Distribution, specific detection and the pathogenesis variation of Calonectria pseudonaviculata isolates, causal agent of boxwood blight disease, in Hyrcanian forest of Iran

During recent years outbreak of box blight trees causing sudden leaf abscission have declined about 40000 hectares boxwood trees in Northern forests of Iran. Calonectria pseudonaviculata is the causal agent of the disease. Distribution of boxwood blight disease was monitored in north forests from June to September of 2014. Sampling locations was included the provinces: Mazandaran (Liresar, Sisangan, Chalandar, Toskatook, Kohnesara, Neka, Sorkhkola, Afrachal, Savadkooh, Chaeebagh, Vachat, Ghaemshahr, Amol, Behshahr), Giulan (Astara, Kalat, Gisoom, Roodsar, Siahkal, Bijarkenar) and Golestan (Bandargaz-Livan sharghi) provinces. The isolates were identified based on morphological characteristics at first, then molecular identification confirmed morphological identification using previously designed species-specific primer ITS113F and ITS4. The pathogenesis variation of Hyracanian isolates was tested in green houses using representative 35 isolates. Boxwood blight fungal agent was isolated from all surveyed areas. Morphological criteria distinguishing this species from close Cylindrocladium species including cylindrical, rounded at both ends, 1-septate, 48- 68 (47.3) ×4- 6 (4.8) µm of conidia along with the long stipe (90-14 µm) which was hyaline, extension terminating in a broadly ellipsoid papillated vesicle with the widest part at the middle resulted in to identify all isolates as Cylindocladium pseudonoviculatum. ITS113F and ITS4 primers amplified the expected 250 bp region in all Calonectria pseudonaviculata isolates specifically. Comparing the pathogenicity of 35 isolates on Buxus sempervirens subsp. Hyrcana saplings revealed pathogenicity degree variation among Hyricanian Cylindocladium pseudonoviculatum divided in 15 groups were scattered in three provinces.