FIRST REPORT OF Diplodia seriata, THE CAUSAL AGENT OF CANKER DISEASE OF APPLE TREES IN IRAN

During the surveys of apple trunk and branch diseases in Urmia orchards, a pycnidial fungus was frequently isolated from Golden Delicious cultivar trees of over 15 years old with symptoms of stem and branch cankers, branch dieback, bark scaling and discoloration (Fig. 1-A in Farsi section). Purification of the isolated fungus was carried out by hyphal tip method. Formation of asexual fruiting bodies of the fungus was induced by the use of pine needles. The purified fungi were cultured on 2 percent water agar medium and then, sterilized pine needles were placed at different parts of the medium. Inoculated Petri plates were kept for four weeks at 25 °C under near-ultraviolet light (near-UV) with 12:12 h photoperiod (Pavlic et al. 2008). Different macro- and micro- morphological characters such as color and growth characteristics of the colony, color, shape and dimensions of conidia, conidiogenous cells and pycnidia were studied. Also, the ITS region of one selected isolate was amplified and sequenced using the ITS1 and NL-4 primers. Based on morphological and ITS sequence analysis, the fungus was identified as Diplodia seriata. The results of Blast search showed high similarity (100 percent) of the sequenced isolate with the isolates of D. seriata deposited in GeneBank. Descriptive characters of the species are: Colony diameter after four days at 25 °C is 75 to 80 mm; colony color is at first white, then turns olive green to olive gray and finally after two weeks becomes dark; colony with regular margins, aerial mycelium dense and thick; conidiomata pycnidial, 380 × 260 µm in diameter, solitary or aggregated, globose to ovoid, dark brown to black, immersed, partially erumpent when mature (Fig. 1-B in Farsi section); conidiogenous cells hyaline, smooth, cylindrical, swollen at the base, 7-12 × 3-5 µm, proliferating at the same level to produce periclinal thickening, or proliferating percurrently, giving rise to 2-3 annelations; conidia at first aseptate, thick-walled with smooth outer surface and verruculose inner surface clearly, ovoid to cylindrical, apex obtuse, base truncate or rarely rounded, hyaline, becoming pale brown and soon darkening before release from the pycnidia and becoming 1-septate (Fig. 1-C in Farsi section), rarely 2 or 3 septate after discharge, (12-) 20.6-22.02 (-31) × (7.5-) 9.6-10.05 (-12) µm, L/W = 2.2[H1] . Pathogenicity test was carried out on potted 2 years old seedlings and fruits of apple Golden Delicious cultivar based on completely randomized design. In seedlings, eight weeks postinoculation, symptoms of bark darkening and canker were seen in inoculated areas (Fig. 1-D in Farsi section). In fruits, six days after inoculation, the brown rot symptoms were seen around the inoculated area (Fig. 1-E in Farsi section), progressing into the fruit flesh. No symptoms were developed in the controls. Reisolation of the inoculated fungus were done from the newly infected tissues and Koch's postulates were fulfilled. All the tested isolates were pathogenic on fruits and seedlings. To our knowledge, this is the first report of isolation and pathogenicity confirmation of D. seriata from apple trees in Iran.