International Journal of Environment, Agriculture and Biotechnology (IJEAB) Vol-4, Issue-4, Jul-Aug- 2019

https://dx.doi.org/10.22161/ijeab/4445 ISSN: 2456-1878

Effect of different media on mycelium growth of

Sclerotium rolfsii Sacc. invitro condition
Prem Pandey1 and Shankar P. Gaire2
1
Agriculture and Forestry University (AFU), Rampur, Chitwan, Nepal
2
Texas A&M AgriLife Research center, Beaumont, TX, U.S.A.
Corresponding author Email: [email protected]

Abstract— An invitro experiment was conducted to study the effect o f different culture media on mycelial growth of
Sclerotium rolfsii Sacc. in Plant pathology Laboratory of Agriculture and Forestry University, Nepal. The
pathogenic isolate of S. rolfsii was isolated from symptomatic tomato and maintained in PDA. Seven different
culture media viz. Potato Dextrose Agar (PDA), Chickpea Dextrose Agar (ChDA), Bean Dextrose Agar (BDA),
Carrot Dextrose Agar (CDA), Papaya Dextrose Agar (PpDA), Czapek Dox Agar (CzDA) and Sabouraud’s Dextrose
Agar (SDA) were prepared and 5-mm diameter mycelial plugs from the margins of 3-days old S. rolfsiiculture was
transfer in the middle of each media plate. The radial mycelial growth was measured at 2 days interval for 14 days.
Chickpea Dextrose Agar (ChDA) had significantly higher mycelia growth, which was superior than Potato dextrose
agar (PDA). Papaya fruit, carrot root, chickpea grain and bean grain can be the common cheap source of carbon in
industrial development of culture media.
Keywords— Media, S. rolfsii, Mycelium, Carbon, isolate.

I. INTRODUCTION potato dext rose agar, V-8 juice agaretc whereas synthetic
Sclerotium rolfsiiSacc. is a necrotrophic soilborne med ia contain defined amounts of carbohydrates, nitrogen,
plant pathogen forming sclerotia as a survival structure and vitamin sources (eg: Czapek-Dox medium).
distributed worldwide. It causes various symptoms like Potato dextrose agar (PDA) is most co mmonly
seedling damping-off, root rot, collar rot, and stem rot in used natural media for the culture of fungi. Most lab used
more than 500 p lant species commonly legu mes, crucifers, med ia are not cost effective at a large-scale, so industrialists
and cucurbits (Barnett and Hunter, 1972). Because of have started to use several co mmon cheap sources of
prolific gro wth of S. rolfsii and ability to produce persistent carbon. So, the objective of this research was to identify
sclerotia, it is contributing in high degree of economic alternative natural fungal med ia using substrate with cheap
losses (Mahen et al., 1995).Under conducive conditions it source of carbon.
can causes 55-95% mortality of the crop at seedling stage
(Gurha and Dubey, 1982). In fection on plants by the II. MATERIALS AND METHODS
pathogen is governed and triggered by the initial Media preparation
recognition phenomenon. Establishment of the pathogen in Five natural media: Potato Dextrose Agar(PDA),
host depends upon some kind of similarity between two Chickpea Dext rose Agar(ChDA), Carrot Dext rose
interacting partners (De Vay & Adler, 1976). Agar(CDA), Bean Dextrose Agar(BDA), Papaya Dext rose
The capacity of fungi to utilize the available Agar(PpDA) and two synthetic media: Saubourds Dextrose
nutrients, of which carbohydrates are the major ones Agar(SDA),Czepado x Dextrose Agar(CzDA) were
determine the ability o f fungi to grow in different med ia. So prepared. 200 g of potato pieces, chickpeagrains, carrot
it was observed that the growth rate varies with different pieces, bean grains, papaya fruit pieceswere weighed
carbon sources. Fungal media containing high carbohydrate individually for their respective media v iz. PDA, Ch DA,
source, nitrogen source is required for the growth of fungi at CDA, BDA, PpDArespectively and boiled in 500 ml of
pH range of 5 to 6, and a temperature range from 15 to water for 10 minutes and filtered with muslin cloths. We
37˚C. Fungal culture media are natural and synthetic. added 20 g o f dext rose and 20g of agar in each media and
Natural media are co mposed of natural substrates like added distilled water to make the final volu me of 1000 ml.

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International Journal of Environment, Agriculture and Biotechnology (IJEAB) Vol-4, Issue-4, Jul-Aug- 2019
https://dx.doi.org/10.22161/ijeab/4445 ISSN: 2456-1878

The synthetic media were prepared as manufactural sulphate, 0.5 g of Potassium chloride, 0.01 g of Ferrous
instruction. SDA was prepared by mixing 40 g of dext rose, nitrate, 15 g of agar in 1000 ml of distilled water. The
10 g of Peptone and 15 g of Agar in 1000 ml o f distilled med ia were autoclaved for 121°C for 20 minutes at 15 psi
water and CzDA by 30 g of Sucrose, 2 g of sodium n itrate, pressure.
1 g of Dipotassium phosphate, 0.5 g of Magnesium

Table 1: Composition of different natural culture media

Ingredients PDA ChDA PpDA BDA CDA
Pieces/grain 200 g 200 g 200 g 200 g 200 g
Dextrose 20 g 20 g 20 g 20 g 20 g
Agar 20 g 20 g 20 g 20 g 20 g
Distilled water 1000 ml 1000 ml 1000 ml 1000 ml 1000 ml

Isolation of S. rolfsii III. RESULTS AND DISCUSSION

The pathogen, S. rolfsii was isolated from the Mycelial growth
stems of infected to mato plants grown in Chit wan, Nepal by Although the mycelial growth of S. rolfsiiin
tissue segment method (Rangaswami and Mahadevan, papaya dextrose agar was significantly lower than potato
1999) onPDA. Small p ieces of tissue of about 1 cm fro m dextrose agar up to 6 days of incubation, the mycelial
infected collar region with some healthy tissue were cut growth was at par in both growth media after 8-DAI (Tab le
with sterile blade. The pieces were surface sterilized with 2). In chickpea dextrose agar mediu m, the mycelial growth
1% sodium hypochlorite solution for 1 minute followed by was consistently superior over the 14-DAI (Tab le 2). This
three subsequent wash with sterilized distilled water. These result showed that chickpea dext rose agar, papaya dextrose
pieces were kept onto PDA med iu m in Petri d ishes. Plates agar and bean dextrose agar can be used as alternative
were incubated at 27 ± 2°C for 2 day. The pathogen was nutrient source for the growth of S. rolfsii.
identified as S. rolfsii based on its mycelial and sclerotia l Since sugars are the principle constituents of
characteristics (Barnett and Hunter, 1972). papaya with total content of 48.3% sucrose, 29.8% glucose
Experimental setup and 2% fructose (Chan and Kwok, 1975; Gomez et al.,
The experiment was conducted in Plant Pathology 2006) and it also contains sodium, potassium, Magnesium,
laboratory of Agriculture and Forestry University (AFU), Calcium, Iron and other vitamins which encourages the
Chit wan, Nepal. Appro ximately 20-ml of autoclaved med ia growth of pathogen.
was poured in each petri plate (9-cm diameter). Each plate Chickpea contains on an average 23% of protein,
was inoculated with a 5-mm d iameter mycelial p lugs fro m 64% carbohydrate, 6% crude fibre and 3% ash. It also has
the margins of 3-days old culture grown on PDA. Plates high mineral content like phosphorous, calcium,
were then incubated at 27±20 C with 12-hour photoperiod magnesium, iron and zinc etc. these nutrients are capable of
for t wo weeks. The rad ial mycelial growth was measured in supporting fungal growth (Junakali AK et al., 2009).
every 2 days interval for 14 days. The experiment was Kan A et al. (2010) reported that chickpea has
replicated four times. natural antibacterial activ ity against gram negative bacteria
Data analysis like Staphylococcus aureus, Bacillus substilis and
Analysis of variance (ANOVA) was conducted Enterococcus faecalis. Hence the natural antibacterial
using R version 3.6.0. Significant differences between property in Chickpea dextrose agar has additional benefits
treatments were determined using a Duncan multiple range as media more selective for fungi and less prone to bacterial
test (DMRT) at p=0.05. contamination.

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International Journal of Environment, Agriculture and Biotechnology (IJEAB) Vol-4, Issue-4, Jul-Aug- 2019
https://dx.doi.org/10.22161/ijeab/4445 ISSN: 2456-1878

Table 2: Effect of different growth media on the mycelial growth of the pathogen S. rolfsiiSacc.
Treatments 2 DAI 4 DAI 6 DAI 8 DAI 10 DAI 12 DAI 14 DAI
Papaya dextrose agar 1.25cd 1.70d 2.47cd 3.22bcd 6.10b 8.10a 9.00a
Chickpea dextrose agar 2.45a 3.35a 4.50a 5.37a 7.30a 7.95a 8.77ab
Potato dextrose agar 2.17ab 3.02ab 3.50b 3.97b 5.52b 7.35ab 7.87abc
Bean dextrose agar 1.75bc 2.65bc 3.32b 3.80bc 5.87 b 6.97ab 7.57bcd
Sabouraud dextrose agar 1.05d 1.62d 2.10d 2.65d 4.45 c 6.12bc 6.90cd
Carrot dextrose agar 0.92d 1.07e 1.30e 1.55e 3.85c 5.05cd 6.47d
Czapek Dox Agar 1.97ab 2.32c 2.95bc 3.05cd 3.82c 4.20d 4.55e
F test *** *** *** *** *** *** ***
SEM 0.119 0.139 0.237 0.239 0.529 0.821 0.613
LSD0.001 0.025 0.027 0.035 0.035 0.053 0.066 0.052
CV (%) 20.8 16.6 16.9 14.5 13.7 13.8 10.7

9
8
7
6 PpDA
5
4 ChDA
3 PDA
2
BDA
1
0 SDA
CDA
CzDA

Fig.1: Mycelial growth of S. rolfsii in different culture media at various days after incubation

Mycelium characteristics on different media

The myceliu m grown in chickpea dext rose agar (ChDA) had bright white color and Papaya Dextrose Agar (PpDA) had
dull white co lor. Ch ickpea dext rose agar (ChDA), Bean Dext rose Agar (BDA) had condensed myceliu m whereas uncondensed in
Carrot Dextrose Agar (CDA).
Table 3: Comparison of mycelium characters of S.rolfsii on different growth media
SN Media Growth Colony color Appearance
1 PpDA Initially slowand very fast in later dull white Cottony dull white
2 ChDA Moderate Bright white Dense cottony white
3 PDA Moderate Cottony white Medium Cottony white
4 BDA Moderate Light white Condensed cottony
5 SDA Slow to moderate Bright white Moderately condensed
6 CDA Slow to fast Bright white Uncondensed cottony
7 CzDA Slow Extra white Wavy like, condensed at
margins, aggregated

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International Journal of Environment, Agriculture and Biotechnology (IJEAB) Vol-4, Issue-4, Jul-Aug- 2019
https://dx.doi.org/10.22161/ijeab/4445 ISSN: 2456-1878

b growth of S. rolfsiiin different culture media at 6 DAI

Fig.2: Preparation of culture media (a) and mycelail

IV. CONCLUSION Fruit and its Relation to Sweet Taste. Journal of Food
Potato dextrose agar (PDA) is most co mmonly Sciences, 67(1), 442-447.
[10] Rangaswami, G., M ahadevan, A. 1999. Diseases of crop
used fungal med ia. As an alternative to PDA, Papaya
plants in India. Prentice Hall of India Pvt. Ltd., New Delhi.
dextrose agar (PpDA), Ch ickpea dextrose agar (ChDA ) and
6079 Pp.
Bean dextrose agar (BDA) can use for the culture of sterile
fungi like S. rolfsii, Papaya, ch ickpea and bean can be the
cheap source of carbon for manufacturing the fungal culture
med ia. Further study should be done to know the
sporulation of fungi in these natural media.

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