First Report of Yellow Rot Caused by Physarum galbeum in Grifola frondosa in China.

Abstract

Grifola frondosa (Dicks.) Gray, named "Maitake" in Japan, is mainly cultivated in China, Japan and Korea as a rare delicacy (Park et al. 2015). G. frondosa is a medicinal and edible mushroom that can enhance the human immunology system. In recent years, the production of G. frondosa has increased in China due to its high economic value and as a source of livelihood for small scale farmers. From August to September 2017, a serious slime mold disease was observed on G. frondosa under greenhouse conditions in Qingyuan County, Lishui city, Zhejiang Province, China. Incidence was 10 to 30% in most surveyed mushroom greenhouses, sometimes more than 80% in mushroom greenhouses without proper management. The disease reduced G. frondosa production by 10% on average, and over 80% in severe cases. Slime mold disease usually appeared after irrigation, the kelly plasmodia migrate firstly from the root of fruiting body to stem and finally to pileus, then the infected parts became soft and putrid with slime on the surface. Additionally, many other organisms grow on decayed fruiting bodies, such as bacteria, fungi, and insects. The disease can spread rapidly through soil to adjacent fruiting bodies resulting in yield reduction. Samples were collected and cultures were isolated by transferring diseased fruiting bodies with yellow green plasmodia onto 2% water agar medium. Plasmodia were purified through aseptically transferring their edge segment to a new sterile 2% water agar medium, and this procedure was repeated three or four times to free the isolate from contaminating organisms. Purified plasmodia were then placed on the solid bacteriological test medium (SGM), containing glucose, peptone, yeast extract, mineral salts and hematin used in the axenic culture of Physarum polycephalum (Daniel et al. 1964), to verify bacteria presence. Plasmodia were also induced to form sporocarps. Voucher specimens were deposited in the Fungarium of Jiangxi Academy of Agricultural Sciences (FJAAS-M0001) and the Herbarium of the Mycology, Engineering Research Center of Edible and Medicinal Fungi, Chinese Ministry of Education, Jilin Agricultural University (HMJAU-M1561). Sporocarps were stalked, globose to discoid, golden-yellow, 0.9-1.8 mm in height, 0.28-0.55 mm in diameter. Hypothallus was small, thin, orange. Stalks were subulated, about twice to thrice the diameter of the sporotheca, bright orange below, yellow above, furrowed. Peridium was weak, thin, and plated with yellow calcareous flakes. Capillitium was a small meshed, persistent net of tubules with small and yellow angular lime nodes. Spores were globose, free, dark brown to black in mass, purplish brown in transmitted light, 8-10 μm in diameter, smooth under light microscopy. Irregular spinulose spores showed clusters of small warts that are conspicuous under scanning electron microscopy. Plasmodia were yellow green. The 18S ribosomal RNA gene was amplified with primer SMNUR101/NS4 (Rusk et al. 1995; White et al. 1990). The 18S rRNA gene sequence was submitted to GenBank (OP373728) and an 18S rRNA gene phylogenetic tree of Physarum obtained by maximum likelihood analysis (ML) and Bayesian inferences (BI) of 23 taxa and 1,608 aligned positions was produced. Based on sporocarps morphological characteristics, plasmodial cultural traits, and the sequence of 18S rRNA, the slime mold was identified as Physarum galbeum. A pathogenicity test was performed by gently inoculating a 12 mm diameter circinal patch of SGM with plasmodia on three healthy fruiting bodies of G. frondosa. All treatments were cultured in a mushroom-growing room with temperature 24 to 29 ℃ and relative humidity of 87 to 96%. Three fruiting bodies inoculated with a 12 mm diameter SGM served as controls. All fruiting bodies inoculated with plasmodia showed the same symptom. No symptoms developed on the controls. The pathogen was consistently reisolated from the symptomatic fruiting bodies of G. frondosa and confirmed to be P. galbeum based on cultural, morphological and molecular characteristics, thus fulfilling Kock's postulates. This is the first report of P. galbeum causing yellow rot disease on cultivated G. frondosa.