First Report of Ewingella americana Causing Bacterial Brown Rot Disease on Cultivated Needle Mushroom (Flammulina velutipes) in China

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Abstract

Needle mushroom (Flammulina velutipes) is one of the most industrially cultivated edible fungi in China. Its production is constrained by multiple abiotic and biotic stresses. Bacterial infections are one of its major biotic stressors. In June 2016, diseased samples of needle mushroom (about 5 to 10% infection) were collected from Gaorong mushroom company (125.24°E, 44.20°N) in Changchun, Jilin Province. Symptoms initially appeared as water-soaked lesions on the fruiting bodies with a color change from white to light brown. Rotten lesions gradually expanded with a slight foul smell. Finally, all lesions collapsed, and the infected stipes fell. Isolations were made from 30 infected mushroom samples. Approximately 1 g of infected tissue was sterilized in 75% alcohol for 30 s, rinsed three times with sterile distilled water, and crushed in sterilized 1.5-ml tubes containing 1 ml of sterile distilled water. The suspensions of the crushed needle mushroom were streaked on the nutrient agar (NA) medium. All the plates were incubated at 28°C for 2 days, and 30 bacterial colonies were obtained from the 30 samples. All colonies of pure cultures were round, convex, semitransparent, and smooth on NA medium. The bacterial cells were gram negative, nonfluorescent, short rods, 1.5 to 1.8 × 0.8 to 1.0 µm, and periflagellate. The physiological properties and enzyme activities were determined using the API 20E system and conventional methods (Reyes et al. 2004). The results obtained for the cells were as follows: positive for catalase and nitrate reduction; negative for oxidase, H₂S production, and indole production; glucose, fructose, and lactose were used as the sole carbon source but not maltose, raffinose, and saccharose. Polymerase chain reaction amplifications of the 16S rRNA and gyrB genes were performed using the primers 27F/1492R and gyrB-UP1s/gyrB-UP2sr. The 16S rRNA and gyrB sequenced results of three strains B6-1 (MF460299, MG821158), B4-1 (MF522252, MG821157), and B2-1 (MF522251, MG821156) showed 99% similarity with Ewingella americana in GenBank (KT031278 and HE585220, KF308476). Finally, we confirmed the pathogen was E. americana based on morphological, biochemical, physiological, and molecular characteristics (Holt et al. 1994). Three independent pathogenicity tests were conducted by dropping bacterial suspensions using the three isolates (approximately 10⁸ CFU/ml) on 30 healthy cultivated needle mushrooms, with sterile distilled water as a control. All needle mushrooms were incubated in a mushroom chamber at 12°C with 90% relative humidity and blue light. After 48 h, all the mushrooms inoculated with the bacterial suspension showed brown rot symptoms similar to the original disease. We consistently reisolated the same pathogenic bacteria from infected needle mushrooms and confirmed by 16S rRNA analysis. E. americana is an opportunistic cross-kingdom pathogen causing stipe necrosis on Agaricus bisporus in Egypt (Madbouly et al. 2014), infecting oak tree in Thailand, and causing pneumonia in humans (Doonan et al. 2016; Ryoo et al. 2005). To our knowledge, this is the first report of E. americana causing brown rot disease on needle mushroom in China. Rapid identification methods, effective monitoring, and control programs are needed to prevent further prevalence and the expansion of the known host range.