MATERIALS AND METHODS:

Description of the study area:

The present study focused on the area in and around Thanjavur district (Fig.1). The study area is situated in Tamilnadu state (Lat. 11^°10– 11^° 30N and Long. 78^°15 – 78^°30E) with the significant features of evergreen forests and also it was a less explored ecosystem for the investigation of Azospirillum population.

Collection of soil samples (Bashan and Wolowelsky, 1987): For the enumeration of Azospirillum, soil samples were collected by aseptic manner at a depth of 5-10 cm according to the V - shaped method, at thirty different locations in and around the Thanjavur district. From each site, five samples were collected and pooled together and considered as one sample. The soil samples were brought to the laboratory and kept in the refrigerator for further process.

Morphological characteristics of Azospirillum isolates:

Gram staining:

Thin smear of the isolates were made on the clean glass slides and heat fixed. Then the smears were stained by crystal violet for 60 seconds and then washed with distilled water, followed by flooded with mordant of Gram’s iodine. After 60 seconds, the slides were washed with distilled water and decolourized with 95% ethyl alcohol. After decolourization, the smear was rinsed with distilled water and counterstained with safranin for 60 seconds. Then, the slide was rinsed with distilled water, air dried and examined microscopically.

Size:

Ocular micrometer was calibrated after putting inside the eyepiece by superimposing the graduations of ocular micrometer over graduations of the stage micrometer, which was accomplished by rotating the ocular lens, and by determination of the number of ocular divisions coinciding with the number of divisions on stage micrometer. The calibration factor for one ocular division is calculated for particular combination of objective and ocular lens used by applying the formula:

One division of ocular micrometer =

Number of divisions on stage micrometer

X 10

No. of division on ocular micrometer

Then the ocular micrometer can be used to determine the size of Azospirillum by terms of length and determined the size using the following formula:

Size in (µm) = No. of ocular divisions X Calibration factor of the objective occupied from micro organisms used.

Determination of swarming motility:

Swarming movement was observed in soft nutrient agar (0.2% agar). The soft nutrient agar was prepared. The isolates were inoculated into the centre of the medium and incubated for 48 h at 32^°C. Results were observed and the surface growth was recorded.

Physiological characteristics of Azospirillum isolates:

Effect of temperature on the growth of Azospirillum:

The isolates were inoculated on Nfb medium and incubated at various temperatures (27, 32, 40, 45 and 50^°C) for 48 h. After incubation, the growth was observed and the results were recorded.

Effect of pH on the growth of Azospirillum:

For pH stability, the isolates were inoculated on Nfb medium with varied pH like 5.5, 6.0, 6.8, 7.0 and 7.5 adjusted with 0.1N HCl 0.1N NaOH. The plates were incubated at 32^°C for 48 h and the growth was recorded.

Biochemical characteristics of Azospirillum isolates:

Utilization of carbon sources:

Regarding carbon utilization, five different carbon sources namely mannitol, sucrose, fructose, lactose and succinate were dissolved in 2mM phosphate buffer and precipitated. They were filtered using membrane filter (bacterial filter G-2) and added separately on the sterile Nfb medium (without malate) and transferred to the test tubes. A loop full culture of the isolates were inoculated and incubated at 32^°C for 48 h. After incubation, the growth rate was measured by spectrophotometer.

Indole test:

Tryptone broth was prepared and Azospirillum isolates were inoculated at 32ºC for 24 h. After incubation the Indole production was tested with Kovac’s reagent. Red colour ring formation indicates positive reaction, whereas yellow colour ring indicates negative result.

Methyl red test:

The Azospirillum isolates were grown in MR-VP broth at 32ºC for 24 h. After that methyl red indicator was added. Red colouration of the broth indicated positive reaction. Yellow colour development indicated negative result.

Voges Proskauer test:

The Azospirillum isolates were cultured in MR-VP broth at 32^°C for 24 h, VP reagent I and II were added in the tubes. Development of red colour indicated positive reaction and the yellow colour development indicates negative result.

Citrate utilization test:

The isolates were inoculated on Simmon’s citrate agar slants and incubated at 32^°C for 48 h. Changes of colour from green to blue indicated positive reaction, whereas no colour change indicated negative result.

Starch hydrolysis test:

The isolates were streaked on starch agar plates and incubated at 32^°C for 24 h. After incubation Lugol’s iodine solution was added and allowed for few minutes. A positive test indicated by the formation of zone of clearance in the medium around the colonies.

Urease test:

The isolates were inoculated in Christensen’s urea agar slants and incubated at 32ºC for 48 h. Changes of colour from yellow to pink indicated positive result.

Effect of biotin:

Biotin requirement was tested in semisolid Nfb medium containing NH₄Cl (0.5 g/L). The pH was adjusted to 6.8 with NaOH media without biotin was prepared. Cultures grown in Nfb liquid medium supplemented with biotin (100mg/l ) and also inoculated in without biotin medium and incubated at 32°C for 48 h. The growth rate was observed and the results were tabulated.

Effect of NaCl:

Nfb semisolid broth was prepared along with 3% of NaCl. All the 30 isolates were separately inoculated into the medium and incubated for 48 h at 30^°C. After incubation, the growth was observed and the results were tabulated.

RESULT AND DISCUSSION:

Diversity of Azospirillum:

For the isolation of Azospirillum spp., Nfb semisolid medium was used. After 24 h incubation, the Nfb semi-solid medium showed white colored pellicle (Plate I). Appearance of pellicle formation on Nfb semi-solid medium indicated successful isolation of Azospirillum. The pellicles were transferred into Nfb plates. After 48 h white, merged colonies were observed on the medium. Typical white or pink, often wrinkled colonies were picked out and transferred into Nfb semi-solid medium. A total number of 30 morphologically distinct Azospirillum isolates were isolated and tabulated. For enumeration of population density, the number of colonies on the plates was counted in the range of 68 to 210 colonies. The highest population density was observed in sandy loamy soil at Thirubuvanam. The lowest population density was observed in sandy clay loamy soil at Thiruvalanjuli (Table 1; Fig.2).

Azospirillum spp. are members of the a - subclass of proteobacteria. Azosprilla have worldwide distribution and occur in large numbers (upto10^-7/g) in rhizosphere soils and associated with the roots, stems and leaves of a large variety of plants. Members of the genus Azospirillum fix nitrogen under microaerophilic conditions, and are frequently associated with root and rhizosphere, these are known as associated diazotrophs. Sen (1929) made one of the earliest suggestions that the nitrogen content of cereal crops could be met by the activity of associated nitrogen fixing bacteria namely Azospirillum. This organism came into focus with the work of Dobereiner from Brazil ( Dobereiner et al., 1976), followed closely by reports from India (Lakshmi-Kumari et al., 1976; Kavimandan et al., 1978; Tilak and Murthy, 1981). After establishing in the rhizosphere azospirilla, usually but not always, promote the growth of plants (Okon, 1985; Tilak and Subba Rao, 1987; Bashan and Holguin, 1997b). Despite their N₂ fixing capability, the increase in yield is mainly attributed to improved root development due to the production of growth promoting substances and consequently increased rates of water and mineral uptake (Dewan and Subba Rao, 1979; Okon and Kapulnik, 1986; Fallik et al., 1994). Azospirilla proliferated in the rhizosphere of numerous plant species and the genus Azospirillum now contains seven species A. brasilense (Tarrand et al., 1978), A. lipoferum (Tarrand et al., 1978), A. amazonense (Magalhaes et al., 1983), A. halopraferens (Reinhold et al., 1987), A. irakense (Khammas and Kaiser, 1991), A. dobereinerae and A. largimobile (Eckert et al., 2001).

Fig. 2. Population density of Azospirillum isolated from 30 different soils of Thanjavur District

Morphological characteristics:

In microscopic observation, the cell shape was observed using Gram staining technique. All the 30 isolates were Gram negative. Among them, 25 isolates were rod in shaped (TA01, TA03 TA04, TA05, TRA01, TRA02, OA01, OA02, OA03, OA04, KA02, KA03, TMA01, TMA02, PA01, PA02, PA04, PA05, PA07, PA08, PTA01, PTA02, PTA03, PTA04 and PRA01), whereas three isolates were vibroid in shaped (TA02, TA06 and KA01) and two isolates were curved rod shaped (PA03 and PA06) (Table 2).

The cell size was measured by micro metrically. The large cell size was observed in strains PA03 and PTA04 (1.2µm) followed by 1.0µm (OA04 and KA01) 0.9µm (TA02, TA06, TRA02, OA02, KA02, PA02, PA04, PA05, PTA02, PTA03 and PRA01), 0.7µm (PA08 and PTA01) and 0.6µm (TA01, TA03, TA04, TA05, TRA01, OA01, OA03, KA03, TMA01, TMA02, PA01, PA06 and PA07) (Table 2).

Among 30 isolates, 18 isolates (TA02, TRA01, TRA02, OA01, OA02, KA02, KA03, TMA01, TRA02, PA01, PA03, PA04, PA05, PA06, PA07, PTA01, PTA02, PTA03, PTA04 and PRA01) were developed white colored colonies on basal minimal salt (BMS) agar medium Whereas, 9 isolates (TA01, TA03, TA05, TA06, OA03, OA04, KA01, PA02 and PA08) were developed pink colored colonies. In contrast, only one strain TA04 was produced pale yellow colored colonies (Table 2).

Among them, 6 isolates (TA03, TA04, TA05, TMA01, PA05 and PA08) were developed pale yellow coloured colonies whereas, 23 isolates (TA01, TA02, TA06, TRA01, TRA02, OA01, OA02, OA03, OA04, KA01, KA02, KAO3, TMA02, PA01, PA03, PA06, PA07, PTA01, PTA02, PTA03, PTA04 and PRA01) developed white colored, flat, elevated, round and raised colonies. In contrast only one strain PA02 was produced pale brown colored colonies (Table 2).

In swarming motility experiment, all the isolates were motile. The longest swarming movement 12mm was achieved by the strain OA04, followed by 10mm (TA05, OA03, TMA02 and PA03), 9mm (TA06, PA02, PA06 and PTA01) 8mm (TRA01 and PA04), 7mm (TMA01 PA01 and PTA02), 6mm (TA01, TA02, KA01, PA05, PA07 and TA03), 5mm (TA04, OA01, OA02,KA02, KA03, PA08 and PRA01) and 4mm (TA03, TRA02 and PTA04) (Table 2).

In the present study, among the 30 isolates studied, 25 isolates were rod shaped, 3 vibroid in shape and 2 curved rod shaped cells were recorded. Three different types of Azospirillum strains were isolated from the roots of Kallar grass (Reinhold et al., 1986). Strain ER15 was motile, vibroid to S-shaped, Gram-negative rod and growth was appeared as a subsurface pellicle in N-free semisolid SM medium, as typical for Azospirillum spp. Whereas, the strain ER8, Gram-negative, motile, vibroid to helical but mainly S-shaped, with the size of 0.3 by 2 to 6 µm. In the present study, all the isolates were sensitive to tetracycline (1.0 to 11.0mm), whereas 29 isolates were sensitive to chloramphenical and streptomycin, except the isolates TRA02 and PRA01 are resistant to chloramphenicol and streptomycin respectively. However, only 13 isolates were sensitive to ampicillin and the remaining 17 isolates were resistant. Similarly, among the azospirilla isolates, A10 showed higher level of resistance to all the six tested antibiotics (Thakuria et al., 2004).

Table 1. Population density of Azospirillum isolates from different locations of Thanjavur Dt.

S. No.	Name of the Taluk	Sampling location	No. of Population X 10^-5dilution CFU/g of soil
1	Thanjavur	Vallam	87
2		Kurungulam	100
3		Punnainallur	95
4		Nanjikottai	87
5		Soorakkottai	115
6		Pudupattinam	78
7	Thiruvaiyaru	Vellamperambur	125
8		Rajagiri	100
9		Pinnaiyur	135
10	Orathanadu	Vadacherri	78
11		Ambalapattu	88
12		Ulur	205
13	Kumbakonam	Darasuram	98
14		Thiruvalanjuli	68
15		Sakkottai	78
16	Thiruvidaimaruthur	Narasingampettai	82
17	Thiruvidaimaruthur	Kanjanur	79
18	Papanasam	Saliyamangalam	85
19		Poondi Papanasam	98
20		Nallavanniyankudikadu	140
21		Raramuthirakottai	168
22		Thirupuvanam	210
23		Ukkaadai	100
24		Neikkunnam	98
25		Arunthavapuram	88
26	Pattukottai	Madhukkur	75
27		Alathur	89
28		Sendankadu	100
29		Thittakudi	128
30	Peravurani	Peravurani	126

Table 2. Morphological and cultural characteristics of Azospirillum isolates

S. No.	Strain No.	Cell shape	Cell size (µm)	BMS Medium	Potato agar Medium	Swarming movement (mm)
1	TA01	Rod	0.6	Pink	White, flat	6
2	TA02	Vibriod	0.9	White	White, elevated	6
3	TA03	Rod	0.6	Pink	Pale yellow	4
4	TA04	Rod	0.6	Pale yellow	Pale yellow	5
5	TA05	Rod	0.6	Pink	Pale yellow	10
6	TA06	Vibriod	0.9	Pink	White	9
7	TRA01	Rod	0.6	White	White, flat	8
8	TRA02	Rod	0.9	White	White, flat	4
9	OA01	Rod	0.6	White	White raised margin	5
10	OA02	Rod	0.9	White	White round	5
11	OA03	Rod	0.6	Pink	White round	10
12	OA04	Rod	1.0	Pink	White raised	12
13	KA01	Vibriod	1.0	Pink	White elevated	6
14	KA02	Rod	0.9	White	White, flat	5
15	KA03	Rod	0.6	White	White, round	5
16	TMA01	Rod	0.6	White	Pale yellow	7
17	TMA02	Rod	0.6	White	White	10
18	PA01	Rod	0.6	White	White, round	7
19	PA02	Rod	0.9	Pink	Pale brown	9
20	PA03	Curved	1.2	White	White raised	10
21	PA04	Rod	0.9	White	White	8
22	PA05	Rod	0.9	White	Pale yellow	6
23	PA06	Curved	0.6	White	White round	9
24	PA07	Rod	0.6	White	White elevated	6
25	PA08	Rod	0.7	Pink	Pale yellow	5
26	PTA01	Rod	0.7	White	White round	9
27	PTA02	Rod	0.9	White	White elevated	7
28	PTA03	Rod	0.9	White	White, flat	6
29	PTA04	Rod	1.2	White	White, flat	4
30	PRA01	Rod	0.9	White	White elevated	5

Table 3. Physiological characteristics of Azospirillum isolates

S. No.

Strain No.

Effect of temperature (°C)

Effect of pH

5.5

6.0

6.8

7.0

7.5

TA01

+++

TA02

+++

TA03

+++

TA04

+++

TA05

+++

TA06

+++

TRA01

+++

TRA02

+++

OA01

+++

OA02

+++

OA03

+++

OA04

+++

KA01

+++

KA02

+++

KA03

+++

TMA01

+++

TMA02

+++

PA01

+++

PA02

+++

PA03

+++

PA04

+++

PA05

+++

PA06

+++

PA07

+++

PA08

+++

PTA01

+++

PTA02

+++

PTA03

+++

PTA04

+++

PRA01

+++

+++ = Good growth; ++ = Normal growth; + = Slow growth; - = No growth

Table 4. Biochemical characteristics of Azospirillum isolates

S. No.

Strain

No.

Utilization of carbon sources

Indole production

Methyl red

Voges Proskaur

Citrate utilization

Starch hydrolysis

Urease production

Biotin

Growth at 3%

NaCl

Mannitol

Sucrose

Fructose

Lactose

Succinate

TA01

TA02

TA03

TA04

TA05

TA06

TRA01

TRA02

OA01

OA02

OA03

OA04

KA01

KA02

KA03

TMA01

TMA02

PA01

PA02

PA03

PA04

PA05

PA06

PA07

PA08

PTA01

PTA02

PTA03

PTA04

PRA01

+ = Positive; - = negative

All the 30 isolates were identified to species level based on their morphological, cultural, biochemical and physiological characteristics. Nine isolates (TA02, TA04, OA02, KA03, TMA02, PA01, PA04, PA06 and PTA01) were identified to be A. halopraferens, eight isolates (TRA01, TRA02, OA01, KA02, TMA02, PA05, PTA3 and PTA04) were A. amazonense, six isolates (OA03, OA04, KA01, PA02, PA03 and PA04) were A. brasilense, four isolates (TA01, TA03, TA05 and TA06) were A. lipoferum and the remaining three isolates (PA07, PTA02 and PRA01) were A. irakense.

Morphological phylogeny:

Based on the morphological and biochemical characteristics, the phylogeny was constructed by neighbour-joining tree method. The phylogenetic tree resulted to 4 main clades I, II, III and IV. The clade I consist of two strains A. brasilense and A. halopraferens, clade II consist of 3 strains A. lipoferum, A. amazonense and A. halopraferens. This clade I and II was dominated by A. lipoferum. The clade IV consists of A.irakense, A. amazonense, A. halopraferens and A. brasilense. As in the phenogram, clade II and clade III were originated from the branch of clade IV. The clade IV was consisting of 3 strains A. amazonense halopraferens, irakense. The clade IV was dominated by A. amazonense. The clade II, III and IV were originated from the clade I they can be designated as the sister clades and the morphotypes of the clade I can be considered as the origin of the morphological evolution for all other morphotypes (Fig.3).

The present study has been measured nitrogen fixation ability of Azospirillum spp. by micro kjeldahl method in nitrogen-free semisolid medium with malate. Altogether, 30 isolates tested, only 28 isolates were able to fix nitrogen. Among them, only 10 isolates (PA03, PTA03, PA04, TA01, OA04, TA05, PTA04, TRA01, PA05 and TA02) were able to produce the highest amount of nitrogen (from 11.0 to 15.06 mg ‘N’/kg). The two isolates namely KA03 and PA07 could not able to fix nitrogen. Okon et al. (1977) and Nelson and Knowles (1978) reported for A. brasilense about 5,000 nmol of C₂H₄ mg protein^-1 h^-1 in cell free extracts and in liquid malate medium. Haahtela et al. (1983) has also studied been the maximum specific nitrogenase activity of Escherichia agglomerans was only 10% and of Klebsiella penumoniae and A. lipoferum 50% in nitrogen-free semisolid medium with malate or sucrose.

The variability of the nitrogenase activity of Azospirillum has been observed previously in vitro by Han and New (1998), with ARA varying from 0 to 155 nmol of C₂H₄ mg protein-1h-1, in pure cultures of A. lipoferum and A. brasilense obtained from soils of different regions. In another study, the nitrogenase activity of Azospirillum isolates ranged from 17.6 to 49.6 nmol C2H4ml^-1h^-1 being higher in cultures of A. brasilense than for A. lipoferum isolates (Mascarua – Esparza et al., 1998). In the present study, large differences in the nitrogen fixing of Azospirillum isolates (0 to 15.6 mg N’ fixed / g) was recorded.

Acetylene reduction by S. lipoferum with glucose, in combination with a small amount of carbon starter compound such as malate or yeast extract, has reported by Day and Dobereiner (1976). Growth and activity probably stopped after the carbon starter compound was depleted, and no further increase in absorbance was observed during 2 weeks of incubation (Okon et al., 1976). Schroder (1932) has been reported good growth on glucose for 3 of 12 isolates of S. lipoferum. In the present study, among the 30 isolates, only 10 isolates showed good growth and acetylene reduction with malate as sole carbon source.

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Received on 30.12.2010

Modified on 25.01.2011

Accepted on 27.02.2011

Research J. Science and Tech. 3(2): March-April. 2011: 85-94