Development of TaqMan Real-Time PCR protocols for simultaneous detection and quantification of the bacterial pathogen ralstonia solanacearum and their specific lytic bacteriophages

Ralstonia solanacearum is the causal agent of bacterial wilt, one of the most destructive diseases of solanaceous plants, affecting staple crops worldwide. The bacterium survives in water, soil, and other reservoirs, and is difficult to control. In this sense, the use of three specific lytic R. solanacearum bacteriophages was recently patented for bacterial wilt biocontrol in environmental water and in plants. To optimize their applications, the phages and the bacterium need to be accurately monitored and quantified, which is laborious and time-consuming with biological methods. In this work, primers and TaqMan probes were designed, and duplex and multiplex real-time quantitative PCR (qPCR) protocols were developed and optimized for the simultaneous quantification of R. solanacearum and their phages. The quantification range was established from 108 to 10 PFU/mL for the phages and from 108 to 102 CFU/mL for R. solanacearum. Additionally, the multiplex qPCR protocol was validated for the detection and quantification of the phages with a limit ranging from 102 targets/mL in water and plant extracts to 103 targets/g in soil, and the target bacterium with a limit ranging from 103 targets/mL in water and plant extracts to 104 targets/g in soil, using direct methods of sample preparation

Ralstonia solanacearum Facing Spread-Determining Climatic Temperatures, Sustained Starvation, and Naturally Induced Resuscitation of Viable but Non-Culturable Cells in EnvironmentalWater

Ralstonia solanacearum is a bacterial phytopathogen affecting staple crops, originally from tropical and subtropical areas, whose ability to survive in temperate environments is of concern under global warming. In this study, two R. solanacearum strains from either cold or warm habitats were stressed by simultaneous exposure to natural oligotrophy at low (4°C), temperate (14°C), or warm (24°C) temperatures in environmental water. At 4°C, the effect of temperature was higher than that of oligotrophy, since R. solanacearum went into a viable but non-culturable (VBNC) state, which proved to be dependent on water nutrient contents. Resuscitation was demonstrated in vitro and in planta. At 14°C and 24°C, the effect of oligotrophy was higher than that of temperature on R. solanacearum populations, displaying starvation-survival responses and morphological changes which were stronger at 24°C. In tomato plants, starved, cold-induced VBNC, and/or resuscitated cells maintained virulence. The strains behaved similarly regardless of their cold or warm areas of origin. This work firstly describes the natural nutrient availability of environmental water favoring R. solanacearum survival, adaptations, and resuscitation in conditions that can be found in natural settings. These findings will contribute to anticipate the ability of R. solanacearum to spread, establish, and induce disease in new geographical and climatic areas

Genomic analysis of the first european bacteriophages with depolymerase activity and biocontrol efficacy against the phytopathogen Ralstonia solanacearum

Ralstonia solanacearum is the causative agent of bacterial wilt, one of the most destructive plant diseases. While chemical control has an environmental impact, biological control strategies can allow sustainable agrosystems. Three lytic bacteriophages (phages) of R. solanacearum with biocontrol capacity in environmental water and plants were isolated from river water in Europe but not fully analysed, their genomic characterization being fundamental to understand their biology. In this work, the phage genomes were sequenced and subjected to bioinformatic analysis. The morphology was also observed by electron microscopy. Phylogenetic analyses were performed with a selection of phages able to infect R. solanacearum and the closely related phytopathogenic species R. pseudosolanacearum. The results indicated that the genomes of vRsoP-WF2, vRsoP-WM2 and vRsoP-WR2 range from 40,688 to 41,158 bp with almost 59% GC-contents, 52 ORFs in vRsoP-WF2 and vRsoP-WM2, and 53 in vRsoP-WR2 but, with only 22 or 23 predicted proteins with functional homologs in databases. Among them, two lysins and one exopolysaccharide (EPS) depolymerase, this type of depolymerase being identified in R. solanacearum phages for the first time. These three European phages belong to the same novel species within the Gyeongsanvirus, Autographiviridae family (formerly Podoviridae). These genomic data will contribute to a better understanding of the abilities of these phages to damage host cells and, consequently, to an improvement in the biological control of R. solanacearum

Genetic diversity reflects geographical origin of Ralstonia solanacearum strains isolated from plant and water sources in Spain

The characterization and intraspecific diversity of a collection of 45 Ralstonia solanacearum strains isolated in Spain from different sources and geographical origins is reported. To test the influence of the site and the host on strain diversity, phenotypic and genotypic analysis were performed by a polyphasic approach. Biochemical and metabolic profiles were compared. Serological relationship was evaluated by Indirect-ELISA using polyclonal and monoclonal antibodies. For genotypic analysis, hrpB and egl DNA sequence analysis, repetitive sequences (rep-PCR), amplified fragment length polymorphism (AFLP) profiles and macrorestriction with XbaI followed by pulsed field gel electrophoresis (PFGE) were performed.The biochemical and metabolic characterization, serological tests, rep-PCR typing and phylogenetic analysis showed that all analysed strains belonged to phylotype II sequevar 1 and shared homogeneous profiles. However, interesting differences among strains were found by AFLP and macrorestriction with XbaI followed by PFGE techniques, some profiles being related to the geographical origin of the strains. Diversity results obtained offer new insights into the biogeography of this quarantine organism and its possible sources and reservoirs in Spain and Mediterranean countries.Keywords: Bacterial wilt · potato · soil · PFGE · AFL

Exploring new roles for the rpoS gene in the survival and virulence of the fire blight pathogen Erwinia amylovora

Erwinia amylovora causes fire blight in economically important plants of the family Rosaceae. This bacterial pathogen spends part of its life cycle coping with starvation and other fluctuating environmental conditions. In many Gram-negative bacteria, starvation and other stress responses are regulated by the sigma factor RpoS. We obtained an E. amylovora rpoS mutant to explore the role of this gene in starvation responses and its potential implication in other processes not yet studied in this pathogen. Results showed that E. amylovora needs rpoS to develop normal starvation survival and viable but nonculturable (VBNC) responses. Furthermore, this gene contributed to stationary phase cross-protection against oxidative, osmotic, and acid stresses and was essential for cross-protection against heat shock, but nonessential against acid shock. RpoS also mediated regulation of motility, exopolysaccharide synthesis, and virulence in immature loquats, but not in pear plantlets, and contributed to E. amylovora survival in nonhost tissues during incompatible interactions. Our results reveal some unique roles for the rpoS gene in E. amylovora and provide new knowledge on the regulation of different processes related to its ecology, including survival in different environments and virulence in immature fruits

Caracterización genómica y filogenética de bacteriófagos líticos de Ralstonia solanacearum con capacidad de biocontrol en agua medioambiental y en planta

Ralstonia solanacearum, agente etiológico de la marchitez bacteriana, es una de las bacterias fitopatógenas más devastadoras. Los virus bacteriófagos (fagos) que la infectan pueden ser agentes prometedores de control biológico que permitan agrosistemas sostenibles. Tres fagos líticos de R. solanacearum, vRsoP-WF2, vRsoP-WM2 y vRsoP-WR2, aislados de agua de distintos ríos españoles han demostrado su capacidad de biocontrol del patógeno tanto en agua medioambiental como en planta, siendo necesaria su caracterización genómica para profundizar en el conocimiento de su biología. En este trabajo, se secuenciaron y realizaron análisis bioinformáticos de los genomas completos de los tres fagos, así como análisis filogenéticos con una selección de fagos activos frente a R. solanacearum y a la especie fitopatógena estrechamente relacionada R. pseudosolanacearum

Distribución de Brenneria spp. en la Comunidad Valenciana y especies forestales a las que afecta

El género Brenneria agrupa especies bacterianas que producen chancros con lesiones necróticas y exudados en plantas leñosas. En España, en los últimos años, se han identificado varias Brenneria sp. como responsables de chancros bacterianos en quercíneas (B. quercina), nogales (B. nigrifluens y B. rubrifaciens) y chopos (Brenneria sp.). En la Comunidad Valenciana también se han detectado focos de B. quercina en encinas de algunos parajes de importancia medioambiental, pero todavía se dispone de escasa información acerca de la incidencia de esta bacteriosis en nuestra Comunidad. Sin embargo, no existen referencias de la presencia de Brenneria spp. en nogal o chopo en la Comunidad Valenciana, aunque no se puede descartar su presencia o introducción a través de material vegetal. El objetivo del presente trabajo ha sido conocer la incidencia de dichas bacteriosis en nuestra Comunidad. Para ello, se han continuado las prospecciones en masas de quercíneas de distintos orígenes geográficos de la Comunidad Valenciana iniciadas en un estudio anterior, y se han centrado en aquellas zonas o especies del género Quercus en las que la bacteria no había sido detectada previamente. Además, se han iniciado prospecciones en otras especies forestales de interés para nuestra Comunidad, como Juglans regia y Populus spp

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 transmitted by the medfly. Our experimental results show: i) E. amylovora can survive for at least 8 days inside the digestive tract of the medfly and until 28 days on its external surface, and ii) medflies are able to transmit the bacteria from inoculated apples to both detached shoots and pear plants, being the pathogen recovered from lesions in both cases. This is the first report on E. amylovora internalization and survival in/on C. capitata, as well as the experimental transmission of the fire blight pathogen by this insect. Our results suggest that medfly can act as a potential vector for E. amylovora, and expand our knowledge on the possible role of these and other insects in its life cycle

Bacterias antagonistas de Erwinia amylovora aisladas de microbiota asociada a plantas: cribado y caracterización

El control de las enfermedades bacterianas, como el fuego bacteriano de las rosáceas causado por Erwinia amylovora, requiere la búsqueda de estrategias de gestión eficaces y eco-sostenibles, como la utilización de agentes de biocontrol. En este trabajo presentamos un cribado de aislados bacterianos, recuperados de diversos nichos asociados a las plantas y orígenes geográficos, como una primera etapa de un programa de búsqueda y selección de agentes de biocontrol de E. amylovora. Se obtuvo una colección de aislados, con los que se evaluó su antagonismo in vitro frente a E. amylovora y su capacidad de reducir la incidencia y severidad del fuego bacteriano ex vivo, tanto en frutos inmaduros como en brotes, así como el retraso en la aparición de los mismos

ECOPHAGE: Combating Antimicrobial Resistance Using Bacteriophages for Eco-Sustainable Agriculture and Food Systems

The focus of this meeting was to discuss the suitability of using bacteriophages as alternative antimicrobials in the agrifood sector. Following a One Health approach, the workshop explored the possibilities of implementing phage application strategies in the agriculture, animal husbandry, aquaculture, and food production sectors. Therefore, the meeting had gathered phage researchers, representatives of the agrifood industry, and policymakers to debate the advantages and potential shortcomings of using bacteriophages as alternatives to traditional antimicrobials and chemical pesticides. Industry delegates showed the latest objectives and demands from consumers. Representatives of regulatory agencies (European Medicines Agency (EMA) and Spanish Agency of Medicines and Health Products (AEMPS)) presented an update of new regulatory aspects that will impact and support the approval and implementation of phage application strategies across the different sectors