First Report of <i>Coniella granati</i> (syn. <i>Pilidiella granati</i> ) Causing Postharvest Fruit Rot on Pomegranate in Albania

Pomegranate (Punica granatum L., Lythraceae, Myrtales) is widely distributed in various natural vegetation types of Albania, and the country might even be one of the centres of its domestication (Xhuveli 2012). In recent years, commercial pomegranate production in Albania has increased substantially, now accounting for approximately 25% of the country’s subtropical fruit production sector (Institute of Statistics Albania 2024). In a survey on post-harvest diseases, fruits with crown rot symptoms leading to total fruit decay were examined. Symptomatic fruits of the cultivars Acco and Wonderful were collected from warehouses in the Fier region (40°52'18.6"N 19°26'51.0"E) in November 2022 (Fig. 1A, B). After surface disinfection, small pieces of fruit lesions (ca. 3 × 3 mm) were excised with a sterile scalpel. Samples were plated on potato dextrose agar (PDA) in 90 mm Petri dishes and incubated at 24°C for 3–5 days. Emerging mycelia were then transferred to oatmeal agar (OA) or PDA plates, where they formed fast-growing cultures, reaching 60 mm diameter after 10 days, and covered the entire plate after 14 days. Colonies on PDA were olivaceous and yellow with irregular concentric rings and black conidiomata. On OA, colonies displayed minute black conidiomata centrally, sparse white aerial mycelia towards the edge, arranged in concentric rings (Fig. 1C, D). On OA, conidiomata superficial; conidiophores measured 12.5 μm ± 1.3 in length, 2.3 μm ± 0.3 wide at the base (n = 15); conidia hyaline, ellipsoid to fusiform, 12.3 μm ± 0.7 long and 3.5 μm ± 0.3 wide (n=50) (Fig. 1E–G). The morphological characteristics were consistent with Coniella granati (Sacc.) Petr. & Syd. (Alvarez et al. 2016). For molecular characterization, DNA was extracted from pure cultures, and ITS, partial LSU rDNA, and partial tef1α loci were amplified using the primers ITS1F/ITS4, LR0R/LR5, EF1-728F/EF1-986R, respectively, and sequenced. Sequences were assembled and edited in Sequencher v5.4.6, deposited in GenBank (ITS: PP968905–10, LSU: PX205251–55, tef-α: PX275353–57), and queried against the NCBI nucleotide collection. The obtained sequences of the five isolates were identical for ITS and LSU, and highly similar (differing by up to three base pairs) for the tef1 gene loci. BLAST query using the sequences obtained from isolate 6 revealed up to 100% homology with ITS, LSU, and tef1-α from C. granati, e.g. CBS 814.71 in GenBank (MH860368, AF408380, KX833682, respecively). A phylogenetic tree was reconstructed using a comprehensive sampling of Coniella spp. and Bayesian inference as implemented in MrBayes 3.2. All five analyzed isolates clustered within the C. granati group (Fig. 2) and together with the BLAST results and morphological characterization confirmed the fungus to be C. granati. Koch’s postulates were fulfilled by using all five isolates by inoculating 0.5 ml conidial suspension (106/ml) collected from a 14-day-old culture of C. granati into two healthy fruits each using a sterile syringe. Controls were inoculated with sterile water. All fruits were incubated at 23°C. The inoculated C. granati conidia induced soft rot within 9 d, and a complete fruit rot after 17 d. Control fruits displayed no disease symptoms (Fig. 1H-K). The pathogen was re-isolated from symptomatic fruits and confirmed as C. granati using morphological and molecular identification. To our knowledge, this study represents the first scientific confirmation of C. granati as a causal agent of fruit rot in pomegranate in Albania.