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

Abstract

Lettuce plants (Lactuca sativa) of the romaine cv. “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, 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 roots, tissues were surface sterilized for 1 min in 1.5% NaOCl, washed twice with sterilized distilled water, and plated onto PDA with streptomycin sulfate (0.5 g per liter). Plates were incubated at 25°C for 3 to 5 days. Fusarium colonies transferred to PDA and SNA agar media (Garibaldi et al. 2004) for morphological identification, and identified as F. oxysporum based on morphology on SNA (Leslie and Summerell 2006). Macroconidia were straight to slightly curved, with one septum and spores measuring (15.0-) 19.1 (-25) × (3.1-) 4.1 (-5.0) µm (n=30), or two septa and spores measuring (20-) 22.8 (-27.5) × (4.8-) 4.9 (-5.0) µm (n=30). Microconidia were borne on short monophialides in false heads, were ovoid to reniform and (7.5-) 11.5 (-15.0) × (2.5-) 3.3 (-5.0) µm (n=30). Chlamydospores were mostly single, terminal and intercalary, measuring (7.5-) 10.8 (-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 EF1-α sequences were identical and one, corresponding to isolate Fm1, was deposited in GenBank (Accession Number 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://www.westerdijkinstitute.nl/fusarium/) exhibited identical homology. Specific primers Hani3’ and Hanilatt3rev (Pascuali et al. 2007) produced a 183 bp product specific for Fol 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 cvs., Romano Odessa and Chiquina, were grown in a mix of peat and sterilized perlite (3:1 v:v) in 500 cc pots (Garibaldi et al. 2004). Fifteen-day-old plants of each cv. were inoculated either by irrigation with spores and 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, cv., 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 to the control plants. Control plants remained asymptomatic. The fungus was 100% re-isolated and identification confirmed as described (morphologically and 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 was first described in Italy (Garibaldi et al. 2002).