Advances in Microbiology, 2012, 2, 368-374 Published Online September 2012 (
Some Characteristics of a Plant Growth Promoting
Enterobacter sp. Isolated from the Roots of Maize
Frank Ogbo*, Julius Okonkwo
Department of Applied Microbiology & Brewing, Nnamdi Azikiwe University, Awka, Nigeria
Email: *
Received July 12, 2012; revised August 12, 2012; accepted August 20, 2012
Some properties of an E nter obacter sp. isolated from the roots of maize are described. Isolation was carried out using
the semisolid enrichment culture technique and subsequent plating, both on nitrogen free biotin medium. Morphological,
biochemical and phylogenetic characterization using the MicroSeq 16S rDNA technique were employed in identi-
fication of isolate, which was revealed to be closest matched at 99.4% with Enterobacter asburiae. The isolate pos-
sessed properties of plant growth promoting bacteria. Thus, it produced indole-3-acetic, plant polymer hydrolyzing en-
zymes, pectinase and cellulase as well as ammonia in vitro. The isolate grew well in the presence of both 3% NaCl and
10 µg of streptomycin. In plate bioassays, isolate promoted the germination of both maize and rice seeds as well as root
and lateral root growth resulting in weight increases of seedlings over their controls. Experiments to quantify ability of
isolate to promote plant growth was performed using hydroponics solutions and as appropriate, an inoculum of the iso-
late. Pot experiments were also employed. Results from these studies showed that isolate enhanced nitrogen accumula-
tion and significantly (P < 0.05), improved the growth of maze seedlings over controls. Isolate has potential for utilize-
tion as inocula for sustainable production of cereals.
Keywords: Cereals; Plant Growth Promoting Enterobacter
1. Introduction
Plant growth promoting rhizobacteria (PGPR) achieve
their effect by diverse mechanisms as recently reviewed
by Lugtenberg and Kamilova [1]. The supply of nutrients
such as nitrogen is improved through various mecha-
nisms of biological nitrogen fixation (BNF), while phos-
phate and potassium are made available through solubi-
lization of their insoluble forms, often abundant in the
soil. PGPR may also provide plants with essential vita-
mins. Other mechanisms employed to promote plant
growth include plant growth regulating effects (phyto-
hormones), both positive and negative, induced systemic
resistance to microbial pathogens and siderophore pro-
duction aiding plant nutrition by chelation. These organ-
isms have also been reported to enhance plant growth
through miscellaneous beneficial effects such as osmotic
adjustment, stomatal regulation and modification of root
morphology, which may lead to enhanced uptake of
minerals and alteration of nitrogen accumulation and
metabolism [1].
Research on BNF, particularly the Rhizobium-legume
symbiosis has made significant progress. Biofertilizer
technologies for the increased agricultural production of
legumes based on these research findings are today con-
sidered essential ingredients for sustainable agriculture.
However, cereal crops like rice, maize and wheat remain
by far the world’s most important sources of food, both
for direct human consumption and indirectly, as inputs to
livestock production [2]. According to Charpentier and
Oldroyd [3], nitrogen fixing cereals would be the break-
through necessary to underpin sustainable food produc-
tion for 9 billion people, the projected population of the
world by 2050.
Research efforts employing molecular biology and
genetic manipulation techniques are ongoing to induct
nodular symbiosis between non-legumes on the one hand
and the rhizobia and other free-living atmospheric nitro-
gen fixers on the other. While this procedure appears to
be in the future, recommendable BNF technologies for
some cereals, such as rice have been achieved using
various rhizospheric bacteria, particularly members of
the genus Azospirillum. Several biofertilizer formulations,
for example, Mazospirflo-2TM (Soygro Ltd, South-Af-
rica), containing Azospirillum brasilense or combinations
of genera such as Azospirillum, Azotobacter, Pseudomo-
nas, Zoogloes as in BiopowerTM (Nuclear Institute for
Biotechnology and Genetic Engineering, Pakistan), are
being marketed successfully today. Nonetheless, the pro-
*Corresponding author.
opyright © 2012 SciRes. AiM
blem of low efficiency of these inoculants persists, re-
quiring often, supplementation with inorganic fertilizer.
As a consequence, the use of this class of microbial in-
oculants in the field has not been extensive, underscoring
the need for sustained search for more efficient organ-
We report here, some characteristics of an Enterobac-
ter sp., which promoted the germination and growth of
maize and rice seedlings under laboratory and green
house conditions. This organism has the potential for
direct exploitation or the contribution of genes for the
engineering of hybrids suitable for the formulation of
biofertilizers for cereals.
2. Materials and Methods
2.1. Isolation of Rhizosphere Bacteria
The roots of maize (Zea mays) plants in their vegetative
stage of growth (about two month-old), were harvested
from a farm on our campus at Awka, Nigeria (6.22N;
7.07E). Roots were returned to the laboratory in sterile
polyethylene bags and adherent soil removed by rinsing
in serial baths of tap water. The isolation of Azospirillum
sp. was the original purpose of this study and thus the
semisolid enrichment culture technique as compiled by
Bashan et al. [4], was employed with minor modifica-
tions and performed as follows. Washed roots were cut
into 3 cm segments and disinfected by soaking in 70 %
ethanol for 5 min, in 6.25 % sodium hypochlorite for 10
min, followed by several rinses in sterile distilled water.
Intact root pieces (0.5 to 1.0 cm) were then placed into
tubes of semisolid (0.05%) nitrogen free biotin medium
(NFb) and incubated without shaking for 5 days at 30˚C.
This medium was composed (g/l) of: DL-malic acid 5;
KOH 4; K2 HPO4 0.5; MgSO4·7H2O 0.2; CaCl2 0.02;
NaCl 0.1; FeSO4·7H2O 0.5; Agar 5 g and (mg/l) of:
NaMoO4·2H2O 2; MnSO4·H2O 10. The medium also
contained 2 ml of 0.5 % solution of bromothymol blue in
95% ethanol and had a final pH of 6.8.
White pellicles from tubes, which showed this charac-
teristic feature, were subcultured onto solid semi selec-
tive, Congo red-NFb medium for Azospirillum [5]. This
medium was similarly composed as the enrichment me-
dium, above but contained 2% agar and additionally,
0.05 g/l yeast extract and 15 ml/1 of 1:400 aqueous solu-
tion of Congo-red. The later was autoclaved separately
and added just before plates were poured.
Red to scarlet colonies suggestive of Azospirillum was
purified by repeated streaking on same medium. One
isolate, MR5, was selected for further studies on the basis
of its exceptional luxuriant growth on Congo red-NFb
medium when compared with the other isolates. This
isolate was stored on nutrient agar slopes at 4˚C during
the period of the study.
2.2. Morphological and Biochemical
Characterization of Selected Isolate
The shape, dimensions and motility of PGPR, in addition
to the range of carbohydrate sugars they utilize are im-
portant determinants of their ability to colonize plant
roots. Therefore, the colony and then, the cellular mor-
phology of pure cultures of the isolate were determined
by light microscopy. Some standard biochemical tests for
Gram negative bacterial genera including catalase, O/F
of D-glucose and hydrolysis of gelatin were performed.
The ability of the isolate to utilize various carbohydrates
such as the disaccharide, sucrose, the trisaccharide, raf-
finose, the pentose, xylose, the alcohol, mannitol and the
polysaccharide starch, were also determined by growing
isolate in minimal salts medium comprised, NH4H2PO4 1
g; KCl 0.2 g; MgSO4·7H2O 0.2 g; Agar 10 g, Distilled
water 1 L and 4 ml 0.2% solution of bromothymol blue.
The medium was supplemented with separately sterilized,
1% (wt/vol) of the appropriate carbon substrate.
2.3. Phylogenetic Characterization and
Identification of Isolate
The isolate was identified by the Bacterial 500bp Stan-
dard technique using the MicroSeq 16S rDNA kit at the
National Collections of Industrial and Marine Bacteria
Ltd, Ferguson Building, Craibstone Estate, Bucksburn,
Aberdeen. AB21 9YA (NCIMB). The 16SrDNA se-
quence analysis was carried out using the NCIMB Ltd.
internal work instructions; WI-NC-58 rev 6, 138 rev 4,
147 rev 13, 149 rev 8, 191 rev 2, 198 rev 0, 214 rev 1,
215 rev 3, 226 rev 0 and 253 rev 1. The resulting se-
quence data were aligned and compared with the 16S
rRNA sequence database of the NCIMB Microseq Data-
base and the public data base European Molecular Biol-
ogy Laboratory (EMBL) in Heidelberg, Germany.
2.4. Determination of the Ability of Isolate to
Promote Plant Growth
There are no efficient high-throughput assays systems
for the selection of superior strains of plant growth pro-
moting bacteria. A combination of bioassay methods
were employed for this purpose during this work. The
modified plate bioassay [6] for the ability to promote the
germination and growth of seedlings was done as fol-
lows. Maize seeds, Zea mays (T2BR Comp 2-W), kindly
provided by the International Institute for Tropical Ag-
riculture, Ibadan, Nigeria (IITA) and rice (FARO 44),
purchased locally were employed for this test. Seeds
were selected on the basis of their uniformity in weight
and surface sterilized with 95% ethanol and 2.5% so-
dium hypochlorite, then washed several times with ster-
ile distilled water before each test. Growth of the seed-
Copyright © 2012 SciRes. AiM
lings were quantified by measurement of root and shoot
Plant polymer hydrolyzing activities using cellulase
and pectinase were qualitatively determined using plate
assays. For the cellulase assay, NFb plates supplemented
with 0.25% carboxymethyl cellulose (CMC) were inocu-
lated with isolate. After incubating for 48 h at 30˚C, the
plates were overlaid with 1 mg ml–1 Congo red solution
for 30 min [7]. Congo red solution was then poured off
followed by washing the surface of the plate with 1 M
NaCl solution. Pectinase activity was determined by
growing isolates on yeast extract pectin medium, com-
posed g/L–1 distilled water of; Pectin 5.0; KH2PO4 4.0;
Na2HPO4 6.0; yeast extract 1.0 and agar 18.0. After in-
cubation at 30˚C for seven days, iodine-potassium iodide
solution (1.0 g iodine, 5.0 g potassium iodide and 330 ml
H2O) was added to detect clearance zones [8].
Indole-3-acetic acid (IAA) production was determined
by the method of Loper and Scroth [9], while ammonia
and hydrogen cyanide production were determined re-
spectively by methods described by Cappucino and Sher-
man [10] and Lock [11].
Isolate was screened for tolerance for 3% NaCl by
streaking two lines in a cross fashion across the surface
of nutrient agar plates in which NaCl concentration was
adjusted to 3%. Controls were set up on plates without
added salt. Streptomycin resistance was determined by
routine disk (10 µg) diffusion assay on nutrient agar.
To determine the ability of the isolate to enhance ac-
cumulation of nitrogen in p lanta, and to compare its effi-
ciency to promote plant growth against the NPK fertilizer,
the following experiment was set up using the com-
pletely randomized design. Maize seeds were grown in
hydroponics solutions, which lacked or contained the
mineral nutrients nitrogen (N), phosphorus (P) and po-
tassium (K) and as appropriate an inoculum of the test
organism composed of 107 cfu/ml washed cells. Treat-
ments were as follows: T0. Distilled water (DW), T1. DW
+ Inoculum, T2. DW + P + K, T3. DW + N + P + K, and
T4. DW + Inoculum + P + K. All treatments were repli-
cated five times. Solutions containing P + K were com-
posed of 125 mg/L of K2HPO4 (250 ppm, K and 100 ppm,
P), while the N + P + K solutions contained additionally,
391 mg/L NH4NO3 (230 ppm, N).
Maize seeds, selected and sterilized as has been de-
scribed earlier, were allowed to germinate and grow on
the hydroponics solutions for a period of 14 days. The
plants were carefully harvested and roots and shoots
were separated. These were weighed separately, and re-
sults obtained were subjected to Analyses of Variance
(ANOVA). Shoots from the DW and the DW + Inoculum
treatments were subsequently dried to constant weight at
50˚C - 60˚C in an air-circulating oven and their nitro-
gen contents per 100 mg of plant material determined by
Kjeldahl digestion [12].
2.5. Effect of Inoculation of Isolate on Growth of
Maize Seedlings in Pot Experiments
Soil for this experiment was sandy loam in nature with a
pH of 4.5 and contained 1.112 g/Kg total N. About 10 kg
of soil was thoroughly mixed and distributed in 1 Kg
portions into polyethylene bags. The variety of maize
already described was used as test plant. The experiment-
tal design was the completely randomized design with
five replicates. Treatments were: T0 control (No inocula-
tion) and T1 (Seed inoculated by soaking in culture of
isolate containing about 107 cfu/ml washed cells for one
hour. The plants were placed in a green house and wa-
tered regularly for 45 days. Plants were carefully up
rooted at the end of this period, washed clean of sand
particles and dried at 50˚C for 72 h. The weights of their
shoots and roots were recorded. The results were sub-
jected to analysis of variance (ANOVA) and the Tukey
HSD (0.05) test.
3. Results
3.1. Morphological and Biochemical
Characteristics of Isolate
Colonies on Congo red-NFb medium were luxuriantly
growing, 3 - 5 mm diameter, pink colonies with a raised
elevation after 72 h incubation at 30˚C. Edges of the
colonies usually became irregular with time. On nutrient
agar, 30˚C/48 h, the isolate grew as 3 - 4 mm mucoid
colonies, with a convex elevation and entire edges. On
prolonged incubation the organism swarmed actively,
covering the surface of the agar plate. Microscopy re-
vealed isolate as Gram negative motile short rods. Bio-
chemical characteristics of the isolate are given in Table 1.
Table 1. Some biochemical characteristics of isolate.
Test Result
Catalase +
Oxidase -
Citrate utilization +
Fermentation/oxidation of glucose +
Sucrose utilization +
Raffinose +
Xylose +
Mannitol +
Starch hydrolysis +
Gelatinase -
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3.2. Phylogenetic Characteristics and
Contrary to expectation, phylogenetic characterization of
isolate MR5 revealed that it was not an Azospirillum. The
“10 top hits” from the comparison of its 16S rRNA se-
quence data using the NCIMB Microseq Database is
shown in Table 2. This revealed that isolate MR5 or
NCOO1927-MR5 as designated by the NCIMB was most
closely matched with Enterobacter asburiae (99.4%).
This isolate also matched Enterobacter sp. and Entero-
bacter cloacae, ATTC 13047 at 98.97 %, respectively on
this database. However, comparison of this sequence
with the EMBL database produced a 100 % match with
both Enterobacter sp. and the type strain Enterobacter
cloacae, ATTC 13047.
Figure 1 shows the phylogenetic relatedness of En-
terobacter sp.-NCOO1927-MR5 to some type strains and
other similar bacteria constructed using the neighbor
joining technique and the NCIMB database. This figure
suggests a closer relationship between this isolate and
Enterobacter asburiae than with Enterobacter cloacae.
3.3. Results of Tests for Ability of Isolate to
Promote Plant Growth
The isolate promoted the germination of both maize and
rice seeds in the plate bioassays. Inoculated maize show-
ed 100% germination, compared to 80% in the controls,
while inoculation improved germination of rice seeds
from 70% in the controls to 90% in the tests. Inoculation
with our isolate promoted root growth, including lateral
root development of tested seedlings, resulting in weight
increases over their controls at the average rates of 43%
for maize and 37% for rice. Similar increases were ob-
served for shoot weights, 35% in maize and 40% in rice.
The isolate produced the plant polymer hydrolyzing
enzymes, cellulase and pectinase and 20 µg/ml of indole
acetic acid. It also produced ammonia, but failed to pro-
duce hydrogen cyanide. Isolate also grew well in nutrient
agar containing 3% NaCl and showed resistance to 10 µg
Shoots of maize seedlings grown in the presence of
inoculum showed a higher N accumulation than shoots
grown without it (Table 3). Furthermore, growth of maize
seedlings, measured as means of weights of shoots and
roots, were comparable in the DW + N + P + K and the
DW + Inoculum + P + K treatments but differed signify-
cantly from seedlings grown in the DW and the DW + P
+ K treatments at P < 0.05.
3.4. Effect of Inoculation of Isolate on Growth of
Maize Seedlings in Pot Experiments
Means of shoot weights for maize seedlings, 0.56 ± 0.10
for the control and 0.818 ± 0.08 for test were signifi-
cantly different at P < 0.05. Even though mean root
Table 2. Ten closest matches with Enterobacter sp.-NCOO1927-MR5 (MicroSeq 500).
Sequence Name % Match Sequence Name % Match
Enterobacter asburiae 99.4 Enterobacter cancerogenus 98.92
Enterobacter hormaechei 99.27 Enterobacter kobei 98.92
Enterobacter pyrinus 99.03 Leclercia adecarboxylata 98.83
Enterobacter cloacae cloacae 98.97 Klebsiella pneumoniae pneumoniae 98.63
Enterobacter cloacae dissolvens 98.97 Citrobacter youngae 98.6
Specimen: NC001927_MR5
Leclercia adecarboxylata
Citrobacter youngae
Enterobacter hormaechei
Enterobacter pyrinus
Enterobacter asburiae
Klebsiella pneumoniae pneumoniae A
Enterobacter cancergenus
Enterobacter cloacae dissolvens ATCC = 23373
Enterobacter cloacae cloacae ATCC = 13047
Enterobacter kobei
Figure 1. Phylogenetic tree showing relatedness of Enterobacter sp.-NCOO1927-MR5 to similar bacteria.
Table 3. Nitrogen composition and mean weights of shoots and roots of maize seedlings grown in hydroponics solutions con-
taining different combinations of mineral elements and inoculum of isolate.
Treatments DW DW + Inoculum DW + P + K DW + N + P + K DW + Inoculum + P + K
N Composition of shoot 0.49% 0.55% - - -
Mean root weight 0.068a ± 0.05 0.208b ± 0.07 0.144a ± 0.06 0.28b ± 0.07 0.248b ± 0.09
Mean shoot weight 0.23 a ± 0.05 0.454a ± 0.04 0.342a ± 0.12 0.668b ± 0.34 0.608b ± 0.08
weight of control seedlings, 1.03 ± 0.09 was not statistic-
cally different from that of test roots, 1.34 ± 0.27, there
was nevertheless, an increase of about 30.1% in growth
of root material.
4. Discussion
The definitive identification of Enterobacter sp.-NCOO-
1927- MR5 reported by the NCIMB was Enterobacter
sp. This is because the 16SrDNA sequence of this isolate
matched different species on the different databases,
NCIMB and the public EMBL consulted. In terms of
DNA-relatedness, Grimont and Grimont [13] have de-
scribed E. cloacae as heterogeneous with many groups.
These authors in fact stated that E. asburiae belongs to
one of the E. cloacae groups. Further studies are needed
to elucidate the identity of this isolate and indeed, the
ambiguities in the taxonomy of the Enterobacter. For the
rest of this report, this isolate is simply designated as
Enterobacter sp.-NCOO1927-MR5.
Enterobacter sp.-NCOO1927-MR5, shared many mor-
phological and biochemical characteristics with earlier
isolates of E. asburiae [14,15]. Notable among these
characteristics are their motility, production of, cellulase
and pectinase enzymes as well as the ability to utilize a
wide range of sugars. Flagellar motility in bacteria is an
important factor in the colonization of plant roots, espe-
cially their interiors [16]. Cellulase and pectinase en-
zymes act as virulence factors for pathogenic bacteria of
plants and are believed to be involved in the invasion of
host plants by PGPR, as reported for E. asburiae JM22
[17]. The possession of these characteristics may have
contributed to the ability of this isolate in colonizing
roots of tested seedlings.
Plants are known to exude a wide range of substances
[18]. The ability of Enterobacter sp.-NCOO1927-MR5
to utilize a wide range of sugars, may contribute to the
success of this species in adaptation to roots of different
plants. Generally, the Enterobacter genus is one of the
most common genera of bacteria isolated as plant en-
dorhizosphere bacteria. They have been found as en-
dorhizosphere bacteria in maize, rice, cotton, cucumber,
common bean, broccoli and sweet potato, to list but a
few [19-22].
The efficiency of PGPR is critically dependent, among
others, on their ability to tolerate saline conditions pre-
valent in many soils, as well as resist numerous antibiot-
ics produced by competing flora. As has been variously
reported [21,22], Enterobacter sp.-NCOO1927-MR5 was
shown in this study to tolerate 3% NaCl as well as resist
inhibition by the antibiotic, streptomycin. The production
of ammonia is frequently reported for PGPR, a process
most probably resulting from the deamination (ammoni-
fication) of the amino acids present in the peptone used
for this assay. It has been suggested that ammonia may
have a role in antagonism against competing flora, par-
ticularly the fungi [22,23].
Some of the Enterobacter asburiae isolated earlier
were characterized as human pathogens [24] and there
had been expression of concerns for safety of human
health. However, the majority of strains reported recently
has been isolated from environmental sources and have
been studied for their various beneficial activities. Such
activities include the promotion of plant growth [21,25,26],
biocontrol of plant diseases [22,23] and even the conver-
sion of carbohydrate compounds in acid hydrolysates of
hemicellulose into ethanol and other fermentation prod-
ucts [15]. These reports have raised the profile of E. as-
buriae as an industrial microorganism.
Enterobacter sp.-NCOO1927-MR5 demonstrated very
good potentials for the promotion of plant growth. Infec-
tion and promotion of the growth of both maize and rice
seeds in the plate bioassays indicate cross-fection abili-
ties. Lack of host specificity is a good attribute for mi-
crobial inocula, which may thus be used for the cultiva-
tion of a wide range of crops. Zakria et al. [21] earlier
reported that Enterobacter sp. strain 35 isolated from
sugarcane successfully colonized and promoted growth
of Brassica oleracea (broccoli), a dicot. The production
of high levels of the plant growth hormone (auxin), IAA
in vitro is noteworthy. Recent findings have revealed that
auxin biosynthesis plays essential roles in many devel-
opmental processes in plants including gametogenesis,
embryogenesis, seedling growth, vascular patterning, and
flower development [27]. IAA production is a major tool
employed by PGPR.
Nitrogen is the most critical of the three major ele-
ments required for plant growth. The lack of a significant
difference in growth of maize seedlings in the hydropon-
ics solutions containing DW and DW + P + K (Table 3)
Copyright © 2012 SciRes. AiM
is in agreement with this claim. The relatively higher
concentration (per 100 mg of dry plant material) of nitro-
gen in maize seedlings grown with inoculum compared
with those grown without it indicate that Enterobacter
sp.-NCOO1927-MR5 enhanced nitrogen accumulation in
maize as been reported earlier for E. asburiae [26]. Lack
of facilities did not permit the demonstration of nitro-
genase activity or the presence of nif genes in this isolate.
It is noteworthy however, that E. asburiae, is reported to
lack the nif genes [20,26] but may contribute nitrogen to
its host plant by enhancing its uptake. E. cloacae on the
other hand, is reported to possess these genes [28,29].
Enhancement of nitrogen uptake or its fixation, as well as
other mechanisms discussed earlier would probably ac-
count for the significant difference, at P < 0.05 between
weights of inoculated and uninoculated maize seedlings
during this study (Table 3). Furthermore, the lack of a
significant difference in growth of maize seedlings in
hydroponics solutions containing DW + N + P + K and
DW + Inoculum + P + K suggest comparable efficiency
in plant-growth-promoting activity of our isolate with
nitrogen chemical fertilizers.
Improvement in growth rate of maize in pot experi-
ments following inoculation with our isolate is compara-
ble with earlier reports. Morales-García et al. [22] re-
ported that maize plants inoculated with Enterobacter sp.
UAPS03001 showed statistically significant greater bio-
mass than the controls under environmental chamber
conditions. In field experiments, this species was re-
ported to have also, significantly improved total kernel
biomass. Zakria et al. [21], have shown that under glass-
house conditions, Brassica oleracea (broccoli), a dicot
plant inoculated with Enterobacter sp. strain 35 had a
significantly greater fresh weight than uninoculated
plants. Rogers et al. [26] obtained similar results under
field conditions with hybrid poplar (a short rotation en-
ergy crop), after plants were inoculated with Enterobac-
5. Conclusion
Results obtained during this study show that Enterobac-
ter sp.-NCOO1927-MR5 has abilities to promote growth
of maize. Mechanisms employed for this purpose in-
cluded enhancement of accumulation of nitrogen and the
production of IAA among others. This organism there-
fore has the potential to promote growth of cereals. It is
noteworthy that members of the genus Enterobacter are
currently in use for biocontrol purposes. Isolation or en-
gineering of species combining these two characteristics
will enhance the sustainable production of cereals. The
taxonomy of the Enterobacter desires further attention to
enable a more precise evaluation of the industrial and
agricultural potentials of this genus.
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