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Stress caused by drought and salinity may compromise growth and productivity of olive (Olea europaea L.) tree crops. Several studies have reported the use of beneficial rhizobacteria to alleviate symptoms produced by these stresses, which is attributed in some cases to the activity of 1-aminocyclopropane-1-carboxylic acid deaminase (ACD). A collection of beneficial olive rhizobacteria was in vitro screened for ACD activity. Pseudomonas sp. PICF6 displayed this phenotype and sequencing of its genome confirmed the presence of an acdS gene. In contrast, the well-known root endophyte and biocontrol agent Pseudomonas simiae PICF7 was defective in ACD activity, even though the presence of an ACD-coding gene was earlier predicted in its genome. In this study, an unidentified deaminase was confirmed instead. Greenhouse experiments with olive ‘Picual’ plants inoculated either with PICF6 or PICF7, or co-inoculated with both strains, and subjected to drought or salt stress were carried out. Several physiological and biochemical parameters increased in stressed plants (i.e., stomatal conductance and flavonoids content), regardless of whether or not they were previously bacterized. Results showed that neither PICF6 (ACD positive) nor PICF7 (ACD negative) lessened the negative effects caused by the abiotic stresses tested, at least under our experimental conditions., This research was funded by the Spanish Ministerio de Economía, Industria y Competitividad/Agencia Estatal de Investigación, grant number AGL2016-75729-C2-1-R, and Junta de Andalucía (Consejería de Economía, Innovación y Ciencia), grant number P12-AGR-0667, both co-financed by the European Regional Development Fund (ERDF).

This study aimed to disentangle the structure, composition, and co-occurrence relation-ships of the banana (cv. Dwarf Cavendish) root endophytome comparing two phenological plant stages: mother plants and suckers. Moreover, a collection of culturable root endophytes (>1000) was also generated from Canary Islands. In vitro antagonism assays against Fusarium oxysporum f.sp. cubense (Foc) races STR4 and TR4 enabled the identification and characterization of potential biocontrol agents (BCA). Eventually, three of them were selected and evaluated against Fusarium wilt of banana (FWB) together with the well-known BCA Pseudomonas simiae PICF7 under controlled conditions. Culturable and non-culturable (high-throughput sequencing) approaches provided concordant information and showed low microbial diversity within the banana root en-dosphere. Pseudomonas appeared as the dominant genus and seemed to play an important role in the banana root endophytic microbiome according to co-occurrence networks. Fungal communities were dominated by the genera Ophioceras, Cyphellophora, Plecosphaerella, and Fusarium. Overall, significant differences were found between mother plants and suckers, suggesting that the phe-nological stage determines the recruitment and organization of the endophytic microbiome. While selected native banana endophytes showed clear antagonism against Foc strains, their biocontrol performance against FWB did not improve the outcome observed for a non-indigenous reference BCA (strain PICF7)., This study was supported by Project ‘MUSA’ (Microbial Uptakes for Sustainable management of major bananA pests and diseases), funded by the EU’s Horizon 2020 Research and Innovation Program (Grant Agreement Identifier: 727624).

Verticillium dahliae is the etiological agent of Verticillium wilt of olive. The virulence of Defoliating V. dahliae isolates usually displays differences and high plasticity. This work studied whether an epigenetic mechanism was involved in this plasticity. An inverse correlation between virulence and DNA methylation of protein-coding genes was found. A set of 831 genes was selected for their highly consistent inverse methylation profile and virulence in the five studied isolates. Of these genes, ATP-synthesis was highly represented, which indicates that the more virulent D isolates are, the more energy requirements they may have. Furthermore, there were numerous genes in the protein biosynthesis process: genes coding for the chromatin structure, which suggests that epigenetic changes may also affect chromatin condensation, many transmembrane transporter genes, which is consistent with denser compounds, traffic through membranes in more virulent isolates, a fucose-specific lectin that may play a role in the attachment to plant cell walls during the host infection process, and pathogenic cutinases that facilitate plant invasion and sporulation genes for rapid spreading alongside plants. Our findings support the notion that differences in the virulence of the Defoliating V. dahliae isolates may be controlled, at least to some extent, by an epigenetic mechanism.

[Background] Verticillium wilt of olive (VWO) is caused by the soilborne fungal pathogen Verticillium dahliae. One of the best VWO management measures is the use of tolerant/resistant olive cultivars. Knowledge on the olive-associated microbiome and its potential relationship with tolerance to biotic constraints is almost null. The aims of this work are (1) to describe the structure, functionality, and co-occurrence interactions of the belowground (root endosphere and rhizosphere) microbial communities of two olive cultivars qualified as tolerant (Frantoio) and susceptible (Picual) to VWO, and (2) to assess whether these communities contribute to their differential disease susceptibility level., [Results] Minor differences in alpha and beta diversities of root-associated microbiota were detected between olive cultivars regardless of whether they were inoculated or not with the defoliating pathotype of V. dahliae. Nevertheless, significant differences were found in taxonomic composition of non-inoculated plants’ communities, “Frantoio” showing a higher abundance of beneficial genera in contrast to “Picual” that exhibited major abundance of potential deleterious genera. Upon inoculation with V. dahliae, significant changes at taxonomic level were found mostly in Picual plants. Relevant topological alterations were observed in microbial communities’ co-occurrence interactions after inoculation, both at structural and functional level, and in the positive/negative edges ratio. In the root endosphere, Frantoio communities switched to highly connected and low modularized networks, while Picual communities showed a sharply different behavior. In the rhizosphere, V. dahliae only irrupted in the microbial networks of Picual plants., [Conclusions] The belowground microbial communities of the two olive cultivars are very similar and pathogen introduction did not provoke significant alterations in their structure and functionality. However, notable differences were found in their networks in response to the inoculation. This phenomenon was more evident in the root endosphere communities. Thus, a correlation between modifications in the microbial networks of this microhabitat and susceptibility/tolerance to a soilborne pathogen was found. Moreover, V. dahliae irruption in the Picual microbial networks suggests a stronger impact on the belowground microbial communities of this cultivar upon inoculation. Our results suggest that changes in the co-occurrence interactions may explain, at least partially, the differential VWO susceptibility of the tested olive cultivars., Supported by grant AGL2016-75729-C2-1-R from the Spanish Ministerio de Economía, Industria y Competitividad/Agencia Estatal de Investigación, and co-financed by the European Regional Development Fund (ERDF).

It is well-known that different plant species, and even plant varieties, promote different assemblages of the microbial communities associated with them. Here, we investigate how microbial communities (bacteria and fungi) undergo changes within the influence of woody plants (two olive cultivars, one tolerant and another susceptible to the soilborne fungal pathogen Verticillium dahliae, plus wild Holm oak) grown in the same soil but with different management (agricultural versus native). By the use of metabarcoding sequencing we determined that the native Holm oak trees rhizosphere bacterial communities were different from its bulk soil, with differences in some genera like Gp4, Gp6 and Solirubrobacter. Moreover, the agricultural management used in the olive orchard led to belowground microbiota differences with respect to the natural conditions both in bulk soils and rhizospheres. Indeed, Gemmatimonas and Fusarium were more abundant in olive orchard soils. However, agricultural management removed the differences in the microbial communities between the two olive cultivars, and these differences were minor respect to the olive bulk soil. According to our results, and at least under the agronomical conditions here examined, the composition and structure of the rhizospheric microbial communities do not seem to play a major role in olive tolerance to V. dahliae., This research was funded by grants AGL2016-75729-C2-1-R from the Spanish Ministerio de Economía, Industria y Competitividad and PID2019-106283RB-I00 from Spanish Ministerio de Ciencia e Innovación/Agencia Estatal de Investigación, and co-financed by the European Regional Development Fund (ERDF).

Trabajo presentado en el Congress of Microbiology and Biotechnology (Microbiotec 19), celebrado en Coimbra del 5 al 7 de diciembre de 2019., Up to date, the bacterial and fungal microbial communities from the olive (Olea europaeaL.) root systems have not been simultaneously studied. In this work, we show that microbial communities from the olive root endosphere are less diverse than those from the rhizosphere. But more relevant was to unveil that olive belowground communities are mainly shaped by the genotype of the cultivar when growing under the same environmental, pedological and agronomic conditions. Furthermore, Actinophytocola, Streptomyces and Pseudonocardia are the most abundant bacterial genera in the olive root endosphere, Actinophytocola being the most prevalent genus by far. In contrast, Gp6, Gp4, Rhizobium and Sphingomonas are the main genera in the olive rhizosphere. Canalisporium, Aspergillus, Minimelanolocus and Macrophominaare the main fungal genera present in the olive root system. Interestingly enough, a high proportion of so far unclassified fungal sequences at class level were detected in the rhizosphere. From the belowground microbial profiles here reported, it can be concluded that the genus Actinophytocolamay play an important role in olive adaptation to environmental stresses. Moreover, the huge unknown fungal diversity suggests that there are still some fungi with important ecological and biotechnological implications that have yet to be discovered.

The use of biological control agents (BCAs) is of interest within an integrated management strategy of Verticillium wilt of olive (VWO) caused by the soil-borne fungus Verticillium dahliae Kleb. Previous studies have shown that the root/rhizosphere of healthy olive plants is an important reservoir of microorganisms displaying biocontrol activity against VWO (i.e., Pseudomonas strains PICF7 and PIC141). Moreover, these BCAs are already adapted to the ecological niche where they are deployed. Three novel bacteria (strains PIC28, PIC73 and PIC167) from nursery-produced olive plants were in-depth characterized using a previously implemented approach consisting of in situ isolation, in vitro antagonism tests, in planta bioassays, phenotypic and metabolic characterization, genome analyses and in silico identification of traits involved in plant-bacteria interactions, and multi-locus sequence analyses. All strains displayed in vitro growth inhibition of different olive pathogens and biocontrol effectiveness against Verticillium dahliae, with strain PIC73 being the most effective BCA. Strains PIC73 and PIC167 were identified as Paenibacillus polymyxa (Prazmowski) Ash et al. and Paenibacillus terrae Yoon et al., respectively. Strain PIC28 belongs to the Bacillus genus. Some of these Bacillales members showed in vitro compatibility with previously characterized BCAs (Pseudomonas spp. strains) also originating from the olive rhizosphere, paving the way for the future development of tailored bacterial consortia effective against VWO., Supported by grants P12-AGR-667 (Junta de Andalucía) and RECUPERA 2020 (MINECO-CSIC agreement), both co-financed by ERDF from the EU.

The use of biological control agents (BCA), alone or in combination with other management measures, has gained attention over the past decades, driven by the need to seek for sustainable and eco-friendly alternatives to confront plant pathogens. The rhizosphere of olive (Olea europaea L.) plants is a source of bacteria with potential as biocontrol tools against Verticillium wilt of olive (VWO) caused by Verticillium dahliae Kleb. A collection of bacterial isolates from healthy nursery-produced olive (cultivar Picual, susceptible to VWO) plants was generated based on morphological, biochemical and metabolic characteristics, chemical sensitivities, and on their in vitro antagonistic activity against several olive pathogens. Three strains (PIC25, PIC105, and PICF141) showing high in vitro inhibition ability of pathogens' growth, particularly against V. dahliae, were eventually selected. Their effectiveness against VWO caused by the defoliating pathotype of V. dahliae was also demonstrated, strain PICF141 being the rhizobacteria showing the best performance as BCA. Genotypic and phenotypic traits traditionally associated with plant growth promotion and/or biocontrol abilities were evaluated as well (e.g., phytase, xylanase, catalase, cellulase, chitinase, glucanase activities, and siderophore and HCN production). Multi-locus sequence analyses of conserved genes enabled the identification of these strains as Pseudomonas spp. Strain PICF141 was affiliated to the “Pseudomonas mandelii subgroup,” within the “Pseudomonas fluorescens group,” Pseudomonas lini being the closest species. Strains PIC25 and PIC105 were affiliated to the “Pseudomonas aeruginosa group,” Pseudomonas indica being the closest relative. Moreover, we identified P. indica (PIC105) for the first time as a BCA. Genome sequencing and in silico analyses allowed the identification of traits commonly associated with plant-bacteria interactions. Finally, the root colonization ability of these olive rhizobacteria was assessed, providing valuable information for the future development of formulations based on these strains. A set of actions, from rhizosphere isolation to genome analysis, is proposed and discussed for selecting indigenous rhizobacteria as effective BCAs., Supported by grants P12-AGR-667 (Junta de Andalucía) and RECUPERA 2020 (MINECO-CSIC agreement), both cofounded by ERDF from the EU.

[Background and aims] The olive root endophyte Pseudomonas fluorescens PICF7 is an effective biocontrol agent of Verticillium wilt of olive (VWO). Colonization of olive roots either by strain PICF7 or by Verticillium dahliae triggers differential systemic transcriptomic responses, many of them related with defense-related genes. The aims were to develop an olive split-root system for assessing VWO development and biocontrol effectiveness of strain PICF7 in plants with a divided root architecture, and for evaluating systemic defense responses during this tripartite interaction when strain PICF7 and V. dahliae are spatially separated., [Methods] An olive split-root system was generated and disease development, biocontrol effectiveness and systemic genetic responses in these plants upon strain PICF7 and V. dahliae colonization were compared to those reported and observed in olive plants grown under standard conditions (single pots). Specific defense-related genes, previously identified during PICF7- and/or V. dahliae-olive root interactions were selected and their expression patterns assessed in above-ground tissues by real-time qPCR analyses., [Results] Symptoms of VWO developed similarly both in split-root and single-root plants. However, even though PICF7 triggered systemic defense responses in aerial tissues prior to the infection by V. dahliae, effective biocontrol was not observed under these experimental conditions. While most of studied genes showed similar expression patterns along time in both systems (i.e. split root and single pot), some of them (e.g. the caffeoyl-O-methyltransferase coding gene) varied depending on whether strain PICF7 and V. dahliae were spatially separated or shared the same compartment., [Conclusions] A successful split-root system was generated to investigate genetic events taking place during the tripartite interaction olive-V. dahliae-P. fluorescens PICF7. VWO biocontrol by strain PICF7 must rely on mechanisms other than induction of systemic resistance responses. The expression pattern of specific defense-related olive genes depended on whether or not the biocontrol agent and the pathogen share the same root/soil region., Research Supported by grant P12-AGR667 from Junta de Andalucía (Spain) and grants AGL2009-07275 and AGL2011-30137 from the Spanish MICINN/MINECO, all co-financed by the European Regional Development Fund (ERDF) of the European Union (EU).

Verticillium wilt of olive (VWO) is one of the most serious biotic constraints for this tree crop. Our knowledge of the genetics of the tolerance/resistance to this disease is very limited. Here we show that tolerance of the cv Frantoio relies on both basal and early pathogen-induced differential transcriptomic responses. A comparative transcriptomic analysis (RNA-seq) was conducted in root tissues of cvs Frantoio (VWO-tolerant) and Picual (VWO-susceptible). RNA samples originated from roots of inoculated olive plants during the early infection stages by Verticillium dahliae (highly virulent, defoliating pathotype). A huge number of differentially expressed genes (DEGs) were found between 'Frantoio' and 'Picual' (27 312 unigenes) in the absence of the pathogen. Upon infection with V. dahliae, 'Picual' and 'Frantoio' plants responded differently too. In the early infection stages, four clusters of DEGs could be identified according to their time-course expression patterns. Among others, a pathogenesis-related protein of the Bet v I family and a dirigent-like protein involved in lignification, and several BAK1, NHL1, reactive oxygen species stress response and BAM unigenes showed noticeable differences between cultivars. Tolerance of 'Frantoio' plants to VWO is a consequence of a complex and multifaceted process which involves many plant traits.

Low temperature severely affects plant growth and development. To overcome this constraint, several plant species from regions having a cool season have evolved an adaptive response, called cold acclimation. We have studied this response in olive tree (Olea europaea L.) cv. Picual. Biochemical stress markers and cold-stress symptoms were detected after the first 24 h as sagging leaves. After 5 days, the plants were found to have completely recovered. Control and cold-stressed plants were sequenced by Illumina HiSeq 1000 paired-end technique. We also assembled a new olive transcriptome comprising 157,799 unigenes and found 6,309 unigenes differentially expressed in response to cold. Three types of response that led to cold acclimation were found: short-term transient response, early long-term response, and late long-term response. These subsets of unigenes were related to different biological processes. Early responses involved many cold-stress-responsive genes coding for, among many other things, C-repeat binding factor transcription factors, fatty acid desaturases, wax synthesis, and oligosaccharide metabolism. After long-term exposure to cold, a large proportion of gene down-regulation was found, including photosynthesis and plant growth genes. Up-regulated genes after long-term cold exposure were related to organelle fusion, nucleus organization, and DNA integration, including retrotransposons., This work was supported by grant AGR-5948 from Junta de Andalucía (Consejería de Economía, Innovación y Ciencia) and Ministerio de Ciencia e Innovación. Funding to pay the Open Access publication charges for this article was provided by the University of Jaén.

During the last decades we have witnessed growing public concern on the abuse/misuse of agrochemicals to control plant pathogens. The fact that some relevant phytopathogens (for instance, the soil-borne fungus Verticillium dahliae Kleb.) are very difficult to control by methods alternative to chemical-based products, has urged researchers to seek effective measures within integrated disease management frameworks. Biological control, alone or in combination with other approaches, emerges as one of the most promising alternatives to confront plant pathogens in a sustainable, environment-friendly strategy. Effectiveness of biological control agents (BCA) largely depends on colonization and persistence capabilities in the ecological niches (e.g. root and/or rhizosphere) where their benefits are expected to be deployed. As a consequence, due to BCA-host specificity (or co-adaptation) the search of potential BCAs in their target environments seems an appropriate strategy. This chapter describes the isolation, identification and characterization of indigenous antagonist bacteria from the olive rhizosphere that can be eventually exploited as BCA against relevant pathogens affecting this woody crop, with emphasis on V. dahliae. The approach here implemented could be of interest for other pathosystems involving trees and soil-borne pathogens.

The colonization process of Olea europaea by the defoliating pathotype of Verticillium dahliae, and the in planta interaction with the endophytic, biocontrol strain Pseudomonas fluorescens PICF7 were determined. Differential fluorescent protein tagging was used for the simultaneous visualization of P. fluorescens PICF7 and V. dahliae in olive tissues. Olive plants were bacterized with PICF7 and then transferred to V. dahliae-infested soil. Monitoring olive colonization events by V. dahliae and its interaction with PICF7 was conducted using a non-gnotobiotic system, confocal laser scanner microscopy and tissue vibratoming sections. A yellow fluorescently tagged V. dahliae derivative (VDAT-36I) was obtained by Agrobacterium tumefaciens-mediated transformation. Isolate VDAT-36I quickly colonized olive root surface, successfully invaded root cortex and vascular tissues via macro- and micro-breakages, and progressed to the aerial parts of the plant through xylem vessel cells. Strain PICF7 used root hairs as preferred penetration site, and once established on/in root tissues, hindered pathogen colonization. For the first time using this approach, the entire colonization process of a woody plant by V. dahliae is reported. Early and localized root surface and root endophytic colonization by P. fluorescens PICF7 is needed to impair full progress of verticillium wilt epidemics in olive.

Verticillium wilt of olive (VWO) is caused by the vascular pathogen Verticillium dahliae. One of the best VWO management measures is the use of tolerant cultivars; however, our knowledge on VWO tolerance/resistance genetics is very limited. A transcriptomic analysis was conducted to (i) identify systemic defense responses induced/repressed in aerial tissues of the tolerant cultivar Frantoio upon root colonization by V. dahliae, and (ii) determine the expression pattern of selected defense genes in olive cultivars showing differential susceptibility to VWO. Two suppression subtractive hybridization cDNA libraries, enriched in up-regulated (FU) and down-regulated (FD) genes respectively, were generated from “Frantoio” aerial tissues. Results showed that broad systemic transcriptomic changes are taking place during V. dahliae-“Frantoio” interaction. A total of 585 FU and 381 FD unigenes were identified, many of them involved in defense response to (a)biotic stresses. Selected genes were then used to validate libraries and evaluate their temporal expression pattern in “Frantoio.” Four defense genes were analyzed in cultivars Changlot Real (tolerant) and Picual (susceptible). An association between GRAS1 and DRR2 gene expression patterns and susceptibility to VWO was observed, suggesting that these transcripts could be further evaluated as markers of the tolerance level of olive cultivars to V. dahliae., Research supported by grant AGL2009-07275 from Spanish MICINN/MINECO and P12-AGR667 from Junta de Andalucía (Spain), both co-financed by the ERDF of EU.

Research was supported by Projects P08-AGR-03635 (Ministry of Agriculture and Fisheries, Government of Andalusia, Spain) and AGL 2011-30137 (Spanish Ministerio de Ciencia e Innovacion, co-financed by FEDER of the EU).

Trabajo presentado en la XIII Meeting of the IOBC Working Group "Biological Control of Fungal and Bacterial Plant Pathogens", celebrada en Uppsala del 15 al 18 de junio de 2014., Pseudomonasjluorescens PlCF7 is a native and effective biocontrol agent against Verticillium wilt of olive. Moreover, strain PICF7 endophytically colonizes olive root tissues. Previous work demonstrated that PICF7 is able to trigger a broad range of defense responses in olive roots . To elucidate whether systemic responses are induced in above-ground organs u pon colonization of olive roots by strain PICF7, aerial olive tissues ofplants grown under non-gnorobiotic conditions v,:ere collected at different time points after PJCF7 inoculation. A suppression subtractíve hybridization cDNA library, enriched in up-regulated genes, was generated. A set of 376 ESTs (99 contigs and 277 singlets) were thus identified, many ofthem related to plant defense responses. Five ofthese ESTs were chosen to carry out time-course, quantitative real-time PCR experiments aiming to (i) validate the índuction of selected genes, and (ii) assess their expression patterns along time (1-15 days). Inductíon of LOX2, CAT, ACO and PAL genes was confirmed for some ofthe analyz.ed time points. Fmihermore, different transcription factors were also up-regulated (i .e.,jetf, bHL H, WRKYs), as previously reported in roots. Results confirmed that root colonization by PICF7 does not only trigger defense responses in roots but al so has the ability to mount a wide array of systemíc defense responses in stems and leaves. Thís sheds light on how olive plants respond to colonization of a bacteria! root endophyte and how induced defense response can contríbute to the biocontrol activity of strain PICF7., Funded by P07-CVI-02624 (Junta de Andalucía, Spain), co-financcd by ERDF (EU).

Fusarium wilt, caused by Fusarium oxysporum f. sp. dianthi (Fod), is the most important carnation disease worldwide. The knowledge of the diversity of the soil population of the pathogen is essential for the choice of suitable resistant cultivars. We examined the genetic diversity of Fod isolates collected during the period 1998-2008, originating from soils and carnation plants in the most important growing areas in Spain. Additionally, we have included some Fod isolates from Italy as a reference. Random amplified polymorphic DNA (RAPD) fragments generated by single-primer PCR were used to compare the relationship between isolates. UPGMA analysis of the RAPD data separated Fod isolates into three clusters (A, B, and C), and this distribution was more related to aggressiveness than to the race of the isolates. The results obtained in PCR amplifications using specific primers for race 1 and race 2, and SCAR primers developed in this work, correlated with the molecular groups previously determined from the RAPD analysis, and provided new molecular markers for the precise identification of the isolates. Results from successive pathogenicity tests showed that molecular differences between isolates of the same race corresponded with differences in aggressiveness. Isolates of races 1 and 2 in cluster A (R1I and R2I isolates) and cluster C (R1-type isolates) were all highly aggressive, whereas isolates of races 1 and 2 in cluster B (R1II and R2II isolates) showed a low aggressiveness profile. The usefulness of the molecular markers described in this study has been proved in double-blind tests with Fod isolates collected in 2008. Results from this work indicate a change in the composition of the Spanish Fod population over time, and this temporal variation could be related to the continuous change in the commercial carnation cultivars used by growers. This is the first report of genetic diversity among Fod isolates in the same race. © 2011 KNPV., This research was supported by grant AGL 2006–03252 from the Spanish Ministry of Science and Innovation.

15 pages: 2 figures; 3 tables, A comprehensive survey on the prevalence and incidence of Verticillium wilt of olive in Turkey has been conducted over 6 years (2003–2008). Vegetative compatibility group (VCG) assessment and PCR-based molecular pathotyping were used to evaluate the distribution of the defoliating (D) and nondefoliating (ND) pathotypes of Verticillium dahliae in surveyed areas. Pathogen prevalence was 35% of all olive orchards inspected and incidence of the disease reached 3.1%. VCG1A was predominant (29.3%) and infected all major cultivars grown in Turkey. The other two VCGs detected (2A and 4B) were of minor relevance (4.9% and 0.9%, respectively). Disease incidence caused by VCG1A infections was higher (ranging from 1.1% to 6.9%) than that caused by VCG2A and VCG4B in 10 provinces (Manisa, Aydin, Kahramanmaras, Izmir, Mugla, Kilis, Denizli, Gaziantep, Mardin and Balikesir). However, VCG2A and 4B were more prevalent (and responsible for higher disease incidence) than VCG1A in three provinces (Hatay, Osmaniye and Bursa). Finally, VCG1A isolates were found in all provinces except Canakkale, and simultaneous presence of the three VCGs was only verified in Hatay province. An artificial inoculation bioassay (19 representative V. dahliae isolates included) revealed that VCG1A (13) isolates as a group were more aggressive and caused defoliation, whereas VCG2A (5) and VCG4B (1) isolates induced milder symptoms. Within a VCG group, virulence varied among isolates infecting the same olive cultivar and this virulence was also related to the differential susceptibility of the cultivars (‘Manzanilla’, ‘Ayvalik’ and ‘Gemlik’) tested. Molecular pathotyping allowed the identification of D (VCG1A) and ND (VCG2A/4B) pathotypes, which correlated with results from pathogenicity tests. Remarkably, the V. dahliae VCG1A/D pathotype population infecting olive in Turkey was molecularly different from that one previously identified in Spain., The Scientific and Technical Research Council of Turkey

11 pages; 2 tables; 1 figure, A degree of genetic diversity may exist among Verticillium dahliae isolates within vegetative compatibility groups (VCGs) that bears phytopathological significance and is worth investigating using molecular tools of a higher resolution than VCG characterization. The molecular variability within and among V. dahliae VCGs was studied using 53 artichoke isolates from eastern-central Spain, 96 isolates from cotton, 7 from cotton soil, and 45 from olive trees in countries of the Mediterranean Basin. Isolates were selected to represent the widest available diversity in cotton- and olive-defoliating (D) and -nondefoliating (ND) pathotypes, as well as for VCG. The VCG of 96 cotton and olive isolates was determined in this present study. Molecular variability among V. dahliae isolates was assessed by fluorescent amplified fragment length polymorphism (AFLP) analysis and by polymerase chain reaction (PCR) assays for DNA fragments associated with the D (462 bp) and ND (824 bp) pathotypes, as well as a 334-bp amplicon associated with D pathotype isolates but also present in some VCG2B isolates. Isolates from cotton were in VCG1A, VCG1B, VCG2A, VCG2B, and VCG4B and those from olive trees were in VCG1A, VCG2A, and VCG4B. Artichoke isolates included representatives of VCG1A, VCG2A, VCG2B (including a newly identified VCG2Ba), and VCG4B. AFLP data were used to generate matrixes of genetic distance among isolates for cluster analysis using the neighbor-joining method and for analysis of molecular variance. Results demonstrated that V. dahliae isolates within a VCG subgroup are molecularly similar, to the extent that clustering of isolates correlated with VCG subgroups regardless of the host source and geographic origin. VCGs differed in molecular variability, with the variability being highest in VCG2B and VCG2A. For some AFLP/VCG subgroup clusterings, V. dahliae isolates from artichoke grouped in subclusters clearly distinct from those comprising isolates from cotton and olive trees. In addition, VCG2B isolates from artichoke formed two distinct clusters that correlated with PCR markers of 334 bp (VCG2B334) or 824 bp (VCG2B824). Artichoke isolates in the VCG2B334/2β334 cluster were molecularly similar to isolates of VCG1A. The molecular difference found among artichoke isolates in VCG2B correlates with virulence of isolates to artichoke and cotton cultivars demonstrated in a previous study., Grants AGL2000-1444 and AGL2003- 00503 from Comisión Interministerial de Ciencia y Tecnología (CICYT) of Spain, and “Verticilosis del olivo” from Fundacion R. Areces.