Identification and characterization of Alternaria alternata causing leaf spot of olive tree (Olea europaea) in Turkey

Abstract During 2015, 2016 surveys, unusual leaf spot symptoms were observed on Gemlik and Ayvalik varieties of olive trees in various orchards in Antalya province in the western Mediterranean region of Turkey. The symptoms appeared as powdery or mycelium-like spots with diameters ranging from 3 to 7 mm on the leaves and stems. The symptoms were observed only in the inner portion of the olive leaves or branches of the older trees, but found on every portion of the leaves of younger trees. The fungus was successfully isolated from the leaves and cultured in vitro . The colonies were white-grey airy mycelium with the mixture of green; and light dark brown inner zones which change to light dark brown; green in the later period of growth. Dark brown conidia in chains ranged from 7 to 45.9 μm in length were observed under microscopic examination. Molecularly, the isolates were subjected to both conventional and Real-time Polymerase Chain Reaction (PCR) using AaltFor/AaltRev, AAF2/AAR3 and ITS2/ITS4 primers which produced approximately 184, 346 and 560 bp, respectively for conventional PCR. The isolates also tested positive with SYBR Green dye and AaltFor/AaltRev primer pair in the Real-time PCR test. The isolates showed high similarity based on the ITS region sequence and the beta tubulin gene among the monophyletic clade of the A. alternata in the inner group and that of those of GenBank, ranged from 95 to 100% for all the three primers tested. The prevalence of the disease was estimated between 40 and 80% in three of the orchards. Pathogenicity of the fungus also produced a positive result on the two-year-old Gemlik and Ayvalik olive seedling varieties, confirming their pathogenic status. This is the first report implicating A. alternata as the causal agent of leaf spot disease of olive trees in Turkey.