- AMRITA B, S KIRTHANA, KAVYA R AND W. JABEZ OSBORNE*
ABSTRACT- Plant Growth Promoting Rhizobacteria (PGPR), are heterogeneous group of bacteria that can be found in the rhizosphere and in association with roots that enhance the quality of plant growth directly or indirectly. Siderophores are low molecular weight iron chelators which convert insoluble Fe3+ to soluble Fe2+ ions. For the isolation of siderophore producing bacteria, sugarcane rhizosphere soil was collected, and 10-5 and 10-6 dilutions were plated. VIT AKK-1 and VIT AKK-2 were checked for its PGPR activities. Siderophore isolation was carried out using modified CAS-agar plate method and quantification of catacholate and hydroxymate siderophore was performed. The sid-gene responsible for the production of siderophore were amplified using a gradient PCR.
Keywords: PGPR, Siderophore, Catacholate, Hydroxymate, PCR, sid-gene.
Plant growth promoting rhizobacteria (PGPR) in representation with a wide variety of soil bacteria when grown in association with a host plant, results in the stimulation of growth of their host. The direct promotion by the PGPR traits entails either providing the plant with a plant growth promoting substances that are been synthezied by the bacteria or by facilating the uptake of certain plant nutrients from the environment. The mechanism by which the PGPR may promote plant growth may include: (i) the ability to produce or to change the concentration of plant growth regulators like indole acetic acid.(ii) solubilisation of mineral phosphates and other nutrients. In addition to these PGPR traits, the PGPR bacterial strains must be rhizosphere competent that is should be able to survive and colonize in the rhizospheric soil.
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The term siderophore was coined by Lankford in 1973 and was used as a term to describe low molecular weight molecules that bind ferric iron and involved in the receptor specific iron transport into the bacteria. Siderophore was derived from a Greek term meaning “iron carrier” Ishimaru 1993). This is an appropriate term because the siderophore binds iron with an extremely high affinity. Many bacteria and fungi are capable of producing more than one type of siderophore or have more than one iron-uptake multiple siderophores (Neilands1981) . Siderophores are classified on the basis of the chemical functional groups they use to chelate iron: (1) Catecholate-type (phenolate) siderophores bind Fe3+ using adjacent hydroxyl groups of catechol rings. Enterobactin, also known as enterochelin, is produced by a number of bacteria including E. coli and is the classic example of a catechol-type siderophore (Fig. 1A) (O. Brien & Gibson, Pollack et al.)[4,5] (2) Hydroxamate-type siderophores Fe3+ is chelated using nitrogen atoms of thiazoline and oxazoline rings in this type (Crosa and Walsh 2002). Ferrichrome is the classic hydroxamate-type siderophore (Fig. 1B).
for iron by both the plant and the bacterial cells because of the synthesis of several essential iron containing proteins. (Guerinot 1994, Lodwig et al. 2003)[8,9].
Thus, the present study was undertaken to assess the isolation of siderophores from the soil of sugarcane. And to assess the effect of micronutrients especially iron on sugarcane growth and nutrient uptake.
Fig.1 Representative Siderophore Structures. A) Enterobactin (catechol-type) B) Ferrichrome (hydroxamate-type).
Iron is one of the major micro-nutrients which is essential for the plant growth. It plays a role in the primary process of photosynthesis. One of the growth-limiting factor for the majority of microorganisms are iron. (Archibald 1983) . Rhizobia which are root nodule bacteria, when in a symbiotic association with their host plant face difficulty in scavenging iron from their host environment, since there is a high demand
MATERIALS AND METHODS:
1.1 Sampling Location:
For the isolation of siderophore producing bacteria, sugarcane soil was collected from a sugarcane field opposite VIT University, Vellore.
1.2 Sample Collection:
The sugarcane rhizosphere soil was collected from the sugarcane paddy field following a random sampling procedure. The samples were collected using sterile polyethylene bags and were processed immediately.
For the isolation of the siderophore producing bacteria from sugarcane soil, two different procedures were used.
2.1 Isolation of the siderophore producing bacteria using Luria Agar media:-
For the isolation of siderophore producing bacteria using Luria Agar media, 100mL of LB agar was prepared in 250mL Erlenmeyer flask. Serial dilution of sugarcane soil was performed and 10-5 and 10-6 dilutions were platted onto LA plates and incubated at 37oC for 24 hours. Obtained colonies were purified and maintained using LA plates.
2.2 Direct isolation of the siderophore producing bacteria using CAS Agar media:-
- Preparation of CAS indicator solution:
In 50mL of distilled water 60.5mg of chromauzoral S, 10mL of Fe(III) solution (13.5mg FeCl3,41.6µL conc. HCl in 50mL distilled water), 72.9mg CTAB in 40mL were added together to make up a final volume of 100mL. The dark blue coloured solution was then autoclaved.
- Preparation of Basal Agar Media:
For the Basal Agar medium 3% of 3-N-morpholino propane sulfonic acid (MOPS), 0.05g NaCl, 0.03g KH2PO4, 0.01g NH4Cl, 0.5g L-Asparagine were dissolved in 88mL distilled water , adjusted to pH of 6.8. Then 1.5g Agar agar was added to the above solution and autoclaved.
- Preparation of CAS Agar plates:
Both the Basal Agar medium and Indicator solution were cooled to 50oC. 2mL glucose and 10mL indicator solution were added together and the CAS plates were prepared.
3. Screening of obtained isolate for PGPR traits
The isolated bacteria VIT AKK-1 and VIT AKK-2 were screened for the PGPR traits.
3.1 Phosphate solubilising
The isolates were inoculated in 150mL Pikovskaya’s Agar plate (1.6gm Pikovskaya’s Broth and 1.0 gm Agar agar) and incubated for 4days at 28oC.
3.2 Indole Acetic Acid production
The isolates were inoculated in NB-M26 media and incubated for 24 hours in the shaker at 28oC. Next, 10mL Minimal Salt media supplemented with 5mM L-Tryptophan was prepared and 100µL of the inoculums was inoculated and incubated in the shaker for 48 hours. Finally, after centrifugation at 10,000 rpm for 10 minutes a 2mL supernatant is obtained to which few drops of Salkoski reagent is added and left for 25 minutes an checked for cherry ring formation.
Siderophores are produced only under low iron concentrations or iron-restricted media. Modified Fiss Minimal media is used for this purpose. Stock chemical reagents prepared are preserved at 4oC.
3.3.1 Preparation of Modified Fiss Minimal media:-
The chemicals reagents are prepared, autoclaved and preserved at 4oC:-
0.5g KH2PO4 supplemented with 0.5g Asparagine in 99mL of distilled water to which 5mL of D-glucose, 40µL of MnSO4, 100µL of MgSO4µL, 500µL of ZnCl2 and 150µL of FeSO4 is added. The total volume is made up to 100mL and pH adjusted to 6.8. The isolates are inoculated in the media and left at the rotator shaker for 24 hours at 28oC. Sideropore detection assays are next followed.
3.3.2 Assay for Catecholate Siderophore:-
CAS Agar does not detect the type of siderophore produced. Catechol combines with HNO3 to give yellow colour which is intensified to orange-red in presence of excess of NaOH.
1mL of 0.5 M HCl, Nitrite-molybdate reagent (10g sod. Nitrite, 10g sod. Molybdate in 100mL distilled water), 1M NaOH(4g NaOH dissolved in distilled water made up to 100mL) are added to 1mL of culture supernatant. The assay was incubated at room temperature for 5 minutes for colour development.
3.3.3 Assay for Hydroxamate Siderophore:-
2.5mL Atkin’s reagent(0.1771g Fe(ClO4)3 dissolved in 100mL distilled water and 1.43mL perchloric acid) is added to 0.5mL culture supernatant and incubated at room temperature for 5 minutes.
a) Siderophore producing bacteria using LB agar media:
The dilution 10-5 and 10-6 that was plated onto LA plates was incubated at 37°C for 24hrs. On observation we obtained colonies showed positive results, giving isolates VIT AKK-1, VIT AKK-2.
b) Detection of siderophore producing bacteria using CAS agar media:
For the initial detection of siderophore, VIT AKK-1, VIT AKK-2, VIT AKK SID-3, VIT AKK SID-1, VIT AKK SID-2, VIT AKK SID-3 and VIT AKK SID-4 were grown in modified fiss minimal medium containing low iron(0.5µM) conditions. After 24hour of incubation turbity was seen in all tubes except VIT AKK-1.
Fig.2: detection of siderophore, VIT AKK SID-1 ,VIT AKK SID -2, VIT AKK SID -3 and VIT AKK SID-4 on CAS agar plate.
2. Screening of obtained isolate for PGPR traits:
The isolated bacteria VIT AKK-1 and VIT AKK-2 were screened for the following PGPR traits:
a) Phosphate solubilisation:
The plant growth is often limited by insufficient phosphate availability. In the present study isolate VIT AKK-1 showed clear hallow zone around their colonies on pikovskaya’s agar medium after 4 days of incubation.
Fig.3 isolate VIT AKK-1 showing positive result for phosphate solubilisation.
b) IAA (Indole Acteic Acid):
The isolates VIT AKK-1 and VIT AKK-2 on incubation for 48 hours in MSM medium showed negative results.
c) Siderophore detection:
Further results on siderophore detection are awaited.
For majority of the organism, iron is a growth-limiting factor. Although it is present in abundant, its availability is less because it is present in insoluble iron oxy-hydroxide polymer under some aerobic conditions at biological pH . On the roots of the leguminous plant, the rhizobia can induces nitrogen fixing nodules. The plant can also produce some components of respiration and leg-haemoglobin (both contain iron). Due to the competition with its host and largely due to the iron content of the bacterial nitrogenise complex, the demand for iron is more and as a result the bacteria synthesize and secrete siderophores to overcome the iron deficiency.
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The low solubility of common phosphate such as Ca3(PO4)2, hydroxyapatite and aluminium phosphate causes low phosphate availability in agricultural soil. The zone produced around the colonies is due to the presence acidity of medium and release of soluble phosphates. IAA is known to have dual role in influencing the plant growth, which involves in the bio-control in association with glutathione-s-transferases in the defence related plant reactions and inhibits the germination of spore and growth of mycelium of different pathogenic fungi. The present study showed negative results for IAA production.
Initially our goal was to investigate the siderophore-producing capabilities of VIT AKK-1, VIT AKK-2, VIT AKK SID-1, VIT AKK SID -2, VIT AKK SID -3 and VIT AKK SID-4 on CAS agar plate. The initial detection of siderophore production was confirmed using the CAS assay, which demonstrates the production of siderophore under iron-deficient conditions.
In conclusion iron uptake studies using radiolabeled siderophore will be useful to characterize the kinetics of tranport.
Future studies on VIT AKK SID strains could include purification and identification of the outer membrane receptor proteins, as well as characterization of the hydroxamate-type siderophore.
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