Isolation and Characterization of Endophytic Microbiome From Indigenous Maize (Zee Mays) Variety of Manipur and Its Impact On Biological Control

International Journal of Human Genetics Medical Biotechnology and Microbiological Studies
ISSN (Online) 2319-1732 : Volume 1 , Issue 3 , December 2012

11
http://www.ijhgmbms.info/article/IJHGMBMSV1I302.pdf

Isolation and characterization of Endophytic microbiome from indigenous maize (Zea mays)
variety of Manipur and its impact on Biological control

Sarangthem Indira Devi* and Momota Potshangbam
Microbial Resources Division,
Institute of Bioresources & Sustainable Development (IBSD),
An autonomous research institute, Department of Biotechnology,
Govt. of India, Takyelpat, Imphal-795001, India

Corresponding Author:
Sarangthem Indira Devi
Scientist, Microbial Resources Division,
Institute of Bioresources & Sustainable Development (IBSD),
An autonomous research institute, Department of Biotechnology,
Govt. of India, Takyelpat, Imphal-795001, India
Email: sidevi1@yahoo.co.in, Tel. 09436892491

Abstract

Reflection on food productivity with the ever
growing demand along with frequent food crisis
scenario around the globe added to serious concern
for continuous exploitation on beneficial microbes
for wide range of applications in the field of
agriculture, medicine and industries. Keeping this in
view, maize which is one of the important cereal
crops grown worldwide was chosen for the isolation
of endophytes owing to its high adaptability and wide
tolerance to varying climatic conditions. A total of
306 endophytic isolates were obtained from different
tissue segments of 18 healthy indigenous maize plants
using different growth media. Bacterial genera were
most frequently isolated, followed by Yeast and
Fungi. Among all the isolates Bacillus sp.,
Pseudomonas sp. and Fusarium sp. were found as
common dominating endophytes. The isolates were
studied for their multiple plant growth promotion and
biocontrol activity such as production of -1,3-
glucanase, chitinase, protease, cellulase and
siderophore. Phosphate utilization, growth on N
2
free
medium and Amylase activity were also evaluated.
Antagonistic activity was tested against commonly
found fungal pathogens in cereal crops, such as
Pyricularia oryzae, Rhizoctonia solani, Sclerotium
oryzae and Pyithium ultimum. Mz10, T-0, Mz30/156,
N116y, C128 and D167 exhibit multiple functions
and were potentially active against the tested
phytopatogens.

Key words: Endophyte, Maize, Economic, Biocontrol,
Agriculture.

1. INTRODUCTION
Plant associated beneficial microbes lives in
varying relation with the host, they provide nutrients
to the associated microbes and in turn get benefited
from microbes by promoting plant growth, increase
yield, vigor tolerance to list of biotic (pathogenic
microbes, insects, pest, parasites, nematode etc.) and
abiotic stresses (drought, temperature, pH, salinity,
flood, wound, cut or injured part, nutrient deprived,
etc). Endophytes are microbes that can be found
living inside plant tissues, where they can live
commensally or execute beneficial functions for the
host [1].The study of plant microbiome is indeed
essential for wide coverage applications and active
metabolites from endophytes is of potential
contribution. Host of endophytes is also a niche to
phytopathogens and hence endophytes capability as a
biocontrol agent against microbial plant pathogens,
insects, nematode and plant pest is encourage.
Endophytes are categorise into three functional
groups mainly, plant growth promoters, biocontrol
[2]and plant stress homeo regulating microorganisms
[3] that can directly or indirectly facilitate the plant
growth in optimal, biotic or abiotic stress condition.
The opportunity to find new endophytic
microorganisms of potential properties among the
diversity of plants in different ecosystem is
captivating and may end up with answer to most
interpretation and combat food crisis worldwide.
Maize is one among the most demanding
economicaly valued cereal crops worldwide and each
portion of the crop is utilized for various purposes

12

and production such as food, fodder, raw materials,
as source to fuel, starch, oil, proteins, sweeteners,
range of beverages etc. Adding to its versatile
adaptability and wide range of growth conditions, it
is preferably the target host to study its associated
endophytes. Adding, the increase concern over the
deteriorating production scenario worldwide due to
various biotic and abiotic causes and factors demands
contributing study for precautionary measures and
control from numerous diseases of maize. About 50-
80% of maize lost is reported to be cause by fungi
alone during storage [4]. Endophytic isolates from
Maize collected from different districts were studied
for various potential activities. The observation on
occurrence frequency of isolates, records higher
bacterial species compared to fungal and yeast. The
most frequently isolated members of the endophytic
bacterial community in maize are Enterobacter spp.
(members of the gamma subclass of the class
Proteobacteria [gamma-proteobacteria]),followed by
the beta-proteobacterial Burkholderia spp) [5]. High
Nitrogen fertilization is required for cereal crops,
especially maize and therefore increasing the fixation
throufh biological process and reducing the N-
chemical fertilization [6]. The present study focuses
on the isolation of beneficially potential endophyte
that could confer broad host spectrum of study
referring to the specificity and non-specificity
category and the overall application of the isolates be
it medical, agro industries or commercial industries.
2. METHODOLOGY
2.1. COLLECTION AND PROCESSING
OF SAMPLE

Healthy and symptomless maize plants of different
growth stages were collected from various districts of
Manipur during 2011, considering the differences in
environmental conditions referring to weather,
landscape and air quality with the essential data of
references. The freshly collected samples were
process within 24-48hours of collection.

2.2. SURFACE STERILIZATION AND
ISOLATION OF ENDOPHYTES
The freshly collected samples were washed
thoroughly under running water to removed adhering
soil and associated unwanted particles. For isolation
and sterility effectiveness, a modified protocol from
[7]) was followed. Leaves, stems and roots were
sectioned into appropriate segments, soaked in sterile
distilled water, drained and surface sterilized by
immersing on 90% ethanol for 2mins, rinse with 5%
of Tween 20 for few minutes and treated with
aqueous solution of Sodium hypochlorite (4%
available chlorine) for 5mins. The samples were rinse
once with sterile distilled water and retreated with
70% alcohol for 1min, followed by 8 successive
rinses with sterile distilled water and dried in aseptic
condition. For direct plate impression of the sterilize
tissue segments, the outer portion and cover was
aseptically removed and very thin slice of each tissue
segments were plated in triplicates followed by the
traditional microbiological methods, spread plate and
pour plate technique. The tissues were homogenized
using sterile mortar and pestle, concentrated and
diluted samples of 10
2
,10
3
and 10
4
dilutions

were
plated on PDA, SDA with chloramphenicol,
Czapekdox agar, MEA, CMA, LB, BHI, TSA,
YMCR, OMA (HiMedia) and incubated at 30C for
7-21days or more till the observation of growth, fast
growing colonies were purified and remove to
encourage and observed the growth of other slow
growing endophytes especially fungus. The isolated
cultures were purified, maintained and deposited at
IBSD, Microbial Resource Division. The
macroscopic and microscopic features were studied
based on the colony morphology and pattern
observed on different media, spore types, hyphae and
related characteristics (Fig.1).

3. GROWTH ASSAY ON NITROGEN
FREE BASAL MEDIA

Burks nitrogen free media [8] and Norris glucose
nitrogen free media (HiMedia) were used to assess the
growth of the isolates. The growth characteristics
observation was made based on the colony growth
pattern in terms of luxurious mycelium growth to thin
and tinge mycelium spread on the media.

4. STUDY OF ANTAGINISM IN VITRO

The endophyte isolates were evaluated for
antagonistic activity against widely prevailing
pathogens of cereal crops viz. Pyricularia oryzae,
Rhizoctonia solani, Sclerotium oryzae and Pyithium
ultimum obtained from Indian Type Culture
Collection, New Delhi. Antagonistic activity was
checked using dual culture plate assay [9]. Fungal
discs (5mm) of pathogen and test organism was
inoculated at opposite sides with partition gap of 3cm
approximately and incubated at 30C2C. The Plates
incubated with pathogen was served as control. The
inhibition percentage was calculated using the
following formula [10].

13

Inhibition % = C T X 100
C

Here, C represents the growth diameter of the
pathogen in the control plate and T represents the
pathogen diameter on the dual.
5. EVALUATION OF HYDROLYTIC
ENZYMATIC ACTIVITIES
5.1. Protease production assay
Pure fungal isolates were inoculated on Skim milk
agar (HiMedia) and incubated at 30C2C. Visible
clearance around the colony qualitatively indicates
protease activity.
5.2. Phosphate solubilization assay
Pikovskya agar was used with addition of 0.5%
calcium phosphate dibasic (HIMEDIA).The test
cultures were inoculated and incubated at 30C2C.
The presence of halo zone or clearance around the
colony after a week or more days of incubation
qualitatively ensures phosphate solubilization potential
of the isolate.
5.3. Chitinase production assay
Chitinase activity was determined by following the
modified methods given by Gupta et al, 1995 [11].
Plates were inoculated and incubated at 30C2C for
4-5 days and formation of clearance zone around the
colony was observed.
5.4. Production of -1, 3-glucanase
Carboxy methyl cellulose agar incorporated with
laminarin (Sigma) was used for plate assay detection
according to the modified method of [12]. Plates were
incubated at 30C2C for 4-5 days. The activity was
determined after treating the colony with 0.1% congo
red dye for 8min and destaining it twice with 1N NaCl
followed by 1 N NaOH for 15mins respectively. A
clearance around the colony indicates the production
of the enzyme.
5.5. Siderophore production assay
The fungal isolates were inoculated on Chrome
azurol S (CAS) agar medium and incubated at
30C2C for 4-5 days following modified protocol
[13]. The colonies turning yellow with zone formation
were considered Siderophore producing and
determined based on the intensity of the zone and
color.
5.6. Cellulase production assay
The activity was determined following a modified
protocol [14] .Inoculated plates were incubated at
30C2C for 4-5 days, followed by flooding the
colony with 1% iodine solution incorporated with 2%
potassium iodide for few seconds and drained off.
Formation of clear halo zone surrounding the colony
indicates qualitative enzyme production.
5.7. Amylase production assay
Glucose Yeast peptone agar with 1.8% soluble
starch was used to determined amylase activity
modified [15]. The plates were inoculated and
incubated at 30C2C for 4-5 days. The plates were
flooded with 1% Iodine in 2% potassium hydroxide.
Clear halo zone formation around the colony indicates
amylase production.

Fig1 Endophytic colony in different culture media
(above) & colony morphology of N188 (2) and D50A
(1) below.
6. STATISTICAL ANALYSIS
2
1

14

One-way analysis of variance (ANOVA) was
performed to determine the data means and standard
error for the enzymatic and biocontrol activity of the
endophytic isolates. The difference was considered
significant at p<0.05.
7. RESULTS AND DISCUSSION
Endophytic microorganisms lives asymptomatically
within plant tissues [16] and colonized the internal
plant and are recognized as a potential biocontrol
agents [17]. Endophytic microbes covering bacteria,
fungi and yeasts were isolated from indigenous
healthy maize crops on different growth stages
following effective surface sterilizarion protocol. A
total isolates of 106 from leaf, 98 from Stem and 102
from root were recovered.

Many factors affect the structure and species
composition of the microbial communities that
colonizes roots, stems and leaves [18]. Endophytic
communities vary specially in the plant or may be
dependent on the interaction with the other
endophytic or pathogenic microorganisms [19].
Relative colonization frequency of representative
endophytes was calculated by the formula given by
[20]. The rate of colonization observe in the study
demonstrated higher in leaf tissues for fungal and
yeast isolates while bacterial colonization was more
in stem tissues (Fig.2). Some of the frequent fungal
genera recovered are Fusarium, Penicillium,
Aspergillus, Trichoderma, Acremonium etc., bacterial
genera includes Pseudomonas, Bulkholderia,
Enterobacter, Bacillus, the most dominating yeast
isolates belongs to the genera Galactomyces,
Rodotorula and Candida sp.

In this study, a large number of morphologically
different bacterial and fungal isolates were obtained.
Antifungal activity was tested for all the isolates
against four common phytopathogens of cereal crops
and produces promising results. Among all the
bacterial isolates Mz10 was active only against
Sclerotium oryzae whereas T-0 was active against all
the tested fungi and has widest range of antifungal
spectra of activity (Fig.2) and isolates N156 and
Mz30/156 was equally active against Pyithium
ultimum. Most of the test organisms were active
against Sclerotium oryzae followed by Pyricularia
oryzae and Pyithium ultimum. Isolates activity
against Rhizoctonia solani was moderate (Fig.3).

Microorganisms of biocontrol activity by
endophytes generally involved competition [20].
Production of bacteria and funal metabolites such as
siderophore, HCN, antibiotics or extracellular
enzymes such as protease, chitinase, cellulose etc.
confirms the antifungal activity profile in these
organisms [21]. The isolates were screened for the
production of hydrolytic enzyme and in vitro
antagonistic potential. Among all the tested bacterial
endophytes, T-0, was found to be very active in the
entire assay with good activity of phosphate
solubilization and protease activity. Yeast isolate
N148y and N116y were found to be comparatively
good (Table.1).

Enzyme assay of the selected endophytic fungal
isolates showed potential results. The isolate C128
and D167 were positive for all the assays with high to
very high activity for -1,3-glucanase and cellulase
respectively, while D167 showed promising chitinase
activity. The other test organisms including D56A,
N188, D51A and D40 were comparatively active.
Growth pattern on nitrogen free basal media was
recorded with the abundance to thin scanty mycelium
spread (Table 2). All the isolates were capable of
growing at the basal media free of nitrogen
supplement and hence more potency was
characterized by the abundant growth of the
endophytes, owing to the colony morphology. D40
and C128 grows well in NFB compared to all.

The result on antagonism test ensures the
functional capability of the isolates against the
pathogens. All the isolates were less active to
Pyricularia oryzae, Except for D51A which controls
all the tested pathogens equally. On the other hand,
Scleritium Oryzae and Pythium ultimum was readily
control by all the endophytes (Fig.4). Therefore,
majority of the endophytes were potential in
expressing beneficial activities when comes to
adaptation and hence exploiting the plant microbe
interaction for better protection of plant is essential.

0
10
20
30
40
Bacteria
Yeast
Fungus
Fig 2 Relative occurence of endophytes
recovered from tissues
Leaf
Stem
Root

15

Fig 3

Antagonistic activity of bacterial and yeast
endophytic isolate against Pyricularia oryzae,
Rhizoctonia solani, Sclerotium oryzae and Pyithium
ultimum.

0

20

40

60

80

100

A
c
t
i
v
i
t
y

(
m
m
)

Isolates

Antagonistic activity of endophytic bacterial & yeast
isolates

S.O

P.O

R.S

P.U

Fig 4 Antagonistic activity of endophytic fungal
isolate against Pyricularia oryzae, Rhizoctonia
solani, Sclerotium oryzae and Pyithium ultimum
0
20
40
60
80
100
N52 C128 D51A N188 D170 D380 D167 D40 D50A D56A
A
c
t
i
v
i
t
y

(
m
m
)
Isolates
Antagonistic activity of endophytic fungal
isolates
P.O S.O P.U R.S
Table I: In vitro enzyme assays and growth on NFB of bacterial endophytes:.
Sl.
no
.
Isolate
Code
Phosphate
-activity
Protease Chitinase Amylase Beta-1,3
Glucanase
Cellulase
Sidero-
phore
activity
Growth
on NFB
1. N119y 2.15 0.08 10.53 0.08 2.55 0.05 0.55 0 .02 - - + +
2. N148y 6.1 0.11 5.1 0.05 4.66 0.33 1.55 0.028 0.23 0.02 -
+ +
3. N119sy 5.16 0.21 8.4 0.05 4.23 0.14 0.32 0.012 - 0.2 .005 - +
4. N116y 3.06 0.03 8.06 0.03 5.16 0.08 1.12 0.014 0.25 0.01 - + +
5. N124 3.06 0.12 12.4 0.02 4.23 0.12 0.18 0.003 - - - +
6. T-0 9.23 0.03 9.46 0.03 3.43 0.12 0.52 0.014 0.23 .005 1.0 0.02 ++ ++
7. Mz27/
10
1.86 0.4 6.22 0.05 1.96 0.03 0.31 0.012 - - + +
8. D42 7.93 0.12 11.3 0.05 5.33 0.33 - 0.18 0.01 - - +
9. D183 2 0.05 9.11 0.06 2.33 0.12 0.2 0.016 - 0.3 0.014 + +
10. D139 2.05 0.03 10.25 0.05 1 0.05 0.85 0.005 - - - +
11. Mz183 4.93 0.12 9.36 0.03 2.56 0.13 0.22 0 .003 - - ++ +
12. D98 2.13 0.08 10.16 0.08 2.53 0.06 0.3 0. 003 - - - +
13. Mz30/
156
6.7 0.14 3.52 0.05 2.60 0.08 1.65 0.015 - - + +
14. Mz10 3.2 0.1 7.55 0.05 1.90 0.05 0.600.02 - 0.28 003 ++ +
15. N156 3.3 0.1 8.65 0.05 1.1 0.05 0.550.02 - - + +
Values are average of three replicates (zone of inhibition in mm; n=3, Mean SE).
(Note: ++, good activity; +, moderate; -, no activity; y- Yeast)

16

Table

2:

Biochemical activities of

endophytic fungal isolates

Isolate code

A

B

C

D

E

F

G

NFB

N52

-

+++

++

-

-

-

+

+

D51A

-

+

+

-

++

+

++

+

N188

+

+

+

+

+++

+

-

+

D170

+

+

+

-

+

++

-

+

D380

+

+

-

+

-

+

++

+

D40

+

++

-

+

++

+

++

++

D50A

-

++

++

-

-

++

+

+

D56A

-

+

+

+

+++

+++

+++

+

D167

+

+++

+

++

++

+++

++

+

C128

+

+

+

+

++

+++

+

++

Note: -, no activity; +, moderate activity; ++, high activity; +++, very high activity.

(A-Phosphate activity, B-Chitinase, C-Siderophore, D-Protease, E--1,3-glucanase, F-Cellulase, G-

amylase)

8. CONCLUSION
Endophytic microbes live non-pathogenically inside
their hosts where they perform a number of beneficial
functions for the plant, including nutrient acquisition,
plant hormone production and antagonism to
pathogens [22]. The endophytes were first believe to
have colonized in algal intertidal zones, or may have
begun to colonize plants 500-700 million years ago
when molecular studies estimate the first plants came
on land [23]. Endophytes being a basis to numerous
benefits in terms of agriculture, bioremediation agent,
therapeutic and production of many more industrially
useful enzymes are widely studied organisms owing
to multiple applications The present study however
enlightened the underlying world of maize endophyte
and its possible application as plant protective bio-
agents. Further in-depth research is still needs to be
continued.
ACKNOWLEDGEMENTS
The authors express humble gratitude to the
Department of Biotechnology (DBT), Government of
India, for assisting financial support.
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Isolation and Characterization of Endophytic Microbiome From Indigenous Maize (Zee Mays) Variety of Manipur and Its Impact On Biological Control

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ISSN (Online) 2319-1732 : Volume 1 , Issue 3 , December 2012

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