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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. [Figure: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

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.” [Figure: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Ralstonia solancearum causes bacterial wilt disease on diverse plant hosts. R. solanacearum cells enter a host from soil or infested water through the roots, then multiply and spread in the water-transporting xylem vessels. Despite the low nutrient content of xylem sap, R. solanacearum grows very well inside the host, using denitrification to respire in this hypoxic environment. R. solanacearum growth in planta also depends on the successful deployment of protein effectors into host cells via a type III secretion system (T3SS). The T3SS is absolutely required for R. solanacearum virulence, but it is metabolically costly and can trigger host defenses. Thus, the pathogen's success depends on optimized regulation of the T3SS. We found that a byproduct of denitrification, the toxic free-radical nitric oxide (NO), positively regulates the R. solanacearum T3SS both in vitro and in planta. Using chemical treatments and R. solanacearum mutants with altered NO levels, we show that the expression of a key T3SS regulator, hrpB, is induced by NO in culture. Analyzing the transcriptome of R. solanacearum responding to varying levels of NO both in culture and in planta revealed that the T3SS and effectors were broadly upregulated with increasing levels of NO. This regulation was specific to the T3SS and was not shared by other stressors. Our results suggest that R. solanacearum may experience an NO-rich environment in the plant host and that this NO contributes to the activation of the T3SS during infection. [Graphic: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

ABSTRACT 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.

We share whole genome sequences of six strains from the Ralstonia solanacearum species complex, a diverse group of Betaproteobacteria that cause plant vascular wilt diseases. Using single-molecule real-time technology, we sequenced and assembled full genomes of Rs5 and UW700, two phylotype IA-sequevar 7 (IIA-7) strains from the southeastern United States that are closely related to the R. solanacearum species type strain, K60, but were isolated >50 years later. Four sequenced strains from Africa include a soil isolate from Nigeria (UW386, III-23), a tomato isolate from Senegal (UW763, I-14), and two potato isolates from the Madagascar highlands (RUN2474, III-19 and RUN2279, III-60). This resource will support studies of the genetic diversity, ecology, virulence, and microevolution of this globally distributed group of high-impact plant pathogens.[Graphic: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

ABSTRACT Bacteriophages put intense selective pressure on microbes, which must evolve diverse resistance mechanisms to survive continuous phage attacks. We used a library of spontaneous Bacteriophage Insensitive Mutants (BIMs) to learn how the plant pathogen Ralstonia solanacearum resists the virulent lytic podophage phiAP1. Phenotypic and genetic characterization of many BIMs suggested that the R. solanacearum Type II Secretion System (T2SS) plays a key role in phiAP1 infection. Using precision engineered mutations that permit T2SS assembly but either inactivate the T2SS GspE ATPase or sterically block the secretion portal, we demonstrated that phiAP1 needs a functional T2SS to infect R. solanacearum. This distinction between the static presence of T2SS components, which is necessary but not sufficient for phage sensitivity, and the energized and functional T2SS, which is sufficient, implies that binding interactions alone cannot explain the role of the T2SS in phiAP1 infection. Rather, our results imply that some aspect of the resetting of the T2SS, such as disassembly of the pseudopilus, is required. Because R. solanacearum secretes multiple virulence factors via the T2SS, acquiring resistance to phiAP1 also dramatically reduced R. solanacearum virulence on tomato plants. This acute fitness trade-off suggests this group of phages may be a sustainable control strategy for an important crop disease. IMPORTANCE Ralstonia solanacearum is a destructive plant pathogen that causes lethal bacterial wilt disease in hundreds of diverse plant hosts, including many economically important crops. Phages that kill R. solanacearum could offer effective and environmentally friendly wilt disease control, but only if the bacterium cannot easily evolve resistance. Encouragingly, most R. solanacearum mutants resistant to the virulent lytic phage phiAP1 no longer secreted multiple virulence factors and had much reduced fitness and virulence on tomato plants. Further analysis revealed that phage phiAP1 needs a functional type II secretion system to infect R. solanacearum, suggesting this podophage uses a novel infection mechanism.

ABSTRACT 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 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.

The soilborne pathogen Ralstonia solanacearum causes a lethal bacterial wilt disease of tomato and many other crops by infecting host roots, then colonizing the water-transporting xylem vessels. Tomato xylem sap is nutritionally limiting but it does contain some carbon sources, including sucrose, trehalose, and myo-inositol. Transcriptomic analyses revealed that R. solanacearum expresses distinct catabolic pathways at low cell density (LCD) and high cell density (HCD). To investigate the links between bacterial catabolism, infection stage, and virulence, we measured in planta fitness of bacterial mutants lacking specific carbon catabolic pathways expressed at either LCD or HCD. We hypothesized that early in disease, during root infection, the bacterium depends on carbon sources catabolized at LCD, while HCD carbon sources are only required later in disease during stem colonization. A R. solanacearum ΔiolG mutant unable to use the LCD-catabolized nutrient myo-inositol was defective in tomato root colonization, but after it reached the stem this strain colonized and caused symptoms as well as wild type. In contrast, R. solanacearum mutants unable to use the HCD-catabolized nutrients sucrose (ΔscrA), trehalose (ΔtreA), or both (ΔscrA/treA), infected roots as well as wild-type R. solanacearum but were defective in colonization and competitive fitness in midstems and had reduced virulence. Further, xylem sap from tomato plants colonized by ΔscrA, ΔtreA, or ΔscrA/treA R. solanacearum mutants contained twice as much sucrose as sap from plants colonized by wild-type R. solanacearum. Together, these findings suggest that quorum sensing specifically adapts R. solanacearum metabolism for success in the different nutritional environments of plant roots and xylem sap.

ABSTRACT Ralstonia solanacearum, which causes bacterial wilt disease of many crops, requires denitrifying respiration to survive in its plant host. In the hypoxic environment of plant xylem vessels, this pathogen confronts toxic oxidative radicals like nitric oxide (NO), which is generated by both bacterial denitrification and host defenses. R. solanacearum has multiple distinct mechanisms that could mitigate this stress, including putative NO-binding protein (NorA), nitric oxide reductase (NorB), and flavohaemoglobin (HmpX). During denitrification and tomato pathogenesis and in response to exogenous NO, R. solanacearum upregulated norA, norB, and hmpX. Single mutants lacking ΔnorB, ΔnorA, or ΔhmpX increased expression of many iron and sulfur metabolism genes, suggesting that the loss of even one NO detoxification system demands metabolic compensation. Single mutants suffered only moderate fitness reductions in host plants, possibly because they upregulated their remaining protective genes. However, ΔnorA/norB, ΔnorB/hmpX, and ΔnorA/hmpX double mutants grew poorly in denitrifying culture and in planta. It is likely that the loss of norA, norB, and hmpX is lethal, since the methods used to construct the double mutants could not generate a triple mutant. Functional aconitase activity assays showed that NorA, HmpX, and especially NorB are important for maintaining iron-sulfur cluster proteins. Additionally, plant defense genes were upregulated in tomatoes infected with the NO-overproducing ΔnorB mutant, suggesting that bacterial detoxification of NO reduces the ability of the plant host to perceive the presence of the pathogen. Thus, R. solanacearum’s three NO detoxification systems each contribute to and are collectively essential for overcoming metabolic nitrosative stress during denitrification, for virulence and growth in the tomato, and for evading host plant defenses. IMPORTANCE The soilborne plant pathogen Ralstonia solanacearum (Rs) causes bacterial wilt, a serious and widespread threat to global food security. Rs is metabolically adapted to low-oxygen conditions, using denitrifying respiration to survive in the host and cause disease. However, bacterial denitrification and host defenses generate nitric oxide (NO), which is toxic and also alters signaling pathways in both the pathogen and its plant hosts. Rs mitigates NO with a trio of mechanistically distinct proteins: NO-reductase (NorB), predicted iron-binding (NorA), and oxidoreductase (HmpX). This redundancy, together with analysis of mutants and in-planta dual transcriptomes, indicates that maintaining low NO levels is integral to Rs fitness in tomatoes (because NO damages iron-cluster proteins) and to evading host recognition (because bacterially produced NO can trigger plant defenses).

Ralstonia solanacearum causes bacterial wilt disease, leading to severe crop losses. Xylem sap from R. solanacearum-infected tomato is enriched in the disaccharide trehalose. Water-stressed plants also accumulate trehalose, which increases drought tolerance via abscisic acid (ABA) signaling. Because R. solanacearum-infected plants suffer reduced water flow, we hypothesized that bacterial wilt physiologically mimics drought stress, which trehalose could mitigate. We found that R. solanacearum-infected plants differentially expressed drought-associated genes, including those involved in ABA and trehalose metabolism, and had more ABA in xylem sap. Consistent with this, treating tomato roots with ABA reduced both stomatal conductance and stem colonization by R. solanacearum. Treating roots with trehalose increased xylem sap ABA and reduced plant water use by lowering stomatal conductance and temporarily improving water use efficiency. Trehalose treatment also upregulated expression of salicylic acid (SA)-dependent tomato defense genes; increased xylem sap levels of SA and other antimicrobial compounds; and increased bacterial wilt resistance of SA-insensitive NahG tomato plants. Additionally, trehalose treatment increased xylem concentrations of jasmonic acid and related oxylipins. Finally, trehalose-treated plants were substantially more resistant to bacterial wilt disease. Together, these data show that exogenous trehalose reduced both water stress and bacterial wilt disease and triggered systemic disease resistance, possibly through a Damage Associated Molecular Pattern (DAMP) response pathway. This suite of responses revealed unexpected linkages between plant responses to biotic and abiotic stress and suggested that R. solanacearum-infected plants increase trehalose to improve water use efficiency and increase wilt disease resistance. The pathogen may degrade trehalose to counter these efforts. Together, these results suggest that treating tomatoes with exogenous trehalose could be a practical strategy for bacterial wilt management.

Xanthomonas species, Pseudomonas syringae and Ralstonia species are bacterial plant pathogens that cause significant yield loss in many crop species. Generating disease-resistant crop varieties can provide a more sustainable solution to control yield loss compared to chemical methods. Plant immune receptors encoded by nucleotide−binding, leucine−rich repeat (NLR) genes typically confer resistance to pathogens that produce a cognate elicitor, often an effector protein secreted by the pathogen to promote virulence. The diverse sequence and presence/absence variation of pathogen effector proteins within and between pathogen species usually limits the utility of a single NLR gene to protecting a plant from a single pathogen species or particular strains. The NLR protein Recognition of XopQ 1 (Roq1) was recently identified from the plant Nicotiana benthamiana and mediates perception of the effector proteins XopQ and HopQ1 from Xanthomonas and P. syringae respectively. Unlike most recognized effectors, alleles of XopQ/HopQ1 are highly conserved and present in most plant pathogenic strains of Xanthomonas and P. syringae. A homolog of XopQ/HopQ1, named RipB, is present in most Ralstonia strains. We found that Roq1 confers immunity to Xanthomonas, P. syringae, and Ralstonia when expressed in tomato. Strong resistance to Xanthomonas perforans was observed in three seasons of field trials with both natural and artificial inoculation. The Roq1 gene can therefore be used to provide safe, economical, and effective control of these pathogens in tomato and other crop species and reduce or eliminate the need for traditional chemical controls.

Virulence assays are powerful tools to study microbial pathogenesis in vivo. Good assays track disease development and, coupled with targeted mutagenesis, can identify pathogen virulence factors. Disease development in plants is extremely sensitive to environmental factors such as temperature, atmospheric humidity, and soil water level, so it can be challenging to standardize conditions to achieve consistent results. Here, we present optimized and validated experimental conditions and analysis methods for nine assays that measure specific aspects of virulence in the phytopathogenic bacterium Ralstonia solanacearum, using tomato as the model host plant.

Plants produce hydroxycinnamic acid (HCA) defense compounds to combat pathogens, such as the bacterium Ralstonia solanacearum. We showed that an HCA degradation pathway is genetically and functionally conserved across diverse R. solanacearum strains. Further, a feruloyl-CoA synthetase (Δfcs) mutant that cannot degrade HCA was less virulent on tomato plants. To understand the role of HCA degradation in bacterial wilt disease, we tested the following hypotheses: HCA degradation helps the pathogen i) grow, as a carbon source; ii) spread, by reducing HCA-derived physical barriers; and iii) survive plant antimicrobial compounds. Although HCA degradation enabled R. solanacearum growth on HCA in vitro, HCA degradation was dispensable for growth in xylem sap and root exudate, suggesting that HCA are not significant carbon sources in planta. Acetyl-bromide quantification of lignin demonstrated that R. solanacearum infections did not affect the gross quantity or distribution of stem lignin. However, the Δfcs mutant was significantly more susceptible to inhibition by two HCA, namely, caffeate and p-coumarate. Finally, plant colonization assays suggested that HCA degradation facilitates early stages of infection and root colonization. Together, these results indicated that ability to degrade HCA contributes to bacterial wilt virulence by facilitating root entry and by protecting the pathogen from HCA toxicity.

ABSTRACT The PhcA virulence regulator in the vascular wilt pathogen Ralstonia solanacearum responds to cell density via quorum sensing. To understand the timing of traits that enable R. solanacearum to establish itself inside host plants, we created a ΔphcA mutant that is genetically locked in a low-cell-density condition. Comparing levels of gene expression of wild-type R. solanacearum and the ΔphcA mutant during tomato colonization revealed that the PhcA transcriptome includes an impressive 620 genes (>2-fold differentially expressed; false-discovery rate [FDR], ≤0.005). Many core metabolic pathways and nutrient transporters were upregulated in the ΔphcA mutant, which grew faster than the wild-type strain in tomato xylem sap and on dozens of specific metabolites, including 36 found in xylem. This suggests that PhcA helps R. solanacearum to survive in nutrient-poor environmental habitats and to grow rapidly during early pathogenesis. However, after R. solanacearum reaches high cell densities in planta, PhcA mediates a trade-off from maximizing growth to producing costly virulence factors. R. solanacearum infects through roots, and low-cell-density-mode-mimicking ΔphcA cells attached to tomato roots better than the wild-type cells, consistent with their increased expression of several adhesins. Inside xylem vessels, ΔphcA cells formed aberrantly dense mats. Possibly as a result, the mutant could not spread up or down tomato stems as well as the wild type. This suggests that aggregating improves R. solanacearum survival in soil and facilitates infection and that it reduces pathogenic fitness later in disease. Thus, PhcA mediates a second strategic switch between initial pathogen attachment and subsequent dispersal inside the host. PhcA helps R. solanacearum optimally invest resources and correctly sequence multiple steps in the bacterial wilt disease cycle. IMPORTANCE Ralstonia solanacearum is a destructive soilborne crop pathogen that wilts plants by colonizing their water-transporting xylem vessels. It produces its costly virulence factors only after it has grown to a high population density inside a host. To identify traits that this pathogen needs in other life stages, we studied a mutant that mimics the low-cell-density condition. This mutant (the ΔphcA mutant) cannot sense its own population density. It grew faster than and used many nutrients not available to the wild-type bacterium, including metabolites present in tomato xylem sap. The mutant also attached much better to tomato roots, and yet it failed to spread once it was inside plants because it was trapped in dense mats. Thus, PhcA helps R. solanacearum succeed over the course of its complex life cycle by ensuring avid attachment to plant surfaces and rapid growth early in disease, followed by high virulence and effective dispersal later in disease.

ABSTRACT Plants use the signaling molecule salicylic acid (SA) to trigger defenses against diverse pathogens, including the bacterial wilt pathogen Ralstonia solanacearum. SA can also inhibit microbial growth. Most sequenced strains of the heterogeneous R. solanacearum species complex can degrade SA via gentisic acid to pyruvate and fumarate. R. solanacearum strain GMI1000 expresses this SA degradation pathway during tomato pathogenesis. Transcriptional analysis revealed that subinhibitory SA levels induced expression of the SA degradation pathway, toxin efflux pumps, and some general stress responses. Interestingly, SA treatment repressed expression of virulence factors, including the type III secretion system, suggesting that this pathogen may suppress virulence functions when stressed. A GMI1000 mutant lacking SA degradation activity was much more susceptible to SA toxicity but retained the wild-type colonization ability and virulence on tomato. This may be because SA is less important than gentisic acid in tomato defense signaling. However, another host, tobacco, responds strongly to SA. To test the hypothesis that SA degradation contributes to virulence on tobacco, we measured the effect of adding this pathway to the tobacco-pathogenic R. solanacearum strain K60, which lacks SA degradation genes. Ectopic addition of the GMI1000 SA degradation locus, including adjacent genes encoding two porins and a LysR-type transcriptional regulator, significantly increased the virulence of strain K60 on tobacco. Together, these results suggest that R. solanacearum degrades plant SA to protect itself from inhibitory levels of this compound and also to enhance its virulence on plant hosts like tobacco that use SA as a defense signal molecule. IMPORTANCE Plant pathogens such as the bacterial wilt agent Ralstonia solanacearum threaten food and economic security by causing significant losses for small- and large-scale growers of tomato, tobacco, banana, potato, and ornamentals. Like most plants, these crop hosts use salicylic acid (SA) both indirectly as a signal to activate defenses and directly as an antimicrobial chemical. We found that SA inhibits growth of R. solanacearum and induces a general stress response that includes repression of multiple bacterial wilt virulence factors. The ability to degrade SA reduces the pathogen’s sensitivity to SA toxicity and increases its virulence on tobacco.

Plant root border cells have been recently recognized as an important physical defense against soil-borne pathogens. Root border cells produce an extracellular matrix of protein, polysaccharide and DNA that functions like animal neutrophil extracellular traps to immobilize pathogens. Exposing pea root border cells to the root-infecting bacterial wilt pathogen Ralstonia solanacearum triggered release of DNA-containing extracellular traps in a flagellin-dependent manner. These traps rapidly immobilized the pathogen and killed some cells, but most of the entangled bacteria eventually escaped. The R. solanacearum genome encodes two putative extracellular DNases (exDNases) that are expressed during pathogenesis, suggesting that these exDNases contribute to bacterial virulence by enabling the bacterium to degrade and escape root border cell traps. We tested this hypothesis with R. solanacearum deletion mutants lacking one or both of these nucleases, named NucA and NucB. Functional studies with purified proteins revealed that NucA and NucB are non-specific endonucleases and that NucA is membrane-associated and cation-dependent. Single ΔnucA and ΔnucB mutants and the ΔnucA/B double mutant all had reduced virulence on wilt-susceptible tomato plants in a naturalistic soil-soak inoculation assay. The ΔnucA/B mutant was out-competed by the wild-type strain in planta and was less able to stunt root growth or colonize plant stems. Further, the double nuclease mutant could not escape from root border cells in vitro and was defective in attachment to pea roots. Taken together, these results demonstrate that extracellular DNases are novel virulence factors that help R. solanacearum successfully overcome plant defenses to infect plant roots and cause bacterial wilt disease.

Ralstonia solanacearum race 3 biovar 2 (R3bv2) is an economically important soilborne plant pathogen that causes bacterial wilt disease by infecting host plant roots and colonizing the xylem vessels. Little is known about R3bv2 behavior in the host rhizosphere and early in bacterial wilt pathogenesis. To explore this part of the disease cycle, we used a novel taxis-based promoter-trapping strategy to identify pathogen genes induced in the plant rhizosphere. This screen identified several rex (root exudate expressed) genes whose promoters were upregulated in the presence of tomato root exudates. One rex gene encodes an assembly protein for a high affinity cbb3-type cytochrome c oxidase (cbb3-cco) that enables respiration in low-oxygen conditions in other bacteria. R3bv2 cbb3-cco gene expression increased under low-oxygen conditions, and a cbb3-cco mutant strain grew more slowly in a microaerobic environment (0.5% O2). Although the cco mutant could still wilt tomato plants, symptom onset was significantly delayed relative to the wild-type parent strain. Further, the cco mutant did not colonize host stems or adhere to roots as effectively as wild type. These results suggest that R3bv2 encounters low-oxygen environments during its interactions with host plants and that the pathogen depends on this oxidase to help it succeed in planta.

Ralstonia solanacearum genes that are induced during tomato infection suggested that this pathogen encounters reactive oxygen species (ROS) during bacterial wilt pathogenesis. The genomes of R. solanacearum contain multiple redundant ROS-scavenging enzymes, indirect evidence that this pathogen experiences intense oxidative stress during its life cycle. Over 9% of the bacterium's plant-induced genes were also upregulated by hydrogen peroxide in culture, suggesting that oxidative stress may be linked to life in the plant host. Tomato leaves infected by R. solanacearum contained hydrogen peroxide, and concentrations of this ROS increased as pathogen populations increased. Mutagenesis of a plant-induced predicted peroxidase gene, bcp, resulted in an R. solanacearum strain with reduced ability to detoxify ROS in culture. The bcp mutant caused slightly delayed bacterial wilt disease onset in tomato. Moreover, its virulence was significantly reduced on tobacco plants engineered to overproduce hydrogen peroxide, demonstrating that Bcp is necessary for detoxification of plant-derived hydrogen peroxide and providing evidence that host ROS can limit the success of this pathogen. These results reveal that R. solanacearum is exposed to ROS during pathogenesis and that it has evolved a redundant and efficient oxidative stress response to adapt to the host environment and cause disease.

Background. Ralstonia solanacearum is an economically important plant pathogen with an unusually large host range. The Moko (banana) and NPB (not pathogenic to banana) strain groups are closely related but are adapted to distinct hosts. Previous comparative genomics studies uncovered very few differences that could account for the host range difference between these pathotypes. To better understand the basis of this host specificity, we used RNAseq to profile the transcriptomes of an R. solanacearum Moko strain and an NPB strain under in vitro and in planta conditions.Results. RNAs were sequenced from bacteria grown in rich and minimal media, and from bacteria extracted from mid-stage infected tomato, banana and melon plants. We computed differential expression between each pair of conditions to identify constitutive and host-specific gene expression differences between Moko and NPB. We found that type III secreted effectors were globally up-regulated upon plant cell contact in the NPB strain compared with the Moko strain. Genes encoding siderophore biosynthesis and nitrogen assimilation genes were highly up-regulated in the NPB strain during melon pathogenesis, while denitrification genes were up-regulated in the Moko strain during banana pathogenesis. The relatively lower expression of oxidases and the denitrification pathway during banana pathogenesis suggests that R. solanacearum experiences higher oxygen levels in banana pseudostems than in tomato or melon xylem.Conclusions. This study provides the first report of differential gene expression associated with host range variation. Despite minimal genomic divergence, the pathogenesis of Moko and NPB strains is characterized by striking differences in expression of virulence- and metabolism-related genes.

ABSTRACT Genomic data predict that, in addition to oxygen, the bacterial plant pathogen Ralstonia solanacearum can use nitrate (NO3−), nitrite (NO2−), nitric oxide (NO), and nitrous oxide (N2O) as terminal electron acceptors (TEAs). Genes encoding inorganic nitrogen reduction were highly expressed during tomato bacterial wilt disease, when the pathogen grows in xylem vessels. Direct measurements found that tomato xylem fluid was low in oxygen, especially in plants infected by R. solanacearum. Xylem fluid contained ~25 mM NO3−, corresponding to R. solanacearum's optimal NO3− concentration for anaerobic growth in vitro. We tested the hypothesis that R. solanacearum uses inorganic nitrogen species to respire and grow during pathogenesis by making deletion mutants that each lacked a step in nitrate respiration (ΔnarG), denitrification (ΔaniA, ΔnorB, and ΔnosZ), or NO detoxification (ΔhmpX). The ΔnarG, ΔaniA, and ΔnorB mutants grew poorly on NO3− compared to the wild type, and they had reduced adenylate energy charge levels under anaerobiosis. While NarG-dependent NO3− respiration directly enhanced growth, AniA-dependent NO2− reduction did not. NO2− and NO inhibited growth in culture, and their removal depended on denitrification and NO detoxification. Thus, NO3− acts as a TEA, but the resulting NO2− and NO likely do not. None of the mutants grew as well as the wild type in planta, and strains lacking AniA (NO2− reductase) or HmpX (NO detoxification) had reduced virulence on tomato. Thus, R. solanacearum exploits host NO3− to respire, grow, and cause disease. Degradation of NO2− and NO is also important for successful infection and depends on denitrification and NO detoxification systems. IMPORTANCE The plant-pathogenic bacterium Ralstonia solanacearum causes bacterial wilt, one of the world's most destructive crop diseases. This pathogen's explosive growth in plant vascular xylem is poorly understood. We used biochemical and genetic approaches to show that R. solanacearum rapidly depletes oxygen in host xylem but can then respire using host nitrate as a terminal electron acceptor. The microbe uses its denitrification pathway to detoxify the reactive nitrogen species nitrite (a product of nitrate respiration) and nitric oxide (a plant defense signal). Detoxification may play synergistic roles in bacterial wilt virulence by converting the host's chemical weapon into an energy source. Mutant bacterial strains lacking elements of the denitrification pathway could not grow as well as the wild type in tomato plants, and some mutants were also reduced in virulence. Our results show how a pathogen's metabolic activity can alter the host environment in ways that increase pathogen success.

An 8× draft genome was obtained and annotated for Ralstonia solanacearum race 3 biovar 2 (R3B2) strain UW551, a United States Department of Agriculture Select Agent isolated from geranium. The draft UW551 genome consisted of 80,169 reads resulting in 582 contigs containing 5,925,491 base pairs, with an average 64.5% GC content. Annotation revealed a predicted 4,454 protein coding open reading frames (ORFs), 43 tRNAs, and 5 rRNAs; 2,793 (or 62%) of the ORFs had a functional assignment. The UW551 genome was compared with the published genome of R. solanacearum race 1 biovar 3 tropical tomato strain GMI1000. The two phylogenetically distinct strains were at least 71% syntenic in gene organization. Most genes encoding known pathogenicity determinants, including predicted type III secreted effectors, appeared to be common to both strains. A total of 402 unique UW551 ORFs were identified, none of which had a best hit or >45% amino acid sequence identity with any R. solanacearum predicted protein; 16 had strong (E < 10-13) best hits to ORFs found in other bacterial plant pathogens. Many of the 402 unique genes were clustered, including 5 found in the hrp region and 38 contiguous, potential prophage genes. Conservation of some UW551 unique genes among R3B2 strains was examined by polymerase chain reaction among a group of 58 strains from different races and biovars, resulting in the identification of genes that may be potentially useful for diagnostic detection and identification of R3B2 strains. One 22-kb region that appears to be present in GMI1000 as a result of horizontal gene transfer is absent from UW551 and encodes enzymes that likely are essential for utilization of the three sugar alcohols that distinguish biovars 3 and 4 from biovars 1 and 2.

Swimming motility allows the bacterial wilt pathogen Ralstonia solanacearum to efficiently invade and colonize host plants. However, the bacteria are essentially nonmotile once inside plant xylem vessels. To determine how and when motility genes are expressed, we cloned and mutated flhDC, which encodes a major regulator of flagellar biosynthesis and bacterial motility. An flhDC mutant was non-motile and less virulent than its wild-type parent on both tomato and Arabidopsis; on Arabidopsis, the flhDC mutant also was less virulent than a nonmotile fliC flagellin mutant. Genes in the R. solanacearum motility regulon had strikingly different expression patterns in culture and in the plant. In culture, as expected, flhDC expression depended on PehSR, a regulator of early virulence factors; and, in turn, FlhDC was required for fliC (flagellin) expression. However, when bacteria grew in tomato plants, flhDC was expressed in both wild-type and pehR mutant backgrounds, although PehSR is necessary for motility both in culture and in planta. Both flhDC and pehSR were significantly induced in planta relative to expression levels in culture. Unexpectedly, the fliC gene was expressed in planta at cell densities where motile bacteria were not observed, as well as in a nonmotile flhDC mutant. Thus, expression of flhDC and flagellin itself are uncoupled from bacterial motility in the host environment, indicating that additional signals and regulatory circuits repress motility during plant pathogenesis.

The phytopathogenic bacterium Ralstonia solanacearum requires motility for full virulence, and its flagellin is a candidate pathogen-associated molecular pattern that may elicit plant defenses. Boiled extracts from R. solanacearum contained a strong elicitor of defense-associated responses. However, R. solanacearum flagellin is not this elicitor, because extracts from wild-type bacteria and fliC or flhDC mutants defective in flagellin production all elicited similar plant responses. Equally important, live R. solanacearum caused similar disease on Arabidopsis ecotype Col-0, regardless of the presence of flagellin in the bacterium or the FLS2-mediated flagellin recognition system in the plant. Unlike the previously studied flg22 flagellin peptide, a peptide based on the corresponding conserved N-terminal segment of R. solanacearum, flagellin did not elicit any response from Arabidopsis seedlings. Thus recognition of flagellin plays no readily apparent role in this pathosystem. Flagellin also was not the primary elicitor of responses in tobacco. The primary eliciting activity in boiled R. solanacearum extracts applied to Arabidopsis was attributable to one or more proteins other than flagellin, including species purifying at approximately 5 to 10 kDa and also at larger molecular masses, possibly due to aggregation. Production of this eliciting activity did not require hrpB (positive regulator of type III secretion), pehR (positive regulator of polygalacturonase production and motility), gspM (general secretion pathway), or phcA (LysR-type global virulence regulator). Wild-type R. solanacearum was virulent on Arabidopsis despite the presence of this elicitor in pathogen extracts.

The bacterial wilt pathogen Ralstonia solanacearum produces three extracellular polygalacturonases (PGs): PehA, PehB, and PehC. All three PGs hydrolyze pectin's polygalacturonic acid backbone, but each releases different reaction products. PehA and PehB contribute significantly to pathogen virulence, probably by facilitating root invasion and colonization. To determine the collective contribution of PGs to virulence and saprophytic survival, we cloned, characterized, and mutated the R. solanacearum pehC gene, which encodes a distinctive monogalacturonate-releasing exo-PG. The virulence of a pehC mutant on tomato was indistinguishable from that of its wild-type parent; thus, this exo-PG alone does not contribute significantly to wilt pathogenesis. Unexpectedly, a completely PG-deficient triple pehA/B/C mutant was slightly more virulent than a pehA/B mutant. PehC may degrade galacturonide elicitors of host defense, thereby protecting the pathogen from plant antimicrobial responses. A galacturonate transporter gene, exuT, is immediately downstream of pehC and the two genes are co-transcribed. It has been hypothesized that galacturonic acid released by PGs from plant cell walls nourishes bacteria during pathogenesis. To separate the pectolytic and nutrient-generating roles of the PGs, we made an exuT mutant, which still produces all three isozymes of PG but cannot uptake PG degradation products. This exuT mutant had wild-type virulence on tomato, demonstrating that metabolism of galacturonic acid does not contribute significantly to bacterial success inside the plant.

While most strains of the plant pathogenic bacterium Ralstonia solanacearum are tropical, the race 3 biovar 2 (R3bv2) subgroup attacks plants in cooler climates. To identify mechanisms underlying this trait, we compared the transcriptional profiles of R. solanacearum R3bv2 strain UW551 and tropical strain GMI1000 at 20°C and 28°C, both in culture and during tomato pathogenesis. 4.2% of the ORFs in the UW551 genome and 7.9% of the GMI1000 ORFs were differentially expressed by temperature in planta. The two strains had distinct transcriptional responses to temperature change. GMI1000 up-regulated several stress response genes at 20°C, apparently struggling to cope with plant defenses. At the cooler temperature, R3bv2 strain UW551 up-regulated a cluster encoding a mannose-fucose binding lectin, LecM; a quorum sensing-dependent protein, AidA; and a related hypothetical protein, AidC. The last two genes are absent from the GMI1000 genome. In UW551, all three genes were positively regulated by the adjacent SolI/R quorum sensing system. These temperature-responsive genes were required for full virulence in R3bv2. Mutants lacking lecM, aidA, or aidC were each significantly more reduced in virulence on tomato at 20°C than at 28°C in both a naturalistic soil soak inoculation assay and when they were inoculated directly into tomato stems. The lecM and aidC mutants also survived poorly in potato tubers at the seed tuber storage temperature of 4°C, and the lecM mutant was defective in biofilm formation in vitro. Together, these results suggest novel mechanisms, including a lectin, are involved in the unique temperate epidemiology of R3bv2.

We previously identified a locus that regulates production of polygalacturonase (PG), an extracellular plant cell wall-degrading enzyme important in bacterial wilt of plants caused by Ralstonia (Pseudomonas) solanacearum. The DNA sequence of this locus, called pehSR, was determined and two consecutive open reading frames (ORFs) of 1,905 and 1,680 bp were identified. The amino acid sequences predicted to be encoded by these ORFs are similar to those of regulators of pilin synthesis in Pseudomonas aeruginosa and Myxococcus xanthus and to a regulator of flagellin synthesis and adhesion in P. aeruginosa, as well as to other two-component regulators of the NtrB/C subfamily. pehSR mutants produced negligible levels of endo-PG activity, while exo-PG activity was reduced by 50%. Northern (RNA) blot analysis showed that PehSR regulates endo-PG expression at the transcriptional level. pehSR mutants grew normally in culture and in planta but were dramatically reduced in virulence; this loss of virulence was substantially greater than that observed for endo-PG structural gene mutants, suggesting that pehSR regulates additional factors important in virulence. Although pehSR mutants were essentially nonmotile, like the wild-type strain, multiple copies of pehSR conferred motility on the bacterium. Reporter gene studies indicated that pehSR expression increased when bacteria grew in plant tissue, and that the pehSR locus was itself negatively regulated by the global virulence gene regulator PhcA.

ABSTRACT During bacterial wilt of tomato, the plant pathogen Ralstonia solanacearum upregulates expression of popS, which encodes a type III-secreted effector in the AvrE family. PopS is a core effector present in all sequenced strains in the R. solanacearum species complex. The phylogeny of popS mirrors that of the species complex as a whole, suggesting that this is an ancient, vertically inherited effector needed for association with plants. A popS mutant of R. solanacearum UW551 had reduced virulence on agriculturally important Solanum spp., including potato and tomato plants. However, the popS mutant had wild-type virulence on a weed host, Solanum dulcamara, suggesting that some species can avoid the effects of PopS. The popS mutant was also significantly delayed in colonization of tomato stems compared to the wild type. Some AvrE-type effectors from gammaproteobacteria suppress salicylic acid (SA)-mediated plant defenses, suggesting that PopS, a betaproteobacterial ortholog, has a similar function. Indeed, the popS mutant induced significantly higher expression of tomato SA-triggered pathogenesis-related (PR) genes than the wild type. Further, pretreatment of roots with SA exacerbated the popS mutant virulence defect. Finally, the popS mutant had no colonization defect on SA-deficient NahG transgenic tomato plants. Together, these results indicate that this conserved effector suppresses SA-mediated defenses in tomato roots and stems, which are R. solanacearum’s natural infection sites. Interestingly, PopS did not trigger necrosis when heterologously expressed in Nicotiana leaf tissue, unlike the AvrE homolog DspEPcc from the necrotroph Pectobacterium carotovorum subsp. carotovorum. This is consistent with the differing pathogenesis modes of necrosis-causing gammaproteobacteria and biotrophic R. solanacearum. IMPORTANCE The type III-secreted AvrE effector family is widely distributed in high-impact plant-pathogenic bacteria and is known to suppress plant defenses for virulence. We characterized the biology of PopS, the only AvrE homolog made by the bacterial wilt pathogen Ralstonia solanacearum. To our knowledge, this is the first study of R. solanacearum effector function in roots and stems, the natural infection sites of this pathogen. Unlike the functionally redundant R. solanacearum effectors studied to date, PopS is required for full virulence and wild-type colonization of two natural crop hosts. R. solanacearum is a biotrophic pathogen that causes a nonnecrotic wilt. Consistent with this, PopS suppressed plant defenses but did not elicit cell death, unlike AvrE homologs from necrosis-causing plant pathogens. We propose that AvrE family effectors have functionally diverged to adapt to the necrotic or nonnecrotic lifestyle of their respective pathogens.

ABSTRACT Plant xylem fluid is considered a nutrient-poor environment, but the bacterial wilt pathogen Ralstonia solanacearum is well adapted to it, growing to 108 to 109 CFU/g tomato stem. To better understand how R. solanacearum succeeds in this habitat, we analyzed the transcriptomes of two phylogenetically distinct R. solanacearum strains that both wilt tomato, strains UW551 (phylotype II) and GMI1000 (phylotype I). We profiled bacterial gene expression at ~6 × 108 CFU/ml in culture or in plant xylem during early tomato bacterial wilt pathogenesis. Despite phylogenetic differences, these two strains expressed their 3,477 common orthologous genes in generally similar patterns, with about 12% of their transcriptomes significantly altered in planta versus in rich medium. Several primary metabolic pathways were highly expressed during pathogenesis. These pathways included sucrose uptake and catabolism, and components of these pathways were encoded by genes in the scrABY cluster. A UW551 scrA mutant was significantly reduced in virulence on resistant and susceptible tomato as well as on potato and the epidemiologically important weed host Solanum dulcamara. Functional scrA contributed to pathogen competitive fitness during colonization of tomato xylem, which contained ~300 µM sucrose. scrA expression was induced by sucrose, but to a much greater degree by growth in planta. Unexpectedly, 45% of the genes directly regulated by HrpB, the transcriptional activator of the type 3 secretion system (T3SS), were upregulated in planta at high cell densities. This result modifies a regulatory model based on bacterial behavior in culture, where this key virulence factor is repressed at high cell densities. The active transcription of these genes in wilting plants suggests that T3SS has a biological role throughout the disease cycle. IMPORTANCE Ralstonia solanacearum is a widespread plant pathogen that causes bacterial wilt disease. It inflicts serious crop losses on tropical farmers, with major economic and human consequences. It is also a model for the many destructive microbes that colonize the water-conducting plant xylem tissue, which is low in nutrients and oxygen. We extracted bacteria from infected tomato plants and globally identified the biological functions that R. solanacearum expresses during plant pathogenesis. This revealed the unexpected presence of sucrose in tomato xylem fluid and the pathogen’s dependence on host sucrose for virulence on tomato, potato, and the common weed bittersweet nightshade. Further, R. solanacearum was highly responsive to the plant environment, expressing several metabolic and virulence functions quite differently in the plant than in pure culture. These results reinforce the utility of studying pathogens in interaction with hosts and suggest that selecting for reduced sucrose levels could generate wilt-resistant crops.

The Ralstonia solanacearum species complex includes R. solanacearum, R. syzygii, and the Blood Disease Bacterium (BDB). All colonize plant xylem vessels and cause wilt diseases, but with significant biological differences. R. solanacearum is a soilborne bacterium that infects the roots of a broad range of plants. R. syzygii causes Sumatra disease of clove trees and is actively transmitted by cercopoid insects. BDB is also pathogenic to a single host, banana, and is transmitted by pollinating insects. Sequencing and DNA-DNA hybridization studies indicated that despite their phenotypic differences, these three plant pathogens are actually very closely related, falling into the Phylotype IV subgroup of the R. solanacearum species complex. To better understand the relationships among these bacteria, we sequenced and annotated the genomes of R. syzygii strain R24 and BDB strain R229. These genomes were compared to strain PSI07, a closely related Phylotype IV tomato isolate of R. solanacearum, and to five additional R. solanacearum genomes. Whole-genome comparisons confirmed previous phylogenetic results: the three phylotype IV strains share more and larger syntenic regions with each other than with other R. solanacearum strains. Furthermore, the genetic distances between strains, assessed by an in-silico equivalent of DNA-DNA hybridization, unambiguously showed that phylotype IV strains of BDB, R. syzygii and R. solanacearum form one genomic species. Based on these comprehensive data we propose a revision of the taxonomy of the R. solanacearum species complex. The BDB and R. syzygii genomes encoded no obvious unique metabolic capacities and contained no evidence of horizontal gene transfer from bacteria occupying similar niches. Genes specific to R. syzygii and BDB were almost all of unknown function or extrachromosomal origin. Thus, the pathogenic life-styles of these organisms are more probably due to ecological adaptation and genomic convergence during vertical evolution than to the acquisition of DNA by horizontal transfer.

Ralstonia solanacearum, which causes bacterial wilt of diverse plants, produces copious extracellular polysaccharide (EPS), a major virulence factor. The function of EPS in wilt disease is uncertain. Leading hypotheses are that EPS physically obstructs plant water transport, or that EPS cloaks the bacterium from host plant recognition and subsequent defense. Tomato plants infected with R. solanacearum race 3 biovar 2 strain UW551 and tropical strain GMI1000 upregulated genes in both the ethylene (ET) and salicylic acid (SA) defense signal transduction pathways. The horizontally wilt-resistant tomato line Hawaii7996 activated expression of these defense genes faster and to a greater degree in response to R. solanacearum infection than did susceptible cultivar Bonny Best. However, EPS played different roles in resistant and susceptible host responses to R. solanacearum. In susceptible plants the wild-type and eps(-) mutant strains induced generally similar defense responses. But in resistant Hawaii7996 tomato plants, the wild-type pathogens induced significantly greater defense responses than the eps(-) mutants, suggesting that the resistant host recognizes R. solanacearum EPS. Consistent with this idea, purified EPS triggered significant SA pathway defense gene expression in resistant, but not in susceptible, tomato plants. In addition, the eps(-) mutant triggered noticeably less production of defense-associated reactive oxygen species in resistant tomato stems and leaves, despite attaining similar cell densities in planta. Collectively, these data suggest that bacterial wilt-resistant plants can specifically recognize EPS from R. solanacearum.

Multidrug efflux pumps (MDRs) are hypothesized to protect pathogenic bacteria from toxic host defense compounds. To study these hypothesis mutations are created in the Ralstonia solanacearum acrA and dinF genes which encode putative MDRs in the broad host range plant pathogen.