Occurrence of green mold disease on Dictyophora rubrovolvata caused by Trichoderma koningiopsis

Dictyophora rubrovolvata is an important edible mushroom that is widely cultivated in China. In 2019, a serious rot disease on D. rubrovolvata was observed in a mushroom production facility located in Ce Heng County, Southwest of Guizhou Province, China. The causal agent was iden-tified as Trichoderma koningiopsis by amplification and sequencing of the internal transcribed spacer (ITS) region, the translation elongation factor 1-alpha (EF-1α) gene, and the RNA polymerase II subunit (RPB2) gene followed by phylogenetic analysis. Koch’s postulates were confirmed by a pathogenicity test that was conducted with healthy D. rubrovolvata , including re-isolation and identification. To our knowledge, this is worldwide the first report of T. koningiopsis as a pathogen on D. rubrovolvata causing green mold disease.

In addition to crop production that provides the basic nutritional resources for humans, mushrooms have been widely cultivated in Asia for their nutritional value as well as for medicinal applications for over 2000 years (Wang et al. 2020). Dictyophora rubrovolvata M. Zang, D.G. Ji & X.X. Liu, a saprophytic fungus which belongs to the Phallaceae family, is commonly known as "Zhu Sun" (bamboo fungi) in Chinese and "Kinugasatake" in Japanese. The use of this fungus as an edible mushroom can be traced back to the Tang Dynasty in China (Wang et al. 2018) . Due to its high nutritional, medical, and economic value, D. rubrovolvata is currently one of the main edible mushrooms commercially grown in Guizhou Province, China (Ye et al. 2016). It has a unique "umbrella-like" appearance and a crispy structure making it more and more popular in Chinese cuisine. Additionally, various polysaccharides with beneficial properties to health were found to be naturally present in D. rubrovolvata (Wang et al. 2018). This has recently attracted broader attention from consumers and caused an increased demand for traditional functional food. In March, 2019, a serious occurrence of rot disease on D. rubrovolvata was observed in Ceheng County, Guizhou Province, China. Green mold and wilt symptoms appeared on the surface of D. rubrovolvata during the early stages of cultivation (four months), causing direct death or preventing it from forming a sporocarp (Fig. 1A). The disease incidence was 60%-70% on the affected 5.33-ha growing area, causing a serious economic loss. This study was carried out to identify the causal agent of green mold on D. rubrovolvata and to provide relevant background for its sustainable management.
A total of 12 samples with symptomatic green mold and wilt symptoms were collected from D. rubrovolvata at different locations of a mushroom production facility located in Ceheng County, Southwest of Guizhou Province, China (20°02′59" N, 105°36′33" E). The mushrooms were  Observations were made with a Nikon Ni-E microscopic system (Nikon Inc, Melville NY). Measurements of conidiophores and conidia (n = 50) were performed using the software Nikon NIS Elements AR 4.50. The colonies of the fungal cultures were light green with a regular round shape at the early stage, and became dark green with fluffy hyphae after ten days, while the underside of the colony appeared pale yellow (Fig. 1B). Conidiophore branches arose at right angles, and primary branches arose singly or in pairs. Conidia were produced on the top of the phialides, with ellipsoidal to oblong shape, (2.1-) 3.5 (-5.4) × (1.4-) 2.8 (-3.8) μm (Fig. 1C). Morphological characteristics of the isolates matched the description of the genus Trichoderma (Bissett 1991). Genomic DNA of four representative isolates was extracted according to the manufacturer's instructions (Biomiga Fungal DNA Extraction Kit; CA, USA). Then, PCR amplifications were performed with primers ITS1 (5'-TCC GTA GGT GAA CCT GCG G-3') and ITS4 (5'-TCC TCC GCT TAT TGA TAT GC-3') for the internal transcribed spacer (ITS) region (White et al. 1990), primers EF1-728F (5'-CAT CGA GAA GTT CGA GAA GG-3') and TEF1LLErev (5'-GCC ATC CTT GGA GAT ACC AGC-3') for the translation elongation factor 1-alpha (EF-1α) gene (Carbone and Kohn 1999), and primers RPB25F (5'-GAY GAY MGWG ATC AYT TYG G-3') and RBP2-7CR (5'-CCC ATR GCT TGY TTR CCC A-3') for the RNA polymerase II subunit (RPB2) gene (Liu et al. 1999). The PCR amplification was carried out in a 25 μl reaction mixture containing 1 μl of DNA sample, 12.5 μl 2 × SanTaq PCR Mix (Sangon Biotech, Shanghai, China), 1 μl of each primer (100 µM), and 9.5 μl nuclease-free water, The PCR conditions were as follows: initial denaturation at 94 ℃ for 3 min, then denaturation at 94 ℃ for 30 s, annealing for 45 s with the corresponding temperatures (55 ℃ for ITS, 56 ℃ for EF-1α, and 55 ℃ for RPB2), extension at 72 ℃ for 1 min, followed by 35 cycles, then a final extension for 3 min. The obtained sequences were compared with other DNA sequences in the GenBank (NCBI) database. BLAST searches of the sequenced fragments resulted in the best match to T. koningii type specimen isolate ATCC 64,262 (ITS region: 99.81% identity to accession NR_138456), to T. koningiopsis strain UNISS 17b-36a (EF1-α: 98.75% identity to accession EF488124.1), and to T. koningiopsis strain GJS 97-273 (RPB2: 98.31% identity to accession FJ442795.1). Representative sequences of the sequenced DNA regions were deposited in GenBank (ITS region: MN108134; EF1-α: MN135988; RPB2: MT038997) and are included in Supplementary File 1 (FASTA format). In addition, a phylogenetic tree was constructed with MEGA 7 based on ITS region and EF1-α gene sequences in order to confirm that the representative isolate CXYL has a high genetic similarity to Trichoderma koningiopsis species (Fig. 2).
Koch's postulates were met to confirm the pathogenicity of this isolate on D. rubrovolvata. Briefly, spore suspensions of T. koningiopsis CXYL were prepared from onemonth-old colonies in 0.05% Tween buffer, and adjusted to a concentration of 1 × 10 6 conidia/mL. The suspension (500 μL for each treatment) was directly sprayed onto disease-free D. rubrovolvata grown in a greenhouse, and six mushrooms were inoculated in total. In addition, six D. rubrovolvata were spray-inoculated with 0.05% Tween buffer as a control. After 15 days, green mold appeared on all inoculated mushrooms. In contrast, no symptoms appeared in the control group that was treated with pathogen-free buffer. Pure cultures of T. koningiopsis Fig. 2 Phylogenetic analysis of the ITS region and EF1-α gene sequences of T. koningiopsis CXYL from this study and reference sequences using the maximum likelihood method (1000 bootstrap iterations). Bootstrap values are provided next to the respective branches 1 3 CXYL were isolated from D. rubrovolvata and verified by molecular analysis.
Similar to other Trichoderma spp., T. koningiopsis strains are considered as potential biocontrol agents against plant disease, as well as plant growth promoters (López et al. 2019;Yu et al. 2020). However, Trichoderma spp. are also common pathogens on mushrooms (Choi et al. 2010;Colavolpe et al. 2015;Kim et al. 2019). They can seriously influence the yield and quality of mushrooms during their cultivation, and thus significantly reduce the economic value in largescale productions. For instance, T. koningiopsis strain DC3 was reported as a destructive pathogen on Pleurotus eryngii (Kim et al. 2013). To our knowledge, this is the first report of T. koningiopsis as a pathogen on D. rubrovolvata causing green mold disease. Due to the high nutritional, medical, and economic value of D. rubrovolvata (Sun et al. 2017;Wang et al. 2020), the identification of the causal agent provides a relevant background for disease management in the future. In addition, the identification adds news insights related to the host range of T. koningiopsis, as it raises a serious biosafety concern when applied in biocontrol practices.

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Ethical approval This article does not contain any studies with human participants or animals performed by any of the authors.

Declaration of competing interest
The authors declare that they have no conflict of interest.
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