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The Pharma Innovation Journal 2022; 11(4): 14-19

ISSN (E): 2277- 7695

ISSN (P): 2349-8242

NAAS Rating: 5.23

TPI 2022; 11(4): 14-19

© 2022 TPI

www.thepharmajournal.com

Received: 18-01-2022

Accepted: 28-02-2022

Ravikumar G Vaniya

Department of Plant Pathology,

N. M. College of Agriculture,

Navsari Agricultural University,

Navsari, Gujarat, India

Pushpendra Singh

Department of Plant Pathology,

College of Agriculture, NAU,

Waghai, Gujarat, India

AJ Deshmukh

Department of Plant Pathology,

College of Agriculture, NAU,

Waghai, Gujarat, India

Corresponding Author:

Ravikumar G Vaniya

Department of Plant Pathology,

N. M. College of Agriculture,

Navsari Agricultural University,

Navsari, Gujarat, India

Effect of different cultural media on growth of different

isolates of Sclerotium rolfsii Sacc. causing stem rot of

Indian bean

Ravikumar G Vaniya, Pushpendra Singh and AJ Deshmukh

Abstract Sclerotium rolfsii Sacc. is one of the most important soil-borne plant pathogen which causes severe loss

at the time of seedling development. It also causes stem rot or collar rot in several crops and wild plants.

In this present investigation five different cultural solid and liquid media viz., Potato dextrose agar and

broth, Host extract agar and broth (Indian bean), Czapek’s agar and broth, Richard’s agar and broth and

Sabouraud’s agar and broth were analyzed for in vitro mycelial growth and dry mycelial weight of

different eight isolates of S. rolfsii collected from different location. The cultural characteristics indicated

that the excellent mycelial growth and dry mycelial weight were observed on different semi-synthetic

media viz., Potato dextrose agar and broth followed by Host leaf extract agar medium and broth (Indian

bean), Czapek’s dextrose agar medium and broth, Richard’s Agar medium and broth. Sabouraud’s Agar

medium and broth recorded comparatively less growth and dry mycelial weight of different eight isolates

of S. rolfsii.

Keywords: Sclerotium rolfsii Sacc. Indian bean, different cultural media, mycelial growth and dry

mycelial weight

1. Introduction

Indian bean (Lablab purpureus L.) belonging to family Fabaceae, is one of the most ancient

among the cultivated crops and is presently grown throughout the tropical regions in Asia,

Africa and America. The Indian bean crop is affected by many fungal diseases viz.,

anthracnose (Colletotrichum lindemuthianum Briosi and Cavara), rust (Uromyces

appendiculatus Pers), powdery mildew (Erysiphe polygoni Weltzien), ashy blight and charcoal

rot (Macrophomina phaseolina Tassi), bacterial diseases viz., bacterial blight (Xanthomonas

phaseoli Dowson) and viral disease viz., bean common mosaic virus (BCMV) and yellow

mosaic virus (BYMV) (Bose et al., 2001) [1] and alternaria leaf spot of dolichos bean caused

by Alternaria sp. (Maheshwari and Singh, 1998) [12]. Among all of the soil borne diseases,

stem rot caused by Sclerotium rolfsii Sacc. Is gaining a serious status due to its high severity.

This disease is also referred as Sclerotium blight, Sclerotium wilt, Southern blight, Southern

stem rot and white mold. S. rolfsii is a soil-borne pathogen that predominantly distributed in

the tropics, sub-tropics and other warm temperate regions of the world causing root rot, stem

rot, wilt and foot rot on more than 500 plant species including almost all the agricultural and

horticultural crops (Aycock, 1996) [2]. S. rolfsii causing stem rot or collar rot or wilt is a well

known polyphagous, ubiquitous and non-target pathogen. It is one of the most destructive soil

inhabiting pathogen and causes heavy loss to the crops in both kharif and rabi season. It is

common where high temperatures exist during the rainy season. The present study on effect of

different cultural media on growth of different isolates of S. rolfsii causing stem rot of Indian

bean was carried out and results thus obtained were presented hereunder.

2. Materials and Methods

2.1 Collection and isolation of diseased samples

Indian bean plant showing the typical symptoms of stem rot were collected from Pulses

research station, Navsari Agricultural University, Navsari. The infected portion of plant parts

were taken for isolation. The isolation of fungus was done by following standard tissue

isolation technique. The infected pieces were cut into small bits and then washing in running

water. These bits were surface sterilized with 0.1% Mercuric chloride (HgCl2) solution for one

minute followed by washing in distilled sterile water, then aseptically transferred to PDA plate

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fungal growth hyphal tip was used for further purification.

Same procedure was followed to obtain the pure culture of S.

rolfsii from all locations.

Table 1: Details of collected different isolates are given below.

Sr. No. District Taluka Village Designation

1. Surat

Olpad Kudiyana SrKUD

2. Olpad Kuvad SrKUV

3.

Navsari

Jalalpore N.A.U, Eru SrERU

4. Jalalpore Kharsad SrKHR

5. Chikhli Peladi bhervi SrPEL

6. Chikhli (Khergam) Jamanpada SrJAM

7. Bharuch

Amod Bhimpura SrBHM

8. Amod (Vagra) Ghamnad SrGHM

2.2 Cultural studies

2.2.1 Growth characteristics of different isolates of S.

rolfsii on different solid media

The growth characteristics of different isolates of S. rolfsii

were studied on different five solid media viz., Potato

dextrose agar, Richards’s agar, Czapek’s agar, Sabouraud’s

agar and Host leaf extract agar. All the media were sterilized

at 1.1 kg/cm2 pressure for 15 minutes. To carry out the study,

20 ml of each of the medium was poured in 90 mm Petri

plates. Such petri plates were inoculated with 5 mm disc cut

from periphery of actively growing seven day old culture of

the individual isolate grown on PDA in petri plate and

incubated at 27±1 oC. Each treatment was replicate 4 times.

Observations were taken when the pathogen covered

complete Petri plate in any one of the media tested. The

colony diameter was recorded by averaging the radial growth

of the colony in 2 directions.

2.2.2 Growth characteristics of different isolates of S.

rolfsii on different liquid media

The liquid media used were same as that of solid media

except agar was not added to the liquid media composition

and preparation were same for both solid and liquid media. 20

ml of the media was poured in to each 100 ml conical flask

and were sterilized at 1.1 kg/cm2 pressure for 15 minutes.

Each of the flasks was inoculated aseptically with five mm

culture disc obtained from the growing periphery of seven day

old culture of the individual isolate grown on PDA in Petri

plate and incubated at 27±1 oC for 10 days as maximum

growth was observed on 10th day.

The mycelial mat was filtered through Whatman no.42 filter

paper disc of 12.50 cm diameter dried to constant weight at 60

°C in an electrical oven prior to filtration. The mycelial mat

on the filter paper was washed thoroughly with the distilled

water to remove any salts likely to be associated with the

mycelium and dried to a constant weight in an electrical oven

at 60°C, cooled in desiccators and weighed immediately on an

analytical balance. The weight of the dry mycelium was

recorded for respective isolate and the data were statistically

analyzed.

3. Results and Discussion

The results obtained from the present investigation are

summarized below:

3.1 Mycelial growth of different isolates of S. rolfsii on different solid media: All the different eight isolates exhibited greater variation with respect to utilization of various nutrient substances. The cultural characteristics of the eight isolates of S. rolfsii were studied on five different solid media. For many of the isolates semi - synthetic media supported better growth than the synthetic media. Among 5 media tested potato dextrose agar supported better growth followed by Host leaf extract agar, Czapek’s agar and Richard’s agar while, least growth was recorded in Sabouraud’s agar. On the other hand isolates exhibited minor variation with regards to their growth on these media presented in Table 02, Plate 01, 02, 03 and Figure 01. For all the eight isolates of S. rolfsii, it is evident from the data presented in Table 02 that S. rolfsii preferred Potato Dextrose Agar medium for better growth. Colony diameter was observed significantly superior on Potato Dextrose Agar medium 85.25 mm (SrKUV), 89.50 mm (SrERU), 82.00 mm (SrKHR), 84.00 mm (SrPEL), 78.25 mm (SrJAM), 86.00 mm (SrBHM), 80.25 mm (SrKUD) and 83.00 mm (SrGHM) after 4 days of inoculation; it’s followed by Host leaf extract agar medium, Czapek’s dextrose agar medium, Richard’s Agar medium. Sabouraud’s Agar medium recorded comparatively less growth of S. rolfsii 64.50 mm (SrKUV), 64.75 mm (SrERU), 59.75 mm (SrKHR), 60.00 mm (SrPEL), 64.00 mm (SrJAM), 62.50 mm (SrBHM), 60.50 mm (SrKUD) and 63.00 mm (SrGHM) after 4 days of inoculation. Similar results were also obtained by Ayed et al., (2018) [3] studied the effects of different culture media were evaluated on S. rolfsii mycelial growth, sclerotial production. Kushwaha et al., (2019) [11] examined total of seven media were used for studying the growth of S. rolfsii in Lentil and Archana et al., (2019) [1] also proved the effect of nine different cultural media like Potato dextrose agar, Host extract agar (Chilli), Oat meal agar, Carrot agar, Plane agar, Richards’s agar, Czapek’s agar, Elliot’s agar, Sabouraud’s agar on mycelial growth of S. rolfsii causing Root rot of chilli.

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Fig 1: Mycelial growth of different isolates of S. rolfsii on different solid media.

Table 2: Effect of different solid media on different isolates of S. rolfsii causing stem rot Indian bean

Sr.

No. Solid media

Surat district Navsari district Bharuch district

Mycelial

growth

4 DAI

(mm) SrKUD

Mycelial

growth

4 DAI

(mm) SrKUV

Mycelial

growth

4 DAI

(mm) SrERU

Mycelial

growth

4 DAI

(mm) SrKHR

Mycelial

growth

4 DAI

(mm) SrPEL

Mycelial

growth

4 DAI

(mm) SrJAM

Mycelial

growth

4 DAI

(mm) SrBHM

Mycelial

growth

4 DAI (mm)

SrGHM

01 Potato Dextrose

Agar 80.25 85.25 89.50 82.00 84.00 78.25 86.00 83.00

02 Richard’s Agar 78.50 76.25 77.25 73.00 73.50 71.75 77.00 71.50

03 Czapek’s Agar 83.25 80.75 82.75 78.50 79.75 76.00 81.50 74.50

04 Sabouraud’s Agar 60.50 64.50 64.75 59.75 60.00 64.00 62.50 63.00

05 Host leaf extract

Agar 82.00 84.00 82.00 80.25 79.25 74.50 83.00 80.75

S Em. ± 1.28 1.16 1.38 1.70 1.44 1.40 1.18 1.25

CD (p= 0.05) 3.91 3.54 4.21 5.18 4.38 4.27 3.59 3.82

CV (%) 3.34 2.97 3.49 4.56 3.82 3.85 3.03 3.37

DAI: Days after Inoculation

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5.2 Dry mycelial weight of different isolates of S. rolfsii on

different liquid media

The isolates of S. rolfsii were grown on five different liquid

media and dry mycelia weight was recorded after ten days of

incubation. The results indicated that, the dry mycelial weight

was significantly varied among all the isolates on these liquid

media presented in Table 03 and plate 04, 05 and figure 02.

Every living organism has a definite growth pattern in which

it attains a maximum growth and declines thereafter. In the

present study the fungus attained maximum growth on 10th

day of incubation in potato dextrose broth. This was taken as

maximum peak period for further experimental studies.

For all the eight isolates of S. rolfsii, it is evident from the

data presented in Table 03 that S. rolfsii preferred Potato

Dextrose broth media for better growth of mycelial disk of

pathogen. Dry mycelial weight was observed significantly

superior on Potato Dextrose broth medium 253.75 mg

(SrKUV), 250.50 mg (SrERU), 243.50 mg (SrKHR), 255.00

mg (SrPEL), 210.25 mg (SrJAM), 236.50 mg (SrBHM),

261.25 mg (SrKUD) and 220.75 mg (SrGHM) after 10 days

of inoculation; it’s followed by Host leaf extract broth

medium, Czapek’s dextrose broth medium, Richard’s broth

medium. Sabouraud’s broth medium recorded comparatively

less growth of S. rolfsii 175.25 mg (SrKUV), 179.25 mg

(SrERU), 186.00 mg (SrKHR), 174.50 mg (SrPEL), 161.25

mg (SrJAM), 168.25 (SrBHM), 171.50 mg (SrKUD) and

159.00 mg (SrGHM) after 10 days of inoculation.

Similar findings were obtained by Banakar et al., (2017) [16]

studied cultural variability in S. rolfsii causing foot rot of

tomato according to mycelial and sclerotial characters which

observed on different liquid media. These cultural studies

showed the maximum dry mycelial weight of fungus observed

in potato dextrose broth (750mg). Kushwaha et al., (2019) [11]

examined total of seven liquid media were used for studying

the growth of S. rolfsii in Lentil. Their results reported that

potato-dextrose medium was most suitable for mycelial

growth and sclerotia production of S. rolfsii.

Fig 2: Dry mycelial weight of different isolates of S. rolfsii on different liquid media

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The Pharma Innovation Journal http://www.thepharmajournal.com Table 3: Dry mycelial weight of different isolates of S. rolfsii on different liquid media

Sr.

No.

liquid media

Surat district Navsari district Bharuch district

Dry

Mycelial

weight 10

DAI (mg

SrKUD

Dry

Mycelial

weight 10

DAI (mg)

SrKUV

Dry

Mycelial

weight 10

DAI (mg)

SrERU

Dry Mycelial

weight 10 DAI

(mg) SrKHR

Dry Mycelial

weight 10 DAI

(mg) SrPEL

Dry Mycelial

weight 10 DAI

(mg) SrJAM

Dry Mycelial

weight 10 DAI

(mg) SrBHM

Dry Mycelial

weight 10 DAI

(mg) SrGHM

01 Potato Dextrose Broth 261.25 253.75 250.50 243.50 255.00 210.25 236.50 220.75

02 Richard’s Broth 216.75 212.00 201.50 215.75 217.25 170.50 209.50 184.50

03 Czapek’s Broth 233.75 225.25 223.75 224.25 228.00 192.75 217.75 202.25

04 Sabouraud’s Broth 171.50 175.25 179.75 186.00 174.50 161.25 168.25 159.00

05 Host leaf extract Broth 239.75 233.00 239.75 232.00 242.75 197.50 223.25 202.50

S Em. ± 1.77 1.88 2.34 2.20 2.16 2.81 1.53 2.37

CD (p= 0.05) 5.36 5.66 7.07 6.63 6.51 8.49 4.64 7.14

CV (%) 1.58 1.71 2.14 1.99 1.93 3.02 1.45 2.44

4. Conclusion

The cultural characteristics of all the eight isolates of S. rolfsii

were studied on five different solid media. Among five media

tested potato dextrose agar supported better growth followed

by Host leaf extract agar, Czapek’s agar and Richard’s agar.

While, least growth was recorded in Sabouraud’s agar.

The isolates of S. rolfsii were grown on five different liquid

media and dry mycelia weight was recorded after ten days of

incubation. The results indicated that, the dry mycelial weight

was significantly varied among all the isolates on these liquid

media. Potato dextrose broth supported the better growth of

all isolates followed by host leaf extract broth, Czapek’s broth

and Richard’s broth. While, least growth was recorded on

Sabouraud’s broth. The present investigation strongly suggest

that the there is a morphological and cultural variation on

different solid and liquid media.

5. References

1. Archana TS, Pankaj B, Jagtap SD and Patil BS. Effect of

different cultural media on growth of S. rolfsii Sacc.

Causing root rot of chilli. International Journal of Current

Microbiology and Applied Sciences. 2019;8(2):3019-

3024.

2. Aycock R. Stem rot and other diseases caused by S.

rolfsii. North Carolina agricultural college, Experimental

Sustainable technology Bulletin, 1996, 174-202.

~ 19 ~

The Pharma Innovation Journal http://www.thepharmajournal.com 3. Ayed F, Jabnoun-Khiareddine H, Aydi Ben Abdallah R,

Daami-Remadi M. Effect of temperatures and culture

media on S. rolfsii mycelial growth, sclerotial formation

and germination. Journal of Plant Pathology

Microbiology. 2018;9:429.

4. Basamma Naik K, Madhura C, Manjunath L. Cultural

and physiological studies on S. rolfsii causing sclerotium

wilt of potato. International Journal of Plant Sciences.

2012;7(2):216-19.

5. Bharathi D, Narayana Swamy H. Influence of different

culture media on growth of S. rolfsii causing stem rot of

tuberose (Polianthes tuberosa L.). Environment &

Ecology. 2017;35(3):2598-2601.

6. Bose TK, Kabir J, Das P, Joy PP. Tropical Horticulture,

Pub. Naya Prakash, Calcutta. 2001;2:167.

7. Chaurasia S, Chaurasia AK, Chaurasia S, Chaurasia S.

Factors affecting the growth and sclerotial production in

S. rolfsii causing foot rot of brinjal. Indian Journal of

Fundamental and Applied Life Sciences. 2013;3(2):73-

84.

8. Divya Bharathi AR, Benagi VI. Cultural and

morphological variability among the isolates of S. rolfsii

Sacc. causing wilt complex disease of betel vine (Piper

betle L.). International Journal of Current Microbiology

and Applied Sciences. 2018;7(6):1014-1019.

9. Ghevariya TV, Patel PR. Effect of light and ph on the

growth of S. rolfsii in vitro on collar rot of Indian bean.

International Journal of Current Microbiology and

Applied Sciences. 2019;8(10):1268-1274.

10. Jabbar Sab, Nagaraja A, Mallikarjun, Manu TG.

Variability among the S. rolfsii Sacc. Isolates from

southern karnataka. International Journal on Agricultural

Sciences. 2014;5(2):229-236.

11. Kushwaha SK, Kumar S, Chaudhary B, Sahu R. Effect of

different media, pH and temperature on growth and

sclerotia formation of S. rolfsii Sacc. Causing collar rot of

lentil. Chemical Science Review and Letters.

2019;8(29):01-05.

12. Maheshwari SK, Singh DV. Control of Alternaria leaf

spot of dolichos bean by foliar application of fungicides,

Annuals of plant protection Science. 1998;6(2):191-193.

13. Manu T, Nagaraja A, Manjunatha S. Morphological and

cultural variability among the S. rolfsii isolates. Journal

of Pharmacognosy and Phytochemistry. 2018;7(1):904-

907.

14. Misra AP, Haque SQ. Factors affecting the growth and

sclerotial production in S. rolfsii Sacc. Causing storage

rot of potato. 1962;56(03):157-168.

15. Reddi Kumar M, Madhavi Santhoshi MV, Giridhara

Krishna T and Raja Reddy K. Cultural and morphological

variability S. rolfsii isolates infecting groundnut and its

reaction to some fungicidal. International Journal of

Current Microbiology and Applied Sciences. 2014;

3(10):553-561.

16. Sahana N, Banakar VB, Sanath Kumar, Thejesha AG.

Morphological and cultural Studies of S. rolfsii Sacc.

Causing foot rot disease of tomato. International Journal

of Current Microbiology and Applied Sciences.

2017;6(3):1146-1153.

17. Sivakumar T, Sanjeevkumar K, Balabaskar P. Variability

in S. rolfsii Sacc. Causing stem rot of ground nut.

Bulletin of Environment, Pharmacology and Life

Sciences. 2016;(02):92-99.

18. Swetha GS, Hegde YR. Studies on variability and

management of S. rolfsii Sacc. Causing wilt of Stevia.

M.Sc. (Agri.) Thesis, University of Agricultural science,

Dharwad, 2011.

19. Zape AS, Gade RM, Ravindra Singh. Physiological

studies on different media, pH and temperature on S.

rolfsii isolates of soybean. Scholarly Journal of

Agricultural Science. 2013;2(6):238-241.