RFLP and Rapd Analyses in the Identification and Differentiation of Isolates of the Leaf Spot Fungus Corynespora cassiicola

Abstract

Genetic variabilty in isolates of the fungal plant pathogen Corynespora cassiicola cultured from pawpaw, mimosa and thyme hosts was assessed using restriction fragment length polymorphism (RFLP) analysis of internal transcribed spacer (ITS) regions of ribosomal DNA and random amplified polymorphic DNA (RAPD) analysis of total fungal DNA. Strains of Corynespora could be distinguished from a member of the closely-related genus Helminthosporium on the basis of amplified ITS fragment size, but could not be typed individually as the ITS regions of all isolates exhibited identical size and restriction endonuclease digestion pattern. However, RAPD profiles generated by 14 decamer primers of arbitrary sequence did reveal significant differences between some of the C. cassiicola isolates and succeeded in differentiating all but two of the strains. Cluster analysis of 218 amplified DNA fragments showed that the five isolates could be placed into three groups that correspond with their host origin and morphological characteristics. The use of these molecular techniques will be extended to assess intra-specific variation in C. cassiicola isolates from rubber trees in Sri Lanka, where highly pathogenic strains present a serious threat to the natural rubber industry.