Characterization of Ralstonia Solanacearum Phylotype II Sequevar 4 as a record on Pathogen associated with Moko Disease of Banana in Peninsular Malaysia

Abstract

Moko Disease caused by Ralstonia Solanacearum race 2 biovar 1 (R.Solanacearum R2Bv1) is a major disease affecting Banana (Musa spp.) production. Although local reports suggested that this disease is wide spreading in Malaysia, characterization of R. Solanacearum strains associated with Moko Disease in this country has not been done. This study was conducted to isolate, identify and characterize R.Solanacearum R2Bv1 of Moko-causing strains in Peninsular Malaysia. During March 2011 to June 2012, 170 banana plants associated with Moko Disease and 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. All 197 strains produced fluidal colonies that were white to pink coloration after incubation at 24 to 48 hours at 29°C on Kelman's TZC agar medium and were divided into two defined colony type, the B and SFR types. These strains appeared as Gram-negative rods after Gram-stain, and positive for potassium Hydroxide (KOH), Kovacs Oxidase, Catalase and Lipase activity on Tween 80 solution tests. In biovar determination, only 30 strains displayed characteristics of Biovar 1 R. Solanacearum, which was negative for utilization of Disaccharides and Hexose Alcohols. Tobacco hypersensitivity assay revealed all strains elicited hypersensitive response (HR) at 12 h after infiltration, suggesting that they were of race 2. In Pathogenicity study, all 30 strains were virulence towards three Moko most affected local Banana cultivars namely Musa Paradisiaca cv. Nipah, Musa paradisiaca cv. Tanduk and Musa acuminate cv. Berangan cultivars with diverse degrees of virulence; most virulent, moderately virulent and least virulent with strain NS-N1 as the most virulent, while strains Ked-KN4 and Ked-KN5 were classified as the least virulent. Musa ParadiSiaCa cv. Nipah (ABB triploid) significantly exhibited the highest degree of severity to R. solanacearum, followed by Musa Paradisiaca cv. Tanduk (AAB triploid) and Musa acuminate cv. Berangan (AAA triploid). Moreover, statistical results revealed there were between geographical origins of strains and their severity, with the most and the lowest severity was related to strains from Johor and Negeri Sembilan. Partial 16S rDNA sequence analyses disclosed that all 30 strains of R. solanacearum biovar 1 were clustered to the published R. solanacearum biovar 1 related to Moko-causing strains from the Philippines (MOD5 and R633) with 91% Bayesian posterior probability support and completely different from Ralstonia syzygii (R. syzygii, S444E), Blood Diease Bacterium (T520) and the outgroup strain, Xanthomonas spp. (55485). These findings provide constructive documentations on R. Solanacearum R2Bv1 in Malaysia, since banana has been identified as the second most important commercial fruit crop with a high economic value in this country.