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Tomato Root Transformation Followed by Inoculation with Ralstonia Solanacearum for Straightforward Genetic Analysis of Bacterial Wilt Disease
Published on: March 11, 2020
Biovar of Ralstonia solanacearum Causing Bacterial Wilt of Tomato in Nigeria
1Crop Protection Division, National Horticultural Research Institute, PMB 5432, Idi-Ishin, Ibadan, Nigeria.
Abstract:
A survey of southwestern Nigeria showed an outbreak of wilt disease caused by Ralstonia solanacearum in 80% of tomato (Lycopersicon lycopersicum) fields in the production area of Ogun State (7°15'N, 3°25'E) in June 1996. Subsequent surveys conducted in Edo (6°45'N, 5°30'E), Delta (5°15'N, 5°45'E), Lagos (6°30'N, 3°40'E), Oyo (8°40'N, 3°30'E), and Osun (7°50'N, 4°E) states between May and November 1998 identified 60 to 80% infected fields per state. Observations made at the experimental plots of the National Horticultural Research Institute (NIHORT) at Ibadan (7°23'N, 2°50'E) also showed similar infections. Affected plants exhibited initial wilting of terminal leaves followed (within 2 days) by sudden and permanent wilt. For further identification of the causal organism, 10 tomato plants showing wilt symptoms were collected from each of five fields in the vegetable blocks of the NIHORT at Ibadan and 20 farmers' fields in Ogun State. The 10 plants per field were thereafter bulked as one composite sample. Creamy bacterial sap from these samples was plated on tetrazolium chloride media, and plates were incubated at 30°C for 48 h (2). Colonies that were fluidal and white with pink centers were used for biovar determination. Basal media was prepared to include one of three disaccharides (cellobiose, lactose, or maltose) or three hexose alcohols (dulcitol, mannitol, or sorbitol). A loopful of bacterial cells of all 25 isolates was inoculated individually to each of the six media. Cultures were incubated at 30°C for 28 days and monitored daily for color changes. The pathogenicity of the 25 isolates was tested using 10 4-week-old seedlings each of eggplant cv. black beauty, tomato cv. Ibadan local, sweet pepper cv. California wonder, and potato cv. Kufri. Each inoculum was prepared by adjusting the concentration to 107 CFU/ml with a colorimeter at a wavelength of 600 nm (optical density of approximately 0.3). Plants were inoculated by pouring 10 ml of inoculum around the base of each plant. Ten uninoculated seedlings of each cultivar served as the control. Plants were assessed for wilt severity 30 days after inoculation. All isolates utilized the three disaccharides and three hexose alcohols, and according to Hayward's classification, all isolates were biovar 3 (1). Furthermore, the isolates caused rapid wilting of all four test plants. R. solanacearum was easily reisolated from the vascular bundles of the test plants. To our knowledge, this is the first report of this biovar of R. solanacearum affecting tomato crops in Nigeria. References: (1) A. C. Hayward. J. Appl. Bacteriol, 27:265, 1964 (2) A. Kelman. Phytopathology 44:693, 1954.
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