First Report of Gnomoniopsis fragrariae causing leaf spots on strawberry in Italy

Strawberry (Fragaria × ananassa Duch.) is widely cultivated in Italy. During May-June 2022, mild symptoms of an unknown leaf spot disease appeared on 5-10% of June-bearing strawberry (cv. Elodì) plants transplanted in July 2021 in a commercial farm located in the province of Cuneo, North Italy. During September-November 2022, the symptoms appeared also on 10-15% of the plants transplanted in July 2022. The disease was scattered throughout the field, large 600 m2, both on new and senescent leaves. Fungicides (sulphur, Tiovit Jet; penconazole, Topas 10 EC) were applied to the plants according to integrated pest management during the growing period. The disease symptoms were purplish to brown necrotic leaf spots up to 1-3 mm in diameter and chlorotic leaf margins. Black lesions were occasionally observed on the petioles, appearing as small necrotic or larger elongated lesions causing leaf death. Peritechia were observed in planta after about 4 months from sampling and measured (144 to 239 μm and 200 to 291 μm, n = 10). Diseased leaves and petioles from about 10 plants were surface disinfested for 1 min in 1% NaClO, rinsed with sterile water and plated on potato dextrose agar (PDA) amended with 25 mg streptomycin sulphate/liter. A fungus with white cottony colonies was repeatedly recovered and maintained in pure culture on PDA. Biguttulate conidia with rounded ends were measured (4.3 to 8.0 μm and 1.2 to 2.9 μm, average 6.1× 2.3 μm, n = 50) from 21-day old colonies grown in PDA at 22°C and 12 h photoperiod. According to colony and conidia morphology, the isolate was identified as Gnomoniopsis sp. (Walker et al., 2010). The fungal DNA was extracted from a pure culture of one isolate selected as a representative (code FR2-22), by using the E.Z.N.A. Fungal DNA Mini Kit (Omega Bio-Tek, Darmstadt, Germany). The identification was carried out by amplifying and sequencing the internal transcribed spacer (ITS) region and the partial translation elongation factor 1-α (TEF) gene using the primers ITS1/ITS4 and EF-728F/EF2 (Udayanga et al., 2021), respectively. The purified PCR products were sequenced at the BMR Genomics Centre (Padova, Italy) obtaining 551bp (ITS) and 652bp (TEF) sequences deposited in GenBank (Accession nos. OQ179950 and OQ190173, respectively). A BLASTn search of both sequences revealed to be 100% identical to the ITS and TEF loci of Gnomoniopsis fructicola sequences of the isolates VPRI_15547 and CBS 275.51 deposited in GenBank with accession Nos. MT378345 and MT383092. The pathogenicity of the isolate FR2-22 was assessed in two trials by biological tests (3 replicates with 1 plant per replicate/pot) in two greenhouse compartments, kept at temperature 20-24°C and at humidity 80-90%. Healthy leaves of forty-day-old strawberry plants (cv. Elodì) were sprayed with 1-5 x106 conidia/ml obtained from the FR2-22 isolate grown on PDA at 25°C for 20 days. The control (water-sprayed plants) was kept in the same conditions. Small leaf spots similar to the symptoms previously observed in the farm were observed 15 days post inoculation. Furthermore, 30 to 40% of leaves developed symptoms similar to those observed in the field after 25-40 days, while the control remained health. The same fungal isolate was repeatedly reisolated from the affected leaves and petioles and identified based on TEF sequencing. Gnomoniopsis fragariae comb. nov., designed as new name for Gnomoniopsis fructicola (Udayanga et al., 2021), has previously been reported on Fragaria × ananassa plants in Australia and in the USA (Farr and Rossman, 2023). To the best of our knowledge, this is the first report of G. fragariae on strawberry in Italy. The impact of the disease caused by this pathogen could be of high importance in the future of strawberry production in Italy. Healthy propagation material and strict disease management practices in nurseries is a requirement to avoid disease epidemics.