2.2.44
PCR-RFLP ANALYSIS OF THE HRP GENES OF RALSTONIA SOLANACEARUM, AGENT OF BACTERIAL WILT

S POUSSIER, P VANDEWALLE and J LUISETTI

CIRAD-FLHOR, laboratoire de Phytopathologie, 97410 Saint-Pierre, La Réunion, France

Background and objectives
Ralstonia solanacearum is a soil pathogen responsible for the bacterial wilt, one of the most important and widely spread bacterial diseases of various crops (tomato, potato, tobacco, banana, peanut,etc.) in tropical, subtropical, warm, and temperate regions of the world. The disease is recorded on several-hundred species belonging to more than 50 plant families. The species R. ;solanacearum is a complex taxonomic group in which strains display an important diversity at different levels (host range, biochemical, serological, genetic characteristics). To organize the species, several subspecific systems of classification are used. Thus, the species is divided into five races, six biovars and 46 RFLP groups. Moreover, American strains (Americanum division ; biovars 1, 2, N2) are distinct from Asiatic strains (Asiaticum division; biovars 3, 4, 5). The objective of this study is to analyse the diversity within R. ;solanacearum using a different method, PCR-RFLP, and consequently to compare it with previous results. The variability within the hrp gene cluster [1], responsible for the hypersensitive response or disease, is explored.

Materials and methods
Eleven pairs of primers were selected from the nucleotide sequence of the hrp gene cluster of the strain GMI 1000 (biovar 3) of R. ;solanacearum. The amplified DNA fragments considered to be specific to R. ;solanacearum were digested by restriction endonucleases. Only enzymes which led to different RFLP patterns were selected. We performed the PCR-RFLP on 120 strains belonging to the first four biovars of R. ;solanacearum and isolated from different hosts on the five continents. Clustering is based on an ascendent hierarchical classification (STATLAB software).

Results and conclusions
Six different amplificates are considered to be specific to R. ;solanacearum. However, this specificity remains to be confirmed with strains belonging to two closely related species, Pseudomonas celebense and P. ;syzygii. Different restriction patterns among strains were observed with eight restriction endonucleases. The data analysis led to distribution of the 120 strains into eight PCR-RFLP clusters.

The exploration of the hrp gene cluster, with the PCR-RFLP technique, confirms the large variability within R. ;solanacearum. Based on these results, biovar 2 strains constitute a single homogeneous cluster whereas biovar 1 strains show a rather large diversity (five clusters). Biovar 3 strains appear to be less variable compared to biovar 1 strains (2 clusters, 5 subclusters). The biovar 4 strains are mixed together with the biovar 3 ones in the same cluster. Some clusters are significantly associated with host range (Musa sp., tomato) or geographical origin (United-States, Africa). Furthermore, the Americanum and Asiaticum divisions are confirmed with the major exception that one cluster including biovar 1 strains isolated from some African countries (Reunion Island, Madagascar, Zimbabwe and Angola) seems to belong to the Asiaticum division rather than to the Americanum one as all other biovar 1 strains do.

Reference
1. Boucher CA, Gough CL, Arlat M, 1992. Annual Review of Phytopathology 30, 443-61.