sustainability
Review
Trichoderma: Advent of Versatile Biocontrol Agent, Its Secrets
and Insights into Mechanism of Biocontrol Potential
Nazia Manzar 1,† , Abhijeet Shankar Kashyap 2, *,† , Ravi Shankar Goutam 3,4 ,
Mahendra Vikram Singh Rajawat 1 , Pawan Kumar Sharma 1, *, Sushil Kumar Sharma 5
and Harsh Vardhan Singh 1
1
2
3
4
5
*
†
Citation: Manzar, N.; Kashyap, A.S.;
Goutam, R.S.; Rajawat, M.V.S.;
Sharma, P.K.; Sharma, S.K.; Singh,
H.V. Trichoderma: Advent of Versatile
Biocontrol Agent, Its Secrets and
Insights into Mechanism of
Biocontrol Potential. Sustainability
2022, 14, 12786. https://doi.org/
10.3390/su141912786
Academic Editor: Gustavo Santoyo
Received: 30 June 2022
Accepted: 7 September 2022
Published: 7 October 2022
Publisher’s Note: MDPI stays neutral
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Copyright: © 2022 by the authors.
Plant Pathology Lab, ICAR-National Bureau of Agriculturally Important Microorganisms,
Maunathbhanjan 275103, India
Molecular Biology Lab, ICAR-National Bureau of Agriculturally Important Microorganisms,
Maunatbhanjan 275103, India
School of Life Sciences, Jawaharlal Nehru University, New Mehrauli Road, New Delhi 110067, India
Department of Biochemistry, Institute of Cell Differentiation and Aging, College of Medicine,
Hallym University, Chuncheon 24252, Korea
ICAR-National Institute of Biotic Stress Management, Baronda 493225, India
Correspondence: abhijeet4497@gmail.com (A.S.K.); pawan111200@gmail.com (P.K.S.)
These authors contributed equally to this work.
Abstract: Trichoderma is an important biocontrol agent for managing plant diseases. Trichoderma
species are members of the fungal genus hyphomycetes, which is widely distributed in soil. It can
function as a biocontrol agent as well as a growth promoter. Trichoderma species are now frequently
used as biological control agents (BCAs) to combat a wide range of plant diseases. Major plant diseases have been successfully managed due to their application. Trichoderma spp. is being extensively
researched in order to enhance its effectiveness as a top biocontrol agent. The activation of numerous
regulatory mechanisms is the major factor in Trichoderma ability to manage plant diseases. Trichodermabased biocontrol methods include nutrient competition, mycoparasitism, the synthesis of antibiotic
and hydrolytic enzymes, and induced plant resistance. Trichoderma species may synthesize a variety
of secondary metabolites that can successfully inhibit the activity of numerous plant diseases. GPCRs
(G protein-coupled receptors) are membrane-bound receptors that sense and transmit environmental
inputs that affect fungal secondary metabolism. Related intracellular signalling pathways also play
a role in this process. Secondary metabolites produced by Trichoderma can activate disease-fighting
mechanisms within plants and protect against pathogens. β- Glucuronidase (GUS), green fluorescent
protein (gfp), hygromycin B phosphotransferase (hygB), and producing genes are examples of exogenous markers that could be used to identify and track specific Trichoderma isolates in agro-ecosystems.
More than sixty percent of the biofungicides now on the market are derived from Trichoderma species.
These fungi protect plants from harmful plant diseases by developing resistance. Additionally, they
can solubilize plant nutrients to boost plant growth and bioremediate environmental contaminants
through mechanisms, including mycoparasitism and antibiosis. Enzymes produced by the genus
Trichoderma are frequently used in industry. This review article intends to provide an overview update
(from 1975 to 2022) of the Trichoderma biocontrol fungi, as well as information on key secondary
metabolites, genes, and interactions with plant diseases.
Keywords: Trichoderma; antibiosis; exogenous marker genes; secondary metabolites; biochemical
defense; induced systemic resistance
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4.0/).
1. Introduction
It is anticipated that there will be 9.1 billion people living on the planet by the year
2050, according to current population projections. As a consequence of this, there needs to
be a seventy percent increase in the amount of food that is produced by agriculture in order
Sustainability 2022, 14, 12786. https://doi.org/10.3390/su141912786
https://www.mdpi.com/journal/sustainability
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to sustainably feed the rising population throughout the world. Challenges, such as global
warming and environmental pollution, have pushed plants toward various forms of biotic
and abiotic stress, which has caused significant reductions in yield. This will lead to issues of
food shortages for future generations [1]. Plant diseases play a direct role in the destruction
of agricultural crops. The loss was estimated in fruit crops (78%), vegetables (54%), and
cereals (32%) [2]. Specifically, disease caused by soil-borne pathogens is destructive and
spreads quickly, causing a huge economic loss of important crops. The application of
pesticides was considered an excellent solution to manage soil-borne pathogens, but their
excessive use has led to the development of pathogen resistance to fungicides. However,
it was realized that applying these pesticides is harmful to the environment and unsafe
for human health [3]. It also kills the non-target organisms present in the soil and also
fertilizers were used extensively around the globe since the Green Revolution, due to the
high subsidies. However, extensive fertilizer use exacerbates soil degradation and causes
yield stagnation and, as a result, threatens food security and soil sustainability, especially
in developing countries, such as India [4]. Hence, the utility of certain microorganisms
that can antagonize the target pathogen has been explored. Bioagents use a host-specific
pathogen, a microbial antagonist, to inhibit diseases and control weed populations [5,6].
Biocontrol agents comprise fungi, bacteria, viruses, nematodes, and protozoa [7]. Plants
treated with biocontrol agents may become more vigorous, robust, healthier, and yield
more than untreated plants. Bioagents are microbial agents that suppress the growth of
other microorganisms associated with them. Bioagents can hinder the life process of other
phytopathogens, including fungi, bacteria, viruses, and other microorganisms. Biocontrol
fungi are mostly saprophytic. They improve plant growth and development, enable the
plant to resist abiotic stresses, improve nutrient uptake from the soil, and reduce the
effect of plant diseases. A fungus genus called Trichoderma can be found as saprotrophs,
mycoparasites, degrading the cell wall components of harmful pathogens by producing
chitinase or cellulase enzyme soil inhabitants and plant symbionts [8]. Different strains of
Trichoderma account for approximately ninety percent of all fungal biocontrol agents used
to combat deleterious microorganisms [9]. In 1794, soil and decomposing organic matter
were used to isolate Trichoderma for the first time [10]. Trichoderma is currently the source
of more than 60% of the world’s most effective bio-fungicides [11]. Trichoderma has been
broadly studied among biocontrol agents due to its ability to antagonize plant pathogenic
species. Trichoderma is a widely distributed ubiquitous hypomycetus fungi occurring nearly
in all soil types and root ecosystems, especially in those rich in organic matter. Trichoderma,
considered a potent biocontrol agent, can be attributed to the following characteristics: high
reproductive capacity, ability to survive even in stressed conditions, efficiency in utilization
of nutritional capacity to alter the rhizosphere, aggressiveness against phytopathogenic
fungi, helps in plant growth promotion, induced defense mechanism as well as providing
plants several secondary metabolites [12], enzymes [13], and PR proteins [14]. Because of
these characteristics, the genus Trichoderma can be found in a diverse environment with a
significant population density. The most common species of Trichoderma as biocontrol agents
are Trichoderma virens, Trichoderma viride, and Trichoderma harzianum [15]. The potential of
Trichoderma as a biocontrol agent in plant disease management was first recognized in the
early 1930s and, in subsequent years, control of many diseases has been reported [16]. Root
rot disease, fruit rot, damping-off, wilt, and other common plant diseases can be managed
by Trichoderma spp. In recent studies [17], it has been observed that Trichoderma spp.
release secondary metabolites that inhibit the growth of plant pathogens and encourage
the growth of plants [18]. In addition, in the plant Trichoderma spp. interaction, they
efficiently modulate root architecture and enhance the length of lateral and primary roots,
which results in the feasibility of nutrient uptake by the plant [19]. Numerous species of
Trichoderma under soil microbiome can produce enzymes that degrade the target pathogenic
fungi or produce several toxic compounds, which restrict the pathogens. The antagonistic
properties of these are based on multiple mechanisms. Trichoderma strains exert biocontrol
action against fungal phytopathogens, either indirectly or by competing for nutrients and
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space, mycoparasitism, production of antibiotics, induction of resistance in host plants,
and promoting plant growth [18,20]. Mycoparasitism is a process in which fungi primarily
locate target hyphae by probing with persistently synthesized cell wall degrading enzymes
(CWDEs) paired with precise detection of cell wall pieces produced from target fungi.
This cell wall degrading enzyme complex produced from Trichoderma spp. is composed of
chitinases, β-glucanases, cellulases, and proteases. These enzymes can decompose the cell
wall of phytopathogens, making it possible for hyphae to penetrate, colonise, and activate
myoparasitism in plants [21,22]. Chitinases are potent enzymes that showed the highest
antagonistic potential against plant diseases [23]. In order to obtain a new formulation for
its efficient biocontrol activity, these metabolites can either be overproduced or mixed with
the suitable biocontrol strain [24]. Excellent results have been attained with T. virens and
metalaxyl against Pythium ultimum infecting cotton [25]. This has resulted in commercial
production of Trichoderma species for the protection of several plant diseases and growth
enhancement of several crops in India, Israel, New Zealand, and Sweden. In 1997, it was
shown that Trichoderma colonisation of roots could minimize foliar pathogen symptoms.
This discovery gave rise to the term "induced systemic resistance" (ISR), which is also
used to refer to all types of induced resistance, both local and systemic. [26]. Trichoderma
research over the following two decades was dominated by IDR as a plant disease control
method [27]. Though synergistic actions in these processes may occur, the degree of
disease suppression caused by mycoparasitic activity is greater than that caused by IDR or
antibiosis [28].
Due to a lack of reliable characteristics, morphological and cultural characteristics
alone have proven difficult to use to distinguish individuals within the genus Trichoderma.
The effectiveness of various biochemical and molecular approaches was assessed to distinguish among isolates of the genus Trichoderma. Information was also collected on ITS
sequence data, RAPD, or RFLP to differentiate between morphologically indistinguishable
isolates [29]. According to a new taxonomic approach, individuals of the genus Trichoderma
are currently identified by combining cultural, biochemical, morphological, and molecular characteristics. The accuracy of sequence and RAPD data in determining Trichoderma
phylogeny is also assessed. The ITS region of sequence data was the most reliable for
predicting the phylogeny of morphologically distinct species, while RAPD and RFLP data
were the most beneficial for predicting the overall phylogeny of morphologically similar
strains [30–34].
The compilation of basic information about the biocontrol potential of Trichoderma
strain and their capabilities to colonise, persist, and disseminate under environmental
conditions is vital in the journey of bioformulation development [35]. Orr and Knudsen
2004 developed a method to monitor the variation in biomass of biocontrol agents in native
soil by using GFP and GUS labeled T. harzianum ThzID1-M3 strain, an environment in
which the presence of local bacteria and fungus impedes the use of conventional methods
for determining biomass [36]. It was shown that epifluorescence microscopy tracking
of GFP-labeled Trichoderma harzianum was a helpful tool in identifying biocontrol-active
hyphal biomass from inactive conidia and chlamydospores, which were measured using
plate counts. The GUS and GFP marker genes were intended to distinguish between
BCA and native Trichoderma populations. Genes that produce a green fluorescent protein
(gfp), hygromycin B phosphotransferase (hygB), and β-glucuronidase (GUS) are examples
of exogenous markers that can be utilized to genetically change BCAs in order to track
and analyze various strains of Trichoderma on field crops [37–39]. This review presents a
compilation of studies regarding molecular characterization and the mechanism of Trichoderma species as biocontrol agents, along with a detailed description of genes involved in
biocontrol activity, enzyme production, and secondary metabolites/bioactive compounds
produced by Trichoderma and findings that discover the potentiality of Trichoderma spp. as a
plant growth promoter agent are discussed in this review.
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2. Insight on Trichoderma and Its Mechanism
2.1. Morphological Studies on Trichoderma
Trichoderma produces numerous spores with varied shades of color, such as white
green, light green, dark green to dull green [40], and on the backside of the plate, colony
color varies from yellow, buff, amber, or uncolored [41]. It proliferates; therefore, it is
also characterized by the radial growth rate. Most of the Trichoderma spp. produces
chlamydospores. Phialide lengths vary from 3.5 to 10.0 × 1.3 − 3.3 µm and the shape of
phialides is flask shaped, but the length of phialides varies considerably depending on
the species [42]. T. harzianum Rifai, Trichoderma virens [43] von Arx, Beih, and T. viride
Pers.:Fr [44,45] were reported as the best effective BCAs against plant pathogenic fungi.
T. harzianum is an assemblage of species defined by the presence of short side branches
on the conidiophore, short inflated phialides, and smooth and small conidia. Based on
the strains and characteristics studied, T. harzianum has been classified into three, four,
or five subspecific groupings [46]. Gams and Meyer [47] neotypified T. harzianum and
divided it into two main groups based on molecular analysis: T. harzianum sensu lato (s.l.)
and T. viride-T. atroviride complex. Th1 and T. inhamatum are some of the most prevalent
strains utilised as BCAs [48] in T. harzianum s.l. [49,50]. Colombian T. harzianum isolates
were formerly categorised as T. inhamatum due to the lack of sterile appendages on the
conidiophores and the globose shape of the conidia [51]. Th1 and T. inhamatum were
previously thought to be two separate species due to physical distinctions and two base
sequence changes in the ITS1 gene. As per molecular data, Th2 and Th4 strains are still
not BCAs and are likewise distinct from T. harzianum s.l. T. viride-T. atroviride complex
isolates, with rapidly darkening conidia identified by particular restriction fragment length
polymorphism (RFLP) pattern. T. viride is a genus of bacteria having globose or subglobose
to ellipsoidal warted conidia, most of which generate antibiotics and have a coconut-like
odor. For many years, the morphology of T. viride was unknown until two forms of
conidial ornamentation were identified [52]. Recent research has shown that T. viride is a
paraphyletic group and a combined morphological and genomic approach has confirmed
the redefinition of T. viride types I and II in two species. Type I includes the genuine
T. viride species in addition to the Hypocrea rufa anamorph and strains of T. atroviride
and T. koningii [53]. Ovoidal instead of globose conidiation, as well as darker and rapid
conidiation, characterise the new species T. asperellum [53,54].
2.2. Molecular Studies on Trichoderma
The advent of molecular tools for investigations in fungal taxonomy prompted research in the mid-1990s to re-assess the morphology-based taxonomy in Trichoderma. For the
species of bioagents that share common ecology or morphology, rDNA sequencing is a valuable tool to differentiate such species within particular groups of strains or isolates [55–57].
These can also be distinguished by randomly amplified polymorphic DNA (ISSR)—PCR,
restriction fragment length polymorphisms in mitochondrial and ribosomal DNA, and
sequence analysis of ribosomal DNA. Ribosomal DNA sequence analysis, RAPD, and
RFLP [30,32,32–34,58–60] are effective methods to distinguish isolates from the same genera. RAPD helps decide species diversity by molecular identification and characterization
of the potent biocontrol agents [61]. The laboratories of G.J. Samuels (Beltsville, MD, USA),
T. Borner (Berlin, Germany), and C.P. Kubicek (Vienna, Austria) collaboratively pioneered
a revision of Bissett’s section Longibrachiatum. They combined the use of molecular markers
(ITS1 and ITS2 sequence analysis, RAPD), physiological (isoenzyme analysis) and phenetic
characters, and also, for the first time, included an analysis of potential teleomorphs of the
Trichoderma spp. from this section [46,51,57,62–65].
The total number of phylogenetically recognised species in the Trichoderma and
Hypocrea genus reached over one hundred in the year 2006 [64,65]. Many reports of
new species of Trichoderma and Hypocrea are listed in Table 1. Misidentifications of particular species have happened in some cases, particularly in earlier publications, such as
the name Trichoderma harzianum, which has been used to describe various species [66].
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However, it is difficult to safely fix these inaccuracies analysing the strains that were
first utilised. As a result, we will summarise the findings using the originally published names. In recent years, the safe identification of novel species has been substantially facilitated by creating an oligonucleotide barcode known as TrichoKEY and a specialised matching search engine known as TrichoBLAST. These tools are available online
at www.isth.info accessed on 5 June 2022 [67,68]. Detailed documentations of the genus
Trichoderma/Hypocrea have been produced due to ongoing efforts to elucidate the diversity
and geographical occurrence of Trichoderma/Hypocrea [69–80]. The Index Fungorum (http:
//www.indexfungorum.org/Names/Names.asp accessed on 5 April 2022) now includes
165 records for Trichoderma and 471 different names for Hypocrea species. Currently, the International Subcommission on Trichoderma/Hypocrea compiled a list of 104 species that have
been characterised at the molecular level (http://www.isth.info/biodiversity/index.php accessed on 20 April 2022). Even this, a huge proportion of the potential Hypocrea strains and
an even larger portion of the Trichoderma strains, for which sequences have been deposited
in GenBank, have not yet been identified [67] and must be investigated further. Trichoderma
taxonomy has been greatly improved by the use of molecular phylogenetic markers and
phylogenetic study of the many Trichoderma species is still an active research area.
2.2.1. Universal Marker-Based Identification
ITS is a reliable marker for the barcoding of fungal DNA. In environmental samples,
ITS of the ribosomal DNA region is used to evaluate fungal diversity. The favored DNA
barcoding marker is the ITS region of nuclear ribosomal DNA for molecular identification
of mixed templates of single taxa [81]. The most promising method is molecular analysis
of the ITS region of rDNA for identification of species [82]. Based on DNA sequence
analysis and phylogenetic studies, 290 species of Trichoderma have been identified and
characterised [83]. In eukaryotes, variable sequences with the larger subunit or smaller
unit of rRNA genes are suitable for analysis of the subgeneric relationship. In many of
the plant pathogenic fungal genera, the phylogenetic relationship is determined by the
D2 region of the LSU [84]. Bisset concluded that T. pseudokoningii, T. parceramosum, T.
citrinoviride, and T. longibrachiatum were allocated under Longibrachiatum section based
onmorphological data [85,86]. Rehner and Samuels and Samuels conducted a phylogenetic
analysis of Trichoderma ITS region sequences and reported that T. virens showed similarity
with T. harzianum, it is distinct from Gliocladium, and the above result supported the Vonrox
and Bisset taxonomic studies [87]. The DNA oligonucleotide barcode method is a quick
method through which Trichoderma and Hypocrea can be identified [88]. Oligonucleotide
barcode combines many oligonucleotides (hallmarks), mainly distributed among the rDNA
region of ITS1 and 2 sequences. The genus species hallmark was defined as oligonucleotide
barcodes that are consistent in all Trichoderma and Hypocrea rDNA regions of ITS1 and ITS2
and differ in related fungal genera. Harmosa et al. described 16 Trichoderma harzianum
Rifai strains and 1 T. viride strain previously recognised as T. harzianum Rifai. A certain
level of polymorphism was found in hybridizations using a mitochondrial DNA probe.
Three different lengths of ITS and four sequence types were confirmed by sequencing
ITS1 and ITS2 [89]. Manzar et al., 2020 reported that tef-1α gene and ITS1 and ITS4 gene
sequence analysis were able to identify and differentiate 20 Trichoderma isolates. Based
on the sequence analysis of tef-1α and ITS1 and ITS4 gene, these isolates were divided
into two species, with 19 isolates belonging to T. asperellum and 1 isolate belonging to
T. harzianum [90]. Oskiera et al. identified 104 strains of Trichoderma based on the sequences
of translation elongation factor 1 alpha (tef-1α), internal transcribed spacers 1 and 2 regions,
as T. simmonsii, T. harzianum, T. atroviride, T. lentiforme, and T. virens. Thus, the comparative
nucleotide sequence analysis of ITS1 and ITS4 and tef-1α gene provides better resolution to
distinguish different Trichoderma species from the sorghum rhizosphere [91].
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2.2.2. DNA-Fingerprinting Techniques
Many DNA-fingerprinting techniques, such as polymerase chain reaction (PCR), Southern hybridization, and restriction enzyme digestion (RED), are used by scientists to locate
strain-specific DNA strands. These methods generate band patterns that can be compared.
Trichoderma species can be identified in agro-conditions using species-specific primers.
In order to distinguish between T. harzianum biotypes Th2 and Th4, a species-specific
primer-based test was devised by Samuels et al., and then redescribed as T. aggressivum
f. europaeum and T. aggressivum f. aggressivum [72]. For Schlick and colleagues, they used
DNA fingerprinting to demonstrate that oligonucleotides as hybridization probes could
differentiate distinct T. harzianum patent-protected strains. All strains could be distinguished from one another using fingerprint patterns [92]. The first approach to separate a
commercially available biocontrol strain from the other Trichoderma strains was established
by Zimand et al. and it was used to identify T. harzianum T-39 as a BCA against B. cinerea
All of the Trichoderma strains studied could be distinguished using a set of 10 mer primers in
a random amplified polymorphic DNA (RAPD). It is clear that this idea has many benefits
beyond morphological feature approaches [93]. The technique can be completed in less
time and with less DNA. To follow the distribution and longevity of T. atroviride C65 on
kiwifruit leaves, Dodd et al. (2004) used an isolate-specific RFLP marker in association with
a dot-blot test [94].
Cross dot-blot hybridization with UP-PCR amplification products investigated T. polysporum, G. roseum, T. hamatum, T. viride, T. virens, and T. koningii isolates to find commonalities
among the strains. The findings show that UP-PCR and ITS-ribotyping could be useful
for identifying various species within a single species [95]. Using random, minisatellite,
and microsatellite primers, Fanti et al. fingerprinted two strains of Trichoderma, which
were potent against Cytospora canker of peach in the environment. The M13 minisatellite
demonstrated that each strain has its own fingerprint. The antagonists were collected four
months after being applied to the soil under the canopy of peach trees; however, they were
not recovered from the soil itself and, after a year, they were no longer there [96]. Although
there is a wide variety of PCR-based fingerprinting techniques, not all of them are strain
specific. These procedures are also very easy to utilise. Both a lower annealing temperature
and a shorter primer length can have an adverse effect on the precision of their results [97].
On the other hand, strain-specific fragments may be utilised as monitoring markers in
accordance with SCARs (sequence-characterised-amplified regions) [98–102].
2.2.3. OMICS Approaches in the Service of Trichoderma Monitoring
Biofertilisers and biocontrol agents developed from Trichoderma spp. are commonly
utilised in agriculture (BCAs) [103]. Information about Trichoderma strains’ ability to
colonise and stay in natural environments is critical for developing a useful Trichoderma
strain for a BCA [104]. Trichoderma strains released as BCAs into the environment must be
monitored for their destiny, behavior, and population dynamics. A need for the registration
of new biocontrol agents based on Trichoderma is the capability to reliably assess and
monitor the strain that has been released, as well as to monitor the changing population
dynamics of that strain over time [105]. As a consequence, it is becoming increasingly
important to differentiate naturally occurring Trichoderma populations found in agricultural areas from newly introduced Trichoderma strains [106]. In classic microbiological
methods, colony forming units (CFU) can be estimated by diluting samples using Trichoderma-selective or semi-selective media [107]. A sample collected for dilution plating
from the environment does not distinguish between indigenous strains of the Trichoderma
population’s existence in the environment and artificially introduced ones by the experimenter. For this, morphological or physiological characteristics are insufficient to identify
the colonies [107]. The omics approach is a promising tool and is used for monitoring
and differentiating the potent biocontrol agent activity, which is artificially introduced
from the native biocontrol agent that is already present in the soil [108]. T. harzianum was
monitored and quantified using a primer set and a TaqMan probe for the ITS region [109].
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ITS copies were quantified using real-time PCR. The ITS copy number and the fungus
biomass were found to have a 0.76 correlation. According to scientists, real-time PCR data
can be used to quantify fungi in soil samples. T. harzianum could be detected and measured
in soils and other organic materials using primers and probes designed for pure fungus
cultures. Beaulieu et al. utilized this method to monitor T. harzianum populations in green
compost and peat, which worked well [109,110]. Cultivation-dependent Trichoderma species
identification and observation were described by Hagn, et al. using ITS-based primers.
Arable soil samples were used to create a library of clones. Its results demonstrated that the
primer set selected to amplify the Trichoderma gene encompassed a wide range of species
relevant to biocontrol [111]. Trichoderma-specific primers targeting the ITS region were
produced by Meincke et al. (2010) to study Trichoderma diversity. On the other hand, the
reverse primer binds to an area of ITS2 that is still polymorphic, making it impossible
to identify many species [112]. The ITS-based metabarcoding technique by Friedl and
Druzhinina eliminated this problem by amplifying the full diagnostic ITS1 and 2 regions
from all members of the genus, using six reverse primers and the forward primer ITS5. This
culture-independent PCR-based method demonstrated the limited Trichoderma diversity
in the soil of a riparian forest. Two types of Trichoderma populations can be incorrectly
estimated using PCR methods: active and inactive hyphae and mycelia, as well as dormant
conidia and spores [113]. Furthermore, the isolation of total RNA, its reverse transcription,
and the subsequent detection of cDNA allow for the identification of active Trichoderma
communities by transcriptomic techniques.
Geistlinger et al. employed Touchdown PCR to track and quantify T. virens using
simple sequence repeats (SSRs). There were 12 distinct loci for which primers were created.
According to findings, this species lives as an endophyte in the roots of tomato plants.
Numerous strains of T. virens had their fungal biomass quantified in plant tissues and
co-colonization of the roots has also been discovered [99]. Utilizing cleaved amplified
polymorphic sequence (CAPS) markers, the genetic diversity of T. atroviride isolates was
investigated [114]. Following amplification of RAPD regions, the amplicons were digested
to restrict enzyme digestion with BslI, DraI, and TaqI. T. atroviride was distinguished
from other species by three CAPS markers, which can be used to assess and monitor
T. atroviride, particularly in environmental specimens. Meena et al. provided a strategy for
detecting T. harzianum and T. hamatum species. SCAR primers were created based on the
sequence of species-specific RAPD fragments [115]. Perez et al. developed a procedure to
monitor the growth and colonisation of T. harzianum in experimental communities similar
to the method described earlier [100]. Because of the pervasive and diversified nature
of T. harzianum, scientists did not suggest using this method for detecting the species in
complicated ecological samples such as soil.
Using microarray technology, it is possible to investigate Trichoderma populations in
natural surroundings, which track gene expression changes. To monitor active Trichoderma
populations, new field-portable microarray analysis systems are now available [101]. These
systems provide data on microbial community composition, dynamics, and the physiological status of Trichoderma populations in soil. When conducting comparative transcriptome
analyses in vitro or in soil microcosm systems, researchers can use the transcriptomic data
from Trichoderma–fungus [102] and Trichoderma–plant interactions to choose target genes
for microarray monitoring. After using Trichoderma as a biocontrol agent, a metatranscriptome investigation of agricultural habitats can reveal significant alterations in the active
microbiome. On the other hand, this method is time consuming and expensive due to the
need for high-throughput sequencing and computing systems.
Now, it is easy to select Trichoderma strains, develop BCA implementation strategies, and establish monitoring strategies for detecting specific cells, secreted proteins, or
secondary metabolites; hence, volumes of information are available on the metabolome,
proteome, and secretome of Trichoderma strains from multiple species. Because each Trichoderma species produces a unique peptaibiome, non-ribosomally produced peptaibols could
be used to build mass spectrometry-based, species-specific monitoring systems [116].
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2.2.4. Exogenous Marker Genes
Utilizing exogenous markers, such as glucuronidase (GUS), green fluorescent protein
(gfp), hygromycin B phosphotransferase (hygB), or producing genes, certain Trichoderma
strains in agroecosystems might be identified and tracked [117–145]. Orr and Knudsen employed the gfp-labeled and GUS, T. harzianum strain ThzID1-M3 to quantify the
biomass changes in a fungal BCA in unsterilised soil. Indigenous bacterial and fungal
populations in unsterilised soil interfere with biomass determination procedures. GUS
and gfp were used to distinguish between the BCA-introduced marker genes and the
indigenous Trichoderma populations [146,147]. It was demonstrated that epifluorescence
microscopy could discriminate active hyphal biomass useful for biological control among
dormant chlamydospores and conidia counted by plate counts while observing gfp-labeled
T. harzianum [148]. Propiconazole-resistant Trichoderma harzianum TF3 is antagonistic to
Botrytis cinerea and can survive at high population densities in tomato, grapevine, and
phylloplane. This strain was changed to be resistant to hygromycin B using high-voltage
electric pulses and the vector pHAT (an offspring of pAN7-1), which was then used to
select strains with high levels of resistance [149]. When grown on tomato phylloplane, all
transformants outperformed the wild-type strain, thrived for 2 weeks in the presence of
hygromycin B or propiconazole, and were mitotically stable after many passages without
selection pressure.
Utilising cucumber plants cultivated in sphagnum peat, Green and Jensen evaluated the population trends and durability of the transformed T. harzianum strain T3a. A
T. harzianum strain inoculated into the rhizosphere was studied for the first time using
the GUS marker to track its existence, population structure, and behaviors [150]. pAN7-l
and pNOM102 plasmids were used by Bowen et al. to transform the T. harzianum strain
M1057 against Sclerotium rolfsii [59]. Two transformants had mitotic stability and their
growth rates matched wild-type ones. The co-transformant mitotic stability and ability
to colonise Sclerotinia sclerotiorum resting sclerotia in soil were studied [129]. T. harzianum
ThzID1-M3 strain was found to colonise about 60% of the sclerotia, suggesting that the
strains can be co-transformed with GUS and gfp to assess and monitor specific strains
introduced into soil [151]. ThzID1-M3 significantly reduces S. sclerotiorum colonisation
in soil by inhibiting the growth of fungivorous and other nematode populations when
applied to soil. Fungivorous nematodes seek out nutrient-rich areas in the soil. A larger
hyphal net produced by a Trichoderma strain and fed to the soil as a pellet formulation
attracts worms by providing food. To release into the environment, various countries,
including those in the European Union, require authorization [152]. Therefore, it is crucial
to monitor distinct, genetically unaltered biocontrol Trichoderma strains in agricultural
contexts utilising molecular techniques based on endogenous DNA markers.
3. Mechanisms of Trichoderma
Trichoderma spp. uses numerous antagonistic strategies against plant diseases. These
include antibiosis, mycoparasitism, competition for nutrients and space, stimulation of
plant growth, and induced plant defense mechanisms (Figure 1) [113].
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Figure 1. Biocontrol mechanism used by Trichoderma against plant pathogens and promoting
healthy crop.
3.1. Mycoparasitism
Mycoparasitism is an essential antagonistic character of Trichoderma, which is responsible for its efficiency against the plant pathogenic fungi [153]. The complex process of
mycoparasitism entails various events. In a biotrophic mycoparasitism, the hyperparasite
relies on the host fungus and obtains nutrition from it via haustoria without causing the
host cell to die out. Equilibrium is maintained between the host and the mycoparasitic
fungus (136). For commercial biocontrol purposes, these species-specific interactions may
be essential but are unlikely to be utilized due to the need for host mycelium as a substrate
for synthesizing hyperparasite. This category of hyperparasites is significantly better suited
for commercial use as MBCA compared to biotrophic hyperparasites since they can grow
on artificial substrates in large quantities, allowing for mass production.
Trichoderma and Clonostachys are the most explored mycoparasites. Most antagonistic
isolates from these genera exhibit a wide spectrum of plant pathogenic hosts among their
antagonistic isolates. CWDEs (cell wall degrading enzymes) and antimicrobial secondary
metabolites are typically used in tandem to kill their hosts, which develop structures for
attachment and infection [121–125]. Chitinolytic enzymes produced from T. harzianum,
such as endochitinase, β-1, 3 glucanase, and chitobiosidase, are more effective against
, suchfungus
as endochitinase,
β
plant pathogenic
than chitinolytic
enzymes isolated from plants or bacteria. The
concentration of cell wall degrading enzymes (CWDEs) viz. chitinase and β-1, 3-glucanase
produced by Trichoderma isolate was high. The cellulase and protease amounts were low
and the polygalacturonase produced by the pathogen was significantly reduced. The
mixture of endochitinase or β 1-3 glucanase showed antifungal activity by lysis of spore cell
walls, thallus, and hyphal tips and combining chitobiosidase and endochitinases inhibited
the plant pathogenic fungal spore germination and hyphal lysis more than singly [126].
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After recognising the host, complicated signaling triggers the production of these lytic
enzymes, which are not constitutive [124]. Recognizing the fungal host triggers transcriptional reprogramming and the development of “molecular weapons”, such as CWDEs (cell
wall degrading enzymes) that are used in host attack and lysis. The host releases oligosaccharides and oligopeptides that are then recognised by Trichoderma receptors and function
as inducers due to the early activities of cell wall degrading enzymes (CWDEs) [124]. Mycoparasites that are necrotrophic can cause more excellent permeability and disintegration
of host cell walls, leading to the death of the host. Several Trichoderma mycoparasitismrelated gene families are upregulated during mycoparasitism, including ech42 and prb1.
Trichothecene, produced by T. harzianum gene tri5, inhibits protein and DNA synthesis,
limiting pathogen growth. Trichothecene has a phytotoxic effect on Fusarium species [127].
Trichoderma virens TvBgn2 and TvBgn3 genes release a cell wall disintegrating enzyme,
which aids in biocontrol activities [128]. T. harzianum mycoparasitic activity has been
boosted by the cloning and expression of genes from five T. harzianum isolates encoding
chitinase (chit42), N-acetyl-ß-D-glucosomidase (exc1, and exc2), β-glucanase (bgn 13.1),
and protease (prb1) (T 30, 31, 32, 57, and 78) [154]. A gene aids mycoparasitic activity from
T. harzianum CECT 2413 that provides adhesion to hydrophobic surfaces and shields plant
cells from R. solani infections, according to previous research [155]. The T. atroviride G protein component, responsible for degrading pathogenic fungi cell walls, produces chitinase
and other antifungal compounds through the Tga1 gene [156]. B. cinerea and Phytophthora
capsici were found to have a synergistic effect on Trichoderma atroviride transcription of genes
involved in cell wall breakdown [156]. The genes identified from these biocontrol agents
have been discovered to have an essential role in biocontrol activity and their detailed
function is discussed in Table 1.
Table 1. List of genes involved in biocontrol activity and enzyme production.
S.No.
Gene
Protein
Function
Reference
1
endoglucanase I (EG I)
Endoglucanase
cellulose hydrolysis
[157]
2
erg1
Squalene epoxidase
activation of plant defense system
[145]
3
tri3
Trichothecene
O-acetyltransferase TRI3
trichodermin biosynthesis
[149]
4
tri4
Cytochrome P450
monooxygenase
trichodermin biosynthesis
[149]
5
tri5
Trichodiene synthase
trichothecene biosynthesis
[100]
6
tri6
Trichothecene biosynthesis
transcription regulator TRI6
transcriptional activator of genes involved
in harzianum A (HA) biosynthesis
[150,158]
7
tri11
Trichothecene C-4 hydroxylase
trichodermin biosynthesis
[149]
8
tri22
Cytochrome P450
monooxygenase
trichothecene biosynthesis
[151]
9
tri10
Trancription factor
regulation of trichothecene
biosynthetic genes
[152]
10
tri11
Trichothecene C-4 hydroxylase
trichodermin biosynthesis
[149]
11
ACEI
Repressor protein
cellulase biosynthesis
[153]
12
AceII
Transcription factor
cellulase biosynthesis
[154,159]
13
CbhI
Cellobiohydrolase I
cellulase hydrolysis
[155]
14
cbhII
Cellobiohydrolase II
cellulase hydrolysis
[148]
15
bgl1
β-glucosidase
cellulose hydrolysis
[156]
16
prb1
Basic proteinase
Mycoparasitism
[157]
17
ech42
Endochitinase
Mycoparasitism
[160]
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Table 1. Cont.
S.No.
Gene
Protein
Function
Reference
18
chit33
Endochitinase 33
Mycoparasitism
[161]
19
chit42
Chitinase 42
biocontol activity against fungus
[162]
20
cre1
Carbon catabolite repressor
Mycoparasitism
[163,164]
21
xyr1
Xylanase regulator 1
systemic resistance induction in plants
[165,166]
22
Rce1
Transcriptional repressor protein
regulation of cellulase biosynthesis
[167,168]
23
nag1
N-acetylglucosaminidase
essential for chitinase induction by chitin
[169,170]
24
egl1
β-1,4-Endoglucanase
Mycoparasitism
[171]
25
tvsp1
Extracellular serine protease
Mycoparasitism
[172,173]
26
Sm1
Cerato-platanin protein
activation of plant defense mechanisms
[174]
27
Sm2
Cerato-platanin protein
activation of plant defense system
[175,176]
28
SS10
Subtilisin-like protease
Broad-spectrum antifungal activity
[177]
29
SA76
Aspartic protease
Biocontrol activity against fungus
[178]
30
SL41
Serine protease
biocontrol activity against fungus
[179]
31
Tas-acdS
ACC deaminase
Plant root growth-promotion
[180]
32
TgaA
G-protein α subunit
mycoparasitism against Sclerotium rolfsii
[181,182]
33
TmkA
mitogen-activated protein kinase
mycoparasitism against Sclerotium rolfsii
[183]
34
ThPG1
Endopolygalacturonase
plant defense induction by T. harzianum
[184,185]
35
TvPG2
Endopolygalacturonase
Induction of Plant Systemic Resistance
[186]
36
ThPTR2
Di/tri-peptide transporter
Mycoparasitic process
[187]
37
tac1
Adenylate cyclase
Growth, germination, mycoparasitism
and secondary metabolism
[188]
38
Vel1
VELVET protein
Regulator of biocontrol as well as
morphogenetic traits in Trichoderma virens
[189]
39
PPT1
4-phosphopantetheinyl
transferase
Role in antibiosis and induction of SA and
camalexin-dependent plant
defense responses
[190]
40
Taabc2
ABC Transporter
Membrane Pump
Role in antagonism and biocontrol against
Pythium ultimum and Rhizoctonia solani
[191]
41
epl1
Eliciting plant response-like
protein
Modulation of Systemic Disease
Resistance in SolanumLycopersicum
[192]
42
epl2
Eliciting plant
response-like protein
Trichoderma mediated promotion of
plant protechion
[176]
43
Thctf1
Transcription factor
Production of secondary metabolites and
in the antifungal activity of T. harzianum
[193]
44
Pgy1
Proline-glycine-tyrosine-rich
protein
Role in antagonism against soil-borne
pathogens of plants
[194]
45
Ecm33
GPI-anchored cell wall protein
Role in antagonism against soil-borne
pathogens of plants
[194]
46
Pac1
Transcription factor
Role in antifungal activity of
Trichoderma harzianum
[195]
47
Tvbgn3
Beta-1,6-glucanase
Mycoparasitism
[174]
48
tvhydii1
Class II hydrophobin
Mycoparasitism and
plant-fungus interaction
[196]
49
Ste12
Transcription factor
Mycoparasitism
[197]
50
LaeA
Methyltransferase protein
Trichoderma atroviride defense
and parasitism
[198]
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3.2. Competition of Ecological Niche
For maintaining the dynamics of the microbial population, where microorganisms
belong to the same community and share the exact physiological needs, the availability of
nutrients is limited and, therefore, nutrient competition is essential for their survival. The
Trichoderma is a ubiquitous fungus, which is present throughout the world in agricultural
and natural soils due to its excellent competitive capability. Trichodema can compete with
pathogens in plants for nutritional sources, viz. nitrogen, carbon, and iron and acts as
a biocontrol agent against soil-borne plant pathogens. The rhizosphere competence of
Trichoderma strains enables them to colonise the root surface and compete with other microorganisms for nutrients secreted by roots in rhizospheric soil [199]. The term rhizosphere
competence was coined by Ahmad and Baker in 1987 [200]. Trichoderma spp. not only
controls many soil-borne pathogens but also promotes plant growth [201,202]. Trichoderma
spp. releases siderophores that sequester iron ions, thereby making them unavailable to the
pathogen. It has been established that plant pathogens produce low binding siderophore
coefficients or fewer siderophores as compared to Trichoderma. Iron is present in the Fe3+
state and it is not available for the growth of microorganisms because Ferric ion is not
soluble. Trichoderma spp. produces siderophores, which chelate Fe3+ and receptor protein
of microbial membrane recognise the complex of siderophore–Fe. Thus, the siderophore–
Fe3+ complex makes Fe unavailable to the microorganisms, including plant pathogens
present in the rhizosphere, ultimately resulting in suppressing pathogenic infection and,
hence, Trichoderma acts as a biological control, contributing to managing the plant pathogen
colonisation [203,204]. This shows the importance of competition for nutrients between
Trichoderma and pathogenic fungi. Trichoderma is more competitive than other rhizospheric
microorganisms because it can mobilise and take up nutrients from the soil [202]. T35
strain of T. harzianum, due to its rhizosphere competence, outcompetes Fusariumoxysporum
f. sp. melonis for nutrients and can colonise the rhizosphere well, resulting in decreased
concentration of nutrients and rhizospheric space for colonization of F. oxysporum f. sp.
meloni [205].
In addition, Trichoderma has an advantage over many other soil microbes due to its
ability to mobilise and take up soil nutrient sufficiently. Competition for nutrients is the
primary mechanism utilised by T. harzianum against Fusarium oxysporum f. sp. melonis.
Microorganisms, such as Trichoderma spp., mediate the solubility of nutrients in the soil
and make them available at the root surface. Soil microorganisms cause changes in soil pH,
a result of which being that equilibrium of many chemical and biochemical reactions is
modified [206]. There is scant evidence that Trichoderma can enhance the bioavailability of
insoluble or sparingly soluble elements viz. P, Fe, Mn, Cu, and Zn. However, results have
been yielded by previous research [207–209]. Significant increases in concentrations of P,
Fe, Mn, Cu, Zn, and Na in roots of cucumber inoculated with Trichoderma were reported
by Yedidia et al. [210]. Fiorentino et al. demonstrated that T. virens GV41 improved the
growth of lettuce and rocket and enhanced nutrient uptake by them and, hence, is the best
performing microbial biostimulant. It enhanced the use efficiency of N and favored the
uptake of native soil N by both the crops. The uptake of N by roots was enhanced under lowN-availability conditions. It has been revealed that Trichoderma acts as a nutrient solubiliser.
However, the detailed effects of Trichoderma inoculation remain to be deciphered [211].
3.3. Antibiosis
Trichoderma strains produce low-molecular-weight volatile or nonvolatile antibiotics or
diffusible compounds that interact and restrict the growth of deleterious plant pathogenic
fungi. This process is called antibiosis. The metabolites viz. antibiotics, mycotoxins, and
phytotoxins produced by Trichoderma helped in antagonism by either antibiosis or competition or through hyperparasitism. The fungus releases, viz. glucanases, chitobioses, and
chitinase enzymes, or antibiotics, such as viridin, gliotoxin, or peptaibols [88]. Trichoderma
produces a broad range of secondary metabolites, such as trichodermin, gliotoxin, viridin,
and peptide antibiotics, which is described in detail in Table 2 [212]. Antibiosis plays a
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vital part in managing Pythium ultimum and Rhizoctonia solani, which causes the damping
of zinnias. Gliotoxin antibiotic produced by biocontrol agent Gliocladium virens prevents
R. solani and P. ultimum growth by affecting the membrane and the leakage of metabolites
from the respective pathogen [213]. Antibiotics, including gliovirin, viridin, and massilactone, and toxic nonvolatile and volatile metabolites, such as tricholin, alamethicins, and
harzianic acid, are produced by Trichoderma strains [214]. Mendoza et al. observed that in
an interaction between the Trichoderma spp. HTE815 strain and M. phaseolina, the antibiosis
phenomenon is involved. There is formation of an intermediate band without growth
between colonies. The culture medium changes color and growth of inhibition zone exist
due to secondary metabolite excretion. Filizola et al. presented a study on antibiosis and
degree of antagonism and reported that the fungi belonging to Trichoderma genus could
potentially prevent the growth of the Fusarium strain [201]. This indicates the specificity
between the antagonist and the potential phytopathogen determined by various genes
and genetic factors that interact with the environment. G˛ebarowska et al. investigated
the volatile secondary metabolites and biometric parameters from coriander (Coriandrum
sativum L.) inoculated with T. harzianum strain T22 and T. asperellum strain. The treatment
with liquid suspension spores of Trichoderma increased the yield of essential oil by about
36% without affecting the composition of essential oils, leaving it to the upper limits of
pharmacopoeial standards [215]. Moreover, the treatment with Trichoderma spp. limited
plant pathogenic fungi belonging to the genus Fusarium. Volatile compounds and proteins
secreted by Trichoderma strongly inhibited the growth of bacterial isolates. This observation
clarifies that Trichoderma significantly modifies rhizosphere bacterial communities due to
the fumigant’s nature of volatile compounds [216]. Gliovirin that is produced by P strains
of T. virens is potent against P. ultimum. The Q strains of T. virens also produce gliotoxin
that is effective against Rhizoctonia solani [217]. Antimicrobial compounds produced by T.
koningii SMF2 have been shown to be active against Gram-positive bacterial and fungal
plant pathogens. Trichokonin VI, VII, and VIII are the peptaibols that make up the metabolites. Over a broad pH range and at different temperatures, the Trichokonins remain stable
and biologically active [218]. The secondary metabolites produced by the T. harzianum
strains T22 and T39 have been investigated. They identified and analysed the chemicals
T39 butenolide, harzianopyridone, and harzianopyridone. T39 butenolide and harzianopyridone demonstrated strong activity against G. graminis var. tritici, even at low doses [219].
The mutant of T. virens, deficient in mycoparasitism and antibiotic activity, retained its
biocontrol efficacy equivalent to that of the parent strain against Pythium ultimum and
R. solani, causing cotton seedling disease [220]. However, it was assumed that this was
due to the synergistic effect of enzymes and antibiotic compounds [163]. Several authors
have reported the involvement of lytic enzymes in biocontrol agent activity with cellulose
and chitin degradation characteristics in Trichoderma spp. [126]. Trichoderma spp. produces
extracellular metabolites, which are nonvolatile diffusible chemicals that have an inhibitory
impact against fungal pathogens, such as Colletotrichum graminicola. Shorter main roots are
a result of higher pyrone concentrations, as shown by Garnica-Vergara et al. [221]. Used
concentrations are not always present in nature. Additionally, Lee et al. discovered no
association between 6-PP generation and growth stimulation [222]. Gliovirin suppresses
the Phytophthora and P. ultimum activity, but it is ineffective in managing the diseases caused
by Bacillus thuringenesis, Rhizopus arrhizus, and Rhizoctonia solani [223].
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Table 2. List of secondary metabolites/bioactive compounds produced by Trichoderma.
Serial Number
Compound (Secondary
Metabolites)
Biological Activity
Produced By
References
1.
Trichorzianin TA
Trichorzianin TB
Antifungal
T. harzianum
[224]
1.
Trichorzins TVB I, II, IV
Antifungal
T. virens
[224]
2.
Harzianopyridone
Antifungal, Plant growth
Regulator
T. harzianum
[225]
Antifungal
T. harzianum
[224]
6-pentyl-α-pyrone
Antifungal,
Antimicrobial,
Plant growth Regulator
T. harzianum
T. koningii
T. viride
[224]
5.
6-pentyl-2H-pyran-2-one
Antifungal, anti-nematode and
plant growth-promoting in
tomato and Arabidopsis
thaliana
T. atroviride
T. harzianum
T. koningii
T. viride
[221]
6.
6-pent-1-enyl-α-pyrone
Antifungal
T. harzianum
T. viride
[226]
7.
Massoilactone- δ-decenolactone
Antifungal
Trichoderma spp.
[224]
8.
Koninginin E, B, A
Antifungal, Plant growth
Regulator
T. harzianum
T. koningii
[227]
9.
Koniginin D
Hydroxykoninginin B
Seco-koninginin
Antifungal, Plant growth
Regulator
T. harzianum
[224]
10.
Koninginin C
Antifungal, Plant growth
Regulator
T. koningii
[28]
11.
3,4-dihydroxycarotene
Antifungal
T. virens
[224]
12.
Lignoren
Antifungal
Antibacterial
T. lignorum
[224]
13.
Trichodermin
Antifungal
Antitrichomonal
Mycotoxin
T. polysporum
T. sporulosum
T. virens
T. reesei
[224]
14.
Harzianum A
Antifungal
T. harzianum
[224]
15.
Mycotoxin T2
Antifungal
Mycotoxin
T. lignorum
[224]
16.
Ergokonin A
Antifungal
T. koningii
T. viride
T. longibrachiatum
[141]
17.
Ergokonin B
Antifungal
T. koningii
T. viride
[224]
18.
Viridin
Antibiotic
Inhibitor Fungal
Spore germination
Phytotoxic
T. viride
T. virens
T. koningii
[224]
19.
Dermadin (U21, 963)
Antimicrobial
T. koningii
T. viride
[130]
20.
Cellulases
Degrade cellulose during root
colonization to penetrate the
plant tissue
T. reesei
[153]
21.
Compactin
Act as Cholestrol lowering agent
T. longibrachiatum
T. pseudokoningii
[132]
3.
4.
HarzianolideDehydroharzianolide
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Table 2. Cont.
Serial Number
Compound (Secondary
Metabolites)
Biological Activity
Produced By
References
22.
5-Hydroxyvertinolide
Fungal antagonist
T. longibrachiatum
[133]
23.
Gliovirin
Antimicrobial
T. virens
[217]
24.
Bisvertinolone
Antifungal
T. longibrachiatum
[134]
25.
Fleephilone
Inhibitory action against Virion
T. harzianum
[135]
26.
Harziphilone
Cytotoxicity against murine
tumor cell line M-109
T. harzianum
[135]
27.
Trichodimerol
Inhibit tumor necrosis factor in
human monocytes.
T. longibrachiatum
[136]
28.
Trichocaranes A,B,D
Growth inhibitor of etiolated
wheat coleoptiles
T. virens
[137]
29.
Viridepyronone
Fungal antagonist
T. viride
[138]
30.
T22azaphilone
Antifungal
T. harzianum
[219]
31.
T39butenolide
Antifungal
T. harzianum
[219]
32.
Emodin
Antimicrobial and antineoplastic
agent
T. viride
[139]
33.
Trichosetin
Antibiotic
T. harzianum
[141]
34.
Trichoderma mide B
Displays cytotoxicity against
HCT-116 human colon
Carcinoma
T. virens
[141]
35.
Wortmannolone
Inhibitor of the
phosphatidylinsitol 3-kinase
T. virens
[141]
36.
Virone
Inhibitor of the
phosphatidylinsitol 3-kinase
T. virens
[141]
37.
Heptelidic acid
Activity against Plasmodium
Falciparum
T. virens
T. viride
[141]
38.
Indole-3-acetic acid
(IAA)
Growth and development
Regulator
T. atroviride
T. virens
[140]
39.
Indole-3-acetaldehyde
Control root growth in
Arabidopsis thaliana
T. atroviride
T. virens
[140]
40.
Indole-3
Carboxaldehyde
Induces adventitious root
formation in Arabidopsis
thaliana
T. atroviride
T. virens
[141]
41.
Ferricrocin
Required in the competition of
iron in the rhizosphere
T. atroviride
T. virens,
T. reesei
[142]
42.
Gliotoxin
Required in the competition of
iron in the rhizosphere
T. hamatum
T. viride
T. virens
[228]
43.
Cyclonerodiol
Antifungal
T. harzianum
T. koningii
[153]
44.
Pachybasin
Antifungal
T. harzianum
[144]
45.
Trichovirin II
Induction of resistance in
cucumber plants
T. virens
[228]
46.
Alamethicin
Induction of plant defense in
lima and pathogen resistance
T. viride
[228]
47.
Coprogen B
Solubilize iron unavailable for
the plan
Trichoderma spp.
[143]
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Table 2. Cont.
Serial Number
Compound (Secondary
Metabolites)
Biological Activity
Produced By
References
48.
Harzianic acid
Antimicrobial, plant growth
Regulator
T. arundinaceum,
T. harzianum
[145]
49.
cis- and
trans-ß-ocimene
Induce expression of JA defense
responses-related genes in
Arabidopsis. Thaliana
T. virens
[229]
50.
ß-Myrcene
Regulates the expression of
genes (abiotic and biotic
stresses)
T. virens
[230]
51.
Abscisic acid (ABA)
Regulates stomatal aperture in
Arabidopsis thaliana
T. atroviride,
T. virens
[231]
52.
Ethylene (ET)
Regulates cell differentiation
and defense responses
T. atroviride
[227]
53.
Trichokonin VI
Inhibits primary root growth in
Arabidopsis thaliana
T. longibrachiatum
[232]
54.
Glu(OMe)18-alamethicin F50 (2)
Anti-tumor
T. arundinaceum
[233]
55.
trichobrevin BIII-D
Anti-tumor
T. arundinaceum
[233]
56.
bisabolan-1,10,11-triol
Antibacterial
Growth inhibitoring
T. asperellum
[234]
57.
12-nor-11-acetoxybisabolen3,6,7-triol
Antibacterial
Growth inhibitoring
T. asperellum
[234]
58.
Dechlorotrichodenone-C
Antibacterial
Growth inhibitoring
T. asperellum
[234]
59.
3-hydroxytrichodenone C
Antibacterial Growth
Inhibitoring
T. asperellum
[234]
60.
3β , 5α,
9α-trihydroxyergosta-7,22-dien6-one
Antifungal
T. asperellum
T. harzianum
Trichoderma spp.
[235]
61.
Isoechinulin A
Antimicroalgal
T. koningiopsis
[236]
62.
Echinuline
Antimicroalgal
T. koningiopsis
[236]
63.
Fructigenine A
Antimicroalgal
T. koningiopsis
[236]
64.
Cyclopenol
Antibacterial
T. koningiopsis
[236]
65.
Wickerol A
Nematicidal
T. koningiopsis
[236]
66.
Sorbicillin
Antibacterial
T. longibrachiatum
[237]
67.
10,11-dihydrocyclonerotriol
Antifungal
T. longibrachiatum
[238]
68.
Sohirnone A
Antifungal
T. longibrachiatum
[238]
69.
Trichokonin A
Antiviral
Anti-tumor
Antimicrobial
Plant resistance
T. longibrachiatum
[239]
70.
Atrichodermone A,B,C
Cytotoxic
Anti-inflammatory
T. atroviride
[240]
71.
Cerebroside A, D
Antibacteria
T. saturnisporum
Trichoderma spp.
[239]
72.
Lignoren
Antibacterial
T. atroviride S361
T. citrinoviride
T. lignorum
[235]
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Table 2. Cont.
Serial Number
Compound (Secondary
Metabolites)
Biological Activity
Produced By
References
73.
Catenioblin C
Antifungal
T. atroviride
T. longibrachiatum
[241]
74.
Trichocarotin E,H
Antimicroalgal
T. virens
[237]
75.
Trichocitrin
Antimicroalgal
T. citrinoviride
[242]
76.
Nafuredin
Antimicroalgal
Antibacterial
T. citrinoviride
Trichodermasp
[235]
77.
Chromone
Antifungal
T. virens
[237]
78.
Aspochalasin D, J, I
Cytotoxic
T. gamsii
[241]
79.
Epicycloneodiol oxide
Antibacterial
T. harzianum
T. koningiopsis
[235]
80.
cycloneodiol oxide
Antibacterial
T. harzianum
T. koningiopsis
[235]
81.
ZSU-H85 A
Antiviral
Trichoderma spp.
[243]
82.
Trichokindin I, II, III, IV, V,VI, VII
Bioinducer
T. harzianum
[244]
83.
2,5-cyclohexadiene-1,4-dione2,6-bis(1,1-dimethylethyl)
Antifungal
Trichoderma spp.
T-33
[245]
84.
N-2′ -hydroxy-3′ E-octadecenoyl1-o-β-D-glucopyranosyl-9methyl-4E,8E-sphingadiene
Antifungal
Trichoderma spp. 09
[246]
85.
Tyrosol
Anti-tumor
Hyperplasia-inhibitory
T. harzianum
T. spirale
[235]
86.
Trichoderma ketone A
Antifungal
T. koningi
[235]
87.
Pyridoxatin
Antibiotic
Trichoderma spp.
MF106
[247]
88.
3-(3-oxocyclopent-1enyl)propanoic
acid
Antibacterial
Trichoderma spp.
YLF-3
[248]
89.
Oxosorbicillinol
DPPH-radical-scavenging
Trichoderma spp.
USF-2690
[56]
90.
α-acetylorcinol
Growth inhibitoring
Trichoderma spp.
Jing-8
[249]
91.
Daidzein
Antibacterial
Trichoderma spp.
YM311505
[238]
3.4. Biochemical and Molecular Defense Response Induced by Trichoderma
A type of active resistance, known as induced systemic resistance, depends on plant
anatomy, such as structural and biochemical barriers. Different physical and biochemical
alterations that are indicative of the development of plant-induced systemic resistance
occur in plants that are co-cultivated with diverse Trichoderma strains. Callose deposition,
cell wall thickening, and tylose development in xylem vessels or cork layers are examples
of structural alterations [250]. The accumulation of local and systemic reactive oxygen
species (ROS), as well as an increase in the production of signaling molecules and secondary
metabolites, such as phytoalexins and PR proteins, was among the biochemical reactions
seen in plants. In analyses of experimental Trichoderma strain–plant–pathogen systems,
these substances are regarded as indicators of the defense response in plants [251–255].
Hydrogen peroxide (H2 O2 ), an ROS implicated in numerous defensive mechanisms, such
as cell wall expansion and lignification, is given a lot of attention. The chemical transmits
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signals and has the potential to activate calcium channels and peroxidases in cell walls [250].
Oxidative burst enzymes, such as NADPH oxidases attached to the plasma membrane,
peroxidases bound to the cell wall, and apoplastic amine oxidases, can either directly
or indirectly generate H2 O2 . It is crucial to note that the accumulation of ROS was not
accompanied by cell damage processes, as indicated by the concentration of lipid peroxides. This suggests the activation of mechanisms that suppress the production of hydroxyl
radicals (OH) as well as the use of ROS by enzymes, such as peroxidase or catalases, to
initiate a biochemical defense response [253,256]. Application with Trichoderma induces
the synthesis and accumulation of enzymes, as well as a range of secondary metabolites
and signaling molecules, such as salicylic acid (SA), ethylene (ET), and jasmonic acid (JA)
(SA) [250,251]. Ethylene and Jasmonic acid are the signaling molecules of ISR, which
ultimately are responsible for producing different defense enzymes, such as polyphenol oxidase, peroxidase, catalase, β1-3glucanase, β1-4-glucanase, N-acetylglucosaminidases, and
chitinases [106,107]. Trichoderma strains, after colonization, penetration, and establishment
inside the root tissue or attachment with the root tissue, lead to an array of enzymatic and
morphological changes inside the host plant that finally help in the production of defensive
enzymes and, lastly, end with the induction of induced systemic resistance in the plant [108].
Elicitation of ISR response in plants begins with recognising specific components from
microbial cell surfaces, known as microbe-associated molecular patterns (MAMPs) by
plant receptors [257]. MAMPs from various biocontrol agents have been linked with ISR
(Baker et al., 2007; Van Wees et al., 2008). MAMP responses start with generation ion
fluxes, reactive oxygen species (ROS), nitric oxide, and ethylene (ET) and later involve the
accumulation of callose and the biosynthesis of antimicrobial substances. The first MAMP
identified from Trichoderma was ET-inducing xylanase (Xyn2/Eix consisting of five surfaceexposed amino acids), eliciting plant defense responses in tomato and tobacco (Rotblat et al.,
2002). Trichoderma-activated cellulases also trigger defence response by activating ET and
SA pathways (Matinez et al., 2001). Trichoderma proteins involved in root colonization, such
as swollen in TasSwo, also trigger defense response in cucumbers (Brotman et al., 2008).
Another protein, endopolygalacturonase ThPG1, stimulates the resistance response in
Arabidopsis (Moran-Diez et al., 2009). During the colonisation of maize and cotton roots by
Trichoderma atroviride and Trichoderma virens, protein Ep11 and Sm1 accumulated in hyphae
and act as MAMPs, respectively. Root colonisation by Trichoderma leads to the systemic
alteration in the proteome, transcriptome, and MAMP interaction in leaves. Trichoderma
interaction with plants leads to elicitor’s production. These elicitors induce the production
of signaling molecules, such as jasmonic acid (JA) and ethylene (ET), bind the receptors,
and after a series of enzymatic reactions, lead to the induction of defensive genes inside the
host plant. These defensive genes are directly responsible for plant pathogen suppression
and strengthen the morphological and biochemical barrier of the plants [109]. In vitro
inoculation of Trichoderma isolates against Sclerotium rolfsii, Colletotrichum gloeosporioides,
and C. capsica inhibits mycelial growth and significantly increases the chitinase and β-1,
3-glucanase activities [39]. This enabled them to promote the plant growth and stimulate defense system against plant pathogens. JA/ET-dependent pathways elicit ISR by Trichoderma
and trigger priming responses in plants, such as other beneficial microbes. The interaction
of Trichoderma with the plant is dynamic. During this interaction, overlapping the expression of defense-related genes of the JA/ET and SA pathways may occur [102]. It depends
on the strains of Trichoderma, the concentrations used, the plant material, the developmental
stage of the plant, and the time of interaction. The Trichoderma produces plant hormones ET
and IAA. These hormones play a vital part in interconnecting development of plants and
their defense responses [103]. The Trichoderma is endowed with genes whose expression
in plants helps them manage diseases and imparts resistance to stressed environmental
conditions. The experimental evidence shows that interactions of Trichoderma with plants
have traits in common with other beneficial microbial associations and they also show their
particular lifestyle characteristics of Trichoderma spp. However, more investigations are
required to decipher the signaling transduction pathways of defense and development.
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These pathways result from Trichoderma–plant interactions in the presence of biotic and
abiotic stresses [102]. Biotrophs trigger the SA route, whereas necrotrophic pathogens
stimulate both the ET and JA pathways [177]. In this defense, PDF1.2 (Plant defensin 1.2),
Thi2.1 (Thionin), or Chib (Chitinase B) are commonly used as marker genes [235]. The
systemic acquired resistance (SAR) mediated by salicylic acid (SA) results in the expression
of pathogenesis-related genes (PR) [236]. These signaling pathways are tightly connected,
allowing for fine control of resource allocation between plant development and response to
environmental stress agents. To avoid or actively decrease defense barriers, plant enemies
frequently modify the underlying network of cross-modulating channels. The necessity to
prioritise the response against a specific type of biotic stress has been demonstrated. Then,
to comprehend plant defense responses against pests and in the context of a beneficial
microbes, molecular pathways must be studied at the metaorganism level. Such research
will shed light on the co-evolutionary mechanisms that shape pest and disease communities
on plants and provide useful information for creating new pest and pathogen control tactics
that mimic and alter plant defensive responses (Figure 2).
Figure 2. Trichoderma-induced resistance in host plant.
3.5. Regulatory Mechanisms Triggering the Defense of Trichoderma
Much research has been conducted on the signal transduction pathways that trigger
the genes involved in biocontrol and mycoparasitism. These signal transduction pathways include heterotrimeric G-protein signaling, mitogen-activated protein kinase (MAPK)
cascades, and the cAMP pathway. These pathways are all interconnected with one another [258]. The MAP-kinase TVK1, which has been identified in T. virens, including its
orthologs in T. asperellum (TmkA) and T. atroviride (TMK1), is particularly important in
the regulation of signaling processes that target output pathways important for successful
biocontrol [259,260]. T. atroviride tmk1 deletion results in increased antifungal activity and
resistance against Rhizoctonia solani but decreased mycoparasitic activity [261]. According to
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this study, T. virens TVK1 deficiency significantly boosts the fungus biocontrol efficacy [259].
As a result, even though deletion of the corresponding genes reduces mycoparasitic efficacy,
the mutant strain biocontrol skills are improved [259].
In terms of the action of the heterotrimeric G-protein signaling pathway in Trichoderma spp., two genes have been studied so far in terms of biocontrol-related mechanisms:
the class I (adenylate cyclase inhibiting) G-alpha subunits TGA1 of T. atroviride and TgaA
of T. virens, as well as the class III (adenylate cyclase activating) G-alpha subunits TGA3
of T. atroviride and GNA3 of T. reesei. TGA1 is known to regulate coiling around host
hyphae as well as the synthesis of antifungal metabolites. The absence of TGA1 causes
host fungus to develop more slowly [262,263]. TgaA has been linked to a host-specific
role, similar to that seen with MAP-kinases [264]. On the other hand, TGA3 is essential
for biocontrol, as the absence of the corresponding gene led to the development of nonpathogenic strains [265]. Because it has been hypothesised that mycoparasitism can be
favorably affected by the constitutive activation of GNA3 in T. reesei, a similar mechanism
is at work on this fungus [266]. These findings are consistent with the analysis of cAMP
signaling components, which show that cAMP has a beneficial function in biocontrol [267].
Efforts were made to identify characteristics among each of these genes and enzymes
that were regulated upon the interaction of Trichoderma with a pathogen. These characteristics could be used to differentiate efficient biocontrol strains isolated from nature that were
less effective [268,269]. However, the effectiveness of standardised marker gene assays for
the assessment of the potential biocontrol strains will not be known until after additional
and more in-depth research has been conducted.
3.6. Plant Growth Promotion
A plant’s root system is colonised by microbial organisms, which simultaneously
protect the plant from soil-borne diseases and stimulate growth [270]. These positive
plant–microbe interactions frequently occur in the rhizosphere, enhancing plant growth
or aiding the plant in resisting abiotic or biotic stresses [271]. Trichoderma spp. multiplies
in the rhizosphere, forming a mutual association that naturally enhances plant nutrition
and growth. It can colonise roots, boosting plant nutrition, growth, and development, as
well as abiotic stress resistance. Plant growth is typically attributed to an indirect effect
of plant disease management when biological control agents are used. T. harzianum was
also found to enhance the concentration of trace and essential elements, such as Zn, Fe,
Cu, Mn, Cu, Ca, Mg, P, N, K, and Na, in the shoots and roots of cucumber and tomato
seedlings [272]. It has the ability to produce a number of phytohormones, siderophores,
and phosphate-solubilizing enzymes [273]. Phytohormones enhance the absorptive surface
of plant roots by stimulating root development. It has been established that plant antimicrobial chemicals produced by Trichoderma can stimulate plant growth. Isolated compounds,
such as Trichocereus A-D, Harzianopyridone, koninginins, 6PP, cyclonerodiol, harzianic
acid (HA), and harzianolide, that helped plant growth promotion are dependent on the
concentration of compounds. Cerinolactone, a novel secondary metabolite identified from
Trichoderma cerinums, was found to have a beneficial impact on tomato seedling growth
3 days after treatment. Similarly, the iron-binding properties of HA and iso-harzianic acid
reported in T. harzianum metabolites are known to enhance plant growth [274]. T. virens
and T. atroviride have also produced indole-3-acetic acid-related indoles (IAA-related indoles). The incorporation of L-tryptophan into Trichoderma liquid cultures increased the
IAA-related indoles production. This finding suggested that Trichoderma production of
IAA-related indoles may be one of the processes used by the fungus to enhance plant
development and secondary root number, resulting in increased biomass in Arabidopsis [275]. Trichoderma plant-growth-promoting action could be due to these pathways [16].
Trichoderma spp. application results in enhanced vegetative growth on various crop plants.
Trichoderma spp. interactions with plants resulted in increased resistance against plant
diseases [276]. Increased productivity, biomass, nutrient uptake, and stress tolerance are
signs of improved plant growth [277]. Trichoderma isolates from the rhizosphere of the
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mangrove Avicennia marina solubilize P from insoluble Ca3 (PO4 )2 —an extracellular phytase activity and acidic phosphatase had been exclusively enhanced in the presence of
Ca3 (PO4 )2 [278]. Furthermore, it has been found that using Trichoderma spp. in a consortium
improves the physical strength and durability of the plant’s cell wall in the presence of cell
wall degrading plant pathogenic fungi [279,280]. The improvement in root growth is likely
the result of one or more mechanisms, including an increase in the rate of carbohydrate
photosynthetic activities, a higher rate of plant growth regulation, an increase in rooting
depth, and, thus, greater tolerance to drought environments [281]. When there are rich
inorganic soil substrates, such as bioorganic fertilisers, Trichoderma spp. is more successful
in colonising and boosting plant growth [282].
4. Conclusions and Future Prospects
The Trichoderma—plant—pathogen interaction is a multi-dependent and dynamic
system. A thorough understanding of Trichoderma mechanisms with plants and pathogens
can significantly improve the efficacy of their actions. Trichoderma employs a wide range
of complex direct and indirect biocontrol mechanisms to protect itself from biotic stresses,
such as pathogenic microbes (bacteria, fungi, and nematodes). This review explains why
Trichoderma spp. has earned its well-deserved track record as a powerful plant growth
promoter, with the added benefit of enhancing localised and systemic resistance in plants.
This is because Trichoderma spp. is capable of producing a broad range of antibiotic substances and synthesising so many secondary metabolites, each of which has the possibility
to parasitise a broad range of pathogenic fungi inside the rhizosphere. Trichoderma elicitors and effectors are recognised by plant receptors, which begin the signaling process
and govern the genetic composition of the host. This provides the foundation for these
symbionts to trigger the defensive metabolism in their host. To verify a database for the
responsible and long-term usage of Trichoderma, it is necessary to examine the ecological
impact of extensive applications of biocontrol agents and their secondary metabolites.
Because of this, Trichoderma genomes are a valuable source of gene candidates for creating transgenic plants resistant to both biotic and abiotic stresses. Finally, in the age of a
green economy focused on protecting both human health and the environment, the use of
Trichoderma species should be encouraged as a viable alternative to pesticides in light of
the information presented in this research. Additionally, the current, thorough, developed,
and yet affordable, quick, and successful ways of detecting and evaluating antagonists,
integrating multiple mechanisms of action with cascade reactions, must be developed and
used in Trichoderma research. The research will also focus on determining the risk of using
BCAs based on Trichoderma, as well as their toxicity and ecotoxicity, not only in vitro but
also in natural farming practices in vitro and in situ, before they are commercialised as
biostimulations, biocontrols, or bioremediation preparations. In conclusion, it can be said
that more research must be conducted to know the mechanism of Trichoderma spp. in detail
so that potent Trichoderma biofungicides can flourish in any type of environment. The social
anxiety of implementing new antifungal and antibacterial microbes into the environment
must be overcome.
Author Contributions: Conceptualization, writing—original draft preparation, N.M.; Writing—
review and editing, A.S.K. and N.M.; writing—original draft preparation, investigation, A.S.K., N.M.,
M.V.S.R. and R.S.G.; supervision, funding acquisition, H.V.S., S.K.S. and P.K.S. All authors have read
and agreed to the published version of the manuscript.
Funding: This research was funded by ICAR-NBAIM, Maunath Bhanjan, Uttar Pradesh.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
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Acknowledgments: The authors extend their gratitude to the Indian Council of Agricultural Research,
New Delhi, ICAR-National Bureau of Agriculturally Important Microorganisms, Maunath Bhanjan,
U.P India, for providing financial assistance during the study. The authors wish to express their
sincere thanks to Faheem Ahamad and Pusparaj for assistance during the time period of these studies.
Conflicts of Interest: The authors declare no conflict of interest.
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