229

AGRIVITA Journal of Agricultural Science. 2020. 42(2): 229–242

AGRIVITA
Journal of Agricultural Science
www.agrivita.ub.ac.id

Mycoparasitic Activity of Indigenous Trichoderma virens Strains Against Mungbean

Soil Borne Pathogen Rhizoctonia solani: Hyperparasite and Hydrolytic Enzyme
Production
Alfi Inayati1*), Liliek Sulistyowati1), Luqman Qurata Aini1) and Eriyanto Yusnawan2)
1)
Department of Plant Pest and Disease, Faculty of Agriculture, Universitas Brawijaya, East Java,
Indonesia
2)
Indonesian Legumes and Tuber Crops Research Institute, Indonesian Agency for Agricultural Research
and Development, East Java, Indonesia

ARTICLE INFO ABSTRACT

Keywords: Rhizoctonia solani is one of the harmful pathogens on mungbean,
Cellulase which is very challenging to be controlled. T. virens has been studied
Chitinase intensively and has great potency to control R. solani through
Hydrolytic enzyme mycoparasitism. Seven strains of T. virens isolated from various
Hyperparasite rhizospheres were tested for their mycoparasitic potential by observing
T. virens their hyperparasitism and the production of hydrolytic enzymes. All
strains showed the ability to suppress the growth of R. solani on dual
Article History: culture assay as well as on culture filtrate test with the inhibition ability
Received: December 31, 2019 from 43.8 to 68.6% on the dual culture assay and from 22.2 to 71.1% on
Accepted: May 6, 2020 the culture filtrate assay. Inter-fungal interaction, which was observed
by an electron microscope, showed hyperparasitic action of T. virens
*) Corresponding author:
against R. solani involved the formation of the knob-like structure
E-mail: [email protected]
followed by the growth of Trichoderma hyphae inside host mycelia,
coiling, lysed cell wall, and swollen of mycelial tips. Mycoparasitism of
T. virens also correlated with the synthesis of hydrolytic enzymes, such
as cellulase and chitinase, which influenced the overall hyperparasitic
ability of T. virens against the pathogen. Based on in vitro assay, the Tv3
strain proposed as a promising strain to control R. solani due to its high
growth inhibition and relatively high cellulase and chitinase productionse.

INTRODUCTION high survival capability (Veena, Priya, Raheesa,

& Divya, 2014), a broad range of hosts (Zheng et
Soil-borne pathogen is one of the most
al., 2013), and genetically different groups which
devastating pathogens on mungbean seedling.
Several phytopathogenic fungi such as Rhizoctonia are sensitive to different pesticides (Carling, Baird,
solani, Sclerotium rolfsii, Fusarium sp., and Gitaitis, Brainard, & Kuninaga, 2002).
Phytophthora sp., have been reported associating There are only a few reports about the
with damping-off, root rot, and wilting on mungbean successful controls of R. solani using chemical
crops in Indonesia (Rahayu, 2016). R. solani fungicides in Indonesia (Djunaedy, 2008; Muis,
becomes an important pathogen in legumes crop 2007). Biological agents are increasingly considered
due to its aggressiveness and was able to devastate to reduce the negative effects of intensive
soybean plantation in swampland in South fungicide applications to the environment (Meena,
Kalimantan (Rahayu, 2014), and infected more than Swapnil, Zehra, Aamir, et al., 2017). However, the
20% of mungbean germplasm collections cultivated effectiveness of bio-control agents often lowers than
in dry season 2014. The control and management the chemical fungicides. The use of integrated bio-
of this pathogen are very difficult since R. solani has control agents such as Trichoderma sp. with some
ISSN: 0126-0537 Accredited First Grade by Ministry of Research, Technology and Higher Education of The Republic of Indonesia,
Decree No: 30/E/KPT/2018

Cite this as: Inayati, A., Sulistyowati, L., Aini, L. Q., & Yusnawan, E. (2020). Mycoparasitic activity of indigenous
Trichoderma virens strains against mungbean soil borne pathogen Rhizoctonia solani: Hyperparasite and hydrolytic
enzyme production. AGRIVITA Journal of Agricultural Science, 42(2), 229–242. https://doi.org/10.17503/agrivita.
v0i0.2514
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Alfi Inayati et al.: Mycoparasitic of Trichoderma virens against Rhizoctonia solani.........................................................

bactericides (oxytetracycline and streptomycin process (Monfil & Casas-Flores, 2014; Vipul et al.,
sulphate or agrimycin) can be expected to increase 2015). Other enzymes such as chitinases, β-1,3
its effectiveness to control pathogens (Soesanto glucanases, β-1,6-glucanases, α-1,3-glucanases,
et al., 2018). Trichoderma sp. has been known for and proteases were also reported having strong
its great potency as promising bio-control agents antifungal activities against plant pathogens
to suppress the growth of soil-borne pathogens (Contreras-Cornejo, Macías-Rodríguez, Herrera-
including R. solani due to its ideal characters such Estrella, & López-Bucio, 2014; Wu et al., 2017).
as fast-growing ability, wide adaptation to various The ability to produce hydrolytic enzymes is a
types of soil and temperature range, good root crucial property for mycoparasitic fungi. Therefore,
colonizer, and survival ability on extreme conditions an efficient hydrolytic strain is necessary to be
(Mohiddin, Khan, Khan, & Bhat, 2010; Zehra, Dubey, explored for developing a promising bio-control
Meena, & Upadhyay, 2017). Moreover, Trichoderma agent to control soil-borne pathogens.
sp. has various action modes directly and indirectly Recently, many studies have been focused
in controlling pathogenic fungi (Nakkeeran, on the development of new approaches for
Vinodkumar, Priyanka, & Renukadevi, 2018). controlling the pathogens by utilizing local or
Trichoderma suppress pathogen growth through indigenous microorganisms commonly found. T.
competition, antibiosis, production of cellulases virens is one of the most common species found
and other hydrolytic enzymes (i.e proteases, β-1,3 around the rhizosphere of various crops in East
glucanases, and chitinase), and mycoparasitsm. Java besides T. asperellum and T. harzianum
Trichoderma sp. is also able indirectly to suppress (data not shown). T. virens has been studied
pathogens by inducing plant resistance and promote intensively for its potential biological control (Angel
plant growth (Nakkeeran, Vinodkumar, Priyanka, & et al., 2016; Angel, Sundram, Ping, Yusof, & Ismail,
Renukadevi, 2018). Mycoparasitism of Trichoderma 2018) and mycoparasitism has been reported as
species is a complex process as they involved both the most common strategies to control pathogen
mechanical and enzymatically actions (Qualhato et (Strakowska, Błaszczyk, & Chełkowski, 2014). T.
al., 2013; Viterbo & Horwitz, 2010). Mycoparasitism virens also reported induced systemic resistance
of Trichoderma sp. to control R. solani is classified that effective against a broad range of pathogens
into necrotrophic mycoparasites where the parasite ( Wang, Borrego, Kenerley, & Kolomiets, 2020).
highly depends on saprophytic growth and caused Our previous study showed that seven indigenous
destructive effects (Mukherjee et al., 2012). T. virens strains from forty indigenous Trichoderma
Initially, Trichoderma recognizes its host by isolates were effective to suppress the growth of
signals producing by the pathogens, one of which different soil-borne pathogens such as Rhizoctonia
is the production of lectin (Monfil & Casas-Flores, solani (R.s1), R. solani (R.s2), and Fusarium sp. on
2014). Mycoparasite then penetrates the host cell dual culture assay (Yusnawan, Inayati, & Baliadi,
wall which can be both through physical damaging 2019). The volatile organic compounds produced
and enzymatic processes. Direct penetration by indigenous T. virens also inhibited the growth
through the formation of special structures such of R. solani through its antifungal activity ((Inayati,
as appressoria or papillae-like structure has been Sulistyowati, Aini, & Yusnawan, 2019). During the
recognized as the most common form of growth study, there was variation in inhibition capability
suppression from Trichoderma species besides among T. virens strains against a certain pathogen,
the formation of hooked branches and coiling for example, strains Tv6 showed high antagonist
around host hyphae (Viterbo & Horwitz, 2010). activity on dual culture assay, however not showed
Those processes are influenced by the production growth inhibition on R. solani growth on double
of hydrolytic enzymes which helped the host cell plate assay indicating a specific action mode from T.
wall degradation and the production of antibiotics virens strains on their mycoparasitism. Therefore,
(Mukherjee et al., 2012). this study aimed to identify potential mycoparasitism
Chitin and β-1,3 glucan are the main of indigenous T. virens against R. solani through
compounds of fungal cell wall including R. solani. their physical and chemical processes as a basis
During mycoparasitism, Trichoderma use cellulase, for further studies of selected indigenous T. virens
chitinase, and glucanases for the enzymatic as a promising bio-control agent on mungbean.
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MATERIALS AND METHODS Scanning Electron Micrograph (SEM)

observation was conducted by growing both
The research was conducted at the Centre
Trichoderma and R. solani on a thin layer of PDA
laboratory of Indonesian Legumes and Tuber Crops
at microscope slides. After the glass was covered
Research Institute (ILETRI), Malang from August
by mycelia of both Trichoderma and pathogen, the
to November 2018. All T. virens strains and R.
object was prepared for SEM observation.
solani isolates were obtained from ILETRI’s culture
collection as isolated by Yusnawan, Inayati, & Determination of Hydrolytic Enzyme: Cellulases
Baliadi (2019). and Chitinases on Agar Media
The production of cellulase was determined
Inhibition Growth Test on Dual Culture and by Congo red plate assay method (Syed, Riyaz-
Culture Filtrate Assay Ul-Hassan, & Johri, 2013; Zehra, Dubey, Meena,
Dual culture assay was carried out using a & Upadhyay, 2017). T. virens was grown on yeast
method performed by F. Zhang et al. (2016). A 5 extract peptone agar medium supplemented with
mm disk of T. virens and R. solani were transferred 0.2% and 0.5% (v/v) of carboxymethylcellulase
to PDA media and placed in the opposite position, (CMC) and 0.2% congo red. Mycelia of T. virens was
then incubated for 5 days. R. solani grown on PDA cultured on the center of the plate and incubated
without Trichoderma was used as the control. for 3 days. The cellulase activity was indicated
The inhibition percentage was calculated by the as a clear zone around the inoculated media after
following formula: staining with 1% Congo red solution and washing
with 1 M NaCl.
...............................……..1)
Chitinase production was observed according
to Agrawal & Kotasthane (2012) with minor
Where: I, R1, and R2 are growth inhibition, radial modifications. The medium (g/l) consisted of 0.3 g
growth of pathogen without Trichoderma, and radial of MgSO4.7H2O, 3.0 g of (NH4)2SO4, 2.0 g of KH
growth of pathogen challenged with Trichoderma, PO4, 1.0 g of citric acid monohydrate, 20 g of agar,
2
respectively. 4.5 g of colloidal chitin and 0.15 g of bromocresol
Growth inhibition of R. solani by non-volatile purple; pH 4.7. A 5 mm of fresh culture plug of
metabolites on fungal filtrate produced by T. virens the T. virens was inoculated into the medium and
was carried out using the protocol described by incubated at room temperature. Positive chitinase
You et al. (2016). Two mycelial disks (5 mm) taken production will change the color of the medium from
from active-growing colonies were inoculated in yellow to purple.
a flask containing 100 ml potato dextrose broth
(PDB) and then incubated for 7 days. Filtrate for Enzymatic Activity Assay
the antagonistic test was prepared by filtering the For the production of cellulases, T. virens was
Trichoderma supernatant through Whatman filter cultured on the same medium for the plate assay
paper and then incubated on a water bath at 60°C without agar. The filtrate from liquid culture was
for 30 minutes before unsolidified PDA was added used as a crude extract. The CMC-ase activity was
at the ratio of v/v. Pure PDB was added to PDA determined by measuring the amount of reducing
at the same ratio, also prepared as the control. A sugar liberated from CMC using 3,5-dinitro salicylic
mycelial block of R. solani was then inoculated on acid (DNS) method (Potprommanee et al., 2017).
PDA plates for 5 days. The percentage of pathogen The reaction mixture was prepared by mixing 0.5 ml
growth inhibition was calculated using the formula: of crude extract and 0.5 ml of CMC (1%) dissolved
in 0,1M phosphate buffer, pH 7.0. The mixture
............................…….............2) was then incubated at 50°C for 20 minutes and
the reaction was stopped by adding 2.5 ml of DNS
reagent. The absorbance values were measured at
Where: C and R are the growth radius of R. 575 nm. One unit (U) of the enzyme activity was
solani in the control treatment and growth radius defined as the amount of enzyme that released 1
of R. solani in media supplemented with T. virens μmol of glucose per minute.
filtrate, respectively.
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Alfi Inayati et al.: Mycoparasitic of Trichoderma virens against Rhizoctonia solani.........................................................

Chitinase from T. virens was produced on RESULTS AND DISCUSSION

liquid media, as proposed by Agrawal & Kotasthane
Hyperparasites Potential of T. virens
(2012). Chitinase activity was determined using a
Competition for space and nutrients were the
colorimetric method (Modification from Qualhato
most common mode of hyperparasitic actions of
et al., 2013). The reaction mixtures contained 1 ml
Trichoderma species on dual culture assay. Rapid
enzyme solution and 1 ml 0.5% colloidal chitin in
radial growth of T. virens in agar media caused
phosphate buffer saline (0.1 M). After incubation at
the reduction and inhibition of pathogen growth
37°C for 15 minutes, a 2 ml DNS was added and
which was determined by the larger area occupied
the reaction was maintained at 100°C for 5 minutes.
by the colonies of Trichoderma on the plate. Our
The amount of reducing sugar was determined
study showed that all seven strains of T. virens
at 540 nm. One unit (U) of enzyme activity was
had hyperparasitic ability to reduce the growth of
determined as the amount of enzyme required to
R. solani starting from 3 days of incubation (doi)
release 1 mmol N-acetylglucosamine in 1 hour at
with different inhibition capabilities (Table 1).
37°C.
The Tv3 strain showed that the highest inhibition
Statistical Analysis percentage was at 3 doi followed by Tv7 and Tv2.
Analysis of variance of data from dual However, at 5 doi, the inhibition growth of Tv3
culture, filtrate, and hydrolytic enzyme assays was strain decreased while other strains indicated the
calculated using Microsoft Excel software. Least increasing pattern, even though Tv3 still performed
Significant Difference (LSD) was performed on 0.05 the strongest inhibition effect. All T. virens strains
confidence level. The robust analysis of principal grew over the plates after 5 days of incubation
component (PCA) was calculated using R-studio displayed the domination of T. virens over R.
statistical environment R version 3.5.3 (RStudio solani’s growth. (Fig. 1). However, Tv6 and Tv7
Team, 2015). strains showed the inhibition growth was less than
50% which represented low potential inhibition to
control pathogenic fungi of R. solani.
Table 1. Growth inhibition of R. solani by T. virens

Dual culture assay* Culture filtrate*

Strain Day 3 Day 5
Day 3 Day 5
15% 30% 40% 15% 30% 40%
Tv1 40.3 bcd
51.2 b
18.3 bc
35.0 b
48.8 b
22.2 bc
29.4 a
30.6b
Tv2 41.6bc 52.6b 28.0a 20.0d 25.0c 29.4a 27.8a 22.2b
Tv3 74.0 a
68.6 a
15.9 c
36.3 b
37.5 bc
21.1 cd
28.9 a
28.9b
Tv4 30.2e 54.1b 26.8a 50.0a 51.3b 22.2bc 21.7a 23.3b
Tv5 32.5cde 51.1b 26.8a 43.8a 55.0b 26.1abc 21.7a 71.1a
Tv6 31.9 de
43.8 bc
20.7 bc
27.5 c
75.0 a
15.0 d
27.2 a
38.3b
Tv7 44.2b 49.6c 23.2ab 47.5a 48.8b 27.8ab 30.6a 38.3b
Remarks: *Numbers in the same column followed by the same letter are not significantly different based on the LSD
test at α = 0.05
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Tv5

Rs Tv3

Rs

Fig. 1. The domination of T. virens (Tv) growth over R. solani (R.s) on dual culture assay

Culture filtrate assay showed that all strains Li, & Zhang, 2019; Meena, Swapnil, Zehra, Dubey,
had the capacity to suppress pathogen growth. & Upadhyay, 2017). At least four toxins have been
It was observed that culture filtrate at a minimum isolated from T. virens ITC-4777 i.e. gliotoxin,
concentration of 15% (v/v) had an inhibitory effect dimethyl gliotoxin, viridian, and viridiol which were
on pathogen growth. This study showed that there effective to control soil-borne pathogens such as R.
was a positive correlation between the increase of bataticola, M. phaseolina , Phytium deharyanum, P.
filtrate concentration and pathogen suppression, aphanidermatum, S. rolfsii, and R. solani (Li, Li,
even though; this trend did not always correlate to & Zhang, 2019). Other studies also reported that
the time of exposure (Fig. 2). Tv6 strain showed T. harzianum and T. viride produced D-Glucose,
the most significant growth inhibition at 3 doi at a 6-O-α-D-galactopyranosyl- and 17-Octadecynoic
concentration of 40% (v/v) in which the filtrate could acid which had antimicrobial activities (Meena,
inhibit R. solani growth by up to 75%. However, Swapnil, Zehra, Dubey, & Upadhyay, 2017).
the inhibitory effect decreased to 38.3% at 5 doi. Mycoparasitic behavior of T. virens to
The decrease of inhibitory effect at 5 doi was also pathogenic fungi of R. solani was observed at the
observed on other strains except for Tv5. The Tv5 cellular level which was conducted at 3 doi using
showed an increase in inhibitory effect from 55% at an electron microscope. Microscopic inhibitory
3 doi to 71.7% at 5 doi. In contrast to the result from effects of T. virens on dual culture and culture
dual culture assay, Tv3 strain showed the lowest filtrate assay showed that the exposure of T. virens
inhibition growth on culture filtrate assay and so did caused morphological alteration and abnormality
Tv2. This result suggested that each strain had an of the R. solani hyphae (Fig. 3). The SEM figures
action mode to inhibit the growth of the pathogen. showed that the first inter-fungal interaction from the
Trichoderma spp. has been reported in many hyperpasatic action was physical contact between
studies to secrete non-volatile metabolites which T. virens and its host of R. solani. T. virens started
diffused into the liquid medium. The metabolites growing parallel to the host hyphae continuing
had the ability to inhibit the growth of pathogens. with the penetration of knob-like structure from
More than 300 non-volatile compounds produced Trichoderma to the host (Fig. 3a). R. solani’s hypha
by Trichoderma sp. which had numerous functions was wider compared to that of T. virens which had
including antifungal and antimicrobial activities (Li, thin and dense hyphae.
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Alfi Inayati et al.: Mycoparasitic of Trichoderma virens against Rhizoctonia solani.........................................................

These structures facilitated the penetration production of antibiotics involved in hyperparasitism

process as mentioned by Dennis & Webster (1971) of Trichoderma sp. (Mukherjee et al., 2012; Smitha,
which wider host hyphae are more susceptible Finosh, Rajesh, & Abraham, 2014; S. Zhang, Gan,
and easier to be penetrated by thinner and solid & Xu, 2014).
hyperparasite hyphae. T. virens then grew inter-
Determination of Hydrolytic Enzymes Produced
cellularly inside the host hyphae (Fig. 3b), or tightly
by T. virens Strains
coiling of the host (Fig. 3c). Mycoparisitism of T.
T. virens produced lytic enzymes that
virens also appeared as the degradation of the cell
could help penetration and inhibition to the host.
wall of host mycelia (Fig. 3d), and abnormal growth
This current study showed that there were lyses
of the host such as swollen on mycelial tips (Fig.
on the mycelial host who was associated with
3e). These processes indicated that necrotrophic
the production of cellulase and other hydrolytic
mycoparasitism of T. virens was preceded by
enzymes, namely chitinase. The observation of
the destruction of host both intercellular and
enzymes related to mycoparasitism in this study
extracellular. Coiling, lyses and abnormal growth
was conducted by plate assay representing a
of host hyphae were influenced by direct contact
qualitative approach and a colorimetric method to
and affected by biochemical stimulus during
examine the activity and the number of enzymes
hyperparasitism. Moreover, many studies proved
produced by the microorganism.
the presence of hydrolytic enzyme and the

K 15% 30% 40% K 15% 30% 40%

(a) (b)

Fig. 2. The R. solani growth inhibition by T. virens filtrate (K = control treatment, R. solani without T. virens
filtrate) (a) growth inhibition capability of Tv3 strain in different filtrate concentration, from the highest (right)
to the lowest (left), (b) growth inhibition capability of Tv4 strain in different filtrate concentration, from the
highest (right) to the lowest (left)
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Alfi Inayati et al.: Mycoparasitic of Trichoderma virens against Rhizoctonia solani.........................................................

(a) (b) (c)

(d) (e)

Fig. 3. Scanning electron micrographs (SEM) shows hyphal interaction between R. solani and T. virens on
dual culture plate assay: (a) knob like-structure penetrates host mycelia, (b) T. virens penetrates and grow
intercellularly in host cell, (c) coiling hyphae, (d) lysis and (e) shrinkage and swollen tips of host hyphae

Cellulase conditions may affect the cellulase production. The

In vitro assay showed that all T. virens cellulase production increased with the increase in
strains had the ability to produce cellulase, one the incubation period (Olaniyi & Oyesiji, 2015). In
of the important enzymes responsible for cell wall this study, T. virens isolates had fully grown over
degradation. The clearing zone formed around the the agar plates after 5 days making the difficulty to
culture presented cellulase activity from T. virens be observed further for the cellulase activity. The
to break down cellulose in the medium (Fig. 4a). study of the specific basal medium composition
The diameter of the clearing zone formed around appropriate for cellulase assay for T. virens may be
all strains was relatively small, ranging from 5.83 needed to obtain better results. Other factors such
to 16.67 mm, indicating low cellulase activities of as optimum time of exposure, inoculum size, pH
all strains). Two isolates of Tv2 and Tv3 produced value, and optimum temperature also important to
relatively stable enzyme activities at two times of be optimized since all those factors influence the
examination at 3 days of incubation. Another study cellulase activity of Trichoderma (El-Hadi, El-Nour,
conducted by Shahriarinour, Abd Wahab, Ariff, & Hammad, Kamel, & Anwar, 2014; Strakowska,
Mohamad (2011) showed that T. viride isolated from Błaszczyk, & Chełkowski, 2014).
palm trees displayed a larger diameter of clearing Cellulolytic enzymes were also produced by
zone up to 31 mm at 14 days of incubation.The T. virens when the fungi were cultured in liquid media
different exposure time of T. virens to the substrate, supplemented with CMC as cellulase substrate.
strains, age of culture, and other environmental The histogram generated from this study showed
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Alfi Inayati et al.: Mycoparasitic of Trichoderma virens against Rhizoctonia solani.........................................................

that all T. virens strains were able to produce 0.63), showing hyperparasitic behavior of T. virens
CMC-ase, the enzyme responsible for hydrolyzing correlated with the activity of cellulase. However,
cellulase, the essential compound of R. solani cell no positive correlation was observed between
wall (Fig. 4b). The amount of cellulase produced cellulase production and activity in liquid media
by Trichoderma on liquid media was measured as and the pathogen suppression on culture filtrate
the release of reducing sugar (glucose) from the assay. Numerous studies reported a positive
substrate. Analysis of variance of cellulase activity association of lyses on host hyphae with the activity
showed that there was no significant difference of cellulase. However, the differences of methods
in cellulase production among strains as well as and techniques employed leading to the different
cellulase concentration (Table 2). All strains showed cellulase activity performance (Florencio, Couri, &
a similar capability to synthesize cellulase in the Farinas, 2012). Our study showed that T. virens was
media. The cellulase enzyme activity ranged from thought to perform higher cellulase activity when
2.82 U/ml to 3.25 U/ml and the cellulase production cultured on liquid media which contained CMC as
varied between 17.0 μg/ml to 19.5 μg/ml. Strain of a substrate compared to the medium containing
Tv4 showed the lowest celullase activity while Tv6 glucose (culture filtrate). As mention by Abou-Taleb,
strain showed the highest cellulase activity. Mashhoor, Nasr, Sharaf, & Abdel-Azeem (2009),
Cellulolytic activity of T. virens on plate the addition of CMC can induce CMC-ase cellulase
assay had a positive correlation with the inhibition production.
of R. solani growth on dual culture assay (R =

a) b)

Fig. 4. (a) The celullase activity of T. virens on yeast extract peptone agar media supplemented with 0.2%
of CMC at 3 days of incubation (b) Diameter of clearing zone represents cellulase produced by T. virens
strains

Table 2. Cellulase activity of T. virens strains

Strains Cellulase concentration (μg/ml glucose) Cellulase activity (U/ml)

Tv1 18.0 ± 0.4ns 3.00 ± 0.07ns
Tv2 18.3 ± 1.7 3.04 ± 0.29
Tv3 17.0 ± 0.3 2.83 ± 0.04
Tv4 17.0 ± 0.5 2.82 ± 0.08
Tv5 18.9 ± 1.4 3.15 ± 0.24
Tv6 19.5 ± 1.6 3.25 ± 0.26
Tv7 17.2 ± 0.5 2.86 ± 0.09
Remarks: ns = not significantly difference
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Chitinase strains could change the color of the medium,

The chitinolytic activity of Trichoderma in although every strain required different time to
chitin-containing media and bromocresol purple initiate the chitinase activity (Table 3). Analysis of
showed the breakdown of chitin into N-acetyl variance of chitinase activities indicated that the
glucosamine (Qualhato et al., 2013). This reaction production of chitinase was significantly influenced
was represented by the change of media around by the time of incubation and the strains. Strains
the colony from yellow to purple. All strain tested of Tv4 and Tv5 showed low chitinase activity for 2
showed positive chitinase activity since a day of days of incubation and took longer time to hydrolyze
incubation. The difference in color development chitin from the medium, in contrast with Tv3 strains
and the intensity of the purple color zone showed which showed rapid and intensive color changes
the various levels of chitinase synthesized among since the first day of incubation.
strains (Fig. 5). After 3 days of incubation, most

Tv1 Tv2 Tv3 Tv4 Tv5 Tv6 Tv7

Fig. 5. The chitinase activity assay of T. virens strains on media supplemented with colloidal chitin (0.5%).
(A) The diameter of the purple color zone at 2 days of incubation, (B) The diameter of the purple color zone
at 3 days of incubation, and (C) The diameter of the purple color zone at 4 days of incubation. Tv1 – Tv7 =
strains T. virens No. 1 - 7
Table 3. Chitinase activity on plate assay determined by purple zone diameter

Strains 2 Days 3 Days 4 Days

Tv1 1.30 ± 0.17 c
4.00 ± 0.44 bc
7.23±0.06 a
Tv2 1.67 ± 0.45 bc 5.27 ± 0.06 b 7.17±0.06 a
Tv3 2.77 ± 0.25 a 7.03 ± 1.07 a 7.20 ±0.10 a
Tv4 0.00± 0.00 e
2.07± 0.98 e
6.50±0.10 b
Tv5 0.63± 0.12 d 3.33 ± 0.15 d 4.70 ±0.10 c
Tv6 1.47 ± 0.42 bc 4.57 ± 0.06 bc 7.18 ±0.03 a
Tv7 1.90± 0.26 b
4.67± 0.06 bc
7.20a ± 0.10
Remarks: Numbers in the same column followed by the same letter are not significantly different based on the LSD
test at α = 0.05
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Alfi Inayati et al.: Mycoparasitic of Trichoderma virens against Rhizoctonia solani.........................................................

This current study also showed a positive besides the defensive action from pathogens.
correlation between the chitinase activity (purple Hyperparasitic action such as coiling and
zone diameter) and the inhibition growth of the penetration of hyphae suggested the action mode
pathogen in dual culture assay at 3 days of incubation preceded the production of the lytic enzyme. This
(R2 = 0.67). The Tv3 strains which displayed the was proved by the formation of a knob-like structure
most intensive and larger purple color zone had the and parallel growth of Trichoderma hyphae prior
highest inhibition growth of R. solani on dual culture to penetration and coiling the host hyphae at 24
assay. This result demonstrated that chitinase was hours of incubation. However, some strains like Tv2
considered to have an important contribution to showed early lytic enzymes activity which allows
mycoparasitism of T. virens. This also in line with them to degraded host cell walls and parasite them
a study conducted by Baek, Howell, & Kenerley for their own growth. The researcher’s previous
(1999) which showed significantly decreased in study showed that volatile organic compounds
the bio-control activity of T. virens strains KO in produced by T. virens strains could inhibit the
which their chitinase genes (cht42) were disrupted growth of R. solani up to 72% (Inayati, Sulistyowati,
compared to the wild-type strains. Aini, & Yusnawan, 2019) which supported the fact
Chitinase was also synthesized by T. that other compounds of sesquiterpenes and fatty
virens on liquid medium supplemented with chitin acids involved in the mycoparasitism of T. virens.
substrate from colloidal chitin. Concentration and Overall, the mycoparasitism variable showed
chitinase activity from all 7 strains are shown in insignificant direct single-factor comparison.
Fig. 6. The amount of chitinase synthesized was Therefore, the principal component analysis (PCA)
not different among strains. The chitinolytic activity was performed to help the interpretation of the
was expressed as the concentration of N- acetyl data variability. PCA analysis showed that T. virens
glucosamine (Gl-Nac) (mg/ml) released in media strains widespread across the PCA chart, thus
supplemented with colloidal chitin per minute confirming the high natural variability of T. virens
(Agrawal & Kotasthane, 2012). The concentration mycoparasitism. These differences were related to
of Gl-Nac released ranged from 0.61 mg/ml to 1.12 the inhibition growth and the production of hydrolytic
mg/ml while the chitinase activity was recorded from enzymes. Two principal components (PC1 and
0.28 to 0.51 U/minute. PC2) explained 57.62% of the data variability
This present study showed the complex which confirmed the mycoparasitism of T. virens
mycoparasitism process of T. virens against R. was influenced by both direct growth inhibition as
solani. R solani has a unique character because well as hydrolytic enzyme production (Fig. 7). Four
besides having cell wall which contains chitin, it also out of the seven strains (Tv2, Tv3, Tv4, and Tv5)
produces cell wall-degrading enzymes including separated along PC1 quadrant indicating that direct
polygalacturonase (Cattelan, Hartel, & Fuhrmann, growth inhibition through hyperparasitism on dual
1999), polymethyl-galacturonase (PMG), cellulase culture as well as through non-volatile compounds
(Cx) and β-glucosidase which are potential to inhibit produced on culture filtrate, and the production of
the growth of its antagonist such as Trichoderma cellulase and chitinase were the major factor of
(Xue, Zhou, Li, Xiao, & Fu, 2018). On the other the T. virens mycoparasitism strains against R.
hand, T. virens was one of the potential Trichoderma solani. PC1 had a positive correlation with growth
species which is very efficient in degrading cell walls inhibition on dual culture assay, the production of
of cellulases (Strakowska, Błaszczyk, & Chełkowski, cellulase on plate agar, and chitinase activity, and
2014). This fact was difficult to conclude whether the fairly correlated negatively with inhibition growth
suppression of pathogen growth directly correlated on culture filtrate. Therefore, PC2 had a positive
with the mycoparasitic properties from antagonistic correlation with the growth suppression on culture
fungi. For example, strains Tv2 which showed filtrate at a concentration of 30% and the production
the highest chitinase activity, had relatively high of chitinase on plate assay, however, having negative
suppression ability, on the other hand, Tv3 which correlation with the production of cellulase which was
showed relatively low chitinase activity performed shown by the characteristic of mycoparasitism of
stronger growth inhibition. This suggested that there Tv1, Tv7, and Tv6 strains. Explanation of variability
were other antifungal compounds produced by T. on this principal component could be related to the
virens which were also involved in mycoparasitism variability of T. virens mycoparasitism.
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Fig. 6. Chitinase concentration releases in media and chitinase activity of T. virens strains

Fig. 7. Principal Component Analysis (PCA) of T. virens mycoparasitism against R. solani

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An important finding of this study was the activity of Trichoderma virens 159C involved
indigenous strains of T. virens isolated from several in biocontrol assay of Ganoderma boninense.
rhizosphere crops cultivated in East Java had Journal of Oil Palm Research, 30(1), 83–93.
potential biocontrol agents through its mycoparasitic https://doi.org/10.21894/jopr.2017.0009
action. Although the enzyme productions were not Angel, L. P. L., Yusof, M. T., Ismail, I. S., Ping, B. T. Y.,
directly correlated with the growth suppression, its Mohamed Azni, I. N. A., Kamarudin, N. H., &
showing synergistic action with the hyperparasitic Sundram, S. (2016). An in vitro study of the
ability such as physical destruction, which antifungal activity of Trichoderma virens 7b and
increased the potency of T. virens to control R. a profile of its non-polar antifungal components
released against Ganoderma boninense.
solani. The maximum inhibition by selected strains
Journal of Microbiology, 54, 732–744. https://
of T. virens was due to hyperparasitism as well doi.org/10.1007/s12275-016-6304-4
as the production of both volatile and non-volatile
antimicrobial compounds. T. virens mycoparasitism Baek, J. M., Howell, C. R., & Kenerley, C. M. (1999).
against pathogenic fungi of R. solani, showed the The role of an extracellular chitinase from
Trichoderma virens Gv29-8 in the biocontrol of
synergistic actions of both hyperparasitism and
Rhizoctonia solani. Current Genetics, 35, 41–
the production of cell wall degrading enzymes, 50. https://doi.org/10.1007/s002940050431
especially cellulase and chitinase.
Carling, D. E., Baird, R. E., Gitaitis, R. D., Brainard, K.
A., & Kuninaga, S. (2002). Characterization
CONCLUSION of AG-13, a newly reported anastomosis
T. virens strains had different mycoparasitic group of Rhizoctonia solani. Phytopathology,
ability against pathogenic fungi of R. solani. 92(8), 893–899. https://doi.org/10.1094/
PHYTO.2002.92.8.893
Synergism in the production of hydrolytic enzyme
productions with the hyperparasitic ability is the Cattelan, A. J., Hartel, P. G., & Fuhrmann, J. J.
mode of action of T. virens for controlling R. solani. (1999). Screening for plant growth-promoting
Tv3 strain which had the highest growth inhibition rhizobacteria to promote early soybean growth.
and showed high cellulase and chitinase activities Soil Science Society of America Journal,
63(6), 1670–1680. https://doi.org/10.2136/
could be promoted as a promising strain to control
sssaj1999.6361670x
R. solani.
Contreras-Cornejo, H. A., Macías-Rodríguez, L.,
ACKNOWLEDGEMENT Herrera-Estrella, A., & López-Bucio, J. (2014).
The 4-phosphopantetheinyl transferase of
The author would like to thank Indonesian Trichoderma virens plays a role in plant protection
Agency for Agricultural Research and Development against Botrytis cinerea through volatile organic
for funding this research. compound emission. Plant and Soil, 379, 261–
274. https://doi.org/10.1007/s11104-014-2069-x
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