First Report of Nut Rot Caused by Botryosphaeria dothidea on Almond in Italy

: Almond (Prunus dulcis) is an important nut crop widely grown in the Mediterranean region, including Italy. In September 2021, almonds cv. Tuono showing dark lesions affecting the hull were collected in Villar San Costanzo (Piedmont, Northwestern Italy). The occurrence of symptoms in the orchard was estimated at 50% incidence. Two samples, each consisting of 50 fruits, were collected from the affected orchard. Small sections taken from the margins of the lesions were surface disinfected with 1% sodium hypochlorite for 1 min, rinsed in sterile water, dried on sterile filter paper, and placed on potato dextrose agar (PDA, VWR International, Leuven, Belgium), amended with streptomycin sulfate (25 mg/l) to inhibit bacterial growth. Plates were incubated at 25°C for 7 days under 12-h photoperiod. Botryosphaeria-like fungi were isolated with a frequency of 60%. Two representative isolates (21-06-F1A; 21-06-F4) were transferred onto new PDA plates to obtain pure cultures. Fungal colonies initially appeared white, then gradually turned dark grey and black in reverse as the colony aged. Abundant aerial mycelium was produced. Globose black pycnidia were produced on water agar supplemented with sterile pine needles (PNA; Smith et al. 1996) after 30 days of incubation at 25 ± 1°C under 12-h photoperiod. Conidia were one-celled, hyaline, elliptical, aseptate, 17.46 to 27.05 μm (average 23.51) long and 5.70 to 9.40 μm (average 7.48) wide (n = 50). Morphologically, the causal agent was identified as Botryosphaeria sp. Genomic DNA was extracted from the isolates using the E.Z.N.A. Fungal DNA mini kit (Omega Bio-Tek, Norcross, GA, USA) according to manufacturer instructions. The rDNA internal transcribed spacer (ITS), the partial translation elongation factor 1-alpha gene (tef-1α) and the partial beta-tubulin gene (tub2) were amplified and sequenced using primers ITS1/ITS4 (White et al. 1990), EF1-728F/EF1-986R (Carbone and Kohn 1999), Bt2a/Bt2b (Glass and Donaldson 1995), respectively. BLAST analysis showed 100% identity of the obtained ITS sequences (GenBank accession nos. PP859471, PP859472), tub2 sequences (PP889527, PP889528) and tef-1α sequences (PP889529, PP889530) with those of the ex-type strain of B. dothidea (CBS 115476). The maximum likelihood method based on combined sequences of ITS, tef-1α and tub2 was performed, and both isolates clustered with high bootstrap support values (96) with the ex-type strain of B. dothidea. Pathogenicity was tested on fruits of almond cv Tuono inoculated on tree in a commercial orchard. Ten fruits were randomly chosen and inoculated for each isolate. Inoculation was performed by removing a piece of hull with a sterile cork borer (6 mm diameter) and applying mycelium plugs of the same diameter, taken from 7-day-old colonies grown on PDA, upside-down onto the wounds. Each fruit was inoculated with one mycelium plug and sealed with parafilm. Control fruits were inoculated with sterile PDA plugs. Dark lesions with softening pulp developed on inoculated almonds after 9 days. The mean diameter of the lesions was 3.89 ± 0.80 cm on almonds inoculated with the strain 21-06-F1A and 3.08 ± 0.56 cm on fruits inoculated with the strain 21-06-F4. The pathogen was successfully reisolated and morphologically identified as B. dothidea, fulfilling Koch's postulates. No symptoms were found on control fruits. Botryosphaeria dothidea was previously reported to affect other nut crops, causing nut rot on walnut (Li et al. 2023) and panicle and shoot blight on pistachio (Gusella et al. 2022). To the best of our knowledge, this is the first report of B. dothidea causing nut rot on almond in Italy.