First Report of Anthracnose Caused by Colletotrichum acutatum on Olive Fruits in Pakistan.

Abstract

Anthracnose symptoms on olive (Olea europaea) fruits cv. "Gamlick" were found in farmer orchards in Chakwal, Punjab, Pakistan (32° N and 72° E), with an average prevalence of 59%. Fruit symptoms start as thin, black, sunken lesions with a watery appearance that grow in diameter and coalesce into a large sunken soft zone. Lesions on mature fruit become noticeable in 5 to 6 days after infection, if temperatures are favorable (28°C). On the fruit lesion, orange conidial masses in dispersed or concentric circle arrangement can appear. Fragments (5 mm) were taken from the margins of fruit lesions and surface-sterilized with 70% ethanol (1 min) and 1% NaClO (2 min), cleaned with sterile purified water, blotted dry, and plated on potato dextrose agar (PDA) in Petri dishes. The petri plates were incubated at 27°C. A fungus was consistently isolated, and thirty-five isolates were characterized. Aerial mycelia from olive isolates Colonies were compact, initially white or cream white, then grey, and eventually dark grey, with conidium masses forming in the middle. Mycelium is branched, septate, and hyaline. Conidia are hyaline, aseptate, fusiform, or often cylindrical, with obtuse apices and tapering bases. Their mean size was 8.5µm in length and 3.0 µm in width. Based on morphological features, the fungus was tentatively identified as Colletotrichum acutatum (Agosteo G.E., 2010). The identification was confirmed by amplification and sequencing of a representative isolate's internal transcribed region (ITS), Beta- tubulin region (TUB2), Actin region (ACT), and Glyceraldehyde 3-phosphate dehydrogenase region (GAPDH) with the primers ITS1/ITS4 (Gardes & Bruns 1993), TUB4/TUB5 (Woudenberg et al. 2009), ACT1/ACT3 (Carbone & Kohn 1999) and GDF1/GDR1 (Guerber et al. 2003). BLAST analysis revealed 100% identity for ITS, GAPDH and ACT and 99% identity for TUB, between the sequences of the olive fruit isolate (GenBank Accessions MW647502, MZ436968, MZ714412 and MW810331, respectively) and sequences of C. acutatum reference isolates (GenBank Accessions GO613492, KF975660.1, MT274752.1 and MH547616 respectively). Phylogenetic analysis based on ITS, GAPDH and TUB regions of the olive fruit isolates and reference isolates of various Colletotrichum species using the MEGA X software program confirmed the isolate from olive was C. acutatum. The fungal isolate was deposited as a living culture in the Barani Agricultural Research Institute's fungal culture collection center (BACA.9381). Pathogenicity tests were conducted with this isolate by placing a 20 µl drop of a conidial suspension (3 × 107conidia ml-1) on five healthy olive cv. Gemlik fruits. As a control, five non-inoculated olive fruits were used. Fruits were placed at a temperature of 27°C with artificial light and a photoperiod of 12 hours. Anthracnose symptoms developed only on inoculated fruits after seven days of inoculation. The fungus was re-isolated from symptomatic fruits, and its identity was confirmed through morphological characteristics, thus verifying Koch's postulates. To the best of our knowledge, this is the first report of C. acutatum infecting olive fruits in Chakwal region of Pakistan.