First Report of Coniella fragariae Causing Leaf Spot on Strawberry ( Fragaria x ananassa ) in Spain

Abstract

In 2024 fall, symptoms of leaf blade and petiole spots were observed on strawberry plants 'Rociera FNM' and 'Marisma FNM'. In 450 randomly selected plants from 3 nurseries of Segovia province (Spain) 3.5% of incidence was recorded. Lesions on the leaf were reddish-brown with yellowish halos and became necrotic. Dark brown lesions with defined edges were noted on petioles. Affected tissue sections were excised, rinsed with 0.1% Tween 20, submerged in 70% ethanol (20 s), followed by 1% NaOCl (60 s), and then placed onto potato dextrose agar (Condalab, Madrid, Spain) with 50 mg/l of streptomycin sulphate. Plates were incubated at 28°C under darkness. From 6 crowns and 2 petioles 8 isolates (Di 3 to Di 10) were obtained from different pycnidia via single conidia isolation. Colonies had petal-shaped margins. Hyphae were initially hyaline, later developing an orange coloration in the center of the colony, and black conidiomata were visible two weeks post-incubation. Conidiomata were ellipsoidal to globose. Single-celled conidia were brown, ellipsoidal with slightly mucronate apices, narrowly truncated bases and smooth walls. This morphology was consistent with Coniella sp. as described by Álvarez et al. (2016). Isolates were stored in 50% glycerol solution at -80ºC. Genomic DNA was extracted from the 8 selected isolates. The internal transcribed spacer (ITS) region, translation elongation factor 1-alpha (TEF1), and large subunit ribosomal RNA (LSU) were amplified using the primer pairs ITS1/ITS4 (White et al. 1990), EF728/EF986 (Carbone and Kohn 1999), and LR0R/LR7 (Chethana et al. 2017), respectively. Sequence alignments performed in MEGA v11.0 (Tamura et al. 2021) showed that all isolates were identical across the three analyzed loci. Sequences of Di6 and Di10 isolates were deposited in GenBank. BLASTn analyses revealed that ITS (PX663172, PX663173), TEF1 (PX666006, PX666007), and LSU (PX663628, PX663629) sequences showed 100% identity with C. fragariae strain CBS 198.18 for ITS and 99% and 100% identity with strain CBS 167.84 for TEF1 and LSU, respectively. A concatenated phylogenetic tree was constructed with representative Coniella species and outgroup sequences from NCBI GenBank (Chethana et al., 2017) and confirmed the identity. Pathogenicity test was conducted using isolates Di6 and Di10. Inoculation with each isolate was performed by spraying 25 mL of a conidial suspension (5 x 104 conidia/mL) onto 5 plants of 'Rociera FNM'. Control plants were sprayed with sterile water. The assay was carried out twice. After inoculation, plants were covered with a plastic bag and kept in darkness for two days. Growth chamber was maintained at 25±2°C with 12-h photoperiod. Identical symptoms to the initially observed appeared on inoculated plants 6 days post-inoculation. Both isolates exhibited comparable severity (proportion of symptomatic leaf area) 20 days after inoculation. An assay was also performed on 'Marismas FNM', giving the same results. Fungal colonies re-isolated from the lesions had the same morphology and ITS, TEF-1 and LSU sequences of the inoculated isolates, thereby fulfilling Koch's postulates. In Switzerland and Great Britain C. fragariae has previously been isolated from strawberry plants (Rigotti et al. 2003; Jones and Baker, 2007). To our knowledge, this is the first report of C. fragariae on strawberry in Spain. Monitoring of the pathogen is essential since it can spread through asymptomatic plants into strawberry’s nurseries and fields.