First report of Alternaria alternata causing leaf spot on Digitalis purpurea in Italy. A. Garibaldi, G. Gilardi, and S. Matic, Centre of Competence for Innovation in the Agro-Environmental Sector (AGROINNOVA), Largo Paolo Braccini 2, 10095 Grugliasco, Italy; and M. L. Gullino, AGROINNOVA and DISAFA, Largo Paolo Braccini 2, 44, 10095 Grugliasco, Italy
Digitalis purpurea (family Scrophulariaceae) is a biennial species used in low maintenance gardens. In summer 2018, extensive necrosis symptoms were observed on leaves of 5-month-old plants of D. purpurea plants in a private garden near Biella (northern Italy). The initial symptoms appeared as small light brown, circular spots on the leaves, subsequently becoming irregular, expanded to dark brown lesions surrounded by a chlorotic halo. Lesions progressively enlarged in diameter and covered entire leaf surface. The disease incidence interested 30 to 50% out of 100 plants grown in the garden at temperatures between 18 to 25°C. 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/L of streptomycin sulphate, 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 non-branched 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. On the basis of this morphological characteristics the fungus was identified as Alternaria sp. (Simmons 2007). Genomic DNA was isolated from a pure culture of a single isolate (18) with the the E.Z.N.A.® Fungal DNA Mini Kit (Omega Bio-Tek, Darmstadt, Germany). The internal transcribed spacer (ITS) region of rDNA of this isolate was amplified by using the primers ITS1/ITS4 (White et al., 1990). The PCR product was sequenced at the BMR Genomics Centre (Padova, Italy) and obtained the 502-bp sequence (GenBank accession no. MK185236). A BLASTn search of the sequence showed 100% identity with Alternaria alternata, A. tenuissima, A. brassicae (GenBank accession numbers MG208004, MG012286 and MG250601, respectively) and other Alternaria species. Therefore, the β-tubulin (tub2) gene portion of the isolate 18 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 number MK193863) showed 100% identity with the reference sequence KU512287 of Alternaria alternata (Fr. : Fr.) Keissl 1912 isolated from Campanula medium. To confirm Koch’s postulates, pathogenicity tests were performed by spraying leaves of 1-month–old healthy potted D. purpurea plants, with 5 ml of a spore suspension of the isolate at 105 conidia/ml. Non inoculated plants were sprayed with sterile
water as controls. Three plants were used to be inoculated and three plants were control in each test. Plants were covered with plastic bags for 5 days after inoculation and maintained 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 non-symptomatic. From observed lesions, Alternaria sp. was consistently reisolated showing identical morphological characteristics as the original used for inoculation. The presence of Alternaria photistica on D. purpurea was reported in UK (Simmons 2007). To our knowledge, this is the first report of Alternaria alternata infecting D. purpurea plants in Italy as well as worldwide (Farr and Rossman, 2018).
References:
Farr, D. F., and Rossman A.Y. 2018. Fungal Databases, U.S. National Fungus Collections, ARS, USDA. Retrieved September 18, from https://nt.ars-grin.gov/fungaldatabases.
O’Donnell, K. and Cigelnik, E. 1997. Mol Phyl Evol 7:103–116. Peever T.L. et al. 2004. Mycologia 96:119-134.
Simmons, E. G. 2007. Alternaria: An Identification Manual. CBS Fungal Biodiversity Centre, Utrecht, Netherlands.
White, T. J. et al. 1990. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA.
