First report of stem rot caused by a Pythium cluster B2a species on lettuce in Italy

In July 2014, symptoms of crown and root rot were observed on lettuce (Lactuca sativa) grown in a commercial field near Fidenza (44°52′0″ N, 10°3′0″ E), Emilia-Romagna, Italy. The first symptoms developed 20 to 25 days after transplanting on 30 to 40% of the plants, when daily temperatures ranged from a minimum of 22 to a maximum of 30°C. Affected plants were stunted, showing lower leaf chlorosis, softening of the base of the stem, and root necrosis, and eventually collapsed. Tissue fragments of 1 mm2 were excised from the root lesions of 10 symptomatic plants, dipped in a solution containing 1% sodium hypochlorite for 30 s, rinsed in sterile water, and plated on potato dextrose agar (PDA) and an oomycete selective medium BNPRA (Masago et al. 1977). Plates were incubated under constant fluorescent light at 22 ± 1°C for 5 days. Pythium sp. was consistently isolated from diseased root tissue obtaining at least one isolate from each of the 10 plants. All isolates showed a similar growth rate/day of 11.7 ± 0.2 mm on potato carrot agar (PCA) at 24°C. Py11-14 and Py12-14, selected as representative isolates, produced on PCA filamentous and terminal sporangia. Oogonia were 14.5 to 26.8 × 15.7 to 24.8 µm, subglobose, and terminal. Oospores were 11.2 to 22.7 µm and aplerotic. Antheridia were monoclinous, 1 to 4 per oogonia (Spencer 2005). The internal transcribed spacer (ITS) region of rDNA of both isolates was amplified using primers ITS1/ITS4. BLAST analysis of the 845 bp (Py11-14) and 849 bp (Py12-14) fragments showed a 99% identity to the rDNA ITS region of Pythium dissotocum strain CBS 166.68 (GenBank accession no. AY598634). However, P. dissotocum belongs to a large species complex, known as Pythium Cluster B2a, which also includes P. coloratum, P. diclinum, P. cf. dictyosporum, P. lutarium, P. sp. ‘Group F,’ and P. sp. tumidum, which are all indistinguishable based on morphology and ITS sequence data (Robideau et al. 2011). Isolates Py11-14 and Py12-14 were thus identified as a Pythium Cluster B2a sp. The sequences of Py11-14 and Py12-14 were deposited in GenBank under accession numbers KY495893 and KY495894, respectively. ITS sequence analysis of eight additional isolates confirmed that all isolates belong to Pythium Cluster B2a. Pathogenicity tests were conducted on lettuce cv. Elisa using 10 to 15 seeds/pot sown in five pots containing a steam-disinfested organic peat substrate (70% black peat, 30% white peat, pH 5.5 to 6, N 110 to 190 mg/liter, P2O5 140 to 230 mg/liter, K2O 170 to 280 mg/liter), infested with wheat and hemp kernels colonized with the strains of Pythium at a rate of 1 g/liter (Garibaldi et al. 2015). The same number of seeds were sown in uninfested substrate. Plants were kept in a greenhouse at 22 to 26°C, and 20 days after inoculation the inoculated plants showed reduced growth, yellow leaves, and root rot and necrotic lesions on the base of stems. At least 28 to 43% of the plants died. All the noninoculated plants remained healthy. The pathogenicity test was carried out twice. A Pythium Cluster B2A sp. was reisolated from inoculated plants based on morphological and ITS sequence data. To our knowledge, this is the first report of the presence of a Pythium Cluster B2a sp. on lettuce in Italy. The importance of the disease, although at present limited, could increase in areas where lettuce is intensively grown.