Preliminary Screening for Possible Presence of Ralstonia Solanacearum

Ralstonia solanacearum is categorized under gram-negative bacteria. It is a soil-born pathogenic bacterial and the causal agent for the widespread disease known as bacterial wilt. This pathogenic bacteria is broadly distributed in several regions such as tropical, sub-tropical, and a few of the temperate regions. The pathogenic bacteria, Ralstonia solanacearum has a very broad range of host species and hence, it able to infects economically important species in many plant families. The target of the host species and geographically distribution of R. solanacearum basically depends on the strains race and to certain degree it also affected by the biovar of the pathogen. R. solanacearum is a heterogenous species with great phenotypic and genetic diversity and the current race and biovar system may be inadequate for identification. This project studies the sequence analysis of the intergenic spacer (ITS) in the rRNA operon between 16s and 23s RRNA gene in four isolates. In this study, Kelman’s tetrazolium chloride (TZC) agar was used to isolate R. solanacearum. Soil samples were obtained from several regions in west peninsular Malaysia. Putative red colonies grown on the TZC agar were cultured and used for morphological and biochemical tests which included catalase test, potassium hydroxide test and Gram-staining. The positive results obtained from catalase test indicated that R. solanacearum is able to produce enzyme catalase. A 3% H2O2 was used in this test and there was copious bubbles formed. Based on Gram staining, R. solanacearum is a gram-negative bacterium. The KOH test was used to supplement the gram staining. A 3% diluted alkali solution was used to the cell walls of R. solanacearum causing the mixture to become viscous. DNA was extracted and the ITS region between 16s and 23s rRNA genes was amplified using eubacterial universal primers. The amplified PCR products was purified and sequence. Surprisingly, the sequence results showed that the organisms studied turned out to be Acinetobacter spp.