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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
Bacterial Wilt of Solanaceae Caused by Ralstonia solanacearum Race 1 Biovar 3 in Mali
A T Thera1, B J Jacobsen2, O T Neher3
1Programme Fruits et Legumes, Institut d'Economie Rurale, BP 258, Rue, Mohamed V, Bamako, Republique du Mali.
Abstract:
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.
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