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European Journal of Applied Sciences – Vol. 12, No. 2
Publication Date: April 25, 2024
DOI:10.14738/aivp.122.16831
Gebrehana, Z. G., Misgana, M., & Anbessa, B. (2024). Symbiotic Response of Field Grown Soybean Varieties to Rhizobia Inoculant
in Western Ethiopia. European Journal of Applied Sciences, Vol - 12(2). 309-321.
Services for Science and Education – United Kingdom
Symbiotic Response of Field Grown Soybean Varieties to Rhizobia
Inoculant in Western Ethiopia
Zerihun Getachew Gebrehana
Assosa Agricultural Research Center,
Ethiopian Institute of Agriculture Research, Assosa, Ethiopia
Mathewos Misgana
Assosa Agricultural Research Center,
Ethiopian Institute of Agriculture Research, Assosa, Ethiopia
Bakala Anbessa
Assosa Agricultural Research Center,
Ethiopian Institute of Agriculture Research, Assosa, Ethiopia
ABSTRACT
Rhizobium inoculation is a widely adopted practice for legume crops like soybeans,
facilitating the formation of N2-fixing symbiotic associations. Effective rhizobia
strains have been used as rhizobial inoculants to maximize nitrogen fixation,
resulting in adequate yields. However, the effectiveness of these inoculants varies
depending on soybean varieties, with beneficial effects observed on nodulation and
grain yield. A study evaluated the potential of elite Rhizobia strains (Mar-1495, SB- 12, and SB-14) on soybean nodulation, growth, and seed yield across different
soybean varieties (Gishema, Wello, and Belessa-95) under farmer field conditions
in different cropping seasons. The combined ANOVA results showed no significant
interaction between soybean variety and Rhizobia inoculant on growth and yield.
However, there was a notable influence of the year on all parameters measured,
reported for each experimental season. Rhizobia inoculation notably improved
root nodulation, growth, and seed yield in soybean varieties. Mar-1495
demonstrated a substantial increase in growth and yield, with a 44.6% yield
increment compared to the control. On the other hand, SB-12 exhibited poorer
performance. Variation in response to Rhizobium inoculation was observed
between locations, likely due to soil fertility differences, mostly soil N and P.
Overall, using compatible and effective rhizobial inoculants like MAR-1495 and SB- 14 can enhance nodulation, growth, and seed yield, particularly in the Gishema and
Belessa-95 soybean varieties.
Keywords: Rhizobium inoculation, nodulation, variety, legume, Nitisols.
INTRODUCTION
Soybean (Glycine max L.) is one of the world’s most important legumes with high-quality
protein (40%) and oil (20%) around the world [1]. Its ability to grow symbiotically on low
nitrogen (N) soils, point to its continued status as the most valuable grain legume in the world
[2]. In Ethiopia, soybean is recently grown in high rainfall areas, in west and southwestern parts
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among smallholder farming systems as an important food and cash crop [3] along with haricot
bean [4]. Legumes like soybean have evolved to host N2 fixing bacteria, known as rhizobia, in
specialized organs called root nodules and they supply nutrients to rhizobia that fix N2 gas from
the atmosphere into reduced forms that are supplied to the legume [5]. In view of this,
biological nitrogen fixation (BNF), a renewable N fertilizer source, holds great promise for
smallholder farmers and constitutes one of the potential solutions and plays a key role in
sustainable grain legumes production in sub-Saharan Africa [6]. Many studies provide
estimates of the contribution of BNF to aboveground soybean biomass (including both grain
and shoots), which, on average accounts for about 60 % of total soybean N uptake [7].
Therefore, N2 fixing soybean crops are agronomically and economically important in organic
cropping systems which could also influence the increase in sustainable agricultural practice
[8].
Low soil fertility often characterized by low soil N availability is often the major factor resulting
in decreased crop plant yields in soils of Ethiopia [9,10]. Especially in western Ethiopia, this low
soil fertility associated with conventional farming practice allow grain legumes to grown in
sever soil conditions [11]. On the other hand, improving Rhizobium-legume symbiosis is
essential that could help smallholder farmers to maintain their soil quality thereby increase
crop productivity. However, the use of rhizobium inoculants for improvement in N-fixation and
productivity of grain legumes is still in the developing stage in most parts of sub-Saharan Africa
[12], including Ethiopia. In Ethiopia, inoculation of soybean with Rhizobial inoculants is not
common practice, but could provide an option to increase grain yields in low nitrogen acidic
soils [13]. However, there is increasing evidence to suggest that inoculation using Rhizobium
increase nodulation, growth, seed and biomass yield of soybean [14] and chickpea [15].
Therefore, Rhizobium-legume association can be manipulated, through inoculation under N- limiting field conditions, to enhance crop production. Inoculation with compatible and
appropriate rhizobia may be necessary where a low population of native rhizobial strains
predominates and is one of the solutions which grain legume farmers can use to optimize yields
[6].
Generally, legume productivity in an agricultural field may be improved by inoculation with
selected highly effective N2-fixing root nodule bacteria [16]. Several studies have already been
reported an improve in soybean yield and yield components using Rhizobium inoculation as
compared to non-inoculated seed [12,17,18]. According to Graham et al. [19] identifying
appropriate and effective legume-Rhizobium combinations that can be readily adopted by
farmers with immediate demonstrable benefits to ensure adoption should be future research
agenda in African farming systems, including Ethiopia. A continuous and coordinated selection
of the most effective combinations of host and microbial symbionts is a prerequisite for
profitable and sustainable agricultural practice [20,21]. Although Wolde-Meskel [22] reported
most soils naturally harbor native rhizobial strains that can nodulate and fix atmospheric N2 in
Ethiopia soils, symbiotically efficient rhizobia nodulating to soybean is scanty. The
performance of symbiotic nitrogen fixation is however influenced by among soybean variety,
rhizobia strains and environment condition [23]. Moreover, Rhizobium-Soybean symbiosis is
highly host-specific and hence evaluation of effective strains to enhance nitrogen fixation and
yield of soybean varieties in smallholder farmers of Ethiopia is crucial. Our study aimed to
evaluate soybean varieties response to elite rhizobial inoculants focus on growth performance
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Gebrehana, Z. G., Misgana, M., & Anbessa, B. (2024). Symbiotic Response of Field Grown Soybean Varieties to Rhizobia Inoculant in Western Ethiopia.
European Journal of Applied Sciences, Vol - 12(2). 309-321.
URL: http://dx.doi.org/10.14738/aivp.122.16831
and seed yields of different soybean varieties across smallholder farms (two sites) representing
different soil fertility status in two cropping seasons.
MATERIAL AND METHODS
Study Areas
The study was carried out under field conditions at Assosa and Bambasi districts for two
consecutive cropping years, 2015 and 2016. Assosa is located about 670 km west of Addis
Ababa, the capital city of Ethiopia. It is a capital city of ‘Benishangul Gumuz’ Region State of
Ethiopia and lies on altitude of 1,480 m above sea level, and located at 09°58’41.7” N,
034°38’09.5” E coordinates. While Bambasi is located 45 km far from Assosa town
(administrative town of the region) at an elevation range of 1300 to 1470 m.a.s.l and located at
10°02’17” N, 034°54’24” E. The dominant soil types at Assosa and Bambasi districts are Dystric
Nitosols, which are poor to medium in organic matter and poor nutrients as a result of the long
cropping history without replenishment of nutrients. The farmer’s fields in the two locations
were selected based on management history (previously had with cereal plantation) and had
no history of rhizobia inoculation.
Soil Physico-Chemical Analysis
Composite soil samples were collected from 0 to 20 cm depth using a soil auger from each site
2 weeks before planting for laboratory physico-chemical analysis. Particle size distribution was
analyzed using Bouyoucos hydrometer method [38]. Soil pH was measured in the supernatant
suspension of 1:2.5 soil and water mixture using a pH meter. Soil organic carbon was
determined using the Walkley and Black, chromic acid wet oxidation method [39]. Total N of
the soils was determined through digestion, distillation, and titration procedures of the Micro- Kjeldahl method as described by Nelson and Sommers [40]. Available phosphorus was
measured using Bray II method [41]. Cation Exchange Capacity (CEC) was determined by
leaching the soil using ammonium acetate buffered at pH 7 [42] and after extraction, the
remaining filtrate was measured for exchangeable K using a flame photometer.
Soil Analysis and Climatic Condition of Sites for The Experiment
The locations where the experiment was conducted were tend to differ in fertility status (Table
4). Both of the experimental sites had sandy clay loam in texture. However, the average soil pH
for Assosa and Bambasi were 5.38 and 5.92, respectively, which are characteristic of weathered
soils, Nitisols. The average organic carbon (OC) and total nitrogen (N) were relatively high in
Bambasi than Assosa.
Although soil N value was tending low at Assosa, but still both parameter’s value falls in the
moderate range for both sites according to the ratings suggested by Tekalign et al. [24]. In
addition, soil available P (Bray-II) was less than 20 ppm and rating low for Assosa in accordance
with Horneck et al. [25], while moderate for Bamabasi. Similarly, cation exchange capacity
(CEC) and exchangeable K were low according to Horneck et al. [25] for Assosa and moderate
for Bambasi. Generally, the fertility status especially average total N and available P at Assosa
were low and maybe not optimal for crop production could be characterized as poor compared
to Bambasi (Table 1).
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Table 1: Some selected physico-chemical properties of soils for the experimental sites
before planting
Type of soil analysis Location
Assosa Bambasi
Soil particle size (texture) Silt clay loam Clay loam
pH (1:2.5, soil to water) 5.38 5.92
OC (%) 1.96 2.14
Total N (%) 0.12 0.19
Bray-II available P (mg kg-1
) 13.85 21.23
Exchangeable K (g kg-1
) 7.27 18.95
CEC (meq 100g-1
) 16.33 21.54
The locations were existed on the same agro-ecology zones (we consider both locations had
relatively similar precipitation), but they were different in soil fertility status. However,
differences in seasonal distributions of rainfall and temperature in the experimental locations
influenced soybean growth. The mean monthly temperature ranged from 19.5 to 26.5oC for 2015
cropping year and from 18.0 to 25.0oC for 2016. The average of monthly air temperature in
2015 experimental year tends to be higher (>20°C) than that of 2016. In 2016 cropping season,
the temperature tends to be followed a linear pattern, optimal for soybean growth. Whereas,
total amount of precipitation during the growing period in 2016 was much higher than 2015.
In the experimental site, the mean monthly rainfall ranged from 12-195 mm in 2015 and 10-
245 mm in 2016. The number of rain days in a month varied more in 2015 (some erratic rainfall
during the cropping season), while in 2016 a constant pattern of rainfall distribution was
observed (Fig. 1).
Fig. 1: Climatic condition during different cropping seasons in the study area
Experimental Design and Treatments
The study consisted of a factorial combination of three strains of Rhizobium (MAR-1495, SB-12
and SB-14) along with one uninoculated treatment and three varieties of soybean (Wello,
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Gebrehana, Z. G., Misgana, M., & Anbessa, B. (2024). Symbiotic Response of Field Grown Soybean Varieties to Rhizobia Inoculant in Western Ethiopia.
European Journal of Applied Sciences, Vol - 12(2). 309-321.
URL: http://dx.doi.org/10.14738/aivp.122.16831
Gishema and Belessa-95). All Rhizobial isolates were obtained from Holeta Agricultural
Research Center, soil microbiology laboratory. The experiment was laid out in a randomized
complete block design (RCBD) with three replications. A starter dose of nitrogen at 18 kg N ha- 1 was applied in the form of urea in each plot, whereas Triple super phosphate (TSP) fertilizer
was applied (near the root zone to control P- fixation in these acidic soils) to all the plots at the
rate of 46 kg P2O5 ha-1. The size of each plot was 4.2m × 3.0m (12.6m2). The plots were kept
0.5m apart with 1m spacing between blocks. Soybean seeds for the three varieties were
selected based on size and healthiness (able to shoot). Then the seeds were weighed on balance
and all the weighed seeds were surface sterilized by soaking them first with 70 % (v/v) ethanol
for 10 seconds and 4 % (v/v) sodium hypochlorite (NaOCl) solution for five minutes and later
washed five times with sterilized water as indicated in Somasegaran and Hoben [43]. Each
strain was applied at the rate of 10g peat-based powder inocula per 1kg of seed (Solomon et al.,
2012). Seeds were moistened in sugar solution (10%) before application of inoculums to get a
thin uniform coating of inoculums on seeds immediate before sowing. The sugar slurry was
gently mixed with dry seed and then with Carrier-based inoculant so that all the seeds received
a thin coating of the inoculant. The inoculated and uninoculated seeds of the varieties were then
planted at a spacing of 5cm between plants and 60cm between rows making 7 rows per plot.
Soybean Varieties and Rhizobium Strains Used
Released and registered soybean varieties were obtained from crop research, Assosa
agricultural research center, EIAR, Ethiopia. Three soybean varieties such as Belessa-95 (PR- 149), a late maturing variety and Gishema (PR-149-(26)) and Wello (TGX-1895-33F), a medium
maturing variety [44] were selected to compare their symbiotic performance to rhizobia
inoculation. Commercial rhizobial inoculants such as Mar-1495, SB-12 and SB-14 were
obtained from Holeta agricultural research center, EIAR, Ethiopia.
Data Collection
Nodulation (nodule number and dry weight) and shoot dry weight data were collected at the
mid-flowering stage of soybean. Five representative plants were randomly taken from the
second border row on each side of the plot for nodule number, nodule dry weight, and shoot
dry weight and plant height measurements. Five plants were sampled randomly at
physiological maturity from each plot, pods were counted for all the five plants, and the average
value was reported as number of pods per plant. The number of seeds per pod was determined
from 20 pods randomly sampled from the five sample plants. Soybean plants from the central
five rows were harvested, the moisture was adjusted to 10% seed moisture level and weighed
to determine the seed yield.
Statistical Analysis
The analysis of variance was carried out using general linear model (GLM) procedure provided
by SAS statistical software version 9.4. Combined analysis of variance between years was
computed with years considered random, whereas genotype and inoculation were considered
fixed. All main effects and their interactions were determined via F- tests and means were
separated using least significant difference (LSD) (p < 0.05). Combined analysis across locations
and cropping seasons was performed. Data were analyzed separately for both cropping years.
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RESULTS
Combined ANOVA Analysis
Combined ANOVA showed that year, year x Rhizobial strain and variety interaction significantly
(P < 0.05) affected yield of soybean. On the other hand, both main effect of Rhizobial strain as
well as Rhizobial strain by variety interaction effect showed non-significant on nodulation,
growth and yield of soybean (Table 2). However, the effect of the year was more dominant than
all the other factors as the years differentiated in terms of mean seasonal rainfall and
temperature. Since year showed highly significant for all parameters recorded, therefore
nodulation, growth and soybean yield data were analyzed and reported for each cropping year.
Table 2: Combined ANOVA for effect of soybean varieties inoculated with different
strain of Rhizobium on nodulation, growth and yield of soybean at Assosa and Bambasi
districts in 2015 and 2016 cropping season
Source of variation NN
(No/plt.)
NDW
(g/plt.)
SDW
(g/plt.)
PH
(cm)
PPP SPP GY
(kg/ha)
Loc 4.13NS 0.13NS 3.14NS 1.02NS 658.8* 43.2NS 147.4NS
Year 68.6*** 5.63* 43.7*** 464.9*** 413.3*** 17.4*** 20.4***
Loc x Year 0.19NS 0.36NS 3.94NS 42.5*** 0.21NS 0.29NS 0.53NS
Rhizobial (R) 1.22NS 1.85NS 0.29NS 1.17NS 0.83NS 0.84NS 2.04NS
Loc x R 2.40NS 5.82NS 0.26NS 1.08NS 1.30 NS 3.06NS 0.78NS
Year x R 17.9*** 7.15*** 6.95*** 0.69NS 9.99*** 1.29NS 5.46**
Loc x Year x R 0.37NS 0.77NS 1.45NS 0.54NS 3.22* 0.31NS 1.24NS
Variety (V) 0.21NS 0.08NS 1.33NS 0.71NS 1.45NS 1.90NS 45.3*
Loc x V 13.0NS 0.72NS 2.16NS 0.92NS 3.52 NS 0.40NS 0.83NS
Year x V 33.1** 3.88* 2.23NS 12.0*** 4.88** 1.11NS 0.16NS
Loc xYear x V 0.11NS 1.20NS 0.08NS 0.97NS 1.04NS 4.04* 2.19NS
R x V 0.84NS 2.14NS 3.34NS 0.39NS 0.40NS 1.03NS 1.50NS
Loc x R x V 0.39NS 0.46NS 0.76NS 0.86NS 0.98NS 0.55NS 2.55NS
Year x R x V 1.18NS 0.77NS 0.38NS 2.26NS 3.24** 0.66NS 1.05NS
Loc xYear x R x V 0.43NS 0.42NS 1.14NS 0.90NS 2.37* 0.77NS 1.69NS
CV(%) 49.29 52.88 23.07 8.20 19.12 6.94 20.08
NN, nodule number; NDW, nodule dry weight; SDW, shoot dry weight; PH, plant height; PPP, pod per plant; SPP,
seed per pod; SY, seed yield, NS, non-significant (p > 0.05) *significant (p < 0.05), **highly significant (p < 0.001),
***extremely significant (p < 0.0001).
Nodulation and Growth of Soybean Varieties
Analysis of variance has shown that locations did not significantly (p < 0.05) differ in nodulation
performance during 2015 cropping season. However, significantly an improved nodule dry
weight was obtained at Bambasi than Assosa during 2016 cropping season. On the other hand,
growth of soybean was not significantly affected by location of the experiment in 2016, while
significantly an improved SDW and plant height was obtained at Bambasi than Assosa in 2015.
Except SDW, nodulation parameters and plant height were not significantly influenced by
variety of soybean used in our experiment in 2015. Variety Gishema had significantly higher
shoot biomass than Wello and Belessa-95 (Table 2). In 2016 cropping season, variety positively
affected nodule dry weight and growth performance but had no significant effect on nodule
number. The highest nodule and shot dry weight as well as average plant height in 2016 was
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Gebrehana, Z. G., Misgana, M., & Anbessa, B. (2024). Symbiotic Response of Field Grown Soybean Varieties to Rhizobia Inoculant in Western Ethiopia.
European Journal of Applied Sciences, Vol - 12(2). 309-321.
URL: http://dx.doi.org/10.14738/aivp.122.16831
observed in Gishema variety, followed by Belessa-95 and Wello. Rhizobia inoculation did not
significantly improve shot dry weight and plant height in both seasons. But nodulation was
significantly affected by Rhizobia inoculation in 2016 cropping season. Thus, all Rhizobia strains
significantly improved nodule number and dry weight of soybean than the uninoculated
soybean plants (Table 3).
Table 3: Effect of soybean variety and Rhizobial strain on nodulation and growth of
soybean at Assosa and Bambasi districts in 2015 and 2016 cropping years
Treatment Year 2015 Year 2016
NN
(No/plt.)
NDW
(g/plt.)
SDW
(g/plt.)
PH
(cm)
NN
(No/plt.)
NDW
(g/plt.)
SDW
(g/plt.)
PH
(cm)
Location
Assosa 15.41 0.536 8.04b 65.85b 44.11 0.333b 6.88 53.68
Bambasi 23.87 0.479 10.75a 80.45a 46.41 0.455a 7.56 53.60
LSD NS NS 2.47 5.17 NS 0.098 NS NS
Variety (V)
Gishema 18.89 0.456 9.94a 73.08 49.59 0.470a 7.44a 57.54a
Wello 22.28 0.535 9.16b 73.78 38.06 0.333b 6.38b 48.25c
Belessa-95 17.74 0.531 9.07b 72.61 48.14 0.380ab 7.84a 55.25b
LSD NS NS 0.74 NS NS 0.09 0.981 1.19
Rhizobial strain (R)
Uninoculated 10.16b 0.309 9.01 73.30 9.69b 0.115b 5.57 52.05
MAR-1495 23.94a 0.685 9.88 73.30 63.86a 0.546a 7.66 55.00
SB-12 25.04a 0.563 9.43 71.85 55.75a 0.431a 7.88 52.50
SB-14 19.40ab 0.472 9.27 74.17 52.76a 0.471a 7.79 54.50
LSD 9.72 NS NS NS 19.84 0.268 NS NS
F-test value
Loc 0.50NS 0.04NS 9.27* 61.37** 0.36NS 10.75* 3.82NS 0.01NS
Rhizobium (R) 9.82* 1.91NS 3.64NS 0.58NS 32.18** 10.74* 5.50NS 2.10NS
Loc x R 0.61NS 2.46NS 0.51NS 1.14NS 0.87NS 3.06* 2.96* 0.01NS
Variety (V) 3.07NS 0.37NS 15.18* 0.17NS 3.54NS 18.98* 23.11* 21.20***
Loc x V 0.31NS 1.33NS 0.06NS 1.96NS 0.78NS 0.27NS 0.44NS 0.01NS
R x V 0.71NS 5.35* 0.89NS 0.33NS 6.44* 2.24NS 0.86NS 27.8***
CV (%) 59.92 60.93 26.15 6.89 43.00 35.65 16.45 9.96
Inoculation of Rhizobial Strains on Agronomic Parameters of Soybean Varieties
Analysis of variance showed soybean yield and yield components significantly (p < 0.05) differ
between two locations in both cropping seasons (Table 4). Soybean yield had shown
significantly higher performance at Bambasi than Assosa. Soybean seed yield was significantly
affected by soybean varieties in both growing seasons and Gishema and Belessa-95 soybean
varieties had significantly higher seed yield than Wello. Strong responses to inoculation were
obtained in both cropping seasons and inoculation of soybean using Rhizobial strains
significantly increased pod per plant and seed yield of soybean. Inoculation of SB-14 showed
significantly high yield and increased seed yield by 27.8 % over non-inoculated treatments in
2015 cropping season. While in 2016 cropping season, inoculation of soybean using MAR-1495
significantly resulted in the highest seed yield which increased yield by 44.6% over non- inoculated control (Table 4). However, the results showed that the interaction effects between