First Report of Phoma herbarum Causing Leaf Spot of Purple Coneflower (Echinacea purpurea) in Northern Italy

Purple coneflower (Echinacea purpurea L.), belonging to the Asteraceae family, is increasingly grown as an ornamental plant in private and public gardens and also as a medicinal herb and for the cut-flower market. From June to October 2018, symptoms of an unknown leaf spot were observed on 6-month-old plants, grown in a private garden located near Biella 45°36′00″N 8°03′00″E (northern Italy) at temperatures between 20 and 28°C. Symptoms appeared typically on older leaves of 10 to 15% of the plants grown in the garden. Symptoms began as circular brown leaf spots (1 to 2 mm in diameter) that gradually enlarged to 30 to 40 mm in diameter, changing from circular to elliptical or irregular. Lesions were generally surrounded by a red halo, and scattered black specks appeared in the center of the spots. No pycnidia were observed on lesions. Isolations were made from the margins of the necrotic lesions that developed on E. purpurea, placing surface-sterilized tissue pieces on potato dextrose agar (PDA) amended with 25 mg/liter of streptomycin sulfate. One fungus developed with 60% frequency. A 15-day-old culture of the isolate 43-2 grown on PDA showed black pycnidia, 56 to 167 μm diameter, with masses of hyaline, elliptical conidia measuring 3.2 to 6.5 (average 4.2) and 1.2 to 1.7 (average 1.3) μm. On the basis of its morphological characteristics, the fungus was identified as Phoma sp. (Boerema et al. 2004). Genomic DNA from isolate 43-2 was obtained using an E.Z.N.A. Fungal DNA Mini Kit (Omega Bio-Tek, Darmstadt, Germany). The polymerase chain reaction (PCR) was performed using primers ITS1/ITS4 to amplify the internal transcribed spacer, the intergenic region between 28S and 18S sequences of the ribosomal RNA, including the 5.8S sequence. The PCR product was purified and sent for sequencing to BMR Genomics (Padova, Italy). BLASTn analysis (Altschul et al. 1997) of the 507-bp sequence showed 100% identity with the corresponding sequence in GenBank for Phoma herbarum (GenBank accession no. JQ936277). The sequence has been deposited in GenBank with accession number MK396763. A pathogenicity test was carried out twice by spraying a suspension of 10⁶ conidia/ml of isolate 43-2 on leaves of 60-day-old E. purpurea plants, which were then maintained at up to 90% relative humidity for 5 days. Plants (four per treatment) were kept in a glasshouse at temperatures from 19 to 22°C. Lesions similar to those seen in the garden with a typical red halo developed on inoculated older leaves beginning 11 days after inoculation, whereas control plants remained healthy. The same fungus was consistently reisolated from the lesions in both tests (GenBank accession no. MK396764). To our knowledge this is the first report of P. herbarum on E. purpurea in Italy as well as worldwide (Farr and Rossman 2019).