Genomic analysis of eight native plasmids of the phytopathogen Pseudomonas syringae

Comunicación a conferenciaThe pPT23A family of plasmids (PFPs) appears to be indigenous to the plant pathogen Pseudomonas syringae and these plasmids are widely distributed and widely transferred among pathovars of P. syringae and related species. PFPs are sources of accessory genes for their hosts that can include genes important for virulence and epiphytic colonization of plant leaf surfaces. Further understanding of the evolution of the pPT23A plasmid family and the role of these plasmids in P. syringae biology and pathogenesis, requires the determination and analysis of additional complete, closed plasmid genome sequences. Therefore, our main objective was to obtain complete genome sequences from PFPs from three different P. syringae pathovars and perform a comparative genomic analysis. In this work plasmid DNA isolation, purification by CsCl-EtBr gradients, and sequencing using 454 platform, were used to obtain the complete sequence of P. syringae plasmids. Different bioinformatic tools were used to analyze the plasmid synteny, to identify virulence genes (i.e. type 3 effectors) and to unravel the evolutionary history of PFPs. Our sequence analysis revealed that PFPs from P. syringae encode suites of accessory genes that are selected at different levels (universal, interpathovar and intrapathovar). The conservation of type IVSS encoding conjugation functions also contributes to the distribution of these plasmids within P. syringae populations. Thus, this study contributes to unravel the genetic basis of the role of PFPs in different P. syringae lifestyles.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Isolation, characterization and selection of bacterial isolates from a suppressive soil with beneficial traits to plants

Backgrounds This study focused on the characterization and selection of bacterial strains obtained from a suppressive soil displaying antifungal activity against the soilborne phytopathogenic fungi Rosellinia necatrix. Bacterial profile from this suppressive soil were first obtained by 16S rRNA gene sequencing, revealing a significant increase in the bacterial class Gammaproteobacteria, especially in some antagonistic representatives of Pseudomonas spp. Objectives To obtain and characterize a collection of 246 bacterial isolates obtained from this suppressive soil, in order to identify new strains with antifungal activity against fungal phytopathogens. Methods To obtain the bacterial collection, we performed an isolation on a selective medium for Pseudomonas-like microorganisms. Further characterization tests were used in order to analyse the bacterial collection, including identification of the general metabolic profile of glucose, the profiling of antifungals produced, including both the putative production of antifungal compounds and lytic exoenzymes, and the evaluation of traits related with beneficial effects on plants. Conclusions A final selection of representative strains resulted in antifungal isolates belonging to the genus Pseudomonas, but also some representatives of the genera Serratia and Stenotrophomonas. These selected strains were tested for plant protection by an in vivo experiment using avocado and wheat plants challenged by the pathogen R. necatrix, showing all of them an antifungal ability and plant disease protection. Pseudomonas-like strains isolated from suppressive soils constitute an excellent source for novel microbial biocontrol agents against soilborne fungal pathogens. This work was supported by grant AGL2014-52518-C2-1-R. Carmen Vida and Sandra Tienda are supported by a PhD fellowship from the FPI program of the Spanish Government.This work was supported by grant AGL2014-52518-C2-1-R. Carmen Vida and Sandra Tienda are supported by a PhD fellowship from the FPI program of the Spanish Government; Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

El enemigo de mi enemigo es mi amigo

Plants health is influenced by the characteristics of the plant, the environment, specially the soil, and the microbiome associated with them. The study of the interaction networks of the different components of the microbiome can provide strategies for the development of a sustainable agriculture as well as being environmental friendlyLa salud de las plantas se ve influenciada por las características de la propia planta, el medio ambiente, especialmente el suelo, y el microbioma asociado a las mismas. El estudio de las redes de interacción entre los componentes del microbioma puede proporcionar estrategias para el desarrollo de una agricultura sostenible a la par de respetuosas con el medio ambiente

Comparative genomic analysis of native pseudomonas syringae plasmids belonging to the ppt23 a family reveals their role in p. Syringae epiphytic and pathogenic lifestyles

Backgrounds The pPT23A family of plasmids (PFPs) appears to be indigenous to the plant pathogen Pseudomonas syringae and these plasmids are widely distributed and widely transferred among pathovars of P. syringae and related species. PFPs are sources of accessory genes for their hosts that can include genes important for virulence and epiphytic colonization of plant leaf surfaces. Objectives Further understanding of the evolution of the pPT23A plasmid family and the role of these plasmids in P. syringae biology and pathogenesis, requires the determination and analysis of additional complete, closed plasmid genome sequences. Therefore, our main objective was to obtain complete genome sequences of PFPs from three different P. syringae pathovars and perform a comprehensive comparative genomic analysis. Methods In this work plasmid DNA isolation, purification by CsCl-EtBr gradients, and sequencing using 454 platform, were carried out to obtain the complete sequence of P. syringae plasmids. Different bioinformatic tools were used to analyze the plasmid synteny, to identify virulence genes (i.e. type 3 effectors) and to unravel the evolutionary history of PFPs. Conclusions Our sequence analysis revealed that PFPs from P. syringae encode suites of accessory genes that are selected at different levels (universal, interpathovar and intrapathovar). The conservation of type IVSS encoding conjugation functions also contributes to the distribution of these plasmids within P. syringae populations. Thus, this study contributes to unravel the genetic bases of the role of PFPs in different P. syringae lifestyles. This work was supported by grants Proyecto de Excelencia, Junta de Andalucía (P07-AGR-02471; P12-AGR-1473) and by Michigan State University AgBioResearch.This work was supported by grants Proyecto de Excelencia, Junta de Andalucía (P07-AGR-02471; P12-AGR-1473) and by Michigan State University AgBioResearch; Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

A novel gene cluster involved in high tolerance to copper compounds has been detected in Pseudomonas syringae pv. syringae

Pseudomonas syringae pv. syringae (Pss), is the most polyphagous bacterium of the P. syringae complex, based on its wide host range, that severely affects woody and herbaceous plants. Pss has been described as the causal agent of bacterial apical necrosis (BAN) on mango crops, an important threat that drastically reduces the mango fruit yield in the Mediterranean region. Copper compounds have traditionally been used as standard bactericides to manage woody plant diseases, being the spraying of copper compound known as Bordeaux mixture (BM), the conventional treatment for controlling BAN disease. The extensive use of copper compounds can lead to many problems, and among them, the emergence of copper-resistant strains is one of the most serious. Pss strains isolated from mango trees harbour at least, two different genetic structures (copABCD and copABCD modified) encoded by plasmids that are involved in copper detoxification. Determination of the minimum inhibitory concentration of copper, together with a phylogenetic distribution analysis performed in a collection of Pss strains isolated from mango trees revealed a phylogenetic group of Pss strongly associated with high levels of copper resistance. Genome mining and functional characterization of one Pss strain present in this group have uncovered a novel gene cluster of chromosomal location that could be playing a major role in high levels of copper resistance during the epiphytic survival of Pss on mango tree surfaces.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Análisis comparativo de dos colecciones de pseudo monas syringae pv. syringae asociadas a mango

Pseudomonas syringae es una bacteria ubicua a nivel ambiental. Presenta una taxonomía compleja en la que se han descrito más de 60 patovares que causan enfermedad en muchas especies de plantas diferentes. Entre los patovares más importantes se encuentra el pv. syringae debido a su amplio rango de hospedador, y al arsenal de mecanismos de virulencia que posee. A principios de los años 90, se describió a Pseudomonas syringae pv. syringae (Pss) como el agente causal de la necrosis apical del mango (NAM). Pss aisladas de mango muestran características importantes para su biología, tanto en sus mecanismos de virulencia como para el fitness epifítico. En este trabajo, se plantea conocer en profundidad cómo han evolucionado las poblaciones de Pss a lo largo del tiempo sobre el mangoUniversidad de Málaga. Campus de Excelencia Internacional Andalucía Tech. Este trabajo ha sido financiado por ayuda de la Consejería de Economía, Innovación y Ciencia, Junta de Andalucía (Pl2-AGR-1473), cofinanciados con fondos FEDER (UE

Pseudomonas syringae pv. syringae Associated With Mango Trees, a Particular Pathogen Within the “Hodgepodge” of the Pseudomonas syringae Complex

The Pseudomonas syringae complex comprises different genetic groups that include strains from both agricultural and environmental habitats. This complex group has been used for decades as a “hodgepodge,” including many taxonomically related species. More than 60 pathovars of P. syringae have been described based on distinct host ranges and disease symptoms they cause. These pathovars cause disease relying on an array of virulence mechanisms. However, P. syringae pv. syringae (Pss) is the most polyphagous bacterium in the P. syringae complex, based on its wide host range, that primarily affects woody and herbaceous host plants. In early 1990s, bacterial apical necrosis (BAN) of mango trees, a critical disease elicited by Pss in Southern Spain was described for the first time. Pss exhibits important epiphytic traits and virulence factors, which may promote its survival and pathogenicity in mango trees and in other plant hosts. Over more than two decades, Pss strains isolated from mango trees have been comprehensively investigated to elucidate the mechanisms that governs their epiphytic and pathogenic lifestyles. In particular, the vast majority of Pss strains isolated from mango trees produce an antimetabolite toxin, called mangotoxin, whose leading role in virulence has been clearly demonstrated. Moreover, phenotypic, genetic and phylogenetic approaches support that Pss strains producers of BAN symptoms on mango trees all belong to a single phylotype within phylogroup 2, are adapted to the mango host, and produce mangotoxin. Remarkably, a genome sequencing project of the Pss model strain UMAF0158 revealed the presence of other factors that may play major roles in its different lifestyles, such as the presence of two different type III secretion systems, two type VI secretion systems and an operon for cellulose biosynthesis. The role of cellulose in increasing mango leaf colonization and biofilm formation, and impairing virulence of Pss, suggests that cellulose may play a pivotal role with regards to the balance of its different lifestyles. In addition, 62-kb plasmids belonging to the pPT23A-family of plasmids (PFPs) have been strongly associated with Pss strains that inhabit mango trees. Further, complete sequence and comparative genomic analyses revealed major roles of PFPs in detoxification of copper compounds and ultraviolet radiation resistance, both improving the epiphytic lifestyle of Pss on mango surfaces. Hence, in this review we summarize the research that has been conducted on Pss by our research group to elucidate the molecular mechanisms that underpin the epiphytic and pathogenic lifestyle on mango trees. Finally, future directions in this particular plant–pathogen story are discussed

Aislamiento de cepas de Pseudomonas syringae pv. syringae asociadas a mango para su uso en análisis evolutivos y epidemiológicos

Comunicación oralLa necrosis apical del mango (NAM) es una enfermedad que se ha observado en el litoral andaluz desde la implantación de este cultivo, y cuyo agente causal es la bacteria Pseudomonas syringae pv. syringae (Pss). Esta enfermedad aparece principalmente asociada a climas con inviernos frescos y húmedos, tal y como ocurre en la cuenca mediterránea, donde, además de en España, se ha descrito en otros países (Israel, Portugal, Italia, Egipto), así como en otras áreas de cultivo con clima similar, como el Noroeste de Australia. Las cepas de Pss aisladas de mango muestran características importantes para su biología, tanto a nivel de virulencia como de fitness epifítico. En trabajos previos se han descrito diferentes genes implicados en el desarrollo de los síntomas de la enfermedad, así como en aumentar la capacidad de la bacteria de sobrevivir y persistir sobre el tejido vegetal; el operón mbo implicado en la producción de mangotoxina, los genes copABCD o cusCBA, implicados en la capacidad de resistencia al cobre, o el operón wss, responsable de la producción de celulosa. Por otro lado, gracias a análisis filogenéticos, se ha podido agrupar a todas las cepas de Pss aisladas de mango y productoras de mangotoxinas en el filotipo I. El objetivo de este trabajo es el análisis fenético comparado de cepas de Pss aisladas de mango de las diferentes zonas de estudio (España, Portugal, Italia, Australia), antes del año 2000 (colección I) y disponibles en nuestro laboratorio, con aislamientos actuales (2016-2018, Colección II). Este estudio se ha iniciado con la caracterización y selección previa de cepas representativas de cada una de las colecciones, para iniciar un abordaje en detalle y comparación de los diferentes atributos.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Contribution of mangotoxin to the virulence and epiphytic fitness of Pseudomonas syringae pv. syringae

Mangotoxin is an antimetabolite toxin that inhibits ornithine acetyl transferase, a key enzyme in the biosynthetic pathway of ornithine and arginine and recently reported in strains of Pseudomonas syringae pv. syringae (Pss) isolated from mango. Since symptoms on mango tissues are very difficult to reproduce, in this study the role of mangotoxin in Pss virulence was addressed by analyzing the in planta growth and development of disease symptoms on tomato leaflets. Inoculation experiments were carried out following several procedures using the wild-type strain Pss UMAF0158, two Tn5-mutant derivative strains defective in mangotoxin production, and their complemented derivative strains in which mangotoxin production is restored. The ability of the mangotoxin-defective mutants to grow in planta was similar, and their epiphytic survival on the tomato leaf surface identical to the wild-type and complemented strains. However, both the disease index data of incidence and the severity of necrotic symptoms indicated that mangotoxin-defective mutants were less virulent, indicating that mangotoxin is a virulence factor. Furthermore, competition experiments showed that the survival values of the wild-type strain were slightly but significantly higher than those of the mangotoxin-defective mutants, suggesting that mangotoxin production would improve the epiphytic fitness of Pss. [Int Microbiol 2009; 12(2):87-95

A Psl-like polysaccharide has a key role in the biofilm architecture of two plant-associated Pseudomonas

Comunicación congresoThe Psl polysaccharide has only been studied in Pseudomonas aeruginosa, and its role in bacteria that interact with plants is still unknown. On the one hand, P. syringae is a model of study of plant-pathogen interactions. On the other hand, P. chlororaphis is a biocontrol agent of plant-fungal diseases, as the white root rot caused by Rosellinia necatrix. In silico analysis have let us identify in both species a genomic region which is ortholog to the Psl-encoding region of P. aeruginosa PAO1. We constructed mutants of this region in both strains and analysed its phenotype in biofilm formation, both in static microwell plates and dynamic flow-cell chamber experiments. The results revealed a different and an important role of this polysaccharide in the biofilm architecture of both strains. Furthermore, in P. syringae this Psl-like polysaccharide has a very important role in swarming motility. All these striking phenotypes led us further study the implication of this polysaccharide in the lifestyles of both models of plant-bacteria interaction.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech. AGL2017-83368-C2-1-