OPTIMIZATION OF RAPD-PCR FINGERPRINTING TO ANALYZE GENETIC-VARIATION WITHIN POPULATIONS OF FUSARIUM-OXYSPORUM F SP CUBENSE

Genetic variation among 11 isolates of Fusarium oxysporum f.sp, cubens e (FOG) Nas analysed by random amplification of polymorphic DNA using the polymerase chain reaction (RAPD-PCR). The isolates represented thr ee of the four FOC races and the seven vegetative compatibility groups (VCGs) known to occur in Australia. Isolates of F. oxysporum f.sp. cu bense were also compared to isolates of F. oxysporum f.sp. gladioli, F . oxysporum f.sp. zingiberi, F., oxysporum f.sp. lycopersici, F. monil iforme, Aspergillus niger and Colletotrichum gloeosporioides. DNA was extracted from fungal mycelium and amplified by RAPD-PCR using one of two single random 10-mer primers; the primer sequences were chosen arb itrarily. The RAPD-PCR products were separated by polyacrylamide gel e lectrophoresis producing a characteristic banding pattern for each iso late. The genetic relatedness of the F. oxysponrm f.sp. cubense isolat es was determined by comparing the banding patterns generated by RAPD- PCR. This RAPD-PCR analysis revealed variation at. all five levels of possible generic relatedness examined. F. oxysporum f.sp. cubense coul d very easily be distinguished from the other fungi, and the three rac es and five VCGs of F. oxysporum f.sp. cubense could also be different iated. Within F. oxysporum f.sp. cubense, each isolate was scored for the presence or absence of each band (50 different bands were produced for primer SS01 and 59 different bands for primer RC09) and these dat a were clustered using the UPGMA method (unweighted pair-group method, arithmetic average). UPGMA cluster analysis of the data generated by primer SSO1 revealed two distinct clusters. One cluster contained race 4 isolates (VCGs 0120, 0129 and 01211) and the other cluster containe d both race 1 (VCGs 0124, 0124/5 and 0125) and race 2 isolates (VCG 01 28). Similar results were obtained with primer RC09. The banding patte rns for each isolate were reproducible between experiments. These resu lts indicated that RAPD-PCR was a useful method for analysing genetic variation within F. oxysporum f.sp. cubense. Some of the advantages of this technique were that it was rapid, no sequence data were required to design the primers and no riadioisotopes were required.