*5.2.3 Biological nitrogen fixation (BNF)*

*Microorganisms*

Howie *et al*. [105] hypothesized two phase processes in which bacterium can attach itself to the plant and soil. In the phase I, bacteria on the seed are attached to the emerging root tip where they are passively transported into the soil. During root growth, some bacteria cells remain associated with the tip while others are left behind on the older parts of the root and the rhizosphere. In phase II, bacteria deposited along the root zone multiply and form microcolonies in nutrient-rich microsites, where they compete with indigenous microflora in order to avoid displacement. Both phases occur simultaneous on different root parts [106]. Root colonization can be influenced by both biotic (such as genetic traits of the host plant and the colonizing organism) and abiotic (such as soil humidity, growth substrate, soil and rhizosphere pH and temperature) factors. Changes in plant physical and chemical composition in the rhizosphere can strongly influence root colonization and competence [107]. Root exudates and mucilage-derived nutrients attract beneficial and neutral bacteria as well as harmful bacteria allowing

In developing a sustainable crop production system, the use of plant growth promoting rhizosphere has played a potential role [109] though its mechanisms of enhancing plant growth and yields have not been fully understood [110]. PGPR play an important role in plant growth through different mechanisms [111]. The relationship of PGPR differs with different host plants. Rhizospheric and endophytic relationships are the two major classes of PGPR relationships. PGPRs that colonize root surfaces or superficial intercellular spaces of the host plant forming root nodules are known to have Rhizospheric relationships. A microbe belonging to the genus *Azospirillum* is the dominant species in the rhizosphere [112]. PGPRs that inhabit and grow within the apoplastic spaces of the host plants are known to have endophytic relationships [107]. Some researchers have shown that inoculation of plants with PGPR help in increasing their nutrient contents [113, 114] and resistivity to pathogens [115, 116]. PGPR colonize plant by interacting with the host plant thus enhancing its nutrient uptake by fixing nitrogen biologically, increasing the availability of nutrients in the rhizosphere, inducing increases in the root surface area, enhancing other beneficial symbioses of the host, and combining the modes of action [107]. PGPR help to solubilize mineral phosphates and other nutrients, stabilize soil aggregates, improve soil structure and organic matter content, and increase plant resistivity to stress. It retains more soil organic nitrogen and other nutrients in the plant–soil system, thereby reducing their need for nitrogen and

them to colonize and reproduce in the rhizosphere [108].

phosphorus fertilizer and enhancing the release of nutrients.

population of harmful ones in the rhizosphere [111].

In addition to increasing plant nutrient contents, PGPR capable of producing phytohormones produce hormones such as cytokinins, ethylene, gibberellins, auxins and abscisic acid. Some of the bacterial genera belonging to the PGPR produce indole-3-acetic acid (IAA), a compound belonging to auxins which promote plant growth. Some PGPR function as a sink for 1-aminocyclopropane-1-carboxylate (ACC), the immediate precursor of ethylene in higher plants, by hydrolyzing it into –ketobutyrate and ammonia, thereby promoting root growth by lowering indigenous ethylene levels in the micro-rhizo environment [117]. In different ecosystems, bacteria can also play a core role in the composition of plant communities by specifically acting on certain plant species and also participating in key environmental processes. In addition to increasing plant nutrient content, it is capable of increasing the population of other beneficial microorganisms and controlling the

*5.2.2 Plant growth promoting rhizobacteria (PGPR)*

**70**

The process of fixing nitrogen biologically by soil microbes is an economically attractive and ecologically sound method to reduce external nitrogen input and enhance the quality and quantity of internal resources [118]. Soil microbe can be considered as a living component of soil organic matter because the biomass comprises all soil organisms with a volume approximately less than 5 × 103 μm3 apart from plant tissue [119]. This process accounts for 65% of nitrogen that are currently used in agriculture, and will continuously be of importance in the sustenance of crop production systems in the future [120]. In most terrestrial ecosystems, BNF is their largest source of new nitrogen [121]. The rates of BNF in tropical forests (15 to 36 kg N/ha/yr) are higher than/similar to their temperate counterparts (7–27 kg N/ ha/yr), which are subjected to strong nitrogen limitation [122]. In the tropics, diazotrophs could have been favored because they receive enough quantity of nitrogen to maintain higher extracellular phosphatase activity, which is prerequisite for overcoming phosphorus limitation and also they have optimum temperature for their activities [123]. Important biochemical reactions of BNF occur mainly through symbiotic relationship of N2-fixing microbes (especially bacteria) with legumes that convert atmospheric nitrogen (N2) into ammonia (NH3) [124].

Symbiotic and non-symbiotic microorganisms in the soil rhizosphere can assist in fixing atmospheric nitrogen in crops and non-crop plants. Over the years, it has been accepted generally that legumes (and the non-legumes genus *Parasponia*) are exclusively nodulated by member of the *Rhizobiaceae* Family in the -proteobacteria, which includes the genera *Bradyrhizobium, Sinorhizobium, Azorhizobium, Mesorhizobium* and *Rhizobium* [125]. Recently, other species of -proteobacteria such as *Methylobacterium*, *Blastobacter denitrificans, Devosia* have been reported to nodulate *Crotalaria, Aeschynomene indica* and *Neptunia natans*, respectively [126–128]. *Ralstonia taiwanensis* and *Burkholderia spp.* belonging to the β-proteobacteria have been found in the nodules of some tropical legumes [129, 130].

Generally, PGPR are classified as biofertilizers, biopesticides and phytostimulators [131]. The biofertilizers help to promote plant growth by supplying nutrients to the host, and these include *Allorhizobium spp., Pseudomonas fluorescens, Rhizobium spp.* and *Trichoderma spp.* (e.g. *T. asperellum* and *T. hamatum*) [132]. The symbiotic association of Rhizobacteria with soil introduces 50–70 × 106 tons of nitrogen into agricultural soils thus reducing the use of inorganic fertilizers [133]. The phytostimulators produce phytohormones such as indole acetic acid, gibberellin and cytokinins which alter root architecture and promote plant development [134] and these include *Bacillus, Azospirillum, Azotobacter, Enterobacter, Pantoea, Pseudomonas, Streptomyces* and *Rhizobium spp.* The biopesticides inhibit the proliferation of pathogen and help in plant growth, and these include *Pseudomonas spp.*, *Streptomyces spp.* and *Bacillus spp.* (e.g. *B. subtilis*) [135]. In addition to these three groups, there are other PGPRs that induce tolerance in plants to abiotic stress. Those in this group include *Paenibacillus polymyxa, Achromobacter piechaudii* and *Rhizobium tropici* [136].

The nitrogen fixed by symbiotic *Rhizobia* in legumes can be beneficial to associated non-leguminous crops through direct transfer of biologically fixed nitrogen to cereals growing in intercrops [137] or to subsequent crops rotated with symbiotic leguminous crops [138]. In many low input grassland systems, the grasses depend on the nitrogen fixed by their legume counterparts for their nitrogen nutrition and protein synthesis, which is much needed for forage quality in livestock production [117]. *Rhizobium* and *Bradyrhizobium* species of *Rhizobia* produce molecules such

as auxins, abscisic acids, riboflavin, cytokinins, vitamins and lipochitooligosaccharides that promote plant growth in addition to fixing atmospheric nitrogen [139]. Other PGPR traits of *Rhizobia* and *Bradyrhizobia* assist in the production of phytohormones [140], release of siderophore [141], solubilization of inorganic phosphorus [142] and also act as antagonist against plant pathogenic microbes [143]. In the study of Kennedy *et al.* [144], a several number of non-symbiotic PGPR significantly increase the vegetative growth and grain yield of C3 and C4 plants such as rice, maize, wheat, cotton and sugarcane due to their interactions. The application of *Azotobacter* increased the yield of rice, cotton and wheat [145, 146]. In a field trial experimental study, Tran Van *et al*. [147] used *Burkholderia vietnamiensis* to inoculate rice and found out that it significantly increased the grain yields up to 8 t/ha. It has been reported that the species belonging to genus *Burkholderia* can produce substances that are antagonistic to nematodes [148].
