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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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