Ralstonia Solanacearum Species Complex Strains Research Articles

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.

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.

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.

The Ralstonia pseudosolanacearum effector RipE1 is recognized at the plasma membrane by NbPtr1, the Nicotiana benthamiana homologue of Pseudomonas tomato race 1.

The bacterial wilt disease caused by soilborne bacteria of the Ralstonia solanacearum species complex (RSSC) threatens important crops worldwide. Only a few immune receptors conferring resistance to this devastating disease are known so far. Individual RSSC strains deliver around 70 different type III secretion system effectors into host cells to manipulate the plant physiology. RipE1 is an effector conserved across the RSSC and triggers immune responses in the model solanaceous plant Nicotiana benthamiana. Here, we used multiplexed virus-induced gene silencing of the nucleotide-binding and leucine-rich repeat receptor family to identify the genetic basis of RipE1 recognition. Specific silencing of the N. benthamiana homologue of Solanum lycopersicoides Ptr1 (confers resistance to Pseudomonas syringae pv. tomato race 1) gene (NbPtr1) completely abolished RipE1-induced hypersensitive response and immunity to Ralstonia pseudosolanacearum. The expression of the native NbPtr1 coding sequence was sufficient to restore RipE1 recognition in Nb-ptr1 knockout plants. Interestingly, RipE1 association with the host cell plasma membrane was necessary for NbPtr1-dependent recognition. Furthermore, NbPtr1-dependent recognition of RipE1 natural variants is polymorphic, providing additional evidence for the indirect mode of activation of NbPtr1. Altogether, this work supports NbPtr1 relevance for resistance to bacterial wilt disease in Solanaceae.

Development and Validation of E-Probes with the MiFi System for Detection of Ralstonia solanacearum Species Complex in Blueberries

The blueberry industry, a fast-growing industry in Florida, is threatened by bacterial wilt, caused by the Ralstonia solanacearum species complex (RSSC). In 2016, distinct RSSC populations were found in Florida causing disease in blueberry. Currently, there are no tools that discriminate between RSSC populations pathogenic to blueberry and other RSSC strains. Early detection of specific RSSC strains is critical, and the Microbe Finder (MiFi) web application is a sensitive and specific tool used to detect known target pathogens in sequenced samples. MiFi is used to develop e-probes and subsequently query sample metagenomes with them. We report the development and validation of e-probes for three different RSSC populations pathogenic to blueberry using the MiFi platform. We also present preliminary data to assess the feasibility of a cost-effective integrated diagnostics system, MiFi, in combination with MinION, a pocket-sized, portable sequencer. The e-probes were validated in silico, generating simulated metagenomes; in vitro by using blueberry DNA spiked with pathogen DNA; and in vivo with soil and stem samples. We tested several methods of DNA extraction for soil and blueberry stems and several MinION libraries with or without barcodes. MiFi blueberry e-probes effectively identified target pathogens at a very specific level with high sensitivity. The MiFi system combined with MinION sequencing using Flongle cells was optimized to produce results in one day. Further optimization and tests can improve greatly the cost effectiveness and flexibility of this system by adding e-probes to test for new blueberry strains and many other pathogens affecting blueberry in one test. [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 .

The phc Quorum-Sensing System in Ralstonia solanacearum Species Complex.

Ralstonia solanacearum species complex (RSSC) strains are devastating plant pathogens distributed worldwide. The primary cell density-dependent gene expression system in RSSC strains is phc quorum sensing (QS). It regulates the expression of about 30% of all genes, including those related to cellular activity, primary and secondary metabolism, pathogenicity, and more. The phc regulatory elements encoded by the phcBSRQ operon and phcA gene play vital roles. RSSC strains use methyl 3-hydroxymyristate (3-OH MAME) or methyl 3-hydroxypalmitate (3-OH PAME) as the QS signal. Each type of RSSC strain has specificity in generating and receiving its QS signal, but their signaling pathways might not differ significantly. In this review, I describe the genetic and biochemical factors involved in QS signal input and the regulatory network and summarize control of the phc QS system, new cell-cell communications, and QS-dependent interactions with soil fungi.

Molecular identification of Ralstonia pseudosolanacearum causing bacterial wilt in tomatoes from Da Nang by using Colony PCR

Ralstonia solanacearum species complex (RSSC) is the main pathogen causing bacterial wilt disease in tomatoes. This study applied the colony polymerase chain reaction (PCR) technique to rapidly screen and select RSSC strains from isolated bacteria of diseased tomatoes. This method directly used bacterial colonies on the Petri plate as templates to amplify with RSSC’s specific and multiplex primers. The results showed that the Vietnamese isolates were identified as R. pseudosolanacearum, phylotype I. Phylogenetic analysis of the 16S-23S rDNA sequencing also confirmed these results. Therefore, this is the first report to recognize R. pseudosolanacearum phylotype I as the cause of bacterial wilt disease in tomatoes from Danang.

Molecular Epidemiology of Ralstonia pseudosolanacearum Phylotype I Strains in the Southwest Indian Ocean Region and Their Relatedness to African Strains.

The increasing requirement for developing tools enabling fine strain traceability responsible for epidemics is tightly linked with the need to understand factors shaping pathogen populations and their environmental interactions. Bacterial wilt caused by the Ralstonia solanacearum species complex (RSSC) is one of the most important plant diseases in tropical and subtropical regions. Sadly, little, outdated, or no information on its epidemiology is reported in the literature, although alarming outbreaks are regularly reported as disasters. A large set of phylotype I isolates (n = 2,608) was retrieved from diseased plants in fields across the Southwest Indian Ocean (SWIO) and Africa. This collection enabled further assessment of the epidemiological discriminating power of the previously published RS1-MLVA14 scheme. Thirteen markers were validated and characterized as not equally informative. Most had little infra-sequevar polymorphism, and their performance depended on the sequevar. Strong correlation was found with a previous multilocus sequence typing scheme. However, 2 to 3% of sequevars were not correctly assigned through endoglucanase gene sequence. Discriminant analysis of principal components (DAPC) revealed four groups with strong phylogenetic relatedness to sequevars 31, 33, and 18. Phylotype I-31 isolates were highly prevalent in the SWIO and Africa, but their dissemination pathways remain unclear. Tanzania and Mauritius showed the greatest diversity of RSSC strains, as the four DAPC groups were retrieved. Mauritius was the sole territory harboring a vast phylogenetic diversity and all DAPC groups. More research is still needed to understand the high prevalence of phylotype I-31 at such a large geographic scale.

Discovery of Cyclic Lipopeptides Ralstopeptins A and B from Ralstonia solanacearum Species Complex and Analysis of Biosynthetic Gene Evolution.

Ralstonia solanacearum species complex (RSSC) strains are plant pathogens that produce lipopeptides (ralstonins and ralstoamides) by the polyketide synthase-nonribosomal peptide synthetase (PKS-NRPS) enzyme hybrid. Recently, ralstonins were found to be key molecules in the parasitism of RSSC to other hosts, Aspergillus and Fusarium fungi. The PKS-NRPS genes of RSSC strains in the GenBank database suggest the production of additional lipopeptides, although it has not been confirmed to date. Here, we report the genome-driven and mass-spectrometry-guided discovery, isolation, and structural elucidation of ralstopeptins A and B from strain MAFF 211519. Ralstopeptins were found to be cyclic lipopeptides with two amino acid residues less than ralstonins. The partial deletion of the gene encoding PKS-NRPS obliterated the production of ralstopeptins in MAFF 211519. Bioinformatic analyses suggested possible evolutionary events of the biosynthetic genes of RSSC lipopeptides, where intragenomic recombination may have occurred within the PKS-NRPS genes, reducing the gene size. The chlamydospore-inducing activities of ralstopeptins A and B, ralstonins A and B, and ralstoamide A in the fungus Fusarium oxysporum indicated a structural preference for ralstonins. Altogether, we propose a model for the evolutionary processes that contribute to the chemical diversity of RSSC lipopeptides and its relation to the endoparasitism of RSSC in fungi.

Validating Methods To Eradicate Plant-Pathogenic Ralstonia Strains Reveals that Growth In Planta Increases Bacterial Stress Tolerance.

Plant-pathogenic bacteria in the Ralstonia solanacearum species complex (RSSC) cause highly destructive bacterial wilt disease of diverse crops. Wilt disease prevention and management is difficult because RSSC persists in soil, water, and plant material. Growers need practical methods to kill these pathogens in irrigation water, a common source of disease outbreaks. Additionally, the R. solanacearum race 3 biovar 2 (R3bv2) subgroup is a quarantine pest in many countries and a highly regulated select agent pathogen in the United States. Plant protection officials and researchers need validated protocols to eradicate R3bv2 for regulatory compliance. To meet these needs, we measured the survival of four R3bv2 and three phylotype I RSSC strains following treatment with hydrogen peroxide, stabilized hydrogen peroxide (Huwa-San), active chlorine, heat, UV radiation, and desiccation. No surviving RSSC cells were detected after cultured bacteria were exposed for 10 min to 400 ppm hydrogen peroxide, 50 ppm Huwa-San, 50 ppm active chlorine, or temperatures above 50°C. RSSC cells on agar plates were eradicated by 30s of UV irradiation and killed by desiccation on most biotic and all abiotic surfaces tested. RSSC bacteria did not survive the cell lysis steps of four nucleic acid extraction protocols. However, bacteria in planta were more difficult to kill. Stems of infected tomato plants contained a subpopulation of bacteria with increased tolerance of heat and UV light, but not oxidative stress. This result has significant management implications. We demonstrate the utility of these protocols for compliance with select agent research regulations and for management of a bacterial wilt outbreak in the field. IMPORTANCE Bacteria in the Ralstonia solanacearum species complex (RSSC) are globally distributed and cause destructive vascular wilt diseases of many high-value crops. These aggressive pathogens spread in diseased plant material and via contaminated soil, tools, and irrigation water. A subgroup of the RSSC, race 3 biovar 2, is a European and Canadian quarantine pathogen and a U.S. select agent subject to stringent and constantly evolving regulations intended to prevent pathogen introduction or release. We validated eradication and inactivation methods that can be used by (i) growers seeking to disinfest water and manage bacterial wilt disease outbreaks, (ii) researchers who must remain in compliance with regulations, and (iii) regulators who are expected to define containment practices. Relevant to all these stakeholders, we show that while cultured RSSC cells are sensitive to relatively low levels of oxidative chemicals, desiccation, and heat, more aggressive treatment, such as autoclaving or incineration, is required to eradicate plant-pathogenic Ralstonia growing inside plant material.

Ralstonia Strains from Potato-Growing Regions of Kenya Reveal Two Phylotypes and Epidemic Clonality of Phylotype II Sequevar 1 Strains.

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.

Identification and characterization of resistance quantitative trait loci against bacterial wilt caused by the Ralstonia solanacearum species complex in potato.

The online version contains supplementary material available at 10.1007/s11032-022-01321-9.

Comprehensive Analysis Reveals the Genetic and Pathogenic Diversity of Ralstonia solanacearum Species Complex and Benefits Its Taxonomic Classification.

Ralstonia solanacearum species complex (RSSC) is a diverse group of plant pathogens that attack a wide range of hosts and cause devastating losses worldwide. In this study, we conducted a comprehensive analysis of 131 RSSC strains to detect their genetic diversity, pathogenicity, and evolution dynamics. Average nucleotide identity analysis was performed to explore the genomic relatedness among these strains, and finally obtained an open pangenome with 32,961 gene families. To better understand the diverse evolution and pathogenicity, we also conducted a series of analyses of virulence factors (VFs) and horizontal gene transfer (HGT) in the pangenome and at the single genome level. The distribution of VFs and mobile genetic elements (MGEs) showed significant differences among different groups and strains, which were consistent with the new nomenclatures of the RSSC with three distinct species. Further functional analysis showed that most HGT events conferred from Burkholderiales and played a great role in shaping the genomic plasticity and genetic diversity of RSSC genomes. Our work provides insights into the genetic polymorphism, evolution dynamics, and pathogenetic variety of RSSC and provides strong supports for the new taxonomic classification, as well as abundant resources for studying host specificity and pathogen emergence.

Meta-analysis of the Ralstonia solanacearum species complex (RSSC) based on comparative evolutionary genomics and reverse ecology.

Ralstonia solanacearum species complex (RSSC) strains are bacteria that colonize plant xylem tissue and cause vascular wilt diseases. However, individual strains vary in host range, optimal disease temperatures and physiological traits. To increase our understanding of the evolution, diversity and biology of the RSSC, we performed a meta-analysis of 100 representative RSSC genomes. These 100 RSSC genomes contain 4940 genes on average, and a pangenome analysis found that there are 3262 genes in the core genome (~60 % of the mean RSSC genome) with 13 128 genes in the extensive flexible genome. A core genome phylogenetic tree and a whole-genome similarity matrix aligned with the previously named species ( R. solanacearum , R. pseudosolanacearum , R. syzygii ) and phylotypes (I–IV). These analyses also highlighted a third unrecognized sub-clade of phylotype II. Additionally, we identified differences between phylotypes with respect to gene content and recombination rate, and we delineated population clusters based on the extent of horizontal gene transfer. Multiple analyses indicate that phylotype II is the most diverse phylotype, and it may thus represent the ancestral group of the RSSC. We also used our genome-based framework to test whether the RSSC sequence variant (sequevar) taxonomy is a robust method to define within-species relationships of strains. The sequevar taxonomy is based on alignments of a single conserved gene (egl). Although sequevars in phylotype II describe monophyletic groups, the sequevar system breaks down in the highly recombinogenic phylotype I, which highlights the need for an improved, cost-effective method for genotyping strains in phylotype I. Finally, we enabled quick and precise genome-based identification of newly sequenced RSSC strains by assigning Life Identification Numbers (LINs) to the 100 strains and by circumscribing the RSSC and its sub-groups in the LINbase Web service.

Biological and Molecular Characterization of a Jumbo Bacteriophage Infecting Plant Pathogenic Ralstonia solanacearum Species Complex Strains.

A jumbo phage infecting Ralstonia solanacearum species complex strains, designated RsoM2USA, was isolated from soil of a tomato field in Florida, United States, and belongs to the family Myoviridae. The phage has a long latent period of 270 min and completed its infection cycle in 360 min with a burst size of approximately 32 particles per cell. With a genome size of 343,806 bp, phage RsoM2USA is the largest Ralstonia-infecting phage sequenced and reported to date. Out of the 486 ORFs annotated for RsoM2USA, only 80 could be assigned putative functions in replication, transcription, translation including 44 tRNAs, and structure with the main structural proteins experimentally confirmed. Phylogenetic analyses placed RsoM2USA in the same clade as Xanthomonas phage XacN1, prompting a proposal of a new genus for the two jumbo phages. Jumbo phage RsoM2USA is a lytic phage and has a wide host range, infecting each of the three newly established Ralstonia species: R. solanacearum, R. pseudosolanacearum, and R. syzygii, and significantly reduced the virulence of its susceptible R. solanacearum strain RUN302 in tomato plants, suggesting that this jumbo phage has the potential to be developed into an effective control against diseases caused by R. solanacearum species complex strains.

A high-throughput virulence screening method for the Ralstonia solanacearum species complex

Ralstonia solanacearum species complex strains are the causative agents for wilting diseases of many plants, including the economically important brown rot of potato. We developed a high-throughput virulence screen that is implemented in 96-well microtiter plates using seedlings grown in soft water agar to save space, effort, and resources. Nicotiana glutinosa was determined to be the most effective host for this assay, and we confirmed bacterial growth and systemic spread in inoculated seedlings. In our assay, N. glutinosa seeds were sown quickly and easily on top of individual water agar wells of a 96-well plate by pipetting out desired number of seeds in an aqueous suspension. They were inoculated on the same day by first touching a bacterial colony with an autoclaved toothpick and then stabbing the toothpick into the center of the water agar well. Such inoculation method resulted in inocula above a threshold of 2 × 104 CFU per well achieving consistent virulence results and enabling reduction of inoculum preparation efforts to facilitate high-throughput screening. Our assay is suitable for forward genetic screening of a large number of strains, isolates or mutants for disease symptoms under both cool (20 °C) and warm (28 °C) temperature conditions before detailed studies can be narrowed down to a manageable number of desired candidates. Our virulence screen method provides a valuable tool for future work in understanding genetics of virulence of Rssc, especially cool virulence of the highly regulated race 3 biovar 2 group of R. solanacearum, leading toward development of effective control strategies.

Evaluation of tomato, eggplant and pepper accessions for resistance to Ralstonia solanacearum species complex (RSSC) strains from Louisiana

Bacterial wilt (BW), caused by Ralstonia solanacearum species complex (RSSC), is a major disease affecting tomato and other solanaceous crops produced in Louisiana and worldwide. The most effective and economic management strategy for BW is the use of crop genetic resistance. However, regional genetic variation among RSSC strains has made stable and durable resistance elusive. A collection of tomato, eggplant and pepper (TEP) accessions that responds to globally diverse strains in the RSSC was screened for resistance to six RSSC strains, belonging to phylotypes I and II, from Louisiana, United States (US). Wilt severity was quantified from six days up to 25 days post-inoculation (DPI). Wilting increased most rapidly to 10 DPI and was generally more severe with Phylotype II strains. However, wilt response profiles depended on the accession-strain combination. Differences in aggressiveness, as measured by the area under the disease progress stairs, were observed among the phylotype I and II strains on TEP. Wilting responses were the most severe with the tomato accessions. Hawaii 7996, TML46, CLN1463 and R3034 were highly resistant to all phylotype I strains, but Hawaii 7996 displayed the best resistance to phylotype II strains. Remarkably, eggplant accession Ceylan SM 164 was resistant to all six strains. Eggplant accessions SM6, Surya, and AG91–25, as well as pepper accessions CA8 and MC4, were moderately to highly resistant to all six strains. Although resistance spectrum and levels of these tomato, eggplant and pepper accessions need to be confirmed by further experiments, our results already indicate that these accessions are potential BW-resistant rootstock candidates for grafting to susceptible market-driven tomato, eggplant and pepper produced in this US state.

Whole genome characterization of strains belonging to the Ralstonia solanacearum species complex and in silico analysis of TaqMan assays for detection in this heterogenous species complex

Identification and classification of members of the Ralstonia solanacearum species complex (RSSC) is challenging due to the heterogeneity of this complex. Whole genome sequence data of 225 strains were used to classify strains based on average nucleotide identity (ANI) and multilocus sequence analysis (MLSA). Based on the ANI score (>95%), 191 out of 192(99.5%) RSSC strains could be grouped into the three species R. solanacearum, R. pseudosolanacearum, and R. syzygii, and into the four phylotypes within the RSSC (I,II, III, and IV). R. solanacearum phylotype II could be split in two groups (IIA and IIB), from which IIB clustered in three subgroups (IIBa, IIBb and IIBc). This division by ANI was in accordance with MLSA. The IIB subgroups found by ANI and MLSA also differed in the number of SNPs in the primer and probe sites of various assays. An in-silico analysis of eight TaqMan and 11 conventional PCR assays was performed using the whole genome sequences. Based on this analysis several cases of potential false positives or false negatives can be expected upon the use of these assays for their intended target organisms. Two TaqMan assays and two PCR assays targeting the 16S rDNA sequence should be able to detect all phylotypes of the RSSC. We conclude that the increasing availability of whole genome sequences is not only useful for classification of strains, but also shows potential for selection and evaluation of clade specific nucleic acid-based amplification methods within the RSSC.

Contrasting genetic diversity and structure among Malagasy Ralstonia pseudosolanacearum phylotype I populations inferred from an optimized Multilocus Variable Number of Tandem Repeat Analysis scheme.

The Ralstonia solanacearum species complex (RSSC), composed of three species and four phylotypes, are globally distributed soil-borne bacteria with a very broad host range. In 2009, a devastating potato bacterial wilt outbreak was declared in the central highlands of Madagascar, which reduced the production of vegetable crops including potato, eggplant, tomato and pepper. A molecular epidemiology study of Malagasy RSSC strains carried out between 2013 and 2017 identified R. pseudosolanacearum (phylotypes I and III) and R. solanacearum (phylotype II). A previously published population biology analysis of phylotypes II and III using two MultiLocus Variable Number of Tandem Repeats Analysis (MLVA) schemes revealed an emergent epidemic phylotype II (sequevar 1) group and endemic phylotype III isolates. We developed an optimized MLVA scheme (RS1-MLVA14) to characterize phylotype I strains in Madagascar to understand their genetic diversity and structure. The collection included isolates from 16 fields of different Solanaceae species sampled in Analamanga and Itasy regions (highlands) in 2013 (123 strains) and in Atsinanana region (lowlands) in 2006 (25 strains). Thirty-one haplotypes were identified, two of them being particularly prevalent: MT007 (30.14%) and MT004 (16.44%) (sequevar 18). Genetic diversity analysis revealed a significant contrasting level of diversity according to elevation and sampling region. More diverse at low altitude than at high altitude, the Malagasy phylotype I isolates were structured in two clusters, probably resulting from different historical introductions. Interestingly, the most prevalent Malagasy phylotype I isolates were genetically distant from regional and worldwide isolates. In this work, we demonstrated that the RS1-MLVA14 scheme can resolve differences from regional to field scales and is thus suited for deciphering the epidemiology of phylotype I populations.

Quorum Sensing Inhibition Attenuates the Virulence of the Plant Pathogen Ralstonia solanacearum Species Complex.

Strains of Ralstonia solanacearum species complex (RSSC) cause "bacterial wilt" on a wide range of plant species and thus lead to marked economic losses in agriculture. Quorum sensing (QS), a bacterial cell-cell communication mechanism, controls the virulence of RSSC strains by regulating the production of extracellular polysaccharide (EPS) and secondary metabolites, biofilm formation, and cellular motility. R. solanacearum strain OE1-1 employs (R)-methyl 3-hydroxymyristate (3-OH MAME) as a QS signal, which is synthesized by the PhcB methyltransferase and sensed by the PhcS/PhcRQ two-component system. We describe the design, synthesis, and biological evaluation of inhibitors of the phc QS system. Initial screening of a small set of QS signal analogues revealed that methyl 3-hydroxy-8-phenyloctanoate, named, PQI-1 (phc quorum sensing inhibitor-1), inhibited biofilm formation by strain OE1-1. To improve its inhibitory activity, the derivatives of PQI-1 were synthesized, and their QS inhibition activities were evaluated. PQIs-2-5 evolved from PQI-1 more strongly inhibited not only biofilm formation but also the production of ralfuranone and EPS. Furthermore, RNA-Seq analysis revealed that the PQIs effectively inhibited QS-dependent gene expression and repression in strain OE1-1. On the other hand, the PQIs did not affect the canonical QS systems of the representative reporter bacteria. These antagonists, especially PQI-5, reduced wilting symptoms of the tomato plants infected with strain OE1-1. Taken together, we suggest that targeting the phc QS system has potential for the development of chemicals that protect agricultural crops from bacterial wilt disease.