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Life Science Archives (LSA)

ISSN: 2454-1354
Volume 1; Issue - 1; Year 2015; Page: 28- 34

Research Article
BIOCONTROL OF Aspergillus flavus IN GROUNDNUT
R. Sanskriti, Vijayalakshmi, R. Neelam, P. Ezilrani and J. Godwin Christopher*,
School of Biosciences and Technology, VIT University, Vellore 632014, Tamil Nadu, India.
Abstract
The groundnut is particularly valued for its protein content. Groundnuts contain more protein than
meat and about two and a half times more than eggs. Aspergillus flavus is a predominant fungus which
causes yellow mold of groundnut seedlings. Aspergillus flavus cause extensive economic losses either by
destroying the plant or by contaminating peanut kernels with the aflatoxins. Groundnuts grown under
drought stress may also be predisposed to subsequent aflatoxin in contamination during harvest, handling, or
storage. Pseudomonas fluorescens work as a antagonist and inhibits the pathogenicity of A. flavus on
groundnut. P. fluorescens were isolated from garden soil samples and selected for their growth inhibition of
A. flavus. In this study, the antifungal activity of Pseudomonas fluorescens against A. flavus in groundnut
was examined in laboratory and field trials. Inoculation of Pseudomonas fluorescens on groundnut resulted
in significant reduction of seed infection by A. flavus, it also reduces the yellowish black spot from the leaves
of groundnut. This results suggest that application of this bacteria is effective in reducing fungal population
in groundnut. By the utilization of P. fluorescens as the antagonist of A. flavus, health of the groundnut plant
can be maintained. So disease free healthy seeds can be produced and disease propagation can be prevented.
Hence this investigation will be eco-friendly and useful in increasing productivity as well as storability of
Groundnut seeds.
Article History

Received : 07.02.2015
Revised : 20.02.2015
Accepted : 27.02.2015
1. Introduction

The cultivation of groundnut has its origin

from South America and spread to Brazil,
Southern Bolivia and North-western Argentina.
Groundnut was introduced by the Portuguese from
Brazil to West Africa and then to south-western
India. India is the second largest producer of
groundnuts (6.25 million metric tonnes 2008-09)
after China (14.30 million metric tonnes 2008-09).
Groundnut is the largest oilseed in India in terms
of production, it accounted for 35.99 per cent of
the oilseeds production of the country during
2007-08. Gujarat is the largest producer by
contributing 25 per cent of the total production

* Corresponding author: J. Godwin Christopher

Tel.: +91-9976605776
E-mail: godwinj@vit.ac.in

Key words: Groundnut, Aspergillums flavus

Pathogen) and Pseudomonas fluorescence
(antagonist).
followed by Tamil Nadu (22.48%), Andhra
Pradesh (18.81%), Karnataka (12.64%) and
Maharashtra (10.09%) during 2006-07. The other
important states where it is grown are Madhya
Pradesh, Rajasthan, Uttar Pradesh and Punjab.
Groundnut contains on an average 40.1 per
cent of fat and 25.3 per cent of protein and is a
rich source of calcium, iron and vitamin B
complex like thiamine, riboflavin, niacin and
vitamin. It is used not only as a major cooking
medium for various food items but also for
manufacture of soaps, cosmetics, shaving creams
and lubricants. Important pathogens of the
groundnut are Fusarium solani, Fusarium
oxysporum,
Macrophomina
phaseolina,
Rhizoctonia solani, Sclerotium rolfsii, Aspergillus

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J. Godwin Christopher /Life Science Archives (LSA), Volume 1, Issue 1, Page 28 to 34, 2015

niger, A. flavus etc (Singh and Oswalt, 1992).

During May 1984, a seedling disease of groundnut
was observed in a commercial groundnut farm in
Atascosa County, Texas (Subrahmanyam et al.,
1987). The A. flavus groups of fungi are
facultative parasites. They invade plant tissues
directly or attack tissues that have been
predisposed by environmental stresses such as dry
weather or damages caused by insects, nematodes,
natural cracking, and harvest equipment (Pettit,
1984). A. flavus produces a toxin called aflatoxin
which is carcinogenic to humans and animals
(Thirumala Devi et al., 2002). The health of the
consumers as well as the enhancing the economy
of the farmers the groundnut breeding program
should be carried out for aflatoxin resistance and
thereby increasing the quality of the groundnut.
According to Liang et al. (2006) and Cotty and
Jaime (2007) derived that removal of
contamination by aflatoxin is a multi-stage
process.
Pseudomonas fluorescens: They are obligate
aerobes, motile, non- pathogenic saprophytes.
They are readily available in soil, water and plant
surface and have simple nutritional requirement.
They are readily amenable to various genetic
engineering technique. P. fluorescens have been
extensively employed for control of various
pathogen
like
.A
flavus,
Fusarium,
Helminthosporium etc. They are very useful in
controlling the disease due to the release of
various secondary metabolites like antibiotics,
siderophore and hydrogen cyanide (Trujillo et al.,
2007).
Aspergillus flavus: The A. flavus is a
facultative fungus. They invade plant tissues
directly or attack tissues that have been
predisposed by environmental stresses such as dry
weather or damages caused by insects, nematodes,
natural cracking, and harvest equipment (Pettit,
1984). The yellow mold fungus, Aspergillus
flavus, is commonly found in the seed of both
rotten and apparently healthy pods of groundnut.
Many strains of this fungus are capable of
producing aflatoxins that render the seed
unacceptable due to high toxicity for human or
animal consumption (Reddy and McDonald,
1983). Aflatoxin contamination in groundnut can

29

occur in the stems of seedlings, pods, and seeds.

The fungus is capable of invading groundnut seeds
before harvest, during postharvest drying, and
during storage. Aflatoxins are carcinogenic and
produced by the Aspergillus group of fungi that
have been identified as B1, B2, G1, and G2 (Pettit,
1984). Yellow mold first appears on groundnut
cotyledons after the emergence of seedlings.
Necrotic spots become covered with masses of
yellow-group spore heads of the A. flavus group of
fungi. Fungus toxins are translocated throughout
the seedling in the transpiration stream. Infected
plants generally become stunted with symptoms of
vein clearing chlorosis on the leaflets. Such
seedlings lack a secondary root system condition
known as "aflaroot." Yellow-green Aspergillus
colonies develop on over mature and damaged
seeds and pods (Singh and Oswalt, 1992).
This study was focused on the antagonistic
property of Pseudomonas fluorescens against A.
flavus. The first part of this study was done in
laboratory. Then the next part is in the field. The
result of this study proves that P. fluorescens
inhibit the growth of A. flavus. Thus it can be used
as a biocontrol agent against yellow mold in
groundnut.
2. Methodology
2.1. Isolation of
groundnut seeds

Aspergillus flavus

from

Groundnut seeds were taken, washed in

running tap water for 10 min., surface disinfected
by immersion in sodium hypochloride solution
containing trace of tween 80 for 5 min. and placed
on Czapez-Dox agar (Difeo laboratories, Detroit,
MI) supplemented with Rose Bengal and
streptomycin (bacterial inhibitor). The plates were
incubated at 25 C in dark for 4 days.
Then LPCB staining of isolated fungi was
performed. After that the isolated fungi was subcultured on PDA slant for mass propagation.
2.2. Isolation of Pseudomonas fluorescens from
soil
Soil samples were taken in a sterile
polythene bag from VIT garden 5 gm of Soil
sample was serially diluted till 10-7 dilution.
Spread plate was done in the dilutions of 10-5, 10-6

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J. Godwin Christopher /Life Science Archives (LSA), Volume 1, Issue 1, Page 28 to 34, 2015

and 10-7. The plates were incubated at room

temperature for 24 -48 hr. The colony
morphologies were noted and preliminary tests
were performed. The isolate was sub-cultured on
media and following tests were performed.
Preliminary tests of Gram staining,
capsular staining and hanging drop technique was
done. Followed by different biochemical tests
which include Indole production, Citrate
utilization, Catalase test, Urease test, Arginine
hydrolysis, Esculin hydrolysis, Mannitol motility
test (As per the methodology of (Rajan and
Christy, 2010).
2.3. Antifungal
fluorescens

activity

of

Pseudomonas

2.3.1. Well Diffusion Method

PDA plates were prepared and suspension
of A. flavus is mixed in PDA media and was
poured in the plates. After solidification well were
punched with the help of sterile borer. P.
fluorescens was inoculated in nutrient broth and
kept for incubation at room temperature for 24 48 hrs and was centrifuged at 6000 rpm for 20
min. High concentrated cells were pipetted and
was poured into the well. These plates were
incubated at room temperature for 48-72 hrs.
(Rajan and Christy, 2010).

30

2.3.3. Control of Aspergillus flavus

Groundnut plant which is infected by
A. flavus is directly treated with suspension of
P. fluorescens.
3. Results
3.1. Isolation of Aspergillus flavus
A. flavus which were grow and isolated
from groundnut seeds are confirmed by LPCB
staining. The growth on 4th day is shown in Fig 1. A. flavus was the predominant fungus followed
by A. niger and A. fumigatus. In general, more
fungi were isolated from surface sterilized seed
then from unsterilized seed. The process of surface
sterilization could have removed or diluted the
fungicide present on the seed. Thus, enabling
more fungi to grow out of the seed. A. flavus was
isolated from seed surface in higher frequencies
then other fungi, suggesting that A. flavus
infection was deeply embedded in the seed
(Subrahmanyam et al., 1987).

2.3.2. Field Trials

For the field study two different methods
were adopted to infect the groundnut plant with A.
flavus:
Method 1: A. flavus was grown on PDA slants for
weeks at 25 C in the dark and conidial suspension
were prepared in sterile distilled water containing
trace of tween 80. Seeds were surface sterilized
with sodium hypochlorite (0.52%) for 20 seconds.
Then the seeds were coated with A. flavus
suspension for 5min, air dried for a period of
overnight and planted in plasticbags.

Fig 1: Growth of A. flavus from surface

disinfected seeds on Czapek dox agar

Method 2: Suspension of A. flavus was mixed

with soil, then groundnut seeds were planted in
soil.

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J. Godwin Christopher /Life Science Archives (LSA), Volume 1, Issue 1, Page 28 to 34, 2015

31

Fig 2: LPCB staining shows conidiophores of Aspergillus flavus

3.2. Isolation of Pseudomonas fluorescens
Pseudomonas fluorescens were isolated
from garden soil were confirmed by performing
several preliminary tests and biochemical tests.
The morphological characteristics of the strain by
preliminary test is shown in Table - 1 and result
obtained by various biochemical parameters is
shown in Table - 2. The growth of
P. fluorescens on Kings B medium is shown in
Fig.3. The aim was to isolate P. fluorescens which
showed antifungal activity against A. flavus.
Subsurface garden soil was used for the isolation
of the bacterium. Serial dilution was prepared till
10-7, thereby decreasing the bacterial load with
each subsequent dilution. Selective medium for
the growth of Pseudomonas spp., Kings B
medium was used. Spread plate technique, was
employed for the isolation of the bacteria. The
incubation temperature is 28-30 C. The colony
morphology were noted down, after the incubation
period was over. Preliminary test comprising the
various staining technique such as Gram staining
and Capsular staining were carried out. Hanging
drop experiment was carried out to check for the
motility of the organism. The Gram negative rod
shape, motile bacterial colonies obtained, were
sub-cultured on Pseudomonas agar medium and
were subjected to a series of biochemical tests.

Fig - 3: Showing colonies of Pseudomonas spp

on Kings B medium
Table - 1: Preliminary test
Experiment
Gram staining
Capsular staining
Hanging drop
motility test

Observation
Pink
colour,
gram
negative rod shape
White colours rod against
dark black background
Movement
of
the
organism was observed.

Table - 2: The isolate was observed for the various biochemical parameters. The results obtained were
as summarized below
S.No.
Test
Result
1
Indole production
(-)
2
Citrate utilization
(+)
3
Catalase activity
(+)
4
Urease activity
(+)
5
Arginine hydrolysis
(+)
6
Esculin hydrolysis
(+)
7
Mannitol motility test
(+)
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J. Godwin Christopher /Life Science Archives (LSA), Volume 1, Issue 1, Page 28 to 34, 2015

3.3. Antifungal activity

The result of the well diffusion method
showed zone of inhibition (Fig.4). The
Pseudomonas fluorescens shows inhibitory
activity against Aspergillus flavus. The inhibition
is thought to be because of the production of
secondary metabolites or some antimicrobial
compounds (Anjaiah et al., 2005). The process of
centrifugation helped in breaking open the
bacterial cells and the release of antimicrobial
compound (Gulfeshan, 2010).

32

for invasion. A. flavus invasion of pods are

generally high when the crop was subjected to
drought stress during pod development. The
disease incidence was higher when the fungus was
inoculated into the soil then when the fungus was
applied to the seeds. Yellow mold first appears on
groundnut cotyledons after the emergence of
seedlings. Necrotic spots become covered with
masses of yellow green spore heads of A. flavus
group of fungi. Fungal toxins are translocated
throughout the seedlings in the transpiration
stream. Infected plants generally become stuned
with symptoms of vein clearing chlorosis on the
leaflets. Such seedlings lack a secondary root
system, a condition known as aflaroot. Yellowgreen Aspergillus colonies develop on over mature
and damaged seeds and pods

Fig - 4: Showing Zone of inhibition by

Pseudomonas fluorescens against Aspergillus
flavus
3.4. Field study
Groundnut seeds that were grown over the
soil mixed with A. flavus positively infected all the
plants with yellow mold disease (Fig. 5). The
symptoms were seen right from the cotyledon
stage. Necrotic lesions were seen (Fig. 6). The
lesions on the cotyledons were covered with
conidia of pathogen. Plants were stunted and
chlorotic. Leaflets were small, had pointed tips,
and vein-clearing. Development of the root system
was also poor. Some of the infected plants
eventually recovered from the disease and
produced normal foliage. Similar symptoms were
reported by El-Khadem (1968). Yellowish black
spots (Fig.7) were observed on the leaves of
groundnut plant. These symptoms developed after
2 weeks. The reason for such symptoms of yellow
mold was attributed due to aflatoxin produced by
the fungus at the infection site (Chohan and
Gupta, 1968). A. flavus can invade groundnut pods
at any stage of crop development but the post
harvest conditions are often specially favourable

.
Fig - 5: Showing positive infection of A. flavus
on groundnut plant

Fig - 6: Showing necrotic lesions on the

cotyledons of groundnut seed

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33

4. References
1)

Fig 7: Showing yellowish black spot on leaves of

groundnut plant
3.5. Biocontrol by Pseudomonas fluorescens
After treatment with P. fluorescens,
diameter of yellowish black spot which was
developed on the groundnut leaves was found to
be reduced shown in Table - 3. Biological control
is a recent trend in disease management. P.
fluorescens have been found to be the most
important in the area of biological control. P.
fluorescens has the ability to solubilize phosphate
in vitro and to produce several antibiotics with
high specificity against several microorganism.
Table - 3: Reduction in the diameter of spot
present on groundnut leaves
S. No

Day

1
2
3
4

1st day
7th day
14th day
21st day

Diameter of the
spot (mm)
5
No change
4.6
4.3

The mechanism behind the biocontrol of disease

needs more understanding. It was assumed to be
because of the production of secondary
metabolites. Hence, P. fluorescens act as
biocontrolling agent which inhibits the growth and
reduces the activity of A. flavus.
Acknowledgment
We thank VIT University for the help and
support for extending necessary facility to carry
out this research work.

Anjaiah, V., Thakur, R. P., & Koedam, N.

2005. Evalution of bacteria and Trichoderma
for biocontrol pre-harvest seed infection by
Aspergillus flavas in groundnut. Biocontrol
Science and Technology, 16(4), 431-436.
2) Chohan, J. S., & Gupta, V. K. 1968. Aflaroot,
a new disease of groundnut caused by
Aspergillus flavus link ex fries. The Indian
Journal of Agricultural Sciences, 568-570.
3) Cotty, P., & Jaime-Garcia, R. 2007.
Influences of climate on aflatoxin producing
fungi
and
aflatoxin
contamination.
International Journal of Food Microbiology,
119, 109115.
4) El-Khadem, M. 1968. Die Bedeutung von
Aflatoxinen fur die durch Aspergillus flavus
verursachte keimlingskrankheit der erdnub.
Journal of phytopathology, 218-231.
5) Gulfeshan, R. 2010. Isolation and
characterization of a biocontrol agent from
soil and testing its efficacy against various
pathogens. M.Sc. Thesis, VIT University, 1467.
6) Liang, X. Q., Luo, M., Guo, B. Z. 2006.
Resistance mechanisms to A. flavusinfection
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7) Pettit, R. E. 1984. Yellow mold and aflatoxin.
In Compendium of Peanut Diseases. 35-36.
8) Rajan, S., & Christy, R. S. 2010.
Experimental procedures in life sciences.
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10) Singh, F., & Oswalt, D. L. 1992. Major
diseases of groundnuts. International Crops
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11) Subrahmanyam, P., Smith, D. H., Raber, R.
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100, 97-102.
12) Thirumala Devi, K., Mayo, M. A., Hall, A.J.,
Craufurd, P. Q., Wheller, T. R., Waliyar, F.,

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34

Subrahmanyam, A., Reddy, D. V. R. 2002.

Development and application of an indirect
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