Iron Decontamination
All glasswares used in the present study were soaked overnight in 6 M hydrochloric acid (HCl) and thoroughly rinsed with distilled water (DW) to remove any traces of iron.
Sample collection and isolation of rhizospheric bacteria
Rhizospheric soil sample of Eragrostis cynosuroides plant was collected from road side (devoid of any fertilizer) of Danapur, Patna, Bihar, India (25º 34’ 56.2” N, 85º 2’ 37.06” E). The plant was uprooted with the help of trowel for the collection of rhizospheric soil. The soil sample was collected and transferred in sterile ziplock polythene bags to the laboratory for further study and processed within three hours. The soil suspension was prepared by adding 1 g of soil sample to 10 mL sterile DW and diluted up to 10-6 dilution and spread on to nutrient agar media (NAM) and incubated at 30±2 °C for 24 h. Bacterial colonies appear on plates were purified; sub cultured repeatedly to get axenic culture and preserved at 4 °C in NAM for further use. The isolates were designated as DR1, DR2, DR3, DR4, DR5, DR6 and DR7. All the experiments were carried out in triplicates.
Siderophore production
Preparation of Chrome Azurol S medium
The Modified Chrome Azurol Sulfonate (CAS) agar plate19was prepared by mixing two solutions given below:
Solution 1: It was prepared by dissolving 60.5 mg CAS in 50 ml de-ionized water and mixing it with a 10 ml Fe3+ solution (containing 1 mmol L-1 FeCl3.6H2O in 10 mmol L-1 HCl).
Solution 2: It was prepared by dissolving 72.9 mg of hexa decyl trimethyl ammonium bromide (HDTMA) in 40 ml de-ionized water resulting in a dark blue solution.
Both solutions (solution 1 and solution 2) were autoclaved separately and mixed slowly. The final mixture of 100 ml volume was added to 900 ml of succinate agar medium (pH 7). After solidification of the media on petri plates, 24 h old bacterial isolates were inoculated and incubated at 30±2 °C for 24-72 h. Formation of orange halo zone from dark blue color around the colonies was indicative positive result.
Qualitative screening of siderophore production
24 h old bacterial isolates was spot inoculated on CAS agar plates (CAS dye + Succinate medium containing K2HPO4 (6 g), KH2PO4 (3 g), (NH4)2SO4 (1 g), MgSO4.7H2O (0.2 g), succinic acid (4 g)) and incubated for 24-72 h. Change in color of the medium surrounding the test colony from blue to orange halo, indicated positive test for siderophore production.
Quantitative Assay of siderophore production (CAS- Shuttle Assay)
It was carried out by CAS shuttle assay19. The isolates were cultured in succinate medium and incubated in shaker incubator at 120 rpm and 30±2 °C for 24 h. The fermented broth was centrifuged at 10,000 rpm for 10 minutes at 4 ºC. An aliquot of 0.5 ml of supernatant (cell-free extract) was mixed with 0.5 ml of CAS solution. The resulting color obtained was measured after 20 min of incubation at the wave length of 630 nm, using the UV-VIS spectrophotometer (Systronics, Ahmedabad, India), referring the uninoculated CAS solution as blank. The percentage of siderophore units (SU) was estimated as the proportion of CAS color shifted using the formula:
% siderophore units = [(Ar - As)/ Ar] ×100
Where,
Ar - absorbance of reference (CAS assay solution + uninoculated media) and
As - absorbance of the sample (CAS assay solution + cell-free supernatant).
Phenotypic and genotypic characterization of isolates
The most efficient isolate was further characterized on the basis of its morphological, cultural and biochemical characteristics as per the Bergey's Manual of Systematic Bacteriology. The potential strain was identified by 16S rRNA gene sequence analysis. The sequence will be submitted to National Center for Biotechnology Information (NCBI) for accession number. PCR based 16S rRNA gene amplification and sequencing of the isolated bacterium was carried out using universal primers at Xcelris lab Ltd, Ahmedabad, Gujarat, India.
Characterization of siderophores
The characterization of the siderophore as catechol or hydroxamate types was carried out as follows:
Hydroxamate type of Siderophore (Tetrazolium salt test):
A pinch of tetrazolium salt and 1-2 drops of 2 N NaOH was added to 0.1 ml supernatant of the test culture. Instant appearance of a red to deep-red color was indicative of presence of hydroxamate siderophores2.
Catecholate type of Siderophore (Arnow’s Test):
In this assay 1 ml of cell-free supernatant was mixed with 1 ml of 0.5 M HCl and 1 ml of nitrate molybdate to turn the mixture solution yellow. Further, 1 ml of 1 M NaOH was added, mixed and incubated for 5 min at room temperature, resulting in red color formation. The color was stable for 1 hour and the absorbance was measured at 510 nm using a UV-VIS spectrophotometer20.
Detection of siderophores by Thin Layer Chromatography (TLC)
The culture supernatant of siderophore producer strain was spotted on 10×20 mm silica gel plates and allowed to dry. The plates were run in an n-butanol: acetic acid: distilled water (12:3:5) solvent system until the solvent front reached the top. Thereafter it was dried and 0.1 M FeCl3 (prepared in 0.1 N HCl) was sprayed. Appearance of a wine-colored spot indicated a hydroxamate-type siderophore, while that of a dark gray spot indicated catechol-type siderophore21.
Optimization of physicochemical parameters for siderophore production
The biological production of siderophores is governed by several environmental factors like growth medium, temperature, pH, incubation time, carbon sources, nitrogen sources etc. In the present study, the optimization experiments were initiated by evaluating the optimum nutrient medium for siderophore production. The three different nutrient media tested in the current study were Nutrient broth, JNFb- broth and Succinate broth. The siderophore production was monitored by using 50 ml medium each, separately inoculated with 0.25 ml of 24 h old culture, incubated at 37 °C in shaker incubator (120 rpm for 24 h).
The optimization of other physicochemical parameters for production of siderophores was studied by varying one parameter at a time, while keeping the others constant. These varying parameters included, incubation time (24 h, 48 h, 72 h, 96 h, 120 h), temperature (25 °C, 30 °C, 35 °C, 40 °C), and pH (5, 6, 7, 8, 9, 10). In addition, the effect of 0.1 % solution of different carbon sources (glucose, sucrose, fructose, lactose, mannitol) and nitrogen sources (urea, sodium nitrate, ammonium sulphate) were also studied on siderophore production. The bacterial isolates were inoculated in the succinate medium and the estimation was done on the above mentioned quantitative assay.
Pot experiment
In order to evaluate the potential of selected isolate in coriander plant, a pot experiment was conducted in a growth chamber at the Department of Botany, Patna University. The coriander seeds purchased from local market (Bakarganj, Patna, Bihar, India) were surface sterilized by exposing to 2-3 % of NaOCl followed by 70 % ethanol solution for 3 min followed by rinsing with autoclaved DW, at least for three times10. Sterilized seeds were soaked in autoclaved DW for 24 h at room temperature inside closed petri dishes. Further seeds were transferred in bacterial suspension (108 cfu ml-1) at 30 ºC for 6 h and sown in the pot having sterile soil (by autoclaving at 15 lbs/121 °C for 3 h) to a depth of 5 mm as a test (inoculated seeds) and control (uninoculated seeds)10. Sterile water was used for maintaining moisture in the pots as per requirements and observed for seed germination, root length and shoot length with respect to control and after two weeks, plants were harvested, roots were washed free of soil and shoot and root lengths were measured. The germination percentage was calculated. After one week, seedling vigour was recorded in terms of root and shoot length with the help of a measuring scale. Each treatment was carried out in three replications. Also number of normal and abnormal seedlings and dead seeds were counted. Germination percentage was determined by the following formula-
Germination (%) = Normal seedlings / Total number of seed taken × 100
The entire plant was dried in an oven at 72 ºC for 48 h and fresh weight and dry weight were recorded as seedling growth parameter. Total biomass was calculated after deducting the dry weight from wet weight. The plants involved in our study comply with institutional guidelines.
Statistical Analysis
The data obtained were statistically analyzed by using software (SPSS 16.0), and graphically represented as the mean ± standard deviation (n=3).