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Ralstonia solanacearum species complex (RSSC) is a phytopathogenic bacterial group that causes bacterial wilt in several crops, being potato (Solanum tuberosum) one of the most important hosts. The relationship between the potato plant ionome (mineral and trace elements composition) and the resistance levels to this pathogen has not been addressed until now. Mineral content of xylem sap, roots, stems and leaves of potato genotypes with different levels of resistance to bacterial wilt was assessed in this work, revealing a positive correlation between divalent calcium (Ca) cation concentrations and genotype resistance. The aim of this study was to investigate the effect of Ca on bacterial wilt resistance, and on the growth and virulence of RSSC. Ca supplementation significantly decreased the growth rate of Ralstonia pseudosolanacearum GMI1000 in minimal medium and affected several virulence traits such as biofilm formation and twitching motility. We also incorporate for the first time the use of microfluidic chambers to follow the pathogen growth and biofilm formation in conditions mimicking the plant vascular system. By using this approach, a reduction in biofilm formation was observed when both, rich and minimal media, were supplemented with Ca. Assessment of the effect of Ca amendments on bacterial wilt progress in potato genotypes revealed a significant delay in disease progress, or a complete absence of wilting symptoms in the case of partially resistant genotypes. This work contributes to the understanding of Ca effect on virulence of this important pathogen and provides new strategies for an integrated control of bacterial wilt on potato. IMPORTANCE: Ralstonia solanacearum species complex (RSSC) includes a diverse group of bacterial strains that cause bacterial wilt. This disease is difficult to control due to pathogen aggressiveness, persistence, wide range of hosts, and wide geographic distribution in tropical, subtropical, and temperate regions. RSSC causes considerable losses depending on the pathogen strain, host, soil type, environmental conditions, and cultural practices. In potato, losses of $19 billion per year have been estimated for this pathogen worldwide. In this study, we report for the first time the mineral composition found in xylem sap and plant tissues of potato germplasm with different levels of resistance to bacterial wilt. This study underscores the crucial role of calcium (Ca) concentration in the xylem sap and stem in relation to the resistance of different genotypes. Our in vitro experiments provide evidence of Ca's inhibitory effect on the growth, biofilm formation, and twitching movement of the model RSSC strain R. pseudosolanacearum GMI1000. This study introduces a novel element, the Ca concentration, which should be included into the integrated disease control management strategies for bacterial wilt in potatoes.

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The bacterial wilt disease caused by Ralstonia pseudosolanacearum presents a notable economic risk to a variety of crucial crops worldwide. During preliminary isolation of this phytopathogen, several colonies of other saprophytic bacteria may be mistaken with it. So, the present study aims to address this issue by proposing the application of immunogenic proteins, particularly flagellin (FliC), to enable a rapid and early identification of bacterial wilt. In this study, a novel approach is unveiled for the early detection of R. pseudosolanacearum. The study exploits the immunogenic attributes of flagellin (FliC), by generating polyclonal antibodies against recombinant FliC within model organisms-rabbits and mice. The efficacy of these antibodies is meticulously assessed through discerning techniques, including DAS-ELISA and Western blot analyses, which elucidate their remarkable specificity in identifying various R. pseudosolanacearum strains. Furthermore, the introduction of antibody-coated latex agglutinating reagents offers an additional layer of confirmation, substantiating the feasibility of establishing a laboratory-based toolkit for swift screening and unambiguous identification of the bacterial wilt pathogen. This study presents a significant stride toward enhancing early diagnostic capabilities, potentially revolutionizing agricultural practices by safeguarding crop yield and quality through proactive pathogen detection and mitigation strategies.

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Bacterial wilt, caused by the Ralstonia solanacearum species complex (RSSC), is the most destructive potato disease in Kenya. Studies were conducted to (i) determine the molecular diversity of RSSC strains associated with bacterial wilt of potato in Kenya, (ii) generate an RSSC distribution map for epidemiological inference, and (iii) determine whether phylotype II sequevar 1 strains exhibit epidemic clonality. Surveys were conducted in 2018 and 2019, in which tubers from wilting potato plants and stem samples of potential alternative hosts were collected for pathogen isolation. The pathogen was phylotyped by multiplex PCR and 536 RSSC strains typed at a sequevar level. Two RSSC phylotypes were identified, phylotype II (98.4%, n = 506 [sequevar 1 (n = 505) and sequevar 2 (n = 1)]) and phylotype I (1.6%, n = 30 [sequevar 13 (n = 9) and a new sequevar (n = 21)]). The phylotype II sequevar 1 strains were haplotyped using multilocus tandem repeat sequence typing (TRST) schemes. The TRST scheme identified 51 TRST profiles within the phylotype II sequevar 1 strains with a modest diversity index (HGDI = 0.87), confirming the epidemic clonality of RSSC phylotype II sequevar 1 strains in Kenya. A minimum spanning tree and mapping of the TRST profiles revealed that TRST27 '8-5-12-7-5' is the primary founder of the clonal complex of RSSC phylotype II sequevar 1 and is widely distributed via latently infected seed tubers. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

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Filamentous bacteriophages contain a single-stranded DNA genome and have a peculiar lifestyle, since they do not cause host cell lysis, but establish a persistent association with the host, often causing behavioral changes, with effects on bacterial ecology. Over the years, a gradual reduction in the incidence of bacterial wilt has been observed in some fields from Brazil. This event, which has been associated with the loss of pathogenicity of Rasltonia spp. isolates due to infection by filamentous viruses of the inovirus group, is widely reported for Ralstonia spp. Asian isolates infected by inoviruses. In an attempt to elucidate which factors are associated with the phenomenon reported in Brazil, we investigated one isolate of R. solanacearum (UB-2014), with unusual characteristics for R. solanacearum, obtained from eggplant with mild wilt symptoms. To verify if the presence of filamentous bacteriophage was related to this phenotype, we performed viral purification and nucleic acid extraction. The phage genome was sequenced, and phylogenetic analyses demonstrated that the virus belongs to the family Inoviridae and was named as Ralstonia solanacerarum inovirus Brazil 1 (RSIBR1). RSIBR1 was transmitted to R. pseudosolanacearum GMI1000, and the virus-infected GMI1000 (GMI1000 VI) isolate showed alterations in phenotypic characteristics, as well as loss of pathogenicity, similarly to that observed in R. solanacearum isolate UB-2014. The presence of virus-infected UB-2014 and GMI1000 VI plants without symptoms, after 3 months, confirms that the infected isolates can colonize the plant without causing disease, which demonstrates that the phage infection changed the behavior of these pathogens.

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Ralstonia pseudosolanacearum GMI1000 (Rpso GMI1000) is a soil-borne vascular phytopathogen that infects host plants through the root system causing wilting disease in a wide range of agro-economic interest crops, producing economical losses. Several features contribute to the full bacterial virulence. In this work we study the participation of light, an important environmental factor, in the regulation of the physiological attributes and infectivity of Rpso GMI1000. In silico analysis of the Rpso genome revealed the presence of a Rsp0254 gene, which encodes a putative blue light LOV-type photoreceptor. We constructed a mutant strain of Rpso lacking the LOV protein and found that the loss of this protein and light, influenced characteristics involved in the pathogenicity process such as motility, adhesion and the biofilms development, which allows the successful host plant colonization, rendering bacterial wilt. This protein could be involved in the adaptive responses to environmental changes. We demonstrated that light sensing and the LOV protein, would be used as a location signal in the host plant, to regulate the expression of several virulence factors, in a time and tissue dependent way. Consequently, bacteria could use an external signal and Rpsolov gene to know their location within plant tissue during the colonization process.

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The first report of Ralstonia mannitolilytica bacteremia in Peru is presented. The patient was a pediatric cancer patient with a long-term central venous access device. For the diagnosis, the MicroScan Walk Away 96 automated system was used. 16S rDNA was amplified by conventional PCR, and the bacterial genus and species were identified by genetic sequencing. In addition, the bacterial resistance profile to major antimicrobials was determined. The article discusses the need to actively monitor Ralstonia mannitolilytica, especially in hospital areas of immunocompromised patients.

Se presenta el primer reporte de una bacteriemia por Ralstonia mannitolilytica en Perú. Se trata de un paciente pediátrico con cáncer que porta un dispositivo de acceso venoso central de larga duración. Para establecer el diagnóstico, se utilizó el sistema automático MicroScan Walk Away 96. Se amplificó el rADN 16S mediante PCR convencional y se identificó el género y la especie bacteriana mediante secuenciación genética. Además, se determinó el perfil de resistencia bacteriana a los principales antimicrobianos. El artículo discute la necesidad de monitorizar activamente la presencia de Ralstonia mannitolilytica, especialmente en áreas hospitalarias de pacientes inmunodeprimidos.

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Abstract Objective: to describe an experience in identification and control of an outbreak of Ralstonia spp. in a renal unit. Material and Method: an epidemiological investigation of a hospital outbreak in 2 sites and extramural service of a renal unit. The investigation included patients who presented fever or chills, during or after dialysis, and who had positive blood culture for Ralstonia spp. Results: Of 769 hemodialysis patients, 124 were identified with bacteremia by Ralstonia spp.; of these, 98.4% had catheter access and 1.6% had fistula. The overall attack rate was 16.1% and the case fatality rate was 0.8%. Environmental cultures were taken and drugs and devices were tracked. Several cultures were taken of the prefilled heparin following the methods described in the International Pharmacopoeia. However, it was the technique of microbial isolation recommended by experts that enabled the isolation of the microorganism and confirmed the source. Conclusions: The outbreak described exceeded the number of patients affected documented in literature. It was caused by a contaminated batch of heparin. Evidence is provided of a recommended by expert technique used for the isolation of Ralstonia spp. in order to achieve control of outbreaks in a timely manner, minimizing clinical, economic, and social impact.

Resumen Objetivo: describir la experiencia en la identificación y control de un brote por Ralstonia spp. en una unidad renal. Material y Método: investigación epidemiológica de brote hospitalario en 2 sedes y servicio extramural de una unidad renal. Se incluyeron pacientes que presentaron fiebre o escalofrío, durante o después de la terapia dialítica, y que tuvieran hemocultivo positivo para Ralstonia spp. Resultados: De los 709 pacientes para hemodiálisis, se identificaron 124 con bacteriemia por Ralstonia spp., 98,4% tenían acceso por catéter. La tasa de ataque global fue del 16,1% y la tasa de letalidad 0,8%. Se realizaron cultivos ambientales y trazabilidad de medicamentos y dispositivos, pero ante la presencia de casos extramurales la hipótesis fue redireccionada. La heparina prellenada había sido cultivada en varias oportunidades siguiendo la metodología de la farmacopea internacional. Sin embargo, la técnica de aislamiento microbiano recomendada por expertos fue la que permitió aislar el microorganismo y confirmar la fuente. Conclusiones: El brote que se describe excedió el número de pacientes documentados en la literatura y fue causado por un lote contaminado de heparina. Se aporta evidencia de una técnica recomendada por expertos utilizada para el aislamiento de Ralstonia spp. a fin de lograr el control de brotes de manera oportuna, minimizando el impacto clínico, económico y social.

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Biofouling causes major problems and economic losses to marine and shipping industries. In the search for new antifouling agents, marine bacteria with biosurfactants production capability can be an excellent option, due to the amphipathic surface-active characteristic that confers antimicrobial and antibiofilm activities. The aim of this study was to evaluate the antifouling activity of biosurfactants producing marine bacteria from the Gulf of California. The cell free culture supernatant (CFCS) of Bacillus niabensis (S-69), Ralstonia sp. (S-74) (isolated from marine sediment) and of B. niabensis (My-30) (bacteria associated to the sponge Mycale ramulosa) were screened for production of biosurfactants (using hemolysis and drop collapse test, oil displacement and emulsifying activity). The toxicity and antifouling activity were evaluated against biofoulers (bacteria forming biofilm and macrofoulers) both in laboratory and field assays. The results indicate that all bacteria were biosurfactant producers, but the higher capability was shown by B. niabensis (My-30) with high emulsifying properties (E24) of 71%. The CFCS showed moderate toxicity but were considered non-toxic against Artemia franciscana at low concentrations. In the antifouling assay, the CFCS of both strains of B. niabensis showed the best results for the reduction of the biofilm formation (up 50%) against all Gram-positive bacteria and most Gram-negative bacteria with low concentrations. In the field assay, the CFCS of B. niabensis (My-30) led to the reduction of 30% of biofouling compared to the control. The results indicate that the biosurfactant produced by B. niabensis (My-30) has promising antifouling activity.

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The extensive genetic diversity of Ralstonia solanacearum, a serious soil-borne phytopathogen, has led to the concept that R. solanacearum encompasses a species complex [R. solanacearum species complex (RSSC)]. Insertion sequences (ISs) are suggested to play an important role in the genome evolution of this pathogen. Here, we identified and analysed transposable elements (TEs), ISs and transposons, in 106 RSSC genomes and 15 Ralstonia spp. We mapped 10 259 IS elements in the complete genome of 62 representative RSSC strains and closely related Ralstonia spp. A unique set of 20 IS families was widespread across the strains, IS5 and IS3 being the most abundant. Our results showed six novel transposon sequences belonging to the Tn3 family carrying passenger genes encoding antibiotic resistance and avirulence proteins. In addition, internal rearrangement events associated with ISs were demonstrated in Ralstonia pseudosolanacearum strains. We also mapped IS elements interrupting avirulence genes, which provided evidence that ISs plays an important role in virulence evolution of RSSC. Additionally, the activity of ISs was demonstrated by transcriptome analysis and DNA hybridization in R. solanacearum isolates. Altogether, we have provided collective data of TEs in RSSC genomes, opening a new path for understanding their evolutionary impact on the genome evolution and diversity of this important plant pathogen.

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Bacterial wilt-causing Ralstonia threaten numerous crops throughout the world. We studied the population structure of 196 isolates of Ralstonia solanacearum and 39 isolates of Ralstonia pseudosolanacearum, which were collected from potato- and tomato-growing areas in 19 states of Brazil. Regardless of the species, three groups of isolates were identified. One group encompassed R. pseudosolanacearum isolates. The other two groups comprise isolates of R. solanacearum (phylotype II) split according to geographic regions, one made of isolates from the North and Northeast and the other made of isolates from the Central, Southeast, and South regions (CSS). Among the isolates collected in CSS, those from tomato were genetically distinct from the potato isolates. The genetic variability in the population of R. pseudosolanacearum was lower than that of R. solanacearum, suggesting that the former was introduced in Brazil. Conversely, the high genetic variability of R. solanacearum in all regions, hosts, and times supports the hypothesis that this species is autochthonous in South America, more precisely in Brazil and Peru. For R. solanacearum, higher variability and lower migration rates were observed when tomato isolates were analyzed, indicating that the variability is caused mainly by the differences of the local, native soil population. The North subpopulation was distinct from all others, possibly because of differences in environmental features of this region. The proximity of some geographic regions and the movement of potato tubers could have facilitated migration and therefore low genetic differentiation between geographic regions. Finally, geography, which also influences host distribution, affects the structure of the population of R. solanacearum in Brazil. Despite quarantine procedures in Brazil, increasing levels of trade are a threat to biosecurity, and these results emphasize the need for improving our regional efforts to prevent the dispersal of pathogens.

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