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Indian Journal of Biotechnology
Vol 12, January 2013, pp 20-31
Plant growth promoting rhizobacteria mediated improvement of health status
of tea plants
U Chakraborty*, B N Chakraborty, A P Chakraborty, K Sunar and P L Dey
Immuno-Phytopathology Laboratory, Department of Botany, University of North Bengal, Siliguri 734 013, India
Plant growth promoting rhizobacteria (PGPR) have immense potential application in sustainable agriculture as
ecofriendly biofertilizers and biopesticides. The present study was undertaken to explore the potential of such
microorganisms from the rhizosphere of tea [Camellia sinensis (L.) O. Kuntze] for the overall improvement in growth and
productivity of tea, which is the most important crop of this region. Isolation and testing of bacteria for PGPR activities
revealed that a large number of them showed such activities. Of which three were selected for various studies. The selected
bacteria were Bacillus amyloliquefaciens, Serratia marcescens and B. pumilus. These bacteria showed positive PGPR traits
in vitro, such as, phosphate solubilization, siderophore production, antagonism to pathogens and IAA production. 16S rDNA
sequencing of the bacteria was done and their phylogenetic relationships determined. Under in vivo conditions, the PGPR
enhanced the seedling growth of tea varieties in the nursery as well as in the field. Plant growth promotion was determined
in terms of increase in number of leaves, their biomass and number of shoots. In order to determine the tolerance of bacteria
to insecticides, in vitro tests were conducted, which indicated that PGPR could tolerate more than 100 times the
concentration applied in the field. Sustainability of the applied bacteria in soil was tested by PTA-ELISA and Dot
immunobinding assay using polyclonal antibodies raised against the PGPR. Certain bioformulations of the PGPR in talc
powder, saw dust and rice husk also been prepared and their viability tested. The bacteria showed good survivability even up
to 9 months of storage. Application of the PGPR led to enhancement in activities of defense related enzymes, such as,
phenyl alanine ammonia lyase, peroxidase, chitinase and β-1,3-glucanase, in tea leaves. Total phenols also increased
quantitatively. It is evident from the present study that application of PGPR in the soil lead to biopriming of the plants
through induced systemic resistance and other mechanisms.
Keywords: Bacillus amyloliquefaciens, B. pumilus, Camellia sinensis, carrier based bioformulation, growth promotion,
Serratia marcescens
Introduction Plant growth promoting rhizobacteria (PGPR), first
defined by Kloepper and Schroth1, include those
bacteria that are able to aggressively colonize plant
roots and stimulate plant growth when applied to
roots, tubers and seeds. PGPR have been reported to
directly enhance plant growth by a variety of
mechanisms like fixation of atmospheric nitrogen that
is transferred to the plant, production of siderophores
that chelate iron and make it available to the plant
root, solubilization of minerals, such as, phosphorus,
and synthesis of phytohormones2. However, in field
conditions, the above traits may not be sufficient to
account for the observed growth promotion. The
biochemical or physiological changes induced in the
host that are activated by the PGPR also lead to plant
growth promotion and develop resistance capacity in
the host against pathogens. Previous studies have
shown that Serratia proteamaculans 1-102 promotes
soybean—bradyrhizobia nodulation and growth, but
the mechanism is unknown3. ISR (induced systemic
resistance) could be a potential mechanism by which
PGPR demonstrate biological disease control4.
Isolates of Bacillus megaterium have also been
reported to produce antibiotics against several fungal
pathogens5.
Tea [Camellia sinensis (L.) O. Kuntze] is one of
the most important plantation crops in Darjeeling and
Dooars regions, which are the tea growing regions of
West Bengal, India. Productivity of tea plantations is
decreasing and one of the reasons for this has been
attributed to the continuous use of huge quantities of
chemicals. In recent years, application of hexaconazole
(RS)-2-2(2,4-dichlorophenyl)-1-(1,H-1,2,4-triazol-1-1-
yl) hexan-zol, an ergosterol biosynthesis inhibitor, has
been common practice in tea cultivation areas6. Hence,
there is a pressing need in tea industry for utilizing
either biological produce completely or reducing the
_______________
*Author for correspondence:
Mobile: +91-9002002096; Fax: +91-353-2699001
E-mail: chakrabortyusha@hotmail.com
CHAKRABORTY et al: RHIZOBACERIA MEDIATED HEALTH IMPROVEMENT IN TEA PLANTS
21
use of chemicals by supplementing with biological
produce as integrated management practices.
Combined application of low dose of insecticide and
PGPR would be beneficial not only for plant growth
but also for reduction of insect pest attack. One of the
common means of application of bacterial inoculants
to soil is in the form of bioformulations. Viability of
inoculum in an appropriate formulation for a certain
length of time is important for commercialization of
the technology7. Previous reports are also available
where Bacillus bioformulations could survive upto
one year or more in several bioformulations8. Carrier-
based preparations of two PGPR, viz., B. subtilis and
Pseudomonas corrugata, developed in five
formulations were also evaluated for their growth
promotion, rhizosphere colonization and viability
under storage9. A study using talc based formulation of
Ochrobactrum anthropi was also reported where its
survival was determined every month up to a period of
12 months10
. Though different bacterial species have
been investigated as biological control agents, the
knowledge concerning the behaviour of these
bacterial strains as antagonists and their genetic
analysis is essential for effective use and the
commercialization. The advent of molecular biology
in general and the PCR reaction in particular have
greatly facilitated genomic analysis of microorganisms,
which provide enhanced capability to characterize and
classify strains and facilitate research to assess the
genetic diversity of populations11
.
The present study was undertaken with an
objective of determining the plant growth promoting
potential of three bacteria isolated from tea
rhizosphere to determine their mechanisms of action
and to determine whether these could be applied as
bioformulations in tea rhizosphere.
Materials and Methods
Isolation of Microorganisms
Initially several microorganisms were isolated from
different rhizospheric soil of tea gardens and screened
for in vitro PGPR activities. Three bacterial strains
showed positive responses in in vitro PGPR tests. They
were also preliminarily identified on the basis of
morphological, microscopic and biochemical
characterization, and finally identity of these three
strains were confirmed from the Plant Diagnostic and
Identification Services, UK and also by 16S rDNA
sequencing. The three strains were B.
amyloliquefaciens TRS 6, B. pumilus BRHS/T-84 and
S. marcescens TRS 1.
Morphological and Scanning Electron Microscopic Studies
Pure cultures of the three isolates were streaked on
NA plates for colony development. The individual
colonies were examined for shape, size, structure of
colonies and pigmentation. The Gram reactions of the
isolates were determined following procedure
outlined by Buchanan and Gibbon12
. Gram
positive/negative reactions and shape of cells
observed were recorded. Scanning electron
micrographs of all three isolates were also recorded.
Molecular Characterization and Identification
Isolation of Genomic DNA and Amplification of 16S rDNA by PCR
Genomic DNA was extracted from 24-h-old culture
following the method of Stafford et al13
with
modifications. DNA was precipitated from the aqueous
phase with chilled ethanol (100%) and pelleted by
centrifuging at 12000 rpm for 15 min, followed by
washing in 70% ethanol and centrifugation. The pellets
were air dried and suspended in TE buffer pH 8. After
further purification, DNA was quantified
spectrophotometrically and the quality analyzed in
0.8% agarose gel.
For ITS-PCR amplification, DNA was amplified
by mixing the template DNA (50 ng) with the
polymerase reaction buffer, dNTP mix, primers and
Taq polymerase. Polymerase chain reaction (PCR)
was performed in a total volume of 100 µL,
containing 78 µL deionized water, 10 µL 10× Taq
polymerase buffer, 1 µL of 1U Taq polymerase
enzyme, 6 µL 2 mM dNTPs, 1.5 µL of 100 mM
reverse and forward primers and 3.5 µL of 50 ng
template DNA. For amplification of the ITS region of
B. altitudinis isolate, the primer pair, Forward primer:
5'-AGAGTRTGATCMTYGCTWAC-3' and Reverse
primer: 5'-CGYTAMCTTWTTACGRCT-3', was
used. The PCR was programmed with an initial
denaturing at 94oC for 5 min, followed by 30 cycles
of denaturation at 94oC for 30 sec, annealing at 61
oC
for 30 sec and extension at 70oC for 2 min and the
final extension at 72oC for 7 min in a Primus 96
advanced gradient Thermocycler. The PCR product
(20 µL) was mixed with loading buffer (8 µL)
containing 0.25% bromophenol blue, 40% w/v
sucrose in water, and then loaded in 2% agarose gel
with 0.1 % ethidium bromide for examination with
horizontal electrophoresis. The PCR product was sent
for sequencing to Genie, Bangalore, India.
16 S rDNA Sequence Analysis
The sequenced PCR product was aligned with ex-
type isolates’ sequences from NCBI GenBank for
INDIAN J BIOTECHNOL, JANUARY 2013
22
identification as well as for studying phylogenetic
relationship. The evolutionary history was inferred
using the UPGMA method14
. The evolutionary
distances were computed using the Maximum
Composite Likelihood method15
and these are
represented in the units of number of base substitutions
per site. Codon positions included were
1st+2nd+3rd+noncoding. All positions containing gaps
and missing data were eliminated from the dataset
(complete deletion option). Phylogenetic analyses were
conducted in MEGA-416
. 16S rDNA of all the three
bacterial isolates were aligned to study the range of
homology present in the conserved regions following
the ClustalW algorithm17
using the Bioinformatic tool
BioEdit. Combinations and percentage of occurrence of
different nucleotide in the entire sequence of
B. pumilus, B. amyloliquifaciens and S. marcescens
were calculated using the bioinformatics algorithm
from the website: http://www.ualberta.ca/~stothard/
javascript/dna_stats.html.
Plant Material
Seedlings (18-month-old) of five tea varieties, viz.,
TV-18, TV-23, TV-25, TV-26 and T-17, were
selected for experimental purpose. The selected tea
seedlings were maintained in 12˝ earthenware pots in
nursery and also in the experimental field. The tea
seedlings were watered regularly for proper
maintenance.
Evaluation of PGPR and Antagonistic Activities of Bacterial
Isolates In Vitro
Phosphate Solubilization
Primary phosphate solubilizing activity of all the
three isolates was detected by allowing the bacteria to
grow in a selective medium, i.e., Pikovskaya’s agar
18.
The appearance of transparent halo zone around the
bacterial colony indicated the phosphate solubilizing
activity of the bacterium.
Siderophore Production
Production of siderophore was detected by standard
method using blue indicator chrome azurol S (CAS)19
.
IAA Production
For detection and quantification of IAA, the selected
bacterial cells were grown for 24 to 48 h in high C/N
ratio medium. Tryptophane (0.1 mM) was added in
order to enhance indole acetic acid (IAA) production
by the bacterium. Production of IAA in culture
supernatant was assayed by Pillet-Chollet method20
.
Antagonism
In vitro antagonism of bacteria to fungal pathogens
was determined by paired culture on nutrient agar
medium21
.
In Vitro Tolerance to Insecticides
As integrated management practices were
attempted, it was essential to determine whether the
applied bacterium could tolerate the concentration of
insecticide sprayed on the leaves and which enter into
the soil as fall out. For in vitro tests, the bacterium
was allowed to grow at much higher concentrations of
insecticides, viz., Acephate (organophosphate
insecticide), Confidor (systemic neuroactive
insecticide, class - Imidacloprid) and Ethion 50EC
(organophosphorus insecticide and miticide), by
adding these to the liquid medium after autoclaving.
Preparation of Inoculum and In Vivo Application
To prepare the bacterial inoculum, initially
bacterium was cultured in nutrient broth medium
(Himedia, M002-100G, ingredients: peptic digest of
animal tissue, 5.00 g/L; sodium chloride, 5.00 g/L; beef
extract, 1.50 g/L; yeast extract, 1.50 g/L, final pH at
25°C, 7.4±0.2) and was allowed to grow properly, with
shaking at 37°C at 120 rpm for 48 h. At the end of the
log phase, bacterial culture was centrifuged at 10,000
rpm for 15 min and the supernatant was discarded,
selecting the bacterial pellet. Pellet was scraped into
sterile distilled water. The aqueous suspension was
diluted as necessary to maintain the bacterial
concentration at 108 cfu/mL. The aqueous suspension
was then applied as a soil drench, @ 100 mL/plant to
the rhizosphere of tea plants 1 month after
transplantation. Three applications were done, each at
an interval of 1 month.
Growth of Tea Plants
Growth promotion was studied in terms of increase
in number of leaves, their biomass and number of
shoots. Plants were grown under natural conditions of
light and temperature (30±2°C). For each treatment 10
replicates were taken and average of the 10 replicate
plants were analysed. Biochemical Analyses
All the biochemical analyses were performed from
treated as well as control tea leaves after 72 h of
treatment.
Enzyme Assay
Peroxidase (POX, EC1.11.1.7.), chitinase (CHT, EC
3.2.1.14), phenyl alanine ammonia lyase (PAL, EC
CHAKRABORTY et al: RHIZOBACERIA MEDIATED HEALTH IMPROVEMENT IN TEA PLANTS
23
4.3.1.5) and β-1,3-glucanase (β-GLU, EC 3.2.1.39)
were extracted and assayed following methods
described by Chakraborty et al22
, Boller and Mauch23
,
Bhattacharya and Ward24
and Pan et al25
, respectively.
Phenolics
Phenols were extracted and estimated from leaf
samples by the method of Mahadevan and Sridhar26
.
Quantification of total phenol was done by a standard
of caffeic acid.
Preparation of Bioformulation
For development of bioformulations using talc
powder, saw dust or rice husk, 10 g of carboxy methyl
cellulose (CMC) was mixed with 1 kg of the substrate
and pH was adjusted to 7.0 by adding calcium
carbonate. It was then sterilized for 30 min in two
consecutive days. To this sterilized substrate 400 mL of
bacterial inoculum containing 3×109
cfu mL-1
were
added and mixed well under sterile condition. The
mixture was then dried under shade to bring moisture
to less than 20%. The formulation was packed in milky
white colour polythene bags, sealed and stored at room
temperature for future use.
Determination of In Vitro Survivability
Viability of bacterium in the formulations was
checked by dilution-plate technique using nutrient agar
medium. The plates were incubated at 37°C and
survivability was expressed in the form of cfu (colony
forming unit). The viability test was carried out with
the fresh as well as stored preparations. Viability was
checked at an interval of one month till nine months.
The data was expressed in log form for analysis.
Sustainability of Bacterium in Soil Detected by Immunoformats
Polyclonal antibodies were raised against the
bacterium in white, male rabbits27
. Before immunization,
normal serum was collected from rabbit. Following
injection schedule with antigens, blood samples were
collected and kept at 37ºC for 1 h for clotting, followed
by centrifugation at 5000 rpm for 10 min at room
temperature and IgG was purified from the serum28
.
Sustainability of the applied bacterium in soil was tested
by PTA-ELISA29
and Dot immunobinding assay30
using
polyclonal antibodies raised against the PGPR.
Results
Microscopic Observation
Morphological observation of B. amyloliquefaciens,
B. pumilus and S. marcescens showed that both
B. amyloliquefaciens and B. pumilus were rod shaped,
Gram +(ve) with wavy cell margin, rough surface and
opaque nature in density. Both the isolates also
produced endospores. S. marcescens was small rod
shaped, Gram –(ve) with smooth cell margin, smooth
surface, opaque nature in density. S. marcescens also
produced red pigment, prodigiosin when maintained in
culture at 30ºC for 5-6 d.
Scanning Electron Microscopy
Scanning electron micrographs also confirmed the
structure of bacteria—B. amyloliquefaciens was
larger, rod shaped (size, 2 µm and width, 8.5 mm) and
B. pumilus was also rod shaped (size, 2 µm and width,
8.8 mm); whereas S. marcescens was much smaller
rod shaped bacterium (size, 1 µm and width, 8.7 mm). rDNA Sequence Analysis for Identification
The BLAST query of 16S rRNA sequence of the
selected isolates against GenBank database confirmed
their identity, where isolate TRS-1 was identified as
S. marcescens, BRHS/T-84 as B. pumilus and TRS-6
as B. amyloliquefaciens,. The sequences have been
deposited in NCBI, GenBank database under the
accession Nos. JN983127.1, JNO20963.1 and
JQ765580.1 for B. amyloliquefaciens, S. marcescens
and B. pumilus, respectively. Evaluation of PGPR Activities of Isolates In Vitro
All three isolates were tested for different PGPR
activities as described in ‘Materials and Methods’.
Results revealed that all three isolates could solubilize
phosphate and produce siderophore. The secretion of
IAA into the medium was also confirmed by
quantification (Table 1).
Table 1—In vitro PGPR characteristics of B. amyloliquefaciens,
S. marcescens and B. pumilus
Characteristics B. amyloliquefaciens S. marcescens B. pumilus
Phosphate
solubilisation
+ + +
Siderophore
production
+ + +
Protease
production
+ + +
Chitinase
production
- + -
HCN production - - -
Volatile
production
+ + +
IAA production + + +
+ = Activity present ; - = Activity absent
INDIAN J BIOTECHNOL, JANUARY 2013
24
In Vitro Antagonistic Test
Antagonism of the isolates was also tested against
Phellinus noxius, Poria hypobrunnea and
Sphaerostilbe repens in both solid and liquid medium
in vitro. Results revealed that isoltes inhibited test
pathogens significantly (Table 2).
In Vitro Test for Insecticide Tolerance by Isolates
All the three isoltes could tolerate Acephate up to
1250 mg/mL concentration and Confidor up to 1000
µL/mL concentration in the medium. The isolates also
tolerated 3 tested insecticides, viz., Contaf 5E, Calixin
and Ethion 50 EC, at much higher concentrations to
that applied in the field.
Growth of Tea Seedlings
Growth promotion was studied in terms of increase
in height, number of branches and leaf dry mass in
comparison to control, both in field and potted
conditions. Percentage increase in height and number
of branches were recorded in all tested five varieties of
tea plants in field conditions after application of three
isolates separately as a soil drench (Figs 1 & 2).
Among the three isolates, B. amyloliquefaciens showed
Fig. 1 (A & B)—Effect of bacterial application on growth of five
varieties of tea in field in terms of % increase in height after 6 (A)
and 12 (B) months.
Table 2—In vitro antagonistic tests of bacteria against test
pathogens.
Test fungi Diameter of fungal
growth after 7 d
(cm)
Zone of
inhibition
(cm)
% inhibition
Fomes lamaoensis 8.2±0.23* - -
F. lamaoensis+
S. marcescens
2.1±0.22 1.8±0.08 74.4±2.98
F. lamaoensis+
B. amyloliquefaciens
1.5±0.09 2.2±0.21 81.7±2.65
F. lamaoensis+
B. pumilus
1.4±0.08 2.0±0.23 82.0±2.67
Poria hypobrunnea 8.5±0.20 - -
P. hypobrunnea+
S. marcescens
3.5±0.04 1.6±0.09 58.8±2.24
P. hypobrunnea+
B. amyloliquefaciens
1.9±0.19 1.6±0.07 77.6±2.86
P. hypobrunnea+
B. pumilus
2.0±0.16 1.5±0.06 77.5±2.85
Spherostilbe repens 8.5±0.34 - -
S. repens+
S. marcescens
5.2±0.54 0.9±0.09 38.8±2.34
S. repen +
B. amyloliquefaciens
3.2±0.34 2.0±0.21 55.3±1.90
S. repens+B. pumilus 2.8±0.31 1.6±0.17 55.3±1.90
*Data shown are average of 3 replicates
Fig. 2 (A & B)—Effect of bacterial application on growth of five
varieties of tea in field in terms of % increase in number of
branches after 6 (A) and 12 (B) months.
CHAKRABORTY et al: RHIZOBACERIA MEDIATED HEALTH IMPROVEMENT IN TEA PLANTS
25
comparatively better response in growth promotion,
followed by B. pumilus and S. marcescens. Changes in
dry mass of leaves from potted plants were also
determined after the treatments (Table 3).
Defense Enzymes and Phenolics
When biochemical tests were performed to evaluate
the changes brought about by S. marcescens,
B. amyloliquefaciens and B. pumilus, it was observed
that there was considerable increase in the activities of
all four defense enzymes (POX, PAL, CHT & β-GLU)
tested (Figs 3 & 4). Total and o-phenols also increased
significantly in plants following bacterial application
compared to the untreated control plants (Fig. 5).
In Vitro Survivability of Bacteria in Bioformulations
The viability of B. amyloliquefaciens, B. pumilus and
S. marcescens in formulations was tested during the
storage period of 9 months at 1 month interval. Results
revealed that both B. amyloliquefaciens and B. pumilus
could survive in the range of 6.1× log10 cfu/mL in
bioformulations of saw dust and rice husk, and 7.0×
log10 cfu/mL in talc powder; whereas S. marcescens
could survive in the range of 7.12×, 7.11× and 7.1× log10
cfu/mL in saw dust, rice husk and talc powder
formulations, respectively.
Fig. 3 (A & B)—Peroxidase (A) and phenyl alanine ammonia lyase
(B) activities in leaves of tea varieties grown in soil treated with
PGPRs .
Fig. 4 (A & B)—β-1,3-glucanase (A) and chitinase (B) activities
in leaves of tea varieties grown in soil treated with PGPRs.
Table 3—Changes in biomass of tea leaves of potted tea plants (2
months after application of bacteria)
Tea
varieties
Treatment Fresh wt
(g)
Dry wt taken
after 7 d
(g)
Control 04.5±1.5* 2.4
B. amyloliquefaciens 15.0±1.4 7.4
S. marcescens 17.0±2.1 8.0
TV-18
B. pumilus 14.0±2.2 6.5
Control 09.0±1.0 4.0
B. amyloliquefaciens 25.0±1.4 10.5
S. marcescens 19.0±1.1 5.0
TV-23
B. pumilus 21.0±1.3 8.5
Control 10.0±1.0 6.5
B. amyloliquefaciens 18.0±1.2 8.8
S. marcescens 19.0±1.1 8.0
TV-25
B. pumilus 22.0±2.0 13.0
Control 02.0±0.5 1.3
B. amyloliquefaciens 15.0±1.0 4.3
S. marcescens 15.0±1.4 9.0
TV-26
B. pumilus 13.0±1.1 5.2
Control 06.0±1.0 4.3
B. amyloliquefaciens 20.0±1.7 8.5
S. marcescens 22.0±1.2 10.1
T-17
B. pumilus 23.0±1.6 11.5
CD (P=0.05) (Treatments)
(Varieties)
2.929
3.275
2.731
3.054
*Mean of leaves from 10 plants
INDIAN J BIOTECHNOL, JANUARY 2013
26
Survival of Applied Bacteria in Rhizosphere
Survival of bacteria in soil following application
was determined immunologically using the PAb
raised against the bacteria. ELISA and DIBA were
done immediately and 6 month after application to the
soil. Tests revealed that the bacteria could survive and
multiply in the rhizosphere (Table 4).
Phylogenetic Analysis
The sequenced PCR product was aligned with ex-
type isolate sequences from NCBI GenBank for
identification as well as for studying phylogenetic
relationship with other ex-type sequences. Multiple
alignment parameters were used (gap penalty=10 &
gap length penalty=10). The use of ClustalW
determines that once a gap is inserted, it can only be
removed by editing. Therefore, final alignment
adjustments were made manually in order to remove
artificial gaps. The optimal tree with the sum of
branch length (=0.62735888) is shown in Fig. 6. The
percentage of replicate trees, in which the associated
taxa clustered together in the bootstrap test (500
replicates), are shown next to the branches31
. The tree
is drawn to scale with branch lengths in the same
units as those of the evolutionary distances used to
Fig. 5 (A & B)—Total (A) and o-dihydroxy phenolic (B) contents
in leaves of tea varieties grown in soil treated with PGPRs.
Table 4—ELISA and DIBA values of rhizosphere soil antigens reacted with PAb of S. marcescens, B. amyloliquefaciens and B. pumilus
ELISA
A405 values*
DIBA
Colour intensity of dots**
Antigens from
rhizosphere of
Treatment
0 d
(±SE)
180 d
(±SE)
0 d 180 d
TV-18 Control 0.254±0.02 0.245±0.07 + +
S.marcescens 1.258±0.18 1.305±0.12 ++++ ++++
B. amyloliquefaciens 1.400±0.63 1.36±0.04 ++++ ++
B. pumilus 1.410±0.60 1.39±0.06 ++++ ++
TV-23 Control 0.321±0.03 0.330±0.04 + +
S. marcescens 1.432±0.04 1.532±0.18 ++++ ++++
B. amyloliquefaciens 1.244±0.03 1.345±0.63 ++++ ++++
B. pumilus 1.200±0.05 1.350±0.61 ++++ ++
TV-25 Control 0.287±0.05 0.312±0.03 + +
S. marcescens 1.356±0.08 1.422±0.08 ++++ ++++
B. amyloliquefaciens 1.248±0.04 1.240±0.12 ++++ ++
B. pumilus 1.257±0.03 1.241±0.12 ++++ ++
TV-26\ Control 0.341±0.03 0.368±0.08 + +
S. marcescens 1.568±0.16 1.630±0.18 ++++ ++++
B. amyloliquefaciens 1.096±0.18 1.083±0.05 ++++ ++
B. pumilus 1.095±0.19 1.088±0.03 ++++ +
T-17 Control 0.286±0.01 0.302±0.02 + +
S. marcescens 1.643±0.07 1.654±0.11 ++++ ++++
B. amyloliquefaciens 1.132±0.02 1.308±0.01 ++++ ++++
B. pumilus 1.134±0.01 1.309±0.04 ++++ +++
*Average of 3 replicates; PAb dilution: 1:1000; Alakaline phosphatase dilution:1:10,000.
**+ = Light pink; ++ = Pink; +++ = Bright pink; ++++ = Pinkish red.
Difference between ELISA values of control and treated are significant at P=0.01 (Student’s t test) in all cases.
CHAKRABORTY et al: RHIZOBACERIA MEDIATED HEALTH IMPROVEMENT IN TEA PLANTS
27
infer the phylogenetic tree. The evolutionary distances
were computed using the Maximum Composite
Likelihood method15
and are in the units of the
number of base substitutions per site. Codon positions
included were 1st+2nd+3rd+noncoding. All positions
containing gaps and missing data were eliminated
from the dataset (Complete deletion option). There
were a total of 125 positions in the final dataset.
Phylogenetic analyses were conducted in MEGA416
.
Phylogenetic analysis of all the three bacterial isolates
showed that B. amyloliquefaciens TRS-6 is closely
related to B. pumilus BRHS/T-84 and are placed
nearer to each other in the phylogenetic tree, whereas
S. marcescens TRS-1 is distantly related to both the
isolates (Fig. 6).
Multiple sequence alignment revealed that there
were regions in the sequences which were not similar
and, hence, gaps were introduced in these regions.
Presence of regions with similar sequences indicated
relationships among the three isolates (Fig. 7).
However, comparative analysis of the combinations
and percentage of occurrence of different nucleotide
in the entire sequence of all the three isolates revealed
that these isolates have unique nucleotide composition
pattern, which was not identical to each other
although a similar 16 S rDNA region was analyzed
for all the three isolates (Table 5).
Discussion In the present study, 3 bacterial isolates, S.
marcescens, B. amyloliquefaciens and B. pumilus,
showed in vitro characteristics of PGPR, such as,
phosphate solubilization, siderophore production and
IAA production. They were also antagonistic to a
large number of fungi in vitro. Further, these
3 bacterial isolates were tested on five varieties of tea
in nursery and field grown plants for their plant
growth promoting activities. They were applied as
aqueous suspension to non-sterile soil with the natural
rhizosphere microflora. Application of the bacteria
resulted in significant increase in growth, measured in
terms of height, leaf numbers and dry mass of leaves
both in potted and field grown plants. Since
insecticides are commonly applied in tea gardens,
potential PGPR should have the ability to tolerate
such insecticides in the soil. Hence tolerances of all
three bacteria to different concentrations of
insecticides were tested in vitro. All three bacteria
could tolerate more than 100 times the insecticide
concentrations applied in the field. Earlier, S.
marcescens NBR11213 was reported to induce plant
growth promotion and biological control of foot and
root rot of betelvine caused by Phytophthora
nicotianeae32
. In a study conducted on groundnut33
,
B. firmis GRS123, B. megaterium GPS 55 and
P. aeruginosa GPS 21 promoted seedling emergence,
root length, shoot length, dry weight and pod yield.
Ability of B. megaterium to promote growth in
Lactuca sativa alone or in combination with
arbuscular mycorrhiza was also reported previously34
.
In order to determine whether the bacteria could
induce systemic resistance (ISR) in tea plants,
accumulation of defense related enzymes and
phenolics were studied. Results revealed that the three
PGPR isolates also enhanced activities of defense
related enzymes, such as, peroxidase, chitinase,
Fig. 6—The phylogenetic analyses conducted using the UPGMA
method among the isolates of B. pumilus, B. amyloliquefaciens
and S. marcescens with other ex-type strains obtained from NCBI
GeneBank database by MEGA4.1 software. The optimal tree with
the sum of branch length (=0.62735888) is shown. The percentage
of replicate trees in which the associated taxa clustered together in
the bootstrap test (500 replicates) is shown next to the branches.
INDIAN J BIOTECHNOL, JANUARY 2013
28
phenylalanine ammonia lyase and β-1,3-glucanase, as
well as total and o-dihydroxy phenols along with an
increase in isomers of catechins. The induction of
defense enzymes, phenols and lignin was also
reported in rice by P. fluorescens against R. solani35
.
Antibiotic-producing P. chlororaphis strains DF190
and PA23, B. cereus strain DFE4 and B.
amyloliquefaciens strain DFE16 were tested for
elicitation of ISR and direct antibiosis for the control
of blackleg disease of canola caused by fungal
pathogen, Leptosphaeria maculans. It was reported
that tested bacteria controlled the disease in canola36
.
For easy handling, it is necessary to pack such
bacteria in inert materials, which can also be
packaged and stored. Hence, three bioformulations
were prepared and bacterial survivability was
determined. The bacteria survived in these
bioformulations for more than nine months.
Sustainability of the applied bacteria was also tested
immunologically by ELISA and the bacteria were
found to survive well in the rhizosphere. Talc based
formulations of B. subtilis and P. fluorescens, either
alone or mixed and along with or without chitin and
neem amendments, were also used for reducing root
rot incidence of chillies along with plant growth
promotion37
.
In the last decade, 16S rDNA sequencing has
played a pivotal role in the accurate identification of
bacterial isolates and the discovery of novel bacteria.
In our previous study38
, we have demonstrated that
analysis of aligned rDNA sequences is a reliable
clustering strategy for identification purposes in a
variety of taxonomic groups and systemic level. The
study showed that it is also applicable in analyzing
Fig 7—16S rDNA sequence alignments of B. pumilus, B. amylolequifaciens and S. marcescens. The conserved regions of the gene are
demonstrated in different colour.
CHAKRABORTY et al: RHIZOBACERIA MEDIATED HEALTH IMPROVEMENT IN TEA PLANTS
29
much shorter DNA sequences from a single gene,
which is going to be the fundamental block in the
massive rDNA database. In our present investigation,
internal transcribed spacer (ITS) regions have been
used successfully to confirm the identity of the
bacterial isolates. The phylogenetic analysis further
confirmed that the strains are phylogenetically related
to the other respective ex-type strains obtained from
NCBI GenBank database. Phylogenetic analysis
conducted among the three groups of isolates revealed
that B. amyloliquefaciens TRS-6 is closely related to
B. pumilus BRHS/T-84 and are placed nearer to each
other in the phylogenetic tree, whereas S. marcescens
TRS-1 has shown to be distantly related to both the
isolates. Comparative analysis of the combinations
and percentage of occurrence of different nucleotides
in the entire sequences of all the three isolates further
confirms their genetic relatedness among each other.
Different pattern in the nucleotide compositions in the
same type of gene for individual isolates proves the
uniqueness of the conserved sites designated to each
species. In the broader context, taxon-selective
amplification of ITS regions, which has now become
a common approach in molecular identification
strategies, can be successfully used to identify diverse
group of microorganisms in a short time.
The overall results of the present study have shown
that all three isolates have potential as plant growth
promoters to increase the growth of tea plants in
experimental plot. Increase in growth was associated
with phosphate solubilization, defense enzymes as
well as increased accumulation of phenolics.
Viabilities of the isolates in bioformulations of talc,
saw dust and rice husk were also examined. In
comparison to S. marcescens, bioformulations of
B. amyloliquefaciens and B. pumilus were more useful
Table 5—Comparision of nucleotide status of rDNA sequences of B. pumilus, B. amyloliquifaciens and Serratia marcescens
B. pumilus-BRHS/T-84
(JQ765580)
1453 sequences,
starting "GGGGGGGGGG"
B. amyloliquefaciens TRS-6
(JN983127)
985 sequences,
starting "GCGCAGGGAA"
S. marcescens-TRS-1
(JN020963)
1364 sequences,
starting "CGGGGAGGAA"
Pattern
Times found Percentage Times found Percentage Times found Percentage
G 461 31.73 322 32.69 433 31.74
A 362 24.91 243 24.67 345 25.29
T 287 19.75 194 19.70 276 20.23
C 343 23.61 226 22.94 310 22.73
Gg 144 9.92 101 10.26 139 10.20
Ga 116 7.99 80 8.13 110 8.07
Gt 97 6.68 68 6.91 87 6.38
Gc 103 7.09 73 7.42 97 7.12
Ag 114 7.85 79 8.03 102 7.48
Aa 101 6.96 68 6.91 101 7.41
At 52 3.58 34 3.46 63 4.62
Ac 95 6.54 62 6.30 79 5.80
Tg 103 7.09 70 7.11 102 7.48
Ta 60 4.13 47 4.78 61 4.48
Tt 62 4.27 40 4.07 52 3.82
Tc 62 4.27 37 3.76 60 4.40
Cg 99 6.82 71 7.22 90 6.60
Ca 85 5.85 48 4.88 73 5.36
Ct 76 5.23 52 5.28 74 5.43
Cc 83 5.72 54 5.49 73 5.36
g,c 804 55.33 548 55.63 743 54.47
a,t 649 44.67 437 44.37 621 45.53
INDIAN J BIOTECHNOL, JANUARY 2013
30
in field application due to the formation of endospores
by bacilli. ELISA values and intensity of dots proved
that three isolates could also survive and multiply in
the rhizosphere even after 6 months of application.
Acknowledgement
Financial help received from the Department of
Biotechnology, Ministry of Science and Technology,
Government of India, New Delhi is gratefully
acknowledged.
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