First Report of leaf spots caused by Diaporthe eres on Viburnum opulus in Italy

European cranberry (Viburnum opulus), Caprifoliaceae family, is an easy to grow shrub, suitable for small gardens, producing white inflorescences followed by decorative red berries. In September 2023, leaf spots were observed on three 10-year-old plants of V. opulus grown in a private garden located in Campiglia Cervo (Biella province, northern Italy). Brown spots expanded and affected a large part of the leaf surface, forming necrotic areas with irregular, indistinct margins. Affected tissues desiccated. The disease compromised the aesthetic value of the plants. Fifteen affected leaves were immersed in a solution of sodium hypochlorite (1%) for 30 seconds, then rinsed in sterile water. Several small fragments were excised from the margin of the affected tissues and plated on PDA (Potato Dextrose Agar) amended with streptomycin sulphate (25 mg/L). Fungal colonies were coarse and radiate, at first whitish, then greyish with brown shades. Pure colonies grown on PDA, under a light/dark regime (13h/11h), produced stromata that exuded cream-white drop-like conidial mass with two types of conidia. Alpha conidia were unicellular, ellipsoidal to fusiform, and measured 5.4 to 8.7 × 1.9 to 3.5 µm (mean: 6.9 × 2.6 µm, n = 50); beta conidia were unicellular, filiform, curved, and measured 20.1 to 29.6 × 1.0 to 3.2 µm (mean: 25.1 × 1.4 µm, n = 50). DNA from isolate 23-35-5 was extracted from pure culture using the E.Z.N.A. Fungal DNA Mini Kit (Omega Bio-Tek). Beta-tubulin (TUB), calmodulin (CAL) and translation elongation factor 1-alpha (EF1-α) regions were amplified using the primers: Bt-2a/Bt-2b (Glass et al. 1995); CAL-228F/CAL-737R (Udayanga et al. 2014) and EF1-728F/ EF1-986R (Carbone et al. 1999). Sequences of 491 (TUB), 356 (CAL), and 323 (EF1-α) base pairs were obtained (GenBank accessions: PV289771, PV289769, PV289770, respectively). BLASTn analysis of these sequences showed 100 % (TUB), 100% (CAL), and 100% (EF1-α) identity with Diaporthe eres (GenBank accessions PQ001653.1, MZ423156.1, MG281548.1, respectively). Therefore, the fungal causal agent of the disease observed on V. opulus was identified as Diaporthe (anamorph Phomopsis) eres, in agreement with the morphological characteristics of the isolates. For the pathogenicity test, the isolate 23-35-5 was grown on PDA, and from the cultures, a conidial suspension was adjusted to a final concentration of 1.6 × 105 CFU/ml. The inoculum was sprayed on the leaves of three 10-year-old plants of V. opulus grown outdoors. Three branches for each inoculated plant (about 10 leaves per branch) were closed in moistened bags for 10 days. Three healthy control plants of V. opulus were sprayed with sterile water, and their branches were maintained in the same environmental conditions. About 20 days after inoculation, the first symptoms of infection appeared on leaves from which several fungal colonies with morphological characteristics identical to the inoculated pathogen were re-isolated. No symptoms appeared in the controls. Diaporthe eres has been reported as the causal agent of leaf spots observed on V. odoratissimum var. awabuki (Wan et al. 2023). To our knowledge, this is the first report of D. eres on V. opulus in Italy, and worldwide. This work provides useful information for monitoring the incidence and diagnosis of this disease whose management could become a serious problem, particularly in public gardens. The susceptibility of Viburnum spp. to D. eres should be tested to identify resistant species to manage this disease.