First Report of Shoot Dieback on Apple Caused by Diaporthe nobilis in China

Abstract

In May 2017, apple trees (Malus pumila Mill.) with shoot dieback were observed in Laiyang (36.97°N, 120.71°E), Shandong Province, China. Disease symptoms included twig and shoot dieback and black necrotic lesions. In the later stage, numerous black pycnidia were observed on the surface of necrotic bark. Small pieces (4 to 5 mm) of necrotic tissues from different apple trees were disinfected for 1 min in 75% ethanol, rinsed thrice with sterile distilled water, and plated onto potato dextrose agar (PDA) with 0.05% lactic acid at 25°C. Eight isolates were obtained, and colonies from all isolates were white. Abundant pycnidia developed on PDA for 3 weeks at 25°C, which produced α-conidia measuring 6.2 to 8.9 × 2.2 to 3.2 μm and β-conidia measuring 18.5 to 27.6 × 1.1 to 2.0 μm (n = 50). α-Conidia were one-celled, hyaline, fusiform, and biguttulate, whereas β-conidia were one-celled, hyaline, and filiform. To identify these isolates to species, total DNA was extracted from the monoconidial isolates. The internal transcribed spacer region (ITS), a partial sequence of β-tubulin gene (TUB), translation elongation factor 1-α gene (TEF1), and calmodulin (CAL) were amplified and 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 sequences (GenBank accession nos. MK522125 for ITS, MK540236 for TUB, MK540238 for TEF1, and MK540240 for CAL) were aligned using BLASTn in GenBank, showing 99.4 to 100% with the sequences of ITS (KC343147), TUB (KC344120), TEF1 (KC343872), and CAL (KC343388) from the ex-epitype strain of Diaporthe nobilis complex (Gomes et al. 2013). The identity of the isolates was proved by maximum likelihood and Bayesian inference with a combined dataset of ITS, TUB, TEF1, and CAL sequences. The isolates were nested within the clade that includes D. nobilis with 98% bootstrap support and 1.0 Bayesian inference. The pathogenicity of three isolates (SDAp1, SDAp2, and SDAp3) was evaluated on 2-year-old shoots using a wound-inoculation method on living apple trees (cv. ‘Fuji’). Fifteen shoots were wounded (3 mm diameter, 2 mm deep) with a scalpel by cutting into the bark and inoculated with 10 μl of spore suspension (10⁶ α-conidia/ml). Wounds were then sealed with Parafilm. The same number of wounded shoots was inoculated with sterile water as a control. The pathogenicity test was conducted three times independently. Necrotic lesions (2.8 ± 0.5 cm in length) developed around the inoculation points after 3 weeks of inoculation. No disease symptoms developed on the control shoots. The pathogen was successfully reisolated from 93.3% of the inoculated shoots. In China, D. nobilis has been reported as a new pathogen of postharvest rot of blueberry (Yu et al. 2018) and shoot canker of chestnut (Zhang et al. 2018). To our knowledge, this is the first report of D. nobilis causing shoot dieback on apple in China. This report is important for the new research aiming at management of apple shoot disease.