First Report of Root and Crown Rot Caused by Pestalotiopsis clavispora (Neopestalotiopsis clavispora) on Strawberry in Spain.

Strawberry (Fragaria x ananassa Duch.) is one of the most important crops in Huelva (southwestern Spain) with 7,300 ha cultivated in 2012 yielding 275,300 tonnes (98% of the total Spanish production). At the beginning of the 2013-2014 season, a low incidence of mortality (~1%) in strawberry plants cultivated at Huelva Province was observed. Symptoms consisted of discoloration or brown necrotic areas in crown and root. To isolate the causal agent, crowns and roots were surface disinfected for 2 min in 1% sodium hypochlorite, rinsed twice in sterile distilled water, air dried in a laminar-flow cabinet, and plated on potato dextrose agar (PDA). Pure cultures of the fungus were grown on PDA at 25°C with a 12-h photoperiod for 7 to 10 days. Fungal colonies were white and cottony, edge undulate, circular (upper side), and pale luteous (reverse side). The acervuli (avg. 280 μm) were black, concentric, and appeared throughout the entire plate after 10 days. Conidia were fusiform to ellipsoid and five-celled (average 22.7 × 7.7 μm) (n = 100). Apical and basal cells were hyaline, whereas the third and fourth cells from the base were darker (brown) than the second cell from base (pale brown). Conidia had one basal appendage (average 9.5 μm) and two to four apical appendages (avg. 25.9 μm). On the basis of colony and conidia morphology, isolates were identified as Pestalotiopsis clavispora(G.F. Atk.) Steyaert, recently renamed as Neopestalotiopsis clavispora (Maharachchikumbura et al. 2014). The identification as P. clavispora was confirmed by amplifying and sequencing of three genetic regions: ITS [(ITS1, 5.8S, ITS2) (GenBank Accession No. KU096879), β-tubulin (KU096880), andtef1 gene (KU096881) from three isolates (Maharachchikumbura et al. 2012). BLAST analysis showed in all cases 98 to 99% identity with P. clavispora [KM402033.1 (ITS); KC537822.1 (β-tubulin); and KM402034.1, JX399044, and KC537815.1 (tef1)]. Pathogenicity assays were carried out in a randomized complete block design with three blocks of 12 plants each, divided among three isolates and a control. Three isolates were compared with noninoculated controls on three strawberry cultivars: ‘Florida Radiance,’ ‘Sabrina,’ and ‘Splendor.’ The roots of three strawberry transplants per isolate were dipped for 4 min in a conidial suspension (1•105 conidia/ml) from 10-day-old PDA cultures and planted in pots (14-cm diameter) filled with sterilized soil. Control plants were dipped in sterilized 0.01% Tween-20. Plants were held at 25°C and 40/70% relative humidity (day/night) in a growth chamber with a 16-h photoperiod. After 3 months of incubation, the mortality of the inoculated plants was: 7.4, 7.4, and 11.1% for Florida Radiance, Splendor, and Sabrina, respectively. No mortality was observed in the control plants. About 23.4% and 25.9% of the inoculated plants showed more than 75% of root rot and crown rot, respectively. The experiment was repeated twice. All isolates showed similar pathogenicity. P. clavispora was consistently reisolated from symptomatic plants. P. clavisporahas been reported causing canker and dieback on blueberry in Chile (Espinoza et al. 2008) and Uruguay (González et al. 2012). P. longisetula has been reported as the cause of strawberry fruit rot in Egypt (Embaby 2007), but to our knowledge, this is the first report of P. clavispora causing crown and root rot of strawberry worldwide. © 2016 The American Phytopathological Society.