**2. Mechanisms of plant growth promotion**

## **2.1. Biological nitrogen fixation**

PGPR improve plant growth by multiple mechanisms. A well-established mechanism is the biological nitrogen fixation (BNF), as described in extensive literature available on diazotrophic association in wheat and subsequent addition of nitrogen to the ecosystem [24], contributing to the total N<sup>2</sup> requirement of wheat [25–27]. Nitrogen fixation is considered to be a direct plant growth-promoting trait and the nitrogen-fixing rhizobacteria provide an alternative source to inorganic nitrogen fertilizers.

*Azospirillum* is a kind of nitrogen-fixing bacterium that lives in close association with plants in the rhizosphere. Its beneficial effects on wheat yields in both greenhouse and field conditions have been reported [28, 29]. Balandreau found that *Azosprillum lipoferum* inoculation increased yield around 1.8 t/ha and wheat grain by up to 30% [30, 31]; Okon and Labandera-Gonzalez by inoculation with *Azospirillum brasilense* [31]. In an earlier study, Boddey et al. were unable to observe fixed N in wheat from similar organisms [32]. Further, Ruppel and Merbach investigated the dinitrogen-fixing ability strain of *Pantoea agglomerans* and *Azospirillum* spp. and in hydroponic experiments with wheat found that bacterial strain inoculation affected plant growth, by nitrogen uptake and the amount of biologically fixed dinitrogen. In this sense, when *Azospirillum* brasilense is inoculated using seed inoculation, it increases the productivity of wheat [33–35]. *P. agglomerans*, as a diazotroph, is able to fix molecular N<sup>2</sup> with wheat [36]. Ruppel et al. reported *P. agglomerans* to be superior strain for winter wheat, reporting a grain yield increase for different wheat cultivars ([37], also in Ref. [38]). Moreover, a nitrogen-fixing *P. agglomerans* Lma2 was isolated from wheat rhizosphere, it was found to have the ability to produce IAA, siderophores and solubilize P, and growth performance of plant was significantly better in the presence of salt [39].

*Acinetobacter* strains also possessed BNF properties, siderophore and ammonia production as well as mineral solubilization. Rana et al. reported a positive correlation of BNF potential of *Providencia* spp. AW4 and *Brevundimonas diminuta* AW7 strains with panicle weight and plant height in wheat, indicating the enhancing plant growth role of BNF [40].
