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 (VCG2B(334)) or 824 bp (VCG2B(824)). Artichoke isolates in the VCG2B(334)/2beta(334) 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.

SummaryThe 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.

Pseudomonas fluorescens PICF7, a native olive root endophyte and effective biocontrol agent (BCA) against Verticillium wilt of olive, is able to trigger a broad range of defense responses in root tissues of this woody plant. In order to elucidate whether strain PICF7 also induces systemic defense responses in above-ground organs, aerial tissues of olive plants grown under non-gnotobiotic conditions were collected at different time points after root bacterization with this endophytic BCA. A suppression subtractive hybridization (SSH) cDNA library, enriched in up-regulated genes, was generated. This strategy enabled the identification of 376 ESTs (99 contigs and 277 singlets), many of them related to response to different stresses. Five ESTs, involved in defense responses, were selected to carry out time-course quantitative real-time PCR (qRT-PCR) experiments aiming to: (1) validate the induction of these genes, and (2) shed light on their expression pattern along time (from 1 to 15 days). Induction of olive genes potentially coding for lipoxygenase 2, catalase, 1-aminocyclopropane-1-carboxylate oxidase, and phenylananine ammonia-lyase was thus confirmed at some time points. Computational analysis also revealed that different transcription factors were up-regulated in olive aerial tissues (i.e., JERF, bHLH, WRKY), as previously reported for roots. Results confirmed that root colonization by this endophytic bacterium does not only trigger defense responses in this organ but also mounts a wide array of systemic defense responses in distant tissues (stems, leaves). This sheds light on how olive plants respond to the “non-hostile” colonization by a bacterial endophyte and how induced defense response can contribute to the biocontrol activity of strain PICF7.

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.

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.

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 oliveassociated 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.

The bacterial and fungal communities from the olive (Olea europaea L.) root systems have not yet been simultaneously studied. We show in this work 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 Macrophomina are the main fungal genera present in the olive root system. Interestingly enough, a large number of as yet unclassified fungal sequences (class level) were detected in the rhizosphere. From the belowground microbial profiles here reported, it can be concluded that the genus Actinophytocola may play an important role in olive adaptation to environmental stresses. Moreover, the huge unknown fungal diversity here uncovered suggests that fungi with important ecological function and biotechnological potential are yet to be identified.