Occurrence of Severe Outbreak of Calyx-End Rot Associated with Botrytis cinerea in Malus × domestica cv. Cripps Pink During Harvest in the Maule Region, Chile

Chile, with 36,063 ha cultivated, leads the export of fresh apples in the Southern Hemisphere. The Maule region (35°25′ S) is the major area of apple production in Chile, with the most hectares of the late harvest cultivar Cripps Pink (www.odepa.cl, 2016). During the Cripps Pink harvest, between mid-April and early May of the 2015–16 growing season, an average of 86 mm of rainfall accumulated was registered. Unusual symptoms of calyx-end rot and signs of gray mold were observed from 15 days before harvest until harvest on apple trees cv. Cripps Pink sampled in orchards (Yerbas Buenas, Longaví, and San Clemente counties), with a incidence of 0.1 to 0.2%. The incidence of calyx-end rot from fruit harvested in 10 counties in the Maule region increased to up to 2.0% after 60 days of storage at 0°C in three packinghouses. The symptoms observed during harvest, included a watery soft rot, light brown to dark brown, starting at the calyx-end area and in severe cases affected the entire fruit. The rot tissue was spongy and not separable from healthy tissue. The sporulation present was profuse with grayish spore masses. Pathogen isolation from 271 symptomatic fruits was performed as described by Díaz et al. (2016). Pure cultures from the most prevalent fungus isolated (white colonies) were obtained from hyphal tips of 2-day-old culture in PDA. Colonies on PDA (n = 144 isolates) were white to gray with septate mycelium and conidiophores were free, erect, and branched after 5 days at 20°C. The conidia were unicellular, hyaline to slightly colored, smooth-walled, ellipsoid to ovoid, and measured 7.3 to 11.2 × 6.5 to 9.7 µm (n = 40). After 30 days at 20°C on PDA, sclerotia were large, elongated or spherical, melanized, and black. According to cultural and morphological characteristics, the fungal pathogen was identified as Botrytis cinerea Pers. (Xiao 2014). This identification was confirmed using BAP04 and BAP48 isolates by sequence analysis of the glyceraldehyde-3-phosphate dehydrogenase (G3PDH), heat-shock protein 60 (HSP60), and DNA-dependent RNA polymerase subunit II (RPB2) genes. BLAST and phylogenic analyses of B. cinerea isolates show 100% homology with B. cinerea strain B05.10 (Staats et al. 2005). Sequences of BAP04 and BAP48 isolates were deposited in GenBank (KY940437–KY940438 for G3PDH, KY951939–KY951940 for HSP60, and KY951955–KY951956 for RPB2). Pathogenicity tests were performed using the same two B. cinerea isolates on mature apples cvs. Cripps Pink, Fuji, and Granny Smith. Fruits (n = 60 apples) were surface disinfested (1% NaOCl, 60 s), injured with sterile hypodermic needle and inoculated with 20 μl of conidia suspension (10⁶ conidia/ml) in the middle of each fruit. Other disinfested fruits (n = 60) were injured with a sterile cork borer and inoculated with a mycelium plug (5 mm diameter). The two isolates induced rot lesions that on average 21.8 and 28.7 for conidia and 20.6 to 35.9 mm for mycelium after 7 days at 20°C. Wounded but noninoculated apples used as controls remained healthy. B. cinerea was reisolated from 100% of the symptomatic fruits, fulfilling Koch’s postulates. To our knowledge, this is the first occurrence of an outbreak of calyx-end rot caused by B. cinerea in apple fruits in Chile during harvest. Previously, B. cinerea has been reported as important pathogen affecting apples in Washington State in the U.S.A. (Xiao and Kim 2008).