Role of non rhizobial rhizobacteria and rhizobia on the nodule development in legumes

Abstract

In rhizobia legume symbiosis, the role of signaling molecules like plant flavonoids, Nod factors, plant hormones and polysaccharides involved in the signaling process have been explicitly studied. Despite the highly specific exchange of signals between the symbionts, presence of non-rhizobial bacteria in root nodules has been reported. However, there is no experimental evidence showing how rhizobacteria breach host specificity and enter root nodule. One of the objectives of the present study was aimed at understanding how non rhizobial rhizobacteria enter and colonize legume root nodules. To test the hypothesis that rhizobacteria invade the infection thread (IT) formed by host specific rhizobia, fluorescently tagged native nodulating rhizobia Ensifer adhaerens (Rfp), predominant rhizobacteria Pseudomonas fluorescens (Gfp) and microaerobic Klebsiella pneumoniae (Cfp) were inoculated to V. radiata seedlings. Root hair infection monitored using confocal microscopy at 5 days post inoculation confirmed that Pseudomonas fluorescens and Klebsiella pneumoniae invaded the root hair only when co-inoculated with E. adhaerens. The inoculated tagged strains were recovered from nodule at 50 days post inoculation and nodule occupancy by test rhizobacteria was confirmed through confocal laser scanning microscopy and 16S rDNA sequencing. A total of 26 bacterial and 1 fungal endophyte were isolated from surface sterilized, field grown V. radiata root nodules on Plate count agar (PCA), Nutrient agar (NA), Congo red Yeast extract Mannitol agar (CRYMA) and Potato Dextrose Agar (PDA). 16S rDNA / ITS nucleotide sequence of endophytes was submitted to NCBI (GeneBank). Bacterial endophytes distributed in 8 different genera were screened qualitatively and quantitatively for plant growth promoting (PGP) activities that included phosphate solubilization, IAA and siderophore production. Most bacterial endophytes had one of the PGP trait while the fungal endophyte (Macrophomina phaseolina) lacked all PGP traits. Endophytes M1, M10 and M15 were most influential in improving Seedling Vigor Index. Three endophytes having multiple PGP traits with maximum siderophore production: 46.77 μgml-1 (Bacillus anthracis; M1), IAA production: 10.81 μgml-1 (Paenibacillus taichungensis; M10) and phosphate solubilization: 134.483 μgml-1 (Paenibacillus xylanilyticus; M15) significantly increased root length (RL), shoot length (SL), number of lateral roots (NLR) and plant dry weight (DW) when inoculated/co inoculated with E. adhaerens to V. radiata in a small field trial. M10 inoculation produced longest RL while M1 when coinoculated with E. adhaerens produced highest SL and NLR. M1 inoculation or coinoculation was most effective in improving dry weight. Nod factors are the key determinants of host specificity and exopolysaccharides also play a vital role. Rhizobial Nod factors are synthesized by the cooperative action of nodA, nodB and nodC while nodZ, involved in addition of fucose to Nod factors usually on C-6 of the reducing GlcNAc (N-Acetylglucosamine) residue, may play subtle roles in defining host range of nodulation, exoZ1 involved in acetyl modifications exert symbiotically important effects through succinoglycan. Therefore, as an attempt to extend the host range, nodZ and exoZ1 from S. fredii NGR 234 were cloned in E. adhaerens. The transformed E. adhaerens was inoculated and screened for inducing nodulation on hosts of S. fredii NGR 234 like Glycine max, Sesbania rostrata and Macroptelium siratro etc. Besides Nod factors and flavonoids, plant hormones play vital role in legume rhizobia symbiosis. The positive effect of Auxin, Brassinosteroids and Cytokinin on nodulation is known, however, reports on the effect of exogenous application of plant hormone combination on IT progression and nodulation are scarce. Therefore, this objective was aimed at monitoring the effect of exogenous application of two morphogens 6-benzyl-amino-purine (6-BAP, c) and Indole-3-acetic acid (IAA, a) on rhizobial IT progression and number of nodules in V. radiata. 0.5 μM 6-BAP, 1 and 10 mgL-1 IAA individually increased number of infections per centimeter of lateral roots and in combination, these concentrations were most promising for IT progression. At 20 μM, 6-BAP produced knotty, atypical and twisted IT’s whereas 10 mgL-1 IAA favored IT progression. Among combinations of hormone concentrations used, c0.5a1 and c0.5a10 significantly increased number of infections per centimeter of lateral root over control. Combined application of 6-BAP (0.5-20 μM) and IAA (0.1-10 mgL-1) had significant effect on nodule numbers compared to control. 0.5 μM 6-BAP treated plants produced highest number of root nodules (102) over inoculated control (61). Increase in 6-BAP concentration beyond 0.5 μM decreased the number of root nodules linearly. All the tested concentrations of IAA significantly increased nodule numbers however, 10 mgL-1 was the most effective in increasing nodule number (by 40%) over control. Among the various combinations of hormones, plants treated with c0.5a10 produced highest number of nodules (124) compared to control (66). Combination of IAA (a0.1, a1) with 6-BAP (c0.5, c1) was most effective as it significantly increased nodule numbers followed by c5a0.1 which produced higher number of nodules (76) over control (66). Exogenous application of the combination of 6-BAP and IAA positively affected rhizobial IT progression and enhanced nodulation in V. radiata. Irrespective of the positive influence on growth, the availability of applied conventional plant growth regulators (e.g., 6-benzylaminopurine) to crops remain inadequate, as they are eliminated by oxidation, chemical degradation, volatilization, leaching and microbial degradation. Controlled release of plant growth hormones enhances effectiveness, increases stability, maintains the required dosage and sustains the supply for a prolonged duration, minimizing the negative effects of overdosage and reducing the required frequency of application. Therefore, 6-BAP formulated with Montmorillonite (MMT) was further entrapped in Alginate (AL) to form composite beads. The incorporation of 6-BAP in MMT/AL hybrids was characterized by powder X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), and thermal analysis. In vitro experiments revealed controlled release of 6-BAP from MMT/AL for over 170 h at pH 7.0-7.4 in contrast to pristine 6- BAP which was released within 48 h of application. The sustained release of 6-BAP from single application of 6-BAP-MMT/AL enhanced expression of the GmRIC1 gene 7 days post application and achieved level of expression of GmRIC1 which otherwise was obtained with daily dosage of pristine 6-BAP upto 5 days.