UJMR, Volume 5 Number 2, December, 2020, pp 17 - 25 ISSN: 2616 - 0668

https://doi.org/10.47430/ujmr.2052.003
Received: 2nd November, 2020 Accepted: 16th November, 2020

Isolation and Enumeration of Bradyrhizobium Species Dwelling In the Root

Nodules of Soybean Plant
Ishaq, Z., Machido, D.A. and Atta*, H.I.
Department of Microbiology, Faculty of Life Sciences, Ahmadu Bello University, Zaria, Kaduna
State, Nigeria.
*Correspondence author: hiatta@abu.edu.ng
Abstract
Biological Nitrogen fixation is one of the important aspects of organic agriculture gaining
considerable attention globally. Information about the number of viable indigenous
Bradyrhizobia in soils planted with legumes and their capacity to nodulate is a valuable tool in
developing strategies to improve biological nitrogen fixation. Such strategies could potentially
lead to increased soybean yields at low cost. This study was conducted to isolate and
enumerate Bradyrizobium species dwelling in the root nodule of soybean plant using
Bradyrhizobium japonicum selective medium (BJSM). Twenty (20) strains of Bradyrizobium
species were isolated from the root nodules of soybean plants harvested from Ahmadu Bello
University farm site, located at Bomo district of Sabongari local government area, Kaduna
State, Nigeria. This was achieved using the streak method of isolation on BJSM. Ninety percent
(18) of these isolates were confirmed as Bradyrizobium species using the plant infection test as
they were able to nodulate the roots of soybean plants. The enumeration of the indigenous
Bradyrizobium species gave a count ranging from 2.07x105 - 4.0x106 CFU/mL. Thus, the number
of Bradyrhizobia obtained in the soil of this study is sufficient to achieve satisfactory results
on nodulation and nitrogen fixation.
Key words: Soybean, Bradyrhizobium species, Nodulation, Nitrogen fixation.

INTRODUCTION as a highly versatile and multipurpose

Soybean [Glycine max (L.) Merril.] is a agricultural product that has about three
leguminous plant of the pea family that grows hundred and sixty-five (365) applications in the
in tropical, subtropical, and temperate formulation of both human, animal foods and
climates. It was domesticated in the 11th other industrial uses. Therefore, it has a great
century BC around northeast of China and it is potential of improving the nutritional status
believed to have been introduced to Africa in and welfare of poor people in Nigeria (Omotayo
the 19th century by Chinese traders along the et al., 2007; Dianda, 2014).
east coast of Africa (IITA, 2009). It was Bradyrhizobia are bacteria that are members of
introduced to Nigeria in 1908 in Ibadan, Oyo the group, alpha Proteobacteria. They are gram
State (Omotayo et al., 2007). negative rods with a single subpolar or polar
This plant is one of the most economically flagellum. They are slow-growing common soil-
important legumes that is cultivated worldwide dwelling bacteria that can form symbiotic
(Hungria and Mendes, 2015). In 2007, more relationships with leguminous plant such as
than 216 million tons of soybeans were soybean where they fix nitrogen in exchange
produced worldwide, of which Africa has about for carbohydrates from the plant (Berrada and
1.5 million. Nigeria is the largest producer of Fikri-Benbrahim, 2014).
soybean in sub-Saharan Africa (SSA), followed Biological nitrogen fixation (BNF) is the process
by South Africa (IITA, 2009). where atmospheric nitrogen is converted into
Soybean as described by (Omotayo et al., 2007) plant usable forms such as ammonia by nitrogen
is known as “miracle bean” or the “golden fixing organisms using a complex enzyme
bean” because it is a cheap, protein-rich grain system known as nitrogenase (Kim and Rees,
with a high protein content of about 40% which 1994; Bishnoi, 2015). This process is an
is superior to all other plant foods as it has a efficient source of fixed nitrogen that plays an
good balance of the essential amino acids important role in land remediation (Mohammadi
(Belewu and Belewu, 2007; Hungria and et al., 2012). It is as well an economically
Mendes, 2015). The seeds also contain about beneficial and environmentally friendly
20% oil on a dry matter basis which is about 85% alternative to replace the high regimens of
unsaturated and cholesterol-free (Dugje et al., chemical fertilizers used in the present day
2009) making it the most important crop for agriculture (Adesemoye et al., 2009; Saturno et
producing edible oil. Generally, it is regarded al., 2017).
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 UJMR, Volume 5 Number 2, December, 2020, pp 17 - 25 ISSN: 2616 - 0668
The symbiotic association between leguminous of each of the five soil subsamples were pooled
plants and rhizobia has the greatest together and mixed thoroughly as one
quantitative impact on the nitrogen cycle. The composite soil sample; the sample was then
process of symbiotic nitrogen fixation supplies sundried for one day, crushed and passed
50-60% of soybean nitrogen demands through the mesh of a 2mm sieve for
(Salvagiotti et al., 2008) but may reach up to physicochemical analysis. The sieved sample
94% for high yields as it requires large amounts was stored in plastic bag at room temperature
of nitrogen (Rodrigues-Navarro et al., 2011; until required for further analysis.
Kinugasa et al., 2012). Sample of river sand was also collected from
A common approach to improve symbiotic river Kubani flowing through Ahmadu Bello
nitrogen fixation and legume productivity has University, Zaria in September, 2018. Sample
been the reliance on superior or very effective was obtained using a sterile hand trowel,
exotic Bradyrhizobia strains as inoculants placed in clean sampling bags and transported
(Emmanuel et al., 2008) since many soils are to the laboratory in the Department of
deficient in the number and quality of Microbiology, Ahmadu Bello University, Zaria.
indigenous rhizobia to enhance biological The river sand collected was dried under the
nitrogen fixation (Abdullahi et al., 2013). In sun, passed through the mesh of a 4mm sieve
order to address the problem of for planting and sterilized by autoclaving
Bradyrhizobium inocula as regards to the lack (Imrana, 2017).
of local production and difficulties in the Determination of Physicochemical Properties
importation of inoculants in the 1970s, the of the Soil
soybean-breeding program at the International The soil particle size was determined by the
Institute of Tropical Agriculture (IITA), Nigeria, Hydrometer method and the soil texture was
developed soybean genotypes, designated TGx classified using the United States Department
(tropical glycine cross) to nodulate effectively of Agriculture (USDA) soil textural class
with indigenous Bradyrhizobium spp. (Okalebo et al., 2002). Soil pH was measured in
populations (Santos et al., 2019). However, a 1:2.5 soil: water ratio using a pH meter as
nitrogen deficiency and poor yields will be described by Okalebo et al. (2002). Total
experienced whenever Bradyrhizobia that Nitrogen was determined by the Kjeldahl
effectively nodulate this soybean genotypes are method (Machido, 2010); available phosphorous
not available in adequate numbers (Chianu et by the Bray and Kurtz P-1 method (Okalebo et
al., 2011). As such, obtaining sufficient al., 2002); organic carbon by the method of
knowledge on the population characteristics of Walkley-Black (Okalebo et al., 2002) and water
indigenous Bradyrhizobial populations in soils as holding capacity by the method described by
well as determining their nodulation capacity Pawar and Shah (2009). Exchangeable bases
will be valuable for developing strategies to were extracted with ammonium acetate
improve BNF and thus increase soybean yields solution 1N (NH4OAc) (Okalebo et al., 2002),
at low cost (Abaidoo et al., 2007). Imrana exchangeable potassium and sodium were
(2017) reported nodulation of promiscuous determined using flame photometer (Okalebo
soybean with indigenous Bradyrhizobia in the et al., 2002) while exchangeable calcium and
Northern Guinea savannah zone of Nigeria. This magnesium were determined by the titration
study was, therefore, aimed at isolating and method (Pawar and Shah, 2009). The cationic
enumerating Bradyrhizobium species dwelling exchange capacity was determined by 1N
in the root nodules of soybean plant. ammonium acetate saturation method (Pawar
and Shah, 2009) and the exchangeable acidity
MATERIALS AND METHODS was by the titration method (Okalebo et al.,
Collection of Soil Sample 2002).
Soybean plants were harvested from five sites Determination of Total Viable Counts of
without history of inoculation (one plant from Bradyrhizobia in the Soil Sample
each site) on Ahmadu Bello University farm This was carried out following the pour plate
located at Bomo district of Sabongari local technique as described by Woomer et al.
government area, Kaduna state, Nigeria. This (2011). Ten grams of the soil sample was
was done in September, 2018 as this month is weighed into conical flask containing 90mL of
the harvesting period of soybean in Nigeria. The sterile distilled water. The sample was mixed
samples were placed in sterile sampling bags with the diluent by shaking thoroughly and a
and transported at ten o’clock in the morning ten-fold dilution was made up to dilution of 10-
6
to the laboratory in the Department of . Using a sterile pipette an aliquot of 1mL was
Microbiology, Ahmadu Bello University Zaria. transferred aseptically from each of the
The soil attached to each of the harvested dilutions to sterile petri dish in duplicate. An
plants was gently removed by shaking and 500 g already prepared sterile molten medium (15
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 UJMR, Volume 5 Number 2, December, 2020, pp 17 - 25 ISSN: 2616 - 0668
mL) (Bradyrhizobium japonicum selective The presumptive Bradyrhizobial isolates were
medium) (Tong and Sadowsky, 1994) was grown in sterile Erlenmeyer flasks (50 mL)
poured into each inoculated petri dish and containing 25 mL of freshly prepared yeast-
swirled gently for even mixing. The agar was mannitol broth. The inoculated culture media
allowed to set and the dishes were incubated in were incubated at room temperature on a
an inverted position at 28oC for 10days in an rotary shaker for a period of three (3) days
incubator. (Woomer et al., 2011).
Isolation of Indigenous Bradyrhizobium Planting was done in the screen house at the
species from the Root Nodules of Soybean Department of Microbiology, Ahmadu Bello
Plant University, Zaria. Plastic cups with a depth of
Bradyrhizobium japonicum selective medium 10.7 cm and diameter 6.2 cm, were punctured
(BJSM) was used for the isolation. Healthy at the base with three or more holes and used
nodules were selected from the harvested for planting the seeds. A Whatman filter paper
plants and surface sterilized by immersing them was placed at the bottom of each cup to
in 95% ethanol for 10 seconds, transferred to 3% prevent excess water from draining out. Four
(v/v) solution of sodium hypochlorite for 2 min hundred grams of autoclaved soil was added to
and rinsed six times with sterile distilled water. surface sterilized cups and saturated with
After surface sterilization, noodles were sliced sterile plant nutrient solution prior to sowing
open with a sterile razor blade and isolate were seeds. Three equally spaced holes were made
made from those with characteristic pink or red in the soil to a depth that will accommodate
interior (Gyogluu et al., 2018). They were seeds one centimetre below the surface. Pre-
crushed with a pair of blunt tipped forceps in germinated seeds were picked with sterile
5ml of normal saline in a sterile petri-dish. One forceps and one seed in each hole were placed
loopful of the nodule suspension was streaked with the radicle entering first (Woomer et al.,
onto plates of BJSM and the plates were 2011; Imrana, 2017).
incubated at 28oC for 10days in an incubator Nitrogen-free nutrient solution (20 mL) was
(Tong and Sadowsky, 1994). Small white supplied to each plant once every week and the
colonies that grew after ten days were plants were replenished with sterile distilled
aseptically subcultured onto the surface of water on a daily basis. Uninoculated controls
freshly prepared Yeast Extract Mannitol Agar with nitrogen (positive control) and without
(YEMA) slants and stored at room temperature nitrogen (negative control) were included, for
for further use (Woomer et al., 2011). These the uninoculated control with nitrogen,
presumptive Bradyrhizobial isolates based on nitrogen was applied as 5mL of 0.05 %
their cultural properties were subjected to potassium nitrate (KNO3) (w/v) solution once
plant infection test (nodulation test) explained every week (Howienson and Dilworth, 2016).
in the next subheading below as it is indicated At eleven days after planting, the plants were
that an isolate cannot be properly regarded as inoculated with the culture yeast-mannitol
Bradyrhizobium until its identity had been broth using a fresh pipette for each isolate. At
confirmed through nodulation test on soybean 30days after planting, harvesting was carried
(Imrana, 2017). out. The soil was washed off the plants using a
Authentication of the Isolates as gentle stream of water and the presence or
Bradyrhizobium species absence of nodules was noted and recorded
Healthy seeds of a soybean variant (TGX1448- accordingly (Woomer et al., 2011).
2E) of similar size and colour were selected for
pre-germination. Seeds were surface sterilized RESULTS
by immersing them in 95% alcohol for 10 Physicochemical properties of the soil
seconds to remove waxy material and trapped The physicochemical analysis of the soil in this
air. They were immersed in 3% sodium study indicated that the soil is sandy loamy in
hypochlorite solution for 3 minutes in a large texture and slightly acidic (6.44). For the
sterile petri-dish; rinsed six times with sterile exchangeable cations, the level of potassium
distilled water and left in the water for four (0.072 Meq/100g) was found to be very low,
hours until they were fully imbibed (Woomer et sodium (0.696 Meq/100g) and magnesium (1.67
al., 2011). The seeds were again rinsed twice Meq/100g) were moderate and the calcium
with sterile distilled water and pre-germinated (2.60 Meq/100g) content was low. The total
by transferring them aseptically with forceps to Nitrogen (0.034%) and organic carbon (0.379%)
the surface of large sterile petri-dish containing were found to be low while the available
moistened cotton wool and incubated at room phosphorus (11.46 mg/kg) was at moderate
temperature until the radicles were 0.5 to 1.0 level.
cm long for easy transfer during planting
(Woomer et al., 2011).
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 UJMR, Volume 5 Number 2, December, 2020, pp 17 - 25 ISSN: 2616 - 0668
The exchangeable acidity (0.50 Meq/100g) was Occurrence and Confirmation of
low and the cationic exchange capacity (2.86 Bradyrhizobium species in soil sample
Meq/100g) was very low (Table 1). These A total of twenty (20) strains (four from each of
ratings are according to United States the five plants) presumptively identified as
department of agriculture standard (USDA, Bradyrhizobium species based on their cultural
1993). properties were isolated. Colonies were small
Population of Bradyrhizobium species in the and whitish in colour on Bradyrhizobium
soil sample japonicum selective medium (BJSM) (plate I).
The plate count of the soil sample revealed an Ninety percent (18) of the twenty (20) isolates
average population of 2.1x106 CFU/mL ranging tested positive to nodulation (plate II) while
from 2.07x105 - 4.0x106 CFU/mL (Table 2). the rest ten percent (2) tested negative (Table
3).

Plate I: Colonies of Bradyrhizobium sp. isolated in tis study growing on the surface of
Bradyrhizobium japonicum selective medium

Plate II: Nodules formed in roots of soy bean plant after inoculation with Bradyrhizobium sp.
isolated in this study.
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 UJMR, Volume 5 Number 2, December, 2020, pp 17 - 25 ISSN: 2616 - 0668
Table 1: Physicochemical properties of soil used for isolation of Bradyrhizobia
Property Value Ratings (USDA Standard)
Very low Low Moderate high
Particle size distribution (%)
Clay (%) 5
Silt (%) 38
Sand (%) 57
Textural class Sandy
loam
pH(H2O)1:2.5 6.44
Water holding capacity (%) 31.2
Total Nitrogen (%) 0.034 - 0-0.15 0.15-0.20 > o.20
Organic Matter (%) 0.653 - < 2.0 2.0-3.0 > 3.0
Organic Carbon (%) 0.379 - < 1.0 1.0- 1.5 > 1.5
Available P (mg/kg) 11.46 - 0-10 10-20 > 20
Exchangeable Bases
(Meq/100g)
K 0.072 < 0.2 0.2 -0.3 0.3-0.6 0.6-1.2
Na 0.696 < 0.1 0.1-0.3 0.3-0.7 0.7-2.0
Ca 2.60 <2 2-5 5-10 10-20
Mg 1.67 < 0.3 0.3-1.0 1.0-3.0 3.0-8.0

Exchangeable Acidity (H++Al3+) 0.50 < 0.5 0.5-1.0 1.0-1.5 1.5-2.5

(Meq/100g)
Cation Exchange Capacity 2.86 <6 6-12 12-25 25-40
(Meq/100g)
Source of ratings: (USDA, 1993).

Table 2: Population of Bradyrhizobium in the soil

Source of Inoculants Population of Bradyrhizobium
Range (CFU/mL) Mean (CFU/mL)
Soil 2.07x105-4.0x106 2.10x106

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 UJMR, Volume 5 Number 2, December, 2020, pp 17 - 25 ISSN: 2616 - 0668
Table 3: Capacity of the Isolates to Form Nodules on Soybean (TGX1448-2E).
Isolate Code Nodulation ability
AF1 +
AF2 +
AF3 +
AF4 +
AF5 +
AF6 +
AF7 +
AF8 -
AF9 +
AF10 +
AF11 +
AF12 -
AF13 +
AF14 +
AF15 +
AF16 +
AF17 +
AF18 +
AF19 +
AF20 +
PC -
NC -
Key: AF-------Ahmadu Bello University Farm, AF1-AF20------Isolates obtained from the root nodules
of harvested plants, PC---------Plant fertilized with KNO3, NC-------- Plant without inoculant nor
fertilized, + ----------Positive to nodulation, - -----------Negative to nodulation
moderate magnesium content, low potassium
DISCUSSION level and low to medium range of CEC.
The soil’s textural class of sandy loam may
have accounted for its low clay and silt The soil was found to be low in total nitrogen
content. This class of soil texture enables high and organic carbon but moderate in available
rate of water infiltration and moisture loss phosphorus. Machido et al. (2011) indicated low
through evaporation and erosion resulting to level of organic carbon, total nitrogen and
the low level of water holding capacity phosphorous as a common feature of the soils
recorded (Umeri et al., 2017). The soil pH was found in the semi-arid zone of Nigeria, hence
6.44, this slight acidity of the soil is a feature their inherent low fertility. The sandy texture
common to the soils of the savannah zone and of the soil could have also played a role in the
it is within the pH range (5.5-7.0) that is washing away of nutrients such as Nitrogen and
optimum for the release of plant nutrient Phosphorus. This finding is supported by the
(Sharu et al., 2013). This pH value is also work of Imrana (2017) who reported a sandy
favourable for the growth and survival of the loam soil texture, low content of organic
indigenous population of Bradyrhizobium spp. carbon and total nitrogen in the soil samples
(Howieson and Dilworth, 2016). The addition of from Zaria, Kaduna State. Abubakar and Ado
ammonium (NH4) fertilizer and the (2016) also reported a slightly acidic to
decomposition of organic matter could result in moderately acidic pH, low carbon and nitrogen
the acidic nature of soils. This is because the content in the soils across three agro-ecological
hydrogen ions produced by these processes zones of Nigeria (Northern Guinea, Southern
displace calcium, magnesium and potassium Guinea and Sudan Savannah zones). The low
from the surface of the soil particles that are level of cationic exchange capacity (CEC) can
then leached from the upper regions of the soil be related to the inherent sandy nature of the
profile by water moving downward through the soil containing a low weatherable mineral
soil especially soil of high sand fraction (Umeri reserve necessary for nutrient recharge and a
et al., 2017). This accounts for the low content small capacity for carbon storage (Shehu et al.,
of potassium and calcium in the soil of this 2018). These ratings are according to USDA
study. The moderate level of sodium and (1993).
magnesium contents is in line with the work of
Adekiya et al. (2019) who also reported a

22
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 UJMR, Volume 5 Number 2, December, 2020, pp 17 - 25 ISSN: 2616 - 0668
The soil’s average Bradyrhizobial count of cannot be over emphasized as it is indicated
2.1x106 CFU/ mL which range from 2.07x105 to that an isolate cannot be properly regarded as
4.0x106 CFU/mL is against the work of Ojo et Bradyrhizobium until its identity had been
al. (2015) who reported Rhizobial counts of confirmed through nodulation test on soybean
4x106 – 1.4x107 cells/g of soil in three different (Imrana, 2017). Ninety percent of the twenty
locations in the rain forest zone of Nigeria using (20) isolates tested positive to nodulation while
the plant infection most probable number MPN the remaining ten percent tested negative
method. However, it has been indicated that a (Isolate number AF8 and AF12). This supports
soil population density of at least 103 the work of Chibeba et al. (2017) where eighty-
Bradyrhizobium japonicum cells g-1 is required seven (87) out of one hundred and five (105)
for maximizing nodule numbers on seedling tap isolates obtained from soybean nodules
roots, and efficient N2 fixation to satisfy the collected in Mozambique were able to nodulate
Nitrogen demand of soybean (Atieno et al., both promiscuous and non-promiscuous
2012). Phanuel et al. (2015) has also reported cultivars. It has been indicated by Tong and
that in the evaluation of effect of Sadowsky (1994), that there is no direct
Bradyrhizobium japonicumm population on correlation between the nodulation ability of
nodulation in some Ghanaian soils, the soil with an isolate and its ability to grow on the
the highest Bradyrhizobial count of 6.0x103 cells selective medium. This was obtained in the
/g of soil also produced the highest number of work that soybean-nodulating Rhizobium fredii
nodules. Thus, the Bradyrhizobial population was unable to grow on the medium since the
observed in the soil of this study is sufficient to medium is selective for Bradyrhizobium species
achieve satisfactory results on nodulation and but 98% of the randomly selected colonies
nitrogen fixation. recovered from a soil sample on BJSM were
A total of twenty (20) isolates were obtained able to nodulate soybean plants (Tong and
from the root nodules of the plants using Sadowsky, 1994). The ten percent non-
Bradyrhizobium japonicum selective medium nodulating isolates obtained could be
(BJSM). Since the development BJSM by Tong opportunistic bacteria that found their way into
and Sadowsky (1994), several researchers such the nodules during the root infection process
as Pongdet et al. (2015), Rouws et al. (2014), and have the ability to utilize the medium
among others have reported its use. The need (Ibanez et al., 2009). They could also be other
to confirm the identity of the isolates as species of Bradyrhizobium that do not nodulate
Bradyrhizobia through plant infection test soybeans.
CONCLUSION twenty (20) isolates were obtained from the
The number of Bradyrhizobia in the soil of this root nodules of the plants and 90% (18) of
study ranged from 2.07x105- 4.0x106 CFU/mL these isolates were able to cause nodule
with an average of 2.10x106 CFU/mL, a total of formation in soybean plants.

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