First report of Botrytis cinerea on Hydrangea paniculata in Italy

Hydrangea paniculata (Panicle hydrangea) is an ornamental plant in the Saxifragaceae family and is used in gardens for its massive upright flowers developing from midsummer to autumn. In summer 2016, an unknown leaf spot was observed on H. paniculata grown in gardens in Valle Cervo near Biella (northern Italy) at 850 m (45°36′00″N, 8°03′00″E). Leaves of 20-year-old plants showed small necrotic spots (4 mm diam.) surrounded by a chlorotic halo, turning progressively brown to black. Lesions often coalesced, generating larger necrotic areas 3 to 8 cm in diameter. Necrosis developed particularly in correspondence of flower dropping of common evening primrose (Oenothera biennis), grown in the same garden close to H. paniculata. About 20 to 30% of the leaves from 20 plants showed symptoms, and up to 60% at 15 to 25°C. Affected leaves were washed in 1% sodium hypochlorite for 60 s, rinsed in sterilized water, and dried on sterile paper. Thirty small pieces were then cut from symptomatic tissues and placed on potato dextrose agar (PDA) amended with streptomycin sulfate (25 mg/liter). After 6 days at 21 to 23°C, a fungus producing abundant gray aerial mycelium was consistently isolated (70% frequency). The isolations have been repeated on symptomatic plants between August and October getting a total of five fungal isolates with the same morphological characteristics. The selected isolate IT11 showed branched conidiophores (981 to 1,080 µm long × 8.86 to 11.42 µm wide) with enlarged apical cells and smooth, ovoid to elongate, light ash-colored, unicellular conidia, 10.3 to 17.3 (avg. 12.4) µm × 6.6 to 8.8 (avg. 7.6) µm. Sclerotia were not present. These characteristics allowed identification as Botrytis cinerea (Ellis 1971). DNA was extracted from one selected monoconidial isolate (IT11) by using the E.Z.N.A. Fungal DNA Mini Kit (Omega Bio-Tek). The analysis of ITS region of rDNA was amplified using primers ITS1/ITS4 (White et al. 1990), and sequenced at the BMR Genomics Centre (Padua, Italy). A BLASTn search of the ITS rDNA sequence of isolate IT11 (464 bp) was 100% identical to that of several isolates of Botryotinia fuckeliana causing gray mold on different hosts (golden berry, pea, oregano, and sweet basil; KP462722, KC683713, KT921335, and KP025968, respectively; Gümrükcü et al. 2016). The accession no. assigned for the obtained nucleotide sequence is KX987862. To confirm pathogenicity, 15 leaves of 10-month-old healthy plants of H. paniculata were sprayed with a conidial suspension (1 × 105 conidia/ml) of IT11 strain grown for 10 days in PDA (20 ml/plant). Eight flowers from O. biennis plants were also sprayed with the same conidial suspension and were immediately placed in contact with the same number of healthy leaves. Control plants and flowers were sprayed with sterilized water only. After inoculation, plants were covered with plastic bags for 5 days to reach 80 to 95% RH and kept in a glasshouse at 21 to 26°C. Three plants per treatment were used. Extensive leaf necrosis developed quickly when flowers were used as an inoculum source for leaf infection, while small leaf spots developed 7 days after the inoculations of the conidial suspension. Ten days after the inoculation, all the artificially inoculated plants showed symptoms similar to those observed in the garden interested. Pathogenicity test was carried out twice showing the same results. Controls remained healthy. B. cinerea, with the same morphology and sequences as described above, was reisolated from affected leaf tissues, while no colonies developed from noninoculated controls. To our knowledge, this is the first report of B. cinerea on H. paniculata in Italy. The pathogen has been previously reported on H. paniculata in the U.S., Canada, and Poland (Farr and Rossman 2016). At present, the disease has been observed only in few gardens, but its importance might increase due to the widespread use of this plant for low-maintenance gardens in hilly and mountainous areas.