Molecular-based detection and analysis of population structure of Mycosphaerella fijiensis, the causal pathogen of black sigatoka of banana

One of the major activities scheduled during the first year of implementation of the project was the establishment of a collection of Mycosphaerella fijiensis isolates obtained from Black Sigatoka infected banana leaves collected from different varieties of banana from different banana growing regions in the country. A total of 762 M. fijiensis isolates were obtained from nine provinces in Luzon (Laguna, Batangas, Cavite, Mindoro, Pangasinan, Nueva Viscaya, Quirino, Isabela, and Cagayan), three provinces in Visayas (Leyte, Cebu and Capiz) and eight provinces/places in Mindanao (Davao City, Davao del Norte, Davao del Sur, Bukidnon, North Cotabato, Compostella Valley, Agusan del Norte, and Agusan del Sur). Majority (76%) of the isolates have been maintained in V8 juice agar in microcentrifuge tubes for short term/temporary storage and in sterile filter paper disks for long term storage. Cultural characterization (colony color, shape, margin and elevation) of 490 (64%) of the isolates has been done. The colony characteristics of the isolates are consistent with those that were reported in the literature. Morphological characterization of the anamorph, Psedocercospora fijiensis, has been initiated but was suspended because of the very slow growth of the isolates that it takes about three weeks before the relevant morphological structures (shape and color and conidia and thickening of the hilum of the basal cell) can be absorbed. In order to fulfill the Kock's Postulates and to confirm the identity of the isolates, pathogenecity test of selected isolates from Luzon on banana cultivars Lakatan and Cavendish was attempted. Three inoculation methods were tried, namely, spraying the surface of banana leaves with conidial suspension, brushing the leaf surface with mycelial fragments using a paint brush and taping mycelial plug on the leaf surface, which was either previously scratched with a blade (abraded) or not scratched (unabraded). The inoculated plants were incubated in a makeshift mist chamber for one week and then transferred to the greenhouse until symptom development. Of the three methods, taping of mycelial plugs on the leaf surface was able to induce symptoms of black leaf streak or Black Sigatoka. Stage 3 symptom was seen on the unabraded leaf while stage 6 symptom was observed on the abraded leaf. For PCR [polymerase chain reaction] detection of M. fijiensis, five pairs of primers designed by previous workers (Johanson and Jeger, 1993, Nakyanzi, 2002, Anzanlou et. al, 2007) were screened against a subset of M. fijiensis isolates in the collection as well as other fungal pathogens of banana. Along with the screening of primers, PCR conditions were also optimized for each of the primers. Direct colony PCR, which makes use of fungal mycelia as source of template DNA, was used for primer screening. Direct colony PCR reduces the time required for molecular detection of pathogens as DNA extraction, which takes hours to do, is not required. Of the five primers screened, the primer pair MFactF/ACTR, designed by Arzanlou et al (2007) based on the sequence of the action gene of M. fijiensis and specific for the fungal pathogen, was selected because of its better ability to discriminate between M. fijiensis and M. musicola, another ascomycetous fungus causing Yellow Sigatoka. The diagnostic band of the expected size (500 kg) was seen only with the 490 M. fijiensis isolates tested and was not observed with the other banana fungal pathogens tested (M. musicola, Fusarium oxysporum f. sp. cubense, Fusarium sp. causing crown rot of banana Phyllosticta sp., Pestalozzia sp.). Determination of the utility of PCR using the selected primer in the direct detection of M. fijiensis from infected plant samples. PCR was done using total DNA extracted from infected and healthy banana leaf tissues by the conventional CTAB method or by using a plant total DNA extraction and Invitrogen's Purelink Plant Total DNA Purification Kit. Genomic DNA extraction has been done from 373 isolates from Luzon, Visayas and Mindanao. Based on the results of DNA fingerprinting, the genetic diversity of subpopulations of M. fijiensis will be assessed using established formula for estimating genetic diversity.