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

2018 ◽  
Vol 10 (3) ◽  
pp. 886-893
Author(s):  
Kalavati Teli ◽  
H. M. Shweta ◽  
M. K. Prasanna Kumar ◽  
Bharath Kunduru ◽  
B. S. Chandra Shekar

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.

Plant Disease ◽  
2010 ◽  
Vol 94 (3) ◽  
pp. 372-372 ◽  
Author(s):  
A. T. Thera ◽  
B. J. Jacobsen ◽  
O. T. Neher

Ralstonia solanacearum (Smith) Yabuuchi et al. causes bacterial wilt worldwide on a wide range of plant species. In Mali, the disease is commonly found on potato (Solanum tuberosum L.), tomato (Lycopersicon esculentum var. esculentum L.), pepper (Capsicum annuum L.), eggplant (Solanum melongena L.), tobacco (Nicotiana tabacum L.), and peanut (Arachis hypogaea L.). Determination of race and biovar is critical for development of potato seed certification programs for management of the disease. Isolates (25) of R. solanacearum were obtained from wilting potato, pepper, eggplant, tobacco, and tomato plants collected from fields near Baguineda, Sonityeni, Sotuba, Sikasso, and Kolikoro. Isolations were made from bacterial streaming by dilution plating on triphenyl tetrazolium chloride medium (TZC) (2). Characteristic colonies were selected and identified by ELISA or Immunostrips (Pathoscreen Rs, Agdia Inc., Elkhart, IN). These isolates were used in host range studies and hypersensitivity (HR) tests on tobacco (cv. xanthi) (3) and tested for their ability to produce acids on Ayers basal media amended with disaccharide and hexose alcohol carbon sources (1). These isolates caused characteristic wilt 40 days postinoculation on greenhouse-grown tobacco (cv. Xanthi), peanut (cv. 4610), and tomato (cv. Roma VF) plants when stems of five plants of each host were syringe inoculated with 0.1 ml of a 1 × 109 CFU/ml of bacteria. Plants inoculated with sterile distilled water remained symptomless and R. solanacearum was reisolated from infected plants on TZC and identified with Immunostrips. All HR tests were negative. Infection of peanut, tobacco, and tomato and the results of the HR tests indicated that all isolates were Race 1 and no significant variation was noted between isolates. Acid was produced from the hexose alcohols: mannitol, sorbitol, and dulcitol; and the disaccharides: cellobiose, lactose, and maltose. This indicated that all isolates were biovar 3, the same as a known Race 1 strain from tobacco (MSU Plant Pathology teaching collection) (1). To assess relative distribution of R. solanacearum, 20 soil samples collected from potato fields in the vicinity of Baguineda, Kati, Koulikoro, and Sikasso were placed in pots (30 × 25 cm) under shade cloth at the IER Station in Sotuba and planted with 30-day-old tobacco plants. After 90 days, infected plants (35 to 100% infection) were found in all soils. Infected plants exhibited classical wilt symptoms and tested positive for R. solanacearum infections as confirmed by Immunostrip tests. Six of nine surface water samples taken near potato fields in Baguineda, Sikasso, Mopti, and Koulikoro tested positive for the presence of R. solanacearum by an Agdia Inc. enrichment kit and ELISA. A weed, Commelina forskalaei (Vahl), collected by Farako creek near Sikasso tested positive in the Immunostrip test even though no symptoms were obvious. No attempt was made to characterize the race, biovar, or phylotype of the soil, water, and weed isolates. To our knowledge, this is the first time that the race and biovar of R. solanacearum from Mali has been reported and the wide distribution of this pathogen in Malian soils and surface water has been demonstrated. It is significant that we did not detect Race 3 biovar 2, which is subject to quarantine and biosecurity regulations. References: (1) A. C. Hayward. J. Bacteriol. 27:265, 1964. (2) A. Kelman. Phytopathology 44:693, 1954. (3) J. Lozano and L. Sequeira. Phytopathology 60:833, 1970.


Plant Disease ◽  
2012 ◽  
Vol 96 (6) ◽  
pp. 904-904 ◽  
Author(s):  
P. Li ◽  
X. X. Wu ◽  
Z. Y. Wang ◽  
H. H. Ho ◽  
Y. X. Wu ◽  
...  

Yacon (Smallanthus sonchifolius) is an important cash crop in Yunnan Province, China. In 2003, yacon was introduced to Yunnan province as a novelty root crop and as an experimental source of natural sugars; now more than 15 provinces cultivate the crop. Yunnan is one of the major yacon producing areas of China, with up to 10,000 ha yielding up to 50,000 t of yacon, which is nearly half of the production in China. In April and May 2010, bacterial wilt of yacon was observed in the fields of Lion Mountain of Wuding County, Yunnan Province, China. In 2011, the disease occurred in approximately 1 ha of yacon, resulting in 10% crop loss in that area. The initial symptoms observed were irregular, black, necrotic lesions on leaf margins. After 4 to 7 days, leaves became totally necrotic, plants wilted, and black stripes were observed on plant stems. Within 2 to 3 weeks, more than 70% of leaves within the crop were wilted. Subsequently, the plants died and stems became brittle. When dead plants were pulled from the soil, tubers were found to have turned black. When diseased stems and/or petioles were cut with a sterile sharp knife or razor blade, bacterial ooze appeared on the cut ends. High populations of morphologically uniform bacteria were isolated from the diseased plants by conventional methods. When cultured on TZC (2,3,5-Triphenylte tetrazolium chloride) agar medium (3), colonies were large, elevated, fluidal, and entirely white with a pale red center. The isolated bacterium was gram-negative, grew aerobically, and did not form endospores. The cells were 0.5 to 0.7 × 1.5 to 2.0 μm and nonencapsulated. Ralstonia solanacearum was identified and confirmed as the pathogen on the basis of morphological and physiological characteristics, pathogenicity test, and 16S rDNA sequence analysis (1,4). The nucleotide sequence is available in GenBank (Accession No. HQ176322.1). The pathogenic strain belonged to race 1 and biovar 3 according to the pathogenicity and carbohydrate utilization tests (2). Koch's postulates were tested in the greenhouse, with 10 plants inoculated per species. Plants were inoculated with 15 μl of cell suspension containing 106 to 107 CFU ml–1 deposited into the third axilla with a capillary tube. The bacteria could infect tomato, pepper, tobacco, potato, common sage (Salvia dugesii Fernald), and patchouli, and caused typical symptoms of wilt and black lesions, but could not infect leaves of swamp mahogany (Eucalyptus robusta Smith), stramonium (Dature stramonium Datura L.), ginger, or maize. To our knowledge, this is the first report of yacon as a host of R. solanacearum. Since the pathogen has a wide host range, monitoring of the vegetation in and around yacon fields should be implemented as a mandatory management measure to prevent disease spread. References: (1) C. A. Boucher et al. J. Bacteriol. 169:5626, 1987. (2) A. C. Hayward. J. Appl. Bacteriol. 27:265, 1964. (3) A. Kelman. Phytopathology 44:693, 1954. (4) W. G. Weisburg et al. J. Bacteriol. 173:697, 1991.


Plant Disease ◽  
2012 ◽  
Vol 96 (1) ◽  
pp. 143-143 ◽  
Author(s):  
M. Cadavid ◽  
J. C. Ángel ◽  
J. I. Victoria

Symptoms of sugarcane orange rust were first observed in July 2010 on sugarcane (interspecific hybrid of Saccharum L. species) cv. CC 01-1884 planted in the La Cabaña Sugar Mill, Puerto Tejada, Colombia. Morphological features of uredinial lesions and urediniospores inspected with an optical microscope and scanning electron microscopy were distinct from common rust of sugarcane caused by Puccinia melanocephala Syd. & P. Syd., revealing spores identical morphologically to those described for the fungus P. kuehnii (Kruger) E. Butler, causal agent of sugarcane orange rust (1,3). Uredinial lesions were orange and distinctly lighter in color than pustules of P. melanocephala. Urediniospores were orange to light cinnamon brown, mostly ovoid to pyriform, variable in size (27.3 to 39.2 × 16.7 to 21.2 μm), with pronounced apical wall and moderately echinulate with spines evenly distributed. Paraphyses, telia, and teliospores were not observed. Species-specific PCR primers designed from the internal transcribed spacer (ITS)1, ITS2, and 5.8S rDNA regions of P. melanocephala and P. kuehnii were used to differentiate the two species (2). The primers Pm1-F and Pm1-R amplified a 480-bp product from P. melanocepahala DNA in leaf samples with symptoms of common rust. By contrast, the primers Pk1-F and Pk1-R generated a 527-bp product from presumed P. kuehnii DNA in leaf samples with signs of orange rust, confirming the identity as P. kuehnii. The Centro de Investigación de la Caña de Azúcar de Colombia (Cenicaña) started a survey of different cultivars in nurseries and experimental and commercial fields in the Cauca River Valley and collected leaf samples for additional analyses. Experimental cvs. CC 01-1884, CC 01-1866, and CC 01-1305 were found to be highly susceptible to orange rust and were eliminated from regional trials, whereas commercial cvs. CC 85-92 and CC 84-75, the most widely grown cultivars, were resistant. With the discovery of orange rust of sugarcane in Colombia, Cenicaña has incorporated orange rust resistance in the selection and development of new cultivars. To our knowledge, this is the first report of P. kuehnii on sugarcane in Colombia. Orange rust has also been reported from the United States, Cuba, Mexico, Guatemala, Nicaragua, El Salvador, Costa Rica, Panama, Ecuador, and Brazil. References: (1) J. C. Comstock et al. Plant Dis. 92:175, 2008. (2) N. C. Glynn et al. Plant Pathol. 59:703, 2010. (3) E. V. Virtudazo et al. Mycoscience 42:167, 2001.


Agriculture ◽  
2019 ◽  
Vol 9 (7) ◽  
pp. 157 ◽  
Author(s):  
Namisy ◽  
Chen ◽  
Prohens ◽  
Metwally ◽  
Elmahrouk ◽  
...  

Bacterial wilt, caused by Ralstonia solanacearum, is highly diverse and the identification of new sources of resistance for the incorporation of multiple and complementary resistance genes in the same cultivar is the best strategy for durable and stable resistance. The objective of this study was to screen seven accessions of cultivated eggplant (Solanum melongena L.) and 40 accessions from 12 wild relatives for resistance to two virulent R. solanacearum strains (Pss97 and Pss2016; phylotype I, race 1, biovar 3). The resistant or moderately resistant accessions were further evaluated with Pss97 in a second trial under high temperatures (and also with Pss2016 for S. anguivi accession VI050346). The resistant control EG203 was resistant to Pss97, but only moderately resistant to Pss2016. One accession of S. sisymbriifolium (SIS1) and two accessions of S. torvum (TOR2 and TOR3) were resistant or moderately resistant to Pss97 in both trials. Solanum anguivi VI050346, S. incanum accession MM577, and S. sisymbriifolium (SIS1 and SIS2) were resistant to Pss2016 in the first trial. However, S. anguivi VI050346 was susceptible in the second trial. These results are important for breeding resistant rootstocks and cultivars that can be used to manage this endemic disease.


2018 ◽  
Vol 36 (2) ◽  
pp. 173-177 ◽  
Author(s):  
Maurício Rossato ◽  
Thais R. Santiago ◽  
Carlos Alberto Lopes

ABSTRACT In Brazil, the bacterial pathogens Ralstonia solanacearum and R. pseudosolanacearum cause substantial losses by inducing bacterial wilt on several solanaceous crops; R. pseudosolanacearum is the main species associated with peppers (Capsicum sp.). To verify the bacterial wilt reaction on Capsicum peppers commercialized in the Federal District (DF), fruits of several genotypes within this genus were collected from six different fairs distributed in the satellite cities of Gama, Sobradinho and Guará. Seedlings with four true leaves derived from seeds extracted from such fruits were root inoculated with 108 CFU/mL with a representative isolate of R. pseudosolanacearum (race 1, biovar 3, phylotype I, sequevar 18). The evaluated species were: Capsicum frutescens (‘pimenta-malagueta’), Capsicum baccatum var. pendulum (‘pimenta-dedo-de-moça’) and C. chinense (‘pimenta-de-bode’ red and yellow, ‘pimenta-cumarí-do-Pará’, ‘pimenta-biquinho’, ‘pimenta-habanero’ and ‘pimenta-de-cheiro’). Not all species were found in all six fairs. The reaction to bacterial wilt was variable and species-dependent. From 26 evaluated genotypes, none presented an immune-like response, 10 were considered resistant and 16 susceptible based on wilt incidence (Scott-Knott, 5%). Four Capsicum baccatum accesses were positioned in the resistant group, whereas 14 out of 18 of C. chinense were susceptible. Capsicum frutescens showed variable reactions. These results contribute to indicate cultivation of specific groups of pepper according to the presence of the pathogen in the soil.


2006 ◽  
Vol 74 (8) ◽  
pp. 4519-4529 ◽  
Author(s):  
Kelley M. Hovis ◽  
Martin E. Schriefer ◽  
Sonia Bahlani ◽  
Richard T. Marconi

ABSTRACT It has been demonstrated that Borrelia hermsii, a causative agent of relapsing fever, produces a factor H (FH) and FH-like protein 1 (FHL-1) binding protein. The binding protein has been designated FhbA. To determine if FH/FHL-1 binding is widespread among B. hermsii isolates, a diverse panel of strains was tested for the FH/FHL-1 binding phenotype and FhbA production. Most isolates (23/24) produced FhbA and bound FH/FHL-1. Potential variation in FhbA among isolates was analyzed by DNA sequence analyses. Two genetically distinct FhbA types, designated fhbA1 and fhbA2, were delineated, and type-specific PCR primers were generated to allow for rapid differentiation. Pulsed-field gel electrophoresis and hybridization analyses demonstrated that all isolates that possess the gene carry it on a 200-kb linear plasmid (lp200), whereas isolates that lack the gene lack lp200 and instead carry an lp170. To determine if FhbA is antigenic during infection and to assess the specificity of the response, recombinant FhbA1 (rFhbA1) and rFhbA2 were screened with serum from infected mice and humans. FhbA was found to be expressed and antigenic and to elicit a potentially type-specific FhbA response. To localize the epitopes of FhbA1 and FhbA2, truncations were generated and screened with infection serum. The epitopes were determined to be conformationally defined. Collectively, these analyses indicate that FH/FHL-1 binding is a widespread virulence mechanism for B. hermsii and provide insight into the genetic and antigenic structure of FhbA. The data also have potential implications for understanding the epidemiology of relapsing fever in North America and can be applied to the future development of species-specific diagnostic tools.


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