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Chapter
Inoculant Formulation and
Application Determine Nitrogen
Availability and Water Use
Efficiency in Soybean Production
Canon E.N. Savala, David Chikoye and Stephen Kyei-Boahen
Abstract
Inoculation of suitable rhizobia enhances biological nitrogen fixation in soybean
production and are economically viable for use among smallholder farmers due to its low
price over inorganic commercial fertilizer blends. In Mozambique, inoculants are
available in liquid or solid form (powder/peat or granular). Field studies were conducted
in 2017 and 2018 seasons in three agroecologies (Angonia, Nampula and Ruace) in
Mozambique to evaluate the performance of inoculants when applied directly to soil and
on seed before planting. Data on nodulation, plant growth, nitrogen fixed, 13C isotope
discrimination related water use efficiency, yield and yield components were analyzed in
Statistical Analysis System® 9.4. Nodulation, yield, and yield components were signifi-
cant for the different application methods, and solid form tended to be better than liquid
form. The nitrogen derived from atmosphere (%Ndfa) were 45.3%, 44.2% and 43.6%
with a yield of 2672, 1752 and 2246 kg ha
1 for Angonia, Nampula and Ruace, respec-
tively. Overall, inoculants applied on soil or seed increase the amount of biologically fixed
nitrogen and has the potential of improving soybean productivity in Mozambique.
Keywords: carbon isotope, nodulation, promiscuous, soybean, rhizobia, water use
efficiency, yield
1. Introduction
1.1 Inoculation history in Africa (Mozambique)
Soybean production in Mozambique is gaining pace through land area expansion at
the expense of other crops mainly driven by lucrative prices and the unsatisfied
market demand particularly the poultry industry [1]. However, climate change
effects, low soil fertility and poor crop management keep yield below the world
average. Some farmers are seeking solutions to these challenges by adopting region
adapted improved varieties, use of soil amendments such as organic manures and
inoculant application to improve nitrogen availability. Nitrogen is the most limiting
nutrient in soybean production due to its high uptake by plants, vulnerability to
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Soybean - Recent Advances in Research and Applications
leaching, denitrification and removal through crop harvest [2]. Inoculation of rhizobia
enhances biological nitrogen fixation (BNF) in soybean production and is economi-
cally viable for use among smallholder farmers due to its low price over inorganic
commercial fertilizer blends [3, 4]. Likewise, soybean producers have the quest to
improve yield which necessitates inoculation with effective rhizobial strains [5–7].
Inoculation improves soybean yield and increases crop resilience to climatic changes
effects across Africa such as drought incidences experienced in Mozambique through
better water use efficiency (WUE) [8]. Although many African countries currently
produce inoculant that is effective for both promiscuous and non-promiscuous
soybean varieties and other legumes like beans, cowpea, and groundnuts [9],
Mozambique as a country lacks the capacity and facilities for local production.
However, production volumes of these inoculants seldom satisfy in-country or
regional demand warranting importation of supplementary stocks from as far as south
America [10]. Unfortunately, produced inoculants fail to reach smallholder farmers in
Africa on time due to logistic constrains linking production and distribution. Devel-
opment of promiscuous soybean varieties, capable of fixing nitrogen with indigenous
rhizobia [11] offer a promising solution to improving BNF. In addition, advancement
in research has led to isolation of promising indigenous rhizobia that establish symbi-
otic association with soybean [12, 13]. The research was based on the notion that
African soils have indigenous rhizobia strains capable of colonizing soybean root.
Unfortunately, isolated indigenous rhizobia strains are yet to be commercialized
despite performing better than or like the well-known USDA 110 strain. Commercial
production in solid or liquid form of identified indigenous rhizobia strains is necessary
to improve their efficiency since naturally they occur in low populations in the soil
coupled with low efficacy as effective nitrogen fixers.
Inoculants can be packaged in liquid, peat, or granular forms. Only the liquid or
peat/powder forms of inoculants are found in Mozambique with the latter being more
abundant and easier to handle among producers. Both forms of inoculants can be
applied on seed or directly on soil before planting. Although both forms of inoculants
improve yield, variations in the amount tend to occur due to other factors such as
viability, storage and environment especially soil moisture in a specific site [14]. In
many cases, seed yield inoculated with liquid formula seldom gives better than the peat
inoculants. Liquid inoculants offer limited protection to the rhizobia hence survivability
can be a challenge in sub-optimal conditions [15–17] while peat carriers provide more
protection to the live cells to a limited extend as it is still important to plant the seed or
cover the soil soon after application. Bacterial cells survival on the seed or soil in
Mozambique could mainly be affected by desiccation and high temperatures [18].
The most common inoculant application method in Mozambique is on seed although
there exists a potential for soil application especially among the large-scale commercial
soybean producers who have the capacity to mechanize farm operations.
1.2 Plant nitrogen uptake
Soybeans acquire N from either BNF or soil and sometimes inorganic N fertilizer if
applied. Maximum N demand in soybean occurs between the R3 and R5 stages of
development [19]. Proportions of N absorbed from these sources differ with the
cropping system and management. Since BNF is an energy consuming process, soy-
bean will not invest in it where either the soil or fertilizer N is adequate. On the other
hand, unavailability of N from any of the sources during plant growth will result in N
translocation from other parts of the plant such as leaves to the grain, which
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diminishes the photosynthesis thus reducing yield potential [20]. Soybean plant N
derived from BNF leads to improved productivity. Nitrogen availability in soybean
production can be enhanced through inoculation. Inoculating soybean with liquid or
peat based effective rhizobia strains promotes nodulation and plant growth that con-
tribute to increased yield. Through BNF, soybean can satisfy between 50% and 60% of
its nitrogen requirement [21]. Farmers in Mozambique rarely apply external inorganic
fertilizer on soybean. Therefore, the N sources of soybean production is either soil or
BNF where inoculants are applied, or effective indigenous rhizobia strains exist in the
soil. More so, where inoculants are applied, there exists no means to quantify the
amount of N fixed in the fields other than the yield obtained. Benefits of BNF are
higher when phosphorus fertilizer is applied in addition to rhizobia inoculation on
soybean [5] or cowpea [3] in Mozambique.
1.3 Carbon isotope discrimination, water use efficiency and yield
Carbon is released from the plant through the leaves as CO2 during transpiration.
Likewise, water is lost from the plant by the same process through the stomata.
Transpiration is important in plants as it facilitates mass-flow movement of nutrients
from the roots to the above ground parts. This process is inversely correlated to
availability of soil moisture content hence affecting plant WUE [22]. WUE is the ratio
of plant dry matter production against the water used over a period. It can also be
defined at a point in time as the ratio between the rate of carbon fixation and the rate
of transpiration. 13C isotope discrimination is used to determine a fraction of carbon
isotope during CO2 uptake and fixation and related to WUE that is an important
physiological character as an indicator of plant adaptability to drought conditions
through the functioning of the stomata [23]. It is strongly linked to the ratio of the
intercellular and atmospheric concentration of CO2 (Ci/Ca) associated with stomatal
conductance and chloroplast affinity for CO2 [24]. Therefore, the intercellular and
atmospheric CO2 ratio theoretically links WUE to 13C isotope discrimination. These
relationship is useful in breeding for selection of high transpiration efficiency, and
increased and grain yield in soybean as demonstrated with wheat [25]. Kumar et al.
[26] demonstrated a positive relationship between grain yield and 13C isotope dis-
crimination and a negative one to transpiration efficiency. Since transpiration is
inverse to WUE the increase in 13C isotope discrimination and WUE lead to increase
in grain yield. In essence, 13C isotope discrimination offer a promise to selection of
criterion for high yielding drought adapted varieties. Therefore, in our study, we sort
to understand how liquid or solid inoculant affect soybean WUE and yield. Earlier
studies have reported that inoculation improves yield as it leads to more available N
from the BNF process. However, the yield increase varies with soybean varieties and
type of inoculant especially nitrogen availability even if similar strains are used [8].
The objective of this study was to evaluate soybean WUE and yield response to liquid
or solid inoculants applied to soil and on seed before planting.
2. Materials and methods
2.1 Site selection and description
Field studies using soybean variety ‘Safari’ (SeedCo. material) were conducted in
2017 and 2018 growing seasons at three locations, Nampula 15.2741° S, 39.3150° E,
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Soybean - Recent Advances in Research and Applications
365 m above sea level (m a.s.l.), Angonia 14.5473° S, 34.1873° E, 1224 m a.s.l. and
Ruace 15.2345° S, 36.6887° E, 772 m a.s.l. in Mozambique. New fields previously under
maize for two growing periods were used for each season. According to the Soils Atlas
of Africa, the predominant soil type at the sites in Nampula is Haplic Lixisols while in
Angonia and Ruace are Chromic Luvisols [27]. Ten soil samples were taken from 0 to
30 cm soil layer using a soil auger in a W pattern across the field for the trial before
plowing or harrowing. Soils from each site were combined into a composite sample
and four subsamples drawn for chemical and particle-size analysis (Table 1). The pH
was determined using a high impedance voltmeter on 1:2 soil–water suspension. Total
N was determined using The Kjeldahl method, P by Olsen’s method, and K plus other
bases by ICP-OES after extraction with Mehlich 3.
2.2 Inoculant sources and preparation
Two inoculants were sourced from Novozymes BioAg (Cell-Tech® liquid and Cell-
Tech® peat) in Saskatoon, SK Canada and Soygro (Soyflo-liquid and Soycap-powder) in
Potchefstroom South Africa. According to the manufacturers’ specifications, the inocu-
lants contained 2  109 cells/ml or cells/g of Bradyrhizobium diazoefficiens formerly
known as Bradyrhizobium japonicum [28] USDA110 strain for Cell-Tech® and USDA122
strain for Soygro. The Cell-Tech® liquid inoculant was applied at the rate of 1900 ml/ha
(3.8 ml/20 m2 plot) while the Cell-Tech® peat was 2.32 kg/681 kg seed (170.5 g/50 kg
seed/ha). On the other hand, the Soyflo-liquid and Soycap-powder were applied at
2000 ml/ha (4.0 ml/20 m2 plot) and (250 g/50 kg seed/ha) respectively.
2.2.1 Seed application
Liquid inoculants required for 2 kg soybean seed were weighed and diluted with
100 ml of distilled water before applying on seed in a plastic bag. The seeds were then
mixed well for the surfaces to be fully coated with the inoculant. For the solid-based
Location Angonia Gurue Nampula
pH 6.4 6.2 6.6
P (ppm) 25.0 44.8 0.3
K (ppm) 122.8 421.0 90.4
Ca (ppm) 772.8 1755.0 800.5
Mg (ppm) 165.5 301.8 113.0
Na (ppm) 29.4 17.9 29.3
EC (dS/cm) 0.059 0.057 0.050
CEC (cmolc/kg) 6.6 15.0 6.0
N (%) 0.09 0.15 0.13
Sand (%) 64.0 56.8 63.2
Silt (%) 6.6 12.1 2.0
Clay (%) 29.4 31.1 34.8
Table 1.
Soil characteristics at the experimental sites’ soils.
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Inoculant Formulation and Application Determine Nitrogen Availability and Water Use…
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inoculants, the seeds were weighed into a plastic bag then moist with water for
Cell-Tech® peat or Mollyflo for the Soycap-powder. Seeds were then mixed well in the
plastic bag until all the surfaces were coated with a film of water or Mollyflo. Then
respective quantities of solid-based inoculants added and mixed well to cover the
surfaces of all the seeds. All the preparations were done under shade and the seeds
planted within 2 h of mixing with the inoculant.
2.2.2 Soil application
Volumes of inoculants to be applied on soil per plot were measured using a syringe
into 2 l hand sprayers before adding 1 l of distilled water. The mixture was then agitated
gently to equally distribute the inoculant cells in the water. Later the mixture was
sprayed into open seed furrows followed immediately with seed placement and covering
with soil. To apply the solid-based inoculants onto soil, quantities of respective plot
inoculants were weight and mixed with 100 g moist fine sieved (1 mm sieve) soil in a
wide mouth plastic container with a lid. Then soil and inoculant were mixed thoroughly
by shaking. The lid was then perforated using a hot nail to open many holes like a
saltshaker. This mixture was then applied in open furrow followed by immediate plant-
ing of seeds and covering with soil. To avoid scorching of the rhizobia strains to death in
the sun, immediately planting the seeds and covering with soil is recommended.
2.3 Experimental layout
A disc plow was used for land preparation followed by two passes of harrowing.
Both seasons’ experiments were planted between 16 and 24 December depending on
the onset of rains in each site. Experimental treatments were formulated by
combining the two inoculants, their formula (liquid or solid) and place of application
(seed or soil) plus a control (no amendment). These resulted in nine treatments
that were layered out in a Randomized Complete Block Design (RCBD). A non-
promiscuous soybean variety Safari was planted in plots of 20 m2 in four replications.
Plots consisted of seven rows of 8 m in length, 0.50 m row-spacing and 0.1 m between
plants within rows. During establishment of the trials, similar treatments were planted
by one person for all the four replicates to avoid contamination. Planting and weeding
(twice) were done by hand at site-specific scheduling. The experiment was conducted
under rainfed conditions for both seasons with no external water supply through
irrigation. Pests were controlled once at beginning of flowering using 100 ml
of Cypermethrin (200 g active ingredient/l) and 50 ml of Lambda Cyhalothrin
(50 g active ingredient/l) applied using 15 l knapsack sprayer.
2.4 Data collection
Data on nodulation, plant growth, nitrogen fixed, 13C related WUE, yield and yield
components were collected. At R3 (flowering to podding) growth stage when pods
had reached 10
12 mm long at one of the four uppermost nodes on main stem, five
randomly selected soybean plants were excavated using a hoe and spade from each
plot ensuring that all the roots were recovered. All the soil was washed out of the roots
and all nodules plucked out carefully by hand. The nodules were counted and later
placed in envelopes before drying in an oven at 60°C for 48 h to determine nodule dry
weight. Plant biomass were also dried in an oven at 60°C until constant dry weight
was achieved. Later the biomass was ground to pass through a 2-mm mesh sieve for
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Soybean - Recent Advances in Research and Applications
plant tissue N analysis stable light isotope ratio mass spectrometer. At maturity, 10
plants were randomly selected and harvested for determination of pod density and
seed weight. Pods from each plot were threshed manually and grain yield was deter-
mined. The moisture content of grain samples from each plot was measured using
Farmex MT-16 grain moisture Tester (AgraTronix LLC, Streetsboro, Ohio, USA) and
grain yield in kg ha
1 was adjusted to 13% moisture content. Above-ground plant
biomass from whole plots were sun-dried to 10% moisture content for 10 days to
determined harvest biomass weight.
2.4.1 Measurement of shoot N and C isotopes
The isotopic analyses of 15N and 13C were performed at the Mammal Research
Institute, University of Pretoria, Pretoria, South Africa using a Stable Light Isotope
Laboratory on a Flash EA 1112 Series coupled to a Delta V Plus stable light isotope ratio
mass spectrometer via a ConFlo IV system (Thermo Fischer, Bremen, Germany).
Aliquots of 1.2 mg were weighed into toluene pre-cleaned tin capsules. During the
analysis, a standard (Merck Gel: 13
δ C = 
20.57 15
‰, δ N = 6.8‰, C% = 43.83, N
% = 14.64) and a blank sample were run after every 12 samples. The air nitrogen was
used as the reference isotope values for nitrogen. The 15N natural abundance
expressed as the δ (delta) notation is the‰ deviation of the 15N natural abundance of
the sample from atmospheric N2 (0.36637 atom % 15N) was calculated [29] with the
analytical precision values used being <0.2 1
‰ for 3
δ C and < 0.2‰ for 15
δ N.
h  i

  
  
 
15 5
δ N ¼ 15N=14N 1
– N=14N = 15N=14N  1000 (1)
plant atm atm
The percentage N derived from the legume (%Ndfa) was determined using [30]:


  
 
%Ndfa ¼ 15
δ N 15
ref–δ Nplant = 15
δ Nref–Bvalue  100 (2)
Where, 15
δ Nref is the mean 15N natural abundance of the collected reference plants
(maize), 15Nleg is the
15N natural abundance of soybean, and the B value is the 15N natural
abundance of the test legume wholly dependent on N2 fixation for its N nutrition. The
Bvalue replaces atmospheric N2 as it incorporates the isotopic fractionation associated with
N2 fixation. The B value used for estimating %Ndfa in this study was 
0.72‰ [29, 31,
32]. The amount of N-fixed was calculated based on the method established by [33].
N 
 fixed ¼ ð%Ndfa=100Þ  legume biomass N (3)
Where legume biomass N refers to the N content of plants shoots.
2.4.2 Carbon assimilation and water use efficiency
To perform the 13C/12C isotopic analysis, the plants shoots were weighed (sub-
sampled) into tin capsules and analyzed on a mass spectrometer as described for the
15N/14N isotopic analysis. Shoot C content was calculated by relating plant %C to the
biomass of the plant.
Shoot C content ¼ %C shoot biomass per plant (4)
Reference carbon isotope values were the Vienna Pee-Dee Belemnite (PDB).
Change in 13C (∆13C) was calculated as follows
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Inoculant Formulation and Application Determine Nitrogen Availability and Water Use…
DOI: http://dx.doi.org/10.5772/intechopen.102639

  
 
13
∆ C ¼ 13 1
δ C 3
atm 
 δ C 13
plant = 1þ δ Cplant : (5)
Where 13
δ Catm is 13C change in atmospheric CO2 (
8) and 13
δ Cplant in plant material.
The relationship between carbon fixation and stomatal conductance in soybean at
R3 stage was determined based on the model linking the isotope discrimination ( 13
∆ C)
to plant and atmospheric 13C [34]. A linear relationship was used to relate the isotope
discrimination to plant physiological properties.
13
∆ C ¼ aþ ðb–aÞ=ðCi=CaÞ: (6)
Where a is the discrimination against 13CO2 during CO2 diffusion through the
stomata (a = 44‰), b is the discrimination associated with carboxylation (b = 27‰),
and Ci and Ca are the intercellular and atmospheric ambient CO2 concentrations
respectively. According to Fick’s law (1855) that states ‘the rate of diffusion of a
substance across unit area (such as a surface or membrane) is proportional to the
concentration gradient’. Then Movement of CO2 can be expressed as;
A ¼ gCO2ðCi=CaÞ (7)
Since the ratio of leaf conductance to water vapor is 1.6 g CO2, and therefor change
in 13C can be related to the A/gH2O ratio as follows:

 
 
13
∆ C ¼ aþ ðb–aÞ 1
 1:6 A=Ca g gH2O (8)
WUE defined as the ratio of the fluxes of net photosynthesis and conductance for
water vapor (A/E) which indicates carbon assimilated per unit of water umol mol
1)
[35]. Therefore, water-use efficiency at growth level (WUEg) 
 biomass accumulated
over water transpired (g C kgH2O

1) was calculated as:

  
WUEg ¼ Ca b
 13
∆ C =1:6ðb–aÞ (9)
2.5 Data analysis
Analyses of variance (ANOVAs) were performed using PROC GLM in Statistical
Analysis System (SAS)® 9.4 [36]. First a combined analysis across locations and
cropping seasons was performed. Since location and season effects were dominant,
the two variables were combined to form environment. Secondly, a factorial ANOVA
was performed, to evaluate the effects of environment, treatment, and their interac-
tions. Environments effects were considered random and were significant for all the
variables [37] while the treatments factors were fixed effects for each environment.
Means were determined for treatments, and comparisons done using Tukey adjust-
ment at p ≤ 0.05 significance level based on the standard error of means (SEM) [36].
3. Results
3.1 Nodulation
Formation of nodules is an indicator of BNF through the symbiotic relationship of
soybean plant and the inoculant strains. Data on nodule count and dry weight per
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Soybean - Recent Advances in Research and Applications
plant were collected for both crown and lateral nodules. There were no significant
differences (p ≤ 0.05) in the nodule count and dry weight between treatments, sites
and their interactions for both crown and lateral nodules. It was however evident that
crown nodules of inoculated soybean averaging at 20.4 nodules plant
1 were more
than lateral nodules at 18.6 nodules plant
1 against the check of 3.4 nodules plant
1
and 3.2 nodules plant
1 respectively. Total nodule counts, and weight combined both
crown and lateral nodules were significant between treatments at Angonia in 2017,
Ruace in 2017 and Ruace in 2018 (Tables 2 and 3). Angonia and Ruace sites are in well
suited high potential soybean production agroecologies while Nampula site is in a low
to marginal production region.
In Angonia and Ruace in 2017, nodule counts were lowest for the uninoculated
soybean and the nodule count per plant was observed to be the highest from seed
inoculated soybean with Soycap-powder (Table 2). Comparable nodules were formed
for inoculated soybean at Ruace in 2018 except for Soycap-powder soil application. A
common trend was observed between manufacturers/source liquid and solid inocu-
lants regardless of the application on soil or seed. The liquid inoculants had numeri-
cally lower nodules formed than the solid (peat or powder) based. Generally, liquid
based inoculants averaged at 36.5, 37.5 and 41.2 versus 56.3, 56.2 and 45.8 nodules
plant
1 for Angonia 2017, Ruace 2017 and Ruace 2018 respectively. Except for Ruace
2018 with 50.2 and 36.8 nodules plant
1 for seed and soil inoculant application, mean
number of nodules formed between the two inoculation methods were not different
for the other environments. The total number of nodules formed per plant were
significantly higher (p ≤ 0.05) for the inoculated soybean in all the sites at 46.4, 46.9
and 43.5 than the uninoculated plants at 9.0, 8.5 and 11.1 nodules plant
1 (Table 2).
Similar trends of nodules plant
1 were also observed for the nodule dry weight
(mg plant
1). Inoculated soybean had heavier nodules than the uninoculated ones
averaging at 206.9, 218.8 and 249.7 mg plant
1 versus 33.5, 36.6 and 69.9 mg plant
1
for Angonia 2017, Ruace 2017 and Ruace 2018 respectively (Table 3). It was also
noted that the dry weight per nodule at Ruace in 2018 was higher than at Angonia and
Treatment (inoculant application) Angonia 2017 Ruace 2017 Ruace 2018
Control 9.0d 8.5b 11.1c
Seed Cell-Tech liquid 36.6bc 42.6a 48.1ab
Seed Cell-Tech peat 52.6abc 57.5a 60.5a
Seed Soyflo-liquid 38.8bc 38.3a 36.0ab
Seed Soycap-powder 63.9a 58.6a 56.1ab
Soil Cell-Tech liquid 37.9bc 34.9a 42.9ab
Soil Cell-Tech peat 53.4abc 54.4a 37.8ab
Soil Soyflo-liquid 32.8c 34.2a 37.6ab
Soil Soycap-powder 55.4ab 54.4a 28.7bc
%CV 10.3 13.2 14.7
p-Value <0.0001 <0.0001 0.0001
The subscripts signify statistical differences at p<0.05. Same letters indicate no differences while different letters show
significance in the treatments within the season.
Table 2.
Nodule count per plant of inoculated soybean.
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Treatment (inoculant application) Angonia 2017 Ruace 2017 Ruace 2018
Control 33.5d 36.6b 69.0b
Seed Cell-Tech liquid 134.3cd 174.1a 247.0ab
Seed Cell-Tech peat 259.4ab 247.3a 275.1ab
Seed Soyflo-liquid 155.4bc 176.6a 228.0ab
Seed Soycap-powder 294.3a 295.7a 310.7a
Soil Cell-Tech liquid 147.0bc 165.1a 249.5a
Soil Cell-Tech peat 238.9abc 255.3a 228.3ab
Soil Soyflo-liquid 169.6bc 180.1a 239.0a
Soil Soycap-powder 256.7ab 256.7a 220.5ab
%CV 28.8 26.9 35.6
p-Value <0.0001 <0.0001 0.0127
The subscripts signify statistical differences at p<0.05. Same letters indicate no differences while different letters show
significance in the treatments within the season.
Table 3.
Nodule weight (mg) per plant of inoculated soybean.
Ruace 2017 for all the treatments. The average weight per nodule was Angonia 2017
(4.3 mg nodule
1), Ruace 2017 (4.6 mg nodule
1) and Ruace 2018 (6.0 mg nodule
1).
The heaviest weight per nodule was from soybean that were inoculated with Soycap
powder applied on the soil at 7.7 mg nodule
1 in Ruace 2018. As observed for the
nodule counts, significantly heavier nodules (p ≤ 0.05) were obtained when Soycap-
powder inoculant was applied on seed which gave 294.3, 295.7 and 310.7 mg plant
1 of
dry nodule weight at Angonia 2017, Ruace 2017 and Ruace 2018 respectively
(Table 3). Application of the inoculants in liquid form had lighter nodules for all the
sites at 151.5, 174.0 and 240.9 mg plant
1 against using inoculants in solid form with
262.3, 263.7, and 258.6 correspondingly. From the contrast analysis, nodule dry weight
had a likelihood of increasing over the uninoculated by 173.4 mg plant
1 in Angonia
2017, 181.9 mg plant
1 in Ruace 2017 and 180.4 mg plant
1 in Ruace 2018 when using
inoculant either as liquid or in solid form. There was a strong correlation between
number of nodules and dry weight in all the environments with the coefficients
ranging between 0.92 and 0.96 (Table 4). This suggests that variation in the nodule
dry weight attributed to nodule count was between 85.1% at Angonia 2018 to 91.6% at
Ruace 2017.
Environment Correlation coefficient Significance level
Angonia 2017 0.926 <0.0001
Nampula 2017 0.935 <0.0001
Ruace 2017 0.957 <0.0001
Angonia 2018 0.922 <0.0001
Ruace 2018 0.938 <0.0001
Table 4.
The correlation between nodule count and nodule dry weight of soybean.
9
Soybean - Recent Advances in Research and Applications
3.2 Nitrogen uptake in non-promiscuous soybean safari
Nitrogen is important in soybean production. Soybean has the ability of obtaining
nitrogen from the atmosphere through BNF. The proportion of nitrogen derived from
the atmosphere denoted as %Ndfa by soybean used as an indicator of nitrogen fixed
through BNF. The %Ndfa was as low as 3.8% for control treatment in Angonia 2017 to
as high as 69.8% for soybean that were inoculated with Cell-Tech liquid inoculant at
Ruace 2018 (Figure 1). Our study showed that inoculating soybean seed with Soycap-
powder could derive as high as 50.8% of the nitrogen from the atmosphere across the
environments compared to 14.1% for the uninoculated soybean. The proportion of N
derived from the atmosphere significantly varied with treatment for each environ-
ment. Therefore, the highest %Ndfa was 44.0% for soil Cell-Tech peat in Nampula
2017, 46.9% for seed Soycap-powder in Angonia 2017, 66.4% for seed Soyflo-liquid
and 69.8% for soil Cell-Tech liquid inoculant at Ruace 2018. In each environment, %
Ndfa due to inoculation was significant (p ≤ 0.05) between the treatments resulting in
average %Ndfa of 27.5% for Nampula 2017, 36.4% for Angonia 2017, 59.1% for
Angonia 2018 and 47.1% for Ruace 2018 (Figure 1). Consequently, the proportion of
N derived from the atmosphere was higher at Angonia in 2018.
Nitrogen uptake associated to BNF by the Safari variety per hectare was also
calculated across the seasons for each site. Inoculating soybean increased the
amount of plant N uptake at all the three sites. Plant N uptake was highest at Angonia
with 235 kg N ha
1, followed by Ruace with 150 kg N ha
1 and at Nampula with
137 kg N ha
1 for the inoculated soybean against the uninoculated counterparts at
113 kg N ha
1, 46 kg N ha
1 and 98 kg N ha
1 correspondingly for all the sites
(Table 5). Different treatments had significantly high amount of plant N uptake at
Figure 1.
Proportion of nitrogen derived from the atmosphere (%Ndfa).
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Inoculant Formulation and Application Determine Nitrogen Availability and Water Use…
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Treatment (inoculant application) Nampula Angonia Ruace
Control 98b 113c 46d
Seed Cell-Tech liquid 110ab 181abc 112c
Seed Cell-Tech peat 137ab 313a 129bc
Seed Soyflo-liquid 110ab 261ab 144ab
Seed Soycap-powder 136ab 213abc 144ab
Soil Cell-Tech liquid 149ab 221abc 194a
Soil Cell-Tech peat 154ab 178bc 171ab
Soil Soyflo-liquid 134ab 202abc 120bc
Soil Soycap-powder 158a 307ab 188ab
%CV 24.1 32.1 18.8
p-Value 0.0257 0.0519 <0.0001
The subscripts signify statistical differences at p<0.05. Same letters indicate no differences while different letters show
significance in the treatments within the season.
Table 5.
Amount of plant nitrogen derived from BNF (kg ha
1) by soybean in 2018 growing season following inoculant
application.
each site. The highest plant N uptake was 158 kg N ha
1 at Nampula, 307 kg N ha
1 at
Angonia for soil Soycap-powder and 194 kg N ha
1 for soil Cell-Tech liquid at Ruace
when averaged across the seasons. Like the nodulation data, the amount of plant N
uptake per ha for liquid based inoculant was numerically lower than the solid form at
every application method (seed or soil) at Nampula. Since the form of inoculant also
affected the amount of plant N uptake per ha at each site, solid-based inoculants
resulted in more N absorbed by the plant than liquid-based at 146 vs. 126, 253 vs. 216
and 158 vs. 143 kg N ha
1 for Nampula, Angonia and Ruace respectively (Table 5).
3.3 13C isotope discrimination and water use efficiency
Water-use efficiency at growth level (WUEg), an indicator of biomass accumula-
tion over water transpired was calculated based on the assimilation of carbon at R3
growth stage. Before the calculations, the C:N ratio of plant biomass was also deter-
mined. Our data indicate that no significant differences existed for the C:N ratio
values across the treatments with an average of 13.6 (data not presented). Similarly,
13C isotope discrimination (a fraction of carbon isotope of soybean leaves during CO2
uptake and fixation) was not significant with an average of 20.1‰ across treatments
within environments except for Ruace 2018 where seed Cell-Tech peat inoculant had
the lowest significant (p ≤ 0.05) value of 19.7‰ than the other treatment (Figure 2).
The highest numerical 13C isotope discrimination at Ruace 2018 was soil Soyflo-liquid
application with 20.93‰. Like the 13C isotope discrimination, WUEg was not signifi-
cant among the treatments within each environment averaging at 11.8 g C kgH2O

1
except at Ruace in 2018 where seed Cell-Tech peat inoculant had the highest signifi-
cant (p ≤ 0.05) value of 12.0 g C kgH2O

1 (Figure 2). TheWUEg average ranged from
11.6 g C kgH2O

1 at Nampula 2017 to 13.3 g C kgH2O

1 at Angonia 2018. There were
also no significant differences in applying either of the inoculants on seed or soil with
a mean of 11.0 g C kgH2O

1. However, application of the inoculant in a solid form
11
Soybean - Recent Advances in Research and Applications
Figure 2.
Relationship between 13C isotope discrimination and WUE in Ruace 2018 growing season.
resulted in a numerically higher WUEg of 11.1 g C kgH2O

1 against the liquid coun-
terpart with 10.6 g C kgH2O

1. There is an inverse relationship between the 13C
isotope discrimination and WUEg (Figure 2). A treatment with higher isotope dis-
crimination had a corresponding lower WUEg value. For instance, soybean inoculated
with seed Cell-Tech peat inoculant has an isotope discrimination value of 20.89‰
which corresponded to WUEg of 12.0 g C kgH2O

1.
3.4 Soybean yield
Inoculation treatment yield was determined within each environment. Significant
differences (p ≤ 0.05) were observed between treatments in all environments except
for Ruace 2017 (p-value = 0.9851) with a mean yield of 2186 kg ha
1 and Angonia 2018
(p-value = 0.0883) averaging at 2572 kg ha
1 (Figure 3). However, in these two
environments, mean yield of the inoculated soybean 2248 kg ha
1 at Ruace 2018 and
2413 kg ha
1 at Angonia 2018 were significantly higher than the uninoculated with
1685 and 1756 kg ha
1 respectively. In Nampula 2017, seed Soycap powder gave the
highest significant yield of 2194 kg ha
1 over the uninoculated production of
978 kg ha
1 representing over 2.3-fold increase in yield due to inoculation (Figure 3).
Soybean production increased in the second season at the Nampula site. In Nampula
2018, the highest yield at 2059 kg ha
1 was 81%more than the uninoculated treatment
with 1140 kg ha
1. Soybean yielded better in Angonia and Ruace sites that are in high
soybean production potential agroecologies. For instance, in Angonia 2017 environ-
ment, the highest statistical yield was from soil Soycap powder at 3439 kg ha
1 against
a check of 1646 kg ha
1 while in Ruace 2018 was from Cell-tech liquid inoculant
applied on the seed before planting with 2684 kg ha
1 compared to the uninoculated
fields with 1439 kg ha
1 (Figure 3). From the data, we deduce that applying inoculant
in solid form either on seed or soil was better than using the liquid formulation. Mean
yield of solid over the liquid inoculants in the different environments were Nampula
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Inoculant Formulation and Application Determine Nitrogen Availability and Water Use…
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Figure 3.
Yield of inoculated soybean at three experimental sites of Nampula, Angonia and Ruace in 2017 and 2018
growing seasons.
2017 (1907 > 1580 kg ha
1), Angonia 2017 (3103 > 2437 kg ha
1), Nampula 2018
(1838 > 1683 kg ha
1) and Ruace 2018 (2445 > 2380 kg ha
1).
Contrast analysis of yield on whether to apply inoculant or not and using which
placement (seed or soil) were conducted at p ≤ 0.05 (Table 6). Inoculation increased
yield in all the environments except Angonia 2018. Yield increase in 2017 due to
inoculation was 82% in Nampula, 68% in Angonia and 35% in Ruace (Table 6).
During the second season of 2018 inoculation increased yield from 1140 to
1760 kg ha
1 in Nampula and 1439 to 2413 kg ha
1 in Ruace. Generally, across all the
environments, it was advantageous to apply inoculant on seed than the soil (Table 6).
The differences in yield due to the inoculant form (liquid or solid), source (Cell-Tech
or Soygro) and placement were also determined through contrast analysis (Table 7).
Soygro inoculants performed statistically better than Cell-Tech counterparts in
Nampula and Angonia 2017. In the same locations, solid-based inoculants enhanced
yield more than the liquid-based application. Results also show that it is more benefi-
cial to apply inoculants on the seed that the soil directly. For instance, 1868 and
2817 kg ha
1 yield obtained from applying inoculant on seed was more than soil
placements with 1620 and 2725 kg ha
1 in Nampula 2017 and Angonia 2017 respec-
tively (Table 7).
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Soybean - Recent Advances in Research and Applications
Contrasts Nampula p-Value Angonia p-Value Ruace p-Value
2017
Control 978 1646 1685
Control vs. inoculant 1779 <0.0001 2770 <0.0001 2270 0.0251
Control vs. seed 1903 <0.0001 2817 <0.0001 2285 0.0268
Control vs. soil 1655 0.0004 2724 <0.0001 2254 0.0350
Contrasts Nampula p-Value Ruace p-Value
2018
Control 1139 1439
Control vs. inoculant 1753 0.0047 2413 0.0005
Control vs. seed 1821 0.0029 2469 0.0005
Control vs. soil 1684 0.0137 2357 0.0014
Table 6.
Yield gains of inoculation and inoculant application place (seed or soil).
Contrasts Nampula p-Value Angonia p-Value
Cell-Tech 1584 2662
Cell-Tech vs. Soygro 1904 <0.0001 2878 0.0501
Liquid 1580 2437
Liquid vs. peat 1907 <0.0001 3103 <0.0001
Seed 1868 2817
Seed vs. soil 1620 0.0002 2725 0.3894
Table 7.
Yield of soybean due to source, grade, and placement of inoculant in 2017 season.
4. Discussions
4.1 Nodulation and plant nitrogen uptake
Inoculation increased the number of nodules and dry weight. Inoculants have been
shown to increase the number of nodules per plant in soybean production regardless
of the source and stage of plant growth at application ranging from planting time to
V6 [38]. Use of the inoculants with compatible rhizobia strain for non-promiscuous
varieties [39, 40] and availability of right strain resident rhizobia for promiscuous
genotypes [41] leads to formation of more nodules in soybean. In our study, on
average, the number of nodules increased by 5.1 times in Angonia 2017, 5.5 times in
Ruace 2017 and 3.9 times in Ruace 2018 due to inoculation with liquid and solid
inoculants either in seed or direct soil application. Solid based inoculants had high
number of nodules and dry weight than the liquid inoculants. Our results corroborate
with the findings from a study conducted in the Eastern Region of the south of
Vietnam where nodulation of the liquid inoculants was less than the peat-based
inoculants for similar rhizobia strains [15, 42]. Solid based inoculants better protect
the rhizobia strains from harsh environmental conditions hence leading to increased
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viability than the liquid inoculants. In addition, solid carrier inoculants attach better
onto the seed during inoculation. Also, our data indicated that although crown nodules
were fewer in number than the lateral nodules, individual nodules of the former were
heavier than the later. It has been reported that crown nodules can account for up to
82% and above of the total nodule count or dry weight in soybean [43]. Crown
nodules from our study accounted for 41.7–64.0% of the total nodule dry weight.
More crown nodules are formed early in the season following inoculation than the
lateral nodules that are formed later after development of lateral roots.
Sources of nitrogen for soybean in our study were either BNF or absorption from
soil. The BNF process was enhanced by introduction of compatible rhizobia strain
through inoculation. More nitrogen was fixed from the atmosphere for inoculated
soybean in Angonia and Ruace relative to Nampula. Nampula lies in a semi-arid region
of Mozambique with frequent incidences of drought leading to low soil moisture. High
temperatures, drought and low soil moisture has been shown to reduce the effective-
ness of rhizobia in BNF process leading to low nodulation hence reduced %Ndfa [44].
In Angonia and Ruace the large share of plant N was from the atmosphere
representing as high as 69.8%. Other studies have reported high percentages of plant
N in soybean to be associated with atmospheric nitrogen though BNF [45, 46]. As
earlier indicated, plant N uptake associated with BNF varies with the biotic factors
such as soybean and rhizobia characteristics as well as abiotic factors largely con-
trolled by the environment and management. Due to the differences in the interaction
levels of these factors, variations were observed in the amount of N uptake by soybean
[47]. For instance, soybean in Angonia a more humid environment, absorbed more N
from the atmosphere than Nampula site that is in a drier ecology. A similar trend of N
fixed in wet versus drier environment was reported on farmer’s fields in humid Dowa
(88.9 kg N ha
1) and drier Salima location (47.1 kg N ha
1) in Malawi [48]. Soil
moisture that depends on the rainfall amount has been reported to greatly affect
amount of N fixed. The amount of N uptake was determined at R3 growth stage in
soybean. This growth stage falls within the peak N demand period of flowering to
podding in soybean production. Like the amount of N derived from the atmosphere,
plant tissue N was enhanced by inoculation [14]. Soybean had accumulated as high as
307 kg N ha
1 in Angonia. These findings are like those reported for inoculated TGx
1660-19F soybean with 306 kg N ha
1 at Mokwa in the southern Guinea savanna of
Nigeria [49]. Although we did not monitor plant N over the growing season, the
amount of N in plant tissue varies with the growth stage due to the translocations that
occur between plant parts.
4.2 13C isotope discrimination and water use efficiency
Both 13C isotope discrimination and WUEg were not significant among the treat-
ments within each environment except at Ruace in 2018. This suggests that these two
parameters measured at the R3 stage were not dependent on the application of the
inoculant. Like our findings at R3 growth stage in soybean, Zhao et al. [50] also
reported that no significant difference existed in C isotope discrimination and
corresponding WUEg at wheat harvest time. Also, Yang et al. [51] reported no clear
significance differences in the amount of carbon isotope composition among C3 plants
in the Yellow River region in China. For the case of Ruace in 2018 a negative relation-
ship was observed between 13C isotope discrimination and WUEg. Values of
13C
isotope discrimination generally decrease with reductions in water availability.
Reduced water availability leads to a decline in transpiration rate hence increased
15
Soybean - Recent Advances in Research and Applications
water-use efficiency [22, 35, 52]. Also earlier reported was a negative relationship
between 13C isotope discrimination in wheat at tillering stage and WUE of above
ground biomass measured over the seedling to tillering period [50]. The change in 13C
isotope discrimination in relation to the environment may differ with plant growth
stages due to variation in physiological processes within the plant that define its
functionality requirements [53]. Since we measured 13C isotope discrimination and
WUEg at one stage for all the treatments, the likelihood of soybean functionality
being comparable was high and more dependent on the environment. Therefore, 13C
isotope discrimination can be used to determine differences in WUEg of different
soybean growth stages rather than a variation associated to inoculation at a single
stage [54].
4.3 Soybean yield
Inoculation increased yield in all the three sites between an average of
602
1124 kg ha
1. Our results agree with a study conducted in 2013 and 2014 in the
same locations using storm a non-promiscuous variety that recorded an increase of
523
989 kg ha
1 [6]. These results of yield increase due to inoculation also confirms
previous report [5, 8] where inoculation alone led to higher soybean yield that
uninoculated. Although numerical average increase in yield due to inoculation was
higher in Angonia and Ruace than Nampula across the seasons, percent rise in pro-
duction was higher at Nampula 620
766 kg ha
1 (65%) than Angonia 918
1124
(60%) and Ruace 602
974 (52%). Chibeba et al. [6] reported and increased of 47% in
yield of inoculated over the uninoculated soybean variety storm. Association between
the introduced rhizobia strain and soybean was enhanced in the humid environments
of Angonia and Ruace than the drier Nampula. Adequate moisture is required to take
full advantage of the BNF process in inoculated soybean. The numerical rise in yield is
also a pointer to the earlier reported enhanced nodulation in the inoculated soybean
regardless of the placement on seed or soil. Across the sites, average soybean yield of
1440 kg ha
1 for the uninoculated fields is above the Mozambique national average of
1216 kg ha
1 [55, 56]. Therefore, use of inoculation in this study indicated that
soybean yield can be increased by 1052 kg ha
1 over the national average figure. Our
study observed that inoculant application on seed (2308 kg ha
1) gave higher yield
than soil application (2228 kg ha
1) agrees with the report by [57] where seed inocu-
lation 2842 kg ha
1 was greater than 2678 kg ha
1 for soil inoculation on planting line.
Seed inoculation plus good adhesive agent and proper mixing of the seeds in the bag
enables better distribution of the rhizobia cells per seed-grain. As a result, the rhizobia
cells remain close to the seed and can attach to the root as soon as it germinates leading
to better nodulation and BNF process that promote increased yield production. Seed
inoculation led to a difference in yield was also noted between the liquid and solid
inoculants. Solid inoculants (peat or powder) gave higher yield of 2389 kg ha
1 than
the liquid inoculant 2147 kg ha
1 across the environments. Similar results where solid
inoculants gave higher yields than liquid inoculants were reported from a study
comparing the two forms of inoculants in Vietnam on promiscuous soybean varieties
where identical rhizobia strains of in peat inoculant outyielded the liquid counterparts
between 40 and 60 kg ha
1 [42]. These results demonstrates that farmers in Mozam-
bique have a basket of inoculation options to choose from in enhancing soybean yield
on their fields. However, selection of suitable inoculant should be made with consid-
eration of environmental site conditions especially soil moisture availability over the
growing season and the easiness of application.
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5. Conclusions
Inoculation improved soybean nodulation by increasing the number of nodule
count and its dry weight. Increase in nodulation could be associated to improved
soybean productivity through high plant N uptake and yield. Nitrogen uptake and
yield increased with application of inoculants. Farmers in Mozambique are likely to
produce more soybean through using of solid cased inoculants applied on the seed
than the liquid inoculants plus soil application. Although WUEg related to 13C isotope
discrimination at R3 stage did not vary with inoculation, it is recommended that
further study be conducted to determine cumulative WUE of the whole plant for the
complete growing season while segregating for different growth stages. This could
offer information on how to time soybean planting to take advantage of shifting
growing seasons characteristics due to climate change. As such, soybean varieties
could be selected for adaptability and resilience in specific agroecologies based on
carbon assimilation, WUE and plant N uptake that affect yield. Data on inoculation
and 13C isotope discrimination could be utilized by breeders in selection of high
yielding soybean varieties adapted to drought conditions like those found in Mozam-
bique. The varieties developed would have high transpiration efficiency and WUE.
Acknowledgements
The authors greatly acknowledge financial support from the Consortium of Inter-
national Agricultural Research Centers (CGIAR) through the Research Program on
Grain Legumes and Dryland Cereals (CRP-GLDC) and United States Agency for
International Development (USAID) through Feed the Future Mozambique Improved
Seeds for Better Agriculture (SEMEAR) project in Mozambique. Thanks to the IITA
technical staff at Angonia, Nampula and Ruace stations in Mozambique for managing
the trials and collecting of field-related data.
Conflict of interest
The authors declare that the research was conducted in the absence of any com-
mercial or financial benefits that could be construed as a potential conflict of interest.
17
Soybean - Recent Advances in Research and Applications
Author details
Canon E.N. Savala1*, David Chikoye2 and Stephen Kyei-Boahen1
1 International Institute of Tropical Agriculture (IITA), Nampula, Mozambique
2 IITA-Zambia, Lusaka Province, Zambia
*Address all correspondence to: c.engoke@cgiar.org
© 2022TheAuthor(s). Licensee IntechOpen. This chapter is distributed under the terms
of theCreativeCommonsAttribution License (http://creativecommons.org/licenses/
by/3.0),which permits unrestricted use, distribution, and reproduction in anymedium,
provided the original work is properly cited.
18
Inoculant Formulation and Application Determine Nitrogen Availability and Water Use…
DOI: http://dx.doi.org/10.5772/intechopen.102639
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