Ralstonia Solanacearum Species Complex Research Articles

Calcium modulation of bacterial wilt disease on potato.

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.

Pathogenic and Comparative Genomic Analysis of Ralstonia pseudosolanacearum Isolated from Casuarina.

Casuarina equisetifolia is crucial in protecting coastal regions of China against typhoon attacks, but has faced a substantial challenge due to wilt disease caused by pathogens of the Ralstonia solanacearum species complex (RSSC). Although the initial outbreak of Casuarina wilt in 1970s was effectively controlled by disease-resistant C. equisetifolia varieties, the disease has recently re-emerged in coastal regions of Guangdong. In this study, we report the isolation, characterization, and comparative genomic analysis of 11 RSSC strains from diseased C. equisetifolia at various locations along the coast of Guangdong. Phylogenomic analysis showed that the strains were closely related and clustered with phylotype I strains previously isolated from peanuts. Single-gene based analysis further suggested these strains could be derived from strains present in Guangdong since the 1980s, indicating a historical context to their current pathogenicity. Casuarina-isolated strains exhibited notably higher virulence against C. equisetifolia and peanuts than representative RSSC strains GMI1000 and EP1, suggesting host-specific adaptations which possibly contributed to the recent outbreak. Comparative genomic analysis among RSSC strains revealed a largely conserved genome structure and high levels of conservation in gene clusters encoding extracellular polysaccharides biosynthesis, secretion systems, and quorum sensing regulatory systems. However, we also found a number of unique genes in the Casuarina-isolated strains that were absent in GMI1000 and EP1, and vice versa, pointing to potential genetic factors underpinning their differential virulence. These unique genes offer promising targets for future functional studies. Overall, our findings provide crucial insights into the RSSC pathogens causing Casuarina wilt in Guangdong, guiding future efforts in disease control and prevention.

RSSC-Lineage Multiplex PCR Assay Detects and Differentiates Ralstonia solanacearum, R. pseudosolanacearum, R. syzygii, and the R3bv2 Subgroup

Bacterial wilt strains in the Ralstonia solanacearum species complex (RSSC) pose serious threats to economically important crops worldwide. In 2014, Safni et al. proposed the reclassification of the RSSC into three genomospecies: R. solanacearum (Rsol), R. pseudosolanacearum (Rpseu), and R. syzygii (Rsyz). The revision requires the proper identification of strains for diagnostic and epidemiological studies. In response, we developed the inexpensive and user-friendly RSSC-Lineage Multiplex PCR, which effectively detects plant-pathogenic Ralstonia strains in general and also distinguishes between Rpseu, Rsol, Rsyz, and the high-security Select Agent “race 3 biovar 2” subgroup of Rsol, also known as the phylotype IIB-1 potato brown rot pandemic lineage. Genomes were retrieved from the NCBI GenBank database and screened for unique gene regions using OrthoMCL and other comparative genomic approaches. Specific primers were designed for each genomospecies, Ralstonia in general, and “race 3 biovar 2.” AT-rich flaps were added at the 5ʹ position of each primer to optimize the reaction thermodynamics. The specificity was tested in silico using the NCBI GenBank genome database and an in-house database. The in vitro specificity and accuracy of the tool was validated with 113 representative Ralstonia strains and 24 strains from other genera. The assay is highly specific, generating neither false positives nor false negatives. Primer set detection limits ranged from 10 to 100 pg. The assay also detected and differentiated strains from naturally and artificially inoculated plant hosts. This tool is highly specific, reliable, and economical for culture characterization, diagnostics, surveys, quarantine decisions, and epidemiological studies. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Genetic Diversity of Ralstonia solanacearum Causes Tobacco Bacterial Wilt in Fujian Province and Identification of Biocontrol Streptomyces sp.

Tobacco bacterial wilt is a highly destructive soil-borne disease caused by Ralstonia solanacearum species complex (RSSC), exhibiting a significant risk to global flue-cured tobacco cultivation, resulting in substantial economic loss. Here, 77 isolates were collected from covering three prominent flue-cured tobacco cultivation areas in Fujian, China (Nanping, Sanming, and Longyan) in 2021 and 2022. The isolated strains were classified through phylotype-specific multiplex polymerase chain reaction (Pmx-PCR) and physiological tests. The analysis showed that all the strains were associated with phylotype Ⅰ, race 1, and biovar Ⅲ. Subsequent phylogenetic analysis using partial egl gene sequences classified the 77 isolates into 5 distinct sequevars, 13, 15, 16, 17, and 34. Notably, a remarkable predominance of sequevar 15 was observed in Fujian Province. while sequevar 16 was first reported on tobacco in China which was identified in other plants, expanding the understanding of its host range and distribution in the country. Additionally, a Streptomyces strain extracted from the rhizosphere soil of tobacco was found to inhibit the growth of multiple sequevars of tobacco R. solanacearum, indicating its broad-spectrum antagonistic properties. Furthermore, pot experiments showed that strain St35 effectively controlled tobacco bacterial wilt. The isolate St35 was conclusively identified as Streptomyces gancidicus according to the morphological and genetic features. In summary, the present study demonstrated the genetic diversity and distribution of tobacco R. solanacearum strains in Fujian Province of China, as well as the identification of a candidate biological control agent for the management of tobacco bacterial wilt.

Characterization of Ralstonia pseudosolanacearum Diversity and Screening Tomato, Pepper, and Eggplant Resistance to Manage Bacterial Wilt in South Asia

In South Asia, bacterial wilt pathogens in the Ralstonia solanacearum species complex (RSSC) impose major constraints on eggplant, tomato, and pepper production. To improve the efficacy of bacterial wilt management, the goals of this study were to (i) conduct a survey of RSSC pathogens in Bangladesh and Nepal, (ii) characterize the genetic diversity of these isolates, and (iii) screen 37 tomato, eggplant, and pepper accessions for resistance to six representative isolates from South Asia. We isolated 99 isolates from Bangladesh and 20 isolates from Nepal and determined that all are phylotype I isolates of the Ralstonia pseudosolanacearum species. We sequenced and assembled draft genomes for 25 isolates. Phylogenomic analyses suggest that there is a wide diversity of endemic phylotype I isolates in South Asia and possible introductions of two clonal phylotype I lineages into Bangladesh and Nepal. We contextualize our newly described isolates based on prior reports of RSSC diversity in South Asia and global reports of RSSC pathogens on eggplant and pepper. Greenhouse trials revealed multiple tomato, eggplant, and pepper accessions that exhibit promising levels of resistance to six phylotype I isolates from South Asia. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

CRISPRals: a web database for assessing CRISPR defense system in Ralstonia solanacearum species complex to avoid phage resistance.

CRISPR has been widely characterized as a defense system against phages and other invading elements in bacteria and archaea. A low percentage of Ralstonia solanacearum species complex (RSSC) strains possess the CRISPR array and the CRISPR-associated proteins (Cas) that would confer immunity against various phages. In order to provide a wide-range screen of the CRISPR presence in RSSC, we analyzed 378 genomes of RSSC strains to find the CRISPR locus. We found that 20.1%, 14.3%, and 54.5% of the R. solanacearum, R. pseudosolanacearum, and R. syzygii strains respectively possess the CRISPR locus. In addition, we performed further analysis to identify the respective phages that are restricted by the CRISPR arrays. We found 252 different phages infecting different strains of RSSC, by means of the identification of similarities between the protospacers in phages and spacers in bacteria. We compiled this information in a database with web access called CRISPRals (https://crisprals.yachaytech.edu.ec/). Additionally, we made available a number of tools to detect and identify CRISPR array and Cas genes in genomic sequences that could be uploaded by users. Finally, a matching tool to relate bacteria spacer with phage protospacer sequences is available. CRISPRals is a valuable resource for the scientific community that contributes to the study of bacteria-phage interaction and a starting point that will help to design efficient phage therapy strategies.

Comparative genomics and host range analysis of four Ralstonia pseudosolanacearum strains isolated from sunflower reveals genomic and phenotypic differences.

Bacterial wilt caused by Ralstonia solanacearum species complex (RSSC) is one of the devastating diseases in crop production, seriously reducing the yield of crops. R. pseudosolanacearum, is known for its broad infrasubspecific diversity and comprises 36 sequevars that are currently known. Previous studies found that R. pseudosolanacearum contained four sequevars (13, 14, 17 and 54) isolated from sunflowers sown in the same field. Here, we provided the complete genomes and the results of genome comparison of the four sequevars strains (RS639, RS642, RS647, and RS650). Four strains showed different pathogenicities to the same cultivars and different host ranges. Their genome sizes were about 5.84 ~ 5.94Mb, encoding 5002 ~ 5079 genes and the average G + C content of 66.85% ~ 67%. Among the coding genes, 146 ~ 159 specific gene families (contained 150 ~ 160 genes) were found in the chromosomes and 34 ~ 77specific gene families (contained 34 ~ 78 genes) in the megaplasmids from four strains. The average nucleotide identify (ANI) values between any two strains ranged from 99.05% ~ 99.71%, and the proportion of the total base length of collinear blocks accounts for the total gene length of corresponding genome was all more than 93.82%. Then, we performed a search for genomic islands, prophage sequences, the gene clusters macromolecular secretion systems, type III secreted effectors and other virulence factors in these strains, which provided detailed comparison results of their presence and distinctive features compared to the reference strain GMI1000. Among them, the number and types of T2SS gene clusters were different in the four strains, among which RS650 included all five types. T4SS gene cluster of RS639 and RS647 were missed. In the T6SS gene cluster, several genes were inserted in the RS639, RS647, and RS650, and gene deletion was also detected in the RS642. A total of 78 kinds of type III secreted effectors were found, which included 52 core and 9 specific effectors in four strains. This study not only provided the complete genomes of multiple R. pseudosolanacearum strains isolated from a new host, but also revealed the differences in their genomic levels through comparative genomics. Furthermore, these findings expand human knowledge about the range of hosts that Ralstonia can infect, and potentially contribute to exploring rules and factors of the genetic evolution and analyzing its pathogenic mechanism.

Draft genomes of two phylotype I and II Ralstonia solanacearum species complex (RSSC) isolates causing bacterial wilt in tomato plants from Costa Rica.

Bacteria from RSSC hold agricultural significance as they are the causal agents of bacterial wilt. Here, we report the draft genomes of two bacteria extracted from vascular tissues of infected tomato plants. Isolate RALF-MA was classified as Ralstonia pseudosolanacearum (phylotype I) and RALSA-MA as Ralstonia solanacearum (phylotype II).

Molecular identification of the causal agent of bacterial wilt in tomato plants in southwestern Paraná

Bacterial wilt, caused by phytopathogenic species of the genus Ralstonia, is one of the main diseases affecting the tomato crop. The Ralstonia solanacearum species complex occurs as a result of variants being widely diverse in terms of adaptation to different climatic conditions, host variations, and aggressiveness, which complicate disease control recommendations. In this study, we employed molecular methods to analyze 26 R. solanacearum isolates collected from tomato plants cultivated in the southwest region of Paraná, Brazil. Isolates were obtained from plants exhibiting wilt symptoms in a protected cultivation system and in an open field. The specific primers 759/760 confirmed the isolates as part of the Ralstonia solanacearum complex, and Nmult primers were used to identify the phylotype. Variability analysis using BOX-PCR with the BOX-A1R primer on 19 isolates revealed molecular diversity. All 26 isolates were confirmed as Ralstonia solanacearum, belonging to phylotype II. Comparison of genomic DNA band patterns amplified by BOX-PCR indicated molecular variability, forming eight groups at a similarity level of 0.63. These results confirm the prevalence of R. solanacearum phylotype II in southwestern Paraná. This information aids decision-making in disease management and contributes to breeding efforts aiming at the development of resistant cultivars.

Taxonomic profiling of Nasutitermes takasagoensis microbiota to investigate the role of termites as vectors of bacteria linked to ironwood tree decline in Guam.

The ironwood tree (Casuarina equisetifolia, family Casuarinaceae), an indigenous agroforestry species in Guam, has been threatened by ironwood tree decline (IWTD) since 2002. Formation of bacterial ooze by the wilt pathogen from the Ralstonia solanacearum species complex and wetwood bacteria (primarily Klebsiella species) has been linked to IWTD. In addition, termite infestation of trees was statistically associated with IWTD. Termites are known carriers of a diverse microbiome. Therefore, we hypothesized that termites could be vectors of bacteria linked to IWTD. To investigate the potential role of termites as pathogen vectors, we employed next-generation 16S rRNA gene sequencing to describe the bacteria diversity of Nasutitermes takasagoensis (Family Termitidae) workers collected from 42 ironwood trees of different disease stages in Guam in association with tree-, plot-, and location-related factors. Nasutitermes takasagoensis workers account for the majority of termite infestations of ironwood trees. The bacterial phyla composition of N. takasagoensis workers was typical for wood-feeding higher termites consisting mainly of Spirochaetes and Fibrobacteres. However, Ralstonia species were not detected and Klebsiella species were rare even in termites collected from trees infected with Ralstonia and wetwood bacteria. Feeding experiments suggested that termites prefer to consume wood with low pathogen content over wood with high pathogen load. Termites were able to ingest Ralstonia but Ralstonia could not establish itself in healthy termite bodies. We concluded that N. takasagoensis workers are not vectors for Ralstonia spp. or the bacterial endophytes associated with wetwood (Klebsiella, Pantoea, Enterobacter, Citrobacter, and Erwinia) that were previously observed in IWTD-infested trees. The bacterial diversity in termite samples was significantly influenced by various factors, including Tree Health, Site Management, Plot Average Decline Severity, Proportion of Dead Trees in the Plot, Proportion of Trees with Termite Damage in the Plot, Presence of Ralstonia, and Altitude.

Genome Resource Announcement of 3 Phylotype I and 14 Phylotype II Ralstonia solanacearum Species Complex Isolates from South America

The Ralstonia solanacearum species complex is a group of gram-negative bacteria that cause lethal wilting on a broad range of plant species. Ralstonia pathogens were responsible for the Moko disease epidemic in plantains, bananas, and Heliconia sp. in Central and South America in the 1960s. To investigate the genomic diversity of Ralstonia strains isolated from Central and South America during this epidemic, we conducted Illumina whole-genome sequencing on 17 Ralstonia isolates. Our phylogenetic analysis showed that three of the isolates were phylotype I strains and 14 isolates were phylotype IIB strains. This genome announcement provides new genomic resources to investigate pathogen evolution, epidemiology, and virulence. [Formula: see text] The author(s) have dedicated the work to the public domain under the Creative Commons CC0 “No Rights Reserved” license by waiving all of his or her rights to the work worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law, 2023.

The Ralstonia Research Community Rejects the Proposal to Classify Phylotype I Ralstonia into the New Species Ralstonia nicotianae

The Ralstonia solanacearum species complex is a group of globally important plant pathogens. Bacteria in this very large and genetically diverse group all colonize the xylem elements of angiosperm plants and cause high-impact wilting diseases of many crops. Because they threaten economic and food security, several R. solanacearum species complex subgroups are strictly regulated as quarantine pests. Biologically meaningful and consistent nomenclature is essential for organisms that have major economic and regulatory importance, such as plant-pathogenic Ralstonia. There are currently three species of Ralstonia wilt pathogens: R. pseudosolanacearum (corresponding to two phylogenetic groups that are described in the literature as phylotypes I and III), R. solanacearum (phylotypes IIA, IIB, and IIC), and R. syzygii (phylotype IV, containing three subspecies: subsp. syzygii, subsp. celebensis, and subsp. indonesiensis). A recent paper proposed reclassifying phylotype I as a new species named “ Ralstonia nicotianae.” The purpose of this commentary is to register our objection to the taxon “ Ralstonia nicotianae.” [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY 4.0 International license .

Differentiation of Cool-Virulent Strains in the Ralstonia solanacearum Species Complex by Melt Curve of a DNA Fragment from Effector Gene Rips1

The Ralstonia solanacearum species complex ( Rssc) causes bacterial wilt diseases across hundreds of plant species. Phylogenetic analysis of DNA sequences has allowed the grouping of Rssc strains into phylotypes based on genetic relatedness. Although specific phenotypes are associated with corresponding phylotypes, the molecular bases for these differences are not always well understood. The complex trait of cool-virulence that allows several members of phylotype II to cause disease at temperatures below 25°C is one of these poorly understood traits. Here, we report that a 96-bp DNA fragment from a gene encoding the type III secretion system effector protein RipS1, which was recently reported to play a role in the cool-virulence phenotype, has a melting temperature that varies based on the strain of origin. By testing 81 Rssc strains with melt-curve analysis, we found a bimodal distribution with clear separation between the group of strains with a melting temperature below 87.5°C that includes phylotypes I, III, and IV strains and the group with a melting temperature above 87.5°C that includes all 35 tested select agent race 3 biovar 2 (phylotype IIB sequevars 1 and 2) and seven phylotype IIB sequevar 4 strains, previously known to possess cool-virulence capability, with a few exceptions. This provides further support that sequence differences in this effector differentiate cool-virulent strains from non-cool-virulent strains and show potential for use as a marker in continued studies of the cool-virulence phenotype. [Formula: see text] The author(s) have dedicated the work to the public domain under the Creative Commons CC0 “No Rights Reserved” license by waiving all of his or her rights to the work worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law, 2023.

Loop-Mediated Isothermal Amplification Assays for Rapid Detection of Ralstonia solanacearum Species Complex and Phylotype I in Solanaceous Crops

The Ralstonia solanacearum species complex (RSSC) causes bacterial wilt diseases, which affect a wide range of plant hosts, including Solanaceae, and is of major economic importance. Point-of-care (POC) techniques such as loop-mediated isothermal amplification (LAMP) enable the rapid and sensitive detection of plant pathogens and can be deployed directly in the field. For fast and reliable diagnosis on site, we optimized an RSSC-LAMP assay and developed a real-time LAMP assay targeting the phylotype I. Both LAMPs were highly specific, yielding negative results for a wide collection of nontarget strains and positive results for all target strains, except for two particular mulberry strains for the phylotype I assay. These results were supported by an extensive in silico analysis performed on 6,105 genomes of Burkholderiaceae. The two LAMP assays displayed high sensitivity on pure suspensions, with a detection limit at 103 and 104 CFU/ml for phylotype I and the RSSC, respectively. These thresholds correspond to theoretical quantities of as low as 5 and 50 CFU per reaction. Two simplified extraction methods were successfully used to detect the pathogen from different Solanaceae samples. We demonstrated that LAMP assays were operable on solanaceous crops during field surveys and varietal evaluation trials as rapid POC diagnostics tools, which can improve the disease management and control of the RSSC. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

A Mutation of a Putative NDP-Sugar Epimerase Gene in Ralstonia pseudosolanacearum Attenuates Exopolysaccharide Production and Bacterial Virulence in Tomato Plant.

Ralstonia solanacearum species complex (RSSC) is a soil borne plant pathogen causing bacterial wilt on various important crops, including Solanaceae plants. The bacterial pathogens within the RSSC produce exopolysaccharide (EPS), a highly complicated nitrogen-containing heteropolymeric polysaccharide, as a major virulence factor. However, the biosynthetic pathway of the EPS in the RSSC has not been fully characterized. To identify genes in EPS production beyond the EPS biosynthetic gene operon, we selected the EPS-defective mutants of R. pseudosolanacearum strain SL341 from Tn5-inserted mutant pool. Among several EPS-defective mutants, we identified a mutant, SL341P4, with a Tn5-insertion in a gene encoding a putative NDP-sugar epimerase, a putative membrane protein with sugar-modifying moiety, in a reverse orientation to EPS biosynthesis gene cluster. This protein showed similar to other NDP-sugar epimerases involved in EPS biosynthesis in many phytopathogens. Mutation of the NDP-sugar epimerase gene reduced EPS production and biofilm formation in R. pseudosolanacearum. Additionally, the SL341P4 mutant exhibited reduced disease severity and incidence of bacterial wilt in tomato plants compared to the wild-type SL341 without alteration of bacterial multiplication. These results indicate that the NDP-sugar epimerase gene is required for EPS production and bacterial virulence in R. pseudosolanacearum.

Analysis of changes in bacterial diversity in healthy and bacterial wilt mulberry samples using metagenomic sequencing and culture-dependent approaches.

Mulberry bacterial wilt is a serious destructive soil-borne disease caused by a complex and diverse group of pathogenic bacteria. Given that the bacterial wilt has been reported to cause a serious damage to the yield and quality of mulberry, therefore, elucidation of its main pathogenic groups is essential in improving our understanding of this disease and for the development of its potential control measures. In this study, combined metagenomic sequencing and culture-dependent approaches were used to investigate the microbiome of healthy and bacterial wilt mulberry samples. The results showed that the healthy samples had higher bacterial diversity compared to the diseased samples. Meanwhile, the proportion of opportunistic pathogenic and drug-resistant bacterial flora represented by Acinetobacter in the diseased samples was increased, while the proportion of beneficial bacterial flora represented by Proteobacteria was decreased. Ralstonia solanacearum species complex (RSSC), Enterobacter cloacae complex (ECC), Klebsiella pneumoniae, K. quasipneumoniae, K. michiganensis, K. oxytoca, and P. ananatis emerged as the main pathogens of the mulberry bacterial wilt. In conclusion, this study provides a valuable reference for further focused research on the bacterial wilt of mulberry and other plants.

First Report of Bacterial Wilt of Eggplant (Solanum melongena) Caused by Ralstonia pseudosolanacearum in Mexico.

Bacterial wilt caused by the Ralstonia solanacearum species complex (RSSC) is a major disease of solanaceous crops worldwide. In May 2022, symptoms of wilting, yellowing, and reduced growth were observed on eggplant (Solanum melongena) cv. Barcelona in a commercial greenhouse located in Culiacán, Sinaloa, Mexico. The disease incidence was recorded up to 30%. Sections of stems from diseased plants showed discoloration of the vascular tissue and the pith. Colonies with typical RSSC morphology were isolated from five eggplant stems on Petri plates containing casamino acid-peptone-glucose (CPG) medium supplemented with 1% 2,3,5-triphenyltetrazolium chloride (TZC), and incubated at 25°C for 48-h (Schaad et al. 2001; Garcia et al. 2019). On CPG medium + TZC, white and irregular colonies with pinkish centers were observed. On King's B medium, mucoid and white colonies were produced. The strains were Gram-negative in the KOH test and were nonfluorescent on King's B medium. Strains were positive using commercial Rs ImmunoStrip® (Agdia, USA). For molecular identification, DNA was extracted, and the partial endoglucanase gene (egl) was amplified by PCR and sequenced using the primer pair Endo-F/Endo-R (Fegan and Prior 2005). BLASTn searches showed 100% identity with available sequences of R. pseudosolanacearum from Musa sp. in Colombia (MW016967) and from Eucalyptus pellita in Indonesia (MW748363, MW748376, MW748377, MW748379, MW748380, MW748382). To confirm the bacterial identity, DNA was amplified with the primers 759/760 (Opina et al. 1997) and Nmult21:1F/Nmult22:RR (Fegan and Prior 2005) to generate 280 and 144-bp amplicons for RSSC and phylotype I (= R. pseudosolanacearum), respectively. A phylogenetic analysis was performed using the Maximum Likelihood method and the strain was distinguished as R. pseudosolanacearum sequevar 14. The strain (CCLF369) is currently preserved in the Culture Collection of the Research Center for Food and Development (Culiacán, Sinaloa, Mexico) and the sequence was deposited in GenBank (accession number OQ559102). Pathogenicity tests were performed by injection of 20-μl of a bacterial suspension (108 CFU/ml) at the base of the stem of five eggplants cv. Barcelona. Five plants inoculated with sterile distilled water were used as control. Plants were kept in a greenhouse at 28/37°C (night/day) for 12 days. All inoculated plants exhibited wilting, chlorosis, and necrosis of leaves between 8 and 11 days after inoculation, whereas control plants remained asymptomatic. The bacterial strain was only isolated from symptomatic plants and confirmed to be R. pseudosolanacearum using the molecular techniques mentioned above, fulfilling Koch´s postulates. Ralstonia pseudosolanacearum has been previously reported to cause bacterial wilt of tomato in Sinaloa, Mexico (García-Estrada et al. 2023); however, to our knowledge, this is the first report of R. pseudosolanacearum infecting eggplant in Mexico. Further studies on epidemiology and management strategies for this disease are required on vegetable crops in Mexico.

Ralstonia solanacearum: An Arsenal of Virulence Strategies and Prospects for Resistance.

The group of strains constituting the Ralstonia solanacearum species complex (RSSC) is a prominent model for the study of plant-pathogenic bacteria because of its impact on agriculture, owing to its wide host range, worldwide distribution, and long persistence in the environment. RSSC strains have led to numerous studies aimed at deciphering the molecular bases of virulence, and many biological functions and mechanisms have been described to contribute to host infection and pathogenesis. In this review, we put into perspective recent advances in our understanding of virulence in RSSC strains, both in terms of the inventory of functions that participate in this process and their evolutionary dynamics. We also present the different strategies that have been developed to combat these pathogenic strains through biological control, antimicrobial agents, plant genetics, or microbiota engineering.

Influence of insertion sequences on population structure of phytopathogenic bacteria in the Ralstonia solanacearum species complex.

Ralstonia solanacearum species complex (RSSC) is a destructive group of plant pathogenic bacteria and the causative agent of bacterial wilt disease. Experimental studies have attributed RSSC virulence to insertion sequences (IS), transposable genetic elements which can both disrupt and activate host genes. Yet, the global diversity and distribution of RSSC IS are unknown. In this study, IS were bioinformatically identified in a diverse collection of 356 RSSC isolates representing five phylogenetic lineages and their diversity investigated based on genetic distance measures and comparisons with the ISFinder database. IS phylogenetic associations were determined based on their distribution across the RSSC phylogeny. Moreover, IS positions within genomes were characterised and their potential gene disruptions determined based on IS proximity to coding sequences. In total, we found 24732 IS belonging to eleven IS families and 26 IS subgroups with over half of the IS found in the megaplasmid. While IS families were generally widespread across the RSSC phylogeny, IS subgroups showed strong lineage-specific distributions and genetically similar bacterial isolates had similar IS contents. Similar associations with bacterial host genetic background were also observed with IS insertion positions which were highly conserved in closely related bacterial isolates. Finally, IS were found to disrupt genes with predicted functions in virulence, stress tolerance, and metabolism suggesting that they might be adaptive. This study highlights that RSSC insertion sequences track the evolution of their bacterial hosts potentially contributing to both intra- and inter-lineage genetic diversity.

First Report of Ralstonia pseudosolanacearum Causing Wilt Disease in Tomato (Solanum lycopersicum L.) Plants from Mexico.

Mexico produces more than four million tons of tomato fruits and ranks tenth worldwide. In February 2022, tomato plants in a greenhouse in Culiacan, Sinaloa State, were affected by wilt diseases with an incidence of 20% and irreversible wilt and death of the infected plants (severity up 70%). When cut stems from affected plants, a reddish to brown discoloration of the vascular system was observed and these were disinfected with 1% NaClO for 5 min and then placed in a humid chamber. Characteristic milky-white exudate was obtained. From that exudate, irregular, mucoid, and white colonies with pink centres were obtained on casamino peptone glucose (CPG) plates supplemented with 1% 2,3,5-triphenyl 15 tetrazolium chloride (TZC); these characteristics are typical of the Ralstonia solanacearum species complex (RSSC) (Garcia et al., 2019). Identification of the pathogen was done by PCR using specific primer pairs reported by Paudel et al. (2022), RssC-wF3 (5'-TATATATCCTCGACTTTTCCATGAAGCTGTG-3') - RssCwR3 (5'-CTATATATATACCCCACTTGTTGAGGAACTG-3') and Rpseu-wF5 (5'-TTTTATTTTTTTGGTGTCCGGGCCAAGATAG-3') - Rpseu-wR5 (5'- TTATATTACTCGAACGTGCTGCAAAACCACT-3'), which amplified fragments of 162 and 251 bp for RSSC and Ralstonia pseudosolanacearum, respectively. Additionally, 759 (5'-GTCGCCGTCAACTCACTTTCC-3') - 760 (5'-GTCGCCGTCAGCAATGCGGAATCG-3') (Opina, et al., 1997) and Nmult21:1F (5'-CGTTGATGAGGCGCGCAATTT-3') - Nmult22:RR (5'- TCGCTTGACCCTATAACGAGTA-3') (Fegan and Prior, 2005) were used to generate 282 and 144 bp amplicons for RSSC and phylotype I, respectively. Subsequen to making the specific detection, the representative strain ClnMx was used to generate a sequence for the endoglucanase (egl) gene for separation into sequevars by using the primers Endo-F (5'- ATGCATGCCGCTGGTCGCCGC-3') and Endo-R (5'-GCGTTGCCCGGCACGAACACC-3'), which amplified a fragment of 750 bp (Fegan et al., 1998). The egl sequence (GenBank Access ON542479) showed 100% identity with the well-defined R. pseudosolanacearum sequevar 14, which was isolated from tomato plants from Senegal (UW763, I-14 GenBank Access CP051174) (Steidl et al., 2021), as well as, the strain MAFF 301070 (GenBank Access AB508612) from Japanese tomato. For pathogenicity tests, four 1-month-old tomato plants were infected using an insulin syringe that contained a pure bacterial suspension with approximately 2x108 CFU/mL. For each plant, 20 µL was infiltrated into the axil of the third upper leaf, and for untreated controls, tomato plants were infiltrated with sterile water. All plants were kept at 28°C under greenhouse conditions. Symptoms resembling those observed in the field were observed in inoculated plants six days after inoculation, and the plant pathogen was recovered on TZC medium. To confirm the bacteria identification a PCR using the specific primer pairs mentioned early was carried out. In contrast, water-treated control plants remained healthy. Koch's postulates were carried out twice with similar results. Ralstonia solanacearum species complex (RSSC) causes severe economic losses in many countries of the world because of their capability to infect a wide range of host plants, including potato, tomato, eggplant, tobacco, and, banana, among others. Ralstonia pseudosolanacearum has been reported to cause tomato wilt disease mainly on the Afro-Eurasian continent in areas such as Senegal, Cambodia, and Japan (Klass et al., 2019). To our knowledge, this is the first report of R. pseudosolanacearum causing bacterial wilt diseases in tomato plants from Mexico and because, the control of this bacteria is a challenge by the long survival time in soil, water, and infected plant tissues, the identification of this important pathogen could provide relevant information for developing management strategies.