Strains Of Ralstonia Solanacearum Research Articles

A Ralstonia solanacearum Strain from Guatemala Infects Diverse Flower Crops, Including New Asymptomatic Hosts Vinca and Sutera, and Causes Symptoms in Geranium, Mandevilla Vine, and New Host African Daisy (Osteospermum ecklonis)

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.

DIVERSITY OFRALSTONIA SOLANACEARUMPOPULATIONS AFFECTING TOBACCO CROPS IN NORTH CAROLINA

One hundred eighty four strains of Ralstonia solanacearum isolated in 2007 and 2008 from 11 tobacco fields in North Carolina were evaluated for genotypic diversity and aggressiveness. All strains were race 1, biovar 1, and belonged to phylotype II. Genetic diversity of the strains was assessed with the use of repetitive sequence–based polymerase chain reaction. DNA primers (REP, ERIC, and BOX) were used to generate genomic fingerprints. Both REP and BOX revealed 3 patterns: Ar, Cr, and Dr and Ab, Cb, and Db, respectively. Five patterns were identified with ERIC-PCR. Pattern Ae was found in 80% of the strains collected. Pattern Be was in 4%, pattern Ce in 13%, pattern De in 2%, and pattern Ee in 1% of the strains collected. Cluster analyses showed that the strains were 88% similar and constitute a rather homogeneous group. Aggressiveness of strains was evaluated on 3 tobacco cultivars with different levels of resistance to bacterial wilt. Overall, aggressiveness depended on the field from which the strains...

Genomic characterization of Ralstonia solanacearum phage ϕRS138 of the family Siphoviridae.

ϕRS138, a bacteriophage of the family Siphoviridae that lyses Ralstonia solanacearum, was isolated. The genomic DNA of ϕRS138 was 41,941 bp long with a GC content of 65.1 % and contained 56 putative open reading frames. The ϕRS138 genome could be divided into three regions based on similarities to other genomes: (1) a region containing genes encoding a putative transcriptional regulator and an integrase, similar to the prophage genes in Ralstonia solanacearum K60-1; (2) a region encoding proteins related to structural modules and virion morphogenesis, similar to genes in the Pseudomonas phages of the family Siphoviridae; and (3) a region highly similar to the genomes of other Ralstonia solanacearum strains.

Tracking and identification of antibacterial components in the essential oil of Tanacetum vulgare L. by the combination of high-performance thin-layer chromatography with direct bioautography and mass spectrometry

Two tansy (Tanacetum vulgare L.) essential oils were obtained by steam distillation of the capitula with subsequent liquid–liquid extraction (oil 1) or with use of an auxiliary phase for the trapping of the steam components (oil 2). These oils were investigated against Bacillus subtilis F1276, B. subtilis spizizenii (DSM 618), Xanthomonas euvesicatoria, Pseudomonas syringae pv. maculicola, Ralstonia solanacearum strain GMI1000 and Aliivibrio fischeri, using the coupling of high-performance thin-layer chromatography to direct bioautography (HPTLC-DB). Using this method with the potato and tomato pathogen R. solanacearum is shown for the first time. Due to the advanced extraction process, oil 2 was richer in components and provided more inhibition zones. The main bioactive components were identified by scanning HPTLC-Direct Analysis in Real Time mass spectrometry (HPTLC-DART-MS) and solid-phase microextraction gas chromatography electron impact MS (SPME-GC-EI-MS) as cis- and trans-chrysanthenol as well as trans-chrysanthenyl acetate. cis-Chrysanthenol exhibited antibacterial effects against all tested bacteria, whereas trans-chrysanthenol inhibited B. subtilis, R. solanacearum and A. fischeri. trans-Chrysanthenyl acetate was an inhibitor for X. euvesicatoria, R. solanacearum and A. fischeri. Although HPTLC-DART-MS resulted in a comparable fragmentation, the ionization characteristics and the recorded mass spectra clearly showed that DART is a softer ionization technique than EI. It is also more affected by ambient conditions and thus prone to additional oxidation products.

A duplex PCR assay for the detection of Ralstonia solanacearum phylotype II strains in Musa spp.

Banana wilt outbreaks that are attributable to Moko disease-causing strains of the pathogen Ralstonia solanacearum (Rs) remain a social and economic burden for both multinational corporations and subsistence farmers. All known Moko strains belong to the phylotype II lineage, which has been previously recognized for its broad genetic basis. Moko strains are paraphyletic and are distributed among seven related but distinct phylogenetic clusters (sequevars) that are potentially major threats to Musaceae, Solanaceae, and ornamental crops in many countries. Although clustered within the Moko IIB-4 sequevar, strains of the epidemiologically variant IIB-4NPB do not cause wilt on Cavendish or plantain bananas; instead, they establish a latent infection in the vascular tissues of plantains and demonstrate an expanded host range and high aggressiveness toward Solanaceae and Cucurbitaceae. Although most molecular diagnostic methods focus on strains that wilt Solanaceae (particularly potato), no relevant protocol has been described that universally detects strains of the Musaceae-infecting Rs phylotype II. Thus, a duplex PCR assay targeting Moko and IIB-4NPB variant strains was developed, and its performance was assessed using an extensive collection of 111 strains representing the known diversity of Rs Moko-related strains and IIB-4NPB variant strains along with certain related strains and families. The proposed diagnostic protocol demonstrated both high accuracy (inclusivity and exclusivity) and high repeatability, detected targets on either pure culture or spiked plant extracts. Although they did not belong to the Moko clusters described at the time of the study, recently discovered banana-infecting strains from Brazil were also detected. According to our comprehensive evaluation, this duplex PCR assay appears suitable for both research and diagnostic laboratories and provides reliable detection of phylotype II Rs strains that infect Musaceae.

Real-time duplex applications of loop-mediated AMPlification (LAMP) by assimilating probes.

Isothermal nucleic-acid amplification methods such as Loop-Mediated isothermal AMPlification (LAMP) are increasingly appealing alternatives to PCR for use in portable diagnostic system due to the low cost, weight, and power requirements of the instrumentation. As such, interest in developing new probes and other functionality based on the LAMP reaction has been intense. Here, we report on the development of duplexed LAMP assays for pathogen detection using spectrally unique Assimilating Probes. As proof of principle, we used a reaction for Salmonella enterica as a model coupled with a reaction for λ-phage DNA as an internal control, as well as a duplexed assay to sub-type specific quarantine strains of the bacterial wilt pathogen Ralstonia solanacearum. Detection limits for bacterial DNA analyzed in individual reactions was less than 100 genomic equivalents in all cases, and increased by one to two orders of magnitude when reactions were coupled in duplexed formats. Even so, due to the more robust activity of newly available strand-displacing polymerases, the duplexed assays reported here were more powerful than analogous individual reactions reported only a few years ago, and represent a significant advance for incorporation of internal controls to validate assay results in the field.

A Multiplex PCR Assay to Detect and Differentiate Select Agent Strains of Ralstonia solanacearum.

Ralstonia solanacearum race 3 biovar 2 strains are considered select agents by the U.S. government because they are not endemic to the United States and have the potential to cause brown rot in our potato production fields. Simple and accurate methods are needed for quick identification prior to more discriminating but time-consuming verification methods. We developed a multiplex PCR assay that identifies R. solanacearum species complex strains, signals whether the strain detected is a select agent, and controls for false negatives associated with PCR inhibition or unsuccessful DNA extractions in one reaction. We identified unique sequences of non-phage-related DNA for the R. solanacearum species complex strains, and for select agent strains, using in silico genome subtraction. We also designed and included an internal plant DNA control assay. Our multiplex PCR assay correctly identified 90 R. solanacearum species complex strains and 34 select agent strains, while not recognizing five out-group bacterial species. Additionally, the multiplex PCR assay facilitated the detection of plant DNA and R. solanacearum from infected tomato, potato, geranium, and tobacco plants. Our rapid, accurate, and reliable detection assay can help government officials make timely and appropriate recommendations to exclude this bacterium from the United States.

Biocontrol of potato wilt by selective rhizospheric and endophytic bacteria associated with potato plant.

Ralstonia solanacearum is the causative agent of wilt disease in plants, which constitutes a severe problem to agricultural crops, particularly for potato production in Madagascar. The present study focuses on the isolation, in vitro and in vivo assays of potential rhizospheric and endophytic bacteria associated with healthy potato plant, capable to inhibit the growth of Ralstonia solanacearum for controlling potato bacterial wilt. A total of 77 bacteria strains were isolated from six soil rhizospheric samples and six vegetal material samples of healthy potatoes in the district of Antsirabe II. Forty of them were telluric actinomycetes, 25 were endophytic actinomycetes and 12 were fluorescent Pseudomonas spp. An additional 30 phytopathogenic isolates were obtained from six rhizopsheric soil samples of diseased potatoes. Morphological, cultural, biochemical characterization and molecular identification with the Ralstonia solanacearum specific primers 759/760 revealed that 24 of the pathogenic isolates belong to the Ralstonia solanacearum species, biovar two; the causal agent of potato bacterial wilt. Isolates from healthy plants were, then, examined in vitro and in vivo for their antagonistic activity against Ralstonia solanacearum strain for their potential to improve potato plant growth. In vitro antagonism of actinomycete and Pseudomonas isolates against Ralstonia solanacearum development was performed using agar diffusion technique, while in vivo tests were conducted under greenhouse conditions. Ten antagonistic strains including two Pseudomonas, four telluric actinomycetes, and four endophytic actinomycetes inhibited the tested Ralstonia strain. Four strains, E7, E13 (endophytic actinomycete from root potatoes), S25 (telluric actinomycetes) and P7 (fluorescent Pseudomonas), showed high antagonistic activity against the pathogen with zones of inhibition from 23 to 40 mm. Of the fours strains tested in greenhouse, E7 significantly reduced (p < 0.05) the percentage of Ralstonia solanacearum that infected plants by 72.04%. The isolates E13 and S25 have also been demonstrated to improve plant growth by increase of plant height to 44.63% and 44.84%, fresh weight to 68.75% and 75.85% and dry weight to 86.17% and 115.42%, respectively compared with non-treated control. Morphological and cultural characterization of these three active isolates showed that they belong to the genus Streptomyces. The antagonism of these isolates against Ralstonia solanacearum according to in vitro and in vivo tests results, along with their high efficiency as regards the improvement of plant development, suggests that these three actinomycete strains E7, E13 and S25 could be useful for biocontrol of potato bacterial wilt.

Variation of Resistance to Different Strains of Ralstonia Solanacearum in Highland Tropics Adapted Potato Genotypes

Ten potato genotypes with field resistance to bacterial wilt were evaluated for resistance to five strains of Ralstonia solanacearum belonging to biovars 1, 2A, 2T and 3 (phylotypes I and II) under greenhouse conditions. The plants were inoculated by pouring bacterial suspension into the soil and wilt incidence was recorded weekly. Stems and tubers of symptomless plants were tested for latent infection using post-enrichment NCM-ELISA. The analysis of variance of the percentage of infected plants indicated significant differences among potato genotypes and strains of R.solanacearum. Complete resistance to all tested strains was not observed in any of the potato genotypes. However, CIP 394895.7 was the most resistant genotype, showing hardly any wilting and low level of latent infections. The use of potato varieties with partial resistance like this genotype in combination with crop rotation, healthy seed and sanitation practices could significantly reduce losses due to bacterial wilt.

Evaluation of Portable DNA-Based Technologies for Identification of Ralstonia solanacearum Race 3 Biovar 2 in the Field

Abstract. We have previously reported several new technologies for selective and sensitive detection of quarantine race 3 biovar 2 strains of the bacterial wilt pathogen Ralstonia solanacearum, including new highly specific PCR and LAMP primers, immunomagnetic separation (IMS) for improved isolation of the pathogen from contaminated materials, and two handheld platforms to implement the LAMP-based assay in the field. Here we report on field testing of these new technologies in a naturally infested potato field in the highlands of Guatemala and compare the results to those obtained by standard reference methods, including plating on SMSA media, use of immunostrips, and quantitative PCR. The new LAMP-based assay was demonstrated to be equivalent to quantitative PCR, as sample classifications using the respective methods were absolutely consistent when directly assaying soil and plant tissue extracts. Isolation of DNA from the extracts using IMS enabled more sensitive detection in samples that otherwise appeared negative. These results suggest that the new LAMP tools are indeed viable approaches for rapid, selective, and sensitive field-based detection of race 3 biovar 2 strains of Ralstonia solanacearum, and that IMS is a useful tool for enhancing the isolation and detection of target pathogens in the field.

Moko Disease-Causing Strains of Ralstonia solanacearum from Brazil Extend Known Diversity in Paraphyletic Phylotype II.

The epidemic situation of Moko disease-causing strains in Latin America and Brazil is unclear. Thirty-seven Ralstonia solanacearum strains from Brazil that cause the Moko disease on banana and heliconia plants were sampled and phylogenetically typed using the endoglucanase (egl) and DNA repair (mutS) genes according to the phylotype and sequevar classification. All of the strains belonged to phylotype II and a portion of the strains was typed as the Moko disease-related sequevars IIA-6 and IIA-24. Nevertheless, two unsuspected sequevars also harbored the Moko disease-causing strains IIA-41 and IIB-25, and a new sequevar was described and named IIA-53. All of the strains were pathogenic to banana and some of the strains of sequevars IIA-6, IIA-24, and IIA-41 were also pathogenic to tomato. The Moko disease-causing strains from sequevar IIB-25 were pathogenic to potato but not to tomato. These results highlight the high diversity of strains of Moko in Brazil, reinforce the efficiency of the egl gene to reveal relationships among these strains, and contribute to a better understanding of the diversity of paraphyletic Moko disease-causing strains of the R. solanacearum species complex, where the following seven distinct genetic clusters have been described: IIA-6, IIA-24, IIA-41, IIA-53, IIB-3, IIB-4, and IIB-25.

A phcA− marker-free mutant of Ralstonia solanacearum as potential biocontrol agent of tomato bacterial wilt

A phcA− mutant of Ralstonia solanacearum strain ZJ3721 was created in a marker-free method. Expression of virulence-associated genes such as xpsR, egl, tek and epsE was significantly suppressed in the phcA− mutant. The ability of the mutant to control tomato bacterial wilt was evaluated by potting experiment. Results showed that application of mutant with wild type (WT) at the same time only delayed the development of wilt for about one day and the population of WT in tomato rhizosphere soil was nearly 70-fold higher than that of the mutant, resulting in a 90% disease incidence at last, as high as that of control. If the phcA− mutant was applied three days earlier than WT pathogen, tomato wilt disease incidence was only 6%, 80% lower than that of control and population of WT was about 0.5-fold as much as that of mutant. Under hydroponic conditions, phcA− mutant significantly triggered the expressions of genes in salicylic acid pathway but inhibited the expressions of genes in jasmonic acid (JA) and ethylene (ET) pathways. The expressions of PR-1a and GluA genes (salicylic acid pathway) in phcA− mutant were 66-fold and 7.5-fold higher than in WT pathogen after three days of inoculation.

Loop-mediated isothermal amplification of specific endoglucanase gene sequence for detection of the bacterial wilt pathogen Ralstonia solanacearum.

The increased globalization of crops production and processing industries also promotes the side-effects of more rapid and efficient spread of plant pathogens. To prevent the associated economic losses, and particularly those related to bacterial diseases where their management relies on removal of the infected material from production, simple, easy-to-perform, rapid and cost-effective tests are needed. Loop-mediated isothermal amplification (LAMP) assays that target 16S rRNA, fliC and egl genes were compared and evaluated as on-site applications. The assay with the best performance was that targeted to the egl gene, which shows high analytical specificity for diverse strains of the betaproteobacterium Ralstonia solanacearum, including its non-European and non-race 3 biovar 2 strains. The additional melting curve analysis provides confirmation of the test results. According to our extensive assessment, the egl LAMP assay requires minimum sample preparation (a few minutes of boiling) for the identification of pure cultures and ooze from symptomatic material, and it can also be used in a high-throughput format in the laboratory. This provides sensitive and reliable detection of R. solanacearum strains of different phylotypes.

Proteomic comparison of Ralstonia solanacearum strains reveals temperature dependent virulence factors

BackgroundRalstonia solanacearum, the causal agent of bacterial wilt, is a genetically diverse bacterial plant pathogen present in tropical and subtropical regions of the world that infects more than 200 plant species, including economically important solanaceous crops. Most strains of R. solanacearum are only pathogenic at temperatures between 25 to 30°C with strains that can cause disease below 20°C considered a threat to agriculture in temperate areas. Identifying key molecular factors that distinguish strains virulent at cold temperatures from ones that are not is needed to develop effective management tools for this pathogen. We compared protein profiles of two strains virulent at low temperature and two strains not virulent at low temperature when incubated in the rhizosphere of tomato seedlings at 30 and 18°C using quantitative 2D DIGE gel methods. Spot intensities were quantified and compared, and differentially expressed proteins were sequenced and identified by mass spectrometry (MS/MS).ResultsFour hundred and eighteen (418) differentially expressed protein spots sequenced produced 101 unique proteins. The identified proteins were classified in the Gene Ontology biological processes categories of metabolism, cell processes, stress response, transport, secretion, motility, and virulence. Identified virulence factors included catalase (KatE), exoglucanase A (ChbA), drug efflux pump, and twitching motility porin (PilQ). Other proteins identified included two components of a putative type VI secretion system. We confirmed differential expression of 13 candidate genes using real time PCR techniques. Global regulators HrpB and HrpG also had temperature dependent expression when quantified by real time PCR.ConclusionsThe putative involvement of the identified proteins in virulence at low temperature is discussed. The discovery of a functional type VI secretion system provides a new potential virulence mechanism to explore. The global regulators HrpG and HrpB, and the protein expression profiles identified suggest that virulence at low temperatures can be partially explained by differences in regulation of virulence factors present in all the strains.

Identification of Ralstonia solanacearum Isolated from a New Host : Cosmos caudatus in Indonesia

The evolutionary dynamic of pathogenic microorganism might change pathogen-host interaction including the establishment of pathogen colonization in a new host. The information of the new host of devastating pathogens will serve a basis for their control method. An unknown bacterial wilt disease attacking wild cosmos (Cosmos caudatus) crop in Pacet-Cianjur, Indonesia was suspiciously caused a novel strain of Ralstonia solanacearum. The research was carried out to investigate the causal agent, characteristics and its grouping based on the morphological, physiological and biochemical properties and the relationship of the pathogen with the hosts. The results showed the incidence of bacterial wilt on wild cosmos was epidemiologically caused by the species complex of pathogen R. solanacearum. Based on the symptoms, morphological, physiological, biochemical and pathogenicity evaluations, the strain classified into group 7, race 1 and biovar 3 and group 7. This was the first report of the incidence of R. solanacearum hosted naturally in wild cosmos in Indonesia.

栃木県産ナス科青枯病菌Ralstonia solanacearumのナス属植物に対する病原性とPCR法による菌群判別の可能性の検討

Strains of Ralstonia solanacearum from eggplant and tomato fields in Tochigi Prefecture separated into four distinct groups (I, II, IV and one unknown) based on differences in pathogenicity to four species of Solanum plants. In repetitive sequence-based polymerase chain reaction (rep-PCR) DNA fingerprinting to discriminate the strains belonging to each pathogenicity group, two characteristic bands were identified. One was universally amplified from all tested strains, and the other was amplified only from strains in groups I, II and V. Based on the DNA sequences of the bands, we designed two more PCR primer sets (RsUp-F and RsUp-R, and RsDwn-F and RsDwn-R) that discriminated the strains in group I, II and V from those in III and IV.

Involvement of HLK effectors in Ralstonia solanacearum disease development in tomato

A multigene family encoding HLK effectors is well conserved in all Ralstonia solanacearum strains. The HLK family contains three paralogous members. We constructed single, double, and triple deletion mutants of hlk genes in R. solanacearum strain OE1-1 and characterized HLK effectors by comparing the phenotypes of the strains. HLK effectors exhibited some functional redundancy. While the triple deletion mutant was significantly impaired in virulence on tomato and multiplied less efficiently than the wild type in the leaves, the mutant was as virulent as the wild type on eggplant and tobacco.

Application of variable-number tandem-repeat typing to discriminate Ralstonia solanacearum strains associated with English watercourses and disease outbreaks.

Variable-number tandem-repeat (VNTR) analysis was used for high-resolution discrimination among Ralstonia solanacearum phylotype IIB sequevar 1 (PIIB-1) isolates and further evaluated for use in source tracing. Five tandem-repeat-containing loci (comprising six tandem repeats) discriminated 17 different VNTR profiles among 75 isolates from potato, geranium, bittersweet (Solanum dulcamara), tomato, and the environment. R. solanacearum isolates from crops at three unrelated outbreak sites where river water had been used for irrigation had distinct VNTR profiles that were shared with PIIB-1 isolates from infected bittersweet growing upriver of each site. The VNTR profiling results supported the implication that the source of R. solanacearum at each outbreak was contaminated river water. Analysis of 51 isolates from bittersweet growing in river water at different locations provided a means to evaluate the technique for studying the epidemiology of the pathogen in the environment. Ten different VNTR profiles were identified among bittersweet PIIB-1 isolates from the River Thames. Repeated findings of contiguous river stretches that produced isolates that shared single VNTR profiles supported the hypothesis that the pathogen had disseminated from infected bittersweet plants located upriver. VNTR profiles shared between bittersweet isolates from two widely separated Thames tributaries (River Ray and River Colne) suggested they were independently contaminated with the same clonal type. Some bittersweet isolates had VNTR profiles that were shared with potato isolates collected outside the United Kingdom. It was concluded that VNTR profiling could contribute to further understanding of R. solanacearum epidemiology and assist in control of future disease outbreaks.

Global gene expression of rhizobacteria-silicon mediated induced systemic resistance in tomato (Solanum lycopersicum) against Ralstonia solanacearum

Transcriptome analysis of tomato (Solanum lycopersicum) stem tissue was performed to elucidate gene expression analysis in plant treated with silicon and/or rhizobacteria strain Bacillus pumilis following inoculation with Ralstonia solanacearum strain ToUdk2 (race 1,phylotype 1 originated from Thailand). A total of 174 genes were differentially regulated of which 113 were up-regulated and 61 down-regulated functional categorization of the regulated genes revealed that most of the up-regulated genes are more involved in signal transduction, defence, protein synthesis and metabolism, while a large proportion of down regulated genes were more involved in photosynthesis, lipid metabolism. In plant treated with silicon a higher number of defence related genes were regulated than those treated with rhizobacterium B. pumilis. During the simultaneous application of the two elicitors five genes were down regulated, while they were up-regulated when they were applied separately, suggesting that an antagonistic interaction occurred between genes mediated by ethylene-jasmonate and salicylate pathways. Therefore, separate application of each elicitor is recommended as best strategy for the induction of resistance against bacterial wilt in tomato, with silicon being a stronger inducer than rhizobacteria.

Pathogenic characters of Japanese potato strains of Ralstonia solanacearum

Ralstonia solanacearum (Rs) strains in phylotypes I and IV isolated from potato in Japan were investigated for pathogenicity on potato, tomato, eggplant, Solanum integrifolium, tobacco, groundnut, and pumpkin. The strains were divided into 17 types based on differences in their pathogenicity on the tested plants. Particularly, the pathogenicity of most phylotype I strains on eggplant was distinctly different from that of the phylotype IV strains. When nine potato varieties (included two breeding lines) were inoculated with several Rs strains, phylotype IV strains were highly virulent on the breeding lines that are regarded as resistant to phylotype I strains.