Morchella is a genus of edible and medicinal fungi with a global distribution and economic value. Artificial cultivation of Morchella has risen to 8,000 hectares in China in last 5 years (Zhao et al. 2020). In the early March of 2021, a peculiar stipe spot disease was first detected on Morchella importuna at a manufacturing base in Yangxian county (33°02' N, 108°11' E), Shaanxi, China. Several dark brown patches were observed on the surface of M. importuna fruiting bodies, with approximately 40% of the fruiting bodies infected at a late stage of production. This symptom typically manifests in March to May each year during the growing season of M. importuna. Following infection of the fruiting body, a light brown spot (2 to 4 mm) appears at the base of the stipe when the fruiting body is about 3 cm long. The spot progressively darkens and proceeds to cover the large stipe of the fruiting body. At 5 to 7 days after the appearance of the first spot, infected young fruiting bodies soften and decay. Temperature (>20℃) or humidity (80 to 90%) increases the likelihood of disease. To isolate the causal agent, the edge of a lesion was carefully scraped, inoculated into potato dextrose agar (PDA), and then incubated in the dark at 24±2°C (Bensch et al. 2010). The colonies were grayish-green in front, green to black on the reverse, and grew as concentric rings after 14 days of culture. The conidiophore was thin, branched with warty ramoconidia, and 2.5 to 4.7 µm long by 1.5 to 2.0 µm wide (length×width, mean=0.72, n=150). The ramoconidia were cylindrical and long at 14.3 to 28.7 µm by 6.4 to 10.7 µm (length×width, mean=2.52, n=150); the secondary ramoconidia were narrower and rod-shaped at 3.8 to 4.9 µm by 2.4 to 3.6 µm (length×width, mean=0.19, n=150). These morphological features were consistent with Cladosporium scabrellum (Bensch et al. 2012). Genomic DNA was extracted from mycelia of the pathogen using an Ezup column fungal genomic DNA extraction kit (Sangon Biotech, Shanghai, China). To confirm the identity of the pathogen (C. scabrellum), the genomic fragments for the internal transcribed spacer (ITS), translation elongation factor 1 alpha (EF-1α) and actin (ACT) gene of the isolate were amplified by PCR (Carbone et al. 1999; Walker et al. 2016; Xu et al. 2021). The resultant sequence was uploaded to the GenBank database with accession number (GeneBank ID of gene ITS, EF-1α, ACT are MZ736660, OQ982380, OQ982381, respectively). BLAST results showed >99% identity with those of C. scabrellum(GeneBank ID of gene ITS, EF-1α, ACT are HM148195, HM148440, HM148685, respectively). Phylogenetic analysis was performed using the maximum-likelihood (ML) method and Bayesian phylogenetic inference (BI) analyses in MEGA7 software (Kumar et al., 2018). For pathogenicity testing, 20 naturally occurring M. importuna fruiting bodies in an artificial climate chamber were randomly selected, inoculated by spraying with a conidial suspension of the pathogen (1×106 conidia/ml sterile water), and incubated at 20°C and 90 to 95% humidity. On the third day after inoculation, a yellow-brown spot appeared around the grafting sites, the spot gradually darkened and began to cover the stipe; the infection rate was 80 to 85%. Ten control fruiting bodies of M. importuna sprayed with sterile water grew normally and were symptomless. The pathogen was reisolated from infected fruiting bodies and confirmed as C. scabrellum by morphology and ITS, EF-1α, ACT sequence. To our knowledge, this is the first report of M. importuna stipe spot disease caused by C. scabrellum. According to a farmer in Yangxian, these spots rapidly spread every year, drastically reducing the production and market value of Morchella. This is a major problem for producers of this edible mushroom.
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