First Report of Pleurostoma richardsiae Associated with Twig and Branch Dieback of Olive Trees in Spain.

Abstract

Pleurostoma richardsiae has been described as an olive tree pathogen causing decline and brown wood streaking (Carlucci et al., 2013). A survey was carried out in plots under olive cultivation (Olea europaea L., cv. Picual; 10 year-old plants) at La Garrovilla, (Spain) in September 2020, in which a putative Verticillium wilt had been visually diagnosed. In Plot 1 (2.6 ha; 741 plants), 20.4% of the plants exhibited wilt, foliar browning and leaf drop, twig, and branch dieback. While the level of incidence in plots 2 (4.8 ha; 1368 plants), 3 (3.20 ha; 912 plants), and 4 (1.85 ha; 527 plants) was 25.0%, 19.5%, and 42.9% respectively, which meant for that harvest an average reduction in olive production, and an economic loss, of 30.2%. Three trees from each plot were uprooted and analyzed. Five out of 12 intriguingly showed brown streaking under the bark extending from the root system and ascending up the trunk, a symptom that is never associated with Verticillium dahliae wich does not produce necrosis and cankers in the wood (López-Escudero and Mercado-Blanco, 2011). Samples from the 5 tree trunks showing necrosis were taken to the lab and surface sterilized. Small pieces of discolored wood were placed onto malt extract agar plates containing chloramphenicol (0.25 g/L) and incubated for 21 days at 25°C in darkness. The growing fungal colonies were then transferred to potato dextrose agar (PDA). Isolates were identified by micromorphological characteristics, according to Vijaykrishna et al. (2004), as P. richardsiae. Colonies on PDA were cottony, brown with whitish edge, and produced abundantly two types of conidia: brown (spherical or subglobose), or hyaline (allantoids to cylindrical) that appeared on septated and inconspicuous phialides respectively. Identification was confirmed by amplification and sequencing of the internal transcribed spacer (ITS) region using ITS1/ITS4 primers (White et al., 1990), and partial sequencing of the β-tubulin gene using T1 (O'Donnell and Cigelnik, 1997) and Bt2b (Glass and Donaldson, 1995) primers. ITS sequence showed a 99.82% identity with that of P. richardsiae IFM51337 (CBS406.93 type strain; GenBank AB364703.1), whereas β-tubulin sequence exhibited a 99.77% identity with P. richardsiae CBS406.93 β-tubulin gene (GenBank MT501304.1). ITS and β-tubulin sequences were deposited in GenBank (MZ519916 and MZ542764 respectively). The P. richardsiae isolate has been deposited in the Spanish Type Culture Collection (CECT 21196). Pathogenicity tests were conducted on 1-year old potted olive plants cv. Picual, maintained in a growth chamber at 25ºC and 12-h dark/12-h light. Twelve plants were inoculated in a wound made in the stem with a scalpel, and mycelial plug (5 mm diameter) from 15-day-old PDA plates were inserted into the wound. Another set of 12 plants were inoculated with sterile agar plugs and used as negative control plants. Four months after inoculation, 66% of the plants inoculated with mycelia plugs, showed wilting, necrosis under the bark, or even had died. P. richardsiae was successfully reisolated from necrotic areas in 75% of the plants inoculated with mycelia plugs. A total of 10 reisolates were identified as P. richardsiae by the above molecular techniques to confirm Koch's postulates. No symptoms were observed in the negative control plants and the pathogen was not re-isolated from them either. To our knowledge, this is the first report of P. richardsiae associated with twig and branch dieback of olive trees in Spain.