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|a First report of Stagonosporopsis pogostemonis causing leaf spot on cauliflower in Italy Wassim Habib1, Mariangela Carlucci2, Roberto Fasano3, Franco Nigro1,2 † 1 Centre of Research, Experimentation and Training in Agriculture (CRSFA) - Basile Caramia, via Cisternino 281, Locorotondo, 70010 Bari, Italy 2 Department of Soil, Plant and Food Sciences, University of Bari - Aldo Moro, via Amendola 165A, 70126 Bari, Italy 3 Bioricerche, Phytopathological Lab, via Pio XI 30, Bellizzi, 84092 Salerno, Italy † Corresponding author: F. Nigro. Email: franco.nigrouniba.it Keywords: Phoma-like, Multi-locus gene sequencing, Brassica oleracea L. var. botrytis, Campania. In September 2022, a severe foliar disease was observed on 2-3-week-old 'Corsaro' and 'Parthenon' seedlings of cauliflower (Brassica oleracea L. var. botrytis) grown under greenhouses in a nursery in Caserta province, Campania (Italy), with an incidence of 90% on about 150,000 plants. Affected plants showed numerous small, irregular, and depressed lesions bordered by a chlorotic halo on leaves that become necrotic, and later drop out giving a shot-hole effect (Fig. 1). Isolations were performed on leaf portions from 30 symptomatic samples which were surface-sterilised by dipping in 70 % ethanol for 30s, then in a sodium hypochlorite solution (1%) for 30s, and rinsed two times in distilled sterilised water. Tissue fragments were left to dry on sterile filter paper and then seeded on potato dextrose agar (PDA) amended with 0.5 g/L of streptomycin sulphate (Sigma-Aldrich S.R.L, Steinheim, Germany). After 6 to 8 days of incubation at 24 ± 1°C, colonies revealed a consistent growth of a Phoma-like fungus resembling Stagonosporopsis genus. On PDA, one-week-old colonies were dark olivaceous green to brown with white regular margin (Fig. 2B). Pycnidia (32-160 µm × 30-95 µm) were subglobose, and conidia (2.7-4.8 µm × 1-2.7 µm) ellipsoidal, with rounded ends, aseptate, with two polar guttules (Fig. 2C). All isolates had similar morphological features, therefore further analyses were performed on one representative strain (CRSFA.753.22). The multilocus phylogenetic approach using Maximum likelihood method and Tamura-Nei model on rDNA-ITS, TUB and RPB2 sequences (accession numbers: OQ318550, OQ326503, OQ326504) including reference strains of Stagonosporopsis (Dong et al. 2021) demonstrated that CRSFA.753.22 clustered with Stagonosporopsis pogostemonis Luo, Huang & Manawas holotype (ZHKUCC 21-0001) with 91% bootstrap support. ITS and RPB2 sequences had 100% BLAST match to ZHKUCC 21-0001, whereas TUB sequence had 99.69%. Pathogenicity tests were performed on 20 healthy 'Corsaro' plantlets. Leaves were surface sterilized with 70% alcohol and wounded with a sterile tip of a needle. A suspension of conidia and mycelial fragments, obtained from a 10-days-old colony of the strain CRSFA.753.22, was sprayed on the leaf surfaces at a distance of 20 cm. Five controls plants were inoculated with sterile distilled water. Plants were then covered with plastic bags and kept on the shelf in a growth chamber at 25° C, with a 12-h photoperiod. The first symptoms developed on leaves five days post-inoculation as irregular small brown spots, that were gradually expanded. Leaf tissues showed chlorosis which evolved into necrosis (Fig. 2A). Stagonosporopsis pogostemonis was consistently re-isolated from all diseased leaves, but not from control plants, thus fulfilling Koch's postulates. In Italy, Stagonosporopsis species have been reported as severe plant pathogens (Garibaldi et al. 2022; Guarnaccia et al. 2022), and this is the first report of the species S. pogostemonis in the country. References Dong, Z.Y., et al. 2021. Pathogens 10:1093. doi: 10.3390/pathogens10091093 Garibaldi, A., et al. 2022. Plant Pathol. J. 104:1157. doi: 10.1007/s42161-022-01138-7 Guarnaccia, V., et al. 2022. Plant Pathol. J. 104:1491. doi: 10.1007/s42161-022-01197-w
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