Abstract: Cultural and nutritional conditions were optimized for Indole Acetic Acid (IAA) production by Rhizobium spp. isolated from root nodules of Crotalaria juncea, C. retusa, C. laburnifolia, C. verrucosa and C. alata. The isolates produced maximum amount of IAA after 72 h of incubation and at 2.5 mg mL-1 L-tryptophan concentration. The effect of different carbon and nitrogen sources on IAA production were also studied and it revealed that, mannitol and L-glutamic acid were the best promoters for IAA production. Addition of cell wall affecting agents increased the IAA production over controls. Among the five isolates of Crotalaria species, maximum amount of IAA was produced by isolate from C. retusa. The compound from Rhizobium sp. from C. retusa was extracted, purified and structurally confirmed as IAA.
INTRODUCTION
The Plant Growth Promoting Rhizobacteria (PGPR) are involved in the production of diverse microbial metabolites like siderophores, plant growth enhancement through Indole Acetic Acid (IAA) production and HCN production (Ahmad et al., 2005). Rhizobia are the first group of bacteria, which are attributed to the ability of PGPR to release IAA that can help to promote the growth and pathogenesis in plants (Mandal et al., 2007).
Crotalaria is one of the largest genera of Fabaceae with more than 500 species commonly occurring in diverse climatological situations (Allen and Allen, 1981). Some Crotalaria species are of great agronomic interest, since they are used as green manure to improve soil fertility or control nematode populations in infested soils (Sy et al., 2001). Very few studies on IAA production by root nodule symbionts associated with this species. The objective of this study was therefore to investigate the IAA production by Rhizobium isolates of most commonly occurring Crotalaria species (C. juncea L., C. laburnifolia L., C. retusa L., C. verrucosa L. and C. alata Buch. Ham.) and regulation of various factors such as incubation time, tryptophan status along with carbon sources, nitrogen sources and cell wall affecting agents.
MATERIALS AND METHODS
Organism and Growth Conditions
The study was conducted in the Microbiology Department of Acharya Nagarjuna
University, India, in January 2007. The symbionts were isolated from the root
nodules of Crotalaria species according to Vincent (1970) using Yeast
Extract Mannitol Agar (YEMA) medium. Identification of the isolates was carried
out on the basis of morphological, cultural and biochemical characteristics
in YEM broth by standard method (Holt et al., 1994).
For IAA production, axenic cultures of the bacteria were grown in 100 mL Erlenmeyer flasks containing 25 mL of yeast extract mineral medium (Skerman, 1959) with 1% mannitol and 0.01% CaCl2 at pH of 7.0 for 72 h (optimum time for IAA production). Bacterial growth was determined using colorimeter taking Optical Density (OD) at 540 nm.
Estimation of IAA Production
The IAA in cell free supernatant was estimated colorimetrically by the method
adopted from Gordon and Weber (1951).
Effect of incubation period was studied by inoculating Rhizobium isolates separately into L-tryptophan supplemented medium and incubated for 168 h at 30±2°C. The samples were withdrawn every 24 h and, the growth and IAA were estimated.
Different concentrations of L-tryptophan (0.5, 1.5, 2.5, 3.0 mg mL-1) were added to the basal medium to find out the maximum IAA production. Different carbon sources were also added to the tryptophan supplemented basal medium omitting mannitol. Then the different chemicals were added individually to the tryptophan supplemented basal medium having most suitable carbon and nitrogen source. The individual effect of the chemicals on IAA production was also measured.
Extraction of IAA
The Rhizobium sp. isolated from C. retusa was inoculated into
200 mL of YEM medium with most suitable substance and incubated at 28±2°C
for 3 days on rotary shaker. After incubation, the IAA was extracted according
to the method described by Sinha and Basu (1981).
Purification and Detection of IAA
Partial purification of IAA from crude extract was done by using silica
gel column chromatography (22X5 cm) and fractions were collected with solvent
system using ethyl acetate and hexane (20:80 v/v). Each fraction (10-20 μL)
was tested on Thin Layer Chromatography (TLC) with solvent system (ethyl acetate
and hexane, 2:8) and then developed with Salkowski reagent (Morales et al.,
2003).
Mass Spectrometry
The fraction that gave positive result with Salkowski reagent was collected
and concentrated and analyzed by Liquid Chromatography-Mass Spectrometry (Agilent
1100 series, USA) equipped with Mass Selective Detector (MSD).
Statistical Analysis
The data were statistically analyzed by ANOVA (two way classification technique)
using Statistica software.
RESULTS AND DISCUSSION
Based on the morphological, cultural and biochemical characteristics, the isolates were identified as Rhizobium species. The identification was done following Bergey’s Manual (Jordan, 1984). The Rhizobium isolates from Crotalaria species produced IAA after 24 h of incubation and reached maximum at 72 h and then decreased (Fig. 1). The decrease in IAA level might be due to the release of IAA degrading enzymes as reported earlier in Rhizobium sp. from Cajanus cajan (Datta and Basu, 2000). Among the five isolates of Crotalaria species, the isolate from C. retusa produced maximum amount of IAA (39.0 μg mL-1) after 72 h.
| Fig. 1: | Growth and IAA production by Rhizobium isolates from Crotalaria species |
| Fig. 2: | Effect of different concentrations of L-tryptophan on growth and IAA production by Rhizobium isolates from Crotalaria species. |
The effect of different concentrations of L-tryptophan revealed that the maximum growth and IAA production were observed in all the isolates at 2.5 mg mL-1 L-tryptophan concentration (Fig. 2). In earlier reports, the Rhizobium sp. isolated from root nodules of Dalbergia lanceolaria also produced high amount of IAA at 2.5 mg mL-1 L-tryptophan concentration (Ghosh and Basu, 2002), while the Rhizobium sp. from root nodules of Roystonia regia produced maximum amount of IAA at 3 mg mL-1 L-tryptophan concentration (Basu and Ghosh, 2001).
Replacement of 10 different carbon sources (1 %) in the basal medium revealed that the Rhizobium isolates vary in their utilization and production of IAA. The isolates produced more amount of IAA when mannitol was used as carbon source (Table 1). But, Rhizobium sp. from Cajanus cajan produced maximum amount of IAA in glucose containing medium as reported earlier (Datta and Basu, 2000). The production of IAA was minimum when rhamnose, ribose, mannose and sorbitol were used as carbon source. But, ribose was reported to be the best promoter at 1.5% concentration by the Rhizobium species isolated from Psophocarpus tetragonolobus (Bhattacharya and Basu, 1992). Statistically significant was occurred in between Rhizobium spp. and in between carbon sources.
Effect of different nitrogen sources (0.1%) was studied by replacing yeast extract in the original YEM medium supplemented with L-tryptophan. It revealed that the inorganic nitrogen sources like KNO3, (NH4)2SO4 and NaNO3 supported maximum IAA production in all isolates, while amino acid glycine as additional nitrogen source reduced growth and IAA production (Table 2). Some amino acids were shown earlier to inhibit IAA production by Rhizobium meliloti (Garcia-Rodriguez et al., 1981) due to inhibition of conversion of tryptophan to IAA.
| Table 1: | Effect of carbon sources on growth and indole acetic acid production by Rhizobium isolates from Crotalaria species |
| *: Significant at 1% (Between Rhizobium sp.: Fc = 4.7, Ft = 2.0; Between carbon sources: Fc = 12.8, Ft = 2.6) | |
| Table 2: | Effect of nitrogen sources on growth and indole acetic acid production by Rhizobium isolates from Crotalaria species |
| *: Significant at 1% (Between Rhizobium sp.: Fc = 4.5, Ft = 2.1; Between nitrogen sources: Fc = 30.2, Ft = 2.6) | |
The Rhizobium isolates from C. retusa, C. laburnifolia and C. alata showed maximum growth and IAA production in the medium amended with L-glutamic acid (44.2, 14.4 and 16.9 μg mL-1). That the L-glutamic acid was the most effective nitrogen source for growth and IAA production were also reported in Rhizobium sp. isolated from root nodules of Cajanus cajan (Datta and Basu, 2000). The effect of different nitrogen sources on IAA production was also found to be statistically significant.
Addition of cell wall affecting agents or surfactant revealed that, all the five isolates produced highest amount of IAA in medium amended with different concentrations of EDTA and SDS. But, penicillin and lysozyme have generally negative effect on IAA production (Table 3). IAA production was maximum at 0.2 μg mL-1 EDTA and 0.1 μg mL-1 SDS concentrations. Among the five isolates, the isolate from C. retusa produced maximum amount of IAA (50.9 μg mL-1) at 0.2 μg mL-1 EDTA concentration. Changes in the cell well or membrane by these agents increased the availability of tryptophan to converting enzyme as well as increase the release of IAA from the cell was reported earlier by Bhattacharya and Basu (1992).
| Table 3: | Effect of cell wall affecting agents on growth and indole acetic acid production by Rhizobium isolates from Crotalaria species |
| * Significant at 1% (Between Rhizobium sp.: Fc = 2.07, Ft = 2.01; Between surfactant sources: Fc = 44.0, Ft = 2.5) | |
The effect of surfactant on IAA production was statistically significant but in different Rhizobium isolates production of IAA was not significant.
As the Rhizobium isolate from C. retusa produced highest amount of IAA (50.9 μg mL-1) in the medium amended with 0.2 μg mL-1 of EDTA, this Rhizobium sp. was selected for characterization of IAA produced.
TLC of chromatogram of purified compound and standard IAA sprayed with Salkowski reagent showed almost the same Rf-values (0.85) with all ithentic JAA, which were identified under UV-light (254 nm).
The fraction that gave positive result with Salkowski reagent was collected and concentrated and analyzed by LC-MS. The pure indole was structurally confirmed as IAA by comparing with standard IAA present in compound library system of LC-MS. The LC-MS of fraction revealed that it has a benzene moiety (m/z 77) in its chemical structure and its molecular weight may be 175.4. From this study, it is clear that all the isolates from Crotalaria sp. were positive for IAA production, but the isolates differ significantly in auxin production depending upon the cultural conditions.