Isolation, identification and molecular characterization of Ralstonia solanacerum isolates collected from Southern Karnataka
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Abstract
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.
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Keywords
Bacterial wilt, Biovar, Phylotype, Race, Ralstonia solanacearum
References
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Ahmed, N. N., Islam, R. M., Hossain, M. A., Meah, M. B. and Hossain, M. M. (2013). Determination of races and biovars of Ralstonia solanacearum causing bacterial wilt disease of potato. J. Agril. Sci., 5(6):86-93
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Avinash, P. and Umesha, S. (2013). Identification and genetic diversity of bacterial wilt pathogen in Brinjal. Archi. Phytopathol. and Plant Prot., 29(4):1-9.
Bradbury, J. F. (1970). Isolation and preliminary study of bacteria from plants. Rev.Pl. Pathol., 49: 213-218
Buddenhagen, I. W., Sequeira, L. and Kelman, A. (1962). Designation of races in Pseudomonas solanacearum. Phytopathol., 52: 726.
Chandrashekara, K. N., Prasanna Kumar, M. K., Deepa, M., Vani, A. and Khan, A. N. A. (2012). Prevalence of races and biotypes of Ralstonia solanacearum in India. J. Pl. Prot. Res., 52(1): 53-58.
Deepa, J., Girija, D., Sally, K., Mathew, P. A., Nazeem, T. D. and Sukumara Varma. (2003). Detection of Ralstonia solanacearum race 3 causing bacterial wilt of solanaceous vegetables in kerala, using random amplified polymorphic DNA (RAPD) analysis. J. Trop. Agric., 41: 33-37.
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He, L. Y., Sequeira, L. and Kelman, A. (1983). Characteristics of strains of Pseudomonas solanacearum from China. Pl. Dis., 67: 1357- 1361.
Makhlouf, A. H. and Hamedo, A. H. (2013). Suppression of bacterial wilt disease of tomato plants using some bacterial strains. J. Life Sci., 10(3): 1732-1741.
Opine, N., Tavner, F., Hollway, G., Wang, J. F., Li, T. H., Maghirang, R., Fegan, M., Hayward,A. C., Krishnapillai, V., Hong, W. F., Holloway, B. W. and Timmis , J. N. (1997). A novel method for development of species and strain specific DNA probes and PCR primers for identifying Bhurkholdria solanacearum. Asia Pac. J. Mol. Bio., 5: 19-30.
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Suslow, T. V., Schroth, M. N. and Isaka, M. (1982). Application of a rapid method for Gram differentiation of plant pathogenic and saprophytic bacteria without staining. Phytopathol., 72: 9117-918.
Venkatesh and Khan, A. N. A. (2000). Prevalence of races/biotypes of Ralstonia solanacearum and management of bacterial wilt of potato. In: Potato, Global Research and Development - 452-455. Indian Potato Association, Shimla.
Venkatesh (2000). Biology, ecology and integrated management of bacterial wilt of potato caused by Ralstonia solanacearum. Ph. D. Thesis, Univ. Agric. Sci., Bangalore.
Yabuuchi, E., Kosako, Y., Yano, I., Hotta, H. and Nishiuchi, Y. (1995). Transfer of two Burkholderia and an Alcaligenes species to Ralstonia gen. nov.: proposal of Ralstoniapicketii (Ralston, Palleroni & Doudoroff 1973) comb. nov.Ralstonia solanacearum (Smith, 1896) comb. nov. and Ralstoniaeutropha (Davis, 1969). Microbiol. Immuno., 897-904.
Garcia, A. L., Lima, W. G., Souza, E.B., Michereff, S. J. and Mariano, R. L. R. (2013). Characterization of Ralstonia solanacearum causing bacterial wilt in bell pepper in the state of Pernambuco, Brazil. Journal of Plant Pathology. 95: 237–245.
Gilson, G., Costa, L. and Rodrigues L. M. R.( 2012). Characterization of Ralstonia Solanacearum strains from Brazil using molecular methods and pathogenicity tests , Journal of Plant Pathology, 94: 505–516.
Vanitha, S. C., Niranjana. S. R., Mortensen, C. N., and Umesha, S. (2009). Bacterial wilt of tomato in Karnataka and its management by Pseudomonas fluorescens. Biocontrol. 54: 685–695.
Agrios, G. N. (1997). Plant Pathology. 4th ed. San Diego, California: Academic Press, Inc.409:431- 433
Ahmed, N. N., Islam, R. M., Hossain, M. A., Meah, M. B. and Hossain, M. M. (2013). Determination of races and biovars of Ralstonia solanacearum causing bacterial wilt disease of potato. J. Agril. Sci., 5(6):86-93
Anonymous (1957). Manual of Microbial Methods: (Ed.). Society of American Bacteriologists, McGraw, New York, 315.
Avinash, P. and Umesha, S. (2013). Identification and genetic diversity of bacterial wilt pathogen in Brinjal. Archi. Phytopathol. and Plant Prot., 29(4):1-9.
Bradbury, J. F. (1970). Isolation and preliminary study of bacteria from plants. Rev.Pl. Pathol., 49: 213-218
Buddenhagen, I. W., Sequeira, L. and Kelman, A. (1962). Designation of races in Pseudomonas solanacearum. Phytopathol., 52: 726.
Chandrashekara, K. N., Prasanna Kumar, M. K., Deepa, M., Vani, A. and Khan, A. N. A. (2012). Prevalence of races and biotypes of Ralstonia solanacearum in India. J. Pl. Prot. Res., 52(1): 53-58.
Deepa, J., Girija, D., Sally, K., Mathew, P. A., Nazeem, T. D. and Sukumara Varma. (2003). Detection of Ralstonia solanacearum race 3 causing bacterial wilt of solanaceous vegetables in kerala, using random amplified polymorphic DNA (RAPD) analysis. J. Trop. Agric., 41: 33-37.
Dhital, S. P., Thaveechai, N. and Shrestha, S. K. (2001). Characteristics of Ralstonia solanacearum strains of potato wilt disease from Nepal and Thailand. Nepal Agric. Res. J., Vol. 4(5): 42-47.
Fegan, M. and Prior, P. (2005). How complex is the Ralstonia solanacearum species complex? In: Allen C, Prior P, Hayward AC, editors. Bacterial wilt: the disease and the Ralstonia solanacearum species complex. St. Paul, MN: APS. 405–414.
French, E. R. (1996). Integrated control of bacterial wilt of potato. In: Bacterial Wilt Training Manual. International Potato Centre, Lima, Peru. Section 3: 4.
Hayward, A. C. (1964). Characteristics of Pseudomonas solannacearum. J. Appl. Bacteriol., 27:265-271.
Hayward, A. C. (1991). Biology and epidemiology of bacterial wilt caused by Pseudomonas solanacearum. Ann. Rev. Phytopathol., 29: 65- 87
He, L. Y., Sequeira, L. and Kelman, A. (1983). Characteristics of strains of Pseudomonas solanacearum from China. Pl. Dis., 67: 1357- 1361.
Makhlouf, A. H. and Hamedo, A. H. (2013). Suppression of bacterial wilt disease of tomato plants using some bacterial strains. J. Life Sci., 10(3): 1732-1741.
Opine, N., Tavner, F., Hollway, G., Wang, J. F., Li, T. H., Maghirang, R., Fegan, M., Hayward,A. C., Krishnapillai, V., Hong, W. F., Holloway, B. W. and Timmis , J. N. (1997). A novel method for development of species and strain specific DNA probes and PCR primers for identifying Bhurkholdria solanacearum. Asia Pac. J. Mol. Bio., 5: 19-30.
Poussier, S., Trigalet-Demery, D., Vandewalle, P., Goffinet, B., Luisetti, J. and Trigalet, A. (2000). Genetic diversity of Ralstonia solanacearum as assessed by PCR-RFLP of the hrp gene region. AFLP and 16S rRNA sequence analysis, and identification of an African subdivision. Microbiology.146:1679-1692
Prasanna Kumar, M. K. (2004). Molecular characterization of the strains of Ralstonia solanacearum (Yabuuchi), ecology and integrated management of bacterial wilt of tomato. Ph.D. Thesis, Univ. Agric. Sci., Bengaluru, 235pp.
Prasanna Kumar, M. K., Chandrashekara, K. N., Deepa, M., Vani, A. and Khan, A. N. A. (2012). Finger printing of Ralstonia solanacearum isolates by Rep-PCR and RAPD. Pest Man. in Hortl. Ecosys., 18(2): 179-187.
Sagar, V., Jeevalatha, A., Sarita Mian, Chakrabarti, S. K., Gurjar, M. S., Arora, R. K., Sharma, S., Bakade, R. R. and Singh, B. P. (2014). Potato bacterial wilt in India caused by strains of phylotype I, II and IV of Ralstonia solanacearum. Eur J Plant Pathol., 138(1). Doi: 10.1007/s10658-013-0299-z.
Schaad, N.W. (1992). Laboratory guide for the identification of plant pathogenic bacteria, 2nd ed. American phytopathol., 138pp.
Shahbaz, M. U., Mukhtar, T., Ul-Haque, M. I. and Begum, N. (2015). Biochemical and serological characterization of Ralstonia solanacearum associated with chilli seeds from Pakistan. Int. J. Agric. Biol., 17(1):31-40.
Smith, E. F. (1896). A bacterial disease of the chilli, eggplant and Irish potato (Bacillus solanacearum) Nov. Sp., U. S. Dept. Agric. Div. Veg., Physiol. Path. Bull., 12: 1-28.
Stevenson, W.R., Loria, R., Franc, G.D. and Weingartner, D.P. Eds. (2001). Compendium of Potato Diseases, 2nd Ed. APS Press, St. Paul, MN.
Sujeet Kumar, Kedarnath, N. H., Rohini, I.B., Rangaswamy, K.T., Ramanjini Gowda, P.H. and Raghavendra Achari. (2017). Isolation and characterization of Ralstonia solanacearum causing bacterial wilt of Solanaceae crops. Int.J.Curr. Microbiol.App. Sci., 6(5): 1173-1190. doi: https://doi.org/10.20546/ijcmas.2017.605.128
Suslow, T. V., Schroth, M. N. and Isaka, M. (1982). Application of a rapid method for Gram differentiation of plant pathogenic and saprophytic bacteria without staining. Phytopathol., 72: 9117-918.
Venkatesh and Khan, A. N. A. (2000). Prevalence of races/biotypes of Ralstonia solanacearum and management of bacterial wilt of potato. In: Potato, Global Research and Development - 452-455. Indian Potato Association, Shimla.
Venkatesh (2000). Biology, ecology and integrated management of bacterial wilt of potato caused by Ralstonia solanacearum. Ph. D. Thesis, Univ. Agric. Sci., Bangalore.
Yabuuchi, E., Kosako, Y., Yano, I., Hotta, H. and Nishiuchi, Y. (1995). Transfer of two Burkholderia and an Alcaligenes species to Ralstonia gen. nov.: proposal of Ralstoniapicketii (Ralston, Palleroni & Doudoroff 1973) comb. nov.Ralstonia solanacearum (Smith, 1896) comb. nov. and Ralstoniaeutropha (Davis, 1969). Microbiol. Immuno., 897-904.
Garcia, A. L., Lima, W. G., Souza, E.B., Michereff, S. J. and Mariano, R. L. R. (2013). Characterization of Ralstonia solanacearum causing bacterial wilt in bell pepper in the state of Pernambuco, Brazil. Journal of Plant Pathology. 95: 237–245.
Gilson, G., Costa, L. and Rodrigues L. M. R.( 2012). Characterization of Ralstonia Solanacearum strains from Brazil using molecular methods and pathogenicity tests , Journal of Plant Pathology, 94: 505–516.
Vanitha, S. C., Niranjana. S. R., Mortensen, C. N., and Umesha, S. (2009). Bacterial wilt of tomato in Karnataka and its management by Pseudomonas fluorescens. Biocontrol. 54: 685–695.
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Isolation, identification and molecular characterization of Ralstonia solanacerum isolates collected from Southern Karnataka. (2018). Journal of Applied and Natural Science, 10(3), 886-893. https://doi.org/10.31018/jans.v10i3.1747
