Phylotype-specific Multiplex PCR Research Articles

Identification, genetic diversity, and pathogenicity of Ralstonia pseudosolanacearum causing cigar tobacco bacterial wilt in China.

Ralstonia pseudosolanacearum, previously known as R. solanacearum species complex (RSSC) phylotypes I and III, is a plant pathogenic bacterium causing significant yield losses in economical crops. In the May of 2020 and 2021, cigar tobacco bacterial wilt was first observed in fields in Danzhou, Hainan Province, China. A total of eight bacterial isolates were isolated and identified as R. pseudosolanacearum with race 1, biovar III by 16S rRNA gene sequencing, Biolog, and host identification. The amino acid sequence showed that Hainan strains and 15 R. pseudosolanacearum reference strains from flue-cured tobacco in Shandong and Guizhou Provinces, all belonged to RS1000 type containing the avrA gene, only Guizhou strains also had the popP1 gene. On the basis of phylotype-specific multiplex PCR amplification, mismatch repair gene and endoglucanase gene-base tree, Hainan strains were identified as phylotype I sequevar 70, and showed stronger pathogenic capabilities on three different varieties than those reference strains. This is the first report of cigar tobacco bacterial wilt caused by R. pseudosolanacearum sequevar 70. The results revealed the diversity of RSSC in Nicotiana tabacum in China and provided useful information regarding the epidemiology of cigar tobacco wilt disease, as well as the breeding for disease resistance in local cigar tobacco.

First Report of Bacterial Wilt Caused by Ralstonia solanacearum on Plectranthus amboinicus in Martinique.

Plectranthus amboinicus, commonly known as Gwo ten in the French West Indies (Martinique), is a semi-succulent perennial plant of the Lamiaceae family. This aromatic plant wich is widespread naturally throughout the tropics is of economic importance because of the therapeutic and nutritional properties attributed to its natural phytochemical compounds wich are highly valued in the pharmaceutical industry. In March 2019, wilted P. amboinicus plants intercropped with tomato plants (cv. Heatmaster) in order to reduce the insect-pest damages on tomato, were observed in a field located at the CIRAD experimental station in Lamentin, Martinique (14.663194 N, -60.999167 W). Average disease incidence of 65.74% was recorded on P. amboinicus, in 3 plots with an area of 22.04 m2. The initial symptoms observed were irregular, black, necrotic lesions on leaves. After 10 days, plants wilted and black stripes were observed on stems. Within 4 weeks, more than 50% of plants were fully wilted. Longitudinal stem sections of the wilted plants showed brown vascular discoloration. The cut stems of the wilted plants released a whitish bacterial ooze in water. In all, 108 stem sections were collected, surface disinfected with 70% ethanol and each was crushed in 2 mL of Tris-buffer, then processed for bacterial isolation by plating on modified Semi-Selective Medium from South Africa SMSA (Engelbrecht 1994). Typical Ralstonia solanacearum colonies grew on SMSA medium for 100 of the 108 samples after incubation for 48h at 28°C and were identified as Ralstonia solanacearum using diagnostic PCR with 759/760 primers (Opina et al. 1997). A phylotype-specific multiplex PCR (Fegan and Prior 2005) classified all the strains in R. solanacearum Phylotype IIA. A subset of 11 strains was selected for sequevar identification. All the strains were identified as sequevar I-39 (100% nucleotide identity with strain ANT92 - Genbank accession EF371828), by partial egl sequencing (Fegan and Prior 2005) (GenBank Accession Nos. MT314067 to MT314077). This sequevar has been reported to be widespread in the Caribbean and tropical America on vegetable crops (particularly on tomato), but not on P. amboinicus (Deberdt et al. 2014; Ramsubhag et al. 2012; Wicker et al. 2007). To fulfil Koch's postulates, a reference strain, isolated from diseased P. amboinicus (CFBP 8733, Phylotype IIA/sequevar 39), was inoculated on 30 healthy P. amboinicus plants. A common tomato cultivar grown in Martinique (cv. Heatmaster) was also inoculated on 30 plants with the same bacterial suspension. Three-weeks-old plants of both crops grown in sterilized field soil were inoculated by soil drenching with 20 ml of a calibrated suspension (108 CFU/mL). P. amboinicus and tomato plants drenched with sterile water served as a negative controls. Plants were grown in a fully controlled environment at day/night temperatures of 30-26°C ± 2°C under high relative humidity (80%). The P. amboinicus plants started wilting 9 days after inoculation, and within four weeks 60% of the P. amboinicus plants had wilted. The tomato plants started wilting 5 days after inoculation with 62% of wilted plants within four weeks. R. solanacearum was recovered from all symptomatic plants on modified SMSA medium. No symptoms were observed and no R. solanacearum strains were isolated from negative controls plants. To our knowledge, this is the first report of R. solanacearum causing bacterial wilt on Gwo ten (P. amboinicus) in Martinique. The importance of this discovery lies in the reporting of an additional host for R. solanacearum, which can be associated with other crops as tomato crop in order to reduce the abundance of insect-pests. Further studies need to be conducted to assess the precise distribution of bacterial wilt disease on P. amboinicus in Martinique and to develop a plan of action avoiding its association with R. solanacearum host crops as tomato for reducing epidemic risk.

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

AbstractBacterial 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.

First report of Ralstonia solanacearum causing bacterial wilt of eucalyptus in Ecuador.

In January 2018, eucalyptus trees showing wilt symptoms were identified in a commercial plantation located in the Quevedo Cantón, Los Ríos province, Ecuador. Disease incidence reached 40% of the eucalyptus field and affected plants displayed reddening and wilting of the foliage, leaf drop, branch dieback, and reduced growth, resembling bacterial wilting symptoms (Santiago et al., 2014). Transverse sections made on trunks of wilted trees revealed vascular discoloration of the wood and bacterial ooze. Wood pieces taken from discoloured tissues were surface disinfected and macerated in sterilized distilled water. The resulting suspensions were spread on plates containing triphenyltetrazolium chloride medium (TZC) (Kelman, 1954) and incubated at 28°C for 48 h. Three mucoid white colonies with pinkish centers, characteristic of Ralstonia solanacearum, were purified (Figure S1C). The isolates were Gram negative in the KOH test, non-fluorescent on King's B medium, and formed cream colonies on yeat extract-dextrose-calcium carbonate (YDC) medium. Phylotype-specific multiplex PCR (PMX-PCR) (Fegan and Prior, 2005) and phylogenetic analysis based on partial endoglucanase (egl) gene sequences (Poussier et al. 2000) (Figure S2) identified the isolates as R. solanacearum Phylotype IIB sequevar 51. The isolates were preserved in the COAD culture collection of the Universidade Federal de Vicosa, Brazil (codes COAD 2586, 2587 and 2588) and the corresponding egl DNA sequences were deposited in the GenBank (accession numbers MH350031, MH350032 and MH350033). Pathogenicity tests were conducted with all three isolates. Twenty microliters of bacterial suspensions (108 CFU/mL of saline solution) were injected at the base of the stem of eucalyptus (Eucalyptus urograndis), banana (Musa sp. cv. Maçã) and tomato (Solanum lycopersicum cv. Santa Clara) plants kept in a greenhouse (28 ± 2 °C) (Fonseca et al., 2016). Three plants of each host species were inoculated per bacterial isolate and plants injected with distilled water were used as controls. Necrotic symptoms appeared at the inoculation site on eucalyptus plants after 10 days. Wilt symptoms started at the top and progressed towards the base of the plants (Figure S1A). Tomato plants died within one week, and eucalyptus seedlings died after three weeks. The inoculated banana seedlings and all plants from the control treatments remained asymptomatic. Bacterial ooze was observed on freshly-cut transverse sections made on wilted eucalyptus seedlings (Figure S1B) and typical colonies of R. solanacearum were isolated from inoculated plants, fulfilling Koch's postulates. The correct diagnosis of the pathogen is the first step in the long-term process of developing effective management tools for this disease in Ecuador.

Isolation, identification and molecular characterization of Ralstonia solanacerum isolates collected from Southern Karnataka

Bacterial wilt caused by Ralstonia solanacearum, is the major threat to tomato cultivation in all tomato growing areas of Karnataka. R. solanacearum was isolated from the infected host plants collected from different locations of southern Karnataka. The identity of the isolates was established using morphological, biochemical, and molecular analysis using species specific PCR primers. The race and biovar specificity of pathogen was determined through pathogenicity test on different host plants and the ability of isolates to use carbohydrates, respectively. Phylotype classification was done by phylotype specific multiplex PCR using phylotype specific primers. All the bacterial isolates showed the characteristic creamy white fluidal growth with pink centre on the Tetrazolium chloride medium. Further, the isolates amplified at 280 bp, which confirmed the identity of pathogen as Ralstonia solanacearum. Our results showed that all isolates belonged to Race 1 of the pathogen. Among different isolates obtained, four isolates each were identified to be Biovar III and Biovar IIIA, repectively, while two isolates were identified as Biovar IIIB. All the ten isolates were affiliated to Phylotype I of Ralstonia solanaceraum species complex. These findings may help in devising the management practices for bacterial wilt of tomato in southern Karnataka.

Screening of tomato genotypes against bacterial wilt (Ralstonia solanacearum) and validation of resistance linked DNA markers

Tomato yield losses are mainly caused due to bacterial wilt (Ralstonia solanacearum) throughout the world. The disease management is mainly done through chemicals which lead to health risks. Among non-chemical strategies, development of resistant varieties can be a good alternative. The present study was conducted to screen the fifty seven different tomato genotypes against bacterial wilt using artificial inoculation technique under greenhouse conditions. The plants showing symptoms were examined using ooze test. Morphological and molecular characterization of the bacterial strains isolated from infected plants was carried out and bacterial phylotype I was determined using phylotype-specific multiplex PCR. The bacterial-infected tomato genotypes were categorized into highly resistant, resistant, moderately resistant, moderately susceptible, susceptible, highly susceptible and extremely susceptible lines. Seven tomato genotypes viz.RIL-118, Indam-1004, Arka Samrat, PKM-1, PED, EC-802390, and EC-816105 were found highly resistant to bacterial wilt. These genotypes were also evaluated for plant growth, yield and yield-related traits and fruit quality traits under field conditions. Maximum production (2533 g/plant) was observed for the genotype Arka Rakshak followed by EC816156 (2486 g/plant).while genotype EC815157 (200 g/plant) exhibited lowest production. In addition, fourteen bacterial wilt linked markers were validated for these genotypes where SCAR marker, SCU176-534 was found to be linked with the bacterial wilt resistance significantly. This study will be significant and useful in increasing tomato production and to develop new resistant tomato varieties through marker assisted breeding.

First Report of Bacterial Wilt of Gboma (Solanum macrocarpon) Caused by Ralstonia solanacearum in Benin

Gboma (Solanum macrocarpon) is the major leafy vegetable cultivated in Benin, with many people depending on this crop for their livelihood. In March 2012, wilted Gboma plants without foliar yellowing were observed at three locations (Dogbo, Lokossa, and Athieme) in the southwestern districts. Longitudinal sections of most stems of Gboma showed brown vascular discoloration. The cut stems released whitish bacterial ooze in water. Bacterial colonies on modified semi-selective medium from South Africa SMSA (Engelbrecht 1994) were mucoid and round with red centers with beige peripheries, thus morphologically similar to Ralstonia solanacearum. To fulfill Koch's postulates, 19 different isolates were inoculated on susceptible Gboma cv. Kpinman, grown in sterilized field soil. Plants (10 per isolate) were inoculated by drenching the soil with 40 ml of a bacterial suspension containing 108 CFU/ml. Plants treated in a similar manner with sterile water served as a negative control. Inoculated plants were incubated under a protected shed at about 30°C. By 15 days after inoculation, 100% of the inoculated plants were wilted whereas all control plants remained healthy. R. solanacearum was recovered from all symptomatic plants on modified SMSA medium. In 2014, a survey throughout the Abomey-Calavi area (Atlantic District) identified bacterial wilt of Gboma within all cultivated farms, with incidences ranging from 15 to 75.2%. Thirty newly collected bacterial isolates were identified as Ralstonia solanacearum using ImmunoStrip assays (Agdia Inc., Elkhart, IN, USA), and diagnostic PCR with 759/760 primers (Opina et al. 1997). A phylotype-specific multiplex PCR (Fegan and Prior 2005) classified all strains in R. solanacearum Phylotype I. Partial sequences of the egl and mutS genes were deposited in GenBank under Accession Nos. KP730628 to KP730687. Alignment of these to R. solanacearum reference sequences confirmed that all strains from Abomey-Calavi and two from Dogbo belonged to one unique Sequence Type (ST) within Phylotype I (egl ST 43 (sequevar 31), mutS ST 22). This ST has also been reported to be widespread in the Ivory Coast (N'Guessan et al. 2012), and in South Africa, India, and Reunion Island (Wicker, unpublished data, 2015). This is the first report of R. solanacearum causing bacterial wilt on Gboma in Benin. Further surveys will help to assess the precise distribution of bacterial wilt disease on Gboma in Benin, and allow development of integrated management strategies combining currently available resistant cultivars and rotation with suppressive crops. (Texte integral)

Genetic diversity of Ralstonia solanacearum phylotype II sequevar 4 strains associated with Moko disease of banana (Musa spp.) in Peninsular Malaysia

Moko disease caused by Ralstonia solanacearum (R. solanacearum) is a major disease affecting banana (Musa spp.) production. Although local reports suggested that this disease is widespread in Malaysia, molecular characterization of R. solanacearum strains associated with Moko disease in this country has not yet been done. During March 2011 to June 2012, 170 banana plants associated with Moko disease and the adjacent soil samples were collected in 12 different locations of five outbreak states in Peninsular Malaysia comprising Kedah, Selangor, Pahang, Negeri Sembilan and Johor, with disease incidence exceeding 80 % in some severely affected plantations. A total of 197 strains were identified as R. solanacearum-like colonies since they produced fluidal colonies that were white to pink coloration after incubation at 24 to 48 h at 29 °C on Kelman’s TZC agar medium, appeared as Gram-negative rods, and positive for potassium hydroxide (KOH), Kovacs oxidase, catalase and lipase activity on Tween 80 solution tests. Biovar tests disclosed that only 30 strains displayed characteristics of biovar 1 R. solanacearum associated with Moko disease, which was negative for utilization of disaccharides and hexose alcohols. Pathogenicity assay showed that these 30 strains were virulence towards Musa paradisiaca cv. Nipah explants with diverse degrees of virulence. Phylotype-specific multiplex PCR (Pmx-PCR) revealed all strains belonged to phylotype II displaying a 372 bp amplicon. Phylogenetic analyses of endoglucanase (egl) sequences clustered all 30 strains into phylotype II/4, together with the reference sequences strains from Peru (UW129, UW162 and UW163) and Colombia (UW070). Pooled rep-PCR fingerprinting method defined two major groups; cluster 1 (sub-group A and B) and cluster 2 (sub-group C), with 35 % average similarity coefficient within these two clusters. The sub-groups in cluster 1 were represented by strains from Kedah, Selangor, Negeri Sembilan and Johor; while cluster 2 sub-group was represented exclusively by strains of Pahang. To our knowledge, this is the first description of R. solanacearum phylotype II/4 in Malaysia and the Asian region. Our findings may expand constructive documentation and describe a better understanding on diversity of Malaysian R. solanacearum Moko-causing strains populations, thus will be useful for designing disease control strategies.

First Report of Bacterial Wilt Caused by Ralstonia solanacearum in Ghana, West Africa.

Tomato and pepper plants exhibiting wilt symptoms were collected from fields in seven villages in Northern (Vea, Tono, Pwalugu), Ashanti (Agogo, Akumadan), and Brong Ahafo (Tanoso, Tuobodom) regions of western Ghana in November 2012. The plants were wilted without leaf yellowing or necrosis. Disease incidence was generally low, with less than 20% symptomatic plants observed. Most of the plants collected produced visible bacterial ooze in water in the field. Ooze was plated on 2,3,5-triphenyltetrazolium chloride-amended (TZC) medium. Isolated colonies were fluidal, irregularly round, white with pink centers, gram-negative, and oxidase positive. One strain from each of seven fields was selected for further study. All strains induced a hypersensitive reaction on tobacco. Randomly selected strains SM855-12 and SM857-12 tested positive in R. solanacearum ImmunoStrip assays (Agdia Inc., IN). An end-point PCR assay with primer set 759/760 (3) generated an R. solanacearum-specific 280-bp amplicon for all seven strains. Two of these strains were biovar I and the remaining five were biovar III based on utilization of cellobiose, lactose, maltose, dulcitol, mannitol, and sorbitol. A phylotype-specific multiplex PCR assay that recognizes four geographically linked monophyletic groups within R. solanacearum (1) indicated that one strain (SM855-12) was phylotype III (African origin), whereas the other six were phylotype I (Asian origin). All strains were subjected to repetitive sequence-based PCR (Rep-PCR) with BOXA1R and REP1R/REP2 primers (4). Strain SM855-12 was grouped with the phylotype III reference strain UW 368 and the remaining six strains were grouped with the phylotype I reference strain GMI 1000. A pathogenicity test was performed with bacterial wilt-susceptible tomato line OH7814. Inoculum was prepared from 48-h cultures of strains SM855-12, SM856-12, and SM858-12 grown on casamino acid peptone glucose (CPG) medium at 30°C. Roots of ten 4-week-old tomato plants per strain were drench-inoculated with 5 ml of a 108 CFU/ml bacterial suspension after wounding with a sterile scalpel. Non-inoculated control plants were drenched with 5 ml distilled water after root wounding. Plants were kept in a greenhouse at 25 to 30°C. By 12 days after inoculation, 80 to 100% of inoculated plants were wilted, whereas no symptoms appeared in non-inoculated plants. Bacteria re-isolated from wilted plants were confirmed to be R. solanacearum using techniques mentioned above. Although an association of bacterial wilt with tomato/pepper was mentioned previously (2), to our knowledge, this is the first documented report of bacterial wilt caused by R. solanacearum in Ghana. The presence of Asian strains (phylotype I) may be the result of one or more accidental introductions. Awareness of this disease in Ghana will lead to deployment of management strategies including use of resistant varieties and grafting desirable varieties onto disease-resistant rootstocks.

Phylotype classification of Ralstonia solanacearum biovar 1 strains isolated from banana (Musa spp) in Malaysia

During 2011–2012, 15 bacterial isolates were obtained from wilting banana plants from seven locations in Malaysia. Characterisation of the Malaysian isolates was determined by biovar determination, pathogenicity test, phylotype-specific multiplex PCR (Pmx-PCR) and endoglucanase (egl) gene amplification. Based on the genotype, phenotype and pathogenic characteristics, all isolates were identified as Ralstonia solanacearum. Pmx- and egl-PCRs indicated that all isolates belong to phylotype II of Ralstonia species complex hierarchical classification. The neighbour joining phylogenetic tree of egl sequences also verified the results where the isolates were all clustered into phylotype II, together with the reference sequences strains, UW070 and UW162. Therefore, the results of our study may provide a better understanding on the taxonomy of R. solanacearum species occupying banana plantations in Malaysia. This study is indeed the first report of phylotype II classification of R. solanacearum biovar 1 strains isolated from banana plants in Malaysia.

First Report of Ralstonia solanacearum Race 2 Biovar 1 Causing Moko Disease of Banana in Malaysia.

During March 2011 to June 2012, 50 banana plants of cultivar Musa × paradisiaca 'Horn' with Moko disease symptoms were randomly sampled in 12 different locations of 5 outbreak states in Peninsular Malaysia comprising Kedah, Selangor, Pahang, Negeri Sembilan, and Johor, with disease incidence exceeding 90% in some severely affected plantations. The disease symptoms observed in the infected plants included yellowing and wilting of the oldest leaves, which became necrotic, and eventually led to their dieback or collapse. The pulp of banana fruits also became discolored and exuded bacterial ooze. Vascular tissues in pseudostems were discolored. Fragments from symptomatic plant samples were excised and cultured on Kelman's-tetrazolium salt (TZC) medium. Twenty positive samples produced fluidal colonies that were either entirely white or white with pink centers after incubation for 24 to 48 h at 28°C on Kelman's-TZC medium and appeared as gram-negative rods after Gram staining. They were also positive for potassium hydroxide (KOH), Kovacs oxidase, and catalase tests, but negative for utilization of disaccharides and hexose alcohols, which are characteristics of biovar 1 Ralstonia solanacearum. For the pathogenicity test, 30 μl of 108 CFU/ml bacterial suspension of three selected virulent strains were injected into banana (Musa × paradisiaca 'Horn') leaves explants grown in plastic pots of 1,440 cm3 volume in a greenhouse, with temperature range from 26 to 35°C. Leaves that were infiltrated with sterile distilled water served as a negative control. Inoculations with all isolates were performed in three replications, as well as the uninoculated control leaves explants. The inoculated plants produced the same symptoms as observed on naturally diseased samples, whereas control plants remained asymptomatic. Strain cultures were re-isolated and possessed the morphological and biochemical characteristics as previously described. PCR amplification using race 2 R. solanacearum primers ISRso19-F (5'-TGGGAGAGGATGGCGGCTTT-3') and ISRso19-R (5'-TGACCCGCCTTTCGGTGTTT-3') (3) produced a 1,900-bp product from DNA of all bacterial strains. BLAST searches resulted that the sequences were 95 to 98% identical to published R. solanacearum strain race 2 insertion sequence ISRso19 (GenBank Accession No. AF450275). These genes were later deposited in GenBank (KC812051, KC812052, and KC812053). Phylotype-specific multiplex PCR (Pmx-PCR) and Musa-specific multiplex PCR (Mmx-PCR) were performed to identify the phylotype and sequevar of all isolates (4). Pmx-PCR showed that all isolates belonged to phylotype II, whereas Mmx-PCR showed that they belonged to phylotype II sequevar 4 displaying 351-bp amplicon. Although there were previously extensive studies on R. solanacearum associated with bacterial wilt disease of banana crops in Malaysia, none related to Moko disease has been reported (1,2). The result has a great importance to better understand and document R. solanacearum race 2 biovar 1, since banana has been identified as the second most important commercial fruit crop with a high economic value in Malaysia.

First Report of Bacterial Wilt Caused by Ralstonia solanacearum on Roselle in Taiwan.

Roselle (Hibiscus sabdariffa L.) is a herbaceous plant belonging to the Malvaceae family. Its calyxes are rich in vitamin C and anthocyanins and are used to make roselle drink and hibiscus tea. Roselles are grown in counties of Taitung, Pingtung, and Chiayi in Taiwan. In addition to a few local cultivars, the major cultivar currently grown in Taiwan is Roselle cv. Victor. In April of 2012, a wilt disease appeared on seedlings of a cultivar, Chiada 1, at the Chungpu Township of Chiayi County. Mature plants were free from this disease. Leaves appeared weak and drooping when they were still green, followed by collapse of the whole plant a few days later. Browning of vascular and pith tissues was evident, especially at the base of the stem. A whitish mass of bacteria oozed from the cut end of diseased stems, suggesting that bacteria might be the cause of this disease. A total of 15 bacterial strains were collected. Colonies on tetrazolium chloride medium (3) were round to oval and fluidal, each with a pink or red center after incubation at 30°C for 48 h. When tobacco leaves were infiltrated with these strains, a hypersensitive reaction (HR) typical of phytopathogenic bacteria was induced. All strains produced the expected amplicon (282 bp) after PCR with the Ralstonia solanacearum-specific primer pair, AU759f and AU760r (4). Three hexose alcohols (mannitol, sorbitol, and dulcitol), rather than three disaccharides (lactose, maltose, and cellobiose), were utilized, which suggests R. solanacearum biovar 4 (2). R. solanacearum phylotype I was determined by phylotype-specific multiplex PCR (1). Pathogenicity of the strains was tested on roselle, tomato, pepper, and eggplant. Young plants of the various species were inoculated at the four- to six-leaf stage by soil drenching with 30 ml of bacterial suspension (about 108 CFU/ml). Control plants were inoculated with sterile water. Each treatment comprised eight plants with a single plant in each pot. Plants were incubated in a greenhouse at 25 to 31°C and 56 to 93% humidity. Wilting was observed 4 to 6 days after inoculation, while the control did not wilt. To find the correlation between plant growth stage and resistance to the pathogen, 2-, 3-, 4-, and 5-week-old roselle plants cv. Chiada 1 were transplanted into artificially infested soil. Eight plants in each treatment were planted with a single plant in each pot. The disease incidences for plants of different ages were 75%, 62.5%, 50%, and 12.5%, respectively. This study showed that resistance increases with plant age. Hence, if older seedlings are transplanted, the risk of bacterial wilt of roselle can be reduced. To our knowledge, this is the first report of R. solanacearum on roselle in Taiwan.

Genetic diversity of Ralstonia solanacearum 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).