First Report of Brown Leaf Blight of Shenguyou (Staphylea bumalda) Caused by Alternaria alternata in China

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Staphylea bumalda DC. (Staphyleaceae) is a woody bush distributed widely in China. The tender leaves and blooms of S. bumalda have high nutritional value and have been used as a cough remedy, antidiarrheal, and blood refresher after delivery. Furthermore, the seed oil content and quality are excellent (Yu et al. 2005). During June 2017, brown leaf blight was observed on this plant in Xiaogan, Hubei Province, and the disease incidence was about 30%. The disease could be prevalent in large-scale planting conditions, and it is potentially a large risk for the development of Shenguyou industry. Thus, the identification and detection of this disease are important. The symptoms emerged from the tip of leaves at the early stage and extended to the middle or even the whole leaves subsequently. The blight was brown and caused defoliation of the infected leaves at the end. Five 3- × 3-mm pieces of leaf tissue were excised from the margins of individual lesions from five 2-year-old S. bumalda trees with typical symptoms. Excised tissues were surface disinfested by using 0.1% mercury bichloride for 3 min, rinsed three times with sterilized water, plated on potato dextrose agar, and incubated at 25°C (Robak et al. 2014). Five fungal isolates with consistent morphology were obtained. The colonies were gray white to black in obverse and dark blue in reverse. Hyphae were achromatic, branched, and septated, with a diameter of 2.1 to 4.6 μm. The conidia were 3.5 to 15.8 μm in width and 7.6 to 46.5 μm in length with 2 to 10 transverses and 0 to 3 longitudinal septa (n = 100) (Simmons 2007). DNA was extracted from 100 mg of freeze-dried mycelium following the cetyltrimethylammonium bromide protocol (O’Donnell et al. 2010). The internal transcribed spacer (ITS) region of rDNA and a histone gene were amplified using the primers ITS1/ITS4 and H3-1a/H3-1b (Glass et al. 1995), respectively, and sequenced (GenBank accessions MF614038 and MF991905). These sequences had ≥99% nucleotide identity with the corresponding sequences (KX926578.1 and KY548071.1) of the reference strains of Alternaria alternata in GenBank. Supported by the molecular identification and morphological characteristics (Simmons 2007), the pathogen was identified as A. alternata. Koch’s postulates were fulfilled on 10 healthy leaves of 2-year-old S. bumalda tree in an incubator. Spore suspension (2 ml, 1.0 × 10⁶ conidia/ml) was inoculated onto leaves until runoff by a spray method, and sterilized water was applied on 10 additional leaves as a control. The incubator was set at 25°C and 12-h light/dark alternation with 192 lux. It was observed that the symptoms on the inoculated leaves 14 days postinoculation were similar to the diseased trees in the field, whereas the control plants remained healthy. The fungus was consistently reisolated from the inoculated symptomatic leaves and identified as the same. To our knowledge, this is the first report of S. bumalda brown leaf blight disease caused by A. alternata in China and across the world. Furthermore, our findings extended the host range of the pathogen A. alternata on characteristic plants.