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Brazilian Semiarid Bradyrhizobium Strains Increase Nitrogen Accumulation

and Modulate Physiological Parameters to Enable Superior Grain Production
in Soybean Genotypes

Article in Journal of Soil Science and Plant Nutrition · February 2023

DOI: 10.1007/s42729-023-01186-7

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Journal of Soil Science and Plant Nutrition
https://doi.org/10.1007/s42729-023-01186-7

SHORT COMMUNICATION

Brazilian Semiarid Bradyrhizobium Strains Increase Nitrogen

Accumulation and Modulate Physiological Parameters to Enable
Superior Grain Production in Soybean Genotypes
Luis Borges Rocha1 · Elaine Martins da Costa1 · Ana Clara Martins de Sousa Ribeiro1 ·
Jasmine Ferreira dos Santos1 · Rafael de Souza Miranda1 · Amanda Soares Santos1 ·
Stelamaris de Oliveira Paula‑Marinho1 · Bruno Sousa Figueiredo da Fonseca1 ·
Fatima Maria de Souza Moreira2

Received: 6 September 2022 / Accepted: 11 February 2023

© The Author(s) under exclusive licence to Sociedad Chilena de la Ciencia del Suelo 2023

Abstract
The selection of rhizobia for soybean is a sustainable strategy to increase the yield of this crop. The aim of this study was
to investigate the efficiency of Bradyrhizobium strains for nitrogen fixation and modulation of physiological parameters and
grain production in inoculated soybean genotypes. The experiment was carried out with three soybean genotypes (M8349
IPRO, M8372 IPRO, and Bônus 8579 RSF) and six nitrogen (N) sources (UFLA06-19, UFLA06-22, UFLA06-24, a com-
mercial inoculant, and two controls without inoculation (without and with nitrogen)). The UFLA strains increased nitrogen
accumulation in the shoots of the M8372 IPRO genotype and modulated photosynthetic pigments and soluble carbohydrates
contents in symbiosis with the M8349 IPRO genotype. Strains UFLA06-22 and UFLA06-24 were efficient in grain nitro-
gen accumulation in genotypes M8372 IPRO and M8349 IPRO. Principal component analysis shows the close positioning
of several parameters in soybean genotypes inoculated with these strains. Our results demonstrate the potential of strains
UFLA06-22 and UFLA06-24 for sustainable soybean production.

Keywords Glycine max L. · Plant performance · Rhizobia selection · Symbiosis

1 Introduction biological nitrogen fixation (BNF) has been considered for

Soybean (Glycine max L.) produces more than 100 million
tons of grain annually worldwide for human and animal con-
sumption. Nevertheless, the crop has high nutrient require-
ments, especially nitrogen (N) (Salvagiotti et al. 2021; Zilli
et al. 2021). Therefore, the development of biotechnological
tools is crucial to economically increase grain production,
protect the environment, and create sustainable cropping
systems. Among these technologies, the use of inoculants
that can meet all the nitrogen needs of legumes through
* Elaine Martins da Costa
elaine.costa@ufpi.edu.br
1
Universidade Federal Do Piauí, Campus Professora
Cinobelina Elvas, Av. Manoel Gracindo, Km 1, Planalto
Horizonte, Bom Jesus, PI 64900‑000, Brazil
2 northeastern Brazil, which displayed elevated efficiency in
Departamento de Ciência Do Solo, Universidade Federal de
Lavras/UFLA, Caixa Postal 3037, Lavras, MG 37200‑000,
Brazil
production systems, especially for soybeans.
Legumes inoculated with Bradyrhizobium strains showed
increased N content and accumulation in tissues, a response
closely related to chlorophyll and carotenoid content,
increased photosynthetic efficiency, and accumulation of
soluble carbohydrates, as reported in soybean (Cagide et al.
2018; Mongolo Júnior et al. 2021; Sheteiwy et al. 2021) and
cowpea (Mendes et al. 2020) plants. Inoculation of soybean
with Bradyrhizobium strains can efficiently meet the high
nitrogen demand of soybean plants (Zilli et al. 2021). Stud-
ies focusing on the selection of strains with efficiency in
­N2 fixation with different soybean genotypes are critical for
adequate BNF, N economics, and agricultural production
of soybean.
In a previous study, our research group isolated some
native Bradyrhizobium strains from the semiarid region of
symbiosis with soybean (Monsoy 7980 genotype) under
axenic conditions with nutrient solution (Ribeiro et al.

13
Vol.:(0123456789)

2015). Among these strains, UFLA06-19 and UFLA06-
22 (classified as Bradyrhizobium brasilense by Costa et al.
2020) and UFLA06-24 (classified as Bradyrhizobium sp.
by Ribeiro et al. 2015) showed high efficiency in fixing
­N2 in soybean (FT4280 IPRO genotype) on nonsterile soil
(Rocha et al. 2021). Nevertheless, the evidence of how these
strains have potential for BNF in other soybean genotypes
to be recommended as inoculants remains to be uncovered.
Numerous soybean genotypes are adapted and widely used
in the Brazilian semiarid region, such as M8349 IPRO,
M8372 IPRO, and Bônus 8579 RSF. Thus, research aim-
ing to select Bradyrhizobium strains for inoculation with
semiarid-adapted genotypes is crucial to optimize BNF in
semiarid conditions.
Our working hypothesis was that strains UFLA06-19,
UFLA06-22, and UFLA06-24 promote increased BNF
and modulate physiological and productive performance in
three semiarid-adapted soybean genotypes (M8349 IPRO,
M8372 IPRO, and Bônus 8579 RSF). Inoculation tests, plant
growth, grain production, and some biochemical and physi-
ological indicators were studied to determine the relation-
ship between Brazilian semiarid Bradyrhizobium strains and
soybean performance.
2 Materials and Methods
The experiment was conducted in a greenhouse of the Fed-
eral University of Piauí, Campus Professora Cinobelina
Elvas, in plastic pots (5 ­dm−3) with nonsterile soil (Oxisol)
samples collected in an original forest area (9°13′28.9344″S
and 44°44′44.4192″W), Bom Jesus, Piauí, Brazil. The exper-
iment had a completely randomized design in a 6 × 3 facto-
rial scheme with 6 replicates. The first factor consisted of
three Bradyrhizobium strains (UFLA 06–19, UFLA 06–22,
and UFLA 06–24); a commercial inoculant recommended
for soybean, consisting of strains SEMIA 5079 (B. japoni-
cum) and SEMIA 5080 (B. diazoefficiens); and two nega-
tive controls, one without inoculant or mineral nitrogen (N)
(without N) and the other without inoculant and with N at
300 mg ­dm−3 (with N). The second factor corresponded to
three soybean genotypes adapted to the semiarid region,
M8349 IPRO, M8372 IPRO, and Bônus 8579 RSF.
The soil was corrected by increasing the base saturation
to 60% with limestone. Fertilization with phosphate, potas-
sium, and micronutrients was performed according to the
results of chemical analysis of the soil. The seeds were dis-
infected in 70% alcohol for 30 s, washed with 2% sodium
hypochlorite for 2 min, and then washed with sterile water.
Four seeds per pot were sown, and thinning was performed
5 days after emergence, leaving only 2 plants per pot. When
inoculated with UFLA strains, 1.0 mL of log phase cultures
(approximately 1 × ­108 cells ­mL−1) in liquid 79 medium
13
Journal of Soil Science and Plant Nutrition
(Fred and Waksman 1928) was added per seed. When treated
with commercial inoculants, 1.0 mL of the liquid inocu-
lant was inoculated per seed at a minimum concentration of
approximately 5 × ­109 cells ­mL−1.
At the beginning of flowering, three plants were harvested
to measure nodule number per plant (NN), nodule dry mass
(NDM), shoot dry mass (SDM), shoot nitrogen accumula-
tion (SNA), and relative efficiency (RE). Nodules and shoots
were placed in a forced-air oven at 65 °C until they reached a
constant mass. The N content of the shoots was determined
by the semi-micro Kjeldahl method (Liao 1981), while
the SNA was calculated by multiplying the SDM (g) and
the respective N content in the shoots (%)/100. The rela-
tive efficiency was calculated according to the expression
RE = (SDM of each treatment)*100/(SDM of the treatment
with mineral N). The contents of chlorophyll a, b, and total,
carotenoids, and soluble carbohydrates (SC) were also meas-
ured. Photosynthetic pigments were measured according to
Wellburn (1994), and SC was measured according to Dubois
et al. (1956).
At physiological maturity, three plants were harvested
to determine the number of grains per plot (NG), grain dry
mass (GDM), grain nitrogen content (GNC), grain nitro-
gen accumulation (GNA), and grain crude protein content
(CPC). GNC was determined by the semi-micro Kjeldahl
method (Liao 1981) and used to calculate GNA with the
formula (GDM * GNC)/100. NG was determined by count-
ing the total grains in each plot. Grains were placed in a
forced-air oven at 65 °C until they reached a constant mass.
CPC was determined using GNC and a conversion factor
(6.25) as described by Liao (1981).
Data were classified as normal according to the Shap-
iro‒Wilk test and then subjected to an analysis of variance
using the Sisvar 5.7 statistical analysis system (Ferreira
2019). Means were compared using the Scott‒Knott test
with a probability of 5%. For the variables NN and NDM,
the data were transformed by the square root of (Y + 0.5).
The data were also subjected to multivariate analysis by the
principal component method (PCA) using the SPSS statisti-
cal program.
3 Results and Discussion
There was no interaction between factors for the variables
NN and NDM, but there was an isolated effect of N sources
and genotypes (Table 1). Soybean plants inoculated with
strains UFLA06-19, UFLA06-22, and UFLA06-24 had
higher NN and NDM values than plants inoculated with a
commercial inoculant and the two controls without inoculant
application.
A significant interaction between factors was found
for the variables SDM, RE, SNA, chlorophyll a, b, total,
Journal of Soil Science and Plant Nutrition

Table 1  Mean values of the NN number of nodules and NDM nodule carotenoids, and total soluble carbohydrate contents
dry mass from three soybean genotypes grown with different nitrogen (Table 2). In general, the genotypes M8372 IPRO and Bônus
sources in an Oxisol from a semiarid region
8579 RSF showed the highest SDM and RE values under
Factors NN NDM inoculation with UFLA06-19, UFLA06-22, and UFLA06-
N° ­plant−1 mg ­plant−1 24 compared to the treatments with commercial inoculant,
Sources of N with and without N, except for the commercial inoculant
UFLA06-19 128.11 A 165.25 A in M8372 IPRO. All UFLA strains promoted higher mean
UFLA06-22 156.88 A 181.77 A SNA values than the commercial inoculant and the control
UFLA06-24 121.50 A 132.14 A without N in genotype M8372 IPRO, with SNA increases
Commercial 38.23 B 65.54 B between 27.33 and 32.31% compared to the commercial
Without N 16.39 C 31.00 C inoculant.
With N 0.00 D 0.00 D The accumulation of photosynthetic pigments varied
Genotypes according to UFLA strain, N fertilization, and soybean
M8349 IPRO 87.64 A 85.81 B genotype (Table 2). Nonetheless, in all soybean genotypes,
M8372 IPRO 84.58 A 119.30 A inoculation with UFLA06-24 resulted in increased soluble
Bônus 8579 RSF 58.33 B 82.75 B carbohydrate (SC) content, with levels similar to those in
CV (%) 25.40 27.44 plants treated with a commercial inoculant and higher than
those in controls with and without N, except in the con-
Means followed by the same uppercase letter in columns within nitro- trol with N in genotype M8372 IPRO. Strain UFLA06-22
gen sources and genotypes do not differ statistically by the Scott‒
Knott test (p < 0.05) also stimulated the content of SC in the M8372 IPRO and
Commercial SEMIA 5079 + SEMIA 5080, Without N without inocu-
Bônus 8579 RSF genotypes, showing similar results to strain
lation and without mineral nitrogen, With N without inoculation and UFLA06-24 and the commercial inoculant.
with mineral nitrogen at 300 mg ­dm−3

Table 2  Mean values of SDM shoot dry mass, RE relative efficiency, phyll b, Chl t chlorophyll total, Carot carotenoid, and soluble carbo-
SNA shoot nitrogen accumulation, Chl a chlorophyll a, Chl b chloro- hydrate contents from three soybean genotypes grown with different
nitrogen sources in an Oxisol from a semiarid region
Genotypes Sources of N SDM RE SNA Chl a Chl b Chl t Carot Carbohydrates
g ­plant−1 % mg ­plant−1 µg g ­DM−1 µg g ­DM−1 µg g ­DM−1 µg g ­DM−1 µmol g ­DM−1

M8349 IPRO UFLA06-19 3.28 A b 99.39 A b 72.96 B b 20.03 A a 7.70 D a 27.73 C a 5.04 C a 189.57 C a
UFLA06-22 3.29 A b 99.83 A b 82.08 B a 20.44 A a 9.23 C a 29.67 C a 5.37 B b 200.87 B a
UFLA06-24 3.01 A b 90.15 A b 74.41 B a 20.72 A a 11.74 B a 32.46 B a 5.92 A a 215.65 A a
Commercial 2.90 A b 87.93 A b 83.20 B a 20.30 A a 9.68 C a 29.98 C a 5.10 C a 211.11 A a
Without N 3.02 A a 91.65 A a 75.80 B a 18.84 B b 6.43 D a 25.27 D b 4.71 D b 178. 21 D a
With N 3.31 A a 100.00 A a 154.55 A a 20.40 A a 15.30 A a 35.70 A a 5.65 A a 193.90 C b
M8372 IPRO UFLA06-19 4.34 A a 153.19 A a 95.78 B a 19.86 B a 8.17 B a 28.03 B a 5.27 B a 185.56 B a
UFLA06-22 4.12 A a 146.82 A a 92.18 B a 19.43 B a 7.01 B a 26.45 B b 4.85 C c 207.25 A a
UFLA06-24 3.78 A a 134.57 A a 92.66 B a 21.43 A a 11.80 A a 33.24 A a 5.76 A a 207.45 A a
Commercial 3.72 A a 131.80 A a 72.39 C a 20.28 B a 8.79 B a 29.07 B a 5.33 B a 202.10 A a
Without N 3.19 B a 112.22 B a 73.24 C a 20.26 B a 8.46 B a 28.71 B a 5.17 B a 185.79 B a
With N 2.85 B a 100.00 B a 118.92 A b 20.58 B a 11.72 A b 32.31 A b 5.45 A a 202.24 A a
Bônus 8579 RSF UFLA06-19 3.13 A b 135.12 A a 74.75 B b 18.21 B b 5.63 C b 23.84 B b 4.21 D b 195.80 B a
UFLA06-22 3.10 A b 134.48 A a 77.72 B a 20.11 A a 8.24 B a 28.49 A a 5.71 A a 213.77 A a
UFLA06-24 3.68 A a 158.45 A a 91.08 A a 19.94 A b 7.77 B b 27.70 A b 5.22 B b 213.33 A a
Commercial 2.83 B b 123.99 B a 61.88 B a 16.62 C b 4.54 C b 21.16 C b 3.82 E b 203.19 A a
Without N 2.62 B a 115.76 B a 69.65 B a 19.32 A b 7.16 B a 26.48 A b 4.68 C b 187.12 B a
With N 2.32 B b 100.00 B a 88.89 A c 20.00 A a 9.72 A c 29.71 A c 4.73 C b 185.69 B b
CV% 12.52 13.65 13.60 2.55 11.85 4.54 3.88 3.25

Means followed by the same uppercase letter in the columns and within each soybean genotype and lowercase letter in the columns between the
soybean genotypes do not differ statistically by the Scott‒Knott test (p < 0.05)
Commercial SEMIA 5079 + SEMIA 5080, Without N without inoculation and without mineral nitrogen, With N without inoculation and with
mineral nitrogen at 300 mg d­ m−3

13
 Journal of Soil Science and Plant Nutrition

There was a significant interaction between N sources and
genotypes for all variables evaluated at the end of the plant
cycle (Table 3). UFLA strains were able to increase plant
production (NG and GDM), and this response depended on
the soybean genotype. The M8349 IPRO genotype exhibited
the highest NG when inoculated with strain UFLA06-22.
The regulation of GNC was similar to that of CPC. In all
cases, strains UFLA06-22 and UFLA06-24 promoted the
highest GNC and CPC values for all soybean genotypes
compared to other N sources. The CPC values of plants
inoculated with strains UFLA06-22 and UFLA06-24 were
24 and 32% (M8349 IPRO), 23 and 42% (M8372 IPRO), and
40 and 18% (Bônus 8579 RSF) higher than those of plants
inoculated with N, respectively. Consequently, modulation
of GNC and CPC was directly related to the regulation of
grain nitrogen accumulation (GNA).
The similar and different responses of soybean genotypes
grown with different N sources can be shown in the principal
component analysis (PCA). The first two PCs of the biplot
graph explained 64.1% of the total variance, with 32.6 and
31.5% explained by PC1 and PC2, respectively (Fig. 1). Two
groups were divided. The first group on the right side of
the plot consisted of the inoculation treatments with strains
UFLA06-19, UFLA06-22, and UFLA06-24, with the excep-
tion of Bônus 8579 RSF and M8349 IPRO, which were
inoculated with UFLA06-19 and formed the second group.
The performance of the soybean genotypes inoculated with
UFLA strains in the first group was closely related to NN,
NDM, SDM, RE, SC, NG, GDM, CPC, and GNA.
Selection of Bradyrhizobium strains with high perfor-
mance in ­N2 fixation in symbiosis with different soybean
genotypes is critical for growing this crop to increase yield
and meet population needs. Inoculants can also replace min-
eral nitrogen fertilization and provide ecological benefits
that make farming systems more sustainable. In our study,
we selected Bradyrhizobium strains from the semiarid region
of northeastern Brazil with high efficiency in N­ 2 fixation in
different soybean genotypes adapted to semiarid regions. All
three UFLA strains exhibited elevated NN and NDM values,
demonstrating the competitive capacity of the strains. Con-
sequently, positive responses to inoculations with the three
UFLA strains were observed for SDM, RE, and SNA in the
M8372 IPRO genotype and for SDM and RE in the Bônus
8579 RSF genotype, indicating the efficiency of these strains
in symbiosis with soybean.
Nitrogen is widely required by plants as a component
of several organic compounds and has specific functions in
plant metabolism and yield. Our results clearly demonstrate
that UFLA strains can provide N for photosynthetic pig-
ment biosynthesis and accumulation in soybean plants, a
response that depends on genotype (Table 2). The increased
content of carotenoids could promote photoprotection and

Table 3  Mean values of the Genotypes Sources of N NG GDM GNC GNA CPC
NG number of grains per plant, grain ­plant−1 g ­plant−1 % g ­plant−1 %
GDM grain dry mass, GNC
grain nitrogen content, GNA M8349 IPRO UFLA06-19 127.00 B b 18.29 B a 4.49 C a 82.33 B a 28.09 C a
grain nitrogen accumulation,
UFLA06-22 164.33 A a 22.66 A a 5.43 B b 122.67 A a 33.95 B b
and CPC crude protein content
from three soybean genotypes UFLA06-24 143.00 B a 21.59 A a 5.78 A b 125.00 A a 36.16 A b
grown with different nitrogen Commercial 115.66 C b 17.00 B a 4.44 C a 75.67 B a 27.77 C a
sources in an Oxisol from a Without N 143.00 B a 19.26 B a 4.64 C a 89.66 B a 29.03 C a
semiarid region
With N 83.50 D b 11.83 C b 4.36 C a 51.66 C b 27.28 C a
M8372 IPRO UFLA06-19 169.33 A a 18.82 A a 4.55 C a 86.00 B a 28.46 C a
UFLA06-22 167.66 A a 22.74 A a 5.72 B a 130.00 A a 35.77 B a
UFLA06-24 161.00 A a 21.34 A a 6.50 A a 139.66 A a 40.62 A a
Commercial 126.50 B b 19.64 A a 4.61 C a 90.67 B a 28.84 C a
Without N 116.50 B b 15.08 B b 4.64 C a 70.00 C b 29.03 C a
With N 169.00 A a 21.17 A a 4.56 C a 97.00 B a 28.55 C a
Bônus 8579 RSF UFLA06-19 144.00 A b 21.70 A a 4.09 D b 88.66 B a 25.60 D b
UFLA06-22 138.33 A b 20.18 A a 5.94 A a 126.00 A a 37.71 A a
UFLA06-24 151.33 A a 20.27 A a 5.06 B c 102.66 B b 31.62 B c
Commercial 151.66 A a 17.22 A a 4.72 C a 81.33 B a 29.54 C a
Without N 147.66 A a 21.21 A a 4.73 C a 100.32 B a 29.58 C a
With N 149.00 A a 23.36 A a 4.30 D a 100.45 B a 26.85 D a
CV% 8.62 11.52 3.32 11.72 3.31

Means followed by the same uppercase letter in columns and within each soybean genotype and lowercase
letter in columns between soybean genotypes do not differ statistically by the Scott‒Knott test (p < 0.05)
Commercial SEMIA 5079 + SEMIA 5080, Without N without inoculation and without mineral nitrogen,
With N without inoculation and with mineral nitrogen at 300 mg d­ m−3

13
Journal of Soil Science and Plant Nutrition

Fig. 1  Principal component

analysis (PCA) of the studied
variables for Bônus 8579,
M8349 IPRO, and M8372 IPRO
soybean genotypes subjected to
different nitrogen (N) sources:
inoculated with UFLA 06–19,
UFLA 06–22, and UFLA
06–24; inoculated with com-
mercial inoculant; without N;
and with N. The percent vari-
ance for each PC is specified on
axes X and Y. PCA biplot pre-
sents the loading of variables:
NN number of nodules, NDM
nodule dry mass, SDM shoot
dry mass, RE relative efficiency,
SNA shoot nitrogen accumula-
tion, Chl a chlorophyll a, Chl
b chlorophyll b, Chl t chloro-
phyll total, Carot carotenoids,
carbohydrate, NG number of
grains per plant, GDM grain
dry mass, GNC grain nitrogen
content, GNA grain nitrogen
accumulation, and CPC crude
protein content

provide precursors for the biosynthesis of phytohormones
abscisic acid and strigolactones, regulating cell develop-
ment, which plays an extremely important role in plant per-
formance (Nisar et al. 2015; Sun et al. 2022). Additionally,
the increased chlorophyll content in plants inoculated with
strains UFLA06-22 and UFLA06-24 indicates an enhanced
capacity for photochemical processes and photosynthetic
performance, as previously reported in soybean inoculated
with Bradyrhizobium (Cagide et al. 2018; Sheteiwy et al.
2021). This fact is confirmed by the increased accumulation
of soluble carbohydrates in inoculated plants and biomass
accumulation (Table 2), demonstrating the strong activity
of ­CO2 assimilation processes (Miranda et al. 2021). The
regulation of carbohydrate accumulation appears to be the
result of an integrative network of metabolic adaptations
stimulated by inoculation treatments, leading to improved
plant growth and productivity (Tables 2 and 3, and Fig. 1).
Variables related to production in a crop cycle, such as
the NG and GDM, clearly demonstrate the potential role of
UFLA strains in soybean production (Table 3). The M8372
IPRO genotype responded most strongly to inoculation for the
NG and GDM parameters, in which the three UFLA strains
promoted better grain production than the control without N,
which was associated with improved SDM, RE, and SNA
(Tables 2 and 3). In all cases, soybean genotypes showed rel-
evant nutrient responses under inoculation with UFLA06-22
and UFLA06-24, as evidenced by increased GNC, GNA, and
CPC values (Table 3 and Fig. 1). Therefore, these strains rep-
resent a potential alternative for the economic and sustainable
production of soybeans, especially in semiarid regions.

13

4 Conclusions
We confirmed that Brazilian semiarid Bradyrhizobium
strains (UFLA06-19, UFLA06-22, and UFLA06-24) are
efficient for modulation of physiological parameters and
shoot nitrogen accumulation in symbiosis with the M8372
IPRO soybean genotype. The UFLA06-22 and UFLA06-24
strains are efficient in nitrogen accumulation in grains of the
M8349 IPRO and M8372 IPRO genotypes. These strains
have emerged as potential inoculants for soybean plants.
Acknowledgements We thank the Coordination for the Improvement
of Higher Education Personnel (CAPES) for the scholarship provided
to Luiz Borges Rocha.
Funding The first author received support from the Coordination for
the Improvement of Higher Education Personnel (CAPES) for carrying
out the research.
Declarations
Ethics Approval Not applicable.
Consent to Participate Not applicable.
Consent for Publication Not applicable.
Conflict of Interest The authors declare no competing interests.
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