plant growth promoting rhizobacteria mediated improvement...

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: [email protected]

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


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


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+


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 +


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.


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



TV-23

B. pumilus 21.0±1.3 8.5

Control 10.0±1.0 6.5



TV-25

B. pumilus 22.0±2.0 13.0

Control 02.0±0.5 1.3



TV-26

B. pumilus 13.0±1.1 5.2

Control 06.0±1.0 4.3



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


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. 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. 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.


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.


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.


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


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.

References

1 Kloepper J W & Schroth M N, Plant growth promoting

rhizobacteria on radishes, in Proc 4th Int Conf on Plant

Pathogenic Bacteria, vol 2 (Station de Pathologie Vegetale et

Phytobacteriologie, INRA, Angers, France) 1978, 879-882.

2 Kloepper J W, Plant growth promoting rhizobacterial as

biological control agents, in Soil microbial ecology—

Applications in agricultural and environmental management,

edited by F B Metting, Jr (Marcel Dekker Inc., New York,

USA) 1993, 255-274.

3 Bai Y, Souleimanov A & Smith D L, An inducible activator

produced by a Serratia proteamaculans strain and its soybean

growth promoting activity under green house conditions, J Exp

Bot, 53 (2002): 1495-1502.

4 Kloepper J W, Zehnder G W, Tuzun S, Murphy J F, Wei G et

al, Toward agricultural implementation of PGPR-mediated

induced systemic resistance against crop pests, in Advances in

biological control of plant diseases, edited by W Tang, R J

Cook & A Rovira (China Agricultural University Press,

Beijing) 1996, 165-174.

5 Jung H K & Kim S D, Purification and characterization of an

antifungal antibiotic from Bacillus megaterium KL 39, a

biocontrol agent of red-pepper Phytophthora blight disease,

Korean J Microbiol Biotechnol, 31(2003): 235-241.

6 Chandramouli B, Blister blight of tea: Biology, epidemiology

and management, Annu Rev Plant Pathol, 2 (2003): 145-162.

7 Bashan Y, Inoculants of plant growth promoting rhizobacteria

for use in agriculture, Biotechnol Adv, 16 (1998) 729-770.

8 El-Hassan S A & Gowen S R, Formulation and delivery of the

bacterial antagonist Bacillus subtilis for management of lentil

vascular wilt caused by Fusarium oxysporum f. sp. lentis, J

Phytopathol, 154 (2006) 148-155.

9 Trivedi P, Pandey A & Palni L M S, Carrier-based

preparations of plant growth promoting bacterial inoculants

suitable for use in cooler regions, World J Microbiol

Biotechnol, 21 (2005) 941-945.

10 Chakraborty U, Chakraborty B N, Basnet M & Chakraborty A

P, Evaluation of Ochrobactrum anthropi TRS-2 and its talc

based formulation for enhancement of growth of tea plants and

management of brown root rot disease, J Appl Microbiol, 107

(2009) 625-634.

11 Louws F J, Rademaker J LW & De Brujin F J, The three Ds

off PCR-based genomic analysis of phytobacteria: Diversity

detection and disease diagnosis, Annu Rev Phytopathol, 37

(1999) 81-125.

12 Buchanan R E & Gibbons N E (Ed), Bergey's manual of

determinative bacteriology, 8th edn (Williams & Wilkins Co.,

Baltimore, MD, USA) 1974, 1246.

13 Stafford W H L, Baker G C, Brown S A, Burton S G & Cowan

D A, Bacterial diversity in the rhizosphere of Proteaceae

species, Environ Microbiol, 7 (2005) 1755-1768.

14 Sneath P H A & Sokal R R, Numerical taxonomy:Principles

and practice of numerical taxonomy (W H Freeman & Co.,

San Francisco, USA) 1973.

15 Tamura K, Nei M & Kumar S, Prospects for inferring very

large phylogenies by using the neighbor-joining method, Proc

Natl Acad Sci USA, 101 (2004) 11030-11035.

16 Tamura K, Dudley J, Nei M & Kumar S, MEGA4: Molecular

evolutionary genetics analysis (MEGA) software version 4.0,

Mol Biol Evol, 24 (2007) 1596-1599.

17 Thompson J, Higgins D & Gibson T, CLUSTALW:

Improving the sensitivity of progressive multiple sequence

alignment through sequence weighting, position-specific gap

penalties and weight matrix choice, Nucleic Acids Res, 22

(1994) 4673-4680.

18 Pikovskaya R E, Mobilization of phosphorous in soil in

connection with vital activity of some microbial species,

Microbiologia, 17 (1948) 362-370.

19 Schwyn B & Neiland J B, Universal chemical assay for the

detection and determination of siderophores, Anal Biochem,

160 (1987) 47-56.

20 Dobbelaere S, Croonenberghs A, Thys A, Vande Broek A &

Vanderleyden J, Photostimulatory effects of Azospirillum

brasilense wild type and mutant strain altered in IAA

production on wheat, Plant Soil, 212 (1999) 155-164.

21 Chakraborty U, Chakraborty B N & Basnet M, Plant growth

promotion and induction of resistance in Camellia sinensis by

Bacillus megaterium, J Basic Microbiol, 46 (2006)

186-195.

22 Chakraborty U, Chakraborty B N & Kapoor M, Changes in the

levels of peroxidase and phenylalanine ammonia lyase in

Brassica napus cultivars showing variable resistance to

Leptoshaeria maculans, Folia Microbiol, 38 (1993) 491-496.

23 Boller T & Mauch F, Colorimetric assay for chitinase,

Methods Enzymol, 161 (1998) 430-435.

24 Bhattacharya M K & Ward E W B, Biosynthesis and

metabolism of glyceollin I in soybean hypocotyls following

wounding or inoculation with Phytophthora megasperma f. sp.

glycinea, Physiol Mol Plant Pathol, 31 (1987) 401-409.

25 Pan S Q, Ye X S & Kuc J, A technique for detection of

chitinase, β-1,3-glucanase, and protein patterns after a single

separation using polyacrylamide gel electrophoresis or

isoelectric focussing, Phytopathology, 81 (1991) 970-974.

26 Mahadevan A & Sridhar R, Methods in physiological plant

pathology, 2nd edn (Sivakami Publications, Ambattur, Madras,

India) 1982.

27 Alba A P C & Devay J E, Detection of cross-reactive antigens

between Phytophthora infestans (Mont.) de Bary and Solanum

species by indirect enzyme-linked immunosorbent assay,

Phytopathology, 112 (1985) 97-104.

28 Clausen J, Immunochemical techniques for the identification

and estimation of macromolecules, 3rd edn (Elsevier Science

Ltd., New York, USA) 1988, 64-65.

29 Chakraborty B N, Basu P, Das R, Saha A & Chakraborty U,

Detection of cross reactive antigen between Pestalotiopsis

theae ant tea leaves and their cellular location, Ann Appl Biol,

207 (1995) 11-21.

30 Lange L, Heide M & Olson L W, Serological detection of

Plasmodiophora brassicae by dot immunobinding and


31

visualization of the serological reaction by scanning electron

microscopy, Phytopathology, 79 (1989) 1066-1071.

31 Felsenstein J, Confidence limits on phylogenies: An approach

using the bootstrap, Evolution, 39 (1985) 783-791.

32 Lavania M, Chauhan P S, Chauhan S V, Singh H B &

Nautiyal C S, Induction of plant defense enzymes and

phenolics by treatment with plant growth promoting

rhizobacterial Serratia marcescens NBRII213, Curr

Microbiol, 52 (2006) 363-368.

33 Kishore G K, Pande S & Podile A R, Phylloplane bacteria

increase seedling emergence, growth and yield of field-grown

groundnut (Arachis hypogea L.), Lett Appl Microbiol, 40

(2005) 260-268.

34 Marulanda-Aguirre A, Azcon R, Ruiz-Lozano J M & Aroca R,

Differential effects of a Bacillus megaterium strain on Lactuca

sativa plant growth depending on the origin of the arbuscular

mycorrhizal fungus coinoculated: Physiologic and biochemical

traits, J Plant Growth Regul, 27 (2008) 10-18.

35 Radjacommare R, Ramanathan A, Kandan A, Harish S,

Thambidurai G et al, PGPR mediates induction of

pathogenesis-related (PR) proteins against the infection of

blast pathogen in resistant and susceptible ragi [Eleusine

coracana (L.) Gaertner] cultivars, Plant Soil, 266 (2005) 165-

176.

36 Ramarathnam R, Fernando W G D & Kievit T, The role of

antibiosis and induced systemic resistance, mediated by strains

of Pseudomonas chlororaphis, Bacillus cereus and B.

amyloliquefaciens, in controlling blackleg disease of canola,

BioControl, 56 (2011) 225-235.

37 Bharathi R, Vivekananthan R, Harish S, Ramanathan A &

Samiyappan R, Rhizobacteria-based bio-formulations for the

management of fruit rot infection in chillies, Crop Protect, 23

(2004) 835-843.

38 Chakraborty B N, Chakraborty U, Sunar K, & Dey P L, RAPD

profile and rDNA sequence analysis of Talaromyces flavus and

Trichoderma species, Indian J Biotechnol, 10 (2011) 487-495.

plant growth promoting rhizobacteria mediated improvement...

Documents