Abstract

English walnut (Juglans regia L.) is the second most widely grown fruit in Chile after table grape, with a cultivated area of about 40,800 ha. In recent years, an unusual fruit drop before fruit maturation has become problematic for Chilean walnut growers, and this symptom has become more noticeable in orchards between Biobio and N~ uble regions since 2015. By 2017, during a rainy spring season, severe fruit losses of 54, 85, and 75% were recorded in Negrete, Biobío Region (37◦35905.20S; 72◦28947.20W), Los Angeles, Biobıo Region (37◦22943.80S, 72◦27912.20W), and Chillán, N uble Region (36◦37901.10S, 71◦58906.30W), respectively. Dropped fruit showed necrosis of the apical (stylar) end of the fruit extending inward to inner tissues into the kernel. Xanthomonas arboricola pv. juglandis recurrently affects walnut production in Chile (Moya-Elizondo et al. 2018) and this bacterial pathogen has been associated with apical necrosis symptom and premature fruit drop (Moragrega et al. 2011); however, in this research we focus on determining if fungal pathogens were associated with this damage. These symptoms were consistent with those described for brown apical necrosis (BAN) disease, which causes severe fruit drop and yield losses of over 20% in walnut orchards in Italy (Belisario et al. 2002; Scotton et al. 2015) (Figs. 1 and 2). Ten symptomatic fruits from each orchard were disinfected with sodium hypochlorite ( 1%) for 2 min and rinsed with sterile distilled water. Small pieces of affected tissue were excised from fruits and plated on potato dextrose agar (PDA) supplemented with streptomycin (200 mg/liter). After 4 days, Alternaria and Fusarium species were identified based on conidial morphology and classified to species using molecular techniques. DNA was extracted from five isolates, and internal transcribed spacer (ITS) regions (18SF2 [Daval et al. 2010] and pITS4 primers [Bryan et al. 1995]) and the 18s rRNA small subunit (SSU) rRNA gene (NS1 and NS4 primers [White et al. 1990]) were amplified and sequenced. For Fusarium isolates, the gene for the translational elongation factor 1-alpha (tef1-a) (EF-1 and EF-2 primers [O’Donnell et al. 1998]) was amplified, whereas for Alternaria isolates, the beta tubulin 1 gene (btub1) (Bt1a and Bt1b primers [Glass and Donaldson 1995]) was amplified