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Review
Mycoparasitism as a mechanism of Trichoderma-
mediated suppression of plant diseases
Prasun K. MUKHERJEE
a,
*, Artemio MENDOZA-MENDOZA
b
,
Susanne ZEILINGER
c
, Benjamin A. HORWITZ
d
a
Nuclear Agriculture and Biotechnology Division, Bhabha Atomic Research Centre, Trombay, Mumbai 400085, India
b
Bio-Protection Research Centre, Lincoln University, Lincoln, New Zealand
c
Department of Microbiology, Universit€
at Innsbruck, Technikerstrasse 25, A-6020 Innsbruck, Austria
d
Faculty of Biology, The Technion-Israel Institute of Technology, Haifa 32000, Israel
article info
Article history:
Received 29 April 2021
Received in revised form
29 October 2021
Accepted 8 November 2021
Keywords:
Biological control
Chitinases
Evolution
G-proteins
Glucanases
Glycoside hydrolases
MAP kinases
Mushrooms
Mycoparasitism
Mycorrhizae
Mycotrophy
Secondary metabolism
Signal transduction
Trichoderma
abstract
Trichoderma spp. are widely used as plant disease biocontrol agents in agriculture. Mycopar-
asitism, which is an ancestral trait of Trichoderma, is one of the most important mecha-
nisms of reducing the pathogen inocula. Mycoparasitism is a complex physiological
process that should be viewed in the broad perspective of microbial competition, and in-
volves the production of enzymes and secondary metabolites. Trichoderma spp. have tradi-
tionally been viewed as necrotrophic mycoparasites; however, there are evidences that, at
least in some instances, they behave as hemibiotrophs, causing minor damage to the host
cell wall and having an intracellular existence in the host cell for a significant period. In
this review, we cover different aspects of Trichoderma as mycoparasites, ranging from evo-
lution to genomics and interactions with “non-target” fungi.
ª2021 British Mycological Society. Published by Elsevier Ltd. All rights reserved.
*Corresponding author.
E-mail addresses: prasunmukherjee1@gmail.com,prasunm@barc.gov.in (P. K. Mukherjee), artemio.mendoza@lincoln.ac.nz
(A. Mendoza-Mendoza), Susanne.zeilinger@uibk.ac.at (S. Zeilinger), horwitz@technion.ac.il (B. A. Horwitz).
journal homepage: www.elsevier.com/locate/fbr
fungal biology reviews 39 (2022) 15e33
https://doi.org/10.1016/j.fbr.2021.11.004
1749-4613/ª2021 British Mycological Society. Published by Elsevier Ltd. All rights reserved.
1. Introduction
Trichoderma spp. (Hypocreales, Ascomycota) are widely used
as biological control (biocontrol) agents of plant diseases. His-
torically, Trichoderma-mediated biocontrol research was trig-
gered by the observation that these fungi can parasitize
other (plant pathogenic) fungi (Weindling, 1932). However, it
took 40 more years to demonstrate that Trichoderma can be
used in the field to suppress a fungal plant disease (Wells
et al., 1972). Since then, several publications highlighted myco-
parasitism as an important mechanism of biological control
(Lifshitz et al., 1986;Inbar et al., 1996;Howell, 2002;Steyaert
et al., 2003;Harman et al., 2004b;Xu et al., 2010;John et al.,
2010;Huang et al., 2011). Details of how Trichoderma parasitizes
other fungi were published, including the use of a biomimetic
system (Inbar and Chet, 1992), scanning and transmission
electron microscopy and fluorescence microscopy. Chiti-
nases, b-glucanases, and proteases were established as key
enzymes involved in mycoparasitism (V
azquez-Garcidue~
nas
et al., 1998;Cortes et al., 1998;Carsolio et al., 1999). This was fol-
lowed by the analyses of individual genes, especially those
involved in signal transduction, and then, genome-scale
studies (Druzhinina et al., 2011). In 1997, it was first observed
that Trichoderma colonization of roots could reduce the symp-
toms of a foliar pathogen, a phenomenon termed induced sys-
temic resistance (ISR) (we prefer to use the term induced
defence response or IDR, to encompass all forms of induced
resistance, both local and systemic, ISR or systemic acquired
resistance i.e., SAR) (Pieterse et al., 2014;Bigirimana et al.,
1997). IDR as a mechanism of plant disease control dominated
Trichoderma research in the next two decades (Harman et al.,
2004;Mendoza-Mendoza et al., 2018). Among the three com-
mon modes of disease suppression (mycoparasitism, antibi-
osis and IDR, apart from competition), mycoparasitism is the
most effective in reducing pathogen inoculum load, especially
for soil-borne pathogens, against which Trichoderma spp. are
widely used. The degree of disease suppression by mycopara-
sitic strains is stronger than those mediated by IDR or antibi-
osis (Zeilinger et al., 2016b), although synergistic activities in
these processes could occur. Here, we revisit mycoparasitism
as a mechanism of biocontrol by Trichoderma spp. In addition
to understanding the physiological basis of mycoparasitism
in-depth, the recent developments in genomics have allowed
us to identify new candidate genes for future research and
applications.
2. Mycoparasitism vs. Mycotrophy
A mycoparasitic fungus establishes itself as a parasite on
another fungus, be it an actively growing hypha or a resting
structure, like sclerotia (Fig. 1). The fungus, being parasitized,
is often referred to as host or prey. Mycotrophy refers to where
the nutrients come from, rather than the nature of the inter-
action. Mycotrophs obtain nutrition from fungi, live or dead,
and a mycotrophic fungus is not necessarily a mycoparasite.
Indeed, specialized mycoparasitism may derive from sapro-
trophy on fungal biomass (Druzhinina et al., 2011). Plant path-
ogens may kill host tissue (necrotrophs) or establish a close
interaction with host cells that remain living while supplying
nutrients (biotrophs). Likewise, a mycoparasite can survive as
a biotroph, obtaining nutrients from a living host, or a necrotr-
oph, which kills host cells and obtains nutrients from the dead
biomass (Barnett and Binder, 1973). Contrary to biotrophs,
necrotrophs often (avoiding over-generalization) have a broad
host range. An example of a biotrophic mycoparasite is Ampe-
lomyces quisqualis (Pleosporales) (Haridas et al., 2020), a biocon-
trol agent against powdery mildew fungi. As illustrated using
a GFP-tagged intracellular Ampelomyces strain, the mycopara-
site takes advantage of the host (Podosphaera xanthii; Erysi-
phales) life cycle and dispersal mechanism to spread its own
pycnidiospores (Fig. 2A).
Study of the three-way interaction of the basidiomycete
yeast Anthracocystis (Pseudozyma) flocculosa (Ustilaginales)
with powdery mildew and its barley (Hordeum vulgare) host
revealed another aspect of biotrophic mycoparasitism.
Anthracocystis parasitizes Blumeria (Erysiphales), a barley path-
ogen. The mycoparasite acts, transiently, as a pathogen taking
nutrients from barley, while eventually destroying Blumeria
(Laur et al., 2018). The overall balance of the interaction is
the destruction of the plant pathogen. Although some Tricho-
derma spp. are known as necrotrophs, intracellular parasitic
development has been documented (Rousseau et al., 1996,
and see Fig. 2). This could be thought of, by analogy with plant
pathogens, as a hemibiotrophic stage. It’s evident that the
boundary between biotroph and necrotroph is not completely
sharp, but the Trichoderma-host fungal interaction proceeds
strongly from a phase where intracellular growth can some-
times be observed, toward degradation of the host, even
when an intact host cell wall remains (Fig. 2B, D, E). Arbuscular
mycorrhizal fungus (AMF) symbioses involve most land
plants, including crops (Sawers et al., 2008), and it will be
imperative to balance the combined benefit of Trichoderma
and AMF against negative interactions like the one in
Fig. 2D, E. AMF can promote disease resistance in the host
plant, as shown, for example, in a recent study of rice (Oryza
sativa)(Campo et al., 2020). AMF, however, cannot substitute
for Trichoderma in mycoparasitism of soilborne pathogens.
3. Mycoparasitism in disease suppression
Trichoderma-mediated disease suppression is affected by a
combination of mycoparasitism, antibiosis, IDR and competi-
tive exclusion (Sharma et al., 2017). Trichoderma spp. are fast
colonizers of the spermosphere (seed zone) and rhizosphere
(root zone), which helps exclude invading pathogens when
the biocontrol fungi are applied to seeds or roots. In direct
antibiosis, secondary metabolites or secreted enzymes from
Trichoderma inhibit pathogen growth or germination (Howell,
2003;Viterbo et al., 2001). The metabolites could also
contribute to competition, mycoparasitism and IDR
(Zeilinger et al., 2016a). It is difficult to study the role of myco-
parasitism “in exclusion”, and hence there are few direct
studies on the role of mycoparasitism in plant disease control,
compared to numerous studies in vitro. Weindling (1932) was
the first to vividly describe the mycoparasitic behaviour of T.
virens (then described as T. lignorum)onRhizoctonia solani (Can-
tharellales): “coiling of hyphae, growth in straight or wavy
lines, coagulation of protoplasts, and loss of vacuolated
16 P. K. Mukherjee et al.
structures”. However, later on, the antagonistic activity of this
Trichoderma strain was attributed to a "lethal principle,” subse-
quently identified as gliotoxin (Weindling, 1934;Weindling
and Emerson, 1936), and the efficacy of this strain to suppress
R. solani was demonstrated in pot studies (Weindling and
Fawcett, 1936). Suppression of R. solani and Globisporangium
(Pythium)debaryanum in cucumber (Cucumis sativus) and peas
(Pisum sativum) in pot studies was attributed to the antibiotic
activity of T. virens (Allen and Haenseler, 1935). Several au-
thors studied the ultrastructure of hyphal parasitism using
electron microscopy and fluorescence microscopy (e.g.,
Hashioka, 1973;Ruano-Rosa et al., 2016). Harman et al. (1980)
proposed mycoparasitism to be the mechanism of biocontrol
of Pythium spp. (Pythiales) and R. solani when Trichoderma
hamatum was applied as a seed treatment. This was based
on the fact that T. hamatum, a strong mycoparasite, showed
no antibiosis against these pathogens. Applied to soil, an
isolate of Trichoderma harzianum [please note that the former
metaspecies T. harzianum has been divided into 14 phyloge-
netic species by Chaverri et al. (2015), and subsequently,
several other species have been added (Cai and Druzhinina,
2021). In this review, we have retained the original species
designation as described in the literature] that is mycopara-
sitic on R. solani and S. rolfsii suppressed diseases caused by
these pathogens under field conditions (Elad et al., 1980). The
authors did not discuss whether the biocontrol activity was
due to mycoparasitism alone. A mycoparasitic strain of T.
virens suppressed R. solani-incited disease in white beans (Pha-
seolus vulgaris) in a dose-dependent manner (Tu and Vaartaja,
1981). Application of Trichoderma koningii reduced the inoc-
ulum levels of Sclerotinia sclerotiorum (Helotiales) in soil by
parasitizing the sclerotia (Santos and Dhingra, 1982;
Trutmann and Keane, 1990). Based on a comparative assess-
ment of hyphal parasitism, sclerotial parasitism and antibi-
osis, parasitism of sclerotia was proposed as the principal
mechanism of suppression of S. rolfsii and R. solani by a “P”
(gliovirin-producing) strain of T. virens in soil (Mukherjee
et al., 1995). Using gamma-ray induced mutagenesis, we
recently isolated a mutant (G2) that produces more secondary
metabolites and has genes related to secondary metabolism,
mycoparasitism and plant interactions upregulated
(Mukherjee et al., 2019). In greenhouse experiments, this
mutant showed improved biocontrol against S. rolfsii, over
the wild type strain. Excellent field control of collar rot (S. rolf-
sii) in chickpea (Cicer arietinum) and lentil (Lens culinaris) was
demonstrated over five years, both in “on-farm” trials and in
farmers’ fields (Fig. 3). Howell (1987) generated UV-induced
mutants of T. virens that were deficient in hyphal parasitism
of R. solani; the mutants were as effective as parental type in
biocontrol potential. Similarly, mutants deficient in gliotoxin
biosynthesis were equally effective in biocontrol. These obser-
vations questioned the role of mycoparasitism and antibiosis,
and induced resistance was emphasized (Howell, 1987,2003).
Howell et al. (1987), however, did not consider the role of para-
sitism of the sclerotia in the biocontrol efficacy of T. virens.In
an early report, T. virens (then described as T. lignorum) was
found to parasitize the sclerotia of R. solani (then described
as Corticium sasakii) and Sclerotinia libertiana (obsolete taxon-
omy, probably S. sclerotiorum) in an assay using river sand
(Hino and Endo, 1940). An isolate of T. virens (earlier described
as Gliocladium virens) readily parasitized the hyphae and scle-
rotia of S. sclerotiorum. Extensive hyphae of the mycoparasite
were seen inside the colonized sclerotia, but no conidia were
present (Tu, 1980).
4. Evolution of mycoparasitism in a genomic
perspective
Trichoderma genome sizes are typical for filamentous fungi: T.
atroviride 36.1 and T. virens 39.0 Mbp, somewhat larger than
R. solani
S. rolfsii
F
R
T
R
R
R
R
T
T
T
T
AB
CD
E
Fig. 1 eMycoparasitism of Trichoderma virens on Rhizoctonia solani and Sclerotium rolfsii.(AeD) Hyphal interactions between
T. virens (T) and R. solani (R) involving coiling structures and collapse of the host mycelium. E and F: Growth and sporulation of
T. virens inside the sclerotia of S. rolfsii and R. solani, respectively (SEM). Once Trichoderma colonises the sclerotia, it is able to
conidiate inside the fungal host tissue. Scale bar- A: 5 mm, BeF: 10 mm. Source: AeD(Mukherjee et al., 2012a), with
permission; E and F (Mukherjee et al., 1995), with permission.
Trichoderma-mediated biocontrol mechanism 17
Fig. 2 eMycoparasitic fungus efungal host interactions. (A) GFP-expressing Ampelomyces quisqualis strain RS1(Aq) intra-
cellular to a developing Podosphaera xanthii (Px) conidiophore; scale bar 20 mm; from N
emeth et al. (2019). (B) Trichoderma
virens (Tv) penetrating into hyphae of Rhizoctonia solani (Rs) (A. MendozaeMendoza, unpublished) and (C) in a scanning
electron microscopy (SEM) image (from: beneficial-soil-microorganisms.pdf (bioworksinc.com) with permission); Th [Tri-
choderma afroharzianum (D,E) Trichoderma atroviride (Ta) growing inside a hypha of the arbuscular mycorrhizal fungus (AMF)
Gigaspora gigantea (Gg) (white arrows; scale bar 15 *m); (From Lace et al., 2015). Images (A) and (D, E) are reproduced with
permission via RightsLink.
Fig. 3 eDemonstration of biocontrol of chickpea collar rot (Sclerotium rolfsii) using seed treatment with Trichoderma virens G2
in a farmer’s field (Raipur, India, 2020e21). Photo courtesy: Dr. BP Tripathi and Dr. Anil Kotasthane.
18 P. K. Mukherjee et al.
the weakly mycoparasitic T. reesei at 33 Mbp (Martinez et al.,
2008;Kubicek et al., 2011;https://mycocosm.jgi.doe.gov/
mycocosm/home). The evolutionary success of fungi resides
in their ability to grow indefinitely as hypha, extremely high
metabolic diversity and ability to interact with living organ-
isms (Naranjo-Ortiz and Gabald
on, 2019). Mycoparasitic asso-
ciations are already found in the oldest fungal fossils formed
around 410 mya (Hass et al.,1994), and this association is
widespread among early-diverging fungi; however, the best-
studied mycoparasites belong to the order Hypocreales
(Naranjo-Ortiz and Gabald
on, 2019). Trichoderma spp. are
ubiquitous fungi displaying a remarkable range of lifestyles
and interactions with other fungi, animals and plants that
evolved in the time of the Cretaceous-Palaeogene extinction
event 66 (15) mya (Druzhinina et al.,2011). A main conclu-
sion, comparing the first three sequenced Trichoderma ge-
nomes (T. reesei, T. virens and T. atroviride), was that
mycotrophy was the ancestral lifestyle of the genus
(Kubicek et al., 2011;Schmoll et al., 2016;Karlsson et al.,
2017). This ancient and basic trait allowed further evolution
leading to the ability to colonize newer ecological niches
like dead wood, plants and animals, including immune-
compromised humans (Druzhinina et al., 2011,2018). It is pre-
dicted that Trichoderma evolved from an ancestor with limited
cellulolytic capability that fed on either fungi or arthropods,
and the formation of the genus was accompanied by an un-
precedented extent of lateral gene transfer (LGT). Nearly
half the genes for plant cell wall-degrading enzymes
(CAZymes) were from plant-associated Ascomycota, reflect-
ing its ability to mycoparasitize these fungi. Even though Tri-
choderma spp. can parasitise unrelated fungi (like
basidiomycetes) and oomycetes, LGT is not reported for this
kind of association (Druzhinina et al., 2018). Aiming to under-
stand the evolution of nutritionally generalist lifestyles,
Kubicek et al. (2019) looked at the many recently sequenced
Trichoderma genomes, proposing that the most frequently
sampled species in the databases should be the most success-
ful generalists (readers may refer to this article for a detailed
phylogenomic analysis). Thus, the 12 most common species
were defined by the number of NCBI GenBank entries. Inven-
tory of the genes present in all Trichoderma species (core
genome) but not shared with other fungi may tell what is spe-
cial about Trichoderma: gene families specific to the mycopar-
asites provide a hint for function in the deconstruction of the
host/prey cells. The greatest numbers of genes gained by Tri-
choderma belong to ankyrin-repeat, HET (heterokaryon in-
compatibility) domain, and MFS transporter families. The
families encoding CAZymes, transcription factors and sec-
ondary metabolism-related genes are also expanded relative
to other fungi. The HET domain genes are intriguing because
they might have a role, in addition to heterokaryon incompat-
ibility, in sensing the host fungus (Kubicek et al., 2019). Pecti-
nolytic enzyme families, in contrast, are more typical in size,
perhaps allowing easier coexistence with plants.
Transcriptomics of Trichoderma ininteractionwithother
fungi began with EST libraries predating microarray and
RNASeq platforms (Seidl et al., 2009). Further study by
sequencing again identified gene sets enriched for meta-
bolism during interaction with the host and time-resolved
expression from before contact through direct interaction
(Reithner et al.,2011). Atanasova et al. (2013a) looked at T.
atroviride,T. virens and T. reesei, in interaction with R. solani.
The three species had different strategies, as inferred from
the transcriptome: T. atroviride had a diverse strategy, up-
regulating secondary metabolite biosynthesis, GH16 b-gluca-
nases, proteases and small secreted proteins; T. reesei
increased the expression of genes encoding cellulases and
hemicellulases as well as transporters; while T. virens appar-
ently relied strongly on its toxic secondary metabolite,
expressing mainly the genes related to the biosynthesis of
gliotoxin (Atanasova et al., 2013a). T. harzianum expresses
distinct b-1,3- and b-1,6-glucanases (the latter belonging to
GH families 5 and 30) during co-culture with host fungi and
upon growth in media containing fungal cell walls (Cohen-
Kupiec et al., 1999;De la Cruz et al., 1995;Montero et al.,
2005;Monteiro and Ulhoa, 2006;Gajera, 2012;Troian et al.,
2014). Similarly, Trichoderma asperellum reacted to fungal
plant pathogens, expressing chitinases and b-1,3-
glucanases (Guig
on-L
opez et al., 2015). T. atroviride (strain
T11) is effective against Verticillium dahliae (Glomerellales).
Proteolysis was a prominent process inferred from the tran-
scriptome, with 10 proteases among the 143 up-regulated
genes (Mor
an-Diez et al., 2019). Recent transcriptomics of
the Trichoderma gamsii eFusarium graminearum (Hypocreales)
interaction focused on the sensing phase before contact. T.
gamsii upregulates a ferric reductase to help compete for
iron; the host upregulates transporters and killer toxins
that could help defend against the mycotroph, while, sur-
prisingly (perhaps to strengthen the cell wall before contact),
T. gamsii downregulates chitinolytic enzymes (Zapparata
et al., 2021).
Whether Trichoderma is aggressively attacking other fungi
in the soil or making a home in the outer root layers, promot-
ing plant growth and immunity, its genome must encode the
genes for traits relevant to such lifestyles. Chitinases and pro-
teases should be most important to degrade fungi, while cellu-
lases, pectinases and expansin-like proteins could act, in a
limited way, on plant cell walls to facilitate ingress into roots,
although cellulases might also be useful for the colonisation of
other filamentous microorganisms like oomycetes. Transcrip-
tomic data are available to answer whether different
CAZymes are expressed in interaction with plants or fungi
(Chac
on et al., 2007;Samolski et al., 2009;Mor
an-Diez et al.,
2019;Malinich et al., 2019;Schweiger et al., 2021;Villalobos-
Escobedo et al., 2020). There is a detailed T. hamatum transcrip-
tome in interaction with S. sclerotiorum and lettuce seedlings
(Shaw et al., 2016). Comparing transcriptomes from Tricho-
derma-fungal with Trichoderma-plant interactions, one can
start addressing the general hypothesis that regulated gene
expression permits the extensive host range (mycotroph and
plant mutualist) of Trichoderma.AT. virens gamma-ray
induced mutant (M7) carrying several major deletions pro-
vides a “minimal” genome, allowing growth but not regulated
processes including conidiation, secondary metabolism and,
of relevance here, mycoparasitism (Pachauri et al., 2020).
Thus, the gene sets missing and/or underexpressed in the
mutant M7 provide leads for discovering mycoparasitism-
relevant functions.
Trichoderma-mediated biocontrol mechanism 19
5. Role of cell wall-degrading enzymes
Considering that fungal cell walls are composed of glucans,
chitin and proteins (Gow et al., 2017;Garcia-Rubio et al., 2020;
Ruiz-Herrera and Ortiz-Castellanos, 2019), one can expect
that glucanases, chitinases and proteases degrade these bio-
polymers in mycoparasitism. Among these, those involved
in chitin degradation are the best studied.
5.1. Enzymes involved in chitin and chitosan degradation
Compared to other fungi, the genomes of Trichoderma species
harbour a high number of chitinolytic genes (Ihrmark et al.,
2010;Kubicek et al., 2011,2019) reflecting the importance of
these enzymes in the mycoparasitic lifestyle. In contrast,
few chitinases are encoded in early-diverging fungi, and fungi
associated with plants, animals or the open environment such
as the rumen symbionts Neocallimastigomycota, the endo-
parasite Rozella and the Glomerales arbuscular mycorrhiza
fungi (Goughenour et al., 2021). Fungal chitinases belong to
the GH families 18 and 20. GH18 chitinases are further divided
into subfamilies A, B and C. From the Trichoderma genomes
analyzed in detail so far, the GH18 family of chitinolytic en-
zymes is significantly expanded in T. virens,T. atroviride,T. har-
zianum, T. asperellum, T. gamsii and T. atrobrunneum (Kubicek
et al., 2011;Fanelli et al., 2018). Similarly, the number of chito-
sanases (GH75) is enhanced, with at least five corresponding
genes compared to only one or two in most other fungi
(Kubicek et al., 2011;Karlsson et al., 2017;Kappel et al., 2020).
Endochitinases perform the initial attack on chitin in the
hosts’ cell wall (Klemsdal et al., 2006;Boer et al., 2007). Indeed
many of these genes are expressed during the mycoparasitic
interactions or are induced by fungal cell walls (Garc
ıa et al.,
1994;Steyaert et al., 2004;Seidl, 2008). ECH42 is the most stud-
ied endochitinase and, together with CHT33 (Chit33/CHI18-
12), CHT36 (Chit36/CHI18-15) and the GH20 N-acetyl-b-gluco-
saminidase NAG1, is the most abundant chitinase present un-
der chitinase-inducing conditions (Carsolio et al., 1999).
The chitinous layer of fungal cell walls typically contains a
mixture of chitin and its totally or partly deacetylated deriva-
tive chitosan (Gow et al., 2017). Of six chitin deacetylase genes
encoded in the T. atroviride genome, deletion of cda1 or cda5 led
to mutants with severely impaired mycoparasitic abilities
(Kappel et al., 2020). Chitosan may scavenge reactive oxygen
species produced by the parasitized fungi (Delgado-Jarana
et al., 2006), and chitin deacetylases might allow Trichoderma
to cope with the reactive oxygen burden. Kappel et al. (2020)
furthermore analyzed the role of predicted exo- and endo-
acting chitosanases of T. atroviride. All six chitosanase-
encoding genes were upregulated after host contact and lysis,
i.e., especially during the later stages of the mycoparasitic
interaction and two (cho1 and cho3) also during contact of Tri-
choderma with itself, evidencing, similar to chitinases, a gen-
eral role in cell wall remodelling (Kappel et al., 2020). The
same study further proved the involvement of chitin syn-
thases in cell wall remodelling during T. atroviride mycoparasi-
tism, of which CHS8 emerged as a candidate of particular
interest. This enzyme not only shows similarity to chitin syn-
thases but also to hyaluronan synthases. Such hybrid
synthases may use both UDP-N-acetyl-glucosamine and
UDP-D-glucuronate as substrates. The authors hence specu-
lated that CHS8, in cooperation with CDA1, forms a chitin gly-
copolymer layer protecting the Trichoderma cell wall during
the mycoparasitic interactions (Kappel et al., 2020).
5.2. a- and b-glucanases
Fungal a-1,3-glucanases are classified as GH71 family mem-
bers, and there is only little information about these enzymes
in Trichoderma. The exo-a-1,3-glucanases AGN13.1 and
AGN13.2 are produced explicitly by T. harzianum CECT 2413
and T. asperellum CECT20539,respectively, in the presence of
Botrytis cinerea (Helotiales) cell walls but not chitin (Ait-
Lahsen et al., 2001;Sanz et al., 2004). AGN13.1 exhibited lytic
properties against fungal cell walls and antifungal activity
(Ait-Lahsen et al., 2001). An a-1,3-glucanase MUT1 (MutAp)
with endo-hydrolytic activity has been purified from T. harzia-
num OMZ779 and shown to predominantly release glucose
upon hydrolysis of crystalline 1,3-a-glucan (Guggenheim and
Haller, 1972;Gr€
un et al., 2006). However, no reports on its tran-
scriptional regulation and biological role are available.
b-1,3-glucanases belong to GH families 16, 17, 55, 64, and
81, of which the number of genes coding for GH55 and GH64
family members are expanded in Trichoderma mycoparasites
compared to other fungi (Kubicek et al., 2011;Fanelli et al.,
2018). b-glucanases have been suggested to be especially
important for mycoparasitism of oomycete preys, whose cell
walls mainly contain cellulose and b-1,3- and b-1,6-glucans.
Trichoderma longibrachiatum Rifai CECT2606 mutants with
increased expression of the EGL1 b-1,4-endoglucanase were
better in protecting cucumber seeds in Globisporangium
(Pythium)ultimum (Pythiales) infested soil (Migheli et al.,
1998). Similar results were obtained with T. virens Gv29-8,
where mutants overexpressing the b-1,6-glucanase encoding
gene bgn3 showed enhanced antagonism of G. ultimum, and
strains overexpressing bgn3 and the b-1,3-glucanase gene
bgn2 displayed enhanced pathogen inhibition and protection
of cotton (Gossypium hirsutum) plants against G. ultimum
(Djonovi
cet al., 2006b,2007). Contrary to these reports, glu31
(gluc31/encoding GH16 endo-b-1,3-glucanase) showed differ-
ential expression during interaction with certain host fungi,
but loss of the gene did not affect the mycoparasitic activity;
instead, it impacted the cell wall organization and remodel-
ling and resulted in the differential expression of other GH16
family glycosyl hydrolases (Suriani Ribeiro et al., 2019). Studies
on the function of glucanases in fungal mycoparasites are
scarce. Because these enzymes may be functionally redun-
dant, further efforts, including the (admittedly difficult) gener-
ation of multiple mutants, are needed before the definite role
of glucanases in Trichoderma mycoparasitism can be
appraised.
5.3. Proteases
Like CAZymes, proteases are essential for fungal cell wall
degradation and hence implicated in the antagonistic activity
of Trichoderma mycoparasites, destabilizing the cellular integ-
rity of host fungi and inactivating host-derived enzymes (Elad
and Kapat, 1999). The number of proteases encoded in
20 P. K. Mukherjee et al.
Trichoderma species is comparable to other fungi (Kubicek
et al., 2019); however certain families including S8 subtilisin-
like proteases are expanded in the well-characterized myco-
parasites T. virens and T. atroviride compared to the weakly
mycoparasitic T. reesei (Kubicek et al., 2011). Several protease
genes are differentially regulated during mycoparasitism or
growth on fungal cell walls (Seidl et al., 2009;Geremia et al.,
1993;Viterbo et al., 2004;Su
arez et al., 2007;Troian et al.,
2014). Expression of the gene encoding the neutral metallopro-
tease NMP1 of Trichoderma guizhouense NJAU4742 and its
orthologue in T. harzianum CECT2413 is induced by the pres-
ence of other fungi and by dead fungal biomass, respectively
(Suarez et al., 2004;Zhang et al., 2016). NMP1 further emerged
as important for the antifungal activity and as a critical
enzyme for interaction (including parasitism, predation and
defense) of T. guizhouense with other fungi (Zhang et al.,
2016). The S8 family protease-encoding prb1 of T. atroviride is
among the best characterized mycoparasitism-relevant genes
(Geremia et al., 1993). Overexpression of T. atroviride (previ-
ously T. harzianum)prb1 or its homologue from T. virens
resulted in an increased plant protection against R. solani
(Flores et al., 1997;Pozo et al., 2004). Similarly, protease over-
production obtained via random UV mutagenesis trans-
formed T. harzianum T334 into a more effective antagonist of
plant pathogens (Szekeres et al., 2004). Like ech42,prb1 gene
expression is induced already before contact with the fungal
host (Cortes et al., 1998). Further analyses revealed prb1 tran-
scription in response to nitrogen limitation, which is in accor-
dance with potential binding sites for the ARE1 transcriptional
activator of nitrogen catabolite-repressed genes in its pro-
moter region (Olmedo-Monfil et al., 2002). Accordingly, more
recent transcriptome studies evidenced that the response of
T. atroviride to a fungal host resembles the gene expression
pattern upon nitrogen limitation stress and confirmed an
important role of proteases in mycoparasitism in T. virens
and T. reesei (Seidl et al., 2009;Atanasova et al., 2013b). These
findings led to the hypothesis that the action of proteolytic en-
zymes in the early stages of the mycoparasitic interaction re-
sults in host-derived nitrogenous products that trigger the
activation of mycoparasitism-relevant genes by binding to
respective nitrogen sensors on the Trichoderma cell surface
(Druzhinina et al., 2011).
6. Secondary metabolites and their role in
mycoparasitism
Trichoderma spp. are effective producers of secondary metabo-
lites, which serve functions not only in antagonism (antibiosis
and mycoparasitic attack) by acting as chemical weapons but
also in the interactions with plants as well as, in self-
signalling (Mukherjee et al., 2012a;Zeilinger et al., 2016a). The
numerous secondary metabolites reported for Trichoderma
fungi over the years comprise polyketides, non-ribosomal
peptides, terpenoids, pyrones, etc., and include volatiles and
non-volatiles (Zeilinger et al., 2016a). A recent comparative ge-
nomics study identified 10e25 genes coding for polyketide
synthases (PKS), 12e34 genes encoding non-ribosomal poly-
peptide synthetases (NRPS), and 6e14 genes for terpenoid syn-
thases (TS) in 12 Trichoderma species studied (Kubicek et al.,
2019). These core genes code for backbone-generating en-
zymes that act in discrete biosynthesis pathways for specific
secondary metabolites and are often part of metabolite gene
clusters. Species from the Harzianum/Virens clades contain
the highest number of PKS genes (Kubicek et al., 2019)of
which, however, only two have been functionally character-
ized hitherto.Secondary metabolite production may be spe-
cies- or even strain-specific as exemplified by 6-pentyl-2H-
pyran-2-one/6-pentyl-a-pyrone (6-PP) and other volatile
organic compounds (VOCs).
Transcript levels of pks2 (pksT-2) were upregulated in T.
harzianum 88 upon confrontation with host fungi, and disrup-
tion of the gene impacted the conidial pigmentation of the
fungus (Yao et al., 2016). Similarly, pks4 of T. reesei QM6a has
been shown to provide pigmentation of conidia and teleo-
morph structures and impact the antagonistic activity of T.
reesei by affecting the formation of inhibitory metabolites
and the expression of other PKS-encoding genes (Atanasova
et al., 2013b). Sorbicillinoids are cyclic polyketides bio-
synthesized by a gene cluster comprising two PKS (SOR1 and
SOR2) which is only present in the section Longibrachiatum
(Druzhinina et al., 2016). In direct confrontations, the sorbicil-
linoids supported growth of T. reesei QM6a in presence of
fungal plant pathogens (Derntl et al., 2017). Accordingly, the
sor1 and sor2 genes were strongly upregulated during interac-
tion with R. solani and under conditions of sexual develop-
ment (Atanasova et al., 2013b;Hinterdobler et al., 2019).
Although individual PKS-encoding genes are poorly studied
in Trichoderma mycoparasites, the importance of PKS and
NRPS in antagonism has been further supported by the func-
tional characterization of the 4-phosphopantetheinyl
transferase-encoding gene ppt1 of T. virens Gv29-8. Inactivat-
ing ppt1 in T. virens resulted in mutants with non-pigmented
conidia and a defect in the biosynthesis of non-ribosomal pep-
tides that went along with a loss of the in vitro antagonistic ac-
tivity against phytopathogenic fungi and the ability to induce
plant defense responses (Vel
azquez-Robledo et al., 2011).
Peptaibols, siderophores and epipolythiodioxopipera-
zines (ETPs) are some of the main non-ribosomal peptides
produced by Trichoderma species. Peptaibols can permeabi-
lize cell membranes, and work synergistically with cell wall
degrading enzymes to promote cell disruption (Schirmbock
et al., 1994;Lorito et al., 1996;Shi et al., 2012). Interestingly,
however, peptaibol synthetase genes are downregulated in
T. atroviride,T. virens as well as T. reesei during the mycopar-
asitic interaction with R. solani (Atanasova et al., 2013b)but
upregulated in T. virens during co-cultivation with plant roots
(Viterbo et al.,2007). Nevertheless, even low gene expression
seems to suffice for adequate peptaibol production as shown
by Holzlechner et al. (2016). MALDI mass spectrometry
imaging-based monitoring of secondary metabolites directly
in T. atroviride P1 eR. solani co-cultures revealed a distinct
local release of peptaibols with chain lengths of 11, 18 and
20 residues in both the pre-contact as well as contact stage
of the interaction (Holzlechner et al., 2016). Similarly, T. har-
zianum CCF2714 produced peptaibols with antifungal activity
when co-cultured with Fusarium oxysporum f. sp. conglutinans
(Hypocreales) and simultaneously was able to block the pro-
duction of the mycotoxin beauvericin by the plant pathogen
(Palyzov
aet al., 2019).
Trichoderma-mediated biocontrol mechanism 21
The limited bioavailability of iron in soil provides an
advantage to microbes that produce efficient extracellular
siderophores (Haas et al., 2008). Accordingly, iron competition
has been suggested to contribute to Trichoderma mycoparasi-
tism by suppressing the growth of other microbes (Ben
ıtez
et al., 2004). Vinale et al. (2017) reported siderophore accumula-
tion in a T. harzianum M10 eTalaromyces pinophilus (Eurotiales)
co-culture; nevertheless, direct experimental proof for the
involvement of siderophores in the mycoparasitic interaction
is missing.
Gliotoxin is an NRPS GLI1(GliP)-derived substance of the
ETPs family which is produced by certain T. virens strains,
designated as “Q” strains. GLI1 has been identified in all spe-
cies of section Longibrachiatum and Harzianum/Virens clades
(Kubicek et al., 2019), and a truncated gliotoxin biosynthesis
cluster seems to be quite common in Trichoderma species,
including even those that do not produce this metabolite
(Mukherjee et al., 2012b;Bulgari et al., 2020). Based on the
fungistatic activity of gliotoxin and the up-regulation of the
gliotoxin cluster genes during the mycoparasitic interaction
with R. solani,T. virens gliotoxin producers seem to follow a
“predation by poisoning” strategy during antagonism
(Atanasova et al., 2013a). Interestingly, however, gli1 mutants
retained their mycoparasitic activity against R. solani but
became ineffective as mycoparasites of G. ultimum and S. scle-
rotiorum (Vargas et al., 2014)(Fig. 4).
Volatile organic compounds (VOCs) are secondary metabo-
lites that might contribute to mycoparasitism by inhibiting
fungal hosts (Ait-Lahsen et al., 2001;Cruz-Magalh~
aes, et al.,
2019;Moya et al., 2018). 6-PP is one of the main volatile second-
ary metabolites of T. atroviride.Trichoderma viridescens,T. hama-
tum, and Trichoderma citrinoviride also were reported as 6-PP
producers (Jele
net al., 2014). 6-PP has antifungal and plant
growth-modulating activities. The oxidation of linoleic acid
to 13S-hydroperoxy-9Z,11E-octadecadienoic acid by a
lipoxygenase has, for decades, been postulated as the first
and limiting step in 6-PP production by T. atroviride (Serrano-
Carreon et al., 1993). By deleting the single lipoxygenase-
encoding gene lox1 present in the T. atroviride genome, howev-
er, we recently disproved this assumption by showing that
LOX1 is dispensable for 6-PP biosynthesis (Speckbacher et al.,
2020). The culture supernatant of R. solani significantly
increased 6-PP production of T. atroviride IMI 206040 (Flores
et al., 2019). A recent study revealed that 6-PP disturbs the ho-
meostasis of amino acid metabolism and induces autophagy
in the ginseng root pathogen Ilyonectria (Cylindrocarpon)
destructans (Jin et al., 2020). Besides, 6-PP can inhibit mycotoxin
secretion by F. graminearum (Cooney et al., 2001) and this vola-
tile acts as an important self-signaling compound. 6-PP
emerged as a highly potent chemoattractant and growth-
stimulating cue for T. atroviride P1 which, especially in the
early stages of colony development, represents an important
self-signal that advances expansion and density of the
mycelia required for the mycoparasitic attack (Moreno-Ruiz
et al., 2020).
T. guizhouense produces excessive hydrogen peroxide
(H
2
O
2
) in interfungal interaction with its host fungus Fusarium
odoratissimum (previously F. oxysporum f. sp. cubense race 4)
(Zhang et al., 2019;Pang et al., 2020). The formation of H
2
O
2
is
necessary for the mycoparasitic activity and is triggered by
the contact with a host fungus by a mechanism dependent
on the NADPH oxidase NOX1 and its regulator NOR1 (Zhang
et al., 2019). T. guizhouense produces polyketides (azaphilones)
as a response to the accumulation of H
2
O
2
and these are
necessary for the self-protection against the reactive oxygen
species produced by Trichoderma during the interaction with
F. odoratissimum (Pang et al., 2020).
Moreover, the overexpression of NOX1 in T. harzianum T34
resulted in increased protease, cellulase and chitinase activ-
ities with respect to the parental strain. Surprisingly, the
Fig. 4 eDeletion of gliP attenuates mycoparasitism in Trichoderma virens (Source: Vargas et al., 2014).
22 P. K. Mukherjee et al.
antagonistic activity in confrontation assays against the
oomycete G. ultimum was more effective in the overexpressing
strains than in the wild type, but not in assays against B. cin-
erea or R. solani (Montero-Barrientos et al., 2011), suggesting
the existence of different mechanisms of host protection.
7. Role of small, secreted cysteine-rich
proteins
Around 60% of the proteins secreted by Trichoderma spp. are
represented by CAZymes, small secreted cysteine-rich pro-
teins (SSCPs), and proteins with an unknown functional anno-
tation (Druzhinina et al., 2012;Mendoza-Mendoza et al., 2018).
In Trichoderma, SSCPs are known to be involved in mycopara-
sitism and induction of plant defences (Schmoll et al., 2016;
Guzm
an-Guzm
an et al., 2017;Mendoza-Mendoza et al., 2018;
Romero-Contreras et al., 2019).
Cerato-platanins (CPs) are SSCPs, usually having between
105 and 134 amino acid residues, with no known enzymatic
activity. CPs contain four conserved cysteine residues forming
two disulphide bridges (Gao et al., 2020). CPs are released dur-
ing early stages of development and play roles in inducing sys-
temic resistance in plants (Djonovi
cet al., 2006a); therefore
these CPs are also called eliciting plant response-like proteins
(EPLs) (Gaderer et al., 2014). Trichoderma spp. contain between
three to four EPLs, while EPL1, EPL2 and EPL3 are present in
all genomes analysed by Gao et al. (2020); EPL4 is absent in
some species (Gao et al., 2020).
The EPL1 from T. atroviride binds to various forms of poly-
meric chitin, and it has been suggested that it has a similar
function to expansins, inducing the opening of physical
spaces of the fungal cell wall, such as in chitin polymers
(Frischmann et al., 2013). In S. sclerotiorum interaction, T. har-
zianum requires EPL1 for mycoparasitism-related genes
expression and coiling around the host (Gomes et al., 2015).
Moreover, T. harzianum EPL1 participates in the downregula-
tion of virulence genes (BcBOT) involved in the botrydial
biosynthesis in B. cinerea (Gomes et al., 2017). In mycoparasi-
tism by T. harzianum, EPL might act as a recognition molecule
to identify its host hyphae, and also to protect Trichoderma cell
wall against their secondary metabolites and cell wall degrad-
ing enzymes (Gomes et al., 2017). The effector TAL6 from T.
atroviride (Gruber and Seidl-Seiboth, 2012), and EPL1 in T. har-
zianum together with the cell wall-bound protein QID74 from
T. harzianum have been suggested to be involved in the cell-
wall protection of self, during mycoparasitism. This mecha-
nism could also apply to other mycoparasitic species, and
the mechanism of self-protection needs further in-depth in-
vestigations. Recently, it was reported that polyketides like
azaphilones from T. guizhouense are necessary for the self-
protection against the hyper-production of H
2
O
2
during the
interaction with the host fungus (Pang et al., 2020).
Hydrophobins that are associated with the protection of
filamentous hyphae and coating of the spores for dispersion,
have also been associated with the mycoparasitic activity in
Trichoderma (Guzm
an-Guzm
an et al., 2017). Class II hydropho-
bins have been related to interaction with other fungi. In T.
virens, overexpression of HYD2-1 (TVHYDII1), a class II hydro-
phobin, increased the antagonistic activity against R. solani
AG2 (Guzm
an-Guzm
an et al., 2017). In T. asperellum HFB2-6
was up-regulated when grown in the presence of 1% Alternaria
alternata (Pleosporales) cell wall and 5% A. alternata fermenta-
tion liquid treatments, suggesting a role in mycoparasitism
(Huang et al., 2015). It may be noted that the VEL1 knockout
mutant of T. virens or the radiation-induced M7 mutant col-
onies show loss of hydrophobicity and are also non-
mycoparasitic (Mukherjee and Kenerley, 2010;Pachauri et al.,
2020). Interestingly, the MAPK TVK1 from T. virens regulates
hydrophobin production in an environment-dependent
manner (Mendoza-Mendoza et al., 2007), but its absence
enhanced the production of CAZymes in presence of R. solani
and increased biocontrol activity against R. solani and G. ulti-
mum in cotton plants (Mendoza-Mendoza et al., 2003). More
functional and systematic studies of knockout mutants in
the hydrophobin genes are required to ascertain the role of in-
dividual hydrophobins in mycoparasitism and biocontrol
potential.
8. Regulation of mycoparasitism
Mycoparasitism, which involves communication between
two microbes, is a tightly regulated process involving activa-
tion of signal transduction pathways leading to transcrip-
tional activation/repression of several genes (Karlsson et al.,
2017). Signal transduction mediated by G-protein and the
MAP kinase pathways are well studied in Trichoderma.
Silencing of GPR1 (a GPCR) resulted in the inability of T. atro-
viride P1 to attach to the host fungus R. solani, and to express
mycoparasitism-related genes (a chitinase and a protease);
the mutants were unable to overgrow the host fungus in
confrontation assay (Omann et al., 2012). In one of the earliest
studies, T. atroviride IMI 206040 transformants over-
expressing the G-protein alpha subunit TGA1 or expressing
a constitutively active allele, had enhanced mycoparasitic
coiling, in contrast to the loss of mycoparasitism in silenced
mutants (Rocha-Ram
ırez et al., 2002). Deletion of the gene
for TGA1 (TgaA) abolished mycoparasitism of T. virens IMI
304061 against S. rolfsii, but did not affect the ability to over-
grow, coil around and lyse the hyphae or sclerotia of R. solani;
loss of the second G-protein alpha subunit TGA2 (TgaB) did
not have any phenotypic defects (Mukherjee et al.,2004).
Deletion of tga3 resulted in a loss of formation of
mycoparasitism-related infection structures in T. atroviride
P1 when confronted with R. solani or B. cinerea (Zeilinger
et al., 2005). Deletion of brg1 (Tbrg-1), a novel Ras-GTPase
increased the antagonistic activity of T. virens Gv29-8 against
R. solani,S. rolfsii and F. oxysporum, associated with enhanced
expression of a serine-protease and cht1 (chitinase) genes
(Dautt-Castro et al., 2020). Interestingly, the mutants were
ineffective in protecting tomato seeds and seedlings against
R. solani infection, and behaved like a pathogen, which could
be due to the over-production of gliotoxin. The role of MAPK
signalling in Trichoderma mycoparasitism has been more
extensively studied. Deletion of the gene for TMK1 (TmkA)
in a "P" strain of T. virens resulted in loss of mycoparasitism
and biocontrol in greenhouse against S. rolfsii, but not against
R. solani (Mukherjee et al., 2003;Viterbo et al.,2005). However,
when the same gene was deleted in a "Q" strain of T. virens,
Trichoderma-mediated biocontrol mechanism 23
24 P. K. Mukherjee et al.
the induction of biocontrol-related genes prb1 (serine prote-
ase), nag1 (exochitinase), cht1 (endochitinase) and bgn2
(beta-glucanase) was higher in the mutants in confrontation
with R. solani, promoting the protection of cotton seedlings
(Mendoza-Mendoza et al., 2003). Deletion of the gene for
TMK1 in T. atroviride P1 reduced mycoparasitic activity
against R. solani and B. cinerea, even though the ability to atta-
ch to host hyphae was not affected (Reithner et al., 2007). In-
duction of nag1 and ech42 was higher in the mutant under
chitinase-inducing conditions, the mutants produced higher
amounts of secondary metabolites, including 6-PP, and had
greater ability to protect bean seedlings against R. solani.
Deletion of the gene for TMK1 orthologue TSK1 (Task1) in T.
asperellum T4 resulted in loss of ability of the mutants to para-
sitize R. solani, but enhanced induction of cell-wall degrading
enzymes in confrontation with the host fungus. Tsk1 mu-
tants also produced more 6-PP (Yang, 2017). Similar to
TMK1, loss of TMK2 (TmkB) attenuated mycoparasitism of
T. virens against S. rolfsii, but not against R. solani or Pythium
aphanidermatum (Kumar et al., 2010). It was proposed that
these two MAPKs might share some substrates, like key tran-
scription factors, that are dependent on two phosphorylation
events for their full activity. In addition to osmo-resistance, T.
harzianum HOG1 MAPK is involved in regulating antagonistic
activities against Neocamarosporium (Phoma)betae (Pleospor-
ales) and Colletotrichum acutatum (Glomerellales) (Delgado-
Jarana et al.,2006). Recent findings suggest that TMK3 affects
the polarity-stress adaptation process especially during the
pre-contact phase, while TMK1 regulates contact-induced
morphogenesis at the early-contact phase (Moreno-Ruiz
et al., 2020). STE12 is a transcription factor that is phosphory-
lated by TMK1. Deletion of the ste12 gene in T. atroviride P1
resulted in reduced attachment, mycoparasitic overgrowth
and lysis of R. solani and B. cinerea, but enhanced expression
of ech42 and nag1 (Gruber and Zeilinger, 2014). The mutants
were not evaluated for biocontrol.
Diverse Trichoderma species produce 6-PP (see above); inter-
estingly this secondary metabolite is tightly regulated by
diverse signalling components, including the MAPKs (e.g.,
TMK1, TMK3), G alpha proteins (e.g., TGA1, TGA3), as well
the NADPH oxidases (e.g., NOX1 and NOX2) (Fig. 5). Indeed,
these signalling components differently control the synthesis
of 6-PP, for example in T. atroviride IMI 206040 NOX1 is a nega-
tive regulator of 6-PP while NOX2 positively regulates its pro-
duction (Cruz-Magalh~
aes et al., 2019). In the same Trichoderma
species, TMK3 is a positive regulator of 6-PP production
(Atrizt
an-Hern
andez et al., 2019), while TMK1 negatively regu-
lates either the accumulation or synthesis of 6PP (Reithner
et al., 2007).
Fungal transcription factor PAC1 (PacC) plays a crucial role
in pH sensing. PAC1 silenced mutants of T. harzianum CECT
2413 were unable to overgrow R. solani,Rhizoctonia meloni and
Phytophthora citrophthora (Peronosporales), indicating a role of
pH sensing in mycoparasitism (Moreno-Mateos et al., 2007).
Similarly, deletion of pac1 resulted in a decreased ability of
T. virens IMI 304061 to overgrow R. solani and S. rolfsii in
confrontation assays (Trushina et al., 2013). Interestingly, a
strain expressing a constitutively active allele was as effective
as the wild type strain against R. solani, but lost the ability to
overgrow S. rolfsii.
In a recent study, T. virens PGY1 (a proline, glycine,
tyrosine-rich protein) and ECM33 knockout mutants not only
failed to overgrow S. rolfsii, but the pathogen instead overgrew
the mycoparasite (Bansal et al., 2019). Both the mutants over-
grew R. solani, but at a very slow pace. TCP1 knockout mutants
failed to overgrow S. rolfsii (but S. rolfsii could not overgrow Tri-
choderma, unlike PGY1 and ECM33 mutants) and took longer to
overgrow R. solani colony. However, the mutant was as effec-
tive as the wild type in antagonism against P. aphanidematum.
TCP1 mutants also lost the ability to parasitize the sclerotia of
S. rolfsii (Bansal et al., 2021). It appears that deletion of many
genes impairs mycoparasitism against S. rolfsii, but less likely
so against R. solani and P. aphanidermatum.
Deletion of the gene for T. virens Gv29-8 Velvet complex
protein VEL1 attenuated conidiation, hydrophobicity, second-
ary metabolism and mycoparasitism on R. solani and G. ulti-
mum; the mutants also lost the ability to suppress these
pathogens of cotton seedlings in a growth room test
(Mukherjee and Kenerley, 2010). Deletion of the gene for T.
atroviride P1 LAE1, another Velvet complex protein, resulted
in under-expression of mycoparasitism related genes like pro-
teases and glucanases, and there was attenuation of mycopar-
asitism against A. alternata (Pleosporales), R. solani and B.
cinerea (Aghcheh et al., 2013). Loss of TGF1, a histone acetyl-
transferase (GCN5), resulted in mutants having a slower
growth rate and diminished capacity to grow over R. solani.
Interestingly the ability to coil around the host hyphae was
not affected (G
omez-Rodr
ıguez et al., 2018). Our current under-
standing of the role of signal transduction in Trichoderma
mycoparasitism is summarized in Fig. 5.
9. Interactions with other beneficial fungi:
mycorrhizae and cultivated mushrooms
While the power of Trichoderma to kill other fungi has been
harnessed for suppressing plant pathogenic fungi, whether
these mycoparasites can adversely impact other beneficial
fungi is an obvious question. There are some scattered (often
contradictory) reports and this subject needs to be studied in
integration. Trichoderma viride and Trichoderma polysporum are
strongly antagonistic to mycorrhizal colonization of black
spruce (Picea mariana) seedlings by Laccaria bicolor (Agari-
cales) (Summerbell, 1987). On the other hand, Laccaria laccata
inhibited the growth of T. virens in co-culture. On mycor-
rhizal roots, T. virens conidia germinated sporadically and
produced short germ tubes. Interestingly, the mantle hyphae
of L. laccata grew towards and coiled around the conidia of T.
virens, eventually leading to deformation, breaks in conidial
Fig. 5 eRole of signal transduction in Trichoderma mycoparasitism. (A). Representation of the two evident mycoparasitism-
related morphological changes in Trichoderma: coiling and hyphae penetration. (B). Schematic of how cAMP and MAPK
signaling pathways regulate diverse mycoparasitism steps, including host recognition, coiling around the host hyphae,
production of fungal cell wall degrading enzymes, and production of secondary metabolites.
Trichoderma-mediated biocontrol mechanism 25
walls and partial degradation (Werner et al., 2002). Co-
cultivation of L. bicolor with Trichoderma spp. altered the
VOCs profile of the latter. In direct contact between both
the mycelia, L. bicolor growth was impaired (Guo et al.,
2019). There have been several studies on the interactions
between endomycorrhizae (mainly Glomus spp. (Glomerales))
and Trichoderma. Inoculation of mycorrhizal plants with Tri-
choderma aureoviride produced more plant (marigold)
biomass, while there was no effect in non-mycorrhizal
plants, indicating a synergistic interaction (Calvet et al.,
1993). However, TEM studies suggested that T. harzianum is
an aggressive mycoparasite on Glomus intraradices, prolifer-
ating abundantly on the spore surface and penetrating and
lysing the cell-wall polymers (Rousseau et al., 1996). T. harzia-
num could exploit the dead mycelia, but not the living
biomass of G. intraradices. On the contrary, T. harzianum
growth was adversely affected by the mycorrhizal fungus
(Green et al., 1999). Of the five strains of Trichoderma pseudoko-
ningii tested, four strains inhibited the germination of G. mos-
sae and Gigaspora rosea (Martinez et al., 2004). T. harzianum
enhanced the root colonization by G. mossae, but the mycor-
rhiza decreased the population development of T. harzianum
in/around the roots of cucumber (Chandanie et al., 2009).
Nevertheless, co-inoculation synergistically enhanced plant
growth. T. harzianum utilizes Glomus irregulare as a vehicle
for entry into potato (Solanum tuberosum) roots, by first colo-
nizing the extraradical mycelium of the AM fungus, then
spreading into the intraradical mycelia, before exiting into
root cells; the hyphae of T. harzianum penetrated the mycor-
rhizal hyphae causing protoplasm degradation resulting in
loss of viability (De Jaeger et al., 2010). A synergistic effect
on AM (Glomus spp.) root colonization was observed under
low fertilizer input conditions due to inoculation with T. har-
zianum (Mart
ınez-Medina et al., 2011). T. harzianum disrupted
the AM (Glomus spp.) extraradical hyphae affecting the trans-
location of phosphorus to host plant (De Jaeger et al., 2011).
In vivo imaging demonstrated that T. atroviride parasitized
Gigaspora gigantea (Diversisporales) and Gigaspora margarita
hyphae resulting in cell wall lysis which was not mediated
by chitinases (Lace et al., 2015). Beneficial interactions of T.
atroviride and G. intraradices in the form of shoot and root
dry weight, chlorophyll index and chlorophyll fluorescence
in lettuce (Lactuca sativa), tomato (Solanum lycopersicum)and
zucchini (Cucurbita pepo) was observed under open field con-
ditions (Colla et al., 2015). Poveda et al. (2019) provided evi-
dence that AMF can enter the roots of non-host Brassica
plants with the help of T. harzianum, resulting in improved
plant productivity. Similarly, co-inoculation with T. viride
improved AMF colonization of onion (Allium cepa) roots,
resulting in improving plant biochemical parameters and
mineral nutrient contents (Metwally and Al-Amri, 2020).
Trichoderma spp. are aggressive mycoparasites on several
members of Basidiomycota, and the host range includes
some of the commonly cultivated mushrooms (Williams
et al., 2003;Guthrie and Castle, 2006;Hatvani et al., 2007;
Komo
n-Zelazowska et al., 2007;Marik et al., 2017;Kredics
et al., 2018). However, most of these species are phylogeneti-
cally distinct from the ones that are used for biological control
of plant diseases. Trichoderma aggressivum f. europeum is an
aggressive colonizer of Agaricus bisporus (Agaricales) composts
while Trichoderma pleuroticola and Trichoderma pleurotum can
overgrow Pleurotus (Agaricales) mycelia (Hatvani et al., 2007;
Komo
n-Zelazowska et al., 2007). Genes for a protease, an endo-
chitinase and a b-glucanase were found to be upregulated in T.
aggressivum in co-cultivation with A. bisporus (Abubaker et al.,
2013). Several new Trichoderma species have been isolated that
are associated with mushrooms, e.g., Trichoderma mienum
(Kim et al., 2012) and Trichoderma songyi (Park et al., 2014).
Wang et al. (2016) identified six species, i.e., T. harzianum,T.
atroviride,T. viride,T. pleuroticola,T. longibrachiatum and T.
oblongisporum as pathogens of Lentinula edodes (Agaricales),
one of the most important edible mushrooms in China;
some of these Trichoderma spp. are also used in agriculture
as biocontrol agents. In confrontation assay, T. harzianum,T.
atroviride and T. citrinoviride were antagonistic on the mycelia
of L. edodes (Kim et al., 2016). Exposure of A. bisporus mycelium
to T. aggressivum resulted in an oxidative stress response asso-
ciated with reduced growth of the mushroom (Kosanovic et al.,
2020).
10. Summary and outlook
Trichoderma spp. are being widely used in agriculture across
the world. When it comes to success under field conditions,
it is impossible to single-out a specific mode of action, and
perhaps a combination of traits makes a successful biocontrol
agent under natural conditions (unlike in controlled condi-
tions). More studies via the generation of knockout mutants
in a larger number of genes are required to ascertain the role
of mycoparasitism in the biocontrol of plant pathogens. Over-
expression of fungal virulence factors against other fungi, for
example chitinase ECH42, enhanced mycoparasitism
(Carsolio et al., 1999). Such genes could also be used for the
generation of disease-resistant transgenic plants, for
example, Lorito et al. (1998). Bringing mycoparasitism back to
the center stage of biocontrol studies should likewise renew
interest in developing more such strategies, keeping in mind
the new genetic engineering technologies becoming available.
Trichoderma spp. have been traditionally viewed as necrotro-
phic mycoparasites and most studies have focused on the
degradation of the host cell wall. In this article, we propose
the concept of a “hemi-biotrophic”- like mode of action, and
it appears that the host fungal cell wall is not extensively
damaged during interactions with Trichoderma. Instead, diges-
tion and mobilization of the cellular content seems to be very
relevant. Future research on Trichoderma mycoparasitism
should test whether this is an unusual observation, or a
more general aspect of fungal hosteparasite interactions.
Similarly, how these fungi protect themselves during myco-
parasitic interactions needs to be investigated in depth, and
the co-existence of Trichoderma with other beneficial fungi in
the rhizosphere needs to be optimized in biocontrol settings.
Funding
This research did not receive any specific grant from funding
agencies in the public, commercial, or not-for-profit sectors.
26 P. K. Mukherjee et al.
Declaration of competing interest
The authors declare no conflict of interest.
Acknowledgements
PKM thanks Head, NA&BTD, BARC for encouragement and
support. AM-M recognizes the Bio-Protection Research Centre
and Massey eLincoln and Agricultural Industry Trust, New
Zealand for their support.
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Trichoderma-mediated biocontrol mechanism 33
