Pathogen isolation
Three diseased fruiting bodies of L. decastes were collected from a mushroom cultivation base, Guiyang (106°43′25″ N, 26°43′41″ E), Guizhou Province, China, on June 22nd 2021. Each diseased fruiting body was cleaned with flowing water and disinfect the surface firstly. Secondly, sections with about 0.3 cm square from the diseased fruiting body was cut off and surface sterilized with the following steps: immersed in 95% ethanol for 1 min, washed with ddH2O 2 times, immersed in 75% ethanol for 30 s, and suspensions were spread on a potato dextrose agar (PDA) plate with three duplications and incubated at 25℃ in darkness. The pathogen of each duplicate was re-isolated and purified while the single colonies formed [26]. All cultures were deposited to Culture Collection of the Department of Plant Pathology, College of Agriculture, Guizhou University, China (GUCC).
Pathogenicity tests
All isolates were tested for pathogenicity using 2-3 cm high of the fruiting bodies following a modified protocol of Tian et al. [27], 10 healthy fruiting bodies were inoculated, with sterilized distilled water as control. All treated fruiting bodies were maintained in the same mushroom-growing space, under the conditions (16-18℃, 90-95% relative humidity). The pathogenicity test was assessed over 4 days. Re-isolated were performed from the infected fruiting bodies, and morphological and phylogenetic analysis were done as below. All experiments were conducted triplicate.
Morphological and molecular characterization
For the morphological observations of the colonies, the strains were grown on PDA and 1.5 % malt extract agar (MEA) medium, at 25°C in darkness [15]. The colony characteristics and microscopic morphological characteristics of mycelia, conidiophore and conidia were observed at 3, 5, 10 and 14 days. Conidia were measured from each isolate. The isolates were then identified based on the morphological characteristics of the conidia and conidiophores according to the descriptions from Gams and Hoozemans [28], Rogerson and Samuels [29]. Additionally, the molecular characteristics of the isolates, total genomic DNA was extracted from the colony of the isolates using a CWBIOTECH Plant Genomic DNA Kit (Changping, Beijing, China) following the manufacturer’s protocol. PCR was set up using the following primers for amplification of the different gene regions: the internal transcribed spacer (ITS) region of the rDNA gene cluster were amplified by PCR with primers ITS4/ITS5 [30]. And three protein-coding genes were amplified using the following primers: the partial translation elongation factor 1-α (TEF1-α: EF1-983f/EF1-2218r) [31, 32]; RNA polymerase I second largest subunit (RPB1: cRPB1Af/RPB1Cr) [33]; RNA polymerase II second largest subunit (RPB2: fRPB2-5f/fRPB2-7cR)[34, 15], respectively.
The PCR was conducted in a Applied Biosystems, ProFlex™ PCR (, Waltham, Massachusetts, USA). The PCR reaction was performed with a 50 µL mixture consisting of 3.2 µL of dNTP mix (2.5 mMˑµL-1), 0.2 µL of Taq polymerase (5 UˑµL-1), 2 µL of genomic DNA (50 ngˑµL-1), 4 µL of polymerase buffers (10× µL-1, Takara, Japan), and 2 µL of each primer (25 mM µL-1). Amplification of the ITS region was performed as follows: initial denaturation at 94°C for 5 min , 30 cycles of 30 s at 94°C, 30 s at 50°C, 30 s at 72°C, and with a final extension of 10 min at 72°C. For amplifying the TEF1-α protein-coding genes programming for an initial denaturation at 94℃ for 3 min followed by 35 cycles of 15 s at 94℃, 15 s at 55℃ and extension at 72℃ for 15 s; and RPB1 region: initial denaturation 5 min at 94°C, 30 cycles of 30 s at 94°C, 30 s at 55°C, 30 s at 72°C; for RPB2 region: initial denaturation at 95℃ for 3 min followed by 35 cycles of 15 s at 94℃, 15 s at 52℃ and extension at 72℃ for 30 s; and with the same final extension at 72℃ for 10 min. Electrophoresis was performed on 0.8% agarose gels stained with Gel Green. PCR products were sequenced by the same primers used for amplification by Qingke Biotech (Chengdu) Co., Ltd.
The sequences of ITS, RPB1, RPB2 and TEF1-α genes from representative ex-type strains were selected for phylogenetic analyses and extracted from GenBank using BLAST. The obtained sequences were visualized and aligned using BioEdit [35] and compared against the non-redundant nucleotide collection (nr/nt) sequences present in the NCBI GenBank database using the Basic Local Alignment Search Tool (BLASTn) tool (https://blast.ncbi.nlm.nih.gov/Blast.cgi). As for building the phylogenetic trees, maximum likelihood (ML), maximum parsimony (MP) and Bayesian inference (BI) were performed at the CIPRES web portal [36]. 24 phylogenetically related species of Cladobotryum, as C. asterophorum, C. paravirescens, C. protrusum, C. prurpureum, Hypomyces subiculosus, H. samuelsii, C. tchimbelense, C. heterosporumne, C. indoafricum, C. multiseptatum, H. dactylarioides, H. rosellus, C. rubrobrunnescens, C. tenue, C. mycophilum, C. semicirculare, H. australasiaticus,et al. were used for phylogenetic analyses [15] (Table 1)
Screening of fungicides for prevention and control of cobweb disease causal agent on Lyophyllum decastes
Various fungicide, including Carvacrol (5% SL), Osthol (1% EW), Eugenol (0.3% SL), Propiconazole (25% EC), Triadimefon (20% EC), Trifloxystrobin and tebuconazole (75% WDG), Prochloraz-manganese chloride complex (50% WP), Pyraclostrobin (10% WDG) and Difenoconazole (10% WDG), were selected. Preliminary indoor screening of fungicides for prevention and control of cobweb disease agent on L. decastes: the methodology was modified as appropriate according to Chen et al.[37]. According to the active ingredients, nine kinds of low toxic fungicides were diluted with sterile water to make mother liquor of certain concentration. In order to determine the concentration range of each fungicide, a pre-test was carried out with a concentration gradient of 5 times for each fungicide. According to the volume ratio, the PDA medium containing fungicide was prepared with the amount of mother liquid : PDA =1:9 in a Petri dish with diameter of 9.0 cm. The pathogen filaments which were cultured grown on PDA medium at 25°C in darkness for 4 days were made into cake with a 5 mm hole punch. PDA medium with equal amount of sterile water without fungicide was used as control. The fungus cakes were transferred into the prepared medium, and incubated at 24°C in darkness. In this process, the growth of pathogen was observed to determine the initial concentration of each fungicide. Selecting the fungicide that could inhibit the pathogen and conduct further concentration screening test. According to the pre-test results, each fungicide was diluted into 6 concentration gradients according to the effective components. The method of inoculation and culture for each treatment was the same as above. The diameter of colonies was measured with crisscross method, when colonies in control almost covered the Petri dish. Inhibitory percentage on mycelia growth was calculated after treatment with different concentrations and fungicides. Inhibition of mycelial growth (%) = [(dimeter of mycelium in control -diameter of mycelium in treatment)/dimeter of mycelium in control]x100. Each treatment was repeated three times. The EC50 value of each fungicide was evaluated by using ANOVA and GraphPad Prism 7.0 program (GraphPad Software, La Jolla, CA, USA) in three replicates. The ANOVA was performed as per Duncan’s multiple range test to determine the significant difference (* p < 0.05) [38].