An interactive dashboard for global reports on the Ralstonia solanacearum species complex

ABSTRACTPlant-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 30 s 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.

ABSTRACTStrains of the Ralstonia solanacearum species complex (RSSC), although known as the causative agent of bacterial wilt disease in plants, induce the chlamydospores of many fungal species and invade them through the spores. The lipopeptide ralstonins are the chlamydospore inducers produced by RSSC and are essential for this invasion. However, no mechanistic investigation of this interaction has been conducted. In this study, we report that quorum sensing (QS), which is a bacterial cell-cell communication, is important for RSSC to invade the fungus Fusarium oxysporum (Fo). ΔphcB, a deletion mutant of QS signal synthase, lost the ability to both produce ralstonins and invade Fo chlamydospores. The QS signal methyl 3-hydroxymyristate rescued these disabilities. In contrast, exogenous ralstonin A, while inducing Fo chlamydospores, failed to rescue the invasive ability. Gene-deletion and -complementation experiments revealed that the QS-dependent production of extracellular polysaccharide I (EPS I) is essential for this invasion. The RSSC cells adhered to Fo hyphae and formed biofilms there before inducing chlamydospores. This biofilm formation was not observed in the EPS I- or ralstonin-deficient mutant. Microscopic analysis showed that RSSC infection resulted in the death of Fo chlamydospores. Altogether, we report that the RSSC QS system is important for this lethal endoparasitism. Among the factors regulated by the QS system, ralstonins, EPS I, and biofilm are important parasitic factors.IMPORTANCERalstonia solanacearum species complex (RSSC) strains infect both plants and fungi. The phc quorum-sensing (QS) system of RSSC is important for parasitism on plants, because it allows them to invade and proliferate within the hosts by causing appropriate activation of the system at each infection step. In this study, we confirm that ralstonin A is important not only for Fusarium oxysporum (Fo) chlamydospore induction but also for RSSC biofilm formation on Fo hyphae. Extracellular polysaccharide I (EPS I) is also essential for biofilm formation, while the phc QS system controls these factors in terms of production. The present results advocate a new QS-dependent mechanism for the process by which a bacterium invades a fungus.

The Ralstonia solanacearum species complex (RSSC) is a globally distributed group of gram-negative, soil-borne bacteria that cause wilt disease on a broad range of host plant species. Tomato is a widely cultivated and economically important crop in Tanzania, but bacterial wilt disease caused by the RSSC has emerged to be one of the most devastating diseases in tomato producing regions in Tanzania. To investigate the genomic diversity of RSSC strains isolated from Tanzania, we generated draft genomes of 19 Ralstonia isolates from 5 villages in 4 regions. Our phylogenetic analysis showed that all 19 of the isolates were phylotype I R. pseudosolanacearum strains, of which 16 were sequevar 31, two were sequevar 48, and one was sequevar 46. Life Identification Number (LIN) assignments were determined for all genomes. This genome announcement provides new genomic resources to investigate pathogen evolution, epidemiology, and virulence.

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.

Ralstonia solanacearum species complex (RSSC) pathogens cause destructive plant wilt diseases of a wide variety of crops, leading to significant agricultural losses worldwide. These bacteria rapidly spread through the water-transporting xylem where they grow prolifically and produce abundant biofilm that clogs xylem vessels. To understand RSSC biofilm behavior in planta, we examined their complex fluid mechanics. Rheological analyses revealed that unlike all previously analyzed microbial biofilms, RSSC biofilms are shear-thinning, viscoelastic fluids at physiologically relevant shear forces. To determine which factors confer these unique mechanics, we analyzed biofilms of bacterial mutants with altered biofilm components. Genetic analysis demonstrated that development of the viscous-dominant biofilms required production of EPS-I, an amphiphilic exopolysaccharide that is a major virulence factor for all RSSC pathogens. We show that EPS-I confers "biofilm mobility", which allows wild-type RSSC colonies to passively expand when deformed. Despite its high metabolic cost, bioassays demonstrated that EPS-I production conferred a net fitness benefit where biofilm mobility allowed the pathogen to spread and access more nutrients in complex environments like xylem vessels. The RSSC are a monophyletic lineage of aggressive plant wilt pathogens, and our evolutionary hypothesis testing suggests the origin of the eps biosynthetic gene cluster coincides with the emergence of wilt pathogenesis in the RSSC ancestor. Furthermore, comparative physiological assays demonstrated that biofilm mobility is unique to the RSSC within the genus Ralstonia. In summary, EPS-I production is a key evolutionary innovation that enables RSSC dispersal and virulence by conferring unique biofilm mechanics.

Soilborne Ralstonia solanacearum species complex (RSSC) pathogens disrupt microbial communities as they invade roots and fatally wilt plants. RSSC pathogens secrete antimicrobial toxins using a type VI secretion system (T6SS). To investigate how evolution and ecology have shaped the T6SS of these bacterial pathogens, we analyzed the T6SS gene content and architecture across the RSSC and their evolutionary relatives. Our analysis reveals that two ecologically similar Burkholderiaceae taxa, xylem-pathogenic RSSC and Paracidovorax, have convergently evolved to wield large arsenals of T6SS toxins. To understand the mechanisms underlying genomic enrichment of T6SS toxins, we compiled an atlas of 1,066 auxiliary T6SS toxin clusters ("aux" clusters) across 99 high-quality RSSC genomes. We classified 25 types of aux clusters with toxins that predominantly target lipids, nucleic acids, or unknown cellular substrates. The aux clusters were located in diverse genetic neighborhoods and had complex phylogenetic distributions, suggesting frequent horizontal gene flow. Phages and other mobile genetic elements account for most of the aux cluster acquisition on the chromosome but very little on the megaplasmid. Nevertheless, RSSC genomes were more enriched in aux clusters on the megaplasmid. Although the single, ancestral T6SS was broadly conserved in the RSSC, the T6SS has been convergently lost in atypical, non-soilborne lineages. Overall, our data suggest dynamic interplay between the lifestyle of RSSC lineages and the evolution of T6SSes with robust arsenals of toxins. This pangenomic atlas poises the RSSC as an emerging, tractable model to understand the role of the T6SS in shaping pathogen populations.IMPORTANCEWe explored the eco-evolutionary dynamics that shape the inter-microbial warfare mechanisms of a globally significant plant pathogen, the Ralstonia solanacearum species complex. We discovered that most Ralstonia wilt pathogens have evolved extensive and diverse repertoires of type VI secretion system-associated antimicrobial toxins. These expansive toxin arsenals potentially enhance the ability of Ralstonia pathogens to invade plant microbiomes, enabling them to rapidly colonize and kill their host plants. We devised a classification system to categorize the Ralstonia toxins. Interestingly, many of the toxin gene clusters are encoded on mobile genetic elements, including prophages, which may be mutualistic symbionts that enhance the inter-microbial competitiveness of Ralstonia wilt pathogens. Moreover, our findings suggest that the convergent loss of this multi-gene trait contributes to genome reduction in two vector-transmitted lineages of Ralstonia pathogens. Our findings demonstrate that the interplay between microbial ecology and pathogen lifestyle shapes the evolution of a genetically complex antimicrobial weapon.

Tomato (Solanum lycopersicum), pepper (Capsicum annum), and gboma (Solanum macrocarpon) are major vegetables in Togo, with many people depending on these crops for their livelihood. In December 2018, during the dry season with temperatures between 21°C to 35°C, tomato ('Petomech'), pepper ('Gboyebesse') and gboma (local landrace) showing wilt symptoms without foliar yellowing were collected from two locations, Tchouloum and CECO-AGRO sites in the Sotouboua Prefecture of Togo, ~300 km from the capital city of Lome. Disease incidence ranged between 10% to 50% in multiple fields. Cut stems of most wilting tomato, pepper and gboma plants produced bacterial ooze in water and vascular discoloration was visible in longitudinal stem sections. Ground cut stem tissue tested positive with Rs ImmunoStrips specific to the Ralstonia solanacearum species complex (RSSC) (Agdia Inc., Elkhart, IN, USA). Collected samples were stored at ambient temperature and cultured within 36 hr. Culturing sap from cut stems plated on modified SMSA medium (Engelbrecht 1994) yielded colonies with typical RSSC morphology: slow-growing, irregular, mucoid, and white with red centers. Genomic DNA was extracted from thirteen isolates: two from gboma, five from tomato and six from pepper. The expected 280-bp band was amplified from all 13 genomic DNAs following polymerase chain reaction (PCR) using the 759/760 RSSC-specific primer pair (Opina et al. 1997). PCR with the 630/631 primers, which identify the Race 3 biovar 2 RSSC subgroup, did not yield a product from any Togo isolate (Opina et al. 1997). The phylotype multiplex PCR identified all Togo isolates as belonging to the phylotype I subgroup, also called R. pseudosolanacearum (Prior et al. 2016; Fegan and Prior 2005). Phylotype control DNAs were from strains GMI1000 (phylotype I, Asia), K60 (phylotype II, Americas), CMR15 (phylotype III, Africa), and PSI07 (phylotype IV, Indondesia). Comparative genomic analysis of the partial endoglucanase (egl) gene, amplified with the Endo primer pairs (Poussier et al. 2000), revealed all Togo strains belonged to sequevar 17, a group known to cause bacterial wilt of peanut in China. (Xu et al. 2009). The egl sequences are in NCBI GenBank accessions MT572393 to MT572405. Koch's postulates were completed by inoculating 28-day-old bacterial wilt-susceptible 'Bonny Best' tomato plants by soil soak (Khokhani et al. 2018). Briefly, soil around each unwounded plant was drenched with 50 ml of a 108 CFU/mL suspension of bacteria grown from a single colony. Five plants were inoculated with each of four randomly selected Togo strains. RSSC phylotype I strain GMI1000 served as a positive control and water treated plants as negative controls. Plants were kept in a 28°C growth chamber with a 12 hr photoperiod. All RSSC inoculated plants were fully wilted within a week; symptoms resembled to those observed in the field. Water treated control plants did not wilt. Culturing sap from all inoculated plants on SMSA medium yielded colonies with typical RSSC morphology that tested positive with the Rs ImmunoStrips. This is the first identification of RSSC in Togo. These results will guide development of disease management strategies and regionally appropriate breeding of vegetable lines with resistance to the phylotype I RSSC strains present in Togo.

The Ralstonia solanacearum species complex (RSSC) comprising soil-borne Gram-negative phytopathogenic bacteria causes bacterial wilt diseases of diverse crop plants. Considering that phylotype I strain OE1-1 enters iron-rich roots from iron-deficient soil during an infection of tomato plants, the mechanisms controlling strain OE1-1 gene expression in response to extracellular iron levels should be clarified. In this study, RSSC was revealed to have two ferric uptake regulator homologs (Fur1 and Fur2). Notably, Fur1 and Fur2 cooperatively repress the expression of genes related to siderophores (Fe3+-chelating compounds) as well as the extracellular Fe3+-chelating activity in the presence of sufficient amounts of extracellular Fe2+. Additionally, Fur1 and Fur2 contribute to the virulence of strain OE1-1 in tomato plants. These findings suggest that RSSC uses two Fur proteins to modulate extracellular Fe3+-chelating activities in response to extracellular iron levels to maintain virulence in crop plants.

The Ralstonia solanacearum species complex (RSSC) is composed of several Ralstonia species and strains that are little related and show varied host range and distinct geographic distributions. The RSSC causes wilt disease, and can thus have severe economic consequences for many important crops and ornamental plants. One such is potato (Solanum tuberosum), where infection causes brown rot of the tubers. It is important that symptomatic tubers and plants can be rapidly and easily tested, as exclusion of infected material is a cornerstone of management of bacterial diseases. A suitable method is loop-mediated isothermal amplification, a rapid, DNA-based method that can be used for specific detection of plant pathogens in infected materials. The combination of this loop-mediated isothermal amplification assay for the RSSC with a simple sample preparation method is fit for purpose for identification of this devastating disease in symptomatic tubers and plants. This methodology is rapid and cost efficient, and can be carried out outside of conventional laboratory facilities.

Ralstonia pseudosolanacearum (Rps), which causes bacterial wilt disease of many crops, must integrate environmental signals to successfully transition from soil to its pathogenic niche in host plant xylem tissue. Mutating a gene encoding a putative sensing/signaling protein had little transcriptomic effect on Rps strain GMI1000 in culture. However, when the mutant grew in tomato, over 180 genes were differentially expressed relative to the wild type. The gene was therefore named rprR for Ralstonia plant-responsive regulator. In planta, the ∆rprR mutant dysregulated genes for diverse traits, including stress response, degradation of phenolic compounds, motility, attachment, and production of extracellular polysaccharide (EPS), which is a key bacterial wilt virulence factor. Quantifying Rps EPS by ELISA found increased levels in stems of plants infected with ∆rprR as compared to the wild type. Functional assays showed that ∆rprR is defective in attachment to tomato roots, colonization of tomato stems, and bacterial wilt virulence. In a rich medium, ∆rprR formed biofilm normally, but the mutant formed less biofilm in tomato stem homogenate and in tomato xylem sap under flow. This phenotype correlates with the mutant's altered expression of EPS biosynthetic genes and aberrant extracellular matrix. When grown in tomato stem homogenate, ∆rprR produced 57% more of the bacterial signal cyclic di-GMP (c-di-GMP) than the wild type. This is consistent with the presence, in RprR, of predicted c-di-GMP-modulating domains. Together, these findings reveal that RprR, which is highly conserved across plant pathogenic Ralstonia, modulates several bacterial wilt virulence traits in response to the plant host.IMPORTANCEMembers of the Ralstonia solanacearum species complex (RSSC) cause bacterial wilt, a globally destructive disease of market and subsistence crops. Like other plant-associated microbes, bacteria in the RSSC must integrate a complex array of biotic and abiotic signals to successfully infect plant hosts. All RSSC genomes encode an unusual protein, termed RprR, that contains multiple sensing and signaling domains, including two putative modulators of the secondary messenger c-di-GMP. Deleting RprR in Ralstonia pseudosolanacearum affected many virulence properties, including production of biofilm and exopolysaccharide, and increased intracellular c-di-GMP levels, all in a strictly plant-dependent fashion. While c-di-GMP has been investigated in other plant pathogenic bacteria, this is the first report of its role in the RSSC. Most importantly, rprR was required for Ralstonia to effectively colonize plants and cause wilt disease. Thus, RprR is a plant-responsive sensor-regulator that controls pathogen adaptation to the host environment and virulence.

Following a request from the European Commission, the EFSA Panel on Plant Health performed a pest categorisation of the Ralstonia solanacearum species complex (RSSC), a distinguishable cosmopolitan group of bacterial plant pathogens (including R. solanacearum, Ralstonia pseudosolanacearum and two subspecies of Ralstonia syzygii) of the family Burkholderiaceae. The RSSC causes bacterial wilt in solanaceous crops, such as potato, tomato and pepper, but can also cause wilts in other important food crops such as fruit banana, plantain banana and cassava. The pest survives in the soil, and a number of weed species can also be infected by the pest, often asymptomatically. The RSSC is regulated in Council Directive 2000/29/EC (Annex IAII) (indicated by its former name R. solanacearum, as delimited by Yabuuchi et al.) as a harmful organism whose introduction into the EU is banned. In addition, Council Directive 1998/57/EC (amended by Commission Directive 2006/63/CE) concerns the measures to be taken within EU Member States (MS) against the RSSC to (a) detect it and determine its distribution, (b) prevent its occurrence and spread, and (c) control it with the aim of eradication. The pest is present in several EU MS, but in all cases with a restricted distribution and under official control. New phylotypes of the RSSC could enter the EU primarily via host plants for planting (including seed tubers). The pest could establish in the EU, as climatic conditions are favourable, hosts are common and the pathogen has high adaptability. Spread is mainly via plants for planting. Substantial crop losses in the EU would occur in the presence of RSSC epidemics. The RSSC is regarded as one of the world's most important phytopathogenic bacteria due to its broad geographical distribution, large host range, aggressiveness, genetic diversity and long persistence in soil and water. The list of hosts and commodities for which the pest is regulated is incomplete due to the high diversity of hosts and the lack of knowledge of the complete host range. Moreover, the comparative epidemiology of the different pathogen species has not yet been studied. The criteria assessed by the Panel for consideration of the RSSC as potential quarantine pest are met, while, for regulated non‐quarantine pests, the criterion on the widespread presence in the EU is not met.

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.

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.

Bacterial pathogens in the Ralstonia solanacearum species complex (RSSC) infect the water-transporting xylem vessels of plants, causing bacterial wilt disease. Strains in RSSC phylotypes I and III can reduce nitrate to dinitrogen via complete denitrification. The four-step denitrification pathway enables bacteria to use inorganic nitrogen species as terminal electron acceptors, supporting their growth in oxygen-limited environments such as biofilms or plant xylem. Reduction of nitrate, nitrite, and nitric oxide all contribute to the virulence of a model phylotype I strain. However, little is known about the physiological role of the last denitrification step, the reduction of nitrous oxide to dinitrogen by NosZ. We found that phylotypes I and III need NosZ for full virulence. However, strains in phylotypes II and IV are highly virulent despite lacking NosZ. The ability to respire by reducing nitrate to nitrous oxide does not greatly enhance the growth of phylotype II and IV strains. These partial denitrifying strains reach high cell densities during plant infection and cause typical wilt disease. However, unlike phylotype I and III strains, partial denitrifiers cannot grow well under anaerobic conditions or form thick biofilms in culture or in tomato xylem vessels. Furthermore, aerotaxis assays show that strains from different phylotypes have different oxygen and nitrate preferences. Together, these results indicate that the RSSC contains two subgroups that occupy the same habitat but have evolved divergent energy metabolism strategies to exploit distinct metabolic niches in the xylem. IMPORTANCE Plant-pathogenic Ralstonia spp. are a heterogeneous globally distributed group of bacteria that colonize plant xylem vessels. Ralstonia cells multiply rapidly in plants and obstruct water transport, causing fatal wilting and serious economic losses of many key food security crops. The virulence of these pathogens depends on their ability to grow to high cell densities in the low-oxygen xylem environment. Plant-pathogenic Ralstonia can use denitrifying respiration to generate ATP. The last denitrification step, nitrous oxide reduction by NosZ, contributes to energy production and virulence for only one of the three phytopathogenic Ralstonia species. These complete denitrifiers form thicker biofilms in culture and in tomato xylem, suggesting they are better adapted to hypoxic niches. Strains with partial denitrification physiology form less biofilm and are more often planktonic. They are nonetheless highly virulent. Thus, these closely related bacteria have adapted their core metabolic functions to exploit distinct microniches in the same habitat.

Around 90% of Peru's ginger (Zingiber officinale) production is concentrated in the Junín region, due to the optimal agroecological conditions for its cultivation. In March 2024, fields with ginger plants (cultivar Criollo) in Junín region, provinces Chanchamayo and Satipo, specifically in the cities of Pichanaqui and Satipo respectively, exhibited approximately 40% of plants with severe symptoms of a disease characterized initially by plant yellowing and rapid progressing to necrosis. Affected rhizomes showed dark vascular bundles with milky white exudates upon cutting, while stems displayed vascular necrosis hindering water and nutrient transport, often resulting in plant death. Fifteen plants were sampled and diseased vascular tissues from rhizomes and stems were cultured on nutrient agar (NA) and incubated at 28°C. After 72 h, all isolations resulted in colonies with typical characteristics of Ralstonia solanacearum species complex (RSSC) were produced, with appearing fluid, irregularly round, and creamy white. Three isolates were selected for the identification steps (UNALM-RP01 to 03) were identified by PCR using primers 759/760 (Opina et al. 1997) confirming as RSSC with a 282 bp amplification product. Additionally, isolates were assigned to biovar 3 based on their ability to metabolize three acid-producing disaccharides (maltose, lactose, cellobiose) and three hexose alcohols (mannitol, sorbitol, dulcitol) (Hayward, 1964). Phylotype I was identified by multiplex PCR (primers Nmult) with a 114 bp amplification product (Fegan and Prior 2005). For the identification of the sequevars of the three isolates, DNA was extracted and PCR with primers ENDO-F/R (Ji et al. 2007) were performed to amplify and sequence the partial gene sequence of egl gene with 681 bp in length. The phylogeny by Neighbor joining with 10,000 bootstraps clustered the UNALM isolates along other sequevar 30 of R. pseudosolanacearum. The sequences were deposited in Genbank under accessions PQ213016, PQ213017 and PQ213018. For pathogenicity tests, bacterial colonies of isolate UNALM-RP01 were scraped from the culture media with a sterile needle and introduced into the stems of three 2-month-old ginger plants (cultivar Gigante). The plants subsequently exhibited yellowing seven days post-inoculation. Additionally, the rhizomes showed internal discoloration and bacterial exudation. Three plants were used as a control, which were pierced with a sterilized needle and showed no symptoms. All tested plants were kept in a greenhouse with controlled temperature (20-40 °C) The pathogen was successfully re-isolated from infected plants on NA medium, presenting typical colonies of RSSC and identified via PCR with primers 759/760, fulfilling Koch's postulates. This represents the first case in Peru of ginger plants infected with a Ralstonia species, specifically R. solanacearum phylotype I, corresponding to R. pseudosolanacearum. This species of RSSC and sequevar is known for causing disease in ginger. Its presence in Peru, however, may be the result of the pathogen's introduction, as its geographical origin is associated with Asia (Fegan and Prior 2005). To our knowledge, this is the first report of R. pseudosolanacearum causing ginger wilt disease in Peru. In 2024, an estimated average yield loss of 30% has been attributed to wilt disease in the Junín region, posing a significant threat to cultivation. Urgent and effective disease management strategies are essential to control and mitigate further losses.