**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

leaching, denitrification and removal through crop harvest [2]. Inoculation of rhizobia enhances biological nitrogen fixation (BNF) in soybean production and is economically 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. Development 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 symbiotic 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, soybean 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

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

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 contribute 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 (*C*i/*C*a) 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 discrimination 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,

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.
