2.2.62
ASSESSMENT OF THE AFLP TECHNIQUE FOR THE GENETIC STUDY OF XANTHOMONAS AXONOPODIS PV. MANIHOTIS

S RESTREPO1,2, J TOHME1 and VERDIER1,2

1Centro Internacional de Agricultura Tropical (CIAT), Biotechnology Research Unit, AA 6713, Cali, Colombia; 2 Institut Français de Recherche Scientifique pour le développement en Coopération (ORSTOM), BP5045, 35032 Montpellier, France

Background and objectives
Xanthomonas axonopodis pv manihotis (Xam) is the causal agent of cassava bacterial blight (CBB), a world-wide disease particularly destructive in South America and Africa. CBB is managed essentially through the use of resistant varieties and therefore, it is essential to assess the genetic diversity of the pathogen's populations in order to develop an appropriate disease management strategy. To date, the genetic diversity of Xam was characterized by RFLP analyses using ribotyping and plasmidic and genomic Xam probes [1]. Recently, the analysis of Colombian Xam populations, collected from different ecological zones (ECZ), showed a high degree of genetic variability among strains and the existence of at least one clonal population in the ECZ 5 (high-altitude tropics). AFLP (amplified fragment length polymorphism) is a novel PCR-based technique that offers several advantages in characterizing genetic diversity [2]. Polymorphism is assessed at a large number of independent loci, AFLP markers are neutral, results are reproducible and variation is assessed in any part of the genome. The complexity of the AFLP fingerprint can be optimized by choosing selective bases to add to the primer during PCR amplifications. Our purpose was to assess the AFLP technique for characterizing Xam genetic diversity.

Materials and methods
Six Xam strains were tested with 65 primer combinations. AFLP markers were analysed as described by Vos et al. [2]. Eight combinations were selected according to reproducibility, number of polymorphic bands, and polymorphism detected among Xam strains. In addition, 48 Xam strains originated from different ecozones in Colombia were analysed with the selected combinations.

Results and conclusions
Specific combinations useful in a Xam population study were selected. Results obtained with AFLP are consistent with those obtained by RFLP using a plasmidic fragment as a probe [1]. Some combinations permitted to differentiate Xam strains that were not distinguished by RFLP analyses. Furthermore, AFLP analyses allowed establishing the phylogenetic relationships among Xam strains. Further application of the AFLP technique to the study of Xam will be discussed.

References
1. Restrepo S, Verdier V, 1997. Applied Environmental Microbiology 63, 4427-4434
2. Vos P, Hogers R, Bleeker M, Reijans M, Van de Lee T, Hornes M, Frijters A, Pot J, Peleman J, Kluper M, Zabeau M, 1995. Nucleic Acids Research 23:4407-4414.