Journal of Pharmacognosy and Phytochemistry 2019; 8(5): 1581-1585

E-ISSN: 2278-4136
P-ISSN: 2349-8234
JPP 2019; 8(5): 1581-1585 Isolation and bio-efficacy screening of native
Received: 04-07-2019
Accepted: 06-08-2019 Trichoderma species as a potential biocontrol
Shruthi TH
agents against pomegranate wilt caused by
Department of Plant Pathology,
University of Agricultural
Ceratocystis fimbriata Ellis and Halst
Sciences, Raichur, Karnataka,
India
Shruthi TH, Gururaj Sunkad, Mallesh SB, ST Yenjerappa and
Gururaj Sunkad Mahadevswamy
Department of Plant Pathology,
University of Agricultural
Sciences, Raichur, Karnataka, Abstract
India The main emphasis of the present study was isolation and antagonistic screening of native Trichoderma
isolates against Ceratocystis fimbriata Ellis and Halst. The causal agent of wilt of pomegranate, which
Mallesh SB cause estimated losses up to 30%. Thirty three native Trichoderma spp. were isolated from the
Department of Plant Pathology, rhizosphere soil of healthy pomegranate plants, identified using morphological and microscopic
University of Agricultural characteristics and were evaluated for in vitro antifungal activity against Ceratocystis fimbriata by dual
Sciences, Raichur, Karnataka, culture plate technique. Among the Thirty three isolates maximum per cent inhibition of 93.10 per cent
India was observed in Trichoderma harzianum (PT-11 isolate) followed by Trichoderma viride (PT-10) and
Trichoderma harzianum (PT-4) of about 92.03% and 91.66%, respectively when compared to the all
ST Yenjerappa
other isolates of Trichoderma spp. Based on the dual culture technique, eleven superior native
Department of Plant Pathology,
University of Agricultural
Trichoderma isolates were selected for elucidating volatile metabolites production by paired plate
Sciences, Raichur, Karnataka, method. The inhibition of pathogen mycelium varied depending on the Trichoderma species producing
India volatile metabolites, Trichoderma virense (PT-6) inhibited fungal growth up to 78.84% followed by
Trichoderma harzianum (PT -11) of about78.67% and Trichoderma viride (PT-10) of 71.84%. Among
Mahadevswamy superior eleven isolates, PT-6, PT-10 and PT-11 isolates were used for growth promotion and disease
Department of Plant Pathology, control studies in pot culture experiment under greenhouse. The growth of pomegranate seedlings with
University of Agricultural the antagonist alone and in combination with pathogen was greater than in plants were inoculated with
Sciences, Raichur, Karnataka, pathogen alone. Among all isolates, T. virense (PT-6) showed higher mean shoot and root length (45.70
India cm and 19.04 cm) with lowest disease incidence (20.38%) when compared to highest incidence in
pathogen check (41.20%).

Keywords: Pomegranate, wilt, Ceratocystis fimbriata, Trichoderma spp.

Introduction
Pomegranate (Punica granatum L.) is a vital cash crop of India, is commercially cultivated in
the states of Maharashtra, Karnataka, Andhra Pradesh, Gujarat, Rajasthan and Tamil Nadu. In
recent years, wilt disease caused by Ceratocystis fimbriata Elli. and Halst is becoming a major
threat by adversely affecting the yields of the pomegranate fruit crop (Sharma et al., 2008 and
Sharma, 2009) [23, 24]. The disease was first reported from Nashik district in Maharashtra in
1978 and subsequently from Kaladgi and Kanamadi areas of Karnataka in 1988 (Somasekhara,
1999) [25] and Cudddapa, Andhra Pradesh in 2002. The disease has also been reported from
other countries like Iran (Banihashemi, 1998) [1], China (Huang et al., 2003) [14] and Greece
(Tziros and zavella-Klonari, 2008) [32]. Based on the damage and loss, the disease has been
considered as a nationally important disease in India and lot of emphasis has been given to
tackle this disease problem. However, there is no much research work carried out with regards
to biological control of wilt of pomegranate. Trichoderma sp. is one of the most important
biocontrol agent used for management of different diseases (Harman, 2004) [10]. Trichoderma
spp. are free living fungi that are common in soil and root ecosystems and promote plant
growth (Yedidia, 2001) [36]. Trichoderma spp. are effective in control of soil borne fungal
diseases in several crop plants (Kubicek et al., 2001) [17]. Different isolates of Trichoderma
spp. were identified as biocontrol agents of groundnut stem rot and other soil-borne diseases
Corresponding Author: (Podile and Kishore, 2002) [19]. These free-living fungi are ubiquitous in the soil environment
Shruthi TH
Department of Plant Pathology,
and are being successfully used and commercialized to combat a broad range of
University of Agricultural phytopathogenic fungi (Hjeljord et al., 2000 [12]; Desai et al., 2002 [5]; Fravel, 2005 [9]).
Sciences, Raichur, Karnataka, Trichoderma spp. can directly impact other fungi, after sensing a suitable fungal host,
India Trichoderma sp. responds with the production of antibiotic compounds, formation of

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specialized structures and degradation of the host’s cell wall, Per cent inhibition (I) = C-T/C ×100
followed by the assimilation of its cellular content, a process Where, C- mycelial growth of pathogen in control
known as mycoparasitism (Steyaert et al., 2003 [29]; Benitez et T- Mycelial growth of pathogen in dual plate
al., 2004 [2]). The mechanisms of mycoparasitism, antibiosis
and competition afforded by Trichoderma spp. have been Effect of volatile compounds produced by antagonists on
widely studied (Howell, 2003 [13]; Harman et al., 2004) [10]. the radial growth of C. fimbriata
Application of bio-agents for growth promotion and disease Volatile compound production assay was done by following
management has attracted much attention in the past few the method of Dennis and Webster (1971) [4]. Eleven different
decades, due to serious environmental and human health species of Trichoderma were inoculated in the center of the
problems resulting from the application of chemical pesticides petri plates containing solidified sterilized PDA medium by
(Cook, 1993 [3]; He, 1993) [11]. Chemical fungicides offer a placing 6mm disc (3d old culture) from the margin of the
degree of protection against pathogens, but their adverse actively growing region of Trichoderma spp. and incubated
effect on beneficial soil microorganisms and the environment for 3 days at 28±1 oC. After that the top lid of each petri plate
cannot be ignored. In this context, biocontrol agents appear to was replaced with bottom part of another petri plate with
hold promise in plant growth promotion and disease same size containing PDA medium, duly inoculated with a
management. Using Trichoderma spp. control of soil borne 6mm mycelial disc of the test pathogen after 3 days of
plant pathogens has been reported by many investigators incubation. The pairs of each plate were sealed with parafilm
(Ulkhede, 1992 [33], Ziedan et al., 2005 [37]) but there is little (adhesive tape) and incubated at 28±1 oC. The PDA medium
information was available on the use of Trichoderma spp. as without Trichoderma isolate in the bottom part of petri plate
biocontrol agents against C. fimbriata in pomegranate plants. with respective test pathogen on the upper lid of plate served
The objective of the present investigation was isolation and as control. Three replicates were maintained for each
screening of effective native isolates Trichoderma spp. treatment. The assembly was opened after 72 h. and the
against the wilt pathogen C. fimbriata and elucidation of observations were recorded by measuring colony diameter of
volatile metabolite production mechanism of Trichoderma the test pathogen (in mm) in each plate and that of the control
spp. plates.

Materials and Methods Pot culture technique

Source and isolation of Trichoderma spp. Potato dextrose broth was prepared in 250ml conical flasks.
Trichoderma spp. was isolated from rhizosphere soil by serial Each flask inoculated with different efficient isolates of
dilution technique (Krassilnikov, 1950) [16] on Trichoderma Trichoderma spp., separately and incubated at 28± 2 oC for 10
specific medium (Elad and Chet, 1981) [7]. Ten grams of days. 1kg of soil and 1kg of sand was taken into polythene
healthy rhizosphere soil samples was taken separately and bags and sterilized at 121 oC for 30 min at 15 lbs pressure for
suspended in 90ml of sterile water and stirred well to get 1: 10 two successive days. 9” earthenware pots were taken;
dilution (10-1). One ml from this was transferred to test tubes sterilized sandy soil was added into the pots. The talc based
containing 9ml of sterile distilled water to get 1:100 (10-2) formulation of Trichoderma spp. was prepared according to
dilution. Likewise the dilution was made up to 10: 10000 the method described by Jayarajan and Ramakrishnan (1991)
(10-4). One ml of the final dilution of each sample was [15]
. Nine mm disc of Trichoderma spp. was inoculated into
aseptically transferred into petri plates containing 100 ml potato dextrose broth and incubated at room
Trichoderma specific medium, the plates were incubated at temperature (28± 2 °C) for 5 days. The mycelial mat was
25± 1˚C for 5- 10 days. Fungus was sub cultured on potato mixed with talc powder in 1:2 ratio and shade dried. To this,
dextrose agar slants and allowed grow at 25 ºC for 15 days carboxy methyl cellulose was added at the rate of 0.5 percent
and such slants were preserved in a refrigerator at 4 ºC and as sticker. The product was shade dried to 20 per cent and
sub cultured once in 30 days. packed in polypropylene bags and sealed.
Three healthy rhizosphere soil samples were collected from Three months old pomegranate seedlings were planted in pots
each taluk and totally thirty three representative Trichoderma filled with sandy soil containing C. fimbriata. Different talc
spp. were isolated from five districts surveyed. based formulation of superior isolates of Trichoderma spp.
was applied after 30 days after planting and three replicates
Screening for antagonistic activity of native Trichoderma were maintained for each treatment. Control pots also
spp. isolates maintained without any fungal cultures. For recording wilt
All the thirty five isolates were screened for potential incidence, observations on total number of branches and
antagonistic activity against pathogenic fungus C. fimbriata number of wilted branches in each treatment were recorded.
using dual culture technique. All antagonistic pathogen Later, per cent disease incidence was calculated.
combinations were examined on 20ml of PDA in 9 cm
petriplates, with three replicate plates per treatment. For dual Results
culture technique, a mycelial disc (5mm in diameter), taken Biological control of plant diseases using microbial inoculants
from actively growing 8 day old culture of C. fimbriata and 3 is receiving increased attention as an eco-friendly alternative
days old Trichoderma isolates placed 8cm apart from each to chemical pesticides. Exploitation of plant-microbe
other on the PDA. For control treatments, a 5mm disc of C. interaction in the rhizosphere could promote plant growth and
fimbriata was placed on the PDA medium. The plates were reduces the diseases.
incubated at 28oC for 8 days. Observations on the antagonistic
activities of Trichoderma isolates on C. fimbriata were Isolation and Identification of Trichoderma isolates
determined by measuring the radial growth of pathogen with Native Trichoderma spp. were isolated by serial dilution
fungal antagonistic culture and control. The per cent technique on Trichoderma specific medium (TSM) and
inhibition over the control was calculated by using the incubated for 7 days at 25 ± 2 ºC. After incubation, all the
formula (Vincent, 1947). thirty three isolates showed typical character of greenish
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fungal colony on TSM and produced fungal characters of in PT-11 followed by PT- 10 (92.03%) and PT-4 (91.66%).
Trichoderma under microscope. The Trichoderma isolates Least inhibition of 78.63 per cent was observed in PT-8
were obtained and pure culture of isolates was maintained on followed by PT-2 (80.70%).
PDA slants for further studies.
Table 1: Antagonist activity of native Trichoderma isolates against
Effect of native isolates of Trichoderma species on mycelial C. fimbriata
growth of C. fimbriata in vitro
A total of Thirty three isolates of Trichoderma were obtained Per cent mycelial
Sl. No. Isolate Radial mycelial growth(mm)
from healthy rhizosphere soil of wilt affected orchards. Inhibition*
1 PT-1 16.00 82.20 (65.04)
Among these thirty three isolates only eleven were bioactive
2 PT-2 17.16 80.70 (63.93)
and have been reported in the present study. The isolates
3 PT-3 13.46 85.10 (67.29)
which showed more than 50% of mycelial inhibition were 4 PT-4 7.46 91.66 (73.21)
selected as efficient superior isolates and designated as PT-1, 5 PT-5 9.01 90.00 (71.56)
PT-2… and PT -11. 6 PT-6 10.0 88.83 (70.47)
Thirty three isolates of Trichoderma spp. were screened 7 PT-7 7.16 92.00 (73.57)
against C. fimbriata for mycelial inhibition by dual culture 8 PT-8 19.16 78.63 (62.46)
technique. Zone of inhibition of mycelium (in mm) was 9 PT-9 12.83 85.62 (67.71)
recorded and the per cent inhibition was calculated. 10 PT-10 7.16 92.03 (73.60)
The results revealed that per cent inhibition of mycelial 11 PT-11 6.16 93.10 (74.77)
growth varied greatly among the thirty three isolates. 12 Control 90.00 0.00
However, the per cent inhibition was higher (> 50%) in S.Em± 0.27
eleven isolates which varied from 80.70 to 93.10 per cent. C.D. at 1% 1.06
The results obtained were highly significant between the * Mean of three replications
different isolates (PT-1….PT-11) and also over control. Figure in parenthesis are arcsine value
Maximum per cent inhibition of 93.10 per cent was observed

Plate 1: Antagonistic activity of native Trichoderma spp. isolates against C. fimbriata

Volatile compounds produced by Trichoderma spp. against C. fimbriata

Eleven superior native Trichoderma isolates were tested for in isolate PT-6 (78.84%) followed byPT-11 (78.67%), FP-10
volatile compound production assay in paired plate technique. (71.84%) and lowest concentration of volatile metabolites was
The results revealed that the isolates produced considerable produced by PT-8 (50.00%). All the isolates have shown
amount of volatile metabolites which varied with the isolates. significant difference in mycelial growth inhibition when
Higher concentrations of volatile metabolites were produced compared to the uninoculated control.
Table 2. Production of volatile metabolites from efficient native Trichoderma isolates on mycelial inhibition of C. fimbriata
Sl. No. Isolate Identification Radial mycelial growth (mm) Per cent mycelial inhibition*
1 PT-1 Trichoderma viride 29.60 63.10(52.60)
2 PT-2 Trichoderma virens 31.20 58.80(50.06)
3 PT-3 Trichoderma viride 29.30 60.70(51.17)
4 PT-4 Trichoderma harzianum 20.00 70.00(56.78)
5 PT-5 Trichoderma hamatum 21.60 68.40(55.79)
6 PT-6 Trichoderma virens 11.16 78.84(62.61)
7 PT-7 Trichoderma harzianum 23.76 66.24(54.47)
8 PT-8 Trichoderma virens 40.00 50.00(45.00)
9 PT-9 Trichoderma harzianum 34.13 55.87(48.37)
10 PT-10 Trichoderma viride 18.16 71.84(57.94)
11 PT-11 Trichoderma harzianum 11.33 78.67(62.49)
12. Control - 90 0.00
S.Em± 0.42
C. D. @ 1% 1.26
* Mean of three replications
Figure in parenthesis are arcsine value
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revealed that the application of native Trichoderma spp.

increased shoot and root length of seedlings. Among all
Trichoderma isolates PT-6 showed higher shoot and highest
root growth (45.70 cm and 19.04 cm respectively). Similar
results were recorded by Sundaramoorthy and Balabaskar
(2013) [30] application of native Trichoderma antagonists
through seedling dip and soil application was found effective
in suppressing wilt incidence (15.33 to 25.50%).
Conspicuously, an application of ANR-1 antagonistic fungal
formulation was recorded least wilt incidence (15.33%)
followed by KGI-3 (17.45%) compared to other isolates.

Conclusion
Trichoderma spp. are effective biocontrol agents for a number
of soil borne plant pathogens and plays a major role in
controlling plant diseases (Yedidia et al., 1999) [36].
Plate 2. Volatile metabolites production by efficient Trichoderma
spp. isolates
Trichoderma species are among the most-promising
biocontrol fungi against many fungal plant pathogens. In our
Discussion present study, we have isolated and screened native isolates of
In this preliminary investigation, an attempt was made to Trichoderma spp. against wilt disease of pomegranate caused
screen the antagonistic potential of rhizospheric native by C. fimbriata. Trichoderma isolates are more effective and
bioagents against C. fimbriata, the causal organism of wilt show excellent control of C. fimbriata, responsible for
disease in pomegranate. Earlier reports attribute Trichoderma Pomegranate wilt. These isolates could be exploited for their
spp. as one of the most efficient strains to be evaluated as volatile compound production mechanism. The three superior
biocontrol agent. isolates will be promising bio control agent against wilt and
All the thirty three isolates of Trichoderma spp. were showed plant growth promotion activity in pomegranate seedlings.
antagonism against C. fimbriata. However, only eleven But there is still need to work in future regarding
isolates showed high level of antagonistic activity by biotechnological approaches for effective and eco-feasible
recording more than 50 per cent inhibition of test pathogen. control of wilt disease.
Among the eleven superior isolates, the per cent inhibition of
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