First Report of Alternaria petroselini Causing Leaf Blight of Fennel in Spain

Fennel (Foeniculum vulgare Mill.) is an aromatic herb widely cultivated in Mediterranean areas for culinary and medicinal uses. In 2010, symptoms consisting of leaf blight and necrosis were observed in commercial organic fennel production areas in Valencia Province in east-central Spain. Disease incidence in affected fields was approximately 20%. Symptomatic leaves from four fields were surface disinfected with 0.5% NaOCl for 2 min, and small fragments from necrotic lesions were then plated on potato dextrose agar (PDA) amended with 0.5 g of streptomycin sulfate/liter. After 7 days at 25°C, isolates of the genus Alternaria were consistently isolated. Single conidium cultures were grown on PDA and V8 agar for morphological examination. On both agar media, colonies were dark olive brown without production of pigments. On V8 agar, conidia were solitary, darkly pigmented, and predominantly ovoid-subsphaeroid. Mature conidia were 25 to 59 × 12 to 23 μm with up to six to seven transepta and one to three longisepta. The 5.8S, ITS2, and 28S ribosomal RNA (rRNA) regions were amplified with the primers ITS3 and ITS4 (3) from DNA extracted from the isolate IVIA-A029, and sequenced (GenBank Accession No. JQ240204). The sequence had 100% identity (total score 399, 97% coverage) with that of Alternaria petroselini (Neergard) Simmons strain EGS 09-159 (GenBank Accession No. AF229454.1) (1). Pathogenicity tests were conducted on four 3-month-old fennel plants (cv. Giotto) by spraying a conidial suspension of the fungus (10 ml/plant, 10³ conidia/ml of water). Four control plants were sprayed with sterile, distilled water. Plants were covered with plastic bags and incubated in a growth chamber for 72 h at 25°C. Leaf necrosis was visible on inoculated plants after 4 days, but symptoms were not observed on control plants. The fungus was reisolated from leaf lesions on inoculated plants, but not from leaves of control plants, confirming Koch's postulates. On the basis of the morphological (2), molecular, and pathogenicity data, the disease was identified as Alternaria leaf blight of fennel caused by A. petroselini. To our knowledge, this is the first report of A. petroselini in Spain. References: (1) B. M. Pryor and R. L. Gilbertson. Mycol. Res. 104:1312, 2000. (2) E. G. Simmons. Alternaria: An Identification Manual. CBS Fungal Biodiversity Centre, Utrecht, The Netherlands, 2007. (3) T. J. White et al. Pages 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA, 1990.