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Biocontrol, plant growth promotion and conferring stress tolerance: multifaceted role of Bacillus licheniformis 9555

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In the present study a bacterial strain, Bacillus licheniformis 9555 was isolated from the rhizopheric soil collected from the banks of Hindon river, India. The river is heavily contaminated by toxic pollutants discharged from agricultural fields and industries. B. licheniformis 9555, served as a biocontrol agent and plant growth promoter. The bacteria also showed efficient production of exopolysaccharides (EPS) and siderophore. The treated plants showed increased chlorophyll content as compared to control. The bacteria was able to alleviate environmental stresses in plants as witnessed by reduced levels of stress related compounds like phenol, tannins and proline in Zea mays. The ability of the bacteria to confer stress resistance and promote growth may be the contributing factor that helps plants to grow on polluted banks of Hindon.
I.J.S.N., VOL. 3(4) 2012: 780-783 ISSN 2229 – 6441
780
BIOCONTROL, PLANT GROWTH PROMOTION AND CONFERRING
STRESS TOLERANCE: MULTIFACETED ROLE OF BACILLUS
LICHENIFORMIS 9555
1Vipin Mohan Dan, 2Sandhya Mishra, 2Vasvi Chaudhry, Swati Tripathi, 2Poonam Singh, 2Sumit Yadav,
2Shashank Mishra, 1Ijinu, T. P., 1Varughese George, Ajit Varma3* &2Chandra Shekhar Nautiyal
1Amity Institute for Herbal and Biotech Product Development, Trivandrum-695005, Kerala, India.
2Division of Plant-Microbe Interaction, CSIR-National Botanical Research Institute, Lucknow 226001, India
3Amity Institute of Microbial Technology, Amity University, Sector-125, Noida -201 303, U.P, India.
ABSTRACT
In the present study a bacterial strain, Bacillus licheniformis 9555 was isolated from the rhizopheric soil collected from the
banks of Hindon river, India. The river is heavily contaminated by toxic pollutants discharged from agricultural fields and
industries. B. licheniformis 9555, served as a biocontrol agent and plant growth promoter. The bacteria also showed
efficient production of exopolysaccharides (EPS) and siderophore. The treated plants showed increased chlorophyll content
as compared to control. The bacteria was able to alleviate environmental stresses in plants as witnessed by reduced levels
of stress related compounds like phenol, tannins and proline in Zea mays. The ability of the bacteria to confer stress
resistance and promote growth may be the contributing factor that helps plants to grow on polluted banks of Hindon.
KEYWORDS: Keywords: Bacteria, siderophore, PGPR, chlorophyll.
INTRODUCTION
Microbes are omnipresent and can survive in a wide range
of environmental conditions and also provide a helping
hand to other organisms (plants) to adapt to the stress in an
environment in which they establish. Plant Growth
Promoting Rhizobacteria (PGPR) are microbes specially
designed by nature that harbor growth promotional
benefits for host plant. Plant growth promotion and
biocontrol action is widely studied in Bacillus genera, a
common inhabitant of rhizopshere (Wahyudi et al., 2011).
Colonization of roots by PGPR is the seed to successful
plant-microbe interaction. In certain associations of
microbes with plants, exopolysaccharides (EPS) have a
major role that help bacteria to inhabit the root surface
through specific adhesion, leading to root colonization that
eventually results in biofilm formation (Michiels et al.,
1991; Matthysse et al., 2005; Ramey et al., 2004).
An investigation by the National Institute of Hydrology,
Roorke, India confirmed that the concentrations of toxic
chemicals in Hindon river, Uttar Pradesh, India,
discharged from agricultural fields and industries were
very high than the maximum permissible limits
(http://www.indiawaterportal.org). The microbes surviving
in such toxic levels will have special adaptability qualities
to resist or utilize the toxic substances and at the same
time help host plant to adapt to the toxic environment.
This present study aims to isolate rhizospheric bacteria
from Hindon river bank and to analyze its biocontrol
activity, plant growth promotion property and ability to
confer stress tolerance to host plants.
MATERIALS AND METHODS
Bacterial strain and growth conditions
Microbes were isolated from rhizopshere soil as per the
protocol described by Lu and Huang (2010). The potential
bacterial isolate was deposited in Microbial type culture
collection (MTCC), Institute of Microbial Technology
(IMTECH), Chandigarh, India and it was identified based
on morphological, physiological and biochemical
characteristics.
Analyzing bacterial isolates for Antifungal property
The agar disc method (Hinton and Bacon, 1995) was used
to evaluate in vitro antifungal activity on PDA (Potato
Dextrose Agar). The phytopathogenic fungus, Fusarium
monoliforme (MTCC 1848) was authenticated by
Microbial Type Culture Collection (MTCC). The cultures
of Fusarium oxysporum, Sclerotinia sclerotiorum,
Alternaria solani were procured from Department of
Plant-microbe interaction, National Botanical Research
Institute, Lucknow, India.
Plant growth promotion
Sterile and non-sterile agricultural field soil was used in
different experimental setups. Bacterial inoculums of 9
log10 CFU/ml was thoroughly mixed with soil and
sterilized seeds of maize were sown after 24 h. The glass
house was maintained at 28 ± 2°C, 16 h light/8-hrs dark
with fluorescent light intensity 1000 Lux and relative
humidity 70%. Plants were irrigated with tap water on
alternate days to maintain about 30% soil moisture. After a
period of 30 days plant parameters were recorded and
chlorophyll content was evaluated using the protocol
described by Arnon (1949).
B. licheniformis 9555 was analyzed for phosphate
solubilization (Mehta and Nautiyal 2001), auxin synthesis
(Patten and Glick 1996) siderophore production (Bano and
Musarrat 2003). EPS production was quantified from 72 h
old culture grown in nutrient broth using phenol-sulphuric
acid method (Titus et al., 1995).
Multifaceted role of Bacillus licheniformis 9555
781
Analysis of biocontrol efficiency in protecting plants
One set of cups with sterile soil was inoculated with B.
licheniformis 9555 and two other sets served as control,
one set was for uninoculated sterile soil and the other for
Fusarium moniliforme treatment alone. Four seedlings per
cup were planted and for B. licheniformis treatment the
seeds were soaked in small volume of bacterial culture
prior to planting. After 24 h F. moniliforme spore
suspension with an inoculums size of 3 x106conidia /ml
was used for spraying on seedling. The uninoculated
sterile soil control set was left unsprayed. Germination
percentages were recorded and plant parameters,
chlorophyll content were analyzed after 30 days.
Estimation of Tannin, Phenol and Proline
Tannin and phenol estimation were conducted based on
the protocol by Bray and Thorpe (1954). The approximate
quantity of phenols and tannins were determined by using
standard curves of gallic acid and tannic acid respectively.
Proline content was determined by the method of Bate et
al.(1973).
Statistical analysis
The data procured was subjected to statistical analysis by
least significant difference (LSD) described by Fisher and
Yates (1963) .The differences were considered
significant at P≤0.01and 0.05 levels.
RESULTS AND DISCUSSION
Screening and identification of biocontrol agent
The selection of potential biocontrol agent involved
screening of over 100 bacterial isolates from rhizosphere
soil. One isolate designated NB2 showed effective
antifungal action against all the tested phytopathogenic
fungi (Table I). NB2 was gram positive, catalase positive,
motile, non-endospore forming, rod shaped and aerobic in
nature. The bacterium was identified as Bacillus
licheniformis based on morphological, physiological and
biochemical characteristics by MTCC, with accession
number MTCC 9555.
TABLE I: Zone of inhibition by B. licheniformis 9555.
Phytopathogenic fungi
Zone of inhibition (mm)
Fusarium monoliforme
10
Fusarium oxysporum
10
Alternaria solani
12
Sclerotinia sclerotiorum
9
TABLE II: Plant growth promotion of Zea mays by B. licheniformis 9555 after 30 days in sterile soil and non-sterile soil.
Parameters
Control
B.licheniformis
9555 treatment
LSD at
1%
B .licheniformis
9555 treatment
LSD at 1%
Sterile soil
Non-sterile soil
Shoot length (cm)
29.11.19
37.30.80
2.661
37.20.97
2.862
Root length (cm)
17.10.73
19.60.59
1.759
20.60.68
1.525
Leaf length (cm)
10.90.40
13.20.53
1.239
13.80.36
0.899
Lateral roots
9.20±0.33
12.90.38
1.207
11.30.44
1.086
Stem dry weight(g)
0.090.009
0.140.002
0.0038
0.140.003
0.010
Root dry weight (g)
0.0622±0.001
0.070.002
0.0047
0.070.002
0.0058
Data was calculated for each parameter as mean ± standard error (n = 60).
Plant growth promotion
The plant growth promotion property of the bacteria was
accomplished in both sterile and non-sterile soil setup.
Stem dry weight was increased by 36.73% and 33.33%
over their corresponding controls in sterile and non-sterile
soil respectively (Table II). A significant increase of root
dry weight biomass was also observed in both soil setup.
The non-sterile agricultural field soil experimental setup
gave an idea of possible rhizosphere competence achieved
by the microbe in establishing successful plant association
and survival over indigenous microflora.
B. licheniformis 9555 showed effective siderophore
production. Rhizospheric bacterial isolate, B. megaterium
produced siderophore that served dual function of efficient
plant growth promotion and disease resistance to host
plant (Chakraborty et al., 2006). B. cereus UW 85 isolated
from soil produced siderophore that were efficient growth
promoters of crop plants (Husen 2003). Thus the plant
growth promoting efficiency and biocontrol property of B.
licheniformis 9555 can be equated to the microbe’s
siderophore production capability, but at the same time
other mechanism should still be explored. B. licheniformis
9555 treated plants showed significant increase in
chlorophyll content when compared to the untreated
control plant (Table III).
Extracellular polysaccharide (EPS) production by B.
licheniformis 9555 was assayed to be 746 µg/ml, after 72
h of incubation. While in related studies EPS production
of B. cereus was 498.04 µg/ml (Bragadeeswaran et al.,
2011) and for B. subtilis NCIM 2063 it was 206 µg/ml
(Francis et al., 2009 ). In another study the biofilm
formation of Bacillus subtilis was correlated to its
biocontrol efficiency (Bais et al., 2004). Efficient
production of EPS by B. licheniformis 9555 may have a
significant role in plant- microbe interaction, biocontrol
and tolerance against environmental stress caused by toxic
pollutants.
I.J.S.N., VOL. 3(4) 2012: 780-783 ISSN 2229 – 6441
782
TABLE III. Estimation of chlorophyll, tannin, phenol and proline content after 30 days.
Parameter
Controls
Treatment with B.licheniformis 9555
LSD at
1%
Plant alone
F.monoliforme
Treated
Plant
Plant +
F.monoliforme
Total chlorophyll(mg/g)
22.33±0.450
15.83±0.372
45.11±0.264
42.37±0.303
1.079
Chlorophyll a (mg/g)
19.38±0.380
14.31±0.521
35.81±0.156
33.75±0.375
1.113
Chlorophyll b(mg/g)
2.866±0.127
1.67±0.064
9.09±0.209
8.63±0.367
0.591
Phenol (µg/g)
207.59±2.17
215.43±2.41
72.17±2.257
113.31±1.680
6.62
Tannic acid (µg/g)
178.45±1.48
185.39±1.69
54.55±2.418
89.34±2.857
6.55
Proline (µm/g)
59.52±2.71
60.28±2.15
21.57±1.209
42.48±1.575
5.94
Data was calculated for each parameter as mean ± standard error (n = 12).
TABLE IV: Biocontrol efficiency of B.licheniformis 9555 in protecting plants against F. monoliforme
Parameters
Controls
F. monoliforme
+B.licheniformis
9555 Treated
LSD at
1%
F. monoliforme
Treated
Plant alone
Shoot length (cm)
10.86±1.94
29.52±1.30
29.71±0.72
3.53
Root length (cm)
8.18±1.31
17.07±0.78
19.30±0.33
2.15
Leaf length (cm)
7.95±0.95
11.21±0.45
13.02±0.25
1.78
Lateral roots
6.09±0.94
9.42±0.34
11.80±0.46
1.56
Stem dry weight(g)
0.053±0.009
0.088±0.001
0.099±0.007
0.016
Root dry weight (g)
0.040±0.006
0.0610±0.009
0.0688±0.008
0.0072
Data was calculated for each parameter as mean ± standard error (n = 54).
Biocontrol efficiency in protecting plants
In the control set treated with F. moniliforme 55% of seeds
germinated, while the dual treated (F. moniliforme +B.
licheniformis 9555) and uninoculated control recorded
100% germination. Shoot length, stem dry weight and root
dry weight of the dual treated plant showed a significant
increase of 63.44%, 46.46% and 41.17% respectively,
above the fungus treated control plant (Table
IV).Siderphore production is an efficient biocontrol
mechanism employed by PGPRs. Study by Wahyudi et al.
(2011) reported 12 Bacillus sp from the rhizosphere of
soya bean plants that showed effective antifungal action
through siderphore production. Significant increase in
chlorophyll content was also observed in dual treated
plants when correlated to control groups (Table III).
Estimation of Phenol, Tannin and Proline
B. licheniformis 9555 treated plant had the least content of
stress response compounds while the F. monoliforme
treated plant showed the highest accumulation. The dual
treated (F. monoliforme +B. licheniformis 9555) plant
showed 47.40% decrease in phenol content when set
against the F. moniliforme treated control (Table III). A
statistically evident decrease in tannic acid and proline
content was noted in the B. licheniformis 9555 treated and
dual treated plants when contrasted against both set of
control plants (Table III). In general stress related
compounds accumulate on treatment with PGPRs and
functions in plant defense mechanism (Singh et al., 2002;
Jain et al., 2012). B. licheniformis 9555 treatment did not
induce production of stress related compounds and at the
same time have reduced the stress caused by the green
house environment on the plant. The biocontrol efficiency
of B. licheniformis 9555 has protected the plant from
phytopathogenic infection thereby resulting in low
accumulation of stress related compounds. The mechanism
by which B. licheniformis 9555 confers environmental
stress tolerance to plant might be one of the factors for
survival by plants in highly polluted regions like Hindon
river.
CONCLUSION
B.licheniformis 9555 proved to be an efficient biocontrol
agent as well as good plant growth promoter. Siderophore
production by bacteria can be stated as the reason behind
this bifold property of the bacteria. B.licheniformis 9555 is
a microbe that has evolved in a stressful environment with
toxic chemicals and this survival ability was conferred by
associated plants in reducing environmental stress as
manifested from reduced levels of stress related compound
accumulation in treated Zea mays. The bacteria seem to
have adapted a mechanism that reduces production of
plant stress related defense compounds, but at the same
time confers environmental stress tolerance to plants.
ACKNOWLEDGEMENT
The authors would like to thank Department of Plant-
microbe interaction, National Botanical Research Institute,
Lucknow and Amity University, Noida for providing
laboratory facilities. Special thanks to CSIR, India for
providing SRF.
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Plant growth-promoting rhizobacteria contain Nitrogen-fixing, Phosphorus-solubilizing, Potassium-solubilizing and other bio-active bacteria. Utilization of microbial Nitrogen fixation, Phosphorus-and Potassium-solubilizing activities can facilitate plant growth and avoid the lack of soil nutrients. This study used the plate dilution method to screen and isolate pro-biotic bacteria from the rhizosphere of pine trees, growing on the outskirts of Nanning City, Guangxi Province China. 16S rDNA was sequenced for the bacteria with strong biological activities. A total of 23 Azotobacter strains were isolated from the rhizosphere of Pinus massoniana Lamb. All of them presented Nitrogenase activity. Particularly, strain N4 and N9 had a strong Nitrogenase activity of 260.975 nmol C 2 H 4 /h·L and 149.787 nmol C 2 H 4 /h·L, respectively. Six Phosphorus-solubilizing strains were isolated, among which P3 and P1 had a strong Phosphorus-solubilizing ability of 7.00 and 3.87 µg/ml, respectively. Among the 5 Potassium-solubilizing strains, K6 had a strong Potassium-solubilizing ability of 2.3 µg/ml. The ability of the strains to solubilize Phosphorus and Potassium was significantly correlated with the composition of the culture medium. All the Potassium-solubilizing strains belong to the genus of Burkholderia. The Phosphorus-solubilizing strains include Enterobacter, Pseudomonas and Serratia marcescens. Nitrogen-fixing strains include Enterobacter, Burkholderia, Rhizobium, Erwinia, Agrobacterium and Gamma proteobacterium.
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