First Report of Coniella granati as a Causal Agent of Pomegranate Crown Rot in Southern Italy

Pomegranate (Punica granatum L.), grown in Italy since ancient times, recently became an important crop for the human health benefits associated with fruit consumption. In the summer 2014 and 2015, crown rot symptoms were observed on 20 to 30% of the pomegranate trees (cultivars Wonderful One, Ako, and Mollar de Elche) in 20 different orchards in Apulia (North-East of Bari and South Salento), Basilicata (Metapontum area), and Calabria (Catanzaro province) and, rarely, a fruit rot was also observed. Crown rot-affected plants showed reduced growth, small, yellowish leaves and decline and ultimately death. In seven orchards at least three 2- to 5-year-old plants were sampled, and in two orchards sampling was carried out twice in 2014 and 2015. A brown-black wood discoloration was extensive in longitudinal sections and affected up to 50% of the cross-sectional area. Samples of decayed wood were placed on Water Agar in petri dishes and cultures kept 7 days at 25 ± 1°C in the dark. After 10 days, the pure colonies obtained on potato dextrose agar (PDA) of 33 selected isolates, were 80 to 90 mm in diameter and the mycelium was white to light yellow. Pycnidia were solitary, globose, and brownish to black. Hyphae were septate and conidia were hyaline, one-celled, ellipsoid to fusiform (12.1 to 15.3 × 4.0 to 4.5 μm). All the isolates showed morphological features were consistent with Coniella granati (Sacc.) Petr. & Syd (syn. Pilidiella granati Sacc.) (Van Niekerk et al. 2004). ITS genomic regions were sequenced for molecular identification. DNA was extracted from 3-day-old mycelium of a representative isolate, amplified by PCR using the universal ITS5/ITS4 primers, and the amplicon was sequenced by external service (Macrogen, Seoul, South Korea). BLASTn analysis of the ITS sequence (562 bps) obtained showed 99 to 100% of identity (E value = 0.0, coverage = 86 to 99%) with Pilidiella (= Coniella) granati (i.e., GenBank Accession Nos. JN815312.1 and KT852449.1). Consequently, the pathogen was ascribed to C. granati and its ITS sequence was deposited in GenBank (KU147239). Pathogenicity tests were carried out on 10 replicated 5-month-old potted plants of each of the pomegranate cultivars Wonderful One and Mollar de Elche. Five-millimeter mycelial plugs from the edge of 15-day-old colonies on PDA were placed in artificial wounds (5 mm long and 3 mm in deep) under crown bark and then protected with a layer of parafilm. PDA medium plugs were used as the noninoculated control. Plants were maintained in the greenhouse (25 ± 2°C; 16-h daylight photoperiod). Six months after inoculation, a wood decay extending 3.0 to 3.5 cm from the inoculation site was observed only on inoculated plants. C. granati was always reisolated only from decayed bark of inoculated plants, hence satisfying the Koch’s postulates. The pathogen was recently reported on pomegranate associated with a fruit rot in China, Iran, and Spain, and with a crown rot in Greece (Thomidis and Exadaktylou 2011) and Turkey (Çeliker et al. 2012). As far as we are aware, this is the first report of C. granati in Italy associated with a severe crown rot decline. It can be expected that severe damage will be caused by the pathogen owing to its rapid spreading to new pomegranate orchards. References: Çeliker, N. M., et al. 2012. Australas. Plant Dis. Notes 7:161. 10.1007/s13314-012-0074-6 Thomidis, T., and Exadaktylou, E. 2011. Plant Dis. 95:79. 10.1094/PDIS-07-10-0514 Van Niekerk, J. M., et al. 2004. Mycol. Res. 108:283. 10.1017/S0953756204009268