Early and delayed long-term transcriptional changes and short-term transient responses during cold acclimation in olive leaves

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.Peer reviewe

Cold response in olive tree; A RNAseq study

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.Peer Reviewe

Verticillium wilt of olive in the Guadalquivir Valley (southern Spain): relations with some agronomical factors and spread of Verticillium dahliae

Verticillium wilt of olive (VWO) is now the most destructive olive disease in the Guadalquivir valley in Andalucía (southern Spain). Disease surveys, conducted to assess the association of agronomical and geographical factors with the current spread of the disease, have shown that VWO is widespread in the valley, with a mean disease incidence (DI) in infested plots reaching 20.4%  (9000 inspected trees), but with significant differences among the provinces surveyed (25.7, 23.7 and 12%, for Jaén, Córdoba and Seville, respectively). The DI was significantly higher in irrigated (20.7%) than in dry-farming (18.3%) orchards, and also higher in non-tilled orchards (25.6%) than in regularly-tilled orchards (16.3%). The DI was likewise significantly lower for tree densities above 200 trees ha-1; and it was higher (21.5%) when the orchards were located near areas where other V. dahliae host plants were cultivated, than if the orchards were surrounded by non-host plants (11.9%). Lastly, the DI was significantly higher in plots where the tree were less than 25 year old and in plantations close to the Guadalquivir River (less than 10 km). ‘Picual’ was the cultivar most often affected with the disease, reaching a DI of 41.9% in orchards where this cultivar was grown. Highly virulent defoliating (D) isolates in the plantation surveyed were significantly more common (67.7%) than non-defoliating (ND) isolates (32.3%). These factors could explain the substantial increase in incidence and severity of VWO seen in the valley during the last decade

The biocontrol endophytic bacterium Pseudomonas fluorescens PICF7 induces systemic defense responses in aerial tissues upon colonization of olive roots

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.Research supported by grants P07-CVI-02624 and P12-AGR-667 from Junta de Andalucía (Spain) and AGL2009-07275 from Spanish MICINN/MINECO, both co-financed by The European Regional Development Fund of the European Union.Peer Reviewe

Systemic responses in a tolerant olive (Olea europaea L.) cultivar upon root colonization by the vascular pathogen Verticillium dahliae

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.Peer reviewedPeer Reviewe

Molecular analysis of Spanish populations of Fusarium oxysporum f. sp. dianthi demonstrates a high genetic diversity and identifies virulence groups in races 1 and 2 of the pathogen

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.Peer Reviewe

Genetic responses induced in aerial olive tissues during the interaction in roots with the defoliating pathotype of Verticillium dahliae

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.Peer Reviewe

Respuestas defensivas sistémicas en olivo a la infección por el hongo fitopatógeno Verticillium dahliae

Trabajo presentado en el XXV Congreso Nacional de Microbiología (SEM), celebrado en Logroño del 7 al 10 de julio de 2015.Verticillium dahliae Kleb. es el agente causal de la Verticilosis del olivo (Olea europaea L.) (VO), una de las enfermedades más amenazadoras para este cultivo, especialmente en la cuenca Mediterránea. La actual dispersión de la VO y la severidad de sus ataques, hacen necesario un control integrado. El uso de variedades de olivo resistentes es una de las mejores medidas de control. Diversos estudios se han centrado en la identificación y evaluación del grado de susceptibilidad y tolerancia/resistencia de genotipos de olivo a la VO. Si bien los resultados han proporcionado una información valiosa, el conocimiento de la base genética de la resistencia a VO es aún muy limitado. En el presente trabajo, hemos realizado un análisis transcriptómico para (1) elucidar las respuestas genéticas que se inducen y/o reprimen de forma sistémica (tejidos aéreos) en plantas del cultivar (cv.) tolerante Frantoio cuando sus raíces son inoculadas por un representante del patotipo más virulento (defoliante, D) de V dahliae y (2) determinar el patrón de expresión de algunos de los genes identificados en cvs. de olivo con diferente grado de susceptibilidad al patógeno.Financiado por el proyecto AGL2009-07275 del MICINN/ MINECO, con cofinanciación PEDER de la UE.N

Co-occurrence network analysis unveils the actual differential impact on the olive root microbiota by two Verticillium wilt biocontrol rhizobacteria

Background: Verticillium wilt of olive (VWO), caused by Verticillium dahliae Kleb, is one of the most threatening diseases affecting olive cultivation. An integrated disease management strategy is recommended for the effective control of VWO. Within this framework, the use of biological control agents (BCAs) is a sustainable and environmentally friendly approach. No studies are available on the impact that the introduction of BCAs has on the resident microbiota of olive roots. Pseudomonas simiae PICF7 and Paenibacillus polymyxa PIC73 are two BCAs effective against VWO. We examined the effects of the introduction of these BCAs on the structure, composition and co-occurrence networks of the olive (cv. Picual) root-associated microbial communities. The consequences of the subsequent inoculation with V. dahliae on BCA-treated plants were also assessed. Results: Inoculation with any of the BCAs did not produce significant changes in the structure or the taxonomic composition of the ‘Picual’ root-associated microbiota. However, significant and distinctive alterations were observed in the topologies of the co-occurrence networks. The introduction of PIC73 provoked a diminution of positive interactions within the ‘Picual’ microbial community; instead, PICF7 inoculation increased the microbiota’s compartmentalization. Upon pathogen inoculation, the network of PIC73-treated plants decreased the number of interactions and showed a switch of keystone species, including taxa belonging to minor abundant phyla (Chloroflexi and Planctomycetes). Conversely, the inoculation of V. dahliae in PICF7-treated plants significantly increased the complexity of the network and the number of links among their modules, suggestive of a more stable network. No changes in their keystone taxa were detected. Conclusion: The absence of significant modifications on the structure and composition of the ‘Picual’ belowground microbiota due to the introduction of the tested BCAs underlines the low/null environmental impact of these rhizobacteria. These findings may have important practical consequences regarding future field applications of these BCAs. Furthermore, each BCA altered the interactions among the components of the olive belowground microbiota in idiosyncratic ways (i.e. PIC73 strongly modified the number of positive relations in the ‘Picual’ microbiota whereas PICF7 mostly affected the network stability). These modifications may provide clues on the biocontrol strategies used by these BCAs.This work was supported by the Spanish Ministerio de Ciencia, Innovación y Universidades/Agencia Estatal de Investigación (grant PID2019-106283RB-I00), with the support of EU ERDF funds. Open Access funding provided thanks to the CRUE-CSIC agreement with Springer Natur

Identificación y caracterización de bacterias de la rizosfera de olivo con potencial como agentes de control biológico de amplio espectro

Trabajo presentado en el VI Reunión del Grupo Especializado SEM - Microbiología de Plantas (Sociedad Española de Microbiología), celebrado en Miraflores de la Sierra (Madrid) del 11 al 13 de marzo de 2015.En las últimas décadas la preocupación por los riesgos derivados del uso continuado de productos fitosanitarios de síntesis química para el control de enfermedades de cultivos ha experimentado un notable aumento. Parte de la comunidad científica ha dirigido sus esfuerzos al desarrollo de estrategias de control alternativas y eficaces frente a patógenos de plantas. Entre ellas destacan las basadas en el uso de agentes de control biológico (ACB). Características tales como su bajo riesgo medioambiental, o el potencial que tienen para ser aplicados de forma individual, combinados con otros microorganismos, o en estrategias de control integrado, hacen que tengan un elevado interés agronómico, económico y social. El presente trabajo surge de la necesidad de mejor ar la sanidad de los cultivos usando métodos más sostenibles y de bajo impacto medioambiental. Se proyectó la creación de una colección de bacterias cultivables procedentes de raíces de olivo como base para el desarrollo de bioformulaciones que, utilizadas individualmente o en combinación, resultasen efectivas frente a diversos patógenos y/o presentaran capacidad de promoción del crecimiento vegetal. Se generó una “bacterioteca” compuesta por 189 aislados que han sido caracterizados a nivel: (1) molecular, mediante la secuenciación parcial del gen 16S rDNA; (2) de capacidad antagonista, evaluando su actividad inhibidora frente aislados de patógenos fúngicos (Verticillium dahliae, Rosellinia necatrix, Alternaria alternata, Colletotrichum nymphaeae, Colletotrichum godetiae, Fusarium xysporum f. sp. dianthi y Ganoderma lucidum), del oomiceto Phytophthora cinnamomi, de dos cepas de la bacteria fitopatógena Pseudomonas savastanoi pv. savastanoi, y del ACB Trichoderma harzianum CECT2413; y (3) enzimático y bioquímico, determinando actividades quitinasa, proteasa, fosfatasa, catalasa, o la producción de sideróforos y de 2,3-butanodiol. También se analizó el metabolismo de fuentes de carbono y sensibilidades químicas mediante el uso del sistema Biolog. La clasificación molecular determinó que más del 45% de las bacterias seleccionadas pertenecían al grupo filogenético Proteobacterias, siendo Pseudomonas el género más representado. Ningún aislado fue eficaz frente al 100% de los patógenos evaluados. Sin embargo, la mayoría mostraron antagonismo frente a buen número de ellos, destacando varias cepas del género Paenibacillus. El patógeno más frecuentemente antagonizado fue V. dahliae, mientras que G. lucidum fue el que menos. El ACB T. harzianum fue antagonizado por menos del 20% de aislados. Los resultados mostraron la presencia generalizada de actividades enzimáticas frecuentemente asociadas tanto al control biológico como a la promoción directa del crecimiento vegetal. Así, la actividad catalasa se detectó en el 100% de las bacterias. Por el contrario, solo un 2% de los aislados mostraron actividad fosfatasa. Este hecho, así como la amplia capacidad antagonista detectada, permiten concluir que algunos de los aislados bacterianos identificados y caracterizados son susceptibles de ser utilizados como ACB frente a diversos patógenos en futuras bioformulaciones.N