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

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Wild olive trees, namely oleaster, are considered the ancestor of cultivated olive and a unexplored source of genetic variability that might contain important traits of agronomic and biotechnological interest. The longevity and genetic diversity of
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  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 might contain important traits of agronomic and biotech-nological interest. The longevity and genetic diver-sity of oleasters may have favoured selection of specific and well adapted rhizosphere microbial populations that can constitute unique reservoirs of microbial 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 of Andalusia, southern Spain. To carry out the studywe used a multiphasic approach. First, the occur-rence and diversity of rhizosphere bacteria wasmonitored by a cultivation-independent-approach,using fluorescent terminal restriction fragment length polymorphism (FT-RFLP) analyses of ampli-fied 16S rDNA sequences. FT-RFLP patternsrevealed a high heterogeneity in the composition of the sampled rhizosphere bacterial communities andsuggested the existence of plant genotype-site-specific communities, with each oleaster haven being a unique reservoir of bacterial diversity.Secondly, to investigate the antagonistic potentialof these root-associated bacterial populations, a totalof 675 bacterial isolates obtained from oleaster rhizosphere 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 antagonistic bacteria, a slightly lower proportion (12.9% of total bacteria) were inhabitant of the oleaster rhizospherecompared to that in the endosphere (16.5%). The biotechnological 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%) showed proteolytic, lipolytic, and chitinolytic activity, and produced IAA and siderophores. Finally, analysis of  Plant Soil (2011) 343:329  –  345DOI 10.1007/s11104-011-0721-2Responsible 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.esR. 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 of the 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.Bacterialcommunities.Biodiversity.FT-RFLP. Olea europaea  L. subsp. europaea  var.  europaea . O. europaea  subsp. europaea  var.  sylvestris .Oliveendosphere.Oliverhizosphere,Verticilliumwilt  Introduction Olive is one of the most ancient domestic, cultivated plants (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 oil  producer in the world, where approximately 65% of 2.5 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 and Kadereit  2001). As a result of early domestication andextensive cultivation of olive throughout the Mediter-ranean Basin, the wild-looking forms of   O. europaea subsp.  europaea  presently observed constitute acomplex scenario, potentially ranging from trueoleasters (wild forms present in natural areas) to feralforms (secondary sexual derivatives of the cultivatedclones 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; Zohary and Hopf  1994). The wild forms are genetically and phenotypically 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 cultivars by 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 and Ouazzani 2001; Lumaret et al. 2004; Rubio de Casas et 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 fungus Verticillium dahliae  Kleb. is one of the most important 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 defoliating pathotype (DP) can be lethal to the tree and arecurrently the main threat to olive production inAndalusia (Jiménez-Díaz et al. 2010; Navas-Cortés et 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 of those strategies can be the use of antagonist-mediated biological control, which is considered an alternativeor a supplemental mean of reducing the use of chemicals in agriculture (Berg et al. 2005a; Paulin et  al. 2009; Raaijmakers and Weller  2001). Thus, use of   planting stocks certified free from  V. dahliae  and 330 Plant Soil (2011) 343:329  –  345   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 root rots, 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 against infection by soilborne pathogens (Cook et al. 1995; Landa et al. 2006; Weller et al. 2007). Consequently, the plant root system may constitute an important niche 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 producedirectandindirectbeneficialeffects onplantgrowthandhealth,aswellasthatthestructureandactivitiesofthosecommunities 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 et  al. 2009; Rumberger et al. 2007) have focused on the diversity 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 biotechnological potential of these isolates by in vitro production of indole-1.3-acetic acid, and several antimicrobial com- pounds.Tothe bestofour knowledge,thispresentstudyis the first one aimed to determine the structure anddiversity of bacterial communities inhabiting the rhizo-sphere and roots of wild olives in Andalusia and their  potential as a new source of   V. dahliae  antagonists. Materials and methods Sampling sitesSamples 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 the sampling sites in Cádiz province were good candi-dates for genuine oleasters according to criteriadefined by Lumaret et al. (2004): (i) past and present  climatic 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) past and present isolation (usually more than 10 km) fromareas of cultivated olive trees. Some of those populations 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 of olive trees. The geographic location and altitude of the sampling sites were determined using a global positioning system (GPS) (Table 1, Fig. 1). Climatic classification (J. Papadakis), annual minimum, max-imum, and mean temperature (°C), and annual precipitation (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 samplesSoil and roots samples were taken from three points per tree at 5 to 30 cm depth in the area of the canopy Plant Soil (2011) 343:329  –  345 331   projection (close to the influence of roots; i.e.,rhizosphere soil). A composite sample was obtained per 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 plastic bags loosely tied to ensure sufficient aeration and to prevent 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 of Córdoba (Córdoba, Spain) (Fig. 2).Olive DNA extraction and microsatellite genotypingTo 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 studySample code Olive genotype (age)  a  Province and sampling site GeographiclocationAlt (m)  b Climatic ClassificationM1 W (>200 yr) Cádiz, 4.1 km S Ubrique 36°38 ′ 27.38 ″  N 361 Mediterranean Maritime5°26 ′ 37.53 ″ OM2 W (>200 yr) Cádiz, 1.0 km SW Benaocaz 36°41 ′ 28.09 ″  N 744 Mediterranean Maritime5°25 ′ 35.34 ″ OM3 W (>200 yr) Cádiz, 0.5 km E Villaluenga delRosario36°41 ′ 42.79 ″  N 828 Mediterranean Maritime5°22 ′ 57.22 ″ OLO W (>200 yr) Cádiz, 8.0 Km SW Benalup-CasasViejas36°18 ′ 43.65 ″  N 53 Mediterranean Maritime5°54 ′ 1.14 ″  OLOBA W (>200 yr) Cádiz, 7.4 Km SW Benalup-CasasViejas36°18 ′ 26.64 ″  N 47 Mediterranean Maritime5°52 ′ 56.01 ″  O NAJARA-1 W (>200 yr) Cádiz, 4,8 km NE Vejer de laFrontera36°17 ′ 25.14 ″  N 42 Mediterranean Maritime5°55 ′ 59.64 ″ O NAJARA-2 W (>200 yr) Cádiz, 4,8 km NE Vejer de laFrontera36°17 ′ 24.82 ″  N 43 Mediterranean Maritime5°55 ′ 58.75 ″ OBAETICA W (>200 yr) Cádiz,1.5 km E Vejer de la Frontera 36°14 ′ 32.64 ″  N 14 Mediterranean Maritime5°57 ′ 1.63 ″  OMACO F (>100 yr) Córdoba, 0.6 Km NE MedinaAzahara37°54 ′ 27.53 ″  N 252 Subtropical Mediterranean4°34 ′ 3.52 ″ OLOMCO F-C (>100 yr) Córdoba, 5.9 Km N Córdoba 37°51 ′ 24.54 ″  N 352 Subtropical Mediterranean4°48 ′ 5.32 ″ OEPCO F-C (>100 yr) Córdoba, 4.5 Km NW Córdoba 37°36 ′ 17.65 ″  N 261 Subtropical Mediterranean4° 7 ′ 8.95 ″ O a  Wild (W), feral (F), cultivated (C) status hypothesized according to environmental, historical and demographic criteria  b Altitude above sea level c 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 the provinces of Córdoba and Cádiz, at Andalusia, Southern Spain.The darker green colour corresponds to olive cultivated area inAndalusia332 Plant Soil (2011) 343:329  –  345  in each location (i.e., 11 samples in total). DNA wasextracted from three samples (50 mg each) per sampling 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). The  polymerase chain reaction (PCR) conditions de-scribed by de la Rosa et al. (2002) and the following single 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 SSR   primers were chosen because they were demonstratedthe most informative and reliable and provided thehigher power of discrimination among a number of loci/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 for Research Support (SCAI) of the University of Córdoba sequencing facilities (Córdoba, Spain). Siz-ing of amplification products was done using aROX500 size standard and the software Genmapper 3.7 from Applied Biosystems as described before(Belaj et al. 2007). Extraction of rhizosphere and endosphere bacterialcommunitiesIntact 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 root segments suspended in 20 ml of sterile distilled water in an orbital shaker for 10 min. The suspensions of rhizosphere 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 of rhizosphere suspensions were subjected to consecu-tive centrifugations at 11,000 rpm for 4 min and the pellet was recovered. For root-endophytic bacterial populations, 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  μ  l 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 at 28±1°C and dark conditions for 3 days.Rhizosphere and endosphere suspensions wereserially diluted and plated onto R2A agar (100  μ  l of each dilution series; with three replicates each) andincubated as indicated above. Adequate dilutions Fig. 2  Cluster analysis of combined data from climatic and physicochemical 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 InformationSystem 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 are present are highlightedPlant Soil (2011) 343:329  –  345 333
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