Journal of Research in Microbes
Journal of Research in Microbes An International Online Open Access Publication group
Original Research
Characterization of rhizospheric bacteria Bacillus licheniformis 9555 and analysis of antifungal action Authors:
Vipin Mohan Dan2, Sandhya Mishra1, Vasvi Chaudhry1, Poonam Singh1, Sumit Yadav1, Shashank Mishra1, Swati Tripathi3, Palpu Pushpangadan2, Varughese George2, Ajit Varma3 and Chandra Shekhar Nautiyal1.
ABSTRACT:
Rhizospheric bacteria Bacillus licheniformis 9555 was characterized at optimum conditions for effective growth. The bacteria showed maximum growth at pH 7 and a temperature of 37°C. The most efficient carbon sources were maltose and dextrose and the nitrogen sources were ammonium chloride and ammonium acetate. The microbe produced the enzyme chitinase which was correlated to the antagonistic Institution: potential of bacteria against the phytopathogenic fungus Drechslera oryzae. 1. Division of Plant-Microbe The optimum requirements for effective antifungal action of the bacteria in co-culture Interaction, CSIR-National condition were determined to be 37°C and pH 7.The antifungal action was maintained Botanical Research Institute, in in vitro saline conditions, but a decreasing trend was shown with increasing salinity Lucknow 226001, India in the medium. 2. Amity Institute for Herbal and Biotech Product Development, Trivandrum-695005, Kerala, India.
Keywords: Antifungal, Chitinase, Bacillus, Rhizosphere.
3. Amity Institute of Microbial Technology, Amity University, Sector-125, Noida -201 303, U.P, India.
Corresponding author: Vipin Mohan Dan and Ajit Varma. Email:
[email protected] [email protected] Phone No: (0471) 2432138 +91 120 4392132. Web Address: http://ficuspublishers.com/
documents/MI0017.pdf.
Article Citation: Vipin Mohan Dan, Sandhya Mishra, Vasvi Chaudhry, Poonam Singh, Sumit Yadav, Shashank Mishra, Swati Tripathi, Palpu Pushpangadan, Varughese George, Ajit Varma and Chandra Shekhar Nautiyal. Characterization of rhizospheric bacteria Bacillus licheniformis 9555 and analysis of antifungal action. Journal of Research in Microbes (2012) 1(2): 077-082
Dates: Received: 31 Aug 2012
Published: 30 Oct 2012
© Ficus Publishers. This Open Access article is governed by the Creative Commons Attribution License (http:// creativecommons.org/licenses/by/2.0), which gives permission for unrestricted use, noncommercial, distribution and reproduction in all medium, provided the original work is properly cited.
Journal of Research in Microbes An International Open Access Online Research Journal
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Dan et al.,2012 plant-microbe interaction, National Botanical Research
INTRODUCTION Microbes are organisms that have the ability to
Institute (NBRI), Lucknow, India.
survive in a wide range of environmental conditions and
Optimization of culture conditions
some are genetically designed to support the growth of
Temperature and pH
higher organisms (plants). Genus Bacillus, which is
The ability of bacterial strains to grow at
ubiquitous in soil, is well known for its adaptability to
different temperatures (25ºC to 50ºC) and pH (4 to 10)
rhizospheric environment and can also protect plants
was determined by inoculating 50 ml of Nutrient Broth
against fungal pathogen attack (Asaka and Shoda 1996).
with the initial bacterial population of 4.5 log10 CFU/ml.
Microbes produce a variety of secondary metabolites that
Bacterial growth was studied upto 10 days by
are being used in different areas from agriculture to
determining CFU (Colony forming unit) using serial
medicine (Powell and Jutsum 1993; Dan et al., 2012a).
dilution method.
Among the first successful biocontrol agents used
Utilization of carbon and nitrogen sources
against insects and pathogens were the members of the
To analyze the utilization of carbon compounds,
genus Bacillus. In relation to antifungal action,
the bacterial strain was streaked individually on M9
cell-wall-degrading
as
minimal media, in which glucose was replaced by
β-1,3-glucanases, cellulases, proteases, and chitinases,
various carbon sources and for nitrogen source
are involved in the antagonistic activity of some
ammonium chloride was replaced by various other
biological control agents against phytopathogenic fungi
nitrogenous compounds. For carbon source, glucose
(Kim and Chung 2004). In particular, numerous
served as control while for nitrogen source ammonium
correlations between fungal antagonism and bacterial
chloride was used as control. The plates were incubated
production of chitinases and/or β-1,3-glucanases have
at 37°C in BOD incubator (Composite Lab line, India)
been noted (Lim et al., 1991). In our laboratory we have
and visually observed till the 7th day.
isolated a rhizospheric bacteria B. licheniformis 9555
Analysis of enzyme production
enzymes
(CWDEs),
such
which possess plant growth promotion properties and
B. licheniformis was analyzed for the presence of
also produces siderophores. (Dan et al., 2012b). The
various enzymes like protease, chitinase, lipase and
present study investigates the antifungal potential of
pectinase as per the protocols described by Rondon,
B. licheniformis 9555.
(2000). Microscopic
MATERIALS AND METHODS
analysis
of
antifungal
action
by
co-culture method Phytopathogenic fungus was grown in 50 ml
Microorganisms Bacillus licheniformis 9555 was previously
PDB (Potato dextrose broth) in 200 ml conical flasks at
isolated from rhizopsheric soil, identified by Microbial
30ºC, 120 rpm for two days, by inoculating with agar
type culture collection (MTCC), Institute of Microbial
disc from the fungal culture. After the incubation period,
Technology (IMTECH), Chandigarh. (Dan et al., 2012b).
the
Biolog system (Biolog Inc., Hayward, CA 94545, USA)
4.5 log10 CFU/ml of overnight grown bacterial strain and
was used to confirm the identity of the rhizopsheric
incubated again. Fungal mycelium was taken from the
bacteria (Figure 1). The bacterial strain was maintained
co-cultured broth at different time intervals and stained
on nutrient agar medium. Plant pathogenic fungus
with lacto-phenol cotton blue and observed under light
Drechslera oryzae was procured from the Department of
microscope.
078
fungal
broth
culture
was
inoculated
with
Journal of Research in Microbes (2012) 1(2): 077-082
Dan et al.,2012
Figure1: Dendogram of Bacillus licheniformis 9555 produced at 24 h Biolog reading Optimization
of
parameters
for
antifungal
and 5%) were prepared, adjusted to pH 7 and proceeded
action - Co-culture method
as described above. All treatments and controls were
Temperature, pH and effect of salt
done in triplicates.
50 ml PDB in 200 ml flasks was prepared and
Statistical analysis
maintained at pH 7 and autoclaved. Fungus was grown
The data procured was subjected to statistical
for two days by inoculating with agar disc from the
analysis by Least Significant Difference (LSD) described
fungal culture. After incubation the fungal broth culture
by Fisher and Yates (1963). The differences were
was inoculated with 4.5 log10 CFU/ml of overnight
considered significant at P≤0.01and 0.05 level.
grown bacterial strain. PDB with fungus alone was taken as control. Flasks in triplicates were incubated in
RESULTS AND DISCUSSION
different temperatures (25, 30, 37, 42 and 50ºC) at 180
Members of the genus Bacillus are known to be
rpm for seven days and the dry weight of fungal biomass
highly competent species in the rhizosphere and
was
For
Bacillus licheniformis has been frequently reported in
determining the optimum pH, broth was prepared with
many rhizopshere related research studies (Probanza
sets of different pH (5.5, 6, 6.5, 7and 7.5) and the
et al., 2002). Rhizospheric bacteria B.licheniformis 9555
protocol was followed as given above, the flasks were
showed the most efficient growth at 37°C and pH 7.
incubated at 37ºC. For analyzing the effect of salt PDB
It was observed that the bacterial count reached
containing different salt (NaCl) concentrations (0, 1, 2 ,3
the highest at 48 h with 9.33 Log10 CFU/ml, followed by
measured
and
compared
with control.
Table 1 Optimization of temperature for effective antifungal action by B. licheniformis 9555 by co-culture method. All the controls and treatments were done in triplicates. Temperature(°C)
20 25 30 37 42
Control (D.O* alone) mg±SE/100 ml 522.00±1.52 539.00±1.15 538.33±1.76 528.66±1.85 508.00±1.52
B.licheniformis 9555 + D.O mg±SE/100 ml 300.33±1.45 129.66±0.88 85.00±1.15 62.00±1.45 99.00±0.57
Biomass decrease %
L.S.D* values
42.46 75.94 84.21 88.14 80.51
6.00 4.10 5.88 6.67 4.24
*D.O: Drechslera oryzae; *L.S.D: Least Significant difference; mg: milligram Journal of Research in Microbes (2012) 1(2): 077-082
079
Dan et al.,2012 Table 2 Optimization of pH for effective antifungal action by B. licheniformis 9555 by co-culture method. All the controls and treatments were done in triplicates. Temperature 37°C. pH 5.5 6 6.5 7 7.5
Control (D.O* alone) mg±SE/100 ml) 534.00±3.05 531.00±1.53 526.00±3.055 528.66±1.85 517.00±3.511
B.licheniformis 9555 + D.O mg±SE /100 ml 427.00±2.08 245.66±2.84 85.67±3.48 62.00±1.45 94.30±2.60
Biomass decrease percent 20.00 53.73 83.71 88.14 81.76
L.S.D* values 10.35 9.48 13.17 6.67 12.32
*D.O: Drechslera oryzae; *L.S.D: Least Significant difference; mg: milligram 8.12
Log10 CFU/ml
on
the
10th
day (Fig
2).
Chitin is a major constituent of many fungal cell walls,
Comparatively good growth was recorded at 42°C and
and various workers have demonstrated in vitro lysis of
50°C with the bacterial count registering the highest peak
fungal cell walls by microbial chitinase (Kim and Chung
of 9.21 Log10 CFU/ml (at 48 h) and 7.93 Log10 CFU/ml
2004). Microscopic analysis of the phytopathogenic
(at 48 h) respectively. Maltose and dextrose were found
fungus co-cultured with 9555, revealed lysis of fungal
to be the best carbon sources, while glucose, fructose and
filament cell wall, thus concreting possible chitinase
sucrose performed to be the second best sources. The
action and cell wall lysis. If fungal cells are lysed and
bacterial strain failed to grow in minimal media
cell walls are degraded, then it is usually assumed that,
supplemented with citric acid and arabinose as the sole
for this act cell wall degrading enzymes produced by the
carbon source. Ammonium chloride and ammonium
bacteria are responsible, even though antibiotics may be
acetate were utilized as best nitrogen sources.
produced at the same time (Whipps 2001). In case of
Bacillus licheniformis 9555 was positive for the
B. licheniformis 9555 production of siderophore was
utilization of chitin, by the production of chitinase
earlier reported (Dan et al., 2012b) and this together with
enzyme. The tests for production of other antifungal
chitinase production by the strain can act as a dual
enzymes were not affirmative for the bacterial strain.
weapon against phytopathogenic fungus.
In related studies B. licheniformis isolated from
The optimum temperature and pH for maximal
rhizospheric soil were investigated to be efficient
antifungal action of Bacillus licheniformis 9555 in
producers of chitinase and they also served as antifungal
co-culture condition was determined to be 37°C and
agents (Takayanagi et al., 1991; Trachuk et al., 1996).
pH 7. This optimum environment provided in the
Figure 2 Growth of the B. licheniformis 9555 at different pH (A) at 37°C and at different temperature (B) at pH 7. 080
Journal of Research in Microbes (2012) 1(2): 077-082
Dan et al.,2012 Table 3 Effect of different salt concentrations (percent) on antifungal action of B. licheniformis 9555 at 37°C, pH 7. All the controls and treatments were done in triplicates. Temperature 37°C and pH 7 Salt (NaCl) percent 0 0.5 1 2 3 5 laboratory
Control (D.O* alone) mg±SE/100 ml) 528.00±3.05 528.66±1.85 530.00±1.73 534.00±3.78 533.00±1.52 527.33±1.45
conditions,
restricted
the
B.licheniformis 9555 + D.O mg±SE /100 ml 62.33±1.33 62.00±1.45 118.00±1.76 122.00±2.08 124.33±2.18 147.33± 2.18 biomass
Biomass decrease percent 88.19 88.14 77.73 77.15 76.67 72.06
L.S.D* values 10.86 6.67 7.04 11.82 7.48 7.33
of
increase in salinity. The bacteria can be further
Drechslera oryzae thus giving a “biomass decrease
investigated as a potential biocontrol agent in field trials
percent” of 88.14% below control (Table 1 and 2). Any
and can also be investigated in saline prone agricultural
fluctuation in the optimum pH and temperature, resulted
fields.
in an increase of fungal biomass in treatments signifying a
reduction
in
the
antifungal
action
by
ACKNOWLEDGEMENT
Bacillus licheniformis 9555. At the same time statistical
The author would like to thank all the members
analysis has supported that for all the treatments for
of the Department of Plant-microbe interaction, National
both temperature (20 to 42ºC) and pH (5.5-7.5), the
Botanical Research Institute, Lucknow. The author
reduction in fungal biomass in treatment group
would like to express his sincere gratitude to Dr Chandra
was significant, when compared to their respective
Shekhar Nautiyal, Director, National Botanical Research
controls (Tables 1 and 2). Under saline conditions
Institute for kindly granting permission to avail
Bacillus licheniformis 9555 showed the most efficient
laboratory facilities. Special thanks to CSIR, India for
antifungal action at 0.5% NaCl (Table 3). The successive
providing SRF.
increase in salt concentration caused slight reduction in antifungal action, but the biocontrol action was
REFERENCES
significant. In general, research studies have revealed
Asaka O and Shoda M. 1996. Biocontrol of
that Bacillus sp show predominance in saline soil and
Rhizoctonia solani damping off of Tomato with
also establish their biocontrol and plant growth
Bacillus subtilis RB14. Appl Environ Microbiol.,
promotion properties, in high saline environment
62:4081-4085.
(Egamberdiyeva and Islam, 2008). CONCLUSION Bacillus licheniformis 9555, a rhizospheric bacteria showed effective antifungal action against phytopathogenic fungi Drechslera oryzae, by the production of lytic enzyme chitinase that lysed the fungal
Dan VM, Mishra S, Chaudhry V, Singh P, Yadav S, Mishra S, Ijinu TP, George V, Nautiyal
CS.
2012b.
Biocontrol,
Varma A and plant
growth
promotion and conferring stress tolerance: Multifaceted role of Bacillus licheniformis 9555. International Journal of Science and nature (to be published in Dec’12 issue)
filaments. In co-culture condition, the microbe recorded
Dan VM, Richard V, Nair A, Pushpangadan P,
the most effective antifungal action at 37°C and pH 7.
George V, Varma A and Pillai R. 2012a. Effect of
The bacteria was able to sustain the antifungal activity in
extract of Bacillus megaterium 9554 on oral cancer cell
saline conditions but showed a decreasing trend with
line SCC131. Journal of Research in Biology. 2:523-528.
Journal of Research in Microbes (2012) 1(2): 077-082
081
Dan et al.,2012 Egamberdiyeva D and Islam KR. 2008. Salt-tolerant
properties.
rhizobacteria:
Plant
42:307-315.
physiological
characterization
growth promoting traits and within
ecologically
stressed environments. Plant Bact. Interact. 14:257-280. Fisher RA and Yates F. 1963. Statistical tables for
Canadian
Journal
of
Microbiology.
Whipps JM. 2001. Microbial interactions and biocontrol in the rhizosphere. Journal of Experimental Botany. 52:487-511.
biological, agricultural and medical research. Oliver and Boyd, Hafner press, New York. Kim P and Chung K. 2004. Production of an antifungal protein for control of Colletotrichum lagenarium by Bacillus amyloliquefaciens MET0908. FEMS Microbiol Lett., 234;177-183. Lim
HS,
Kim
YS
and
Kim
SD.
1991.
Pseudomonas stutzeri YPL-1 genetic transformation and antifungal mechanism against Fusarium solani, an agent of plant root rot. Appl. Environ. Microbiol., 57:510-516. Powell KA and Jutsum AR. 1993. Technical and commercial aspects of biological control products. Pestic Sci., 37:315-321. Probanza A, Lucas García JA, Ruiz-Palomino M, Ramos B and Gutierrez MFJ. 2002. Pinus pinea L seedling growth and bacterial rhizosphere structure after inoculation with PGPR Bacillus (B. licheniformis CECT 5106 and B. pumilus CECT 5105). Appl Soil Ecol., 20:75-84. Rondon MR. 2000. Cloning the soil metagenome: a strategy for accessing the genetic and functional diversity of
uncultured
microorganisms.
Appl.
Environ.
Microbiol., 66:2541-2547. Takayanagi
T,
Ajisaki
Submit your articles online at Ficuspublishers.com K,
Takiguchi
Y
and
Shimahara K. 1991. Isolation and characterization of thermostable chitinase from Bacillus licheniformis X-7u. Biochimica et Biophysica Acta. 1078:404-410. Trachuk
LA,
Revina
LP,
Shemyakina
TM,
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Chestukhina GG and Stepanov VM. 1996. Chitinase
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