First Report of Colletotrichum nymphaeae on Olive in Italy

Olive anthracnose is one of the most important diseases of olive worldwide (Moral et al. 2009). Among the Colletotrichum species associated to the disease, some in the complexes C. acutatum and C. gloeosporioides have been reported as the most frequent in Italy (Schena et al. 2014). During the 2017 season, severe losses were reported in several (18) olive orchards (approximately 30 ha) located in the districts of San Daniele, Friuli Venezia Giulia, one of the northernmost olive growing areas in Italy. Drupes from cultivars Leccino and Leccio del Corno, at fruit size about 10% of final size (July), showed no symptoms or small, dark-brown rot lesion in the flesh; sometimes, peduncle was chlorotic, then necrotic, causing an easy detachment of the fruitlets. Symptomatic and asymptomatic fruits fell to the ground, determining a premature fruit drop of 70 to 80% of the fruit. Fruit with or without symptoms were surface sterilized in 2% NaOCl for 5 min, followed by 30 s in 75% ethanol, and then rinsed with sterilized distilled water. Fruitlets were sectioned with a sterile razor, and slices (1 mm thick) were seeded on potato dextrose agar plates amended with streptomycin-sulfate (250 mg/liter). Plates were incubated at 25°C for 8 to 15 days. Developing colonies showed a white to pale olivaceous, gray, aerial mycelium and partly with salmon to orange acervuli. Conidia were hyaline, smooth-walled, aseptate, straight, cylindrical to cylindric-clavate with one end round and the other rounded to acute, measuring 12.1 ± 2 × 5.1 ± 1.9 μm (mean ± SD). Culture and conidial morphology were in concordance with published descriptions of C. acutatum sensu lato (Damm et al. 2012). To confirm the identity, the internal transcribed spacer (ITS), the 28S ribosomal RNA (LSU), and partial sequence of β-tubulin (TUB2) regions of representative isolates were amplified and sequenced. A BLAST search revealed that all the sequences showed 99 to 100% homology with the corresponding sequences of previously identified Colletotrichum nymphaeae (Pass.) isolates in GenBank, and only one base difference in the TUB2 sequence, compared with the type strain (JQ949848). Sequences were deposited in GenBank with accession numbers MG906799 (ITS), MG906796 (LSU), and MH010593 (TUB2). Pathogenicity of C. nymphaeae was investigated using a pure culture of the isolates C290 and C291. Fruit (cv. Leccino) at the veraison stage (100 per isolate), were surface disinfected by dipping in 3.5% NaOCl for 1 min, rinsed in sterile distilled water, and arranged in sterile humid chambers. Fruit were then inoculated by depositing a drop (10 μl) of a spore suspension (104 conidia/ml) on the equatorial surface, which was pierced with a sterilized needle. Fruit treated with sterile distilled water served as a control. After 10 days incubation at 25 ± 1°C, all the inoculated fruit showed typical anthracnose symptoms and lesions with cream to salmon pink acervuli, whereas the controls remained healthy. The species C. nymphaeae was reisolated from the rotted fruit. Pathogenicity tests were repeated twice with the same results. C. nymphaeae has been previously reported to cause severe anthracnose on olive in Portugal (Talhinhas et al. 2005) and on multiple hosts in diverse parts of the world (Baroncelli et al. 2017). C. acutatum sensu stricto and C. godetiae have been reported as dominant species of olive anthracnose in Italy (Mosca et al. 2014); however, to the best of our knowledge, this is the first report of C. nymphaeae causing olive anthracnose in Italy.