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Fungal trunk diseases (FTDs) have been a significant threat to the global stone fruit industry. FTDs are caused by a consortium of wood-decaying fungi. These fungi colonize woody tissues, causing cankers, dieback, and other decline-related symptoms in host plants. In this study, a detailed screening of the fungal microbiota associated with the decline of stone fruit trees in the Czech Republic was performed. The wood fragments of plum and apricot trees showing symptoms of FTDs were subjected to fungal isolation. The partial internal transcribed spacer region, partial beta-tubulin, and translation elongation factor 1-α genes were amplified from genomic DNA extracted from fungal cultures. All isolates were classified, and the taxonomic placement of pathogenic strains was illustrated in phylogenetic trees. The most abundant pathogenic genus was Dactylonectria (31%), followed by Biscogniauxia (13%), Thelonectria (10%), Eutypa (9%), Dothiorella (7%), Diplodia (6%), and Diaporthe (6%). The most frequent endophytic genus was Aposphaeria (17%). The pathogenicity of six fungal species ( Cadophora daguensis , Collophorina africana , Cytospora sorbicola , Dothiorella sarmentorum , Eutypa lata , and E. petrakii var. petrakii ) to four Prunus spp. was evaluated, and Koch's postulates were fulfilled. All tested isolates caused lesions on at least one Prunus sp. The most aggressive species was E. lata , which caused the largest lesions on all four tested Prunus spp., followed by E. petrakii var. petrakii and D. sarmentorum . Japanese plum ( Prunus salicina ) and almond ( P. amygdalus ) were the most susceptible hosts, while apricot ( P. armeniaca ) was the least susceptible host in the pathogenicity trial., Competing Interests: The author(s) declare no conflict of interest.

Neofusicoccum parvum (Pennycook & Samuels) Crous, Slippers & A.J.L. Phillips is a cosmopolitan pathogen causing dieback of multiple diverse woody hosts including highbush blueberry (Vaccinium corymbosum L.). This fungus can survive inside colonized plants without causing any symptoms for several years. Once the endophytic lifestyle is switched to the parasitic one, the symptoms of dieback can rapidly occur (bronze leaves, necroses under the bark, apoplexy) and the plant usually declines within a few weeks (Slipper and Wingfield 2007). In August 2022, blueberry plants displaying symptoms described above were observed in a production orchard located in Hovorany, the Czech Republic. Around 3 % of 1000 observed plants were symptomatic. In order to identify the pathogen, leaves, stems and roots of three diseased plants were collected, sectioned into small pieces (5 × 5 mm), surface sterilized (60 s in 75% ethanol, followed by 60 s in 1% sodium hypochlorite and rinsed three times using sterile distilled water), plated on potato dextrose agar (PDA) supplemented with 0.5 g/liter of streptomycin sulfate (PDAS) (Biosynth, Staad, Switzerland) and incubated at 25°C for 2 weeks at dark. Newly developed mycelia were immediately transferred to fresh PDA plates and purified by single-spore or hyphal-tip isolation. In total 33 fungal isolates were obtained. All the 33 isolates shared similar morphology and resembled Botryosphaeriaceae spp. Colonies on PDA (7 d at 25°C) were felty, white to iron grey in the centre. Conidiomata were observed on sterile pine needles on 2 % water agar (WA) at 25°C under near-UV light after 2 wks (110-220 × 60-175 μm). Conidia (n=30) were cylindrical to ellipsoidal, hyaline, 0(-1)-septate, (3.8-8.1 × 2-3 μm). Two representative isolates were deposited at the Westerdijk Fungal Biodiversity Institute, Utrecht, the Netherlands (CBS 149846 and CBS 149847). The partial internal transcribed spacer (ITS) regions, beta-tubulin gene (tub2) and translation elongation factor 1-alpha (tef) gene were amplified from genomic DNA of both isolates following primers and protocols previously described (Eichmeier et al. 2020). Newly generated sequences were deposited in NCBI GenBank (acc. nos. ITS: OQ376566, OQ376567; tub2: OQ401701, OQ401702 and tef: OQ401699, OQ401700), being >99% identical (ITS 483/484 nt, tub2 426/430 nt and tef 230/232 nt) with the ex-type ITS (AY236943), tub2 (AY236888) and tef (AY236917) sequences of N. parvum strain CMW9081. Phylogenetically, newly obtained isolates grouped with ex-type and another three cultures of N. parvum in the three gene-based phylogenetic tree with strong 98/1.0 (BP/PP) support. To confirm pathogenicity, one-year-old canes of ten two-year-old V. corymbosum plants grown in pots were wounded by a 5 mm diam cork borer, and a 5-mm mycelial plug of a 7-day-old culture of both (CBS 149846 and CBS 149847) strains (five plants per strain) was inserted into the wound. Ten plants were inoculated with sterile PDA plugs and served as controls. Wounds were covered by sterile wet cotton, sealed with Parafilm® and inoculated plants were maintained in a growth chamber at 20 °C with 12/12 h light/dark period. Within two weeks, inoculated shoots changed colour from green to dark brown and exhibited dark necroses under the bark; after one month inoculated plants declined, while controls remained symptomless. The pathogen was reisolated from the inoculated plants with 100 % re-isolation rate, and its identity confirmed by sequencing ITS region. The experiment was repeated. Neofusicoccum parvum causing dieback of highbush blueberry was already reported from Australia, California, Chile, China, Italy, Mexico, Portugal and Uruguay (Rossman et al. 2023). Pecenka et al. (2021) reported a presence of another pathogen - Lasiodiplodia theobromae (Pat.) Griffon & Maubl. from the same plantation. This suggests that stem blight and dieback of highbush blueberry is caused by more than one Botryosphaeriaceae spp. as it was previously proved by Xu et al. (2015). To the best of our knowledge, this is the first report of stem blight and dieback of highbush blueberry caused by N. parvum in the Czech Republic.

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 × 10 6 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.