First Report of Lecanicillium aphanocladii Causing Rot of Morchella sextelata in China

Abstract

Morel mushroom (Morchella spp.) is a rare edible fungus with high nutritional and medicinal value. In China they are cultivated in sandy soils in greenhouses and production of fresh mushrooms reached 10,000 tons in 2019. However, from 2019 to 2020, a serious rot disease with 30% natural incidence was observed on M. sextelata at a mushroom farm in Pinghu (N30°39’, E121°2’), Zhejiang province of China. The symptoms mainly occurred after the first flush in the early February. First, a small white mold-like symptoms appeared on the surface or the pinnacle of pileus. Then the lesion developed to encircle the pileus and spread gradually to the stipe. The lesions expanded rapidly at high temperature (>20 °C) and humidity (>70%). In the final stages of infection, the fruiting bodies became soft with white molds. The pathogen was isolated from the margin of the lesions by plating onto potato dextrose agar (PDA) and incubated at 25 °C in the dark. Colonies on PDA grew fast, reaching 60 mm in 7 days at 25 °C, and were white to cream in color, while the back of colonies appeared red to brick-red gradually. Conidiogenous cell was solitary or in whorls of 2-4, flask-shaped in the beginning, and tapered into a thread-like neck. Conidia were borne at the tips of conidiogenous cells, were oval to sub-globose, and ranged from 1.2-2.0 µm in width and 3.2-4.3 µm in length. All these characteristics were consistent with those of Lecanicillium aphanocladii (Zare R and Gams W. 2001). To confirm the identity of the pathogen (L. aphanocladii strain G1), the genomic fragments for the internal transcribed spacer (ITS) and RNA polymerase II second largest subunit (RPB2) gene of the isolate were amplified by PCR (White et al. 1990; Zhou et al. 2020). The resulting sequence was deposited in GenBank with accession No. OL629617 and No. ON005041, respectively. BLAST results showed >99% identity with those of L. aphanocladii (MG593981.1 and KM283853.1, respectively). Concatenated sequences of the two genes in L. aphanocladii strain G1 were used to conduct a phylogenetic analysis using Bayesian inference (BI) and maxium likelihood (ML) methods in MEGA6 (Tamura et al. 2013). The pathogen was grown in PDB medium at 25 °C, 200 r/min for 14 days, and after which conidial suspension (1×107conidia/mL) was prepared by filtration with four layers of sterile gauze. A pathogenicity test was performed by spraying on ten fruit bodies of M. sextelata and cultured in 20 °C and 90 to 95% relative humidity for 7 days. The test results showed that the pathogen infected the pileus and developed into white mold-like lesion, gradually spread to the stipe, and eventually the whole fruiting body became soft with white molds. The pathogen was re-isolated from infected fruiting bodies and was confirmed to be L. aphanocladii, based on morphological characteristics and the ITS, RPB2 sequence. Meanwhile,the control M. sextelata was sprayed with PDB medium and grew normally without any symptoms. L. aphanocladii has been reported on cultivated fungi such as Agaricus bisporus and A. bitorquis in Europe (Zare & Gams 2001) as well as more recently on Tremella fuciformis in China (Liu et al 2018). To our knowledge, this is the first report of L. aphanocladii causing rot of M. sextelata. According to the disease observation in the farm of Pinghu, this rot disease breaks out and spreads fast, and is getting worse ever year, resulting in a huge loss of yield and commodity value. It is a big concern to producers of this edible fungus.

Epidemic Identification of Fungal Diseases in Morchella Cultivation across China

“…Lecanicillium aphanocladii is a newly reported fungus causing the rot of M. sextelata ascocarps [ 34 ]. However, the pathogen ( L. aphanocladii ) was not detected in this study.…”

“…[ 32 ], Cladobotryum spp. [ 30 ] and L. aphanocladii [ 34 ] were not detected. Spherical or nearly spherical tandem chlamydospores can be easily observed in fungal lesion of morel ascocarps and the pure culture ( Figure 1 H), which was consistent with the morphological characteristics of D. longispora identified earlier [ 31 ].…”

“…With a red and withered stipe, the ascocarp becomes sticky and malodorous after being infected by bacterial diseases, but no specific pathogenic bacteria have been identified. The obvious symptoms of morel fungal disease, particularly common under high temperature and high humidity, are white villous lesions and wilting and malformation of the ascocarps [ 28 , 30 , 31 , 32 , 33 , 34 ]. Approximately 25% of the cultivation area is almost completed damaged after being infected by fungal diseases.…”

“…For M. importuna, cobweb disease was caused by Cladobotryum protrusum [ 30 ], pileus rot by Diploöspora longispora [ 31 ], stalk rot by Fusarium incarnatum — F. equiseti species complex [ 32 ], and both cap and stalk morbidity by Paecilomyces penicillatus [ 33 ], while in M. sextelata rot was caused by Lecanicillium aphanocladii [ 34 ]. In general, quicklime is widely used to sterilize and control pathogens during morel cultivation [ 28 , 29 ].…”

Transcriptomic and Metabolomic Profiles Provide Insights into the Red-Stipe Symptom of Morel Fruiting Bodies

“…It is estimated that 25% of the morel cultivation area has been affected by fungal infections, resulting in severe yield losses [ 11 ]. Several pathogenic fungi that infect morels have been identified, including Cladobotryum protrusum [ 32 ], Diploöspora longispora [ 33 ], the Fusarium incarnatum – equiseti species complex [ 34 ], Paecilomyces penicillatus [ 26 , 35 ], and Lecanicillium aphanocladii [ 36 ], have been identified. Recently, ITS-amplicon sequencing and microscopic examination were performed on morel ascocarp lesions from 32 sites in 18 provinces across China, revealing that D. longispora was a major cause of morel fungus diseases [ 11 ].…”