2.2.109
GENETIC DIFFERENTIATION OF CLAVIBACTER XYLI SUBSP. XYLI STRAINS

D PILLAYl, P SAROOP l, SM BRUMBLEY 2 and B PJLLAYl

lDepartment of Microbiology, University of Durban-Westville, Durban, South Africa; 2 Bureau of Sugar Experiment Stations, Indooroopilly, Queensland Cooperative Research Centre for Tropical Plant Pathology, University of Queensland, Brisbane, Queensland, Australia

Background and objectives
Ratoon stunting disease, caused by Ciavibacter xyli subsp. xyli (Cxx) is one of the most economically important diseases of sugarcane world-wide. Strains of Cxx exhibit little morphological, physiological or pathogenic variation [1]. The objective of the present study was to assess the degree of relatedness among 12 strains of Cxx based on random amplification of polymorphic DNA (RAPD) analysis and on their ribosomal RNA gene restriction patterns (ribotyping).

Materials and methods
PCR amplification of genomic DNA from 14 strains of Cxx was performed using arbitrary 10-mer primers, according to standard RAPD conditions. For ribotyping, chromosomal DNA was prepared, digested with AvaI, BglI and HindII, blotted and hybridized with digoxygenin labelled 16S+23S rRNA from Escherichia coli.

Results and conclusions
RAPD analysis of genomic DNA of all strains tested reproducibly yielded unique polymorphisms for each strain, indicating genetic variability. The degree to which intraspecies variation was expressed varied with each primer used. The complexity of the banding patterns also varied with the different primers, with all primers producing multiple bands. Variation in PCR products obtained by RAPD analysis reflects the sequence variation of RAPD priming sites among the strains investigated. The RAPD profiles generated also indicate a degree of relatedness among certain strains. The unique fingerprints observed were reproducibly obtained and can therefore serve as a means of identification of each strain. RAPD markers common to all strains could also be used to develop Cxxspecific DNA probes. RAPD analysis should also provide a simple tool for epidemiological studies of Cxx, their differentiation, identification and the construction of phylogenetic trees.

With respect to ribotyping, strains of Cxx investigated produced different ribotype patterns for each strain. Ribotype patterns showed certain Cxx strains to be more distinguishable than others. The various ribotypes obtained demonstrated the genetic divergence of the C. xyii subspecies. Some fragments appeared to be common to all strains indicating relatedness. These fragments may be employed in DNA-based assays for the detection of Cxx strains. Ribotyping yielded good genetic markers for intraspecific differentiation of Cxx strains, suggesting that it may be a useful tool for the genotypic differentiation of closely related strains. Genotypic differentiation of Cxx by ribotyping confirms the trends observed in our laboratories using RAPD analysis.

References
1. Davis MJ, Gillespie AG Jr., Vidaver AK, Harris RW, 1984. International Journal of Systematic Bacteriology 34, 107-117.