoDistinct microbialcommunities within the endosphere and rhizosphere ofPopulusdeltoides roots across contrasting soil typesrdquo Applied and Envi-ronmental Microbiology vol 77 no 17 pp 5934ndash5944 2011

[54] B Ma X Lv A Warren and J Gong ldquoShifts in diversity andcommunity structure of endophytic bacteria and archaea acrossroot stem and leaf tissues in the common reed Phragmitesaustralis along a salinity gradient in a marine tidal wetland ofnorthern Chinardquo Antonie van Leeuwenhoek vol 104 no 5 pp759ndash768 2013

[55] J A Vorholt ldquoMicrobial life in the phyllosphererdquo NatureReviews Microbiology vol 10 no 12 pp 828ndash840 2012

[56] R P Ryan S Monchy M Cardinale et al ldquoThe versatilityand adaptation of bacteria from the genus StenotrophomonasrdquoNature Reviews Microbiology vol 7 no 7 pp 514ndash525 2009

[57] AWolf A FritzeMHagemann andG Berg ldquoStenotrophomo-nas rhizophila sp nov a novel plant-associated bacterium withantifungal propertiesrdquo International Journal of Systematic andEvolutionary Microbiology vol 52 no 6 pp 1937ndash1944 2002

[58] C W Bacon and D M Hinton ldquoEndophytic and biologicalcontrol potential of Bacillus mojavensis and related speciesrdquoBiological Control vol 23 no 3 pp 274ndash284 2002

[59] G Berg L Eberl and A Hartmann ldquoThe rhizosphere as areservoir for opportunistic human pathogenic bacteriardquo Envi-ronmental Microbiology vol 7 no 11 pp 1673ndash1685 2005

[60] H L Tyler and E W Triplett ldquoPlants as a habitat for ben-eficial andor human pathogenic bacteriardquo Annual Review ofPhytopathology vol 46 pp 53ndash73 2008

[61] R Perry R Terry L K Watson and E Ernst ldquoIs lavender ananxiolytic drug A systematic review of randomised clinicaltrialsrdquo Phytomedicine vol 19 no 8-9 pp 825ndash835 2012

[62] I Maida A lo Nostro G Pesavento et al ldquoExploring theanti-Burkholderia cepacia complex activity of essential oilsa preliminary analysisrdquo Evidence-Based Complementary andAlternativeMedicine vol 2014 Article ID 573518 10 pages 2014

Evidence-Based Complementary and Alternative Medicine 9

005

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

095

09074

055

099

05

1

081

088

078

1

084

1

071

1

083

1

091

1

099

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 Pantoeaand those of reference type strains Posterior probability values are indicated at the node Nodes are collapsed at 70 probability LS = bacteriaisolated from the stem LL = bacteria isolated from the leaves (see Section 2 for details)

10 Evidence-Based Complementary and Alternative Medicine

05

LL6

LL67

LL81

LL5

LL8

LL29

LS87

LL100

LL82

LL85

LL83

LL7

LT3

LT5

LT67LT56

LL32

LL84

LL57

LL59

LT7

LL30

LL2

LT85

LL31LL1

LT4

LT6

LT8

LT2

08882

0679

0997

09977

05035

0644

0996409961

09758

07238

09846

05929

0504

0704107642

06421

1

06098

05001

05655

06027

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 paraoxydansS000381732 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 genusMicrobacterium and those of reference type strains Posterior probability values are indicated at the node Nodes are collapsed at 70probability LT = bacteria isolated from the rhizosphere LS = bacteria isolated from the stem LL = bacteria isolated from the leaves (seeSection 2 for details)

Evidence-Based Complementary and Alternative Medicine 11

Table 3The 40Burkholderia cepacia complex strains used as targetsin the cross streak experiments

Target strainStrain Species OriginFCF1 B cepacia CFFCF3LMG17588 B multivorans ENVFCF16 B cenocepacia (IIIA) CFJ2315FCF18

B cenocepacia (IIIB) CF

FCF20FCF23FCF24FCF27FCF29FCF30LMG16654C5424CEP511MVPC116 ENVMVPC173LMG19230 B cenocepacia (IIIC) ENVLMG19240FCF38 B cenocepacia (IIID) CFLMG21462FCF41 B stabilis CFFCF42 B vietnamiensis CFTVV75 ENVLMG18941

B dolosa CFLMG18942LMG18943MCI7

B ambifariaENV

LMG19467 CFLMG19182 ENVLMG16670 B anthina ENVFCF43 B pyrrocina CFLSED4 B lata CFLMG24064 B latens CFLMG24065 B diffusa CFLMG23361 B contaminans AILMG24067 B seminals CFLMG24068 B metallica CFLMG24066 B arboris ENVLMG24263 B ubonensis NIAbbreviations CF strains isolated from cystic fibrosis patients AI strainsisolated from animal infection NI strains isolated from nosocomial infec-tion ENV environmental strain

isolates showed similarities with the stress resistantB safensis

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

complex Regardless of the overall low support ofthe tree (typical of Pseudomonas phylogenies) someobservations may be drawn the presence of largeunresolved clusters shows clearly a low divergenceof the isolated colonies implying the existence ofclonal populations Pseudomonas spp isolated fromthe different compartments are intermixed suggest-ing the presence of a continuum rhizosphere-leaves(Pseudomonas is the only genus found in all the 4sample analysed)

32 Inhibition of Burkholderia Cepacia Complex StrainsGrowth by L angustifolia Endophytic Bacteria In order tocheck the ability of L angustifolia bacterial endophytes toantagonize the growth of (opportunistic) human pathogenicbacteria cross-streak experiments were carried out asdescribed in Section 2 using a panel of the 19 randomlychosen different endophytic strains isolated from soil rootsor leafs and phylogenetically assigned to six different gen-era (Bacillus Microbacterium Pantoea Plantibacter Pseu-domonas and Rhizobium) as testers versus 40 Bcc strainsrepresentative of seventeen species (out of eighteen) andwith either environmental or clinical origin with some ofwhich being multidrug-resistant Data obtained are shownin Table 4 The analysis of these data revealed that all thetester strains were able to completely inhibit the growthof Bcc strains including those with a clinical source andthat exhibited multidrug resistance this finding suggestedthat lavender endophytic bacteria are able to synthesize(strong) antimicrobial compounds However tester strainsshowed a different pattern of Bcc growth inhibition eventhose affiliated to the same genus In addition to this thereis no apparent difference in the antimicrobial potentialbetween strains belonging to different plant compartmentsConcerning the sensitivity of Bcc strains to the antimicrobialactivity of lavender endophytes strains belonging to differentspecies exhibited a different sensitivity spectrum However itis quite interesting that strains with clinical origin appearedto be more sensitive to the antimicrobials synthesized byendophytic bacteria than their environmental counterparts(see for instance strains belonging to the species Burkholderiacenocepacia IIIB) (Supplementary Table 3)

4 Discussion

Medicinal plants are stirring the attention of manyresearchers due to the presence of compounds thatconstitute a large fraction of the current pharmacopoeiasNatural products have been the source of most of theactive ingredients of medicines and more than 80 of drugsubstances are natural products or inspired by a naturalcompound including most of the of anticancer and anti-infective agents [50] Lavender plants are grown mainlyfor the production of essential oil which has antisepticand anti-inflammatory properties In spite of the relativeharsh environment for bacterial colonization L angustifoliatissues were found to be rich in bacterial diversity Howeverthe endophytic community isolated from different plant

12 Evidence-Based Complementary and Alternative Medicine

Table4Growth

of40

Burkholderiacepacia

complex

strains

inthep

resenceabsenceo

fend

ophytic

bacterialstrains

isolated

from

lavend

er

Species

Strain

Orig

inLL

1LL

2LL

3LL

4LL

6LL

7LL

9LL

10LS

1LS

2LS

3LS

4LS

5LS

6LR

1LR

2LR

3LR

4LR

5C-

Bcepacia

FCF1

CFminusminusminusminusminusminusminusminus

minusminusminusminusminusminusminusminusminusminusminus

+B

cepacia

FCF3

CF+minusminusminusminusminusminusminusminus

minusminusminusminusminusminusminusminusminusminusminus

+B

multivoran

sLM

G17588

Env

+minusminus

++minus

+minusminus

+minus

+minus

++minus

+minusminus

+minus

+minusminus

+B

cenocepacia

(IIIA

)FC

F16

CFminusminusminusminusminusminusminusminus

minusminusminusminusminusminusminusminusminusminusminus

+B

cenocepacia

(IIIA

)J2315

CF+minusminus

+minus

+minusminusminus

+minusminusminus

+minusminusminusminusminusminusminusminus

+B

cenocepacia

(IIIB)

FCF18

CF+minusminusminusminusminusminusminus

minusminusminusminusminusminus

+minusminusminusminusminus

+B

cenocepacia

(IIIB)

FCF20

CF+

++

++

+minus

++minus

+minus

+minus

+minus

++minus

++minus

++

++

+B

cenocepacia

(IIIB)

FCF23

CF+minusminusminusminusminusminusminus

+minusminusminus

++minusminusminus

+minusminusminusminus

+B

cenocepacia

(IIIB)

FCF24

CF+minusminusminusminusminusminusminus

+minusminus

+minusminusminusminus

+minusminusminusminus

+B

cenocepacia

(IIIB)

FCF27

CF+minusminusminusminusminusminusminus

+minusminusminusminusminusminusminusminusminusminusminus

+B

cenocepacia

(IIIB)

FCF29

CF+minusminus

+minus

+minusminusminus

+minusminusminusminusminusminusminus

+minus

+minusminus

+B

cenocepacia

(IIIB)

FCF30

CF+minusminus

+minus

+minusminusminus

+minus

+minusminusminusminusminusminus

+minus

+minusminus

+B

cenocepacia

(IIIB)

LMG16654

CF+

+minusminus

++minus

+minus

++minus

++minusminus

+minus

+minusminus

+B

cenocepacia

(IIIB)

C5424

CF+

+minusminus

+minusminusminusminus

+minusminusminus

+minusminusminusminus

+minusminus

+minusminus

+B

cenocepacia

(IIIB)

CEP5

11CF

+minusminus

+minusminusminusminus

+minus

+minusminus

+minus

+minusminusminus

+minus

+minusminus

+B

cenocepacia

(IIIB)

MVPC

116

Env

++minusminus

+minus

++minusminus

+minus

+minus

+minus

+minusminus

+minusminus

+minusminus

+minus

+minus

+B

cenocepacia

(IIIB)

MVPC

173

Env

++

++minus

++

+minus

++

++

++

++

++

++minus

+B

cenocepacia

(IIIIC)

LMG19230

Env

++

++

++

+minus

++

++

++

++

++

++

+B

cenocepacia

(IIIC

)LM

G19240

Env

++

++

+minus

+minus

++

+minus

++

++minus

++

++

+minus

+B

cenocepacia

(IIID)

FCF38

CF+minusminusminusminusminusminusminus

minusminusminusminusminusminusminusminusminusminusminus

+B

cenocepacia

(IIID)

LMG21462

CFminusminusminusminusminusminusminusminus

minusminusminusminusminusminusminusminusminusminusminus

+B

stabilis

FCF41

CF+

+minus

++

+minus

+minusminus

++

+minus

++minusminus

++

++minusminus

+B

vietnamien

sisFC

F42

CFminusminusminusminusminusminusminusminus

minusminusminusminusminusminusminusminusminusminusminus

+B

vietnamien

sisTV

V75

Env

++minusminus

+minusminusminusminus

+minusminusminus

++minusminusminus

+minusminus

+minusminus

+B

dolosa

LMG18941

CF+minusminus

+minus

+minus

+minusminus

+minusminusminus

++minusminusminus

+minus

+minus

+minusminus

+B

dolosa

LMG18942

CF+

+minusminus

+minus

+minusminus

++

+minus

++minusminusminus

++minus

++minusminus

+B

dolosa

LMG18943

CF+

+minusminus

++minus

+minusminus

+minus

+minusminus

++minusminusminus

++minus

+minusminus

+B

ambifaria

MCI

7En

v+

+minus

+minus

+minus

+minusminus

++minusminus

++minusminusminus

+minus

+minusminus

+B

ambifaria

LMG1946

7CF

++minus

+minus

+minusminusminus

++minusminus

++minusminusminus

+minus

+minusminusminus

+B

ambifaria

LMG19182

Env

++minus

+minus

+minusminusminus

+minusminus

+minusminusminus

+minus

+minusminusminus

+B

anthina

LMG16670

Env

+minusminusminusminus

+minus

+minusminus

++minusminusminus

+minus

+minusminusminus

+B

pyrrocinia

FCF43

CF+minusminusminusminusminusminusminusminus

minusminus

+minusminusminusminusminusminus

+minusminusminus

+B

lata

LSED

4CF

+minus

+minusminus

+minusminus

+minusminus

++minusminusminus

+minus

+minus

+minusminusminus

+B

latens

LMG2406

4CF

+minus

++minusminus

+minusminus

++minus

++minus

++

++

+minus

+B

diffu

saLM

G24065

CF+

+minus

++minus

+minus

+minus

++minus

++minus

+minus

++

++minus

+B

contam

inan

sLM

G23361

AI

++minus

+minus

+minus

+minus

+minus

++

++

++

+minus

++

++minus

+B

seminalis

LMG24067

CF+

+minus

++minus

++

++

++

++

++

++

++minus

+B

metallica

LMG2406

8CF

++

++minus

++

++

++

++

++

++

++minus

+B

arboris

LMG2406

6En

v+

+minus

++minus

++

+minus

++

++

++minus

+minus

++

++minusminus

+B

ubonensis

LMG24263

NI

+minusminusminusminus

+minusminus

+minus

+minus

++minusminus

+minusminus

+minusminusminus

+Ab

breviatio

nsC

Fstr

ains

isolatedfro

mcysticfi

brosispatie

ntsAIstr

ains

isolated

from

anim

alinfectionNIstr

ains

isolated

from

nosocomialinfectio

nEN

Venvironm

entalstrainLstr

ains

isolatedfro

mtheleaf

Sstr

ains

isolatedfro

mthes

temR

strains

isolatedfro

mther

oot

Symbo

ls+

grow

th+

minusreduced

grow

th+

minusminusveryredu

cedgrow

thminus

nogrow

thC

-Petrid

ishes

containing

onlythetargetstrains

Evidence-Based Complementary and Alternative Medicine 13

compartments showed different levels of diversity withthe 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 theleaf and roots community is already reported in otherspecies [26] even if there are few direct comparisons ofroots and phyllosphere bacterial communities especiallycomparisons using material from the same plants Themicrobial community residing in the phyllosphere is facedwith a nutrient poor and variable environment that ischaracterized by fluctuating temperature humidity and UVradiation and so it is predicted to be more diverse than thatwithin the rhizosphere a more homeostatic environmentThese findings supported also by the similar bacterial titreare in agreement with the idea that the source of inoculumfor the leaf community (except for the vertically transferredvia seeds) may be exclusive (eg aerosol even if the processis not yet clarified Bulgarelli et al 2013) and not related totransmission from the roots Moreover the low diversity ofthe community isolated in the stem dominated by clonally(Table 2) populations of Pseudomonas could be related tothe highly specific main tissue (phloem) present there whichcould provide an homeostatic environment rich in nutrient(sucrose contained in the phloem tissues) but poorer (ifcompared to leaf and roots) in secondary metabolites thatmay promote ecological niche differentiation

Concerning the taxonomic community composition dif-ferent taxonomic profiles were found for endosphere andrhizosphere More in detail the rhizosphere communityshowed quite similar diversity indexes if compared to theroots one but with a different composition as an examplemembers of the phylumActinobacteria are completely absentfrom the root internal tissues that are largely dominatedby Proteobacteria while they are present in the soil Toexplain this finding a two-step model has been proposed[16] soil abiotic properties determine the structure of theinitial bacterial communities that is then modified by rhi-zodeposits with plant roots cell wall features and releasedmetabolites promoting the growth of organotrophic bacteriathereby initiating a soil community shift In the second stepconvergent host genotype-dependent selection [51 52] fine-tunes community profiles thriving on the rhizoplane andwithin plant roots

Moreover endosphere communities were differentbetween plant organs (roots stems and leaves) even if theinternal tissues are dominated as already reported [25 52ndash54] by members of the phylum Proteobacteria an examplewhile rhizobia were isolated from root internal tissues theyare completely absent from stems and leaves (Figures 2 and3) isolates belonging to the genus Pantoea on the contrarywere isolated from aerial parts but not from root tissuesHence it is possible that plant might ldquoselectrdquo specific taxafor entering inside its compartment Such hypothesis issupported also by the pairwise differentiation values thecommunities isolated from the different tissues even those inphysical continuity are in fact strongly differentiated with thestem representing a low diversity compartment separatingthe two high diversity organs roots and leaves In this view itis noteworthy that in lavender only Pseudomonas represents

a ubiquitous and abundant genus of both rhizosphere andendosphere (Figure 3) The analysis of intrageneric diversitypointed out also that Pseudomonas isolates belong to a lowdifferentiated clonal population (Table 2 and Suppl Figure2) suggesting also that this component of the communityeither diffused inside the organs from a radical or foliar entrypoint or established during plants development putativelyfrom the seed inoculum A similar consideration can beproposed for isolates of rhizobia but for the rhizosphere-root internal tissues continuum a low taxonomicallydifferentiated community from the soil entered (increasingits relative abundance) inside the plant organ An entrypoint from the leaves is on the contrary consistent with thedistribution of Pantoea isolates with their strong diversity(Table 2) suggesting a diversification in response to the highvariable leaf environmentThe isolates belonging to the genusStenotrophomonas show an interesting distribution pattern(Figure 3) they are rare in the rhizosphere abundant in theroots and in the leaves and absent in the stem this findingsuggests a different entry point or a negative selection in thestem the second explanation is supported by admixture ofroot and leaves isolates (Figure 4)

The detailed phylogenetic analyses of the isolates enabledus also to putatively ascribe some isolates to already describedendophytic strains that can be targeted for functional charac-terisation many Microbacterium leaves isolates cluster withM hydrocarbonoxydans and M oleivorans two species withcrude oil degrading activity [49] being this metabolic activitythat is found frequently associated with a phyllosphereadapted lifestyle [55] Several isolates cluster with bacteriawith interesting biotechnological or ecological applicationssuch as S chelatiphaga a remarkable species with biotech-nological potential in phytoremediation [56] and S rizophilaa plant associated bacterium with antifungal activity [57] orBacillus mojavensis a bacteriumclosely related to B subtilisand already reported having an endophytic lifestyle andshowing an antagonistic role against plant pathogenic fungi[58] On the contrary many lavender Pseudomonas isolatescluster with known plant pathogen strains highlighting thesometimes subtle difference among pathogenic and endo-phytic lifestyle and with Pantoea agglomerans (and also withlavender isolates clustering withinthe B licheniformis and Bsoronensis groups) a known plant associated bacterium andan opportunistic pathogen in immune-compromised humanindividuals drawing attention to the possible pathogenicaction of endophytic bacteria in humans [59 60]

The overall analysis of the dendrograms points out thatthe characterization of isolates from endophytic communitiescan lead to the identification (as an example for Pantoeaand rhizobia) of not yet described strains that may representuntapped resources of bioactive compounds of agricultural ormedical interest

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

14 Evidence-Based Complementary and Alternative Medicine

context the characterization of cultivable bacterial commu-nities is a fundamental step to this purpose since it paves theway to the possibility to build collections of isolates that maybe phenotypically typed for important traits In our opiniondata obtained in this work (Table 4 and Supplementary Tables2 and 3) offers a preliminary but very promising exampleof the biotechnological potential of bacteria isolated frommedicinal plants In this pilot study in fact the majority ofthe tested endophytes showed to inhibit the growth of several(in some case all) human pathogenic strains belonging tothe Bcc complexes that are known for their resistance totraditional antibiotics These data are particularly interestingif correlated with the recent finding that essential oils fromdifferent medicinal plants are able to strongly (or completely)interfere with the growth of the same Bcc strains [61] and thatbacterial endophytes isolated from plants of the same speciesexhibited the same bioactivity (Maida et al in preparation)It is therefore possible that the therapeutic properties of theessential oil might reside also on bioactive compounds ofmicrobial origin We are completely aware that the determi-nation of the correlation (eventually) existing between thecomposition of bacterial endophytic communities and thebioactivity of essential oils extracted from a medicinal plantwould require the parallel analysis of the bacterial communityand the essential oil activity extracted from the same plantHowever data obtained in this work and those from Maidaet al [62] support this idea

It is also particularly intriguing the idea that the antimi-crobial activity of essential oils may reside on the actionof multiple compounds some of them produced or elicitedby the microbiota limiting the observed rapid evolutionof human pathogenic bacteria resistant to single moleculeantimicrobials

The characterization of the heterotrophic aerobic endo-phytic bacterial community of L angustifolia highlightedthe existence of a diversified community between differentorganstissues that may be related to the coexistence ofdifferent sources of inoculum andor a selection of the com-munities promoted by nutrient sand metabolites availabilityand antagonistic forces Several not previously characterizedstrains have been isolated and molecularly typed confirmingthat the analysis of the bacterial communities inhabitingextreme or unconventional environments may represent aproper strategy for the discovery of untapped sources offunctional biodiversity and bioactive molecule production

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This work was supported by grants from the Ente Cassadi Risparmio di Firenze (Grant 20130657 Herbiome newantimicrobial compounds from endophytic bacteria isolatedfrom medicinal plants) Giovanni Emiliani is financially

supported by the Regione Toscana POR CRO FSE 2007-2013 Asse IV Grant Sysbiofor (CNR-9) Marco Fondi andElena Perrin are financially supported by the FEMSAdvancedFellowship (FAF2012) and the Buzzati-Traverso Foundationrespectively

References

[1] B Reinhold-Hurek andTHurek ldquoLiving inside plants bacterialendophytesrdquo Current Opinion in Plant Biology vol 14 no 4 pp435ndash443 2011

[2] M Rosenblueth and E Martınez-Romero ldquoBacterial endo-phytes and their interactions with hostsrdquo Molecular Plant-Microbe Interactions vol 19 no 8 pp 827ndash837 2006

[3] R P Ryan K Germaine A Franks D J Ryan and DN Dowling ldquoBacterial endophytes recent developments andapplicationsrdquo FEMSMicrobiology Letters vol 278 no 1 pp 1ndash92008

[4] R Cakmakci F Donmez A Aydın and F Sahin ldquoGrowthpromotion of plants by plant growth-promoting rhizobacteriaunder greenhouse and two different field soil conditionsrdquo SoilBiology and Biochemistry vol 38 pp 1482ndash1487 2006

[5] P R Hardoim L S van Overbeek and J D V Elsas ldquoProp-erties of bacterial endophytes and their proposed role in plantgrowthrdquo Trends in Microbiology vol 16 no 10 pp 463ndash4712008

[6] S Compant C Clement and A Sessitsch ldquoPlant growth-promoting bacteria in the rhizo- and endosphere of plantstheir role colonization mechanisms involved and prospects forutilizationrdquo Soil Biology and Biochemistry vol 42 no 5 pp669ndash678 2010

[7] B R Glick ldquoBacterial ACC deaminase and the alleviation ofplant stressrdquo Advances in Applied Microbiology vol 56 pp 291ndash312 2004

[8] B R Glick B Todorovic J Czarny Z Cheng J Duan andB McConkey ldquoPromotion of plant growth by bacterial ACCdeaminaserdquo Critical Reviews in Plant Sciences vol 26 no 5-6pp 227ndash242 2007

[9] S L Doty B Oakley G Xin et al ldquoDiazotrophic endophytesof native black cottonwood and willowrdquo Symbiosis vol 47 no 1pp 23ndash33 2009

[10] B Lugtenberg and F Kamilova ldquoPlant-growth-promoting rhi-zobacteriardquoAnnual Review ofMicrobiology vol 63 pp 541ndash5562009

[11] U F Castillo G A Strobel E J Ford et al ldquoMunumbicinswide-spectrum antibiotics produced by Streptomyces NRRL30562 endophytic on Kennedia nigriscansrdquo Microbiology vol148 no 9 pp 2675ndash2685 2002

[12] Y Igarashi M E Trujillo E Martınez-Molina et al ldquoAnti-tumor anthraquinones from an endophytic actinomyceteMicromonospora lupini sp novrdquo Bioorganic amp Medicinal Chem-istry Letters vol 17 no 13 pp 3702ndash3705 2007

[13] S Shweta J H Bindu J Raghu et al ldquoIsolation of endophyticbacteria producing the anti-cancer alkaloid camptothecinefromMiquelia dentata Bedd (Icacinaceae)rdquo Phytomedicine vol20 pp 913ndash917 2013

[14] T Taechowisan A Wanbanjob P Tuntiwachwuttikul and JLiu ldquoAnti-inflammatory activity of lansais from endophyticStreptomyces sp SUC1 in LPS-induced RAW 2647 cellsrdquo Foodand Agricultural Immunology vol 20 no 1 pp 67ndash77 2009

Evidence-Based Complementary and Alternative Medicine 15

[15] P R Hardoim C C P Hardoim L S van Overbeek and J Dvan Elsas ldquoDynamics of seed-borne rice endophytes on earlyplant growth stagesrdquo PLoS ONE vol 7 no 2 Article ID e304382012

[16] D Bulgarelli K Schlaeppi S Spaepen E V L van Themaatand P Schulze-Lefert ldquoStructure and functions of the bacterialmicrobiota of plantsrdquo Annual Review of Plant Biology vol 64pp 807ndash838 2013

[17] A Mengoni S Mocali G Surico S Tegli and R Fani ldquoFluctu-ation of endophytic bacteria and phytoplasmosis in elm treesrdquoMicrobiological Research vol 158 no 4 pp 363ndash369 2003

[18] A Mengoni F Pini L-N Huang W-S Shu and MBazzicalupo ldquoPlant-by-plant variations of bacterial commu-nities associated with leaves of the nickel hyperaccumulatorAlyssum bertolonii desvrdquo Microbial Ecology vol 58 no 3 pp660ndash667 2009

[19] S Mocali E Bertelli F di Cello et al ldquoFluctuation of bacteriaisolated from elm tissues during different seasons and fromdifferent plant organsrdquo Research in Microbiology vol 154 no2 pp 105ndash114 2003

[20] F Pini A Frascella L Santopolo et al ldquoExploring the plant-associated bacterial communities in Medicago sativa Lrdquo BMCMicrobiology vol 12 article 78 2012

[21] A Mengoni L Cecchi and C Gonnelli ldquoNickel hyperac-cumulating plants and Alyssum bertolonii model systems forstudying biogeochemical interactions in serpentine soilsrdquo inBio-Geo Interactions in Metal-Contaminated Soils E Kothe andA Varma Eds pp 279ndash296 Springer Berlin Germany 2012

[22] S Ikeda T Okubo M Anda et al ldquoCommunity- and genome-based views of plant-associated bacteria plant-bacterial inter-actions in soybean and ricerdquo Plant and Cell Physiology vol 51no 9 pp 1398ndash1410 2010

[23] F Pini M Galardini M Bazzicalupo and A Mengoni ldquoPlant-bacteria association and symbiosis are there common genomictraits in alphaproteobacteriardquoGenes vol 2 no 4 pp 1017ndash10322011

[24] K Aleklett and M Hart ldquoThe root microbiota a fingerprint inthe soilrdquo Plant Soil vol 370 pp 671ndash686 2013

[25] A Beneduzi F Moreira P B Costa et al ldquoDiversity and plantgrowth promoting evaluation abilities of bacteria isolated fromsugarcane cultivated in the South of BrazilrdquoApplied Soil Ecologyvol 63 pp 94ndash104 2013

[26] N Bodenhausen M W Horton and J Bergelson ldquoBacterialcommunities associated with the leaves and the roots of Ara-bidopsis thalianardquo PLoS ONE vol 8 no 2 Article ID e563292013

[27] T F da Silva R E Vollu D Jurelevicius et al ldquoDoes theessential oil of Lippia sidoides Cham (pepper-rosmarin) affectits endophytic microbial communityrdquo BMC Microbiology vol13 no 1 article 29 2013

[28] M E Lucero A Unc P Cooke S Dowd and S SunldquoEndophyte microbiome diversity in micropropagated Atriplexcanescens and Atriplex torreyi var griffithsiirdquo PLoS ONE vol 6no 3 Article ID e17693 2011

[29] D S Lundberg S L Lebeis S H Paredes et al ldquoDefining thecoreArabidopsis thaliana rootmicrobiomerdquoNature vol 487 no7409 pp 86ndash90 2012

[30] H M Cavanagh and J M Wilkinson ldquoBiological activities oflavender essential oilrdquo Phytotherapy Research vol 16 no 4 pp301ndash308 2002

[31] G Woronuk Z Demissie M Rheault and S MahmoudldquoBiosynthesis and therapeutic properties of Lavandula essentialoil constituentsrdquo Planta Medica vol 77 no 1 pp 7ndash15 2011

[32] R Perry R Terry L K Watson and E Ernst ldquoIs lavender ananxiolytic drug A systematic review of randomised clinicaltrialsrdquo Phytomedicine vol 19 pp 825ndash835 2012

[33] S de Rapper G Kamatou A Viljoen and S van VuurenldquoThe in vitro antimicrobial activity of Lavandula angustifoliaessential oil in combination with other aroma-therapeutic oilsrdquoEvidence-Based Complementary and Alternative Medicine vol2013 Article ID 852049 10 pages 2013

[34] T Moon J M Wilkinson and H M Cavanagh ldquoAntiparasiticactivity of two Lavandula essential oils against Giardia duode-nalis Trichomonas vaginalis andHexamita inflatardquo ParasitologyResearch vol 99 no 6 pp 722ndash728 2006

[35] P S Yap S H Lim C P Hu and B C Yiap ldquoCom-bination of essential oils and antibiotics reduce antibioticresistance in plasmid-conferred multidrug resistant bacteriardquoPhytomedicine vol 20 pp 710ndash713 2013

[36] G Brader S Compant B Mitter F Trognitz and A SessitschldquoMetabolic potential of endophytic bacteriardquo Current Opinionin Biotechnology vol 27 pp 30ndash37 2014

[37] P Drevinek and E Mahenthiralingam ldquoBurkholderia ceno-cepacia in cystic fibrosis epidemiology and molecular mecha-nisms of virulencerdquo Clinical Microbiology and Infection vol 16no 7 pp 821ndash830 2010

[38] S Bazzini C Udine and G Riccardi ldquoMolecular approaches topathogenesis study of Burkholderia cenocepacia an importantcystic fibrosis opportunistic bacteriumrdquo Applied Microbiologyand Biotechnology vol 92 no 5 pp 887ndash895 2011

[39] I Maida A lo Nostro G Pesavento et al ldquoExploring theanti-Burkholderia cepacia complex activity of essential oilsa preliminary analysisrdquo Evidence-Based Complementary andAlternativeMedicine vol 2014 Article ID 573518 10 pages 2014

[40] A Mengoni R Barzanti C Gonnelli R Gabbrielli andM Bazzicalupo ldquoCharacterization of nickel-resistant bacteriaisolated from serpentine soilrdquo Environmental Microbiology vol3 no 11 pp 691ndash698 2001

[41] S F Altschul T L Madden A A Schaffer et al ldquoGappedBLAST and PSI-BLAST a new generation of protein databasesearch programsrdquo Nucleic Acids Research vol 25 no 17 pp3389ndash3402 1997

[42] R C Edgar ldquoMUSCLE multiple sequence alignment with highaccuracy and high throughputrdquo Nucleic Acids Research vol 32no 5 pp 1792ndash1797 2004

[43] F Ronquist M Teslenko P van der Mark et al ldquoMrbayes32 efficient bayesian phylogenetic inference and model choiceacross a large model spacerdquo Systematic Biology vol 61 no 3 pp539ndash542 2012

[44] K Tamura D Peterson N Peterson G Stecher M Nei andS Kumar ldquoMEGA5 molecular evolutionary genetics analysisusing maximum likelihood evolutionary distance and max-imum parsimony methodsrdquo Molecular Biology and Evolutionvol 28 no 10 pp 2731ndash2739 2011

[45] F Sievers A Wilm D Dineen et al ldquoFast scalable generationof high-quality protein multiple sequence alignments usingClustal Omegardquo Molecular Systems Biology vol 7 article 5392011

[46] Oslash Hammer D A T Harper and P D Ryan ldquoPAST pale-ontological statistics software package for education and dataanalysisrdquo Palaeontologia Electronica vol 4 no 1 pp 1ndash9 2001

16 Evidence-Based Complementary and Alternative Medicine

[47] M C Papaleo M Fondi I Maida et al ldquoSponge-associatedmicrobial Antarctic communities exhibiting antimicrobialactivity againstBurkholderia cepacia complex bacteriardquoBiotech-nology Advances vol 30 no 1 pp 272ndash293 2012

[48] A lo Giudice V Bruni and L Michaud ldquoCharacterization ofAntarctic psychrotrophic bacteria with antibacterial activitiesagainst terrestrial microorganismsrdquo Journal of Basic Microbiol-ogy vol 47 no 6 pp 496ndash505 2007

[49] A Schippers K Bosecker C Sproer and P SchumannldquoMicrobacterium oleivorans sp nov and Microbacteriumhydrocarbonoxydans sp nov novel crude-oil-degrading Gram-positive bacteriardquo International Journal of Systematic and Evo-lutionary Microbiology vol 55 no 2 pp 655ndash660 2005

[50] J D McChesney S K Venkataraman and J T Henri ldquoPlantnatural products back to the future or into extinctionrdquo Phyto-chemistry vol 68 no 14 pp 2015ndash2022 2007

[51] D K Manter J A Delgado D G Holm and R A StongldquoPyrosequencing reveals a highly diverse and cultivar-specificbacterial endophyte community in potato rootsrdquo MicrobialEcology vol 60 no 1 pp 157ndash166 2010

[52] K Ulrich A Ulrich and D Ewald ldquoDiversity of endophyticbacterial communities in poplar grown under field conditionsrdquoFEMS Microbiology Ecology vol 63 no 2 pp 169ndash180 2008

[53] N R Gottel H F Castro M Kerley et al ldquoDistinct microbialcommunities within the endosphere and rhizosphere ofPopulusdeltoides roots across contrasting soil typesrdquo Applied and Envi-ronmental Microbiology vol 77 no 17 pp 5934ndash5944 2011

[54] B Ma X Lv A Warren and J Gong ldquoShifts in diversity andcommunity structure of endophytic bacteria and archaea acrossroot stem and leaf tissues in the common reed Phragmitesaustralis along a salinity gradient in a marine tidal wetland ofnorthern Chinardquo Antonie van Leeuwenhoek vol 104 no 5 pp759ndash768 2013

[55] J A Vorholt ldquoMicrobial life in the phyllosphererdquo NatureReviews Microbiology vol 10 no 12 pp 828ndash840 2012

[56] R P Ryan S Monchy M Cardinale et al ldquoThe versatilityand adaptation of bacteria from the genus StenotrophomonasrdquoNature Reviews Microbiology vol 7 no 7 pp 514ndash525 2009

[57] AWolf A FritzeMHagemann andG Berg ldquoStenotrophomo-nas rhizophila sp nov a novel plant-associated bacterium withantifungal propertiesrdquo International Journal of Systematic andEvolutionary Microbiology vol 52 no 6 pp 1937ndash1944 2002

[58] C W Bacon and D M Hinton ldquoEndophytic and biologicalcontrol potential of Bacillus mojavensis and related speciesrdquoBiological Control vol 23 no 3 pp 274ndash284 2002

[59] G Berg L Eberl and A Hartmann ldquoThe rhizosphere as areservoir for opportunistic human pathogenic bacteriardquo Envi-ronmental Microbiology vol 7 no 11 pp 1673ndash1685 2005

[60] H L Tyler and E W Triplett ldquoPlants as a habitat for ben-eficial andor human pathogenic bacteriardquo Annual Review ofPhytopathology vol 46 pp 53ndash73 2008

[61] R Perry R Terry L K Watson and E Ernst ldquoIs lavender ananxiolytic drug A systematic review of randomised clinicaltrialsrdquo Phytomedicine vol 19 no 8-9 pp 825ndash835 2012

[62] I Maida A lo Nostro G Pesavento et al ldquoExploring theanti-Burkholderia cepacia complex activity of essential oilsa preliminary analysisrdquo Evidence-Based Complementary andAlternativeMedicine vol 2014 Article ID 573518 10 pages 2014

10 Evidence-Based Complementary and Alternative Medicine

05

LL6

LL67

LL81

LL5

LL8

LL29

LS87

LL100

LL82

LL85

LL83

LL7

LT3

LT5

LT67LT56

LL32

LL84

LL57

LL59

LT7

LL30

LL2

LT85

LL31LL1

LT4

LT6

LT8

LT2

08882

0679

0997

09977

05035

0644

0996409961

09758

07238

09846

05929

0504

0704107642

06421

1

06098

05001

05655

06027

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 paraoxydansS000381732 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 genusMicrobacterium and those of reference type strains Posterior probability values are indicated at the node Nodes are collapsed at 70probability LT = bacteria isolated from the rhizosphere LS = bacteria isolated from the stem LL = bacteria isolated from the leaves (seeSection 2 for details)

Evidence-Based Complementary and Alternative Medicine 11

Table 3The 40Burkholderia cepacia complex strains used as targetsin the cross streak experiments

Target strainStrain Species OriginFCF1 B cepacia CFFCF3LMG17588 B multivorans ENVFCF16 B cenocepacia (IIIA) CFJ2315FCF18

B cenocepacia (IIIB) CF

FCF20FCF23FCF24FCF27FCF29FCF30LMG16654C5424CEP511MVPC116 ENVMVPC173LMG19230 B cenocepacia (IIIC) ENVLMG19240FCF38 B cenocepacia (IIID) CFLMG21462FCF41 B stabilis CFFCF42 B vietnamiensis CFTVV75 ENVLMG18941

B dolosa CFLMG18942LMG18943MCI7

B ambifariaENV

LMG19467 CFLMG19182 ENVLMG16670 B anthina ENVFCF43 B pyrrocina CFLSED4 B lata CFLMG24064 B latens CFLMG24065 B diffusa CFLMG23361 B contaminans AILMG24067 B seminals CFLMG24068 B metallica CFLMG24066 B arboris ENVLMG24263 B ubonensis NIAbbreviations CF strains isolated from cystic fibrosis patients AI strainsisolated from animal infection NI strains isolated from nosocomial infec-tion ENV environmental strain

isolates showed similarities with the stress resistantB safensis

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

complex Regardless of the overall low support ofthe tree (typical of Pseudomonas phylogenies) someobservations may be drawn the presence of largeunresolved clusters shows clearly a low divergenceof the isolated colonies implying the existence ofclonal populations Pseudomonas spp isolated fromthe different compartments are intermixed suggest-ing the presence of a continuum rhizosphere-leaves(Pseudomonas is the only genus found in all the 4sample analysed)

32 Inhibition of Burkholderia Cepacia Complex StrainsGrowth by L angustifolia Endophytic Bacteria In order tocheck the ability of L angustifolia bacterial endophytes toantagonize the growth of (opportunistic) human pathogenicbacteria cross-streak experiments were carried out asdescribed in Section 2 using a panel of the 19 randomlychosen different endophytic strains isolated from soil rootsor leafs and phylogenetically assigned to six different gen-era (Bacillus Microbacterium Pantoea Plantibacter Pseu-domonas and Rhizobium) as testers versus 40 Bcc strainsrepresentative of seventeen species (out of eighteen) andwith either environmental or clinical origin with some ofwhich being multidrug-resistant Data obtained are shownin Table 4 The analysis of these data revealed that all thetester strains were able to completely inhibit the growthof Bcc strains including those with a clinical source andthat exhibited multidrug resistance this finding suggestedthat lavender endophytic bacteria are able to synthesize(strong) antimicrobial compounds However tester strainsshowed a different pattern of Bcc growth inhibition eventhose affiliated to the same genus In addition to this thereis no apparent difference in the antimicrobial potentialbetween strains belonging to different plant compartmentsConcerning the sensitivity of Bcc strains to the antimicrobialactivity of lavender endophytes strains belonging to differentspecies exhibited a different sensitivity spectrum However itis quite interesting that strains with clinical origin appearedto be more sensitive to the antimicrobials synthesized byendophytic bacteria than their environmental counterparts(see for instance strains belonging to the species Burkholderiacenocepacia IIIB) (Supplementary Table 3)

4 Discussion

Medicinal plants are stirring the attention of manyresearchers due to the presence of compounds thatconstitute a large fraction of the current pharmacopoeiasNatural products have been the source of most of theactive ingredients of medicines and more than 80 of drugsubstances are natural products or inspired by a naturalcompound including most of the of anticancer and anti-infective agents [50] Lavender plants are grown mainlyfor the production of essential oil which has antisepticand anti-inflammatory properties In spite of the relativeharsh environment for bacterial colonization L angustifoliatissues were found to be rich in bacterial diversity Howeverthe endophytic community isolated from different plant

12 Evidence-Based Complementary and Alternative Medicine

Table4Growth

of40

Burkholderiacepacia

complex

strains

inthep

resenceabsenceo

fend

ophytic

bacterialstrains

isolated

from

lavend

er

Species

Strain

Orig

inLL

1LL

2LL

3LL

4LL

6LL

7LL

9LL

10LS

1LS

2LS

3LS

4LS

5LS

6LR

1LR

2LR

3LR

4LR

5C-

Bcepacia

FCF1

CFminusminusminusminusminusminusminusminus

minusminusminusminusminusminusminusminusminusminusminus

+B

cepacia

FCF3

CF+minusminusminusminusminusminusminusminus

minusminusminusminusminusminusminusminusminusminusminus

+B

multivoran

sLM

G17588

Env

+minusminus

++minus

+minusminus

+minus

+minus

++minus

+minusminus

+minus

+minusminus

+B

cenocepacia

(IIIA

)FC

F16

CFminusminusminusminusminusminusminusminus

minusminusminusminusminusminusminusminusminusminusminus

+B

cenocepacia

(IIIA

)J2315

CF+minusminus

+minus

+minusminusminus

+minusminusminus

+minusminusminusminusminusminusminusminus

+B

cenocepacia

(IIIB)

FCF18

CF+minusminusminusminusminusminusminus

minusminusminusminusminusminus

+minusminusminusminusminus

+B

cenocepacia

(IIIB)

FCF20

CF+

++

++

+minus

++minus

+minus

+minus

+minus

++minus

++minus

++

++

+B

cenocepacia

(IIIB)

FCF23

CF+minusminusminusminusminusminusminus

+minusminusminus

++minusminusminus

+minusminusminusminus

+B

cenocepacia

(IIIB)

FCF24

CF+minusminusminusminusminusminusminus

+minusminus

+minusminusminusminus

+minusminusminusminus

+B

cenocepacia

(IIIB)

FCF27

CF+minusminusminusminusminusminusminus

+minusminusminusminusminusminusminusminusminusminusminus

+B

cenocepacia

(IIIB)

FCF29

CF+minusminus

+minus

+minusminusminus

+minusminusminusminusminusminusminus

+minus

+minusminus

+B

cenocepacia

(IIIB)

FCF30

CF+minusminus

+minus

+minusminusminus

+minus

+minusminusminusminusminusminus

+minus

+minusminus

+B

cenocepacia

(IIIB)

LMG16654

CF+

+minusminus

++minus

+minus

++minus

++minusminus

+minus

+minusminus

+B

cenocepacia

(IIIB)

C5424

CF+

+minusminus

+minusminusminusminus

+minusminusminus

+minusminusminusminus

+minusminus

+minusminus

+B

cenocepacia

(IIIB)

CEP5

11CF

+minusminus

+minusminusminusminus

+minus

+minusminus

+minus

+minusminusminus

+minus

+minusminus

+B

cenocepacia

(IIIB)

MVPC

116

Env

++minusminus

+minus

++minusminus

+minus

+minus

+minus

+minusminus

+minusminus

+minusminus

+minus

+minus

+B

cenocepacia

(IIIB)

MVPC

173

Env

++

++minus

++

+minus

++

++

++

++

++

++minus

+B

cenocepacia

(IIIIC)

LMG19230

Env

++

++

++

+minus

++

++

++

++

++

++

+B

cenocepacia

(IIIC

)LM

G19240

Env

++

++

+minus

+minus

++

+minus

++

++minus

++

++

+minus

+B

cenocepacia

(IIID)

FCF38

CF+minusminusminusminusminusminusminus

minusminusminusminusminusminusminusminusminusminusminus

+B

cenocepacia

(IIID)

LMG21462

CFminusminusminusminusminusminusminusminus

minusminusminusminusminusminusminusminusminusminusminus

+B

stabilis

FCF41

CF+

+minus

++

+minus

+minusminus

++

+minus

++minusminus

++

++minusminus

+B

vietnamien

sisFC

F42

CFminusminusminusminusminusminusminusminus

minusminusminusminusminusminusminusminusminusminusminus

+B

vietnamien

sisTV

V75

Env

++minusminus

+minusminusminusminus

+minusminusminus

++minusminusminus

+minusminus

+minusminus

+B

dolosa

LMG18941

CF+minusminus

+minus

+minus

+minusminus

+minusminusminus

++minusminusminus

+minus

+minus

+minusminus

+B

dolosa

LMG18942

CF+

+minusminus

+minus

+minusminus

++

+minus

++minusminusminus

++minus

++minusminus

+B

dolosa

LMG18943

CF+

+minusminus

++minus

+minusminus

+minus

+minusminus

++minusminusminus

++minus

+minusminus

+B

ambifaria

MCI

7En

v+

+minus

+minus

+minus

+minusminus

++minusminus

++minusminusminus

+minus

+minusminus

+B

ambifaria

LMG1946

7CF

++minus

+minus

+minusminusminus

++minusminus

++minusminusminus

+minus

+minusminusminus

+B

ambifaria

LMG19182

Env

++minus

+minus

+minusminusminus

+minusminus

+minusminusminus

+minus

+minusminusminus

+B

anthina

LMG16670

Env

+minusminusminusminus

+minus

+minusminus

++minusminusminus

+minus

+minusminusminus

+B

pyrrocinia

FCF43

CF+minusminusminusminusminusminusminusminus

minusminus

+minusminusminusminusminusminus

+minusminusminus

+B

lata

LSED

4CF

+minus

+minusminus

+minusminus

+minusminus

++minusminusminus

+minus

+minus

+minusminusminus

+B

latens

LMG2406

4CF

+minus

++minusminus

+minusminus

++minus

++minus

++

++

+minus

+B

diffu

saLM

G24065

CF+

+minus

++minus

+minus

+minus

++minus

++minus

+minus

++

++minus

+B

contam

inan

sLM

G23361

AI

++minus

+minus

+minus

+minus

+minus

++

++

++

+minus

++

++minus

+B

seminalis

LMG24067

CF+

+minus

++minus

++

++

++

++

++

++

++minus

+B

metallica

LMG2406

8CF

++

++minus

++

++

++

++

++

++

++minus

+B

arboris

LMG2406

6En

v+

+minus

++minus

++

+minus

++

++

++minus

+minus

++

++minusminus

+B

ubonensis

LMG24263

NI

+minusminusminusminus

+minusminus

+minus

+minus

++minusminus

+minusminus

+minusminusminus

+Ab

breviatio

nsC

Fstr

ains

isolatedfro

mcysticfi

brosispatie

ntsAIstr

ains

isolated

from

anim

alinfectionNIstr

ains

isolated

from

nosocomialinfectio

nEN

Venvironm

entalstrainLstr

ains

isolatedfro

mtheleaf

Sstr

ains

isolatedfro

mthes

temR

strains

isolatedfro

mther

oot

Symbo

ls+

grow

th+

minusreduced

grow

th+

minusminusveryredu

cedgrow

thminus

nogrow

thC

-Petrid

ishes

containing

onlythetargetstrains

Evidence-Based Complementary and Alternative Medicine 13

compartments showed different levels of diversity withthe 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 theleaf and roots community is already reported in otherspecies [26] even if there are few direct comparisons ofroots and phyllosphere bacterial communities especiallycomparisons using material from the same plants Themicrobial community residing in the phyllosphere is facedwith a nutrient poor and variable environment that ischaracterized by fluctuating temperature humidity and UVradiation and so it is predicted to be more diverse than thatwithin the rhizosphere a more homeostatic environmentThese findings supported also by the similar bacterial titreare in agreement with the idea that the source of inoculumfor the leaf community (except for the vertically transferredvia seeds) may be exclusive (eg aerosol even if the processis not yet clarified Bulgarelli et al 2013) and not related totransmission from the roots Moreover the low diversity ofthe community isolated in the stem dominated by clonally(Table 2) populations of Pseudomonas could be related tothe highly specific main tissue (phloem) present there whichcould provide an homeostatic environment rich in nutrient(sucrose contained in the phloem tissues) but poorer (ifcompared to leaf and roots) in secondary metabolites thatmay promote ecological niche differentiation

Concerning the taxonomic community composition dif-ferent taxonomic profiles were found for endosphere andrhizosphere More in detail the rhizosphere communityshowed quite similar diversity indexes if compared to theroots one but with a different composition as an examplemembers of the phylumActinobacteria are completely absentfrom the root internal tissues that are largely dominatedby Proteobacteria while they are present in the soil Toexplain this finding a two-step model has been proposed[16] soil abiotic properties determine the structure of theinitial bacterial communities that is then modified by rhi-zodeposits with plant roots cell wall features and releasedmetabolites promoting the growth of organotrophic bacteriathereby initiating a soil community shift In the second stepconvergent host genotype-dependent selection [51 52] fine-tunes community profiles thriving on the rhizoplane andwithin plant roots

Moreover endosphere communities were differentbetween plant organs (roots stems and leaves) even if theinternal tissues are dominated as already reported [25 52ndash54] by members of the phylum Proteobacteria an examplewhile rhizobia were isolated from root internal tissues theyare completely absent from stems and leaves (Figures 2 and3) isolates belonging to the genus Pantoea on the contrarywere isolated from aerial parts but not from root tissuesHence it is possible that plant might ldquoselectrdquo specific taxafor entering inside its compartment Such hypothesis issupported also by the pairwise differentiation values thecommunities isolated from the different tissues even those inphysical continuity are in fact strongly differentiated with thestem representing a low diversity compartment separatingthe two high diversity organs roots and leaves In this view itis noteworthy that in lavender only Pseudomonas represents

a ubiquitous and abundant genus of both rhizosphere andendosphere (Figure 3) The analysis of intrageneric diversitypointed out also that Pseudomonas isolates belong to a lowdifferentiated clonal population (Table 2 and Suppl Figure2) suggesting also that this component of the communityeither diffused inside the organs from a radical or foliar entrypoint or established during plants development putativelyfrom the seed inoculum A similar consideration can beproposed for isolates of rhizobia but for the rhizosphere-root internal tissues continuum a low taxonomicallydifferentiated community from the soil entered (increasingits relative abundance) inside the plant organ An entrypoint from the leaves is on the contrary consistent with thedistribution of Pantoea isolates with their strong diversity(Table 2) suggesting a diversification in response to the highvariable leaf environmentThe isolates belonging to the genusStenotrophomonas show an interesting distribution pattern(Figure 3) they are rare in the rhizosphere abundant in theroots and in the leaves and absent in the stem this findingsuggests a different entry point or a negative selection in thestem the second explanation is supported by admixture ofroot and leaves isolates (Figure 4)

The detailed phylogenetic analyses of the isolates enabledus also to putatively ascribe some isolates to already describedendophytic strains that can be targeted for functional charac-terisation many Microbacterium leaves isolates cluster withM hydrocarbonoxydans and M oleivorans two species withcrude oil degrading activity [49] being this metabolic activitythat is found frequently associated with a phyllosphereadapted lifestyle [55] Several isolates cluster with bacteriawith interesting biotechnological or ecological applicationssuch as S chelatiphaga a remarkable species with biotech-nological potential in phytoremediation [56] and S rizophilaa plant associated bacterium with antifungal activity [57] orBacillus mojavensis a bacteriumclosely related to B subtilisand already reported having an endophytic lifestyle andshowing an antagonistic role against plant pathogenic fungi[58] On the contrary many lavender Pseudomonas isolatescluster with known plant pathogen strains highlighting thesometimes subtle difference among pathogenic and endo-phytic lifestyle and with Pantoea agglomerans (and also withlavender isolates clustering withinthe B licheniformis and Bsoronensis groups) a known plant associated bacterium andan opportunistic pathogen in immune-compromised humanindividuals drawing attention to the possible pathogenicaction of endophytic bacteria in humans [59 60]

The overall analysis of the dendrograms points out thatthe characterization of isolates from endophytic communitiescan lead to the identification (as an example for Pantoeaand rhizobia) of not yet described strains that may representuntapped resources of bioactive compounds of agricultural ormedical interest

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

14 Evidence-Based Complementary and Alternative Medicine

context the characterization of cultivable bacterial commu-nities is a fundamental step to this purpose since it paves theway to the possibility to build collections of isolates that maybe phenotypically typed for important traits In our opiniondata obtained in this work (Table 4 and Supplementary Tables2 and 3) offers a preliminary but very promising exampleof the biotechnological potential of bacteria isolated frommedicinal plants In this pilot study in fact the majority ofthe tested endophytes showed to inhibit the growth of several(in some case all) human pathogenic strains belonging tothe Bcc complexes that are known for their resistance totraditional antibiotics These data are particularly interestingif correlated with the recent finding that essential oils fromdifferent medicinal plants are able to strongly (or completely)interfere with the growth of the same Bcc strains [61] and thatbacterial endophytes isolated from plants of the same speciesexhibited the same bioactivity (Maida et al in preparation)It is therefore possible that the therapeutic properties of theessential oil might reside also on bioactive compounds ofmicrobial origin We are completely aware that the determi-nation of the correlation (eventually) existing between thecomposition of bacterial endophytic communities and thebioactivity of essential oils extracted from a medicinal plantwould require the parallel analysis of the bacterial communityand the essential oil activity extracted from the same plantHowever data obtained in this work and those from Maidaet al [62] support this idea

It is also particularly intriguing the idea that the antimi-crobial activity of essential oils may reside on the actionof multiple compounds some of them produced or elicitedby the microbiota limiting the observed rapid evolutionof human pathogenic bacteria resistant to single moleculeantimicrobials

The characterization of the heterotrophic aerobic endo-phytic bacterial community of L angustifolia highlightedthe existence of a diversified community between differentorganstissues that may be related to the coexistence ofdifferent sources of inoculum andor a selection of the com-munities promoted by nutrient sand metabolites availabilityand antagonistic forces Several not previously characterizedstrains have been isolated and molecularly typed confirmingthat the analysis of the bacterial communities inhabitingextreme or unconventional environments may represent aproper strategy for the discovery of untapped sources offunctional biodiversity and bioactive molecule production

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This work was supported by grants from the Ente Cassadi Risparmio di Firenze (Grant 20130657 Herbiome newantimicrobial compounds from endophytic bacteria isolatedfrom medicinal plants) Giovanni Emiliani is financially

supported by the Regione Toscana POR CRO FSE 2007-2013 Asse IV Grant Sysbiofor (CNR-9) Marco Fondi andElena Perrin are financially supported by the FEMSAdvancedFellowship (FAF2012) and the Buzzati-Traverso Foundationrespectively

References

[1] B Reinhold-Hurek andTHurek ldquoLiving inside plants bacterialendophytesrdquo Current Opinion in Plant Biology vol 14 no 4 pp435ndash443 2011

[2] M Rosenblueth and E Martınez-Romero ldquoBacterial endo-phytes and their interactions with hostsrdquo Molecular Plant-Microbe Interactions vol 19 no 8 pp 827ndash837 2006

[3] R P Ryan K Germaine A Franks D J Ryan and DN Dowling ldquoBacterial endophytes recent developments andapplicationsrdquo FEMSMicrobiology Letters vol 278 no 1 pp 1ndash92008

[4] R Cakmakci F Donmez A Aydın and F Sahin ldquoGrowthpromotion of plants by plant growth-promoting rhizobacteriaunder greenhouse and two different field soil conditionsrdquo SoilBiology and Biochemistry vol 38 pp 1482ndash1487 2006

[5] P R Hardoim L S van Overbeek and J D V Elsas ldquoProp-erties of bacterial endophytes and their proposed role in plantgrowthrdquo Trends in Microbiology vol 16 no 10 pp 463ndash4712008

[6] S Compant C Clement and A Sessitsch ldquoPlant growth-promoting bacteria in the rhizo- and endosphere of plantstheir role colonization mechanisms involved and prospects forutilizationrdquo Soil Biology and Biochemistry vol 42 no 5 pp669ndash678 2010

[7] B R Glick ldquoBacterial ACC deaminase and the alleviation ofplant stressrdquo Advances in Applied Microbiology vol 56 pp 291ndash312 2004

[8] B R Glick B Todorovic J Czarny Z Cheng J Duan andB McConkey ldquoPromotion of plant growth by bacterial ACCdeaminaserdquo Critical Reviews in Plant Sciences vol 26 no 5-6pp 227ndash242 2007

[9] S L Doty B Oakley G Xin et al ldquoDiazotrophic endophytesof native black cottonwood and willowrdquo Symbiosis vol 47 no 1pp 23ndash33 2009

[10] B Lugtenberg and F Kamilova ldquoPlant-growth-promoting rhi-zobacteriardquoAnnual Review ofMicrobiology vol 63 pp 541ndash5562009

[11] U F Castillo G A Strobel E J Ford et al ldquoMunumbicinswide-spectrum antibiotics produced by Streptomyces NRRL30562 endophytic on Kennedia nigriscansrdquo Microbiology vol148 no 9 pp 2675ndash2685 2002

[12] Y Igarashi M E Trujillo E Martınez-Molina et al ldquoAnti-tumor anthraquinones from an endophytic actinomyceteMicromonospora lupini sp novrdquo Bioorganic amp Medicinal Chem-istry Letters vol 17 no 13 pp 3702ndash3705 2007

[13] S Shweta J H Bindu J Raghu et al ldquoIsolation of endophyticbacteria producing the anti-cancer alkaloid camptothecinefromMiquelia dentata Bedd (Icacinaceae)rdquo Phytomedicine vol20 pp 913ndash917 2013

[14] T Taechowisan A Wanbanjob P Tuntiwachwuttikul and JLiu ldquoAnti-inflammatory activity of lansais from endophyticStreptomyces sp SUC1 in LPS-induced RAW 2647 cellsrdquo Foodand Agricultural Immunology vol 20 no 1 pp 67ndash77 2009

Evidence-Based Complementary and Alternative Medicine 15

[15] P R Hardoim C C P Hardoim L S van Overbeek and J Dvan Elsas ldquoDynamics of seed-borne rice endophytes on earlyplant growth stagesrdquo PLoS ONE vol 7 no 2 Article ID e304382012

[16] D Bulgarelli K Schlaeppi S Spaepen E V L van Themaatand P Schulze-Lefert ldquoStructure and functions of the bacterialmicrobiota of plantsrdquo Annual Review of Plant Biology vol 64pp 807ndash838 2013

[17] A Mengoni S Mocali G Surico S Tegli and R Fani ldquoFluctu-ation of endophytic bacteria and phytoplasmosis in elm treesrdquoMicrobiological Research vol 158 no 4 pp 363ndash369 2003

[18] A Mengoni F Pini L-N Huang W-S Shu and MBazzicalupo ldquoPlant-by-plant variations of bacterial commu-nities associated with leaves of the nickel hyperaccumulatorAlyssum bertolonii desvrdquo Microbial Ecology vol 58 no 3 pp660ndash667 2009

[19] S Mocali E Bertelli F di Cello et al ldquoFluctuation of bacteriaisolated from elm tissues during different seasons and fromdifferent plant organsrdquo Research in Microbiology vol 154 no2 pp 105ndash114 2003

[20] F Pini A Frascella L Santopolo et al ldquoExploring the plant-associated bacterial communities in Medicago sativa Lrdquo BMCMicrobiology vol 12 article 78 2012

[21] A Mengoni L Cecchi and C Gonnelli ldquoNickel hyperac-cumulating plants and Alyssum bertolonii model systems forstudying biogeochemical interactions in serpentine soilsrdquo inBio-Geo Interactions in Metal-Contaminated Soils E Kothe andA Varma Eds pp 279ndash296 Springer Berlin Germany 2012

[22] S Ikeda T Okubo M Anda et al ldquoCommunity- and genome-based views of plant-associated bacteria plant-bacterial inter-actions in soybean and ricerdquo Plant and Cell Physiology vol 51no 9 pp 1398ndash1410 2010

[23] F Pini M Galardini M Bazzicalupo and A Mengoni ldquoPlant-bacteria association and symbiosis are there common genomictraits in alphaproteobacteriardquoGenes vol 2 no 4 pp 1017ndash10322011

[24] K Aleklett and M Hart ldquoThe root microbiota a fingerprint inthe soilrdquo Plant Soil vol 370 pp 671ndash686 2013

[25] A Beneduzi F Moreira P B Costa et al ldquoDiversity and plantgrowth promoting evaluation abilities of bacteria isolated fromsugarcane cultivated in the South of BrazilrdquoApplied Soil Ecologyvol 63 pp 94ndash104 2013

[26] N Bodenhausen M W Horton and J Bergelson ldquoBacterialcommunities associated with the leaves and the roots of Ara-bidopsis thalianardquo PLoS ONE vol 8 no 2 Article ID e563292013

[27] T F da Silva R E Vollu D Jurelevicius et al ldquoDoes theessential oil of Lippia sidoides Cham (pepper-rosmarin) affectits endophytic microbial communityrdquo BMC Microbiology vol13 no 1 article 29 2013

[28] M E Lucero A Unc P Cooke S Dowd and S SunldquoEndophyte microbiome diversity in micropropagated Atriplexcanescens and Atriplex torreyi var griffithsiirdquo PLoS ONE vol 6no 3 Article ID e17693 2011

[29] D S Lundberg S L Lebeis S H Paredes et al ldquoDefining thecoreArabidopsis thaliana rootmicrobiomerdquoNature vol 487 no7409 pp 86ndash90 2012

[30] H M Cavanagh and J M Wilkinson ldquoBiological activities oflavender essential oilrdquo Phytotherapy Research vol 16 no 4 pp301ndash308 2002

[31] G Woronuk Z Demissie M Rheault and S MahmoudldquoBiosynthesis and therapeutic properties of Lavandula essentialoil constituentsrdquo Planta Medica vol 77 no 1 pp 7ndash15 2011

[32] R Perry R Terry L K Watson and E Ernst ldquoIs lavender ananxiolytic drug A systematic review of randomised clinicaltrialsrdquo Phytomedicine vol 19 pp 825ndash835 2012

[33] S de Rapper G Kamatou A Viljoen and S van VuurenldquoThe in vitro antimicrobial activity of Lavandula angustifoliaessential oil in combination with other aroma-therapeutic oilsrdquoEvidence-Based Complementary and Alternative Medicine vol2013 Article ID 852049 10 pages 2013

[34] T Moon J M Wilkinson and H M Cavanagh ldquoAntiparasiticactivity of two Lavandula essential oils against Giardia duode-nalis Trichomonas vaginalis andHexamita inflatardquo ParasitologyResearch vol 99 no 6 pp 722ndash728 2006

[35] P S Yap S H Lim C P Hu and B C Yiap ldquoCom-bination of essential oils and antibiotics reduce antibioticresistance in plasmid-conferred multidrug resistant bacteriardquoPhytomedicine vol 20 pp 710ndash713 2013

[36] G Brader S Compant B Mitter F Trognitz and A SessitschldquoMetabolic potential of endophytic bacteriardquo Current Opinionin Biotechnology vol 27 pp 30ndash37 2014

[37] P Drevinek and E Mahenthiralingam ldquoBurkholderia ceno-cepacia in cystic fibrosis epidemiology and molecular mecha-nisms of virulencerdquo Clinical Microbiology and Infection vol 16no 7 pp 821ndash830 2010

[38] S Bazzini C Udine and G Riccardi ldquoMolecular approaches topathogenesis study of Burkholderia cenocepacia an importantcystic fibrosis opportunistic bacteriumrdquo Applied Microbiologyand Biotechnology vol 92 no 5 pp 887ndash895 2011

[39] I Maida A lo Nostro G Pesavento et al ldquoExploring theanti-Burkholderia cepacia complex activity of essential oilsa preliminary analysisrdquo Evidence-Based Complementary andAlternativeMedicine vol 2014 Article ID 573518 10 pages 2014

[40] A Mengoni R Barzanti C Gonnelli R Gabbrielli andM Bazzicalupo ldquoCharacterization of nickel-resistant bacteriaisolated from serpentine soilrdquo Environmental Microbiology vol3 no 11 pp 691ndash698 2001

[41] S F Altschul T L Madden A A Schaffer et al ldquoGappedBLAST and PSI-BLAST a new generation of protein databasesearch programsrdquo Nucleic Acids Research vol 25 no 17 pp3389ndash3402 1997

[42] R C Edgar ldquoMUSCLE multiple sequence alignment with highaccuracy and high throughputrdquo Nucleic Acids Research vol 32no 5 pp 1792ndash1797 2004

[43] F Ronquist M Teslenko P van der Mark et al ldquoMrbayes32 efficient bayesian phylogenetic inference and model choiceacross a large model spacerdquo Systematic Biology vol 61 no 3 pp539ndash542 2012

[44] K Tamura D Peterson N Peterson G Stecher M Nei andS Kumar ldquoMEGA5 molecular evolutionary genetics analysisusing maximum likelihood evolutionary distance and max-imum parsimony methodsrdquo Molecular Biology and Evolutionvol 28 no 10 pp 2731ndash2739 2011

[45] F Sievers A Wilm D Dineen et al ldquoFast scalable generationof high-quality protein multiple sequence alignments usingClustal Omegardquo Molecular Systems Biology vol 7 article 5392011

[46] Oslash Hammer D A T Harper and P D Ryan ldquoPAST pale-ontological statistics software package for education and dataanalysisrdquo Palaeontologia Electronica vol 4 no 1 pp 1ndash9 2001

16 Evidence-Based Complementary and Alternative Medicine

[47] M C Papaleo M Fondi I Maida et al ldquoSponge-associatedmicrobial Antarctic communities exhibiting antimicrobialactivity againstBurkholderia cepacia complex bacteriardquoBiotech-nology Advances vol 30 no 1 pp 272ndash293 2012

[48] A lo Giudice V Bruni and L Michaud ldquoCharacterization ofAntarctic psychrotrophic bacteria with antibacterial activitiesagainst terrestrial microorganismsrdquo Journal of Basic Microbiol-ogy vol 47 no 6 pp 496ndash505 2007

[49] A Schippers K Bosecker C Sproer and P SchumannldquoMicrobacterium oleivorans sp nov and Microbacteriumhydrocarbonoxydans sp nov novel crude-oil-degrading Gram-positive bacteriardquo International Journal of Systematic and Evo-lutionary Microbiology vol 55 no 2 pp 655ndash660 2005

[50] J D McChesney S K Venkataraman and J T Henri ldquoPlantnatural products back to the future or into extinctionrdquo Phyto-chemistry vol 68 no 14 pp 2015ndash2022 2007

[51] D K Manter J A Delgado D G Holm and R A StongldquoPyrosequencing reveals a highly diverse and cultivar-specificbacterial endophyte community in potato rootsrdquo MicrobialEcology vol 60 no 1 pp 157ndash166 2010

[52] K Ulrich A Ulrich and D Ewald ldquoDiversity of endophyticbacterial communities in poplar grown under field conditionsrdquoFEMS Microbiology Ecology vol 63 no 2 pp 169ndash180 2008

[53] N R Gottel H F Castro M Kerley et al ldquoDistinct microbialcommunities within the endosphere and rhizosphere ofPopulusdeltoides roots across contrasting soil typesrdquo Applied and Envi-ronmental Microbiology vol 77 no 17 pp 5934ndash5944 2011

[54] B Ma X Lv A Warren and J Gong ldquoShifts in diversity andcommunity structure of endophytic bacteria and archaea acrossroot stem and leaf tissues in the common reed Phragmitesaustralis along a salinity gradient in a marine tidal wetland ofnorthern Chinardquo Antonie van Leeuwenhoek vol 104 no 5 pp759ndash768 2013

[55] J A Vorholt ldquoMicrobial life in the phyllosphererdquo NatureReviews Microbiology vol 10 no 12 pp 828ndash840 2012

[56] R P Ryan S Monchy M Cardinale et al ldquoThe versatilityand adaptation of bacteria from the genus StenotrophomonasrdquoNature Reviews Microbiology vol 7 no 7 pp 514ndash525 2009

[57] AWolf A FritzeMHagemann andG Berg ldquoStenotrophomo-nas rhizophila sp nov a novel plant-associated bacterium withantifungal propertiesrdquo International Journal of Systematic andEvolutionary Microbiology vol 52 no 6 pp 1937ndash1944 2002

[58] C W Bacon and D M Hinton ldquoEndophytic and biologicalcontrol potential of Bacillus mojavensis and related speciesrdquoBiological Control vol 23 no 3 pp 274ndash284 2002

[59] G Berg L Eberl and A Hartmann ldquoThe rhizosphere as areservoir for opportunistic human pathogenic bacteriardquo Envi-ronmental Microbiology vol 7 no 11 pp 1673ndash1685 2005

[60] H L Tyler and E W Triplett ldquoPlants as a habitat for ben-eficial andor human pathogenic bacteriardquo Annual Review ofPhytopathology vol 46 pp 53ndash73 2008

[61] R Perry R Terry L K Watson and E Ernst ldquoIs lavender ananxiolytic drug A systematic review of randomised clinicaltrialsrdquo Phytomedicine vol 19 no 8-9 pp 825ndash835 2012

[62] I Maida A lo Nostro G Pesavento et al ldquoExploring theanti-Burkholderia cepacia complex activity of essential oilsa preliminary analysisrdquo Evidence-Based Complementary andAlternativeMedicine vol 2014 Article ID 573518 10 pages 2014

Evidence-Based Complementary and Alternative Medicine 11

Table 3The 40Burkholderia cepacia complex strains used as targetsin the cross streak experiments

Target strainStrain Species OriginFCF1 B cepacia CFFCF3LMG17588 B multivorans ENVFCF16 B cenocepacia (IIIA) CFJ2315FCF18

B cenocepacia (IIIB) CF

FCF20FCF23FCF24FCF27FCF29FCF30LMG16654C5424CEP511MVPC116 ENVMVPC173LMG19230 B cenocepacia (IIIC) ENVLMG19240FCF38 B cenocepacia (IIID) CFLMG21462FCF41 B stabilis CFFCF42 B vietnamiensis CFTVV75 ENVLMG18941

B dolosa CFLMG18942LMG18943MCI7

B ambifariaENV

LMG19467 CFLMG19182 ENVLMG16670 B anthina ENVFCF43 B pyrrocina CFLSED4 B lata CFLMG24064 B latens CFLMG24065 B diffusa CFLMG23361 B contaminans AILMG24067 B seminals CFLMG24068 B metallica CFLMG24066 B arboris ENVLMG24263 B ubonensis NIAbbreviations CF strains isolated from cystic fibrosis patients AI strainsisolated from animal infection NI strains isolated from nosocomial infec-tion ENV environmental strain

isolates showed similarities with the stress resistantB safensis

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

complex Regardless of the overall low support ofthe tree (typical of Pseudomonas phylogenies) someobservations may be drawn the presence of largeunresolved clusters shows clearly a low divergenceof the isolated colonies implying the existence ofclonal populations Pseudomonas spp isolated fromthe different compartments are intermixed suggest-ing the presence of a continuum rhizosphere-leaves(Pseudomonas is the only genus found in all the 4sample analysed)

32 Inhibition of Burkholderia Cepacia Complex StrainsGrowth by L angustifolia Endophytic Bacteria In order tocheck the ability of L angustifolia bacterial endophytes toantagonize the growth of (opportunistic) human pathogenicbacteria cross-streak experiments were carried out asdescribed in Section 2 using a panel of the 19 randomlychosen different endophytic strains isolated from soil rootsor leafs and phylogenetically assigned to six different gen-era (Bacillus Microbacterium Pantoea Plantibacter Pseu-domonas and Rhizobium) as testers versus 40 Bcc strainsrepresentative of seventeen species (out of eighteen) andwith either environmental or clinical origin with some ofwhich being multidrug-resistant Data obtained are shownin Table 4 The analysis of these data revealed that all thetester strains were able to completely inhibit the growthof Bcc strains including those with a clinical source andthat exhibited multidrug resistance this finding suggestedthat lavender endophytic bacteria are able to synthesize(strong) antimicrobial compounds However tester strainsshowed a different pattern of Bcc growth inhibition eventhose affiliated to the same genus In addition to this thereis no apparent difference in the antimicrobial potentialbetween strains belonging to different plant compartmentsConcerning the sensitivity of Bcc strains to the antimicrobialactivity of lavender endophytes strains belonging to differentspecies exhibited a different sensitivity spectrum However itis quite interesting that strains with clinical origin appearedto be more sensitive to the antimicrobials synthesized byendophytic bacteria than their environmental counterparts(see for instance strains belonging to the species Burkholderiacenocepacia IIIB) (Supplementary Table 3)

4 Discussion

Medicinal plants are stirring the attention of manyresearchers due to the presence of compounds thatconstitute a large fraction of the current pharmacopoeiasNatural products have been the source of most of theactive ingredients of medicines and more than 80 of drugsubstances are natural products or inspired by a naturalcompound including most of the of anticancer and anti-infective agents [50] Lavender plants are grown mainlyfor the production of essential oil which has antisepticand anti-inflammatory properties In spite of the relativeharsh environment for bacterial colonization L angustifoliatissues were found to be rich in bacterial diversity Howeverthe endophytic community isolated from different plant

12 Evidence-Based Complementary and Alternative Medicine

Table4Growth

of40

Burkholderiacepacia

complex

strains

inthep

resenceabsenceo

fend

ophytic

bacterialstrains

isolated

from

lavend

er

Species

Strain

Orig

inLL

1LL

2LL

3LL

4LL

6LL

7LL

9LL

10LS

1LS

2LS

3LS

4LS

5LS

6LR

1LR

2LR

3LR

4LR

5C-

Bcepacia

FCF1

CFminusminusminusminusminusminusminusminus

minusminusminusminusminusminusminusminusminusminusminus

+B

cepacia

FCF3

CF+minusminusminusminusminusminusminusminus

minusminusminusminusminusminusminusminusminusminusminus

+B

multivoran

sLM

G17588

Env

+minusminus

++minus

+minusminus

+minus

+minus

++minus

+minusminus

+minus

+minusminus

+B

cenocepacia

(IIIA

)FC

F16

CFminusminusminusminusminusminusminusminus

minusminusminusminusminusminusminusminusminusminusminus

+B

cenocepacia

(IIIA

)J2315

CF+minusminus

+minus

+minusminusminus

+minusminusminus

+minusminusminusminusminusminusminusminus

+B

cenocepacia

(IIIB)

FCF18

CF+minusminusminusminusminusminusminus

minusminusminusminusminusminus

+minusminusminusminusminus

+B

cenocepacia

(IIIB)

FCF20

CF+

++

++

+minus

++minus

+minus

+minus

+minus

++minus

++minus

++

++

+B

cenocepacia

(IIIB)

FCF23

CF+minusminusminusminusminusminusminus

+minusminusminus

++minusminusminus

+minusminusminusminus

+B

cenocepacia

(IIIB)

FCF24

CF+minusminusminusminusminusminusminus

+minusminus

+minusminusminusminus

+minusminusminusminus

+B

cenocepacia

(IIIB)

FCF27

CF+minusminusminusminusminusminusminus

+minusminusminusminusminusminusminusminusminusminusminus

+B

cenocepacia

(IIIB)

FCF29

CF+minusminus

+minus

+minusminusminus

+minusminusminusminusminusminusminus

+minus

+minusminus

+B

cenocepacia

(IIIB)

FCF30

CF+minusminus

+minus

+minusminusminus

+minus

+minusminusminusminusminusminus

+minus

+minusminus

+B

cenocepacia

(IIIB)

LMG16654

CF+

+minusminus

++minus

+minus

++minus

++minusminus

+minus

+minusminus

+B

cenocepacia

(IIIB)

C5424

CF+

+minusminus

+minusminusminusminus

+minusminusminus

+minusminusminusminus

+minusminus

+minusminus

+B

cenocepacia

(IIIB)

CEP5

11CF

+minusminus

+minusminusminusminus

+minus

+minusminus

+minus

+minusminusminus

+minus

+minusminus

+B

cenocepacia

(IIIB)

MVPC

116

Env

++minusminus

+minus

++minusminus

+minus

+minus

+minus

+minusminus

+minusminus

+minusminus

+minus

+minus

+B

cenocepacia

(IIIB)

MVPC

173

Env

++

++minus

++

+minus

++

++

++

++

++

++minus

+B

cenocepacia

(IIIIC)

LMG19230

Env

++

++

++

+minus

++

++

++

++

++

++

+B

cenocepacia

(IIIC

)LM

G19240

Env

++

++

+minus

+minus

++

+minus

++

++minus

++

++

+minus

+B

cenocepacia

(IIID)

FCF38

CF+minusminusminusminusminusminusminus

minusminusminusminusminusminusminusminusminusminusminus

+B

cenocepacia

(IIID)

LMG21462

CFminusminusminusminusminusminusminusminus

minusminusminusminusminusminusminusminusminusminusminus

+B

stabilis

FCF41

CF+

+minus

++

+minus

+minusminus

++

+minus

++minusminus

++

++minusminus

+B

vietnamien

sisFC

F42

CFminusminusminusminusminusminusminusminus

minusminusminusminusminusminusminusminusminusminusminus

+B

vietnamien

sisTV

V75

Env

++minusminus

+minusminusminusminus

+minusminusminus

++minusminusminus

+minusminus

+minusminus

+B

dolosa

LMG18941

CF+minusminus

+minus

+minus

+minusminus

+minusminusminus

++minusminusminus

+minus

+minus

+minusminus

+B

dolosa

LMG18942

CF+

+minusminus

+minus

+minusminus

++

+minus

++minusminusminus

++minus

++minusminus

+B

dolosa

LMG18943

CF+

+minusminus

++minus

+minusminus

+minus

+minusminus

++minusminusminus

++minus

+minusminus

+B

ambifaria

MCI

7En

v+

+minus

+minus

+minus

+minusminus

++minusminus

++minusminusminus

+minus

+minusminus

+B

ambifaria

LMG1946

7CF

++minus

+minus

+minusminusminus

++minusminus

++minusminusminus

+minus

+minusminusminus

+B

ambifaria

LMG19182

Env

++minus

+minus

+minusminusminus

+minusminus

+minusminusminus

+minus

+minusminusminus

+B

anthina

LMG16670

Env

+minusminusminusminus

+minus

+minusminus

++minusminusminus

+minus

+minusminusminus

+B

pyrrocinia

FCF43

CF+minusminusminusminusminusminusminusminus

minusminus

+minusminusminusminusminusminus

+minusminusminus

+B

lata

LSED

4CF

+minus

+minusminus

+minusminus

+minusminus

++minusminusminus

+minus

+minus

+minusminusminus

+B

latens

LMG2406

4CF

+minus

++minusminus

+minusminus

++minus

++minus

++

++

+minus

+B

diffu

saLM

G24065

CF+

+minus

++minus

+minus

+minus

++minus

++minus

+minus

++

++minus

+B

contam

inan

sLM

G23361

AI

++minus

+minus

+minus

+minus

+minus

++

++

++

+minus

++

++minus

+B

seminalis

LMG24067

CF+

+minus

++minus

++

++

++

++

++

++

++minus

+B

metallica

LMG2406

8CF

++

++minus

++

++

++

++

++

++

++minus

+B

arboris

LMG2406

6En

v+

+minus

++minus

++

+minus

++

++

++minus

+minus

++

++minusminus

+B

ubonensis

LMG24263

NI

+minusminusminusminus

+minusminus

+minus

+minus

++minusminus

+minusminus

+minusminusminus

+Ab

breviatio

nsC

Fstr

ains

isolatedfro

mcysticfi

brosispatie

ntsAIstr

ains

isolated

from

anim

alinfectionNIstr

ains

isolated

from

nosocomialinfectio

nEN

Venvironm

entalstrainLstr

ains

isolatedfro

mtheleaf

Sstr

ains

isolatedfro

mthes

temR

strains

isolatedfro

mther

oot

Symbo

ls+

grow

th+

minusreduced

grow

th+

minusminusveryredu

cedgrow

thminus

nogrow

thC

-Petrid

ishes

containing

onlythetargetstrains

Evidence-Based Complementary and Alternative Medicine 13

compartments showed different levels of diversity withthe 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 theleaf and roots community is already reported in otherspecies [26] even if there are few direct comparisons ofroots and phyllosphere bacterial communities especiallycomparisons using material from the same plants Themicrobial community residing in the phyllosphere is facedwith a nutrient poor and variable environment that ischaracterized by fluctuating temperature humidity and UVradiation and so it is predicted to be more diverse than thatwithin the rhizosphere a more homeostatic environmentThese findings supported also by the similar bacterial titreare in agreement with the idea that the source of inoculumfor the leaf community (except for the vertically transferredvia seeds) may be exclusive (eg aerosol even if the processis not yet clarified Bulgarelli et al 2013) and not related totransmission from the roots Moreover the low diversity ofthe community isolated in the stem dominated by clonally(Table 2) populations of Pseudomonas could be related tothe highly specific main tissue (phloem) present there whichcould provide an homeostatic environment rich in nutrient(sucrose contained in the phloem tissues) but poorer (ifcompared to leaf and roots) in secondary metabolites thatmay promote ecological niche differentiation

Concerning the taxonomic community composition dif-ferent taxonomic profiles were found for endosphere andrhizosphere More in detail the rhizosphere communityshowed quite similar diversity indexes if compared to theroots one but with a different composition as an examplemembers of the phylumActinobacteria are completely absentfrom the root internal tissues that are largely dominatedby Proteobacteria while they are present in the soil Toexplain this finding a two-step model has been proposed[16] soil abiotic properties determine the structure of theinitial bacterial communities that is then modified by rhi-zodeposits with plant roots cell wall features and releasedmetabolites promoting the growth of organotrophic bacteriathereby initiating a soil community shift In the second stepconvergent host genotype-dependent selection [51 52] fine-tunes community profiles thriving on the rhizoplane andwithin plant roots

Moreover endosphere communities were differentbetween plant organs (roots stems and leaves) even if theinternal tissues are dominated as already reported [25 52ndash54] by members of the phylum Proteobacteria an examplewhile rhizobia were isolated from root internal tissues theyare completely absent from stems and leaves (Figures 2 and3) isolates belonging to the genus Pantoea on the contrarywere isolated from aerial parts but not from root tissuesHence it is possible that plant might ldquoselectrdquo specific taxafor entering inside its compartment Such hypothesis issupported also by the pairwise differentiation values thecommunities isolated from the different tissues even those inphysical continuity are in fact strongly differentiated with thestem representing a low diversity compartment separatingthe two high diversity organs roots and leaves In this view itis noteworthy that in lavender only Pseudomonas represents

a ubiquitous and abundant genus of both rhizosphere andendosphere (Figure 3) The analysis of intrageneric diversitypointed out also that Pseudomonas isolates belong to a lowdifferentiated clonal population (Table 2 and Suppl Figure2) suggesting also that this component of the communityeither diffused inside the organs from a radical or foliar entrypoint or established during plants development putativelyfrom the seed inoculum A similar consideration can beproposed for isolates of rhizobia but for the rhizosphere-root internal tissues continuum a low taxonomicallydifferentiated community from the soil entered (increasingits relative abundance) inside the plant organ An entrypoint from the leaves is on the contrary consistent with thedistribution of Pantoea isolates with their strong diversity(Table 2) suggesting a diversification in response to the highvariable leaf environmentThe isolates belonging to the genusStenotrophomonas show an interesting distribution pattern(Figure 3) they are rare in the rhizosphere abundant in theroots and in the leaves and absent in the stem this findingsuggests a different entry point or a negative selection in thestem the second explanation is supported by admixture ofroot and leaves isolates (Figure 4)

The detailed phylogenetic analyses of the isolates enabledus also to putatively ascribe some isolates to already describedendophytic strains that can be targeted for functional charac-terisation many Microbacterium leaves isolates cluster withM hydrocarbonoxydans and M oleivorans two species withcrude oil degrading activity [49] being this metabolic activitythat is found frequently associated with a phyllosphereadapted lifestyle [55] Several isolates cluster with bacteriawith interesting biotechnological or ecological applicationssuch as S chelatiphaga a remarkable species with biotech-nological potential in phytoremediation [56] and S rizophilaa plant associated bacterium with antifungal activity [57] orBacillus mojavensis a bacteriumclosely related to B subtilisand already reported having an endophytic lifestyle andshowing an antagonistic role against plant pathogenic fungi[58] On the contrary many lavender Pseudomonas isolatescluster with known plant pathogen strains highlighting thesometimes subtle difference among pathogenic and endo-phytic lifestyle and with Pantoea agglomerans (and also withlavender isolates clustering withinthe B licheniformis and Bsoronensis groups) a known plant associated bacterium andan opportunistic pathogen in immune-compromised humanindividuals drawing attention to the possible pathogenicaction of endophytic bacteria in humans [59 60]

The overall analysis of the dendrograms points out thatthe characterization of isolates from endophytic communitiescan lead to the identification (as an example for Pantoeaand rhizobia) of not yet described strains that may representuntapped resources of bioactive compounds of agricultural ormedical interest

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

14 Evidence-Based Complementary and Alternative Medicine

context the characterization of cultivable bacterial commu-nities is a fundamental step to this purpose since it paves theway to the possibility to build collections of isolates that maybe phenotypically typed for important traits In our opiniondata obtained in this work (Table 4 and Supplementary Tables2 and 3) offers a preliminary but very promising exampleof the biotechnological potential of bacteria isolated frommedicinal plants In this pilot study in fact the majority ofthe tested endophytes showed to inhibit the growth of several(in some case all) human pathogenic strains belonging tothe Bcc complexes that are known for their resistance totraditional antibiotics These data are particularly interestingif correlated with the recent finding that essential oils fromdifferent medicinal plants are able to strongly (or completely)interfere with the growth of the same Bcc strains [61] and thatbacterial endophytes isolated from plants of the same speciesexhibited the same bioactivity (Maida et al in preparation)It is therefore possible that the therapeutic properties of theessential oil might reside also on bioactive compounds ofmicrobial origin We are completely aware that the determi-nation of the correlation (eventually) existing between thecomposition of bacterial endophytic communities and thebioactivity of essential oils extracted from a medicinal plantwould require the parallel analysis of the bacterial communityand the essential oil activity extracted from the same plantHowever data obtained in this work and those from Maidaet al [62] support this idea

It is also particularly intriguing the idea that the antimi-crobial activity of essential oils may reside on the actionof multiple compounds some of them produced or elicitedby the microbiota limiting the observed rapid evolutionof human pathogenic bacteria resistant to single moleculeantimicrobials

The characterization of the heterotrophic aerobic endo-phytic bacterial community of L angustifolia highlightedthe existence of a diversified community between differentorganstissues that may be related to the coexistence ofdifferent sources of inoculum andor a selection of the com-munities promoted by nutrient sand metabolites availabilityand antagonistic forces Several not previously characterizedstrains have been isolated and molecularly typed confirmingthat the analysis of the bacterial communities inhabitingextreme or unconventional environments may represent aproper strategy for the discovery of untapped sources offunctional biodiversity and bioactive molecule production

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This work was supported by grants from the Ente Cassadi Risparmio di Firenze (Grant 20130657 Herbiome newantimicrobial compounds from endophytic bacteria isolatedfrom medicinal plants) Giovanni Emiliani is financially

supported by the Regione Toscana POR CRO FSE 2007-2013 Asse IV Grant Sysbiofor (CNR-9) Marco Fondi andElena Perrin are financially supported by the FEMSAdvancedFellowship (FAF2012) and the Buzzati-Traverso Foundationrespectively

References

[1] B Reinhold-Hurek andTHurek ldquoLiving inside plants bacterialendophytesrdquo Current Opinion in Plant Biology vol 14 no 4 pp435ndash443 2011

[2] M Rosenblueth and E Martınez-Romero ldquoBacterial endo-phytes and their interactions with hostsrdquo Molecular Plant-Microbe Interactions vol 19 no 8 pp 827ndash837 2006

[3] R P Ryan K Germaine A Franks D J Ryan and DN Dowling ldquoBacterial endophytes recent developments andapplicationsrdquo FEMSMicrobiology Letters vol 278 no 1 pp 1ndash92008

[4] R Cakmakci F Donmez A Aydın and F Sahin ldquoGrowthpromotion of plants by plant growth-promoting rhizobacteriaunder greenhouse and two different field soil conditionsrdquo SoilBiology and Biochemistry vol 38 pp 1482ndash1487 2006

[5] P R Hardoim L S van Overbeek and J D V Elsas ldquoProp-erties of bacterial endophytes and their proposed role in plantgrowthrdquo Trends in Microbiology vol 16 no 10 pp 463ndash4712008

[6] S Compant C Clement and A Sessitsch ldquoPlant growth-promoting bacteria in the rhizo- and endosphere of plantstheir role colonization mechanisms involved and prospects forutilizationrdquo Soil Biology and Biochemistry vol 42 no 5 pp669ndash678 2010

[7] B R Glick ldquoBacterial ACC deaminase and the alleviation ofplant stressrdquo Advances in Applied Microbiology vol 56 pp 291ndash312 2004

[8] B R Glick B Todorovic J Czarny Z Cheng J Duan andB McConkey ldquoPromotion of plant growth by bacterial ACCdeaminaserdquo Critical Reviews in Plant Sciences vol 26 no 5-6pp 227ndash242 2007

[9] S L Doty B Oakley G Xin et al ldquoDiazotrophic endophytesof native black cottonwood and willowrdquo Symbiosis vol 47 no 1pp 23ndash33 2009

[10] B Lugtenberg and F Kamilova ldquoPlant-growth-promoting rhi-zobacteriardquoAnnual Review ofMicrobiology vol 63 pp 541ndash5562009

[11] U F Castillo G A Strobel E J Ford et al ldquoMunumbicinswide-spectrum antibiotics produced by Streptomyces NRRL30562 endophytic on Kennedia nigriscansrdquo Microbiology vol148 no 9 pp 2675ndash2685 2002

[12] Y Igarashi M E Trujillo E Martınez-Molina et al ldquoAnti-tumor anthraquinones from an endophytic actinomyceteMicromonospora lupini sp novrdquo Bioorganic amp Medicinal Chem-istry Letters vol 17 no 13 pp 3702ndash3705 2007

[13] S Shweta J H Bindu J Raghu et al ldquoIsolation of endophyticbacteria producing the anti-cancer alkaloid camptothecinefromMiquelia dentata Bedd (Icacinaceae)rdquo Phytomedicine vol20 pp 913ndash917 2013

[14] T Taechowisan A Wanbanjob P Tuntiwachwuttikul and JLiu ldquoAnti-inflammatory activity of lansais from endophyticStreptomyces sp SUC1 in LPS-induced RAW 2647 cellsrdquo Foodand Agricultural Immunology vol 20 no 1 pp 67ndash77 2009

Evidence-Based Complementary and Alternative Medicine 15

[15] P R Hardoim C C P Hardoim L S van Overbeek and J Dvan Elsas ldquoDynamics of seed-borne rice endophytes on earlyplant growth stagesrdquo PLoS ONE vol 7 no 2 Article ID e304382012

[16] D Bulgarelli K Schlaeppi S Spaepen E V L van Themaatand P Schulze-Lefert ldquoStructure and functions of the bacterialmicrobiota of plantsrdquo Annual Review of Plant Biology vol 64pp 807ndash838 2013

[17] A Mengoni S Mocali G Surico S Tegli and R Fani ldquoFluctu-ation of endophytic bacteria and phytoplasmosis in elm treesrdquoMicrobiological Research vol 158 no 4 pp 363ndash369 2003

[18] A Mengoni F Pini L-N Huang W-S Shu and MBazzicalupo ldquoPlant-by-plant variations of bacterial commu-nities associated with leaves of the nickel hyperaccumulatorAlyssum bertolonii desvrdquo Microbial Ecology vol 58 no 3 pp660ndash667 2009

[19] S Mocali E Bertelli F di Cello et al ldquoFluctuation of bacteriaisolated from elm tissues during different seasons and fromdifferent plant organsrdquo Research in Microbiology vol 154 no2 pp 105ndash114 2003

[20] F Pini A Frascella L Santopolo et al ldquoExploring the plant-associated bacterial communities in Medicago sativa Lrdquo BMCMicrobiology vol 12 article 78 2012

[21] A Mengoni L Cecchi and C Gonnelli ldquoNickel hyperac-cumulating plants and Alyssum bertolonii model systems forstudying biogeochemical interactions in serpentine soilsrdquo inBio-Geo Interactions in Metal-Contaminated Soils E Kothe andA Varma Eds pp 279ndash296 Springer Berlin Germany 2012

[22] S Ikeda T Okubo M Anda et al ldquoCommunity- and genome-based views of plant-associated bacteria plant-bacterial inter-actions in soybean and ricerdquo Plant and Cell Physiology vol 51no 9 pp 1398ndash1410 2010

[23] F Pini M Galardini M Bazzicalupo and A Mengoni ldquoPlant-bacteria association and symbiosis are there common genomictraits in alphaproteobacteriardquoGenes vol 2 no 4 pp 1017ndash10322011

[24] K Aleklett and M Hart ldquoThe root microbiota a fingerprint inthe soilrdquo Plant Soil vol 370 pp 671ndash686 2013

[25] A Beneduzi F Moreira P B Costa et al ldquoDiversity and plantgrowth promoting evaluation abilities of bacteria isolated fromsugarcane cultivated in the South of BrazilrdquoApplied Soil Ecologyvol 63 pp 94ndash104 2013

[26] N Bodenhausen M W Horton and J Bergelson ldquoBacterialcommunities associated with the leaves and the roots of Ara-bidopsis thalianardquo PLoS ONE vol 8 no 2 Article ID e563292013

[27] T F da Silva R E Vollu D Jurelevicius et al ldquoDoes theessential oil of Lippia sidoides Cham (pepper-rosmarin) affectits endophytic microbial communityrdquo BMC Microbiology vol13 no 1 article 29 2013

[28] M E Lucero A Unc P Cooke S Dowd and S SunldquoEndophyte microbiome diversity in micropropagated Atriplexcanescens and Atriplex torreyi var griffithsiirdquo PLoS ONE vol 6no 3 Article ID e17693 2011

[29] D S Lundberg S L Lebeis S H Paredes et al ldquoDefining thecoreArabidopsis thaliana rootmicrobiomerdquoNature vol 487 no7409 pp 86ndash90 2012

[30] H M Cavanagh and J M Wilkinson ldquoBiological activities oflavender essential oilrdquo Phytotherapy Research vol 16 no 4 pp301ndash308 2002

[31] G Woronuk Z Demissie M Rheault and S MahmoudldquoBiosynthesis and therapeutic properties of Lavandula essentialoil constituentsrdquo Planta Medica vol 77 no 1 pp 7ndash15 2011

[32] R Perry R Terry L K Watson and E Ernst ldquoIs lavender ananxiolytic drug A systematic review of randomised clinicaltrialsrdquo Phytomedicine vol 19 pp 825ndash835 2012

[33] S de Rapper G Kamatou A Viljoen and S van VuurenldquoThe in vitro antimicrobial activity of Lavandula angustifoliaessential oil in combination with other aroma-therapeutic oilsrdquoEvidence-Based Complementary and Alternative Medicine vol2013 Article ID 852049 10 pages 2013

[34] T Moon J M Wilkinson and H M Cavanagh ldquoAntiparasiticactivity of two Lavandula essential oils against Giardia duode-nalis Trichomonas vaginalis andHexamita inflatardquo ParasitologyResearch vol 99 no 6 pp 722ndash728 2006

[35] P S Yap S H Lim C P Hu and B C Yiap ldquoCom-bination of essential oils and antibiotics reduce antibioticresistance in plasmid-conferred multidrug resistant bacteriardquoPhytomedicine vol 20 pp 710ndash713 2013

[36] G Brader S Compant B Mitter F Trognitz and A SessitschldquoMetabolic potential of endophytic bacteriardquo Current Opinionin Biotechnology vol 27 pp 30ndash37 2014

[37] P Drevinek and E Mahenthiralingam ldquoBurkholderia ceno-cepacia in cystic fibrosis epidemiology and molecular mecha-nisms of virulencerdquo Clinical Microbiology and Infection vol 16no 7 pp 821ndash830 2010

[38] S Bazzini C Udine and G Riccardi ldquoMolecular approaches topathogenesis study of Burkholderia cenocepacia an importantcystic fibrosis opportunistic bacteriumrdquo Applied Microbiologyand Biotechnology vol 92 no 5 pp 887ndash895 2011

[39] I Maida A lo Nostro G Pesavento et al ldquoExploring theanti-Burkholderia cepacia complex activity of essential oilsa preliminary analysisrdquo Evidence-Based Complementary andAlternativeMedicine vol 2014 Article ID 573518 10 pages 2014

[40] A Mengoni R Barzanti C Gonnelli R Gabbrielli andM Bazzicalupo ldquoCharacterization of nickel-resistant bacteriaisolated from serpentine soilrdquo Environmental Microbiology vol3 no 11 pp 691ndash698 2001

[41] S F Altschul T L Madden A A Schaffer et al ldquoGappedBLAST and PSI-BLAST a new generation of protein databasesearch programsrdquo Nucleic Acids Research vol 25 no 17 pp3389ndash3402 1997

[42] R C Edgar ldquoMUSCLE multiple sequence alignment with highaccuracy and high throughputrdquo Nucleic Acids Research vol 32no 5 pp 1792ndash1797 2004

[43] F Ronquist M Teslenko P van der Mark et al ldquoMrbayes32 efficient bayesian phylogenetic inference and model choiceacross a large model spacerdquo Systematic Biology vol 61 no 3 pp539ndash542 2012

[44] K Tamura D Peterson N Peterson G Stecher M Nei andS Kumar ldquoMEGA5 molecular evolutionary genetics analysisusing maximum likelihood evolutionary distance and max-imum parsimony methodsrdquo Molecular Biology and Evolutionvol 28 no 10 pp 2731ndash2739 2011

[45] F Sievers A Wilm D Dineen et al ldquoFast scalable generationof high-quality protein multiple sequence alignments usingClustal Omegardquo Molecular Systems Biology vol 7 article 5392011

[46] Oslash Hammer D A T Harper and P D Ryan ldquoPAST pale-ontological statistics software package for education and dataanalysisrdquo Palaeontologia Electronica vol 4 no 1 pp 1ndash9 2001

16 Evidence-Based Complementary and Alternative Medicine

[47] M C Papaleo M Fondi I Maida et al ldquoSponge-associatedmicrobial Antarctic communities exhibiting antimicrobialactivity againstBurkholderia cepacia complex bacteriardquoBiotech-nology Advances vol 30 no 1 pp 272ndash293 2012

[48] A lo Giudice V Bruni and L Michaud ldquoCharacterization ofAntarctic psychrotrophic bacteria with antibacterial activitiesagainst terrestrial microorganismsrdquo Journal of Basic Microbiol-ogy vol 47 no 6 pp 496ndash505 2007

[49] A Schippers K Bosecker C Sproer and P SchumannldquoMicrobacterium oleivorans sp nov and Microbacteriumhydrocarbonoxydans sp nov novel crude-oil-degrading Gram-positive bacteriardquo International Journal of Systematic and Evo-lutionary Microbiology vol 55 no 2 pp 655ndash660 2005

[50] J D McChesney S K Venkataraman and J T Henri ldquoPlantnatural products back to the future or into extinctionrdquo Phyto-chemistry vol 68 no 14 pp 2015ndash2022 2007

[51] D K Manter J A Delgado D G Holm and R A StongldquoPyrosequencing reveals a highly diverse and cultivar-specificbacterial endophyte community in potato rootsrdquo MicrobialEcology vol 60 no 1 pp 157ndash166 2010

[52] K Ulrich A Ulrich and D Ewald ldquoDiversity of endophyticbacterial communities in poplar grown under field conditionsrdquoFEMS Microbiology Ecology vol 63 no 2 pp 169ndash180 2008

[53] N R Gottel H F Castro M Kerley et al ldquoDistinct microbialcommunities within the endosphere and rhizosphere ofPopulusdeltoides roots across contrasting soil typesrdquo Applied and Envi-ronmental Microbiology vol 77 no 17 pp 5934ndash5944 2011

[54] B Ma X Lv A Warren and J Gong ldquoShifts in diversity andcommunity structure of endophytic bacteria and archaea acrossroot stem and leaf tissues in the common reed Phragmitesaustralis along a salinity gradient in a marine tidal wetland ofnorthern Chinardquo Antonie van Leeuwenhoek vol 104 no 5 pp759ndash768 2013

[55] J A Vorholt ldquoMicrobial life in the phyllosphererdquo NatureReviews Microbiology vol 10 no 12 pp 828ndash840 2012

[56] R P Ryan S Monchy M Cardinale et al ldquoThe versatilityand adaptation of bacteria from the genus StenotrophomonasrdquoNature Reviews Microbiology vol 7 no 7 pp 514ndash525 2009

[57] AWolf A FritzeMHagemann andG Berg ldquoStenotrophomo-nas rhizophila sp nov a novel plant-associated bacterium withantifungal propertiesrdquo International Journal of Systematic andEvolutionary Microbiology vol 52 no 6 pp 1937ndash1944 2002

[58] C W Bacon and D M Hinton ldquoEndophytic and biologicalcontrol potential of Bacillus mojavensis and related speciesrdquoBiological Control vol 23 no 3 pp 274ndash284 2002

[59] G Berg L Eberl and A Hartmann ldquoThe rhizosphere as areservoir for opportunistic human pathogenic bacteriardquo Envi-ronmental Microbiology vol 7 no 11 pp 1673ndash1685 2005

[60] H L Tyler and E W Triplett ldquoPlants as a habitat for ben-eficial andor human pathogenic bacteriardquo Annual Review ofPhytopathology vol 46 pp 53ndash73 2008

[61] R Perry R Terry L K Watson and E Ernst ldquoIs lavender ananxiolytic drug A systematic review of randomised clinicaltrialsrdquo Phytomedicine vol 19 no 8-9 pp 825ndash835 2012

[62] I Maida A lo Nostro G Pesavento et al ldquoExploring theanti-Burkholderia cepacia complex activity of essential oilsa preliminary analysisrdquo Evidence-Based Complementary andAlternativeMedicine vol 2014 Article ID 573518 10 pages 2014

12 Evidence-Based Complementary and Alternative Medicine

Table4Growth

of40

Burkholderiacepacia

complex

strains

inthep

resenceabsenceo

fend

ophytic

bacterialstrains

isolated

from

lavend

er

Species

Strain

Orig

inLL

1LL

2LL

3LL

4LL

6LL

7LL

9LL

10LS

1LS

2LS

3LS

4LS

5LS

6LR

1LR

2LR

3LR

4LR

5C-

Bcepacia

FCF1

CFminusminusminusminusminusminusminusminus

minusminusminusminusminusminusminusminusminusminusminus

+B

cepacia

FCF3

CF+minusminusminusminusminusminusminusminus

minusminusminusminusminusminusminusminusminusminusminus

+B

multivoran

sLM

G17588

Env

+minusminus

++minus

+minusminus

+minus

+minus

++minus

+minusminus

+minus

+minusminus

+B

cenocepacia

(IIIA

)FC

F16

CFminusminusminusminusminusminusminusminus

minusminusminusminusminusminusminusminusminusminusminus

+B

cenocepacia

(IIIA

)J2315

CF+minusminus

+minus

+minusminusminus

+minusminusminus

+minusminusminusminusminusminusminusminus

+B

cenocepacia

(IIIB)

FCF18

CF+minusminusminusminusminusminusminus

minusminusminusminusminusminus

+minusminusminusminusminus

+B

cenocepacia

(IIIB)

FCF20

CF+

++

++

+minus

++minus

+minus

+minus

+minus

++minus

++minus

++

++

+B

cenocepacia

(IIIB)

FCF23

CF+minusminusminusminusminusminusminus

+minusminusminus

++minusminusminus

+minusminusminusminus

+B

cenocepacia

(IIIB)

FCF24

CF+minusminusminusminusminusminusminus

+minusminus

+minusminusminusminus

+minusminusminusminus

+B

cenocepacia

(IIIB)

FCF27

CF+minusminusminusminusminusminusminus

+minusminusminusminusminusminusminusminusminusminusminus

+B

cenocepacia

(IIIB)

FCF29

CF+minusminus

+minus

+minusminusminus

+minusminusminusminusminusminusminus

+minus

+minusminus

+B

cenocepacia

(IIIB)

FCF30

CF+minusminus

+minus

+minusminusminus

+minus

+minusminusminusminusminusminus

+minus

+minusminus

+B

cenocepacia

(IIIB)

LMG16654

CF+

+minusminus

++minus

+minus

++minus

++minusminus

+minus

+minusminus

+B

cenocepacia

(IIIB)

C5424

CF+

+minusminus

+minusminusminusminus

+minusminusminus

+minusminusminusminus

+minusminus

+minusminus

+B

cenocepacia

(IIIB)

CEP5

11CF

+minusminus

+minusminusminusminus

+minus

+minusminus

+minus

+minusminusminus

+minus

+minusminus

+B

cenocepacia

(IIIB)

MVPC

116

Env

++minusminus

+minus

++minusminus

+minus

+minus

+minus

+minusminus

+minusminus

+minusminus

+minus

+minus

+B

cenocepacia

(IIIB)

MVPC

173

Env

++

++minus

++

+minus

++

++

++

++

++

++minus

+B

cenocepacia

(IIIIC)

LMG19230

Env

++

++

++

+minus

++

++

++

++

++

++

+B

cenocepacia

(IIIC

)LM

G19240

Env

++

++

+minus

+minus

++

+minus

++

++minus

++

++

+minus

+B

cenocepacia

(IIID)

FCF38

CF+minusminusminusminusminusminusminus

minusminusminusminusminusminusminusminusminusminusminus

+B

cenocepacia

(IIID)

LMG21462

CFminusminusminusminusminusminusminusminus

minusminusminusminusminusminusminusminusminusminusminus

+B

stabilis

FCF41

CF+

+minus

++

+minus

+minusminus

++

+minus

++minusminus

++

++minusminus

+B

vietnamien

sisFC

F42

CFminusminusminusminusminusminusminusminus

minusminusminusminusminusminusminusminusminusminusminus

+B

vietnamien

sisTV

V75

Env

++minusminus

+minusminusminusminus

+minusminusminus

++minusminusminus

+minusminus

+minusminus

+B

dolosa

LMG18941

CF+minusminus

+minus

+minus

+minusminus

+minusminusminus

++minusminusminus

+minus

+minus

+minusminus

+B

dolosa

LMG18942

CF+

+minusminus

+minus

+minusminus

++

+minus

++minusminusminus

++minus

++minusminus

+B

dolosa

LMG18943

CF+

+minusminus

++minus

+minusminus

+minus

+minusminus

++minusminusminus

++minus

+minusminus

+B

ambifaria

MCI

7En

v+

+minus

+minus

+minus

+minusminus

++minusminus

++minusminusminus

+minus

+minusminus

+B

ambifaria

LMG1946

7CF

++minus

+minus

+minusminusminus

++minusminus

++minusminusminus

+minus

+minusminusminus

+B

ambifaria

LMG19182

Env

++minus

+minus

+minusminusminus

+minusminus

+minusminusminus

+minus

+minusminusminus

+B

anthina

LMG16670

Env

+minusminusminusminus

+minus

+minusminus

++minusminusminus

+minus

+minusminusminus

+B

pyrrocinia

FCF43

CF+minusminusminusminusminusminusminusminus

minusminus

+minusminusminusminusminusminus

+minusminusminus

+B

lata

LSED

4CF

+minus

+minusminus

+minusminus

+minusminus

++minusminusminus

+minus

+minus

+minusminusminus

+B

latens

LMG2406

4CF

+minus

++minusminus

+minusminus

++minus

++minus

++

++

+minus

+B

diffu

saLM

G24065

CF+

+minus

++minus

+minus

+minus

++minus

++minus

+minus

++

++minus

+B

contam

inan

sLM

G23361

AI

++minus

+minus

+minus

+minus

+minus

++

++

++

+minus

++

++minus

+B

seminalis

LMG24067

CF+

+minus

++minus

++

++

++

++

++

++

++minus

+B

metallica

LMG2406

8CF

++

++minus

++

++

++

++

++

++

++minus

+B

arboris

LMG2406

6En

v+

+minus

++minus

++

+minus

++

++

++minus

+minus

++

++minusminus

+B

ubonensis

LMG24263

NI

+minusminusminusminus

+minusminus

+minus

+minus

++minusminus

+minusminus

+minusminusminus

+Ab

breviatio

nsC

Fstr

ains

isolatedfro

mcysticfi

brosispatie

ntsAIstr

ains

isolated

from

anim

alinfectionNIstr

ains

isolated

from

nosocomialinfectio

nEN

Venvironm

entalstrainLstr

ains

isolatedfro

mtheleaf

Sstr

ains

isolatedfro

mthes

temR

strains

isolatedfro

mther

oot

Symbo

ls+

grow

th+

minusreduced

grow

th+

minusminusveryredu

cedgrow

thminus

nogrow

thC

-Petrid

ishes

containing

onlythetargetstrains

Evidence-Based Complementary and Alternative Medicine 13

compartments showed different levels of diversity withthe 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 theleaf and roots community is already reported in otherspecies [26] even if there are few direct comparisons ofroots and phyllosphere bacterial communities especiallycomparisons using material from the same plants Themicrobial community residing in the phyllosphere is facedwith a nutrient poor and variable environment that ischaracterized by fluctuating temperature humidity and UVradiation and so it is predicted to be more diverse than thatwithin the rhizosphere a more homeostatic environmentThese findings supported also by the similar bacterial titreare in agreement with the idea that the source of inoculumfor the leaf community (except for the vertically transferredvia seeds) may be exclusive (eg aerosol even if the processis not yet clarified Bulgarelli et al 2013) and not related totransmission from the roots Moreover the low diversity ofthe community isolated in the stem dominated by clonally(Table 2) populations of Pseudomonas could be related tothe highly specific main tissue (phloem) present there whichcould provide an homeostatic environment rich in nutrient(sucrose contained in the phloem tissues) but poorer (ifcompared to leaf and roots) in secondary metabolites thatmay promote ecological niche differentiation

Concerning the taxonomic community composition dif-ferent taxonomic profiles were found for endosphere andrhizosphere More in detail the rhizosphere communityshowed quite similar diversity indexes if compared to theroots one but with a different composition as an examplemembers of the phylumActinobacteria are completely absentfrom the root internal tissues that are largely dominatedby Proteobacteria while they are present in the soil Toexplain this finding a two-step model has been proposed[16] soil abiotic properties determine the structure of theinitial bacterial communities that is then modified by rhi-zodeposits with plant roots cell wall features and releasedmetabolites promoting the growth of organotrophic bacteriathereby initiating a soil community shift In the second stepconvergent host genotype-dependent selection [51 52] fine-tunes community profiles thriving on the rhizoplane andwithin plant roots

Moreover endosphere communities were differentbetween plant organs (roots stems and leaves) even if theinternal tissues are dominated as already reported [25 52ndash54] by members of the phylum Proteobacteria an examplewhile rhizobia were isolated from root internal tissues theyare completely absent from stems and leaves (Figures 2 and3) isolates belonging to the genus Pantoea on the contrarywere isolated from aerial parts but not from root tissuesHence it is possible that plant might ldquoselectrdquo specific taxafor entering inside its compartment Such hypothesis issupported also by the pairwise differentiation values thecommunities isolated from the different tissues even those inphysical continuity are in fact strongly differentiated with thestem representing a low diversity compartment separatingthe two high diversity organs roots and leaves In this view itis noteworthy that in lavender only Pseudomonas represents

a ubiquitous and abundant genus of both rhizosphere andendosphere (Figure 3) The analysis of intrageneric diversitypointed out also that Pseudomonas isolates belong to a lowdifferentiated clonal population (Table 2 and Suppl Figure2) suggesting also that this component of the communityeither diffused inside the organs from a radical or foliar entrypoint or established during plants development putativelyfrom the seed inoculum A similar consideration can beproposed for isolates of rhizobia but for the rhizosphere-root internal tissues continuum a low taxonomicallydifferentiated community from the soil entered (increasingits relative abundance) inside the plant organ An entrypoint from the leaves is on the contrary consistent with thedistribution of Pantoea isolates with their strong diversity(Table 2) suggesting a diversification in response to the highvariable leaf environmentThe isolates belonging to the genusStenotrophomonas show an interesting distribution pattern(Figure 3) they are rare in the rhizosphere abundant in theroots and in the leaves and absent in the stem this findingsuggests a different entry point or a negative selection in thestem the second explanation is supported by admixture ofroot and leaves isolates (Figure 4)

The detailed phylogenetic analyses of the isolates enabledus also to putatively ascribe some isolates to already describedendophytic strains that can be targeted for functional charac-terisation many Microbacterium leaves isolates cluster withM hydrocarbonoxydans and M oleivorans two species withcrude oil degrading activity [49] being this metabolic activitythat is found frequently associated with a phyllosphereadapted lifestyle [55] Several isolates cluster with bacteriawith interesting biotechnological or ecological applicationssuch as S chelatiphaga a remarkable species with biotech-nological potential in phytoremediation [56] and S rizophilaa plant associated bacterium with antifungal activity [57] orBacillus mojavensis a bacteriumclosely related to B subtilisand already reported having an endophytic lifestyle andshowing an antagonistic role against plant pathogenic fungi[58] On the contrary many lavender Pseudomonas isolatescluster with known plant pathogen strains highlighting thesometimes subtle difference among pathogenic and endo-phytic lifestyle and with Pantoea agglomerans (and also withlavender isolates clustering withinthe B licheniformis and Bsoronensis groups) a known plant associated bacterium andan opportunistic pathogen in immune-compromised humanindividuals drawing attention to the possible pathogenicaction of endophytic bacteria in humans [59 60]

The overall analysis of the dendrograms points out thatthe characterization of isolates from endophytic communitiescan lead to the identification (as an example for Pantoeaand rhizobia) of not yet described strains that may representuntapped resources of bioactive compounds of agricultural ormedical interest

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

14 Evidence-Based Complementary and Alternative Medicine

context the characterization of cultivable bacterial commu-nities is a fundamental step to this purpose since it paves theway to the possibility to build collections of isolates that maybe phenotypically typed for important traits In our opiniondata obtained in this work (Table 4 and Supplementary Tables2 and 3) offers a preliminary but very promising exampleof the biotechnological potential of bacteria isolated frommedicinal plants In this pilot study in fact the majority ofthe tested endophytes showed to inhibit the growth of several(in some case all) human pathogenic strains belonging tothe Bcc complexes that are known for their resistance totraditional antibiotics These data are particularly interestingif correlated with the recent finding that essential oils fromdifferent medicinal plants are able to strongly (or completely)interfere with the growth of the same Bcc strains [61] and thatbacterial endophytes isolated from plants of the same speciesexhibited the same bioactivity (Maida et al in preparation)It is therefore possible that the therapeutic properties of theessential oil might reside also on bioactive compounds ofmicrobial origin We are completely aware that the determi-nation of the correlation (eventually) existing between thecomposition of bacterial endophytic communities and thebioactivity of essential oils extracted from a medicinal plantwould require the parallel analysis of the bacterial communityand the essential oil activity extracted from the same plantHowever data obtained in this work and those from Maidaet al [62] support this idea

It is also particularly intriguing the idea that the antimi-crobial activity of essential oils may reside on the actionof multiple compounds some of them produced or elicitedby the microbiota limiting the observed rapid evolutionof human pathogenic bacteria resistant to single moleculeantimicrobials

The characterization of the heterotrophic aerobic endo-phytic bacterial community of L angustifolia highlightedthe existence of a diversified community between differentorganstissues that may be related to the coexistence ofdifferent sources of inoculum andor a selection of the com-munities promoted by nutrient sand metabolites availabilityand antagonistic forces Several not previously characterizedstrains have been isolated and molecularly typed confirmingthat the analysis of the bacterial communities inhabitingextreme or unconventional environments may represent aproper strategy for the discovery of untapped sources offunctional biodiversity and bioactive molecule production

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This work was supported by grants from the Ente Cassadi Risparmio di Firenze (Grant 20130657 Herbiome newantimicrobial compounds from endophytic bacteria isolatedfrom medicinal plants) Giovanni Emiliani is financially

supported by the Regione Toscana POR CRO FSE 2007-2013 Asse IV Grant Sysbiofor (CNR-9) Marco Fondi andElena Perrin are financially supported by the FEMSAdvancedFellowship (FAF2012) and the Buzzati-Traverso Foundationrespectively

References

[1] B Reinhold-Hurek andTHurek ldquoLiving inside plants bacterialendophytesrdquo Current Opinion in Plant Biology vol 14 no 4 pp435ndash443 2011

[2] M Rosenblueth and E Martınez-Romero ldquoBacterial endo-phytes and their interactions with hostsrdquo Molecular Plant-Microbe Interactions vol 19 no 8 pp 827ndash837 2006

[3] R P Ryan K Germaine A Franks D J Ryan and DN Dowling ldquoBacterial endophytes recent developments andapplicationsrdquo FEMSMicrobiology Letters vol 278 no 1 pp 1ndash92008

[4] R Cakmakci F Donmez A Aydın and F Sahin ldquoGrowthpromotion of plants by plant growth-promoting rhizobacteriaunder greenhouse and two different field soil conditionsrdquo SoilBiology and Biochemistry vol 38 pp 1482ndash1487 2006

[5] P R Hardoim L S van Overbeek and J D V Elsas ldquoProp-erties of bacterial endophytes and their proposed role in plantgrowthrdquo Trends in Microbiology vol 16 no 10 pp 463ndash4712008

[6] S Compant C Clement and A Sessitsch ldquoPlant growth-promoting bacteria in the rhizo- and endosphere of plantstheir role colonization mechanisms involved and prospects forutilizationrdquo Soil Biology and Biochemistry vol 42 no 5 pp669ndash678 2010

[7] B R Glick ldquoBacterial ACC deaminase and the alleviation ofplant stressrdquo Advances in Applied Microbiology vol 56 pp 291ndash312 2004

[8] B R Glick B Todorovic J Czarny Z Cheng J Duan andB McConkey ldquoPromotion of plant growth by bacterial ACCdeaminaserdquo Critical Reviews in Plant Sciences vol 26 no 5-6pp 227ndash242 2007

[9] S L Doty B Oakley G Xin et al ldquoDiazotrophic endophytesof native black cottonwood and willowrdquo Symbiosis vol 47 no 1pp 23ndash33 2009

[10] B Lugtenberg and F Kamilova ldquoPlant-growth-promoting rhi-zobacteriardquoAnnual Review ofMicrobiology vol 63 pp 541ndash5562009

[11] U F Castillo G A Strobel E J Ford et al ldquoMunumbicinswide-spectrum antibiotics produced by Streptomyces NRRL30562 endophytic on Kennedia nigriscansrdquo Microbiology vol148 no 9 pp 2675ndash2685 2002

[12] Y Igarashi M E Trujillo E Martınez-Molina et al ldquoAnti-tumor anthraquinones from an endophytic actinomyceteMicromonospora lupini sp novrdquo Bioorganic amp Medicinal Chem-istry Letters vol 17 no 13 pp 3702ndash3705 2007

[13] S Shweta J H Bindu J Raghu et al ldquoIsolation of endophyticbacteria producing the anti-cancer alkaloid camptothecinefromMiquelia dentata Bedd (Icacinaceae)rdquo Phytomedicine vol20 pp 913ndash917 2013

[14] T Taechowisan A Wanbanjob P Tuntiwachwuttikul and JLiu ldquoAnti-inflammatory activity of lansais from endophyticStreptomyces sp SUC1 in LPS-induced RAW 2647 cellsrdquo Foodand Agricultural Immunology vol 20 no 1 pp 67ndash77 2009

Evidence-Based Complementary and Alternative Medicine 15

[15] P R Hardoim C C P Hardoim L S van Overbeek and J Dvan Elsas ldquoDynamics of seed-borne rice endophytes on earlyplant growth stagesrdquo PLoS ONE vol 7 no 2 Article ID e304382012

[16] D Bulgarelli K Schlaeppi S Spaepen E V L van Themaatand P Schulze-Lefert ldquoStructure and functions of the bacterialmicrobiota of plantsrdquo Annual Review of Plant Biology vol 64pp 807ndash838 2013

[17] A Mengoni S Mocali G Surico S Tegli and R Fani ldquoFluctu-ation of endophytic bacteria and phytoplasmosis in elm treesrdquoMicrobiological Research vol 158 no 4 pp 363ndash369 2003

[18] A Mengoni F Pini L-N Huang W-S Shu and MBazzicalupo ldquoPlant-by-plant variations of bacterial commu-nities associated with leaves of the nickel hyperaccumulatorAlyssum bertolonii desvrdquo Microbial Ecology vol 58 no 3 pp660ndash667 2009

[19] S Mocali E Bertelli F di Cello et al ldquoFluctuation of bacteriaisolated from elm tissues during different seasons and fromdifferent plant organsrdquo Research in Microbiology vol 154 no2 pp 105ndash114 2003

[20] F Pini A Frascella L Santopolo et al ldquoExploring the plant-associated bacterial communities in Medicago sativa Lrdquo BMCMicrobiology vol 12 article 78 2012

[21] A Mengoni L Cecchi and C Gonnelli ldquoNickel hyperac-cumulating plants and Alyssum bertolonii model systems forstudying biogeochemical interactions in serpentine soilsrdquo inBio-Geo Interactions in Metal-Contaminated Soils E Kothe andA Varma Eds pp 279ndash296 Springer Berlin Germany 2012

[22] S Ikeda T Okubo M Anda et al ldquoCommunity- and genome-based views of plant-associated bacteria plant-bacterial inter-actions in soybean and ricerdquo Plant and Cell Physiology vol 51no 9 pp 1398ndash1410 2010

[23] F Pini M Galardini M Bazzicalupo and A Mengoni ldquoPlant-bacteria association and symbiosis are there common genomictraits in alphaproteobacteriardquoGenes vol 2 no 4 pp 1017ndash10322011

[24] K Aleklett and M Hart ldquoThe root microbiota a fingerprint inthe soilrdquo Plant Soil vol 370 pp 671ndash686 2013

[25] A Beneduzi F Moreira P B Costa et al ldquoDiversity and plantgrowth promoting evaluation abilities of bacteria isolated fromsugarcane cultivated in the South of BrazilrdquoApplied Soil Ecologyvol 63 pp 94ndash104 2013

[26] N Bodenhausen M W Horton and J Bergelson ldquoBacterialcommunities associated with the leaves and the roots of Ara-bidopsis thalianardquo PLoS ONE vol 8 no 2 Article ID e563292013

[27] T F da Silva R E Vollu D Jurelevicius et al ldquoDoes theessential oil of Lippia sidoides Cham (pepper-rosmarin) affectits endophytic microbial communityrdquo BMC Microbiology vol13 no 1 article 29 2013

[28] M E Lucero A Unc P Cooke S Dowd and S SunldquoEndophyte microbiome diversity in micropropagated Atriplexcanescens and Atriplex torreyi var griffithsiirdquo PLoS ONE vol 6no 3 Article ID e17693 2011

[29] D S Lundberg S L Lebeis S H Paredes et al ldquoDefining thecoreArabidopsis thaliana rootmicrobiomerdquoNature vol 487 no7409 pp 86ndash90 2012

[30] H M Cavanagh and J M Wilkinson ldquoBiological activities oflavender essential oilrdquo Phytotherapy Research vol 16 no 4 pp301ndash308 2002

[31] G Woronuk Z Demissie M Rheault and S MahmoudldquoBiosynthesis and therapeutic properties of Lavandula essentialoil constituentsrdquo Planta Medica vol 77 no 1 pp 7ndash15 2011

[32] R Perry R Terry L K Watson and E Ernst ldquoIs lavender ananxiolytic drug A systematic review of randomised clinicaltrialsrdquo Phytomedicine vol 19 pp 825ndash835 2012

[33] S de Rapper G Kamatou A Viljoen and S van VuurenldquoThe in vitro antimicrobial activity of Lavandula angustifoliaessential oil in combination with other aroma-therapeutic oilsrdquoEvidence-Based Complementary and Alternative Medicine vol2013 Article ID 852049 10 pages 2013

[34] T Moon J M Wilkinson and H M Cavanagh ldquoAntiparasiticactivity of two Lavandula essential oils against Giardia duode-nalis Trichomonas vaginalis andHexamita inflatardquo ParasitologyResearch vol 99 no 6 pp 722ndash728 2006

[35] P S Yap S H Lim C P Hu and B C Yiap ldquoCom-bination of essential oils and antibiotics reduce antibioticresistance in plasmid-conferred multidrug resistant bacteriardquoPhytomedicine vol 20 pp 710ndash713 2013

[36] G Brader S Compant B Mitter F Trognitz and A SessitschldquoMetabolic potential of endophytic bacteriardquo Current Opinionin Biotechnology vol 27 pp 30ndash37 2014

[37] P Drevinek and E Mahenthiralingam ldquoBurkholderia ceno-cepacia in cystic fibrosis epidemiology and molecular mecha-nisms of virulencerdquo Clinical Microbiology and Infection vol 16no 7 pp 821ndash830 2010

[38] S Bazzini C Udine and G Riccardi ldquoMolecular approaches topathogenesis study of Burkholderia cenocepacia an importantcystic fibrosis opportunistic bacteriumrdquo Applied Microbiologyand Biotechnology vol 92 no 5 pp 887ndash895 2011

[39] I Maida A lo Nostro G Pesavento et al ldquoExploring theanti-Burkholderia cepacia complex activity of essential oilsa preliminary analysisrdquo Evidence-Based Complementary andAlternativeMedicine vol 2014 Article ID 573518 10 pages 2014

[40] A Mengoni R Barzanti C Gonnelli R Gabbrielli andM Bazzicalupo ldquoCharacterization of nickel-resistant bacteriaisolated from serpentine soilrdquo Environmental Microbiology vol3 no 11 pp 691ndash698 2001

[41] S F Altschul T L Madden A A Schaffer et al ldquoGappedBLAST and PSI-BLAST a new generation of protein databasesearch programsrdquo Nucleic Acids Research vol 25 no 17 pp3389ndash3402 1997

[42] R C Edgar ldquoMUSCLE multiple sequence alignment with highaccuracy and high throughputrdquo Nucleic Acids Research vol 32no 5 pp 1792ndash1797 2004

[43] F Ronquist M Teslenko P van der Mark et al ldquoMrbayes32 efficient bayesian phylogenetic inference and model choiceacross a large model spacerdquo Systematic Biology vol 61 no 3 pp539ndash542 2012

[44] K Tamura D Peterson N Peterson G Stecher M Nei andS Kumar ldquoMEGA5 molecular evolutionary genetics analysisusing maximum likelihood evolutionary distance and max-imum parsimony methodsrdquo Molecular Biology and Evolutionvol 28 no 10 pp 2731ndash2739 2011

[45] F Sievers A Wilm D Dineen et al ldquoFast scalable generationof high-quality protein multiple sequence alignments usingClustal Omegardquo Molecular Systems Biology vol 7 article 5392011

[46] Oslash Hammer D A T Harper and P D Ryan ldquoPAST pale-ontological statistics software package for education and dataanalysisrdquo Palaeontologia Electronica vol 4 no 1 pp 1ndash9 2001

16 Evidence-Based Complementary and Alternative Medicine

[47] M C Papaleo M Fondi I Maida et al ldquoSponge-associatedmicrobial Antarctic communities exhibiting antimicrobialactivity againstBurkholderia cepacia complex bacteriardquoBiotech-nology Advances vol 30 no 1 pp 272ndash293 2012

[48] A lo Giudice V Bruni and L Michaud ldquoCharacterization ofAntarctic psychrotrophic bacteria with antibacterial activitiesagainst terrestrial microorganismsrdquo Journal of Basic Microbiol-ogy vol 47 no 6 pp 496ndash505 2007

[49] A Schippers K Bosecker C Sproer and P SchumannldquoMicrobacterium oleivorans sp nov and Microbacteriumhydrocarbonoxydans sp nov novel crude-oil-degrading Gram-positive bacteriardquo International Journal of Systematic and Evo-lutionary Microbiology vol 55 no 2 pp 655ndash660 2005

[50] J D McChesney S K Venkataraman and J T Henri ldquoPlantnatural products back to the future or into extinctionrdquo Phyto-chemistry vol 68 no 14 pp 2015ndash2022 2007

[51] D K Manter J A Delgado D G Holm and R A StongldquoPyrosequencing reveals a highly diverse and cultivar-specificbacterial endophyte community in potato rootsrdquo MicrobialEcology vol 60 no 1 pp 157ndash166 2010

[52] K Ulrich A Ulrich and D Ewald ldquoDiversity of endophyticbacterial communities in poplar grown under field conditionsrdquoFEMS Microbiology Ecology vol 63 no 2 pp 169ndash180 2008

[53] N R Gottel H F Castro M Kerley et al ldquoDistinct microbialcommunities within the endosphere and rhizosphere ofPopulusdeltoides roots across contrasting soil typesrdquo Applied and Envi-ronmental Microbiology vol 77 no 17 pp 5934ndash5944 2011

[54] B Ma X Lv A Warren and J Gong ldquoShifts in diversity andcommunity structure of endophytic bacteria and archaea acrossroot stem and leaf tissues in the common reed Phragmitesaustralis along a salinity gradient in a marine tidal wetland ofnorthern Chinardquo Antonie van Leeuwenhoek vol 104 no 5 pp759ndash768 2013

[55] J A Vorholt ldquoMicrobial life in the phyllosphererdquo NatureReviews Microbiology vol 10 no 12 pp 828ndash840 2012

[56] R P Ryan S Monchy M Cardinale et al ldquoThe versatilityand adaptation of bacteria from the genus StenotrophomonasrdquoNature Reviews Microbiology vol 7 no 7 pp 514ndash525 2009

[57] AWolf A FritzeMHagemann andG Berg ldquoStenotrophomo-nas rhizophila sp nov a novel plant-associated bacterium withantifungal propertiesrdquo International Journal of Systematic andEvolutionary Microbiology vol 52 no 6 pp 1937ndash1944 2002

[58] C W Bacon and D M Hinton ldquoEndophytic and biologicalcontrol potential of Bacillus mojavensis and related speciesrdquoBiological Control vol 23 no 3 pp 274ndash284 2002

[59] G Berg L Eberl and A Hartmann ldquoThe rhizosphere as areservoir for opportunistic human pathogenic bacteriardquo Envi-ronmental Microbiology vol 7 no 11 pp 1673ndash1685 2005

[60] H L Tyler and E W Triplett ldquoPlants as a habitat for ben-eficial andor human pathogenic bacteriardquo Annual Review ofPhytopathology vol 46 pp 53ndash73 2008

[61] R Perry R Terry L K Watson and E Ernst ldquoIs lavender ananxiolytic drug A systematic review of randomised clinicaltrialsrdquo Phytomedicine vol 19 no 8-9 pp 825ndash835 2012

[62] I Maida A lo Nostro G Pesavento et al ldquoExploring theanti-Burkholderia cepacia complex activity of essential oilsa preliminary analysisrdquo Evidence-Based Complementary andAlternativeMedicine vol 2014 Article ID 573518 10 pages 2014

Evidence-Based Complementary and Alternative Medicine 13

compartments showed different levels of diversity withthe 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 theleaf and roots community is already reported in otherspecies [26] even if there are few direct comparisons ofroots and phyllosphere bacterial communities especiallycomparisons using material from the same plants Themicrobial community residing in the phyllosphere is facedwith a nutrient poor and variable environment that ischaracterized by fluctuating temperature humidity and UVradiation and so it is predicted to be more diverse than thatwithin the rhizosphere a more homeostatic environmentThese findings supported also by the similar bacterial titreare in agreement with the idea that the source of inoculumfor the leaf community (except for the vertically transferredvia seeds) may be exclusive (eg aerosol even if the processis not yet clarified Bulgarelli et al 2013) and not related totransmission from the roots Moreover the low diversity ofthe community isolated in the stem dominated by clonally(Table 2) populations of Pseudomonas could be related tothe highly specific main tissue (phloem) present there whichcould provide an homeostatic environment rich in nutrient(sucrose contained in the phloem tissues) but poorer (ifcompared to leaf and roots) in secondary metabolites thatmay promote ecological niche differentiation

Concerning the taxonomic community composition dif-ferent taxonomic profiles were found for endosphere andrhizosphere More in detail the rhizosphere communityshowed quite similar diversity indexes if compared to theroots one but with a different composition as an examplemembers of the phylumActinobacteria are completely absentfrom the root internal tissues that are largely dominatedby Proteobacteria while they are present in the soil Toexplain this finding a two-step model has been proposed[16] soil abiotic properties determine the structure of theinitial bacterial communities that is then modified by rhi-zodeposits with plant roots cell wall features and releasedmetabolites promoting the growth of organotrophic bacteriathereby initiating a soil community shift In the second stepconvergent host genotype-dependent selection [51 52] fine-tunes community profiles thriving on the rhizoplane andwithin plant roots

Moreover endosphere communities were differentbetween plant organs (roots stems and leaves) even if theinternal tissues are dominated as already reported [25 52ndash54] by members of the phylum Proteobacteria an examplewhile rhizobia were isolated from root internal tissues theyare completely absent from stems and leaves (Figures 2 and3) isolates belonging to the genus Pantoea on the contrarywere isolated from aerial parts but not from root tissuesHence it is possible that plant might ldquoselectrdquo specific taxafor entering inside its compartment Such hypothesis issupported also by the pairwise differentiation values thecommunities isolated from the different tissues even those inphysical continuity are in fact strongly differentiated with thestem representing a low diversity compartment separatingthe two high diversity organs roots and leaves In this view itis noteworthy that in lavender only Pseudomonas represents

a ubiquitous and abundant genus of both rhizosphere andendosphere (Figure 3) The analysis of intrageneric diversitypointed out also that Pseudomonas isolates belong to a lowdifferentiated clonal population (Table 2 and Suppl Figure2) suggesting also that this component of the communityeither diffused inside the organs from a radical or foliar entrypoint or established during plants development putativelyfrom the seed inoculum A similar consideration can beproposed for isolates of rhizobia but for the rhizosphere-root internal tissues continuum a low taxonomicallydifferentiated community from the soil entered (increasingits relative abundance) inside the plant organ An entrypoint from the leaves is on the contrary consistent with thedistribution of Pantoea isolates with their strong diversity(Table 2) suggesting a diversification in response to the highvariable leaf environmentThe isolates belonging to the genusStenotrophomonas show an interesting distribution pattern(Figure 3) they are rare in the rhizosphere abundant in theroots and in the leaves and absent in the stem this findingsuggests a different entry point or a negative selection in thestem the second explanation is supported by admixture ofroot and leaves isolates (Figure 4)

The detailed phylogenetic analyses of the isolates enabledus also to putatively ascribe some isolates to already describedendophytic strains that can be targeted for functional charac-terisation many Microbacterium leaves isolates cluster withM hydrocarbonoxydans and M oleivorans two species withcrude oil degrading activity [49] being this metabolic activitythat is found frequently associated with a phyllosphereadapted lifestyle [55] Several isolates cluster with bacteriawith interesting biotechnological or ecological applicationssuch as S chelatiphaga a remarkable species with biotech-nological potential in phytoremediation [56] and S rizophilaa plant associated bacterium with antifungal activity [57] orBacillus mojavensis a bacteriumclosely related to B subtilisand already reported having an endophytic lifestyle andshowing an antagonistic role against plant pathogenic fungi[58] On the contrary many lavender Pseudomonas isolatescluster with known plant pathogen strains highlighting thesometimes subtle difference among pathogenic and endo-phytic lifestyle and with Pantoea agglomerans (and also withlavender isolates clustering withinthe B licheniformis and Bsoronensis groups) a known plant associated bacterium andan opportunistic pathogen in immune-compromised humanindividuals drawing attention to the possible pathogenicaction of endophytic bacteria in humans [59 60]

The overall analysis of the dendrograms points out thatthe characterization of isolates from endophytic communitiescan lead to the identification (as an example for Pantoeaand rhizobia) of not yet described strains that may representuntapped resources of bioactive compounds of agricultural ormedical interest

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

14 Evidence-Based Complementary and Alternative Medicine

context the characterization of cultivable bacterial commu-nities is a fundamental step to this purpose since it paves theway to the possibility to build collections of isolates that maybe phenotypically typed for important traits In our opiniondata obtained in this work (Table 4 and Supplementary Tables2 and 3) offers a preliminary but very promising exampleof the biotechnological potential of bacteria isolated frommedicinal plants In this pilot study in fact the majority ofthe tested endophytes showed to inhibit the growth of several(in some case all) human pathogenic strains belonging tothe Bcc complexes that are known for their resistance totraditional antibiotics These data are particularly interestingif correlated with the recent finding that essential oils fromdifferent medicinal plants are able to strongly (or completely)interfere with the growth of the same Bcc strains [61] and thatbacterial endophytes isolated from plants of the same speciesexhibited the same bioactivity (Maida et al in preparation)It is therefore possible that the therapeutic properties of theessential oil might reside also on bioactive compounds ofmicrobial origin We are completely aware that the determi-nation of the correlation (eventually) existing between thecomposition of bacterial endophytic communities and thebioactivity of essential oils extracted from a medicinal plantwould require the parallel analysis of the bacterial communityand the essential oil activity extracted from the same plantHowever data obtained in this work and those from Maidaet al [62] support this idea

It is also particularly intriguing the idea that the antimi-crobial activity of essential oils may reside on the actionof multiple compounds some of them produced or elicitedby the microbiota limiting the observed rapid evolutionof human pathogenic bacteria resistant to single moleculeantimicrobials

The characterization of the heterotrophic aerobic endo-phytic bacterial community of L angustifolia highlightedthe existence of a diversified community between differentorganstissues that may be related to the coexistence ofdifferent sources of inoculum andor a selection of the com-munities promoted by nutrient sand metabolites availabilityand antagonistic forces Several not previously characterizedstrains have been isolated and molecularly typed confirmingthat the analysis of the bacterial communities inhabitingextreme or unconventional environments may represent aproper strategy for the discovery of untapped sources offunctional biodiversity and bioactive molecule production

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This work was supported by grants from the Ente Cassadi Risparmio di Firenze (Grant 20130657 Herbiome newantimicrobial compounds from endophytic bacteria isolatedfrom medicinal plants) Giovanni Emiliani is financially

supported by the Regione Toscana POR CRO FSE 2007-2013 Asse IV Grant Sysbiofor (CNR-9) Marco Fondi andElena Perrin are financially supported by the FEMSAdvancedFellowship (FAF2012) and the Buzzati-Traverso Foundationrespectively

References

[1] B Reinhold-Hurek andTHurek ldquoLiving inside plants bacterialendophytesrdquo Current Opinion in Plant Biology vol 14 no 4 pp435ndash443 2011

[2] M Rosenblueth and E Martınez-Romero ldquoBacterial endo-phytes and their interactions with hostsrdquo Molecular Plant-Microbe Interactions vol 19 no 8 pp 827ndash837 2006

[3] R P Ryan K Germaine A Franks D J Ryan and DN Dowling ldquoBacterial endophytes recent developments andapplicationsrdquo FEMSMicrobiology Letters vol 278 no 1 pp 1ndash92008

[4] R Cakmakci F Donmez A Aydın and F Sahin ldquoGrowthpromotion of plants by plant growth-promoting rhizobacteriaunder greenhouse and two different field soil conditionsrdquo SoilBiology and Biochemistry vol 38 pp 1482ndash1487 2006

[5] P R Hardoim L S van Overbeek and J D V Elsas ldquoProp-erties of bacterial endophytes and their proposed role in plantgrowthrdquo Trends in Microbiology vol 16 no 10 pp 463ndash4712008

[6] S Compant C Clement and A Sessitsch ldquoPlant growth-promoting bacteria in the rhizo- and endosphere of plantstheir role colonization mechanisms involved and prospects forutilizationrdquo Soil Biology and Biochemistry vol 42 no 5 pp669ndash678 2010

[7] B R Glick ldquoBacterial ACC deaminase and the alleviation ofplant stressrdquo Advances in Applied Microbiology vol 56 pp 291ndash312 2004

[8] B R Glick B Todorovic J Czarny Z Cheng J Duan andB McConkey ldquoPromotion of plant growth by bacterial ACCdeaminaserdquo Critical Reviews in Plant Sciences vol 26 no 5-6pp 227ndash242 2007

[9] S L Doty B Oakley G Xin et al ldquoDiazotrophic endophytesof native black cottonwood and willowrdquo Symbiosis vol 47 no 1pp 23ndash33 2009

[10] B Lugtenberg and F Kamilova ldquoPlant-growth-promoting rhi-zobacteriardquoAnnual Review ofMicrobiology vol 63 pp 541ndash5562009

[11] U F Castillo G A Strobel E J Ford et al ldquoMunumbicinswide-spectrum antibiotics produced by Streptomyces NRRL30562 endophytic on Kennedia nigriscansrdquo Microbiology vol148 no 9 pp 2675ndash2685 2002

[12] Y Igarashi M E Trujillo E Martınez-Molina et al ldquoAnti-tumor anthraquinones from an endophytic actinomyceteMicromonospora lupini sp novrdquo Bioorganic amp Medicinal Chem-istry Letters vol 17 no 13 pp 3702ndash3705 2007

[13] S Shweta J H Bindu J Raghu et al ldquoIsolation of endophyticbacteria producing the anti-cancer alkaloid camptothecinefromMiquelia dentata Bedd (Icacinaceae)rdquo Phytomedicine vol20 pp 913ndash917 2013

[14] T Taechowisan A Wanbanjob P Tuntiwachwuttikul and JLiu ldquoAnti-inflammatory activity of lansais from endophyticStreptomyces sp SUC1 in LPS-induced RAW 2647 cellsrdquo Foodand Agricultural Immunology vol 20 no 1 pp 67ndash77 2009

Evidence-Based Complementary and Alternative Medicine 15

[15] P R Hardoim C C P Hardoim L S van Overbeek and J Dvan Elsas ldquoDynamics of seed-borne rice endophytes on earlyplant growth stagesrdquo PLoS ONE vol 7 no 2 Article ID e304382012

[16] D Bulgarelli K Schlaeppi S Spaepen E V L van Themaatand P Schulze-Lefert ldquoStructure and functions of the bacterialmicrobiota of plantsrdquo Annual Review of Plant Biology vol 64pp 807ndash838 2013

[17] A Mengoni S Mocali G Surico S Tegli and R Fani ldquoFluctu-ation of endophytic bacteria and phytoplasmosis in elm treesrdquoMicrobiological Research vol 158 no 4 pp 363ndash369 2003

[18] A Mengoni F Pini L-N Huang W-S Shu and MBazzicalupo ldquoPlant-by-plant variations of bacterial commu-nities associated with leaves of the nickel hyperaccumulatorAlyssum bertolonii desvrdquo Microbial Ecology vol 58 no 3 pp660ndash667 2009

[19] S Mocali E Bertelli F di Cello et al ldquoFluctuation of bacteriaisolated from elm tissues during different seasons and fromdifferent plant organsrdquo Research in Microbiology vol 154 no2 pp 105ndash114 2003

[20] F Pini A Frascella L Santopolo et al ldquoExploring the plant-associated bacterial communities in Medicago sativa Lrdquo BMCMicrobiology vol 12 article 78 2012

[21] A Mengoni L Cecchi and C Gonnelli ldquoNickel hyperac-cumulating plants and Alyssum bertolonii model systems forstudying biogeochemical interactions in serpentine soilsrdquo inBio-Geo Interactions in Metal-Contaminated Soils E Kothe andA Varma Eds pp 279ndash296 Springer Berlin Germany 2012

[22] S Ikeda T Okubo M Anda et al ldquoCommunity- and genome-based views of plant-associated bacteria plant-bacterial inter-actions in soybean and ricerdquo Plant and Cell Physiology vol 51no 9 pp 1398ndash1410 2010

[23] F Pini M Galardini M Bazzicalupo and A Mengoni ldquoPlant-bacteria association and symbiosis are there common genomictraits in alphaproteobacteriardquoGenes vol 2 no 4 pp 1017ndash10322011

[24] K Aleklett and M Hart ldquoThe root microbiota a fingerprint inthe soilrdquo Plant Soil vol 370 pp 671ndash686 2013

[25] A Beneduzi F Moreira P B Costa et al ldquoDiversity and plantgrowth promoting evaluation abilities of bacteria isolated fromsugarcane cultivated in the South of BrazilrdquoApplied Soil Ecologyvol 63 pp 94ndash104 2013

[26] N Bodenhausen M W Horton and J Bergelson ldquoBacterialcommunities associated with the leaves and the roots of Ara-bidopsis thalianardquo PLoS ONE vol 8 no 2 Article ID e563292013

[27] T F da Silva R E Vollu D Jurelevicius et al ldquoDoes theessential oil of Lippia sidoides Cham (pepper-rosmarin) affectits endophytic microbial communityrdquo BMC Microbiology vol13 no 1 article 29 2013

[28] M E Lucero A Unc P Cooke S Dowd and S SunldquoEndophyte microbiome diversity in micropropagated Atriplexcanescens and Atriplex torreyi var griffithsiirdquo PLoS ONE vol 6no 3 Article ID e17693 2011

[29] D S Lundberg S L Lebeis S H Paredes et al ldquoDefining thecoreArabidopsis thaliana rootmicrobiomerdquoNature vol 487 no7409 pp 86ndash90 2012

[30] H M Cavanagh and J M Wilkinson ldquoBiological activities oflavender essential oilrdquo Phytotherapy Research vol 16 no 4 pp301ndash308 2002

[31] G Woronuk Z Demissie M Rheault and S MahmoudldquoBiosynthesis and therapeutic properties of Lavandula essentialoil constituentsrdquo Planta Medica vol 77 no 1 pp 7ndash15 2011

[32] R Perry R Terry L K Watson and E Ernst ldquoIs lavender ananxiolytic drug A systematic review of randomised clinicaltrialsrdquo Phytomedicine vol 19 pp 825ndash835 2012

[33] S de Rapper G Kamatou A Viljoen and S van VuurenldquoThe in vitro antimicrobial activity of Lavandula angustifoliaessential oil in combination with other aroma-therapeutic oilsrdquoEvidence-Based Complementary and Alternative Medicine vol2013 Article ID 852049 10 pages 2013

[34] T Moon J M Wilkinson and H M Cavanagh ldquoAntiparasiticactivity of two Lavandula essential oils against Giardia duode-nalis Trichomonas vaginalis andHexamita inflatardquo ParasitologyResearch vol 99 no 6 pp 722ndash728 2006

[35] P S Yap S H Lim C P Hu and B C Yiap ldquoCom-bination of essential oils and antibiotics reduce antibioticresistance in plasmid-conferred multidrug resistant bacteriardquoPhytomedicine vol 20 pp 710ndash713 2013

[36] G Brader S Compant B Mitter F Trognitz and A SessitschldquoMetabolic potential of endophytic bacteriardquo Current Opinionin Biotechnology vol 27 pp 30ndash37 2014

[37] P Drevinek and E Mahenthiralingam ldquoBurkholderia ceno-cepacia in cystic fibrosis epidemiology and molecular mecha-nisms of virulencerdquo Clinical Microbiology and Infection vol 16no 7 pp 821ndash830 2010

[38] S Bazzini C Udine and G Riccardi ldquoMolecular approaches topathogenesis study of Burkholderia cenocepacia an importantcystic fibrosis opportunistic bacteriumrdquo Applied Microbiologyand Biotechnology vol 92 no 5 pp 887ndash895 2011

[39] I Maida A lo Nostro G Pesavento et al ldquoExploring theanti-Burkholderia cepacia complex activity of essential oilsa preliminary analysisrdquo Evidence-Based Complementary andAlternativeMedicine vol 2014 Article ID 573518 10 pages 2014

[40] A Mengoni R Barzanti C Gonnelli R Gabbrielli andM Bazzicalupo ldquoCharacterization of nickel-resistant bacteriaisolated from serpentine soilrdquo Environmental Microbiology vol3 no 11 pp 691ndash698 2001

[41] S F Altschul T L Madden A A Schaffer et al ldquoGappedBLAST and PSI-BLAST a new generation of protein databasesearch programsrdquo Nucleic Acids Research vol 25 no 17 pp3389ndash3402 1997

[42] R C Edgar ldquoMUSCLE multiple sequence alignment with highaccuracy and high throughputrdquo Nucleic Acids Research vol 32no 5 pp 1792ndash1797 2004

[43] F Ronquist M Teslenko P van der Mark et al ldquoMrbayes32 efficient bayesian phylogenetic inference and model choiceacross a large model spacerdquo Systematic Biology vol 61 no 3 pp539ndash542 2012

[44] K Tamura D Peterson N Peterson G Stecher M Nei andS Kumar ldquoMEGA5 molecular evolutionary genetics analysisusing maximum likelihood evolutionary distance and max-imum parsimony methodsrdquo Molecular Biology and Evolutionvol 28 no 10 pp 2731ndash2739 2011

[45] F Sievers A Wilm D Dineen et al ldquoFast scalable generationof high-quality protein multiple sequence alignments usingClustal Omegardquo Molecular Systems Biology vol 7 article 5392011

[46] Oslash Hammer D A T Harper and P D Ryan ldquoPAST pale-ontological statistics software package for education and dataanalysisrdquo Palaeontologia Electronica vol 4 no 1 pp 1ndash9 2001

16 Evidence-Based Complementary and Alternative Medicine

[47] M C Papaleo M Fondi I Maida et al ldquoSponge-associatedmicrobial Antarctic communities exhibiting antimicrobialactivity againstBurkholderia cepacia complex bacteriardquoBiotech-nology Advances vol 30 no 1 pp 272ndash293 2012

[48] A lo Giudice V Bruni and L Michaud ldquoCharacterization ofAntarctic psychrotrophic bacteria with antibacterial activitiesagainst terrestrial microorganismsrdquo Journal of Basic Microbiol-ogy vol 47 no 6 pp 496ndash505 2007

[49] A Schippers K Bosecker C Sproer and P SchumannldquoMicrobacterium oleivorans sp nov and Microbacteriumhydrocarbonoxydans sp nov novel crude-oil-degrading Gram-positive bacteriardquo International Journal of Systematic and Evo-lutionary Microbiology vol 55 no 2 pp 655ndash660 2005

[50] J D McChesney S K Venkataraman and J T Henri ldquoPlantnatural products back to the future or into extinctionrdquo Phyto-chemistry vol 68 no 14 pp 2015ndash2022 2007

[51] D K Manter J A Delgado D G Holm and R A StongldquoPyrosequencing reveals a highly diverse and cultivar-specificbacterial endophyte community in potato rootsrdquo MicrobialEcology vol 60 no 1 pp 157ndash166 2010

[52] K Ulrich A Ulrich and D Ewald ldquoDiversity of endophyticbacterial communities in poplar grown under field conditionsrdquoFEMS Microbiology Ecology vol 63 no 2 pp 169ndash180 2008

[53] N R Gottel H F Castro M Kerley et al ldquoDistinct microbialcommunities within the endosphere and rhizosphere ofPopulusdeltoides roots across contrasting soil typesrdquo Applied and Envi-ronmental Microbiology vol 77 no 17 pp 5934ndash5944 2011

[54] B Ma X Lv A Warren and J Gong ldquoShifts in diversity andcommunity structure of endophytic bacteria and archaea acrossroot stem and leaf tissues in the common reed Phragmitesaustralis along a salinity gradient in a marine tidal wetland ofnorthern Chinardquo Antonie van Leeuwenhoek vol 104 no 5 pp759ndash768 2013

[55] J A Vorholt ldquoMicrobial life in the phyllosphererdquo NatureReviews Microbiology vol 10 no 12 pp 828ndash840 2012

[56] R P Ryan S Monchy M Cardinale et al ldquoThe versatilityand adaptation of bacteria from the genus StenotrophomonasrdquoNature Reviews Microbiology vol 7 no 7 pp 514ndash525 2009

[57] AWolf A FritzeMHagemann andG Berg ldquoStenotrophomo-nas rhizophila sp nov a novel plant-associated bacterium withantifungal propertiesrdquo International Journal of Systematic andEvolutionary Microbiology vol 52 no 6 pp 1937ndash1944 2002

[58] C W Bacon and D M Hinton ldquoEndophytic and biologicalcontrol potential of Bacillus mojavensis and related speciesrdquoBiological Control vol 23 no 3 pp 274ndash284 2002

[59] G Berg L Eberl and A Hartmann ldquoThe rhizosphere as areservoir for opportunistic human pathogenic bacteriardquo Envi-ronmental Microbiology vol 7 no 11 pp 1673ndash1685 2005

[60] H L Tyler and E W Triplett ldquoPlants as a habitat for ben-eficial andor human pathogenic bacteriardquo Annual Review ofPhytopathology vol 46 pp 53ndash73 2008

[61] R Perry R Terry L K Watson and E Ernst ldquoIs lavender ananxiolytic drug A systematic review of randomised clinicaltrialsrdquo Phytomedicine vol 19 no 8-9 pp 825ndash835 2012

[62] I Maida A lo Nostro G Pesavento et al ldquoExploring theanti-Burkholderia cepacia complex activity of essential oilsa preliminary analysisrdquo Evidence-Based Complementary andAlternativeMedicine vol 2014 Article ID 573518 10 pages 2014

14 Evidence-Based Complementary and Alternative Medicine

context the characterization of cultivable bacterial commu-nities is a fundamental step to this purpose since it paves theway to the possibility to build collections of isolates that maybe phenotypically typed for important traits In our opiniondata obtained in this work (Table 4 and Supplementary Tables2 and 3) offers a preliminary but very promising exampleof the biotechnological potential of bacteria isolated frommedicinal plants In this pilot study in fact the majority ofthe tested endophytes showed to inhibit the growth of several(in some case all) human pathogenic strains belonging tothe Bcc complexes that are known for their resistance totraditional antibiotics These data are particularly interestingif correlated with the recent finding that essential oils fromdifferent medicinal plants are able to strongly (or completely)interfere with the growth of the same Bcc strains [61] and thatbacterial endophytes isolated from plants of the same speciesexhibited the same bioactivity (Maida et al in preparation)It is therefore possible that the therapeutic properties of theessential oil might reside also on bioactive compounds ofmicrobial origin We are completely aware that the determi-nation of the correlation (eventually) existing between thecomposition of bacterial endophytic communities and thebioactivity of essential oils extracted from a medicinal plantwould require the parallel analysis of the bacterial communityand the essential oil activity extracted from the same plantHowever data obtained in this work and those from Maidaet al [62] support this idea

It is also particularly intriguing the idea that the antimi-crobial activity of essential oils may reside on the actionof multiple compounds some of them produced or elicitedby the microbiota limiting the observed rapid evolutionof human pathogenic bacteria resistant to single moleculeantimicrobials

The characterization of the heterotrophic aerobic endo-phytic bacterial community of L angustifolia highlightedthe existence of a diversified community between differentorganstissues that may be related to the coexistence ofdifferent sources of inoculum andor a selection of the com-munities promoted by nutrient sand metabolites availabilityand antagonistic forces Several not previously characterizedstrains have been isolated and molecularly typed confirmingthat the analysis of the bacterial communities inhabitingextreme or unconventional environments may represent aproper strategy for the discovery of untapped sources offunctional biodiversity and bioactive molecule production

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This work was supported by grants from the Ente Cassadi Risparmio di Firenze (Grant 20130657 Herbiome newantimicrobial compounds from endophytic bacteria isolatedfrom medicinal plants) Giovanni Emiliani is financially

supported by the Regione Toscana POR CRO FSE 2007-2013 Asse IV Grant Sysbiofor (CNR-9) Marco Fondi andElena Perrin are financially supported by the FEMSAdvancedFellowship (FAF2012) and the Buzzati-Traverso Foundationrespectively

References

[1] B Reinhold-Hurek andTHurek ldquoLiving inside plants bacterialendophytesrdquo Current Opinion in Plant Biology vol 14 no 4 pp435ndash443 2011

[2] M Rosenblueth and E Martınez-Romero ldquoBacterial endo-phytes and their interactions with hostsrdquo Molecular Plant-Microbe Interactions vol 19 no 8 pp 827ndash837 2006

[3] R P Ryan K Germaine A Franks D J Ryan and DN Dowling ldquoBacterial endophytes recent developments andapplicationsrdquo FEMSMicrobiology Letters vol 278 no 1 pp 1ndash92008

[4] R Cakmakci F Donmez A Aydın and F Sahin ldquoGrowthpromotion of plants by plant growth-promoting rhizobacteriaunder greenhouse and two different field soil conditionsrdquo SoilBiology and Biochemistry vol 38 pp 1482ndash1487 2006

[5] P R Hardoim L S van Overbeek and J D V Elsas ldquoProp-erties of bacterial endophytes and their proposed role in plantgrowthrdquo Trends in Microbiology vol 16 no 10 pp 463ndash4712008

[6] S Compant C Clement and A Sessitsch ldquoPlant growth-promoting bacteria in the rhizo- and endosphere of plantstheir role colonization mechanisms involved and prospects forutilizationrdquo Soil Biology and Biochemistry vol 42 no 5 pp669ndash678 2010

[7] B R Glick ldquoBacterial ACC deaminase and the alleviation ofplant stressrdquo Advances in Applied Microbiology vol 56 pp 291ndash312 2004

[8] B R Glick B Todorovic J Czarny Z Cheng J Duan andB McConkey ldquoPromotion of plant growth by bacterial ACCdeaminaserdquo Critical Reviews in Plant Sciences vol 26 no 5-6pp 227ndash242 2007

[9] S L Doty B Oakley G Xin et al ldquoDiazotrophic endophytesof native black cottonwood and willowrdquo Symbiosis vol 47 no 1pp 23ndash33 2009

[10] B Lugtenberg and F Kamilova ldquoPlant-growth-promoting rhi-zobacteriardquoAnnual Review ofMicrobiology vol 63 pp 541ndash5562009

[11] U F Castillo G A Strobel E J Ford et al ldquoMunumbicinswide-spectrum antibiotics produced by Streptomyces NRRL30562 endophytic on Kennedia nigriscansrdquo Microbiology vol148 no 9 pp 2675ndash2685 2002

[12] Y Igarashi M E Trujillo E Martınez-Molina et al ldquoAnti-tumor anthraquinones from an endophytic actinomyceteMicromonospora lupini sp novrdquo Bioorganic amp Medicinal Chem-istry Letters vol 17 no 13 pp 3702ndash3705 2007

[13] S Shweta J H Bindu J Raghu et al ldquoIsolation of endophyticbacteria producing the anti-cancer alkaloid camptothecinefromMiquelia dentata Bedd (Icacinaceae)rdquo Phytomedicine vol20 pp 913ndash917 2013

[14] T Taechowisan A Wanbanjob P Tuntiwachwuttikul and JLiu ldquoAnti-inflammatory activity of lansais from endophyticStreptomyces sp SUC1 in LPS-induced RAW 2647 cellsrdquo Foodand Agricultural Immunology vol 20 no 1 pp 67ndash77 2009

Evidence-Based Complementary and Alternative Medicine 15

[15] P R Hardoim C C P Hardoim L S van Overbeek and J Dvan Elsas ldquoDynamics of seed-borne rice endophytes on earlyplant growth stagesrdquo PLoS ONE vol 7 no 2 Article ID e304382012

[16] D Bulgarelli K Schlaeppi S Spaepen E V L van Themaatand P Schulze-Lefert ldquoStructure and functions of the bacterialmicrobiota of plantsrdquo Annual Review of Plant Biology vol 64pp 807ndash838 2013

[17] A Mengoni S Mocali G Surico S Tegli and R Fani ldquoFluctu-ation of endophytic bacteria and phytoplasmosis in elm treesrdquoMicrobiological Research vol 158 no 4 pp 363ndash369 2003

[18] A Mengoni F Pini L-N Huang W-S Shu and MBazzicalupo ldquoPlant-by-plant variations of bacterial commu-nities associated with leaves of the nickel hyperaccumulatorAlyssum bertolonii desvrdquo Microbial Ecology vol 58 no 3 pp660ndash667 2009

[19] S Mocali E Bertelli F di Cello et al ldquoFluctuation of bacteriaisolated from elm tissues during different seasons and fromdifferent plant organsrdquo Research in Microbiology vol 154 no2 pp 105ndash114 2003

[20] F Pini A Frascella L Santopolo et al ldquoExploring the plant-associated bacterial communities in Medicago sativa Lrdquo BMCMicrobiology vol 12 article 78 2012

[21] A Mengoni L Cecchi and C Gonnelli ldquoNickel hyperac-cumulating plants and Alyssum bertolonii model systems forstudying biogeochemical interactions in serpentine soilsrdquo inBio-Geo Interactions in Metal-Contaminated Soils E Kothe andA Varma Eds pp 279ndash296 Springer Berlin Germany 2012

[22] S Ikeda T Okubo M Anda et al ldquoCommunity- and genome-based views of plant-associated bacteria plant-bacterial inter-actions in soybean and ricerdquo Plant and Cell Physiology vol 51no 9 pp 1398ndash1410 2010

[23] F Pini M Galardini M Bazzicalupo and A Mengoni ldquoPlant-bacteria association and symbiosis are there common genomictraits in alphaproteobacteriardquoGenes vol 2 no 4 pp 1017ndash10322011

[24] K Aleklett and M Hart ldquoThe root microbiota a fingerprint inthe soilrdquo Plant Soil vol 370 pp 671ndash686 2013

[25] A Beneduzi F Moreira P B Costa et al ldquoDiversity and plantgrowth promoting evaluation abilities of bacteria isolated fromsugarcane cultivated in the South of BrazilrdquoApplied Soil Ecologyvol 63 pp 94ndash104 2013

[26] N Bodenhausen M W Horton and J Bergelson ldquoBacterialcommunities associated with the leaves and the roots of Ara-bidopsis thalianardquo PLoS ONE vol 8 no 2 Article ID e563292013

[27] T F da Silva R E Vollu D Jurelevicius et al ldquoDoes theessential oil of Lippia sidoides Cham (pepper-rosmarin) affectits endophytic microbial communityrdquo BMC Microbiology vol13 no 1 article 29 2013

[28] M E Lucero A Unc P Cooke S Dowd and S SunldquoEndophyte microbiome diversity in micropropagated Atriplexcanescens and Atriplex torreyi var griffithsiirdquo PLoS ONE vol 6no 3 Article ID e17693 2011

[29] D S Lundberg S L Lebeis S H Paredes et al ldquoDefining thecoreArabidopsis thaliana rootmicrobiomerdquoNature vol 487 no7409 pp 86ndash90 2012

[30] H M Cavanagh and J M Wilkinson ldquoBiological activities oflavender essential oilrdquo Phytotherapy Research vol 16 no 4 pp301ndash308 2002

[31] G Woronuk Z Demissie M Rheault and S MahmoudldquoBiosynthesis and therapeutic properties of Lavandula essentialoil constituentsrdquo Planta Medica vol 77 no 1 pp 7ndash15 2011

[32] R Perry R Terry L K Watson and E Ernst ldquoIs lavender ananxiolytic drug A systematic review of randomised clinicaltrialsrdquo Phytomedicine vol 19 pp 825ndash835 2012

[33] S de Rapper G Kamatou A Viljoen and S van VuurenldquoThe in vitro antimicrobial activity of Lavandula angustifoliaessential oil in combination with other aroma-therapeutic oilsrdquoEvidence-Based Complementary and Alternative Medicine vol2013 Article ID 852049 10 pages 2013

[34] T Moon J M Wilkinson and H M Cavanagh ldquoAntiparasiticactivity of two Lavandula essential oils against Giardia duode-nalis Trichomonas vaginalis andHexamita inflatardquo ParasitologyResearch vol 99 no 6 pp 722ndash728 2006

[35] P S Yap S H Lim C P Hu and B C Yiap ldquoCom-bination of essential oils and antibiotics reduce antibioticresistance in plasmid-conferred multidrug resistant bacteriardquoPhytomedicine vol 20 pp 710ndash713 2013

[36] G Brader S Compant B Mitter F Trognitz and A SessitschldquoMetabolic potential of endophytic bacteriardquo Current Opinionin Biotechnology vol 27 pp 30ndash37 2014

[37] P Drevinek and E Mahenthiralingam ldquoBurkholderia ceno-cepacia in cystic fibrosis epidemiology and molecular mecha-nisms of virulencerdquo Clinical Microbiology and Infection vol 16no 7 pp 821ndash830 2010

[38] S Bazzini C Udine and G Riccardi ldquoMolecular approaches topathogenesis study of Burkholderia cenocepacia an importantcystic fibrosis opportunistic bacteriumrdquo Applied Microbiologyand Biotechnology vol 92 no 5 pp 887ndash895 2011

[39] I Maida A lo Nostro G Pesavento et al ldquoExploring theanti-Burkholderia cepacia complex activity of essential oilsa preliminary analysisrdquo Evidence-Based Complementary andAlternativeMedicine vol 2014 Article ID 573518 10 pages 2014

[40] A Mengoni R Barzanti C Gonnelli R Gabbrielli andM Bazzicalupo ldquoCharacterization of nickel-resistant bacteriaisolated from serpentine soilrdquo Environmental Microbiology vol3 no 11 pp 691ndash698 2001

[41] S F Altschul T L Madden A A Schaffer et al ldquoGappedBLAST and PSI-BLAST a new generation of protein databasesearch programsrdquo Nucleic Acids Research vol 25 no 17 pp3389ndash3402 1997

[42] R C Edgar ldquoMUSCLE multiple sequence alignment with highaccuracy and high throughputrdquo Nucleic Acids Research vol 32no 5 pp 1792ndash1797 2004

[43] F Ronquist M Teslenko P van der Mark et al ldquoMrbayes32 efficient bayesian phylogenetic inference and model choiceacross a large model spacerdquo Systematic Biology vol 61 no 3 pp539ndash542 2012

[44] K Tamura D Peterson N Peterson G Stecher M Nei andS Kumar ldquoMEGA5 molecular evolutionary genetics analysisusing maximum likelihood evolutionary distance and max-imum parsimony methodsrdquo Molecular Biology and Evolutionvol 28 no 10 pp 2731ndash2739 2011

[45] F Sievers A Wilm D Dineen et al ldquoFast scalable generationof high-quality protein multiple sequence alignments usingClustal Omegardquo Molecular Systems Biology vol 7 article 5392011

[46] Oslash Hammer D A T Harper and P D Ryan ldquoPAST pale-ontological statistics software package for education and dataanalysisrdquo Palaeontologia Electronica vol 4 no 1 pp 1ndash9 2001

16 Evidence-Based Complementary and Alternative Medicine

[47] M C Papaleo M Fondi I Maida et al ldquoSponge-associatedmicrobial Antarctic communities exhibiting antimicrobialactivity againstBurkholderia cepacia complex bacteriardquoBiotech-nology Advances vol 30 no 1 pp 272ndash293 2012

[48] A lo Giudice V Bruni and L Michaud ldquoCharacterization ofAntarctic psychrotrophic bacteria with antibacterial activitiesagainst terrestrial microorganismsrdquo Journal of Basic Microbiol-ogy vol 47 no 6 pp 496ndash505 2007

[49] A Schippers K Bosecker C Sproer and P SchumannldquoMicrobacterium oleivorans sp nov and Microbacteriumhydrocarbonoxydans sp nov novel crude-oil-degrading Gram-positive bacteriardquo International Journal of Systematic and Evo-lutionary Microbiology vol 55 no 2 pp 655ndash660 2005

[50] J D McChesney S K Venkataraman and J T Henri ldquoPlantnatural products back to the future or into extinctionrdquo Phyto-chemistry vol 68 no 14 pp 2015ndash2022 2007

[51] D K Manter J A Delgado D G Holm and R A StongldquoPyrosequencing reveals a highly diverse and cultivar-specificbacterial endophyte community in potato rootsrdquo MicrobialEcology vol 60 no 1 pp 157ndash166 2010

[52] K Ulrich A Ulrich and D Ewald ldquoDiversity of endophyticbacterial communities in poplar grown under field conditionsrdquoFEMS Microbiology Ecology vol 63 no 2 pp 169ndash180 2008

[53] N R Gottel H F Castro M Kerley et al ldquoDistinct microbialcommunities within the endosphere and rhizosphere ofPopulusdeltoides roots across contrasting soil typesrdquo Applied and Envi-ronmental Microbiology vol 77 no 17 pp 5934ndash5944 2011

[54] B Ma X Lv A Warren and J Gong ldquoShifts in diversity andcommunity structure of endophytic bacteria and archaea acrossroot stem and leaf tissues in the common reed Phragmitesaustralis along a salinity gradient in a marine tidal wetland ofnorthern Chinardquo Antonie van Leeuwenhoek vol 104 no 5 pp759ndash768 2013

[55] J A Vorholt ldquoMicrobial life in the phyllosphererdquo NatureReviews Microbiology vol 10 no 12 pp 828ndash840 2012

[56] R P Ryan S Monchy M Cardinale et al ldquoThe versatilityand adaptation of bacteria from the genus StenotrophomonasrdquoNature Reviews Microbiology vol 7 no 7 pp 514ndash525 2009

[57] AWolf A FritzeMHagemann andG Berg ldquoStenotrophomo-nas rhizophila sp nov a novel plant-associated bacterium withantifungal propertiesrdquo International Journal of Systematic andEvolutionary Microbiology vol 52 no 6 pp 1937ndash1944 2002

[58] C W Bacon and D M Hinton ldquoEndophytic and biologicalcontrol potential of Bacillus mojavensis and related speciesrdquoBiological Control vol 23 no 3 pp 274ndash284 2002

[59] G Berg L Eberl and A Hartmann ldquoThe rhizosphere as areservoir for opportunistic human pathogenic bacteriardquo Envi-ronmental Microbiology vol 7 no 11 pp 1673ndash1685 2005

[60] H L Tyler and E W Triplett ldquoPlants as a habitat for ben-eficial andor human pathogenic bacteriardquo Annual Review ofPhytopathology vol 46 pp 53ndash73 2008

[61] R Perry R Terry L K Watson and E Ernst ldquoIs lavender ananxiolytic drug A systematic review of randomised clinicaltrialsrdquo Phytomedicine vol 19 no 8-9 pp 825ndash835 2012

[62] I Maida A lo Nostro G Pesavento et al ldquoExploring theanti-Burkholderia cepacia complex activity of essential oilsa preliminary analysisrdquo Evidence-Based Complementary andAlternativeMedicine vol 2014 Article ID 573518 10 pages 2014

Evidence-Based Complementary and Alternative Medicine 15

[15] P R Hardoim C C P Hardoim L S van Overbeek and J Dvan Elsas ldquoDynamics of seed-borne rice endophytes on earlyplant growth stagesrdquo PLoS ONE vol 7 no 2 Article ID e304382012

[16] D Bulgarelli K Schlaeppi S Spaepen E V L van Themaatand P Schulze-Lefert ldquoStructure and functions of the bacterialmicrobiota of plantsrdquo Annual Review of Plant Biology vol 64pp 807ndash838 2013

[17] A Mengoni S Mocali G Surico S Tegli and R Fani ldquoFluctu-ation of endophytic bacteria and phytoplasmosis in elm treesrdquoMicrobiological Research vol 158 no 4 pp 363ndash369 2003

[18] A Mengoni F Pini L-N Huang W-S Shu and MBazzicalupo ldquoPlant-by-plant variations of bacterial commu-nities associated with leaves of the nickel hyperaccumulatorAlyssum bertolonii desvrdquo Microbial Ecology vol 58 no 3 pp660ndash667 2009

[19] S Mocali E Bertelli F di Cello et al ldquoFluctuation of bacteriaisolated from elm tissues during different seasons and fromdifferent plant organsrdquo Research in Microbiology vol 154 no2 pp 105ndash114 2003

[20] F Pini A Frascella L Santopolo et al ldquoExploring the plant-associated bacterial communities in Medicago sativa Lrdquo BMCMicrobiology vol 12 article 78 2012

[21] A Mengoni L Cecchi and C Gonnelli ldquoNickel hyperac-cumulating plants and Alyssum bertolonii model systems forstudying biogeochemical interactions in serpentine soilsrdquo inBio-Geo Interactions in Metal-Contaminated Soils E Kothe andA Varma Eds pp 279ndash296 Springer Berlin Germany 2012

[22] S Ikeda T Okubo M Anda et al ldquoCommunity- and genome-based views of plant-associated bacteria plant-bacterial inter-actions in soybean and ricerdquo Plant and Cell Physiology vol 51no 9 pp 1398ndash1410 2010

[23] F Pini M Galardini M Bazzicalupo and A Mengoni ldquoPlant-bacteria association and symbiosis are there common genomictraits in alphaproteobacteriardquoGenes vol 2 no 4 pp 1017ndash10322011

[24] K Aleklett and M Hart ldquoThe root microbiota a fingerprint inthe soilrdquo Plant Soil vol 370 pp 671ndash686 2013

[25] A Beneduzi F Moreira P B Costa et al ldquoDiversity and plantgrowth promoting evaluation abilities of bacteria isolated fromsugarcane cultivated in the South of BrazilrdquoApplied Soil Ecologyvol 63 pp 94ndash104 2013

[26] N Bodenhausen M W Horton and J Bergelson ldquoBacterialcommunities associated with the leaves and the roots of Ara-bidopsis thalianardquo PLoS ONE vol 8 no 2 Article ID e563292013

[27] T F da Silva R E Vollu D Jurelevicius et al ldquoDoes theessential oil of Lippia sidoides Cham (pepper-rosmarin) affectits endophytic microbial communityrdquo BMC Microbiology vol13 no 1 article 29 2013

[28] M E Lucero A Unc P Cooke S Dowd and S SunldquoEndophyte microbiome diversity in micropropagated Atriplexcanescens and Atriplex torreyi var griffithsiirdquo PLoS ONE vol 6no 3 Article ID e17693 2011

[29] D S Lundberg S L Lebeis S H Paredes et al ldquoDefining thecoreArabidopsis thaliana rootmicrobiomerdquoNature vol 487 no7409 pp 86ndash90 2012

[30] H M Cavanagh and J M Wilkinson ldquoBiological activities oflavender essential oilrdquo Phytotherapy Research vol 16 no 4 pp301ndash308 2002

[31] G Woronuk Z Demissie M Rheault and S MahmoudldquoBiosynthesis and therapeutic properties of Lavandula essentialoil constituentsrdquo Planta Medica vol 77 no 1 pp 7ndash15 2011

[32] R Perry R Terry L K Watson and E Ernst ldquoIs lavender ananxiolytic drug A systematic review of randomised clinicaltrialsrdquo Phytomedicine vol 19 pp 825ndash835 2012

[33] S de Rapper G Kamatou A Viljoen and S van VuurenldquoThe in vitro antimicrobial activity of Lavandula angustifoliaessential oil in combination with other aroma-therapeutic oilsrdquoEvidence-Based Complementary and Alternative Medicine vol2013 Article ID 852049 10 pages 2013

[34] T Moon J M Wilkinson and H M Cavanagh ldquoAntiparasiticactivity of two Lavandula essential oils against Giardia duode-nalis Trichomonas vaginalis andHexamita inflatardquo ParasitologyResearch vol 99 no 6 pp 722ndash728 2006

[35] P S Yap S H Lim C P Hu and B C Yiap ldquoCom-bination of essential oils and antibiotics reduce antibioticresistance in plasmid-conferred multidrug resistant bacteriardquoPhytomedicine vol 20 pp 710ndash713 2013

[36] G Brader S Compant B Mitter F Trognitz and A SessitschldquoMetabolic potential of endophytic bacteriardquo Current Opinionin Biotechnology vol 27 pp 30ndash37 2014

[37] P Drevinek and E Mahenthiralingam ldquoBurkholderia ceno-cepacia in cystic fibrosis epidemiology and molecular mecha-nisms of virulencerdquo Clinical Microbiology and Infection vol 16no 7 pp 821ndash830 2010

[38] S Bazzini C Udine and G Riccardi ldquoMolecular approaches topathogenesis study of Burkholderia cenocepacia an importantcystic fibrosis opportunistic bacteriumrdquo Applied Microbiologyand Biotechnology vol 92 no 5 pp 887ndash895 2011

[39] I Maida A lo Nostro G Pesavento et al ldquoExploring theanti-Burkholderia cepacia complex activity of essential oilsa preliminary analysisrdquo Evidence-Based Complementary andAlternativeMedicine vol 2014 Article ID 573518 10 pages 2014

[40] A Mengoni R Barzanti C Gonnelli R Gabbrielli andM Bazzicalupo ldquoCharacterization of nickel-resistant bacteriaisolated from serpentine soilrdquo Environmental Microbiology vol3 no 11 pp 691ndash698 2001

[41] S F Altschul T L Madden A A Schaffer et al ldquoGappedBLAST and PSI-BLAST a new generation of protein databasesearch programsrdquo Nucleic Acids Research vol 25 no 17 pp3389ndash3402 1997

[42] R C Edgar ldquoMUSCLE multiple sequence alignment with highaccuracy and high throughputrdquo Nucleic Acids Research vol 32no 5 pp 1792ndash1797 2004

[43] F Ronquist M Teslenko P van der Mark et al ldquoMrbayes32 efficient bayesian phylogenetic inference and model choiceacross a large model spacerdquo Systematic Biology vol 61 no 3 pp539ndash542 2012

[44] K Tamura D Peterson N Peterson G Stecher M Nei andS Kumar ldquoMEGA5 molecular evolutionary genetics analysisusing maximum likelihood evolutionary distance and max-imum parsimony methodsrdquo Molecular Biology and Evolutionvol 28 no 10 pp 2731ndash2739 2011

[45] F Sievers A Wilm D Dineen et al ldquoFast scalable generationof high-quality protein multiple sequence alignments usingClustal Omegardquo Molecular Systems Biology vol 7 article 5392011

[46] Oslash Hammer D A T Harper and P D Ryan ldquoPAST pale-ontological statistics software package for education and dataanalysisrdquo Palaeontologia Electronica vol 4 no 1 pp 1ndash9 2001

16 Evidence-Based Complementary and Alternative Medicine

[47] M C Papaleo M Fondi I Maida et al ldquoSponge-associatedmicrobial Antarctic communities exhibiting antimicrobialactivity againstBurkholderia cepacia complex bacteriardquoBiotech-nology Advances vol 30 no 1 pp 272ndash293 2012

[48] A lo Giudice V Bruni and L Michaud ldquoCharacterization ofAntarctic psychrotrophic bacteria with antibacterial activitiesagainst terrestrial microorganismsrdquo Journal of Basic Microbiol-ogy vol 47 no 6 pp 496ndash505 2007

[49] A Schippers K Bosecker C Sproer and P SchumannldquoMicrobacterium oleivorans sp nov and Microbacteriumhydrocarbonoxydans sp nov novel crude-oil-degrading Gram-positive bacteriardquo International Journal of Systematic and Evo-lutionary Microbiology vol 55 no 2 pp 655ndash660 2005

[50] J D McChesney S K Venkataraman and J T Henri ldquoPlantnatural products back to the future or into extinctionrdquo Phyto-chemistry vol 68 no 14 pp 2015ndash2022 2007

[51] D K Manter J A Delgado D G Holm and R A StongldquoPyrosequencing reveals a highly diverse and cultivar-specificbacterial endophyte community in potato rootsrdquo MicrobialEcology vol 60 no 1 pp 157ndash166 2010

[52] K Ulrich A Ulrich and D Ewald ldquoDiversity of endophyticbacterial communities in poplar grown under field conditionsrdquoFEMS Microbiology Ecology vol 63 no 2 pp 169ndash180 2008

[53] N R Gottel H F Castro M Kerley et al ldquoDistinct microbialcommunities within the endosphere and rhizosphere ofPopulusdeltoides roots across contrasting soil typesrdquo Applied and Envi-ronmental Microbiology vol 77 no 17 pp 5934ndash5944 2011

[54] B Ma X Lv A Warren and J Gong ldquoShifts in diversity andcommunity structure of endophytic bacteria and archaea acrossroot stem and leaf tissues in the common reed Phragmitesaustralis along a salinity gradient in a marine tidal wetland ofnorthern Chinardquo Antonie van Leeuwenhoek vol 104 no 5 pp759ndash768 2013

[55] J A Vorholt ldquoMicrobial life in the phyllosphererdquo NatureReviews Microbiology vol 10 no 12 pp 828ndash840 2012

[56] R P Ryan S Monchy M Cardinale et al ldquoThe versatilityand adaptation of bacteria from the genus StenotrophomonasrdquoNature Reviews Microbiology vol 7 no 7 pp 514ndash525 2009

[57] AWolf A FritzeMHagemann andG Berg ldquoStenotrophomo-nas rhizophila sp nov a novel plant-associated bacterium withantifungal propertiesrdquo International Journal of Systematic andEvolutionary Microbiology vol 52 no 6 pp 1937ndash1944 2002

[58] C W Bacon and D M Hinton ldquoEndophytic and biologicalcontrol potential of Bacillus mojavensis and related speciesrdquoBiological Control vol 23 no 3 pp 274ndash284 2002

[59] G Berg L Eberl and A Hartmann ldquoThe rhizosphere as areservoir for opportunistic human pathogenic bacteriardquo Envi-ronmental Microbiology vol 7 no 11 pp 1673ndash1685 2005

[60] H L Tyler and E W Triplett ldquoPlants as a habitat for ben-eficial andor human pathogenic bacteriardquo Annual Review ofPhytopathology vol 46 pp 53ndash73 2008

[61] R Perry R Terry L K Watson and E Ernst ldquoIs lavender ananxiolytic drug A systematic review of randomised clinicaltrialsrdquo Phytomedicine vol 19 no 8-9 pp 825ndash835 2012

[62] I Maida A lo Nostro G Pesavento et al ldquoExploring theanti-Burkholderia cepacia complex activity of essential oilsa preliminary analysisrdquo Evidence-Based Complementary andAlternativeMedicine vol 2014 Article ID 573518 10 pages 2014

16 Evidence-Based Complementary and Alternative Medicine

[47] M C Papaleo M Fondi I Maida et al ldquoSponge-associatedmicrobial Antarctic communities exhibiting antimicrobialactivity againstBurkholderia cepacia complex bacteriardquoBiotech-nology Advances vol 30 no 1 pp 272ndash293 2012

[48] A lo Giudice V Bruni and L Michaud ldquoCharacterization ofAntarctic psychrotrophic bacteria with antibacterial activitiesagainst terrestrial microorganismsrdquo Journal of Basic Microbiol-ogy vol 47 no 6 pp 496ndash505 2007

[49] A Schippers K Bosecker C Sproer and P SchumannldquoMicrobacterium oleivorans sp nov and Microbacteriumhydrocarbonoxydans sp nov novel crude-oil-degrading Gram-positive bacteriardquo International Journal of Systematic and Evo-lutionary Microbiology vol 55 no 2 pp 655ndash660 2005

[50] J D McChesney S K Venkataraman and J T Henri ldquoPlantnatural products back to the future or into extinctionrdquo Phyto-chemistry vol 68 no 14 pp 2015ndash2022 2007

[51] D K Manter J A Delgado D G Holm and R A StongldquoPyrosequencing reveals a highly diverse and cultivar-specificbacterial endophyte community in potato rootsrdquo MicrobialEcology vol 60 no 1 pp 157ndash166 2010

[52] K Ulrich A Ulrich and D Ewald ldquoDiversity of endophyticbacterial communities in poplar grown under field conditionsrdquoFEMS Microbiology Ecology vol 63 no 2 pp 169ndash180 2008

[53] N R Gottel H F Castro M Kerley et al ldquoDistinct microbialcommunities within the endosphere and rhizosphere ofPopulusdeltoides roots across contrasting soil typesrdquo Applied and Envi-ronmental Microbiology vol 77 no 17 pp 5934ndash5944 2011

[54] B Ma X Lv A Warren and J Gong ldquoShifts in diversity andcommunity structure of endophytic bacteria and archaea acrossroot stem and leaf tissues in the common reed Phragmitesaustralis along a salinity gradient in a marine tidal wetland ofnorthern Chinardquo Antonie van Leeuwenhoek vol 104 no 5 pp759ndash768 2013

[55] J A Vorholt ldquoMicrobial life in the phyllosphererdquo NatureReviews Microbiology vol 10 no 12 pp 828ndash840 2012

[56] R P Ryan S Monchy M Cardinale et al ldquoThe versatilityand adaptation of bacteria from the genus StenotrophomonasrdquoNature Reviews Microbiology vol 7 no 7 pp 514ndash525 2009

[57] AWolf A FritzeMHagemann andG Berg ldquoStenotrophomo-nas rhizophila sp nov a novel plant-associated bacterium withantifungal propertiesrdquo International Journal of Systematic andEvolutionary Microbiology vol 52 no 6 pp 1937ndash1944 2002

[58] C W Bacon and D M Hinton ldquoEndophytic and biologicalcontrol potential of Bacillus mojavensis and related speciesrdquoBiological Control vol 23 no 3 pp 274ndash284 2002

[59] G Berg L Eberl and A Hartmann ldquoThe rhizosphere as areservoir for opportunistic human pathogenic bacteriardquo Envi-ronmental Microbiology vol 7 no 11 pp 1673ndash1685 2005

[60] H L Tyler and E W Triplett ldquoPlants as a habitat for ben-eficial andor human pathogenic bacteriardquo Annual Review ofPhytopathology vol 46 pp 53ndash73 2008

[61] R Perry R Terry L K Watson and E Ernst ldquoIs lavender ananxiolytic drug A systematic review of randomised clinicaltrialsrdquo Phytomedicine vol 19 no 8-9 pp 825ndash835 2012

[62] I Maida A lo Nostro G Pesavento et al ldquoExploring theanti-Burkholderia cepacia complex activity of essential oilsa preliminary analysisrdquo Evidence-Based Complementary andAlternativeMedicine vol 2014 Article ID 573518 10 pages 2014