Diaporthe rudis Associated with Berry Rot of Postharvest Grapes in Italy.

Grapes for Italian passito wine production are subject to fungal decay during postharvest processing (withering). In January 2018, decayed berries of Vitis vinifera ‘Nosiola’ were observed in bunches stored in a fruit-drying room in Valle dei Laghi, Provincia Autonoma di Trento (Italy). The bunches displayed single or few berries with brown to dark brown discolorations and solitary or rarely aggregated, erumpent, black ascomata in the skin. The incidence of bunches with symptomatic berries was approximately 5%. Twelve small pieces (about 10 mm2) of epidermal and mesocarp berry tissues were sterilized in 0.5% NaOCl for 2 min, rinsed once in sterile distilled water, and then placed on potato dextrose agar (PDA). After 4 days at 25°C in the dark, fungal colonies showed similar morphology resembling cultures of Diaporthe spp. (Udayanga et al. 2014). Single-spore isolation was carried out to obtain pure cultures. Two representatives of these cultures (P19 [= CBS 145104] and P10) were identified by morphological and molecular analysis. On PDA, after 7 days at 25°C, colonies showed flat, entire edges, whitish to grayish, fluffy aerial mycelium. The reverse colony was light brown to gray, with dark brown pigmentation in the center. Mycelial PDA plugs (2.5 mm2) were transferred to alfalfa stems on water agar (15 g of agar/liter) as described by Udayanga et al. (2014). Pycnidia on alfalfa stems were black, globose, and erumpent at maturity. Conidiophores were hyaline, cylindrical, smooth, branched, ampulliform, straight to sinuous, 18 to 40 × 2 to 2.5 μm. Conidiogenous cells were cylindrical, terminal, with slight tapering toward apex (0.5 to 1 μm diameter). α-Conidia were hyaline, smooth, ellipsoidal, biguttulate, 5.7 ± 0.7 μm long, and 2.3 ± 0.4 μm wide (n = 40). β-Conidia were not observed. Species identification was performed by amplification of internal transcribed spacers, translation elongation factor 1-α, histone H3, and β-tubulin gene sequences using ITS1/ITS4, EF728/EF986, CYLH3F/H3-1b, and Bt2a/Bt2b primers, respectively (Guarnaccia et al. 2018). BLAST analysis and phylogenetic analysis, constructed with MEGA 7.0 software using the neighbor-joining method (evolutionary distances using the Tajima–Nei method) from combined multilocus databases, identified the isolates as Diaporthe rudis. The gene sequences were deposited in GenBank (accession nos. MH447355, MH447356, MH457708, MH457709, and MK205427 to MK205430). Pathogenicity testing was performed on 120 healthy partially dehydrated grape berries (cultivar Nosiola) with intact pedicel, surface sterilized by immersion in 0.5% NaOCl and rinsed three times in sterile distilled water. Each berry was inoculated with a 106 conidia/ml suspension of both isolates by piercing using a sterile tip (wounded berry, n = 50) or pipetting onto the berry surface (unwounded berry, n = 50). The control (20 berries) was inoculated with sterile water by piercing and pipetting. All inoculated withered berries were incubated at 25°C for 7 days at 95% relative humidity (RH). High RH values (>90%) are reached during the natural withering process depending on seasonal weather conditions. Disease index (DI), calculated for four disease severity classes in percentage, where DI = [sum (class frequency × score of rating class)]/[(total number of berries) × 4] × 100, of wounded and unwounded berries was approximately 26 and 18%, respectively. The frequent presence of microcracks and wounds on berry skin, owing to dehydration, handling, and insects that favor the fungal colonization, could explain the high DI values observed in unwounded berries. Symptoms on the infected berries were similar to those observed in the fruit-drying room. There were no symptoms on the control berries. The fungus was reisolated from the infected berries, thus completing Koch’s postulates. Although D. rudis has been recovered from asymptomatic and symptomatic grapevine (cane and leaf spots) (Guarnaccia et al. 2018), this is the first report of the fungus causing rot on grape fruit. Recently, D. eres, together with Pestalotiopsis biciliata and Diplodia seriata, has been associated with fruit rot in withered grapes (Lorenzini and Zapparoli 2018). This finding indicates that D. rudis may contribute, together with other pathogenic fungi, to the decay of grapes during the withering. Such decay is a major concern for producers because it could have a negative impact on wine quality.