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Fundamental and Applied Agriculture
Vol. 6(1), pp. 95–106: 2021
doi: 10.5455/faa.46616
AGRICULTURE |ORIGINAL ARTIC LE
Isolation and characterization of plant associated rhizobacteria
for plant growth promoting traits
Mst Julekha Khatun 1, Atiqur Rahman 2*, Quazi Forhad Quadir 2, Md Shafiul Islam
Rion 1, Md Zakir Hossen 2
1Department of Agricultural Chemistry, Bangladesh Agricultural University, Mymensingh, Bangladesh 2202
2Laboratory of Plant Nutrition and Environmental Chemistry, Department of Agricultural Chemistry,
Bangladesh Agricultural University, Mymensingh, Bangladesh 2202
ARTI CL E INFORMATI ON
Article History
Submitted: 25 Jan 2020
Accepted: 27 Mar 2021
First online: 29 Mar 2021
Academic Editor
Sirinapa Chungopast
agrsrnp@ku.ac.th
*Corresponding Author
Atiqur Rahman
atiqur.ac@bau.edu.bd
ABSTRACT
The use of plant growth promoting rhizobacteria (PGPR) in sustainable and
eco-friendly management of plant growth promoting is gaining importance
over the previous decades around the world. In the current research work,
the isolation of the rhizobacteria were done using nutrient agar media fol-
lowing standard protocol for isolation of bacteria. We have isolated and
characterized 32 rhizobacterial isolates from five different plant species and
subjected to N2-fixation, phosphorus solubilization and indole-3-acetic acid
assay to identify potential PGPR. All the 32 rhizobacterial isolates showed at
least one of the three major functionalities; viz. phosphorus solubilization, in-
dole acetic acid production and N
2
fixation; considered for selection of PGPR
when tested in vitro. Among the 32 isolates, 15 produced clear halo zones
surrounding their colonies indicating phosphate solubilization with variable
intensities. Among the fifteen, six bacterial isolates having high phosphate
solubilization index (PSI) proved to be efficient phosphorus solubilizer in
liquid medium. The bacterial isolate MQ2 solubilized maximum (0.697
µ
g
mL
−1
) phosphorus in liquid medium, followed by MQ3 and MQ1. Ten of
the bacterial isolates were able to synthesize indole-3-acetic acid (IAA) in
L-tryptophan supplemented media at varying capacity. The isolate OSn8 pro-
duced highest amount (6.204
µ
g mL
−1
) of IAA followed by MQ5 and OSbr6,
while the lowest amount of IAA (1.268
µ
g mL
−1
) was produced by MQ1. All
the isolated bacteria were tested positive for putative N
2
-fixing ability with
variation among the isolates as indicated by their growth in N
2
-free medium.
Considering the all three tested functionalities, the isolate MQ1 proved to be
the best candidate as potential biofertilizer development. A consortium of
isolated rhizobacteria comprising the best isolates from each category could
be constructed to provide the best benefit to crops for maintaining yield and
quality while decreasing agrochemical inputs.
Keywords:
Plant growth promoting rhizobacteria (PGPR), Phosphorus solu-
bilization, Indole-3-acetic acid, nitrogen fixation
Cite this article:
Khatun MJ, Rahman A, Quadir QF, Rion MSI, Hossen MZ. 2021. Isolation and
characterization of plant associated rhizobacteria for plant growth promoting traits. Fundamental
and Applied Agriculture 6(1): 95–106. doi: 10.5455/faa.46616
1 Introduction
Modern agriculture heavily dependent on agrochemi-
cal including fertilizers and pesticides to manage soil
fertility and pest control, allowing a boast in cropping
intensity and crop production to meet the increasing
demand of food products. But the injudicious use
of these agrochemicals for crop production has be-
come a matter of concern as they decrease soil fertil-
ity and dismantle the environmental integrity (Jilani
Khatun et al. Fundam Appl Agric 6(1): 95–106, 2021 96
et al.,2007;Rahman et al.,2015). Ecological imbalance
fueled by the over-use of agrochemicals also leaves
harmful contaminants and residues in soil water sys-
tems which in turn destroys or destabilizes the soil mi-
crobiome responsible for nutrient mineralization and
recycling (Rahman et al.,2015). Over the years, the
scientists are trying to reduce dependency on agro-
chemicals and looking for an effective alternative/
amendment for crop production. The use of soil in-
habiting microorganisms in crop production is being
considered best options by growing numbers of plant
scientists (Glick,2012;Lwin et al.,2012;Majeed et al.,
2015). Plant growth promoting rhizobacteria (PGPR)
are diverse array of microorganisms which selectively
colonize in the rhizosphere and stimulate the growth
and development of plants (Kloepper,1981;Bashan
et al.,1993). The PGPR enhance the growth and de-
velopment of plants by multiple direct and indirect
mechanisms (Rahman et al.,2010;Backer et al.,2018).
The direct mechanisms by which PGPR imparts their
growth promoting functionalities include production
of phytohormones, nitrogen (N
2
) fixation, mineral
phosphate and zinc solubilization, siderophore pro-
duction etc., while production of antibiotics, extracel-
lular polymeric substances (EPS), induced systemic
resistance and production of defense related enzymes,
competition for space and nutrient are considered as
the principal indirect mechanisms of plant growth
promotion by PGPR (Glick,2012;Rahman et al.,2015;
Majeed et al.,2015;Asha et al.,2015).
Indole-3-acetic acid (IAA) is considered as the
one single molecule in plants which has profound
role on plant growth and development (Patten and
Glick,1996) and considered as one of the direct mech-
anisms of plant growth promoting by rhizospheric mi-
croorganisms. IAA producing rhizobacteria releases
enough auxin in the soil sufficient for developmental
processes of plants and enable plants to fight against
biotic and abiotic stresses (Spaepen et al.,2007;Glick,
2012;Costa-Gutierrez et al.,2020). L-tryptophan de-
pendent IAA production is considered as the main
pathway for bacterial IAA production in rhizosphere
and it is now conformed that many soil microbio-
tas have the capacity to convert minute quantities of
plant-derived L-tryptophan in the rhizosphere to IAA
(Zhao,2010;Rahman et al.,2010). Thus, plant micro-
biologists consider IAA production by rhizobacteria
as one of the criteria for selecting PGPR.
Phosphorus (P) is an important essential plant nu-
trient, deficiency of which limits plant growth and
crop yield seriously. Only a small portion of applied
phosphorus containing fertilizers are up taken by
plants, while the remainder is fixed in most soils.
The PGPR plays an important role in mobilizing the
fixed pool of inorganic phosphates along with organic
phosphate pool (Gaind and Gaur,1989;Khan et al.,
2010;Taher et al.,2019) thereby reducing amount of
phosphatic fertilizer for crop production. PGPR em-
ploys a number of mechanisms including produc-
tion of organic acids and phosphatase enzymes to
solubilize phosphate. Besides IAA production and
phosphate solubilization, N
2
-fixing ability of PGPR is
also considered as one of the important mechanisms
of plant growth promotion and used as criteria for
PGPR selection. N
2
-fixation by rhizobacteria in legu-
minous plants including lentil, soybean etc. are well-
documented and credited to symbiotic N
2
-fixing bac-
teria (Rashid et al.,2009;Islam et al.,2007,2013). N
2
fixation by non-symbiotic rhizobacteria in crops other
than legume is gaining attention (Franche et al.,2008;
Islam et al.,2013;Díez-Méndez and Menéndez,2020).
In Bangladesh, the use of PGPR in crop production is
very limited, partly due to the unavailability of poten-
tial PGPR based biofertilizer to the farmers and also
lack of farmer’s willingness to reduce the use of con-
ventional fertilizers. Therefore, the current research
was designed to isolate rhizobacteria from different
plant sources and their functional characterization
as potential agriculturally important microorganism
that can be used for biofertilizer development.
2 Materials and Methods
Plant samples were collected from different areas of
Agronomy Field Laboratory, and undisturbed cam-
pus soils of Bangladesh Agricultural University, My-
mensingh, Bangladesh. A total of five (5) plant
species comprising eight (8) samples (Table 1) includ-
ing their roots and rhizospheric soil were collected.
After removing extra soil by vigorous shaking, plant
roots were cut off using surface sterilized scissor and
kept in labeled sterile test tubes containing 10 mL of
sterilized distilled water and immediately brought to
the laboratory for isolation of rhizobacteria.
2.1 Isolation of rhizobacteria
The collected plant samples were shaken vigorously
in test tubes for few minutes to mix well. A dilution
series of up to 10
−3
were prepared for each sample
with sterilized distilled water to facilitate bacteria
isolation. Nutrient broth agar (NBA) medium (pH
7.0) was used to isolate the bacteria and prepared ac-
cording to the manufacturer’s instruction (Asha et al.,
2015;Glick,2012). The 10 mL of bacterial suspension
were inoculated into the medium with the help of a
glass spreader and plates were incubated in microbial
incubator for 24 hours at 28
±
2
°C
(Asha et al.,2015).
Then the bacterial colonies were picked with sterile
toothpick based on size, shape and color and repeat-
edly inoculated on NBA media until obtaining a pure
culture of bacterial isolate. Finally, pure colonies of
bacterial isolates were maintained on NBA plates for
regular use and preserved in 30% glycerol for long
time storage at low temperature refrigerator (
−
20
°C
).
Khatun et al. Fundam Appl Agric 6(1): 95–106, 2021 97
Table 1. Plant species collected for isolation of rhizobacteria and location of sampling
Local name Scientific name N†Sampling location
Rice Oryza sativa 3 Bangladesh Agricultural University
Shama Echinochloa crusgalli 1 (24°43010.000N, 90°25039.500E)
Soybean Glycine max 1
Fern Pteris spp. 2
Sushni Shak Marsilea quadrifolia 1
†Number of samples
2.2 Screening of IAA producing bacteria
Modified Winogradsky’s mineral solution was used
as a media for screening of IAA producing rhizobacte-
ria and the media was prepared as described in Rah-
man et al. (2010). The medium was supplemented
with 100 mg L
−1
L-tryptophane and the pH of the
solution was adjusted to 6.0-6.2 with 0.1M HCl and
0.1M NaOH. 30 mL of liquid medium were inocu-
lated with a loopful of overnight grown bacteria and
incubated at room temperature in a horizontal shaker
(JSOS-500 JSR, Korea) at 120 rpm for 72 hours under
dark condition. After 3 days, the culture media were
centrifuged at 10,000 rpm for 10 minutes to obtain
cell free supernatant. IAA production was qualita-
tively and quantitatively determined by Salkowski
reagent method (Rahman et al.,2010). After centrifu-
gation, the supernatant was decanted and pH was
adjusted to 2.5 to 3.0 with 2 M HCl. Then 2 mL of
supernatant and 2 mL of Salkowski’s reagent (2% of
0.5 M FeCl
3
solution in 35% of HClO
4
) were taken in
the test tube and kept in dark condition for 30 min-
utes. Development of pink to reddish color was taken
as the indication of IAA production. Quantitative
determination of IAA was done by the colorimetric
method using UV/VIS spectrophotometer at 535 nm
wavelength (Lwin et al.,2012). A calibration curve
was prepared using standard solution of pure IAA.
2.3 Screening of PSB
Screening of PSB was done using Pikovskaya’s agar
medium (Pikovskaya,1948). The medium was pre-
pared according to Asha et al. (2015) with adjusted
pH at 7.0 before sterilization. Bacterial isolates were
tested in triplicates by plate assay for observing min-
eral phosphate solubilization activities. Each isolate
was inoculated in Pikovskaya’s agar medium contain-
ing tricalcium phosphate and incubated at 28
±
2
°C
for 6 days. A clear halo zone around the bacterial
colony was considered as the indication of mineral
phosphate solubilization (de Freitas et al.,1997). The
diameter of the colony and diameter of the clear halo
zone were measured with scale after 6 days of inoc-
ulation. Finally, phosphate solubilizing index (PSI)
was calculated by method as suggested by Premono
et al. (1996) (Fig. 1).
2.4 Determination of P solubilized by po-
tential rhizobacteria
Quantification of phosphorus solubilized by poten-
tial rhizobacteria was done using Pikovskaya’s broth
medium. The bacteria used in this assay were selected
on the basis of PSI determined in previous assay. The
6 selected bacteria were inoculated into Pikovskaya’s
broth media containing tricalcium phosphate with
sterile inoculating loop and incubated at room tem-
perature for 6 days on horizontal shaker (JSOS-500
JSR, Korea) at 120 rpm. After 6 days, the pH of the me-
dia was recorded using pH meter. Bacterial cultures
were then centrifuged in a falcon tube at 6000 rpm
for 10 minutes and the cell-free supernatant were col-
lected in glass bottle for determination of phosphate
content. Determination of phosphate solubilization
content from the liquid media was done by devel-
oping phosphomolybdate blue complex with stan-
nous chloride (SnCl
2
.2H
2
O). To form molybdophos-
phoric blue complex, SnCl
2
.2H
2
O was used as a re-
ducing agent. Exactly 2 mL of cell free supernatant
was taken in 100 mL volumetric flask. Then sulpho-
molybdic acid and stannous chloride solution were
added 4 mL and 6 drops, respectively. The volume
of the solution was made up to the mark with dis-
tilled water and shaken thoroughly. After shaking,
the solution allowed to stand for 5 minutes for color
development. Finally, a spectrophotometer (Model-
T60, PG Instruments, UK) was used for measuring the
intensity of blue color (absorbance) at 660 nm wave
length along with standard series solutions (Schroth
and Hancock,1982). Finally, the quantity of soluble
phosphate was calculated by the regression equation
of standard curve (Kumar et al.,2012). The values of
phosphate solubilization was expressed in µg mL−1.
2.5 Determination of total titratable acid-
ity of PSB grown liquid medium
For determining titratable acidity, 10 mL of cell-free
supernatant was taken in volumetric flask. Here, 2
drops of the phenolphthalein indicator were added.
This content of the volumetric flask was titrated
against standard 0.1 M sodium hydroxide (NaOH).
During the addition of 0.1 M NaOH, the flask was
Khatun et al. Fundam Appl Agric 6(1): 95–106, 2021 98
Figure 1. Equation for calculating phosphate solubilization index (Premono et al.,1996)
shaken thoroughly. The initial and final readings of
burette were noted down for observing the differ-
ences and calculated the volume of NaOH used. The
percentage of acidity in the PSB grown liquid media
was calculated on the basis of the following relation
(Sadler and Murphy,2010).
W=M×V×192.43
3(1)
where,
W
= weight of acid (g),
M
= molarity of NaOH
used (0.1), and V= volume of NaOH used (L).
TA (%) =W×100
Ws(2)
where,
TA
= % of total acidity, and
Ws
= weight of
sample (g).
2.6 Screening of N2-fixing bacteria
The isolates were grown in modified Winogradsky’s
N-free agar medium (Winogradsky’s medium with-
out tryptophan and yeast extract) to study N
2
-fixing
ability of the bacteria (Hashidoko et al.,2002). Then
the culture plates were kept in microbial incubator
at 28
°C
for 48 hours. Growth of bacterial colonies
on the N-free media were the indication of N2-fixing
ability of rhizobacteria.
3 Results
3.1 Isolated rhizobacteria
A total of Thirty-Two bacterial strains were isolated
from five species of plants depending on morpholog-
ical characteristics and each of them were subjected
to gram reaction and catalase test. Code names were
given to the bacterial strains according to their ori-
gin on the basis of morphological characters (Table 2).
The morphological features of the isolates along with
the results of gram reaction and catalase test are given
in Table 2. Most of the isolated bacteria were whitish
and cream in color with few producing yellow and
red pigments. Gram reaction test revealed that 18 of
the isolated bacteria were Gram positive and the rest
14 were gram negative. None of the isolated bacteria
were catalase positive.
3.2 IAA production
The ability of the isolates to produce IAA were tested
using modified Winogradsky mineral media supple-
mented with L-tryptophan. About 34.71% of the iso-
lated bacteria (10 out of 32) were able to produce IAA
in liquid media in varying quantities as indicated by
pink to reddish pink color development when treated
with Salkowsky’s reagent, while 64.29% failed to do
so (Fig. 2A). Four bacterial isolates assumed to be
strong IAA producer; three of which were isolated
from rice rhizosphere [OS29(3), OSbr(6) and OSn(8)]
samples and one, MQ5 was isolated from Sushni Shak.
The other 6 of the IAA isolates were slight to medium
IAA producer. The rest 18 bacterial isolates produced
no color which was the indication of no IAA pro-
duction (Table 2 and Fig. 2B). The quantity of IAA
produced by bacterial isolates were also determined
using the liquid medium. The quantity of IAA pro-
duced by the rhizobacterial isolates ranged from 1.268
µ
g mL
−1
to 6.204
µ
g mL
−1
in Salkowski reagent posi-
tive isolates. Highest quantity of IAA (6.204
µ
g mL
−1
)
was produced by OSn8 isolated from rice rhizosphere
followed by MQ5 (5.643
µ
g mL
−1
), while the lowest
(1.268
µ
g mL
−1
) was produced strain MQ1 isolated
from Sushni Shak (Table 3).
3.3 Isolates of PSB
All the isolated rhizobacteria were subjected to tri-
calcium phosphate amended medium to assess their
phosphate solubilization capacity. The results of plate
assay revealed that about 46.87% of the rhizobacte-
ria (15 out 32 isolates) were able to produce clear
halo zones surrounding their (Fig. 3B) colonies in-
dicating their ability to scavenge phosphorus from
unavailable sources. While 53.13% of bacteria proved
Khatun et al. Fundam Appl Agric 6(1): 95–106, 2021 99
Table 2.
List of rhizobacteria isolated from different plant sources, their morphological characters and results of
gram reaction
Source Bacteria Colony color Elevation Shape Gram reaction
Oryza sativa (BRRIdhan 29) OS29(1) Whitish Non-raised Round (−)ve
OS29(2) Whitish Non-raised Round (−)ve
OS29(3) Yellow Raised Round (−)ve
Oryza sativa (Bashi Raj) OSbr4 Whitish Raised Round (+)ve
OSbr5 Cream Raised Round (−)ve
OSbr6 Cream Non-raised Irregular (−)ve
OSn7 Whitish Raised Round (+)ve
OSn8 Cream raised Round (−)ve
Echinochloa crusgalli EC1 Dark yellow Non-raised Irregular (+)ve
EC2 Whitish Non-raised Round (+)ve
EC3 Yellow Raised Irregular (−)ve
EC4 Whitish Non-raised Round (+)ve
EC5 Yellow Raised Round (+)ve
ECL1 Cream Raised Round (+)ve
Pteris spp. Fr1 Whitish Non-raised Round (−)ve
Fr2 Whitish Non-raised Irregular (+)ve
Fr3 Cream Non-raised Round (+)ve
Fr4 Whitish Non-raised Round (+)ve
Fr5 Cream Non-raised Irregular (+)ve
Fr6 Cream Raised Irregular (+)ve
Fr7 Cream Non-raised Irregular (+)ve
Glycine max GM1 Whitish Non-raised Round (+)ve
GM2 Cream Non-raised Round (+)ve
Marsilea quadrifolia MQ1 Reddish pink Raised Round (−)ve
MQ2 Cream Non-raised Round (−)ve
MQ3 Cream Raised Round (+)ve
MQ4 Cream Non-raised Irregular (+)ve
MQ5 Cream Raised Round (−)ve
MQ6 Whitish Raised Irregular (−)ve
MQL7 Whitish Raised Round (−)ve
MQL8 Whitish Raised Irregular (−)ve
MQL9 Cream Non-raised Irregular (+)ve
Khatun et al. Fundam Appl Agric 6(1): 95–106, 2021 100
Table 3. Qualitative and quantitative assay for IAA production by rhizobacterial isolates from different plant
sources
Bacteria Visual color Intensity Quantity of IAA (µg/mL)
OS29(1) Colorless − −
OS29(2) Colorless − −
OS29(3) Reddish pink +++ 2.93±0.58
OSbr4 Colorless − −
OSbr5 Colorless − −
OSbr6 Reddish pink +++ 4.55±1.21
OSn7 Light pink ++ 1.85±0.34
OSn8 Reddish pink +++ 6.20±1.89
EC1 Light pink +1.44±0.51
EC2 Light pink +1.30±0.38
EC3 Colorless − −
EC4 Colorless − −
EC5 Colorless − −
ECL1 Colorless − −
Fr1 Colorless − −
Fr2 Colorless − −
Fr3 Colorless − −
Fr4 Colorless − −
Fr5 Colorless − −
Fr6 Light pink +1.67±0.44
Fr7 Colorless − −
GM1 Light pink +1.47±0.48
GM2 Colorless − −
MQ1 Pink ++ 1.27±0.21
MQ2 Colorless − −
MQ3 Colorless − −
MQ4 Colorless − −
MQ5 Reddish pink +++ 5.64±2.12
MQ6 Colorless − −
MQL7 Colorless − −
MQL8 Colorless − −
MQL9 Colorless − −
Development of pink to reddish pink color indicates IAA biosynthesis from L-tryptophan. The sign (
−
) de-
notes IAA non-producer isolate. The sign (
+
) denotes the intensity of IAA biosynthesis by the rhizobacteria;
+++strong producer, ++ medium producer and +low IAA producer isolate.
Khatun et al. Fundam Appl Agric 6(1): 95–106, 2021 101
Figure 2.
IAA biosynthesis from l-tryptophan by bacteria isolated from different plant species. (A): proportion
of IAA producing and non-producing bacteria with their biosynthetic capacity. Reddish color
denotes strong IAA producer, while light pink color denotes low IAA producer isolates. More than
two-thirds of the rhizobacteria did not produce IAA in culture medium. (B). Salkowski reagent
positive isolates develop reddish to light pink color in culture supernatant, an indication of IAA
biosynthesis from l-tryptophan. Colorless tubes represent isolates with no ability to produce IAA
Figure 3. Assay results for nitrogen fixation and plate assay for P solubilization. (A): The putative nitrogen
fixing ability of the rhizobacteria on N-free medium. All the bacteria were able to grow in N-deficient
medium indicating their ability to grow in varying capacity. (B): Representative bacterial isolates
developed clear halo zones surrounding the colony in Pikovskaya’s agar medium containing
tricalcium phosphate, an indication of phosphate solubilization
Khatun et al. Fundam Appl Agric 6(1): 95–106, 2021 102
to be phosphorus non-scavengers. The phosphorus
solubilization capacity of the isolates varies and the
intensity of P solubilization were denoted as level
1 (
+
), level-2 (
++
) and level-3 (
+++
) phosphorus
solubilizer (Table 4). The phosphorus solubilization
index (PSI) of the isolates ranged between 1.1 to 11
for the 15 potential phosphate solubilizing bacteria
(PSB). The highest PSI value (11) was recorded for the
isolate MQ2 followed by MQ1 (PSI 8) both isolated
from Sushni Shak. On the contrary, lowest PSI (1.1)
were recorded for in OSn7, OSn8 and MQ4 isolates
among the P solubilizers (Table 4).
Selected phosphate solubilizing rhizobacteria (6)
were quantitatively evaluated using broth medium.
The bacteria were selected on the basis of maximum
PSI values and supported by the qualitative plate
assay (Fig. 3B). Among the isolates, the highest quan-
tity (0.697
µ
g mL
−1
) of P was solubilized by MQ2
followed by MQ1 and MQ3 (Table 4). On the other
hand, the lowest quantity of P (0.0278
µ
g mL
−1
) was
solubilized by Fr7 which was isolated from fern rhi-
zosphere. The rest four isolates Fr4 (0.072
µ
g mL
−1
),
GM2 (0.038
µ
g mL
−1
), MQ1 (0.260
µ
g mL
−1
) and
MQ3 (0.260
µ
g mL
−1
) showed differential of phos-
phate solubilization in liquid media.
The reduction of pH in culture medium was also
recorded to understand the mechanism of P solubi-
lization. The pH of the culture media for isolate MQ2
dropped to 4.87 followed by MQ3 (pH 5.53). The pH
of the culture media for other isolates studied also
dropped from initial pH (7.0) of the culture media
which corelates to their phosphate solubilizing capac-
ity (Fig. 4A). Titratable acidity was measured for the
determination of total acidity of the PSB liquid media
with rhizobacterial isolates. In this study, titratable
acidity of the six-phosphate solubilizing rhizobacteria
was measured and found that the titratable acidity
was highest (0.055%) for MQ2 isolate which was also
highest P solubilizer (0.6972
µ
g mL
−1
) (Table 4). The
lowest percentage of acidity (0.0064%) was the acidity
was obtained for Fr4 and Fr7 both of which are also
slight P solubilizer (Fig. 4B).
3.4 Screened out N2-fixing bacteria
Growth of bacteria in N-free media was the indication
of N
2
-fixation by the bacteria. All the bacterial isolates
are able to fix N
2
. Among the 32 rhizobacteria about
13 bacteria (Fr3, Fr4, Fr5, Fr6, Fr7, GM1, GM2, EC1,
MQ2, MQ3, MQL7, MQL8, and MQL9) showed high,
11 bacteria [OS29(2), OS29(3), OSbr6, OSn7, OSn8,
EC2, Fr1, Fr2, MQ1, MQ4 and MQ5] showed medium
and 8 bacterial isolates [OS29(3), OSbr4, OSbr5, EC4,
EC5, Fr1, MQ6, and ECL1] showed slight ability to
grow in N-free medium. Accordingly, the isolates
were grouped into low, medium and high N
2
fixer
(Fig. 3A).
4 Discussion
Diverse array of both beneficial and pathogenic mi-
croorganisms inhabits in the plant rhizosphere and
surrounding areas due to the presence of abundant
carbon resources excreted from plant as root exudates.
A total of 32 rhizobacteria were isolated from different
plant species comprising samples of both agronomic
and non-agronomic plant species. Majority (18) of the
rhizobacteria isolated were Gram positive and rest
of them (14) were Gram negative (Table 2). Most of
the isolated bacteria were whitish and cream in color
with few producing yellow and red pigments (Table 2)
shows the bacterial diversity in different plant species.
The screening tests to find out potential plant growth
promoting rhizobacteria revealed that about one third
of the isolated rhizobacteria exhibits at least one plant
of the major plant growth promoting traits i.e., IAA
production, phosphate solubilization or N2-fixation.
L-tryptophan dependent IAA production thought
to be the major biosynthetic pathway for bacteria
(Rahman et al.,2010). Indole-3-acetic acid is the dom-
inant phytohormone produced by rhizobacteria and
implicated in the growth promotion and developmen-
tal processes in plant. The IAA production assay us-
ing 32 isolated bacteria reported that 10 rhizobacterial
isolates can bio-transform L-tryptophan in varying
quantities, between 1.268 and 6.204
µ
g mL
−1
(Table 3),
which is much lower than other previously reported
value (77
µ
g mL
−1
) for IAA production by rhizobac-
teria (Majeed et al.,2015). The variation in rhizobac-
terial IAA production has previously been reported
by bacteria isolated from various plant species like
tomato, rice, maize, fern etc. (Majeed et al.,2015;
Lwin et al.,2012;Asha et al.,2015). Rahman et al.
(2010) found that half of the bacterial isolates among
69 showed positive colour reactions to Salkowski’s
reagent. IAA production by PGPR isolates may vary
from different strains and species; and was addition-
ally influenced by substrate availability, culture con-
ditions and growth stage (Devi et al.,2015). Verma
et al. (2015) also observed IAA producing ability of
rhizobacteria (Pseudomonas spp., Bacillus spp. and
Acinetobacter spp.) isolated from the rhizosphere of
wheat.
Scavenging different nutrient elements via miner-
alization (to phosphate ion) of unavailable sources by
PGPR is considered as one of the most important cri-
teria for selection rhizobacteria for plant growth pro-
motion (Vessey,2003;Islam et al.,2007;Rahman et al.,
2015). In this study, we found 6 bacterial isolates to
be efficient P solubilizer when tricalcium phosphate
was used as a source of phosphorus (Table 4). We
also reported a drop in the pH of culture medium
for the isolates that solubilized and released differ-
ent quantities of phosphorus. Furthermore, percent
of total titratable acidity in culture medium also in
accordance with the results of P solubilization and
Khatun et al. Fundam Appl Agric 6(1): 95–106, 2021 103
Table 4. In vitro qualitative and quantitative analysis of phosphate solubilization by rhizobacteria
Bacterial isolates Intensity of PSB in solid medium PSI after 6 days Conc. of P (µg/mL)
OS29(1) −1.0 −
OS29(2) +1.5 −
OS29(3) −1.0 −
OSbr4 −1.0 −
OSbr5 −1.0 −
OSbr6 −1.0 −
OSn7 +1.1 −
OSn8 +1.1 −
EC1 −1.0 −
EC2 −1.0 −
EC3 +1.3 −
EC4 −1.0 −
EC5 −1.0 −
ECL1 −1.0 −
Fr1 −1.0 −
Fr2 −1.0 −
Fr3 −1.0 −
Fr4 ++ 7.5 0.07±0.00
Fr5 −1.0 −
Fr6 −1.0 −
Fr7 ++ 6.5 0.03±0.00
GM1 −1.0 −
GM2 ++ 3.0 0.04±0.00
MQ1 ++ 8.0 0.26±0.05
MQ2 +++ 11 0.70±0.19
MQ3 +2.5 0.26±0.11
MQ4 +1.1 −
MQ5 +1.25 −
MQ6 −1.0 −
Qualitative analysis was done using Pikovskaya’s agar medium supplemented with tricalcium phosphate as
insoluble source of phosphorus (P). Pikovskaya’s broth medium was used to quantify the concentration of P
released by selected rhizobacterial isolates (based on PSI value >2.0). The failure to produce clear halo zones
around the colony on solid media is indicated by negative sign (
−
). The positive signs (
+
,
++
and
+ + +
) for
solid media indicate the P solubilization ability of the isolates. The negative sign for the concentration of P in
liquid medium indicates that these bacteria were excluded from quantitative P determination due to low PSI
values. Here, (
+
), (
++
) and (
+++
) represent level 1, level 2 and level 3 phosphorus solubilizers, respectively
Khatun et al. Fundam Appl Agric 6(1): 95–106, 2021 104
Figure 4.
Reduction in medium pH due to inoculation of Rhizobacteria (A), and percent total titratable acidity
(B). The results indicates that phosphorus was solubilized by the rhizobacteria due to the production
of low molecular weight organic and inorganic acids
reduction in medium pH (Fig. 4A, B). The mecha-
nism of microbial P solubilization varies among the
bacterial species. In general, microbial phosphorus
solubilization from inorganic pool of soil phospho-
rus is attributed to the production of different low
molecular weight organic and inorganic acids like
gluconic acid and 2-ketogluconic acid (Walpola,2012)
and release of chelating substance and carbonic acids
(Vessey,2003;Oteino et al.,2015).
The ability of N
2
fixation by PGPR is another ma-
jor criterion for selecting rhizobacteria as components
of microbial biofertilizer. Apart from the symbiotic
N
2
fixing rhizobacteria, large number of free living
and non-symbiotic rhizobacteria has been reported
to have the ability to fix atmospheric N
2
and imparts
plant growth promotion (Franche et al.,2008;Xu et al.,
2018). In our study, all the rhizobacterial isolates were
able to grow at varying capacity in N-free Winogrd-
sky’s mineral media, an indication of putative N
2
-
fixation (Asha et al.,2015). Among the isolates, 13
isolates were designated as strong N
2
-fixing bacteria
due to their rapid and vigorous growth in N-free me-
dia (Fig. 3A). The presence of nitrogenase activity in
bacteria is considered as the mechanism of N-fixation
by free-living rhizobacteria (Rilling et al.,2018).
5 Conclusion
The search for multifunctional PGPR is gaining im-
portance around the world to minimize the amount
of chemical agrochemical inputs required for sustain-
able crop production. We isolated and characterized
functionalities of 32 rhizobacteria from different plant
species. We have reported the production of IAA, sol-
ubilization of mineral phosphate and N
2
fixing ability
of the isolated rhizobacteria, all of which are consid-
ered as primary criteria for selecting PGPR. Based on
the criteria studied for selecting potential PGPR, the
isolate MQ1 proved to be the best over other isolated
rhizobacteria and could be considered as potential
PGPR for bioformulation. The application of these
bacteria with plant growth promoting traits can be
used to promote plant growth after evaluation of bio-
functionalities under in vitro and in vivo conditions
and detailed molecular characterization.
Acknowledgments
The authors acknowledge the Ministry of Science
and Technology (MoST), Bangladesh for granting Na-
tional Science and Technology (NST) fellowship to the
first author and BAURES (BAUproject#2018/354 and
RPP-2018-127), Bangladesh Agricultural University
for funding the research work
Conflict of Interest
The authors declare that there is no conflict of inter-
ests regarding the publication of this paper.
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