Genetic diversity of <Emphasis Type="Italic">Ralstonia solanacearum</Emphasis> strains from China

A survey of bacterial wilt in China collected 286 strains of Ralstonia solanacearum from 17 plant species in 13 Chinese provinces to investigate genetic diversity using the biovar (bv.) and phylotype classification schemes. A phylotype-specific multiplex-PCR showed that 198 isolates belonged to phylotype I (bv. 3, 4 and 5) and 68 to phylotype II (bv. 2 and bv. 1). A phylogenetic analysis examined the partial sequence of the egl and hrpB gene of all strains and the genetic diversity of 95 representatives was reported, demonstrating that Chinese strains are partitioned into phylotype I (Asia) and II (Americas). Phylotype I strains (historically typed bv. 3, 4 and 5), had considerable phylogenetic diversity, including 10 different sequevars: seven previously described sequevars 12 to 18 and three new sequevars: 34, 44 and 48. Chinese strains Z1, Z2, Z3, Z7, Pe74 and Tm82 were not genetically distinguishable from the edible ginger reference strain ACH92 (r4-bv. 4) for sequevar 16. This is believed to be the first report of this ginger group in China. All Chinese bv. 2 strains falling into the genetically and phenotypically diverse phylotype II were placed into phylotype IIB sequevar 1 (historically the Andean race3-bv. 2 potato brown rot agent). In both the egl and hrpB sequence-based trees, strains isolated from mulberry were present in two distinct branches found in sequevars 12 and 48 (reference strains R292 and M2, respectively).     

Ralstonia solanacearum as a potato pathogen in Serbia: Characterization of strains and influence on peroxidase activity in tubers

Since 2011, the outbreaks of brown rot caused by Ralstonia solanacearum race 3, biovar 2, phylotype IIB-1 (R3/B2/PIIB-1) have significantly compromised potato production in Serbia. During 6 years of monitoring (2013–2018) among 3,524 potato tuber samples, 344 were found positive for brown rot disease. R. solanacearum R3/B2/PIIB-1 was isolated from seven cultivars among 12 monitored, and in five localities among 17 monitored. Cultivar Lady Claire was found to have the highest disease frequency (31.98%). A total of 78 isolates were identified by R. solanacearum-specific primer pairs (PS-1/PS-2 and OLI-1/Y-2), as well as the following tests: restriction fragment length polymorphism analysis, biovar determination, immunofluorescence, biochemical analysis, and pathogenicity. The genetic composition of 36 selected isolates assessed using multilocus sequence analysis with seven genes (adk, gapA, gdhA, gyrB, ppsA, hrpB, and fliC) showed that all isolates originating from Serbian potato were homogeneous. By using the TCS algorithm of concatenated sequences to get insight into the phylogeography of isolates and other R. solanacearum strains deposited in the NCBI database, we showed that their origin is undetermined. Peroxidase (POD) activity was measured in brown rotted potato tubers. A positive correlation was found between POD activity and disease severity rated on the analysed tubers. In general, POD activity increased by 2–22 times in vascular necrotic tissues compared to non-necrotic ones, and depended on disease severity but not on cultivar. Native polyacrylamide gel electrophoresis analysis of POD profiles resulted in a total of 10 distinct POD isoforms, of which PODs 3–5 were highly intensified in response to R. solanacearum.     

Genetic diversity of Ralstonia solanacearum species complex strains obtained from Guangxi,China and their pathogenicity on plants in the Cucurbitaceae family and other botanical families

Bacterial wilt, caused by the Ralstonia solanacearum species complex (RSSC), is a destructive plant disease in Guangxi, China. However, the diversity of RSSC populations in the area is unknown. To this end, we performed an extensive bacterial wilt survey from 2015 to 2018. Using phylotype-specific multiplex PCR (Pmx-PCR) and an egl-based tree, 189 strains collected from 20 plant species were identified as R. pseudosolanacearum phylotype I, which included 14 sequevars (12, 13, 14, 15, 16, 17, 18, 30, 34, 44, 48, 54, 70, and 71); two strains isolated from potato plants belonged to R. solanacearum phylotype II, sequevar 1. Sequevars 13, 17, and 44 were prevalent in Guangxi, and sequevar 13 dominated the RSSC sequevars of four Cucurbitaceae plants. The susceptibility of different Cucurbitaceae species to bacterial wilt and the host range of 16 representative strains were further tested. Members of the Cucurbita, Momordica, and Luffa genera were susceptible to bacterial wilt, with wilt incidence ranging from 73% to 100%. Most strains were pathogenic to solanaceous plants, mulberry, and ginger plants but not to melon crops; however, the strains from kidney bean, pepper, and Cucurbitaceae plants were highly virulent to melon crops. This is the first comprehensive report on the genetic and host range diversity of the RSSC in Guangxi and the susceptibility of different Cucurbitaceae species to bacterial wilt, which can provide valuable information for the development of bacterial wilt control strategies.     

Validation of four real‐time TaqMan PCRs for the detection of Ralstonia solanacearum and/or Ralstonia pseudosolanacearum and/or Clavibacter michiganensis subsp. sepedonicus in potato tubers using a statistical regression approach

A new DNA extraction method and a new multiplex real‐time TaqMan PCR test for detection of Ralstonia solanacearum, Ralstonia pseudosolanacearum and Clavibacter michiganensis subsp. sepedonicus in asymptomatic potato tubers are presented. This new multiplex PCR and three published TaqMan PCRs for detection of R. solanacearum and/or R. pseudosolanacearum and/or R. syzygii spp. and/or C. michiganensis subsp. sepedonicus were validated using linear regression analysis for estimating the Ct values and its variation at 5 × 10³ bacteria mL^?1. The three published PCRs that have been validated are Massart et al. (2014, detecting R. solanacearum and C. michiganensis subsp. sepedonicus), Weller et al. (1999, detecting R. solanacearum, R. pseudosolanacearum and R. syzygii spp.) and Gudmestad et al. (2009, detecting C. michiganensis subsp. sepedonicus). All tested PCRs were fit for purpose for their target organisms. The PCR tests have different target genes, allowing one of the sets to be used as first screening test and another as second screening test for the detection of R. solanacearum and/or R. pseudosolanacearum and/or C. michiganensis subsp. sepedonicus in asymptomatic potato tubers.     

Multiplex PCR for the identification of Agrobacterium biovar 3 strains

A biovar 3-specific primer set Ab3-F3/Ab3-R4 was designed based on the comparison of sequences of the 16S rDNA region of agrobacteria and related rhizobia for rapid identification of Agrobacterium biovar 3 strains. A 570-bp 16S rDNA fragment was amplified from cell lysates of Agrobacterium biovar 3 strains by polymerase chain reaction (PCR) using Ab3-F3/Ab3-R4 primers. Discrimination of Agrobacterium tumefaciens biovar 3 from Agrobacterium radiobacter biovar 3 and of Agrobacterium biovar 3 strains from other Agrobacterium strains was done simultaneously using multiplex PCR with a mixture of two primer sets (Ab3-F3/Ab3-R4 and VCF3/VCR3) previously designed for the virC region of Ti-plasmid and Ri-plasmid.     

Potato bacterial wilt in India caused by strains of phylotype I, II and IV of Ralstonia solanacearum

Bacterial wilt or brown rot is one of the most devastating diseases of potato caused by a bacterium Ralstonia solanacearum (Smith 1986) Yabuuchi et al. (Microbiol Immunol 39:897–904 1995). Traditionally, R. solanacearum is classified into five races (r) on the basis of differences in host range and six biovars (bvs) on the basis of biochemical properties. Recently using molecular methods, R.?solanacearum has been classified into phylotypes based on the intergenic transcribed sequence of the ribosomal RNA genes 16S and 23S and into sequevars based on the endoglucanase gene (egl) sequence. In the present study, 75 bacterial strains, isolated from wilt infected potatoes from various potato growing regions of India, were classified by traditional and molecular methods. The identity of all the strains was confirmed as R. solanacearum as expected single 280-bp fragment resulted in all the strains following PCR amplification using R. solanacearum specific universal primer pair 759/760. Biovar (bv) analysis, based on utilization of disaccharide sugars and hexose alcohols, categorised the 75 strains into bv2 (78.7 %), 2 T (5.3 %), 3 (5.3 %) and 4 (10.7 %). The phylotype specific multiplex PCR assigned 78.7 % strains to phylotype II, 16.0 % to phylotype I and 5.3 % to phylotype IV. Phylogenetic analysis of egl gene sequences clustered all fifty nine phylotype II (bv2) strains with reference strain IPO1609 (IIB-1), all four phylotype IV (bv2T) strains with reference strain MAFF301558 (IV-8), three phylotype I (bv3) strains with reference strain MAFF211479 (I-30) and all eight phylotype I (bv4) and one phylotype I (bv3) strain with reference strain CIP365 (I-45). The study concluded that the Indian potato strains of R. solanacearum belong to three out of four phylotypes namely: the Asian phylotype I, the American phylotype II, and the Indonesian phylotype IV. This is the first study to address the diversity of R. solanacearum from potato in India using phylotype and sequevar scheme. We also report here for the first time the occurrence of phylotype IV sequevar 8 (bv2T) strain of R. solanacearum causing potato bacterial wilt in mid hills of Meghalaya in India.     

PCR-based specific detection of <Emphasis Type="Italic">Ralstonia solanacearum</Emphasis> race 4 strains

Two primer sets were designed based on the sequence of polymorphic bands that were derived from repetitive sequence-based polymerase chain reaction (rep-PCR) fingerprinting and specifically detected in Ralstonia solanacearum race 4 strains (ginger, mioga, and curcuma isolates). One primer set (AKIF-AKIR) amplified a single band (165bp) from genomic DNA obtained from all mioga and curcuma and some ginger isolates; another set (21F-21R) amplified one band (125bp) from the other ginger isolates. These primer sets did not amplify the bands from genomic DNA of other R. solanacearum strains or of other related bacteria. PCR detection limit for the pathogen was 2 × 10²cfu.The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under accession numbers AB118756 and AB118757     

Sequence Analysis and Detection of Ralstonia solanacearum by Multiplex PCR Amplification of 16S–23S Ribosomal Intergenic Spacer Region with Internal Positive Control

Polymerase chain reaction (PCR) methods for detection and differentiation of Ralstonia solanacearum strains were compared. The 16S–23S rRNA gene ITS sequence data revealed the two main sequence clusters (divisions I and II) of R. solanacearum and further subclusters of division II. Based on this sequence data, primers were designed which differentiated divisions I and II. Furthermore, to improve reliability of the PCR assay for routine detection of R. solanacearum in host plants, a novel multiplex PCR assay was developed in which the pathogen-specific sequences are coamplified with host plant DNA as an internal PCR control (IPC). The assay was validated during routine testing of potato samples submitted in official surveys. Of 4300 samples from 143 cultivars, 13 tested positive in both multiplex PCR and immunofluorescence (IF) assays and could be confirmed by bioassay in tomato seedlings and reisolation of the pathogen. The IPC was successfully amplified from all samples tested. A further 12 samples gave positive IF results which were not confirmed by either the multiplex PCR or tomato bioassay, indicating a greater specificity of the latter two assays.     

Phylogenetic relationships of Ralstonia solanacearum species complex strains from Asia and other continents based on 16S rDNA, endoglucanase, and hrpB gene sequences

The 16S rDNA, endoglucanase, and hrpB genes were partially sequenced for Asian strains of Ralstonia solanacearum spp. complex, including 31 strains of R. solanacearum and two strains each of the blood disease bacterium (BDB) and Pseudomonas syzygii. Additional sequences homologous to these DNA regions, deposited at DDBJ/EMBL/GenBank databases were included in the analysis. Various levels of polymorphisms were observed in each of these DNA regions. The highest polymorphism (approximately 25%) was found in the endoglucanase gene sequence. The hrpB sequence had about 22% poly-morphism. The phylogenetic analysis consistently divided the strains into four clusters, as distinctly shown on the phylogenetic trees of 16S rDNA, hrpB gene, and endo-glucanase gene sequences. Cluster 1 contained all strains from Asia, which belong to biovars 3, 4, 5, and N2. Cluster 2 comprised the Asian strains of R. solanacearum (as biovars N2 and 1) isolated from potato and clove, as well as BDB and P. syzygii. Cluster 3 contained race 3 biovar 2 strains from potato, race 2 biovar 1 strains from banana, and race 1 biovar 1 strains isolated from America, Asia, and other parts of the world. Cluster 4 was exclusively composed of African strains. The results of the study showed the distribution and diversity of the Asian strains, which are present in three of the four clusters. The similarity of Asian strains to those in the other regions was also observed.The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under accession numbers AY464950 to AY465050     

Development of the simultaneous detection of Ralstonia solanacearum race 3 and Clavibacter michiganensis subsp. sepedonicus in potato tubers by a multiplex real-time PCR assay

Clavibacter michiganensis subsp. sepedonicus and Ralstonia solanacearum (Smith) Yabuuchi et al. race 3 are the causal agents of ring-rot and brown-rot of potato respectively. These diseases represent a serious threat to potato production in temperate climates. Both bacteria are listed as A2 pests in the EPPO region and as zero-tolerance quarantine organisms in the European Union. All the detection tests developed so far were only focused on the detection of a single pathogen while the absence of both bacteria has to be certified in the seed tubers. We have therefore developed a new multiplex real-time PCR assay to simultaneously detect both bacteria in a single assay. Additionally, the reliability of this molecular diagnostic test has been improved by the simultaneous amplification of an internal control, corresponding to a potato gene co-extracted from the sample. The polyvalence and the specificity of each set of bacterial primers and probes were evaluated on more than 90 bacterial strains. The limit of detection of this triplex real-time protocol was similar to those observed with other molecular protocols previously developed for the individual detection of one of these bacteria. A concordance of 100 % was obtained in a blind test mimicking the routine application of the technology. In conclusion, this new protocol represents a straightforward and convenient method potentially adapted to primary screening of potato tubers.     

Simultaneous detection by multiplex PCR of the high-temperature-growing Pythium species: P. aphanidermatum, P. helicoides and P. myriotylum

The objective of this study was to develop a multiplex PCR detection method for the high-temperature-growing pathogens Pythium aphanidermatum, P. helicoides and P. myriotylum. Species-specific primer pairs were designed that targeted the rDNA ITS regions. The multiplex PCR was constructed with a universal primer pair for eukaryotes directed at the 18S rDNA as a positive control, in addition to the three species-specific primer pairs. When the multiplex PCR was applied to naturally infested soils, the expected species were reliably identified, suggesting that the method is suitable for the detection of the three Pythium pathogens in environmental samples.     

Überleben von Ralstonia solanacearum Biovar 2 (Rasse 3), dem Erreger der Schleimkrankheit der Kartoffel, in Boden und auf verschiedenen Materialien (Mikrokosmos)

Microcosm studies were carried out to test the survival of Ralstonia solanacearum biovar 2 (race 3) in soil at the permanent wilting point (wp) water content and at field capacity (fc) water content and on various material. Soils were placed at permanent ?5°C, 4°C, 15°C and 20°C and weekly fluctuating ?10/0/+10°C and the material at 5, 15 °C, 20°C with relative humidity (rh) uncontrolled or at constant 10% or 90%. In soil, survival was clearly dependent on temperature independent of water content. At 20°C Ralstonia solanacearum could be reisolated up to 364 days, at 15°C up to 290 days, at 4°C up to 209 days and at fluctuating temperatures (?10/0/+10°C) only up to 18 days. The lower the temperature, the more the population declined. At 15°C and 20°C appr. 10⁷ cfu/g soil were detected after 100 days, whereas at ?5°C only 10² cfu/g soil were detected after only 18 days. The pathogen was longer detectable in sandy-clay loam than in lighter sandy soil. It could be longer reisolated at wilting point and the populations did not decline as rapidly as at field capacity. Ralstonia solanacearum could best survive on material surfaces like rubber, plastic and varnished metal with maximum survival of 40 days at 5°C and 10% rh. In general there is a low risk of Ralstonia solanacearum overwintering under European climatic conditions when the fields are cleared of plant debris and the soil is frozen. Contamined material surfaces pose the risk of pathogen transmission to healthy tubers.     

Detection of <Emphasis Type="Italic">Agrobacterium vitis</Emphasis> by PCR using novel <Emphasis Type="Italic">virD2</Emphasis> gene-specific primers that discriminate two subgroups

Tumour tissue samples were collected from vines grown in various regions of Italy and other parts of Europe and extracted for detection of Agrobacterium vitis. Fifty strains were isolated on agar plates and screened by PCR with consensus primers from the virD2 gene. They were confirmed as A. vitis with a species-specific monoclonal antibody. The isolates were further analyzed by PCR for their opine synthase genes and ordered into octopine, nopaline and vitopine strains. Primers designed on the octopine synthase gene did not detect octopine strains of Agrobacterium tumefaciens. For quantitative PCR, virD2 fragments were sequenced: two classes of virD2 genes were found and two primer sets designed, which detected octopine and nopaline strains or only vitopine strains. For simultaneous identification of all opine-type strains, multiplex real-time PCR with either primer pair and SYBR Green was performed: the combined sets of primers gave signals with DNA from any A. vitis strain. Specificity of the new primers for real-time PCR was evaluated using several unidentified bacterial isolates from grapevines and other plant species. An elevated level of non-specific background was observed when the combined primer sets were used in multiplex PCR assays. The real-time PCR protocol was also used to detect A. vitis cells directly from grapevine tumours; avoiding direct isolation procedures a sensitivity in the range of one to ten cells per assay was found. Inhibition of the PCR reaction by plant material was overcome by treating tumour extracts with a DNA purification kit as a step for the isolation of nucleic acids.     

Diversity of Brazilian biovar 2 strains of Ralstonia solanacearum

Ralstonia solanacearum is responsible for bacterial wilt disease. Specific and accurate identification of this pathogen is essential for protection of susceptible crops as well as breeding resistant varieties. Historically, R. solanacearum has been classified into biovars based on the use of sugar and alcohol as carbon sources, into races based on its ability to infect different hosts, more recently into phylotypes based on the intergenic transcribed sequence of the ribosomal RNA genes 16S and 23S and into sequevars based on the endoglucanase gene (egl) sequence. Race 3 biovar 2 (R3Bv2) is widespread in South and Central America, and in Brazil it is present in all potato-producing regions as the most prevalent strain. In this study, we classified 53 Brazilian R. solanacearum biovar 2 (Bv2) strains by traditional and molecular methods. PCR with specific primers confirmed all 53 bacterial strains as belonging to the R. solanacearum species complex, and all were classified as biovar 2A or 2T based on acidification of sugars and alcohols. Multiplex phylotype PCR assigned all strains to phylotype II. Phylogenetic analysis of egl sequences showed that most Bv2 strains from Brazil analyzed in this study did not cluster with known sequevars and are less clonal than the R3Bv2 strains reported for other countries. This is the first study to address the diversity of a collection of Brazilian R. solanacearum strains using the phylotype and sequevar classification scheme.     

Specific Detection of Biovars of Ralstonia solanacearum in Plant Tissues by Nested-PCR-RFLP

A sensitive and specific assay, based on a Nested-PCR-RFLP protocol, was developed for the detection of biovars of Ralstonia solanacearum, the causal agent of bacterial wilt. Oligonucleotide primer pairs were selected within the hrp gene region. Specific amplification of the hrp fragments was obtained for all R. solanacearum strains and also for two closely related species, Pseudomonas syzygii and the blood disease bacterium. No amplification was observed for a wide range of other bacterial species, including R. pickettii and Burkholderia cepacia. Digestion with HindII provided four distinct restriction profiles specific to biovars or groups of biovars of R. solanacearum: one for biovar 1 strains originating from the Southern part of Africa, one for American biovar 1 and biovars 2 and N2 strains, one for biovars 3 and 4 strains, and one for biovar 5 strains. When applied to either pure culture or infected plant tissues, Nested-PCR allowed detection as low as 10³cfu ml^–1, which corresponds to 1cfu per reaction. Amplification was partially or completely inhibited by compounds contained in plant extracts (potato plant and potato tuber, tomato, tobacco, eggplant, pepper and Pelargonium asperum). A combined PVPP/BSA treatment prior to amplification permitted reliable Nested-PCR detection of R. solanacearum strains in plant samples. Nested-PCR-RFLP, assessed with isolates from Reunion Island but also applicable to any R. solanacearum strain, provides a wide range of possible uses for identification, detection and epidemiological investigations.     

Bacterial wilt disease: Host resistance and pathogen virulence mechanisms

Bacterial wilt (BW), caused by Ralstonia solanacearum, is one of the most destructive bacterial diseases of Solanaceous species worldwide. The species infects plants in more than 200 species and 50 families and was ranked second in a list of the top 10 most scientifically and economically important bacterial plant pathogens [1]. The molecular mechanisms underlying resistance and the functions of R. solanacearum effectors are beginning to be uncovered, and much remains to be discovered. In this mini-review, we provide a summary of host resistance and R. solanacearum virulence mechanisms, with a focus on tomato.     

Vine cuttings as possible initial inoculum sources of Ralstonia solanacearum race 1 biovar 4 on vegetable sweet potato in fields

Ralstonia solanacearum, which consists of five races/biovars, is considered a “species-complex” and is an important phytopathogen that causes wilt disease in more than 200 plant species. R. solanacearum race 1 biovar 4 (R1bv4) has caused yield losses of 30–80 % in the vegetable sweet potato (VSP) in the last decade in Taiwan. To identify the source of the initial inoculum of R1bv4 in VSP fields, soil and cuttings from these fields were examined from 2009 to 2010. The results of the investigation indicated that the population of R1bv4 was generally distributed throughout the natural soil of VSP fields at a density ranging from 1.3?×?10² to 9.5?×?10⁵ cfu/g soil; however, the incidence of bacterial wilt was not significantly associated with the density of the R1bv4 population in soils (R²?=?0.084). In contrast, densities of R1bv4 ranging from 2.3?×?10³ to 5.9?×?10⁵ cfu/g tissue were detected in the vine tissue of asymptomatic plants in the fields. Additional experiments demonstrated that R1bv4-free VSP cuttings without visible symptoms planted in infested soils in the greenhouse setting could carry approximately 3.1?×?10⁵ R1bv4 cfu/g tissue, which suggests the existence of a latent period for R1bv4 in VSP plants. The results of a BIO-PCR analysis showed that R1bv4 was detected in 2.0 to 98.0 % of the VSP cuttings used for propagation in fields; in addition, the percentage of VSP cuttings carrying R1bv4 and the incidence of bacterial wilt in fields were positively correlated (R²?=?0.909). The inoculation experiments conducted in greenhouses and in fields showed that the cutting inoculum (CI) contributed more to the incidence of bacterial wilt in VSP plants than the soil inoculum (SI). In the field experiments conducted in 2010, an incidence of disease of 27.1 to 38.5 % was detected in healthy field cuttings 8 months after transplantation; in contrast, the incidence of disease in field cuttings carrying R1bv4 was 49.0 to 68.8 %. The incidence of disease was significantly lower in healthy cuttings than in cuttings carrying R1bv4 (p?=?0.05).     

Application of matrix-assisted laser desorption ionization time-of-flight mass spectrometry for rapid and accurate identification of <Emphasis Type="Italic">Ralstonia solanacearum</Emphasis> and <Emphasis Type="Italic">Ralstonia pseudosolanacearum</Emphasis>

Ralstonia solanacearum “species complex” (RSSC) represents soil-borne plant pathogenic bacteria, consisting of diverse and widespread strains that cause bacterial wilt on a wide range of host plants. A recent polyphasic taxonomic study has divided the RSSC into three bacterial species; Ralstonia pseudosolanacearum (phylotypes I and III), Ralstonia solanacearum (phylotype II) and Ralstonia syzygii (phylotype IV). Currently, standard identification of RSSC in plant health laboratories mainly relies on performance of two tests that are based on a different principle. However, these tests are inadequate to precisely discriminate among the three bacterial species in the RSSC. The accurate identification of each of the three bacterial species in the RSSC requires additional molecular tests, including a phylotype determination. These methodologies are labor-intensive, time consuming and rather impractical for routine identification purposes in a plant health laboratory. We explored the potential for an accurate identification of R. pseudosolanacearum (phylotypes I and III) and R. solanacearum (phylotype II) in RSSC, upon implementation of the MALDI-TOF MS tool, and after the creation and validation of an in-house database supplementing the commercial database and covering the entire known genetic diversity in RSSC. MALDI-TOF MS is an emerging approach for identification of bacterial plant pathogens and has been shown to be robust and reproducible. Additionally, when compared to the conventional microbial identification methods it is shown to be less laborious and less expensive. Validation data demonstrated that our in-house database (Mass Spectra Profiles, MSPs) was very specific resulting in the rapid and accurate identification of Ralstonia solanacearum (phylotype II), and Ralstonia pseudosolanacearum (phylotypes I and III). Additionally, no false positive results were obtained with our in-house database for other related Ralstonia sp., such as the R. picketii isolate PD 3286, or for the Pseudomonas syringae and Pseudomonas spp. isolates.     

Molecular typing of Japanese strains of Ralstonia solanacearum in relation to the ability to induce a hypersensitive reaction in tobacco

The genetic diversity of 120 Ralstonia solanacearum strains isolated from a variety of host plants across Japan was assessed on the basis of hypersensitive response (HR) in tobacco leaves and phylogenetic analyses of endoglucanase gene egl, hrpB, and gyrB. Phylogenetic analysis of egl revealed that only three strains belonged to phylotype IV, and 117 strains belonged to phylotype I. Partial sequences of HrpB were identical among phylotype I strains except for one strain. Analyses using the partial nucleotide sequences of the gyrB and egl gene fragments grouped phylotype I strains into 11 gyrB and 8 egl types, respectively, whereas analyses using the partial amino acid sequences of GyrB and Egl grouped phylotype I strains into 4 GyrB and 5 Egl types, respectively. Using multilocus sequence typing of GyrB and Egl, we identified 10 unique sequence types within the Japanese phylotype I strains. Strains belonging to the GyrB42 or GyrB66 type caused wilt in tobacco, and strains belonging to GyrB2 or GyrB9 type elicited HR, demonstrating that HR induction in tobacco is genetically differentiated in the Japanese strains of R. solanacearum.     

Performance of real‐time PCR and immunofluorescence for the detection of Clavibacter michiganensis subsp. sepedonicus and Ralstonia solanacearum in potato tubers in routine testing

This paper describes a comparison study of test methods and supports the use of real‐time polymerase chain reaction (PCR) for the detection of Clavibacter michiganensis subsp. sepedonicus and Ralstonia solanacearum in potato tubers in routine testing. These 2 bacteria are quarantine organisms under European Union (EU) regulatory control and testing for (latent) infections of these bacteria in seed potatoes is mandatory. Real‐time PCR tests were performed on 276 routine potato tuber samples, including samples infected with either C. michiganensis subsp. sepedonicus or R. solanacearum, and the performance of these real‐time PCR tests was compared with that of immunofluorescence (IF). Real‐time PCR tests, using different primer sets and extraction and PCR protocols, proved to be sensitive and specific for the detection of C. michiganensis subsp. sepedonicus and R. solanacearum in potato tubers in routine testing, and performed at least as well as IF. Real‐time PCR is a good addition to the detection protocols as laid down in EU regulations (EU Council Directives 2006/56/EC and 2006/63/EC).