Removal of bacterial plant pathogens in columns filled with quartz and natural sediments under anoxic and oxygenated conditions.

Irrigation with surface water carrying plant pathogens poses a risk for agriculture. Managed aquifer recharge enhances fresh water availability while simultaneously it may reduce the risk of plant diseases by removal of pathogens during aquifer passage. We compared the transport of three plant pathogenic bacteria with Escherichia coli WR1 as reference strain in saturated laboratory column experiments filled with quartz sand, or sandy aquifer sediments. E. coli showed the highest removal, followed by Pectobacterium carotovorum, Dickeya solani and Ralstonia solanacearum. Bacterial and non-reactive tracer breakthrough curves were fitted with Hydrus-1D and compared with colloid filtration theory (CFT). Bacterial attachment to fine and medium aquifer sand under anoxic conditions was highest with attachment rates of max. katt1 = 765 day-1 and 355 day-1, respectively. Attachment was the least to quartz sand under oxic conditions (katt1 = 61 day-1). In CFT, sticking efficiencies were higher in aquifer than in quartz sand but there was no differentiation between fine and medium aquifer sand. Overall removal ranged between < 6.8 log10 m-1 in quartz and up to 40 log10 m-1 in fine aquifer sand. Oxygenation of the anoxic aquifer sediments for two weeks with oxic influent water decreased the removal. The results highlight the potential of natural sand filtration to sufficiently remove plant pathogenic bacteria during aquifer storage.

An immunochromatographic test strip for on-site detection of Ralstonia solanacearum in tobacco leaves and soil.

Tobacco bacterial wilt is one of the most devastating soil-borne diseases in tobacco-producing regions worldwide. It is often responsible for significant economic losses during tobacco production. A rapid, specific, and high-throughput on-site detection method is important for plant disease management. In this study, monoclonal antibody 3H3 and polyclonal antibody 0344 specific for Ralstonia solanacearum were used to prepare a colloidal gold-based immunochromatographic test strip (ITS). Under optimal conditions, the detection limit of the ITS was 105 CFU/mL. The ITS was able to detect different R. solanacearum strains collected from Shandong, Yunnan, Guizhou, and Sichuan provinces in China. Moreover, the ITS was highly specific for R. solanacearum, with no cross-reactivity with Alternaria alternata (Fries) Keissler, Pseudomonas syringae pv. angulata, and P. syringae pv. tabaci. Furthermore, R. solanacearum-spiked tobacco leaves and soil were used to evaluate the matrix interference of the developed ITS, which indicated the test strip was unaffected by leaf size or soil abundance.

Transboundary introduction of potato-infecting Ralstonia solanacearum in the Andaman Islands revealed by multilocus sequence typing: A potential threat to island agriculture.

Wilting of potato plants with an incidence of 20-30 % was observed for the first time in the agricultural farms of Andaman Islands, India. The infected plants showed wilting syndrome that included downward drooping of leaves, yellowing, and collapse of the entire plants. Characteristic milky-white exudate from the infected stem indicated bacterial etiology of the disease. Upon streaking onto 2, 3, 5 triphenyl-tetrazolium chloride amended nutrient medium, the bacterial exudate yielded characteristic creamy-white, fluidal, irregular colonies with the pink center. Upon inoculation, the randomly picked bacterial colonies, AN_PRSGr and AN_PRSCh, representing the two locations, incited wilt symptoms on one-month-old potato plants. The host range studies revealed that the isolates were pathogenic on tomato and eggplant but non-pathogenic to chili and Solanum torvum (wild eggplant). The 16S rRNA gene sequencing and the Ralstonia-specific PCR test confirmed the identity of AN_PRSGr and AN_PRSCh as Ralstonia solanacearum. Intra-species level classification revealed their identity as strains of race 1, biovar 3, and phylotype-I. Multilocus sequence typing (MLST)-based in-depth sequence alignment for phylogenetic analysis revealed the isolates AN_PRSGr and AN_PRSCh clustered with two mainland race 1/biovar 3/phylotype-I isolates of Kerala, India. However, the allelic profile-based goeBURST-analysis placed them as singletons in the global collection of Ralstonia solanacearum, conforming intra-racial/ intra-phylotype diversity within race 1/biovar3/phylotype-I strains. The molecular characterization.

Development of Recombinase Polymerase Amplification Combined with Lateral Flow Detection Assay for Rapid and Visual Detection of Ralstonia solanacearum in Tobacco.

Bacterial wilt caused by Ralstonia solanacearum is a serious soilborne disease that results in severe losses to tobacco (Nicotiana tabacum) production in China. In this study, a novel RPA-LFD assay for the rapid visual detection of R. solanacearum was established using recombinase polymerase amplification (RPA) and lateral-flow dipstick (LFD). The RPA-LFD assay was performed at 37°C in 30 min without complex equipment. Targeting the sequence of the RipTALI-9 gene, we designed RPA primers (Rs-rpa-F/R) and an LF probe (Rs-LF-probe) that showed high specificity to R. solanacearum. The sensitivity of RPA-LFD assay to R. solanacearum was the same as that in conventional PCR at 1 pg genomic DNA, 103 CFU/g artificially inoculated tobacco stems, and 104 CFU/g artificially inoculated soil. The RPA-LFD assay could also detect R. solanacearum from plant and soil samples collected from naturally infested tobacco fields. These results suggest that the RPA-LFD assay developed in this study is a rapid, accurate molecular diagnostic tool with high sensitivity for the detection of R. solanacearum.

High genomic diversity of novel phages infecting the plant pathogen Ralstonia solanacearum, isolated in Mauritius and Reunion islands.

Bacterial wilt caused by the Ralstonia solanacearum species complex (RSSC) is among the most important plant diseases worldwide, severely affecting a high number of crops and ornamental plants in tropical regions. Only a limited number of phages infecting R. solanacearum have been isolated over the years, despite the importance of this bacterium and the associated plant disease. The antibacterial effect or morphological traits of these R. solanacearum viruses have been well studied, but not their genomic features, which need deeper consideration. This study reports the full genome of 23 new phages infecting RSSC isolated from agricultural samples collected in Mauritius and Reunion islands, particularly affected by this plant bacterial pathogen and considered biodiversity hotspots in the Southwest Indian Ocean. The complete genomic information and phylogenetic classification is provided, revealing high genetic diversity between them and weak similarities with previous related phages. The results support our proposal of 13 new species and seven new genera of R. solanacearum phages. Our findings highlight the wide prevalence of phages of RSSC in infected agricultural settings and the underlying genetic diversity. Discoveries of this kind lead more insight into the diversity of phages in general and to optimizing their use as biocontrol agents of bacterial diseases of plants in agriculture.

Host adaptation and microbial competition drive Ralstonia solanacearum phylotype I evolution in the Republic of Korea.

Bacterial wilt caused by the Ralstonia solanacearum species complex (RSSC) threatens the cultivation of important crops worldwide. We sequenced 30 RSSC phylotype I (R. pseudosolanacearum) strains isolated from pepper (Capsicum annuum) and tomato (Solanum lycopersicum) across the Republic of Korea. These isolates span the diversity of phylotype I, have extensive effector repertoires and are subject to frequent recombination. Recombination hotspots among South Korean phylotype I isolates include multiple predicted contact-dependent inhibition loci, suggesting that microbial competition plays a significant role in Ralstonia evolution. Rapid diversification of secreted effectors presents challenges for the development of disease-resistant plant varieties. We identified potential targets for disease resistance breeding by testing for allele-specific host recognition of T3Es present among South Korean phyloype I isolates. The integration of pathogen population genomics and molecular plant pathology contributes to the development of location-specific disease control and development of plant cultivars with durable resistance to relevant threats.

Competition for iron drives phytopathogen control by natural rhizosphere microbiomes.

Plant pathogenic bacteria cause high crop and economic losses to human societies1-3. Infections by such pathogens are challenging to control as they often arise through complex interactions between plants, pathogens and the plant microbiome4,5. Experimental studies of this natural ecosystem at the microbiome-wide scale are rare, and consequently we have a poor understanding of how the taxonomic and functional microbiome composition and the resulting ecological interactions affect pathogen growth and disease outbreak. Here, we combine DNA-based soil microbiome analysis with in vitro and in planta bioassays to show that competition for iron via secreted siderophore molecules is a good predictor of microbe-pathogen interactions and plant protection. We examined the ability of 2,150 individual bacterial members of 80 rhizosphere microbiomes, covering all major phylogenetic lineages, to suppress the bacterium Ralstonia solanacearum, a global phytopathogen capable of infecting various crops6,7. We found that secreted siderophores altered microbiome-pathogen interactions from complete pathogen suppression to strong facilitation. Rhizosphere microbiome members with growth-inhibitory siderophores could often suppress the pathogen in vitro as well as in natural and greenhouse soils, and protect tomato plants from infection. Conversely, rhizosphere microbiome members with growth-promotive siderophores were often inferior in competition and facilitated plant infection by the pathogen. Because siderophores are a chemically diverse group of molecules, with each siderophore type relying on a compatible receptor for iron uptake8-12, our results suggest that pathogen-suppressive microbiome members produce siderophores that the pathogen cannot use. Our study establishes a causal mechanistic link between microbiome-level competition for iron and plant protection and opens promising avenues to use siderophore-mediated interactions as a tool for microbiome engineering and pathogen control.

Antibacterial activity of three essential oils and some monoterpenes against Ralstonia solanacearum phylotype II isolated from potato.

The present study describes the possibility of using some essential oils and monoterpens as bioagents against the growth of Ralstonia solanacearum, a causal bacterium of potato brown rot disease. Eight isolates of the bacterium were recovered from infected potato tubers, showing typical symptoms of the disease, Isolates were identified as R.solanacearum phylotype II, based on biochemical and physiological characteristics, as well as, at the molecular level through PCR analysis. Three essential oils extracted from Corymbia citriodora (leaves), Cupressus sempervirens (aerial parts), and Lantana camara (aerial parts) were evaluated for their antibacterial activity against eight isolates of R. solanacearum phylotype II. Results demonstrated that L. camara essential oil (concentration 5000 µg/mL) had the highest effects against the RsMo2, RsSc1 and Rs48, with inhibition zone (IZ) values of 17.33, 16.33, and 17.50 mm, respectively, also against Rs2 (IZ 14.33 mm), and RsIs2 (IZ 16 mm). C. citriodora oil showed the highest activity against RsBe2 (IZ 14 mm), RsFr4 (IZ 13.66 mm) and RsNe1 (IZ 13.66 mm). Gas Chromatography-Mass Spectrometry (GC-MS-FID) analyzed the chemical composition of these essential oils. It was proved that L. camara leaves contains mainly trans-caryophyllene (16.24%) and α-humulene (9.55%), in C. citriodora oil were α-citronellal (56.55%), α-citronellol (14.89%), and citronellol acetate (13.04%), and in Cup. sempervirens aerial parts were cedrol (22.17%), and Δ3-carene (18.59%). Five monoterpenes were evaluated against the most resistance Ralstonia isolate RsFr5 to the three studied essential oils and found that limonene had the highest effect against it compared with the lowest thymol. The results proved the strong bio effects of the essential oil from L. camara leaves as a natural product contained monoterpenes that can inhibit the growth of tested R. solanacearum phylotype II isolates.

Comparative Evaluation of LAMP, qPCR, Conventional PCR, and ELISA to Detect Ralstonia solanacearum in Kenyan Potato Fields.

Bacterial wilt caused by Ralstonia solanacearum is considered among the most damaging diseases of potato in Sub-Saharan Africa and the most significant biotic constraint of potato production alongside late blight. Unlike late blight, which can be managed by chemical means, R. solanacearum can only be managed through cultural methods and clean seed. Laboratory testing to certify seed before planting is required to confirm the absence of the pathogen in Kenya. A loop-mediated isothermal amplification (LAMP) assay was developed using the UDP-(3-O-acyl)-N-acetylglucosamine deacetylase gene (IpxC) to screen seed potato for R. solanacearum strains. The assay was assessed using DNA extracted from R. solanacearum and other soil and potato pathogens to demonstrate specificity and sensitivity. The LAMP assay was validated using field samples from different potato growing regions of Kenya collected over two growing seasons and compared with established nucleic acid and protein-based assays. The IpxC LAMP assay was found to be specific and sensitive to R. solanacearum, detecting as low as 2.5 pg/µl of R. solanacearum DNA. Of the 47 potentially infected field samples collected, both IpxC LAMP and quantitative polymerase chain reaction (PCR) detected R. solanacearum DNA in 90% of the samples, followed by conventional PCR (86%) and ELISA (75%). This IpxC LAMP assay is a promising diagnostic tool to rapidly screen for R. solanacearum in seed potato with high sensitivity in Kenya. Copyright © 2019 The Author(s). This is an open access article distributed under the CC BY 4.0 International license .

Phylogenetic Assignment of Ralstonia pseudosolanacearum (Ralstonia solanacearum Phylotype I) Isolated from Rosa spp.

During the last two years, greenhouse cultivation of rose (Rosa spp.) in the Netherlands has been challenged by an uncommon bacterial disease. Affected plants suffered from chlorosis, stunting, wilting, and necrosis. The bacterial isolates obtained from the different Rosa spp. cultivars were all identified as phylotype I, sequevar 33 from the 'Ralstonia solanacearum species complex' (RSSC), actually reclassified as Ralstonia pseudosolanacearum. The work in this paper considers the genetic diversity and the phylogenetic position of 129 R. pseudosolanacearum isolates from the outbreak. This was assessed by AFLP based on four different primer combinations and MLP using partial sequences of the egl, mutS, and fliC genes. The AFLP revealed identical profiles for all the isolates, irrespective of their association with Rosa sp. propagating material, Rosa spp. plants for cut flowers, or water used in the different greenhouse cultivations. These AFLP profiles were unique and diverged from profiles of all other reference isolates in the RSSC included. Furthermore, MLP on egl, fliC, and mutS gene sequences clearly demonstrated that all R. pseudosolanacearum isolates clustered in phylotype I, as a distinct monophyletic group. Interestingly, this monophyletic group also included phylotype I strain Rs-09-161 from eggplant (Solanum melongena), isolated in 2009 in India. AFLP and MLP were both efficient in revealing the genetic divergence from the RSSC isolates included. The phylogenetic tree constructed from the AFLP profiles was, in general, in agreement with the one obtained from MLP. Both phylogenetic trees displayed a similar clustering, supported by high posterior probabilities. Both methodologies clearly demonstrated that the R. pseudosolanacearum isolates from Rosa spp. grouped in a monophyletic group inside phylotype I, with a particular correspondence to a strain present in India, as revealed in MLP.

Phylotype and sequevar of Ralstonia solanacearum which causes bacterial wilt in Curcuma alismatifolia Gagnep.

Bacterial wilt of Curcuma alismatifolia (Patumma) caused by Ralstonia solanacearum is a major disease affecting the quality of rhizome exports. Traditionally, R. solanacearum is classified into five races based on differences in host range and six biovars based on biochemical properties. Recently a classification scheme based on phylotypes and sequevars was presented by the scientific community as a tool for determining phylogenetic relationships within R. solanacearum. This study used traditional and molecular methods to identify R. solanacearum strains from Patumma. All the strains were identified as biovar 4. A phylotype-specific multiplex PCR-based phylotyping of all the isolates detected the phylotype I-specific amplicon of 144 bp and the R. solanacearum-specific 281 bp amplicon. Phylogenetic analyses of endoglucanase (egl) sequences clustered all three strains of Patumma into phylotype I, sequevar 48 with reference strains M2 and M6. The study determined that the R. solanacearum strains from Patumma belong to biovar 4, phylotype I that originated from Asia, and sequevar 48. SIGNIFICANCE AND IMPACT OF THE STUDY: Phylotype and sequevar of Ralstonia solanacearum were associated with geographic region and geographic distribution. This is the first study to identify phylotype and sequevar of R. solanacearum from Patumma in Chiang Mai, Thailand. This will be useful for study of disease epidemiology and could help management for control of bacterial wilt diseases in this host.

Matrix approach to the simultaneous detection of multiple potato pathogens by real-time PCR.

AIM: Create a method for highly sensitive, selective, rapid and easy-to-use detection and identification of economically significant potato pathogens, including viruses, bacteria and oomycetes, be it single pathogen, or a range of various pathogens occurring simultaneously. METHODS AND RESULTS: Test-systems for real-time PCR, operating in the unified amplification regime, have been developed for Phytophthora infestans, Pectobacterium atrosepticum, Dickeya dianthicola, Dickeya solani, Ralstonia solanacearum, Pectobacterium carotovorum, Clavibacter michiganensis subsp. sepedonicus, potato viruses Y (ordinary and necrotic forms as well as indiscriminative test system, detecting all forms), A, X, S, M, potato leaf roll virus, potato mop top virus and potato spindle tuber viroid. The test-systems (including polymerase and revertase) were immobilized and lyophilized in miniature microreactors (1·2 µl) on silicon DNA/RNA microarrays (micromatrices) to be used with a mobile AriaDNA® amplifier. CONCLUSIONS: Preloaded 30-reaction micromatrices having shelf life of 3 and 6 months (for RNA- and DNA-based pathogens, respectively) at room temperature with no special conditions were successfully tested on both reference and field samples in comparison with traditional ELISA and microbiological methods, showing perfect performance and sensitivity (1 pg). SIGNIFICANCE AND IMPACT OF THE STUDY: The accurate, rapid and user-friendly diagnostic system in a micromatrix format may significantly contribute to pathogen screening and phytopathological studies.

Antigenic and pathogenicity activities of Ralstonia solanacearum race 3 biovar 2 molecularly identified and detected by indirect ELISA using polyclonal antibodies generated in rabbits.

Eight molecular-characterized isolates of Ralstonia solanacearum from potato belonging to race 3 biovar 2, their virulence were evaluated on potato cv. Lady Rosette, tomato cv. Strain B, eggplant cv. Balady and pepper cv. Balady and showed high virulence on potato and tomato, and lower virulence on eggplant and pepper. A laboratory study conducted to produce polyclonal antibodies against the potato brown rot bacterium; R. solanacearum cells were generated in female New Zealand white rabbits. A modification were made on the technique of indirect enzyme-linked immunosorbent assay (ELISA) to improve the sensitivity of detection, including antigenic and sensitivity to R. solanacearum race 3 biovar 2 isolates. Determination of the optimum period to collect the antiserum (including, polyclonal antibodies) showed that the best collection dates were at 14, 3 and 7 days, in that order. The efficiency of the antiserum was compared among 42 isolates that cause potato brown rot disease; our polyclonal antiserum (14 days) reacted positively with all tested isolates at a dilution of 1:6.4 × 103. Data indicated the different reactions of eight R. solanacearum isolates at various dilutions (1:1.6 × 103 to 1:5.12 × 106) at 14 days against polyclonal antiserumat a concentration of approximately 1 × 108 CFU/mL and we found the lowest detection level by the indirect ELISA technique was 106 CFU/mL. Finally we recommended the reasonable sensitivity results of the ELISA technique to detect the bacterial pathogen given than the cost of this technique if much lower than that of other expensive molecular techniques.

Comparison of Prokaryotic and Eukaryotic Communities in Soil Samples with and without Tomato Bacterial Wilt Collected from Different Fields.

Biocontrol agents (BCA) effectively suppress soil-borne disease symptoms using natural antagonistic prokaryotes or eukaryotes. The main issue associated with the application of BCA is that disease reduction effects are unstable under different field conditions. In order to identify potentially effective BCA among several fields, we compared prokaryotic and eukaryotic communities in soil with and without tomato bacterial wilt from three different fields, each of which had the same field management and similar soil characteristics. Soil samples were collected from three fields and two depths because bacterial wilt pathogens were present in soil at a depth greater than 40 cm. We classified soil samples based on the presence or absence of the bacterial phcA gene, a key gene for bacterial wilt pathogenicity and tomato disease symptoms. Pyrosequencing of the prokaryotic 16S rRNA gene and eukaryotic internal transcribed spacer region sequences showed that the diversity and richness of the communities mostly did not correlate with disease symptoms. Prokaryotic and eukaryotic community structures were affected more by regional differences than the appearance of disease. Several prokaryotes and eukaryotes were more abundant in soil that lacked disease symptoms, and eight prokaryotes and one eukaryote of this group were commonly detected among the three fields. Some of these taxa were not previously found in disease-suppressive soil. Our results suggest that several prokaryotes and eukaryotes control plant disease symptoms.

General phytoplasma detection by a q-PCR method using mycoplasma primers.

Phytoplasmas and mycoplasmas are bacteria belonging to the class Mollicutes. In this study, a fine tuning of quantitative polymerase chain reaction (qPCR) with a universal mycoplasma primer pair (GPO3F/MGSO) targeting the 16S rRNA gene was carried out on phytoplasmas. The dissociation curves of DNAs from Catharanthus roseus phytoplasma-infected micropropagated shoots and from phytoplasma field-infected plant samples showed a single peak at 82.5 °C (±0.5) specifically detecting phytoplasmas belonging to several ribosomal groups. Assay specificity was determined with DNA of selected bacteria: 'Candidatus Liberibacter solanacearum', Xylella fastidiosa, Ralstonia solanacearum and Clavibacter michiganensis. No amplification curves were observed with any of these tested bacteria except 'Ca. L. solanacearum' that was amplified with a melting temperature at 85 °C. Absolute quantification of phytoplasma titer was calculated using standard curves prepared from serial dilutions of plasmids containing the cloned fragment GPO3F/MGSO from European stone fruit yellows phytoplasma. Phytoplasma copy number ranged from 106 to 103 according with the sample. The sensitivity evaluated comparing plasmid serial dilutions resulted 10-6 for conventional PCR and 10-7 for qPCR. The latter method resulted therefore able to detect very low concentrations of phytoplasma in plant material.

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

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Genome sequencing of four strains of Phylotype I, II and IV of Ralstonia solanacearum that cause potato bacterial wilt in India

Abstract Ralstonia solanacearum is a heterogeneous species complex causing bacterial wilts in more than 450 plant species distributed in 54 families. The complexity of the genome and the wide diversity existing within the species has led to the concept of R. solanacearum species complex (RsSC). Here we report the genome sequence of the four strains (RS2, RS25, RS48 and RS75) belonging to three of the four phylotypes of R. solanacearum that cause potato bacterial wilt in India. The genome sequence data would be a valuable resource for the evolutionary, epidemiological studies and quarantine of this phytopathogen.

Ralstonia solanacearum Differentially Colonizes Roots of Resistant and Susceptible Tomato Plants.

Ralstonia solanacearum is the causal agent of bacterial wilt and infects over 200 plant species in 50 families. The soilborne bacterium is lethal to many solanaceous species, including tomato. Although resistant plants can carry high pathogen loads (between 105 and 108 CFU/g fresh weight), the disease is best controlled by the use of resistant cultivars, particularly resistant rootstocks. How these plants have latent infections yet maintain resistance is not clear. R. solanacearum first infects the plant through the root system and, thus, early root colonization events may be key to understanding resistance. We hypothesized that the distribution and timing of bacterial invasion differed in roots of resistant and susceptible tomato cultivars. Here, we use a combination of scanning electron microscopy and light microscopy to investigate R. solanacearum colonization in roots of soil-grown resistant and susceptible tomato cultivars at multiple time points after inoculation. Our results show that colonization of the root vascular cylinder is delayed in resistant 'Hawaii7996' and that, once bacteria enter the root vascular tissues, colonization in the vasculature is spatially restricted. Our data suggest that resistance is due, in part, to the ability of the resistant cultivar to restrict bacterial root colonization in space and time.

Genome sequencing of four strains of Phylotype I, II and IV of Ralstonia solanacearum that cause potato bacterial wilt in India.

Ralstonia solanacearum is a heterogeneous species complex causing bacterial wilts in more than 450 plant species distributed in 54 families. The complexity of the genome and the wide diversity existing within the species has led to the concept of R. solanacearum species complex (RsSC). Here we report the genome sequence of the four strains (RS2, RS25, RS48 and RS75) belonging to three of the four phylotypes of R. solanacearum that cause potato bacterial wilt in India. The genome sequence data would be a valuable resource for the evolutionary, epidemiological studies and quarantine of this phytopathogen.

New Multilocus Variable-Number Tandem-Repeat Analysis (MLVA) Scheme for Fine-Scale Monitoring and Microevolution-Related Study of Ralstonia pseudosolanacearum Phylotype I Populations.

Bacterial wilt caused by the Ralstonia solanacearum species complex (RSSC) is considered one of the most harmful plant diseases in the world. Special attention should be paid to R. pseudosolanacearum phylotype I due to its large host range, its worldwide distribution, and its high evolutionary potential. So far, the molecular epidemiology and population genetics of this bacterium are poorly understood. Until now, the genetic structure of the RSSC has been analyzed on the worldwide and regional scales. Emerging questions regarding evolutionary forces in RSSC adaptation to hosts now require genetic markers that are able to monitor RSSC field populations. In this study, we aimed to evaluate the multilocus variable-number tandem-repeat analysis (MLVA) approach for its ability to discriminate genetically close phylotype I strains and for population genetics studies. We developed a new MLVA scheme (MLVA-7) allowing us to genotype 580 R. pseudosolanacearum phylotype I strains extracted from susceptible and resistant hosts and from different habitats (stem, soil, and rhizosphere). Based on specificity, polymorphism, and the amplification success rate, we selected seven fast-evolving variable-number tandem-repeat (VNTR) markers. The newly developed MLVA-7 scheme showed higher discriminatory power than the previously published MLVA-13 scheme when applied to collections sampled from the same location on different dates and to collections from different locations on very small scales. Our study provides a valuable tool for fine-scale monitoring and microevolution-related study of R. pseudosolanacearum phylotype I populations.IMPORTANCE Understanding the evolutionary dynamics of adaptation of plant pathogens to new hosts or ecological niches has become a key point for the development of innovative disease management strategies, including durable resistance. Whereas the molecular mechanisms underlying virulence or pathogenicity changes have been studied thoroughly, the population genetics of plant pathogen adaptation remains an open, unexplored field, especially for plant-pathogenic bacteria. MLVA has become increasingly popular for epidemiosurveillance and molecular epidemiology studies of plant pathogens. However, this method has been used mostly for genotyping and identification on a regional or global scale. In this study, we developed a new MLVA scheme, targeting phylotype I of the soilborne Ralstonia solanacearum species complex (RSSC), specifically to address the bacterial population genetics on the field scale. Such a MLVA scheme, based on fast-evolving loci, may be a tool of choice for field experimental evolution and spatial genetics studies.