First Report of Colletotrichum acutatum and C. fragariae Causing Bitter Rot of Apple in Uruguay.

Almost 50% of deciduous fruit produced in Uruguay are apples and bitter rot is ubiquitous in the apple-production regions in Uruguay. In rainy and hot seasons (25 to 32°C by day), severe outbreaks of bitter rot occur. In summer 2010, when apple rot incidence reached 70% in some orchards, fruit with typical symptoms of bitter rot were collected from orchards in the south-central region, the main apple-production area. Symptoms included 0.5 to 6.0 cm in diameter, circular, sunken, light brown-to-brown lesions on the fruit surface that contained black, pinhead-sized fruiting structures that produced orange-to-brown conidial masses under high relative humidity. Each lesion progressed to the core of the fruit in a V-shaped pattern. Single-conidial isolates from lesions were examined morphologically (3), and based on sequences of the internal transcribed spacer (ITS) rDNA determined using ITS1/ITS4 primers (4), three species were identified: Colletotrichum acutatum with white-to-pale orange colonies and one-celled, hyaline, fusiform to cylindrical conidia that averaged 14.5 (9.3 to 17.8) × 5.0 (6.9 to 4.0) μm (isolates C11 and C18, GenBank Nos. JN413081 and JN413082, respectively); C. fragariae with white-to-pale gray and/or dark gray colonies and one-celled, hyaline, cylindrical to fusiform conidia that averaged 20.5 (14.3 to 22.9) × 6.0 (4.6 to 7.6) μm (isolate C15 and C37, GenBank Nos. JN413083 and JN413084, respectively); and C. gloeosporioides with white-to-pale gray or gray colonies and one-celled, hyaline, cylindrical to fusiform conidia that averaged 16.5 (13.1 to 20.3) × 6.5 (3.7 to 7.6) μm (isolates C5 and C29, GenBank Nos. JN413079 and JN413080) when grown on potato dextrose agar (PDA) at 25°C. To confirm pathogenicity, two isolates of each Colletotrichum spp. were inoculated onto mature, asymptomatic fruit of cv. Pink Lady (eight fruit per isolate). Each fruit was surface disinfested with 70% ethanol, wounded with a sterile needle, and inoculated with 10 μl of a spore suspension (5 × 10 conidia/ml) of the appropriate isolate. Eight control fruit were each inoculated with 10 μl of sterile water. Inoculated fruit and the control fruit were placed in plastic bags (eight fruit per bag) and incubated at 25°C. Symptoms (sunken, brown lesions each with a V-shaped pattern extending to the core) developed on all inoculated fruit 2 to 4 days after inoculation. No lesions were observed on control fruit. When fungi were reisolated from lesions of inoculated fruit onto PDA and incubated at 25°C, colony and conidial morphology were identical to those of the original isolates, confirming Koch's postulates. This study confirms a previous report of C. gloeosporioides causing bitter rot on apple in Uruguay (1). C. acutaum and C. gloeosporioides are known to cause bitter rot on apple. C. fragariae has traditionally been associated with strawberry and recently with other host plants (2), but not with bitter rot of apple. To our knowledge, this is the first report of bitter rot of apple caused by C. fragariae, and the first report of this disease caused by C. acutatum and C. fragariae in Uruguay. References: (1) S. García. Page 49 in: Guía Para el Manejo Integrado de Plagas y Enfermedades en Frutales. INIA Las Brujas, Canelones, 1998. (2) S. J. MacKenzie et al. Plant Dis. 92:1432, 2008. (3) B. C. Sutton. The Coelomycetes. CAB International Publishing, New York, 1980. (4) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, 1990.