First report of Alternaria alternata causing leaf spot on Digitalis purpurea in Italy

Digitalis purpurea (family Scrophulariaceae) is a biennial species used in low maintenance gardens. In summer 2018, extensive leaf necrosis infected 30 to 50% out of 100 D. purpurea plants grown in a private garden near Biella (northern Italy) at temperatures between 18 and 25°C. The initial symptoms appeared as small, light brown, circular spots on the leaves, subsequently expanding to irregular dark brown lesions surrounded by a chlorotic halo. Lesions progressively enlarged in diameter and covered the entire leaf surface. Small fragments (1 to 2 mm) of symptomatic leaf tissues were surface sterilized in 1% sodium hypochlorite for 1 min, rinsed in sterile water, plated on potato dextrose agar (PDA) amended with 25 mg/liter of streptomycin sulfate, and incubated at 22°C. A single fungus was isolated with 90% frequency after 4 days. The colonies of a 10-day-old monoconidial culture of the representative isolate 18 plated on potato carrot agar (PCA) were gray to dark blackish and showed aerial mycelium with concentric rings. Multicellular, obclavate to obpyriform, brown conidia originated from nonbranched conidiophores, singly or in chains (8 to 11 conidia), measuring 10.3 to 41.0 µm (average 25.7 µm, n = 40) in length and 4.2 to 12.5 µm (average 9.4 µm, n = 40) in width, with 0 to 2 longitudinal and 1 to 5 transverse septa. When present, the beak measured 1.9 to 8.7 µm. The fungus was identified as Alternaria sp. (Simmons 2007). Genomic DNA was isolated from isolate 18 and the internal transcribed spacer (ITS) region of rDNA was amplified by using the primers ITS1/ITS4 (White et al. 1990). The 502-bp PCR product was sequenced at the BMR Genomics Centre (Padova, Italy) and submitted at GenBank as accession no. MK185236. A BLASTn search of the sequence showed 100% identity with Alternaria alternata (GenBank accession no. MG208004) and other Alternaria species. Therefore, the β-tubulin (tub2) gene was also amplified by using the primers T1/β-tub-2 (O’Donnell and Cigelnik 1997; Peever et al. 2004). A BLASTn search of the 975-bp sequence (GenBank accession no. MK193863) showed 100% identity with sequence KU512287 of A. alternata (Fr.: Fr.) Keissl 1912. Pathogenicity tests were performed by spraying leaves of 1-month-old healthy potted D. purpurea plants, with 5 ml at 105 conidia/ml. Noninoculated plants were sprayed with sterile water. In each test, six plants were inoculated and three control plants were sprayed with sterile water. Plants were covered with plastic bags for 5 days after inoculation at 20 to 26°C in a greenhouse. The pathogenicity test was repeated once. The first Alternaria lesions were observed on leaves 10 days after inoculation, while control plants remained nonsymptomatic. From observed lesions, Alternaria sp. was consistently reisolated showing identical morphological characteristics as the original used for inoculation. The presence of A. photistica on D. purpurea was reported in the U.K. (Simmons 2007). To our knowledge, this is the first report of A. alternata infecting D. purpurea plants in Italy as well as worldwide.