INTERNATIONAL JOURNAL OF PLANT PROTECTION

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VOLUME 12 | ISSUE 2 | OCTOBER, 2019 | 127-131

IJPP

RESEARCH PAPER DOI : 10.15740/HAS/IJPP/12.2/127-131

In vitro antagonistic activity of Trichoderma species against

important soil borne pathogens
N.N. Sohaliya*, D.M. Pathak and J.R. Pandya
Department of Plant Pathology, College of Agriculture, NAU, Campus Bharuch, Maktampur (Gujarat) India

ARITCLE INFO ABSTRACT

Received : 08.07.2019 The rhizospheric soil samples were collected from different cultivated agricultural fields
Revised : 01.09.2019 from Bharuch and Narmada districts and the mycoflora were isolated by serial dilution
Accepted : 15.09.2019
plate technique. Total eight isolates of Trichoderma viride, Trichoderma harzianum
KEY WORDS : and Trichoderma longibrachiatum were isolated on Potato Dextrose Agar medium.
Antagonistic activity, Soil borne The green coloured colonies were identified by comparing with taxonomic key. They
pathogens were purified by single spore isolation method and maintained on PDA slants at 40 C in
the refrigerator at Department of Pl.Pathology, NMCA, NAU, Navsari. Soil borne
Pathogenic fungi viz., Sclerotium rolfsii, Macrophomina phaseolina and Fusarium
oxysporum were isolated from the respective diseased plants during field survey in
Navsari Agricultural University farm, Navsari. The antagonistic efficacy against test
pathogen was evaluated by dual culture plate technique. Among all 8 Trichoderma
isolates, The Trichoderma harzianum NCJD8 isolate has showed 24.17 mm mycelial
growth with 73.15 per cent inhibition of Sclerotium rolfsii, where in case of
Macrophomina phaseolina, Minimum mycelial growth (32.67 mm) of test pathogen
was recorded in T. longibracheatum NCJD2 isolate with 63.70 per cent inhibition which
was statistically at par with T. viride NCJD6 (34.50 mm) with 61.67 per cent inhibition
and when it comes to Fusarium oxysporum, T. harzianum NCJD5 showed minimum
mycelial growth and highest per cent growth inhibition (75.56%) with 22.00 mm colony
diameter of the pathogen after seven days of incubation which was statistically at par
with isolate T. harzianum NCJD1 (72.96%) with 24.33 mm colony diameter.
How to view point the article : Sohaliya, N.N., Pathak, D.M. and Pandya, J.R. (2019). In vitro
antagonistic activity of Trichoderma species against important soil borne pathogens. Internat. J.
*Corresponding author: Plant Protec., 12(2) : 127-131, DOI : 10.15740/HAS/IJPP/12.2/127-131, Copyright@ 2019:
Email : dmpathak@nau.in Hind Agri-Horticultural Society.

INTRODUCTION organisms have developed resistance against chemical

Injudicious use of pesticide in agriculture leads to fungicides (Gaigole et al., 2011). Fungicides pose serious
environmental pollution, causing hazardous effects to both hazards to health and environment. This emphasized an
environment and food quality. Many pathogenic micro- alternative method to control fungal diseases. Bio-control

HIND AGRICULTURAL RESEARCH AND TRAINING INSTITUTE

 N.N. Sohaliya, D.M. Pathak and J.R. Pandya
of plant pathogen is an ecofriendly, safe approach that
utilizes antagonistic micro-organisms as a potential means
of disease control. Trichoderma is a non-pathogenic bio-
control agent having antagonistic properties against many
plant pathogens in various degrees (Dennis and Webster,
1971a). To determine the antagonistic property of
Trichoderma spp. against Sclerotium rolfsii,
Macrophomina phaseolina and Fusarium oxysporum,
isolates were compared on a medium and at temperature
where antagonist and pathogen both can grow well in
the laboratory. The present study was undertaken, to
find out the bio-control efficacy of Trichoderma spp.
against above mentioned pathogens.
MATERIAL AND METHODS
Isolation of antagonist:
The rhizospheric soil samples were collected from
different cultivated agricultural fields from Bharuch and
Narmada districts and the mycoflora were isolated by
serial dilution plate technique (Johnson and Curl, 1972).
Total eight isolates of Trichoderma viride, Trichoderma
harzianum and Trichoderma longibrachiatum were
isolated on Potato Dextrose Agar medium. The green
coloured colonies were identified by comparing with
taxonomic key. They were purified by single spore
isolation method and maintained on PDA slants at 40 C
in the refrigerator.
Isolation of pathogenic fungi:
Pathogenic fungi viz., Mp was isolated from the
diseased plant of sesame, Sr from infected plant of Indian
bean and Fo from wilt infected pigeonpea root during
field survey in Navsari Agricultural University farm,
Navsari of Gujarat State, India (Aneja, 2003). Parts of
plants with symptoms of infection were surface sterilised
by immersion in 0.1% sodium hypochlorite for 30
seconds and then rinsed thoroughly with sterile distilled
water three times. They were transferred to potato
dextrose agar (PDA) medium in petri plates and
incubated at 26 ± 2 0 C for seven days. They were
purified by single spore isolation method and maintained
on PDA slants at 40 C in the refrigerator.
Dual culture plate technique:
Trichoderma spp. were evaluated against
Sclerotium rolfsii, Macrophomina phaseolina and
Fusarium oxysporum by the dual culture plate technique
Internat. J. Plant Protec., 12(2) Oct., 2019 : 127-131
128 HIND AGRICULTURAL RESEARCH AND TRAINING INSTITUTE
(Dennis and Webster, 1971b). The antagonistic efficacy
against test pathogen was evaluated on PDA medium.
Both pathogen and antagonists were grown on PDA
plates separately for 5 days. Mycelial discs of 5 mm in
diameter of antagonist was excised from the edge of an
actively growing culture plate and inoculated opposite to
the pathogenic fungi in the same plate 2 cm away from
the edge similarly. For each treatment three replicates
were maintained and incubated at 27 ± 20 C. Control
plates were maintained for test pathogen in triplicate.
Both, antagonist and test pathogen were placed
equidistant from the periphery so that they would get
equal opportunity for their growth. After the incubation
period, the radial growth of Sr, Mp and Fo in control, as
well as in treatment plate was measured and the per
cent inhibition was calculated using the formula
(Edgington et al., 1971).
C-T
I x 100
C
where I = Percentage inhibition of radial growth of
pathogen (%),
C = Radial growth of the pathogen (mm) in control,
T = Radial growth of the pathogen (mm) in treatment
The mycelial mats from zone of interaction in dual
culture plate between pathogen and antagonist were
placed on glass slide. The glass slides were stained with
lacto phenol cotton blue (HiMedia) to improve the
visibility of the hyphae and then observed under a light
microscope (CH20i Olympus, India). The hyphal
interaction between the mycelia of opposite colonies was
studied.
RESULTS AND DISCUSSION
Isolates of Trichoderma spp. were evaluated for
their antifungal activity against Sclerotium rolfsii,
Macrophomina phaseolina and Fusarium oxysporum.
Among all 8 Trichoderma isolates, The Trichoderma
harzianum NCJD8 isolate has showed 24.17 mm
mycelial growth with 73.15 per cent inhibition of
Sclerotium rolfsii, where in case of Macrophomina
phaseolina, Minimum mycelial growth (32.67 mm) of
test pathogen was recorded in T. longibracheatum
NCJD2 isolate with 63.70 per cent inhibition which was
statistically at par with T. viride NCJD6 (34.50 mm)
with 61.67 per cent inhibition and when it comes to
Fusarium oxysporum, T. harzianum NCJD5 showed
minimum mycelial growth and highest per cent growth
 In vitro antagonistic activity of Trichoderma species against important soil borne pathogens

Colony of Trichoderma Trichoderma harzianum NCJD8 + Sclerotium rolfsii

T. longibracheatum NCJD2 + Macrophomina phaseolina T. harzianum NCJD5 + Fusarium oxysporum

Table 1: Antagonistic effect of Trichoderma isolates against S. rolfsii (Indian bean stem rot)
Sr. No. Isolates ACD (mm) PIMG
1. T. harzianum strain NCJD8 24.17 73.15
2. T. harzianum strain NCJD1 46.67 48.15
3. T. longibracheatum strain NCJD4 29.33 67.41
4. T. longibracheatum strain NCJD2 45.00 50.00
5. T. harzianum strain NCJD5 40.67 54.81
6. T. viride strain NCJD6 31.17 65.37
7. T. longibracheatum strain NCJD7 38.17 57.59
8. T. viride strain NCJD3 43.17 52.04
9. Control 90 -
S.E.± 0.13 1.09
C.D. (P=0.05) 0.39 3.28
C.V.% 3.71 3.79

Internat. J. Plant Protec., 12(2) Oct., 2019 : 127-131

HIND AGRICULTURAL RESEARCH AND TRAINING INSTITUTE
129
 N.N. Sohaliya, D.M. Pathak and J.R. Pandya

Table 2: Antagonistic effect of Trichoderma isolates against M. phaseolina (Chickpea root rot)
Sr. No. Isolates ACD (mm) PIMG
1. T. harzianum strain NCJD8 42.17 53.15
2. T. harzianum strain NCJD1 43.17 52.04
3. T. longibracheatum strain NCJD4 41.50 53.89
4. T. longibracheatum strain NCJD2 32.67 63.70
5. T. harzianum strain NCJD5 40.33 55.19
6. T. viride strain NCJD6 34.50 61.67
7. T. longibracheatum strain NCJD7 38.17 57.59
8. T. viride strain NCJD3 41.67 53.70
9. Control 90 -
S.E.± 0.11 0.88
C.D. (P=0.05) 0.33 2.64
C.V.% 3.03 3.13

Table 3: Antagonistic effect of Trichoderma isolates against F. oxysporum f.sp. udum (Pigeonpea wilt)
Sr. No. Isolates ACD (mm) PIMG
1. T. harzianum strain NCJD8 26.84 70.18
2. T. harzianum strain NCJD1 24.33 72.96
3. T. longibracheatum strain NCJD4 25.70 71.44
4. T. longibracheatum strain NCJD2 28.17 68.70
5. T. harzianum strain NCJD5 22.00 75.56
6. T. viride strain NCJD6 26.50 70.56
7. T. longibracheatum strain NCJD7 25.67 71.48
8. T. viride strain NCJD3 37.17 58.70
9. Control 90 -
S.E.± 0.11 0.76
C.D. (P=0.05) 0.33 2.27
C.V.% 3.03 2.31

inhibition (75.56%) with 22.00 mm colony diameter of
the pathogen after seven days of incubation which was
statistically at par with isolate T. harzianum NCJD1
(72.96%) with 24.33 mm colony diameter.
Overall experimental results clearly indicated that
all the isolates proved effective against the all the
pathogens tested. Maximum average inhibitory effect
was found with isolate TVMs (T. viride) against all three
pathogens viz., F. oxysporum f.sp. udum, S. rolfsii and
M. phaseolina which was followed by THSh2, TLS,
TLMa, THMo, TLD, THSh1 and TVH. In general, the
results of isolate wise efficacy showed that isolate THMo
(T. harzianum) was found comparatively superior with
an average mycelial growth inhibition of 75.56 per cent
as compared to the rest. The variation of different isolates
in their efficacy against the fungal pathogens might be
due to different levels of secondary metabolites produced
by different isolates. The results of the present study
also indicated that the effect of bio-control agents may
be specific and hence more study on this aspect is
required. The present results are in agreement with the
earlier results obtained by Gurha (2001); Pan and Bhagat
(2007); Vishwanath et al. (2008) and Madhusudan et
al. (2010).
Conclusion:
Plant diseases caused by pathogenic fungi constrain
the yields. In agriculture, farmers still depend on the use
of chemical fungicides to control plant diseases.
However, misuse of these synthetic chemicals cause
hazardous to both environment and health. The
alternative method for replacement of chemical
fungicides has led to the use of biological control agents.
Biocontrol of soil borne pathogens is met by the
introduction of micro-organisms. Micro-organisms that
grow in the rhizosphere are ideal for use as biocontrol
agents. Our studies proved that Trichoderma spp. have
the potential to control F. oxysporum f.sp. udum, S.

Internat. J. Plant Protec., 12(2) Oct., 2019 : 127-131

130 HIND AGRICULTURAL RESEARCH AND TRAINING INSTITUTE
 In vitro antagonistic activity of Trichoderma species against important soil borne pathogens

rolfsii and M. phaseolina in vitro to the extent of 75.56
per cent, 73.15 and 63.70 per cent, respectively. The
potential use of these biocontrol agents can be improved
by isolation, formulation and application methods,
particularly in the field.
REFERENCES
Aneja, K.R. (2003). Experiments in Microbiology, Plant
Pathology and Biotechnology, 4th ed., New Age International
(P) Ltd.
Dennis, C. and Webster, J. (1971a). Antagonistic properties
of species- groups of Trichoderma II. Production of volatile
antibiotics. Transactions British Mycological Society, 57 :
41–43.
Dennis, C. and Webster, J. (1971b). Antagonistic properties
of species groups of Trichoderma I. Production of non-volatile
antibiotics. Transactions British Mycological Society, 57 :
25–39.
Edgington, L.V., Khew, K.L. and Barrron, G.L. (1971).
Fungitoxic Spectrum of Benzimidazole Compounds.
Phytopathol., 61 : 42 - 44.
Gaigole, A.H., Wagh, G.N. and Khadse, A.C. (2011). Antifungal
activity of Trichoderma spesies against soil borne pathogen.
Asiatic J. Biotechnol. Resourc., 4 : 461-465.
Gurha, S.N. (2001). Effect of some Trichoderma spp. on the
growth of different isolates of Fusarium oxysporum f.sp. ciceri
in vitro. Annals Plant Protec. Sci., 9(2) : 332-334.
Johnson, L.F. and Curl, E.A. (1972). Methods for research on
the ecology of soil borne plant pathogens, Burgress
Publishing Co, Minneapolis, pp.v+247.
Madhusudan, P., Gopal, K., Haritha, V., Sangale, U.R. and
Rao, S.V.R.K. (2010). Compatability of Trichoderma viride
with fungicides and efficiency against Fusarium solani. J.
Plant Dis. Sci., 5 : 23-26.
Pan, S. and Bhagat, S. (2007). Antagonistic potential of
Trichoderma and Gliocladium spp. from West Bengal. J.
Mycology & Plant Pathol., 37(2) : 235-243.
Vishwanath, K., Gopal, K. and Gopi, V. (2008). Isolation of
potential Trichoderma spp. Associated with dry root rot
infected acid lime (Citrus aurantifolia Swingle). J. Plant
Disease Sci., 3 (2) : 165-168.

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P-ISSN: 2349–8528
E-ISSN: 2321–4902
IJCS 2019; 7(2): 1029-1030
© 2019 IJCS
Received: 16-01-2019
Accepted: 20-02-2019
AR Khunt
Department of Plant Pathology,
College of Agriculture, Junagadh
Agricultural University,
Junagadh, Gujarat, India
LF Akbari
Professor and Head, Department
of Plant Pathology, College of
Agriculture, Junagadh
Agricultural University,
Junagadh, Gujarat, India
GJ Goswami
Department of Plant Pathology,
College of Agriculture, Junagadh
Agricultural University,
Junagadh, Gujarat, India
NN Patel
Subject matter Specialist (Plant
Protection), Krishi Vigyan
Kendra, Kutch-I, Gujarat, India
NN Sohaliya
Department of Plant Pathology,
College of Agriculture, Junagadh
Agricultural University,
Junagadh, Gujarat, India
Correspondence
AR Khunt
Department of Plant Pathology,
College of Agriculture, Junagadh
Agricultural University,
Junagadh, Gujarat, India
International Journal of Chemical Studies 2019; 7(2): 1029-1030
Preliminary study on the effect of temperature in
uredospore germination of wheat leaf rust
(Puccinia triticina Eriks.)
AR Khunt, LF Akbari, GJ Goswami, NN Patel and NN Sohaliya
Abstract
Leaf rust, caused by the fungus Puccinia triticina, has been one of the major foliar diseases of wheat.
Temperature and moisture are the key climatic factors which interact with leaf rust infection and the rate
of disease development. To know the effect of different temperature on rust uredospore germination;
various temperature i.e. 5, 10, 15, 20, 25, 30 and 35°C were set in a B.O.D. incubator. The studies on
effect of temperature on germination of uredospores revealed that maximum per cent spore germination
with 6 hours of incubation, was recorded in case of temperature range of 20-25°C. The mean maximum
spore germination was recorded at 20°C (92.95%) which was followed by at 25°C (87.65%).
Keywords: Puccinia triticina, leaf rust, temperature, uredospore germination and wheat
Introduction
Wheat (Triticum aestivum L.) is one of the most important food crops and is a staple food for
over one third of the world’s population. More of the earth’s surface is covered by wheat than
with any other food crop. Wheat is a widely grown cereal in climates varying from temperate,
irrigated to dry, high rainfall, warm humid to dry and cold. As a C3 plant, wheat is capable of
thriving in cool environments (Acevedo et al., 2006) [1]. One of the major constraints of
production in the country is occurrence of different diseases. Out of various diseases of wheat
rust diseases i.e. Black rust, Brown rust and Yellow rust are the most significant which have
continued to ravage this crop since ancient times. Among the three wheat rusts, brown rust
(Puccinia triticina Eriks.) is the most widely distributed and prevalent all over the country
(Bhardwaj et al., 2006) [4]. All rusts are obligate pathogens of living tissue and thus require a
host as a “green bridge” in order to survive until the next growing season (Staples, 2003) [8].
Temperature is the most recognized environmental factor that is able to affect spore
germination. Infection at temperatures between 16-27°C causes significant yield losses, by
reducing kernels number, quality, and weight (Agrios, 2004) [2]. As the temperature moves
outside this range, development is either slowed or becomes dormant until the temperature
moves back within the required range. At optimum temperature range urediospores germinate
and infect leaves within 6 to 8 hours after landing on the plant surface. Environmental impact
on plant disease is difficult to determine in the field due to complicating factors such as
multiple climatic variables, varying disease levels and perhaps most importantly pathogen-
environment interactions so that in present investigation, effect of different temperature on
uredospore germination was carried out in laboratory condition.
Mythology
The experiment was carried out for studying the effect of temperature on germination of
uredospore. A drop of distilled water was applied on a clean microscopic slide and kept in
moistened Petri dishes. The uredospores were collected from fresh uredosori from infected
plants of wheat. The uredospores were put with the help of sterilized fine brush in the drop of
water on microscopic slide and three microscopic slides were prepared for each treatment. The
slides were incubated at each of the temperature point viz., 5, 10, 15, 20, 25, 30 and 35oC for 6
hours in B.O.D. incubators. Spores with germtubes longer than the spore diameter were
considered as germinated and the levels of germination were represented in per cent from each
of the slide in microscopic filed. A light microscope at 40x objective lens was used to examine
~ 1029 ~
International Journal of Chemical Studies
germination of uredospore. The statistical analysis was carried
out using standard method. Per cent uredospore germination
was calculated by following formula (Anusha et al., 2018) [3].
A References
Per cent spore germination = --------- X 100
B physiology, development. Laboratory of soil plant water
Where,
A = No. of uredospores germinated;
B = No. of uredospores observed.
Results and Discussion
The results (Table 1) of studies on the effect of temperature
on germination of uredospores of P. triticina after 6 hours
indicated that germination varies in percentage depending on
temperature. Uredospore germination was maximum with 6
hours of incubation, in the temperature range of 20-25°C. The
mean maximum spore germination was recorded at 20°C
(92.95%) which was followed by at 25°C (87.65%). It was
statistically superior over all the tested temperatures. The
lowest germination was recorded at 5°C (10.36%) and 35°C
(6.93%). Thus, studies indicated that the optimum
temperature for uredospore germination was 20- 25°C. The
minimum temperature for germination under the conditions
used was between 0 and 5°C, the optimum between 20 and
25°C, and the maximum between 30 and 35°C and these
similar values were reported by Roelfs et al. 1992 [7]. There is
evidence that low and high temperatures affect plant disease.
The maximum number of P. striijormis urediniospores
germinated between 8-12°C was recorded by Vallavieille-
Pope et al. (1995) [10]. According to Kadvani (2012) [5] the
highest urediniospore germination of pear millet rust was
observed at 20 0C temperatures after 12 hours of incubation
and also spore germination was maximum within 24hr of
incubation, in temperature range of 20-30°C while the mean
maximum spore germination was recorded at 25°C that was
studied by Utpal et al. (2015) [9]. According to Mederick
(1972) [6] minimum temperature for germination of
urediospores of P. sorghi on water agar was between 2 and
5°C, optimum between 10 and 25°C, and maximum between
30 and 35°C.
Table 1: Effect of different temperature on spore germination of
uredospores of P. triticina in vitro
Temperature oC Per cent mean germination of urediospore
5 18.78** (10.36)*
10 35.08 (33.03)
15 51.04 (60.47)
20 74.60 (92.95)
25 69.42 (87.65)
30 31.55 (27.37)
35 15.26 (6.93)
S.Em. ± 0.75
C.D. at 5 % 2.27
C.V. % 3.07
**Data were transformed (Arcsine) prior to analysis
*Data given in parentheses are retransformed values
Conclusion
On basis of ongoing discussion, it can be concluded that
uredospore germination of Puccinia triticina causing rust in
wheat was maximum with 6 hours of incubation, in the
temperature range of 20-25°C. This is most favourable
condition for infection and diseases development. As
temperature moves outside this range, development of disease
either slowed or becomes dormant until the temperature
~ 1030 ~
moves back within the required range. This information will
be helpful in formulating management strategy under climate
change.
1. Acevedo E, Silva P, Silva H. Growth and wheat
relations faculty of agronomy and forestry science
university of chile, Chile, 2006, 1-47.
2. Agrios GN. Plant Pathology. Department of Plant
Pathology, University of Florida, Elsevier Academic
Press, Amsterdam. 2004, 565.
3. Anusha A, Mahesh YS, Mesta RK, Basavarajappa MP.
Patil SN, Babu AG. Effect of different sugar solutions,
temperature and relative humidity on uredospore
germination of Cerotelium fici (Cast.) Arth. causing rust
disease on fig (Ficus carica Linn.). International Journal
of Current Microbiology and Applied Sciences. 2018;
7(11):3558-3565.
4. Bhardwaj SC, Prashar M, Kumar S, Datta D. Virulence
and diversity of Puccinia triticina on wheat in India
during 2002-04. Indian Journal of Agricultural Science.
2006; 76:302-306.
5. Kadvani DL. Management of pearl millet rust (Puccinia
substricta Ell. & Barth. Var. indica Ramachar &
Cumm.). Thesis submitted to Junagadh Agricultural
University, Junagadh, 2012.
6. Mederick FM, Sackston WE. Effects of temperature and
duration of dew period on germination of rust
urediospores on corn leaves. Canadian Journal of Plant
Science. 1972; 52:551-557.
7. Roelfs AP, Huerta-Espino J, Marshall D. Barley stripe
rust in Texas. Plant Disease, 76: 538.
8. Staples RC. A novel gene for rust resistance. Trends in
Plant Science. 1992; James Currey Ltd, Oxford, 2003,
151.
9. Utpal D, Harlapurl SI, Dhutraj DN, Dibakar P, Pawar
DV. Effect of different temperature levels and time
intervals on germination of uredospores of Puccinia
sorghi. African Journal of Microbiology Research. 2015;
9(19):1299-1303.
10. Vallavieille-Pope C, Huber L, Leconte M, Goyeau H.
Comparative effects of temperature and interrupted
periods on germination, penetration, and infection of
Puccinia recondita f. sp. tritici and P. striifornis on wheat
seedlings. The American Phyto pathological Society.
1995; 85(4):409-415.
International Research Journal of Multidisciplinary Scope (IRJMS), 2021; 2(1): 46-49
2020 Iquz Galaxy Publisher, India.

ORIGINAL ARTICLE | ISSN (O): 2582 – 631X DOI: 10.47857/irjms.2021.v02i01.049

Isolation, Screening and Characterization of Cellolytic

Bacteria from Agricultural Fields of Narmada District
Alpesh Bhimani1* and Nikunj Sohaliya2
*1Department of Agricultural Microbiology, Navsari Agricultural University, Navsari. 2Department of Plant Pathology, Navsari
Agricultural University, Navsari

ABSTRACT
The aim of this presented work is to elucidate the screening and characterization of cellulolytic bacteria from soil. Soil
samples from different areas of agricultural fields of Narmada district were used for the screening of cellulolytic
bacteria by serial dilution and pore plate method. Isolates were additionally characterized by morphological and
biochemical tests. Out of forty nine isolates were selected on the basis of clear zone produced 7mm ≥. These five
potential isolates were further screened for cellulolytic activity among which three isolates AII3, AI3 and CIII5
exhibited promising activity of cellulase in agar plate assay. Isolation, screening and characterization of isolates for
cellulolytic activity provided appreciated and novel enzymes for the alteration of cellulolytic waste into valuable
compost.
Key words: Cellulolysis, Bacteria, Agricultural Waste, Characterization.

INTRODUCTION glycosidic bonds in a cellulose chain. β-1, 4-

Cellulose is a homopolysaccharide of glucose
residues linked with β-1, 4-glycosidic linkage.
Copious accessibility of cellulose makes it
striking substrate for producing many
industrially important metabolites. Desolately,
most of the cellulosic biomass is often disposed
of by burning. This is not limited to developing
countries alone, but is measured a
comprehensive phenomenon. With the help of
cellulolytic enzyme system, cellulose can be
altered to glucose which is a multi-valued
product, in a considerably economical and
biologically favourable process. Cellulose
degradation is mainly the bio-process
performed by the cellulases enzymes. Cellulase
enzyme system includes three classes of soluble
extracellular enzymes: 1, 4-β-exoglucanase, 1, 4-
β-endoglucanase and β-glucosidase.
Exoglucanase is essential for breaking of the
non-reducing end of a cellulose chain and
breaking of the elementary fibrils from the
crystal-like cellulose. Endoglucanase is
accountable for randomly breaking of β- 1-4,
_________________________________________________________________________________________________________________________
glucosidase hydrolyses water-soluble
cellodextrin and cellobiose to glucose (1-2). Only
the interaction of this three enzymes makes the
complete hydrolysis of cellulose to glucose (3–5)
or mineralized to H2O and CO2. Microbial system
is best suitable source for cellulase system
extraction found in the gut of organisms living in
cellulosic substrates as their major food. Insects
like bookworm (Lepidoptera) and termites
(Isopteran) are found to have important
symbiotic microflora in their digestive track
responsible for Cellulolysis (6–7).
Cellulosic activities have been reported in
many microorganisms including many fungal
and bacterial strains both anaerobic and
aerobic. Chaetomium, Fusarium, Myrothecium,
Trichoderma. Aspergillus, Penicillium and so
forth, are some of the accounted fungal species
accountable for cellulolysis. Cellulose degrading
bacterial species include Clostridium,
Trichonympha, Bacteroides, Actinomycetes,
Ruminococcus albus, succinogenes,
Methanobrevibacter ruminantium and
Butyrivibrio fibrisolvens, (8, 9).

*Address of Correspondence: Bhimani Alpesh, Dept. of Agricultural Microbiology, Navsari Agricultural

University, Navsari – 396421, Gujrat, India. E-mail- bhimani4@gmail.com

(Received 18 January 2021; 20 January 2021; Accepted 28 January 2021)

Alpesh and Nikunj
Cellulase has been used in several industrial
processes such as biofuels alike bioethanol (10,
11), triphasic biomethanation (12); plant waste
and agricultural management (13,14); ligand
binding and chiral separation studies (15) due
to its massive applicability.
This work focusses on the isolation of
cellulolytic bacteria from soil of different
organic farms of agricultural universities and
explore their cellulose degrading ability.
MATERIALS AND METHODS
Sample collection
The soil samples (10gm) were collected from
agricultural fields of Narmada District were
screened for the isolation of cellulose degrading
bacteria. With the help of sterile spatula soil
samples were collected from surface and 4, 8
and 12 cm depth. Sterile polythene plastic bags
were used for sampling. The samples were then
brought to the laboratory for microbiological
analysis. To minimize saprophytic developments
isolation of all the samples were done within 3-4
h of collection.
Isolation of Bacteria from Soil
Cellulolytic bacteria were isolated from soil by
using culture enrichment and pore plate
Cellulolytic index =
Morphological characterization
isolates
Bacterial isolates were characterized by their
morphological and physiological features.
Macro-morphological characteristics were study
by observing colony morphology, gram’s
staining of isolates was performed and slides
were examined under binocular optical
microscope to observe the cellular morphology.
RESULTS AND DISCUSSIONS
Sample Collection and Isolation
Cellulose is the major fraction of organic
carbon in soil and main structural
component of plants. Soil microbes, are
responsible for recycling of this organic carbon
to the environment (14). Cellulosic material
degradation is a complex process and requires
involvement of microbial cellulolytic enzymes.
Habitations where cellulosic substrates present
are the preeminent sources for isolation of
International Research Journal of Multidisciplinary Scope (IRJMS), Volume 2, Issue 1: 2021
Original article
methods. The Modified Han’s (MH) medium
along with Carboxymethylcellulose as sole
carbon source was used for this purpose (16).
The medium used for cellulolytic bacteria
contains CMC 1.88 g, sodium citrate 0.5 g,
KH2PO4 2.0 g, K2HPO4 7.0 g, MgSO4.7H2O 0.1 g,
(NH4)2SO4 1.0 g, Agar 10 g, Congo red 0.20 g,
PH 7.0, Distilled water 1 lit. The plates were
incubated for 2-3 days at 37 ̊C and observed for
clear zone around the colony. To visualize the
hydrolysis zone the plates were flooded with an
aqueous solution of 1% Congo red for 15 min
(9).To visualize clear zone formed by cellulase
positive strains the plates were distained using
1M NaCl solution. For identification and
cellulase production the bacterial colonies
having clear zone were selected. Further
bacterial strains were purified by repeated
streaking. The purified colonies were preserved
at 4 ̊C.
Primary screening of isolates by agar
plate assay
Cellulose degradation activity was analysed as a
diameter of clear zone in the
Carboxymethylcellulose congored plate.
Following formula was used to calculate
cellulolytic index. (11)
(Diameter of zone –Diameter of Bacterial colony)
-------------------------------------------------------------------------
Diameter of Bacterial colony
of cellulose degrading bacteria (15). Several
bacterial species have been discovered which
have capacity to alter cellulose to simple sugars
(16) but requirement for anew isolated
cellulolytic microorganisms still continues (17).
With the aim of isolation of cellulolytic bacteria
from soil, total seven soil samples (A1I, A1II,
A1III) and (A1I, A1II, C1I, D1I) were collected.
Cellulose degrading bacteria were enriched and
isolated by basal salt media (BSM), which
contained, NaNO3 2.5 g; KH2PO4 2 g; MgSO4 0.2
g; NaCl 0.2 g; CaCl2.6H2O 0.1 g supplemented
with cellulosic substrate CMC Na salt as a sole
carbon source and nutrient agar. After dilution
plate technique, well isolated colonies showing
distinct colony morphology were selected and
further purified by sector streaking. Altogether,
49 isolates were obtained in pure culture and
were preserved at 4 ºC temperature for further
47
studies.
Page
Alpesh and Nikunj Original article

Morphological and biochemical They all gives positive reactions for Oxidase,
characteristics of cellulolytic Catalase, Gelatinase, Vogues Proskauer,
Amylase, citrate utilization test and Negative
bacterial isolates reactions for methyl red and Indole test,. All
Five cellulolytic bacteria were isolated from soil. isolates ferment glucose, fructose & sucrose but
All bacterial isolates are Gram positive, rod not mannitol & lactose.
shaped, motile and endospore forming bacteria.

Table 1: Morphological and cultural Characteristics of bacterial isolates

Morphological Characteristics Cultural Characteristics

Sr.
Isolates Grams Shape Arrange Shape/ Margin/Ed
no Colony size Elevation
staining ment Form ge
1 BI2 G (+) Rod Chain Intermediate Round Erose Umbonate
2 AII3 G (+) Rod Chain Intermediate Round Erose Umbonate
3 AI3 G (+) Rod Chain Intermediate Irregular Undulate Raised
4 CIII5 G (+) Rod Single Intermediate Round Entire Umbonate
5 DI1 G (+) Rod Single Intermediate Round Undulate Pulvinate

Screening of isolates by agar plate
assay
Cellulolytic activity of bacterial isolates was
based on clear zone of degraded CMC area
around the colony. Cellulolytic activity test
showed that isolate AII3, AI3 and CIII5 has the
largest cellulolytic index (4.0, 2.0 & 2.0) isolate
BI2 & isolate DI1 has the smallest cellulolytic
index (0.9 & 0.4) respectively. Based on
cellulolytic index and growth isolate AII3, AI3
and CIII5 were potential isolates.

Table 2: Cellulolytic activity of bacterial isolates

Isolates No. CMCase Zone Ratio (cm)

BI2 0.9
AII3 2
AI3 4
CIII5 2
DI1 0.4

CONCLUSION REFERENCES
According to this study, our isolation, screening, 1. Shewale JG. “Glucosidase: its role in cellulase
morphological and cultural identification synthesis and hydrolysis of cellulose”. International
methods were quick and efficient for allowing us Journal of Biochemistry. 1982; 14: 435–443.
2. Woodward J and Wiseman A. “Fungal and
to identify several good cellulase producing
other β-glucosidases: their properties and
bacteria from a wide variety of samples. applications.” Enzyme and Microbial Technology.
Isolation of naturally occurring cellulase 1983; 4: 73–79.
producing bacteria from the environment is 3. Ryu and Mandels M. “Cellulases:
important for degradation of agricultural waste. biosynthesis and applications.” Enzyme and Microbial
All of our positive isolates may be an integral Technology. 1980; 2: 91–102.
part of future work. It develops good cellulases 4. Samdhu and Bawa. “Improvement of
or produce efficient cellulase producing systems cellulase activity in Trichoderma.” Applied
such as microbial consortia which can be used Biochemistry and Biotechnology. 1992; 34: 175–192.
5. Wood TM. “Synergism between enzyme
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of such beneficial enzymes. Biochemistry. 1989; 260: 37–43.
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Entomology. 2004; 49: 71–92.

There is no conflict of interest in this present
7. Saxena, Bahadur, Varma. “Cellulose and
research paper. This research work is not a part hemicelluloses degrading bacteria from termite gut
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of any other studies and it is our original work.

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and mould soils of India.” The Indian Journal of
Microbiology. 1993; 33: 55–60.
8. Milala, Shugaba, Gidado, Ene, Wafar.
“Studies on the use of agricultural wastes for cellulase
enzyme production by A. niger.” Journal of
Agriculture and Biological Science. 2005; 1: 325–328.
9. Schwarz WH. “The cellulosome and cellulose
degradation by anaerobic bacteria.” Applied
Microbiology and Biotechnology. 2001; 56: 634–649.
10. Ekperigin MM. “Preliminary studies of
cellulase production by Acinetobacter anitratus and
Branhamella sp.” African Journal of Biotechnology.
2007; 1: 28–33.
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Apiwatanapiwat. “Bioethanol production from
enzymatically saccharified sunflower stalks using
steam explosion as pretreatment.” Proceedings of
World Academy of Science, Engineering and
Technology. 2009; 37:140–143.
12. Chakraborty N, Sarkar GM, and Lahiri HC.
“Cellulose degrading capabilities of cellulolytic
bacteria isolated from the intestinal fluids of the silver
cricket.” Environmentalist. 2000; 20: 9–11.
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13. Lu JW, Wang HT, Nie YF. “Effect of
inoculating flower stalks and vegetable waste with
ligno-cellulolytic microorganisms on the composting
process.” Journal of Environmental Science and
Health. 2004; 39: 871– 887.
14. Mswaka and Magan. “Wood degradation,
and cellulase and ligninase production, by Trametes
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indigenous forests of Zimbabwe.” Mycological
Research. 1998; 102: 1399– 1404.
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“Progress curve as a means for functional
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16. Kasana RC, Salwan R, Dhar H, Dutt S, Gulati
A. A rapid and easy method for the detection of
microbialcellulases on agar plates using gram's
iodine. Current Microbiology. 2008; 5: 503-507.
17. Brander JR, Gillings MK, Nevalainen, MH.
Qualitative assessment of hydrolytic activities in
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International Research Journal of Multidisciplinary Scope (IRJMS), 2021; 2(1): 50-53
2020 Iquz Galaxy Publisher, India.

ORIGINAL ARTICLE | ISSN (O): 2582 – 631X DOI: 10.47857/irjms.2021.v02i01.050

Isolation of Soil Mycoflora from Agricultural Fields of

Narmada District
Alpesh Bhimani1* and Nikunj Sohaliya2
*1Department of Agricultural Microbiology, Navsari Agricultural University, Navsari. 2Department of Plant Pathology, Navsari
Agricultural University, Navsari

ABSTRACT
The soil samples were collected from different fields of Narmada District. Soil samples were collected in two different
zones i.e. rhizoplane and rhizosphere. Then collected samples were inoculated on Potato Dextrose Agar (PDA)
medium which was supplemented by suitable antibiotics such as Chloramphenicol by adopting soil dilution method
and soil plate method. The present study was conducted to find out the fungal diversity in agricultural fields in
Narmada. A total of 154 colonies were isolated. From this investigation, 14 species of fungi belongs to 6 different
genera were isolated and identified whereas 23 strains left unknown. Identification and characterization of the soil
mycoflora were done with the help of authentic manuals of soil fungi.
Key Words: Micro Fungi, Culture Media, Isolation, Fungal Diversity, Narmada.

INTRODUCTION have been considered as measures of functional

Soil contain large amount of microbial diversity
which can interact with the plants resulting in
useful effect or in harmful consequences. Among
all microbes fungi are an important component
of the soil microbiota (1) and they are present as
mycelia fragments, rhizomorphs or as spores.
They are playing significant role in soil and plant
nutrition. Some fungi are saprophytic means
they lives on dead and decaying organic matter,
so breaking it down and converting to available
forms to higher plants by excreting a wide range
of degraded enzymes that attack virtually any
organic material. These types of degrading
activities make fungi important participants in
recycling natural/agricultural waste in our
environment. But sometimes unfortunately their
degrading activity also results in the undesired
growth of fungi that degrade useful materials
(2). Fungi grow on diverse habitats in nature
and are cosmopolitan in nature. In laboratory
condition they can isolated on specific culture
medium for cultivation, preservation,
microscopic examinations and different
biochemical and physiological characterization.
The diversity richness of a microbial community
and relative abundance of individual species
_________________________________________________________________________________________________________________________
*Address of Correspondence: Bhimani Alpesh, Dept. of Agricultural Microbiology, Navsari Agricultural
University, Navsari – 396421, Gujrat, India. E-mail- bhimani4@gmail.com
(Received 18 January 2021; 20 January 2021; Accepted 28 January 2021)
activities of the group in the particular habitat
(3).
Micro-organisms like fungi, bacteria,
actinomycetes and other organisms colonize in
different type of habitats and different
substrates. Besides producing diseases, they
play important role in plant health and
productivity. Two important terms viz.,
Rhizosphere and Rhizoplane are necessary to
consider while learning with soil micro-
organisms. The Rhizosphere is a micro-
ecological zone in direct proximity of plant
roots. It is functionally defined as the particulate
matter and microorganisms that cling to roots
after being gently shaken in water. The
rhizosphere is a metabolically busier, faster
moving, more competitive environment than the
surrounding soil while The Rhizoplane is the
region around the root epidermis and outer
cortex where soil particles, bacterial structures
and fungal hyphae adhere. There are more
microbes in the rhizoplane than in the
rhizosphere. The diversity of the microbial
population is determined by counting the
number of colony forming units (CFUs). By
spreading the extracted soil
Alpesh and Nikunj
microorganisms across agar and counting the
number of individual group of microorganisms,
the CFUs can be determined. Endophytic fungi
and bacteria means which lives within the cells
of the roots are not considered a part of the
rhizoplane.
MATERIALS AND METHODS
Soil sample were collected from different
location of Narmada district in Gujarat, India.
Potato Dextrose Agar (PDA) medium was used
for isolation of different groups of fungi which
influence the vegetative growth and colony
morphology. (PDA was prepared by using
extract from 250g of potato boiled and filtered,
dextrose 20g, agar 15g and distilled water
1000ml) (2). The pH was maintained at 5.5-6 as
it is optimum for the growth and sporulation of
majority of fungi.
Soil sample were collected from a depth
of 15cm with the help of a sterilized cork borer
as majority of fungi are microscopic and shows
vast variation in quantitatively and qualitatively
aspects with change in sites of collection and at
different depths. The collected soil was emptied
into sterilized polyethylene bags. Each sample
bag was labeled appropriately by indicating the
site of collection, time, date and place of
collection. Then samples were taken to the
laboratory using sterilized cellophane bags (2).
Isolation of fungi from the soil
samples
The soil dilution (4) and soil plate method (5) on
media like Potato Dextrose Agar (PDA) used as
isolation techniques.
1. Soil Dilution Plate Method (4): soil
dilutions were made by adding 1g of soil
from each sample in 10ml of sterile distilled
water. Dilutions of 10-3, 10-4 and 10-5 were
used to isolate fungi to avoid over population
of the fungal colonies. 1ml of the suspension
from each concentration was added to sterile
Petri dishes with three replication of each
dilution, containing sterilized Potato
Dextrose Agar medium. Chloramphenicol
% Contribution =
International Research Journal of Multidisciplinary Scope (IRJMS), Volume 2, Issue 1: 2021
Original article
was added to the medium for preventing
bacterial growth, before pouring into Petri
plates. The plates were then incubated at
27±2 oC for 4-7 days. Micro-organisms were
easily isolated because they have formed
surface colonies that were well dispersed.
2. Soil Plate Method (5):
About 0.005g of soil sample was scattered on
the bottom of sterile Petri dish and molten
cooled (40-45 oC) agar medium PDA was
added. Then it was rotated gently to disperse
the soil particles evenly in the agar medium.
Then plates were incubated at 27±2 oC for 4-
5 days. One isolate of each fungal genus from
each soil sample were selected by random for
further sub-culturing and experiments. The
pure cultures were maintained on Potato
Dextrose Agar Slants.
Identification of the Fungi
Normally identification of the fungal species can
be carried out by morphological characteristics
of the colony and microscopic examinations (6).
The colony growth like length and width of the
colony, the presence or absence of aerial
mycelium, the color and other pigment
production were the macro morphological
characters evaluated. Although molecular
methods continue to improve and become more
rapidly available, microscopic observation and
cultural character remain commonly used and
important tools for identification of fungal
species (6) .The fungi were identified with the
help of standard procedure and relevant
literature (7, 8). Inoculating needle was flamed
over the burning burner. Then using that needle,
a small portion of the growth from the culture
plate was transferred into the drop of lacto-
phenol in cotton blue on the slide. The specimen
was teased carefully using needle to avoid
squashing and over-crowding of the mycelium
(2). The specimens were observed under the
microscope for microscopic identification.
Statistical Analysis
The population density was expressed in terms
of Colony Forming Unit (CFU) per gram of soil
with dilution factors. The percent contribution
of each isolate was calculated by
Total No. of CFU of an individual Species
× 100
Total No. of CFU of all sps
Where, CFU – Colony Forming Unit
51
Page
Alpesh and Nikunj
RESULTS AND DISCUSSION
Soil micro-organisms act as essential
determinants of plant community variety and
productivity (9). Different environmental factors
such as the soil pH, moisture, temperature,
organic carbon and nitrogen play an important
role in the distribution of mycoflora. The soil
mycoflora in four different village of Narmada
districts were observed. Soil dilution plate and
soil plate methods were used for the isolation of
fungi during this study. Total number of fungal
colonies isolated on Petri plates containing PDA
Figure 1: Different colonies of Fungi on PDA
These fungi are the major decomposers of dead
organic waste and have important role in
recycling of nutrients in natural and modified
ecosystems (11). All four soil samples from four
different villages were examined for fungal
diversity. The experiment resulted in presence
of 14 species of fungi were identified and
characterized. The maximum fungal species
belonged to Deuteromycotina (127 species) and
Zygomycotina (4 species) and 23 colonies were
left unknown on the plates containing PDA
medium. Due to its simple formulation and
efficient to support wide range of fungal growth,
PDA medium is the most commonly used culture
medium and it was stated to be the best media
for mycelia growth by several workers (12, 13).
Characterization of the isolates were up to genus
level and to the species level was made by using
the macro-morphological and micro-
morphological characters by use of authentic
manuals of soil fungi.
Our findings were similar to those
isolated by Rasheed et al. (14). Aspergillus
species particularly like A.flavus and A.niger,
Penicillium and Rhizopus were isolated only
from the soil whereas Trichoderma species,
International Research Journal of Multidisciplinary Scope (IRJMS), Volume 2, Issue 1: 2021
Original article
medium were 154. As mentioned earlier, soil
dilution plate (10) and soil plate method (5)
were used for the isolation of fungi during this
study. A large number of species and colonies
were isolated on soil plates than on dilution
plates and further the total number of species
isolated decreased with increased dilutions of
the samples. The purification of the culture was
carried out by using culturing of the hyphal tips
and then transferred to fresh agar slants of PDA
medium.
Alternaria alternata, Curvularia lunata and
Fusarium species were obtained from both soil
and plant parts. In our study, among the
obtained fungal isolates the genera Aspergillus
and Penicillium were dominant on media used.
The most common isolates among them viz., A.
clavatus, A. flavus, A. fumigatus, A. nidulans,
A.niger, A. restrictus, A. terreus, Curvularia
clavata, C. lunata, Fusarium oxysporium, F. solani,
Penicillium Chrysogenum, P. frequentens, P.
funiculosum, Rhizopus stolonifer, Trichoderma
harzianum, T.viride, T. virens, T. longibracheatum
were isolated and characterized. The percent
contributions of different soil mycoflora of all
four villages were evaluated.
CONCLUSION
In the present experiment the soil sample of
four different villages of Narmada districts viz;
Sagai, Mohbi, Mal and Samot were studied for
detection of the fungal diversity. Among the
isolates Aspergillus and Penicillium were
dominant in all agricultural fields of all areas
52
mentioned due to high sporulation and
production of bacterial antibiotics from the
Page
Penicillium species and production of different
types of toxic materials from the Aspergillus
Alpesh and Nikunj Original article

species may prevent the growth of other fungal 5. Warcup JH. The soil plate method for
species. Trichoderma species were also isolated isolation of fungi from soil. Nature. Lond. 1950;
from these soil samples which show organic 166: 117-118.
richness of soil. This study was conducted as a 6. Diba K, kordacheh P, Mirhendi SM,
effort to understand the soil microbial diversity Rezaie S, Mahmoudi M. Identification of
in the agricultural fields of Narmada district as Asergillus species using morphological
soil microflora not only plays an important role characteristics. Pal J Med sci. 2007; 23: 867-872.
in decomposition and contribute to 7. Gilman JC. A Manual of soil fungi, 2nd
biogeochemical cycling but also are responsible Indian Edition, Biotech Books, Delhi, 2001.
for the prevalence of diseases in the crop fields 8. Nagamani A, Kumar IK and
and availability of some minerals and nutrients. Manoharachary C. Hand Book of Soil Fungi, I.K.
International Publishing House Pvt Ltd, New
CONFLICT OF INTEREST Delhi, India. 2006.
There is no conflict of interest in this present 9. Wardle DA, Bardgett RD, Klironomos JN,
research paper. This research work is not a part Setala H, Van der Putten WH, Wall DH.
Ecological linkages between above ground and
of any other studies and it is our original work.
below ground biota. Science. 2004, 304: 1629-
1633.
REFERENCES 10. Waksman SA. Principles of Soil
Microbiology. Williams and Wilkins Co.
1. Ainsworth GC and Bisby GR. Dictionary Baltimore, Md. 1927.
of the fungi. Commonwealth Mycological 11. Gadd GM. Mycotransformation of
Institute. 1995; 445. organic and inorganic substrates. Mycologist.
2. Aina VO, Adewuni AAJ, Hauwa Haruna 2004; 18: 60-70.
and Amina Zaraki. Isolation and identification of 12. Maheshwari SK, Singh DV, Sahu AK.
fungi associated with the deterioration of Effect of several nutrient media, pH and carbon
painted wall surfaces within Kaduna sources on growth and sporulation of Alternaria
polytechnic. Asian Journal of Medical Sciences. alternate. J Mycopathol Res. 1999; 37: 21- 23.
2011; 3: 250-253. 13. Saha A, Mandal P, Dasgupta S, Saha D.
3. Kjoller A, Struwe S. Microfungi in Influence of Culture Media and Environmental
Ecosystems.,fungal occurrence and activity in factors on mycelia growth and sporulation of
litter and soil. Oikos. 1982; 39: 389-422. Lasiodiplodia theobromae (Pat.). Griffon and
4. Waksman SA. A method for counting the Maubl. J Environ Biol. 2008; 29: 407-410.
number of fungi in the soil. J Bact. 1922; 7: 339- 14. Rasheed S, Dawar S, Ghaffar A. Location
341. of fungi in groundnut seed. Pak J Bot. 2004; 36:
663-668.

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International Research Journal of Multidisciplinary Scope (IRJMS), Volume 2, Issue 1: 2021

 Int.J.Curr.Microbiol.App.Sci (2018) 7(6): 3515-3518

International Journal of Current Microbiology and Applied Sciences

ISSN: 2319-7706 Volume 7 Number 06 (2018)
Journal homepage: http://www.ijcmas.com

Original Research Article https://doi.org/10.20546/ijcmas.2018.706.412

Management of Sorghum [Sorghum bicolor (L.) Moench] Seed Mycoflora by

Means of Bio-agents in vitro

Sonal Vaja1*, Nikunj Sohaliya1 and Bipin Vahunia1

1Department of Plant Pathology, N. M. College of Agriculture, Navsari Agricultural

University, Navsari, Gujarat 396 450, India

*Corresponding author

ABSTRACT

Keywords Sorghum [Sorghum bicolor (L.) Moench] is a vital life-sustaining food crop for human
being as well as for livestock in many parts of world. In India, Maharashtra,
Sorghum, Seed Karnataka, Andhra Pradesh, Madhya Pradesh, Gujarat and Tamil Nadu are major sorghum
mycflora, Bio- growing states. Therefore, investigation was undertaken during 2016 at N. A. U., Navsari
control agents, in on isolation of sorghum seed infecting fungi, (Colletotrichum sp., Fusarium moniliforme,
vitro Alternaria alternata, Curvularia lunata, Macrophomina sp., Aspergillus niger and A.
Article Info flavus.) and symptomatology induced by these seed infecting fungi, affect on seed quality,
loss of seed germination and seedling vigour. Therefore seeds are treated with bio-control
Accepted: agents. All the tested bio-control agents were found significantly superior in inhibiting the
25 May 2018 mycelial growth of the pathogen over control. Among them seed treated with
Available Online:
10 June 2018
Trichoderma viride recorded highest seed germination (83.00%) and shoot length (7.10
cm). The maximum root length recorded in Trichoderma harzianum (6.90 cm) and
maximum seedling vigour index recorded in Pseudomonas fluorescens (1137.60).

Introduction mold fungi which grow on the seed

Sorghum is known to suffer from more than
30 fungal diseases (USDA, 1960). Important
seed borne fungal diseases recorded on
sorghum are stalk rot, target spot, stalk rot/
anthracnose/red leaf, seed rot/stalk rot,
seedling blight/charcoal rot and covered
smut/grain smut (Richardson, 1990). Among
them, the grain infecting molds have become a
major constraint of early maturing high
yielding hybrids and improved varieties that
are grown during the rainy season (Thakur et
al., 2006). These losses in potential yield,
substratum produce mycotoxins which are
hazardous to man and animals (Halt, 1994).
Seed is the most important input for crop
production. Pathogen free healthy seed is
urgently needed for desired plant populations
and good harvest. Though the regular
fungicides used for seed treatment are found
to inhibit growth of the seed borne pathogens
and their role in improvement of seed quality
is poorly understood (Raju et al., 1999). Seed
treatment for controlling plant diseases has
been termed as the “pain less method” for
farmers. In under developing country like

3515
 Int.J.Curr.Microbiol.App.Sci (2018) 7(6): 3515-3518
India, it is all the more important since we
cannot pay the heavy costs of spraying and
dusting. Biological control of plant pathogens
using antagonistic microorganisms is a vital
area of plant pathological research in the
present day strategy of avoiding
environmental pollution. In the present study
efforts were made to develop strategies for the
management of seed borne fungi and to
enhance the germination and seedling vigour.
Therefore, the present investigation was
undertaken to find out the mycoflora
associated with the seeds of sorghum and to
evaluate the efficiency of bio-agents against
seed mycoflora and seed germination of
sorghum.
Materials and Methods
Experimental location
The research experiment was conducted in
Department of Plant Pathology, N. M. College
of Agriculture, Navsari agricultural
University, Navsari during 2015-16.
Determine the antagonistic activity of
different bio-control agents against Sorghum
seed mycoflora by blotter paper method
Isolation of pathogen
Detection of fungi associated with sorghum
seeds were carried out by taking 100 seeds
through standard blotter paper method and
PDA plate method. Ten seeds each of non-
surface sterilized and surface sterilized by 1%
Sodium hypochloride (NaOCl) solution for
one minute were placed at equal-distance on
three layers of properly moistened sterilized
blotters and petri plates. These were incubated
under 12/12 hr alternating light and dark
period at 25 ± 2º C. Developing fungal growth
on each of the seed were observed regularly
and identified by microscopic observations.
Isolation was carried out by inoculating the
detected mycoflora by standard agar plate
3516
method. These cultures were further purified
by single spore isolation method. These pure
culture isolates were maintained on PDA
slants in refrigerator at 5 ± 2oC temperature.
The streptomycin was added after autoclaving
the media to avoid bacterial contamination.
Efficacy of bio-control agents against
Sorghum seed mycoflora in vitro
The following materials were used during the
present investigations:
Bio-control agents
Trichoderma viride Pers, ex. grey NAU
isolate, Trichoderma harzianum Rifai. NAU
isolate, Trichoderma koningii Oudem,
Pseudomonas fluorescens Migula NAU isolate
and Bacillus subtilis Ell NAU isolate.
Methods
Different bio-agents were evaluated to check
their effect on germination and vigour index
of seeds inoculated with isolated fungi. For
this healthy seeds of sorghum variety GJ-42
were inoculated with the mixture of all
isolated fungus by soaking the seeds into
mixed spore suspension of fungi and then
treated with all respective bio-agents. These
treated seeds were evaluated by paper towel
method (Khare, 1996) and incubated at 27 ± 2º
C for 10 days. After ending of incubation
period observations were recorded as number
of germinated seeds, shoot length and root
length to calculating vigour index and
germination percentage.
Results and Discussion
In vitro testing of bio-control agents
The results presented that Different bio-agents
(Table 1) were tested to check their effect on
seed germination and seedling health of
 Int.J.Curr.Microbiol.App.Sci (2018) 7(6): 3515-3518

sorghum seeds variety GJ-42 inoculated with

mixture of all isolated fungi. Data presented in
the Table 1 and graph revealed significant
effect of all bio-agents on seed germination
shoot length, root length and seedling vigour
index. Overall, bio-agents recorded 23.64 to
50.91, 45.83 to 97.22 and 56.61 to 98.28 %
increase in seed germination, shoot length and
root length, respectively over control.
Seed treated with Trichoderma viride recorded
highest seed germination (83.00%) which was
at par with Trichoderma harzianum (80.00%)
and Trichoderma koningii (77.00%). Whereas,
in Pseudomonas fluorescens and Bacillus
subtilis, recorded 72.00 and 68.00 % seed
germination, respectively.

Table.1 Screening of known antagonists as bio-agents to control of sorghum

seed borne fungi in vitro
Treatment Conc. Seed Increase in seed Shoot length Increase in Root Increase in Seedling
germination germination (cm)* shoot length length root length Vigour Index
(%)* over control over control (cm)* over control (SVI)
(%) (%) (%)

Trichoderma viride Pers, ex. 0.4% 83.00 50.91 7.10 97.22 6.65 91.09 880.70
Grey NAU isolate
Trichoderma harzianum Rifai. 0.4% 80.00 45.45 6.95 93.06 6.90 98.28 1108.00
NAU isolate
Trichoderma koningii Oudem 0.4% 77.00 40.00 6.73 86.81 6.23 78.88 998.70
Pseudomonas fluorescens 0.5% 72.00 30.91 6.30 75.00 6.60 89.66 1137.60
Migula NAU isolate
Bacillus subtilis Ell NAU 0.5% 68.00 23.64 5.25 45.83 5.45 56.61 770.50
isolate
Absolute control - 55.00 0.00 3.60 0.00 3.48 0.00 389.50
S. Em ± 2.59 - 0.11 - 0.12 - 28.54
CD 0.05% 7.70 - 0.34 - 0.37 - 84.78
CV % 7.15 - 5.98 - 6.63 - 9.16
*Average of three repetitions and 25 seeds each repetition

Graph.1 Screening of known antagonists as bio-agents on sorghum seed germination, shoot

length, root length and seedling vigour index in vitro

3517
 Int.J.Curr.Microbiol.App.Sci (2018) 7(6): 3515-3518
The result in term of shoot and root length
with seedling vigour index, all the treatments
showed larger shoot length, root length and
seedling vigour index as compared to control.
Seed bio-priming with T. viride recorded
maximum shoot length (7.10 cm) which was
at par with T. harzianum (6.95 cm).
The maximum root length recorded in T.
harzianum (6.90 cm) which was at par with T.
viride (6.65 cm). The maximum seedling
vigour index recorded in Pseudomonas
fluorescens (1137.60) which was at par with
T. harzianum (1108.00). Similarly, B. subtilis
recorded 5.25 cm, 5.45 cm and 770.50, shoot
length, root length and seedling vigour index,
respectivrly and P. fluorescens recorded 6.30
cm shoot length and 6.60 cm root length as
compared to control (3.60 cm, 3.48 cm and
389.50).
From this study, it is clear that Trichoderma
harzianum and Trichoderma viride were
found effective in reducing the seed
mycoflora in Sorghum. Our result are
harmony with earlier worker Elad et al.,
(1986) that Trichoderma harzianum inhibited
linear growth and microsclerotia production
of M. phaseolina in vitro and also
Alagarsamy and Sivaprakasam (1988) found
that Trichoderma viride was capable of
checking the growth of M. phaseolina in
vitro.
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How to cite this article:
Sonal Vaja, Nikunj Sohaliya and Bipin Vahunia. 2018. Management of Sorghum [Sorghum bicolor
(L.) Moench] Seed Mycoflora by Means of Bio-agents in vitro. Int.J.Curr.Microbiol.App.Sci.
7(06): 3515-3518. doi: https://doi.org/10.20546/ijcmas.2018.706.412
3518
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