First Report of Colletotrichum acutatum Causing Bitter Rot on Apple in Italy.

Italy could be considered the main apple-producing country in the European Union. Italian apple (Malus domestica L. Borkh.) production is estimated at approximately 2.1 million tons and encompasses a wide range of cultivars, harvested from August to November. Colletotrichum acutatum, which causes severe losses to strawberry production, was a regulated organism for all European countries until 2008, when it was removed from the EPPO quarantine pathogen list because of its wide distribution in strawberry-production areas. During the growing season of 2010, fungi were isolated from apple fruits exhibiting bitter rot symptoms after 4 months of storage in several packinghouses in the Emilia Romagna Region. The apples belonged to the Golden Delicious, Granny Smith, Pink Lady and Crisp Pink cultivars. Lesions on the fruit surface were circular and 1 to 3 cm in diameter. When lesions enlarged, they became sunken with relatively firm rotten tissues. The fungal fruiting structures, acervuli, were distributed sparsely or densely on old lesions, and under humid conditions, they discharged an orange conidial mass. Conidia observed with a light microscope appeared hyaline and fusiform, 8 to 16 × 2.5 to 4 μm, with two pointed ends or one rounded end. The fungal isolates were grown on potato dextrose agar (PDA) plates incubated at 25°C. After 7 days, colonies were white, becoming gray to pale orange, and when viewed from the reverse side, the color ranged from pink to reddish orange. Both cultural and morphological characteristics of the pathogen were similar to those described for C. acutatum J.H. Simmonds (3), which is responsible for bitter rot of apple (2). Koch's postulates were performed with one representative isolate from each host by artificial inoculation of 30 healthy apples from the cultivars listed above. Fruit surfaces were disinfected with 70% ethanol, wounded with a sterile needle, and then inoculated with 20 μl of a spore suspension (10 conidia ml) prepared from a 15-day-old culture on PDA. Inoculated fruits were sealed in a plastic bag and incubated at 25°C for 10 days. In 92% of fruits, symptoms appeared 10 days later, forming lesions with cream-to-salmon pink fruiting structures. The fungus was reisolated onto PDA from the lesions on the inoculated apples. After 7 days of incubation, the colonies and the morphology of conidia were the same as those of the original isolates. The tests were performed on all four cultivars with similar results. The PCR analysis, carried out using universal primers ITS1 and ITS4 (4) directly from single-spore-derived mycelium (1), resulted in an amplification product with 100% sequence homology with C. acutatum isolate AB626881 from GenBank database. Considering the results obtained, to our knowledge, this is the first report of C. acutatum in Italy causing bitter rot on apple. The disease is common in practically all countries where apples are commercially grown and since the losses could be severe under prolonged warm and wet weather conditions, C. acutatum could represent a serious issue for the Italian apple industry. References: (1) M. Iotti and A. Zambonelli. Mycol. Res. 110:60, 2006. (2) A. L. Jones et al. Plant Dis. 80:1294, 1996. (3) B. C. Sutton. Page 1 in: Colletotrichum: Biology, Pathology and Control. Brit. Soc. Plant Pathol. Oxon. UK 1992. (4) T. J. White et al. Page 315 in: PCR Protocols. A Guide to Methods and Applications. Academic Press, San Diego, CA, 1990.