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Int. J. Curr. Res. Biosci. Plant Biol. 2015, 2(6): 101-106
Manish Kumar et al. (2015) / Int. J. Curr. Res. Biosci. Plant Biol. 2015, 2(6): 101-106
101
Original Research Article
Biocontrol Activity of Some Potent Methylotrophs Isolated from Bhitarkanika
Mangrove Sediment
Manish Kumar1, Alok K. Srivastava*1 and Akhilesh K. Pandey2
1National Bureau of Agriculturally Important Microorganisms (NBAIM), Mau, U.P., India
2Department of Biosciences, Rani Durgavati Vishwavidyalaya (RDVV), Jabalpur, M.P., India
*Corresponding author.
A b s t r a c t
K e y w o r d s
Methylotrophs are C1 carbon compound utilizing group of bacteria. These
subpopulations of bacteria were isolated from the sediment sample of
Bhitarkanika mangrove ecosystem. A total 50 isolates were obtained at the
universal media plate specific for methylotrophs, containing methanol as a
source of carbon and energy. All the isolates were subjected to screening for
the antagonistic activity against fungal pathogen Macrophomina phaseolina.
The test performed resulted a few of them were designated as potent inhibitor
of this rot causing fungal pathogen. The effect of methylotrophic isolate on the
fungal mycelia was clearly observed after scanning electron imaging of the
interface of fungus and bacteria. Therefore, study indicates the
biotechnological potential of methylotrophs isolated from mangrove sediment.
Biocontrol
Carbon
Mangrove ecosystem
Methylotrophs
Introduction
Mangrove ecosystem is recognized as a natural
resource of importance to mankind by virtue of its
efficacy and aesthetic value. The mangrove ecosystem
is one of the most fertile ecosystem of tropical and
subtropical regions of the world. Such type of dynamic
mangrove ecosystem supports numerous diversified
soil micro and macro organisms. Soil organisms of
mangrove, especially microbial flora, play a very
important and crucial role in the degradation of
mangrove foliage, which is continuously shed and
decomposed in the mangrove base (Kumar, 2000). A
total of 4827 km2 mangrove forests in India is reported
which is 0.1% of total geographical area of India and
5% of the world’s mangrove vegetation (Kathiresan
and Rajendran, 2005). Bhitarkanika mangrove
ecosystem, located at the junction of the Brahmani and
Baitarni rivers, is the second largest Indian mangrove
ecosystem (next to Sundarban, West Bengal). Because
of its rich biodiversity, it is regarded as one of the top
ranking mangrove forests of the world in terms. High
biological species diversity is observed in this
mangrove which is evaluated among one of the best in
the world (Thatoi et al., 1999).
Bhitarkanika mangrove is deputed as a Ramsar
wetland of international importance in year 2002.
International Journal of Current Research in
Biosciences and Plant Biology
ISSN: 2349-8080 Volume 2 Number 6 (June-2015) pp. 101-106
www.ijcrbp.com
Int. J. Curr. Res. Biosci. Plant Biol. 2015, 2(6): 101-106
Manish Kumar et al. (2015) / Int. J. Curr. Res. Biosci. Plant Biol. 2015, 2(6): 101-106
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Because of richness in carbon and reduced carbon
along with other nutrient content, the mangrove
ecosystem harbors variant microbial communities
which can adapt to the saline condition of this niche.
Mangrove forest is environments subject to significant
degradation by anthropogenic activities. In coastal
area, interfacing the continents and the oceans makes it
substantially important in the prediction for
biotechnological applications. A number of bacterial
groups like phosphate solubilizers, methanogens,
methane oxidizers, nitrogen fixers, cellulose
decomposers, nitrifiers, archaea, denitrifiers, iron
oxidizers, sulphur oxidizers and iron reducers are
generally inhabitating this mangrove forest (Holguin et
al., 2001).
Macrophomina phaseolina (Tassi) Goid is a well
known seed damaging, soil borne pathogen, infecting a
number of plant species throughout the world (Kunwar
et al., 1986; Mihail and Taylor, 1995, Srivastava and
Singh, 1990). Many diseases like damping off,
seedling blight, collar rot, stem rot, charcoal rot and
root rot are caused by this fungus under favorable
condition in numerous economically important crop
plants. The root-rot caused by Macrophomina among
the fungal diseases, prevails to be a challenging
assignment in terms of management. It is prevalent
sub-tropical and tropical climate, in arid and
particularly in the areas with low rainfall, high
temperature and it is distributed worldwide (Raut and
Bhombe, 1984). To eradicate pathogenic fungi,
numerous disease management methods have been
employed to resist. Apart from target organism,
several beneficial organisms are also killed by
pesticides. In soil and environment their toxic forms
persist (Hayes and Laws, 1991).
Due to increment in awareness of humankind for the
ecosystem and environment, a remarkable shift from
synthetic materials to bio-products was observed.
Against several kinds of pests fungi constitute an
extensive group of bioagents. Fungi such as
Trichoderma, Gliocladium can minimize, suppress and
may inhibit the parasitism of pathogens like Fusarium
sp., Rhizoctonia sp., Sclerotium sp. (Rajappan and
Ramaraj, 1999; Hadar et al., 1979; Papavizas and
Lewis, 1989; Murmanis et al., 1988; Tu, 1991; Kim
and Roh, 1987). The present investigation is,
however, design in a way to investigate efficacy of
some methylotrophic species against pathogenic M.
phaseolina.
Materials and methods
Sampling site and sample collection
Geographically Bhitarkanika is located between 20°4'-
20°8'N Latitudes and 86°45'- 87°50' Longitudes. The
samples were collected in the month of March from
top 4 cm soil profile of sediment where most of the
microbial activity takes place, and thus where most of
the bacterial population is concentrated. Total five
composite samples were made from Gupti (BN1),
Habalganda (BN2), Mahisamunda (BN3), Dangmal
(BN4) and Kalibhanjadian (BN5). Samples were taken
in replicates. Soil samples were collected by using
clean, dry and sterile polythene bags along with sterile
spatula, marking pen, rubber band and other
accessories. The site selection was done by taking care
of the point where widely varying characteristics as
possible with regard to the organic matter, moisture
content, and particle size and color of soil and to avoid
contamination as far as possible. Samples were stored
in iceboxes and transported to the laboratory where
they were kept in refrigerator at -20°C until analysis.
Isolation and enumeration
The samples were taken for the serial dilution up to
104 dilution, 0.2 ml of each dilution were inoculated in
duplicate plates of the NMS (Nitrate mineral salt)
media containing 0.1% methanol as carbon substrate
for the isolation of methylotrophs by the spread plate
technique (Joshi et al., 2008). After incubation all
plates incubated at 30ºC in the incubator for 5-6 days.
After incubation the population was recorded for each
sample. Cycloheximide was used as antifungal agent
in plates. A total 50 pure isolates of methylobacteria
have been isolated by streak plate method.
Biocontrol activity against fungal pathogen
The pathogen used in the present study was obtained
from DSR (Directorate of seed research), Mau, U.P.,
India. The fungal pathogen used in present
investigation was maintained on PDA (Potato dextrose
agar) plates. In vitro antagonistic activity of
methylotrophic isolates against M. phaseolina was
studied in dual culture technique by following the
method by (Kucuk and Kivanc, 2003; Dennis and
Webster, 1971). Control plate was grown individually
on PDA plate after incubated. Three replications were
maintained for each treatment. Three times replication
Int. J. Curr. Res. Biosci. Plant Biol. 2015, 2(6): 101-106
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was done for each treatment with incubation of
28±2°C. After 8 days of incubation, percent growth of
antagonists, pathogen and zone of inhibition is
recorded. Antagonistic activity of methylobacteria was
tested in which a 5 mm mycelia agar disc from
pathogen plate was placed on the centre of Petriplate
containing PDA. Then plates were incubated at 28°C
overnight. Methylotrophic cultures were then streaked
3cm away from the disc of M. phaseolina. Control
containing plates inoculated only with test pathogen.
The formula for the calculation of percent growth
inhibition (PGI):
% inhibition = KR-R1/KR × 100
Where KR is the distance between point of inoculation
to the colony margin on the control plates, and R1 is
the distance of fungal growth from the point of
inoculation to the colony margin on the treated plates
in the direction of the antagonists (Korsten and De
Jager, 1995). The percent growth inhibition scale
ranges from 0 to 4, where 0= no growth inhibition; 1=
1-25% growth inhibition, 2=26-50% growth inhibition;
3=51-75% growth inhibition; 4=76-100% growth
inhibition. After 7 days of incubation, the zone of
inhibition (distance between pathogen and are of
antagonist) was recorded (Zivkovic et al., 2010).
Scanning electron microscopy
The interaction of the test fungi with antagonistic
isolates was studied by scanning electron microscopy
(SEM). The hyphae from the interaction zone was
transferred on glass coverslip, then fixed with 1.5%
gluteraldehyde and dehydrated with graded series of
ethanol washes followed by drying in desiccator (Yuan
and Crawford 1995). Samples were affixed to SEM
stubs using carbon tape followed by thin coating with
gold: palladium (60:40) and examined by SEM (JEOL,
JSM-62804).
Results
Soil properties and enumeration of methanol
oxidizing methylobacteria
Five composite sediment samples were subjected to
the estimation of physical properties in which the
range of pH was 7.4 to 8.0 while the electrical
conductivity ranges from 4.04 dSm-1 to 6.67 dSm-1
with organic carbon from 0.7 to 1.5 unit.
Enumeration of culturable, aerobic, methylotrophic
bacteria was carried out by standard spread plate count
dilutions using specific media. The population of
methylotrophs ranged from 5 -300 x 104 g-1 soil at
different sampling sites. The bacterial diversity found
in the isolations from the five different locations
revealed to be statistically different (p<0.05). At
location BN1, the number of bacteria had a log value
of 4.69, location BN3 and BN5 had a value of 5.69
while at location BN2 and BN4 the value was 6.47,
maximum among different sites (Table 1).
Table 1. Enumeration of methylobacteria population at different sampling sites.
Sample
Sample type
Sampling site
CFU
Log CFU
BN1
Sediment
Gupti
5 × 104
4.69 ± 0.33
BN2
Sediment
Habalganda
300 × 104
6.47 ± 0.37
BN3
Sediment
Mahisamunda
50 × 104
5.69 ± 0.46
BN4
Sediment
Dangmal
300 × 104
6.47 ± 0.48
BN5
Sediment
Kalibhanjadian
50 × 104
5.69 ± 0.49
However, no significant differences were observed
between samples collected from different locations.
From these soil samples, a total of 50 isolates were
randomly selected for their antagonistic activity
against pathogenic fungus. The methylotrophic
population was higher in the sediment sample from
Dangamal and Habalganda district. Methylotrophs
with different morphology and pigmentation were
observed from this ecosystem. All of the isolates
were aerobic, catalase, urease positive and weakly
oxidase positive. The halotolerant ability of the
isolate was observed and NaCl tolerance ranges
from 100 mM to 550 mM in most of the
methylobacteria.
Biocontrol activity against pathogenic fungus M.
phaseolina and SEM
In this screening, some isolates were found potential to
antagonize the pathogen at considerable level ranging
from 25.45- 54.65% inhibition. Antagonistic potential
of some selected methylobacteria was concluded and
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validated by restriction and inhibition of pathogen
growth by showing zone of inhibition towards the
antagonists as shown in photo-plates of dual culture
plate assay (Fig. 1). The isolates NBMS37 and
NBMS48 showed maximum antagonism (54.65% and
51.23% respectively) followed by isolates NBMS19,
NBMS5, NBMS33, NBMS11 and NBMS9. SEM
images of the interface of pathogenic fungus and
methylobacteria reveals that fungal growth is inhibited
by methylobacteria (Fig. 2). The growth of fungus is
relatively checked by inhibiting the dispersal of
hyphae in the plate. The observation revealed the
disruption of the fungal hyphae by the activity of
methylobacteria.
Fig.1: Biocontrol activity of the methylotrophic isolates against M. phaseolina.
Fig. 2: Scanning electron micrographs of bicontrol
activity of isolate NBMS48 against pathogenic fungus
M. phaseolina. Picture indicates the ruptured fungal
mycelia surrounded by methylotrophic bacterial cells
(NBMS37).
Discussion
Biological control is considered as an environmental
friendly strategy to minimize and eradicate the crop
damage caused by plant pathogens. Pesticide use by
the general public and governmental agencies are
increasing concerns now a day, severely restricting the
availability and use of several important pesticides. In
vitro antagonistic activity of methylotrophic isolates
against pathogen was estimated and observed in dual
culture technique by following the method of Kucuk
and Kivanc (2003).
Differential biocontrol ability among the antagonists
was noticed against M. phaseolina. Seven days of
incubation represent various degrees of mycelial
growth inhibition of Macrophomina. The intermingled
contact zone of methylotrophs and pathogenic fungus
was cut and was observed under the electron
microscopy. The electron micrograph of the
intermingled region shows the collapse, shrinkage and
disruption of cytoplasm of the fungal mycelia with the
healthy methylotrophic cells around mycelia. Baker
and Cook (1979) have reported that the mycelial walls
and septal walls may be digested by the enzymes
produced or antibiotic formation that inhibit growth or
cause endolysis. The isolates NBMS37 and NBMS48
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were found to be most potent in reducing and
inhibiting the growth of pathogen. The hypothesis of
antibiosis, i.e. the secretion of extracellular toxic
metabolites is supported by the results obtained, led to
the fact that the main mode of action by which
methylotrophs exhibit its biocontrol potential
(Hajlaoui and Belanger, 1993).
In many disease control programs, fungicides as
standards have been used, been increasingly regulated.
Research efforts with pathogens have indicated that
most of the biocontrol organisms either did not control
the pathogen or they did not perform as well as
selected fungicides. Reduced carbon utilizing
microbes, Methylotrophs are a physiologically
interesting group of bacteria that preferentially utilize
methanol and other reduced one carbon compounds
such as formate and formaldehyde as sole source of
carbon and energy give a good biocontrol activity
against phytopathogens like M. phaseolina, Fusarium
oxysporum and Sclerotium rolfsii (Poorniammal et al.,
2009). In this study the bacterial isolates with
methylotrophic potential were subjected to check their
activity against phytopathogen like M. phaseolina.
Few isolates like NBMS5, NBMS37 and NBMS48
were giving very considerable antagonistic activity
against pathogenic fungi while others were not
showing antagonism. The scanning electron
micrograph clears the image of interface of bacteria
and fungi in which the fungal mycelia were disrupted
and growth was inhibited by the healthy bacterial
community present.
Acknowledgement
Authors are thankful to ICAR (Indian council of
agricultural research) for financial assistance and
laboratory facilities. Authors are also thankful to the
Director, NBAIM and HOD, Department of
Biosciences, RDVV, Jabalpur.
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