Vol. 7(45), pp.

5154-5158, 14 November, 2013

DOI: 10.5897/AJMR10.198
ISSN 1996-0808 ©2013 Academic Journals African Journal of Microbiology Research
http://www.academicjournals.org/AJMR

Full Length Research Paper

Microbacterium arborescens AGSB sp. nov., isolated

from the rhizosphere of sand dune plant,
Ipomoea pes caprae
Aureen L Godinho* and Saroj Bhosle
Department of Microbiology, Goa University, Taleigao Plateau, Goa-403206, India.
Accepted 8 October, 2013

Phenotypic and phylogenetic studies were performed for the facultative alkalophile from the
rhizosphere of Ipomoea pes caprae, a plant growing on coastal sand dunes. The isolate was Gram
positive and showed optimum growth at pH 10.5. Chemotaxonomic analysis revealed that the isolate
contained type B1 peptidoglycans with L-lysine as the diamino acid; rhamnose and galactose were the
cell wall sugars and belonged unambiguously to the genus Microbacterium. The major menaquinones
were MK-11 and MK-12. The 16S rDNA sequence of the Microbacterium arborescens isolate was
deposited in the GenBank with an accession number DQ287961. The phylogenetic and phenotypic
distinctiveness of the strain indicates it as a novel Microbacterium sp., named as M. arborescens
AGSB.

Key words: facultative alkaliphile, Microbacterium arborescens AGSB, Coastal sand dune vegetation, Ipomoea
pes caprae, 16S rRNA sequencing.

INTRODUCTION

The sand dune ecosystem is a stressed habitat with only predominant facultative alkaliphile from the rhizosphere
certain type of vegetation surviving in this ecosystem, of Ipomoea pes caprae that exhibited orange, pigmented
one such plant is Ipomoea pes caprae which is com- colonies and gram positive, non sporing regular rods
monly found on coastal dunes. Although a nutrient which has previously not been reported. Further, the
limiting ecosystem, in the rhizosphere, plant litter con- isolate was identified using polyphasic taxonomic tools
tributes to humus and organic matter on which the including chemotaxonomic and 16S rRNA sequencing.
microbiological communities survive. Few studies on the Based on the results, a new species is proposed as
bacterial species present in coastal dunes have been Microbacterium arborescens AGSB.
published. The isolates obtained in this study have been
identified as Acinetobacter, Pseudomonas, Paenibacillus,
MATERIALS AND METHODS
Microbacterium, Agrobacterium Chryseobacterium and
Pseudomonas (Park et al., 2005, 2006; Leveau et al., Strain, cultivation and maintenance
2009; Godinho and Bhosle, 2010; Muthezhilan et al.,
2012; Gaonkar et al., 2012). Earlier studies on isolates The strain chosen for this study was a predominant isolate from
coastal sand dunes; it was isolated from the rhizosphere of
from this ecosystem have shown their ability to produce
Ipomoea pes caprae sand dune vegetation by serial dilution method
exopolymers which aid in sand aggregation and stabilize of the rhizosphere sand and then plating on polypeptone yeast
the dunes (Godinho and Bhosle, 2009). A potent extract glucose agar (PPYG) medium, pH 10.5. The plates were
exopolymer producing isolate was selected for identifying incubated for 2 days at 30°C on polypeptone yeast extract glucose
it to genus level. We report here the characteristics of a agar (PPYG) medium containing (g/l): peptone, 5; yeast extract, 1.5;

*Corresponding author. E-mail: aureengomes@gmail.com.

 Godinho and Bhosle 5155

Figure 1. Scanning Electron Micrograph of Microbacterium arborescens AGSB.

disodium hydrogen phosphate, 1.5; sodium chloride, 1.5; proteinase K inactivation at 80°C for 10 min. The reaction mixture
magnesium chloride,0.1; agar, 15, glucose (10%); sodium was centrifuged at 15,000 rpm at 4°C for 15 min. The supernatant
carbonate (10%); pH 10.5 (Horikoshi, 1987). Biomass for containing genomic DNA was directly used as template in PCR
chemotaxonomic analysis was obtained by growing the strain reaction. PCR amplification of almost full length 16S rRNA gene
aerobically in PPYG without agar (Polypeptone yeast extract was carried out with eubacteria specific primer set 16F27N
glucose broth) on an orbital shaker at approximately 160 rpm for 2 and16R1525XP, in a 25 µl final reaction volume, containing about
days before harvesting by centrifugation (10,000 rpm for 20 10 ng of genomic DNA, 1X reaction buffer, 0.4 mM (each)
min).The cells were washed twice with sterile distilled water and deoxynucleoside triphosphates (Invitrogen), 0.5 U of DNA
freeze dried. Polymerase (New England Labs, UK) and the final volume was
made 25 µl by adding sterile nuclease free water. The PCR was
performed in an Automated Gene Amp PCR system 9700 thermal
Morphological, physiological and biochemical characterization cycler (Applied Biosystems, Foster City, USA) under the following
conditions. The amplification conditions were as follows: 94°C for 1
Cell morphology was determined by phase contrast microscopy and min (denaturation), 55°C for 1 min (annealing), 72°C for 1.30 min
electron microscopy (Figure 1), motility by the hanging drop (elongation), and 72°C for 10 min final elongation. Expected PCR
method, biochemical characteristics were determined by the product of around 1.5 kb was checked by electrophoresis of 5 µl of
method described (Takeuchi and Yokota, 1994; Zhang et al., 2010; the PCR product on 1% agarose gel in 1X TBE buffer and stained
Shivakumar, 2012). with ethidium bromide (0.5 µg/ml). The PCR product was
precipitated by PEG-NaCl (20%PEG in 2.5MNaCl) precipitation at
37°C for 30 min. The reaction mixture was centrifuged at 12,000
Chemotaxonomic methods rpm for 30 min at room temperature. The supernatant was
discarded and the pellet was washed twice with 70% ethanol. After
Preparation of cell walls and determination of peptidoglycan drying the pellet, it was resuspended in 5 µl of sterile nuclease free
structure was carried out by the methods described by Schleifer water. One microliter (50 ng) of purified 16S rRNA PCR product
and Kandler (1972). Menaquinones were extracted and analysed was sequenced by 16S rRNA specific primer that is 16F27N, 530F
as described by Komagata and Suzuki (1987), Collins (1985) and and 16R1525XP.
Minnikin and Goodfellow (1985). Polar lipids were extracted and
analyzed by Thin Layer Chromatography according to Komagata
and Suzuki (1987). Phylogenetic analysis

Purified double stranded PCR fragments were directly sequenced

DNA extraction, PCR amplification and sequencing using BIG DYE Terminator cycle sequencing ready reaction kit
(v3.1) in ABI PRISM 3730 Genetic Analyzer (Applied Biosystems,
A single isolated colony of the selected bacterial cultures was taken USA). The sequences were compared with the 16S rDNA
from agar plate and suspended in 50 µl of colony lysis solution. The sequences available in the public databases from a BLAST search,
reaction mixture was incubated at 55°C for 15 min followed by and identified to the generic level.
5156 Afr. J. Microbiol. Res.

Table 1. Significant characteristics of the isolate Microbacterium

arborescens AGSB.

Characteristic M. arborescens AGSB

Colour of the colony Orange
Motility Positive
Hydrolysis
Gelatin Positive
Starch Negative
H2S production Positive
VP test Negative
Arginine dihydrolase Negative
Assimilation
Arabinose Positive
N-acetyglucosamine Positive
Malate Positive
Citrate Positive
Phenyl acetate Negative
Fumarate Positive
Propionate Negative
Acid from
Glucose Positive
Cell wall diamino acid Lysine
Major menaquinone acid MK-11,12

The 16S rDNA sequences were aligned using CLUSTALX environment. There have been no reports so far of a
(ftp://ftp.ebi.ac.uk/pub/software/clustalw2) with Microbacterium facultative alkaliphile from coastal sand dune ecosystem.
nucleotide sequences derived from GenBank. The trees were
In the present study, an attempt was made to sequence
constructed using the neighbour-joining method. The PHYLIP
package (Felsenstein, 1993) was used to generate trees with the the 16S rDNA in order to study its relationship to other
four algorithms and the trees were viewed using the TREEVIEW species of Microbacterium. The phylogenetic analysis of
package (Page, 1996; Felsenstein, 1981, 1985). Tree topologies the 16S rDNA sequence was accompanied by PCR
were evaluated by bootstrap analysis of the neighbour-joining tree amplification of approximately 1500 base pairs using
using the original dataset and 1000 bootstrap datasets. universal primers. The resulting PCR segments were
sequenced and optimally aligned and phylogenes were
constructed using alogorithms available in the PHYLIP
RESULTS AND DISCUSSION site of phylogenetic analysis programs. Phylogenetic tree
was constructed by neighbour joining method (bootstrap
The phenotypic characteristics of the strain are in agree- method) as shown in Figure 2. The sequences were
ment with the placement of the strain in the genus deposited in the GenBank and the extent of similarities
Microbacterium. The strain showed biochemical charac- with other Microbacterium strain were determined. The
teristics as described in Table 1. The specific charac- phylogenetic tree showed the strain to be 97% similar to
teristics which indicated that the isolate belongs to the Microbacterium sp.MSCB7. The genus Microbacterium
genus Microbacterium are hydrolysis of gelatin, hydrogen was established to accommodate a diverse collection of
sulphide production, assimilation of malate, citrate, n- gram positive non spore forming rods isolated during
acetylglucosamine, fumarate and arabinose. Chemo- studies on lactic acid producing bacteria. Extensive
taxonomic analysis of the isolate revealed the presence phylogenetic studies have recently resulted in
of the amino acid lysine and sugars rhamnose and amalgamation of the genera Microbacterium and
galactose in the cell wall, unsaturated menaquinones Aureobacterium into a redefined genus Microbacterium
MK-11 and MK-12 while polar lipids present were (Takeuchi and Hatano, 1998)
diphosphatidylglycerol and phoshatidylinositol. Based on The isolate characterized in the study is a bacteria
these results and the identification in Bergey’s Manual of which can tolerate a high alkaline pH and survive in
Systematic Bacteriology, this isolate has been designated stressed conditions where the moisture holding capacity
to genus Microbacterium. The present isolate is a of the sand is minimal. Interestingly, this isolate was
facultative alkaliphile and is present in highly stressed found to produce large quantities of exopolysaccharide
 Godinho and Bhosle 5157

Bacterium rJ6 AB021324

M.arborescens DQ287961
41
M.sp.MSCB-7 EF103204
7
M.sp.MAS133 AJ251194
100
M.arborescens DSM20754 X77443
100
M.imperiale DSM20530 X77442

M.arborescens AB007421

100 Unculturedbacterium SSmCB08-6

100
M.arborescens SE14 AY649756
29 100
Unculturedactinomycete BD4-12
2
Unculturedactinobacterium W4 A
100
7
M.sp.oralclone AV005b AF385527
TRICHOTOMY
M.sp.CNJ743PL04 DQ448707
100
M.imperiale AB007414
100
M.sp.Atl-19 EF028128

M.sp.Ellin174 AF409016
10
M.ginsengisoli AB271048
1 19
M.hominis DSM12509 AM181504

M.arborescens kr1 AY238940

24
M.lacticum H193 EF204396
1
M.schleiferi Y17237
31
M.sp.KV-488 AB234027

M.arborescens D21339
20
Unculturedbacterium ODP-69B-02
39
M.sp.IMCC1739 DQ664254
0.1

Figure 2. Unrooted tree showing the phylogenetic relationships of Microbacterium

arborescens sp. nov. and members of the genus Microbacterium based on 16S rDNA
sequences. The tree, constructed using the neighbour-joining method, boot strap values,
expressed as percentage of 1000 replications, are given at the branching points.

(Godinho and Bhosle, 2009) which perhaps supports its Gaonkar T, Nayak PK, Garg S, Bhosle S (2012). Siderophore producing
bacteria from a sand dune ecosystem and the effect of sodium
adherence and survival in this otherwise stressed eco- benzoate on siderophore production by a potential isolate. Sci. World
system. Based on these results, we report the isolation and J. 1-8
characterization of a new facultative alkaliphile M. Godinho A, Ramesh R, Bhosle S (2010). Bacteria from sand dunes of
arborescens AGSB. Goa promoting growth in Eggplant. World J. Agric. Sci. 6(5):555-564.
Godinho AL, Bhosle S (2009). Sand aggregation by exopolysaccharide
producing Microbacterium arborescens AGSB. Curr. Microbiol.
REFERENCES 58:616-621.
Horikoshi K (1987) Alkaliphiles: Genetic Properties and Applications of
Collins DM (1985). Analysis of Isoprenoid quinones. Methods Microbiol. Enzymes, Kodansha, Springer publication.
18:329-365. Komagata K, Suzuki K (1987). Lipid and cell wall analysis in bacterial
Felsenstein J (1981). Evolutionary trees from DNA sequences: a systematics. Methods Microbiol. 19:160-207.
maximum likelihood approach J. Mol. Evol. 17(6):36. Leveau HJJ, Stéphane Uroz S, Boe W (2009).The bacterial genus
Felsenstein J (1985). Confidence limits on phylogenies: an approach Collimonas: mycophagy, weathering and other adaptive solutions to
using the bootstrap. Evolution 39:783-79. life in oligotrophic soil environments. Environ. Microbiol.12:281-292.
Felsenstein J (1993). Phylogeny Inference Package (PHYLIP). Version Minnikin ED, Goodfellow M (1985). Chemical methods in bacterial
3.5. University of Washington, Seattle. pp. 8-76. systematics. The society for applied bacteriology technical series (No
5158 Afr. J. Microbiol. Res.

10). Academic Press. Takeuchi M, Hatano K (1998). Union of the genera Microbacterium
Muthezhilan R, Sindhuja BS, Jaffar Hussain A, Jayaprakashvel M Orla-Jensen and Aureobacterium Collins et al. in a redefined genus
(2012). Efficiency of plant growth promoting rhizobacteria isolated Microbacterium. Int. J Syst. Bacteriol. 48:739-747.
from sand dunes of Chennai coastal area. Pak. J. Biol. Sci. 15:795- Takeuchi M, Yokota A (1994). Phylogenetic analysis of the genus
799. Microbacterium based on 16srRNA gene sequences. FEMS
Page RDM (1996). TREEVIEW: An application to display phylogenetic Microbiol. Lett. 124:11-16.
trees on personal computers. Comput. Appl. Biosci. 12: 357-358. Zhang W, Zhu H, Yuan M, Yao Q, Tang R, Lin M, Yang S, Li Z, Chen M
Park MS, Se RJ, Lee MS, Kyoung O, Kim Do JO, Lee KH, Kim SB, Bae (2010). Microbacterium radiodurans sp. nov., a UV radiation-resistant
KS (2005). Isolation and characterization of bacteria associated with bacterium isolated from soil. Int. J. Syst. Evol. Microbiol. 60(11):2665-
two sand dune plant species, Calystegia soldanella and Elymus 2670.
mollis. J. Microbiol. 43(3):219-227.
Park MS, Se RJ, Lee MS, Kyoung O, Kim Do JO, Lee KH, Kim SB, Bae
KS (2006). Chryseobacterium soldanellicola sp. nov. and
Chryseobacterium taeanense sp. nov., isolated from roots of sand-
dune plants Int. J. Syst. Evol. Microbiol. 56:433-438.
Schleifer KH, Kandler O (1972). Peptidoglycan types of bacterial cell
walls and their taxonomic implications. Bacteriol. Rev. 36:407-477.
Shivakumar S (2012).Accumulation of poly (3-hydroxybutyrate) by
Microbacterium barkeri DSM 20145. Turk. J. Biol. 36:225-232.