Strains Of Ralstonia Solanacearum Research Articles

Involvement of avirulence genes avrA and popP1 of Japanese Ralstonia solanacearum strains in the pathogenicity to tobacco

We investigated the involvement of two avirulence genes, avrA (ripAA in unified nomenclature) and popP1 (ripP1 in unified nomenclature), of Japanese Ralstonia solanacearum strains in the pathogenicity to tobacco. One virulent strain OE1-1 and four hypersensitive response (HR)-eliciting strains, 8107, MAFF 211471, MAFF 211496, and MAFF 301520, were used. While 8107 and MAFF 211471 contain popP1, other three strains do not. When popP1 of strain 8107 was transferred into the virulent strain OE1-1, the transconjugant strain had significantly reduced virulence but did not become a HR-eliciting strain. We deleted avrA and/or popP1 from the HR-eliciting strains; all deletion mutants still elicited a HR and did not become virulent to tobacco, although leaf lesion appearance was delayed on infection with avrA mutants. These results indicate that although avrA and popP1 could be functional as avirulence determinants, other unidentified factors are necessary for full virulence or HR elicitation by Japanese R. solanacearum strains.

Genetic diversity reflects geographical origin of Ralstonia solanacearum strains isolated from plant and water sources in Spain.

The characterization and intraspecific diversity of a collection of 45 Ralstonia solanacearum strains isolated in Spain from different sources and geographical origins is reported. To test the influence of the site and the host on strain diversity, phenotypic and genotypic analysis were performed by a polyphasic approach. Biochemical and metabolic profiles were compared. Serological relationship was evaluated by Indirect-ELISA using polyclonal and monoclonal antibodies. For genotypic analysis, hrpB and egl DNA sequence analysis, repetitive sequences (rep-PCR), amplified fragment length polymorphism (AFLP) profiles and macrorestriction with XbaI followed by pulsed field gel electrophoresis (PFGE) were performed. The biochemical and metabolic characterization, serological tests, rep-PCR typing and phylogenetic analysis showed that all analysed strains belonged to phylotype II sequevar 1 and shared homogeneous profiles. However, interesting differences among strains were found by AFLP and macrorestriction with XbaI followed by PFGE techniques, some profiles being related to the geographical origin of the strains. Diversity results obtained offer new insights into the biogeography of this quarantine organism and its possible sources and reservoirs in Spain and Mediterranean countries.

Ralfuranones contribute to mushroom-type biofilm formation by Ralstonia solanacearum strain OE1-1.

After invasion into intercellular spaces of tomato plants, the soil-borne, plant-pathogenic Ralstonia solanacearum strain OE1-1 forms mushroom-shaped biofilms (mushroom-type biofilms, mBFs) on tomato cells, leading to its virulence. The strain OE1-1 produces aryl-furanone secondary metabolites, ralfuranones (A, B, J, K and L), dependent on the quorum sensing (QS) system, with methyl 3-hydroxymyristate (3-OH MAME) synthesized by PhcB as a QS signal. Ralfuranones are associated with the feedback loop of the QS system. A ralfuranone productivity-deficient mutant (ΔralA) exhibited significantly reduced growth in intercellular spaces compared with strain OE1-1, losing its virulence. To analyse the function of ralfuranones in mBF formation by OE1-1 cells, we observed cell aggregates of R.solanacearum strains statically incubated in tomato apoplast fluids on filters under a scanning electron microscope. The ΔralA strain formed significantly fewer microcolonies and mBFs than strain OE1-1. Supplementation of ralfuranones A, B, J and K, but not L, significantly enhanced the development of mBF formation by ΔralA. Furthermore, a phcB- and ralA-deleted mutant (ΔphcB/ralA) exhibited less formation of mBFs than OE1-1, although a QS-deficient, phcB-deleted mutant formed mBFs similar to OE1-1. Supplementation with 3-OH MAME significantly reduced the formation of mBFs by ΔphcB/ralA. The application of each ralfuranone significantly increased the formation of mBFs by ΔphcB/ralA supplied with 3-OH MAME. Together, our findings indicate that ralfuranones are implicated not only in the development of mBFs by strain OE1-1, but also in the suppression of QS-mediated negative regulation of mBF formation.

Molecular and biological characterization of ϕRs551, a filamentous bacteriophage isolated from a race 3 biovar 2 strain of Ralstonia solanacearum.

A filamentous bacteriophage, designated ϕRs551, was isolated and purified from the quarantine and select agent phytopathogen Ralstonia solanacearum race 3 biovar 2 strain UW551 (phylotype IIB sequevar 1) grown under normal culture conditions. Electron microscopy suggested that ϕRs551 is a member of the family Inoviridae, and is about 1200 nm long and 7 nm wide. ϕRs551 has a genome of 7929 nucleotides containing 14 open reading frames, and is the first isolated virion that contains a resolvase (ORF13) and putative type-2 phage repressor (ORF14). Unlike other R. solanacearum phages isolated from soil, the genome sequence of ϕRs551 is not only 100% identical to its prophage sequence in the deposited genome of R. solanacearum strain UW551 from which the phage was isolated, but is also surprisingly found with 100% identity in the deposited genomes of 10 other phylotype II sequevar 1 strains of R. solanacearum. Furthermore, it is homologous to genome RS-09-161, resulting in the identification of a new prophage, designated RSM10, in a R. solanacearum strain from India. When ORF13 and a core attP site of ϕRs551 were either deleted individually or in combination, phage integration was not observed, suggesting that similar to other filamentous R. solanacearum ϕRSM phages, ϕRs551 relies on its resolvase and the core att sequence for site-directed integration into its susceptible R. solanacearum strain. The integration occurred four hours after phage infection. Infection of a susceptible R. solanacearum strain RUN302 by ϕRs551 resulted in less fluidal colonies and EPS production, and reduced motilities of the bacterium. Interestingly, infection of RUN302 by ϕRs551 also resulted in reduced virulence, rather than enhanced or loss of virulence caused by other ϕRSM phages. Study of bacteriophages of R. solanacearum would contribute to a better understanding of the phage-bacterium-environment interactions in order to develop integrated management strategies to combat R. solanacearum.

Three Draft Genome Sequences of the Bacterial Plant Pathogen Ralstonia solanacearum, Isolated in Georgia.

ABSTRACTRalstonia solanacearum, the causative agent of bacterial wilt, is a devastating bacterial plant pathogen with a wide range of hosts. We report here the first draft genome sequences for three strains of Ralstonia solanacearum isolated from infected potato, tomato, and pepper plants in Georgia.

Distribution, pathological and biochemical characterization of Ralstonia solanacearum in Benin

In 2006 and 2007, 75 strains of Ralstonia solanacearum were collected from wilting tomato, pepper and eggplant in Benin. The distribution and the incidence of tomato bacterial wilt in the field were assessed by counting wilted tomato plants on 3 plots of 50m2 per field. The isolated bacterial strains, including the reference strain, were identified using ELISA, pathogenicity test and carbohydrate oxidation. Bacterial wilt is widely distributed in Benin and was found in five of the eight agro-ecological zones (AEZ) of Benin, which correspond to eight of the 12 districts of Benin. The disease was more severe in ferralitic soil (AEZ V), in valleys and lowlands (AEZ IV) and in highlands (AEZ I). The incidence of tomato bacterial wilt was up to 71%. No R. solanacearum strains were isolated from AEZ II, AEZ VII and AEZ VIII. Strains identified as R. solanacearum were more widely distributed in the south than the center and the north of Benin. Based on biochemical characteristics, Beninese R. solanacearum strains were grouped into biovar I/race1 and biovar III/race 1.

Genetic diversity of Ralstonia solanacearum strains causing bacterial wilt of solanaceous crops in Myanmar

In 2013 and 2014, an extensive survey of bacterial wilt in Myanmar was performed, and 70 strains of Ralstonia solanacearum (Rs) were collected from wilting plants of tomato, potato, chili and eggplant. Myanmar Rs strains were characterized by traditional and molecular methods. Polymerase chain reaction (PCR) test using Rs-specific primer set amplified one specific band (281-bp) from template DNA of all strains. Pathogenicity tests on the four solanaceous plants differentiated the strains into six pathogenic groups. Biovar determination tests showed that biovar 3 strains predominated (63%) among all Rs strains. Biovar 4 strains (7%) were obtained from both tomato and chili strains, whereas biovar 2 (30%) strains were isolated only from potato. Multiplex-PCR analysis indicated that tomato, eggplant and chili strains belonged to phylotype I, whereas potato strains comprised phylotype I and phylotype II. Strains in phylotype I, which was suggested to have originated from Asia, were the most prevalent in all surveyed areas. Phylogenetic analysis based on the endoglucanase (egl) gene sequences revealed that Myanmar strains partitioned into two major clusters that corresponded to phylotype I and II. Strains in phylotype I were further divided into seven subclusters, each corresponding to a distinct sequevar (15, 17, 46, 47, 48, unknown 1 or unknown 2). All strains in phylotype II belonged to sequevar 1. This is the first comprehensive report of the presence of diverse Rs strains in Myanmar.

Genetic diversity of Ralstonia solanacearum strains from Mexico associated with Moko disease

The Ralstonia solanacearum species complex is one of the most destructive bacterial plant pathogens for many crops. Cavendish banana is an important staple food which is severely impacted by the bacterial wilt Moko disease, caused by R. solanacearum race 2. In Mexico, banana plantations comprise 75,000 ha, supporting the economy of more than 100,000 families. Moko disease is present in the states of Chiapas and Tabasco, which are responsible for >60% of the total banana production. Each year, thousands of diseased plants are eradicated there because of Moko disease. The goal of this study was to genotype and characterizes Mexican Moko strains. Most of the strains were pathogenic to banana (PB), but two strains were found to be unable to develop wilt symptoms (NPB). A phylogenetic tree based on egl placed all Mexican strains in phylotype IIA, sequevar 6. Mexican Moko strains share identical egl sequence with strains from Central America and Caribbean countries, supporting the Caribbean origin proposed for sequevar 6 by some authors. The Mexican Seq6 nonpathogenic to banana (6NPB) and pathogenic (6 PB) strains were clustered apart in sister clades, in hrpB and pga phylogenetic trees, suggesting that this lineage is under divergence in Mexico. This is the first report worldwide of race 2 NPB strains genotyped in sequevar 6, and extends the currently known phenotypic diversity of R. solanacearum.

Foliar Applications of Acibenzolar-S-Methyl Negatively Affect the Yield of Grafted Tomatoes in Fields Infested with Ralstonia solanacearum.

Three field experiments were conducted in Florida from 2012-2014 to assess the impact of acibenzolar-S-methyl (ASM), a systemic acquired-resistance inducer, applied as foliar spray or through drip-irrigation lines, on bacterial wilt incidence and yield of grafted tomatoes. The experiments were conducted in a field with race 1, biovar 1 strain of Ralstonia solanacearum, causal agent of tomato bacterial wilt. In all three experiments, the susceptible tomato variety BHN 602, grafted onto a resistant rootstock BHN 998, was compared with nongrafted BHN 602, treated with or without foliar applications of ASM and with grafted plants treated with foliar applications of ASM. In two experiments, an additional treatment of drip applications of ASM on grafted and nongrafted plants was evaluated. Grafting alone or in combination with drip applications of ASM (178.6 μM) significantly reduced disease incidence and increased total marketable yield relative to nongrafted treatments. There were no significant differences between grafted plants with or without drip ASM applications in terms of bacterial wilt incidence or total marketable yield. However, we demonstrate for the first time that foliar ASM applications on grafted plants negatively affects the total marketable yield compared with drip ASM applications on grafted plants or nontreated grafted control.

Genetic and Pathogenic Diversity of Ralstonia solanacearum Causing Potato Brown Rot in China

Causing potato brown rot, Ralstonia solanacearum (R. solanacearum) strains are reported as one of the most destructive bacteria to potato (Solanum tuberosum L.) in China. In this study, 113 strains were isolated from potato, collected in the four major agroecological zones in China. The study showed that 102 strains belonged to the phylotype IIB sequevar 1 (race 3 biovar 2). The 11 remaining strains belonged to the phylotype I, sequevar 13, 17, 18, 16 or 14 M, a new sequevar closely related to sequevar 14. Thirty-four strains were further characterized according to their virulence at low temperature on three wild potato species. IIB-1 strains all belonged to high and moderate virulence, while others belonged to the low virulence group, which had limited pathogenicity.

Involvement of ralfuranones in the quorum sensing signalling pathway and virulence of Ralstonia solanacearum strain OE1-1.

The soil-borne, plant-pathogenic Ralstonia solanacearum strain OE1-1 produces and secretes methyl 3-hydroxymyristate (3-OH MAME) as a quorum sensing (QS) signal, which contributes to its virulence. A global virulence regulator, PhcA, functioning through the QS system, positively regulates the expression of ralA, which encodes furanone synthase, to produce aryl-furanone secondary metabolites, ralfuranones. A ralfuranone-deficient mutant (ΔralA) is weakly virulent when directly inoculated into tomato xylem vessels. To investigate the functions of ralfuranones, we analysed R. solanacearum transcriptome data generated by RNA sequencing technology. ΔralA expressed phcB, which is associated with 3-OH MAME production, and phcA at levels similar to those in strain OE1-1. In addition, ΔralA exhibited down-regulated expression of more than 90% of the QS positively regulated genes, and up-regulated expression of more than 75% of the QS negatively regulated genes. These results suggest that ralfuranones affect the QS feedback loop. Ralfuranone supplementation restored the ability of ΔralA cells to aggregate. In addition, ralfuranones A and B restored the swimming motility of ΔralA to wild-type levels. However, the application of exogenous ralfuranones did not affect the production of the major exopolysaccharide, EPS I, in ΔralA. Quantitative real-time polymerase chain reaction assays revealed that the deletion of ralA results in the down-regulated expression of vsrAD and vsrBC, which encode a sensor kinase and a response regulator, respectively, in the two-component regulatory systems that influence EPS I production. The application of ralfuranone B restored the expression of these two genes. Overall, our findings indicate that integrated signalling via ralfuranones influences the QS and virulence of R.solanacearum.

Characterization and evaluation of Bacillus amyloliquefaciens strain WF02 regarding its biocontrol activities and genetic responses against bacterial wilt in two different resistant tomato cultivars.

Bacillus amyloliquefaciens strain WF02, isolated from soil collected at Wufeng Mountain, Taiwan, has siderophore-producing ability and in vitro antagonistic activity against bacterial wilt pathogen. To determine the impact of plant genotype on biocontrol effectiveness, we treated soil with this strain before infecting susceptible (L390) and moderately resistant (Micro-Tom) tomato cultivars with Ralstonia solanacearum strain Pss4. We also compared the efficacy of this strain with that of commercial Bacillus subtilis strain Y1336. Strain WF02 provided longer lasting protection against R. solanacearum than did strain Y1336 and controlled the development of wilt in both cultivars. To elucidate the genetic responses in these plants under WF02 treatment, we analyzed the temporal expression of defense-related genes in leaves. The salicylic acid pathway-related genes phenylalanine ammonia-lyase and pathogenesis-related protein 1a were up-regulated in both cultivars, whereas expression of the jasmonic acid pathway-related gene lipoxygenase was only elevated in the susceptible tomato cultivar (L390). These results suggest that WF02 can provide protection against bacterial wilt in tomato cultivars with different levels of disease resistance via direct and indirect modes of action.

Identification and analysis of differentially expressed genes in Solanum lines in response to challenge with a Ralstonia solanacearum phylotype I strain

Identification of differentially expressed genes is a direct approach to understand the molecular basis of physiological processes. In this study, the GeneFishing™ PCR technique was used to identify genes which were differentially expressed in two Solanum lycopersicum lines, the resistant Lycopersicon cerasiforme and a susceptible cultivar known as Quatre carrées, after inoculation with a phylotype I strain of Ralstonia solanacearum. With twenty annealing control primers (ACPs), nineteen differentially expressed genes (DEGs) were identified, 17 of which were from the L. cerasiforme line and 2 from the Quatre carrées cultivar. Six DEGs (DEG1, DEG5, DEG8, DEG12, DEG15 and DEG18) were chosen for further studies based on the blast results. Primers were designed for the six DEGs and quantitative real-time PCR was carried out to confirm the differential expression. Expression of the six DEGs was also tested in Solanum commersonii, the Solanum tuberosum cultivar, Spunta and the S. lycopersicum lines MST 32/1, CRA 66 and Hawaii 7996. Level of expression was closely related to the phenotype of the plants, in terms of susceptibility and resistance to the pathogen. The resistant lines L. cerasiforme, S. commersonii, CRA 66 and Hawaii 7996 always had higher levels of gene expression than the moderately resistant MST 32/1 cultivar and the susceptible Quatre carrées and Spunta cultivars.

Simplified recovery process of Ralstonia solanacearum-synthesized polyhydroxyalkanoates via chemical extraction complemented by liquid-liquid phase separation

Poly (3-hydroxybutyrate) (P(3HB)) is the most studied thermoplastic biopolymer belonging to the polyhydroxyalkanoate (PHA) family, the main features of which include rapid biodegradability and biocompatibility. The bioplastic recovery process is an important step during production and can directly influence the characteristics of PHAs. However, more efficient methods for the production of P(3HB) are necessary to make it economically viable. The aim of the present study was to improve the standard, chloroform-based, extraction step for the recovery of P(3HB). The polymer was produced using a Ralstonia solanacearum strain. The following parameters were improved in the recovery process: heating time, separation method (filtration or liquid-liquid phase separation), biomass state (fresh or dry cell concentrate) and the solvent:biomass ratio. By improving the chemical extraction of P(3HB) we recovered 98% of the cumulative polymer and reduced the heating time by 75%. Furthermore, we improved the separation process and developed an extraction solution that was faster and more economical.

Bacterial Wilt of Tomato (Solanum lycopersicum) in Louisiana is Caused by Phylotype II, Biovar 1 and 3 strains of Ralstonia solanacearum

Bacterial Wilt of Tomato (<i>Solanum lycopersicum</i>) in Louisiana is Caused by Phylotype II, Biovar 1 and 3 strains of <i>Ralstonia solanacearum</i>

In vitro screening of plant growth promoting rhizobacteria to control bacterial wilt (Ralstonia solanacearum) of tomato (Lycopersicon esculentum)

Ralstonia solanacearum is the causative agent of bacterial wilt that causes considerable damages in the yield of various crop plants. The intent of the study was to evaluate potential of bacterial antagonists to suppress bacterial wilt disease development and evaluate the role of the strains as plant growth-promoting rhizobacteria (PGPR) in tomato. One hundred rhizosphere bacterial isolates were screened against virulent strain of Ralstonia solanacearum . After in vitro screening 10 antagonistic strains designated PR3, PR17, PR26, 1NAB15, 1NAB20, 3NAA1, 3NAA2, 2CBA2, 2CBA4, 2CBA18 showed antagonistic effect by producing inhibition zone supressing the growth of R. Solanacearum . The isolate PR3 showed the highest inhibition zone measuring 33.3mm whereas 1NAB15 showed the lowest zone measuring 10mm. The present study, therefore, suggests that the use of PGPR isolates which showed the antagonistic activity can be used as inoculants/ bioantagonists might be beneficial for the control of bacterial wilt of tomato in field studies.

Evaluation of the antimicrobial activity of 9-oxo-agerophorone against soil borne pathogens

9-Oxo-agerophorone is a cadinene sesquiterpene, isolated from the leaves of Eupatorium adenophorum. This study evaluated the antimicrobial activity of 9-oxo-agerophorone against three strains of Ralstonia solanacearum and four pathogenic fungi, with an emphasis on the growth and morphogenesis of fungi at scanning electron microscopy level. The determination of minimal inhibitory concentrations demonstrated that 9-oxo-agerophorone exhibited moderate bactericidal activity against R. solanacearum strains. However, antifungal assays showed that 9-oxo-agerophorone has strong toxicity against Fusarium oxysporum, Bipolaris sorokiniana, Fusarium proliferatum, and Alternaria tenuissima with median lethal concentrations ranging from 0.476 to 0.357 mg ml−1 after 4 days incubation on potato dextrose agar medium. The main changes observed by scanning electron microscopy after 9-oxo-agerophorone treatment were fungal cell wall shrinkage, loss of conidia, formation of short branches in F. oxysporum and F. proliferatum, collapse, broken cell walls, and increased hyphae diameter in A. tenuissima. The tested treatments were also effective in inhibiting spore germination of Fusarium species. The results indicated that 9-oxo-agerophorone is a potential candidate as a phytotherapeutic agent for soil-borne fungal diseases.

Draft Genome Sequence of Ralstonia solanacearum Strain Rs-T02, Which Represents the Most Prevalent Phylotype in Guangxi, China.

Ralstonia solanacearum strain Rs-T02 was originally isolated from a bacterial wilt of tomato plant in Nanning City of Guangxi Province, China. It represents the most prevalent phylotype in Guangxi. Here, we present the draft genome sequence of this strain, which comprises 5,225 genes and 5,976,011 nucleotides with an average G+C content of 66.79%. There are 968 different genes between this isolate and the previously reported genome sequence of Ralstonia solanacearum GMl l000 (race l, biovar 3, phylotype I), and the genome sequence information of this isolate may be useful for comparative genomic studies to determine the genetic diversity in this species.

Evaluation of antibacterial and antifungal properties of 9-oxo-10,11-dehydroageraphorone extracted from Eupatorium adenophorum

9-Oxo-10,11-dehydroageraphorone (ODA) is a cadinene sesquiterpene isolated from Eupatorium adenophorum. The antimicrobial activity of ODA was tested in vitro against four fungal strains and four bacterial strains. The fungitoxicity of ODA was evaluated by percentage radial growth inhibition using the poisoned food technique. ODA inhibited Fusarium oxysporum more than the other tested strains, giving EC50 values of 0.325 mg ml−1 at 48 h and 0.365 mg ml−1 at 96 h. Tests also revealed that ODA inhibits the germination of Fusarium oxysporum spores, reducing germination by 97 % at 0.5 mg ml−1 concentration. The minimum inhibitory concentrations (MIC) of ODA against four tested strains of Ralstonia solanacearum ranged from 0.25 to 1 mg ml−1 using a micro-well dilution method. In addition, our scanning electron microscopy study revealed previously unreported, pronounced effects of ODA on Fusarium oxysporum, Bipolaris sorokiniana, Fusarium proliferatum and Alternaria tenuissima, including marked shrinkage in the mycelia of all tested strains, swelling at the ends of Bipolaris sorokiniana mycelia and inhibition of spore production in the strains of Bipolaris sorokiniana and Fusarium proliferatum, providing firm evidence of the toxicity of ODA to these pathogenic fungi.

The vascular plant-pathogenic bacterium Ralstonia solanacearum produces biofilms required for its virulence on the surfaces of tomato cells adjacent to intercellular spaces.

The mechanism of colonization of intercellular spaces by the soil-borne and vascular plant-pathogenic bacterium Ralstonia solanacearum strain OE1-1 after invasion into host plants remains unclear. To analyse the behaviour of OE1-1 cells in intercellular spaces, tomato leaves with the lower epidermis layers excised after infiltration with OE1-1 were observed under a scanning electron microscope. OE1-1 cells formed microcolonies on the surfaces of tomato cells adjacent to intercellular spaces, and then aggregated surrounded by an extracellular matrix, forming mature biofilm structures. Furthermore, OE1-1 cells produced mushroom-type biofilms when incubated in fluids of apoplasts including intercellular spaces, but not xylem fluids from tomato plants. This is the first report of biofilm formation by R. solanacearum on host plant cells after invasion into intercellular spaces and mushroom-type biofilms produced by R. solanacearum in vitro. Sugar application led to enhanced biofilm formation by OE1-1. Mutation of lecM encoding a lectin, RS-IIL, which reportedly exhibits affinity for these sugars, led to a significant decrease in biofilm formation. Colonization in intercellular spaces was significantly decreased in the lecM mutant, leading to a loss of virulence on tomato plants. Complementation of the lecM mutant with native lecM resulted in the recovery of mushroom-type biofilms and virulence on tomato plants. Together, our findings indicate that OE1-1 produces mature biofilms on the surfaces of tomato cells after invasion into intercellular spaces. RS-IIL may contribute to biofilm formation by OE1-1, which is required for OE1-1 virulence.