*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


### **Table 1.**

*Soil characteristics at the experimental sites' soils.*

### *Inoculant Formulation and Application Determine Nitrogen Availability and Water Use… DOI: http://dx.doi.org/10.5772/intechopen.102639*

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 planting 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 nonpromiscuous soybean variety Safari was planted in plots of 20 m<sup>2</sup> 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 1012 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

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 determined. 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�<sup>1</sup> 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: <sup>δ</sup>13C = �20.57‰, <sup>δ</sup>15N = 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‰ for δ13C and < 0.2‰ for δ15N.

$$\mathbf{\dot{\delta}}^{15}\mathbf{N} = \left[ \left( \mathbf{\dot{\epsilon}}^{15}\mathbf{N} / ^{14}\mathbf{N} \right)\_{\text{plant}} - \left( ^{15}\mathbf{N} / ^{14}\mathbf{N} \right)\_{\text{atm}} \right) / \left( ^{15}\mathbf{N} / ^{14}\mathbf{N} \right)\_{\text{atm}} \right] \times 1000 \,\tag{1}$$

The percentage *N* derived from the legume (%Ndfa) was determined using [30]:

$$\text{\textbullet Ndf\'a} = \left( \left( \text{\textbullet N}\_{\text{ref}} \text{--} \text{\textbullet}^{15} \text{N}\_{\text{plant}} \right) / \left( \text{\textbullet}^{15} \text{N}\_{\text{ref}} \text{--} B\_{\text{value}} \right) \right) \times \mathbf{100} \tag{2}$$

Where, δ15Nref 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 *B*value 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].

$$N-\text{fixed} = (\text{\textquotedbl{}}\text{\textquotedbl{}}\text{Ndf\textquotedbl{}}/\text{100}) \times \text{legume biomass N} \tag{3}$$

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 (subsampled) 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.

$$\text{Short C content} = \% \text{C} \times \text{shot biomass per plant} \tag{4}$$

Reference carbon isotope values were the Vienna Pee-Dee Belemnite (PDB). Change in 13C (Δ13C) was calculated as follows

*Inoculant Formulation and Application Determine Nitrogen Availability and Water Use… DOI: http://dx.doi.org/10.5772/intechopen.102639*

$$
\Delta^{13}\mathbf{C} = \left(\delta^{13}\mathbf{C}\_{\text{atm}} - \delta^{13}\mathbf{C}\_{\text{plant}}\right) / \left(\mathbf{1} + \delta^{13}\mathbf{C}\_{\text{plant}}\right). \tag{5}
$$

Where <sup>δ</sup>13Catm is 13C change in atmospheric CO2 (�8) and <sup>δ</sup>13Cplant 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 (Δ13C) to plant and atmospheric 13C [34]. A linear relationship was used to relate the isotope discrimination to plant physiological properties.

$$
\Delta^{13}\mathbf{C} = a + (b\text{-}a)/(\mathbf{C}\_{\text{i}}/\mathbf{C}\_{\text{a}}).\tag{6}
$$

Where *a* is the discrimination against 13CO2 during CO2 diffusion through the stomata (*a* = 4�4‰), *b* is the discrimination associated with carboxylation (*b* = 27‰), and *C*<sup>i</sup> and *C*<sup>a</sup> 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;

$$A = \mathcal{g}\_{\text{CO2}}(\mathcal{C}\_{\text{i}}/\mathcal{C}\_{\text{a}}) \tag{7}$$

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:

$$
\Delta^{13}\mathbf{C} = a + (b \text{--} a) \left( \mathbf{1} - \mathbf{1}.6 \left( \mathbf{A} / \mathbf{C}\_{\mathbf{a}} \, \mathbf{g} \, \mathbf{g} \, \mathbf{H}\_{2} \, \mathbf{O} \right) \tag{8}
$$

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�<sup>1</sup> ) [35]. Therefore, water-use efficiency at growth level (WUEg) � biomass accumulated over water transpired (g C kgH2O�<sup>1</sup> ) was calculated as:

$$\text{WUE}\_{\mathfrak{g}} = \mathbb{C}\_{\mathfrak{a}} \left[ \left( b - \Delta^{13} \mathbb{C} \right) / \mathbf{1.6} (b \mathfrak{a} - a) \right] \tag{9}$$

### **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 interactions. 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 adjustment 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

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<sup>1</sup> were more than lateral nodules at 18.6 nodules plant<sup>1</sup> against the check of 3.4 nodules plant<sup>1</sup> and 3.2 nodules plant<sup>1</sup> 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 inoculants regardless of the application on soil or seed. The liquid inoculants had numerically 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<sup>1</sup> for Angonia 2017, Ruace 2017 and Ruace 2018 respectively. Except for Ruace 2018 with 50.2 and 36.8 nodules plant<sup>1</sup> 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<sup>1</sup> (**Table 2**).

Similar trends of nodules plant<sup>1</sup> were also observed for the nodule dry weight (mg plant<sup>1</sup> ). Inoculated soybean had heavier nodules than the uninoculated ones averaging at 206.9, 218.8 and 249.7 mg plant<sup>1</sup> versus 33.5, 36.6 and 69.9 mg plant<sup>1</sup> 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


*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.*

*Inoculant Formulation and Application Determine Nitrogen Availability and Water Use… DOI: http://dx.doi.org/10.5772/intechopen.102639*


*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<sup>1</sup> ), Ruace 2017 (4.6 mg nodule<sup>1</sup> ) and Ruace 2018 (6.0 mg nodule<sup>1</sup> ). The heaviest weight per nodule was from soybean that were inoculated with Soycap powder applied on the soil at 7.7 mg nodule<sup>1</sup> in Ruace 2018. As observed for the nodule counts, significantly heavier nodules (*p* ≤ 0.05) were obtained when Soycappowder inoculant was applied on seed which gave 294.3, 295.7 and 310.7 mg plant<sup>1</sup> 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<sup>1</sup> 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<sup>1</sup> in Angonia 2017, 181.9 mg plant<sup>1</sup> in Ruace 2017 and 180.4 mg plant<sup>1</sup> 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.


#### **Table 4.**

*The correlation between nodule count and nodule dry weight of soybean.*

### **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 Soycappowder 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 environment. 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<sup>1</sup> , followed by Ruace with 150 kg N ha<sup>1</sup> and at Nampula with 137 kg N ha<sup>1</sup> for the inoculated soybean against the uninoculated counterparts at 113 kg N ha<sup>1</sup> , 46 kg N ha<sup>1</sup> and 98 kg N ha<sup>1</sup> 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).*

*Inoculant Formulation and Application Determine Nitrogen Availability and Water Use… DOI: http://dx.doi.org/10.5772/intechopen.102639*


*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<sup>1</sup> ) by soybean in 2018 growing season following inoculant application.*

each site. The highest plant N uptake was 158 kg N ha<sup>1</sup> at Nampula, 307 kg N ha<sup>1</sup> at Angonia for soil Soycap-powder and 194 kg N ha<sup>1</sup> 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<sup>1</sup> for Nampula, Angonia and Ruace respectively (**Table 5**).
