World J Microbiol Biotechnol (2014) 30:719–725

DOI 10.1007/s11274-013-1488-9

ORIGINAL PAPER

Molecular characterization and identification of plant growth

promoting endophytic bacteria isolated from the root nodules
of pea (Pisum sativum L.)
Mohsin Tariq • Sohail Hameed • Tahira Yasmeen •

Mehwish Zahid • Marriam Zafar

Received: 20 May 2012 / Accepted: 30 January 2013 / Published online: 26 September 2013
Ó Springer Science+Business Media Dordrecht 2013

Abstract Root nodule accommodates various non-nodu- most efficient plant growth promoting agents, MSP9 and
lating bacteria at varying densities. Present study was planned MSP10, were phylogenetically identified by 16S rRNA gene
to identify and characterize the non-nodulating bacteria from sequence analysis as Ochrobactrum and Enterobacter,
the pea plant. Ten fast growing bacteria were isolated from the respectively, with 99 % similarity. It is suggested the potential
root nodules of cultivated pea plants. These bacterial isolates endophytic bacterial strains, Ochrobactrum sp. MSP9 and
were unable to nodulate pea plants in nodulation assay, which Enterobacter sp. MSP10, can be used as biofertilizers for
indicate the non-rhizobial nature of these bacteria. Bacterial various legume and non-legume crops after studying their
isolates were tested in vitro for plant growth promoting prop- interaction with the host crop and field evaluation.
erties including indole acetic acid (IAA) production, nitrogen
fixation, phosphate solubilization, root colonization and bio- Keywords Pisum sativum L.  Plant growth
film formation. Six isolates were able to produce IAA at promoting bacteria  16S rRNA sequencing  Nodules
varying level from 0.86 to 16.16 lg ml-1, with the isolate
MSP9 being most efficient. Only two isolates, MSP2 and
MSP10, were able to fix nitrogen. All isolates were able to Introduction
solubilize inorganic phosphorus ranging from 5.57 to
11.73 lg ml-1, except MSP4. Bacterial isolates showed con- Rhizobia are soil bacteria, recognized by the character of
siderably better potential for colonization on pea roots. Isolates infecting roots of legume (Fabaceae) plants and develop-
MSP9 and MSP10 were most efficient in biofilm formation on ing an outgrowth on root, called nodule, which are the
polyvinyl chloride, which indicated their potential to withstand small factories of atmospheric nitrogen fixation. In the past,
various biotic and abiotic stresses, whereas the remaining rhizobia were considered to include the bacteria belonging
isolates showed a very poor biofilm formation ability. The to genera Rhizobium, Azorhizobium, Bradyrhizobium,
Ensifer and Mesorhizobium. Recently, it is found that rhi-
zobia is a paraphyletic group, fall into three classes of
proteobacteria-alpha, beta and gamma-proteobacteria
M. Tariq  S. Hameed  T. Yasmeen  M. Zahid  M. Zafar
(Angus and Hirsch 2010; Benhizia et al. 2004).
Microbial Physiology Laboratory, National Institute for
Biotechnology and Genetic Engineering (NIBGE)/ PAEC, Root nodules also accommodate various non-nodulating
Islamabad, Pakistan bacteria having definite influence on the survival, nodulation
and grain yield of crop and their densities are reported to be
M. Tariq (&)  T. Yasmeen
very high (Mishra et al. 2009; Tariq et al. 2012). These
Government College University Faisalabad (GCUF),
Allama Iqbal Road, Faisalabad, Pakistan endophytic bacteria live inside the nodule tissues without
e-mail: mruaf@hotmail.com; mohsintariq@gcuf.edu.pk substantially harming or gaining benefit other than shelter
(Kobayashi and Palumbo 2000). Such bacteria are relatively
M. Zafar
protected from the competitive, high-stress environment of
Centre of Agricultural Biochemistry and Biotechnology
(CABB), University of Agriculture Faisalabad, Faisalabad, soil and promote plant growth by producing plant growth
Pakistan promoting substances (Aravind et al. 2012; Patel et al. 2012).

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Furthermore, these bacteria act synergistically with rhizobia Nodulation assay

to improve nodulation and nitrogen fixation (Duangpaeng
et al. 2012). Non-nodulating bacteria for the first time iso- Nodulation assays were performed according to Ma et al.
lated from nodules of legume plant were identified as (2003) with some modifications. All the potential nodule
Agrobacterium radiobacter (Beijerinck and Delden 1902). endophytic bacteria were grown individually in YEM broth,
Non-nodulating bacteria isolated from nodules of various and maintained at 106 c.f.u. ml-1 in sterile water. Rhizobium
legume plants include Inquilinus, Pantoea, Escherichia, leguminosarum bv. viciae PS-I and PS-II were used as positive
Bosea, Phyllobacterium, Sphingomonas, Pseudomonas, control for nodulation. Seeds of pea cv. Meteore were surface
Agromyces, Microbacterium, Paenibacillus, Aerobacter, sterilized with 0.1 % mercuric chloride for 5 min and washed
Agrobacterium, Chryseomonas, Curtobacterium, Erwinia, extensively with sterile water. Surface sterilized seeds were
Flavimonas, Sphingomonas, Methylobacterium, Blastob- allowed to germinate in dark room at 25 ± 2 °C on moist
acter, Devosia, Rhodopseudomonas, Paracraurococcus, filter paper kept in sterile Petri plates containing 15 seed.
Phyllobacterium, Ochrobactrum, Cupriavidus, Herbaspir- Germinated seedlings (1 cm) were treated with inocula of
illum, Pseudomonas, Enterobacter, Leclercia, Ochrobac- each endophytic bacteria, separately, and transplanted in the
trum, Starkeya, Azotobacter, Azospirillum, Ornithinicoccus, sterilized magenta boxes containing vermiculite-perlite (ratio
Bacillus (Sturz et al. 1997; Selvakumar et al. 2008; Tariq 1:1 v/v) and incubated at 30 ± 2 °C (day) and 20 ±2 °C
et al. 2012). (night) for a light photoperiod of 16 h per day (Fraile et al.
The present study was aimed to isolate the non-nodu- 1988). Plants were watered with quarter strength nitrogen free
lating endophytic bacteria from the root nodules of pea Hoagland solution (Arnon and Hoagland 1940). After
plants and characterize in vitro for plant growth promoting 4 weeks, plants were observed for nodulation.
properties. Using 16S rRNA gene sequence analysis, we
also studied the taxonomic position of these non-nodulat- Characterization of endophytic bacteria for plant
ing endophytic bacteria. growth promoting attributes

Bacterial isolates were also studied in vitro for plant

Materials and methods growth promoting properties including indole acetic acid
(IAA) production, nitrogen fixation, solubilization of
Isolation of nodule endophytic bacteria phosphate, root colonization and biofilm formation.
Detection of IAA production by bacterial strains was
Pea plants grown in the field of National Institute for carried out by growing cultures in YEM broth supple-
Biotechnology and Genetic Engineering (NIBGE), Faisal- mented with tryptophan (100 mg l-1) for 1 week. Quali-
abad, Pakistan, were sampled for bacterial isolation at tative estimation of IAA was assessed by Fe-HClO4 and
nodulation stage after 4 weeks. Roots were washed thor- Fe-H2SO4 reagents producing pink color (Gordon and
oughly using sterile distilled water to remove adhering soil. Weber 1951). IAA was quantified by ethyl acetic acid
About 10 nodules were detached from the roots with a oxidation method (Tien et al. 1979) using HPLC with
sterilized forceps. The intact and undamaged nodules were Turbochom software (Perkin Elmer USA).
immersed in ethanol for 30 s and transferred to a 3 % Nitrogenase activity was examined by acetylene reduc-
solution of calcium hypochlorite for 5 min under aseptic tion assay (Hardy et al. 1968) on a gas chromatograph
conditions. Nodules were rinsed in five changes of sterile (Thermoquest, Trace G.C. Italy) using Porapak Q column
water. Surface sterilization of nodules was ensured by and H2-flame ionization detector. Nitrogen fixation ability
incubating nodules on yeast extract mannitol (YEM) agar of bacterial strains was assessed by inoculating single
(Vincent 1970) plate for 72 h at 28 ± 2 °C. Nodules were colony in 5 ml semisolid nitrogen free media (NFM) (Okan
crushed to prepare a suspension in 20 ll sterile water in a et al. 1977) in 15 ml vials and incubated at 28 ± 2 °C for
Petri dish. One loop full of nodule suspension was streaked 48 h. Acetylene (10 % v/v) was injected to the vials and
on YEM plates containing Congo red and incubated at incubated for 16 h at room temperature. Gas samples
28 ± 2 °C until the appearance of single colonies, which (100 ll) from these vials were analyzed on a gas chro-
were selected after 24 h of showing different morphotypes matograph as described by Hameed et al. (2004).
and subcultured until the purity of cultures was confirmed For phosphate solubilization study, a single colony of
(Marsudi et al. 1999). Pure cultures were stored in 20 % each bacterial culture grown on YEM plate was streaked on
glycerol at -80 °C. Cell shape and size were observed under to Pikovskaya’s agar plates containing tricalcium phos-
light microscope by taking a drop of bacterial culture sus- phate (Pikovskaya 1948) and incubated at 25 ± 2 °C for
pension in saline. Gram’s reaction was performed according 7–10 days. The plates were analyzed for clear phosphate
to Vincent (1970). solubilization zone around the colonies. Phosphate

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solubilization was quantified by Phospho-molybdate blue and Doly 1979). The primers used for amplification of full
color method (Nair et al. 2007) using spectrophotometer length 16S rRNA gene were universal primer P1 (forward
(Campec M350 Double Beam). primer, 50 -CGGGATCCAGAGTTTGATCCTGGTCAGA
ACGAACGCT-30 ) and P6 (reverse primer, 50 -CGGGATCCT
Root colonization assay ACGGCTACCTTGTTACGACTTCACCCC-30 ), which
correspond to E. coli positions 8–37 and 1,479–1,506,
Potential of bacterial isolates to colonize pea roots was also respectively and amplifies 1,500 bp fragment (Tan et al.
studied. Ten-day-old plants were harvested and the roots were 1997). Each 25 ll of PCR reaction mixture contained 2.5 ll
cut into 1.5 cm segments. Pieces of uniform shape and size 109 PCR buffer, 2 ll MgCl2, 1 ll dNTPs (2.5 mM), 1 U of
were placed into the 96-wells of a microtiter plate. 200 ll of Taq Polymerase (Promega), 1 ll of each primer (100
bacterial culture maintained at OD600 = 0.2 were added to the ng ll-1) and 1 ll template DNA (12.5 ng ll-1). Reaction
wells and the plates were incubated at 28 °C for 24 h for fast mixture (25 ll) prepared for 16S rRNA gene amplification,
growing and 72 h for slow growing bacteria. After the incu- was initially denatured at 94 °C for 2 min followed by 25
bation period, the root pieces were removed from the cultures, cycles consisting of denaturation at 94 °C for 1 min, primer
washed with sterile water, and then added to 1 ml sterile annealing at 52 °C for 1 min and primer extension at 72 °C
water. Bacterial biofilms were removed from the root surface for 3 min and finally extension at 72 °C for 20 min in a
and dispersed in sterile water by vigorous shaking. An aliquot thermal cycler. The amplified 16S rRNA gene was ligated in
(100 ll) of the dispersed preparation was plated on YEM agar TA cloning vector pTZ57R/T (Fermentas), which has
and counted after 5 days as c.f.u. 0.1 mg-1 root. 2,886 bp length. 30 ll ligation reaction was prepared in sterile
water with 1.5 ll T4 DNA ligase, 3 ll ligation Buffer, 3 ll
Biofilm formation assay pTZ57R/T vector (Fermentas), 3 ll of PEG 4000 and 4 ll
amplified DNA in 1.2 ml tube. Ligation was performed
Biofilm formation was studied on a abiotic surface by a overnight in water bath at 16 °C. Plasmids were transformed
microtiter plate assay according to Fujishige et al. (2006) chemically in E. coli TOP10 and extraction of recombinant
with some modifications. The bacterial cultures were grown plasmids was carried out using GeneJET Plasmid Miniprep
upto an optical density at k600 nm (OD600) = 2.0 in YEM Kit (Fermentas) and clones were confirmed by restriction
broth, pelleted by centrifugation at 8,000 rev. min-1 for analysis using EcoRI and BamHI (Fermentas). During
2 min, and washed with sterile distilled water. The cells molecular studies, amplified products and clones were
were resuspended in the same medium and maintained at resolved on 1 % agarose gel and GeneRulerTM 1 kb ladder
OD600 = 0.2. An aliquot (150 ll) of bacterial cell suspen- #SM0313 was used as DNA size marker. Clones were
sion was added to individual wells in a 96-well polyvinyl sequenced on ABI Prism 3100 Genetic Analyzer (Hitachi,
chloride (PVC) plate (Fisher, USA). YEM alone was used Japan) using Big Dye Terminator v 1.1 Cycle Sequencing Kit.
as the control. The plates were covered with plastic lids and The gene sequences were compared with others in sequence
incubated at 28 °C for 24 h for fast growing and 72 h for databases using NCBI BLAST software (Altschul et al. 1990)
slow growing bacteria. After the incubation period, the at http://www.ncbi.n1m.nih.gov/blast/Blast.cgi.
medium was removed and the wells were washed with
sterile water. The plates were allowed to dry and the wells
were treated with 150 ll of 0.001 % crystal violet for Results
15 min. The excess of dye was removed and the wells were
washed with sterile water. The retained stain was solubi- Isolation of endophytic bacteria from root nodules
lized with 150 ll of 95 % ethanol and amount of dye was
quantified by plate reader at absorbance of 570 nm. A total of ten bacterial isolations were obtained on the basis
of colony morphology from the nodules sweet pea grown in
Statistical analysis the fields of NIBGE, Faisalabad, Pakistan. Colony and cell
morphological characters were highly variable as presented
The data were analyzed statistically using MSTAT soft- in Table 1. Bacterial isolates were also tested for Gram
ware. Analysis of variance was computed and means were reaction. Out of ten isolates, two were gram positive while
compared employing the LSD test (Steel and Torrie 1980). rests of the bacterial isolates were gram negative (Table 1).

Phylogenetic analysis of nodule endophytic bacteria Nodulation assay

Total genomic DNA of potential bacterial strains MSP9 and All the nodule endophytic bacteria were initially tested for
MSP10 was isolated by the alkaline lysis method (Birnboim plant infectivity assay for nodulation. They were able to

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Table 1 Morphological characteristics of nodule endophytic bacteria Five bacterial isolates were able to produce IAA at
isolated from sweet pea nodule varying level, ranged from 0.86 to 16.16 lg ml-1. Isolate
Isolates Colony morphology Cell Gram MSP9 was most efficient in IAA production by producing
morphology reaction 16.16 lg ml-1 (Table 2). Bacterial isolates MSP2 and
MSP10 were found positive for nitrogenase activity in
MSP1 Round medium, smooth, creamy Small rods -ve
acetylene reduction assay by reducing 58.9 ± 3.4 and
MSP2 Round small, smooth, light Small rods -ve
yellow 348 ± 15.6 n mole C2H2 h-1 mg-1 protein, respectively.
MSP3 Small round, smooth, greenish Small rods -ve Phosphate solubilization data indicated that all the bacterial
yellow isolates were able to solubilize phosphate except MSP4.
MSP4 Large round, smooth, creamy Small rods ?ve Phosphate solubilization efficiency ranged from 5.57 to
MSP5 Round medium, wavy, dark Medium -ve 11.73 lg ml-1 (Table 2).
creamy rods Further, these bacterial isolates were checked for
MSP6 Small, wavy, creamy Small rods ?ve legume root surface colonization. All the isolates showed
MSP7 Small, smooth, yellow Small rods -ve very high colonization on root surface of their respective
MSP8 Round medium, wavy, dark Small rods -ve hosts, which was statistically different. MSP9 was found to
creamy be most efficient colonizing bacteria (Fig. 1). Similarly,
MSP9 Round medium, smooth, creamy Very small -ve equally efficient biofilm formation ability on the abiotic
rod
surface (PVC) was also observed. Bacterial isolates
MSP10 Round medium, smooth, light Small rod -ve established a significantly different biofilm. Bacterial iso-
brown
lates MSP9 and MSP10 showed high efficiency in biofilm
formation on abiotic surface (Fig. 2).
Table 2 Comparison of plant growth promoting attributes of pea
nodule endophytic bacterial isolate Molecular identification of potential bacterial strains
Isolate IAA ARA Phosphate
production (n mole C2H2 solubilization Two bacterial strains were selected for phylogenetic iden-
(lg ml-1) reduced h-1mg-1 (lg ml-1) tification on the basis of plant growth promoting potential.
protein) Phylogenetic identification was performed using 16S rRNA
gene sequencing technique. DNA fragments of *1,500 bp
MSP1 1.38 ± 0.27d – 6.8 ± 0.33c
were amplified using primers P1 and P6 set (Fig. 3a). The
MSP2 – 58.9 ± 3.4b 11.73 ± 0.26a
DNA fragments were cloned in vector pTZ57R/T. When
MSP3 _ – 9.3 ± 0.43b
the clones were restricted with EcoRI and BamHI, DNA
MSP4 3.39 ± 0.33c – –
fragment of *2,900, 900 and 600 bp were observed,
MSP5 – – 5.57 ± 0.36c
which revealed that 16S rRNA gene fragments had the site
MSP6 – – 9.33 ± 0.36b
of either EcoRI or BamHI (Fig. 3b). 16S rRNA gene
MSP7 1.64 ± 0.29d – 9.2 ± 0.36b
sequences of the potential strains were compared with
MSP8 4.13 ± 0.3b – 6.85 ± 0.12c
others in sequence databases using NCBI BLAST software.
MSP9 16.16 ± 0.27a – 11.73 ± 0.27a
MSP9 showed 99 % similarity to different species of genus
MSP10 0.86 ± 0.12e 348 ± 15.6a 6.83 ± 0.18c
Ochrobactrum, while bacterial strains MSP10 showed
LSD 0.05 0.425 26.9 1.37 99 % similarity with the Enterobacter. 16S rRNA gene
Means are followed by the standard error sequences of MSP9 and MSP10 were deposited at NCBI
Mean sharing same letter in column do not differ significantly GenBank under the accession numbers JF313266 and
JF313267, respectively.

reinfect roots, but did not develop root nodule on their host,
sweet pea. Moreover, PS-I and PS-II (Rhizobium legu- Discussion
minosarum bv. viciae), which was used as positive control,
developed nodules on pea plants. Plant growth promoting bacteria is a group of soil bacteria
which promote plant growth by developing a positive
Characterization for plant growth promoting attributes relationship with roots, endophytically or in the rhizo-
sphere. Endophytic bacteria reside inside plant tissue and
All the bacterial endophytes were tested for plant growth can be isolated from surface sterilized plant tissues
promoting properties including IAA production, nitrogen (Garbeva et al. 2001). Root nodules of legume accommo-
fixation and phosphate solubilization (Table 2). date a large number of non-nodulating plant growth

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80000
a
70000
b
60000 c

cfu/0.1 mg root .
cd cd
50000 de cde de
e
40000
f
30000

20000
10000
0
MSP1 MSP2 MSP3 MSP4 MSP5 MSP6 MSP7 MSP8 MSP9 MSP10
Isolates

Fig. 1 Colonization efficiency of nodule endophytic bacteria on pea roots. Each value is plotted as the mean ± SE (n = 3). Mean sharing same
letter in column do not differ significantly. LSD 0.05 = 7,699

0.8

0.7 a

0.6
Absorbance at 570nm

0.5 b

0.4

0.3

0.2 c cd cd
d cd
0.1 e
e e
0
MSP1 MSP2 MSP3 MSP4 MSP5 MSP6 MSP7 MSP8 MSP9 MSP10
Isolates

Fig. 2 Biofilm formation efficiency of nodule endophytic bacteria on abiotic surface (PVC). Each value is plotted as the mean ± SE (n = 16).
Mean sharing same letter in column do not differ significantly. LSD 0.05 = 0.0354

Fig. 3 a Amplified fragments Ladder Control MSP9 MSP10 Ladder Control MSP9 MSP10 GeneRuler 1kb
of 16S rRNA gene.
b Confirmation of 16S rRNA
gene cloning into vector
pTZ57R/T by restriction
analysis

a b

promoting bacteria along with rhizobia (Mishra et al. and identified as A. radiobacter in the clover plant. Agro-
2009). Occurrence of bacteria other than rhizobia in root bacterium spp. has also been reported to found in the
nodule was first reported by Beijerinck and Delden (1902) nodules of chick pea (Hameed et al. 2004). In this study,

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we isolated ten fast growing bacteria from the nodules of intercellular space between the cortex and endoderm of
pea. These fast growing nodule endophytic bacteria were roots (Gough et al. 1997). In case of legumes, it is specu-
further tested for nodulation assay and it is found they were lated that non-nodulating endophytic bacteria might get
able to reinfect roots, but were unable to nodulate pea entrapped into the pocket of root hair during the process of
plant. These results strengthen the literature on the occur- curling and finally enter the nodule primordial where it
rence of non-rhizobial bacteria in the root nodules (Taurian may multiply in number, but this area further need to be
et al. 2012). Such a type of non-rhizobial bacteria within studied.
nodule establish a synergistic interaction with the rhizobia Considering all the findings, this is the first report on the
and promote plant growth (Bansal 2009). occurrence of Ochrobactrum and Enterobacter spp. in the
Generally, endophytic bacteria have better plant growth nodules of pea plant, which showed enormous potential for
promotion abilities to a certain extent than bacteria plant growth promotion. Furthermore, it is suggested that
restricted to the rhizoplane and rhizosphere (Dong et al. these bacterial strains can be used as biofertilizers alone or
2003). Therefore, non-rhizobial endophytic bacteria were in combination with other plant growth promoting bacteria
isolated from nodules, which could have plant growth for legumes and non-legume crops after field evaluation.
promoting potential. IAA production, nitrogen fixation,
phosphate solubilization, biofilm formation and root colo-
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