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First Report of Colletotrichum acutatum Causing Anthracnose on Olives in Greece

Iliadi, M. K., Tjamos, E. C., Antoniou, P. P., Tsitsigiannis, D. I.
Plant disease 2018 v.102 no.4 pp. 820
Colletotrichum acutatum, DNA, Olea europaea, acidity, anthers, anthracnose, autumn, branches, calyx, conidia, corolla, cultivars, culture media, death, defoliation, discoloration, disease severity, fruit drop, fruits, fungi, genes, groves, humidity, internal transcribed spacers, leaves, microscopy, necrosis, oils, olives, orchards, pathogenicity, pathogens, peroxide value, photoperiod, pistil, plant rots, polymerase chain reaction, sequence analysis, sodium hypochlorite, spraying, tissues, trees, winter, Greece
Anthracnose is the main disease of olive fruit (Olea europaea L.) that is caused by different species of Colletotrichum spp. primarily belonging to two complexes, C. acutatum sensu lato (s.l.) and C. gloeosporioides s.l. (Cacciola et al. 2012; Damm et al. 2012; Schena et al. 2014). Observations in January 2015 in >20 olive groves in the Aitoloakarnania region (West-Central Greece) showed severe symptoms of mummified olive fruits in about 50% of trees, in table olive cultivar Kalamon. In March 2015, severe brown discoloration of inflorescences with 40 to 50% disease severity was observed in >15 orchards of cultivar Koroneiki in the island of Zakynthos (West Greece). In autumn 2015, mature olive fruits showed typical anthracnose symptoms with dark necrotic lesions and rot with abundant orange conidial masses that resulted in premature fruit drop or mummification. Symptoms appeared also on tree twigs and leaves, leading to necroses, severe defoliation, and branchlet death. The disease also affected the oil quality by increasing the acidity and the peroxide number in oil-producing varieties. Since then, autumn and winter heavy rainfalls resulted in extensive spread of anthracnose in West Greece and Peloponnese in 2016, causing severe losses in several olive cultivars. Stereoscopic and microscopic observations showed acervuli on fruits, anthers, pistils, petals, and sepals of flowers after 2 to 3 days incubation time under high humidity. The causal agent was isolated directly from infected pistils and fruits in potato dextrose agar, and microscopic examinations showed acervuli with typical conidia of the genus Colletotrichum that were subcylindrical with rounded ends, straight, hyaline, and aseptate, 10.8 to 18.1 µm long (mean = 14.5 µm) and 3.4 to 4.7 µm wide (mean = 4.1 µm) (n = 60 conidia). To identify the fungal species, DNA from two single-spore isolates from fruits and flowers was extracted, and six genes were amplified (ITS, GAPDH, CHS-1, HIS3, ACT, and TUB2) using the primers reviewed in Damm et al. (2012). PCR products were sequenced, and BLAST analysis showed 100% identity to C. acutatum for both isolates (GenBank accession nos. KY305483 [ITS1-5.8-ITS2], MF979822 [CHS-1], MF979823 [HIS3], MF979824 [GAPDH], MF979825 [TUB2], and MF979826 [ACT]). Then, pathogenicity tests were carried out to confirm the ability of C. acutatum isolates to cause disease. Fruits and leaves were surface disinfected with 0.1% NaClO for 3 min and rinsed with ddH₂O. Artificial inoculation of three different isolates from fruits and flowers was performed by spraying with a conidia suspension (10⁶ conidia/ml) in five olive fruits and five tree leaves per isolate in cultivars Kalamon and Koroneiki (Gomes et al. 2012; Talhinhas et al. 2009). Control fruits and leaves were treated with sterilized water. After inoculation, olive fruits and leaves were enclosed in plastic boxes and kept at 26°C with a 12-h photoperiod. First rot symptoms and formation of acervuli of the pathogen were initiated 3 days after inoculation. Eight days postinoculation, all treatments exhibited symptoms similar to those observed in olive orchards (extensive fruit rot and leaves with necrotic lesions), and C. acutatum was reisolated from the symptomatic olive tissues, confirming their identity and Koch’s postulates. Neither symptom was observed in control plants, nor were positive fungal isolations obtained. To our knowledge, this is the first report of C. acutatum causing fruit rot and flower and leaf necroses on olive trees in Greece.