First Report of Colletotrichum fioriniae and C. godetiae Causing Apple Bitter Rot in Slovenia.

Bitter rot is a common disease of apple (Malus domestica Borkh.) in several apple growing regions worldwide. The disease affects the fruit pre-harvest in orchards and/or post-harvest in storage, resulting in considerable economic losses. The most frequently reported causal agents belong to Colletotrichum acutatum and C. gloeosporioides species complexes. Apple bitter rot was observed in Slovenia in 2011 and 2012 at harvest in three commercial apple orchards near Ljubljana. Symptoms were observed on approximately 10% of fruits in the orchard, and included circular, brown, slightly sunken necrotic lesions on fruit surface. When placed in high humidity, acervuli with orange conidial masses developed on the lesions. Eleven isolates were obtained from symptomatic apples by culturing pieces of necrotic tissue on potato dextrose agar (PDA). Single spore isolates were prepared and grown at 21°C and a 12-h photoperiod for 10 days. Total DNA was extracted and fragment of the β-tubulin-2 gene (exons 2-6) was amplified with primer pair T1 (3) and Bt-2b (2), and was sequenced (Macrogen, The Netherlands). BLAST analysis showed that six isolates had 100% identity to C. fioriniae (Marcelino & Gouli) R.G. Shivas & Y.P. Tan (4) including the ex-holotype strain CBS 128517. The remaining isolates showed 100% identity to sequences reported for C. godetiae Neerg. (1), including the ex-type strain CBS 133.44. One sequence of each species was deposited in GenBank (KJ650030 and KJ650029). Morphological characteristics of two isolates per species were recorded after 10 days of incubation on PDA at 21°C. C. fioriniae colonies were white to light gray on the upper side and brownish pink to vinaceous with black spots on reverse. Average growth rate was 56.5 mm in 7 days. Conidia were cylindrical to fusiform, pointed at both ends, and measured 7.5 to 16.7 μm (mean 13.1 μm) × 3.5 to 4.9 μm (mean 4.1 μm). C. godetiae colonies were light gray on the upper side and gray with black spots on reverse. Average growth rate was 48.5 mm in 7 days. Conidia were cylindrical to fusiform, pointed at one or both ends, and measured 7.9 to 18.1 μm (mean 12.2 μm) × 3.4 to 4.9 μm (mean 4.2 μm). Pathogenicity of both species was tested by inoculating surface-sterilized 'Gala' and 'Golden Delicious' apples with 10 μl of a conidial suspension (10 conidia ml) obtained from single spore cultures. Five mature apples were wound-inoculated with one isolate of each species and sterile distilled water was used for controls. After 10 days of incubation at 23°C and 100% relative humidity, symptoms, identical to those observed initially, developed around the inoculation point, while controls showed no symptoms. C. fioriniae and C. godetiae were re-isolated from inoculated apples. To our knowledge, this is the first report of apple bitter rot associated with C. fioriniae and C. godetiae in Slovenia. The disease can pose a threat to our apple production and cause substantial losses during storage. References: (1) U. Damm et al. Stud. Mycol. 73:37, 2012. (2) N. L. Glass and G. Donaldson. Appl. Environ. Microbiol. 61:1323, 1995. (3) K. O'Donnell and E. Cigelnik. Mol. Phylogenet. Evol. 7:103, 1997. (4) R. G. Shivas and Y. P. Tan. Fungal Divers. 39:111, 2009.