First Report of Erwinia persicina Causing Pink Disease in Flammulina velutipes (Enoki Mushroom) in China

From the paper

Abstract

Flammulina velutipes is one of the main commercial mushrooms widely cultivated in China. In March 2018, pink infection (0.5% incidence) was observed on fruiting bodies of F. velutipes growing in an industrial cultivation unit in Zhangzhou, China. Initially, light pink patches (1 to 1.5 cm) were observed on the surface of fruiting bodies. Within 2 days, patches became large and dark pink, and infection permeated inner tissues of fruiting bodies. However, infected fruiting bodies were able to grow well. For the isolation of causative agent(s), internal tissue samples of the infected stipes (0.2 × 0.5 cm) were suspended in sterile distilled water, and 100 µl of suspension was spread onto potato dextrose agar (PDA) and Luria Bertani (LB) media and incubated at 25°C for 2 days. Bacterial colonies were observed on both media plates. Purified colonies were light beige, convex, and circular with smooth margins, and they produced a pink diffusible pigment on PDA but not on LB. The bacterial cells were gram negative, rod-shaped, and motile. No capsules or endospores were observed. Amplified 16S rDNA (1,502 bp; MH362699) of six randomly selected colonies using universal primers 27f/1492r exhibited 99% identity with Erwinia persicina (= E. persicinus) strain NBRC102418 (NR_114078). Based on morphological and 16S rDNA similarities among purified bacterial colonies, a representative strain (strain P) was randomly selected for further identity confirmation. Partial sequences of gpd (474 bp, MK035572) and recA (546 bp, MK035571) genes with primers GPD1F/GPD1R (Geider et al. 2009) and RECA1/REC2c (Waleron et al. 2002), respectively, also exhibited 99 to 100% similarities with E. persicina (CP022725; DQ859883) strains. Phylogenetic analysis showed that strain P formed a separate clade with E. persicina strains. The biochemical tests for strain P were performed via vtk2 GN test kit (20 CARDS, bioMérieux, Marcy-l’Etoile, France) and E. persicina NBRC102418 as a positive control. Data analyses showed that strain P was in accordance with strain NBRC102418. The ability to ferment d-mannose, d-sorbitol, and sucrose also confirmed strain P as E. persicina (Moretti et al. 2011). Pathogenicity tests were performed by bacterial suspensions (1 × 10⁹ CFU/ml) sprayed onto the surface of young F. velutipes fruiting bodies and incubating at 12°C and 90% relative humidity. The typical strain NBRC102418 was used as a positive control. Pathogenicity tests were performed twice, and three bottles of fruiting bodies were inoculated each time. After 5 days of incubation, pink infection was observed on fruiting bodies that were separately inoculated by strain P and NBRC102418. The disease symptoms were similar to those observed on the original samples. The control, fruiting bodies sprayed with sterile distilled water, remained asymptomatic. E. persicina has been reported as a pathogen affecting several plant species (Gálvez et al. 2015) and has also been isolated from the urine of a patient who presented with a urinary tract infection (O’Hara et al. 1998). However, to the best of our knowledge, this is the first report of E. persicina causing pinkish disease in F. velutipes worldwide.