Molecular Identification of Cochliobolus sativus, the Causal Agent of Wheat Spot Blotch Disease, Using ITS Sequencing and its Phylogenetic Relationships with other Cochliobolus Species

The causal agent of spot blotch is the fungus Cochliobolus sativus which is one of the most important wheat pathogens worldwide. Among several wheat samples showing typical spot blotch symptoms collected from different regions of north of Iran, eight samples were selected as representative and the fungal pathogens were subsequently isolated and characterized using morphological, pathological and molecular approaches. Morphological characteristics of these isolates were determined using illustrated fungal keys demonstrating that the observed symptoms were due to wheat infection to C. sativus. Pathogenicity tests showed that isolated C. sativus were able to cause typical spot blotch symptoms on Bolani as the susceptible cultivar. Sequencing results revealed that ITS regions of Iranian isolates were identical to that of the C. sativus reference isolate. Phylogenic relationships of Cochliobolus spp. were investigated using different methods including genetic distance, maximum parsimony and maximum likelihood showing that all phylogenetic tree patterns were similar and Iranian isolates were clustered with the C. sativus reference isolate. Cochliobolus spp. were clustered in two major groups containing some minor groups. Estimated genetic distance showed that C. eleusines is the most related species to C. sativus, while C. kusanoi and C. austaliensis were the most distanced species to C. sativus. This study revealed that spot blotch is a widespread disease in the north of Iran and, therefore, to control the increasing spread of the disease, the resistance for this disease should be considered in breeding programs in this region.