First Report of Cercospora cf. flagellaris on Soybean (Glycine max L.) in Italy

Soybean (Glycine max L.) is widely cultivated in northeastern Italy, especially in Friuli-Venezia Giulia and Veneto. Since 2013, during early seedling development, necrotic lesions have appeared on upper leaves, spreading downward and causing up to 25% defoliation in the field. Purplish discoloration was observed on petioles, stems, and pods. Seeds showed pale to dark purple blotches on the seed coat, from small spots to full coverage. These symptoms are typical of Cercospora leaf blight (CLB) and Purple Seed Stain (PSS). In 2018-2019, symptomatic tissues were analyzed. Small fragments of infected stems and pods from five different fields (ten plants/site), were surface-sterilized in 1% sodium hypochlorite for 10 min, rinsed with sterile distilled water, and plated onto Potato Dextrose Agar (BD Difco™) with streptomycin sulphate (300 mg/L). Plates were incubated in the dark at 25°C for 7 days. Seeds were disinfected similarly, but for 1 minute. Single-spore cultures produced deep purple mycelium on the lower surface and grey on the upper. Stromata were absent or poorly developed. Conidiophores were straight, pale to dark brown, 120–180 × 3.0–5.5 µm; conidia were solitary, hyaline, slightly curved, and measured 40–250 × 2.5–4.5 µm (n=25). Eight representative isolates from Friuli-Venezia Giulia, three from pods (CREA-CI24, CREA-CI25, CREA-CI26) and five from seeds (C-N7, C-170-1, C-203-4, C-203-5, C-212), underwent multilocus molecular analysis (Groenewald et al., 2012). DNA was extracted and internal transcribed spacer region (ITS), calmodulin (CAL), and histone H3 (HIS3) were amplified (White et al., 1990; Carbone and Kohn, 1999; Crous et al., 2004) and Sanger sequenced. Sequences were deposited in GenBank (ITS: MK989496–98, PX092092–96; CAL: MK991296–98, PX091946–50; HIS3: MK991293–95, PX091951–55). MegaBLAST analysis showed 99% to 100% identity with reference sequences of C. cf. flagellaris (ITS: JX143614, CAL: JX142867, HIS3: JX142621). Phylogenetic analysis using concatenated sequences and 63 references confirmed clustering within C. cf. flagellaris. Pathogenicity tests (Cai & Schneider, 2005) were conducted with strains CI24, CI25, and CI26, in triplicate. Sterile soybean leaves on living plants were sprayed with conidial suspension (10⁴ conidia/mL) and incubated at 25°C under moist conditions. After 8–10 days, necrosis and blight symptoms developed as in the field. No symptoms were seen in controls. Reisolation from symptomatic inoculated leaves confirmed C. cf. flagellaris as the causal agent, based on morphology and ITS sequences. The symptoms observed in this study align with Cercospora-related diseases (CLB and PSS). Previously attributed to C. kikuchii, recent studies (Soares et al., 2015; Albu et al., 2016) showed that C. cf. flagellaris and C. cf. sigesbeckia are also involved. Fernandes et al. (2025) reported that C. cf. flagellaris causes milder symptoms on young soybean leaves than other Cercospora spp., consistent with our observations. To our knowledge, this is the first report of C. cf. flagellaris on soybean in Italy. Soybean covers about 300,000 ha in the country, mainly in northeastern valleys, and is a key food and feed crop. This finding expands known Cercospora diversity associated with CLB and PSS in Italy and highlights the need for further studies on pathogen distribution, epidemiology, cultivar susceptibility, and management strategies to sustain soybean production.