First Report of f. sp. Race 4 Causing Lettuce Wilt in Spain.

Spain is the fourth largest lettuce-producing country in the world and the leading European producer. Much of the production, mainly grow in open field, is dedicated to export with a value of 887 million U.S. dollars per year. In summer 2021 wilting symptoms were observed in a commercial field crop on butterhead lettuce 'Amible' in Albacete (Castilla-La Mancha, Spain). Approximately 15% of plants were affected, but losses were even more severe on subsequent crops. Vascular tissue of affected plants showed a brown to red discoloration. Sections of infected vascular tissue (3 to 5 mm long) were surface sterilized in 70% ethanol for 30 s, washed three times with sterile water, and plated on potato dextrose agar (PDA) amended with streptomycin sulfate (100 mg/liter). From 5-day-old cultures typical pale cream to purplish mycelia with microconidia, macroconidia, and chlamydospores of Fusarium oxysporum were observed. Microconidia were abundant on carnation leaf agar and measured 6.1 to 9.2 μm (mean 7.1 ± 0.7 μm; n=50)). Macroconidia were sparse, three-septate, straight to slightly curved, 23.3 to 34.8 × 4 to 5.2 µm (mean 31.5 ± 2.8 × 4.2 ± 0.3 µm; n=50). Chlamydospores were terminal and intercalary, rough walled, and measured 7.2 to 10.1 μm (mean 9.5 ± 0.6 μm; n=50) µm. DNA was extracted from three single-spore isolates using the protocol of Querol et al. (1992) and the translation elongation factor 1-α gene (TEF) was sequenced with exTEF-F/FUexTEF-R primers as described by Taylor et al. (2016). All TEF sequences (GenBank accession no. OP903519) were identical. In BLAST analyses, the isolates showed 100% identity to the corresponding region of Fusarium oxysporum f. sp. lactucae (FOL) race 4 (MK059958). All Spanish isolates were identified as FOL race 4 using a race-specific polymerase chain reaction (PCR) with the primers FPUF/FPUR (Gilardi et al. 2017), and a previously identified FOL race 4 isolate Fus 1.01 as a positive control. Pathogenicity tests were conducted to confirm the positive result of the race 4-specific PCR and to complete Koch's postulates. Three differential lettuce cultivars ('Costa Rica No. 4', 'Banchu Red Fire', and 'Romana Romabella 30 CN') provided by Rijk Zwaan (The Netherlands) were inoculated with three Spanish isolates (Al1A1, Al1D, Al2B) and the Fus 1.01 isolate used as FOL race 4 positive control (Claerbout et al., 2018). Roots of 3-week-old plants (five replicates per treatment) were dipped in a spore suspension (1 × 106 conidia/ml) for 10 min before transplanting into 250-ml pots with sterile substrate. Non-inoculated control plants were dipped in sterile water for 10 min. The experiment was carried out twice. Inoculated lettuce seedlings were planted and maintained in a growth chamber (25°C day, 18°C night). Plants were slightly watered every other day. After 21 days, wilting was observed in the cultivars 'Costa Rica No. 4' and 'Romana Romabella 30 CN'. Moreover, taproots were cut longitudinally, and vascular browning was observed in the taproot. No discoloration could be observed in the taproot of 'Banchu Red Fire' plants, coinciding with the result of isolate Fus 1.01 used as FOL race 4 positive control. Non-inoculated control plants remained healthy and vascular browning was not observed. In both experiments, F. oxysporum f.sp. lactucae was consistently reisolated using PDA medium. These results confirmed that the isolates Al1A1, Al1D and Al2B were FOL race 4. This race has recently been identified in The Netherlands (Gilardi et al. 2017), Belgium (Claerbout et al. 2018), United Kingdom, Ireland (Taylor et al. 2019) and Italy (Gilardi et al. 2019) and could become a serious threat to Spain lettuce production.