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First Report of Pleurostomophora richardsiae Causing Branch Dieback and Collar Rot of Olive in Istria, Croatia

During 2016 and 2017, a survey for olive (Olea europaea L.) diseases was carried out in Istria, Croatia. Samples were collected from trees showing wilt, decline, and branch dieback and were analyzed in the laboratory for the presence of potential pathogens. The fungus Pleurostomophora richardsiae (Nannf.) L. Mostert, W. Gams and Crous was found in four out of 23 orchards inspected. It was isolated from six trees in two orchards, from cankers and purple-brown streaking on olive twigs and branches after placing fragments of diseased tissue onto carrot piece agar. The same fungus was also isolated in two orchards from four declining trees showing necrotic areas beneath the bark of root collars and lower part of the trunk. P. richardsiae was identified by morphology of pure cultures, conidiophores, phialides, and two types of conidia (Schol-Schwarz 1970 ; Vijaykrishna et al. 2004) after transferring colonies on potato dextrose agar (PDA) and incubation for 25 days in darkness. Colonies on PDA were cottony, brown with whitish edge, gray-brown in reverse, abundantly producing two types of conidia on phialides: spherical brown conidia (2.1-μm diameter) and hyaline allantoid to reniform conidia (5.4 to 6 × 2 μm). Two isolates (PI 2-17 and ZT 1/17) were selected to confirm species identification by amplification and sequencing of the internal transcribed spacer (ITS) region using ITS1/ITS4 primer pair (White et al. 1990). Sequences showed 99% identity with P. richardsiae strain CBS406.93 (GenBank accession no. AB364703) and were deposited in GenBank (MH014999 and MH015000). Both Croatian isolates were selected to assess their pathogenicity to olive plants. Pathogenicity tests were performed on 2-year-old potted plants of local olive cultivar Buza maintained in a growth chamber set on 18/22°C and 12-h dark/12-h light regime. Five microliters of conidial suspensions (106 conidia/ml) of selected isolates was injected in 2-mm wounds made in basal, middle, and upper part of the trunk of each plant. Another set of plants was inoculated by placing mycelial plugs (5-mm diameter) from 10-day-old colonies on PDA into circular wounds made on the same positions described above. Twelve plants in total were inoculated: three with conidial suspension and three with mycelial plugs for each fungal isolate. Twelve control plants were inoculated with sterile water and noncolonized PDA plugs. Four months after inoculation, four out of 12 P. richardsiae-inoculated seedlings wilted above the inoculation point. Two plants wilted completely, and two plants wilted from the middle inoculation points. Necrosis under the bark developed in 25 out of 36 P. richardsiae-inoculation points (69%). Necrosis developed on all 18 points inoculated with mycelial plugs and on seven out of 18 points inoculated with conidial suspensions. Necrotic area length beneath the bark ranged from 17 to 44 mm. The fungus was reisolated from necrotic areas in 24 out of 25 attempted reisolations (96%). Wilting or necrosis was not recorded on control plants, and the pathogen was not reisolated. This is the first report of P. richardsiae associated with dieback and collar rot of olive in Croatia, although P. richardsiae was previously reported as a pathogen of olive in southern Italy (Carlucci et al. 2013). Presence of P. richardsiae in Croatia indicates that this olive pathogen might be more broadly distributed in European olive-growing regions than previously reported.