First Report of Shoot Canker on Chestnut Caused by Diaporthe nobilis in Shandong Province of China.

Abstract

Chinese chestnut (Castanea mollissima) is an economic tree widely cultivated for nut production. In May 2015, for the first time, canker and severe twig dieback were observed in the chestnut orchards in Yantai, Shandong Province, China. The cankers appeared on shoots, twigs, and branches with sunken reddish-brown/tan symptoms. The shoots died above the infected area when girdled. However, fruit rot was not observed. In later summer, numerous small black pycnidia were formed along the lenticels on the surface of diseased bark with curly slim and pale-yellow spore-tendrils being released from the pycnidia after rainfall. To identify the causal agents, tissue fragments (5 mm²) were excised from the canker lesions, and 10 isolates were obtained on potato dextrose agar (PDA) (Bai et al. 2015). These isolates formed creamy white mycelia with concentric rings on PDA. Black pycnidia developed in culture and produced two types of conidia: hyaline, fusiform-elliptic to oblong-elliptic alpha conidia and hyaline, filiform, slightly curved or hooked beta conidia. The size of alpha conidia varied from 7.5 to 12.5 × 2.1 to 2.8 μm, and that of beta conidia ranged from 26.8 to 30.2 × 1.2 to 1.7 μm. Based on morphological characteristics, these 10 isolates were close to Diaporthe nobilis CBS 587.79 (Gomes et al. 2013). To confirm the identification, DNA was extracted from the mycelium of three monoconidial isolates (CHNut1, CHNut2, and CHNut3). The internal transcribed spacer region (ITS), a partial sequence of calmodulin (CAL), histone H3 gene (HIS), translation elongation factor 1-α gene (TEF1), and β-tubulin gene (TUB) were amplified and sequenced using primers ITS1/ITS4, Bt2a/Bt2b, H3-1a/H3-1b (Glass and Donaldson 1995), EF1-728F/EF1-986R, and CAL-228F/CAL-737R (Carbone and Kohn 1999), respectively. The consensus sequences (GenBank accession nos. MG920017 for ITS, MG926648 for CAL, MG926649 for TEF1, MG926650 for TUB, and MG926651 for HIS) were aligned using BLASTn in GenBank showing high homology (99, 100, 99, 100, and 100%) with the sequences of ITS (KC343153), CAL (KC343395), TEF1 (KC343879), TUB (KC344121), and HIS (KC343637) from D. nobilis complex (Gomes et al. 2013). The ITS sequence obtained in this study showed 99% similarity with that of the strain Ch1 (JF812647), which caused stem canker on cherry in China (Wang et al. 2011). Pathogenicity of the three isolates was performed in the field and under laboratory conditions. Briefly, 30 stems from 5-year-old chestnut trees were wounded with a scalpel on the phloem tissues and inoculated with an inoculum suspension (10⁶ alpha conidia/ml), which were then wrapped with moist cheesecloth and sealed with Parafilm. The wounded phloem tissues of 10 stems were inoculated with sterile distilled water as noninoculated controls. After 2 weeks, necrotic lesions (32 to 55 mm in diameter) developed on inoculated shoots, which resembled the symptoms on infected chestnut shoots in the field. In contrast, control plant tissues did not show any disease symptoms. The pathogen was reisolated from symptomatic shoots and was confirmed as D. nobilis based on morphological and molecular analysis. The experiment was conducted three times. To our knowledge, this is the first report of D. nobilis causing shoot canker and dieback on chestnut in China, and even worldwide. This study provides important information for necessitating new disease-management practices.