First report of Coniella granati causing leaf spot of pomegranate (Punica granatum L.) in Hungary.

Pomegranate (Punica granatum L.), the hystoric fruit and ornamental crop native to Iran and North India is widely planted in the Mediterranean and became popular in the house gardens of northest parts of Europe (Fernandez et al. 2014) including Hungary. In August 2020 necrotic black lesions and serious defoliation were observed on 60% of 1-3 year old pomegranate trees (cv. Wonderful) in a horticultural nursery near Gödöllo, Hungary (47°36'00.9"N 19°21'26.5"E). Symptoms started as small irregular dark brown spots on the leaves, which later increased in size (2.6 ± 0.9 mm). Ultimately, the entire leaf turned yellow, defoliation resulted in damage on (6) - 8 - (15)% of the leaves. Then, black pycnidia with unicelled, elliptical to fusiform, colourless conidia (Avg. 50 conidia: 2.4 - (3.6) - 3.9 × 10.2 - (13,1) - 17.9 µm) developed on the surface. These morphological features matched those described earlier by Van Niekerk et al. (2004) and Alvarez et al. (2016) for C. granati. Conidia from pycnidia were directly transferred to potato dextrose agar (PDA) by sterile needle. The plates were incubated at 24°C in the dark. Light yellow colonies with whitish aerial mycelia and later black globose pycnidia were observed. Mass of conidia oozed from pycnidia after 15 days of incubation. Pathogenicity tests were carried out on 1-year-old potted P. granatum trees (cv. Wonderful) with 5 replicates in the greenhouse. Ten, randomly selected leaves were inoculated per plant. 7-mm mycelial plugs from the edge of 10-day-old colonies were placed directly on disinfested (2% NaOCl solution, than sterile distilled water) leaves. The plants were covered with plastic film for 3 days after inoculation (26±3°C and 87±3% relative humidity). Pathogenicity was also tested on nonwounded, surface-disinfested fruits by mycelial plugs in 3 × 3 replicates. Inoculated fruits were placed in large grass vessels for 15 days (24±2°C and 80±5% relative humidity). Uncolonized, sterile PDA plugs were used as controls in both cases. Dark brown legions developed after 9-12 days on the plants in the greenhouse. On pomegranate fruits, the fungus colonized the fruit after 7-8 days, followed by fruit rot. In some cases, after 2 weeks pycnidia developed on the skin surface. No decay were present on control leaves or fruits. The pathogen was reisolated from all infected tissues and identified as C. granati, thus fulfilling Koch's postulates. For molecular identification, total genomic DNA of the isolate was extracted from the growing margins of colonies on PDA and partial sequence of internal transcribed spacer (ITS) and translation elongation factor 1-alpha (tef1) were amplified by PCR using primers described by Alvarez et al. (2016). Sequence data of the Hungarian isolate of the ITS region (GenBank acc. no. MW581953) showed 99.8% identity (559 bp out of 560 bp) with C. granati sequences deposited in GeneBank (Acc. nos. MH860368, MH855389 and KX833582). Considering tef1 sequence of the Hungarian isolate (OM908764) obtained had complete identity with other published C. granati isolates (KX833676, KX833682). C. granati has been previously reported on pomegranate from Europe (Palou et al. 2010, Pollastro et al. 2016). Based on morphological and molecular studies, this is the first record of C. granati in Hungary. The economic importance of this disease in currently limited in Hungary due to pomegranate is rather an ornamental crop, however, the first cultivation trials have been already started. There is a risk that the spread of the pathogen began with the infected propagating material, as a result the disease may outbreak anywhere in the country.