Identification and fungicides sensitivity evaluation of the causal agent of cobweb disease on Lyophyllum decastes in China

Abstract

Background: Cobweb disease is a fungal disease that often occurs in the cultivation and production of edible fungi, which can harm a variety of cultivated edible fungi and cause serious losses. Cobweb disease is considered to be one of the four most serious fungal diseases in edible fungi. Symptoms suspected of cobweb disease were found during the cultivation of Lyophyllum decastes mushrooms. The objective of our study was to identify the cobweb pathogen and screen out the effective fungicides, so as to provide a reference for the comprehensive prevention and control of velvet mushroom cobweb disease.
Results: The causal agent for this cobweb disease was isolated from symptomatic samples and was found to be Cladobotryum mycophilum based on morphological characteristics, phylogeny (ITS, RPB1, RPB2 and TEF1-α) and the cultural characteristics of two isolates on PDA and MEA medium. Results of pathogenicity tests also supported the conclusion that C. mycophilum is the pathogen responsible for this condition. The antibacterial effect of Prochloraz-manganese chloride complex, Trifloxystrobin and tebuconazole, and Difenoconazole among the tested fungicides is remarkable, with EC50 being 0.076 μg/mL, 0.173 μg/mL and 0.364 μg/mL respectively. These fungicides have good control effect, low toxicity, and have good application potential on L. decastes.
Conclusion: First report of cladobotryum mycophilum causing cobweb disease of Lyophyllum decastes in China.

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Identication and fungicides sensitivity evaluation of the
causal agent of cobweb disease on Lyophyllum
decastes in China
Keqin Peng
Guizhou University
Meiling Lin
Guizhou University
Xiaoxiao Yuan
Guizhou University
Changtian Li
Jilin Agricultural University
Xiangyu Zeng
Guizhou University
Yu Li
Jilin Agricultural University
Fenghua Tian (  fhtian@gzu.edu.cn )
Guizhou University
Research Article
Keywords: Koch's postulates, Antibacterial effect, Hypomyces odoratus, Multi-gene phylogenetic tree
Posted Date: October 14th, 2022
DOI: https://doi.org/10.21203/rs.3.rs-2128611/v1
License:   This work is licensed under a Creative Commons Attribution 4.0 International License.  Read Full
License

Page 2/15
Abstract
Background: Cobweb disease is a fungal disease that often occurs in the cultivation and production of edible
fungi, which can harm a variety of cultivated edible fungi and cause serious losses. Cobweb disease is considered
to be one of the four most serious fungal diseases in edible fungi. Symptoms suspected of cobweb disease were
found during the cultivation of
Lyophyllum decastes
mushrooms. The objective of our study was to identify the
cobweb pathogen and screen out the effective fungicides, so as to provide a reference for the comprehensive
prevention and control of velvet mushroom cobweb disease.
Results: The causal agent for this cobweb disease was isolated from symptomatic samples and was found to be
Cladobotryum mycophilum
based on morphological characteristics, phylogeny (ITS,
RPB1
,
RPB2
and
TEF1-α
) and
the cultural characteristics of two isolates on PDA and MEA medium. Results of pathogenicity tests also
supported the conclusion that
C. mycophilum
is the pathogen responsible for this condition. The antibacterial
effect of Prochloraz-manganese chloride complex, Trioxystrobin and tebuconazole, and Difenoconazole among
the tested fungicides is remarkable, with EC50 being 0.076 μg/mL, 0.173 μg/mL and 0.364 μg/mL respectively.
These fungicides have good control effect, low toxicity, and have good application potential on
L. decastes
.
Conclusion: First report of
cladobotryum mycophilum
causing cobweb disease of
Lyophyllum decastes
in China.
Background
Lyophyllum decastes
(Fr.) Singer, also known as antler mushroom, is a precious edible and medicinal mushroom
(Fig.1A). It is belongs to Basidiomycetes, Agaricomycetes, Agaricales, Lyophyllaceae. In Japan, there is a saying
of “Smell then
Tricholoma matsutake
, eat then
Lyophyllum decastes
”, and in Europe it has the reputation of “Fried
chicken mushroom” [1]. In China,
L. decastes
is mainly distributed in Liaoning, Jilin, Heilongjiang, Jiangsu,
Qinghai, Sichuan, Guizhou, Yunnan and Xinjiang province [2]. Its fruiting body is delicate and refresh with high
nutritional and medical value.
L. decastes
is rich in protein as well as an essential amino acid, polysaccharide
(LDS), which has a series of important effects such as anti-tumor, hypoglycemic, hypolipidemic and antioxidant
[3, 4]. According to statistics of China Edible Mushroom Association, the yeild of
L. decastes
in China has
increased rapidly since 2015, and has reached 21,600 tons in 2019.
At present, cobweb disease is a common disease of edible mushroom. Its typical symptom is having cobweb-like
mycelium on the surface of fruit bodies at the initial stage [5, 6]. In the middle and late stage, the mycelium cover
the whole fruiting body and wrapped it, resulting in the decay of fruiting bodies and massive conidia spread
rapidly casuing extensive harm. Cobweb disease, brown spot disease (caused by
Verticillium
spp.), green mold
disease (
Trichoderma
spp.) and brown rot disease (caused by
Hypomyces perniciosus
) are considered to be the
four most serious fungal diseases on many mushroom, such as
Flammulina velutipes
,
Pleurotus eryngii
var.
tuoliensis
,
Lentinula edodes
,
P. eryngii
,
Agaricus bisporus
and so on [5, 7]. Cobweb disease has seriously hindered
the development of mushroom industry in China. It has occurred on mushroom such as
A. bisporus
,
P. eryngii
,
Coprinus comatus
and
Ganoderma lingzhi
, which has a great impact on agricultural economy and mushroom
farmers' income [8–11]. Cobweb disease on mushrooms also occurs in different regions and countries. Cobweb
disease caused by
C. mycophilum
and
C. varium
was reported on
A. bisporus
,
P. eryngii
and
F. velutipes
in Korea
[12]. In the same period,
C. mycophilum
also caused cobweb disease of
A. bisporus
in Castilla La Mancha, Spain
[13, 14] reported a cobweb disease on
A. bisporus
caused by
C. mycophilum
, which is becoming more and more
serious in the cultivation of edible mushroom in South Africa. So it is urgent to study the prevention and control of

Page 3/15
cobweb disease, as there are so many kinds of pathogens and a wide range of harm on mushroom cultivation. To
prevent disease outbreaks, attention should be paid to improving the disease resistance of varieties, ensuring the
vitality and purity of spawn, cleanliness of culture rooms, soil disinfection and standard operation in the process
of cultivation. However, cobweb disease on mushroom is common [15], and there is no simple and effective
means to control cobweb disease, resulting in a high risk of this disease. To tackle disease occurrence, fungicides
are often applied as preventive treatments for extensive outbreaks [16].
In 2021, the author conducted disease investigation in the cultivation area of
L. decastes
in Baiyun District,
Guiyang, Guizhou, China, and found a widespread disease suspected of cobweb disease. The disease is highly
contagious and destructive that led to almost no harvest in two mushroom sheds, which poses a serious threat to
the cultivation and production of
L. decastes
. The isolated pathogen was identied by combining the
morphological characteristics and phylogenetic analysis, and its pathogenicity was veried according to Koch's
rule. Furthermore, antibacterial effect of several fungicides were analyzed by mycelial growth rate method on the
pathogen. Thus, the results of this study will provide a reference for the comprehensive prevention and control the
disease on
L. decastes
.
Results
Disease symptoms identication
The symptoms of this disease were obvious in the middle and late growth stage of cultivation, and tended to be
aggravated with the increase of the number of fruiting tides. The pathogen was rst appeared on the base of the
stalk of the covering soil or fruit body.Initially, the roots of the fruiting bodies were covered with white, coarse and
cobweb-like mycelia,and then spread along the stalk to the cap. After that, the white occulent mycelia of the
pathogen would quickly cover the surrounding soil and fruiting bodies.Finally, the fruiting bodies were
rotten,shrink, while making it dark brown and rancid,covered with massive of conidia, which can spread rapidly
by air ow to adjacent ones(Fig. 1B-D). The disease spread rapidly in the whole shed, resulting in the abnormal
growth of the mushroom and failure of harvest.
Pathogenicity results
A total number of 12 isolates were obtained from the diseased fruiting bodies, among which the strain
2021062102-1 and 2021062102-3 were pathogenic. Pathogenicity results showed that cobweb disease
symptoms were visible 2 days after inoculation (Fig. 1F). Filaments similar to white hairs were produced at the
inoculation point, and then gradually spread around, with clear symptoms developing subsequently at 2 days post
inoculation. These symptoms resulting from articial inoculation were similar to those observed in the eld. The
control was asymptomatic (Fig. 1E). The pathogens were consistently re-isolated from the infected fruiting bodies
of
L. decastes
fullling Koch’s postulate, and were conrmed to be consistent with the inoculated strain by
morphological characteristics.
Morphological description
Colonies grow rapidly on a 90 mm PDA plate and covering the petri dish after 3 days at 25℃; reverse initially
yellowish ochraceous turning roseous or brownish red in 10 d (Fig. 2A-D). The aerial mycelium of the colony were
lush and cotton-like, with massive conidia. Colonies grow slowly on a 90 mm MEA medium and grew all over the
culture dish at 25℃ for 10 days, produced a large number of dense conidia (Fig. 2E-H). Conidiophores straight,

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hyaline, branching profuse, irregular, tips simple, 24.5-37.6 × 4.0-6.7 μm (n=30) (Fig. 2I-J). Conidia hyaline, mostly
ellipsoidal, 0-3 septate, bases rounded, slightly constricted at the septum, 17.3-27.2 × 7.9-10.4 μm (n=50) (Fig. 2K-
N). According to the morphological characteristics, the isolate (2021062102-1) was identied as
Cladobotryum
mycophilum
.
Phylogenetic analyses
The ITS-rDNA,
RPB1
,
RPB2
and
TEF1-α
genes of two isolates were amplied and sequenced with primers
ITS4/ITS5,
cRPB1Af/RPB1Cr
,
RPB2-5f/RPB2-7cR
and
EF1-983f/EF1-2218r
, respectively. Sequences of the two
isolates (GUCC202106:2021062102-1, GUCC202107:2021062102-3) were identical, and DNA sequenceswere
deposited in GenBank (ITS, OK285275 OK285276;
RPB2
, OK458561 OK458562;
TEF1-α
, OK448484
OK448458;
RPB1
, OK513067 OK513068). A multigene phylogenetic tree, inferred by the ML (Maximum-Likelihood)
method based on the concatenated ITS-
RPB1-RPB2
-
TEF1-α
sequences, conrmed the multiple isolates as
C.
mycophilum
. According to comprehensive identication of the phylogenetic analysis, morphological
characteristics and cultural characteristics,the isolates were identied as
C.mycophilum
(Fig.3). The results were
similar to morphological identication.
Effect of different fungicides on the cobweb disease pathogen of
Lyophyllum decastes
The effect of nine fungicides on radial growth of thepathogen was studied to screen out which fungicides are
highly effective against the pathogen.The average radial growth of fungus was signicantly affected by different
fungicides. The results of the nine fungicides screening showed that in PDA medium, the inhibition effect of all
the fungicide was good. Among them,Prochloraz-manganese chloride complex (50% WP)were the most effective
in controlling the pathogen,with EC50 being 0.076 μg/mL.Trioxystrobin and tebuconazole (75% WDG) has the
secondeffective in controlling the pathogen, with EC50 being 0.173 μg/mL.Furthermore, theeffective in
controlling the pathogenof Difenoconazole (10% WDG) was better, with EC50 being 0.364 μg/mL. Among them,
the inhibitory effect of carvacrol (5% SL) was slightly worse than that of other fungicides (Table 2).
Discussion
The cobweb disease has been reported in all mushroom-growing countries around the world, which causes heavy
economic losses, especially in the mid-1990s [21, 22]. In the late 1980s, the disease rarely occurred in cultivated
edible fungi, and even if it occurred, it was easily controlled by fungicides. However, the use of fungicides
gradually developed resistance to pathogens and eventually caused the arachnid epidemic in Ireland and the
United Kingdom in the 1990s, where the annual production of
Agaricus bisporus
decreased by nearly 40% [8–11].
C. mycophilum
has a wide host range that has been reported on
A. bisporus
,
Albatrellus
sp.,
Lactarius mitissimus
,
L. mitissimus
cf.
vellereus
,
Russula
sp.,
Coniophora
sp.,
Megacollybia platyphylla
,
Inocybe
sp.,
Armillaria mellea
,
Lycoperdon pyriforme
,
P. eryngii
,
P. ostreatus
,
G. lingzhi
[11–12, 19–20, 23]. The disease is prevalent especially in
A. bisporus
. Gea et al. [24] and Kim et al [25]. The prevention and control of the disease is mainly based on the
principle of "Prevention rst, comprehensive prevention and control", strictly control the production of each link,
prevent the invasion of pathogens, if the disease after the application of chemical pesticides for prevention and
control. In terms of prevention and control, we can create conditions suitable for edible fungi but not conducive to
the growth and development of pathogens by controlling nutritional and environmental conditions, and we can
also control the breeding and harm of hybrid bacterium by comprehensively using various prevention and control
methods, so as to ensure the yield and quality of
Lyophyllum decastes
. However, it is a strategy to screen and use

Page 5/15
effective fungicides in a targeted way.Attempted to study the prevention and control agents for
C. mycophilum
.
But it has not been reported on
L. decastes
. This is the rst report of
C. mycophilum
causing cobweb disease on
cultivated
L. decastes
in the world.
Conclusions
L. decastes
is an important edible and medicinal mushroom mainly cultivated in Shandong, Jiangsu, Fujian,
Guangdong, Hubei and Guizhou Provinces, China. About 1 million sticks per year is being cultivated in Guizhou
province. In June 2021, cobweb disease appeared on fruiting bodies of
L. decastes
in Guiyang, Guizhou Province,
with 3-5% incidence rate, bringing great reduction yield. Initially, the roots of the fruiting bodies were covered with
white, coarse and cobweb-like mycelia, and the cap and stipe were rapidly affected. Finally, the fruiting bodies
were rotten,dark brown, rancidand covered with massive of conidia, which can spread rapidly by air ow to
adjacent ones. Unlike the disease on
A. bisporus
, no symptoms of cap spotting were seen on the fruit bodies of
L.
decastes
[17-20].Two samples with typical symptoms which were collected from the location.Two strain
2021062102-1 and 2021062102-3 were pathogenic by Koch’s postulate. On the basis of the phylogenetic,
morphological andculturalcharacteristics analysis, the causal agent was introduced herein as
C. mycophilum
.
As species of
Cladobotryum
grow much faster than mushrooms, they spreads very fast in the fruiting body stage,
which will cause serious economic losses. Therefore, the control of the cobweb disease should be applied at early
as possible during the cultivation, whereas nine fungicides with recommend concentration were selected for the
primary screening experiment. Afterwards, according to the preliminary screening results, different gradient
treatments with appropriate concentrations carried out for the fungicides were applied in the culture media with
three replicates to perform linear regression analysis and determine the half maximal effective concentration
(EC50) values.Prochloraz-manganese chloride complex (50%     WP), Trioxystrobin and tebuconazole (75%
WDG), and Difenoconazole (10% WDG)were indicated to be effective fungicides among the nine candidates to
control the pathogen, with EC50 being 0.076 μg/mL, 0.173 μg/mL and 0.364 μg/mL respectively. These fungicides
have good control effect, low toxicity, and have good application potential on
L. decastes
. Management of the
disease requires an integrated approach, among which cultural practices, physical and biological prevention and
control must be emphasized for delaying the development of resistance and maintaining ecacy which directly
impacts yield.
Methods
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 owing water and disinfect the surface rstly. 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 puried 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

Page 6/15
All isolates were tested for pathogenicity using 2-3 cm high of the fruiting bodies following a modied 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 identied 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 amplication of the different gene
regions: the internal transcribed spacer (ITS) region of the rDNA gene cluster were amplied by PCR with primers
ITS4/ITS5 [30]. And three protein-coding genes were amplied 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 mMµL-1), 0.2 µL of Taq
polymerase (5 UµL-1), 2 µL of genomic DNA (50 ngµL-1), 4 µL of polymerase buffers (10× µL-1, Takara, Japan),
and 2 µL of each primer (25 mM µL-1). Amplication 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 nal 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 nal 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 amplication 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.

Page 7/15
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), Trioxystrobin 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
modied 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 laments 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 signicant difference (* p < 0.05) [38].
Declarations
Data Analysis
All statistical analyses were conducted in MS Excel and SPSS statistics (version 19.0) software.The ANOVA was
performed as per Duncan’s multiple range test to determine the signicant difference (* p < 0.05). Figures were
generated usingGraphPad Prism 7.0 program.
Author details
1Department of Plant Pathology, College of Agriculture, Guizhou University, Huaxi 550025, Guiyang, Guizhou,
China. 2Engineering Research Center of Chinese Ministry of Education for Edible and Medicinal Fungi,Jilin
Agricultural University,Nanguan 130118,Changchun, Jilin,China.3 Institute of Edible Mushroom, Guizhou
University, Huaxi 550025, Guiyang, Guizhou, China.
Ethics approval and consent to participate
Not applicable.

Page 8/15
Consent for publication
Not applicable.
Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on
reasonable request.
Competing interests
The authors declare that they have no competing interests
Funding
This work was supported by the Science and Technology Projects of Guizhou Province Cultivation [grant number
Support of QKH [2021] General 199]; Natural Science Research Projects of Guizhou Education Department [grant
number QKH-KY[2021]054]; Major Special Characters of QianKeHe [grant number QKH[2019]3005-1]; and Science
and Technology Innovation Team of Guizhou Province [grant number QKH-PTRC [2020]5001].
Authors' contributions
Keqin Peng: Data curation, Investigation,Writing - original draft. Meiling Lin: Data curation, Software. Xiaoxiao
Yuan: Investigation, Methodology. Changtian Li: Supervision, Writing - review and editing. Xiangyu Zeng:
Methodology,Software. Fenghua Tian: Conceptualization, Funding acquisition, Supervision, Visualization, Writing
- review and editing. Yu Li: Conceptualization, Resources, Validation.
Acknowledgments
The authors would like to thank Guizhou Kaidong Technology Co., LTD for providing collection and research
materials.
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Tables
Table 1. Materials of
Cladobotryum
species used in phylogenetic analyses

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Species Strain GenBank accession number
ITS
RPB2 TEF-1a RPB1
C. asterophorum
CBS 676.77 FN859395 FN868649 FN868712 FN868776
C. paravirescens
TFC 97-23 FN859406 FN868660 FN868724 FN868787
C. protrusum
CBS 118999 FN859408 FN868662 FN868726 FN868789
C. protrusum
FSU 5877 FN859411 FN868665 FN868729 FN868792
C. prurpureum
CBS 154.78 FN859415 FN868669 FN868733 FN868796
Hypomyces samuelsii
G.J.S. 96-41 FN859448 FN868702 FN868766 -
C. tchimbelense
TFC 201146 FN859419 FN868673 FN868737 FN868800
C. heterosporumne
CBS 719.88 FN859398 FN868653 FN868716 FN868780
C. indoafricum
FSU 5807 FN859399 FN868654 FN868717 FN868781
C. multiseptatum
CBS 472.71 FN859405 FN868659 FN868723 FN868786
Hypomyces dactylarioides
CBS 141.78 FN859429 FN868683 FN868748 FN868809
Hypomyces rosellus
TFC 99-229 FN859441 FN868695 FN868759 FN868820
C. rubrobrunnescens
CBS 176.92 FN859416 FN868670 FN868734 FN868797
C. tenue
CBS 152.92 FN859420 FN868674 FN868738 FN868801
C. mycophilum
TFC 200102 FN859433 FN868687 FN868752 FN868813
C. mycophilum
TFC 98-25 FN85943 FN868688 FN868753 FN868814
C. mycophilum
TFC 05-93 FN859436 FN868690 FN868755 FN868816
C. semicirculare
CBS 705.88 FN859417 FN868671 FN868735 FN868798
Hypomyces australasiaticus
TFC 03-8 FN859428 FN868681 FN868746 FN868807
Hypomyces khaoyaiensis
G.J.S. 01-304 FN859431 FN868685 FN868750 -
Hypomyces armeniacus
TFC 02-86/2 FN859424 FN868678 FN868742 FN868804
C. cubitense
TFC 2007-13 AM779857 FN868652 FN868715 FN868779
Hypomyces gabonensis
TFC 201156 FN859430 FN868684 FN868749 FN868810
Hypomyces aurantius
TFC 95-171 FN859425 FN868679 FN868743 FN868805
Hypomyces lactiuorum
TAAM 170476 FN859432 EU710773 FN868751 FN868812
Hypomyces subiculosus
TFC 97-166 FN859452 FN868706 FN868770 FN868829
C. penicillatum
CBS 407.80 FN859407 FN868661 FN868725 FN868788
Table 2.The virulence effect of nine kinds of fungicides on the pathogen

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Fungicides Treatment concentration ug mL-1 Toxicity regression
equation EC50/(ug
mL-1) Correlation
coecient
T1 T2 T3 T4 T5
Carvacrol (5%
SL) 500.00 100.00 20.00 4.00 0.80 y=3.0897x+2.4323 6.777 0.9711
Osthol (1% EW) 50.00 10.00 2.00 0.40 0.08 y=1.0215x+4.3536 4.294 0.9619
Eugenol (0.3%
SL) 30.00 6.00 1.20 0.24 0.05 y=2.0136x+5.0131 0.985 0.9725
Propiconazole
(25% EC) 5.00 2.50 1.25 0.63 0.31 y=1.6649x+5.4195 0.560 0.9163
Triadimefon
(20% EC) 5.00 2.50 1.25 0.63 0.31 y=1.0460x+4.8275 1.462 0.9979
Trioxystrobin
and
tebuconazole
(75% WDG)
1.39 0.35 0.09 0.02 0.01 y=1.3942x+6.0633 0.173 0.9572
Prochloraz-
manganese
chloride
complex (50%
WP)
1.00 0.33 0.11 0.04 0.01 y=0.7724x+5.8659 0.076 0.9759
Pyraclostrobin
(10% WDG) 2.40 1.20 0.60 0.30 0.15 y=1.4208x+4.7917 1.402 0.9805
Difenoconazole
(10% WDG) 10.00 1.00 0.10 0.01 0.00 y=0.8038x+5.3524 0.364 0.9688
Figures

Page 14/15
Figure 1
Wild symptoms of causing cobweb disease on
Lyophyllum decastes
and pathogenicity tests of
Cladobotryum
mycophilum
(2021062102-1). (A), Healthy fruiting bodies of
L. decastes.
(B-C), Rotten fruiting bodies at late stage
of the disease. (D), White anamorph spread over
L. decastes
. (E), Pathogenicity tests, day 2 after inoculation,
control, asymptomatic. (F), Pathogenicity tests, day 2 after inoculation of
C. mycophilum
2021062102-1,
diseased.
Figure 2
Morphology characterization of
Cladobotryum mycophilum
(2021062102-1). (A-D), Colony morphology on PDA
medium at 25℃. A: after 3 days; B: after 5 days; C: after 10 days; D: after 14 days. (E-H), Colony morphology on
MEA medium at 25℃. E: after 3 days; F: after 5 days; G: after 10 days; H: after 14 days. (I-J), Conidiophores cells
straight, hyaline, branching profuse, tips simple, Bar=10 μm. (K-N), Conidia, with 0-3 septa, Bar=10 μm.

Page 15/15
Figure 3
Multi-gene phylogenetic tree based on combined ITS
, RPB1
,
RPB2
,
TEF1-α
sequences. ML (maximum likelihood)
and MP (maximum parsimony) bootstrap values greater than 50% are reported above the branches, BI (Bayesian
inference) values > 0.90 are shown next to topological nodes and separated by “/”. Bootstrap values < 50% and BI
values < 0.90 are labeled with “-”. The tree was rooted to
Cladobotryumpenicillatum
CBS 407.80.