Abstract: The rhizosphere soil and roots of maize (Zea mays L.) plants were collected from plain land of Islamabad, from Durgai and from the Shringal Campus of Malakand University. Azospirillum was isolated, identified and characterized by physiological and biochemical tests using QTS (Quick Testing System) miniatured identification system from the 24 h old bacterial culture grown on Lb (liquid broth) and NFM (nitrogen free medium) suspended in saline solution. The rhizosphere and roots of maize plants collected from plain land of Islamabad at the altitude of 800 m contained Azospirillum lipoferum whereas both the roots and rhizosphere of maize plants collected from Durgai at the altitude of 3500 m and Shringal campus of Malakand University at the altitude of 4000 m contained Azospirillum brasilense.
INTRODUCTION
Since its rediscovery by J. Dobereiner and her collaborator in 1970 the species Azospirillum has gained the reputation of being the most studied plant-associative bacterium. Apart from direct agricultural application, Azospirillum is an excellent model for genetic studies of plant-associative bacteria in general. As the most researched associative bacterium. Azospirillum has also been used in production of Poly B-hydroxybutyrate (PHB) for medical use, degradation of pollutants, vitamin production and purification of residual water are slowly being introduced into this field.
Two species of the genus Azospirillum lipoferum and A. brasilense were identified by DNA/DNA homology experiments with a large number of strains.
Azospirillum species occur in soil and are enriched in root surface of many different plants. A. lipoferum and A. brasilense could be isolated from 30-90% of soil samples collected all over the world (Dobereiner et al., 1995; Del Gallo and Fabbre 1991).
The beneficial effects of Azospirillum on plants are enhanced when coinoculated with other microorganism. Apparently, coinoculation allows plants to have a more balanced nutrition and the absorption of Nitrogen and phosphorus and other mineral nutrients are improved. Combination with both Rhizobia and Azospirillum gave the highest yield (Hassouma at al., 1994).
Azospirillum can produce in vitro the phytohormones IAA, gibberellins, cytokinins, (Tien et al., 1979; Bashan, 1991) and ethylene. Sometime external application of synthetic hormones initiated the positive effect of Azospirillum on root development morphology. Nitrogen fixation was the first mechanism proposed to explain improved plant growth following inoculation with Azospirillum. Present investigation is aimed to isolate, characterize and identify Azospirillum species from the roots and rhizosphere of maize plants collected from 3 different altitudes.
MATERIALS AND METHODS
Fresh maize soil from the rhizosphere and maize roots were collected from Islamabad (plain land, altitude 800 m), Durgai (altitude 3500 m) and Shringal campus of Malakand University( altitude 4000 m). The soil samples were shaken vigorously for 10-15 min in a test tube with hand. The soil suspension was centrifuged. Then serial dilution were made for the supernatant. Three vials of LB (liquid broth) and three vials of NFM were inoculated with diluted soil extracted from series of maize rhizosphere soil. After inoculation these vials were incubated at 30°C for 48-72 h. Then a thin layer of pellicle was observed. For rapid multiplication, the culture was grown in liquid media of LB and NFM and these LB and NFM media were inoculated from the vials that contain growth in a thin layer of pellicle.
After inoculation these media were kept in shaker at 28°C for 24 h. These media of LB and NFM were processed for QTS.
Physiological and biochemical tests were performed using QTS (24) miniaturized identification system (DESTO laboratories Karachi Pakistan) following the method of MacFaddin (1980).
| Table 1: | QTS (Quick Test System) for Azospirillum collected from rhizosphere of maize plants from three different places |
The bacterial cultures (24 h old) grown on LB (liquid broth) and NFM (nitrogen free medium)plates were suspended in saline solution.
Oxidase test was performed according to Steel (1961). The catalase test was performed according to the procedure of MacFaddin (1980).
Petri plates and vials containing the growth in the form of pellicles and colonies were observed under microscope. A very characteristic pellicle appeared that grow rapidly in to dense white undulated pellicles. These pellicles were easily recognized against blue medium. Simultaneously petri plates contain discrete colonies that differ in appearance on each of the agar plate culture of LB and NFM. The vials and petri plates containing microorganisms growth were observed under the microscope. Azospirillum species were very motile characterizing wiggling rods. After observation of growth of Azospirillum species, from first culture further subculture were made, in order to obtain pure colonies for reconviction of more transparent growth of Azospirillum species. This observation was repeated for all the rhizosphere of maize plants of different altitudes.
For isolation of Azospirillum from roots of maize plants, the roots were washed in tap water to remove adhering soil particle, thereafter surface sterilized in 10% chlorox and successively washed in sterile water and the roots were crushed in sterile water. The suspension was centrifuged and serial dilutions were made as described earlier.
QTS 24 is based on 25 biochemical tests in addition to cytochromes oxidase test and results revealed that the isolates obtained from rhizosphere of maize collected from plain land of Islamabad differ from those of isolates collected from Durgai and Shringal Campuses in biochemical tests of ONPG, Sodium malonate, ornithine decarboxylase, acid from glucose, nitrate reductase, acid from mannitol and acid from sucrose (Table 1). These results were compared with Bergeys Manual of Determinative Bacteriology (Holt et al., 1994).
RESULTS AND DISCUSSION
According to the Bergeys Manual of Determinative Bacteriology (Holt et al., 1994), on the basis of biochemical test results (Table 2) indicated that the roots and rhizosphere of maize plant, collected from the plain land of Islamabad at the altitudes of (800 m) contained Azospirillum lipoferum, rhizosphere of maize plants collected from Durgai at the altitude of 3500 m and Shringal campus of Malakand University at the altitude of 4000 m contained Azospirillum brasilense.
| Table 2: | QTS (Quick Test System) showing difference between Azospirillum lipoferum and Azospirillum brasilense and the selected biochemical reaction from the QTS test are represented for showing the differences in the identification of Azospirillum lipoferum and Azospirillum brasilense from maize root |