First Report of Diaporthe nobilis Causing Postharvest Rot of Blueberry in Shandong Province, China

Abstract

Blueberry (Vaccinium L.) has become increasingly popular in China over the last several years owing to its high nutritional value. During June 2015, a postharvest fruit rot was observed on blueberry cultivar Duke cultivated in Qingdao, Shandong, China, and stored 7 days at 4°C after harvesting. Infected fruits showed soft and rotted lesions, covered with grayish or white mycelium under high humidity. To identify the causal agent of the disease, tissues were excised from surface-sterilized fruits and cultured on potato dextrose agar (PDA). Fungal colonies developed from all pieces of diseased tissues. Most isolates were identified morphologically and molecularly as Botrytis cinerea, Penicillium expansum, and Alternaria alternata, the important postharvest fungal pathogens of blueberry in China (Dai et al. 2016). Seven previously unidentified isolates were obtained, and all had white mycelia on PDA. After incubation for 3 weeks at 25°C on PDA, abundant pycnidia developed and produced hyaline, fusiform, biguttulate α-conidia and one-celled, hyaline, filiform β-conidia. The α-conidia dimensions were 5.9 to 8.7 × 2.0 to 3.4 μm (average 7.3 × 2.6 μm) and β-conidia were 16.5 to 27.8 × 0.9 to 1.9 μm (average 22.3 × 1.3 μm, n = 50). Morphological characteristics suggested the identity of the fungal isolates to be Diaporthe nobilis. To confirm identification, DNA was extracted from the mycelium of three representative isolates (SDB1, SDB2, and SDB3). The rDNA internal transcribed spacer (ITS), a partial sequence of β-tubulin gene (TUB), translation elongation factor 1-α gene (TEF1), and calmodulin (CAL) were sequenced using primers ITS1/ITS4 (White et al. 1990), Bt2a/Bt2b (Glass and Donaldson 1995), EF1-728F/EF1-986R, and CAL-228F/CAL-737R (Carbone and Kohn 1999), respectively. The consensus sequences (GenBank accession nos. MG557846 for ITS, MG557847 for TUB, MG557848 for TEF1, and MG557849 for CAL) showed highly similar (99, 100, 99, and 99%) with the sequences of ITS (KJ609006), TUB (KC344121), TEF1 (KC343874), and CAL (KC343395) from D. nobilis (Gomes et al. 2013). Pathogenicity tests were performed on 120 mature surface-disinfested blueberry (cv. Duke) fruits with a conidial suspension (10⁶ α-conidia/ml) of the three isolates. Sixty fruits were stab-inoculated to a depth of 3 mm using a sterile needle. For each isolate, 20 wounded and 20 unwounded fruits were inoculated with a 10-μl droplet. Forty fruits with or without wounding were treated with sterile water to create controls. Fruits were incubated in a chamber with a 12-h photoperiod at 25°C and 80% humidity. After 7 days, typical symptoms of postharvest rot with softened areas 3.7 to 6.4 mm in diameter were observed at wound sites. Experiments were conducted three times, and the pathogen was reisolated each time and confirmed as D. nobilis based on morphological and molecular analysis. Noninoculated and nonwounded fruits showed no symptoms. To our knowledge, this is the first report of D. nobilis causing blueberry postharvest rot in China. To prevent the spread of blueberry rot diseases, more care should be taken to avoid wounding during or after harvest.