First Report of Brown Rot Caused by Monilinia fructicola on Apple in Italy.

Abstract

Monilinia fructicola (G. Wint.) Honey, the causal agent of brown rot, is one of the most important fungal pathogens of stone fruit but may also affect pome fruits. The pathogen is common in North America, Oceania, South America, and Asia. It is a quarantined pathogen in Europe (3), but was recently detected in apple from the Czech Republic, Germany, and Serbia (1,2,4). In January 2012, during a survey for fungal postharvest pathogens, stored apple (Malus domestica Borkh.) belonging to the cultivars Gala and Pink Lady showing brown rot symptoms were observed in the Emilia Romagna region, Italy. Typical decay spots were circular and brown, tending toward black. Decayed tissues remained firm, and numerous grayish pustules containing spores appeared on rotted areas. The pathogen was isolated on V8 juice agar and culture plates were incubated at 25°C in darkness for 5 days. A conidial suspension was spread on malt extract agar and single spores were selected. The colonies were morphologically identified as M. fructigena. Two colonies developing a gray mass of spores in concentric rings with the reverse side black were further studied by molecular tools. The colony margins were even and the conidia were one-celled, limoniform, hyaline, and 12.1 to 17.4 × 8.4 to 11.2 μm. The ribosomal ITS1-5.8S-ITS2 region was PCR-amplified from genomic DNA obtained from mycelium using primers ITS1 and ITS4. A BLAST search in GenBank revealed the highest similarity (99%) to M. fructicola sequences (GenBank Accession Nos. HQ893748.1 and FJ515894.1). Pathogenicity was confirmed using surface-sterilized mature 'Gala' apples, wounded with a sterile needle, and inoculated with an isolate conidial suspension (103 spores/ml). A 20 μl droplet was placed in the wound; control fruits received sterile water without conidia. After 5 days of incubation at 20°C in plastic containers with high humidity, typical symptoms of brown rot developed on inoculated fruits, while control fruits remained symptomless. The fungus isolated from inoculated fruit exhibited the same morphological and molecular features shown by the original isolates. To our knowledge, this is the first report of the fungus M. fructicola on apple in Italy. Further studies are necessary to determine geographic distribution, prevalence and economic importance of this quarantine organism in Italy.