International Journal of Agriculture and Crop Sciences.

Available online at www.ijagcs.com

IJACS/2013/6-18/1246-1252
ISSN 2227-670X ©2013 IJACS Journal

Biocontrol ability of Trichoderma

Shahzad Munir1, Qaiser Jamal1, Kalsoom Bano2, Sikandar Khan Sherwani3, Tasveer
Zahra Bokhari4, Tariq Ahmed Khan5,RashidAzim Khan1, Abdul Jabbar1, Muhammad
Anees1
1. Department of Microbiology, Kohat University of Science and Technology, Kohat, Khyber Pakhtunkhwa,
Pakistan
2. Department of Microbiology, Quaid-i-Azam University Islamabad, Pakistan
3. Department of Microbiology, Federal Urdu University of Arts, Science and Technology, Karachi, Pakistan
4. Institute of Pure and Applied Biology, BZU- Multan
5. Attock Refinery, Islamabad, Pakistan

Corresponding Author email: shazid_10@yahoo.com

ABSTRACT: In the world, the traditional agricultural practices are affected by various problems such as
diseases, pests, drought, decreased soil fertility; certain alternative plant diseases control methods and
due to use of hazardous chemical pesticides, pollution and global warming. There should be biological
controls that have the ability to solve these problems. Fungi and bacteria are the various types of
biological control agents involved in biocontrol activity. Among them, fungi assigned to genus
Trichoderma plays a major role in controlling the fungal plant diseases. The different Trichoderma spp.
are known to produce diverse kind of enzymes which have a significant role in biocontrol activity like cell
wall degradation, hyphal growth, and antagonistic activity against plant pathogens.Trichoderma spp. are
free-living cosmopolitan species interacting with roots, soil and foliar environments. Trichoderma species
are rapidly growing, good competitors competing with other microorganisms. Furthermore, they inhibit or
degrade pectinases and other enzymes that are essential for plant-pathogenic fungi. The present review
is focused on the biocontrol ability of the Trichoderma species.
Keywords:Trichoderma, Biocontrol, Phytopathogens

Trichoderma
Persoon was the first who described the genus Trichodermain the year 1794. Persoon reported three
species, out of which, only one was accepted. However, Persoon’s classification was doubtful and contradictory
(Rifai, 1969). Rifai (1969) made the first real attempt. He produces a feasible classification system of the genus
Trichoderma. The system was based on species morphology as well as on the concept of species aggregates. He
defined nine aggregate species of Trichoderma using his classification system. The most detailed morphological
studies of genus Trichoderma based on key morphological characteristics carried out by Bissett (1984, 1991). In
his studies, he identified four sections of Trichoderma (Trichoderma, Pachybasium, Longibrachiatum, and
Hypocreanum). Lieckfeldtet al., (1998) also mentioned the use of molecular techniques for identifying the species.
They stated that morphological characteristics for identifying Trichodermaspecies, is being progressively replaced
by molecular tools, which provide a more reliable form of species identification. Samuels (1996) performed
advance study of the genus Trichoderma. He developed molecular methodologies based techniques for
classification of the genus, which greatly improved understanding of the genus especially at species level.
Druzhininaet al., (2005) described the study of species within the genus Trichoderma on by the use of molecular
tools. They described the development of an oligonucleotide barcode important approach to the identification of
Hypocrea/ Trichodermaspecies known as TrichOKEY. The respective application software is available at
www.isth.info.Harman et al., (2004) conducted a research about specie identification of Trichoderma on both
morphological and molecular basis and they reported 75 identified species of Trichoderma.

Morphology ofTrichoderma
The morphology of Trichoderma was first time described by Rifai (1969).Trichoderma is a septate fungus
and produces highly branched conidiophores with a conical or pyramidal outline. He further described that
Trichodermaspecies form floccose or tufted colonies of various colors (white, yellow, green), which can be used to
Intl J Agri Crop Sci. Vol., 6 (18), 1246-1252, 2013

identify and differentiate about various species of the genus. Gams and Bissett (1998) conducted their research on
morphology of Trichoderma. They described that Trichoderma species are characterized by rapid growth, mostly
bright green conidia and a repetitively branched conidiophore structure. Species of the genus produce a broad
array of pigments from bright greenish-yellow to reddish in color, although some are also colorless. Similarly,
conidial pigmentation varies from colorless to various green shades and sometimes also gray or brown. Other than
pigmentation, species identification within the genus is difficult because of the narrow range of variation of the
simplified morphology in Trichoderma.

Ecology ofTrichoderma
Trichodermaspp. comprises a fast-growing group that appears to be extremely common in agricultural
lands, salt marshes, desert soils and almost all climatic zones. They further stated that saprophytic nature of
Trichodermameans that the fungi are commonly present in the soil’s top horizons (Danielson and Davey 1973
a)Trichodermaspp. can be detected in soils by smell, the coconut odor associated with certain of them being due to
the volatile 6-pentyl-a-pyrone (Kikuchi et al., 1974 and Collins and Halim, 1972). In 2010 Schmoll et al. conducted
his latest research on ecology and habitat of Trichoderma species. They declared Trichoderma species as highly
successful colonizers of their habitats, which is reflected both by their efficient utilization of the substrate at hand as
well as their secretion capacity for antibiotic metabolites and enzymes. Under all these conditions, they respond to
their environment by regulation of growth, conidiation, enzyme production, and hence adjust their lifestyle to current
conditions, which can be exploited for the benefit of mankind.

Morphological Characterization ofTrichoderma

The genus Trichodermacan be identified when grown on selective media (Gams and Bissett 1998).
Colonies have key characteristics that can be used to identify them as Trichodermaincluding growth pattern, growth
speed, odor and color. Bissett (1991a);Rifai (1969); Samuels (1996) stated that this morphology based technique is
not very accurate especially if identification to species level is required. They further stated that the most common
approach to determine individual species is to use the light microscope to study morphological characters such as
the branching pattern of conidiophore, the conidiophore apex elongation and shape (coiled, straight or undulate),
the phialides shape, structure and size and the conidial shape. Samuels (1996) mentioned limitations of light
microscopy, because the boundaries of the Trichodermagenus are ill defined and the existence of both anamorph
and teleomorph stages only increase the complexity of Trichoderma species identification. Some species of
Trichoderma can show great variation in its morphology that can easily lead to misidentification and most people do
not have the experience required to accurately identify Trichodermaspecies. Morphological key software is
available at www.isth.info. (Hagnet al., 2002)

Molecular Characterization of Trichoderma

The concept of analysis of isozymes (protein markers) by electrophoresis is frequently used to estimate
genetic variation between Trichoderma isolates and therefore, provides identification at the intraspecies level was
introduced by Zamir& Chet in 1985.Wuczkowski et al. (2003) investigated the occurrence and genetic diversity of
Trichoderma, forty-six strains at the species level by analysis of morphological characters, by sequence analysis of
their internal transcribed pacer regions 1 and 2 (ITS1 and 2) of the rDNA cluster and in some cases, a fragment of
the translation elongation factor 1alpha (tef1) gene, and RAPD-analysis.
The application of reverse dot-blot method for rapid identification of Trichodermaspecies from soil samples
was showed by Zhou et al., 2001. The principle of this method is based on the binding of specific poly-dT tailed
oligonucleotides onto a nylon membrane strip. Lubecket al., (2004) described UP-PCR cross-blot hybridization and
reverse dot blot for identification species with in T. viride/atroviride/koningiicomplex. A representative 64 isolates
were verified at the species level by oligonucleotide barcode programTrichoKey v.1.0 and the custom BLAST
server TrichoBLAST, using sequence of the ITS1 and 2 region of the rRNA cluster from longest intron of tef1 gene.
As in previous studies, T. harzianumaccounted for almost half of the diversity; although, in this study, it was
exclusively found in the North, and was predominantly represented by an ITS1 and 2 haplotype, which has so far
been rarely found elsewhere (Zhang et al.2005). Kubicek et al. (2003) used isolates of Trichoderma spp. to assess
the genetic and metabolic diversity of endemic species of Trichoderma. Ninety-six strains were isolated in total, and
identified at the species level analysis of morphological and biochemical characters (Biolog system), and
sequences analysis of their internal transcribed spacer regions 1 and 2 (ITS1 and 2) of the rDNA cluster, using ex-
type strains and taxonomically established isolates of Trichoderma as reference. The data indicate that the
Trichodermaharzianum/ T. hamatumgroup represents species with high metabolic diversity and partially unique
metabolic characteristics.

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Sequence data, obtained from the ITS1 region of rDNA and a fragment of the translation elongation factor
1 (tef1) gene, and were used in a phylogenetic analysis. (Hermosa et al.2004).Phylograms showing similar
topologies were generated using alignments containing the ITS1 region or a portion of the tef1 gene. 21 distinct
ITS1 sequence type and 17 distinct tef1 type were identified among the 69 isolates. More than 50% of the potential
biocontrol strains were grouped within Trichoderma sect. pachybasium; of these, 81% were grouped within the
cluster that included the ex-type strains of T. harzianumand T. hamatum, and 16% were grouped with T. virens.
Within T.sect.Trichoderma, included 36% of the 69 strains, 56% were grouped with T. asperellum, and 24% with
T.viride, T. atrovirideor T. koningii. Only 10% of the strains studied were located in T. sect. longibrachiatum.
Trichoderma from rice fields in four provinces of Philippines using rDNA-ITS1 analysis and universally primed
polymerase chain reaction (UP-PCR) were characterized by (Cumagun et al. 2000). Two groups were clearly
distinguishable in the basis of length and restriction pattern of the internal transcribed spacer (ITS) region of the
ribosomal DNA and UP-PCR banding profile using UP primer, L45. The isolates comprising the largest group were
very similar with respect to their UP-PCR banding profiles and were assigned to T. harzianumrifai following
morphological identification of four of the isolates.
Molecular characterization of T. virideand T.harzianum based on rDNA Markers and analysis of their PCR-
RAPD profiles were did by B.N. Chakraborty et al., in 2010.Nineteen isolates of Trichodermavirideand
Trichodermaharzianumobtained from rhizosphere soil of plantation crops, forest soil and agricultural fields of North
Bengal region were studied using RAPD and ITS-PCR. The genetic relatedness among eleven isolates of T.
virideand eight isolates of T. harzianum were analyzed with six random primers. RAPD profiles showed genetic
diversity among the isolates with the formation of eight clusters. Analysis of dendrogram revealed that similarity
coefficient ranged from 0.67 to 0.95. ITS-PCR of rDNA region with ITS1 and ITS4 primers produced 600bp
products in all isolates. This result indicated the identification patterns of Trichoderma isolates.

Biodiversity ofTrichoderma
Kullning et al. (2000) and Kubicek et al. (2002), assessing Trichoderma biodiversity of specific geographic
areas, where the most common species was T. harzianum, in contrast in neotropical areas studied by Hoyos-
Carvajal et al. (2009a) was T.asperellum(33% of strains) and T. harzianumthe second most common (27%).
Genetic variation was evident for both species, and two (T. asperellum) or three (T. harzianum) distinct genotypes
were evident in the analysis of tefsequences and metabolic profiles. T.asperellum, which is often isolated from
tropical regions (Druzhinina et al., 2006) could be divided into two groups (A and B), which more recently have
been described as separate species, T. asperellumand T. asperelloidesrespectively (Samuels et al., 2006).
T.asperellumincludes isolates from Brazil, Peru and Colombia originating in soils with poorly degraded materials
such as fallen leaves or crop residuals in colder climate zones. T. asperelloidesincludes strains collected in
Colombia and Ecuador that exhibit a preference for soils and substrates with high organic content, and often
adapted to the rhizosphere of crops in Andean zones. These two species could not be differentiated by
morphological characters or by growth rates, suggesting the development of ecologically or geographically isolated
lineages as has been reported for T. harzianum(Chaverri et al., 2003) and T. koningii(Samuels et al., 2006).
Ninety-six strains of Trichoderma were isolated in total, and identified at the species level by analysis of
morphological and biochemical characters (Biolog system), and by sequence analysis of their internal transcribed
spacer regions 1 and 2 (ITS1 and 2) of the rDNA cluster, using ex-type strains and taxonomically established
isolates of Trichoderma as reference (Kubicek CP., et al 2003). Seventy-eight isolates were positively identified as
T. harzianum/T.inhamatum (37 strains) T. virens(16 strains), T.spirale (8 strains), T. koningii (3 strains), T.atroviride
(3 strains), T. asperellum (4 strains), Hypocreajecorina(anamorph: T. reesei; 2 strains), T.viride (2 strains),
T.hamatum (1 strain), and T.ghanense (1 strain). Analysis of biochemical characters revealed that T. virens, T.
spirale, T. asperellum, T. koningii, H. jecorina, and T. ghanense formed clearly defined clusters, thus exhibiting
species-specific metabolic properties. In biochemical character analysis T. atrovirideand T. viride formed partially
overlapping clusters, indicating that these two species may share overlapping metabolic characteristics. This
behavior was even more striking with T. harzianum/T. inhamatum where genotypes defined on the basis of ITS1
and 2 sequences overlapped significantly with adjacent genotypes in the biochemical character analysis, and four
strains from the same location (Bali, Indonesia) even clustered with species from section Longibrachiatum. The
data indicate that the T. harzianum/T.inhamatum group represents species with high metabolic diversity and
partially unique metabolic characteristics. Nineteen strains yielded three different ITS1/2 sequence types which
were not align able with any known species. They were also uniquely characterized by morphological and
biochemical characters and therefore represents three new taxa of Trichoderma. Sadfi-Zouaoui N., et al (2009)
explained the biodiversity of Trichodermastrains identified at the species level by analysis of their internal
transcribed spacers regions 1 and 2 (ITS1 and ITS2) of the rDNA cluster and (or) a fragment of the translation

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elongation factor 1 (tef1) gene, using an online interactive key for species identification in Trichoderma and ex-type
strains and taxonomically established isolates of Trichoderma as references. At least 2 different species were
observed in each ecosystem. Trichodermaharzianumclade VI and Trichodermalongibrachiatum were present in
forest soils in north Tunisia; Trichodermaatroviride and Trichodermahamatum were found in cultivated fields in
northeast Tunisia; T. harzianum clade VI, a Trichoderma spp. close to the T. harzianum complex, and
Trichodermasaturnisporum were isolated from forest soils in central Tunisia; and T. harzianum clade II and T.
hamatum were present in oasis soils in south Tunisia.

Trichodermaas Plant Growth Enhancer

The species belonging to the genus Trichodermaare avirulent plant symbionts. They can induce localized
or systematic resistance to diseases and their causative pathogens through the release of metabolites. They
further described that the mechanisms involved in induced plant resistance are still poorly understood and to
exploit such applications of Trichoderma, more research is needed (Harman et al., 2004) Ruoccoet al., in 2007
conducted a study about hydrophobins, a class of small cystein-rich proteins that are expressed only by fungi. They
described that one effect that many Trichodermastrains induce is enhanced root development is the hydrophobin
the necessary triggering molecule for this reaction. They further described that a strain of Trichoderma, the T22
hydrophobin induces resistance as well as rooting. Marraet al., (2006) mentioned that numerous benefits of these
Trichoderma species on plant growth and resistance are due changes in the physiology of plants. Trichoderma,
after establishing association with plant, modifies plant’s physiology and introduces such changes which enhances
growth of plant and make it resistant to numerous diseases and disease causing pathogens.

Trichodermaas Biocontrol Agent

Once infection occurs, a zone of chemical interaction develops at the sites where Trichoderma hyphae
interact with host’s hyphae. Within this zone of chemical interaction, the Trichoderma hyphae are walled off by the
plant but are not killed (Harman et al., 2004aHarman &Shoresh 2007).Dix & Webster (1995) defined
mycoparasitism as the direct attack of one fungus on another and can be generally described as direct antagonism.
Chet et al., (1981);Papavizas (1985) described that Trichoderma hyphae identifies and recognize the cell surface of
the pathogen. Then Trichoderma hyphae surround its host and simply grow along the host’s hyphae.
Trichoderma species secrete of specific lytic enzymes which degrade the host cell wall. They further stated
that the main lytic enzymes involved in the degradation of the host cell wall are β-glucanase, chitinase and
proteinases (Chet et al., 1998) Bailey et al., in 2006 reported some strains are endophytes, i.e., they colonize not
only roots, but also the above-ground parts of plants. These are being investigated for control of tropical tree
diseases, such as those affecting cacao. They further mentioned that endophytes provide another aspect for
biocontrol systems using Trichoderma species with additional opportunities, but it is not known at this time whether
endophytic and root-colonizing abilities are the same or separate phenomena. Defense mechanisms of
Trichoderma comprise both enzymatic and chemical weapons, which make Trichoderma species efficient
mycoparasites, antagonists, and biocontrol agents, possessing characteristics that can be exploited by using
Trichoderma or the metabolites secreted by these fungi as biological fungicides to fight plant diseases caused by
pathogenic fungi ( Vinale et al., 2009)
Benitez et al. (2004) mentioned Trichoderma species used for the purpose of biological control. According
to them, after publication of Trichodermalignorum (later found to be T. atroviride) acting as a parasite on other fungi
in 1932 by Weindling, lots of work was done to identify and characterize biologically efficient strains and species of
Trichoderma. Nowadays, the most important species in this field are T. atroviride (in earlier reports sometimes
misidentified as T. harzianum), T. harzianum, T. virens, and Trichodermaasperellum. The genus Trichoderma
comprises a great number of fungl strains that act as biological control agent, the antagonistic properties of which
are based on the activation of multiple mechanisms (Benitezet al. 2004).Trichoderma species act like guards for
their host. The response of Trichoderma to its host involves stress response, response to nitrogen shortage, cross
pathway control, lipid metabolism, and signaling processes. Hence, Trichoderma fulfill all the needs of its host,
playing a vital role in its growth and protection from pathogenic invaders (Seidl et al., 2009b)
Mukherjee and Kenerley (2010) conducted a study to exploit the biocontrol mechanism of T. virens. They
described that an important role in biocontrol of T. virens has been reported for the homolog of the VELVET
proteins, which so far mainly, are known as light-dependent regulatory proteins. Shoresh et al., (2010) described
the beneficial action of Trichoderma species. According to them, the association or useful role of Trichoderma
species is not limited to fighting pathogens only; they have also been shown to be opportunistic plant symbionts,
enhancing systemic resistance of plants and making them able to resist against numerous pathogenic invaders.

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Like many other fungi, Trichoderma species have also been shown to produce a broad array of volatile
organic compounds mentioned a completely new perspective of Trichoderma species. These compounds recently
have received closer attention. Further studies are in progress to know the exact nature and role of these organic
compounds (Stoppacher et al., 2010)

Role ofTrichodermaChitinase Enzymes in Biocontrol

Major cause of biocontrol activity of Trichoderma is concerned with production of chitinases to disintegrate
the cell wall of fungal phytopathogens (Anand and Reddy 2009). Matroudi et al. (2009) tested 30 Trichoderma
isolates and on the basis of maximum level of chitinase and indicated that T. atroviride can be employed in the field
as biological control agents against Sclerotiniasclerotiorum.Woo and Lorito (2007)studied that various species of
Trichoderma belongs to soil microbes that have capability to antagonize the plant pathogens. These biocontrol
species are known to produce different kinds of Cell Wall Degrading Enzymes (CWDEs), hundreds of antibiotic and
lot of bioactive compounds which are still uncharacterized.
Trichoderma spp. produces plentiful biologically active compounds; include CWDEs and the secondary
metabolites that help in reduction of drastic effects of the phytopathogens and also promote positive responses by
the plant (Francesco et al. 2007). These fungi produce mixture of enzymes including β-1, 3 glucanases and
chitinases which are antifungal in nature (Table 1). Both of antifungal enzymes cooperate with each other during
antagonistic activity and also with other related enzymes. The transgenic plant resistant to disease can be
produced from the genes encoding these enzymes and these antifungal enzymes themselves have a role in
biological control (Harman, 2006).Limon et al. in 2004 detected that Trichodermaharzianum is a commonly
dispersed antagonistic fungus generally correlates with the antifungal enzyme chitinases that degrade the fungal
cell walls. The chitinase enzymes produced by T. harzianum have important role in antagonistic activity and
indicated that chitinolytic enzymes results in hydrolysis of chitin containing fungal cell wall.
Marco et al. (2003) found that growth of the plant pathogen Crinipellisperniciosa impaired by the chitinase
enzymes of Trichodermaharzianum.Thrane et al. (2000) studied the two antagonistic Trichodermaspp P1 and T3
that produced different kind of lytic enzymes in liquid culture. The combination of the two fungi resulted less activity
than each of the isolate tested alone and there were no significant differences between these two isolates in their
ability to protect cucumber seedlings against P. ultimum.
The fresh and dried mycelium of the plant pathogenic fungus S. rolfsii was easily hydrolyzed by
thechitinase and β-1, 3-glucanase present in culture filtrate of T. harzianum and these enzymes reduced the
growth of S. rolfsii. (Katatny et al. 2000)

Use of Trichodermain Industry

Kumar et al., (2008) conducted a study about the use of T. reesei as biofuel. According to them as a potent
cellulase producer, research with T. reesei is nowadays particularly focused on improvement of efficiency of the
enzyme produced in order to decrease overall costs of production of bioethanol from cellulosic waste material.
Nevalainen et al., (2005) described that industrial use of filamentous fungi as a producer of heterologous proteins
started more than 20 years ago. Nowadays, T. reesei is one of the most commonly used filamentous fungi for
heterologous protein production.
Trichoderma species are extensively known for their safe industrial scale enzyme production for a longer
period of time, Trichoderma spp. have also been successfully applied for production of food additives and related
food products, possessing high nutritional quality and comparatively less cost (Nevalainen et al., 1994; Blumenthal
2004). Oda et al., in 2009 mentioned that not only enzymes but also metabolites of Trichoderma species are used
as additives. One of the products isolated from T. viride is a chemical with characteristic coconut-like aroma, a 6-
pentyl-α-pyrone with antibiotic properties, the production of which was constantly improved to reach concentrations
of more than 7 g/L in extractive fermentation cultures in T. atroviride nowadays. Wiater et al., (2005) introduced a
new perspective regarding the use of T. harzianum in industry. They described that T. harzianummutanase can be
used in toothpaste to prevent accumulation of mutan in dental plaque.

CONCLUSIONS

Biocontrol of plant pathogens provides an alternative means of reducing the incidence of plant disease
avoiding the negative aspects of chemical controls like pesticides. Trichoderma species are among the most
studied biocontrol agents. Most of the researches on mechanisms which are responsible for the biocontrol activity
of Trichodermaspp. against plant pathogenic microorganisms have led to the purification of cell wall degrading
enzymes that have a role in antagonism. The genes encoding these enzymes should be sequence in order to

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produce organisms with superior biocontrol capabilities and also the production of transgenic plant that showed
resistance to phytopathogens.

Table 1. Nomenclature of chitinolytic enzymes

Chitinase enzymes EC No Mode of action Reference

Endochitinase,poly(1,4-N-acetyl-β-D 3.2.1.14 Random hydrolysis Fisher and Stein

glucosaminide)glycanohydrolase of N-acetyl-β-D (1960)
glucosaminide 1,4-
β-linkages in chitin
and chitodextrins
Exochitinasechitobiasesβ -N-
acetylhexosaminidase, β -N-acetyl- 3.2.1.52 Hydrolysis of Cabezas
Dhexosaminide terminal non- (1989)
N-acetylhexosaminohydrolase reducing N-acetyl-
Dhexosamine
residues
in N-acetyl- β -D-
hexosaminides

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