In-vitro evaluation of fungicides and biocontrol agents ...
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Journal of Pharmacognosy and Phytochemistry 2020; 9(2): 1134-1138
E-ISSN: 2278-4136
P-ISSN: 2349-8234
www.phytojournal.com
JPP 2020; 9(2): 1134-1138
Received: 04-01-2020
Accepted: 06-02-2020
Ramniwas Yadav
Ph.D. Scholar, Department of
Plant Pathology, RARI,
Durgapura, Jaipur, Rajasthan,
India
Sushila Choudhary
Ph.D. Scholar, Department of
Plant Pathology, RARI,
Durgapura, Jaipur, Rajasthan,
India
Kalpana Yadav
Kalpana Yadav, Ph. D. Scholar,
Department of Plant Pathology,
Rajasthan College of Agriculture,
Rajasthan, India
Rajendra Prasad Jat
M.Sc., Department of Plant
Pathology, Rajasthan College of
Agriculture, MPUAT, Udaipur,
Rajasthan, India
Rajpal Yadav
Ph.D. Scholar, Department of
Plant Pathology, RARI,
Durgapura, Jaipur, Rajasthan,
India
Corresponding Author:
Ramniwas Yadav
Ph.D. Scholar, Department of
Plant Pathology, RARI,
Durgapura, Jaipur, Rajasthan,
India
In-vitro evaluation of fungicides and biocontrol
agents against R. solani causing root rot of cotton
in Rajasthan
Ramniwas Yadav, Sushila Choudhary, Kalpana Yadav, Rajendra Prasad
Jat and Rajpal Yadav
Abstract
Root rot pathogens (R. solani) were isolated in majority from diseased cotton plants showing root rot
symptoms collected from farmer’s field during survey. The pathogenicity of recovered isolates of R.
solani were separately confirmed by Koch’s postulates by growing susceptible cotton cultivar “Jai BG-
II” in pathogen inoculated pots also observed incidence and typical symptoms by respective isolates.. In
view of increasing disease incidence in cotton growing areas, attempts were made to evaluate six
(systemic and non-systemic) fungicides viz., Carbendazim, Vitavax power, Azoxistrobin, Rhizolex,
Tebuconazole and Thiram at 50, 100, 250 and 500 ppm concentrations against R. solani in vitro.
Sensitivity of Carbendazim, Vitavax power and Tebuconazole was found more pronounced against R.
solani, whereas Carbendazim was highly effective against R. solani at all the concentrations with 100 per
cent inhibition of mycelial growth. In vitro studies for evaluating comparative efficacy of four
Trichoderma isolates. The efficacy of the T. viride isolates (T-5 and T-3) found to be highly effective
against R. solani pathogens in vitro. However, T. aureoviride was found to be less effective than T.
viride.
Keywords: Rhizoctonia solani, management, fungicides, biocontrol agents, disease incidence etc.
1. Introduction
Cotton (Gossypium spp.) is one of the most important fibers and cash crop belongs to genus
Gossypium of the family Malvaceae. It is originated as a tropical and subtropical perennial
plant, but is produced as an annual crop in many temperate regions around the world. Cotton is
cultivated in about 80 countries in the world in which top five producers are India, China,
Pakistan, USA and Brazil (Anonymous, 2016-17) [2]. Cotton is popularly known as “white
gold” and it is the crop with multiple uses that supplies five basic products viz. - lint, oil, seed,
hulls and linters. Among them the lint is the most important product of cotton plant and
provides much of the high quality fiber for the textile industry.India is the first largest cotton
producer, consumer and exporter in the world, it is cultivated in an area about 11.87 million
hectares with the 30.15 million tons production and 4.32 q ha-1 productivity respectively,
Agriculture statistics at a glance-2016 (2017-a). In Rajasthan total area under cotton
cultivation is around 4.4 lac hectares with production of 13.2 lac tones and productivity of 5.01
q ha-1, Agriculture statistics at a glance-2016 (2017-b).
Most common fungal diseases in cotton are leaf spot and leaf blight caused by (Alternaria
macrospora, Alternaria alternata, Cercospora gossypina, Cochiobolus spicifera, Myrothecium
roridum (Kamal and Moghal, 1968; Jagirdar and jagirdar, 1980; Jiskani 1992 and jaskani
2001) [13, 9, 10, 11]. Anthracnose (Colletotrichum gossypii), Areolate mildew (Cercosporella
gossypii), Ascochyta blight (Ascohyta gossypii) and black root rot (Thielaviopsis basicola),
boll rot is caused by several pathogen including (Ascohyta gossypii, Colletotrichum gossypii,
Fusarium spp., Lasiodiplodia theobromae, Rhizoctonia solani). Under favorable
environmental condition Charcoal rot (Macrophomina phaseolina), Fusarium wilt Fusarium
oxysporum f. sp. vasinfectum), powdery mildew (Leveillula taurica), cotton rust (Puccinia
schedonnardii, Puccinia cacabata), Sclerotium stem rot (Sclerotium rolfsii) and Damping-off
and root rot (Rhizoctonia solani (Silva et al. 1995 and Belot and Zambiasi, 2007) [18, 4]. Seed
treatment with fungicides is the appropriate method for control the seed/soil borne diseases
but, the fungus R. solani is non-spore forming and soil borne, it’s propogules and sclerotia are
evenly distributed in the soil and fungicides are not able to reach at the target point for manage
the disease up to longer period. On other hand overzealous and indiscriminate use of most of
the synthetic fungicides has created different type of environmental and toxicological
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Journal of Pharmacognosy and Phytochemistry http://www.phytojournal.com problems. Attention has been paid towards exploitation of non
hazardous bio control agents or botanical amendment in plant
protection. Therefore, the ultimate aim of research has been
the development of integrated control strategies, as such; use
of alternative methods like eco-friendly botanicals and
biological control with integration of fungicides to manage
this disease.
2. Materials and Methods
2.1 In vitro efficacy of fungicides (Poison food technique)
relative efficacy of different systemic and non-systemic
fungicides was evaluated by using poisoned food technique
(Schmitzs, 1930) [19]. In vitro efficacy of fungicides was tested
against the most virulent/aggressive isolate of R. solani (SGN
Rs-03) with six fungicides viz., Thiram 75% WP (Gupta
Chemicals (p.) Ltd., Mumbai), Vitavax power 75% WP
[carboxin] (Pesticide India Ltd., Udaipur), Bavistin 50% WP
[carbendazim] (BASF India Ltd., Mumbai), Tebuconazole
25.9 w/w [Folicular 250 EC)] (Bayer Crop Science, India
Ltd., Mumbai), Rhizolex 50%WP [tolclophos-methyl]
(Sumitomo chemicals ltd.) and Azoxistrobin 23% SC
[Amistar] (Syngenta ltd.) were tested at four concentrations
i.e. 50, 100, 250 and 500 ppm. Desired quantity of each
fungicide was added separately to sterilized medium, mixed
thoroughly and poured in sterilized Petri dishes and allowed
to solidify. Each plate was inoculated with three mm disc of
fungal culture and incubated at 28±1 0C. The linear growth
after 7 days was measured and per cent inhibition was
calculated according to formula as follows:
Where,
C = Diameter of the colony in control.
T = Diameter of the colony in treatment.
A = Check was also maintained where medium was not
supplemented with any fungicide.
2.2 Efficacy of biocontrol agents on mycelial growth
inhibition of the Rhizoctonia solani (in vitro)
In vitro, screening of bio agents was done by dual culture
technique (Johnson et al., 1959) [12]. The following three
resident and one non-resident biocontrol agents were used
against R. solani SNG Rs-03 isolate viz., Trichoderma viride
T-5, T harzianum, T. aureoviride and non-resident isolate T.
viride T-3 (IARI isolate).
All the biocontrol agents were obtained from Department of
Plant Pathology, Rajasthan College of Agriculture, MPUAT,
Udaipur, India. Single colonies of the isolate were sub-
cultured in PDA and stored in refrigerator to maintain their
genetic purity.
20 ml of PDA medium was poured into sterile Petri plate and
allowed for solidification. Three mm diameter discs from
actively growing colony of pathogen was cut with a sterile
cork borer and placed near the periphery of PDA plate.
Similarly, biocontrol agents were placed on the other side i.e.,
at an angle of 180°. Plates with no antagonists served as
control for the pathogen. The plates were incubated at 28 ±
1°C for seven days. In each treatment three replications were
maintained. The extent antagonistic activity by biocontrol
agent was recorded after incubation period of 7 days by
measuring the growth of the test pathogen in dual culture and
in control plates. The per cent mycelial inhibition zone of
pathogen was calculated using following formula:
Where,
I = Per cent mycelial inhibition zone
C = Growth of fungal plant pathogen in control (mm)
T = Growth of fungal plant pathogen in dual culture plate
(mm)
3. Results and Discussion
3.1 Isolation of the pathogen, Purification and
identification of the pathogen
The disease samples of cotton root rot were also collected
from different villages of six districts during the surveys to
isolate the pathogen. The most of root rot samples yielded
Rhizoctonia, Fusarium spp. and Sclerotinia spp., whereas, in
majority of R. solani colonies were recovered.
To purify R. solani single sclerotia was picked under a stereo-
binocular microscope and using single hyphal tip culturing on
PDA plates. The cultures of different isolates were identified
on the basis of morphological characters of the fungus and
compared with the standard description (Holiday, 1981 and
Mordue, 1988) [7, 16].
3.2 In vitro evaluation of fungicides (poisoned food
technique)
Six fungicides Carbendazim, Vitavax power, Azoxistrobin,
Rhizolex, Tebuconazole, and Thiram were evaluated at four
concentrations viz., 50, 100, 250 and 500 ppm by poisoned
food technique for their effectiveness against R. solani. All
the test fungicides significantly inhibited the mycelial growth
of R. solani at all concentrations.
The Carbendazim was found the most effective that
completely 100 per cent inhibited the mucelial growth of R.
solani at all concentrations (50, 100, 250 and 500 ppm)
followed by Vitavax power was found effective with
inhibition of 95.6, 95.9, 96.1 and 96.9 per cent mycelia
growth of R. solani at 50, 100, 250 and 500 ppm respectively.
Third most effective fungicide was Tebuconazole which
showed 91.4, 94.4, 96.1 and 96.7 per cent growth inhibition at
50, 100, 250 and 500 ppm respectively. It was also observed
that Tebuconazole found at par at 250 and 500 ppm with
Vitavax power, followed by Rhizolex and Azoxistrobin
showed 81.7, 84.4, 88.4, 91.7 and 70.2, 73.3, 76.1, 79.2 per
cent inhibition at 50, 100, 250 and 500 ppm, respectively.
Thiram was found least effective at all concentrations against
R. solani with 19.4, 22.3, 25.2 and 30.8 per cent growth
inhibition of R. solani at 50, 100, 250 and 500 ppm
concentration respectively (Table-1, Fig.-1). Similar results
have been observed by several workers, where fungicides like
Carbendazim, Vitavax and Tebuconazole have been reported
to be effective for control of root rot of cotton (Yadav et al.
2003) [21] and on other crop (Vadhera et al. 1997) [20]; (Meen
and Chatopadhyay 2002) [15]; (Dutta and Kalha 2011) [6];
(Rajendraprasad et al. 2017) [17].
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Journal of Pharmacognosy and Phytochemistry http://www.phytojournal.com Table 1: Comparative efficacy of different fungicides on the growth of R. solani at various concentrations (ppm) in vitro
S. No. Fungicides Mycelial growth (mm)* Per cent growth inhibition*
50 100 250 500 50 100 250 500
1 Carbendazim 0.0 0.0 0.0 0.0 100 (90.0) 100 (90.0) 100 (90.0) 100 (90.0)
2 Vitavax power 4.0 3.6 3.5 2.8 95.6 (77.8) 95.9 (78.4) 96.1 (78.5) 96.9 (79.8)
3 Azoxistrobin 26.8 24.0 21.5 18.7 70.2 (56.9) 73.3 (58.9) 76.1 (60.8) 79.2 (62.9)
4 Rhizolex 16.5 14.0 10.4 7.5 81.7 (64.7) 84.4 (66.8) 88.4 (70.1) 91.7 (73.2)
5 Tebuconazole 7.7 5.0 3.5 3.0 91.4 (72.0) 94.4 (76.4) 96.1 (78.8) 96.7 (79.5)
6 Thiram 72.5 69.9 67.3 62.2 19.4 (26.1) 22.3 (28.1) 25.2 (30.1) 30.8 (33.7)
7 Control 90.0 90.0 90.0 90.0 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0)
SEm± 0.531 0.614 0.134 0.106 0.082 0.078 0.074 0.089
CD (P= 0.05) 1.57 1.82 0.39 0.31 0.24 0.23 0.22 0.26
* Average of four replications; Figures given in parentheses are arcsine √ per cent angular transformed values.
Plate 1: Inhibition of mycelial growth of R. solani by different fungicides
3.3 In vitro evaluation of biocontrol agents (Dual culture
technique)
Efficacy of biocontrol agents T. viride (T-5), T. harzianum
(Th.J. 89-2), T. viride (T-3) and T. aureoviride (Ta-D 91-5)
was studied in vitro against R. solani.
Results indicate that all the biocontrol agents viz., T. viride, T.
harzianum, T. viride (T-3) and T. aureoviride were have
antagonistic activity to the growth of R. solani in vitro.
Maximum and significant high per cent inhibition of R. soalni
growth (84.5 per cent) was recordded by T. viride (T-5) in
dual culture method, followed by T. viride (T-3) 80.0 per cent
while the T. harzianum (Th.J. 89-2) was showed 77.7 per cent
mycelial growth inhibition. Minimum growth inhibition was
recorded by T. aureoviride (Ta-D 91-5) which was 72.3 per
cent (Table-2, Fig.-2). Similar results have been observed by
several workers, where biological control agents like T.
viride, T. harzianum and T. aureoviride have been reported to
be effective for control of root rot caused by R. solani.
(Bunker and Mathur 2001) [5]; Mathur and Gurjar 2002) [14];
(Arya et al. 2017) [3]; (Hassanein 2012) [8].
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Journal of Pharmacognosy and Phytochemistry http://www.phytojournal.com Table 2: Per cent inhibition zone of mycelial growth of R. solani with four isolates of Trichoderma spp. by dual culture technique
Treatments Biocontrol agents Growth of R. solani*
Growth (mm)* Per cent inhibition
T1 Trichoderma viride (T-5) 13.9 84.5 (66.9)
T2 Trichoderma harzianum (Th.J. 89-2) 20.1 77.7 (61.8)
T3 Trichoderma viride (T-3) 18.0 80.0 (63.4)
T4 Trichoderma aureoviride (Ta-D 91-5) 24.95 72.3 (58.3)
T5 Control 90.0 0.0 (0.0)
SEm± 0.699 0.573
CD (P= 0.05) 2.10 1.73
* Average of four replications; Figures given in parentheses are arcsine √ per cent angular transformed values.
Plate 2: Evaluation of fungal biocontrol agent against R. solani (SNG Rs-03) by dual culture technique in vitro
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