Current Research in Microbiology and Biotechnology

Vol. 1, No. 4 (2013): 133-142

Review Article
Open Access

ISSN: 2320-2246

Molecular mechanism of Trichoderma as bio-control

agents against phytopathogen system a review
Harsukh Gajera*, Rinkal Domadiya, Sunil Patel, Mansukh Kapopara, Balubhai Golakiya
Department of Biotechnology, College of Agriculture, Junagadh Agricultural University, Junagadh 362 001, Gujarat, India.

* Corresponding author: Harsukh Gajera, email: harsukhgajera@yahoo.com

Received: 21 May 2013

Accepted: 04 June 2013

Online: 01 July 2013

ABSTRACT
Trichoderma is a genus of asexually reproducing filamentous fungi and widely distributed in the soil, plant
material, decaying vegetation and wood. The antagonistic properties of Trichoderma are based on the activation
of multiple mechanisms. Trichoderma strains act as bio-control agents against fungal phytopathogens either
indirectly or directly. Indirect mechanism comprises competition for nutrients and space, modification of the
environmental conditions, antibiosis and induction of plant defensive mechanisms, however direct mechanism
encompasses mycoparasitism. Some bio-control agents use only one of these strategies but the most successful
bio-control agents like Trichoderma use several of them. These indirect and direct mechanisms may act
coordinately and their importance in the bio-control process depends on the Trichoderma strain, the antagonized
fungus, the crop plant, and the environmental conditions including nutrient availability, pH, temperature and
iron concentration. Activation of each mechanism implies the production of specific compounds and metabolites
such as plant growth factors, pathogenesis related lytic enzymes, siderophores, antibiotics, and carbon and
nitrogen permeases. These metabolites can be either overproduced or combined with appropriate bio-control
strains in order to obtain new formulations for use in more efficient control of plant diseases.

Keywords: Trichoderma, Phytopathogenic fungi, Antibiosis, Lytic enzymes, Induced resistance, Biological
control

INTRODUCTION
Fungi in the genus Trichoderma have been known since
at least the 1920s for their ability to act as bio-control
agents against plant pathogens [1]. Trichoderma sp. are
beginning to be used in reasonably large quantities in
plant agriculture, both for disease control and yield
increases. Recent advances demonstrate that the effects
of Trichoderma on plants, including induced systemic
or localized resistance, are very important [2]. The
most useful strains showed a property of rhizosphere
competence that is, the ability to colonize and grow in
association with plant roots (3). The taxonomies of
these fungi are being revised significantly, and many
new species are being recognized. The taxonomic
classification of Trichoderma are as - Domain:
Eukaryota; Kingdom: Fungi; Division: Ascomycota;
Class: Euascomycetes; Order: Hypocreales; Family:
Hypocreaceae; Genus: Trichoderma.

features of the conidia and phialides help in

differentiation of these species from each other. Apart
from these, two other species, T. asperelum and T.
citrinoviride have been proposed [4].

The genus Trichoderma has important six species;

Trichoderma harzianum, T. koningii, T. longibrachiatum,
T. pseudokoningii, T. viren and T. viride. Morphological

Trichoderma sp. has been studied as biological control

agents against soil borne plant pathogenic fungi [5].
Results from different studies showed that several
strains of Trichoderma had a significant reducing effect
on plant diseases caused by pathogens such as
Rhizoctonia solani, Sclerotium rolfsii, Phythium
aphanidermatium, Fusarium oxysporum, F. culmorum
and Gaeumannomyces graminisvar var. tritici under
greenhouse and field conditions [5-8]. Knowledge
concerning the behavior of these fungi as antagonists is
essential for their effective use because they can act
against pathogens in several ways [9]. Isolates of
Trichoderma harzianum can produce lytic enzymes [10]
and antifungal antibiotics [11, 12] and they can also be
competitors of fungal pathogens [13], and promote
plant growth [14].

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The bio-control agents-Trichoderma can work in

several ways: (1) A bio-control agent may grow faster
or use its food source more efficiently than the
pathogen, thereby crowding out the pathogen and
taking over. This process is called nutrient competition.
(2) A bio-control agent may release a product that
slows down or kills the pathogens in the vicinity of such
a product. This process is called antibiosis. (3) A biocontrol agent may cause a plant to make a product that
discourages or kills the pathogen. This process is called
induced resistance. The plant actually fights back. (4) A
bio-control agent may feed directly on or in a pathogen.
This process is called parasitism. In this way, the
pathogen is destroyed. Some bio-control agents use
only one of these strategies but the most successful biocontrol agents like Trichoderma use several of them
[15]. This article is a general overview of the different
reported
mechanisms
of
bio-control
agents
Trichoderma against phytopathogens.
BIO-CONTROL BY COMPETITIONS
Fungistasis: Few antagonists are usually able to
overcome the fungistatic effect of soil that results from
the presence of metabolites produced by other species,
including plants, and to survive under very extreme
competitive conditions. Trichoderma strains grow
rapidly when inoculated in the soil, because they are
naturally resistant to many toxic compounds, including
herbicides, fungicides and insecticides such as DDT, and
phenolic compounds [16], and because the strains
recover very rapidly after the addition of sub-lethal
doses of some of these compounds. Resistance to toxic
compounds may be associated with the presence in
Trichoderma strains of ABC transport systems [17]. For
this reason, preparations of Trichoderma strains are
very efficient in controlling several phytopathogens,
such as R. solani, P. ultimum or Sclerotium rolfsii, when
alternated with methyl bromide, benomyl, captan or
other chemicals [18].
Competition for nutrients: Starvation is the most
common cause of death for microorganisms, so that
competition for limiting nutrients results in biological
control of fungal phytopathogens [16]. In most
filamentous fungi, iron uptake is essential for viability,
and under iron starvation, most fungi excrete low
molecular weight ferric-iron specific chelators, termed
siderophores, to mobilize environmental iron [19].
Subsequently, iron from the ferri-siderophore
complexes is recovered via specific uptake
mechanisms. In Aspergillus fumigatus and Aspergillus
nidulans, siderophore biosynthesis is negatively
regulated by carbon source [19]. In Ustilago maydis,
gene products related to iron uptake affect the
development of plant disease [20]. Some Trichoderma
isolates produce highly efficient siderophores that
chelate iron and stop the growth of other fungi [5]. For
this reason, soil composition influences the bio-control
effectiveness of Pythium by Trichoderma according to
iron availability. In addition, T. harzianum T35 controls
Fusarium oxysporum by competing for both
rhizosphere colonization and nutrients, with biohttp://crmb.aizeonpublishers.net/content/2013/4/crmb133-142.pdf

control becoming more effective as the nutrient

concentration decreases [21]. Competition has proved
to be particularly important for the bio-control of
phytopathogens such as Botrytis cinerea, the main
pathogenic agent during the pre and post harvest in
many countries [22]. The extraordinary genetic
variability of this fungus makes it possible for new
strains to become resistant to essentially any novel
chemical fungicide on which it is exposed [22]. The
advantage of using Trichoderma to control B. cinerea is
the coordination of several mechanisms at the same
time, thus making it practically impossible for resistant
strains to appear. Among these mechanisms, the most
important is nutrient competition, since B. cinerea is
particularly sensitive to the lack of nutrients.
Trichoderma has a superior capacity to mobilize and
take up soil nutrients compared to other organisms.
The efficient use of available nutrients is based on the
ability of Trichoderma to obtain ATP from the
metabolism of different sugars, such as those derived
from polymers wide-spread in fungal environments:
cellulose, glucan and chitin and others, all of them
rendering glucose [16]. The key components of glucose
metabolism include assimilation enzymes and
permeases, together with proteins involved in
membrane and cell wall modifications. While the role of
the glucose transport system remains to be discovered,
its efficiency may be crucial in competition [23], as
supported by the isolation of a high affinity glucose
transporter, Gtt1, in Trichoderma harzianum CECT
2413 (referred to here as strain 2413). This strain is
present in environments very poor in nutrients, and it
relies on extracellular hydrolases for survival. Gtt1 is
only expressed at very low glucose concentrations, i.e.
when sugar transport is expected to be limiting in
nutrient competition [23]. In fact, glucose uptake is
increased three to four fold in a transformant
derivative that carried an additional copy of the
transporter gene. Only two other genes encoding
glucose transporters have been described in
filamentous fungi [24, 25], one of them in Uromyces
fabae. This basidiomycete has an ATPase and a protoncoupled glucose transport system that is expressed
during infection of Vicia faba. This suggests an
additional, antagonistic role for Gtt1, allowing the
fungus to obtain energy from hydrolyzed polymers and
to transport sugar rapidly into the cells. As a
consequence, transformants able to transport glucose
more rapidly than the wild type [23] and thus it should
be more efficient bio-control agents. This would serve
as a very useful mechanism of nutrient competition
during mycoparasitism interactions.
Promoter analysis of genes related to antagonism in
Trichoderma strains revealed the presence of
consensus sequences for transcription factors
responsible for carbon (CreaA), nitrogen (AreA), Stress
(Msn2/Msn4), pH (PacC) and mycoparasitism (MYC)
regulation, among others [26]. Thus, appropriate
manipulation of these regulators would provide an

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alternative to the isolation of more competitive Biocontrol agents.

positive bacteria Staphylococcus aureus and Bacillus

subtilis.

ANTIBIOSIS
Antibiosis occurs during interactions involving low
molecular weight diffusible compounds or antibiotics
produced by Trichoderma strains that inhibit the
growth of other microorganisms. Most Trichoderma
strains produce volatile and non volatile toxic
metabolites that impede colonization by antagonized
microorganisms. These metabolites are harzianic acid,
alamethicins, tricholin, peptaibols, antibiotics, 6penthyl--pyrone, massoilactone, viridin, gliovirin,
glisoprenins, heptelidic acid [27]. Strains of T. virens
with the best efficiency as bio-control agents are able to
produce gliovirin [28]. Also, the most effective isolates
of T. harzianum against Gaeumannomyces graminis var.
tritici produce pyrone antibiotics, and the success of the
strains was clearly related to the pyrones they
produced.

STIMULATION OF PLANT RESISTANCE AND PLANT

DEFENSE MECHANISMS
Trichoderma-based biofungicides are a reality in
agriculture, with more than 50 formulations today
available as registered products worldwide. Several
strategies have been applied to identify the main genes
and compounds involved in this complex, three-way
cross-talk between the fungal antagonist, the plant, and
microbial pathogens [34]. Woo and his coworkers [34]
have transgenically demonstrated the ability of
Trichoderma sp. to transfer heterologous proteins into
plant during root colonization, and have used green
fluorescent protein and other markers to study the
interaction in vivo and in situ between Trichoderma sp.
and the fungal pathogen or the plant.

The combination of hydrolytic enzymes and antibiotics

results in a higher level of antagonism than that
obtained by either mechanism alone [15, 28].
Synergetic effects between an endochitinase from T.
harzianum and gliotoxin, and between hydrolytic
enzymes and peptaibols on conidial germination of B.
cinerea is well known [29]. A mutant from strain 2413
that had higher levels of extracellular enzymes and of
-pyrone performed better than the wild type in in
vitro confrontation experiments against R. solani and in
assays of grape protection against B. cinerea, both
under repression (only pyrones were produced) and
derepression conditions (enzymes and pyrones were
produced) [30]. Similarly in transformants of strain
2413 that overexpressed chit42 chitinase, bio-control
activity correlated with chitinase production except for
one transformant which was unable to completely
overgrow R. solani and did not produce - pyrone, so
that synergism did not occur [31].
Sequential roles of antibiosis and hydrolytic enzymes
during fungal interactions have been described by
Howell [29]. When combinations of antibiotics and
several kinds of hydrolytic enzymes were applied to
propagules of B. cinerea and F. oxysporum, synergism
occurred, but it was lower when the enzymes were
added after the antibiotics, indicating that cell-wall
degradation was needed to establish the interaction. In
tobacco plants, exogenous applications of peptaibols
trigger a defense response and reduce susceptibility to
tobacco mosaic virus [32]. A peptaibol synthetase from
T. virens has recently been purified, and the
corresponding gene, which has been cloned, will
facilitate studies of this compound and its contribution
to bio-control.
Marfori and his coworkers [33] found that the dual
culture of Trichoderma harzianum and Catharanthus
roseus callus produced an antimicrobial compound
trichosetin with a remarkable activity against the gram-

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The ability of Trichoderma strains to protect plants

against root pathogens has long been attributed to an
antagonistic effect against the invasive pathogen [16]
However, these root fungus associations also stimulate
plant defensive mechanisms. Strains of Trichoderma
added to the rhizosphere protect plants against
numerous classes of pathogens, those that produce
aerial infections, including viral, bacterial and fungal
pathogens, which points to the induction of resistance
mechanisms similar to the hypersensitive response
(HR), systemic acquired resistance (SAR), and induced
systemic resistance (ISR) in plants [2, 17]. At a
molecular level, resistance results in an increase in the
concentration of metabolites and enzymes related to
defensive mechanisms, such as the enzymes
phenylalanine ammonio lyase (PAL) and chalcone
synthase (CHS), involved in the biosynthesis of
phytoalexins (HR response), chitinases and glucanases.
These comprise pathogenesis related proteins (PR)
(SAR response) and enzymes involved in the response
to oxidative stress [35].
Plant genes respond to pathogens and elicitors. For this
reason, plant defense mechanisms do not necessarily
require stimulation by the living organism. The
addition of Trichoderma metabolites that may act as
elicitors of plant resistance, or the expression in
transgenic plants of genes whose products act as
elicitors, also results in the synthesis of phytoalexins,
PR proteins and other compounds, and in an increase in
resistance against several plant pathogens, including
fungi and bacteria [36, 37] as well as resistance to
hostile abiotic conditions (17). Barley expressing
Trichoderma atroviride endochitinase Ech42 showed
increased resistance towards Fusarium infection [20].
Expression of the chitinase Chit42 from T. harzianum in
tobacco and potato plants resulted in transgenic lines
highly tolerant or completely resistant to the foliar
pathogens Alternaria alternata, Alternaria solani and
Botrytis cinerea and to the soil borne pathogen
Rhizoctonia solani [29]. Similar results have been
obtained with the heterologous expression of Chit42 in
strawberry infected with Colletotrichum and with
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Chit42 and a -1, 6 glucanase in melon and tomato

plants. Thus, plant protection seems to result
exclusively from an increase in enzymatic activities.
Activation of defense responses using elicitors could be
a valuable strategy as an alternative to the use of
transgenic plants, to protect plants against pathogens.
Addition of laminarin, a -1,3 glucan, to grapevines
induced several defense genes and reduced infection by
B. cinerea and Plasmopara viticola [38]. Phytophthora
parasitica produces a cell-wall glycoprotein (CBEL) that
has a cellulose-binding domain (CBD) and elicits
defense responses in the host plant. Infiltration of CBEL
into non-host plants also induced defense reactions
[20]. Expression in plants of fungal chitinases with
CBDs, such as Chit42CBD, which already has increased
antifungal activity [31], may result in greater resistance
against phytopathogens. It would be of interest to test
the effect on plants of the addition of chitinases or
glucanases produced by Trichoderma strains with
respect to long-term increases in defense mechanisms,
that is, a SAR like response. Some authors have
described that, when such plants have been in the
presence of Trichoderma, their resistance persisted for
long periods, at least several months [17]. Plants can be
induced to develop enhanced resistance to pathogen

infection by treatment with a variety of biotic inducers.

Resistance induced by elicitors is broad spectrum and
long lasting, but rarely provides complete control of
infection, with many resistance elicitors providing
between 20 and 85% disease control [39]. A similar
effect could take place after the addition of
Trichoderma elicitors, which is particularly important
in protecting fruits against post-harvest infections.
Plant when exposed to pathogens (fungi, bacteria,
viruses or nematodes), it produces low molecular
weight antimicrobial compounds called phytoalexins,
antimicrobial peptides and small proteins (e.g.,
thionins, defensins, hevein like proteins and knottin
like peptides) and up regulate a number of
antimicrobial proteins [40, 41]. These plant proteins
called pathogenesis-related (PR) proteins. Bio-control
agents like Trichoderma produces some elicitors during
plant-pathogen-Trichoderma bio-agent interactions and
cause a plant to make a product like PR proteins. These
proteins discourage or kill the pathogen. This process is
called induced resistance. The plant actually fights
back. There are many of antifungal peptides and
proteins known, with more being discovered almost
daily. Some important families of PR proteins, found in
plants, are listed as Table 1.

Table 1. Recognized families of pathogenesis-related proteins in plants

Families
PR-1
PR-2
PR-3
PR-4
PR-5
PR-6
PR-7
PR-8
PR-9
PR-10
PR-11
PR-12
PR-13
PR-14
PR-15
PR-16
PR-17

Type member
Tobacco PR-1a
Tobacco PR-2
Tobacco P, Q
Tobacco 'R'
Tobacco S
Tomato Inhibitor I
Tomato P69
Cucumber chitinase
Tobacco 'lignin-forming peroxidase'
Parsley 'PR1'
Tobacco 'class V' chitinase
Radish Rs-AFP3
Arabidopsis THI2.1
Barley LTP4
Barley OxOa (germin)
Barley OxOLP
Tobacco PRp27

Properties
Antifungal
-1,3 glucanase
Chitinase type I,II, IV,V,VI,VII
Chitinase type I,II
Thaumatin-like
Proteinase inhibitor
Endoproteinase
Chitinase type III
Peroxidase
Ribonuclease-like
Chitinase, type I
Defensin
Thionin
Lipid-transfer protein
Oxalate oxidase
Oxalate oxidase-like
Unknown

MYCOPARASITISM
It is a process of direct attack of one fungus on another.
It is a very complex process that involves sequential
events, including recognition, attack and subsequent
penetration and killing of the host. Trichoderma sp. may
exert direct bio-control by parasitizing a range of fungi,
detecting other fungi and growing towards them. The
remote sensing is partially due to the sequential
expression of pathogenesis related proteins, mostly
chitinases, glucanases and proteases [17]. The pattern
of induction differs from one Trichoderma strain to
another. It is believed that fungi secrete exochitinases
constitutively at low levels. When chitinases degrade
fungal cell-walls, they release oligomers that induce
exochitinases, and attack begins.

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Gene symbol
Ypr1
Ypr2, [Gns2 ('Glb')]
Ypr3, Chia
Ypr4, Chid
Ypr5
Ypr6, Pis ('Pin')
Ypr7
Ypr8, Chib
Ypr9, Prx
Ypr10
Ypr11, Chic
Ypr12
Ypr13, Thi
Ypr14, Ltp
Ypr15
Yrp16
Yrp17

Reference
[76]
[76]
[77]
[77]
[77]
[78]
[79]
[80]
[81]
[82]
[83]
[41]
[40]
[84]
[85]
[86]
[87]

Morphological Changes
Mycoparasitism involves morphological changes, such
as coiling and formation of appressorium like
structures, which serve to penetrate the host and
contain high concentrations of osmotic solutes such as
glycerol [20]. Trichoderma attaches to the pathogen
with cell wall carbohydrates that bind to pathogen
lectins. Once Trichoderma is attached, it coils around
the pathogen and forms the appresoria. The following
step consists of the production of pathogenesis related
enzymes and peptaibols [29], which facilitate both the
entry of Trichoderma hyphae into the lumen of the
parasitized fungus and the assimilation of the cell wall
content. The significance of lytic enzymes [42] has been
demonstrated by over expression and deletion of the
respective genes.
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Investigation on the responsible signal transduction

pathways of T. atroviride during mycoparasitism have
led to the isolation of key components of the cAMP and
MAP kinase signaling pathways, such as -subunits of G
proteins (G-), which control extracellular enzyme,
antibiotic production and coiling around host hypha
[20]. In Trichoderma, there is biochemical evidence for
the participation of G- in coiling, since an increase in
coiling around nylon fibers was detected after the
addition of activators of G-protein (mastoparan and
fluoroaluminate) [43]. G- gene (tga1) has been
expressed either under the control of its own promoter
or under the control of the promoter of the basic
proteinase prb1 in T. atroviride [44]. Both types of
transformants showed an increase in coiling. Moreover,
the capacity of T. viride overexpressing tga1 to
overgrow Rhizoctonia was enhanced.
Cell wall Degrading Enzymes (CWDEs) : Cellulases,
Poly galacturonases (PG)
The cell wall of either bio-control agents - Trichoderma
or host plant is the first barrier encountered by most
plant pathogens, and thus it must be degraded to allow
their (pathogen) penetration and tissue colonization.
Necrotrophic fungal pathogens degrade the structural
polymers in plant host cell wall and colonized inter
cellular spaces facilitated by the production of extra
cellular cell wall degrading enzymes (CWDEs).
Pectinase and cellulase were breakdown into pectin
and cellulose, the two major polymers that maintain the
firmness and structure of host cell walls. Production of
CWDEs ofter determines the pathogenecity of
necrotrophic pathogen [45].
Among the different pectinolytic enzymes, poly
galacturonase (PG) is the most notable, since it
facilitates the penetration of primery cell walls and
onset the production of other isoenzymes involved in
pectin catabolism. Inhibition of CWDEs production
leads to the reduction in virulence of fungal pathogens
[46]. Several bacterial and fungal bio-control agents
were known to inhibit the in vitro production of fungal
pathogen CWDEs that correlated with their in vitro
efficacy. T. harzianum and Bacillus megaterium
inhibited the production of cutin esterase and different
pectinases which otherwise produced by R. solani. [47].
Inhibition of CWDEs of Botrytis cineres by T harzianum
on bean leaf surface leads to reduced disease incidence
[48]. However, Inhibition of fungal CWDEs production
by the secondary metabolites of the bio-control agents
in the infection courts is one of the important
mechanisms that need detailed investigations. Kishore
and his group [49] found that cell free culture filtrate of
Pseudomonas aeruginosa GSE 18 at dilutions
inhibited the in vitro production of A. niger CWDEs cellulase and PG. This mechanism might have a
significant role in control of collar rot disease by
reducing the virulence of A. niger in the groundnut
rhizosphere.

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Thus, these groups of enzymes are generally produced

by pathogenic fungi to degrade cell wall of host or biocongtrol agents. However, in some cases, cellulases may
also produced by Trichoderma to inhibit the growth of
pathogenic fungi. Trichoderma sp. are known to
produce cellulases [50] which hydrolyse -1,4 glucans.
In accordance, all three Trichoderma harzianum
isolates (39.1, 1051, TVC) were found to produce
substantial amounts of these enzymes [51].
Cellulases(-1,4glucanases),
comprising
cellobiohydrolases, endoglucanases (egl1, egl2) and glucosidases, have not been widely studied for biocontrol purposes, although cellulose is abundant in
oomycetes [52]. Howell [29] obtained transformants
with greater bio-control activity than the wild type
against Pithium ultimun on cucumber seedling. T.
harzianum T3 produces a variety of cellulases, which
make this isolate very effective in the control of P.
ultimun.
Pathogenesis Related (PR) Protein/ Enzyme:
Chitinases
Two isolates of Trichoderma harzianum (39.1 and
1051), which reduce the incidence of witchesbroom
disease caused in cocoa by Crinipellis perniciosa, were
evaluated for their potential to produce chitinolytic
activity in liquid medium [51]. Both Trichoderma
isolates produced and secreted on induction substantial
amounts of chitinolytic enzymes. The chitinase
increased within 72 h. Maximal activity (0.39 U/ml)
was produced by isolate 1051. This activity was 13-fold
higher than those determined for the 39.1 (0.013 U/ml)
strains.
Kucuk and Kivank [53] found that all filtrates of
Trichoderma harzianum T9, T10, T15 and T19 were
effective against plant pathogens Fusarium culmarum,
F. oxysporum, F. moniliforme, Rhizoctonia solani,
Sclerotium rolfsii, Gaeumannomyces graminis var. tritici
and Drechslera sorokiniana. Among these isolates, T.
harzianum T19 showed a wide range of inhibitory
effects on plant pathogens. F. oxysporum was found to
be the most resistant to the filtrates of the strains
above. All isolates showed different behaviors
depending on the physiological tests carried out such as
growth in the presence inhibitory substrates, pH limits
of growth and hydrolysis of gelatin. The chitinase
activity determined from T. harzianum T15 by SDSPAGE was nearly 73 kDa.
Transformants of the bio-control agent Trichoderma
harzianum strain CECT 2413 that overexpressed a 33
kDa chitinase (chit33) were obtained and characterized
[54]. Strain CECT 2413 was cotransformed with the
amdS gene and its own chit33 gene under the control of
pki constitutive promoter from T. reesei. Southern
blotting indicated that the chit33 gene was integrated
ectopically, mostly in tandem. Some transformants
showed the same restriction pattern, indicating
preferable sites of integration. There was no
correlation between the number of integrated copies
and the level of expression of the chit33 gene in the
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transformants. The transformants were more effective

in inhibiting the growth of Rhizoctonia solani as
compared with the wild type.

to test the role of these chitinases in mycoparasitism,

and the 42-kDa chitinase is believed to be a key enzyme
[29].

Regulatuion of the expression of the two major

chitinase genes ech42 and nag1 of the chitinolytic
system of the mycoparasitic bio-control fungus
Trichoderma atriviride (Trichoderma harzianum P1)
was investigated by using reporter system based on the
Aspergillus niger glucose oxidase [55]. nag1 gene
expression was triggered during growth on fungal
(Botrytis cinerea) cell walls and on the chitin
degradation
product
N-acetylglucosamine.
Nacetylglucosamine, di- N-acetylchitobiose or tri- Nacetylchitotriose also induced nag1 gene expression
when added to mycelia pregrown on different carbon
sources. ech42 expression was also observed during
growth of fungal cell walls.

T. virens transformants over expressing Chit42 showed

significantly enhanced bio-control activity compared
with the wild type when assayed against R. solani in
cotton seedlings experiments [29]. Other Trichoderma
sp. transformants over expressing chit42 resulted in
better antagonism than obtained with the wild type
[31, 57]. In greenhouse bio-control tests, however, the
activity of chit42 disruptants did not differ from that of
the wild type [17, 57].

The chitinolytic system of Trichoderma comprises

many enzymes and the list of its components is rapidly
being updated as new enzymes and genes are reported.
Chitinases
are
divided
into
-1,
4
acetylglucosaminidases (GlcNAcases), endochitinases
and exochitinases. Many GlcNAcases and their genesexc1 (nag1), exc2, tvnag1, and tvnag2 from T.
harzianum T25-1, T. atroviride P1 and T. virens Tv29-8
have been described [17, 56]. The 73-kDa Nag1
represents the main GlcNAcase in T. atroviride. Nag1disruption strain lacks chitinase activity, and the
endochitinase chit42 mRNA is absent [17]. This
indicates that nag1 is essential for triggering chitinase
gene expression. The pathogen cell-wall and chitin
induce nag1, but it is only triggered when there is
contact with the pathogen [17, 29, 55, 57].
GlcNAcases chit73 and chit102 were detected in T.
harzianum TM and Trichoderma asperellum [58].
Chit102 triggers the expression of other chitinolytic
enzymes. In addition, strain 2413 produces three extra
cellular endochitinases whose genes, chit33, chit37 and
chit42, have been cloned from this strain. Other genes
coding for Chit42 chitinase - ech42, cht42 and ThEn4
have also been cloned from T. atroviride IMI206040
[57], Gv2908 [29] and P1 [29], respectively. Chit37
shows 89% similarity to Chit36 from T. harzianum TM
at the amino-acid level (17). Chit36 inhibits B. cinerea
spore germination and the growth of both Sclerotium
rolfsii and Fusarium oxysporum [59].

T. harzianum transformants over expressing Chit33

chitinase constitutively inhibited the growth of R. solani
under both repressing and derepressing conditions; the
antagonist tests demonstrated that this chitinase also
has an important role in mycoparasitism [54]. T.
harzianum Rifai TM transformants overexpressing
Chit36 chitinase inhibited F. oxysporum and S. rolfsii
more strongly than the wild type. Moreover, culture
filtrates inhibited the germination of B. cinerea almost
completely [59]. The antagonism of Chit33 and Chit42
transformants has been improved by the addition of a
cellulose-binding domain to the chitinase genes. As a
result, the strains producing the chimeric enzymes
increased their specific chitinase activity [31].
Pathogenesis Related (PR) Protein/ Enzyme: -1,3
Glucanases
It has been shown that -1, 3 glucanases inhibit spore
germination or the growth of pathogens in synergistic
cooperation with chitinases [61, 62, 63] and antibiotics
[17, 56]. Many -1,3-glucanases have been isolated, but
only a few genes have been cloned, e.g. bgn13.1 [61]
and lam1.3 [64] from T. harzianum, and Tv-bgn1 and
Tv-bgn2 [56] from T. virens. However, only strains over
expressing bgn13.1 from T. harzianum have been
constructed. Marco and his coworkers [51] found
glucanolytic enzyme activity maximum in Trichodrma
isolates during 72 to 120 h growth in presence of
specific substrate.

Other genes homologous to chit36 have been cloned

from T. harzianum TM, T. atroviride P1 and T.
asperellum T203. Endochitinases are regulated by a
variety of mechanisms but induction by stress has been
reported for chit33, chit36 and chit42. However, the
induction under mycoparasitic conditions is not clear.
Ech42 is induced prior to any physical contact with R.
solani [60]. Chit33 is expressed only during the contact
phase and not before overgrowing R. solani [36]; and
chit36Y does not need the direct contact of the
pathogen to be expressed. Chit33, chit42 and chit36
have been over expressed in Trichoderma spp. in order

Transformants over expressing bgn13.1 have been

reported to inhibit the growth of B. cinerea, R. solani
and Phytophthora citrophthora. Transformant T28,
which had the highest bgn13.1 glucanase activity under
both repressing and inducing conditions, showed the
highest inhibition of pathogens. Antagonism was higher
against P. citrophthora oomycete with cellulose and
glucans as the main cell-wall components [61] than
against Botrytis or Rhizoctonia [65], which have chitin
and glucan as their main cell-wall components [61]. In
addition, three -1,6-glucanases have been purified
from strain 2413 [37, 61]. bgn 16.2 exhibited antifungal
properties alone or in combination with chitinases [61]
and reduced the growth of B. cinerea and Gibberella
fujikuroi [66]. Transformants producing bgn 16.2
controlled R. solani and B. cinerea growth. Other
hydrolases, such as -1,3 glucanases, have been
purified from strain 2413, and their genes isolated and

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Harsukh Gajera et al. / Curr Res Microbiol Biotechnol. 2013, 1(4): 133-142

overexpressed, which resulted in increased bio-control

activity of the transformant strains [67].
Pathogenesis Related (PR) Protein/ Enzyme:
Proteases
Filamentous fungal cell wall contains lipids and
proteins [68]. It therefore expected that antagonistic
fungi synthesize proteases which may act on the host
cell-wall. The Trichoderma harzianum isolates 1051
and TVC secreted equivalent amounts (about 1.41
U/mL) of proteolytic activity after 72 h of growth.
These activities were significantly higher than that
(0.86 U/mL) produced by T. harzianum strain 39.1
under the same conditions [51].
Bio-control of B. cinerea by T. harzianum has been
attributed in part to the action of proteases produced
by the bio-control agent that inactivate hydrolytic
enzymes produced by this pathogen on bean leaves
[29]. Proteases involved in the degradation of
heterologously produced proteins have been
characterized by Delgado-Jarana et al. [69]. For
example, alkaline protease Prb1 from T. harzianum IMI
206040 has been demonstrated to play an important
role in biological control [61], and prb1 transformants
showed an increase of up to five-fold in the bio-control
efficiency of Trichoderma strains against R. solani.
Protease pra1 from T. harzianum has affinity for fungal
cell-walls [37]. The gene for an extracellular serine
protease (tvsp1) has been cloned from T. virens [70]

and its over expression significantly increased

protection of cotton seedlings against R. solani. This
gene shows great potential in improving bio-control
ability, as serine proteases are effective against
oomycetes [71] and nematodes [29, 72].
Antal et al. [73] screened cold tolerant strains and
found that most of them antagonized phytopathogens
and produced chitinases, -glucosidases and trypsinlike, and chymotrypsin-like proteases, active at low
temperatures. The role of proteases in mycoparasitism
has been reinforced with the isolation of new proteaseoverproducing strains of T. harzianum [74]. Mutants
with new profiles and higher quantities of secreted
proteases were obtained by UV-irradiation. Some of
these mutants have proved to be effective antagonists
against plant pathogenic fungi such as Fusarium
culmurum and R. solani.
Zaldivar and his coworkers [75] found that T.
aureoviride 7-121 strain enhanced in vitro production
of various enzymes cellulases, -1,3 glucanases,
chitinases and proteases. This improvement in
extarcellular enzyme production by the mutant T.
aureoviride 7-121 suggests that it is a suitable strain to
be used for waste cellulose degradation and in
biological control. Lytic enzymes from Trichoderma sp.
in the bio-control of fungal plant pathogens are
summarized in Table 2.

Table 2. Details of lytic enzymes from Trichoderma harzianum that have potential bio-control capability.
S. No.

Gene

MW
kDa

Chitinases
1.
102
2.
73
3.
exc2
73
4.
Excl/nag1
64-69
5.
28
6.
52
7.
ech42
42
8.
chit44
44
9.
cht42
42
10.
ThEn42
42
11.
40
12.
37
13.
chit36
36
14.
chit33
33
15.
31
Glucanases
1.
bgn13.1
78
2.
74
3.
36
4.
17
5.
b16.2
43
6.
lam1.3
110
7.
75
8.
eg11
50
Proteases
1.
prb1
31
a = T. atroviride, b = T. longibrachiatum

Activity

Strain

References

N-acetylgluco-saminidase
N-acetyl glucosaminidase
N-acetylgluco-saminidase
endochitinase
endochitinase
chitobiosidase
endochitinase
endochitinase
endochitinase
endochitinase

TM-39-1
TM-25-1
TM
TM-25-1, PIa
T-189
TM-TY
IMI206040a
CECT2413
GV2908
OIa
PIa
CECT2413, 109
TM
CECT2413
TM, TY

[88]
[88]
[89]
[90]
[91]
[58]
[92]
[93]
[94]
[95]
[17]
[96]
[59]
[96]
[58]

-1,3 endoglucanase
-1,3 endoglucanase
-1,3 endoglucanase
-1,3 endoglucanase
-1,3 endoglucanase
-1,3 endoglucanase
-1,3 endoglucanase

PIa, CECT2413
T24
39.1
CECT2413
CECT2413
T.Y
RUT C30b

[97]
[62]
[51]
[98]
[99]
[64]
[100]
[101]

Alkaline protease

IMI206040a

[102]

SYNERGISM
Synergism among lytic enzymes and between enzymes
and antibiotics suggests formulations to test mixtures

of Trichoderma transformants that produce different

enzymes, in order to improve the antagonistic effects of
bio-control agents on phytopathogenic fungi. T.

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harzianum wild type inhibited the growth rate of B.

cinerea by 30% and transformants expressing either a
-1,3 glucanase, a chitinase, or a -1,6-glucanase
inhibited the growth rate of B. cinerea by 60%.
Transformants were differently combined in order to
test synergism among the enzymes secreted against
several phytopathogens. The combination that
overproduced chitinase and -1,3-glucanase was more
effective than the individual transformants in inhibiting
Rhizoctonia meloni, whereas using other combinations,
the inhibition was not improved [65].
CONCLUDING REMARKS
Optimal application of bio-control agents is possible
only when the operating mechanisms are fully
understood. Research on the mechanisms responsible
for the bio-control exerted by Trichoderma sp. on
phytopathogenic fungi have led to a better
understanding of such mechanisms These tools will
allow the isolation of improved strains and thus of
more efficient formulations to control fungal pathogens
in pre and post harvest periods. The studies of
mycoparasitism also have demonstrated that
Trichoderma produce a rich mixture of antifungal
enzymes, including chitinases and -1,3 glucanases.
These enzymes are synergistic with each other, with
other antifungal enzymes, and with other metabolites.
The genes encoding the enzymes appear useful for
producing transgenic plants resistant to diseases and
the enzymes themselves are beneficial for biological
control and other processes.

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