**3.3 13C isotope discrimination and water use efficiency**

Water-use efficiency at growth level (WUEg), an indicator of biomass accumulation 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 determined. 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 significant among the treatments within each environment averaging at 11.8 g C kgH2O<sup>1</sup> except at Ruace in 2018 where seed Cell-Tech peat inoculant had the highest significant (*p* ≤ 0.05) value of 12.0 g C kgH2O<sup>1</sup> (**Figure 2**). The WUEg average ranged from 11.6 g C kgH2O<sup>1</sup> at Nampula 2017 to 13.3 g C kgH2O<sup>1</sup> 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<sup>1</sup> . However, application of the inoculant in a solid form

**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<sup>1</sup> against the liquid counterpart with 10.6 g C kgH2O<sup>1</sup> . There is an inverse relationship between the 13C isotope discrimination and WUEg (**Figure 2**). A treatment with higher isotope discrimination 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<sup>1</sup> .

### **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<sup>1</sup> and Angonia 2018 (*p*-value = 0.0883) averaging at 2572 kg ha<sup>1</sup> (**Figure 3**). However, in these two environments, mean yield of the inoculated soybean 2248 kg ha<sup>1</sup> at Ruace 2018 and 2413 kg ha<sup>1</sup> at Angonia 2018 were significantly higher than the uninoculated with 1685 and 1756 kg ha<sup>1</sup> respectively. In Nampula 2017, seed Soycap powder gave the highest significant yield of 2194 kg ha<sup>1</sup> over the uninoculated production of 978 kg ha<sup>1</sup> 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<sup>1</sup> was 81% more than the uninoculated treatment with 1140 kg ha<sup>1</sup> . Soybean yielded better in Angonia and Ruace sites that are in high soybean production potential agroecologies. For instance, in Angonia 2017 environment, the highest statistical yield was from soil Soycap powder at 3439 kg ha<sup>1</sup> against a check of 1646 kg ha<sup>1</sup> while in Ruace 2018 was from Cell-tech liquid inoculant applied on the seed before planting with 2684 kg ha<sup>1</sup> compared to the uninoculated fields with 1439 kg ha<sup>1</sup> (**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

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

### **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<sup>1</sup> ), Angonia 2017 (3103 > 2437 kg ha<sup>1</sup> ), Nampula 2018 (1838 > 1683 kg ha<sup>1</sup> ) and Ruace 2018 (2445 > 2380 kg ha<sup>1</sup> ).

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<sup>1</sup> in Nampula and 1439 to 2413 kg ha<sup>1</sup> 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 beneficial to apply inoculants on the seed that the soil directly. For instance, 1868 and 2817 kg ha<sup>1</sup> yield obtained from applying inoculant on seed was more than soil placements with 1620 and 2725 kg ha<sup>1</sup> in Nampula 2017 and Angonia 2017 respectively (**Table 7**).

### *Soybean - Recent Advances in Research and Applications*


### **Table 6.**

*Yield gains of inoculation and inoculant application place (seed or soil).*


**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

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

the rhizobia strains from harsh environmental conditions hence leading to increased 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 effectiveness 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 controlled 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<sup>1</sup> ) and drier Salima location (47.1 kg N ha<sup>1</sup> ) 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<sup>1</sup> in Angonia. These findings are like those reported for inoculated TGx 1660-19F soybean with 306 kg N ha<sup>1</sup> 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.
