Brenneria quercina and Serratia spp. isolated from Spanish oak trees Molecular characterization and development of PCR primers

Brenneria quercina has been reported as one of the causal agents of oak decline in Spain. To investigate the bacterial variability of this pathogen from different Spanish oak forests, a collection of 38 bacterial isolates from seven geographic locations and from different oak species was analysed by sequencing 16S rDNA and rep-PCR fingerprinting. All Spanish isolates of B. quercina were grouped by rep-PCR into a homogenous cluster that differed significantly from B. quercina reference strains from California. 16S rDNA analysis revealed that 34 out of 38 isolates were Brenneria. However, four isolates belonged to the genus Serratia, suggesting that this bacterium could cause cankers in oak t…

Survival Strategy of Erwinia amylovora against Copper: Induction of the Viable-but-Nonculturable State

Copper compounds, widely used to control plant-pathogenic bacteria, have traditionally been employed against fire blight, caused by Erwinia amylovora. However, recent studies have shown that some phytopathogenic bacteria enter into the viable-but-nonculturable (VBNC) state in the presence of copper. To determine whether copper kills E. amylovora or induces the VBNC state, a mineral medium without copper or supplemented with 0.005, 0.01, or 0.05 mM Cu2+ was inoculated with 107 CFU/ml of this bacterium and monitored over 9 months. Total and viable cell counts were determined by epifluorescence microscopy using the LIVE/DEAD kit and by flow cytometry with 5-cyano-2,3-ditolyl tetrazolium chlori…

Erwinia piriflorinigrans sp. nov., a novel pathogen that causes necrosis of pear blossoms

Eight Erwinia strains, isolated from necrotic pear blossoms in València, Spain, were compared with reference strains of Erwinia amylovora and Erwinia pyrifoliae, both of which are pathogenic to species of pear tree, and to other species of the family Enterobacteriaceae using a polyphasic approach. Phenotypic analyses clustered the novel isolates into one phenon, distinct from other species of the genus Erwinia, showing that the novel isolates constituted a homogeneous phenotypic group. Rep-PCR profiles, PCR products obtained with different pairs of primers and plasmid contents determined by restriction analysis showed differences between the novel strains and reference strains of E. amylovo…

Influence of native microbiota on survival of Ralstonia solanacearum phylotype II in river water microcosms.

ABSTRACT Ralstonia solanacearum phylotype II biovar 2 causes bacterial wilt in solanaceous hosts, producing severe economic losses worldwide. Waterways can be major dissemination routes of this pathogen, which is able to survive for long periods in sterilized water. However, little is known about its survival in natural water when other microorganisms, such as bacteriophages, other bacteria, and protozoa, are present. This study looks into the fate of a Spanish strain of R. solanacearum inoculated in water microcosms from a Spanish river, containing different microbiota fractions, at 24°C and 14°C, for a month. At both temperatures, R. solanacearum densities remained constant at the initial…

Choice of CTO scores to predict procedural success in clinical practice. A comparison of 4 different CTO PCI scores in a comprehensive national registry including expert and learning CTO operators

Background We aimed to compare the performance of the recent CASTLE score to J-CTO, CL and PROGRESS CTO scores in a comprehensive database of percutaneous coronary intervention of chronic total occlusion procedures. Methods Scores were calculated using raw data from 1,342 chronic total occlusion procedures included in REBECO Registry that includes learning and expert operators. Calibration, discrimination and reclassification were evaluated and compared. Results Mean score values were: CASTLE 1.60±1.10, J-CTO 2.15±1.24, PROGRESS 1.68±0.94 and CL 2.52±1.52 points. The overall percutaneous coronary intervention success rate was 77.8%. Calibration was good for CASTLE and CL, but not for J-CTO…

Survival of Erwinia amylovora in rain water at low temperatures

E.G. Biosca , R.D. Santander, M. Ordax, E. Marco-Noales, B. Aguila, A. Flores and M.M. Lopez 1 Universidad de Valencia, Departamento de Microbiologia y Ecologia, Avenida Dr. Moliner 50, 46100, Burjassot, Valencia, Spain. 2 Instituto Valenciano de Investigaciones Agrarias (IVIA), Centro de Proteccion Vegetal y Biotecnologia, Carretera Moncada – Naquera, km 4.5, 46113, Moncada,Valencia, Spain. 3 Universidad de Valencia, Servicio Central de Soporte a la Investigacion Experimental, Avenida Dr. Moliner 50, 46100, Burjassot, Valencia, Spain.

Seasonal Variation of Ralstonia solanacearum Biovar 2 Populations in a Spanish River: Recovery of Stressed Cells at Low Temperatures

ABSTRACT The presence of Ralstonia solanacearum biovar 2 in the watercourses of European countries is increasing, but little is known about its ecology in aquatic habitats. The detection of this pathogen in 2000 in one Spanish river led us to study its population density at different locations on the river over a period of 3 years. During 2000 and 2001, the pathogen was recovered at low densities (10 to 80 CFU/ml) by direct plating on modified SMSA agar from water samples at 14°C or higher, but its isolation was usually unsuccessful at temperatures below 9°C. To monitor the pathogen's abundance in winter, we used two liquid selective media for enrichment (at 29 and 35°C) and compared them b…

Survival of Erwinia amylovora in mature apple fruit calyces through the viable but nonculturable (VBNC) state

Aims:  Survival of Erwinia amylovora, causal agent of fire blight in pome fruits and other rosaceous plants, was monitored inside mature apples calyces under some storage conditions utilized in fruit. Methods and Results:  Apple fruit calyces inoculated with two E. amylovora strains and their respective GFP-marked strains were maintained at 26° and 5°C, and the effect of copper treatment was assayed at 0·01 and 0·1 mmol l−1 CuSO4. In nontreated apples at 26°C, part of the population of E. amylovora survived in the ‘viable but nonculturable’ (VBNC) state, whereas at 5°C the majority of the population retained culturability. In copper-treated apples, the whole population adopted the VBNC stat…

Exopolysaccharides favor the survival of Erwinia amylovora under copper stress through different strategies.

Erwinia amylovora causes fire blight, a destructive disease of rosaceous plants very difficult to control. We demonstrated that copper, employed to control plant diseases, induces the "viable-but-nonculturable" (VBNC) state in E. amylovora. Moreover, it was previously reported that copper increases production of its main exopolysaccharide (EPS), amylovoran. In this work, the copper-complexing ability of amylovoran and levan, other major EPS of E. amylovora, was demonstrated. Following this, EPS-deficient mutants were used to determine the role of these EPSs in survival of this bacterium in AB mineral medium with copper, compared to their wild type strain and AB without copper. Tot…

Medfly Ceratitis capitata as Potential Vector for Fire Blight Pathogen Erwinia amylovora: Survival and Transmission

Monitoring the ability of bacterial plant pathogens to survive in insects is required for elucidating unknown aspects of their epidemiology and for designing appropriate control strategies. Erwinia amylovora is a plant pathogenic bacterium that causes fire blight, a devastating disease in apple and pear commercial orchards. Studies on fire blight spread by insects have mainly focused on pollinating agents, such as honeybees. However, the Mediterranean fruit fly (medfly) Ceratitis capitata (Diptera: Tephritidae), one of the most damaging fruit pests worldwide, is also common in pome fruit orchards. The main objective of the study was to investigate whether E. amylovora can survive and be tra…

Exploring diversity among Spanish strains of Erwinia amylovora and possible infection sources

Aims:  We have examined the intraspecific diversity of a collection of 63 Spanish strains of Erwinia amylovora, isolated from 1995 to 2001, to determine whether or not they could be grouped based on phenotypic or genotypic criteria and to investigate the sources of inoculum for fire blight dissemination in Spain. Methods and Results:  Several biochemical and molecular techniques, such as miniaturized API 20E, API 50CH, ATB G-5 and API-ZYM tests, BIOLOG metabolic fingerprinting, PCR ribotyping, pulsed-field gel electrophoresis (PFGE), minisatellite-primed PCR (MSP-PCR), random amplified polymorphic DNA (RAPD) analyses and AFLP were used. We report the first identification in Spain of the PFG…

In silico evaluation of molecular probes for detection and identification of Ralstonia solanacearum and Clavibacter michiganensis subsp. sepedonicus.

Ralstonia solanacerum and Clavibacter michiganensis subsp. sepedonicus are the two most relevant bacterial pathogens of potato for which a large number of molecular diagnostic methods using specific DNA sequences have been developed. About one hundred oligonucleotides have been described and thoroughly tested experimentally. After having compiled and evaluated all these primers and probes in silico to check their specificity, many discrepancies were found. A detailed analysis permitted the recognition of different possible reasons for such discrepancies: sequencing errors in public sequences, wrong supposed specificity (sometimes due to more recent sequences than the oligonucleoticles being…

Survival strategies and pathogenicity of Ralstonia solanacearum phylotype II subjected to prolonged starvation in environmental water microcosms

Survival strategies exhibited over 4 years by Ralstonia solanacearum phylotype (ph) II biovar (bv) 2 in environmental water microcosms were examined. The bacterium is a devastating phytopathogen whose ph II bv 2 causes bacterial wilt in solanaceous crops and ornamental plants. Outbreaks of the disease may originate from dissemination of the pathogen in watercourses, where it has to cope with prolonged nutrient limitation. To ascertain the effect of long-term starvation on survival and pathogenicity of R. solanacearum in natural water microcosms, survival experiments were conducted. Microcosms were prepared from different sterile river water samples, inoculated separately with two European s…

Biocontrol of the Major Plant Pathogen Ralstonia solanacearum in Irrigation Water and Host Plants by Novel Waterborne Lytic Bacteriophages

Three new lytic bacteriophages were found to effectively control the pathogen Ralstonia solanacearum, a quarantine bacterium in many countries, and causative agent of bacterial wilt, one of the most important vascular plant diseases. Bacterial wilt management has been carried out with fluctuating effects, suggesting the need to find alternative treatments. In this work, three lytic phages were isolated from environmental water from geographically distant regions in Spain. They proved to specifically infect a collection of R. solanacearum strains, and some of the closely related pathogenic species Ralstonia pseudosolanacearum, without affecting non-target environmental bacteria, and were abl…

Transcriptional response of Erwinia amylovora to copper shock: in vivo role of the copA gene

Fire blight is a devastating plant disease caused by the bacterium Erwinia amylovora, and its control is frequently based on the use of copper-based compounds whose mechanisms of action are not well known. Consequently, in this article, we investigate the response of E. amylovora to copper shock by a whole-genome microarray approach. Transcriptional analyses showed that, in the presence of copper, 23 genes were increased in expression; these genes were classified mainly into the transport and stress functional categories. Among them, the copA gene was strongly induced and regulated in a finely tuned manner by copper. Mutation of copA, soxS, arcB, yjcE, ygcF, yhhQ, galF and EAM_3469 genes re…

Innovation in bacteriophage-based biocontrol of the plant pathogen ralstonia solanacearum through irrigation water