# Bacterial Indole-3-Acetic Acid Influences Soil Nitrogen Acquisition in Barley and Chickpea
## 1 Introduction  
With rapid and continuing growth of the world's population and the resulting demand in food production, excessive use of chemical fertilizer is degrading the agricultural soil health. Amongst many others, lack of nitrogen is classified as an important abiotic stress factor [1] and its deficiency in soil negatively affects many physiological processes in a plant. To overcome this shortage, different forms of nitrogen are applied to the soil as nitrogen fertilizers. According to an FAO report, the worldwide consumption of nitrogen fertilizer in 2020 was 108.74 million tons, which is expected to reach 111.9 million tons in 2022 [2]. One alternative solution to replace or minimize the use of nitrogen fertilizers is to better harness the biological nitrogen fixation (BNF) in which nitrogenase enzymes from microorganisms can provide fixed nitrogen to plants [3]. However, the practice of using a microbial strategy to fix nitrogen is limited due to a lower output in terms of yield, hence the use of chemical nitrogen sources persists in agriculture [4]. One problem lies in the fact that only around 30% of the applied nitrogen is utilized by the crop plants and the remaining unutilized nitrogen causes severe environmental pollution [5]. Increased nitrogen use efficiency (NUE) could alleviate these environmental impacts and is generallydivided into two phases; a. the nitrogen uptake efficiency (NUpe) of the plant that involves assimilation, and b. nitrogen utilization efficiency (NUte) that involves immobilization of nitrogen [6]. The use of plant growth promoting microorganisms (PGPMs) could be a feasible solution to improve NUE. The few main mechanisms of the rhizosphere that influence N-uptake efficiency are: root size morphology, root N transporter system, and interaction of root-microorganisms such as plant growth-promoting bacteria (PGPB) [7]. Many PGPBs are reported to positively improve root system development by manipulating plant hormones such as auxins, cytokins, or gibberellins [8; 9], and hence the acquisition of some nutrients. NO\({}_{3}\)- and NH\({}_{4}\)+ uptake systems may be enhanced by the interaction with arbuscular mycorrhizal fungi (AMF), PGPB, and some organic acids [7; 10]. The pursuit of BNF, especially in cereals, started long ago with isolation of nitrogen-fixing bacteria from different cereal plants [11]. Few studies have also focused on the appearance of diazotrophs in association with cereal crops and evidence of nitrogen supply [12]. One such example of a bacterial diazotroph is _Klebsiella_. It has been dominant and widely pervasive in the rhizosphere of a variety of plants such as maize [13], wheat [14; 15], and sugarcane [16]. Here we explore the potential of highly ubiquitous _Klebsiella_ as a tool to improve plant NUE. We hypothesized two bacterial mechanisms that might play an important role. First, the nitrogen-fixing ability of the wild type strains, and second, the auxin (IAA) production. The latter will influence the root structure and thus help in acquisition of soil nitrogen (no chemical nitrogen fertilizer supplements in the present case). In proteobacteria, nitrogen fixation genes (_nif_) are generally transcribed by the alternative s\({}^{54}\) RNA-polymerase under nitrogen limiting conditions and regulated by the enhancer binding protein NifA [17]. Auxin production depends on the expression of _ipdC_, coding for thiamindiphosphate-dependent indolepyruvate decarboxylase, which was reported to also be s\({}^{54}\)-dependent in the diazotrophic _Azospirillum brasilense_[18] and the non-diazotrophic _Enterobacter cloace_[19]. To understand which out of the two aforementioned mechanisms has larger impact, the effects of three different strains of _Klebsiella_ (from different origin) on a model monocot _Hordeum vulgar_ (barley) and a dicot legume _Cicer arietinum_ (chickpea) were analyzed.