Bhore et al.

/ JPBMS, 2012, 14 (12)

Available online at www.jpbms.info

Short communication

ISSN NO- 2230 7885

CODEN JPBSCT

JPBMS
JOURNAL OF PHARMACEUTICAL AND BIOMEDICAL SCIENCES

Bacterial Endophytes in Purple Coraltree (Erythrina fusca Lour.) and Their Screening
for Cytokinins
*Bhore Subhash J, PhD1, Tan Yin Yin, MSc1, Komathi V, MSc student1 & Weber JFF, PhD2
1Department

of Biotechnology, Faculty of Applied Sciences, AIMST University, Bedong-Semeling Road, Bedong,

08100, Kedah, Malaysia.
2Research Institute of Natural Products for Drug Discovery (RiND) and Faculty of Pharmacy, MARA University of
Technology (UiTM), Puncak Alam Campus, 42300 Bandar Puncak Alam, Selangor, Malaysia.

Abstract:

Cytokinins are a group of plant growth regulators and known to delay senescence. The isolation, identification and
screening of endophytes for cytokinins are needed to explore their applications in agriculture. The objective of this study
was the isolation and identification of the endophytic bacterial isolates (EBIs) from the leaves of purple coraltree
(Erythrina fusca Lour.) and screening of EBIs broth-extracts for cytokinin-like activity. We isolated eleven (11) EBIs from
E. fusca leaves collected from the five different sites in Peninsular Malaysia. The PCR amplified 16S rRNA gene sequence
based method of bacterial identification was used to identify EBIs. Using cucumber cotyledon greening bioassay (CCGB),
the ethyl acetate (EtOAc) extracts from EBIs-broth were screened for the presence of cytokinin. Consequently, the EBIs
were identified as Bacillus cereus, B. licheniformis, B. megaterium, B. mycoides, B. pumilus, B. subtilis, Pseudomonas
oryzihabitans, and Bacillus anthracis. Six (6) out of 11 EBIs broth extracts showed positive results in CCGB. The EBIs that
shows positive results in CCGB do have potential applications in prolonging the shelf-life of cut-flowers, leafy-vegetables
and fruits.

Key Words: 16S rDNA, Bacterial endophytes, Cytokinin, Erythrina fusca, Plant growth regulators.

Introduction:

The main aim of this study was to isolate and identify the
EBIs from E. fusca leaves and to screen the ethyl acetate
(EtOAc) extracts from respective EBIs broth for cytokinin
using cucumber cotyledon greening bioassay (CCGB). The
isolated and identified bacterial endophytes of E. fusca are
being reported in this paper along with the results of their
screening for cytokinin-like compounds.

The purple coraltree, Erythrina fusca Lour. (Fabaceae) is

an introduced ornamental plant species in South-east Asia.
It is also used for medicinal purpose. For instance, the
root, bark and leaves of this plant are used as an
antipyretic in Thailand. It is known in Malaysia as dadap
or dedap. [1] The seeds and leaves of this plant are a good
Materials and methods:
source of erythrina alkaloids, whereas the stem bark
Erythrina fusca samples were collected from five different
[2-5]
contains avonoids and pterocarpans.
sites in Peninsular Malaysia, i.e. Semeling (Kedah), Kuala
Endophytes are microorganisms (bacteria and fungi) that
Kangsar (Perak), Bentong (Pahang), Putra Jaya (Federal
live in host plant tissues without causing any visible
Territory) and Nilai (Negeri Sembilan). Healthy plant
[6]
The presence of various bacterial
disease symptoms.
leaves were selected to avoid interferences by plant
endophytes and their interaction and association with
pathogens. Collected leaf samples were brought to the
agronomic, medicinal [7] and other plants is reported for a
laboratory within 24 h after collection following guidelines
variety of species; Glycine max, Oryza sativa, [8] Gynura
by Nalini et al. [16] Leaf samples were treated as reported
procumbens [9] and Ocimum sanctum [10] are few examples
in a previous paper. [9]
to quote. Bacterial endophytes may induce some beneficial
Surface
sterilized leaf pieces were aseptically inoculated
effects to the host such as producing antimicrobial
on
Luria
Bertani (LB) agar medium and incubated at 37C
[11]
increasing the host plants resistance to
compounds,
for
18
to
20 h in the dark. Well grown colonies of
[12,
13]
promoting biological
parasites and pathogens,
cultivable
bacterial
endophytes were chosen based on
[14]
and boosting plants growth and
nitrogen fixation
differential
morphological
appearance. The culture
[15]
The
development by releasing plant growth promoters.
conditions
were
identical
to
those stated in a previous
latter effects have several potential applications in a
[9]
communication.
sustainable agriculture. Cytokinins a group of plant
Overnight grown 10 ml cultures of respective EBIs were
growth regulators is known to delay senescence in
centrifuged; and 7 ml of cell-free broth was extracted with
addition to their other biological functions. Endophytes
an equal volume of EtOAc. The solvent was evaporated
that could produce cytokinin-like compounds do have
under reduced pressure and the residues of each sample
potential applications in prolonging the shelf-life of cutwere dissolved in 1 ml absolute ethanol. Finally the
flowers, leafy-vegetables and or fruits. Therefore, this
ethanol was evaporated and the dry extracts were restudy was undertaken to explore bacterial endophytes.
1
Journal of Pharmaceutical and Biomedical Sciences (JPBMS), Vol. 14, Issue 14

Bhore et al. / JPBMS, 2012, 14 (12)

dissolved in the bioassay solution described by Fletcher et

al. [17]
The cells harvested from the above mentioned 10 ml broth
were re-suspended in 50 l sterile distilled water, boiled
for 10 min and centrifuged at 12,000 rpm for 5 min. Five l
lysate was used to amplify conserved region of the 16S
rRNA gene. The amplification (PCR) reactions were
carried out in a total volume of 50 l using Bak11W-F
(forward) [5-AGT TTG ATC MTG GCT CAG-3], and Bak-R
(reverse) [5-GGA CTA CHA GGG TAT CTA AT-3] primer.
The PCR conditions and the concentration of the PCR
components were identical as those reported previously
by our group. [9] The amplified PCR products from all EBIs
were purified with the Wizard SV Gel and PCR Clean up
system kit (Promega Corp.). The purified 16S rDNA
fragments were sequenced using primers used in PCR
amplification of 16S rRNA gene. The sequence of 16S rDNA
fragments from each EBI were blasted against the
collection of non-redundant nucleotide sequence database
of NCBI (http://www.ncbi.nlm.nih.gov/). The isolates
were identified based on hits analysis from Megablast
(highly similar sequences) output.
To screen EtOAc extracts of EBIs-broth, CCGB was used.
The cucumber seeds (Cucumis sativus L., cultivar F1
Cucumber 303) were purchased from Tai Lung Farm Seed
Co Ltd, Taiwan. The methodology for the seeds
germination and CCGB were carried out as described by
Fletcher et al. [17] Briefly, the cucumber seeds were
germinated in plastic trays containing wet tissue paper at
room temperature (~27C 3) in the dark for 6 days. The
cotyledons were then separated from hypocotyls. After
weighing, cotyledons were added in Petri dishes
containing the 10 ml bioassay solution supplemented with
broth extracts of respective EBIs and incubated under
fluorescent tube light for 3.5 hours at 27C 2. After
incubation, the cotyledons were ground with 80% acetone
in a pre-chilled mortar and pestle. The extracted
chlorophyll samples were collected and centrifuged at
4,000 rpm for 10 min. The absorbance of the supernatant
was measured at 663 and 645 nm. The positive controls
were cotyledons treated with 1 ppm (1 mg/l) solutions of
6-benzylaminopurine (BAP), while negative controls were
cotyledons treated with the vehicle only. The CCGB was
carried out in triplicate under the same set of conditions.
The data of the total amount of chlorophyll in cucumber
cotyledons treated with EtOAc extracts of EBIs-broth,

positive control, and negative control were analyzed by

one-way analysis of variance (ANOVA) test. [18]

Results and discussion:

The inoculated leaves discs (from samples collected at 5

different locations in Malaysia) on LB medium produced
139 bacterial colonies in total. White and pale yellow
colored bacterial colonies were observed visibly on the
margins of leaf discs incubated on LB-agar medium.
Eleven bacterial colonies representing the two types of
colonies (white and pale yellow) and the plant material
samples were selected and inoculated in LB medium. The
16S rRNA coding gene fragments were PCR-amplified
using separately prepared lysate of respective isolates.
The size of the amplified 16S rDNA fragments was about
800 bp. The purified 16S rDNA PCR products were
sequenced using the same primers used in the PCR. The
partial 16S rRNA gene sequences from 11 EBIs were
deposited in the GenBank/DDBJ/EMBL gene sequence
database.
Plant organs such as roots, [19] stem, petioles, leaves, [9]
inflorescence, flowers, [20] fruits, seeds, [21] pods, tubers etc.
can be used in isolating the cultivable endophytic bacteria
(and fungi). In this study we have used leaves of the E.
fusca plant from five different locations in Peninsular
Malaysia to isolate their bacterial endophytes in order to
increase the diversity of endophytes that could be isolated.
With an incubation of 18 to 20 h and a non-specific growth
medium for bacteria, the isolation method was favoring
fast growing bacterial species. Actinomycetes, [22] known
to be major bioactive metabolite producers, had little
chance to be isolated in these conditions. This research
work was intended to find out whether some ubiquitous
germs could also be involved in influencing the host
metabolism. Eight bacterial species were identified based
on 16S rRNA gene sequence homology of eleven EBIs
selected on the basis of their macroscopical appearance
(white or yellow colonies) as well as sample origin. The
amplified 16S rRNA gene fragments size was about 800
bp. Based on the primer locations, this size was matching
with the expected size of the PCR product. However, only
the quality sequence length of 16S rRNA gene (from
amplified product) was used in analysis and identification
of respective EBIs as depicted in Table 1.

Table 1: Isolated and identified endophytic bacterial isolates (EBIs) from Erythrina fusca leaves
Length of analyzed
Isolate
Erythrina fusca
No.
16S rRNA gene
Species*
ID
location
(bp)
Efe1

Semeling, Kedah

531

Efe3

Semeling, Kedah

453

Bacillus mycoides

458

Pseudomonas oryzihabitans

742

Bacillus cereus

2
4
5
6
7

Bacillus megaterium

Efe2
Efe4

Semeling, Kedah
Semeling, Kedah

Efe6

Semeling, Kedah (AIMST University)

5.Ery.2

Putra Jaya (Botanical Garden)

4.Ery.1

Kuala Kangsar, Perak

394

Bacillus licheniformis

172

Bacillus subtilis

580

Bacillus subtilis

Similarity %

Accession number#

97

HM104474

99

HM104475

99

HM104476

99

HQ418463

93
97
98

HQ232297
HQ232298
HQ637458

Journal of Pharmaceutical and Biomedical Sciences (JPBMS), Vol. 14, Issue 14

Bhore et al. / JPBMS, 2012, 14 (12)

8

6.Ery.3

Nilai, Negeri Sembilan

748

Bacillus pumilus

10

9.Ery.5

Bentong, Pahang

750

Bacillus pumilus

11

6.Ery.4
1.Ery.6

Nilai, Negeri Sembilan

Semeling, Kedah

741

Bacillus pumilus

756

Bacillus anthracis

99

HQ418464

99

HQ418466

99
99

HQ418465
HQ418467

*Identity of respective EBI based on analysis of their 16S rRNA sequence. #GenBank accession numbers of deposited 16S rRNA gene sequence fragments
from respective EBIs.

The blastn output and its analysis led to the identification

of the 11 cultivable isolated EBIs as Bacillus anthracis, B.
cereus, B. licheniformis, B. megaterium, B. mycoides, B.
pumilus, B. subtilis and Pseudomonas oryzihabitans. In
Genbanks nucleotide database, there are 2942, 21337,
27167, 1301, 4221, 1722, 12205 and 45 nucleotide
sequence entries for various strains of the above species,
respectively
(April
19,
2011,
http://www.ncbi.nlm.nih.gov/genbank/). The GenBank
accession numbers and the length of deposited 16S rRNA
gene sequences of EBIs are shown in the Table 1. Very
limited reports are available on the endophytic roles of
these bacteria, despite the fact that Pseudomonas and
Bacillus species of bacteria are reported as endophytes in
many plant species. [6] Bacillus anthracis is the pathogen of
the anthrax disease; and it is interesting to note that E.
fusca can serve as a host for it. However, Cinnamomum
camphora (Linn.) Presl. is also known to host this
bacterium (GenBank accession no: FJ174595). It is also
found in Tectona grandis L. f., Hylocereus undatus Haw.,
Bambusa sp., Ficus benjamina Linn. and some other plants
leaves (our unpublished findings). Endophytic bacteria are
considered as biocontrol agents of plant diseases; however
the benefits of the isolated EBIs for E. fusca are not clear at
this stage.The results of the CCGB are shown in Figure 1.
Figure 1: Screening of 11 endophytic bacterial isolates (EBIs) isolated
from Erythrina fusca Lour. leaves for cytokinin using CCGB. The ethyl
acetate (EtOAc) extracts were prepared separately from 11 EBIs-cellfree-broth. P.Con, positive control; N.Con, negative control; positive
control is with 1 ppm BAP, and negative control is without BAP and
EtOAc extracts (codes of the EBIs are shown in the Table 1). Bars indicate
the standard deviation, and different letters (a, b and c) indicates that the
total amount of chlorophyll is significantly different from each other
(P>0.05).

cytokinin-like activity. The cucumber cotyledons treated

with extracts of Efe 2, 4.Ery.1, and 1.Ery.6 showed a total
amount of chlorophyll content equivalent to that of the
positive control. True enough, the differences between the
negative controls and tested extracts as well as the
positive controls appear modest. It should be noted that
cucumber cotyledons in positive controls were treated
with a very high dilution of the BAP (1 mg/l). The
experiments were only designed as to explore the
potential of bacterial endophytes from a plant grown for
ornamental purpose and known to be rich in bioactive
chemicals. Further studies carried out for the
identification of the active ingredients will be reported in
due time.

Conclusion:

The hypothesis that endophytes, even belonging to

common species, might have the potential to produce
compounds that acts like cytokinin is therefore vindicated
and provides a basis for further studies on their
applications in agriculture and other sectors of
biotechnology. Such microorganisms could be useful in
agriculture to delay the senescence of cut-flowers, leafyvegetables and fruits.

Acknowledgments:

Authors are grateful to the Ministry of Agriculture and

Agro-Based Industry (MoA), Malaysia for research funding
(Grant Code Number: 05-02-16-SF1001). Authors attest
that there are no conflicts of interest to declare. TYY and
KV collected the samples and carried out the experimental
work. BSJ conceived the study and supervised the
experiments. BSJ and WJFF wrote the manuscript. SJB had
full access to all the data in the study and takes
responsibility for the integrity of the data and the accuracy
of the data analysis. All authors read and approved the
final manuscript.

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the extraction of compounds with appropriate
hydrophilic/lipophilic balance. [24] The results of the CCGB
suggest that 6 out of the 11 EtOAc extracts induce

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Source(s) of support: Malaysias Ministry of Agriculture and Agro-Based Industry (MoA)

(Grant Code Number: 05-02-16-SF1001)
Conflict of interest: - None

Corresponding Author:-

Bhore Subhash J.,

Department of Biotechnology, Faculty of Applied Sciences,
AIMST University, Bedong-Semeling Road, Bedong, 08100, Kedah,
Malaysia, Tel: +60 4 429 8176, Fax: +60 4 429 8109,
Email id:- [email protected]
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