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Biodiversity Data Journal 10: e87697
doi: 10.3897/BDJ.10.e87697
Short Communication
First report of Cladobotryum verticillatum
(Ascomycota, Hypocreaceae) causing cobweb
disease on Paxillus involutus
Xiaoya An , Guohui Cheng , Hanxing Gao , Yang Yang , Dan Li , Changtian Li , Yu Li
‡ Engineering Research Center of Chinese Ministry of Education for Edible and Medicinal Fungi, Jilin Agricultural University,
Changchun, China
§ College of Plant Protection, Shenyang Agricultural University, Shenyang, China
| Environment and Plant Protection Institute, Chinese Academy of Tropical Agricultural Sciences, Haikou, China
Corresponding author: Dan Li (lidan@jlau.edu.cn), Changtian Li (lct@jlau.edu.cn), Yu Li (yuli966@126.com)
Academic editor: Renan Barbosa
Received: 13 Jun 2022 | Accepted: 28 Aug 2022 | Published: 11 Oct 2022
Citation: An X, Cheng G, Gao H, Yang Y, Li D, Li C, Li Y (2022) First report of Cladobotryum verticillatum
(Ascomycota, Hypocreaceae) causing cobweb disease on Paxillus involutus. Biodiversity Data Journal 10:
e87697. https://doi.org/10.3897/BDJ.10.e87697
Abstract
Paxillus, a type of ectomycorrhizal fungi distributed widely in the world, is also an essential
category for researching bioactive substances and pharmacological functions. We
discovered fruitbodies of Paxillus involutus covered in a layer of white mycelium in 2020.
Cladobotryum verticillatum, a pathogenic fungus related to cobweb disease, was isolated
and identified based on morphological and phylogenetic features. Koch's postulates were
used to confirm the pathogenicity. The host range test revealed that C. verticillatum could
cause disease in all examined mushrooms except Ganoderma sichuanense. To our
knowledge, C. verticillatum is a new record species in China and a new pathogen on
Paxillus involutus.
Keywords
Paxillaceae, Hypocreaceae, mycoparasite, cobweb disease
‡,§ §,‡ ‡ | ‡ ‡ ‡
© An X et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Introduction
Paxillus Fr. is a genus in the family Paxillaceae, order Boletes. Its members form typical
ectomycorrhizal structures with a variety of wooden hosts (Wallander and Söderström
1999) and are distributed throughout the Northern Hemisphere in a variety of ecosystems
and habitats (Jargeat et al. 2014). Although it can cause severe anaphylactic reactions
when used improperly in cooking, Paxillus involutus is an important edible mushroom (Dai
et al. 2010, Sayyed and Hussain 2020). Recent studies on Pa. involutus have focused on
the symbiotic mechanism with related trees (Ma et al. 2014) and bioactive substances
(Antkowiak et al. 2003, Mikołajczyk and Antkowiak 2009, Lv et al. 2021). Additionaly, Pa.
involutus has pharmacological functions such as antioxidant, anticancer and antibacterial
activities of its metabolites. It can also disperse blood stasis and dehumidification
(Kalyoncu et al. 2010, Liu et al. 2018, Zhang et al. 2020). However, there have been few
reports of disease on it.
Mycoparasites are an important ecological category that interacts with other fungi
(including parasites and saprobes) (Gams et al. 2004, Sun et al. 2019), particularly the
genus Hypomyces/Cladobotryum (Hypocreaceae, Hypocreales), which can cause
mushroom cobweb diseases (Põldmaa 2000) and cause significant economic losses for
the global edible fungi industry (Bhatt and Singh 2002, Adie et al. 2006). Identification of
Hypomyces/Cladobotryum species depends heavily on the colour of the subicula and
perithecia as well as the characteristics of the ascospores (Zeng and Zhuang 2019).
Discomycetes (Rogerson and Samuels 1985), Boletales (Rogerson and Samuels 1989),
Polyporales (Rogerson and Samuels 1993), and Agaricales (Rogerson and Samuels 1994
), are all possible hosts for the genus. Currently, most of the reports about cobweb disease
occurred in the cultivation process and focused on artificial edible mushrooms. On the
contrary, we paid less attention to cobweb disease in the wild.
In August 2020, we discovered Paxillus involutus basidiocarps covered with a layer of
white mycelium in the Changbai Mountain Biosphere Reserve (CMBR), Jilin Province,
China. Broad-leaved forests with Quercus mongolica and Betula platyphylla as the primary
tree species supported the diseased fruitbodies. Crippled and decaying mushrooms were
collected (42°52′N, 127°81′E). In this paper, we present our findings from natural
infestations of Pa. involutus fruiting structures with strongly sporulating ascomycetous
mycopathogens. We isolated a fungus of C. verticillatum, a pathogen of cobweb disease,
and investigated its morphology and pathogenic potential. The internal transcribed spacer
(ITS), translation elongation factor 1-α (TEF1) and RNA polymerase II subunit (RPB2) were
combined and analysed to confirm the identification. We also conducted infection ability
tests using the fruiting bodies of other basidiomycetous species.
2An X et al
Materials and Methods
Fungal isolation
Diseased fruitbodies were cut into small pieces (5 mm × 5 mm × 5 mm) with a sterilised
scalpel, and infected tissues were immersed in 75% ethanol solution for 45 s before being
rinsed three times with sterilised water. Then, dried surface with sterile filter paper, placed
on Potato Dextrose Agar (PDA) plates containing 100 mg/l streptomycin sulphate,
incubated at room temperature, and transferred the culture to fresh PDA plates when the
fungal hyphae emerged and cultured the plates at 25°C for five days to allow the colonies
to sporulate fully. Use the single spore separation to get the pure cultures following the
method described by Chomnunti et al. (2014). The spore suspension was obtained by
washing the spores with 10 ml of sterile water into the Petri dishes and diluted to a final
concentration of 5×10 conidia/ml using a blood count plate. Then, the prepared spore
suspension (100 μl) was placed uniformly on Petri dishes containing a 2- to 3-mm-thick
layer of 2% water agar (WA) medium (20 g agar powder, 1000 ml water). After being
incubated at 25°C for 12 hours, single colonies were picked on a new PDA plate with a
sterile needle by observation under a microscope, thereby obtaining pure colonies. Store
the strains at 4°C in the Engineering Research Center of Edible and Medicinal Fungi,
Ministry of Education, Jilin Agricultural University (Changchun, Jilin, China).
Morphology
After activating the pathogen, picked some hyphae with the inoculation needle from the
culture and transferred them on a slide aseptically for morphological identification. Mycelial
samples with conidiophores and conidia were observed under a Zeiss Axio Lab A1 light
microscope (Carl Zeiss, Germany) and microscopic observations made with objectives of
10x, 20x, 40x and 100x oil immersion. All measurements and photographs were performed
using a Zeiss Imager A2 microscope with an Axiocam 506 colour camera and integrated
software. Microscopically, the characteristics of 30 conidia and conidiophores from the
isolates were observed. Morphological identification was performed using the Gams and
Hoozemans (1970) and Seifert and Gams (2011) methods.
DNA extraction and PCR amplification
The genomic DNA of the pathogen (C. verticillatum) was extracted from the mycelia of
colonies on PDA. Gene sequences of ITS, TEF1 and RPB2 were amplified by a
polymerase chain reaction (PCR) with the primer pairs of ITS4/ITS5 (White et al. 1990),
EF1-983F/EF1-2218R (Rehner and Buckley 2005) and RPB2-5F/RPB2-7Cr (Liu et al.
1999), respectively. The reaction included an initial denaturation at 95°C for 5 min, followed
by 35 cycles of denaturation at 95°C for 60 s, annealing at 55°C for 60 s for RPB2
(incrementally increasing by 2 s), 54°C for 50 s for ITS, 55°C for 60 s for TEF1, extension
at 72°C for 60 s and a final extension at 72°C for 10 min, using an Applied Biosystems
S1000 Thermal Cycler. PCR products were sent to the Changchun Branch of Sangon
Biotech Co., LTD for sequencing and confirmed by BLAST on NCBI (https://blast.
2
TM
First report of Cladobotryum verticillatum (Ascomycota, Hypocreaceae) causing ... 3
ncbi.nlm.nih.gov/Blast.cgi
). The strains and the NCBI Genbank accession numbers of DNA
sequences used in this work are listed in Suppl. material 1.
Phylogenetic analyses
BLASTn searches with the sequences were performed against NCBI to detect the most
closely-related species (http://www.blast.ncbi.nlm.nih.gov/). Phylogenetic trees were
constructed using ITS, TEF1 and RPB2 sequences, and phylogenetic analyses were
performed with the Maximum Likelihood (ML) and Maximum Parsimony (MP) methods.
Multiple alignments of all present sequences were automatically generated using MAFFT
V. 7.471, and manual improvements were made using BioEdit when necessary (Hall 1999,
Katoh and Standley 2013), and converted to nexus and NEX format through the software
Aliview (Larsson 2014). In the analysis, ambiguous areas were excluded and gaps were
regarded as missing data. The Maximum Parsimony phylogram (Swofford 2003) was
constructed with PAUP 4.0a 167 from the combined sequences of ITS, TEF1 and RPB2,
using 1000 replicates of heuristic search with random addition of sequences and
subsequent tbr (tree bisection and reconnection) branch swapping. Analyses were
performed with all characters treated as unordered and unweighted, with gaps treated as
missing data. Maximum Parsimony bootstrap proportion (MPBP) was used to test the
topological confidence of the resulting sequences with 1000 replications, each with ten
replicates of random addition of taxa. An ML phylogram was constructed with raxmlGUI 2.0
(Edler et al. 2020) with the sequence after alignment. The ML+ Rapid bootstrap program
and 1000 repeats of the GTRGAMMAI model were used to evaluate the bootstrap
proportion (BP) of each branch for constructing the phylogenetic tree.
Koch's postulates and host range test
The experiments were carried out in duplicate to confirm the pathogenicity of the strain
YW, according to Koch's postulates. We found Pa. involutus fruiting bodies in a birch forest
on the campus of Jilin Agricultural University and inoculated them with spore suspension
(50 μl) on caps. We observed the process in the wild and recorded changes in disease
symptoms for ten days. Select the fruiting body with white mycelium for fungal isolation.
Furthermore, the host range tests were investigated by inoculating it on to nine commercial
mushroom species: Pleurotus ostreatus, Hypsizygus marmoreus, Agrocybe aegerita,
Pleurotus geesteranus, Pleurotus citrinopileatus, Flammulina filiformis, Pleurotus
salmoneostramineus, Ganoderma sichuanense and Agaricus bisporus. All mushrooms
were grown on the substrate and kept in the growing station. Mushrooms were inoculated
with one droplet (50 μl) of spore suspension (5 × 10 unit/ml) mixed with Tween 80 on the
upper surface of caps when they reached 3 to 4 cm in diameter (Pl. ostreatus, Pl.
salmoneostramineus, Pl. geesteranus, Pl. citrinopileatus, Aga. bisporus, Hyps. marmoreus,
Agr. aegerita) or stipe (F. filiformis). For G. sichuanense, inoculated the spore suspension
on the solid layer under the pileus. Placed all mushroom bags at 25℃ and kept the air
humidity at 80% –90%. The incident was observed and photographed.
6
4An X et al
Results
Morphological characteristics
Colonies spread, appearing fluffy, lanose, tufted or fine linen, white, with suberect tufts
about 1–2 cm high, at length sinking and fading. Mycelium is branched, septate and
hyaline with rich inclusions. Hyaline conidiophores have one to three septa, are
verticillately or irregularly branched and their carriers are branched into two to five
phialides. Conidia are 10.6–16.2 × 6.6–11.1 µm, one-celled, smooth- and thin-walled,
hyaline, elliptical or elliptical-oblong, with protruding basal scars (Fig. 1). The
characteristics agreed with the description of C. verticillatum offered by Hoog (1978) and
Rogerson and Samuels (1989).
Phylogenetic analyses
The BLAST results showed that the ITS sequence of strain YW was 99.83% similar to
MT237489, the RPB2 sequence was 99.72% similar to FN868678, and the TEF1
Figure 1.
Field symptoms and morphological characteristics of Cladobotryum verticillatum. A Diseased
fruiting bodies in the wild; B Colony on PDA; C Conidiophores with whorled and single
phialides; D-F Tapered conidiogenous cells form singly or in whorls; G Conidiogenous cells; H
Conidia; I, J chlamydospore; K, L Healthy mushrooms in the wild; M-P Mushrooms artificially
inoculated with pathogens at 24 h, 48 h, 72 h and seven days, respectively. Bars: C–G, I, J =
20 μm; H = 10 μm
First report of Cladobotryum verticillatum (Ascomycota, Hypocreaceae) causing ... 5
sequence was 99.02% similar to FN868742, respectively. The dataset for phylogenetic
analyses contained 27 ITS sequences, representing 19 species, choosing Trichoderma
virid as the outgroup taxon. Multi-locus data were concatenated, which comprised 2554
characters with ITS 597 characters, TEF1 888 characters and RPB2 1069 characters.
Estimated base frequencies were as follows: A = 0.233413, C = 0.296140, G = 0.248170
and T = 0.222278; substitution rates AC = 1.489328, AG = 3.647092, AT = 1.111646, CG =
0.925803, CT = 7.920581 and GT = 1.000000. In the resulting tree (Fig. 2), the combined
phylogenetic analyses using ITS, TEF1 and RPB2 showed that our strains were clustered
with the sequences of Hyp. armeniacus (the teleomorph name of C. verticillatum) in a
branch with high statistical support (MPBP/MLBP = 100%/100%). The phylogenetic tree
indicated that the pathogen was Hyp. armeniacus. However, we did not observe any
characteristics of the teleomorph phase. Thus we named it C. verticillatum. The branch of
YW was most related to the clade that contains C. cubitense and C. semicirculare. The MP
and ML trees showed similar topologies with high statistical support values, and the MP
tree was selected as the representative phylogeny (Fig. 2). The bootstrap values (BP) ≥
50% were shown on the branches. The sequences of Pa. involutus have been submitted
on Genbank with accession numbers ITS-OL659295, TEF1-OP243230 and GPD-
OP243231.
Fruiting body infection tests
The pathogenicity test revealed that all the inoculated Pa. involutus exhibited first
symptoms after 24 hours, with taupe lesions appearing on the surface of the gills (Fig. 1).
White hyphae then appeared and spread through the gills of Pa. involutus (Fig. 1). The
pileus and stipe surfaces were covered in fluffy white mycelium that resembled spider
webs 72 hours after inoculation (Fig. 1). After seven days of incubating at 25°C, pathogenic
mycelium formed white spots on the surface of fruiting body, and the casing shrivelled and
wilted (Fig. 1). Ten days later, the gills had decayed and turned black, brown water droplets
had exuded from the collapsed fruiting bodies, and the pathogen's white hypha had
vanished. During this process, we were able to re-isolate and identify the pathogen from
the infected fruiting bodies and obtained the strain YW-F, which was stored at Jilin
Agricultural University. This species was identified as the same as YW, and the ITS, RPB2
and TEF1 sequences have been submitted on Genbank. The accession numbers are
shown in Suppl. material 1.
The strain YW was tested on nine commercial mushroom types and found to be capable of
infecting all but G. sichuanese. After inoculating the spore suspension on the stipes or caps
of fruiting bodies, the hyphae began to grow. Typical cobweb signs, such as small brown
spots, were seen 1–3 days post-inoculation (dpi). The white mycelia were then visible, and
the fruiting bodies were rotting and covered in massive spores after 3-5 days. Finally, the
mushrooms wilted and rotted, mirroring the characteristics of the field sample (Fig. 1).
However, the extent and duration of wilting of edible fungi varied due to host differences
(Fig. 3).
6An X et al
Figure 2.
Maximum Parsimony phylogram reconstructed from the combined sequences of ITS, TEF1
and RPB2, showing the species’ phylogenetic position. Bootstraps above 50% (MP left/ML
right) are given, respectively. The new sequences are shown in bold.
First report of Cladobotryum verticillatum (Ascomycota, Hypocreaceae) causing ... 7
Figure 3.
Disease development on different mushrooms after inoculating Cladobotryum verticillatum. A–
D Pictures of Hypsizygus marmoreus in healthy condition, 1 dpi, 2 dpi and 3 dpi; E–H Pictures
of Agrocybe aegerita in health, 1 dpi, 2 dpi and 3 dpi; I–L Pictures of Flammulina filiformis in
health, 1 dpi, 2 dpi and 4 dpi; M–P Pictures of Pleurotus ostreatus in health, 1 dpi, 2 dpi and 8
dpi; Q–T Pictures of Pleurotus salmoneostramineus in health, 1 dpi, 2 dpi and 7 dpi; U–X
Pictures of Pleurotus citrinopileatus in health, 1 dpi, 2 dpi and 3 dpi; Y–b Pictures of Pleurotus
geesteranus in health, 1 dpi, 2 dpi and 3 dpi; C–f Pictures of Agaricus bisporus in health, 1
dpi, 2 dpi and 3 dpi.
8An X et al
Disease processions on F. filiformis, Aga. bisporus, Agr. aegerita, Hyps. marmoreus and
Pl. citrinopileatus, were usually completed within four days and caused serious damage.
White hyphae were visible on the first day post-inoculation and spread quickly, causing the
mushrooms to become soft and brown. Symptoms of Aga. bisporus and Hyp. marmoreus
were similar, with brown spots and mycelia visible at the inoculation site. Mycelia
eventually covered the cap of Aga. bisporus and spread to the stalk-cap junction,
resembling a spider's web. Pleurotus citrinopileatus, unlike others, had no brown spots on
the cap, but as the hardness decreased or even disappeared, it eventually fell in clusters
and turned brown.
Although Pl. ostreatus, Pl. salmoneostramineus and Pl. geesteranus displayed symptoms
earlier, the progression was slow and prolonged. The hyphae grew on the first day, but no
lesions were visible. Hyphae continued to stretch, causing the mushrooms to stop growing
and atrophy, and the surface of the fruitbodies to become covered in white mycelia. When
inoculated on the primordium or cap, G. sichuanense showed less sensitivity or high
resistance to the pathogen when compared to other edible fungi.
Discussion
Based on morphological and molecular characteristics, we isolated a fungal pathogen from
diseased Pa. involutus and identified it as C. verticillatum. It was originally described by
Heinrich and named by Hughes (1958). Amongst members of the Hypocreales parasitising
agaricomycetes from temperate to tropical latitudes (Põldmaa 2000, Põldmaa and
Samuels 2004), C. verticillatum is often found in temperate regions, Colombia, Europe
(England, France, Germany, Sweden), Canada and the United States (Rogerson and
Samuels 1994), but has never been reported in China before, nor on Pa. involutus.
Hypomyces/Cladobotryum species that live on Polyporales may have a lower host-
selection than others (Tamm and Põldmaa 2013). Cladobotryum arnoldii (Hyp. lithuanicus)
and Hypomyces hyalinus, for example, were strictly host-specific, living on Lactariustor
minosus and Amanita, respectively. Although C. verticillatum occurs mostly on Russula,
Lactarius and Agaricus ( Hughes 1958, Gams and Hoozemans 1970, Põldmaa 2011), it
does not have such strong host specificity or can even grow on the substratum of the
actual host when the hosts are destroyed (Rogerson and Samuels 1993, Põldmaa and
Samuels 1999). The host range test in this study, however, revealed that it could not cause
disease in G. sichuanense. McKay et al. (1999) demonstrated that the anamorphs of
temperate, red perithecial Hypomyces are the causative agents of cobweb disease, which
cause epidemics in mushroom farms. Cladobotryum verticillatum can also cause cobweb
disease on Aga. bisporus, causing a botryte-like disease (verticillium wilt) in edible fungi
(Fletcher and Gaze 2007). Unlike the common pathogenic species of cobweb disease, C.
mycophilum, C. dendroides and C. protrusum, C. verticillatum occasionally produces
sclerotium, and the mycelia dissolve with increased time on PDA. Furthermore, C.
verticillatum does not produce pink or red pigments throughout the course of infection,
remaining white or light ochre yellow. A large number of conidia can only be observed in
the later stage of infection. However, the pathogens for cobweb disease are generally
First report of Cladobotryum verticillatum (Ascomycota, Hypocreaceae) causing ... 9
highly adaptable to a wide range of pH (Grogan and Gaze 2000, Adie 2001, Zhang et al.
2015). Therefore, they can better adapt to a variety of habitats and hosts, thus occupying a
higher ecological niche.
Acknowledgements
We thank the Engineering Research Center of Edible and Medicinal Fungi, Ministry of
Education, Jilin Agricultural University, China, for providing the laboratory and equipment
for the duration of this project. We thank Lan Yao and Jianhua Lv from Jilin Agricultural
University for the sample collection. We thank Yang Wang at Shenyang Agricultural
University for his help in the process of mushroom hunting for supplementary experiments.
Funding program
(i) U20A2046 funded by the National Natural Science Foundation of China, (ii) the
earmarked fund for CARS-20 (Edible Mushroom) funded by the Ministry of Agriculture and
Rural Affairs, PRC, (iii) 1630042022003 funded by the Ministry of Finance of China and (iv)
322QN365 funded by the Natural Science Foundation of Hainan Province, China.
Author contributions
Xiaoya An did the experiment, analysed the data and wrote the manuscript. Guohui Cheng,
Hanxing Gao and Yang Yang collected the sample, isolated the fungi and performed the
phylogeny analysis. Dan Li, Changtian Li and Yu Li conceived and coordinated the study.
All authors contributed critically to the drafts and gave final approval for publication.
Conflicts of interest
The authors declared that they have no conflict of interest.
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Supplementary material
Suppl. material 1: Strains and specimens of Cladobotryum/Hypomyces included in the
phylogenetic analyses
Authors: Xiaoya An
Data type: GenBank accession numbers
Brief description: Accession numbers include details such as locality, isolate numbers of the
sequences used for this study
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First report of Cladobotryum verticillatum (Ascomycota, Hypocreaceae) causing ... 13
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