First report of Colletotrichum spinaciae associated with anthracnose on spinach in Southern Italy

Abstract

Spinacia oleracea L. is an important leafy vegetable crop in Italy, with the Apulia region (Capitanata area) cropping about 800 ha with an annual production of 15.339 tons. During the 2024–2025 growing season, anthracnose-like symptoms were observed on spinach cv. ‘Kangaroo’ in a field in the Capitanata area (41.3501° N, 15.6025° E). Disease incidence ranged 23 to 36%, based on visual assessment of 200 random plants. Initial symptoms on leaves consisted of small circular water-soaked lesions surrounded by a chlorotic halo. Lesions enlarged, becoming tan to dark brown and coalescing into larger necrotic areas with thin papery tissue; acervuli bearing dark setae were visible on mature lesions. Symptomatic leaves were surface sterilized (70% ethanol 20 s, 20% NaOCl 20 s, 70% ethanol 20 s), rinsed three times in sterile distilled water and dried on sterile filter paper. Leaf pieces (0.5–0.8 cm diameter) were placed on potato dextrose agar (PDA; 39 g L⁻¹, Oxoid) amended with streptomycin sulphate (500 ppm). Plates were incubated at 23 ± 2°C in the dark. After 14 days, colonies resembling Colletotrichum sp. were transferred to fresh PDA. Hyphal tips from the colonies, were spread on water agar (20 g L-1). After 24-36 h, hyphal tips were transferred to fresh PDA plates for 7-10 days to obtain pure cultures. Colonies showed irregular margins with dense mycelium, dark olivaceous-grey at the center and cream toward the periphery. Microscopic observation revealed hyaline, aseptate, falcate conidia (16.01–19.7 × 2.9–3.8 µm) produced on conidiophores within acervuli on oatmeal agar (OA; 72.5 g L⁻¹, Fluka). Genomic DNA was extracted from a representative colony (strain SPIN2) following Carlucci et al. (2013). The internal transcribed spacer (ITS) region and partial fragments of actin (act), beta-tubulin (tub2), chitin synthase (chs-1), glyceraldehyde-3-phosphate dehydrogenase (gapdh), and histone H3 (his3) genes were amplified using primers ITS1/ITS4, ACT-512F/ACT-783R, T1/Bt2b, CHS-79F/CHS-354R, GDF1/GDR1, and CYLH3F/CYLH3R, respectively (Damm et al. 2009). Sequences of strain SPIN2 (GenBank acc. nos. PZ091375 (ITS); PZ118972 (act); PZ118971 (tub2); PZ118973 (chs-1); PZ118974 (gapdh); PZ118975 (his3) showed 100% identity with Colletotrichum spinaciae strain CBS 128.57. Maximum parsimony analysis of combined sequences, including reference taxa from the C. dematium species complex (Damm et al. 2009; Liu et al. 2022), confirmed the identification of SPIN2 as C. spinaciae. Pathogenicity was assessed on ten healthy 30-day-old spinach seedlings (cv. ‘Kangaroo’) grown in pots (soil:sand:peat 2:1:1). Plants were sprayed with a suspension of propagules (4 × 10⁵ mL⁻¹) of the SPIN2 strain (collected on OA plates for 14 d) until runoff; ten control plants received sterile distilled water. Plants were maintained in a greenhouse at 25 ± 3°C, 70–80% relative humidity. The experiment was conducted three times. Twelve days after inoculation, dark brown to black circular lesions developed on inoculated plants, whereas control plants remained symptomless. Colletotrichum spinaciae was consistently re-isolated from symptomatic tissues (95%), fulfilling Koch’s postulates. Colletotrichum spinaciae has been previously reported on spinach in Canada (Cerkauskas et al. 1991), North America (Correll et al. 1994), Australia (Washington et al. 2006), and Turkey (Kurt et al. 2016). To our knowledge, this is the first report of C. spinaciae causing anthracnose on S. oleracea in Italy, and can thus form the basis for improved disease scouting and management in spinach crops.