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The degree of genetic diversity of 127 Colletotrichum gloeosporioides isolates from Stylosanthes guianensis genotypes in South America was measured at the molecular level by random amplified polymorphic DNA (RAPD) with nine arbitrary primers of 10 bases, and by restriction fragment length polymorphism (RFLP) with a non-LTR (long terminal repeats) retrotransposon DNA sequence. The RAPD products revealed scorable polymorphism among the isolates, and a total of 80 band positions were scored. Sixty-three of the 127 isolates were clustered into 13 distinct lineages usually correlating with geographic origin. Where isolates from various regions were clustered together, most had identical host genotype origin. The pathogen population sampled from Carimagua, Colombia, a long-time Stylosanthes breeding and selection site, with a savanna ecosystem, was highly diverse. A set of 12 S. guianensis genotype differentials was used to characterize pathogenic variability of 104 isolates and their virulence patterns were grouped into 57 pathotypes. However, when they were tested on four Australian differentials, they grouped into 11 pathotypes. As shown in previous studies, no strict correlations existed between genetic diversity measured by RAPD or RFLP, and pathotype defined by pathogenicity pattern on the differentials. Southern blot analysis of the 127 isolates revealed 23 hybridizing fragments, resulting in 41 fingerprint patterns among the 127 isolates. Relationships between RFLP and RAPD variables were examined using Spearman's Rank Correlation Coefficient, which showed that the two measures of genotypic variation are in agreement.