First Report of Phaeoacremonium minimum causing wood decay in nursery plants of almond in Spain

In May 2015, a survey was conducted to evaluate the phytosanitary status of almond (Prunus dulcis) propagating materials in a commercial nursery in La Rioja Province (northern Spain). Fungal isolation was performed on 7 plants (2-year-old almond ‘Guara’ grafted onto rootstock GF 677) showing collapsed branches, leaf chlorosis, and shoot dieback in approximately 20% of the trees. Black spots and dark streaking of xylem vessels were observed in cross- or longitudinal sections of the stems. Symptomatic branches and stems were collected and wood sections (10 cm long) were cut, washed in running tap water, surface-disinfested for 1 min in a 1.5% sodium hypochlorite solution, and washed twice with sterile distilled water. The sections were split longitudinally, and small pieces of discolored tissues were plated onto malt extract agar (MEA) supplemented with 0.5 g/liter of streptomycin sulfate. Plates were incubated at 25°C in the dark for 14 to 21 days, and all colonies were transferred to potato dextrose agar (PDA). A Phaeoacremonium sp. was consistently isolated from necrotic tissues (more than 50% of the isolation points). Single conidial isolates were obtained and grown on PDA and MEA in the dark at 25°C for 2 to 3 weeks until colonies produced spores (Mostert et al. 2006). Colonies were pale brown on PDA and yellowish white on MEA. Conidiophores were short and usually unbranched, 17 to 41 (mean 28) µm long. Phialides were terminal or lateral, mostly monophialidic. Conidia were hyaline, oblong-ellipsoidal or cylindrical, 3 to 4 (mean 3.9) µm long, 1 to 2 (mean 1.5) µm wide. Based on these characters, the isolates were identified as P. minimum (Tul. & C. Tul.) D. Gramaje, L. Mostert & Crous (Mostert et al. 2006; Gramaje et al. 2015). DNA sequencing of a fragment of the beta-tubulin gene of the isolates BV-008 and BV-009 using primers T1 (O’Donnell and Cigelnik 1997) and Bt2b (Glass and Donaldson 1995) (GenBank Accession Nos. KU094046 and KU094047) matched P. minimum Accession No. HQ605013. Pathogenicity tests were conducted using isolates BV-008 and BV-009. Two-year-old almond trees of cv. Ferragnes grown in pots were wounded in the stem with a 8-mm cork borer. A 8-mm mycelium PDA plug from a two-week-old culture was placed in the wound before being wrapped with Parafilm. Control plants were inoculated with 8-mm noncolonized PDA plugs. There were 10 replicate plants per isolate and ten controls, and the experiment was repeated once. Inoculated plants were immediately planted in a field site in Logroño (La Rioja). Within 4 months, shoots on all Phaeoacremonium-inoculated stems had weak growth with chlorosis of leaves and there was black streaking in the xylem vessels. The vascular necroses that developed on the inoculated plants were 5.4 ± 0.5 cm long (isolate BV-008) and 5.6 ± 0.5 cm long (isolate BV-009), significantly greater than those on the control plants (P < 0.01). Control plants did not show any symptoms. The fungus was reisolated from discolored tissue of all inoculated stems, completing Koch’s postulates. To our knowledge, this is the first report of P. minimum associated with wood decay of almond nursery stock in Spain or any country in the world. Phaeoacremonium minimum is the most common Phaeoacremonium species associated with esca and Petri disease in grapevines, thereby causing untenable economic losses to the grapevine industry worldwide (Mostert et al. 2006). This study demonstrates the risk for the almond industry posed by the potential spread of P. minimum using contaminated planting material.Peer reviewe