A Dose-Response Approach Differentiating Virulence of Mycosphaerella fijiensis Strains on Banana Leaves Uses Either Spores or Mycelia as Inocula

The assessment of virulence of geographically and genetically diverse strains of Mycosphaerella fijiensis and resistance of their Musa hosts poses several challenges. Mycosphaerella fijiensis is slow growing and requires relatively strict culture conditions to produce conidia for inoculum. The size and relatively slow growth rate of banana plants from tissue culture further complicate screening efforts. In addition, relatively little is known about the effect of dose on disease outcome. A reliable and robust virulence assay using weighed fragmented mycelia brushed onto multiple defined areas (5 × 5 cm)of the abaxial surface of leaves of 3 to 6 month-old banana plants was developed. Methods to reduce the time needed for inoculum production and increase the number of assays that could be undertaken over time were developed. Image analysis software was used to measure the percentage of each inoculated leaf section showing black leaf streak symptoms, thereby allowing calculation and statistical analyses of leaf area affected using a dose-response model. The mycelium-based assay differentiated virulence levels of four M. fijiensis strains and revealed that one mutant, in addition to being deficient in conidia production and pigmentation, is of very low virulence. By localising inoculum onto small areas of large leaves, a dramatic increase in the number of strains that can be tested on each leaf and plant was achieved. The standardised method will be of value for evaluating Mycosphaerella pathogen virulence and Musa resistance where direct, quantitative measures during symptom development are necessary without requiring the use of expensive biotechnology-based tools.