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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.

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. [Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

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. [Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Ralstonia solanacearum phylotype II sequevar 1 (RsII-1, formerly race 3 biovar 2) causes tomato bacterial wilt, potato brown rot, and Southern wilt of geranium. Strains in RsII-1 cause wilting in potato and tomato at cooler temperatures than tropical lowland R. solanacearum strains. Although periodically introduced, RsII-1 has not established in the United States. This pathogen is of quarantine concern and listed as a Federal Select Agent. We report a rapidly sequenced (

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.

Detecting and correctly identifying Ralstonia solanacearum in infected plants is important because the race 3 biovar 2 (R3bv2) subgroup is a high-concern quarantine pathogen, while the related sequevar 7 group is endemic to the southeastern United States. Preventing accidental import of R3bv2 in geranium cuttings demands sensitive detection methods that are suitable for large-volume use both onshore and offshore. However, detection is complicated by frequent asymptomatic latent infections, uneven pathogen distribution within infected plants, pathogen viable-but-not-culturable state, and biosecurity laws that restrict transport of R3bv2 strains for diagnosis. There are many methods to detect R3bv2 strains but their relative utility is unknown, particularly in the realistic context of infected plant hosts. Therefore, we compared the sensitivity, cost, and technical complexity of several assays to detect and distinguish R3bv2 and sequevar 7 strains of R. solanacearum in geranium, tomato, and potato tissue in the laboratory and in naturally infected tomato plants from the field. The sensitivity of polymerase chain reaction (PCR)-based methods in infected geranium tissues was significantly improved by use of Kapa3G Plant, a polymerase with enhanced performance in the presence of plant inhibitors. R3bv2 cells were killed within 60 min of application to Whatman FTA(R) nucleic acid-binding cards, suggesting that samples on FTA cards can be safely transported for diagnosis. Overall, culture enrichment followed by dilution plating was the most sensitive detection method (101 CFU/ml) but it was also most laborious. Conducting PCR from FTA cards was faster, easier, and sensitive enough to detect approximately 104 CFU/ml, levels similar to those found in latently infected geranium plants.

A strain of Ralstonia solanacearum was associated with wilting and blight of flowering ornamentals grown from cuttings that originated in Guatemala. This isolate, UW757, could wilt geranium, Osteospermum spp., and mandevilla, but was more aggressively virulent on tomato. Although it rarely caused symptoms, after inoculation UW757 colonized the stems of 11 widely grown ornamental species, reaching mean populations as high as 109 CFU/g stem at the common tropical highland production temperature of 24°C. UW757 belongs to phylotype I, sequevar 14 of the R. solanacearum species complex, not to the Race 3 biovar 2 Select Agent group that also infects geraniums. Whole genome sequencing revealed that UW757 is closely related to several Asian R. solanacearum strains and also to P781, a mandevilla-infecting strain established in Florida. Strains similar to UW757 were isolated from tomato in Guatemala in 2006 and 2015, indicating that this group is common and persistent. This finding enlarges the known host range of R. solanacearum, which was not previously known to infect Osteospermum, Vinca, or Sutera spp. Growers should be aware that a broad range of ornamental plants, including previously unknown hosts, can be latently infected by non-Race 3 biovar 2 strains of R. solanacearum that are also highly virulent on tomato. Accepted for publication 28 April 2016. Published 26 May 2016.

Plants produce hydroxycinnami c acid (HCA) defense com- pounds to combat pathogens, such as the bacterium Ral- stonia solanacearum . We showed that an HCA degradation pathway is genetically and functionally conserved across diverse R. solanacearum strains. Further, a feruloyl-CoA synthetase ( Δ fcs ) mutant that canno t 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 patho- gen i) grow, as a carbon source; ii) spread, by reducing HCA-derived physical barriers; and iii) survive plant anti- microbial compounds. Although HCA degradation enabled R. solanacearum growth on HCA in vitro, HCA degrada- tion was dispensable for growth in xylem sap and root exu- date, 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 Δ fcs mutant was significantly more suscepti- ble to inhibition by two HCA, namely, caffeate and p -couma- rate. 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.

In January 2019, wilted tomato plants were discovered in fields in the Chi Kraeng district of Cambodia, 60 km south of Siem Reap. We observed vascular browning from cut stems from symptomatic plants, which tested positive for Ralstonia solanacearum with the Rs Immunostrip (Agdia, Elkhart, IN). Although bacterial wilt was noted in Cambodia (UK-CAB-International 1977), there has been no official report, and tomato farmers have reported historical losses up to 100% from a disease called sropoun sros (“fresh wilt” or in Khmer). Fluidal, irregular bacterial colonies typical of the R. solanacearum species complex (RSSC) were isolated from cut tomato stems on casamino peptone glucose (CPG) plates amended with 1% 2,3,5-triphenyl tetrazolium chloride (Schaad et al. 2001). To confirm the bacterial identification and to subclassify isolates by phylotype, genomic DNA from eight distinct isolates was amplified with the diagnostic RSSC multiplex PCR primers 759/760, Nmult21:1F, Nmult21:2F, βNmult23:AF, Nmult22:InF, and Nmult22:RR (Fegan and Prior 2005; Opina et al. 1997). Positive controls for each RSSC phylotype were genomic DNA from RSSC strains GMI1000 (phylotype I), K60 (phylotype II), CMR15 (phylotype III), and PSI07 (phylotype IV). Each Cambodian isolate yielded the 280-bp RSSC-specific DNA fragment and the 144-bp phylotype I-specific DNA fragment from this diagnostic multiplex PCR. Therefore, all Cambodian isolates were identified as phylotype I, which was recently renamed R. pseudosolanacearum (Prior et al. 2016; Safni et al. 2014). To perform Koch’s postulates, a single fresh colony from each isolate was cultured overnight in CPG broth at 28°C with 220 rpm shaking, pelleted, and resuspended in sterile water to O.D.₆₀₀ ₙₘ = 0.1 (∼1 × 10⁸ CFU/ml). Unwounded 33-day-old tomato plants (wilt-susceptible cultivar Bonny Best) were inoculated via soil soak with 50 ml of inoculum to a final density of ∼3.1 × 10⁷ CFU/g of soil. Two plants were inoculated per isolate, and four plants were treated with sterile water as negative controls. Fourteen out of 16 tomato plants showed wilting symptoms after 14 days, and all plants were wilted after 21 days. These results are representative of two replicates. No water-treated control plants wilted. R. pseudosolanacearum colonies were successfully reisolated from the wilted tomato plants. Sropoun sros or bacterial wilt has likely been a longstanding problem for Cambodian farmers. Characterizing and reporting bacterial wilt of tomato in Cambodia will be the foundation for future management strategies and the development of tomato varieties specifically resistant to Cambodian R. pseudosolanacearum.

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.

In 2003 and 2004, 15 isolates of Ralstonia solanacearum were obtained from wilting plants of field-grown pepper (Capsicum annuum) in south Florida and from pot-grown hydrangea (Hydrangea paniculata and H. macrophylla) and geranium (Pelargonium × hortorum) in commercial nurseries and retention ponds in north Florida. Diagnostic immunoassays and polymerase chain reaction (PCR) analyses identified all the isolates as R. solanacearum but not race 3 biovar 2. Pathogenicity studies on tomato, pepper, and tobacco revealed that all 15 strains had similar high virulence on tomato and all caused wilting of tobacco, although there were significant differences among the strains in aggressiveness on tobacco. An indigenous Florida tomato strain, race 1 biovar 1 (Rs5), caused no disease on tobacco and little or none on pepper. The three pepper strains were more aggressive than Rs5 or two hydrangea strains on all three pepper cultivars studied. Phylogenetic analysis based on an endoglucanase gene sequence indicated that these strains had three distinct origins. The three pepper strains belonged to phylotype I biovar 3 and clustered with strains from diverse hosts in Asia belonging to sequevar 13. The six geranium strains and four of the hydrangea strains were closely related to strains in sequevar 5, a distinct subcluster of phylotype II biovar 1 strains isolated from the French West Indies and Brazil. Two other biovar 1 strains from hydrangea and strains K60, AW, and Rs5 belonged to sequevar 7 in phylotype II and probably are native to North America. None of the Florida isolates belong to the highly regulated Select Agent race 3 biovar 2 subgroup, according to both the DNA sequence analysis and the biovar phenotypic test results. However, the race 3 biovar 2-specific B2 primers weakly amplified a product from some race 1 biovar 1 strains in real-time PCR, indicating that this assay may give false positives under some conditions. Given the high cost of a misdiagnosis, it seems advisable to use at least two independent diagnostic methods to confirm that a suspect isolate is R. solanacearum R3B2. This is the first report of the presence of R. solanacearum race 1 biovar 3 or phylotype I strains in North America, and the first report confirming R. solanacearum causing natural infection of hydrangea in Florida. Thus, R. solanacearum strains that are quite distinct from presumably indigenous strains are present and can infect diverse hosts in Florida.

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 bacterial wilt pathogen Ralstonia solanacearum causes major agricultural losses on many crop hosts worldwide. Resistance breeding is the best way to control bacterial wilt disease, but the biological basis for bacterial wilt resistance is unknown. We found that R. solanacearum uses an AvrE-family, Type III-secreted effector called PopS to overcome plant defenses and cause disease on tomato. Orthologs of PopS are widely conserved across distinct classes of plant pathogenic bacteria and could provide novel, durable targets for resistance. Accepted for publication 25 September 2013. Published 25 November 2013.

R. solanacearum Race 3 biovar 2, which causes potato brown rot and Southern wilt of geranium, is a high-consequence quarantine pathogen in Europe and North America and causes large crop losses in tropical highlands. This practical review emphasizes prevention, detection, diagnosis, and effective management of diseases caused by this destructive pathogen. Accepted for publication 17 January 2009. Published 13 March 2009.

Ralstonia solanacearum requires a Type III (T3) secretion system for bacterial wilt pathogenesis, but the biological functions of individual effectors remain unknown. During tomato wilt, R. solanacearum expresses popS, which encodes an AvrE-family T3 effector. popS homologs were present in all 17 sequenced R. solanacearum strains, and the phylogeny of popS parallels that of the R. solanacearum species complex, suggesting that PopS is an ancient effector needed for association with plants. We determined that popS is required for full virulence on multiple Solanum crop hosts (susceptible potato and susceptible and quantitatively resistant tomato), but not for virulence on a related epidemiologically relevant weed, S. dulcamara. The popS mutant was also significantly delayed in tomato stem colonization following direct inoculation through cut petioles. AvrE-type effectors in other plant pathogenic bacteria suppress plant defenses triggered by the plant signaling molecule salicylic acid (SA). The popS mutant induced higher expression of SAresponsive tomato PR genes than its wild-type parent. Further, pretreating plant roots with SA exacerbated the popS virulence defect. Finally, PopS was dispensable for bacterial colonization of SA-deficient NahG transgenic tomato plants. These results indicate that this conserved T3 effector suppresses SAmediated defenses in tomato roots and stems, which are the natural infection courts of this soilborne vascular pathogen. (texte integral)