Submitted to: Journal of Microbiological Methods
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/18/2011
Publication Date: 12/10/2011
Citation: Huang, Q., Yan, X., Wang, J. 2011. Improved biovar test for Ralstonia solanacearum. Journal of Microbiological Methods. 88:271-274. Interpretive Summary: Ralstonia solanacearum causes bacterial wilt, a soil-borne vascular disease that is distributed worldwide and attacks over 450 plant species including ornamentals such as geranium. It also limits the production of such economically important crops as tomato, tobacco, potato and banana. R. solanacearum is generally classified into 5 races based on host range and 5 biovars based on carbohydrate utilization. R. solanacearum race 3, biovar 2 causes destructive brown rot of potato, and is a quarantined and select agent pathogen in the U. S. Currently, biovars of R. solanacearum are differentiated by polymerase chain reaction assays using specific primers and biovar tests based on utilization of a carbohydrate panel. Standard biovar tests use bromothymol blue as a pH indicator in 15 ml culture tubes containing 3 to 5 ml of test medium, and take weeks to complete. We improved the biovar test by using phenol red as a pH indicator to produce a color change from red to yellow when the carbohydrate in the medium is utilized. We also conducted the test in small tube strips so less than one twentieth of the medium is needed, which not only saves money and space but also allows the test to be completed in three to four days. The improved biovar differentiation test will help state and federal regulatory officials to make timely decisions to prevent and exclude the brown rot pathogen from becoming established in the U.S.
Technical Abstract: Race 3, biovar 2 strains of Ralstonia solanacearum are quarantined pathogens in Europe and Canada and Select Agent pathogens in the United States. The biovar classification of R. solanacearum strains is based on their biochemical abilities to utilize a carbohydrate panel. The standard biovar test uses bromothymol blue as a pH indicator in 15 ml culture tubes containing 3 to 5 ml of test media, and takes weeks to complete at 24 or 28oC. We improved the biovar test by using phenol red as a pH indicator that changes color at a higher pH when a carbohydrate is utilized. We also conducted the test at 32oC in 0.2 ml of 8-tube strips that reduced the medium needed by at least 20 fold. Using the improved test, biovars of R. solanacearum strains can be determined in 4 days when a panel of seven carbohydrates is used including glucose, trehalose, maltose, cellobiose, mannitol, sorbitol and dulcitol. To differentiate biovars 1, 2, 3 and 4, the test can be further simplified and completed in 3 days using a panel of four carbohydrates containing glucose, trehalose, maltose and dulcitol, significantly saving money, space and time.