Infection Process and Genome Assembly Provide Insights into the Pathogenic Mechanism of Destructive Mycoparasite Calcarisporium cordycipiticola with Host Specificity

Abstract

Calcarisporium cordycipiticola is the pathogen in the white mildew disease of Cordyceps militaris, one of the popular mushrooms. This disease frequently occurs and there is no effective method for disease prevention and control. In the present study, C. militaris is found to be the only host of C. cordycipiticola, indicating strict host specificity. The infection process was monitored by fluorescent labeling and scanning and transmission electron microscopes. C. cordycipiticola can invade into the gaps among hyphae of the fruiting bodies of the host and fill them gradually. It can degrade the hyphae of the host by both direct contact and noncontact. The parasitism is initially biotrophic, and then necrotrophic as mycoparasitic interaction progresses. The approximate chromosome-level genome assembly of C. cordycipiticola yielded an N50 length of 5.45 Mbp and a total size of 34.51 Mbp, encoding 10,443 proteins. Phylogenomic analysis revealed that C. cordycipiticola is phylogenetically close to its specific host, C. militaris. A comparative genomic analysis showed that the number of CAZymes of C. cordycipiticola was much less than in other mycoparasites, which might be attributed to its host specificity. Secondary metabolite cluster analysis disclosed the great biosynthetic capabilities and potential mycotoxin production capability. This study provides insights into the potential pathogenesis and interaction between mycoparasite and its host.

Full text

Fungi
Journal of
Article
Infection Process and Genome Assembly Provide Insights into
the Pathogenic Mechanism of Destructive Mycoparasite
Calcarisporium cordycipiticola with Host Specificity
Qing Liu 1,2, Yanyan Xu 1, Xiaoling Zhang 1, Kuan Li 1, Xiao Li 1,2, Fen Wang 1, Fangxu Xu 3and Caihong Dong 1, *


Citation: Liu, Q.; Xu, Y.; Zhang, X.;
Li, K.; Li, X.; Wang, F.; Xu, F.; Dong, C.
Infection Process and Genome
Assembly Provide Insights into the
Pathogenic Mechanism of Destructive
Mycoparasite Calcarisporium
cordycipiticola with Host Specificity. J.
Fungi 2021,7, 918. https://doi.org/
10.3390/jof7110918
Academic Editors: Chiung-Yu Hung
and Yufeng Wang
Received: 30 September 2021
Accepted: 25 October 2021
Published: 28 October 2021
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
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iations.
Copyright: © 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
1State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences,
Beijing 100101, China; liuqingfungi@gmail.com (Q.L.); xuyanyan@im.ac.cn (Y.X.); zhangxl@im.ac.cn (X.Z.);
likuan@im.ac.cn (K.L.); lixmushroom@gmail.com (X.L.); wangfen@im.ac.cn (F.W.)
2University of Chinese Academy of Sciences, Beijing 100049, China
3Experimental Teaching Center, Shenyang Normal University, Shenyang 110034, China; dzb@synu.edu.cn
*Correspondence: dongch@im.ac.cn
Abstract:
Calcarisporium cordycipiticola is the pathogen in the white mildew disease of Cordyceps
militaris, one of the popular mushrooms. This disease frequently occurs and there is no effective
method for disease prevention and control. In the present study, C. militaris is found to be the only
host of C. cordycipiticola, indicating strict host specificity. The infection process was monitored by
fluorescent labeling and scanning and transmission electron microscopes. C. cordycipiticola can invade
into the gaps among hyphae of the fruiting bodies of the host and fill them gradually. It can degrade
the hyphae of the host by both direct contact and noncontact. The parasitism is initially biotrophic,
and then necrotrophic as mycoparasitic interaction progresses. The approximate chromosome-level
genome assembly of C. cordycipiticola yielded an N50 length of 5.45 Mbp and a total size of 34.51 Mbp,
encoding 10,443 proteins. Phylogenomic analysis revealed that C. cordycipiticola is phylogenetically
close to its specific host, C. militaris. A comparative genomic analysis showed that the number of
CAZymes of C. cordycipiticola was much less than in other mycoparasites, which might be attributed to
its host specificity. Secondary metabolite cluster analysis disclosed the great biosynthetic capabilities
and potential mycotoxin production capability. This study provides insights into the potential
pathogenesis and interaction between mycoparasite and its host.
Keywords:
Calcarisporium cordycipiticola;Cordyceps militaris; white mildew disease; infection process;
genome sequencing; host specificity; necrotrophy; mycotoxin
1. Introduction
Cordyceps militaris (L.) Fr., one of the famous edible and medicinal fungi, has been
widely used as an herbal drug and tonic in East Asia. The fruiting bodies have been
cultivated artificially and it is estimated that the annual value of production of C. militaris is
about 10 billion RMB in China [
1
]. During large-scale cultivation, fungal diseases frequently
occur [
2
] and white mildew disease is one of the damaging diseases of C. militaris (Figure S1),
resulting in a significant reduction in production and economic losses [3].
Calcarisporium cordycipiticola Jing Z. Sun, Cai H. Dong, Xing Z. Liu & K.D. Hyde, was
firstly isolated from the infected fruiting body of C.militaris and belongs to the family
Calcarisporiaceae of the order Hypocreales within the fungal phylum Ascomycota [
4
,
5
]. It
was confirmed that C. cordycipiticola is the pathogen of white mildew disease of C. militaris
by Koch’s postulates [
3
]. Some fungi that are consistently associated with other fungi
are named as fungicolous (or mycophilic) fungi [
6
]. Biological characteristics of these
fungicolous fungi have been reported before [
3
]. White colonies formed on the fruiting
bodies of C. militaris (Figure S1) and a large number of conidia were produced in a short
time, resulting in a rapid spread of the pathogen. There was no information on the infection
process and pathogenic factors, which were crucial for disease prevention and control.
J. Fungi 2021,7, 918. https://doi.org/10.3390/jof7110918 https://www.mdpi.com/journal/jof

J. Fungi 2021,7, 918 2 of 25
The interactions between fungicolous fungi and their hosts have been of considerable
interest for a long time. Fungicolous fungi can infect their hosts via producing specialized
structures such as appressorium and haustorium, and kill hosts through the secretion of
enzymes or antibiotics, or by competition for nutrition and niches [
7
]. As for the fungal
diseases of mushroom, some studies were performed on the pathogens of Agaricus bisporus,
including wet bubble caused by Hypomyces perniciosus [syn: Mycogone perniciosa] [
8
], dry
bubble by Lecanicillium fungicola [
9
], cobweb disease by Cladobotryum dendroides [
10
] and
green mold by Trichoderma aggressivum [
11
]. H. perniciosus produced specific structures and
invaded the hyphae of A. bicospora [
12
]. With electron microscopy and cytohistological
techniques, Zhou found that H. perniciosus infected A. bisporus through the secretion of
cell wall degradation enzymes and mechanical pressure from the appressorium [
12
]. L.
fungicola did not produce a specialized structure, however, spores and hyphae of L. fungicola
adhered to the surface of the host A. bisporus, and then protruded into the hyphae of the
host [
13
]. Whether C. cordycipiticola can produce the specialized structure during infection,
and how it interacted with the host C. militaris are our major concerns.
The parasitic lifestyle of some species of Trichoderma was related to the expansion of
genes encoding fungal cell wall degrading enzymes and secondary metabolites (SMs) [
14
].
Comparative genomic and transcriptomic analyses revealed that there were significant
expansions for protein families of transporters, cell wall degrading enzymes, cytochrome
P450, and SMs in the genome of H. perniciosus, which were essential for the mycopara-
sitism of A. bisporus [
8
,
15
]. It was found that gene families with significant expansion in
C. dendroides were mainly associated with the major facilitator superfamily (MFS) trans-
porter, ankyrin repeats, and NACHT [NAIP (neuronal apoptosis inhibitory protein), CIITA
(MHC class II transcription activator), HET-E (incompatibility locus protein from Podospora
anserina) and TP1 (telomerase-associated protein)] domains, whereas P450 members were
contracted noticeably [
10
]. Transcriptomic analysis of the interactions between A. bis-
porus and L. fungicola indicated that several oxidoreductases, cell wall degrading enzymes,
ATP-binding cassettes (ABC) and MFS transporter proteins may play roles in the pathosys-
tems [
16
]. Omics analysis will be helpful for the identification of pathogenicity-related
genes and the characterization of pathogen-mushroom interactions.
Calcarisporiaceae and Cordycipitaceae are sister families in Hypocreales [
5
]. There is
also much to be learned about the nature and evolution of interactions of Calcarisporium
with host Cordyceps because of the relatively close phylogenetic relationship. Comparative
genomic and phylogenomic analyses will identify the evolutional relationship, and genomic
basis of their differing physiologies.
Mycotoxins are fungal SMs which have been associated with toxic effects to verte-
brates, and mycotoxin contamination of food and feed is a threat to the health of humans
and animals worldwide [
17
]. The safety of C. militaris has been confirmed since the genome
did not contain genes for known mycotoxins [
18
]. Sometimes the fruiting bodies of C.
militaris infected by C. cordycipiticola are mixed with normal C. militaris for sale, so whether
C. cordycipiticola can produce mycotoxins is our concern. The genome data allows us to
make a full view of C. cordycipiticola genes involved in biosynthesis of SMs for comparison
with known mycotoxins such as citrinin, zearalenone, hypothemycin and solanapyrone.
In the present study, the genetic transformation and red fluorescent protein (RFP)
labeling of C. cordycipiticola were developed. The infection process of C. cordycipiticola
to the fruiting bodies of C. militaris was monitored by RFP-labeling, scanning electron
microscopy (SEM) and transmission electron microscopy (TEM). The high-quality genome
of C. cordycipiticola was sequenced by single molecule real-time (SMRT) and Illumina’s
methods. Comparative genomic and SM cluster analyses were performed. It will be helpful
for revealing the interaction between the pathogen and host, prevention and control of
this disease.

J. Fungi 2021,7, 918 3 of 25
2. Materials and Methods
2.1. Strains and Culture Conditions
C. cordycipiticola strain CGMCC 5.2193 was isolated from the diseased fruiting bodies
of C. militaris strain CGMCC 3.16320 in this laboratory and inoculated on Potato Dextrose
Agar (PDA) medium at 25
◦
C. Escherichia coli strain DH5
α
(Tiangen Biotech Co., Ltd.,
Beijing, China) was used for routine sub-cloning and cultured in Luria-Bertani medium at
37
◦
C. Agrobacterium tumefaciens strain AGL-1 (Tiangen Biotech Co., Ltd., Beijing, China)
was used for the introduction of DNA into filamentous fungi and cultured at 28
◦
C in Yeast
Extract and Beef medium supplemented with 50 µg/mL carbenicillin.
2.2. Agrobacterium tumefaciens-mediated Transformation (ATMT) of
Calcarisporium cordycipiticola
For antibiotic sensitivity assays, conidia of C. cordycipiticola were spread on PDA
plates with different concentrations of geneticin, glufosinate-ammonium, bleomycin and
hygromycin B, respectively, and then incubated at 25
◦
C for 7 days. ATMT of C. cordy-
cipiticola was performed following the method of species Calcarisporium arbuscula with
some modifications [
19
]. The concentrations of acetosyringone (AS, 200, 400 and 600
µ
M),
A. tumefaciens AGL-1 (OD
600
0.2, 0.4, 0.6 and 0.8), C. cordycipiticola conidia (10
5
, 10
6
and
107conidia/mL), and days for co-culture (2, 3 and 4 days) were optimized.
2.3. Transformation of RFP-Tagged Calcarisporium cordycipiticola and Microscopy
Plasmid pFGL815-neoR-tubCp-RFP (kindly provided by Professor Xuming Mao, In-
stitute of Pharmaceutical Biotechnology, Zhejiang University) containing resistant genes
to kanamycin and geneticin, and RFP gene was transformed into C. cordycipiticola. Trans-
formants expressing RFP were selected by PCR and fluorescent with ZEISS LSM700 laser
scanning confocal microscopy (LSCM) (Zeiss, Jena, Germany).
The fruiting bodies of C. militaris strain CGMCC 3.16320 were cultivated according to
our procedure [
20
]. Wild type of C. cordycipiticola and transformant expressing RFP were
inoculated on the surface of fruiting bodies, respectively and the infection process was
observed by LSCM after inoculation for 0, 3, 7, 15, 23 and 30 days. The red fluorescence
was detected with an emission filter (excitation wavelength of 555 nm and emission
wavelength of 580 nm). Photographs were taken with a Zeiss AxioCam MRc 5 digital
camera
(Göttingen, Germany)
attached to an Imager A1 microscope (Göttingen, Germany).
Dual culture on PDA plates was performed between C. cordycipiticola labeled by RFP
and C. militaris, which were inoculated at opposite sides of a 9-cm-diameter Petri plate.
Sterile cover glasses were inserted and then incubated at 20
◦
C in darkness for 10 days.
After two colonies contacted each other at about 24–48 h, the cover glasses were pulled out
and observed by LSCM.
For observation by SEM, the samples (0.5 cm in length) after being infected for 3 d
were processed following the method of Nunes et al. [
13
] with modifications. The samples
were fixed in 2.5% glutaraldehyde in 0.05 M phosphate buffered saline (pH 7.2) for 8 h
at 4
◦
C, and then washed with deionized water for 7 min. Samples were then washed
again in cacodylate buffer for 8 min, dehydrated in graded ethanol and dried in a fume
hood using critical point dryers (Autosamdri
®
931, Tousimis, MD, USA) with CO
2
. Finally,
samples were sputter-coated with gold by ion sputter coater (ISC150, SuPro Instruments,
Shenzhen, China
) under vacuum of lower than 1–2 Pa, a voltage of 110 V, frequency of
50/60 Hz, current of 10 mA and deposition time of 60 s and observed under SEM (SU8010,
Hitachi, Tokyo, Japan). Secondary electron imaging mode was used with an accelerating
voltage of 3–5 kV and effective working distance of 8 mm.
For observation by TEM, the samples (0.3 cm in length) being infected for 3 days were
processed following the method of Ayache et al. [
21
] with modifications. The samples were
fixed for 30 min in 2.5% glutaraldehyde and 1% osmium for 2 h. After washing three times
for 10 min in 0.1 M phosphate buffered saline, samples were dehydrated in graded ethanol.
Samples were then infiltrated with resin:70% ethanol (1:1) for 2 h at room temperature, in

J. Fungi 2021,7, 918 4 of 25
fresh pure resin for 1 h and then in fresh pure resin for overnight on an agitating device.
Polymerization was carried out in an oven at 60
◦
C for 24 h. Sections thickness of 20–30 nm
were cut transversely under ultrathin microtome (EM UC7, Leica, Weztlar, Germany).
Finally, the samples were observed under TEM (HT7800, Hitachi, Tokyo, Japan) with an
accelerating voltage of 80 kV and nominal resolution of 0.2 nm.
2.4. Infection of Calcarisporium cordycipiticola to the Other Species of Cordycipitaceae
The synnemata of some species of Cordycipitaceae, including Beauveria bassiana
CGMCC 3.3575, Cordyceps tenuipes DCH268 and Isaria cicadae DCH463, were cultured
following the methods of our previous report [
22
]. C. cordycipiticola strain CGMCC 5.2193
was cultured on PDA plates under dark at 25
◦
C for 7–10 days and discs of mycelia (0.5 cm
in diameter) were cut with a sterile blade. Six pieces of mycelia were inoculated on the
surface of synnemata in each cultivation bottle and cultured for 30 days.
2.5. DNA/RNA Preparation
The single conidium isolation CC01 from C. cordycipiticola strain CGMCC 5.2193
was cultivated in Potato Dextrose Broth at 25
◦
C and 150 rpm for 3 days. Mycelia were
collected from the media by centrifugation at 5310
×g
for 20 min and ground in liq-
uid nitrogen. Genomic DNA was extracted using a modified cetyltrimethylammonium
bromide method [
23
]. For transcriptome analysis, C. cordycipiticola was grown on PDA
medium at 25
◦
C for 7 days. Total RNA was extracted using E.Z.N.A.
™
Plant Kit (Omega,
Stamford, CT, USA).
2.6. Genome Sequencing and Assembly
The C. cordycipiticola genome was sequenced by Illumina and SMRT methods [
24
]
by Nextomics Biosciences Co., Ltd. (Wuhan, China). DNA libraries with 400 bp and
20 kb inserts were constructed. The DNA sample of 400 bp library was quantified using
NanoDrop 2000 (Thermo Fisher Scientific, Waltham, MA, USA), Qubit 3.0 (Invitrogen,
Carlsbad, CA, USA), 2100 Bioanalyzer (Agilent, Santa Clara, CA, USA) and subjected to
paired-ended 150 bp sequencing by Illumina HiSeq X10. The sequencing data (filtered
reads: 5.50 Gbp, sequencing depth: 155
×
) were used to estimate the genome size, repeat
content, and heterozygosity. The 20 kb library was quantified by the same ways, sequenced
by SMRT using PacBic Sequel (Pacific Biosciences of California, Inc. Menlo Park, CA, USA),
and the sequencing data (filtered reads: 5.93 Gbp, sequencing depth: 170
×
) were assembled
by Canu v. 1.7 [
25
] with default parameters. Finally, Illumina reads were used for error
correction and gap filling with SOAPdenovo GapCloser v. 1.12 [
26
]. The completeness
of the assembled genome was evaluated using CEGMA [
27
] and BUSCO v. 3 [
28
]. The
genome was deposited in the NCBI BioProject database https://www.ncbi.nlm.nih.gov/
sra/PRJNA766243, accessed on 15 September 2021) under accession number PRJNA766243.
Transfer RNAs were predicted using tRNAscan-SE 2.0 [
29
], whereas rRNAs and
noncoding RNAs were identified using RNAmmer 1.2 [
30
] and the the Rfam database [
31
].
RNA samples were sequenced by the Illumina method by Biomarker Technology Co.,
Ltd. (Beijing, China). Clean data were used for genome-assisted assembly. Quantification
of gene expression levels was estimated by fragments per kilobase of per million mapped
fragments (FPKM). The initial Illumina reads are available at the Sequence Read Archive
of NCBI under accession number PRJNA766340.
2.7. Assembly and Annotation of Mitogenomes
Blast analysis was performed against C. cordycipiticola CC01 genome data using C.
militaris mitogenome (KP719097) as the query. The mitogenome of C. cordycipiticola was
first annotated automatically using the MFannot tool (http://megasun.bch.umontreal.
ca/cgi-bin/mfannot/mfannotInterface.pl, accessed on 18 October 2020) based on genetic
code, and then individually checked. Comparative analysis of mitochondrial genome was
performed via online software Kalign [32].

J. Fungi 2021,7, 918 5 of 25
2.8. Repetitive Sequences, Transposases, Repeat-Induced Point Mutation and Whole Genome DNA
Methylation Analysis
Tandem repeats were identified by Tandem Repeats Finder v. 4.04 [
33
] by searching
against the Repbase database [
34
]. Transposon elements (TEs) were excavated strictly
using two software, including a de novo software RepeatModeler v. 1.0.11 (http://www.
repeatmasker.org, accessed on 21 October 2020) and database-based software RepeatMasker
v. 4.0.9 [
35
]. All the parameters were set as default. Repeat-induced point mutation (RIP)
index was determined with the software RIPCAL by reference against the non-repetitive
control families [
36
]. The transposons/retrotransposons encoding fragments were classified
by blastp analysis against the Repbase. Whole-genome DNA modification detection and
motif analysis were performed according to the method of Blow et al. [
37
] using the PacBio
SMRT software v. 2.2.3 (http://www.pacb.com, accessed on 23 October 2020).
2.9. Genome Annotations
Protein-encoding genes were annotated by a combination of three independent ab
initio methods, GeneMark v. 4.30 [
38
], SNAP [
39
], and GlimmerHMM [
40
]. Transcriptome
data were incorporated into PASA (program to assemble spliced alignments) to improve
the quality of C. cordycipiticola annotation [
41
]. Functional annotations for all predicted
gene models were made using multiple databases, including TrEMBL, Swiss-Prot, nr,
KEGG, GO, and KOG by blastp with an E-value of
≤
1
×
10
−20
. Domain-calling analyses of
protein-encoding genes were performed using the Pfam database [42] and HMMER [43].
2.10. Functional Annotation of Pathogenicity-Related Genes
The secretomes were identified based on recognizing the signal peptide and no trans-
membrane sequences. Proteins were considered to be secreted proteins if the signal
peptides were identified by two methods, SignalP 5.0 [
44
] and TargetP 2.0 [
45
], and trans-
membrane sequences were not identified by at least one of the methods among
SignalP 5.0,
TMHMM 2.0 [
46
], and WoLF PSORT [
47
]. Effectors were predicted by EffectorP v. 2.0 [
48
].
Candidate pathogenic factors were predicted by sequence alignment against the pathogen-
host interactions (PHI) database v. 3.5, by blastp with an E-value of <1 ×10−20 [49].
2.11. Phylogenomic Analysis
Phylogenomic analysis was performed using 32 fungal genomes with Ustilago maydis
as an outgroup; 631 single-copy orthologs of the 32 fungi were identified using BUSCO v3
and were concatenated into one sequence using Sequence Matrix-Windows 1.7.8 [
50
]. The
sequence alignment was done using MUSCLE v13 [
51
]. The PROTGAMMAILGF model
was selected by ProtTest v. 3.4 [
52
]. A maximum likelihood (ML) phylogenomic tree was
built using the 631 concatenated amino acid sequences with RAxML v. 8 [
53
]. Trees were
figured in Tree v. 1.4.2 [
54
]. Bootstrap values higher than 50% from RAxML were indicated.
The divergence time between species was estimated with the program r8s [
55
] using a
Langley-Fitch model by calibration with the origin of the Ascomycota at 500 to 650 million
years ago (MYA) [56].
2.12. Comparison Analysis of Carbohydrate-Active Enzymes
Carbohydrate-active enzymes (CAZymes) were identified using the dbCAN2 meta
server (http://bcb.unl.edu/dbCAN2/, accessed on 30 October 2020) with HMMER [
43
].
Family-specific HMM profiles were downloaded from dbCAN CAZymes database [
57
].
The executable file hmmscan and the hmmscan-parser script (E-value
≤
1
×
10
−17
and
coverage > 45%) provided by dbCAN were used to generate and extract the searching
results. CAZymes of 13 other fungal species were analyzed with the same methods and
compared with those of C. cordycipiticola.

J. Fungi 2021,7, 918 6 of 25
2.13. Analysis of Genes Involved in Secondary Metabolism
SM clusters were predicted with fungal anti-SMASH 5.0 [
58
]. The core genes were
annotated using stand-alone blast against Swiss-Prot and pfam databases. Domains were
predicted using PKS/NRPS analysis website (http://nrps.igs.umaryland.edu/, accessed
on 20 May 2021) and extracted manually. For phylogenetic analysis, the domain sequences
were aligned by MUSCLE v13 and the tree was generated using a Dayhoff model with
1000 bootstrap replications, and pair-wise deletions for gaps or missing data. A neighbor-
joining (NJ) tree was built by MEGA 7 [
59
] based on the ketoacyl CoA synthase (KS) amino
acid sequences.
2.14. Experimental Design and Statistical Analysis
A flow chart of the whole experimental design was shown in Figure S2. For antibi-
otic sensitivity assays, optimization of ATMT and infection experiments, three technical
replicates were used in a randomized design. The data were analyzed by one-way analysis
of variance (ANOVA). The Tukey’s test was used for comparing more than two datasets,
whereas the Student’s t-test was used for pairwise comparisons. The asterisks above the
error bars represented means that were statistically different at: * p< 0.05; ** p< 0.01;
*** p< 0.001.
Data analyses were completed with SPSS 19.0 (SPSS, Inc., Chicago, IL, USA)
and OriginPro 8.5 (OriginLab Corporation, Northampton, MA, USA). The graph was
constructed using GraphPad Prism 8.0 (GraphPad Software, Inc., San Diego, CA, USA).
3. Results
3.1. Development of ATMT for Efficient Transformation of Calcarisporium cordycipiticola
and RFP-Labeling
Antibiotic susceptibility testing showed that C. cordycipiticola was sensitive to geneticin
and glufosinate-ammonium, and insensitive to bleomycin and hygromycin B (Figure S3).
The high transformation efficiency of ATMT was observed with C. cordycipiticola conidia
concentration of 1
×
10
7
conidia/mL, co-cultured for 3 days, A. tumefaciens OD
600
of
0.8 and AS concentration of 400
µ
M (Figure S4). Transformants expressing RFP were
selected by PCR validation (Figure S5A) and strong red fluorescence was observed by
LSCM (Figure S5B).
3.2. Infection Process of Calcarisporium cordycipiticola to the Fruiting Body of Cordyceps militaris
The visible mycelia of C. cordycipiticola on the fruiting bodies of C. militaris were ob-
served at 3 days post-inoculation (dpi, Figure 1A1,A2) and the section of fruiting bodies of
C. militaris showed as a pale yellow based on the handbook of Kornerup and Wanscher [
60
]
(Figure 1B2), being consistent with the control (Figure 1B1). Then, the mycelia of C. cordy-
cipiticola proliferated on the surface of fruiting bodies of C. militaris and covered them
completely at about 20–30 dpi, and the color of the surface of the fruiting bodies showed
as greyish-yellow at 30 dpi (Figure 1A3–A6). On the contrary, the color of the interior of
the fruiting bodies became deeper gradually and showed as brown with liquid flowing
out at last (Figure 1B3–B6). More and more intense red fluorescence was observed as the
infection progressed (Figure 1C2–C6). The pathogenic hyphae can invade and then fill the
fruiting bodies of C. militaris within 30 days (Figure 1C1–C6,E1–E6).

J. Fungi 2021,7, 918 7 of 25
Figure 1.
Infection process of Calcarisporium cordycipiticola to the fruiting bodies of Cordyceps militaris
revealed by RFP labeling. (
A1
–
A6
), (
B1
–
B6
) and (
C1
–
C6
) the surface, longitudinal and cross sections
of the fruiting bodies of C. militaris after being infected. (
C1
–
C6
), (
D1
–
D6
) and (
E1
–
E6
) observed
by RFP labeling. (
C1
–
C6
) fluorescent micrograph of the infected samples. (
D1
–
D6
) brightfield
micrograph of C1–C6. (
E1
–
E6
) overlay of fluorescent and brightfield micrographs. Scale bar = 2 mm
in A1-B6 and 200 µm in C1–E6.
The dual culture of C. militaris and the RFP transformant of C. cordycipiticola on PDA
plates confirmed that the hyphae of the two species were in a mixed arrangement, and there
was no hyphal intertwinement, no haustorium and appressorium formation (Figure S6).
Neither evident growth inhibition nor mycelia shrinking of C. militaris were observed even
after being cultured for 10 days.
The samples at 3 dpi (Figure 2A) were observed by SEM and TEM. It is easy to
distinguish the hyphae of these two fungi under SEM since the diameter of C. militaris
hyphae is 2.02
±
0.39
µ
m, almost twice of C. cordycipiticola (1.01
±
0.20
µ
m, Figure 2C,D,I).
The two kinds of fungal hyphae were in mixed arrangement (Figure 2C), however, there
was no intertwinement of the hyphae as observed in some species of Trichoderma [
61
].
There were some gaps among hyphae of the fruiting body of C. militaris [
62
] and the
pathogen invaded into these gaps until being full of the entire fruiting bodies (Figure 2B,H).
It was observed that some hyphae of these two fungi can stick together
(Figure 2D–F),
with some sticky substance at the touch point (Figure 2E,F), where some micropores
were produced (Figure 2F,G) with hyphal shrinking (Figure 2E,F). Some hyphae of C.
militaris which were not in touch with C. cordycipiticola directly also became shrinking
(Figure 2D,E).
The haustorium and appressorium were not observed during the infection
(Figures 2D–F and 3).
A small number of pathogenic hyphae can penetrate into the host
hyphae
(Figure 2I).
Conidia entered into the fruiting body along the gaps and germinated,
accelerating the spread of disease (Figure 2H–J).

J. Fungi 2021,7, 918 8 of 25
Figure 2.
Interaction between Cordyceps militaris and Calcarisporium cordycipiticola observed by SEM and RFP labeling.
(
A
) The fruiting body of C. militaris infected by C. cordycipiticola used for SEM observation. (
B
) The pathogen invaded
into the gaps among the hyphae of C. militaris fruiting bodies. (
C
) Mixed arrangement of the two fungal hyphae.
(
D
–
F
) The hyphae of C. militaris and C. cordycipiticola contacted with each other; (
E
–
G
) showed hyphal shrinking of
C. militaris and some micropores. (
H
) The pathogen (hyphae and conidia) invaded into the fruiting bodies along the gaps.
(
I
) A small number of pathogenic hyphae can penetrate into the host hyphae. (
J
) The conidia of C. cordycipiticola in the
fruiting bodies of the host germinated (longitudinal section) observed by RFP labeling. Red and blue arrows indicated
hyphae of C. cordycipiticola and C. militaris, respectively. Yellow arrow indicated that the pathogenic hyphae penetrated into
the host hyphae and green indicated the conidia of C. cordycipiticola. Scale bar = 1 mm in A; 10
µ
m in B, C, H–J; 1
µ
m in D–F
and 100 nm in G.
Under TEM, the cell walls of the pathogen were intact (Figure 3A,C,E) whereas cells
of C. militaris were deformed whether they were in touch or not touch with the hyphae
of the pathogen directly (Figure 3A,C,D,F). The organelles and cytoplasm of the hyphal
cells of C. militaris were dissolved, resulting in hollow hyphal cells even if the hyphae had
not burst (Figure 3B,C). Finally, the cells were broken and died, leading to the wilting of
fruiting bodies.

J. Fungi 2021,7, 918 9 of 25
Figure 3.
Cell morphologies of Cordyceps militaris and Calcarisporium cordycipiticola during infec-
tion observed by TEM. Cross sections of the infected fruiting body of C. militaris (3 dpi) were
shown. (
A
), (
C
) Hyphal cells of C. cordycipiticola and C. militaris. (
E
) Hyphal cell of C. cordycipiticola.
(
B
–
D
,
F
) Hyphal cells of C. militaris after being infected by C. cordycipiticola. Red arrow indicated cells
of C. cordycipiticola, yellow indicated empty hyphal cells of C. militaris without organelles, and blue
indicated the deformed cells. Scale bar = 1 µm.
3.3. Host Specificity of Calcarisporium cordycipiticola
After inoculation on the synnemata of B. bassiana, C. tenuipes, and I. cicadae for 30 days
(Figure 4A–H), the mycelia of C. cordycipiticola cannot proliferate and the inoculation site
remained unchanged. There was no pathogenic sign (Figure 4F–H). However, the surface
of the fruiting bodies of C. militaris has been fully covered by the mycelia of C. cordycipiticola
(Figure 4E). It seemed that the pathogen C. cordycipiticola can only infect C. militaris.

J. Fungi 2021,7, 918 10 of 25
Figure 4.
Infection on different species of Cordycipitaceae. (
A
–
D
) were C. militaris,C. tenuipes,
B. bassiana, and I. cicadae that were cultivated on the wheat media, respectively. (
E
–
H
) are those
infected by C. cordycipiticola for 30 days. Three biological replicates for each species.
3.4. General Features of the Calcarisporium cordycipiticola Genome
In order to investigate the infection mechanism, the genome of C. cordycipiticola was
sequenced by both Illumina and PacBio SMRT technologies. Subreads distribution analyses
confirmed the high quality of the 20 kb library (Figure S7). The resulting assembly yielded
an N50 length of 5.45 Mbp and a total size around 34.51 Mbp (Table 1), which was similar
to Calcarisporium sp. 525 (36.8 Mbp) [
63
] and C. militaris (32.2 Mbp) [
18
], less than that of C.
arbuscula (45.0 Mbp) [64].
Table 1. Genome feature comparison between Calcarisporium cordycipiticola and Cordyceps militaris.
Feature C. cordycipiticola
CGMCC 5.2193 C. militaris Cm01
Size (Mbp) * 34.51 32.2
Coverage (fold) 170×147×
(G + C) percentage (%) 51.7 51.4
N50 (Mbp) 5.45 4.6
Percentage repeat rate 4.04 3.04
Protein-coding genes 10,443 9684
Average gene length (bp) 1692 1742
Percentage of secreted proteins (%) 9.02 8.00
Gene density (no. gene per Mbp) 302 257
tRNA genes 155 136
Pseudogenes 343 102
NCBI accession PRJNA766243 AEVU00000000
* Mbp, mega base pairs.
The sequenced data were assembled into eight scaffolds, an approximate chromosome-
level, with three of these scaffolds containing characteristic telomeric (CCCTAA)n or
(TTAGGG)n repeats on both the 5
0
and 3
0
ends of the sequence, and four having telomeric
repeats on one of the 5
0
or 3
0
ends of the sequence. The three scaffolds with telomeres on
both ends were taken to be fully sequenced chromosomes. The remaining one scaffold was
evaluated as mitochondrion genes and fragments that were not integrated into the genome
(Table S1). The seven assembled scaffolds which ranged in size from 3.3 to 6.2 Mbp were
displayed by circos-plot (Figure 5, Table S1). The related species C. militaris contained seven
chromosomes ranging in size from 1.9 to 8.3 Mbp [
65
]. The mitogenome of C. cordycipiticola

J. Fungi 2021,7, 918 11 of 25
is 25,762 bp (Figure S8), containing 15 common protein-coding genes, two rRNA and
24 tRNA genes, which was highly similar to that of C. militaris (Table S2).
1
Figure 5.
Circos-plot of genome of Calcarisporium cordycipiticola. The Seven scaffolds of C. cordycipiticola were displayed by
circos-plot (Mbp scale). The circos from outside to inside were: (A) Seven chromosomes almost; (B) DNA methylations;
(C) Carbohydrate-active
enzymes; (D) PHI-base genes; (E) Putative effectors; (F) Clusters of secondary metabolites;
(G) GC
content; (H) Duplicated genes. DNA methylations and GC contents were statistical results of 10 kb non-overlapping
windows. The inner lines link duplicated genes.
A total of 10,443 protein-coding genes with an average sequence length of 1,692.74 bp
were predicted, in which the gene density was 302 gene per 1 Mbp. Three-hundred-and
fifty-six tRNA genes and 467 pseudogenes were predicted in the genome which was much
more than that of C. militaris (Table 1). The length of protein-coding genes was 17.7 Mbp
(almost 50%, 17.7 /34.51 Mbp) and each protein-coding gene had about 2.84 exons and
1.84 introns. 99.3% (9612) putative protein-coding genes were supported by RNA-seq data.
Overall, 10,413 (99.71%) of the predicted proteins had known homologues in at least one
functional protein database (Figure S9, Table S3). The completeness of genome assembly
and annotation with single-copy orthologs test suggested a well-completed annotation set,
with 94.7% of the Fungi BUSCOs being present within the RefSeq annotation set (Figure S9).
According to the GO database, 4944 annotated genes were assigned to GO cate-
gories, with the first three as “metabolic process”, “catalytic activity” and “cellular process”
(Table S4).
By mapping to the KEGG database, “global and overview maps” accounted for

J. Fungi 2021,7, 918 12 of 25
the maximum proportion of KEGG annotations. The first three were biosynthesis of amino
acids, biosynthesis of antibiotics and carbon metabolism (Figure S10, Table S5).
Approximately 4.04% of the repeat sequences that included TEs (~2.82%) and tandem
repeats (~1.22%) were identified in C. cordycipiticola genome. The Class I (retrotransposons)
and Class II (DNA transposons) TEs occupied 1.18% and 0.16% of the genome (Figure 6A).
Class I (retrotransposons) mainly included Gypsy and Tad1 and Class II (DNA transposons)
mainly included MULE-MuDR and hAT-Charilie (Figure S11A). C. cordycipiticola exhibited
a little expansion of repeat content compared to some other ascomycetes (Table S6). The
repeat sequence of congeneric fungi, Calcarisporium sp. KF525 and C. arbuscula NRRL 3705,
only accounted for 1.28% and 3.08% of their genome sizes, respectively [
63
,
64
]. It was
reported that two mycoparasite species, Trichoderm atroviride and T. virens, only had 0.49%
and 0.48% repeat sequences [14].
Figure 6.
Repeat elements and DNA methylation sites of Calcarisporium cordycipiticola. (
A
) The
percentage of different types of repetitive sequences in the C. cordycipiticola genome. (
B
) Statistic
analysis of candidate DNA methylation sites from primary sequence of C. cordycipiticola genome.
RIP is a genome defense mechanism in fungi during which duplicated sequences are
mutated from CpA to TpA [
66
]. Sequences that have been subjected to RIP were expected
to have a high TA/AT ratio and low (CA + TG)/(AC + GT) ratio, with
values >0.89
and <1.03, respectively [
36
]. A significant RIP was observed in C. cordycipiticola genome
by the high value of TpA/ApT (2.03) and the low ratio of (CpA + TpG)/(ApC + GpT)
(0.64) (Figure S11B). One homologue (CCOR_04202) of RIP defective-1 of Neurospora crassa
(NCU02034) required for RIP was identified in C. cordycipiticola genome (identity of 39%
and E-value of
3×10−90).
Transcriptome analysis showed that this gene expressed with
FPKM of 0.27, 0.75 and 0.37 for the three repeats in the mycelial sample and there was no
increase during the infection process. It was indicated that there was indeed RIP defense
mechanism in C. cordycipiticola which was not related to the infection.
DNA methylation is involved in many important cell processes, such as genomic
imprinting and gene transcription regulation [
67
]. Using SMRT, 6-methyl-adenosine (m6A)
and 4-methyl-cytosine (m4C) methylation were detected in particular. In total, 85,229 m4C
and 27,110 m6A were identified in the C. cordycipiticola genome. However, the majority
of methylation sites (129,857) were uncategorized (Figure 6B). Interestingly, most of the
categorized DNA methylations are m4C, accounting for 75.9%, whereas m6A only accounts
for 24.1%.
3.5. Phylogenomic and Evolution Analyses
A ML phylogenomic tree was generated based on 631 single-copy orthologs of 32 fungi,
including insect pathogens, plant pathogens, nematode parasites, mycoparasites and sapro-
phytes, with U. maydis as the outgroup (Table S7). The results verified that C. cordycipiticola
was a distinct lineage (Calcarisporiaceae) from other families in Hypocreales, as described
by Sun et al. [5].

J. Fungi 2021,7, 918 13 of 25
The inferred phylogeny illustrated that C. cordycipiticola was evolutionarily close to its
host C. militaris, and they diverged after a split with Trichoderm spp., which was consistent
with the report that B. bassiana and C. militaris diverged after a split with Trichoderma
spp. [68]. C. cordycipiticola diverged before C. militaris (Figure 7).
Figure 7.
Phylogeny of Calcarisporium cordycipiticola and other 30 sequenced Ascomycota genomes. A ML phylogenomic
tree showed the evolutionary relationship of C. cordycipiticola with different fungal species. Bootstrap values were shown
beside the nodes. Life strategies and host preferences were indicated.
The branch of B. bassiana,Cordyceps brongniartii and C. militaris diverged from C.
cordycipiticola around 80.9–62.2 MYA (Figure 7) after or at the cretaceous extinction event
(65 MYA) [
69
]. This phylogeny also reinforced the previous result that the split between
Cordycipitaceae and Clavicipitaceae occurred before Ophiocordycipitaceae diverged from
Clavicipitaceae (Figure 7) [70].
The earliest diverging group in Nectriaceae of Hypocreales comprised primarily plant
pathogenic species, including Fusarium graminearum,Fusarium oxysporum and Nectria haema-
tococca (Figure 7). Comparative genomic studies supported that the ancestral state of
Hypocrea/Trichoderma was mycoparasitic [
14
]. The four nematode parasitic fungi, Drechme-
ria coniospora,Hirsutella minnesotensis,Pochonia chlamydosporia and Purpureocillium lilacinum,
clustered with insect pathogens, indicating that nematode and insect pathogens might
share a common ancestor. The host analysis supported a major transition within the order
from plant hosts/substrates in early diverging lineages to fungal hosts and then to insect
and nematode hosts.
3.6. The Secretome and Potential Eeffectors
Secreted proteins are important for the pathogenicity of parasitic fungi. A total of
942 sequences were identified to encode proteins with signal peptides but not transmem-
brane helices (Table S8). The proportion of genes encoding secreted proteins (9.02%) was
similar to 7–10% in plant pathogens [
71
]. KEGG pathway enrichment analysis indicated
that the putative secreted proteins were mainly involved in primary metabolism, especially

J. Fungi 2021,7, 918 14 of 25
sugar metabolism, including starch and sucrose metabolism (ko00500), amino sugar and
nucleotide sugar metabolism (ko00520), glycosaminoglycan degradation (ko00531), galac-
tose metabolism (ko00052), other glycan degradation (ko00511) and N-glycan biosynthesis
(ko00510) (Figure S12A).
In the fungal plant pathogens, pathogen-secreted effectors play critical roles in facili-
tating the proliferation of pathogens, often by suppressing the host immune system [
72
].
These secretomes were further examined for effector candidates and 189 proteins were
obtained (Table S8) based on the assumption that fungal effectors are small cysteine-rich
proteins with fewer than 400 amino acids and more than four cysteine residues [73].
There were 434 putative pathogenic factors in C. cordycipiticola annotated by the PHI
database (Table S8). KEGG pathway analysis indicated that they were mainly enriched
with MAPK signaling pathway (ko04011), AGE-RAGE signaling pathway in diabetic
complications (ko04933) and ABC transporters (ko02010) (p< 0.001, Figure S12B).
3.7. Carbohydrate-active Enzymes (CAZymes)
CAZymes which can degrade and modify polysaccharides might be required when
C. cordycipiticola degraded the structural polysaccharide armor of the host, such as chitin,
during the course of its parasitism. A detailed examination of the CAZymes of C. cordy-
cipiticola was performed and compared with other fungi, including mycoparasitic fungi
(Clonostachys rosea,Trichoderma atroviride and Trichoderma harziam), nematode parasitic fungi
(H. minnesotensis and P. chlamydosporia), entomopathogenic fungi (B. bassiana,C. militaris
and Ophiocordyceps sinensis) and plant pathogenic fungi (Botrytis cinerea,F. graminearum,
Magnaporthe oryzae and Verticillium dahliae) (Table 2). Four hundred and forty-one CAZymes
were identified in the genome of C. cordycipiticola (Table 2), which was much less than the
other mycoparasitic fungi, C. rosea (656), T. harziam (516) and T. atroviride (473). In general,
the number of CAZymes of mycoparasites (average 522) was a little less than that of plant
pathogenic fungi (average 561) but was much more than that of entomopathogens (average
307, p= 0.019).
There were also significant differences in the spectrum of CAZymes produced by my-
coparasitic fungi. Compared to mycoparasitic fungi, C. rosea,T. atroviride and T. harzianum,
CAZymes candidates of C. cordycipiticola were radically lower in number for each class
except AAs (Auxiliary activity family) (Table 2).
Seventy-seven families containing 211 genes in C. cordycipiticola that encoded glyco-
side hydrolases (GHs) were identified (Table S9). The number of GHs of mycoparasitic
fungi (average 250) was close to the plant pathogens (average 246), and much more than
that of entomopathogenic fungi (average 134, p= 0.012), indicating that GHs were more
important for mycoparasitic and plant pathogenic fungi. The most abundant family for C.
cordycipiticola was GH16 (
β
-glucanases) and GH18 (chitinase) (Table S9), both had 16 encod-
ing genes that might be responsible for the cell wall degradation of the host. Family GH18,
enzymes involved in chitin degradation, was also strongly expanded in Trichoderma [
74
].
Among the 16 GH18 genes, 11 encoded secreted protein (Table S10).
Especially, the most abundant family of CBM (carbohydrate-binding module) was
CBM18s, which was related to the high number of GH18 in this species (Table S11). How-
ever, it was reported there was a low number of GH18 and CBM18s in the mycoparasitic
fungi C. rosea (Tables S9 and S11). The significant contraction of CBM1 (only 4) in C. cordy-
cipiticola was observed compared to the mycoparasitic (average 18) and plant pathogenic
fungi (average 21), and even lower than the average (12) of all the fungi tested. C. cordy-
cipiticola also possessed several families of cellulases (GH6, GH7, GH12 and GH45) and
other enzymes involved in degrading cell walls (GH11, GH30, GH51, GH53, GH62, GH67
and GH115) (Table S9).
Another major class of CAZymes, glycosyltransferases (GTs) which were involved
in the biosynthesis of oligo and polysaccharides, were represented in the genome with
71 members in 34 families (Table S12). These enzymes exhibited less variability in as-
comycetes. A series of carbohydrate esterase (CE)-encoding genes were also detected in

J. Fungi 2021,7, 918 15 of 25
the genomes, including the most abundant sterol esterases (CE10) and cutinases (CE5),
which are virulence factors of some plant pathogens [
68
]. There was only 1 pectate lyase (9
to 25 in plant pathogens).
Table 2. Comparison of CAZymes among fungi with different lifestyles.
Life-Style Species AA CBM CBM1 CE GH GT PL All
mycoparasitic
fungi
C. cordycipiticola 67 26 4 56 211 71 6 441
C. rosea 103 29 13 97 310 73 31 656
T. harzianum 61 49 21 58 245 76 6 516
T. atroviride 51 41 22 43 234 74 8 473
nematode
parasitic fungi
P. chlamydosporia 63 33 9 43 278 86 7 519
H. minnesotensis 54 28 0 32 137 80 2 333
entomopathogenic
fungi
B. bassiana 51 30 3 33 160 79 2 358
C. militaris 38 30 1 30 148 69 3 319
O. sinensis 35 24 0 23 95 64 2 243
plant pathogenic
fungus
F. graminearum 82 42 12 74 237 78 22 547
M. oryzae 100 51 22 72 244 82 6 577
V. dahliae 85 31 28 65 241 69 34 553
B. cinerea 82 38 23 71 260 84 10 568
AA: Auxiliary activity family, CBM: Carbohydrate-binding module, CE: Carbohydrate esterase, GH: Glycoside hydrolase, GT: Glycosyl-
transferase, PL: Polysaccharide lyase.
3.8. Great Biosynthetic Capabilities of Secondary Metabolites and Potential of Mycotoxin
Biosynthesis in Calcarisporium cordycipiticola
Sixty-six SMs were identified in the genome of C. cordycipiticola (Table 3), similar to
60 SMs in marine fungus Calcarisporium sp. KF525 [
63
] and 65 in mushroom-endophytic
fungus C. arbuscula NRRL 3705 [
64
]. However, compared to the sequenced species in
Cordyciptaceae, the number of SMs in C. cordycipiticola (66) was much more than 30 SMs in
C. militaris [
18
], 45 in B. bassiana [
68
]. It was indicated that the fungi of Calcarisporium genus
had more biosynthetic gene clusters and had a large potential for SM production (Table 3).
Table 3. Secondary metabolite clusters in the genome of Calcarisporium cordycipiticola.
Feature
Calcarisporium
cordycipiticola
CGMCC 5.2193
Calcarisporium
arbuscula
NRRL 3705
Calcarisporium
sp. 525
Cordyceps
militaris Cm01
NRPS 14 12 17 8
T1PKS 18 23 24 6
Terpene 4 11 7 4
NRPS-like 12 0 0 6
PKS-NRPS-Hybrid
(NRPS-PKS-Hybrid) 9 7 3 5
other 9 12 9 1
Total 66 65 60 30
The average number of core genes related to SM synthesis in ascomycetes is 48 [
75
].
However, 82 core genes were identified, including 20 type I polyketide synthase (T1PKSs),
17 non-ribosomal peptide synthases (NRPSs), 11 PKS-NRPS hybrids, 2 NRPS-PKS hybrids,
12 putative NRPS-like enzymes, four terpene, and 16 others as described in Table S13.
Forty-four SM clusters have no homologues blasting against the MiBiG database.
Three PKS-NRPS clusters (cluster 8, 40 and 50) were predicted for pyranonigrin E, botrye-
nalol and radicicol, respectively; two NRPS clusters (cluster 6 and 15) for dimethylcoprogen
and aureobasidin A1; one T1pks cluster (cluster 65) for phomopsins, and one terpene clus-
ter (cluster 49) for clavaric acid with 100% of similarity; one T1pks cluster (cluster 46) for
depudecin with 50% of similarity (Table S13).

J. Fungi 2021,7, 918 16 of 25
Blast analysis showed that CCOR_00730 of cluster 6 shared high similarity with
Trichoderm hypoxylon dfcA, which was confirmed to be responsible for biosynthesis of
coprogens and required for the inhibition of the pathogenic fungi F. oxysporum and Mucor
corcinelloides [
76
]. The flanking genes, including the putative AMP-dependent synthetase
/ligase, acetyltransferase, and siderophore transporter, also shared the high similarity with
the biosynthetic gene cluster of amphiphilic coprogens in T. hypoxylon (Figure 8A). This
cluster was widespread in fungi, and it was assumed that cluster of CCOR_00730 was
responsible for the biosynthesis of coprogens in C. cordycipiticola (Table S14).
Figure 8.
The putative biosynthetic gene clusters for coprogen and radicicol in the genome of
Calcarisporium cordycipiticola CGMCC 5.2193. (
A
) Comparison with the coprogen cluster reported in
Metarhizium robertsii ARSEF 23, T. harzianum CBS 226.9 and T. hypoxylon. (
B
) Comparison with the
radicicol cluster reported in P. chlamydosporia. The identity and query cover of each homologue to C.
cordycipiticola CGMCC 5.2193 counterparts were shown.
Five proteins encoding by genes of cluster 50 shared high sequence identity of 59–69%
with the counterparts of Rdc1, Rdc2, Rdc3, Rdc4 and Rdc5 of P. chlamydosporia ATCC 16,683
(Figure 8B) which were confirmed to be responsible for radicicol biosynthesis [
77
]. Radicicol
and analogs have significant antibacterial, antifungal, antiviral, anticancer and antiparasitic
activities, and are also potent heat-shock protein 90 inhibitors [
78
,
79
]. Transcriptome
analysis showed that all the genes of this cluster were expressed, though most were at low
levels (Table S15).
Six proteins encoded by the genes of cluster 8 showed high sequence identity
(31–52%)
with a cluster of Aspergillus niger CBS 513.88 being responsible for pyranonigrins
(Figure S13A) [
80
]. Pyranonigrins are a family of antioxidative compounds that are reported
to be produced by A. niger, all featuring a unique pyrano [2,3-c] pyrrole core structure
which is rarely found in nature [
81
]. Transcriptome data showed that these genes were
moderately expressed in the mycelia (Table S15), indicating C. cordycipiticola might produce
pyranonigrins-related compounds.
Depudecin is an inhibitor of histone deacetylase, similar to HC toxin. CCOR_06814 of
cluster 46 showed the high sequence identity with DEP5 (identity of 54% and E-value of
0) which was confirmed to be involved in depudecin biosynthesis in Alternaria brassicicola
ATCC 96,836 [
82
]. Moreover, the two FAD-dependent monooxygenase of cluster 46 showed

J. Fungi 2021,7, 918 17 of 25
sequence identity with DEP2 (identity of 50% and E-value of 0) and DEP4 (identity of 59%
and E-value of 0) of A. brassicicola ATCC 96,836 (Figure S13B). However, transcriptome
data showed CCOR_06808-CCOR_06814 were barely expressed in the mycelia (Table S15).
Phylogenetic analysis based on the 55 KS domain sequences of PKSs of C. cordycipiticola
(Table S16) and 17 known mycotoxins (Table S17) indicated the groupings of some PKS pro-
teins of C. cordycipiticola with those involved in mycotoxin-producing enzymes
(Figure 9A).
Modular analysis indicated that CCOR_07363 groups with hypothemycin-producing PKS,
CCOR_01813 with solanapyrone, CCOR_07909 with citrinin and CCOR_07359 with zear-
alenone (Figure 9A) have similar domain organizations with the corresponding mycotoxin
producing PKSs except for CCOR_07359 (Figure 9B).
Figure 9.
Phylogenetic and modular analyses of Calcarisporium cordycipiticola polyketide synthases
compared with those involved in the production of mycotoxins. (
A
) A NJ tree showing the re-
lationships of KS domain sequences. HR PKS, highly reducing PKS; PR PKS, partially reduced
PKS; NR PKS, non-reduced PKS. (
B
) Modulation and comparison of C. cordycipiticola PKSs with
those involved in production of mycotoxins. ACP, acyl carrier protein domain; AT, acyltransferase
domain; DH, dehydratase domain; ER, enoyl reductase domain; KR, ketoreductase domain; MT,
methyltransferase domain; TE, thioesterase domain. The different mycotoxin encoding PKSs used
were listed in Table S17.
Further survey showed that CCOR_01813 protein has 41% identity (E value of 0 and
query cover of 99%) with SOL1 which has been confirmed for solanapyrone biosynthe-
sis in Alternaria solani [
83
]. The cluster for solanapyrone included six genes (sol1,sol2,
sol3,sol4,sol5,sol6), and homologous comparison found that the six proteins of the clus-
ter 17 of C. cordycipiticola shared a sequence identity of 25–52% with those of A. solani
(Figure S13C, Table S14).
Transcriptome analysis showed that these genes were highly or
moderately expressed in the mycelia (Table S15), indicating C. cordycipiticola might produce
solanapyrone-related compounds.

J. Fungi 2021,7, 918 18 of 25
For the cluster of CCOR_07363, it was found that core genes CCOR_07359 and
CCOR_07363 shared the identity of 55% and 61% with HPM3 and HPM8 of Hypomyces
subiculosus which were responsible for hypothemycin biosynthesis (Table S14) [
84
]. Besides,
CCOR_07359 and CCOR_07363 proteins shared 54% and 59% identity with PKS13 and
PKS4 for zearalenone biosynthesis in F. graminearum (Table S14). However, the flanking
proteins for both hypothemycin and zearalenone biosynthesis clusters were not found in
C. cordycipiticola.
CCOR_07909 protein had the identity of 52% (E value of 0 and query cover of 99%)
with CitS responsible for citrinin biosynthesis in Monascus ruber [
85
]. Flanking proteins of
the cluster, CCOR_07908 and CCOR_07910, showed high sequence identity with serine
hydrolase (CitA) and oxidoreductase (CitC) of M. ruber (identity 60% and 50%) (Table S14).
Transcriptome analysis showed that these three genes were highly expressed in the mycelia
(Table S15), indicating C. cordycipiticola might produce citrinin-related compounds.
4. Discussion
Mycoparasitism is a lifestyle where a living fungus (host or prey) is parasitized by
and acts as a nutrient source of another fungus (mycoparasite or predator) [
86
], which can
cause devastating diseases of mushrooms in nature and industry by reducing the yield
and quality worldwide if the host is mushroom [
6
]. In the present study, C. cordycipiticola
was found to parasitize and degrade the hyphae of the fruiting body of C. militaris, by
direct contact and noncontact (Figure 2). The approximate chromosome-level genome
was assembled and some characteristics being consistent with mycoparasitic lifestyle
were revealed. We showed specific mycoparasitism between two phylogenetically close
species here.
C. cordycipiticola cannot infect the other species of Cordycipitaceae, including B.
bassiana, C. tenuipes and I. cicadae (Figure 4),indicating the specificity of the host. Un-
til now, C. militaris is known to be the only host of C. cordycipiticola. This strict host
specificity is not common. Species of the genus Trichoderma together with C. rosea, which
are the most studied fungal mycoparasites, have wide host ranges comprising several plant
pathogens [
86
]. For the congeneric fungi, Calcarisporium parasiticum has been observed
to parasitize several species of Physalospora and closely related fungi [
87
]. Scleromitrula
shiraiana is a causal agent of mulberry sclerotial disease with a narrow host range but
includes some species of genus Morus [88].
The fruiting body of C. militaris was consists of prosenchyma and pseudoparenchyma
tissues [
62
]. The prosenchyma tissues are loosely woven and have distinguishable and
typical elongated hyphal cells lying parallel to one another [
89
]. There were some gaps
among the hyphae of the fruiting body as shown in Figures 2and 3, which was consistent
with the observation of Feng et al. [
62
]. C. cordycipiticola can invade into these gaps, filling
the entire fruiting bodies gradually (Figures 1and 2). It was reported that plant stomata are
not only essential for gas exchange with the environment and controlling water loss, but
also as a checkpoint of host immunity and pathogen virulence [
90
]. Here it was suggested
that the gaps among the hyphae of the fruiting body of C. militaris may play similar roles
with the plant stomata, viz promote the respiration of hyphae inside fruiting bodies and
become the channels for C. cordycipiticola to invade.
Some mycoparasites form specialized infection structures such as hook-like, braid-like,
and clamp-like contact structures at host-parasite interfaces [
91
]. Invasive mycoparasites,
such as Trichoderma species [
92
] and C. rosea [
7
] can form appressoria-like infection struc-
tures aiding in penetration. C. parasiticum can form a small ’buffer’ cell at the tip of a
hypha where it contacts the host [
87
,
93
]. In the present study, we have observed the whole
infection process carefully, focusing on the specialized infection structures, however, we
cannot find any (Figures 2and 3). It was only observed that the hyphae of these two fungi
were closely appressed, and then produced some micropores at the interface (Figure 2F,G).
Finally, the hyphal cells of the host were deformed and the protoplasm was discharged,

J. Fungi 2021,7, 918 19 of 25
resulting in the empty cells (Figure 3B–D). During the infection, only a few pathogenic
hyphae were observed to penetrate into the host hyphae (Figure 2I).
Mycoparasitism can be largely categorized into two groups, biotrophic and necrotrophic
parasitisms according to the trophic mechanism [
86
]. Necrotrophic mycoparasites tend
to have a broad host range, with relatively unspecialized parasitic mechanisms, and kill
their host. In contrast, the biotrophic relationships between one fungus and another are
complex, controlled and relatively non-destructive, and often, but not always, with narrow
host ranges that have co-evolved [
94
]. Based on the strict host specificity, the observation
that micropores formed between the appressed hyphae of host and parasite, as well as a
few hyphae that penetrated into the host hyphae and that host cells remained functional
at the early stage of infection, it was proposed that C. cordycipiticola was firstly biotrophic.
However, it turned to necrotrophy as the mycoparasitic interaction progressed. C. cordy-
cipiticola infected the fruiting bodies of C. militaris (Figure 10) and shuttled among the
host hyphae (Figure 10E). The cell of C. militaris was deformed with hyphal shrinking till
plasmatorrhexis. C. cordycipiticola derived nutrients from the disintegrated host, resulting
in the wilting of the fruiting body of C. militaris. In a word, C. cordycipiticola is a destructive
mycoparasite of C. militaris.
Figure 10.
Model of pathogenic process of Calcarisporium cordycipiticola. (
A
) The asexual life cycle of
C. cordycipiticola. (
B
) Fruiting body infected by C. cordycipiticola. (
C
–
E
) Model of colonization and
invasion.
The pathogen C. cordycipiticola is an invasive type, similar to deep fungal infections of
animal [
95
], which can well explain the destructiveness of the disease. Once the symptoms
were visible to the naked eye, the pathogen had already entered the fruiting bodies. Quick
germination of a large number of conidia, the rapid expansion of hyphae [
3
] and intruding
into the fruiting bodies resulted in the spread of disease in a short time.
C. cordycipiticola can be cultured on artificial media besides mycoparasitism, but no
morphologic alteration of C. militaris was observed when the dual culture of C. militaris
and C. cordycipiticola on PDA plates was performed (Figure S6). The same results were
observed with dual culture between A. bisporus and L. fungicola [
96
], A. bisporus and H.
perniciosus [
97
]. It was suggested that the interactions between mushrooms and pathogens
on plates (pythogenesis) are different from infecting on the fruiting bodies (mycoparasites).
C. cordycipiticola may initiate different lifestyles when facing different living environments,
which has also been reported in some mycoparasites such as Saccharomycopsis schoenii [
98
].
Sequencing and analyses of the genomes of mycoparasites Trichoderma spp. [
14
] and
C. rosea [
7
], as well as Tolypocladium ophioglossoides [
99
] and Escovopsis weberi [
100
] in the

J. Fungi 2021,7, 918 20 of 25
order Hypocreales, revealed several gene families with a mycoparasitic-related expansion,
but distinct differences also emerged among the different mycoparasites. The genome size,
structure, and gene content are influenced heavily by natural selection and governed by
the lifestyle and ecological niche of a species [
86
]. The C. cordycipiticola genome size of
34.51 Mbp is similar to genomes of mycoparasites Trichoderma spp. (vary between 31.7
and 39.0 Mbp) and the truffle parasite T. ophioglossoides (31.2 Mbp), but a pronounced
reduction than that of C. rosea (55.18 to 70.7 Mbp) [
7
,
101
]. This reduction in genome size is
accompanied by a lower number of protein-encoding genes (>14,268 in C. rosea and 10,443
in C. cordycipiticola).
Fungal secreted proteins, effectors, cell wall degrading enzymes and SM genes are
known to be under positive selection in pathogenic fungi [
102
]. There were some charac-
teristics for the genome of C. cordycipiticola which were consistent with the mycoparasitic
lifestyle. The proportion of genes encoding secreted proteins (9.02%) is relatively high; the
pathogen-secreted effectors were mainly enriched with amino sugar and nucleotide sugar
metabolism (ko00520); the most abundant family of GHs for C. cordycipiticola was GH16
and GH18 (Table S9), and the most abundant family of CBM was CBM18s (Table S11) etc.
Though abundant CAZymes were identified, the number of CAZymes in the genome
of C. cordycipiticola (441) was much less than that of the other mycoparasitic fungi, C. rosea
(656), T. harziam (516) and T. atroviride (473), especially those for cell wall degradation
(Table 2).
This gene loss may be attributed to its specialization as a parasite of C. militaris. In
the genome of E. weberi, a specific parasite of ant cultivated Leucoagaricus spp., the depletion
of CAZymes was also found [
100
]. There were fewer genes encoding cell wall-degrading
enzymes and effector proteins in the genome of Sclerotinia shiraiana than those of Sclerotinia
sclerotiorum and B. cinerea, which was probably a key factor of the narrow host range
of S. shiraiana [88].
Fungal SMs serve survival functions by supporting the fungus in competition against
other microbes, in self-protection and defense, and have important roles in communication
and interaction with other organisms [
103
]. The number of SMs in C. cordycipiticola (66)
was more than twice of its host C. militaris [
18
]. It was reported that compared with most
other filamentous ascomycetes, the sequenced mycoparasitic Trichoderma species and C.
rosea are especially enriched in SM-related genes [
86
] which serve in chemical warfare
against their competitors and fungal preys and play a crucial role in mycoparasitism. SMs
were also found to play a crucial role in specialized mycoparasite E. weberi, which kills its
ant-cultivated host fungi from a distance, thereby likely involving secreted toxins [
100
].
The same phenomenon occurred during the infection of C. cordycipiticola. In some cases, for
the hyphae of the host which did not contact with those of the pathogens directly, the cell
organelles had been lost even if the cell wall was only partially degraded and deformed
(Figure 3A–C). It was speculated that some diffusible SMs might play an important role
during the infection process. Comparative genomic and transcriptomic analyses suggest
that this fungus may produce coprogens, radicicol, depudecin-related and many other
unknown compounds at the mycelial stage. Among them, coprogenss may be an important
virulence factor of C. cordycipiticola and promote iron absorption during infection, which is
our ongoing project.
The fruiting bodies of C. militaris have been consumed worldwide as a tonic [
1
].
Sometimes the mycelia of C. cordycipiticola may mix with the fruiting bodies for sale and
so the safety is still of concerned. Our genome data suggest that there are some gene
clusters for mycotoxin-production, such as solanapyrone, hypothemycin, zearalenone and
citrinin-related compounds (Tables S14 and S15). Transcriptomic analysis revealed that
at least solanapyrone and citrinin-related compounds are likely to be produced by this
fungus at the mycelial stage because of the high or moderate expression of the encoding
genes. From the safety point of view, the fruiting bodies which have been infected by C.
cordycipiticola should not be consumed.
Phylogenomic analysis based on the single copy orthologs of 32 fungi revealed that
C. cordycipiticola is evolutionarily close to its specific host, C. militaris (Figure 7), which

J. Fungi 2021,7, 918 21 of 25
makes it difficult to study the co-evolution. The divergence time estimation revealed that
the branch of C. militaris diverged from C. cordycipiticola around 80.9–62.2 MYA (Figure 7)
after or at the cretaceous extinction event (65 MYA), being concurrent with the Cretaceous
diversification of fungal-arthropod symbioses supported by the oldest fossil evidence of
animal parasitism by fungi [104]. Our analysis supported the conclusion that the shifts to
mycoparasitism occurred several times during the evolution of hypocrealean fungi [105].
Mycoparasitism generally involves four sequential steps: chemotropism, recognition,
attachment and cell wall penetration, and digestion of host cell content [
106
]. The present
study gave little information on chemotropism and recognition, which are important for
pathogenicity and disease control and will be our next project.
Supplementary Materials:
The following are available online at https://www.mdpi.com/article/
10.3390/jof7110918/s1, Figure S1. Large-scale cultivation of Cordyceps militaris and white mildew
disease. Figure S2. A flow chart of the whole experimental design. Figure S3. Antibiotic sensitivity
assays for Calcarisporium cordycipiticola. Figure S4. Optimization of ATMT in Calcarisporium cordycipiti-
cola. Figure S5. Validation of transformants expressing RFP of Calcarisporium cordycipiticola. Figure S6.
Interaction between Cordyceps militaris and Calcarisporium cordycipiticola in dual culture. Figure S7.
Statistical analysis for high quality reads of Calcarisporium cordycipiticola genome sequencing data.
Figure S8. Calcarisporium cordycipiticola mitochondrion genome. Figure S9. Evidence for gene pre-
diction accuracy in Calcarisporium cordycipiticola. Figure S10. Histogram of KEGG distribution of
predicted proteins of Calcarisporium cordycipiticola. Figure S11. Families of transposase genes and
estimation of RIP. Figure S12. KEGG pathway enrichment analysis of the secreted proteins and
putative pathogenic factors annotated by PHI database in Calcarisporium cordycipiticola. Figure S13.
The putative pyranonigrins, depudecin and solanapyrone biosynthetic gene cluster of Calcarisporium
cordycipiticola. Table S1. Characteristics of eight scaffolds. Table S2. The comparison of mitogenome
between Calcarisporium cordycipiticola and Cordyceps militaris. Table S3. Characteristic of the annotated
Calcarisporium cordycipiticola. Table S4. GO distribution of predicted proteins of Calcarisporium cordy-
cipiticola. Table S5. KEGG category of predicted proteins of Calcarisporium cordycipiticola. Table S6.
Repeat content of Calcarisporium cordycipiticola compared with other ascomycetes fungi Table S7.
Species used in phylogenomic and evolution analyses. Table S8. The secretome, potential effectors,
PHI genes and P450 genes in Calcarisporium cordycipiticola. Table S9. Family of glycoside hydrolases
(GH) in Calcarisporium cordycipiticola compared with parasitic fungi Table S10. Sixteen GH18 genes
in Calcarisporium cordycipiticola. Table S11. Family of carbohydrate-binding module (CBM) in Cal-
carisporium cordycipiticola compared with parasitic fungi. Table S12. Family of glycosyltransferases
(GT) in Calcarisporium cordycipiticola compared with parasitic fungi. Table S13. Core genes of sec-
ondary metabolites (SMs) in Calcarisporium cordycipiticola. Table S14. Comparison of some clusters in
Calcarisporium cordycipiticola with the related fungi. Table S15. Transcript analysis of genes of some
secondary metabolite clusters. Table S16. Domain analysis of 38 PKS in Calcarisporium cordycipiticola.
Table S17. Seventeen known mycotoxins were used for phylogenetic analysis.
Author Contributions:
Conceptualization, C.D. and Q.L.; methodology, Q.L., X.Z. and K.L.; valida-
tion; C.D. and Q.L.; formal analysis, C.D. and Q.L.; investigation, C.D., Q.L. and X.L.; resources, C.D.,
Q.L., F.W., F.X.; writing-original draft preparation, Q.L.; writing-review and editing, C.D., Q.L. and
Y.X.; supervision, C.D.; project administration, C.D.; funding acquisition, C.D. All authors have read
and agreed to the published version of the manuscript.
Funding:
This research was funded by the National Natural Science Foundation of China (grant
number 31872163).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement:
Publicly available datasets were analyzed in this study, which have
been listed in Table S7.
Acknowledgments:
We are grateful to Xuming Mao (Institute of Pharmaceutical Biotechnology,
Zhejiang University, China) who kindly provided plasmid pFGL815-neoR-tubCp-RFP, and Fei Cao
and Jintao Cheng from Mao’s group for helping in genetic system construction. We are also grateful
to Kathryn E. Bushley, from the USDA in Ithaca, NY, for the suggestion on the phylogenomic and

J. Fungi 2021,7, 918 22 of 25
evolution analyses. The authors would like to thank Chunli Li and Jingnan Liang from Institute of
Microbiology, Chinese Academy of Sciences for their instruction on sample process and observation
by SEM and TEM. The authors are grateful to Yanhan Li, from University of California, Davis, for
language improving. We also thank the anonymous reviewers and editors for their helpful comments
and suggestions.
Conflicts of Interest: The authors declare no conflict of interest.
References
1.
Dong, C.H.; Li, W.J.; Li, Z.Z.; Yan, W.J.; Li, T.H.; Liu, X.Z.; Cai, L.; Ceng, W.P.; Chai, M.Q.; Chen, S.J.; et al. Cordyceps industry in
China: Current status, challenges and perspectives-Jinhu declaration for Cordyceps industry development. Mycosystema
2016
,35,
1–15. [CrossRef]
2.
Liu, Q.; Wang, F.; Xu, F.X.; Xu, Y.Y.; Dong, C.H. Study on fungal diseases of artificially cultivated Cordyceps militaris.Mycosystema
2021,40, 1–19. [CrossRef]
3.
Liu, Q.; Wan, J.X.; Zhang, Y.C.; Dong, C.H. Biological characterization of the fungicolous fungus Calcarisporium cordycipiticola, a
pathogen of Cordyceps militaris.Mycosystema 2018,37, 1054–1062. [CrossRef]
4.
Sun, J.-Z.; Dong, C.-H.; Liu, X.-Z.; Hyde, K.D. Calcarisporium cordycipiticola sp. nov., an important fungal pathogen of Cordyceps
militaris. Phytotaxa 2016,268, 135. [CrossRef]
5.
Sun, J.-Z.; Liu, X.-Z.; Hyde, K.D.; Zhao, Q.; Maharachchikumbura, S.; Camporesi, E.; Bhat, J.; Nilthong, S.; Lumyong, S.
Calcarisporium xylariicola sp. nov. and introduction of Calcarisporiaceae fam. nov. in Hypocreales. Mycol. Prog.
2017
,16,
433–445. [CrossRef]
6.
Sun, J.-Z.; Liu, X.-Z.; McKenzie, E.H.C.; Jeewon, R.; Liu, J.-K.J.; Zhang, X.-L.; Zhao, Q.; Hyde, K.D. Correction to: Fungicolous
fungi: Terminology, diversity, distribution, evolution, and species checklist. Fungal Divers. 2019,95, 431–432. [CrossRef]
7.
Karlsson, M.; Durling, M.B.; Choi, J.; Kosawang, C.; Lackner, G.; Tzelepis, G.D.; Nygren, K.; Dubey, M.K.; Kamou, N.; Levasseur,
A.; et al. Insights on the Evolution of Mycoparasitism from the Genome of Clonostachys rosea. Genome Biol. Evol.
2015
,7, 465–480.
[CrossRef]
8.
Li, D.; Sossah, F.L.; Sun, L.; Fu, Y.; Li, Y. Genome Analysis of Hypomyces perniciosus, the Causal Agent of Wet Bubble Disease of
Button Mushroom (Agaricus bisporus). Genes 2019,10, 417. [CrossRef]
9. Banks, A.M.; Aminuddin, F.; Williams, K.; Batstone, T.; Barker, G.; Foster, G.D.; Bailey, A.M. Genome Sequence of Lecanicillium
fungicola 150-1, the Causal Agent of Dry Bubble Disease. Microbiol. Resour. Announc. 2019,8, e00340-19. [CrossRef]
10.
Xu, R.; Liu, X.; Peng, B.; Liu, P.; Li, Z.; Dai, Y.; Xiao, S. Genomic Features of Cladobotryum dendroides, Which Causes Cobweb
Disease in Edible Mushrooms, and Identification of Genes Related to Pathogenicity and Mycoparasitism. Pathogens
2020
,9, 232.
[CrossRef]
11.
Fletcher, J.T.; Gaze, R.H. Mushroom Pest and Disease Control: A Color Handbook; Manson Publishing Ltd. Academic Press: San
Diego, CA, USA, 2008; p. 192.
12.
Zhou, C.Y. The Etiology and Pathogenic Mechanisms of Mycogone perniciosa Causing Wetbubble Disease on Agaricus bisporus.
Ph.D. Thesis, Jilin Agricultural University, Jilin, China, 2014.
13.
Nunes, J.S.; de Brito, M.R.; Zied, D.C.; Leite, E.A.D.G.; Dias, E.S.; Alves, E. Evaluation of the infection process by Lecanicillium
fungicola in Agaricus bisporus by scanning electron microscopy. Rev. Iberoam. Micol. 2017,34, 36–42. [CrossRef]
14.
Kubicek, C.P.; Herrera-Estrella, A.; Seidl-Seiboth, V.A.; Martinez, D.; Druzhinina, I.S.; Thon, M.; Zeilinger, S.; Casas-Flores, S.A.;
Horwitz, B.; Mukherjee, P.K.; et al. Comparative genome sequence analysis underscores mycoparasitism as the ancestral life style
of Trichoderma. Genome Biol. 2011,12, 1–15. [CrossRef]
15.
Yang, Y.; Sossah, F.L.; Li, Z.; Hyde, K.D.; Li, D.; Xiao, S.; Fu, Y.; Yuan, X.; Li, Y. Genome-Wide Identification and Analysis of
Chitinase GH18 Gene Family in Mycogone perniciosa. Front. Microbiol. 2021,11, 596719. [CrossRef]
16.
Bailey, A.M.; Collopy, P.D.; Thomas, D.J.; Sergeant, M.; Costa, A.; Barker, G.; Mills, P.R.; Challen, M.P.; Foster, G. Transcriptomic
analysis of the interactions between Agaricus bisporus and Lecanicillium fungicola. Fungal Genet. Biol.
2013
,55, 67–76. [CrossRef]
17.
Gallo, A.; Ferrara, M.; Perrone, G. Phylogenetic Study of Polyketide Synthases and Nonribosomal Peptide Synthetases Involved
in the Biosynthesis of Mycotoxins. Toxins 2013,5, 717–742. [CrossRef]
18.
Zheng, P.; Xia, Y.; Xiao, G.; Xiong, C.; Hu, X.; Zhang, S.; Zheng, H.; Huang, Y.; Zhou, Y.; Wang, S.; et al. Genome sequence of the
insect pathogenic fungus Cordyceps militaris, a valued traditional chinese medicine. Genome Biol. 2011,12, R116. [CrossRef]
19.
Cao, F.; Cheng, J.-T.; Chen, X.-A.; Li, Y.-Q.; Mao, X.-M. Development of an efficient genetic system in a gene cluster-rich endophytic
fungus Calcarisporium arbuscula NRRL 3705. J. Microbiol. Methods 2018,151, 1–6. [CrossRef]
20.
Zhan, Y.; Dong, C.-H.; Yao, Y.-J. Antioxidant Activities of Aqueous Extract from Cultivated Fruit-bodies of Cordyceps militaris
(L.) Link In Vitro. J. Integr. Plant Biol. 2006,48, 1365–1370. [CrossRef]
21.
Ayache, J.; Beaunier, L.; Boumendil, J.; Ehret, G.; Laub, D. Web sample preparation guide for transmission electron microscopy
(TEM). In Proceedings of the 14th European Microscopy Congress, Aachen, Germany, 1–5 September 2008.
22.
Liu, K.; Wang, F.; Liu, G.; Dong, C. Effect of Environmental Conditions on Synnema Formation and Nucleoside Production in
Cicada Flower, Isaria cicadae (Ascomycetes). Int. J. Med. Mushrooms 2019,21, 59–69. [CrossRef]
23. Doyle, J.J.; Doyle, J.L. A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochem. Bull. 1987,19, 11–15.

J. Fungi 2021,7, 918 23 of 25
24.
Chin, C.-S.; Alexander, D.H.; Marks, P.; Klammer, A.A.; Drake, J.; Heiner, C.; Clum, A.; Copeland, A.; Huddleston, J.; Eichler, E.E.;
et al. Nonhybrid, finished microbial genome assemblies from long-read SMRT sequencing data. Nat. Methods 2013,10, 563–569.
[CrossRef]
25.
Koren, S.; Walenz, B.P.; Berlin, K.; Miller, J.; Bergman, N.H.; Phillippy, A.M. Canu: Scalable and accurate long-read assembly via
adaptivek-mer weighting and repeat separation. Genome Res. 2017,27, 722–736. [CrossRef]
26.
Luo, R.; Liu, B.; Xie, Y.; Li, Z.; Huang, W.; Yuan, J.; He, G.; Chen, Y.; Pan, Q.; Liu, Y.; et al. SOAPdenovo2: An empirically improved
memory-efficient short-read de novo assembler. GigaScience 2012,1, 18. [CrossRef]
27.
Parra, G.; Bradnam, K.; Korf, I. CEGMA: A pipeline to accurately annotate core genes in eukaryotic genomes. Bioinformatics
2007
,
23, 1061–1067. [CrossRef]
28.
Simão, F.A.; Waterhouse, R.M.; Ioannidis, P.; Kriventseva, E.V.; Zdobnov, E.M. BUSCO: Assessing genome assembly and
annotation completeness with single-copy orthologs. Bioinformatics 2015,31, 3210–3212. [CrossRef]
29.
Lowe, T.M.; Chan, P.P. tRNAscan-SE On-line: Integrating search and context for analysis of transfer RNA genes. Nucleic Acids Res.
2016,44, W54–W57. [CrossRef]
30.
Lagesen, K.; Hallin, P.; Rødland, E.A.; Staerfeldt, H.-H.; Rognes, T.; Ussery, D.W. RNAmmer: Consistent and rapid annotation of
ribosomal RNA genes. Nucleic Acids Res. 2007,35, 3100–3108. [CrossRef]
31.
Gardner, P.P.; Daub, J.; Tate, J.G.; Nawrocki, E.P.; Kolbe, D.L.; Lindgreen, S.; Wilkinson, A.C.; Finn, R.D.; Griffiths-Jones, S.; Eddy,
S.R.; et al. Rfam: Updates to the RNA families database. Nucleic Acids Res. 2009,37, D136–D140. [CrossRef]
32.
Lassmann, T.; Sonnhammer, E. Kalign, Kalignvu and Mumsa: Web servers for multiple sequence alignment. Nucleic Acids Res.
2006,34, W596–W599. [CrossRef]
33. Benson, G. Tandem repeats finder: A program to analyze DNA sequences. Nucleic Acids Res. 1999,27, 573–580. [CrossRef]
34.
Bao, W.; Kojima, K.K.; Kohany, O. Repbase Update, a database of repetitive elements in eukaryotic genomes. Mob. DNA
2015
,6,
1–6. [CrossRef] [PubMed]
35.
Chen, N. Using Repeat Masker to Identify Repetitive Elements in Genomic Sequences. Curr. Protoc. Bioinform.
2004
,5, 4–10.
[CrossRef] [PubMed]
36.
Hane, J.K.; Oliver, R.P. RIPCAL: A tool for alignment-based analysis of repeat-induced point mutations in fungal genomic
sequences. BMC Bioinform. 2008,9, 478. [CrossRef] [PubMed]
37.
Blow, M.J.; Clark, T.; Daum, C.G.; Deutschbauer, A.M.; Fomenkov, A.; Fries, R.; Froula, J.; Kang, D.D.; Malmstrom, R.; Morgan,
R.D.; et al. The Epigenomic Landscape of Prokaryotes. PLoS Genet. 2016,12, e1005854. [CrossRef]
38.
Ter-Hovhannisyan, V. Unsupervised and Semi-Supervised Training Methods for Eukaryotic Gene Prediction. Ph.D. Thesis,
Georgia Institute of Technology, Atlanta, GA, USA, 2008.
39. Salamov, A.A.; Solovyev, V.V. Ab initio Gene Finding in Drosophila Genomic DNA. Genome Res. 2000,10, 516–522. [CrossRef]
40.
Allen, J.; Majoros, W.H.; Pertea, M.; Salzberg, S.L. JIGSAW, GeneZilla, and GlimmerHMM: Puzzling out the features of human
genes in the ENCODE regions. Genome Biol. 2006,7, S9. [CrossRef]
41.
Haas, B.J.; Zeng, Q.; Pearson, M.D.; Cuomo, C.A.; Wortman, J.R. Approaches to Fungal Genome Annotation. Mycology
2011
,2,
118–141.
42.
Finn, R.D.; Bateman, A.; Clements, J.; Coggill, P.; Eberhardt, R.Y.; Eddy, S.R.; Heger, A.; Hetherington, K.; Holm, L.; Mistry, J.; et al.
Pfam: The protein families database. Nucleic Acids Res. 2014,42, D222–D230. [CrossRef]
43.
Prakash, A.; Jeffryes, M.; Bateman, A.; Finn, R.D. The HMMER Web Server for Protein Sequence Similarity Search. Curr. Protoc.
Bioinform. 2017,60, 3–15. [CrossRef]
44.
Armenteros, J.J.A.; Tsirigos, K.D.; Sønderby, C.K.; Petersen, T.N.; Winther, O.; Brunak, S.; Von Heijne, G.; Nielsen, H. SignalP 5.0
improves signal peptide predictions using deep neural networks. Nat. Biotechnol. 2019,37, 420–423. [CrossRef]
45.
Emanuelsson, O.; Brunak, S.; Von Heijne, G.; Nielsen, H.A. Locating proteins in the cell using TargetP, SignalP and related tools.
Nat. Protoc. 2007,2, 953–971. [CrossRef]
46.
Sonnhammer, E.L.; von Heijne, G.; Krogh, A. A hidden Markov model for predicting transmembrane helices in pro-
tein sequences. In Proceedings of the 16th International Conference on Intelligent System for Molecular Biology,
Menlo Park, CA, USA, 1–28 July 1998.
47.
Horton, P.; Park, K.-J.; Obayashi, T.; Fujita, N.; Harada, H.; Adams-Collier, C.J.; Nakai, K. WoLF PSORT: Protein localization
predictor. Nucleic Acids Res. 2007,35, W585–W587. [CrossRef]
48.
Sperschneider, J.; Dodds, P.N.; Gardiner, D.M.; Singh, K.B.; Taylor, J.M. Improved prediction of fungal effector proteins from
secretomes with EffectorP 2.0. Mol. Plant Pathol. 2018,19, 2094–2110. [CrossRef]
49.
Urban, M.; Cuzick, A.; Rutherford, K.; Irvine, A.; Pedro, H.; Pant, R.; Sadanadan, V.; Khamari, L.; Billal, S.; Mohanty, S.; et al.
PHI-base: A new interface and further additions for the multi-species pathogen–host interactions database. Nucleic Acids Res.
2017,45, D604–D610. [CrossRef]
50.
Vaidya, G.; Lohman, D.J.; Meier, R. SequenceMatrix: Concatenation software for the fast assembly of multi-gene datasets with
character set and codon information. Cladistics 2011,27, 171–180. [CrossRef]
51.
Edgar, R.C. MUSCLE: Multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res.
2004
,32,
1792–1797. [CrossRef]
52.
Darriba, D.; Taboada, G.L.; Doallo, R.; Posada, D. ProtTest 3: Fast selection of best-fit models of protein evolution. Bioinformatics
2011,27, 1164–1165. [CrossRef]

J. Fungi 2021,7, 918 24 of 25
53.
Stamatakis, A. RAxML version 8: A tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics
2014
,30,
1312–1313. [CrossRef]
54. Rambaut, A. FigTree v. 1.4.3. 2016. Available online: http://tree.bio.ed.ac.uk/software/figtree (accessed on 14 May 2020).
55.
Sanderson, M.J. r8s: Inferring absolute rates of molecular evolution and divergence times in the absence of a molecular clock.
Bioinformatics 2003,19, 301–302. [CrossRef]
56.
Lücking, R.; Huhndorf, S.; Pfister, D.H.; Plata, E.R.; Lumbsch, H.T. Fungi evolved right on track. Mycology
2009
,101, 810–822.
[CrossRef]
57.
Huang, L.; Zhang, H.; Wu, P.; Entwistle, S.; Li, X.; Yohe, T.; Yi, H.; Yang, Z.; Yin, Y. dbCAN-seq: A database of carbohydrate-active
enzyme (CAZyme) sequence and annotation. Nucleic Acids Res. 2018,46, D516–D521. [CrossRef]
58.
Blin, K.; Shaw, S.; Steinke, K.; Villebro, R.; Ziemert, N.; Lee, S.Y.; Medema, M.H.; Weber, T. antiSMASH 5.0: Updates to the
secondary metabolite genome mining pipeline. Nucleic Acids Res. 2019,47, W81–W87. [CrossRef]
59.
Kumar, S.; Stecher, G.; Tamura, K. MEGA7: Molecular Evolutionary Genetics Analysis Version 7.0 for Bigger Datasets. Mol. Biol.
Evol. 2016,33, 1870–1874. [CrossRef]
60. Kornerup, A.; Wanscher, J.H. Methuen Handbook of Colour, 3rd ed.; Eyre Methuen: London, UK, 1978; p. 225.
61.
Zhang, Y.B.; Zhuang, W.Y. First step evaluation of Trichoderma antagonism against plant pathogenic fungi in dual culture.
Mycosystema 2017,36, 1251–1259. [CrossRef]
62. Feng, H.L.; Zhu, H.T. Histological studies on Cordyceps militaris.Acta Mycol. Sin. 1990,9, 1–5. [CrossRef]
63.
Kumar, A.; Sørensen, J.L.; Hansen, F.T.; Arvas, M.; Syed, M.F.; Hassan, L.; Benz, J.P.; Record, E.; Henrissat, B.; Pöggeler, S.; et al.
Genome Sequencing and analyses of Two Marine Fungi from the North Sea Unraveled a Plethora of Novel Biosynthetic Gene
Clusters. Sci. Rep. 2018,8, 1–16. [CrossRef]
64.
Cheng, J.T.; Cao, F.; Chen, X.N.; Li, Y.Q.; Mao, X.M. Genomic and transcriptomic survey of an endophytic fungus Calcarisporium
arbuscula NRRL 3705 and potential overview of its secondary metabolites. BMC Genom. 2020,21, 424. [CrossRef]
65.
Kramer, G.J.; Nodwell, J.R. Chromosome level assembly and secondary metabolite potential of the parasitic fungus Cordyceps
militaris. BMC Genom. 2017,18, 912. [CrossRef]
66. Galagan, J.E.; Selker, E.U. RIP: The evolutionary cost of genome defense. Trends Genet. 2004,20, 417–423. [CrossRef] [PubMed]
67.
Chater-Diehl, E.; Sarah, J.G.; Cytrynbaum, C.; Turinsky, A.L.; Choufani, S.; Weksberg, R. Anatomy of DNA methylation signatures:
Emerging insights and applications. Am. J. Hum. Genet. 2021,108, 1359–1366. [CrossRef]
68.
Xiao, G.; Ying, S.-H.; Zheng, P.; Wang, Z.-L.; Zhang, S.; Xie, X.-Q.; Shang, Y.; Leger, R.J.S.; Zhao, G.-P.; Wang, C.; et al. Genomic
perspectives on the evolution of fungal entomopathogenicity in Beauveria bassiana. Sci. Rep.
2012
,2, 483. [CrossRef] [PubMed]
69.
McElwain, J.C.; Punyasena, S. Mass extinction events and the plant fossil record. Trends Ecol. Evol.
2007
,22, 548–557. [CrossRef]
[PubMed]
70.
Quandt, C.A.; Kepler, R.M.; Gams, W.; Araújo, J.P.M.; Ban, S.; Evans, H.C.; Hughes, D.; Humber, R.; Hywel-Jones, N.; Li, Z.; et al.
Phylogenetic-based nomenclatural proposals for Ophiocordycipitaceae (Hypocreales) with new combinations in Tolypocladium.
IMA Fungus 2014,5, 121–134. [CrossRef] [PubMed]
71.
Cuomo, C.A.; Güldener, U.; Xu, J.-R.; Trail, F.; Turgeon, B.G.; Di Pietro, A.; Walton, J.D.; Ma, L.-J.; Baker, S.; Rep, M.; et al. The
Fusarium graminearum Genome Reveals a Link Between Localized Polymorphism and Pathogen Specialization. Science
2007
,
317, 1400–1402. [CrossRef]
72.
Asai, S.; Shirasu, K. Plant cells under siege: Plant immune system versus pathogen effectors. Curr. Opin. Plant Biol.
2015
,28, 1–8.
[CrossRef]
73.
Marton, K.; Flajšman, M.; Radišek, S.; Košmelj, K.; Jakše, J.; Javornik, B.; Berne, S. Comprehensive analysis of Verticillium
nonalfalfae in silico secretome uncovers putative effector proteins expressed during hop invasion. PLoS ONE
2018
,13, e0198971.
[CrossRef]
74.
Druzhinina, I.S.; Seidl-Seiboth, V.; Herrera-Estrella, A.; Horwitz, B.A.; Kenerley, C.M.; Monte, E.; Mukherjee, P.K.; Zeilinger, S.;
Grigoriev, I.; Kubicek, C.P. Trichoderma: The genomics of opportunistic success. Nat. Rev. Genet. 2011,9, 749–759. [CrossRef]
75.
Wang, X.; Zhang, X.; Liu, L.; Xiang, M.; Wang, W.; Sun, X.; Che, Y.; Guo, L.; Liu, G.; Guo, L.; et al. Genomic and transcriptomic
analysis of the endophytic fungus Pestalotiopsis fici reveals its lifestyle and high potential for synthesis of natural products. BMC
Genom. 2015,16, 1–13. [CrossRef]
76.
Zhang, J.; Qi, L.; Chen, G.; Yin, W.-B. Discovery and genetic identification of amphiphilic coprogen siderophores from Trichoderm
hypoxylon. Appl. Microbiol. Biotechnol. 2021,105, 2831–2839. [CrossRef]
77.
Qin, F.; Li, Y.; Lin, R.; Zhang, X.; Mao, Z.; Ling, J.; Yang, Y.; Zhuang, X.; Du, S.; Cheng, X.; et al. Antibacterial Radicicol Analogues
from Pochonia chlamydosporia and Their Biosynthetic Gene Cluster. J. Agric. Food Chem. 2019,67, 7266–7273. [CrossRef]
78.
Nam, S.; Ga, Y.J.; Lee, J.-Y.; Hwang, W.-Y.; Jung, E.; Shin, J.S.; Chen, W.; Choi, G.; Zhou, B.; Yeh, J.-Y.; et al. Radicicol Inhibits
Chikungunya Virus Replication by Targeting Nonstructural Protein 2. Antimicrob. Agents Chemother. 2021,65. [CrossRef]
79.
Turbyville, T.J.; Wijeratne, E.M.K.; Liu, M.X.; Burns, A.M.; Seliga, C.J.; Luevano, L.A.; David, C.L.; Faeth, S.H.; Whitesell, L.;
Gunatilaka, A.A.L. Search for Hsp90 Inhibitors with Potential Anticancer Activity: Isolation and SAR Studies of Radicicol and
Monocillin I from Two Plant-Associated Fungi of the Sonoran Desert. J. Nat. Prod. 2006,69, 178–184. [CrossRef]
80.
Awakawa, T.; Yang, X.-L.; Wakimoto, T.; Abe, I. Pyranonigrin E: A PKS-NRPS Hybrid Metabolite from Aspergillus niger Identified
by Genome Mining. ChemBioChem 2013,14, 2095–2099. [CrossRef]

J. Fungi 2021,7, 918 25 of 25
81.
Kishimoto, S.; Tsunematsu, Y.; Sato, M.; Watanabe, K. Elucidation of Biosynthetic Pathways of Natural Products. Chem. Rec.
2017
,
17, 1095–1108. [CrossRef]
82.
Wight, W.D.; Kim, K.-H.; Lawrence, C.B.; Walton, J.D. Biosynthesis and Role in Virulence of the Histone Deacetylase Inhibitor
Depudecin from Alternaria brassicicola. Mol. Plant-Microbe Interactions 2009,22, 1258–1267. [CrossRef]
83.
Kasahara, K.; Miyamoto, T.; Fujimoto, T.; Oguri, H.; Tokiwano, T.; Oikawa, H.; Ebizuka, Y.; Fujii, I. Solanapyrone Synthase, a
Possible Diels-Alderase and Iterative Type I Polyketide Synthase Encoded in a Biosynthetic Gene Cluster from Alternaria solani.
ChemBioChem 2010,11, 1245–1252. [CrossRef]
84.
Reeves, C.D.; Hu, Z.; Reid, R.; Kealey, J.T. Genes for the Biosynthesis of the Fungal Polyketides Hypothemycin from Hypomyces
subiculosus and Radicicol from Pochonia chlamydosporia. Appl. Environ. Microbiol. 2008,74, 5121–5129. [CrossRef]
85. He, Y.; Cox, R.J. The molecular steps of citrinin biosynthesis in fungi. Chem. Sci. 2016,7, 2119–2127. [CrossRef]
86.
Karlsson, M.; Atanasova, L.; Jensen, D.F.; Zeilinger, S. Necrotrophic Mycoparasites and Their Genomes. Microbiology
2017
,5, 1–21.
[CrossRef]
87. Barnett, H.L. A new Calcarisporium parasitic on other fungi. Mycologia 1958,50, 497–500. [CrossRef]
88.
Lv, Z.; He, Z.; Hao, L.; Kang, X.; Ma, B.; Li, H.; Luo, Y.; Yuan, J.; He, N. Genome Sequencing Analysis of Scleromitrula shiraiana, a
Causal Agent of Mulberry Sclerotial Disease With Narrow Host Range. Front. Microbiol. 2021,11, 603927. [CrossRef]
89.
Alexopoulos, C.J.; Blackwell, M.; Mims, C.W. Introductory Mycology, 4th ed.; John Wiley and Sons Inc.: Manhattan, NY, USA, 1996;
p. 164.
90.
Zeng, W.; Melotto, M.; He, S.Y. Plant stomata: A checkpoint of host immunity and pathogen virulence. Curr. Opin. Biotechnol.
2010,21, 599–603. [CrossRef]
91.
Kim, S.H.; Vujanovic, V. Relationship between mycoparasites lifestyles and biocontrol behaviors against Fusarium spp. and
mycotoxins production. Appl. Microbiol. Biotechnol. 2016,100, 5257–5272. [CrossRef]
92.
Lu, Z.; Tombolini, R.; Woo, S.; Zeilinger, S.; Lorito, M.; Jansson, J.K. In Vivo Study of Trichoderma -Pathogen-Plant Interactions,
Using Constitutive and Inducible Green Fluorescent Protein Reporter Systems. Appl. Environ. Microbiol.
2004
,70, 3073–3081.
[CrossRef]
93.
Hoch, H.C. Mycoparasitic relationships. III. Parasitism of Physalospora obtuse by Calcarisporium parasiticum. Can. J. Bot.
1977
,
55, 198–207. [CrossRef]
94.
Boddy, L.; Hiscox, J. Fungal Ecology: Principles and Mechanisms of Colonization and Competition by Saprotrophic Fungi.
Microbiol. Spectr. 2016,4, 293–308. [CrossRef]
95.
Grooters, A.M. Deep Fungal Infections. Saunders Manual of Small Animal Practice, 3rd ed.; Elsevier: Amsterdam, The Netherlands,
2006; pp. 435–444.
96.
Dos Santos, T.L.; Belan, L.L.; Zied, D.C.; Dias, E.S.; Alves, E. Essential oils in the control of dry bubble disease in white button
mushroom. Ciência Rural 2017,47. [CrossRef]
97.
Huang, Q.G.; Wang, S.; Zhang, Y. The interactions between Mycogone perniciosa and Agaricus bisporus.Mycosystema
2014
,33,
440–448. [CrossRef]
98.
Junker, K.; Chailyan, A.; Hesselbart, A.; Forster, J.; Wendland, J. Multi-omics characterization of the necrotrophic mycoparasite
Saccharomycopsis schoenii. PLOS Pathog. 2019,15, e1007692. [CrossRef]
99.
Quandt, C.A.; Bushley, K.E.; Spatafora, J.W. The genome of the truffle-parasite Tolypocladium ophioglossoides and the evolution
of antifungal peptaibiotics. BMC Genom. 2015,16, 1–14. [CrossRef]
100.
de Man, T.J.B.; Stajich, J.E.; Kubicek, C.P.; Teiling, C.; Chenthamara, K.; Atanasova, L.; Druzhinina, I.S.; Levenkova, N.; Birnbaum,
S.S.L.; Barribeau, S.; et al. Small genome of the fungus Escovopsis weberi, a specialized disease agent of ant agriculture. Proc.
Natl. Acad. Sci. USA 2016,113, 3567–3572. [CrossRef] [PubMed]
101.
Wang, H.; Dong, Y.; Liao, W.; Zhang, X.; Wang, Q.; Li, G.; Xu, J.-R.; Liu, H. High-Quality Genome Resource of Clonostachys rosea
strain CanS41 by Oxford Nanopore Long-Read Sequencing. Plant Dis. 2021, PDIS12202615A. [CrossRef]
102.
Aguileta, G.; Hood, M.E.; Refrégier, G.; Giraud, T. Chapter 3 Genome Evolution in Plant Pathogenic and Symbiotic Fungi. Adv.
Bot. Res. 2009,49, 151–193. [CrossRef]
103.
Keller, N.P. Translating biosynthetic gene clusters into fungal armor and weaponry. Nat. Chem. Biol.
2015
,11, 671–677. [CrossRef]
104.
Sung, G.-H.; Hywel-Jones, N.L.; Sung, J.-M.; Luangsa-Ard, J.J.; Shrestha, B.; Spatafora, J.W. Phylogenetic classification of
Cordyceps and the clavicipitaceous fungi. Stud. Mycol. 2007,57, 5–59. [CrossRef]
105.
Spatafora, J.W.; Sung, G.-H.; Sung, J.-M.; Hywel-Jones, N.L.; White, J. Phylogenetic evidence for an animal pathogen origin of
ergot and the grass endophytes. Mol. Ecol. 2007,16, 1701–1711. [CrossRef]
106. Harman, G.E.; Kubicek, C.P. Trichoderma and Gliocladium; Taylor and Francis: London, UK, 1998; Volume 2, pp. 243–270.