First report of Fusarium oxysporum f. sp. fragariae race 1 causing Fusarium wilt of strawberry (Fragaria × ananassa) in the Alentejo region of Portugal

In the summer of 2023, within the Alentejo region (Portugal), a new occurrence of a plant disease of strawberry (Fragaria × ananassa) cv. 'Monterey' was observed in a Spring commercial planting. Symptoms consisted of foliar wilting, drying of older leaves, deformed and highly chlorotic leaflets, crown discoloration, plant stunting, and in some cases death. Several outbreak foci, covering nearly half a hectare, were observed within the affected farm, with almost 80% of the plants showing symptoms. Four samples,of 6 plants, were collected from 4 locations within the field. Petiole sections (1 cm) were rinsed with 0.1% Tween 20, submersed in 70% EtOH for 20 s followed by 60 s in 1% NaOCL, and then placed on Komada's medium (Komada, 1975). After incubation at room temperature in the dark for a week, white-colored fluffy mycelia grew profusely from the petioles of all samples. Colony morphology and non-septate, ellipsoidal microconidia (5.7-12.4×2.5-4.3 μm) borne on monophialides, exhibited a resemblance to Fusarium oxysporum (Leslie and Summerell, 2006). Ten single strains were obtained from different plants by single hyphal tip isolation. For molecular confirmation, a portion of the translation elongation factor 1-alpha (EF1α) was amplified by PCR using EF1/EF2 primers (O'Donnell et al., 1998). Additionally, the RNA polymerase subunit RPB2 was amplified as two contiguous fragments via primers and protocols described by O'Donnell et al., 2021. Amplicons were sequenced (GenBank Accession Nos. PP426617 - 426626, PQ058494 - 058513). Using Fusarium-ID and Fusarioid-ID databases, EF1α and RPB2 sequences were found to be more than 99% identical to published F. oxysporum type isolates and Fusarium sp. isolates in the F. oxysporum species complex (Crous et al., 2021; Wang et al., 2022). A specific F. oxysporum f. sp. fragariae (Fof) qPCR assay (Burkhardt et al., 2019) was used to determine if these isolates could be Fof race 1. A Fof race 1 isolate (MAFF305558), negative controls, and water controls were included. All ten isolates and the Fof race 1 control were positive (Ct < 30), while other Fusarium spp. used as negative controls and the water controls did not amplify. Two isolates (F.200 and F.202) and MAFF305558 (positive control) were included in a pathogenicity test on two strawberry cultivars, 'Monterey' (susceptible to race 1) and 'Fronteras' (resistant to race 1) (Dilla-Ermita et al., 2023). Each isolate was included in two independent trials. In each trial, 5 plants per cultivar were inoculated by dipping roots for 10 min in 5 × 106 conidia/mL of 0.1% water agar (WA) or in sterile 0.1% WA for the negative control plants. Each plant was then planted in a pot filled with peat. Pots were placed randomly in random positions in a growth chamber at 28/20°C and 12h photoperiod. After 8 - 10 weeks, the control plants and 'Fronteras' plants remained healthy, while the inoculated plants cv. 'Monterey' were severely wilted and/or dead. Fusarium oxysporum was re-isolated from all symptomatic plants. Recovered isolates were confirmed to be the same as the inoculated ones using the Fof-qPCR, including the same controls as above. This is the first report of Fof race 1 in the Iberian Peninsula. Given that the land was not previously used for strawberry, it is highly probable that the pathogen was introduced with the planting material originated from a Spanish nursery. In conclusion, it is imperative to implement more severe control measures in nurseries to avoid the spread of this race.