Characterization Of Groundnut Rhizobacteria Biology Essay

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Chapter 3

An extensive survey was carried in the farmer's field for the collection of soil and groundnut samples. The groundnut rhizobacteria were isolated from the roots and rhizospheric soil by using dilution plate technique, where Phosphate Buffer Saline (PBS, 1X) was used as saline solution. The bacteria were then grown on nutrient media i.e. Tryptic Soy Agar (TSA) in sterilized petri plates and placed in incubator at 28°C for at least 48 hours. After bacterial growth individual colonies were picked and streaked on plates containing TSA media for purification under sterilized conditions in laminar air flow cabinet. Single colonies were repeatedly re-streaked till the purified cultures were obtained (Ahmed et al., 2007).

3.1.2 Morphological Characterization groundnut rhizobacteria

The purified strains were cultured on solid media for the observation of colony morphological characteristics. Slides of the isolated and purified strains were prepared to study cell morphology (Murray et al., 1994).

3.1.3 Biochemical characterization groundnut rhizobacteria

All isolates were biochemically characterized by gram's reaction, carbohydrate fermentation and for cultural conditions i.e. temperature, pH and NaCl (Cappuccino and Sherman, 1992).

3.1.4 Preparation of DNA Template

DNA template were prepared by picking individual colony with the help of sterilized toothpick and dissolve in 0.2 ml ependorf tube containing 1X Tris EDTA (Ethylenediamine tetra acetic acid). PCR was performed at 95°C to obtain template (Ahmed et al., 2007).

3.1.5 PCR Reaction

The isolated strains were identified using modern techniques of PCR and DNA sequence analysis in Plant Biotechnology Program, NIGAB, NARC. For this purpose, nearly complete 16S rRNA gene sequences of the strains were obtained after PCR amplification of the genes as described by Katsivela et al. (1999) using universal forward and reverse primers: 9F (5'-GAGTTTGATCCTGGCTCAG-3') and 1510R (5'-GGCTACCTTGTTACGA-3'). Reaction mixture (25 µL), prepared for full-length 16S rRNA gene amplification was initially denatured at 94°C for 2 min, followed by 30 cycles consisting of denaturation at 94°C for 2 min; primer annealing at 55°C for 60 sec and primer extension at 72°C for 2 min and finally extension at 72°C for 10 min in a thermo cycler (Ahmed et al., 2007).

3.1.6 Gel Electrophoresis

Amplified PCR products of 16S ribosomal gene were separated on 1% agarose gel in 0.5 x TE (Tris-EDTA) buffer containing 2 µL ethidium bromide (20 mg/mL). λ Hind-III ladder marker was used as a size marker. The gel was viewed under UV light and photographed using gel documentation system (Ahmed et al., 2007).

3.1.7 Characterization of rhizobacteria for PGP traits Production of Indole Acetic Acid (IAA)

IAA production was detected by the modified method as bacterial cultures were grown for 48 hrs -72 hrs on their respective media at 36±2 °C. Fully grown cultures were centrifuged at 3000 rpm for 30min. The supernatant (2ml) was mixed with two drops of orthophosphoric acid and 4ml of the Salkowski reagent (50ml, 35% of perchloric acid, 1ml 0.5M FeCl3 solution). Development of pink color indicated IAA production (Brick et al., 1991). NH3 production

Bacterial isolates were tested for the production of ammonia in peptone water. Freshly grown cultures will inoculated in 10ml peptone water in each tube and incubated for 48-72 hrs at 36 0C. Nessler's reagent (0.5ml) will added in each tube. Development of brown to yellow color was the positive test for ammonia production (Cappuccino and Sherman, 1992).

On the basis of characterization, for the plant growth promoting traits five stains were selected for the pot experiment

3.1.8 Preparation and application of the inocula

Inocula were prepared in the broth culture and applied 1 ml to the pots after the germination of the crop (Sapatnekar et al., 2001).


A pot experiment was carried out on chickpea (cv. DASHT) under cotrolled conditions at PMAS-AAUR during, 2009-10 to investigate the beneficial effect of groundnut rhizobacterial strains on chickpea growth and N2 fixation. Each pot was filled 8 kg soil and 4 seeds were sown in each pot. Strains inocula were prepared in Soil Science lab PGPRs was selected on the basis of their morphological and physiological traits. Experiment was replicated three times in a Complete Randomized Design (CRD).

Detail of Treatments

T1 Control

T2 Media

T3 NCCP-94

T4 NCCP-98

T5 NCCP-99

T6 NCCP- 100

T7 NCCP-101


The soil used for pot experiment was analyzed for following physiochemical properties.

3.2.1 Electrical Conductivity (ECe)

Soil extract was taken from the saturated soil paste and the electrical conductivity of the extract was recorded by using conductivity meter calibrated with 0.01 N KCl solution (Rhoades, 1996).


Air- dried soil (50 grams) was taken into a glass beaker; 100 ml of distilled water was added. The contents was mixed and allowed to stand for 1 hour. After this, the soil pH was measured by using calibrated pH meter with buffer solution of pH 7 and 9 (Thomas, 1996).

3.2.3 Total Organic Carbon (TOC)

Soil sample (2 grams) was taken in separate digestion tubes and 5 ml of potassium dichromate (K2Cr2O7) solution was added and mixed for few seconds. While mixing, 10 ml of sulphuric acid (H2SO4) was added to the tube. After addition of all the acid, further mixing was continued for 30 seconds. Then the tubes were placed in preheated block digester. Exactly, after 30 minutes, tubes was removed, allowed to cool, water was added to half way and mixed. After further cooling, the tubes were filled to graduated mark with water and vortex 3-4 times for thorough mixing. After the settlement of the suspension, the same amount from each digest solution was decanted into centrifuge tube for 15 minutes. Similarly, the standards will also be prepared. Eventually, absorbance for sample and standard solution was measured using spectrophotometer at 610 nm (Heans, 1984).Soil total organic carbon was calculated as under:

mg C

% C = ------------------------ ï‚´ 100

Oven dry soil wt

3.2.4 Nitrate Nitrogen (NO3-N)

Salicylic acid method was used for determination of NO3-N. A 10 gram soil sample was taken into a 250 ml bottle and 20 ml of distilled water was added. The contents was shaken for an hour and filtered through Whatman No. 42 filter paper. A sample of 0.5 ml was taken in test tube and 1 ml of 5 % salicylic acid reagent was added, removed thoroughly, and tube was left for thirty minutes. After this, 10 ml of 4 M NaOH reagent was added to each tube. Contents were mixed thoroughly and were left for an hour for full colour development. Reading was taken by using Spectronic 20 at 410 nm (Mulvaney, 1996).

3.2.5 Available Phosphorus (P)

Five grams soil was taken into 250 ml of Erlenmeyer flask along with 100 ml of 0.5M of NaHCO3 solution. Flasks were shaken for 30 minutes and filtrate was taken. 10 ml of filtrate was added into 50 ml volumetric flask along with 1 ml of 5 N H2SO4. Total volume was made up to 40 ml by addition of distilled water. After this 8-ml of reagent B (Ascorbic acid) was added in order to develop the color. Transmittance was recorded after 10 minutes by using Spectrophotometer (Kuo, 1996).

Table 3.1: Chemical properties of soil before start of experiment



ECe (dS m-1)


Soil pH


Total Organic Carbon (g 100g-1)


Nitrate Nitrogen (ug g-1)


Available P (ug g-1)


Exchangeable K (µg g-1)


Exchangeable Potassium (K)

Five grams soil was taken into 50 ml centrifuge tube, 33 ml of ammonium acetate solution was added into it, and then it was shaked for 5 minutes. The solution was centrifuged until the supernatant liquid and then extract was collected into 100 ml volumetric flask. Then the solution was diluted to 100 ml with ammonium acetate and concentration of K was read by using flame photometer (Helmke and Sparks, 1996).

3.3 Plant analysEs

Plant samples of chickpea were taken at flowering. The samples were dried in oven at 70 oC for 48 hrs and will ground in a Wiley mill and samples were stored in plastic containers and were analyzed for total N.

Digestion for Total Nitrogen (TN)

Ground plant material (0.2 gram) was taken in separate digestion tubes, 4.4 ml of digestion mixture containing selenium powder, lithium sulphate and hydrogen peroxide was added and it was digested for two hours at 360 0C till solution is colorless, 50 ml of water was added and mixed well. After cooling, it was made up to 100 ml and mixed. After the settlement, the clearer solution was used for further analysis of TN calorimetrically (Anderson and Ingram, 1993) Colorimetric Determination of Total Nitrogen (%)

For this purpose 0.1 ml of each standard and sample, 5 ml of reagent containing sodium salicylate, sodium citrate, sodium tartarate and sodium nitroprusside was added. It was mixed well and left for 15 minutes. Then 5 ml of reagent containing a solution of NaOH, water and sodium hypochlorite was added to each test tube and left for one hour for full color development. Absorbance of samples was measured using spectrophotometer at 665 nm.

Plant TN was calculated by the following formula:

TN % = C/W x 0.01

Where C is correctd concentration (μg/ml) and W is weight of sample (g).

3.3.2 Assessment of Nitrogen Fixation by Natural 15N Abundance Technique

Plant samples were dried at 70 0C and ground to fine powder. A sub sample of one gram for both legume and reference non legume (wheat) was sent to Stable Isotope Unit, University of Waikato, Hamilton, New Zealand for analysis of N15 on Mass Spectrometer. The 15N content of the legume and reference plant (wheat) was determined in finally ground 10 mg sample by dry Dumas combustion, followed by isotope ratio mass spectrometry (People et al., 1989).

δ15N= 1000 (R sample - Rair) / Rair

Where R is the ratio of mass 29 / mass 28.

An estimate of nitrogen derived from atmosphere (%Ndfa) was obtained using following equation.

% Ndfa= 100 (x- y)/ (x-z)


x is δ15N of shoot of wheat deriving all N from the soil

y is he δ15N of chickpea shoot

z is the δ15N of chickpea receiving all N from N2 fixation. (Shah et al. 1997).

3.4 Statistical Analysis

The data collected for various parameters was subjected to Analysis of Variance (ANOVA) and the means obtained was compared by LSD at 5 % level of significance (Steel et al., 1997).