First report of pseudopithomyces mori causing leaf spot on mandarin in Italy

In July 2023, a leaf spot-like disease was observed on approximately 15% plants in a mandarin (Citrus reticulata) orchard in Calabria, Southern Italy. The symptomatic leaves displayed irregular, dark brown lesions surrounded by a yellow halo. Fifteen symptomatic leaves from five trees were collected and surface sterilized in 2% NaOCl. Lesion pieces (3 × 3 mm) were plated on potato dextrose agar (PDA) with antibiotics and incubated at 24 °C for 5 days. Three isolates morphologically identical were obtained from different plants, and purified as required. The colonies on PDA were cottony, first hyaline, then salmon pink (front) to orange (reverse). Conidia were ellipsoid, light brown, with 3–4 transverse septa, usually divided by a longitudinal septum, and 23.5 ± 5.5 × 13.69 ± 4.79 μm. The morphological features were consistent with those of Pseudopithomyces sp. (Ariyawansa et al. 2015). Identification was confirmed for the representative strain AP77 by sequencing the internal transcribed spacer (ITS) region and portion of translation elongation factor 1 alpha (TEF1) gene with primers ITS1/ITS4 and EF-983 F/EF-2218R, respectively (Jayasiri et al. 2019). Sequences were deposited in GenBank with accession numbers OR911598 (ITS) and OR921209 (TEF1). BLASTn analyses showed their 99% homology with Pseudopithomyces mori strain MFLUCC 18-1630 (GenBank No. MW183777 and MW063153, respectively). Phylogenetic analyses of the concatenated sequences confirmed the clustering with P. mori strain MFLUCC 18-1630 (Tennakoon et al. 2022). The pathogenicity was tested on 10 healthy citrus leaves, which were slightly injured with a sterile scalpel, inoculated by a drop of suspension (106 conidia/ml) or sterile water (control), and maintained at 24 °C in a humid chamber. Seven days after inoculation, lesions like those observed in the field occurred only on inoculated leaves. The same fungus was reisolated from infected leaves, fulfilling Koch’s postulates. To our knowledge, this is the first report of leaf spot on mandarin leaves caused by P. mori in Italy