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First Report of Red Rot of Jujube Fruit Caused by Botryosphaeria dothidea in China

Ran, L. X., Zhang, M., Shen, H. M.
Plant disease 2018 v.102 no.7 pp. 1458
Botryosphaeria dothidea, Fusarium oxysporum, Xanthomonas arboricola, Ziziphus jujuba, agar, branches, conidia, cooking, cross contamination, culture media, ethanol, flavor, fruit trees, fruits, fungal growth, fungi, genes, humidity, hyphae, internal transcribed spacers, mercuric chloride, mycelium, pathogenicity, pathogens, peptide elongation factors, plant rots, pycnidia, raw fruit, traditional medicine, translation (genetics), vinasse, wines, China, South Korea
Jujube (Zizyphus jujuba Mill.) is an economically important fruit tree widely grown in China and South Korea. Jujube fruit has been commonly used in cooking, wine, and traditional medicine for thousands of years. In recent years, red rot disease has seriously affected the growth and quality of gray jujube fruits (Z. jujuba Mill. var. Huizao) in Xinzheng at 34°16′ to 39′ N and 113°30′ to 54′ E, Henan Province, China. Initial symptoms consisted of small, circular, red spots lacking concentric rings that expanded rapidly, and usually within 2 to 3 days, superficial lesions evolved into soft rot affecting the entire fruit, producing a vinasse flavor. The pathogen was isolated from diseased fruits that were surface disinfected with 70% ethanol for 30 s, followed by 0.1% HgCl₂ for 1 min and rinsed three times in sterile distilled water. Then the skin on the margins of healthy and infected tissue was aseptically peeled off with a scalpel, cut into 3- to 4-mm cubes, placed on water agar, and incubated at 25°C in the dark. Once fungal growth was evident, agar disks with hyphal tips were transferred to potato dextrose agar (PDA) plates and incubated at 25°C in the dark for purification. All fungal colonies were spread over Petri dishes of PDA, and after an overnight incubation, single germinating conidium was transferred to fresh PDA. The pure culture obtained was subjected to morphological characterization. The aerial mycelium was initially white, turning dark gray after 5 to 6 days and black 2 weeks later. The mycelium grew luxuriantly with a velvet appearance. The pycnidia were flask-shaped and averaged 196.9 μm high and 213.3 μm wide. Conidia were hyaline, unicellular, fusoid, and averaged 15.0 to 20.0 × 4.5 to 6.5 μm. These morphological characteristics are similar to those reported by Slippers et al. (2004). To confirm the identity of the fungus, the internal transcribed spacer (ITS) region and partial translation elongation factor 1-alpha (EF1-α) gene were partially sequenced using primers ITS1/ITS4 and EF1-728F/EF1-986R (De Wet et al. 2008). The obtained ITS sequence (GenBank MG827237) and EF1-α sequence (MG850862) showed 99% identity with those of the reference sequences of Botryosphaeria dothidea (AJ938004 and JX462293, respectively). Morphological and molecular results confirmed the fungus as B. dothidea. Pathogenicity of the isolated fungus was evaluated by inoculation of 20 surface-sterilized, detached gray jujube fruits, and the same number of fresh fruits of gray jujube on a healthy tree, with mycelial PDA plugs (5 mm in diameter) placed on both wounded and nonwounded jujube fruits. As a control, wounded and nonwounded fruits were inoculated with sterile PDA plugs. Detached fruits were incubated in plastic boxes under 90% RH at 25°C, and rot symptoms developed on wounded and unwounded inoculated fruits 3 days after incubation, while control fruits remained symptomless. All branches with inoculated fruits were enclosed in a clear plastic bag to maintain humidity and prevent cross contamination. Symptoms similar to the original ones on naturally infected fruits were observed on all wound-inoculated fruits 3 days after inoculation, while no symptoms developed on unwounded inoculated fruits or controls. The same fungus was reisolated from each of the inoculated fruits with symptoms, fulfilling Koch’s postulates, whereas no fungus could be isolated from asymptomatic and control fruits. Based on the results of both morphological and molecular identification, the pathogen of red rot of gray jujube fruits in Xinzheng was identified as B. dothidea (Moug. ex Fr.) Ces. et de Not., which is different from Xanthomonas arboricola, the pathogen causing bacterial fruit rot of jujube in Korea (Myung et al. 2010), and Fusarium oxysporum, which had been reported to cause soft fruit rot disease of gray jujube (Zhang et al. 2013). To our knowledge, this is the first report of B. dothidea causing red rot of gray jujube fruits in China.