First report of Alternaria alternata on chili pepper (Capsicum frutescens) in Italy

Chili (Capsicum frutescens L.), belonging to the family Solanaceae, is widely cultivated in many countries as a vegetable. During summer of 2017, extensive necrosis was observed on leaves of 5-month-old plants grown outdoors in a garden in Biella province in Piedmont (northern Italy). In many cases, small light brown circular spots, becoming irregular, dark brown, surrounded by a chlorotic halo, mainly at the leaf margin were observed. Lesions progressively enlarged from 3 to 8 cm in diameter to encompass the entire leaf surface, and severely affected plants were partially defoliated. Disease incidence was between 40 and 50%. Small pieces of symptomatic leaf tissues were surface sterilized in 1% sodium hypochlorite for 1 min, rinsed in sterile water, and plated on potato dextrose agar (PDA) amended with 25 mg/liter of streptomycin sulfate. After 5 days at 22°C a fungus was consistently isolated. Spore suspensions of isolates 3-18 and 5-18 were distributed on PDA plates (10 μl/plate) to obtain single-spore colonies. Ten-day-old monoconidial cultures of both isolates grown on potato carrot agar medium produced brown septate and generally branched conidiophores. Conidia borne singly or in short chains were multicellular, obclavate to obpyriform, and 16.2 to 46.5 µm (average 30.6 µm) in length and 8.2 to 15.5 µm (average 11.7 µm) in width, with zero to three longitudinal and two to five transverse septa (n = 30). When present, the beak measured 1.2 to 14.3 µm (average 8.5 µm). The fungus was identified on the basis of its morphological characteristics as Alternaria sp. (Simmons 2007). DNA was extracted from isolates 3-18 and 5-18 by using the E.Z.N.A. Fungal DNA Mini Kit (Omega Bio-Tek, Darmstadt, Germany). The internal transcribed spacer (ITS) region of rDNA and the portion of the β-tubulin (tub2) gene of two isolates were amplified using the primers ITS1/ITS4 and T1/β-tub2 (O’Donnell and Cigelnik 1997; Peever et al. 2004; White et al. 1990). The amplicons were sequenced at the BMR Genomics Centre (Padova, Italy). A BLASTn analysis of the sequences showed a 100% identity to the rDNA ITS region of Alternaria alternata (GenBank accession nos. MF167293 and MH879772) and to the tub2 region of A. alternata (HQ413316). The ITS and tub2 sequences were deposited, respectively, to GenBank under accession numbers MH920250 and MH926018 (for 3-18), and MH920251 and MH926019 (for 5-18). Pathogenicity tests were performed by spraying leaves of 2-month-old healthy potted C. frutescens plants with a 105 CFU/ml spore suspension of both isolates. Noninoculated plants sprayed with sterile water served as controls. Ten plants per treatment were used. Plants were covered with plastic bags for 5 days after inoculation and maintained at 24 to 28°C in a greenhouse. The pathogenicity test was repeated. The first lesions developed on leaves 10 days after inoculation, whereas control plants remained nonsymptomatic. Alternaria, morphologically identical to the original isolates, was consistently reisolated from inoculated plants. The presence of A. alternata on C. frutescens was reported in California and China (Farr and Rossman 2018). To our knowledge, this is the first report of the fungus infecting C. frutescens plants in Italy.