First Report of Leaf Spot of Spinach Caused by Pleospora betae in Spain

The production of spinach (Spinacia oleracea L.) in Spain has increased 50% since 2009, mainly due to the commercialization of fresh-cut spinach leaves packaged in modified atmosphere containers. In October 2012, light brown leaf spots 1 to 2 cm in diameter with dark concentric rings were observed in a commercial spinach production area in Valencia Province, Spain. The initial outbreak comprised an area of about 3 ha with a 20% disease incidence. Symptomatic leaves from spinach cv. Apollo were collected in the affected area and were surface disinfected with 0.5% NaOCl for 2 min. Small fragments from lesions were placed onto potato dextrose agar (PDA) amended with 0.5 g streptomycin sulfate/liter. Fungal colonies developed after 3 days of incubation at 23°C from about 90% of the infected tissues plated. Isolates were transferred to oatmeal agar (OA) (1) and water agar (WA) amended with autoclaved pea seeds (2). Plates were incubated for 30 days at 24°C with 13 h of fluorescent light and 11 h of dark for morphological examination. Colonies were olivaceous grey in OA and pycnidia developed in WA were globose to subglobose, olivaceous black, and 100 to 200 μm in diameter. Conidia were globose to ellipsoidal, hyaline, aseptate, and 3.8 to 7.7 × 2.4 to 3.9 μm. Swollen cells were observed. Isolates showed a positive reaction to NaOH (1). Partial 18S, ITS1, 5.8S, ITS2, and partial 28S ribosomal RNA (rRNA) regions were amplified using the primers ITS1 and ITS4 (4) and sequenced from DNA extracted from the isolate designated as IVIA-V004 (GenBank Accession No. KF321782). The sequence had 100% identity (e-value 0.0) with that of Pleospora betae (Berl.) Nevod. (syn. Phoma betae A.B. Frank) representative strain CBS 523.66 (1). Pathogenicity tests were performed twice by inoculating 4-month-old plants of spinach cv. Apollo, table beet (Beta vulgaris L.) cv. Detroit, and Swiss chard (B. vulgaris subsp. cicla) cv. Verde de Penca Blanca. Plants were inoculated by spraying a conidial suspension of isolate IVIA-V004 (10 ml/plant, 10⁵ conidia/ml water) using a manual pressure sprayer. Plants were immediately covered with black plastic bags and incubated in a growth chamber at 23°C. In each experiment, four plants of each host were inoculated with the fungus and four additional plants sprayed with sterile distilled water were used as controls. Plastic bags were removed after 48 h and leaf spots similar to those observed in affected spinach plants in the field were visible on all spinach, table beets, and Swiss chard plants 3 to 5 days after inoculation. No symptoms were observed on control plants. Fungal colonies morphologically identified as P. betae were re-isolated from leaf lesions on inoculated plants, but not from asymptomatic leaves of control plants. To our knowledge, this is the first report of leaf spot caused by P. betae on spinach in Spain, where it was previously described affecting sugar beet (3). The disease reduces the quality of spinach leaves and proper control measures should be implemented. References: (1) G. H. Boerema et al. Phoma Identification Manual, Differentiation of Specific and Infra-Specific Taxa in Culture. CABI Publishing, Wallingford, UK, 2004. (2) O. D. Dhingra and J. B. Sinclair. Basic Plant Pathology Methods, 2nd ed. CRC Press, Boca Raton, FL, 1995. (3) P. Melgarejo et al. Patógenos de Plantas Descritos en España. MARM-SEF, Madrid, 2010. (4) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA, 1990.