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International audience; Abstract The initial greening of angiosperm occurs upon light-activation of photoreceptors that trigger photomorphogenesis followed with the development of chloroplasts. In these semi-autonomous organelles, the construction of the photosynthetic apparatus depends on the coordination of nuclear and plastid gene expression. Here we show that PAP8, as an essential subunit of the plastid-encoded RNA polymerase, is under the control of a regulatory element recognized by the photomorphogenic factor HY5. PAP8 is localized and active in both plastids and the nucleus and particularly essential for the formation of late photobodies. In the albino pap8 mutant, phytochrome-mediated signalling is altered, PIFs are maintained, HY5 is not stabilized, and GLK1 expression is impaired. PAP8 translocates into plastids losing its pre-sequence, interacts with the PEP, and using an unknown route or a retrograde transport, reaches the nucleus where it has the ability to interact with pTAC12/HMR/PAP5. Since PAP8 is required for the phytochrome-B-mediated signalling cascade and the reshaping of the PEP, it may coordinate nuclear gene expression with the PEP-driven chloroplastic gene expression during chloroplast biogenesis.

Additional file 2: Figure S1. Phyla showing significant changes in the bacterial structural (DNA) community of the ‘Picual’ rhizosphere after inoculation with Verticillium dahliae. No/green: non-inoculated; Yes/red: Verticillium dahliae-inoculated.

Additional file 6: Figure S3. Genera showing significant changes in bacterial structural (DNA) community of ‘Frantoio’ (panel a) and ‘Picual’ (panel b) endosphere after inoculation with Verticillium dahliae. No/green: non-inoculated; Yes/red: Verticillium dahliae-inoculated.

Additional file 8: Figure S5. Genera with significantly different changes in rhizosphere of Frantoio and Picual bacterial structural (panels a and c respectively) and functional (panels b and d respectively) communities after inoculation with Verticillium dahliae. No/green: non-inoculated; Yes/red: Verticillium dahliae-inoculated.

Additional file 13: Figure S8. Genera showing significant changes in fungal structural (DNA) and functional (RNA) communities of ‘Frantoio’ (panel a) and ‘Picual’ (panel b) rhizosphere after inoculation with Verticillium dahliae. No/green: non-inoculated; Yes/red: Verticillium dahliae-inoculated.

Additional file 17: Figure S12. Co-occurrence networks of functional (RNA) communities from rhizosphere of both cultivars before and after inoculation.

Trabajo presentado en el Congress of Microbiology and Biotechnology (Microbiotec 19), celebrado en Coimbra del 5 al 7 de diciembre de 2019., Banana (Musa acuminateColla) is a widely cultivated crop in (sub)tropical regions. Fusarium wilt of banana (FWB), caused by the soilborne fungus Fusarium oxysporum f. sp. cubense(Foc), poses a great risk to banana industry worldwide. Pesticides do not represent a sustainable option for its control and other alternatives, such as biological control agents (BCA) are gaining interest. The fact that beneficial endophytes can colonize the same niche than Foc favors them as potential control tools against FWB. We pursued two aims: the unravelling of the banana root endosphere microbiota, and the isolation, identification and characterization of culturable members of this specific niche as potential BCA within a FWB integrated management strategy. A thorough root surface sterilization protocol was implemented to ensure only the handling of banana root endophytes, originated from plants of different farms at Tenerife, La Palma and La Gomera islands. On the one hand, DNA from each sample was purified and the 16S rDNA gene (for bacteria) and the ITS2 region (for fungi) were amplified and sequenced by MySEQ. On the other hand, individual colonies (bacteria and fungi) from ground root tissues showing distinctive morphology when growing in different culturing media were isolated. A collection of indigenous endophytes (>1000) was thus generated (80% and 20% single/pure bacteria and fungi cultures, respectively). Subsequently, in vitro antagonism tests against different Foc races were conducted. More than 100 strains showing antagonism were molecularly identified and a phenotypic characterization was performed to identify traits associated to biocontrol and plant growth promotion. Based on these results the best isolates were selected. Both culturable and non-culturable approaches showed low microbial diversity, particularly within bacterial communities. Only few significant differences in alpha-diversity were found. Concerning beta-diversity, the most relevant significant differences were observed among farms regardless of the island from where they originated. Results point to the fact that banana roots are a good source of potential BCAs against FWB. Biocontrol assays using the selected native endophytes were successfully carried out and their results will be discussed., Supported by Project MUSA (Ref.No: 727624), funded by the EU’s Horizon 2020 Research and Innovation Program.

Trabajo presentado en el Congress of Microbiology and Biotechnology (Microbiotec 19), celebrado en Coimbra del 5 al 7 de diciembre de 2019., Verticillium wilt of olive (VWO), caused by the soilborne fungus Verticillium dahliae Kleb, is a relevant disease affecting olive cultivation in many areas where this tree is cultivated. The use of tolerant/resistant cultivars is considered an efficient control tool of the disease. A greenhouse experiment was set up to: (i) describe the root-associated microbial communities of two olive cultivars (cv.) qualified as tolerant (cv. Frantoio) and susceptible (cv. Picual) to V. dahliae, and (ii) assess whether the belowground-associated microbiome participate in the tolerance/susceptibility level of these cultivars to VWO. Nursery-produced olive plants were grown in pots containing non-sterile ad hoc prepared soil. After acclimation, a group of plants were inoculated with a defoliating (highly-virulent) representative of V. dahliae. A control group of non-inoculated plants was treated just with water. Surface disinfected roots and rhizosphere soil from both groups of plans were sampled at 0, 8, 15, and 30 (four plants per time-point and per cultivar) days after inoculation. DNA and RNA were extracted and sequenced (PCR products of the V3-V4 region of the 16S rRNA gene for bacteria and the ITS2 for fungi) by Illumina MiSeq. Bioinformatic, statistical and network analyses were then performed to analyze the composition, functionality and co-occurrence interactions of the endosphere and rhizosphere microbial communities. The belowground microbial communities of the two cultivars are similar, minor significant differences in diversity and composition of root-associated microbiota were found between olive cultivars regardless they were inoculated or not with the pathogen. Presence of V. dahliaecaused changes mainly in communities’ taxa relative abundance, mostly in the VWO-susceptible cultivar. However, notable differences were found in the communities co-occurrence interactions in response to the pathogen presence. Both cultivars showed changes in the topology of the networks, both at DNA and RNA level, and modifications of the positive/negative edges ratio. A correlation between microbial networks modifications and susceptibility/tolerance to the pathogen was found. Therefore, changes in the microbial communities interactions may explain, at least partially, the differential VWO susceptibility of the tested olive cultivars.

Trabajo presentado en el International Symposium Microbe-assisted Crop Production (miCROPe 2019, Opportunites, Challenges and Needs), celebrado en Viena del 2 al 5 de diciembre de 2019., To cope with the growing world population an increase in the production of the main crops for human nutrition, including rice, is now urgently needed. To achieve this goal, the expensive and polluting chemical fertilizers have already been overused. Nitrogen (N) is one of the primary nutrients limiting plant growth in agriculture. Biological nitrogen fixation (BNF) by diazotrophic bacteria, which reduce atmospheric N to ammonium using nitrogenase enzyme systems, accounts for 30-50% of the total N in crop fields. The area of BNF research has been expanded by the discovery of N-fixing bacterial endophytes in non-nodulating plants. In the last few years a wide diversity of bacteria associated with cereals have shown to possess the nifH gene coding for dinitrogenase reductase. This gene is genetically conserved and thus traditionally used as a marker gene to study the genetic diversity of diazotrophs in nature. To improve plant growth and yield the use of genetically modified diazotrophs or the co-inoculation with nitrogen-fixing and plant growth promoting bacteria has been proposed. We have previously reported that the strain Enterobacter cloacae RCA25-64, engineered to produce and release 36-fold more indole-3-acetic acid (IAA) than the wild type E. cloacae RCA25, showed increased nifH gene expression and nitrogenase activity in liquid cultures and inoculated rice plants. In the present study we analysed the effect of purified IAA on the nitrogen-fixing ability of E. cloacae RCA25. Co-inoculation studies were also carried out to test the ability of different wild type IAA-producing endophytes to enhance the nifH gene expression and nitrogenase activity in E. cloacae RCA25, preventing the use of engineered strains. Our results showed that Herbaspirillum huttiense RCA24 performed best. Improvements in nitrogen-fixation and changes in physiological parameters such as chlorophyll, nitrogen content and shoot dry weight were observed for rice plants (Oryza sativa L. cv. Baldo) co-inoculated with strains RCA25 and RCA24 in a 10:1 ratio. Based on confocal laser scanning microscopy analysis, strain RCA24 was the best colonizer of the root interior and the only IAA producer located in the same root niche occupied by RCA25 cells. Our data highlight that the assessment of location and distribution of the individual microbial components within the host plant tissues is fundamental to select bio-inoculants containing IAA-producer strains able to enhance nitrogen-fixation.

Trabajo presentado en el Congress of Microbiology and Biotechnology (Microbiotec 19), celebrado en Coimbra del 5 al 7 de diciembre de 2019., It is well-known that different plant species, and even plant varieties, promote different assemblages of the microbial communities associated to 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 soilbut with different management (agricultural versus native). By the use of rRNA and ITS Illumina amplicon sequencing we determined that the native Holm-oak trees rhizosphere microbial communities were different from its bulk soil. 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. 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.

Trabajo presentado en el Congress of Microbiology and Biotechnology (Microbiotec 19), celebrado en Coimbra del 5 al 7 de diciembre de 2019., Stress caused by drought and high salinity can affect growth and productivity of olive (Olea europaea L.) trees. The phytohormone ethylene plays essential roles in plants but high levels in response to (a)biotic stresses may cause negative effects. Some rhizobacteria have been investigated for its potential to ease these effects due to the enzyme 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase (ACD). ACD degrades ACC, the immediate ethylene biosynthetic precursor. The aim of this work was to examine whether indigenous olive rhizobacteria showing ACD activity may alleviate salt and drought stress in young olive plants. A collection of 32 previously-isolated olive rhizobacteria was in vitro screened for the presence/absence of ACD activity. The well-characterized beneficial olive root endophyte Pseudomonas simiae (fluorescens) PICF7 showed as defective in ACD activity, although genes phylogenetically related to ACD and putatively coding for a D-cysteine desulfhydrase and an unidentified deaminase are present in its genome. Pseudomonas sp. PICF6 displayed this activity and sequencing of its genome revealed the presence of a true ACD gene. By confocal laser scanning microscope analysis using fluorescently-labelled derivatives of both strains similar olive root colonization patterns were visualized, including evidence of the endophytic behaviour of strain PICF6. Greenhouse experiments were performed in which olive ‘Picual’ plants inoculated either with strain PICF6 or PICF7, or with a combination of both strains, were subjected to drought or salt stress. Different physiological and biochemical parameters (chlorophyll and flavonoids contents, stomatal conductance and spectral plant index) were measured along time and compared to the situation in non- stressed and/or non-bacterized plants. Proline content and stem water potential was also scored in plants subjected to salt and drought stress, respectively. Results showed that neither PICF6 (ACD- positive) nor PICF7 (ACD-negative) were able to lessen the negative effects caused by the abiotic stresses tested, although some of the parameters examined (e.g. stomatal conductance or flavonoid content) showed differences in some cases. In summary, inoculation with strain PICF6 does not help olive plants to cope with salt/drought stress, suggesting that ACD activity does not seem to play any protective role under experimental conditions tested., Research supported by grants P12-AGR-667 and AGL2016-55729-C2-1-R.

Pseudomonas simiae PICF7 is an indigenous inhabitant of the olive (Olea europaea L.) rhizosphere/root endosphere and an effective biocontrol agent against Verticillium wilt of olive (VWO), caused by the soil-borne fungus Verticillum dahliae. This study aimed to evaluate the potential involvement of selected phenotypes of strain PICF7 in root colonization ability and VWO biocontrol. Therefore, a random transposon-insertion mutant bank of P. simiae PICF7 was screened for the loss of phenotypes likely involved in rhizosphere/soil persistence (copper resistance), root colonization (biofilm formation) and plant growth promotion (phytase activity). Transposon insertions in genes putatively coding for the transcriptional regulator CusR or the chemotaxis protein CheV were found to affect copper resistance, whereas an insertion in fleQ gene putatively encoding a flagellar regulatory protein hampered the ability to form a biofilm. However, these mutants displayed the same antagonistic effect against V. dahliae as the parental strain. Remarkably, two mutants impaired in biofilm formation were never found inside olive roots, whereas their ability to colonize the root exterior and to control VWO remained unaffected. Endophytic colonization of olive roots was unaltered in mutants impaired in copper resistance and phytase production. Results demonstrated that the phenotypes studied were irrelevant for VWO biocontrol.

Olive knot disease, caused by Pseudomonas savastanoi pv. savastanoi, is one of the most important biotic constraints for olive cultivation. Pseudomonas fluorescensPICF7, a natural colonizer of olive roots and effective biological control agent (BCA) against Verticillium wilt of olive, was examined as potential BCA against olive knot disease. Bioassays using in vitro-propagated olive plants were carried out to assess whether strain PICF7 controlled knot development either when co-inoculated with the pathogen in stems or when the BCA (in roots) and the pathogen (in stems) were spatially separated. Results showed that PICF7 was able to establish and persist in stem tissues upon artificial inoculation. While PICF7 was not able to suppress disease development, its presence transiently decreased pathogen population size, produced less necrotic tumours, and sharply altered the localization of the pathogen in the hyperplasic tissue, which may pose epidemiological consequences. Confocal laser scanning microscopy combined with fluorescent tagging of bacteria revealed that when PICF7 was absent the pathogen tended to be localized at the knot surface. However, presence of the BCA seemed to confine P. savastanoi at inner regions of the tumours. This approach has also enabled to prove that the pathogen can moved systemically beyond the hypertrophied tissue. Pseudomonas fluorescens PICF7, a natural colonizer of olive roots and effective biological control agent (BCA) against Verticillium wilt of olive, was examined as potential BCA against olive knot disease caused by Pseudomonas savastanoi pv. savastanoi. While PICF7 was not able to suppress disease development, its presence transiently decreased pathogen population size, produced less necrotic tumors, and sharply altered the localization of the pathogen in the hyperplasic tissue. Confocal laser scanning microscopy revealed that when PICF7 was absent the pathogen tended to be localized at the knot surface. However, presence of the BCA seemed to confine P. savastanoi at inner regions of the tumors. This approach has also enabled to prove that the pathogen can moved systemically beyond the hypertrophied tissue. © 2013 The Authors. Published by Society for Applied Microbiology and Blackwell Publishing Ltd., Research supported by grants AGL2009-07275, AGL2011-30343-C02-01 (Spanish MICINN/MINECO), P07-CVI-02624 and P08-CVI-03475 (Junta de Andalucía, Spain), co-financed by ERDF from EU., Junta de Andalucía, Spain

Fox, Ana Romina et al.-- Special Issue on Ecophysiology of Pseudomonas ., A main goal of biological nitrogen fixation research has been to expand the nitrogen-fixing ability to major cereal crops. In this work, we demonstrate the use of the efficient nitrogen-fixing rhizobacterium Pseudomonas protegens Pf-5 X940 as a chassis to engineer the transfer of nitrogen fixed by BNF to maize and wheat under non-gnotobiotic conditions. Inoculation of maize and wheat with Pf-5 X940 largely improved nitrogen content and biomass accumulation in both vegetative and reproductive tissues, and this beneficial effect was positively associated with high nitrogen fixation rates in roots. 15N isotope dilution analysis showed that maize and wheat plants obtained substantial amounts of fixed nitrogen from the atmosphere. Pf-5 X940-GFP-tagged cells were always reisolated from the maize and wheat root surface but never from the inner root tissues. Confocal laser scanning microscopy confirmed root surface colonization of Pf-5 X940-GFP in wheat plants, and microcolonies were mostly visualized at the junctions between epidermal root cells. Genetic analysis using biofilm formation-related Pseudomonas mutants confirmed the relevance of bacterial root adhesion in the increase in nitrogen content, biomass accumulation and nitrogen fixation rates in wheat roots. To our knowledge, this is the first report of robust BNF in major cereal crops., This work was supported by grants PICT-2011-1325, PICT-2014-1397 and PICT-2014-3659 to N.D.A.

Trabajo presentado en el 12th International Verticillium Symposium, celebrado en LjubIjana del 6 al 9 de octubre de 2016., Olive (Olea europaea L.) is an emblematical woody crop in the Mediterranean Basin. Verticillium wilt of olive (VWO) (Verticillium dahliae Kleb.) is considered one of the most threatening biotic constraints throughout its range of cultivation. Its effective control is challenging and requires an integrated disease management framework where biocontrol can play an active role. Pseudomonas fluorescens PICF7 is an endophyte of olive roots exerting effective VWO biocontrol. Our understanding of the V. dahliae-olive-PICF7 interaction has been enhanced by using different '-omic' and microscopy approaches. This tripartite interaction can serve as a study model to unravel mechanisms underlying the multitrophic dialogue established among the three partners. Our research has shown that early colonization of intact olive roots by PICF7 is needed to somehow hamper the pathogen's infection process. Moreover, this bacterium induces local and above-ground gene expression changes, many of them related to defense responses. Likewise, V. dahliae provokes systemic changes upon olive root colonization. Interestingly, some of them occur in both interactions. To assess whether PICF7 is able to mount an effective systemic defense response in the host prior to be challenged by the pathogen, an olive split-root system was developed. The gene expression patterns of selected genes involved in defense responses were examined in above-ground tissues when both microorganisms were spatially separated and sequentially applied. Recently, and by using RNA-seq, a more in-depth transcriptomic analysis of the V. dahliae-olive root interaction at early stages has revealed profound differences depending on the susceptibility level of the olive cultivar involved.

Trabajo presentado en el VI Simposio Nacional de Bacteria Fitopatógenas, el V Simposio Internacional de Bacterias Fitopatógenas (2016) II Simposio de Bacterias Benéficas de las Plantas (2016), celebrados en guadalajara (México) del 22 al 24 de septiembrel de 2016., Endophytes, from the Greek ‘endon’ (inside) + ‘phyton’ (plant), are microorganisms (mainly bacteria and fungi) that can be detected at any moment within the tissues of healthy plants without producing any visible symptom. Phytopathogenic microorganisms, nitrogen-fixing nodule-producing bacteria and mycorrhizal fungi also exhibit endophytism during part of their life cycle, but they will not be considered in this presentation. Moreover, the focus will be on endophytic bacteria. Most of the components of plant-associated microbiomes colonize and persist on the surface of plant tissues/organs or in the rhizosphere, but some are also able to establish themselves as non-deleterious endophytes. It is now generally accepted that all plants harbor an amazing diversity of endophytic communities which play relevant roles, poorly understood though, in the development, growth and health of the host. They also help the plant to cope with diverse types of (a)biotic stresses. Powerful metagenomics and cultureindependent approaches are revealing that the plant inner microbiome is more diverse and complex than the rather partial portrait obtained by traditional culture-dependent methods. It must be emphasized that the ability of a given bacterium to internally colonize plant tissues must be verified by microscopy techniques, along with the demonstration of its capacity to recolonize plant tissues (e.g. from inoculation of disinfected seeds). Therefore, the mere isolation from surface-disinfected tissues is not enough to claim true endophytism., Research supported by grants P07-CVI02624 and P12-AGR667 (Junta de Andalucía, Spain), co-financed by ERDF from the EU.

Pseudomonas fluorescens PICF7 is an indigenous inhabitant of olive (Olea europaea L.) rhizosphere, able to display endophytic lifestyle in roots, to induce a wide range of defence responses upon colonization of this organ and to exert effective biological control against Verticillium wilt of olive (VWO) (Verticillium dahliae). We aimed to evaluate the involvement of specific PICF7 phenotypes in olive root colonization and VWO biocontrol effectiveness by generating mutants impaired in swimming motility (fliI) or siderophore pyoverdine production (pvdI). Besides, the performance of mutants with diminished in vitro growth in potato dextrose agar medium (gltA) and cysteine (Cys) auxotrophy was also assessed. Results showed that olive root colonization and VWO biocontrol ability of the fliI, pvdI and gltA mutants did not significantly differ from that displayed by the parental strain PICF7. Consequently, altered in vitro growth, swimming motility and pyoverdine production contribute neither to PICF7 VWO suppressive effect nor to its colonization ability. In contrast, the Cys auxotroph mutant showed reduced olive root colonization capacity and lost full biocontrol efficacy. Moreover, confocal laser scanning microscopy revealed that all mutants tested were able to endophytically colonize root tissue to the same extent as wild-type PICF7, discarding these traits as relevant for its endophytic lifestyle., This research was funded by Grant AGL2009-07275 from Spanish MICINN/MINECO and Grant P07-CVI-02624 from J. Andalucía (Spain), both co-financed by ERDF of the EU.

The effective management of Verticillium wilts (VW), diseases affecting many crops and caused by some species of the soil-borne fungus Verticillium, is problematic. The use of microbial antagonists to control these pathologies fits modern sustainable agriculture criteria. Pseudomonas fluorescens PICF7 is an endophytic bacterium isolated from olive roots with demonstrated ability to control VW of olive caused by the highly virulent, defoliating (D) pathotype of Verticillium dahliae Kleb. However, the study of the PICF7-V. dahliae-olive tripartite interaction poses difficulties because of the inherent characteristics of woody, long-living plants. To overcome these problems we explored the use of the model plant Arabidopsis thaliana. Results obtained in this study showed that: (i) olive D and non-defoliating V. dahliae pathotypes produce differential disease severity in A. thaliana plants; (ii) strain PICF7 is able to colonize and persist in the A. thaliana rhizosphere but is not endophytic in Arabidopsis; and (iii) strain PICF7 controls VW in Arabidopsis. Additionally, as previously observed in olive, neither swimming motility nor siderophore production by PICF7 are required for VW control in A. thaliana, whilst cysteine auxotrophy decreased the effectiveness of PICF7. Moreover, when applied to the roots PICF7 controlled Botrytis cinerea infection in the leaves of Arabidopsis, suggesting that this strain is able to induce systemic resistance. A. thaliana is therefore a suitable alternative to olive bioassays to unravel biocontrol traits involved in biological control of V. dahliae by P. fluorescens PICF7., This research was funded by grants AGL2009-07275 from Spanish MICINN/MINECO and P07-CVI-02624 and P12-AGR-667 from J. Andalucía (Spain), both co-financed by ERDF of the EU.

Trabajo presentado en la 3rd Annual Meeting of FP1305 Biolink Cost Action (Soil Biological Communities and Aboveground Resilience), celebrado en Roma del 17 al 19 de noviembre de 2015., In recent decades we have witnessed growing public concern on the abuse/misuse of agrochemicals to control plant pathogens. Besides, the fact that some phytopathological agents (for instance, the soil-borne fungus Verticillium dahliae Kleb.) are very difficult to control by alternative methods to those based on 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. In this study, a collection of indigenous culturable bacteria (>300) from olive roots was generated. First selection was performed based on their antagonistic activity against V. dahliae. As a result, some 190 isolates were selected and further (i) tested against relevant plant pathogens (Alternaria alternata, Colletotrichum godetiae, Colletotrichum nymphaeae, Fusarium oxysporum f. sp. dianthi, Phytophthora cinnamomi, Pseudomonas savastanoi pv. savastanoi, and Rosellinia necatrix), (ii) identified molecularly (16S rDNA and gyrB genes), and (iii) screened for the presence of phenotypes usually associated to antagonism and/or plant growth promotion (PGP). Results showed prevalence of Proteobacteria (55.5 %) and Firmicutes (36.5 %) phyla, Pseudomonas (28.6 %) and Bacillus (33 %) being the most abundant genera. Although none of the strains showed effective against all tested pathogens, twelve of them belonging to genera Paenibacillus, Rhizobium, Pseudomonas or Bacillus presented a broad antagonistic activity, together with interesting phenotypic characteristics such as enzymatic activities associated to biocontrol and PGP (e.g. production of siderophore or chitinase activity). These results indicate that some of these bacteria could be good candidates to be used in future bioformulations aiming to improve health and growth of this tree crop., Research supported by grants P12-AGR667 (Junta de Andalucía, Spain) and RECUPERA 2020 (MINECO-CSIC), all co-funded by ERDF from EU.

Trabajo presentado en el International Congress Soil and food, resources for a healthy life (The application of beneficial microorganisms in sustainable agriculture), celebrado en Lasi (Rumanía) del 22 al 24 de octubre de 2015., Endophytes (bacteria and fungi) can be detected at any moment within the tissues of healthy plants without producing any visible deleterious symptom. It is generally accepted that all plants harbour highly diverse endophytic microbiomes playing relevant roles, poorly understood though, for the growth, health, development and fitness of the host. These microbial communities also help the plant to cope with different (a)biotic stresses. Current powerful metagenomics and culture-independent approaches are revealing that endophytic communities are much more diverse and complex than information obtained by traditional culture-dependent methods. Endophytic bacteria penetrate into plant tissues primarily by the roots, although they can also do so through leaves, stems, flowers, or cotyledons. Sorne can move systemically and be localized at distant tissues from the original penetration place. They can enter passively through cracks, wounds or points of emergence oflateral roots. Active penetration appears to take place preferably by the root differentiation and elongation regions, using the intercellular spaces of the root epidermis. Our studies have shown that root hairs play an important role in the endophytic colonization of olive (Oleae europaea L.) roots by strains of Pseudomonas spp. However, the knowledge on bacteria! traits involved in both inner colonization and persistence of plant tissues is scarce. The type IV 'pili: lipopolysaccharide and exopolysaccharides have been confirmed as crucial components. On the contrary, we have demonstrated that 'swimming' motility and production of the siderophore pyoverdine are not involved in the endophytic colonization ability of olive roots by strain Pseudomonas fluorescens PICF7. Comparative analysis of endophytic bacteria genomes is currently offering valuable information to identify bacteria! traits involved in endophytic lifestyle, although data are is still scant and controversia!., Living within plant tissues means that endophytic bacteria are adapted to an environment providing a constant and steady source of nutrients. They are also less exposed to (a)biotic stresses prevailing in the rhizosphere. In return, endophytic bacteria have developed strategies enabling them to overcome, modulate or evade defence responses deployed by the host plant against penetration of microorganisms, thereby being recognized as 'non hostile: Our studies have also shown that colonization and establishment of strain PICF7 in olive roots causes local and systemic transcriptomic changes, many of them related to defensive responses. There is an increasing interest on endophytic bacteria due to numerous applications they may have in agricultura! biotechnology. Among the beneficia! properties associated with endophytic lifestyle, promotion of plant growth and control of diseases have a special significance. Promotion of growth can be due to direct or indirect mechanisms. Direct plant growth promotion can be due to mobilization of (micro )nutrients (biofertilization) or synthesis of phytohormones (phytostimulation). Indirect plant growth promotion is usually a consequence of suppressing the effects caused by deleterious microorganisms. In this case, mechanisms involved can be different: antagonism or direct antibiosis against the pathogen, competition for nutrients and/ or space, or stimulation of defensive mechanisms of the plant host against pathogens attack. Endophytic bacteria can also stimulate plant growth by aiding the host to cope with presence of pollutants or heavy metals (rhizoremediation) or water and saline stresses. Many questions on how, when and why a bacteria! consortium is set as endophytic in any given host plant remain to be elucidated. They can basically be summarized in: (i) What are the driving forces operating to build a certain endophytic community? and (ii) what does the endophytic microbiome do for the host plant? To know the answers to these questions will be helpful to take full advantage of the potential of endophytic bacteria as sustainable and environmentfriendly biotechnological tools in agriculture and forestry., Research supported by grants P07-CVI02624 and P12-AGR667 (Junta de Andalucía, Spain), co-financed by ERDF from the EU.

Trabajo presentado en el 7th IOBC/WPRS Symposium Integrated Protection of Olive Crops, celebrado en Kalamata (Grecia) del 11 al 14 de mayo de 2015., Pseudomonas jluorescens PICF7 and Pseudomonas pulida PICP2 are two effective biocontrol strains against Verticillium wilt (Verticillium dahliae Kleb.) of olive. In addition, both bacteria can colonize endophytically olive roots, even simultaneously. However, our knowledge on bacteria! traits involved in biological control and endophytic lifestyle is scarce. In this study, we have sequenced and annotated the complete genomes of strains PICF7 (1 contig, 6.14 Mbp) and PICP2 (63 contigs, 5.60 Mbp). A comparative bioinformatics analysis was catTied out aiming to identify genes potentially involved in (i) biological control, (ii) plant-bacteria interaction, and (iii) endophytism. The analysis has revealed that, among other determinants frequently associated to biocontrol andlor plant-bacteria interaction, the genome of strain PICF7 harbours the genetic information to encode two type VI (T6SS) and one Type III (T3SS) secretion systems, the T3SS effectors avrE 1 and hopB 1, the siderophores pyoverdine, pyochelin and the hemophore HasAp, diverse adhesion factors, a battery of celldegrading enzymes (protease, cellulase and lipase), as well as detoxifying systems such as CopA, KatB, Dps and Cbb. On the contrary, the genome of strain PICP2 showed, among other differences, no coding region for T3SS. Sorne ofthe phenotypes predicted by genome analysis have been demonstrated by biochemical and microbiological analyses. The availability of both genomes will help to understand both biocontrol activity and the ability to colonize and persist within olive root tissues., Supported by grants P07-CVI-02624, P10-AGR-5797, Pl2-AGR-667 (Junta de Andalucía, Spain), and AGL2011-30343-C02-0l from the MINECO (Spain), all co-funded by ERDF from EU.

Trabajo presentado en el International Symposium Microbe-Assisted Crop Production - Opportunities, Challenges & Needs (MICROPE), celebrado en Viena del 23 al 25 de noviembre de 2015., Pseudomonas fluorescens PICF7 is a natural colonizer of olive (Olea europaea L.) roots. Our previous studies have demonstrated that this strain is an effective biocontrol agent (BCA) against Verticillium wilt of olive (VWO), one of the most important diseases affecting this woody crop caused by the soilborne fungal pathogen Verticillium dahliae. Endophytic lifestyle of strain PICF7 in olive root tissues was demonstrated under different experimenta l conditions as well. Root hairs were shown to play a key role in the endophytic colonization of root tissues by this rhizobacteria. Surface colonization of intact olive roots and endophytic establishment by PICF7 seem to be required for the effective control of VWO. Yet, mechanism(s) underlying PICF7-mediated biocontrol of V. dahliae and endophytism remain poorly understood. Mutant analysis has revealed that bacteria! traits such as siderophore pyoverdine production and swimming motility are needed neither for biocontrol performance nor endophytic lifestyle. On the other hand, colonization of olive roots by PICF7 triggers a broad range of transcriptomic changes, mostly related to defense responses to different (a)biotic stresses. Sorne of these responses occur at both local (roots) and systemic (stems) leve!. The way they are modulated along time could explairi biocontrol effectiveness of strain PICF7 and how olive tissues recognize this bacterium as a lnon-hostilel invader. While systemic defense responses are activated upon olive root colonization1 presence of PICF7 in this organ does not hinder olive knot development caused by Pseudomonas savastanoi pv. savastanoi (Psv), another relevant olive pathogen. However, when the BCA and the pathogenic bacterium are co-inoculated in stems, PICF7 altered both the appearance and anatomy of the tumors as well as the localization of Psv within the hyperplasic tissue. Strain PICF7 is also able to colonize roots of barley (Hordeum vulgare L.), even endophytically. lt also prometes plant growth and increases yield in this Poaceae species. Thus, strain PICF7 seems to be a versatile root endophyte providing benefits to the host which colonizes. The recent release of strain PICF7 genome will undoubtedly help to identify traits involved in biocontrol, endophytism and plant growth promotion., Supported by grants P07-CVI02624 and P12-AGR667 (Junta de Andalucía, Spain) and AGL2012-33264 (Spanish MICINN/MINECO), all co-financed by EU ERDF.

Trabajo presentado en el 7th IOBC/WPRS Symposium Integrated Protection of Olive Crops, celebrado en Kalamata (Grecia) del 11 al 14 de mayo de 2015., Verticillium wilt (Verticillium dahliae Kleb.) is one ofthe main biotic constraints affecting olive trees in the Mediterranean Basin. The difficulty to control this disease has urged researchers to seek effective measures within an integrated management framework. Biological control, alone or combined with other approaches, emerges as one of the most promising altematives to confront this pathogen. In this study, a collection of indigenous culturable bacteria (>300) from olive roots was generated. First selection was performed based on their antagonistic activity against V dahliae. As a result, 189 isolates were selected which were: (i) tested against a battery of relevant olive pathogens (V dahliae, Pseudomonas savastanoi pv. savastanoi, Rosellinia necatrix, Colletotrichum godetiae and Colletotrichum nymphaeae); (ii) identified molecularly; and (iii) screened for the presence of phenotypes usually associated to antagonism and!or plant growth promotion (PGP). Results showed predominan ce of phyla Proteo bacteria and Firmicutes, Pseudomonas and Bacillus being the most abundant genera. Although none of the strains showed effectiveness against all tested pathogens, many of them presented a broad antagonistic activity, particular! y strains of the genus Paenibacillus. Phenotypic characterization revealed enzymati~ activities usually associated to biocontrol . and PGP, such as production of catalase and siderophores, among others. This fact, together with the observed broad antagonist activity allow us to conclude that sorne of these bacteria are good candidates to be used as biological control agents in future bioformulations against diverse relevant olive pathogens., Supported by grants Pl2-AGR667 (Junta de Andalucía, Spain) and RECUPERA 2020 (MINECO-CSIC), all co-funded by ERDF from EU.

Trabajo presentado en el XVII Congreso de la Sociedad Española de Fitopatología, celebrado en Lleida del 7 al 10 de octubre de 2014., Grape powdery mildew, caused by Uncinula necator, is one of the most damaging diseases of Vitis spp. in the drought areas of China. The main measure used to manage this disease is the application of diverse chemical fungicides about 8 times in growing seasons. In order to develop environmentally friendly biocontrol agents (BCAs), twenty two strains from Streptomyces spp. and Pseudomonas spp. were screened for their potential to suppress grape powdery mildew. Conidiospore germination inhibition tests were performed on water agar media mixed with the BCAs. Streptomyces aureofaciens CN122, Streptomyces rimosus CN123, the genetically-modified derivatives of Pseudomonas putida WCS358r WCS358::phl and WCS358::phz, constitutively producing 2,4-diacetylphloroglucinol (DAPG) and phenazine carboxylic acid (PCA), respectively, and Pseudomonas fluorescens strains WCS417r, L1d6, L2d6 and L3d6, were found to significantly inhibit germination of U. necator conidiospores. Based on these results, two types of bioassays were performed. The first was a preventive bioassay in which detached leaves were dipped in a suspension of each beneficial strain for 20 min, surface dried, and followed by the inoculation of the pathogen spore suspension. The second was a curative bioassay in which detached leaves were first inoculated with the spore suspension of the pathogen and then dipped in suspensions of the BCAs. Results from the preventive bioassay showed that all strains tested produced a strong inhibition of spore germination. Thus, effective suppression of grape powdery mildew was observed, and DAPG and PCA produced by the genetically-modified strains could be involved in the control of the disease. On the other hand, none of the bacterial strains tested showed significant control of the disease when applied after pathogen inoculation (curative bioassay). This suggests that effective control may only be expected when the biocontrol strains are applied in a preventative way., This research was supported financially by the Special Fund for Agro-Scientific Research in the Public Interest of P.R. China (201203035).

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., Verticillium wilt of olive tree (Olea europaea L.) (VWO), ca u sed by the soil-borne fungus Verticillium dahliae Kleb., is considered one ofthe most devastatíng diseases affecting this crop in many areas. Preventive measures su eh as the treatment of the root systems at the nursery stage with microbial antagonists emerges as a promising altemative. Species ofthe soil-borne fungus Trichoderma ha ve been widely described as biocontrol agents (BCAs) of severa[ phytopathogenic fungi by means of different mechanisms. In this study we aimed to evaluate the potential of T harzianum CECT 2413 as effective BCA of VWO. Strain CECT 2413 was shown to in vitro overgrow both the bighly-virulent, defoliating (D) and the midly-virulent, non-defoliating (ND) V. dah!iae pathotypes infecting olive. Inhibition of V dahliae growth in cHituring media where strain CECT 2413 had previously grown showed that this ability might be dueto excretion of secondary metabolites or cell wall degrading enzymes. Wben a suspension of conidia of T harzianum CECT 2413 were applied by irrigation to roots of nursery-propagated, three-month-old olives (cv. Picual) prior to the treatment with a D isolate ofthe pathogen, disease symptoms were significantly reduced compared to that observcd in control plants. M oreo ver, increase in the number and size of lea ves in plants treated \vith Trichoderrna was also reported. Biofonnulations ofthis BCA, alone or in combination with other Trichoderma spp. strains, have been recently patented and licensed., Supported by ERDF-­cofinanced grants (AGR-­‐6038, AGR-­‐5948, Consejería de Economía, Innovación y Ciencia, Junta de Andalucía, Spain, and NUTESCA S.L. (Jaén, Spain).

Ponencia presentada en el 11 th International Verticillium Symposium, celebrado en Göttingen (Alemania) del 5 al 8 de mayo de 2013., Verticillium wilt of olive (VWO), caused by Verticillium dahliae Kleb., is one of the most important biotic constrains affecting olive. The incidence and severity of this disease has increased during the last decades and its effective control requires implementation of an integrated disease management strategy, with emphasis on preventive measures. The use of biological control agents (BCA) appears as an excellent before-planting measure since they can be applied during the nursery production stage. Pseudomonas fluorescens PICF7 is a natural inhabitant of olive roots and an effective BCA against V. dahliae. Strain PICF7 displays some traits in vitro which may explain its biocontrol activity. For instance, growth inhibition of V. dahliae and production of salicylic acid and the siderophore pyoverdine have been demonstrated, as well as swimming motility which can be important for rhizosphere colonization. Moreover, PICF7 is able to endophytically colonize olive roots and induces a broad range of defense responses. However, mechanism(s) underlying its biocontrol activity remain mostly unknown. To unravel traits involved in the suppressive effect against VWO, a mutant bank of P. fluorescens PICF7 was generated (>9.000 insertions) by random transposon Tn5 mutagenesis. Phenotypes affected in swimming motility, siderophore production and growth inhibition of V. dahliae were screened. A collection of selected mutants altered in one of these phenotypes were analyzed by nested-PCR to localize the Tn5 insertion site. Adjacent DNA regions to the insertion point were sequenced and compared against available databases. Four mutants were finally chosen to conduct in planta bioassays: ME424, affected in motility (insertion in fliI homologue); ME589, a siderophore mutant with insertion located in a gene coding for a putative pyoverdine nonribosomal peptide synthetase; ME419, showing enhanced growth inhibition of V. dahliae (undetermined Tn5 insertion); and ME1508, a mutant displaying diminished growth inhibition of the pathogen (insertion in a gene encoding a putative sulfite reductase). Biocontrol bioassays using nursery-produced olive plants (cv. Picual) have consistently shown that mutant ME1508 has significantly lost the ability to control V. dahliae (defoliating [D] pathotype). However, the remaining mutants displayed variable biocontrol performance among bioassays. Arabidopsis thaliana has been also used to evaluate these mutants. This model plant was first checked for: i) Verticillium wilt symptoms development upon inoculation with an olive V. dahliae D isolate; ii) successful rhizosphere colonization by strain PICF7; and iii) effective control of the disease by the BCA. Results were similar to those observed in olive plants: mutant ME1508 had significantly lost the biocontrol ability compared to PICF7. Thus, the gene disrupted in ME1508 seems to play an important role in biocontrol of PICF7 against V. dahliae. However, phenotypes such as motility and siderophore production either do not participate in the suppressive effect or are greatly influenced by the experimental conditions used., Research supported by grants AGL2009-07275 from Spanish MICINN/MINECO and P07-CVI-02624 from J. Andalucía (Spain), both co-financed by ERDF of the EU.

Póster presentado en el COST TD801 StatSeq 5th Workshop, celebrado en Helsinki del 24 al 26 de abril de 2013., Low temperature severely affects plant growth and development. Several plant species have evolved an adaptive response, named cold acclimation. In order to study this response in olive tree (Olea europaea), we maintained twomonths- old vegetative propagated olive trees cv. Picual at low temperature (14h at 10ºC with light, 10h at 4ºC at the dark) for 15 days. Control plants were maintained in field growing conditions. Cold stress symptoms were observed after the first 24h as sagging leaf and biochemical stress markers. After 5 days the plants were observed completely recovered. We extracted RNA from leaves of three control plants and the same number from 24h and 10 days cold stressed plants. We pooled RNA and made two cDNA libraries for sequencing. We used Illumina HiSeq 1000 sequencer.

Trabajo presentado en el 11 th International Verticillium Symposium, celebrado en Göttingen (Alemania) del 5 al 8 de mayo de 2013., Verticillium dahliae Kleb is the causal agent of Verticillium wilt of olive (Olea europaea L.) (VWO), one of the most threatening diseases in many areas where this tree is cultivated, particularly in the Mediterranean basin. The current spread of the disease and the severity of its attacks need of an integrated disease management strategy to achieve effective control. The use of resistant olive cultivars is one of the most plausible measures for controlling VWO within such framework. While diverse studies aimed to identify VWO resistant or tolerant genotypes under diverse inoculation conditions have provided some promising results, knowledge on the genetics of resistance to VWO is still very limited. One of our current lines of research aims to shed light on the genetic responses taking place during V. dahliae-olive interaction. In the present study, we have conducted a functional genomics analysis to unravel responses that could be induced in aerial tissues of the resistant olive cultivar Frantoio. We particularly aimed to elucidate whether early systemic responses can be differentially triggered in above-ground tissues upon artificial inoculation of V. dahliae in the root system. A suppression subtractive hybridization cDNA library, enriched in up-regulated genes, was generated from aerial olive tissues of ‘Frantoio’ nursery-produced plants, sampled at different time points along 21 days after root-dip inoculation with a representative isolate of the defoliating pathotype. This strategy has enabled the identification of 612 ESTs (71 contigs and 541 singlets) expressed in aerial tissues during the interaction of V. dahliae with roots. Querying (Blastx) the non-redundant NCBI database allowed the attribution of homologous hits for transcripts (approximately 43.8%) with coding sequences present in genomes of woody plants such as Vitis vinifera, Populus trichocarpa and Ricinus communis. Moreover, 2.3% of up-regulated genes matched to genes already identified in olive. Finally, nearly 24% of detected olive transcripts corresponded to unidentified genes. Computational analysis showed a number of transcripts involved, among other processes, in plant defense response to biotic and abiotic stresses (i.e., formamidase, phosphatase 2c, PR proteins such as thaumatin-like protein belonging to PR-5 family and lipoxygenases), phenylpropanoid biosynthesis (i.e., caffeoyl- O-methyltransferase) or terpenoids and hormones biosynthesis (i.e., acetone-cyanohydrin lyase and malate dehydrogenase). Similarly, different classes of transcription factors such as GRAS1 and WRKYs were shown to be up-regulated in aerial tissues after V. dahliae inoculation in roots. Thus, a broad range of defensive responses seems to be induced in tissues located far away from where the pathogen colonization process is taking place in a resistant olive cultivar., Research supported by grants AGL2009-07275 from Spanish MICINN/MINECO and P07-CVI-02624 from J. Andalucía (Spain), both co-financed by ERDF of the EU.

Póster presentado en el 28th New Phytologist Symposium. Functions and Ecology of the Plant Microbiome, celebrado en Rodas (Grecia) del 18 al 21 de mayo de 2012., Information concerning processes underlying the interaction between beneficial bacteria and woody plants is still very limited and totally absent in the case of olive (Olea europaeaL.). We aimed to elucidate genomic responses occurring during the colonization of olive roots by the indigenous endophyte Pseudomonas fluorescens PICF7, an effective biocontrol agent against Verticillium wilt of olive (VWO). A Suppression Subtractive Hybridization cDNA library, corresponding to up-regulated genes, was generated from roots of olive plants (cv. Arbequina) inoculated with strain PICF7, enabling the identification of some 450 EST sequences differentially expressed during this interaction. Computational analysis showed a number of transcripts involved, among other processes, in plant defence response to different stresses (i.e., PAL, acetone cyanohydrin lyase, lipoxygenase, β-1,3-glucanase, endochitinases or lignin-forming peroxidases). Similarly, diverse transcription factors such as bHLH, GRAS1, ARF2 and SA-responsive WRKYs were shown to be induced in olive roots after PICF7 inoculation. Interestingly, 19% of detected olive transcripts corresponded to unidentified genes. Real-time quantitative PCR confirmed the induction of some of the aforementioned genes. Thus, the beneficial endophytic strain P. fluorescens PICF7 seems to trigger a broad array of defence responses in olive root tissues which can be responsible of its VWO biocontrol activity.

Póster presentado en la 18th International Sunflower Conference, celebrada en Mar de Plata y Balcarce (Argentina), del 27 de abril al 1 de marzo de 2012., One of the most important biotic constraints for sunflower production is the soil-borne, obligate-parasitic oomycete Plasmopara halstedii Farl. Berl. & de Toni, which causes sunflower downy mildew (SDM). Control of SDM by means of biocontrol agents (BCAs) has been poorly investigated so far. Our previous studies have demonstrated that Pseudomonas fluorescens PICF7 is an effective BCA against Verticillium wilt of olive, a disease caused by the soil-borne pathogenic fungus Verticillium dahliae Kleb. Moreover, strain PICF7 was confirmed as endophyte in olive roots. Recently, colonization and persistence in Arabidopsis thaliana roots of strain PICF7 and one spontaneous rifampicin-resistant mutant derivative (PICF7Rf) were assessed, thus confirming the ability of this strain to colonize different plant species. Considering these antecedents, we aimed in this study to determine whether P. fluorescens PICF7: (1) colonize and persist in sunflower roots; (2) promote growth of sunflower at early stages; and (3) control SDM. Hybrid seeds genetically susceptible to SDM were dipped in bacterial suspensions of PICF7 (aprox. 1 x 109 cfu/mL) or PICF7Rf, (aprox. 1 x 109 cfu/mL) or in 10 mM MgSO4•7H2O (control treatment) at 23ºC, 150 rpm for 5h. Seeds of each treatment were germinated and subsequently immersed in a zoosporangial suspension of an isolate of P. halstedii collected in southern Spain in 2010 (2 x 104 zoosporangia/mL) prepared in water for 5-h at 23ºC, 150 rpm. Bioassays were then designed and conducted under controlled growth conditions. Ten pots per treatment were filled with perlite and five sunflower seeds were sown in each pot. Plants were grown at 23ºC, 70-100% relative humidity and a 12h photoperiod (100 µE•m-2•s-1) for 18-25 days. Colonization and persistence of PICF7 and PICF7Rf in roots were assessed at different time points along the bioassays. Root macerates were diluted and sown in King’s B (KB) agar (PICF7) or KB agar amended with rifampicin (PICF7Rf). To assess growth promotion of sunflower seedlings upon bacteria inoculation, the dry-weight of the above-ground part of plants was determined at the end of the experiments. Disease incidence (percentage of diseased plants per pot) and symptoms were scored at 10 days after bacterial inoculation and at the end of the experiments. Results showed that strains PICF7 and PICF7Rf are able to colonize sunflower roots from the very first moment after seed bacterization (107-108 cfu/g) and persist until the end (about three weeks) of the experiments (107cfu/g). On the other hand, no significant differences of dry-weight were found between control and bacteria-inoculated plants. Plants inoculated with the pathogen displayed similar final disease incidences whether they were bacterized (52%, PICF7; 68%, PICF7Rf) or not (64%). Although results showed that Pseudomonas fluorescens PICF7 and PICF7Rf are good colonizers of sunflower roots, neither increase of the above-ground biomass, nor control of SDM were observed under the tested experimental conditions. Root colonization ability by an enhanced green fluorescent protein (EGFP)-tagged derivative of strain PICF7 is currently being evaluated by confocal laser scanning microscopy (CLSM). The aim will be to determine whether the lack of SDM suppression could be explained by a failure of PICF7 to establish as an endophyte in sunflower root tissues. Biological control may offer an environmentally-friendly approach fitting criteria of modern sustainable agriculture and may be a complementary action to other disease control measures. This study is one of the first attempts to evaluate the potential biocontrol effectiveness of a beneficial, naturally-occurring bacteria originating from the same bioclimatic area as sunflower is cultivated., This research was supported by grants AGL2009-07275 and AGL2010-17909 (Spanish Ministerio de Ciencia e Innovación, and P07-CVI-02624 (Junta de Andalucía, Spain), all of them cofinanced by ERDF of the EU.

Verticillium wilt (VW), caused by Verticillium dahliae Kleb., is an important disease in many crops and its effective management has proven difficult. Among the various disease control measures to be implemented, the use of microbial antagonists (biological control agents, BCAs) constitutes an environmentally-friendly approach fitting criteria of modern sustainable agriculture. Pseudomonas fluorescens PICF7 was isolated from root tissues of nursery--propagated olive plants. Selection of this strain was based on in vitro growth inhibition of V. dahliae, colonizing ability of olive roots, endophytic lifestyle, and control of the highly-virulent defoliating (D) pathotype of V. dahliae in olive planting stocks. The mode of action by which PICF7 controls VW in olive is as yet unknown; moreover, to uncover potential biocontrol mechanisms poses additional difficulties in this pathosystem because the target is a tree. Therefore we used the model plant Arabidopsis thaliana to study: i) if PICF7 colonizes the rhizosphere of A. thaliana; ii) disease symptoms caused by V. dahliae in A. thaliana; iii) control of VW by PICF7 in different accessions and mutants of A. thaliana; and iv) if motility, antibiosis and/or siderophores are involved in control of V. dahliae by PICF7. Diverse bioassays were conducted and in all of them both the BCA and the pathogen were introduced in the rhizosphere of A. thaliana. Both D and non-defoliating isolates of V. dahliae caused disease symptoms in A. thaliana. PICF7 colonized and persisted in the rhizosphere of different Arabidopsis accessions and could control the D pathotype in some of them. PICF7 mutants affected in antibiosis significantly lost their ability to control VW in A. thaliana. We conclude that the model plant A. thaliana is useful to unravel interactions between this BCA and V. dahliae.

[EN] The invention relates to the use of strains of Trichoderma fungi for preventing and/or treating infections of plants and/or soils caused by phytopathogenic fungi belonging to the genus Verticillium. Furthennore, the invention relates to new fungal strains belonging to the species Trichoderma harzianum and Trichoderma atroviride, which are likewise resistant to copper and capable of inhibiting the growth of other microorganisms, preferably phytopathogenic fungi. Thus, the use thereof is proposed as a fertilizer and for preventing and/or treating infections of plants and/or soils caused by phytopathogenic microorganisms, preferably the defoliant or non-defoliant pathotypes of Verticillium dahliae, the causative agent of olive Verticillium wilt., [ES] La invención se refiere al uso de cepas de hongos Trichoderma para la prevención y/o tratamiento de infecciones de plantas y/o suelos provocadas por hongos fitopatógenos pertenecientes al género Verticillium. Además, la invención se refiere a nuevas cepas de hongos perteneciente a las especies Trichoderma harzianum y Trichoderma atroviride que son también resistentes a cobre capaces de inhibir el crecimiento de otros microorganismos, preferiblemente hongos, fitopatógenos. Por ello, se propone su uso como fertilizante y para la prevención y/o tratamiento de infecciones de plantas y/o suelos provocadas por microorganismos fitopatógenos, preferiblemente por los patotipos defoliante o no defoliante de Verticillium dahliae, agente causante de la verticilosis del olivo., Barroso Albarracin, Juan Bautista et al., Universidad de Jaén, Nutesca S.L, Fundación Citoliva, Consejo Superior de Investigaciones Científicas, Universidad de Sevilla, A2 Solicitud de patente sin informe sobre el estado de la técnica

Verticillium wilt, caused by Verticillium dahliae Kleb., is a problem in many crops and the disease is difficult to control. Strains of Pseudomonas fluorescens and P. putida were previously isolated from root tissues of olive trees, cv. Picual. Some of them are endophytic and can control the highly-virulent, defoliating (D) pathotype of V. dahliae in olive. One mode of action of disease suppression by fluorescent Pseudomonas spp. is induced systemic resistance (ISR). Pseudomonas spp. strains were tested for ISR in a system using Arabidopsis thaliana and the pathogens P. syringae pv. tomato and Botrytis cinerea. To include V. dahliae in these studies we inoculated A. thaliana Col-0 with several isolates of this pathogen belonging to different vegetative compatibility groups (VCG). Isolate V937I (VCG1A, D pathotype) produced severe symptoms in Arabidopsis, and P. fluorescens PICF7 was able to control the disease caused by this virulent isolate. The use of non-ISR expressing accessions and mutants of A. thaliana will allow to evaluate involvement of ISR in control of Verticillium wilt., The research was supported by grants P07-CVI-02624 from Junta de Andalucía and AGL2009-07275 from Spanish MICINN (co-financed by FEDER of the EU).

Within an integrated disease control strategy to p romote long-term protection against Verticillium wilt of olive ( Olea europaea L.), we aimed to elucidate the genetic and molecul ar processes taking place during the colonization of o live roots by Pseudomonas fluorescens PICF7, an effective biocontrol agent against VWO. SSH methodo logy and transcriptome analysis enabled the identification of putative genes differentially exp ressed such as several enzymes related to biosynthesis of plant hormones (i.e. lipoxygenase, chorismate synthase) and of phenylpropanoids compounds (i.e. phenylalanine ammonia lyase, cinnam yl-alcohol dehydrogenase). Interestingly, among the transcriptionally up- or down-regulated g enes, diverse transcription factors implicated in plant signaling pathways for both biotic and abioti c stimulus response were also identified., This research was supported by grants P07-CVI-02624 from Junta de Andalucía and AGL2009-07275 from Spanish MICINN (co-financed by F EDER of the EU).

Working Group “Integrated Protection of Olive Crops”. Proceedings of the meeting at Cordoba (Spain), 01 – 04 June, 2009., The colonisation process of olive by an enhanced yellow fluorescent protein (EYFP) tagged Verticillium dahliae defoliating (D) pathotype derivative (VDAT-36I), and its in planta interaction with an enhanced green fluor escent protein (EGFP)-tagged derivative of Pseudomonas fluorescens PICF7 have been monitored on/in olive tissues using a nongnotobiotic system, vibratome-tissue sectioning, and confocal laser scanning microscopy (CLSM). Isolate VDAT-36I invaded root cortex and vascular tissues via macro- and microbreakages, and progressed to the aerial parts of the plant through xylem vessel cells. Strain PICF7 used root hairs as preferred penetration site. Early and localised root surface and root endophytic colonisation by P. fluorescens PICF7 is needed to impair full progress of verticillium wilt in olive.

13 pages; 4 figures, 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., Junta de Andalucía, Spain. Grant P07-CVI- 02624 (Proyecto de Excelencia).

11 páginas, 4 figuras, An increasing incidence and distribution of verticillium wilt has occurred in the last few years in newly established olive orchards in southern Spain. This spread of the disease may result from use of Verticillium dahliae-infected planting material. The early in planta detection of the pathogen would aid the implementation of certification schemes for pathogen-free planting material. In this work, a nested polymerase chain reaction (PCR) method was developed for the in planta detection of the nondefoliating (ND) V. dahliae pathotype, aimed especially at nurseryproduced olive plants. For this purpose, specific primers were designed from the sequence of a 1958-bp random amplified polymorphic DNA (RAPD) marker of ND V. dahliae, and a procedure for the extraction of PCR-quality total genomic DNA from infected root and stem tissues of young olive plants was tested and further optimized. Nested PCR assays detected ND V. dahliae in 4- to 14-month-old artificially infected plants of three olive cultivars. The ND-specific PCR product was not amplified from total genomic DNA extracted from olive plants infected with the defoliating V. dahliae pathotype. Detection of the ND pathotype was effective from the very earliest moments following artificial inoculation of olive plants with a V. dahliae conidial suspension. Also, detection was achieved in inoculated, though symptomless, olive plants as well as in plants that were symptomatic but became symptomless by 217 days after inoculation., Comisión Interministerial de Ciencia y Tecnología (CICYT), España. 1FD97-0763-C03-01; Comunidad Europea, contrato no. QLK5-CT1999-01523.