Molecular Detection of Mycosphaerella spp. Involved in Sigatoka Disease of Banana using Species-Specific Primer Sets

The Sigatoka disease complex of banana involves three related ascomycetous fungi viz., Mycosphaerella fijiensis, M. musicola, and M. eumusae. Due to rather similar symptoms on banana, similar teleomorph morphology and problems associated with growth and sporulation in culture, species identification based on classical methods is troublesome. The exact distribution of these three species and their disease epidemiology remain unclear. Furthermore, M. fijiensis and M. eumusae have not been reported from many banana producing countries, so as to be considered as quarantine organisms. Rapid and accurate detection of these three species is essential to adopt and apply proper disease management strategy. In the present study, species-specific primer sets were developed as based on sequence data of actin gene. Species-specific primer combinations were designed with expected amplicon sizes of 500 bp from M. fijiensis, 200 bp from M. musicola, and 630 bp for M. eumusae. Accuracy and efficacy of each of species-primer sets were tested and verified on DNA extracted from pure cultures as well as DNA from naturally infected banana leaves. The sensitivity of the primer sets enabled reliable detection of M. fijiensis DNA as low as 100 pg, whereas for M. musicola and M. eumusae a higher sensitivity was achieved of 1 pg and 10 pg genomic DNA, respectively.