First Report of Trichoderma oblongisporum Causing Green Mold Disease on Lentinula edodes (shiitake) in China

Abstract

Lentinula edodes (shiitake) is well known for its delicious taste and valuable medical functions, and ranked as the second most important mushroom in terms of total world production. In March 2012, a serious green mold epidemic occurred on many mushroom farms in Suizhou County of China. The infected mycelia of L. edodes in cultivated bags became rotten, yellow, wilted, and finally died, with the surfaces of the cultivated bags covered with dark green fungal colonies. At a temperature above 20°C, disease incidence was nearly 100% on some mushroom farms. Three diseased cultivated bags were collected from three different mushroom farms, and two portions at the junction of the diseased and healthy portions of the bag were plated individually on potato dextrose agar (PDA) and incubated at 25°C for 4 days. Following incubation, agar discs cut from the growing front of colonies were inoculated onto fresh PDA and subcultured to obtain putative pathogens. Three purified isolates were all whitish initially, followed by the emergence of greenish conidial clusters at the outer margin of the colony. The underside of the colony appeared pale yellow. The growth rate of the isolates was about 0.95 to 1.02 cm/day in PDA at 25°C. Aerial mycelia were floccose, white, and septate. Chlamydospores were sub-globose to broadly ellipsoidal. Conidiophore branches arose at right angles, and primary branches arose singly or in pairs. Phialides were ampulliform, 3.1 to 6.7 × 2.7 to 4.0 μm, slightly constricted at the base, swollen in the middle, and narrowed abruptly at the apex. Conidia were produced on the top of the phialides with the shape varying from ellipsoidal to oblong, 3.3 to 4.7 × 2.4 to 3.2 μm. These observations were consistent with the description of Trichoderma oblongisporum by Bissett (1). The ITS and partial tef1 were amplified from the three isolates as previously reported (2) and sequenced (KM110064 to KM110069). Nucleotide alignment showed 99% sequence identity (ITS) with two T. oblongisporum isolates (FJ623268 and DQ083020), and 88% similarity (tef1) with T. oblongisporum (AY750884). Neighbor joining tree of ITS and tef1 nucleotide sequences also showed that our three isolates had the closest relationship with the aforementioned three T. oblongisporum strains. To determine pathogenicity, a sawdust substrate was sterilized for 2 h in polyethylene bags and subsequently inoculated separately with three isolates and L. edodes strain Qiu7, which was cultivated widely in Suizhou. When the mycelia of Qiu7 colonized the bags, 10 cm3 of substrate was withdrawn from each bag. The substrate was then exposed to 10 cm3 of mycelium from each pathogen in 10 bags. A parallel inoculation with 10 cm3 of sterilized sawdust substrate without pathogen mycelia was performed as a control. The inoculated cultivated bags were kept at 25°C. After 2 months, all of the mycelia in the bags became wilted and dead, and the cultivated bags became soft, rotten, and covered with dark green fungal colonies. The controls remained uninfected. The symptoms were similar to those observed on mushroom farms. Pathogens re-isolated from all the inoculated culture bags were confirmed to be T. oblongisporum through morphological characteristics, molecular identification, and phylogenetic analysis. To our knowledge, this is the first report of T. oblongisporum causing green mold disease on L. edodes in the world. References: (1) J. Bissett. Can. J. Bot. 69:2398, 1991. (2) N. Sadfi-Zouaoui et al. Can. J. Microbiol. 55:154, 2009.