LAUSR

Clematis L. is one of the largest genera of Ranunculaceae, accommodating over 300 plant species (Wang & Li 2005). They are mostly flowering creepers commonly grown as ornamentals. Clematis leaf spot and wilt is a fungal disease caused by Calophoma clematidina (Thüm.) Q. Chen & L. Cai. Infected plants initially show irregular brown to black leaf spots which later turn into large necroses, usually leading to wilt disease. In June 2021, Clematis plants displaying symptoms described above were observed in three independent nurseries located in three counties (Brno-venkov, Břeclav and Nymburk) in the Czech Republic. Around 60% of 120 inspected plants were symptomatic, including both mother plants and young plants. Leaves, stems and roots of 43 diseased plants originating from the three nurseries were collected, sectioned into small pieces (5 × 5 mm), surface sterilized (60 sec in 75% ethanol, followed by 60 sec in 1% sodium hypochlorite and rinsed three times using sterile distilled water), plated on potato dextrose agar (PDA) and incubated at 25°C for 5 weeks. Newly developed mycelia were immediately transferred to a fresh PDA plates and purified by single-spore isolation. A total of 21 strains morphologically resembled the genus Calophoma. Colonies on PDA (7 d at 25°C) were felty, white to olivaceous/iron grey in the centre. Conidiomata were dark brown, pycnidial, solitary or in groups, (117-220 × 65-170 μm). Conidia were cylindrical to ellipsoidal, hyaline, 0(-1)-septate, (4-8 × 2-3 μm). Two representative isolates were deposited at the Westerdijk Fungal Biodiversity Institute, Utrecht, the Netherlands (CBS 149230 and CBS 149231). The partial internal transcribed spacer (ITS) regions, large ribosomal subunit of the nrRNA gene (LSU), beta-tubulin gene (tub2) and RNA polymerase II second largest subunit gene (rpb2) were amplified from genomic DNA of both isolates following protocols previously described (Spetik et al. 2022). Sequences were deposited in NCBI GenBank (accession nos. ITS: ON107539, ON107540; LSU: ON108575, ON108576; tub2: ON314832, ON314833; rpb2: ON125007, ON125008), being 100% identical with that of the ex-type strain of C. clematidina (CBS 108.79), ITS (NR_135964), LSU (FJ515632), tub2 (FJ427100), and rpb2 (KT389588). Phylogenetically, the two representative isolates formed a fully supported clade with sequences of the ex-type and another culture of C. clematidina in the multigene phylogeny. To confirm Koch's postulates, leaves of ten two-month-old Clematis plants grown in pots were wounded by a needle and inoculated with a conidial suspension (1.0 × 106 conidia ml-1) of both strains (five plants per strain) following Golazar et al. (2011). Ten plants were mock-inoculated with sterile distilled water and served as controls. Within one month, inoculated plants exhibited dark necrotic leaf spots similar to the symptoms observed in the nurseries, while controls remained symptomless. Calophoma clematidina was reisolated from the inoculated plants, and its identity confirmed (ITS, GenBank OP363927). The experiment was repeated. Although known from Europe, this is the first report of Clematis leaf spot and wilt caused by C. clematidina in the Czech Republic. Clematis leaf spot and wilt represents a serious disease in Czech nurseries, with the pathogen present in leaves, stems and roots of Clematis spp.