REMOLIF G., GUARNACCIA V., SPADARO D. 2024 First report of nut rot caused by Botryosphaeria dothidea on almond in Italy Plant Disease in press DOI: 10.1094/PDIS-02-24-0482-PDN
Abstract
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.
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