First Report of Fusarium oxysporum f. sp. lactucae Race 4 on Lettuce in Belgium

Abstract

In Belgium, lettuce (Lactuca sativa L.) is an important crop that is mainly grown in soil in glasshouses. During autumn 2015, wilting symptoms on butterhead lettuce ‘Halewyn’ (Rijk Zwaan, the Netherlands) and an unknown cultivar were observed in two different commercial glasshouses in the Province of Antwerp, Belgium. The disease incidence was around 10 and 20%, respectively, with a disease severity of 9 and 18%. Since 2015 the disease has spread very fast; already 15% of the glasshouse lettuce production area in Flanders (northern part of Belgium) is infested. Dwarf growth and yellowing of the outer leaves were noticed on affected plants, followed by complete wilting and death. The vascular tissue showed a brown to red discoloration. Affected root and leaf tissues were surface-sterilized with 1% NaOCl for 30 s and washed three times with sterile water. The plant tissues were cut into 1 cm² pieces and plated on potato dextrose agar amended with streptomycin sulfate (100 mg/liter) and incubated at room temperature (19 to 22°C). Consistently dense fungal colonies with pale cream to purplish mycelia grew out of the plant tissues. Microconidia, macroconidia, and chlamydospores typical for Fusarium oxysporum were observed. Microconidia from isolates Fus1.01 and Fus1.02, coming from the two different glasshouses, measured respectively 5.99 to 8.64 (mean 6.98) × 2.75 to 4.39 (mean 3.32) µm and 6.75 to 11.50 (mean 8.42) × 2.75 to 4.59 (mean 3.61) µm. Chlamydospores were terminal and intercalary, rough walled, and measured 6.86 to 10.72 (mean 8.25) µm for Fus1.01 and 6.13 to 10.80 (mean 8.55) µm for Fus1.02. Macroconidia were straight to slightly curved with three septa and measured 24.49 to 31.27 (mean 27.27) × 2.93 to 4.42 (mean 3.84) µm for Fus1.01 and 20.91 to 26.09 (mean 22.58) × 3.42 to 4.70 (mean 3.98) µm for Fus1.02. Subsequently, DNA from single-spore cultures (Fus1.01 and Fus1.02) was extracted using the Invisorb Spin Plant Mini Kit (Stratec Molecular). The translation elongation factor 1-α (EF1-α) gene was amplified using primers EF1/EF2 (O’Donnell et al. 1998) and sequenced in both directions by LGC Genomics (Berlin) using Sanger sequencing technology. The EF1-α sequences of both isolates showed 100% similarity with the EF1-α sequence of F. oxysporum f. sp. lactucae strain S1 (accession no. DQ837657) (Mbofung et al. 2007) and were deposited (MG599512 and MG599513). By using specific primers FPUF and FPUR (Gilardi et al. 2016), we could show that both isolates belong to race 4. Moreover, pathogenicity tests with three different lettuce cultivars (‘Costa Rica No. 4’, ‘Banchu Red Fire’, and ‘Romana Romabella 30 CN’) provided by Rijk Zwaan (the Netherlands) were conducted to confirm the positive result with the primers FPUF and FPUR and to complete Koch’s postulates. Roots of 2-week-old lettuce plants were dipped in a 5 × 10⁵ spores/ml suspension, and five plants per cultivar were used. The experiment was carried out twice. Inoculated lettuce seedlings were planted in 100 g of steamed potting substrate and were maintained in a climate room at 24°C. In both experiments, wilting was observed after 4 weeks for the cultivars Costa Rica No. 4 and Romana Romabella 30 CN, but no symptoms could be seen on the cultivar Banchu Red Fire. F. oxysporum was consistently reisolated from all inoculated cultivars. These results are consistent with pathogenicity tests carried out before with two isolates of F. oxysporum f. sp. lactucae race 4 from the Netherlands (Gilardi et al. 2016) and indicate that this new race is also the causal agent of Fusarium wilt on lettuce in Belgium. This report shows that race 4 is spreading fast and imposes a serious risk to other lettuce production areas in Europe.