Earthern and Pot Culture Method to Check the Stability of Marine Azotobacter in Soil

was devoid of the bacterial inoculums. The effects of bacterial inoculums on the growth of plant root, shoot length were measured on the 7th, 14th, 21st day of plant growth.

Growth characters:

1. Percentage of germination

2. Shoot length

3. Root length

Percentage of germination:

The germination rate of all treated and control plant was calculated by using the following formula: (Table 6)

Number of seeds germinated

Percentage of germination = —————————————– × 100

Number of seeds sown

Shoot length:

The shoot length of the plant was measured in centimeter (cm) scale on 7th, 14th, 21st day of sowing from ground level to the shoot tip. (Table 7)

Root length:

The plants were uprooted without disturbing the root system, and then the roots were washed with tap water to remove the soil particles. The length of the root was measured in cm scale. (Table 7)

Biometric analysis; Estimation of chlorophyll :

Weighed 1g of leaf was finely cut in to pieces; tissues were ground to a pulp with the addition of 20ml of 80% acetone. Then centrifuged at 5000 for 5 min and transferred the supernatant to the 100ml volumetric flask. This procedure was repeated until the residue turned colorless. Finally the volume was made to 100ml with 80% acetone. The absorbance was read at 645,663nm against the solvent (80% acetone) blank.( Table 8a and 8b)

RESULT AND DISCUSSION:

Totally 100 samples were collected in marine region of both water and sediments at the intervals of approximately 20 days (Table: 1).

Table :1 The total samples collected from marine region.

samples water sediment

1st time 10 10

2nd time 15 5

3rd time 15 5

4th time 15 5

5th time 15 5

Total 70 30

Out of 70 marine water samples collected, all the 70 samples were showed the presence of Azotobacter, but only 23 marine sediments out of 30 were showed the presence of Azotobacter (Table no: 2).

Table no 2: presence of Azotobacter sp (in percentage).

Source No of samples POSITIVE (Presence of Azotobacter) Percentage (Presence of Azotobacter)

Water 70 70 100

Sediment 30 23 76.6

Azotobacter sp is a gram-negative soil–dwelling organism with a wide variety of metabolic capabilities which includes the ability to fix atmospheric nitrogen by converting it to ammonia. These bacteria possess the highest cellular respiratory rate of any known organism. Their rapid consumption of oxygen allows them to grow well and to fix nitrogen under extreme aeration condition. (Page et al. 1988).

Initial isolation of marine bacteria prefers sea water or 3 % NaCl to fresh water in the medium for growth (ROBERT A. MACLEOD 1965).

The total hardness of water represents primarily the total concentration of calcium and magnesium ions expressed as calcium carbonate. Hardness may range from 0-100 of parts per million. Mg++ to maintain the respiratory activity of cell Azotobacter, an organism stable in water suspension.

Water analysis result showed that the total hardness of water was 20200 ppm and total chloride content was 18273.98 ppm. Zobell and upham define marine bacteria as being bacteria from the sea which on initial isolation required seawater in the medium for growth.

Table: 3 colony morphology of Azotobacter

Media Details

Jenson’s medium Large, circular, mucoid, watery due drop like colonies.

Azotobacter agar medium Small, circular, mucoid and watery colonies in a medium

Burk’s medium Surface Pellicle formation, turbidity indicating the Heavy growth of Azotobacter.

Marine agar medium Small, circular, smooth edged, raised elevated colonies were observed

Table4: characteristics of Azotobacter sp.

Test Result

Gram’s staining Gram negative rod shaped cells were seen

Catalase test Air bubbles were seen

Phase contrast microscopy Motile cells were seen/rarely non-motile cells were seen with different morphology.

The colony morphology of Azotobacter strain is found to be varying based on the selective media used for isolation.

Studies on the rates of nitrogen fixation were greatly enhanced by development of the acetylene reduction assay (Hardly et al., 1968). This assay is based on the fact that nitrogenase enzyme will reduce acetylene to ethylene. The rate of formation of ethylene is a measure of nitrogenase or nitrogen –fixing activity. Ethylene can be conveniently assayed with great sensitivity using a gas chromatography. In this study acetylene reduction was performed and their peak values were noted. Based on this assay the organism was selected for pot culture experiment.

Table: 5 Chemical and nutrient analysis of garden soil

The garden soil was tested for micro and macro elements.

PARTICULARS LEVELS

pH 6.9

Electrical conductivity(dSm-1) 0.446

N(kg/ha) 98

P(Kg/ha) 14.5

K(Kg/ha) 275

Copper(ppm) 0.84

Manganese(ppm) 6.32

Iron(ppm) 8.04

Zinc(ppm) 1.04

pot culture experiment :

Five efficient strains were selected for pot culture experiment based on acetylene reduction assay.

Table 6: Percentage of germination

Result showed 85 percent of germination

pot culture seed germination in %

control 72

pot A 80

pot B 81

pot C 70

pot D 86

pot E 82

Table: 7 shoot and root length

Shoot and root length of the plant were measured, which ranged from 21.4 –

26.3 cm and 7.6-12.2cm respectively.

pot culture Shoot length(cm) Root length(cm)

7th day 14th day 21st day 7th day 14th day 21st day

CONTROL 8.3 18.0 20.0 7.2 9.2 11.2

POT A 8.9 21.0 23.0 7.6 9.6 11.6

POT B 9.5 22.1 24.2 8.5 9.8 11.1

POT C 7.3 19.2 21.4 8.0 9.7 11.5

POT D 9.1 24.4 26.3 8.2 9.8 12.0

POT E 9.2 22.6 25.0 8.5 9.7 12.2

Biometric analysis:

Estimation of chlorophyll by spectrophotometric method:

Table 8a : The optical density value at 645nm ranged from 0.102 – 0.202 OD and 0.266 – 0.562 OD at 663 nm

pot culture OD AT 645 nm OD AT 663 nm

control 0.103 0.302

pot A 0.156 0.423

pot B 0.202 0.562

pot C 0.182 0.522

pot D 0.154 0.455

pot E 0.102 0.266

Table 8a: Estimation of total chlorophyll content:

Pot culture Chlorophyll a Chlorophyll b Total chlorophyll mg/g

control 0.3558 0.0949 0.4502

pot A 0.4952 0.1592 0.6543

pot B 0.6594 0.1995 0.8587

pot C 0.6139 0.1724 0.7862

pot D 0.5364 0.1397 0.6759

pot E 0.3103 0.1091 0.4194

The total chlorophyll ranged from 0.4194-0.8587 mg total chlorophyll/g tissue.

The pot culture experiment results showed that, inoculation with Azotobacter influence the growth of black gram by increasing their shoot and root length and chlorophyll content.

Experiments with Azotobacter cultures and crop plants at the Indian Agricultural Research Institute, New Delhi, lead us to believe that significant increases in growth and yield of wheat, rice and vegetable crops could be obtained in pot trials. Experiment on soil Azotobacter on the growth of maize was carried out by N.A Hegazi(1979) result showed significant increase in the count of Azotobacter in 6 –week- old plant.

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