In autumn 2012, severe branch cankers and diebacks of sweet cherry trees (Prunus avium L.) were observed in orchards located in two different growing areas in Alicante Province (eastern Spain). In affected trees, leaves become dried without defoliation. Sectorial wood necrosis was also observed, occasionally associated with swollen bark and gum exudates. Isolations were made from diseased branches by surface-disinfecting small fragments of symptomatic tissue in 0.5% NaOCl, double-rinsing in sterile water, and plating them onto potato dextrose agar (PDA) amended with 0.5 g liter−1 of streptomycin sulfate. Plates were incubated at 25°C in the dark for 10 days, and all colonies were transferred to PDA. Pink to red colonies with white margins were consistently isolated. All isolates produced hyaline, allantoid to oblong-ellipsoidal conidia, 4 to 6 × 1.5 to 2 μm. The fungus was identified as Calosphaeria pulchella (Pers.: Fr.) J. Schröt (anamorph Calosphaeriophora pulchella Réblová, L. Mostert, W. Gams & Crous) based on morphology (1). Identification of C. pulchella isolates was confirmed by sequence comparison in GenBank database using the internal transcribed spacer region (ITS1-5.8S-ITS2) of the rDNA. Sequences showed 100% identity and 100% query coverage with C. pulchella reference isolate CBS 115999 (EU367451) (2). The ITS sequence of one of the isolates obtained in this study was deposited into GenBank (KJ396346). Two-year-old sweet cherry trees cv. Burlat were inoculated with two representative C. pulchella isolates from different orchards (1701 and 1702). A 5-mm cork borer was used to remove bark, and an agar plug from the growing margin of 20-day-old colonies was placed directly into the fresh wound, mycelium side down. Five trees were inoculated per isolate (five branches per tree) and 25 control branches were inoculated with non-colonized agar plugs. Inoculated tissue was covered with Vaseline and Parafilm to avoid the loss of water. Branches were taken to the laboratory 9 months after inoculation and thoroughly examined for canker development. The length of vascular discoloration was evaluated in each branch and resulting data were statistically analyzed. Length of vascular discoloration on the inoculated branches (6.6 ± 0.7) was significantly longer than in control plants (2.3 ± 0.3) at P < 0.001. Perithecia were neither observed on the artificially inoculated branches nor in the diseased sweet cherry trees from the sampled orchards. C. pulchella was re-isolated from the inoculated branches and no fungi were isolated from discolored tissue of the controls, confirming Koch's postulates. Canker of sweet cherry caused by C. pulchella is responsible for reducing yields and tree longevity in California and South Australia (3). Cultivated area of sweet cherry in Spain is around 25,000 ha. Hence, the potential economic loss from this pathogen could be substantial if left unchecked. To our knowledge, this is the first report of C. pulchella as a pathogen of sweet cherry trees in Spain.
References: (1) M. E. Barr. Mycologia 77:549, 1985. (2) U. Damm et al. Persoonia 20:39, 2008. (3) F. P. Trouillas et al. Plant Dis. 96:648, 2012.
