Detection of banana leaf spot pathogens by PCR 1

Abstract

Two sigatoka leaf‐spot diseases affect banana and plantain: yellow sigatoka and black sigatoka. These are caused by two closely related and morphologically similar species of fungi: yellow sigatoka by Mycosphaerella musicola, and black sigatoka by M. fijiensis. Correct identification of these pathogens is hampered by the ambiguity of symptoms on different cultivars, and the difficulty in successfully isolating the pathogens in pure culture. Subsequent identification relies on sporulation which may also be difficult to induce. A diagnostic test based on the polymerase chain reaction (PCR) was therefore developed for these pathogens. The internally transcribed spacer (ITS1) region of ribosomal DNA (rDNA) of several isolates of M. fijiensis, M. musicola, and M. musae was sequenced, and a 21‐base oligonucleotide primer constructed for each species from a variable region identified in the sequences. These primers were used with primers in the conserved regions of the rDNA to produce a species‐specific product for each of the three species using PCR. The primers also amplified a fungal product from single ascospores placed in the PCR reaction, and from DNA from banana leaf tissue showing disease symptoms. No PCR products were produced when DNA from other species commonly found on banana was amplified. The fungi have been detected in herbarium samples up to 3 years old. The technique is very sensitive, and is able to detect DNA in a 1:20000 dilution of DNA extracted from 50 mg of freeze‐dried fungal mycelium.