First Report of Necrotic Leaf Spot Caused by Plectosphaerella cucumerina on Lamb's Lettuce in Southern Italy.

During spring 2013, lamb's lettuce plants (Valerianella locusta) cv. Calarasi (Rijk Zwaan) in a commercial greenhouse in Sele Valley (Salerno Province, southern Italy) exhibited small, black-brown, irregular spots (1 mm²) that became necrotic, enlarged, and coalesced. The spots were mostly on the tips of leaves, and were surrounded by a yellow halo. The disease was severe under greenhouse conditions of 60 to 90% RH and maximum air temperature of 26°C, and affected up to 70% of the plants. The greenhouses covered an area of ~3,000 ha where many salad species are grown. Tissue fragments were excised from symptomatic leaves, sterilized by sequential dipping in 70% ethanol for 30 s and in 1% NaOCl for 30 s, rinsed in sterilized distilled water, and placed in 9-cm-diameter petri dishes containing potato dextrose agar (PDA) medium amended with streptomycin sulfate (0.1 g/liter). The plates were incubated at 24°C in the dark. A fungus was isolated consistently from infected leaf tissue after 4 days. Each colony was whitish to orange. Mycelium was hyaline, branched, septate, 3 to 4 μm wide, with numerous anastomosis-forming hyphal coils. Conidiophores were solitary, hyaline, smooth, thin-walled, unbranched or rarely irregularly branched. Conidiogenous cells were phialidic, determinate, discrete, smooth, solitary, and formed on hyphal coils. Phialides were aseptate or occasionally 1-septate near the base. Conidia (n = 100) were ellipsoidal, hyaline, smooth, septate or aseptate, and 6.6 ± 0.9 × 2.8 ± 0.4 μm. On the basis of morphological criteria (3), the fungus was ascribed to Plectosphaerella cucumerina (L.) Laterr. (anamorph Plectosporium tabacinum). An aliquot (50 ng) of genomic DNA extracted from mycelium of five cultures obtained by monosporic isolation on PDA was used as template for a PCR reaction with primers ITS5/ITS4, specific for the ITS 5.8S rDNA region of fungi (3). The 500-bp sequences amplified from the five isolates were identical, and the sequence of isolate Val-2 was submitted to GenBank (KF753234). Sequence analysis with BLASTn showed 100% identity of this sequence to the ITS-5.8S rDNA sequences of 11 isolates of P. cucumerina in GenBank. Three isolates were selected for pathogenicity tests on the lamb's lettuce cv. Calarasi. Before planting, seeds were surface-disinfected in 1% NaOCl and rinsed with sterilized distilled water. Plants (35 days old, 30 plants tested/isolate) were grown in 0.7-liter pots filled with a sterilized (autoclaved at 112°C for 1 h on each of two consecutive days) mixture of soil:sand:perlite (2:1:1), and inoculated by spraying the leaves with a spore suspension (10⁶ CFU/ml, ~3 ml applied/plant) of each isolate prepared from 7-day-old cultures on PDA. As a control, five plants were sprayed with sterilized water. All plants were incubated in a growth chamber at 90% RH with a 12-h photoperiod at 24°C. Leaf spots typical of those on the original symptomatic plants appeared 7 to 10 days after inoculation on all inoculated plants. No symptoms were observed on control plants. P. cucumerina was re-isolated only from symptomatic leaves, as described for the original isolations. P. cucumerina has been associated with root and collar roots of some horticultural crops (1), and a leaf spot on Diplotaxis tenuifolia (2), often grown in rotation with lamb's lettuce in southern Italy. To our knowledge, this is the first report of P. cucumerina as a pathogen of V. locusta in Italy or elsewhere. The disease caused economic loss to lamb's lettuce, primarily used in Italy in fresh-cut, mixed salads. References: (1) A. Carlucci et al. Persoonia 28:34, 2012. (2) A. Garibaldi et al. Plant Dis. 96:1825, 2012. (3) T. J. White et al. PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA, 1990.