First Report of Fusarium oxysporum f. sp. lactucae Race 4 Causing Lettuce Wilt in England and Ireland

Abstract

In the UK, lettuce is grown both outdoors and under glass with a value of £171 million p.a. (DEFRA, 2016). Glasshouse production is very intensive with up to six crops per year grown in the same soil. In summer 2016 in Ireland (Dublin County) and summer 2017 in England (Lancashire), wilting symptoms were observed on indoor lettuce (cvs. Skye and Espirando). Initially, approx. 10% of plants were affected but losses were more severe on subsequent crops (approx. 30-60%) with a grower in Ireland reporting up to 100% losses. Initial symptoms were stunting and yellowing (often at leaf margins), followed by wilting and plant death. A brown/black/red discoloration of the vascular tissue was observed. Sections of infected vascular tissue (0.5cm) were surface sterilized in 70% ethanol for 30s, washed twice in sterile water and plated on potato dextrose agar (PDA) containing 20 µg/ml chlorotetracycline. Plates were incubated at 20°C for 4 days and fungal colonies sub-cultured onto fresh PDA. Fungal isolates were obtained from four sites in Ireland and four sites in England, were cream to pink in colour and displayed morphology typical of Fusarium. For isolate IRE1 (Ireland), macroconidia were sparse, 3-septate, straight or slightly curved, 20.9 to 38.1 µm x 4.1 to 5.1 µm (mean 30.8 x 4.6); microconidia were abundant, elliptical or reniform and 4.3 to 17.0 x 3.4 to 5.6 µm (mean 8.6 x 4.5). For isolate LANCS1 (UK), conidia were as described for IRE1 except macroconidia were more abundant and measured 24.0 to 51.7 µm x 4.4 to 5.5 µm (mean 32.7 x 4.7); microconidia measured 5.4 to 14.8 x 2.4 to 5.2 µm (mean 8.8 x 4.3). DNA was extracted from all isolates (rapid method based on PrepMan®Ultra reagent), and the translation elongation factor 1-α (EF1-α) gene sequenced as described by Taylor et al. (2016). All EF1-α sequences (Accession no. MK059958) were identical and were identified as Fusarium oxysporum using BLAST with a 99% match to an isolate from statice (KX822794). All isolates were identified as F. oxysporum f. sp. lactucae (FOL) race 4 using a race-specific PCR (Gilardi et al., 2017). Pathogenicity and race identity were confirmed using the differential lettuce cultivars (Gilardi et al. 2017) Costa Rica No. 4 (CR; resistant to FOL race 1), Banchu Red Fire (BR; resistant to races 2 and 4) and Romana Romabella (RR; resistant to races 1 and 2) (seed supplied by Rijk Zwaan). Plants were inoculated with isolates LANCS1, IRE1 and the FOL race 1 isolate ATCCMYa-3040 from Italy (Gilardi et al. 2017). Roots of 3-week-old plants (8 replicates per treatment) were dipped in a spore suspension (1 x 106 conidia/ml) for 10 minutes before transplanting into 250ml pots. Control plants were dipped in sterile water. Pots were placed in a glasshouse (25°C day, 18°C night) and wilting scored twice weekly. For isolates LANCS1 and IRE1, wilting was observed for CR and RR 11-14 days after inoculation, while for ATCCMYa-3040, wilting was only observed in BR. Plants were cut longitudinally after 38 days and vascular browning was observed in all plants where wilting had occurred. Control plants remained healthy. These results confirmed that the isolates LANCS1 and IRE1 are FOL race 4. Finally, F. oxysporum was consistently re-isolated from infected CR and RR plants and identified as FOL race 4 using the specific PCR hence fulfilling Koch’s postulates. FOL race 4 has recently been identified in another area of the UK (Cambridgeshire) and is a serious threat to UK lettuce production. It is also present in the Netherlands and Belgium.