Effect of maize root exudates on indole-3-acetic acid production by rice endophytic bacteria under influence of L-tryptophan

Introduction

Indole-3-acetic acid (IAA) is one of the most physiologically active phytohormones, and is a product of L-tryptophan metabolism (Yu et al., 2017). 80% of rhizospheric microorganisms naturally yield auxins as secondary metabolites due to the rich supplies of root exudates (Myresiotis et al., 2015). The rhizosphere is a highly selective area for host-microbe interaction, and during their life span few microbes may enter inside the plant tissue and stay without causing any negative symptoms (Santoyo et al., 2016). These indigenous colonizers reside in almost all internal tissues/cells of plant ranging from tissues of the roots to stem, leaf, flower, fruit and seed (Souza et al., 2015).

These endophytes actively or passively facilitate modification of morphology in the plant cell. It is reported by many workers that endophytes promote growth due to favourable adaptations to abiotic or biotic stresses (Nutaratat et al., 2014). Indigenous bacteria also promote plant growth through various mechanisms. These include direct and indirect mechanisms. Direct mechanisms involve various plant growth promoting hormones (i.e. indole-3-acetic acid, gibberelic acid, adenine-type cytokinins, phenylurea-type cytokinins, ethylene, and abscisic acid), solubilizing inorganic phosphate and atmospheric N₂ fixation. However, indirect mechanisms involve production of various antimicrobial chemical, siderophores and lytic enzymes against the plant pathogens (Zhang et al., 2013). The aim of the present study is to analyse the effect of L-tryptophan and maize root exudates on the production of IAA by rice endophytic bacteria.

Methods

Results

In the present study, 5.6 × 10¹ CFU/ml morphologically unambiguous indigenous bacteria were purified and selected for further analysis for phytohormone IAA production ability. Isolates RE1 and RE17 were identified on the basis of Gram stain, biochemical activities and sugar fermentation, as described in Bergey’s manual of determinative bacteriology (Holt et al., 1994) (Table 1). BLAST analysis of the 16S rRNA gene sequence of RE1 and RE17 isolates demonstrated maximum sequence similarity with the P. fluorescens strain ATCC 13525 (99%, Genbank Sequence ID: NR_114476.1) and P. fluorescens strain CCM 2115 (98%, Genbank Sequence ID: NR_115715.1), respectively as shown in phylogenetic tree analysis by using MUSCLE algorithm and PhyML phylogenetic tree creation software (Figure 1).

Figure 1. Phylogenetic tree of bacterial isolates created by using TreeDyn, Tree Rendering software based on MUSCLE alignment data.

(A) BLAST similarity search results and phylogenetic tree for isolate RE1; (B) Phylogenetic tree for isolate RE17.

In the deficiency of L-tryptophan, strain RE17 released considerable levels of indole (0.7µg/.ml) in comparison with RE1 (0.2µg/ml). In the presence of 50µg/ml of L-tryptophan, RE17 produced significantly higher concentrations of indole compared to RE1 (Figure 2). When 200µg/ml of L-tryptophan was added to the medium, RE1 and RE17 produced eight, and three times the concentration of indole produced at 50µg/ml of L-tryptophan concentration, respectively (Figure 2). It has been observed that RE17 has greater IAA production ability than RE1 (Figure 3). Varying levels of IAA were recorded with different concentrations of tryptophan, i.e. 0, 100, 200 and 500 μg/ml by RE1 as shown in Figure 3.

Figure 2. Production of indoles (µg/ml) by RE1 and RE17 at various concentrations of L-tryptophan.

Figure 3. Production of IAA (pmol/ml) by RE1 and RE17 at various concentrations of L-tryptophan

The concentrations of IAA secreted with maize root exudates in growth chamber studies with RE1 and RE17 were significantly higher (2.8 pmol/ml and 3.4 pmol/ml) than that of the control (0.2 pmol/ml), as shown in Figure 4.

Figure 4. IAA concentration in growth pouches inoculated with or without indigenous isolates.

Discussion

Indigenous microbes colonize internal regions of the plant and are present in almost every plant globally (Ji et al., 2010). IAA secretion through endophytes is a beneficial trait leading to plant development (Ahmad et al., 2008). Therefore, current studies examine IAA generating endophytic P. fluorescens strains from rice roots, with many reporting the active role of tryptophan in the production of IAA by plant growth promoting bacteria (Karnwal, 2017; Stachecki et al., 2004). Tryptophan improves IAA biosynthesis in P. fluorescens strains associated with current research, revealing that it might be the precursor for IAA biosynthesis in these bacterial strains (Khan & Bano, 2016). It has been shown that L-tryptophan concentration affects biosynthesis of IAA at significant levels (Ignatova et al., 2015), however, in this study IAA production was between 1.3 and 9.3pmol/ml in both P. fluorescens strains used, with or without tryptophan. Using ELISA based studies, it was revealed that strain RE17 was the best IAA producer in the presence of maize root exudates in growth chamber study.

The rhizosphere is a rich environment for growth of microorganisms, and it generates a great diversity of microbes (Balseiro-Romero et al., 2016; Kierul et al., 2015; Vaishnav et al., 2016). The growth chamber study confirmed the beneficial potential of maize root exudates on IAA secretion. This supports the fact that plant roots produce some chemical substances (root exudates) that support the growth of rhizospheric microorganisms and their colonization (Kierul et al., 2015; Pereira & Castro, 2014). Hence, these strains have a potential of being developed as bio-inoculants.

Data availability

Bacterial isolate sequence data at NCBI:

Pseudomonas fluorescens strain RE1 16S ribosomal RNA gene, partial sequence: GenBank: MF102882.1.

Pseudomonas fluorescens strain RE17 16S ribosomal RNA gene, partial sequence: GenBank: MF103672.1.

Raw data for biochemical analyses and IAA production: http://doi.org/10.17605/OSF.IO/X5Q46 (Karnwal, 2018).