The Initial Development of Soybean Subjected to Co-Inoculation with Azospirillum Brasilense and Bradyrhizobium Japonicum

Nitrogen (N) is classified as an essential element for plants, since it is found in the composition of the most important biomolecules, such as ATP, NADPH, NADH, chlorophyll, proteins and several enzymes (MIFLIN; LEA, 1976; HARPER, 1994; BREDEMEIER; MUNDSTOCK, 2000). 

The main N sources required for plant growth are the soil nitrogen derived from the decomposition of organic matter and rocks, the nitrogen derived from the application of fertilizers, and the nitrogen resulting from the atmospheric nitrogen fixation process (HUNGRIA et al., 1994). There is also the reaction between electrical discharges and N. Such reaction results in nitrate, which is added to the soil (COSTA, 2011).

 As soon as the nitrogen is fixed in the soil, in the form of ammonia or nitrate, a biogeochemical cycle takes place and it makes the nitrogen go through different organic and inorganic forms before it goes back to molecular N (TAIZ; ZEIGER, 2013). The amino acids, ammonia and ammonium resulting from the nitrogen, or released due to organic matter decomposition in the soil, are disputed by plants and microorganisms. Consequently, the plants develop several nitrogen fixation mechanisms such as the symbiosis with fixing bacteria (SIQUEIRA; FRANCO, 1988; FISHER; NEWTON, 2002).

On the other hand, the organisms living near the soil surface are able to fix nitrogen through the decomposition of dead animals and plants. The saprophytic bacteria and several fungal species are the main responsible for dead organic matter decomposition (SIQUEIRA; FRANCO, 1988). These microorganisms use proteins and amino acids, and release the nitrogen excess in the form of ammonium. 

The nitrogen assimilation is one of the several stages in the nitrogen cycle, which encompasses different biosphere N forms and their interconversions. The main sources of nitrogen available for plants are nitrate (NO-3), ammonium (NH4) and atmospheric N biological fixation (PRADO, 2008). 

The biological nitrogen fixation is the most important way to fix the atmospheric nitrogen (N ) into ammonium (TAIZ & 2 ZEIIGER, 2013). Thus, the atmospheric gases also diffuse into the porous space of the soil, and the N is used by the microorganisms living in it. It happens due to nitrogenase enzyme activity, which is able to break the triple bond in N and 2 reduce it to ammonia (HUNGRIA et al., 2001). 

 According to Gitti (2015), the symbiotic association between the soybean roots and the bacteria belonging to genus Bradyrhizobium provides all the nitrogen required to obtain approximately 3,600 kg ha-1 soybean (mean yield). It also provides 20-30 kg ha-1 nitrogen to the successive culture. 

According to Moreira et al. (2010), the biological fixation of atmospheric nitrogen is done by diazotrophic microorganisms which can be free-living, associated with plant species or may establish symbiosis with leguminous plants. This integrated association between leguminous plants and bacteria belonging to the genus Bradyrhizobium occurs through the development of new structures in the root system, called nodes, where the BNF takes place.

Therefore, the symbiosis process benefits both the host plant and the microorganism. The bacterium benefits from products supplied by the host plant, such as photosynthates or organic carbon, whereas the rhizobium provides the plant with the nitrogen compound that is readily available to it (CASSINI et al., 2006). 

The nodulation process starts right after germination since the rhizobium is already in the soil or adhered to the seed due to the inoculation process. Thus, the process is divided in three stages: infection, nodular development, and node activation and functioning (CASSINI et al., 2006). 

The phenomenon known as chemotaxis occurs in the pre-infection process. The root system produces several substances such as carbohydrates, amino acids, as well as phenolic compounds (flavonoids), which comprise a chemical gradient in the rhizosphere and, thus, attract the bacteria to the root surface (STRALIOTTO et al., 2002). 

The activation of the nodulation genes occurs through substances produced by the plant (TAIZ & ZEIIGER, 2013). The rhizobium starts producing compounds known as nod factors after the activation, and it changes the structure of the root system cells. 

After the initial adhesion, the rhizobium dissolves the cell wall of the modified absorbing hair, penetrates the cortical cells, forms a special structure known as infection thread, and multiplies the rhizobial cells within such structure (CASSINI et al., 2006). 

Once the rhizobium reaches the cortical region, it enters the cortical cells, adapts to the new nitrogen fixation function, and forms bacteroids (CASSINI et al., 2006). The first soybean root nodules can be seen 10 to 15 days after the emergence of the plants, according to favorable environmental and management conditions (CÂMARA, 2000). 

The plant growth-promoting bacteria (PGPB) are a group of microorganisms that can be beneficial to plant development due to their ability to colonize the root surface, the rhizosphere, the phyllosphere and the internal tissues of plants (DAVISON, 1988; KLOEPPER et al., 1989). 

These bacteria belong to free-living gram-negative bacterial groups and their versatile carbon and N metabolism makes them competitive during the colonization process (QUADROS, 2009). 

According to Trentini (2010), these bacteria use N sources such as ammonia, nitrate, nitrite, amino acids and molecular nitrogen in their metabolism. According to Barassi et al. (2008), the inoculation with Azospirillum brasilense improves the photosynthetic parameters of the leaves such as chlorophyll content and stomatal conductance, increases the proline content in the shoots and roots, improves the water potential, and increases the water content in the apoplast, the cell wall elasticity, the biomass production, and the plant height. 

According to Bashan et al. (2006), there is increase in several photosynthetic pigments such as chlorophyll a and b, as well as in auxiliary photoprotective pigments such as violaxanthin, zeaxanthin, antheraxanthin, lutein, neoxanthin and beta-carotene, and it results in greener plants and in no water stress. According to Bárbaro et al. (2008), when Azospirillum brasilense was applied to leguminous plants, the beneficial effect from the association with rhizobium have mostly resulted from the bacteria’s ability to produce phytohormone. Thus, it resulted in better root system development and allowed better exploring the soil volume.

 Molla et al. (2001) have found that the genus Azospirillum has the potential to significantly stimulate root growth, even in plants whose roots were subjected to mechanical damage. Thus, this genus may have positive influence on root growth and development, since it not only promotes root growth, but it may also favor the emergence and development of nodes in soybean plants. 

The alternative co-inoculation technique is seen as a mixed inoculation that consists of applying different combinations of microorganisms able to produce a synergistic effect, wherein the productive results obtained from such combination overcome those obtained when microorganisms are individually used (FERLINI, 2006; BÁRBARO et al., 2008).

 According to Cassán et al. (2009), the number of nodes and the rate of nodulated plants were higher in soybean plants subjected to co-inoculation with B. japonicum and A. brasilense. It happened due to the excretion of metabolic products, such as root growth regulating compounds (IAA), by A. brasilense. 

The use of co-inoculation with Bradyrhizobium japonicum and Azospirillum brasilense in soybean has promoted high yield in leguminous plants subjected to water stress [10].