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First Report of Diaporthe eres Causing Twig Canker on Zizyphus jujuba (Jujube) in China

Zhang, Q. M., Yu, C. L., Li, G. F., Wang, C. X.
Plant disease 2018 v.102 no.7 pp. 1458
Diaporthe, Ziziphus jujuba, agar, bark, branches, buds, calmodulin, conidia, cork, culture media, dieback, ethanol, fruit trees, fungi, genes, hyphae, inoculum, internal transcribed spacers, mycelium, nutritive value, orchards, pathogenicity, pathogens, peptide elongation factors, phloem, pycnidia, ribosomal DNA, shoots, shrubs, tubulin, China
Jujube (Zizyphus jujuba Mill.) is a fruit crop native to China that has high nutritional value. It is one of the most common and economically important fruit tree species in Binzhou, Shandong Province, China. In April 2015, canker and twig dieback were observed with 30 to 60% incidence in about 5-year-old jujube orchards in Binzhou. Brown, sunken, elongated, necrotic lesions appeared on the twigs or shoots. The shoots died when the canker lesion girdled and new twigs developing from infected buds usually wilted and died. Samples were collected from three heavily affected orchards. Bark tissues (5 mm²) were disinfected with 75% ethanol for ∼1 to 2 min and rinsed with sterilized water before being placed onto potato dextrose agar (PDA). Fifteen isolates showing white colonies were obtained and three isolates from different orchards were selected for further tests. Black pycnidia with a mass of α-conidia (6.7 to 9.3 × 2.1 to 3.6 μm, n = 40) and β-conidia (17.6 to 28.8 × 1.0 to 1.9 μm, n = 40) were produced after incubation on PDA for 2 weeks at 25°C. α-conidia were hyaline, ellipsoidal to obovoid, and 2-guttulate. β-conidia were hyaline, unicellular, and filiform to hamate. The morphological characteristics are consistent with the description of Diaporthe eres Nitschke (Udayanga et al. 2014). To confirm the identity of the fungus, total DNA was extracted from the mycelium of three selected monoconidial isolates. The ribosomal DNA internal transcribed spacer (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 consensus sequences (GenBank accession nos. MG599554 for ITS, MG599556 for TUB, MG599555 for TEF1, and MG599557 for CAL) were aligned using BLASTn in GenBank obtaining 99% homology with strains of D. eres for ITS (KC343073), 100% for TUB (KC344041), TEF1 (KC343804), and CAL (KC343315) (Gomes et al. 2013). Pathogenicity tests of the three isolates were confirmed by inoculating branches of jujube trees with either conidia or hyphae. Inoculations were done by making round wounds with a cork borer in the phloem of the stem of both living trees and detached shoots. An agar plug (4 × 4 mm) containing 3-day-old cultured hyphae or 10 μl of an inoculum suspension (10⁶ α-conidia/ml) was placed into the wound of 30 two-year-old jujube stems. As controls, wounded tissues on 10 two-year-old stems were treated with either uncolonized PDA or water. All the treatments were wrapped with moist cheesecloth and sealed with Parafilm, and placed in the same environment. After 7 days, necrotic lesions, 15 to 32 mm in diameter, developed on all shoots inoculated with D. eres, whereas no symptoms appeared on the controls. The pathogen, morphologically similar to the inoculated one, was reisolated from symptomatic shoots and the experiment was performed three times. Although D. eres was previously reported on several tree or shrub species, to our knowledge, this is the first report of D. eres causing twig canker on jujube in China, and even worldwide. This study provides important information that will be useful for establishing effective disease-management practices.