Genetic Variation Among Macrophomina phaseolina Isolates as Detected by PCR-Based Techniques

Charcoal rot disease is one of the major soybean disease in the North Central region. The causal agent, Macrophomina phaseolina (Tassi) Goid., is recognized as a highly variable fungal pathogen, and is responsible for economically significant damage to a wide variety of crops worldwide. Molecular variation among forty-nine isolates of M. phaseolina was examined by restriction enzyme analysis of polymerase chain reaction amplified DNA from the internal transcribed spacer region of rDNA. The amplified ITS region was defined by the ITS1 and ITS4 primers. Amplification yielded a single fragment of approximately 700 bp. The amplified fragment contained restriction sites for six out of seven enzymes tested, but restriction analysis revealed no variation among any of the isolates despite their wide geographic distribution, as well as ecological and phenotypic variation; Thus the ITS region of rDNA is highly conserved within M. phaseolina. The forty-nine isolates were also examined with the RAPD technique utilizing twenty different 10-nucleotide primers. A phenetic analysis of 43 isolates was conducted through NTSYS-PC software based on a total of 153 polymorphic RAPD bands. Six legume isolates were separately tested to determine the relationship to host preference. RAPD data revealed a very high degree of genetic variability of M. phaseolina, but the RAPD clusters, in general, do not match the phenotypic clusters. RAPD profiles for certain clusters (e.g. the Zea mays cluster, soil clusters and legume isolates) are consistent with some degree of substrate or host preference within M. phaseolina