REGULAR ARTICLE

Microbial communities associated with the root systemof wild olives (Olea europaea L. subsp. europaea var.sylvestris) are good reservoirs of bacteria with antagonisticpotential against Verticillium dahliae

Sergio Aranda & Miguel Montes-Borrego &

Rafael M. Jiménez-Díaz & Blanca B. Landa

Received: 5 November 2010 /Accepted: 30 January 2011 /Published online: 20 February 2011# Springer Science+Business Media B.V. 2011

Abstract Wild olive trees, namely oleaster, areconsidered the ancestor of cultivated olive and aunexplored source of genetic variability that mightcontain important traits of agronomic and biotech-nological interest. The longevity and genetic diver-sity of oleasters may have favoured selection ofspecific and well adapted rhizosphere microbialpopulations that can constitute unique reservoirs ofmicrobial antagonists of Verticillium dahliae, themain soilborne fungal pathogen of olive worldwide.The objective of this present study was to determinethe structure and diversity of bacterial communitiesin the rhizosphere and endosphere of oleaster from11 havens in Cádiz and Córdoba provinces ofAndalusia, southern Spain. To carry out the study

we used a multiphasic approach. First, the occur-rence and diversity of rhizosphere bacteria wasmonitored by a cultivation-independent-approach,using fluorescent terminal restriction fragmentlength polymorphism (FT-RFLP) analyses of ampli-fied 16S rDNA sequences. FT-RFLP patternsrevealed a high heterogeneity in the composition ofthe sampled rhizosphere bacterial communities andsuggested the existence of plant genotype-site-specific communities, with each oleaster havenbeing a unique reservoir of bacterial diversity.Secondly, to investigate the antagonistic potentialof these root-associated bacterial populations, a totalof 675 bacterial isolates obtained from oleasterrhizosphere and endosphere were screened by dualtesting for inhibition of in vitro growth of the highlyvirulent, olive defoliating pathotype of V. dahliae.Out of 675 tested bacterial isolates, 94 (14%)showed a strong antagonistic activity against adefoliating V. dahliae pathotype. Of the antagonisticbacteria, a slightly lower proportion (12.9% of totalbacteria) were inhabitant of the oleaster rhizospherecompared to that in the endosphere (16.5%). Thebiotechnological potential of those isolates wasassessed by in vitro production of different hydro-lytic enzymes, indole-1.3-acetic acid (IAA), side-rophores, and antimicrobial compounds. Overall,most of bacterial antagonists (58.5 to 78.3%) showedproteolytic, lipolytic, and chitinolytic activity, andproduced IAA and siderophores. Finally, analysis of

Plant Soil (2011) 343:329–345DOI 10.1007/s11104-011-0721-2

Responsible Editor: Peter A.H. Bakker.

S. Aranda :M. Montes-Borrego : R. M. Jiménez-Díaz :B. B. Landa (*)Institute for Sustainable Agriculture,Spanish National Research Council (CSIC),Alameda del Obispo, s/n, P.O. Box 4084,14080 Córdoba, Spaine-mail: blanca.landa@ias.csic.es

R. M. Jiménez-DíazDepartment of Agronomy,Campus de Rabanales-Edificio Celestino Mutis,University of Córdoba,Carretera de Madrid Km 396,14071 Córdoba, Spain

the 16S rDNA gene sequence indicated that most ofthe 94 bacterial antagonists belong to genera Bacil-lus (56.4%), Pseudomonas (27.7%), and Paeniba-cillus (7.4%). Overall, the rhizosphere andendosphere of wild olives were proved as a goodreservoir of bacteria antagonists against V. dahliae.Several of those bacteria showing high and broadantagonism potential may therefore be consideredfor further analyses as promising biocontrol agentsagainst V. dahliae in olive.

Keywords Antagonism . Bacterial communities .

Biodiversity . FT-RFLP.Olea europaea L. subsp.europaea var. europaea .O. europaea subsp.europaea var. sylvestris . Olive endosphere . Oliverhizosphere, Verticilliumwilt

Introduction

Olive is one of the most ancient domestic, cultivatedplants (Zohary and Spiegel-Roy 1975). Olive, occursin two forms, namely wild (Olea europaea subsp.europaea var. sylvestris) and cultivated (Olea euro-paea L. subsp. europaea var. europaea) (Green2002). For millennia, the cultivated form has beenculturally and economically the main oleaginous cropin the Mediterranean Basin, where circa 9.5 millionha of olives are grown accounting for 95% of thecultivated olive area worldwide (Angiolillo et al.1999; Bronzini de Caraffa et al. 2002; Issaoui et al.2008; Sefc et al. 2000). Spain is the largest olive oilproducer in the world, where approximately 65% of2.5 million ha of cultivated olive are in the Andalusiaregion (southern Spain) (CAP-JA 2009; IOOC 2009).

Olive groves dominate the landscape of Andalusiain an impressive monoculture that covers approxi-mately 17% of the total surface of the region. In thisarea, an exceptional presence of forests of wild olives(also known as oleasters) also represents a predomi-nant and distinctive component of the Mediterraneanflora (Rubio de Casas et al. 2002, 2006; Vargas andKadereit 2001). As a result of early domestication andextensive cultivation of olive throughout the Mediter-ranean Basin, the wild-looking forms of O. europaeasubsp. europaea presently observed constitute acomplex scenario, potentially ranging from trueoleasters (wild forms present in natural areas) to feralforms (secondary sexual derivatives of the cultivated

clones or products of hybridisation between cultivatedtrees and nearby oleasters) which occur in secondaryhabitats (i.e., disturbed areas or abandoned fields)(Angiolillo et al. 1999; Lumaret et al. 2004; Zoharyand Hopf 1994). The wild forms are genetically andphenotypically distinct, being more variable than thecultivated varieties or their derivatives feral forms, anaspect that bears important implications for theconservation of the ancient lineages (Lumaret andOuazzani 2001).

In recent years, several studies have focused on thegenetic variation of wild and cultivated olive pop-ulations and their relationships within olive cultivarsby using different molecular markers (e.g., Belaj et al.2002, 2007, 2010; Besnard and Bervillé 2000;Besnard et al. 2007; Breton et al. 2008; Lumaret andOuazzani 2001; Lumaret et al. 2004; Rubio de Casaset al. 2006). In Andalusia, simple sequence repeat(SSR) markers and allozymes analyses have demon-strated that wild olives represent an important sourceof genetic variability and that true wild oleasters stillsurvive in the south of Spain (Belaj et al. 2010;Lumaret et al. 2004; Rubio de Casas et al. 2006).

Olive crop production in the Mediterranean Basinis threatened by several diseases, among whichVerticillium wilt caused by the soilborne fungusVerticillium dahliae Kleb. is one of the mostimportant in many olive-growing areas includingSpain. V. dahliae can be classified into nondefoliatingand defoliating pathotypes according to their viru-lence on olive trees (Jiménez-Díaz et al. 2010; Navas-Cortés et al. 2008). Infections by the defoliatingpathotype (DP) can be lethal to the tree and arecurrently the main threat to olive production inAndalusia (Jiménez-Díaz et al. 2010; Navas-Cortéset al. 2008).

Verticillium wilt in olive must be managed usingan integrated strategy based mainly on preventivedisease control measures since no resistant varietiesand/or effective fungicides are commercially available(Jiménez-Díaz et al. 2010). Therefore, there is a needto develop novel and environmentally-friendly controlstrategies for management of this disease. One ofthose strategies can be the use of antagonist-mediatedbiological control, which is considered an alternativeor a supplemental mean of reducing the use ofchemicals in agriculture (Berg et al. 2005a; Paulin etal. 2009; Raaijmakers and Weller 2001). Thus, use ofplanting stocks certified free from V. dahliae and

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protection of their root system from infection byresidual soilborne or incoming V. dahliae inoculumwith microbial antagonists would be a suitablestrategy for reducing the potential for severe diseasein young olive trees (Mercado-Blanco et al. 2004).

Genes encoding resistance against some of themost widespread soilborne pathogens that cause rootrots, crown rots, damping-off or wilts are oftenlacking in crop species. As an alternative, crops seemto have evolved a strategy of stimulating andsupporting specific groups of indigenous antagonisticmicroorganisms as the first line of defense againstinfection by soilborne pathogens (Cook et al. 1995;Landa et al. 2006; Weller et al. 2007). Consequently,the plant root system may constitute an importantniche of microorganisms that may serve as anunexplored reservoir of plant-pathogen antagonists.Thus, many investigations on bacterial communities inthe rhizosphere soil and roots of many plant specieshave shown that those microorganisms can producedirect and indirect beneficial effects on plant growth andhealth, as well as that the structure and activities of thosecommunities are greatly influenced by the plant species(e.g., Ahn et al. 2007; Berg et al. 2005b; Jung et al.2008; Marilley and Aragno 1999; Mendes et al. 2007;Park et al. 2005). In this context, cultivated and wild-relatives of long-living plant species might constitutean ideal niche for exploring new bacterial isolatesharbouring some of the above properties. Furthermore,the longevity of wild olives may have favouredselection of specific and well adapted rhizospheremicrobial populations that can constitute uniquereservoirs of microbial antagonists of main olive root-associated fungal pathogens, such as V. dahliae.

Few studies (e.g., Hernesmaa et al. 2005; Muleta etal. 2009; Rumberger et al. 2007) have focused on thediversity and structure of microbial populationsassociated with woody plants. The specific objectivesof the present study were: (i) to examine the structureand diversity of bacterial communities in rhizospheresoil and roots of wild olives in Andalusia, southernSpain by culture-independent molecular approachesand to determine if that structure and/or diversity arecorrelated with the genetic diversity in the sampledwild olive populations; (ii) to assess the in vitroantagonistic potential of bacterial isolates obtained byculture-dependent approaches from the rhizosphereand endosphere of wild olives against the DP of V.dahliae; and iii) to identify the selected V. dahliae

antagonists and to characterize the biotechnologicalpotential of these isolates by in vitro production ofindole-1.3-acetic acid, and several antimicrobial com-pounds. To the best of our knowledge, this present studyis the first one aimed to determine the structure anddiversity of bacterial communities inhabiting the rhizo-sphere and roots of wild olives in Andalusia and theirpotential as a new source of V. dahliae antagonists.

Materials and methods

Sampling sites

Samples potentially including true oleaster and feralforms were obtained from 11 sites in Córdoba (threesites) and Cádiz (eight sites) provinces in Andalusiaand used in this study (Table 1; Fig. 1). Several of thesampling sites in Cádiz province were good candi-dates for genuine oleasters according to criteriadefined by Lumaret et al. (2004): (i) past and presentclimatic conditions suitable for wild olive growth(humid and subhumid variants of the thermo-Mediterranean climate with an average minimumtemperature of the coldest month and annual rainfallexceeding 5°C and 450 mm, respectively), and ii) pastand present isolation (usually more than 10 km) fromareas of cultivated olive trees. Some of thosepopulations were in evergreen oak forests located inuneven areas that currently have become naturalreserves. The three sampling sites in Córdoba prov-ince were located in undisturbed habitats, includingdegraded formations and abandoned groves potential-ly containing a mixture of feral forms and cultivars ofolive trees. The geographic location and altitude ofthe sampling sites were determined using a globalpositioning system (GPS) (Table 1, Fig. 1). Climaticclassification (J. Papadakis), annual minimum, max-imum, and mean temperature (°C), and annualprecipitation (mm) of each sampling site wereobtained from SigMapa, Geographic InformationSystem from the Spanish Ministry of “Medio Ambi-ente y Medio Rural y Marino” (http://sig.mapa.es/geoportal/) (Table 1, Fig. 2).

Soil, rhizosphere and root samples

Soil and roots samples were taken from three pointsper tree at 5 to 30 cm depth in the area of the canopy

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projection (close to the influence of roots; i.e.,rhizosphere soil). A composite sample was obtainedper sampling site by pooling the roots and rhizospheresoil from at least five trees from each sampling site.

Samples were immediately stored at 4°C in plasticbags loosely tied to ensure sufficient aeration and toprevent moisture loss until assaying of bacterialcommunity structure. The remaining rhizosphere soilwas air-dried and sieved (2-mm to 5-mm mesh size)prior to soil physicochemical analyses. Physicochem-ical properties of soil from each location includingsoil texture, pH, organic carbon (SOC) and nitrogen(N) content, and cation exchange capacity (CEC)were determined as described before (Álvarez et al.2007) by the official Agroalimentary Laboratory ofCórdoba (Córdoba, Spain) (Fig. 2).

Olive DNA extraction and microsatellite genotyping

To characterize the genetic diversity of oleaster andferal forms in each sampling site, total genomic DNAwas extracted from the composite sample obtainedfrom the pooled roots obtained from all sampled trees

Table 1 Site location and characteristics of oleaster and feral forms sampled at Andalusia, southern Spain used in the study

Sample code Olive genotype (age) a Province and sampling site Geographiclocation

Alt (m)b Climatic Classification

M1 W (>200 yr) Cádiz, 4.1 km S Ubrique 36°38′27.38″N 361 Mediterranean Maritime5°26′37.53″O

M2 W (>200 yr) Cádiz, 1.0 km SW Benaocaz 36°41′28.09″N 744 Mediterranean Maritime5°25′35.34″O

M3 W (>200 yr) Cádiz, 0.5 km E Villaluenga delRosario

36°41′42.79″N 828 Mediterranean Maritime5°22′57.22″O

LO W (>200 yr) Cádiz, 8.0 Km SW Benalup-CasasViejas

36°18′43.65″N 53 Mediterranean Maritime5°54′1.14″ O

LOBA W (>200 yr) Cádiz, 7.4 Km SW Benalup-CasasViejas

36°18′26.64″ N 47 Mediterranean Maritime5°52′56.01″ O

NAJARA-1 W (>200 yr) Cádiz, 4,8 km NE Vejer de laFrontera

36°17′25.14″N 42 Mediterranean Maritime5°55′59.64″O

NAJARA-2 W (>200 yr) Cádiz, 4,8 km NE Vejer de laFrontera

36°17′24.82″N 43 Mediterranean Maritime5°55′58.75″O

BAETICA W (>200 yr) Cádiz,1.5 km E Vejer de la Frontera 36°14′32.64″ N 14 Mediterranean Maritime5°57′1.63″ O

MACO F (>100 yr) Córdoba, 0.6 Km NE MedinaAzahara

37°54′27.53″N 252 Subtropical Mediterranean4°34′3.52″O

LOMCO F-C (>100 yr) Córdoba, 5.9 Km N Córdoba 37°51′24.54″N 352 Subtropical Mediterranean4°48′5.32″O

EPCO F-C (>100 yr) Córdoba, 4.5 Km NW Córdoba 37°36′17.65″N 261 Subtropical Mediterranean4° 7′8.95″O

aWild (W), feral (F), cultivated (C) status hypothesized according to environmental, historical and demographic criteriab Altitude above sea levelc Climatic classification (J. Papadakis) was obtained from SigMapa, Geographic Information System from Spanish Ministry of “MedioAmbiente y Medio Rural y Marino” (http://sig.mapa.es/geoportal/)

Fig. 1 Location of the wild olive havens sampled in theprovinces of Córdoba and Cádiz, at Andalusia, Southern Spain.The darker green colour corresponds to olive cultivated area inAndalusia

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in each location (i.e., 11 samples in total). DNA wasextracted from three samples (50 mg each) persampling site using the ‘G-SpinTM IIp Plant Ge-nomic DNA extraction kit’ (Intron Biotechnology,Korea) and the Fast Prep System Bio 101 (Qbiogene,Illkirch, France) according to Landa et al. (2007). Thepolymerase chain reaction (PCR) conditions de-scribed by de la Rosa et al. (2002) and the followingsingle sequence repeats (SSR) primer pairs were used:ssrOeUA-DCA03, ssrOeUA-DCA09, ssrOeUA-DCA16 and ssrOeUA-DCA18 (Sefc et al. 2000),and UDO99-043 (Cipriani et al. 2002). These SSRprimers were chosen because they were demonstratedthe most informative and reliable and provided thehigher power of discrimination among a number ofloci/alleles for cultivated and wild olives populationstudies (Baldoni et al. 2009; Belaj et al. 2007, 2010).

Amplification products were detected with anautomated DNA multicapilar sequencer (Model3130XL genetic analyzer; Applied Biosystems, UK)at the Unit of Genomics of the Central Service forResearch Support (SCAI) of the University ofCórdoba sequencing facilities (Córdoba, Spain). Siz-ing of amplification products was done using aROX500 size standard and the software Genmapper3.7 from Applied Biosystems as described before(Belaj et al. 2007).

Extraction of rhizosphere and endosphere bacterialcommunities

Intact root systems were shaken gently by hand toremove all but the soil close- and naturally-adheringto the plant root. Pooled root samples were cut into 1-

cm pieces with a sterile scissors to get a uniformsample per location. Suspensions of rhizosphere(including rhizosphere soil and rhizoplane bacteria)were obtained by vigorously shaking 2 g of rootsegments suspended in 20 ml of sterile distilled waterin an orbital shaker for 10 min. The suspensions ofrhizosphere soil were then sonicated (Ultrasons, JPSelecta SA, Barcelona, Spain) for 10 min. Theresulting suspension was considered to contain arepresentative of the rhizosphere bacterial populations(extracted from rhizosphere soil and rhizoplane).

For DNA extraction from rhizosphere, 3 ml ofrhizosphere suspensions were subjected to consecu-tive centrifugations at 11,000 rpm for 4 min and thepellet was recovered. For root-endophytic bacterialpopulations, the root pieces (2 g) were surface-disinfested with a 2% NaOCl for 3 min and washedthree times with sterile distilled water. The disinfestedroot samples were then ground in a mill (Bosh MMB2000, Robert Bosch España, S.A., Madrid, Spain)with 100 ml of sterile distilled water and the resultingsuspensions were considered to contain a representa-tive of the endophytic bacterial community. Toconfirm that superficial disinfestation of roots wassuccessful, 100 μl aliquots of the sterile distilledwater used in the final washed step of root sampleswere plated and spread onto low-nutrient agar R2A(Biolife Italiana S.r.l. Milan, Italy). Plates wereexamined for bacterial growth after incubation at28±1°C and dark conditions for 3 days.

Rhizosphere and endosphere suspensions wereserially diluted and plated onto R2A agar (100 μl ofeach dilution series; with three replicates each) andincubated as indicated above. Adequate dilutions

Fig. 2 Cluster analysis of combined data from climatic andphysicochemical soils characteristics of wild olive locationssampled in the study. All soil properties were estimatedaccording to Álvarez et al. (2007) by the official AgroalimentaryLaboratory of Córdoba (Córdoba. Spain) and climatic character-istic were obtained from SigMapa, Geographic Information

System from Spanish Ministry of “Medio Ambiente y MedioRural y Marino” (http://sig.mapa.es/geoportal/). Bootstrap valuesare indicated in each node. Wild (W), feral (F), cultivated (C)status hypothesized according to environmental, historical anddemographic criteria. Locations where true wild olives arepresent are highlighted

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were selected and bacterial colonies counted after4 days. For each location, 40 or 22 isolates wereselected for the rhizosphere and endosphere bacterialpopulations, respectively. Those isolates were consid-ered representative (most abundant) of the totalbacteria grown in the plate after visual examinationof colony features under a stereomicroscope at 20 to120x (Nikon SMZ1500, Japan).

DNA extraction from rhizosphere bacterialcommunities

Samples of pellets from centrifuged rhizosphere soilsuspensions (approximately 200 mg) were shaken todisrupt soil particles and bacterial cells in theFastPrep-24 Instrument (MP Biomedicals, Inc.France) run at 6.0 m/s for 40 s. Total DNA was thenextracted using the MoBio Ultraclean™ soil DNAisolation kit (MoBio laboratories, Inc; Carlsbad, CA,USA) according to manufacturer instructions.Extracted DNA was eluted in ultrapure sterile water(Sigma, Madrid, Spain), checked for integrity andquality by staining with ethidium bromide andquantified in triplicate using the Quant-iT DNAAssay Kit Broad Range fluorometric assay (Molecu-lar Probes Inc., Leiden, The Netherlands) and aNanodrop spectrophotometer ND-1000 (NanodropTechnologies, Wilmington, DE, USA). DNA concen-tration from rhizosphere samples ranged from 3 to15 ng/μl and was used directly for amplification.

Rhizosphere bacterial community profiling

The structure of bacterial communities in the rhizo-sphere of sampled oleasters and feral forms wasinvestigated by using fluorescent terminal restrictionfragment length polymorphism (FT-RFLP) analysesof amplified 16S rDNA sequences. The 16S rDNAgenes were amplified using primers 8f (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492r (5′-ACGGCTACCTTGTTACGACTT-3′) (Weisburg etal. 1991). Primer 8f was labelled with FAM (6-carboxyfluorescein) for visualizing the terminal re-striction fragments (TRF). Amplifications were car-ried out in a final volume of 25 μl using 2.5 μl 10xMg-free buffer, 2 mM MgCl2, 0.2 mM of each dNTP,1 mM of each primer, 0.5 μg RNase, 0.33 μl ofBiotaqTM polymerase (BioLine, London, UK) and2.5 μl template DNA from rhizosphere samples. PCR

assays were performed in a C1000 Bio-Rad thermalcycler (Bio-Rad, Madrid, Spain). The cycling pro-gram consisted of an initial denaturation step of 95°Cfor 5 min, followed by 30 cycles of 94°C for 1 min,54°C for 45 s and 70°C for 1 min, and a finalextension step at 70°C for 8 min. PCR amplificationproducts were first tested by agarose gel electropho-resis before conducting FT-RFLP analysis.

PCR products were digested in a 10 μl of reactioncontaining 5 units ofMspI or RsaI enzymes (Fermentas,Hanover, MD, USA) and 4 μl of labelled amplicons.Restrictions were performed at 37°C for 3 h followedby an inactivation step at 65°C for 10 min. Fourmicroliters of digested DNA were diluted into 6 μl ofwater for fragment analysis. Terminal restriction frag-ments (TRF) were loaded and separated on a 3130XLgenetic analyzer (Applied Biosystems) at the SCAI,University of Córdoba sequencing facilities. Size offragments were determined using a ROX500 sizestandard, and matrixes containing incidence as wellas peak area data of individual TRF were generated forall samples with Genemapper software. Peaks of lessthan 100 fluorescence units (FU) and 50 bp were notincluded in the analysis to eliminate primer dimers andother small charged molecules. Similarly, moleculesthat were not present in at least two of the threereplicate profiles were disregarded. Also, TRFs thatdiffered by less than 1 bp were clustered, unlessindividual peaks were detected in a reproduciblemanner. TRFs profiles were standardized based onmethods described previously by Dunbar et al. (2001).The relative abundance of each TRF was calculated asthe ratio of the peak area for that TRF to the sum ofpeak areas for all TRFs in the profile and wasexpressed as a percentage.

In vitro screening of antagonistic bacteria

The ability of rhizosphere and endosphere bacterialisolates from oleasters and feral forms to inhibit invitro fungal growth was tested by means of dual-culture assays on Waskman agar containing 5 gproteose-peptone (Panreac, Barcelona, Spain), 10 gglucose (Panreac), 3 g meat extract (Sharlau Chemie,Barcelona Spain), 5 g NaCl (Panreac), and 20 g agar(Difco, Laboratories, Detroit, MI, USA) per liter ofmedium, at pH 6.8. The DP V. dahliae isolate V138(Navas-Cortés et al. 2008) was grown in Czapek-Doxbroth medium (Difco) in and orbital shaker at

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120 rpm and 25±1°C for 14 days in the dark. Conidiaconcentration in the culture was estimated with ahematocytometer and adjusted to 5×106 conidia.ml−1

with sterile distilled water; 100 μl of the conidialsuspension was spread onto the medium and dried for30 min at room temperature. Then, 5 μl of cultures ofbacterial isolates grown in 96-well microplates con-taining Tryptic-soy broth medium (TSA) (Difco) at28±1°C for 24 h were placed onto cultures of V.dahliae and plates were incubated at 28±1°C for5 days. The width of zones of inhibition was recordedaccording to the following rating scale: +++= 10 to15 mm inhibition halo, ++= 5 to 10 mm inhibitionhalo, += 1 to 5 mm inhibition halo and -= no inhibitionhalo. Only bacterial isolates that showed inhibitoryactivity of in vitro growth of V. dahliae were furthercharacterized phenotypically and genotypically.

Production of exoenzymes and secondary metabolitesby bacteria

Bacterial antagonists were tested for chitinolytic, lipo-lytic and proteolytic activities using a semi-quantitativeplate assay (Chernin et al. 1995). Chitinolytic activity(β-1-4-glucosamine polymer degradation) was testedin chitin minimal medium with clearing zones detected5 days after incubation at 30±1°C. Proteolytic activity(casein degradation) was determined from clearingzones in skim milk agar (50 ml sterilized skimmedmilk mixed at 55°C with 50 ml 1/5 TSA and 4% agar)after 5 days of incubation at 30±1°C. Lipolytic activitywas tested using Tween 80 agar (Sigma). The lipolyticactivity was determined by the presence of turbidzones around bacterial colonies growth. Total side-rophore production was assayed under iron-limitedconditions using the universal siderophore assay ofSchwyn and Neilands (1987) after incubation for5 days at 30±1°C. Zones of haloes formation wererecorded as followed: +++= >10 mm halo, ++= 5–10 mm halo, += 1–5 mm halo, and - = no halo. Theability of bacterial isolates to produce indole-3-aceticacid (IAA) was determined using the microplatemethod developed by Sarwar and Kremer (1995).Ability of Pseudomonas spp. isolates to producefluorescent siderophores was tested on King’s Bmedium (King et al. 1954) and incubating at 25±1°Cfor 2 days and determined by visual inspection under366-nm UV light. Also, the potential of Pseudomonasspp. isolates to produce 2,4-dyacetilphloroglucinol was

determined by amplification of phlD gene usingprimers B2BF and BPR4 and PCR conditions previ-ously described (Landa et al. 2002, 2006).

Identification of antagonistic bacteria

All bacterial isolates showing antagonistic activityagainst V. dahliae were identified to genus/specieslevel by sequencing the 16S rDNA gene. Amplifica-tions were performed as described above, andsequences were purified using the PureLink PCRpurification kit (Invitrogen, Carlsbad, CA, USA). Thefull-length of 16S rDNA amplicon (~1,500 bp) wasdirectly sequenced using primers 8F and 1492r atSTABVIDA sequencing facilities (Oeiras, Portugal).Sequences were assembled, and the errors of consen-sus sequences were corrected manually using Bionu-merics 6.1 (Applied Maths, Sint-Martens-Latem,Belgium). Isolates were identified to genus/specieslevel by: (i) the Ribosomal Database Project’s (http://rdp.cme.msu.edu) Naïve Bayesian Classifier (80%confidence threshold), and (ii) the nearest neighbourin the GenBank ‘nt’ database after alignment withreference 16S rRNA gene sequences using theBLAST algorithm according to Altschul et al.(1997). Representative 16S rRNA sequences fromthe different phylum, class, order, family and genus ofbacteria obtained in the study have been deposited inthe GenBank database under accession numbersHQ606092-HQ606105.

Data analyses

Population data were converted to log (CFU g−1) dryweight of fresh weight of root to satisfy assumptionsof the parametric statistical test used. Data wereanalyzed using STATISTIX 9.0 (Analytical Software,St. Paul, MN, USA). Differences in populationdensities among treatments were determined bystandard analysis of variance. Mean comparisonsamong treatments were performed by using Fisher’sprotected least significant difference test at P=0.05.

Data of soil physicochemical and climatic charac-teristics of the sampled sites, and data of TRFs fromrhizosphere bacterial community profiles (averagefrom the three replicates samples per oleaster/feralhaven) were subjected to principal component (PCA)and cluster analyses using the Euclidean distancemeasure. Dendrograms were constructed using the

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unweighted pair-group method with arithmetic mean(UPGMA). All statistical procedures were performedusing Bionumerics 6.1 and SAS (Statistical AnalysisSystem, version 9.1; SAS Institute, Cary, NC, USA).

Diversity statistics were calculated from standard-ized profiles of rhizosphere samples by using thenumber and area of peaks in each profile asrepresentative of the number and relative abundanceof phylotypes, as defined by Dunbar et al. (2000).Phylotype richness (S) was calculated as the totalnumber of distinct TRF sizes (between 50 and500 bp) in a profile. The Shannon-Weiner diversityindex (Shannon and Weaver 1963) was calculated asfollows: H0 ¼ �P

pið Þ log pið Þ, where pi is theproportion of the i-th peak area relative to the sumof all peak areas (Hackl et al. 2004).

An assessment scheme similar to that developed byFaltin et al. (2004) was used to characterize bacterialisolates with high antagonistic potential and tocompare their antagonistic potential according to thesampling location, microhabitat (rhizosphere andendosphere), and species identity. For this purpose, ascore system was used, where points were given foreach of the bacterial traits: up to three pointsaccording to the width of the inhibition zone orproduction of a clearing halo for inhibitory activity ofV. dahliae growth and for production of hydrolyticenzymes, siderophores, auxin, and fluorescence onKing’s medium B. A total number of 21 points waspossible. Then, a relative antagonism score index(RASI) was calculated by referring the score given toeach of the bacterial traits to the maximum scorepossible. A Cluster analysis of phenotypic antagonis-tic traits was performed with the Pearson correlationmoment coefficient and with the UPGMA algorithmmethod as described above.

Results

Characterization of oleaster and feral sites sampledin the study

Combined analysis of soil physicochemical andclimatic characteristics grouped the 11 oleaster/feralsampled sites into three main clusters according totheir geographical location and climatic conditions(Figs. 1 and 2). Olive feral forms sampled in Córdobaprovince were clearly differentiated from true olea-

sters in Cádiz province (Fig. 2). While estimating thegenetic diversity in oleaster and feral forms usingDNA extracted from pooled root samples, we foundreproducible amplifications and discernible allelesonly with the ssrOeUA-DCA03 and UDO99-043markers. However, these results were enough todetermine that each sampled location constituted adifferent olive gene pool, with locations in Cádizprovince showing a higher number of alleles than inCórdoba province (data not shown).

Bacterial community profiling of oleaster/feral havens

Amplifications using the DNA extracted directly fromrhizosphere pellets as template yielded fragments ofapproximately 1,500 bp. Both, fragment length andpeak area were considered as parameters for profilecomparison after standardization of TRF data asdescribed by Hackl et al. (2004). The two restrictionenzymes used to create different fingerprints foreach of the samples in FT-RFLP analysis, MspI andRsaI, consistently yielded 12 to 59 and 10 to 38 ofresolvable peaks, respectively, depending of therhizosphere sample and replication, with MspIgenerating a much higher number of different TRFs(115) as compared to RsaI (54). The phylotyperichness and Shannon-Weiner index of diversityvalues varied depending upon the restriction enzymeused and the site of sampling with no general pattern(Fig. 3).

Cluster (UPGMA) and PCA analyses of com-bined TRFs data analyzed independently for MspIand RsaI, or combined, of the 11 rhizospherecommunities revealed that each oleaster/feral havenconstitutes a unique reservoir of bacterial diversity(Fig. 3). Thus, rhizosphere bacterial communities ofeach oleaster/feral haven appeared to be unique,even though the rhizosphere samples were closed intheir location or had similar physicochemical soilproperties (Fig. 3). Furthermore, bacterial rhizo-sphere communities from feral forms grouped to-gether in same cluster and clearly differed from thoseof real oleasters (Fig. 3).

Culturable bacterial populations from the rhizosphereand endosphere of oleaster and feral forms

In all the sampled sites, the population density ofculturable bacteria in the rhizosphere of oleaster and

336 Plant Soil (2011) 343:329–345

feral forms ranged from log 5.94 to 6.69 cfu root−1.This population density was significantly higher (P<0.05) than that in the endosphere, which ranged fromlog 2.51 to 4.93 cfu g root−1 (Table 2). Bacterialendophytes in EPCO location in Córdoba were belowthe detection limit (Table 2). Also, in all sampledsites rhizosphere bacterial populations were signif-icantly higher (P<0.001) than those found in theendosphere. All samples from Córdoba provinceincluding feral forms harboured significantly lower(P<0.05) rhizosphere and endophytic bacterial pop-ulations (with the exception of M1) compared withthose sampled in Cádiz province, which includedoleaster forms only (Table 2). There was a positiveand significant correlation (r=0.7765; P=0.0049)between population densities of total culturablebacteria inhabiting the rhizosphere and those in theendosphere.

In vitro inhibition of V. dahliae growthand production of exoenzymes and secondarymetabolites

A total of 675 bacterial strains from the rhizosphereand endosphere of oleaster and feral forms werearbitrarily chosen and screened for their ability toinhibit in vitro growth of V. dahliae DP. Out of the675 bacterial isolates, 94 (14%) displayed high levelsof growth inhibition of V. dahliae DP. Of the 94bacterial antagonists, 59.6% were isolated from therhizosphere and 40.4% were endophytic. Overall,12.9% of the total rhizosphere isolates were inhib-ititory to the pathogen compared with those from theendosphere that amounted 16.5%. All samples in therhizosphere and endosphere of oleaster/feral formsyielded isolates with inhibitory activity against V.dahliae DP, with the exception of two endosphere

Fig. 3 Cluster analysis and Principal Component Analysis offluorescent terminal restriction rhizosphere bacterial populationfingerprints from 11 wild olive locations generated by MspI andRsaI enzymes. Three independent amplifications were com-bined before analysis for each location. The unweighted pair-group method using arithmetic averages (UPGMA) algorithmwas applied to the similarity matrix generated from the relativeabundance of each terminal restriction fragment (TRFs) by

using the squared Euclidean distance coefficient. Combinedcluster analysis of MspI + RsaI TRFs patterns correspond to theaverage of both restriction enzymes analyzed independently.Richness (Number of TRFs) and Shannon-Weiner Diversityindex for both restriction enzymes is also included for eachlocation. Cophenetic correlation values are indicated in eachnode. Locations where true wild olives are highlighted

Plant Soil (2011) 343:329–345 337

samples from LOMCO and EPCO sites in Córdobaprovince (Table 2). Interestingly, there was a positiveand significant correlation between the percentage ofantagonists found in the rhizosphere and the endo-sphere (r=0.6956; P=0.0175), as well as between thepopulation density of total culturable bacteria in theendosphere and the proportion of antagonists found inthis niche (r=0.7396; P=0.0093). However, such acorrelation did not occur with the culturable bacteriain the rhizosphere (r=−0.1752; P=0.6064).

Bacterial antagonists from rhizosphere and endo-sphere showed similar production of exoenzymes andsecondary metabolites, with most of them showing

proteolytic (73.3%), lipolytic (76.4%) and chitinolytic(56.8%) activity, and producing IAA (75.3%) andsiderophores (76.0%). Of 26 bacterial isolates identi-fied as Pseudomonas spp. (see below), 76.9%produced pyoverdines on King’s B medium asindicated by fluorescence under UV light, and23.1% were phlD positive (Fig. 4). All isolatesshowed at least three out of the five antagonisticactivities or traits of above, but 21.3% of themexpressed all of them (Fig. 4). The relative antago-nism score index (RASI) ranged from 14.3 to 61.9%among the 94 isolates evaluated. The number ofantagonistic activities and traits or RASI values were

Table 2 Population densities of indigenous culturable bacteria and bacterial isolates antagonistic against Verticillium dahliaerecovered from the rhizosphere and endosphere of wild olives in 11 sites at Andalusia, southern Spain

Root niche Sample Code Bacterial isolatea Population level log(cfu.g−1 root)b

Antagonists (%)

Prefix Number

Rhizosphere M1 RacM1- 1–7, 11–12, 66–70 5.93 de * 50.0

M2 RacM2- 16–18, 22, 73 6.69 a * 20.0

M3 RacM3- 76 6.48 a * 3.3

LO RacLO- 23–28 6.37 ab * 20.0

LOBA RacLOBA- 36–40, 60–64 6.25 abc * 16.7

NAJARA-1 RacNa1- 56–58 6.33 ab * 13.3

NAJARA-2 RacNa2 48–51 6.47 a * 13.6

BAETICA RacBaet- 78–80 6.46 ab * 10.0

MACO RacMACO- 86–90 5.94 cd * 16.7

LOMCO RacLOMCO- 91 6.12 bcd * 3.3

EPCO RacEPCO- 92–94 5.37 e * 10.0

Endosphere M1 EacM1- 9–10, 71–72 4.76 ab 46.7

M2 EacM2- 19–21, 74–75 4.93 ab 33.3

M3 EacM3- 77 4.70 ab 6.6

LO EcLO- 29–35 4.78 ab 46.7

LOBA EacLOBA- 41–47, 65 4.74 a 46.7

Najara-1 EcNa1- 59 4.08 abc 6.7

Najara-2 EacNa2- 52–55 4.77 ab 26.7

Baetica EacBaet- 81–83 3.61 bc 20.0

MACO EacMACO- 84–85 2.51 de 13.3

LOMCO EacLOMCO- – 3.09 cd 0.0

EPCO EacEPCO- – 1.60 e (DL) 0.0

a Bacterial isolates from each location were designated as Racx-y and Eacx-y, for rhizosphere and endosphere root origin, respectively.In the bacteria code, x represents the sample code, and y the isolate numberb Population size of total bacteria were determined by dilution plating onto R2A medium. Means followed by different letters or andasterisk indicate statistically significant differences among locations or between root niches (rhizosphere vs. endosphere), respectively,according to Fisher’s-protected LSD (P=0.05). DL = Population levels were below the detection limit for some replicates.Colonization data were converted to log10 CFU.g

−1 fresh root weight before data analysis

338 Plant Soil (2011) 343:329–345

not correlated with the source of isolates (i.e.,rhizosphere, endosphere, sampling site or nature ofthe sampled plant) (Fig. 4).

Combined cluster analysis of phenotypic antago-nistic traits performed with the Pearson correlationmoment coefficient and the UPGMA algorithmmethod revealed four main clusters at a cluster cutoffvalue of 26.1%, all of them with cophenetic correla-tion higher than 70%. There was no correlationbetween the clustering of bacterial isolates and theirroot source (rhizosphere vs. endosphere) or samplingsite (Fig. 4). Within the four main clusters, there weresubclusters that grouped bacterial isolates accordingto their antagonistic properties and RASI values(Fig. 4).

Identification of antagonistic bacteria

Partial 16S rRNA sequences (about 1,400-bp long)were obtained from all bacterial antagonists of V.dahliae to asses the genetic diversity within them.Analysis of these sequences revealed that fourbacterial phyla were represented within the bacterialantagonist derived from all oleaster and feral havens(Fig. 4, Table 3). The phylum Firmicutes was themost abundant phylogenetic group, with 52 isolates,which accounted for 62.8% of all the examinedbacteria, followed by Proteobacteria with 32 isolates(34.0%). Bacteroidetes and Actinobacteria were lessabundant and accounted only for 2.1% and 1.1% ofthe isolates, respectively.

Bacillus, the major taxon identified, represented56.4% of the total of antagonists followed by Pseudo-monas with 27.7%. All Bacillus spp. and Pseudomo-nas spp. identified were isolated both from therhizosphere and endosphere. While all Bacillus spp.identified were found in comparable proportion in therhizosphere (56.6%) and as root endophytes (43.4%),most of Pseudomonas spp. identified were isolatedfrom the rhizosphere (73.1%) and to a lesser extentfrom the endosphere (26.9%) (Fig. 4, Table 3). Usingthe nearest neighbor in the GenBank ‘nt’ the Bacillusspecies more frequently identified were B. subtilis/B.amyloliquefaciens group followed by B. cereus/B.thuringiensis group (Fig. 4). In the case of Pseudomo-nas, the majority of antagonistic bacterial isolatescould not be assigned to the species level or, if so,belonged to P. fluorescens/P. reactans, and P. brassi-cacearum (Fig. 4). Other antagonists found in minor

proportion (7.4%) were identified as Paenibacilluspolymyxa and were isolated both from the rhizosphereand endosphere. Finally, three isolates of Acinetobacterrhizosphaere (isolates RacM1-1, RacMACO-86 andRacMACO-87) and one of Rhodococcus wratislavien-sis (isolate RacM1-1) were recovered only from therhizosphere; and two isolates of Chryseobacteriumvrystaatense (RacM1-14 and RacM1-15) were foundas root endophytes only.

All the above referred isolates except four wereassigned to the genus level with a 100% confidencethreshold using the Naïve Bayesian Classifier of theRibosomal Database Project’s. The four exceptionisolates (confidence threshold 69–76%), namelyRacM1-70, RacLOBA-47, RacM2-20 and RacM1-2,were identified as Bacillus sp., Rahnella aquatilis,Erwinia sp. and Pseudomonas sp., respectively, usingthe nearest neighbor in the GenBank ‘nt’ (Fig. 4).

There was a degree of correlation between the fourclusters defined on the basis of combined analysis ofphenotypic antagonistic traits and the identity ofbacterial isolates. Thus, cluster I included onlyBacillus spp., except for an isolate each of Paeniba-cillus sp., Pseudomonas sp., and Chryseobacteriumsp., and Cluster II was the most diverse and includedrepresentatives of all the genera identified. On thecontrary Clusters III and IV comprised Pseudomonasspp. only, and Cluster IV grouped all Pseudomonasharboring the phlD gene (Fig. 4).

Discussion

The use of biocontrol agents, including bacteria, iscurrently increasing worldwide as a promising alter-native for the control of soilborne plant pathogens,mainly in sustainable and organic agricultural sys-tems. Soil bacteria are an essential and importantcomponent of the biotic community in natural andcultivated woody trees habitats. Moreover, soil bac-teria are largely responsible for functioning ofecosystems because of their participation in transfor-mation of most nutrients, their profound effect onplant growth and health, and as a source of potentialbiocontrol agents of soilborne plant pathogens. In thiscontext, wild-relatives of long-living trees, such asoleasters, which exhibit high genetic variability, mightconstitute an ideal niche for exploring new bacterialisolates with diverse mechanisms of antagonism

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340 Plant Soil (2011) 343:329–345

against soilborne fungal diseases of olives such asVerticillium wilt.

In our study, results of olive microsatellite geno-typing and of cluster analysis of soil physicochemicaland climatic characteristics indicated that each sam-pled site differed in the genotypes of wild olivespresent, soil type and climate. Accordingly, PCA andcluster analyses of TRFs revealed same main group-ings that those revealed by the climatic and soilcharacteristics of the different ecosystem sampled,with bacterial communities in the rhizosphere ofoleasters in Cádiz province being differentiated in anindependent cluster from those of putative feral formsin Córdoba province. Interestingly, a recent study byBelaj et al. (2010) also revealed patterns of highgenetic variation in oleaster and pointed out thatoleasters at Cádiz province represent a separate genepool of true oleasters, which can also be differentiated

from oleasters in other areas and also from allcultivated olive varieties in Spain.

Plant species, even genotype or varieties, and soiltype are within the most important factors determin-ing the rhizosphere microbial community structure(Landa et al. 2006). In fact, natural plant genotypicvariants of a single species can select for specificmicrobiota consortia as a result of their uniqueexudates profiles (Micallef et al. 2009). In a similarway, different soils show different physicochemicalcharacteristics that influence microbial communitiesby providing a specific habitat for selecting specificadapted microbes and also by influencing plant rootexudation (Garveba et al. 2004; Wang et al. 2008).For example, significant differences in rhizospherecommunity composition have been revealed associat-ed with the genetic characteristics of apple rootstocksas well as soil characteristics (Rumberger et al. 2007).Our results indicate that oleaster havens constituteexcellent reservoirs of bacterial diversity that may bedifferentiated from rhizosphere bacterial communitiesof olive feral forms. However which of severalfactors: plant genotype, soil type or climate is/arethe main driver(s) of the olive bacterial communitystructure has yet to be determined.

Endophytic microorganisms have been studied inseveral host plants (Cocking 2003). However, accord-ing to our knowledge naturally occurring root-endophytic microbial populations of oleasters havenot been studied yet. Several studies indicated that thepopulation density and diversity of endophytes are

Fig. 4 Cluster analysis of eight phenotypic antagonistic traitswith the Pearson correlation moment coefficient and with theunweighted pair-group method using arithmetic averages(UPGMA). Total antagonism score, the relative antagonismscore index (RASI), root origin, location of sampling, and theidentification at the Genus level of the 94 bacterial isolates isindicated. Isolates were identified to genus/species level by theRibosomal Database Project’s (http://rdp.cme.msu.edu) NaïveBayesian Classifier (80% confidence threshold), and the nearestneighbour in the GenBank ‘nt’ database after alignment withreference 16S rRNA gene sequences using the BLASTalgorithm according to Altschul et al. (1997). Copheneticcorrelation values are indicated in each node. (*) = Clustergroups were defined based on a cluster cutoff value of 26.14%

�

Table 3 Taxonomic distribution of 94 bacterial isolates antagonistic against Verticillium dahliae defoliating pathotype recovered fromthe rhizosphere and endosphere of oleaster and feral forms in Andalusia, southern Spain

Phylum Class Order Family Genera Root nichea (%)b

Rhizosphere Endosphere

Firmicutes Bacilli Bacillales Bacillaceae Bacillus 30 23 56.4

Paenibacillaceae Paenibacillus 3 4 7.4

Proteobacteria Gammaproteobacteria Pseudomonadales Pseudomonadaceae Pseudomonas 19 7 27.7

Moraxellaceae Acinetobacter 3 3.1

Enterobacteriales Enterobacteriaceae Rahnella 1 1.1

Erwinia 1 1.1

Actinobacteria Actinobacteria Actinomycetales Nocardiaceae Rhodococcus 1 1.1

Bacteroidetes Flavobacteria Flavobacteriales Flavobacteriaceae Chryseobacterium 2 2.1

a Total number of antagonistic bacterial isolates assigned to each populationb Proportion of the total bacterial antagonists identified derived from rhizosphere and endosphere of the plants

Plant Soil (2011) 343:329–345 341

highly variable traits, depending mainly of thebacterial species and host genotype but also the hostdevelopmental stage and environmental conditions(e.g., Ahn et al. 2007; Marschner et al. 2004; Mendeset al. 2007; Rosenblueth and Martínez-Romero 2006).Our results showed that same antagonistic Pseudo-monas and Bacillus species occur both in therhizosphere and endosphere microenvironments inmost sampled sites. These sites differed in soilphysico-chemical properties and environmental con-ditions, thus suggesting a closely association betweenthose bacteria genera and wild olive genotypes.

In the present study, 675 bacterial isolates wererecovered from rhizosphere and endosphere of wildolives and tested for their inhibitory activity of invitro growth of the DP of V. dahliae in dual cultures.From those, 14% showed a high level of inhibitoryactivity against the pathogen. Lower proportions (5.7to 8.3%) of antagonists from total culturable bacterialpopulations have been found in other studies (e.g.,Berg et al. 2002; Berg and Hallmann 2006). In ourstudy all the oleaster and feral havens sampledharboured rhizosphere and endosphere antagonists,and only in two sites that included potential feralforms was not possible to recover bacterial antago-nists from the endosphere. However, the proportion ofantagonistic bacteria in the total culturable bacteriavaried greatly among sampled sites, ranging from3.3% to 50.0%. As indicated above, the genotype ofwild olives, soil characteristics and climate may affectdifferently to the specific populations of bacterialantagonists present in the rhizosphere or endosphere.

Previous investigations (Berg et al. 2002, 2005b)have shown that plant species, soil type (as deter-mined by different locations) and rhizosphere have astrong influence on the root-associated bacterialantagonists in Verticillium host plants. Thus, therhizosphere effect on the antagonistic bacterial com-munity was shown by an enhanced proportion ofantagonistic isolates, by enrichment of specific am-plified ribosomal DNA restriction analysis types,species and genotypes, and by a reduced diversity inthe rhizosphere in comparison to bulk soil. Further-more, the proportion and composition of populationsof bacterial antagonists associated with potato, oilseedrape and strawberry was shown to be influenced bythe plant species and growth stage. In our study wefound the highest bacterial species diversity in therhizosphere as compared to the endosphere.

Bacillus spp. and Pseudomonas spp. were the mostpredominant bacterial antagonists of V. dahliae DP inthe root environment of oleasters and feral forms.Bacillus spp. colonized both the rhizosphere andendosphere of those plants in similar proportion.Conversely, Pseudomonas spp. were significantlymore abundant in the rhizosphere. Our results areconsistent with those from other investigations thatfound Pseudomonas and Bacillus as main compo-nents of root-associated microbial communities inmany plant species (Donate-Correa et al. 2004;Poonguzhali et al. 2006). These bacteria efficientlycolonize the root surface of plants they are associatedwith and contribute to preventing development ofplant pathogens and improving plant growth (Bergand Hallmann 2006; Correa et al. 2009; Debode et al.2007).

The ability of Pseudomonas and Bacillus spp. toinhibit growth of pathogenic fungi is well known andthese bacteria have been reported to be one of themost frequent beneficial rhizobacteria showing broadantifungal effect (Muleta et al. 2007). In culture-dependent studies, Pseudomonas spp. isolated fromdifferent plant species were the most abundantrhizobacteria showing in vitro antagonism against V.dahliae, with their diversity being both site- andplant-specific (Berg et al. 2002, 2005b). Recently,Muleta et al. (2007, 2009), screened the in vitroantagonistic effect of indigenous cultivable rhizobac-teria associated with Coffea arabica, and found thatmost bacteria showing inhibitory activity againstgrowth of Fusarium spp. were members of Pseudo-monas and Bacillus spp.

The rhizosphere and endosphere communities ofPseudomonas spp. were dominated mainly by un-identified Pseudomonas spp., or P. fluorescens/P.reactans, whereas those of Bacillus spp. weredominated by B. subtilis/B. amyloliquefaciens. Otherantagonists were found in very minor proportion andconsisted of Rhodococcus sp. and Acinetobacter sp.,which occurred only in the rhizosphere, or Erwiniasp., Chryseobacterium sp. and Rahnella sp. that werefound only in the endosphere. A special case was theantagonist Rahnella aquatilis that was found only inone site representing a low proportion of the bacterialcommunity in it. Results of in vitro antagonisticactivity of R. aquatilis against V. dahliae in our studyare in concordance with those reported by Uppal et al.(2008). Moreover, previous studies have reported that

342 Plant Soil (2011) 343:329–345

R. aquatilis strains produce different inhibitory sub-stances with a broad spectrum of activity against anumber of plant pathogenic bacteria and fungi (Calvoet al. 2007; Chen et al. 2009; Jafra et al. 2009).

Multiple biocontrol mechanisms have been impli-cated in the suppression of fungal root diseases bybiocontrol strains of Pseudomonas and Bacillus. Ithas been strongly suggested that the main success of abiocontrol agent is largely attributable to multifunc-tional biocontrol traits (Vassilev et al. 2006). In thisregards, some of the Pseudomonas and Bacillusstrains in our study showing the highest inhibitoryactivity against V. dahliae DP were those harbouringthe larger number of traits tested. Thus, all antago-nistic strains showed at least three antagonisticmechanisms and other important traits (mainlylipolytic and proteolytic activity, and productionof different levels of IAA and siderophores), but ahigh percentage of isolates showed the five of themechanisms tested. Since many of the isolatesshowed different traits, it was difficult to deter-mine which of the mechanism of antagonism areplaying an important role in the in vitro inhibitionof V. dahliae growth. Also, it should be pointed outthat independently of their antagonistic activity,several of the Pseudomonas and Bacillus spp. foundin the oleaster/feral rhizosphere and endosphere areknown to be active in phosphate and nitrogensolubilisation (Cocking 2003). Similarly, strains ofR. aquatilis associated with the rhizosphere ofdifferent crops have also ability to solubilisemineral phosphate and fixate nitrogen. Overall, thatimplies that all these bacteria could have beneficialeffects on the growth of their host plants (Berge etal. 1991; Kim et al. 1997).

In conclusion, we report for the first time a highdiversity of bacteria in the rhizosphere and endo-sphere of oleaster and feral forms of olives, which to alarge extent remains hidden and unexplored andrepresent excellent reservoirs of V. dahliae antagonistsexhibiting different potential biocontrol mechanisms.Future research should be based on exploiting theinherent ability of specific isolates of those bacteria tocolonize the olive rhizosphere and endosphere. Ifdemonstrated, this ability would enhance the potentialuse of those bacteria as biocontrol agents of V. dahliaeor of other olive soilborne plant pathogens for theprotection of the olive root system from infections aswell as promotion of plant growth.

Acknowledgements This research was supported by grantsAGL2008-00344 and HA2008-0014 from ‘Ministerio deCiencia e Innovación’ of Spain and the European Social Fund.S. Aranda was recipient of a PhD grant from ‘Consejo Nacionalde Ciencia y Tecnología’ (CONACYT) México. The authorsthank H. Müller from Graz University of Technology forproviding helpful comments for antagonistic assays and P.Castillo from IAS-CSIC for providing some samples from wildolives. We also thank J.A. Navas-Cortés from IAS-CSIC forproviding helpful comments while reviewing the manuscript.

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