First Report of Fusarium Wilt of Lettuce Caused by Fusarium oxysporum f. sp. lactucae Race 1 in Spain

Lettuce plants (Lactuca sativa) of the romaine cultivar Amible showing wilt symptoms were observed in a 2-ha field located in the Comarca del Noroeste in the region of Murcia, Spain, in August 2017. The incidence of wilted plants was 60%. On affected plants the leaves showed chlorosis, and necrosis also was observed, particularly on the internal younger leaves, with vascular darkening and severe wilting. Small (3- to 4-mm) pieces of necrotic vascular and root tissues were surface sterilized for 1 min in 1.5% NaOCl, washed twice with sterilized distilled water, and plated onto potato dextrose agar (PDA) with streptomycin sulfate (0.5 g per liter). Plates were incubated at 25°C for 3 to 5 days. Fusarium colonies were transferred to PDA and Spezieller Nahrstoffarmer agar (SNA) media (Garibaldi et al. 2004) for morphological identification and were identified as Fusarium oxysporum based on morphology on SNA (Leslie and Summerell 2006). Macroconidia were straight to slightly curved, with one septum and spores measuring (15.0 to) 19.1 (to 25) × (3.1 to) 4.1 (to 5.0) µm (n = 30), or two septa and spores measuring (20 to) 22.8 (to 27.5) × (4.8 to) 4.9 (to 5.0) µm (n = 30). Microconidia were borne on short monophialides in false heads, were ovoid to reniform, and were (7.5 to) 11.5 (to 15.0) × (2.5 to) 3.3 (to 5.0) µm (n = 30). Chlamydospores were mostly single, terminal, and intercalary, measuring (7.5 to) 10.8 (to 12.5) µm (n = 30). The translation elongation factor-1α (EF-1α) gene of 10 representative isolates was sequenced using EF-1/EF-2 primer pairs (O’Donnell et al. 1998). All EF-1α sequences were identical, and one (corresponding to isolate Fm1) was deposited in GenBank (accession no. MN379455). BLASTn comparison showed a 100% homology with the EF-1α sequence of F. oxysporum f. sp. lactucae (KY009874). Comparison of this sequence in the Fusarium ID database (http://fusarium.mycobank.org/) exhibited identical homology. Specific primers Hani3′ and Hanilatt3rev (Pasquali et al. 2005) produced a 183-bp product specific for Fusarium oxysporum f. sp. lactucae race 1, and specific primers for race 4, FPUF and FPUR (Gilardi et al. 2017), showed no amplification. For pathogenicity testing four isolates (Fm1, Fm2, Fm3, and Fm4) were inoculated onto lettuce plants following the protocol by Pasquali et al. (2005). Two lettuce cultivars, Romano Odessa and Chiquina, were grown in a mix of peat and sterilized perlite (3:1 v:v) in 500-cm³ pots (Garibaldi et al. 2004). Fifteen-day-old plants of each cultivar were inoculated either by irrigation with spores or by immersing the roots in a spore suspension (1 × 10⁶ CFU/ml) for each isolate. For the former, plants were irrigated with 5 ml of the conidial suspension, and for the latter roots were immersed in the spore suspension for 30 min. The inoculated plants and the controls (10 replicate plants per isolate, cultivar, and treatment) were kept in growth chambers with a completely randomized design at 26 to 28°C with a 14-h photoperiod per day. The first wilt symptoms appeared 9 days after inoculation, independent of the cultivar, inoculation method, and isolate. Lettuce growth was stunted compared with the control plants. Control plants remained asymptomatic. The fungus was 100% reisolated and identification confirmed as described (morphologically and with EF-1α sequencing), fulfilling Koch’s postulates. To our knowledge, this is the first report of F. oxysporum f. sp. lactucae causing Fusarium wilt of lettuce in Spain. This severe disease poses a substantial threat to the continued production of lettuce in this region; in Europe, it was first described in Italy (Garibaldi et al. 2002).