OPTIMIZING ECOTILLING TECHNIQUE FOR TRACKING FIELD COCHLIOBOLUS SATIVUS POPULATION
M. Jawhar, B.J. Till, A. Albaterni, A. Skiheita, M.I.E. Arabi, Y. Bakri, N. MirAli
Accurate and robust detection and quantification of fungi is essential for diagnosis, modeling and surveillance. Also, direct detection of fungi enables a deeper understanding of natural microbial communities, particularly as a great number of fungi are difficult or impossible to cultivate. Yet, many methods are not suitable for developing countries with varying levels of laboratory infrastructure. Optimized low cost method for screening Cochliobolus sativus, the causal agent of barley spot disease, was established using self-extracted nuclease and agarose gel electrophoresis. Gene-specific primers were designed from the whole fungal genome for use in the Ecotilling assays on 50 isolates collected over the past 20 years throughout the Syrian territory, representing distinct virulence profiles. Gene-specific primer pairs were used to optimize enzymatic mismatch cleavage and polymorphism discovery in three GlutSynth, Carp1 andXYL2 genes.Thirty-seven putative nucleotide polymorphisms were identified. The protocol is rapid, inexpensive and can robustly distinguish pathotypes in haploid species, such as fungi without high informatics load of DNA sequencing. In principle, this strategy can be widely applied to monitor the epidemics of a variety of plant pathogens.