Isolation of Azotobactor sp. and Azospirillum sp. from the rhizosphere soil and root samples

ISOLATION OF AZOTOBACTOR AND AZOSPIRILLUM FROM SOIL AND ROOT SAMPLES

AIM: To isolate the Azotobactor sp. and Azospirillum sp. from the given rhizosphere soil and root samples.

PRINCIPLE:

                Azospirillum live as symbiotic with plants in the rhizosphere. The plant stimulatory effect exerted by Azospirillum has been attributed to several mechanisms, including biological nitrogen fixation and production of plant growth promoting substances. It has been described to the bacterial production of plant growth regulating substances like plant hormones. An increased number of lateral roots and root hairs enlarge the root surface available for nutrients. This results in higher nutrient uptake by inoculated roots and an improved water status of the plant, which in turn could be the main factor for enhancing plant growth. Azospirillum is not only able to fix atmospheric N but also to mineralize nutrients from the soil, to sequester, Fe, to survive to marsh environmental conditions, and can help plants minimize the negative effects of abiotic stresses.

Azotobacter sp, are free-living aerobic bacteria dominantly found in soils, present in alkaline and neutral soils. They are nonsymbiotic heterotrophic bacteria capable of fixing an average 20kg N/ha/year. Besides, it also produces growth promoting substances and is shown to be antagonistic to pathogens. Azotobacter sp. are found in the soil and rhizosphere of many plants and their population ranges from negligible to 104 g-1 of soil depending upon the physico-chemical and microbiological (microbial interactions) properties. Besides, nitrogen fixation, Azotobacter also produces thiamin, riboflavin, indole acetic acid and gibberellins. When Azotobacter is applied to seeds, seed germination is improved to a considerable extent, so also it controls plant diseases due to above substances produced by Azotobacter. The exact mode of action by which Azotobacteria enhances plant growth is not yet fully understood. Three possible mechanisms have been proposed: N2 fixation; delivering combined nitrogen to the plant; the production of phytohormone-like substances that alter plant growth and morphology, and bacterial nitrate reduction, which increases nitrogen accumulation in inoculated plants. Bacteria isolated from soil rhizosphere by using serial dilution on selective media for Azotobacter (LG medium). Isolates were characterized by morphological & biochemically according to Bergey’s Manual method, to shown properties of Azotobacter spp. 

MATERIALS REQUIRED:

All the media components which has been mentioned in the procedure, glasswares such as test tubes, conical flasks, slides, inoculation loops, Staining kits, Microscopes.

PROCEDURE: Bacterial isolation and identification.

AZOTOBACTOR SP.

1.  Different soil samples from the rhizosphere of agricultural crop were transferred to laboratory.

2.  Strategies used for isolation were: (i) Enrichment of Azotobacter strains, one gram from each of the soil samples were added into 100 ml Erlenmeyer flasks containing 20 ml of Azotobacter broth of the following composition; mannitol 20g, K2HPO4 0.8 g, KH2PO4 0.2 g, MgSO4·7H2O 0.5 g, FeSO4·6H2O 0.10 g, CaCO3 20 g, NaMoO4·2H2O 0.05 g supplemented with ZnSO4.7H2O 10 mg, MnSO4.4H2O 1.0 mg and cycloheximide (100μg/ml) per liter (Adjust to pH 7.2). Incubation was at 28°C for 2-5days.

(ii) Isolation was carried out by preparing serial dilutions from enrichment culture followed by streaking and incubation at 28ºC for 2-5 days. All the isolates were subcultured on selective nitrogen-free specific medium Azotobacter Agar plates.

AZOSPIRILLUM SP.

1.      Soils were collected from various crop fields in and around Coimbatore.

2.      One gram of each collected soil sample was suspended in a tube filled with 5ml of half strength of Winogradsky’s N-free mineral medium containing (g/liter): 10 g of glucose, 25 g of KH₂PO₄, 12.5 g of MgSO₄·7H₂O, 12.5 g of NaCl, 12.5 g of FeSO₄·7H₂O, 0.1 g of Na₂MoO₄·2H₂O, 0.38 g of MnSO₄·H₂O, 0.1 g of CaCO₃, which were sterilized separately, and 15 g of agar, pH 7.2 and then grown at 37⁰C in an incubator shaker overnight.

3.      Serial dilution were made and 0.1 ml aliquots (10^-3 -10^-5) were speeded on plate containing the same agar medium.

4.      The plates were incubated for 2 days at 37⁰C and morphologically different colonies appearing on the medium were isolated.

5.      The isolates were characterized for the following traits: color pigment, form elevation margin, diameter, surface, opacity and texture.

6.      Morphology was evaluated under microscopy and motility was tested by observing the spread of the growth in test tubes of semi agar media.

7.      Biochemical characters of bacterial isolates were examined according to methods described in Bergey’s Manual of Systematic Bacteriology.

RESULTS:(Expected results)

Azospirillum sp. the isolates were microscopically observed for their cell shape and gram reaction. The cell shape of all the isolates was spiral; all the isolates were Gram negative and had cork screw movement when observed under microscope.

Azotobactor sp. After incubation of soil sample in Azotobacter selected media, colony has been thoroughly characterized on the basis of colony color, shape and diameter of the colony. Out of them the characteristics features of some selected colony has been summarized  (Table) Most of the bacterial colony isolated are circular (even) in shape and most of the bacterial colony are whitish in colour and the size ranges between 1.0 – 4.0 mm in size (Table) . Others bacterial colonies isolated are spindle (even) in shape and circular (undulated ) and others bacterial colony are translucent white with central black dot yellowish, and creamy .