First Report of Heart Rot of Pomegranate (Punica granatum) Caused by Alternaria alternata in Italy

Pomegranate (Punica granatum L.), native to central Asia, has been cultivated in Italy since ancient times, but has only recently become a commercial fruit crop. In 2008, its cultivation extended 7 ha and in 2013 was over 130 ha. The prevalent cultivar is ‘Wonderful’ and major producing regions are Apulia, Sicily, Sardinia, Calabria, and Latium. In the provinces of Catania and Syracuse (eastern Sicily), in September and October 2013 and 2014, in several commercial orchards of ‘Wonderful’ a fruit disease with an incidence of 1 to 9% was observed. The symptoms consisted of a black rot of arils, which spreads from the calyx area, sometimes confined to part of fruit compartments, while the rind remained unaffected. When fruit was cut open, a dark gray to black mold emerged. Affected mature fruit were lighter in weight but remained firm and attached to the tree canopy. The only external symptom was an asymmetrical shape and a dark colored area of rind corresponding to the interior rot. Isolations from infected tissues on potato carrot agar (PCA) with 500 mg/liter of streptomycin sulfate gave consistently sooty black and turfy fungal colonies identified as Alternaria sp. on the basis of conidia morphology (Simmons 2007). On PCA colonies produced single, suberect conidiophores with an apical cluster of multiple branching chains of conidia. Conidia were olivaceous to brown, mostly ovoid or ellipsoid, with both transverse (1 to 7) and longitudinal (0 to 5) septa and a prominent tapered beak; they measured 9 to 30 × 5 to 12 (average 20.2 × 9.4) μm. To identify the species, the ITS-rDNA and partial TEF1-alpha regions of two representative single-conidium isolates (PhP1 and PhP2) were amplified and sequenced with primers ITS1-ITS4 and EF1-728F and EF1-986R, respectively (Woudenberg et al. 2013). Sequences (GenBank Accession Nos. KP861900, KP861901 for ITS, and KP861902, KP861903 for TEF1-alpha regions, respectively) were identical (100% similarity) to those of ex-epitype CBS 916.96 of A. alternata (Fr.) Keissl (GenBank accessions AF347031 and KC584634). Pathogenicity tests of the isolates PhP1 and PhP2 were performed on detached, ripening fruits of ‘Wonderful’. Fruits were sterilized for 3 min in a 1% NaOCl solution and rinsed in sterile distilled water (SDW). A suspension of 5 × 104conidia/ml was injected (0.5 ml/fruit) into the aril compartments of 10 fruit per isolate with a 23-gauge syringe. Ten control fruit were inoculated with SDW. After 12 days at 25°C, all fruit inoculated with the fungus showed symptoms similar to those observed in naturally infected fruit. Both isolates decayed the arils without affecting rind and compartment membranes. Control fruits showed no symptoms. A. alternata was reisolated only from symptomatic fruits, thus confirming Koch’s postulates. The hearth rot of pomegranate, also known as black rot, was reported in other major pomegranate producing countries, such as Greece, Israel, and California (Ezra et al. 2015; Thomidis 2014). To our knowledge, this is the first report of heart rot in Italy. This disease is a serious concern for expanding pomegranate industry also due to the difficulty in screening infected fruit on the basis of external symptoms.