Characterization Of Groundnut Rhizobacteria Biology Essay

Chapter 3

An extended study was carried in the husbandman ‘s field for the aggregation of dirt and Indian potato samples. The Indian potato rhizobacteria were isolated from the roots and rhizospheric dirt by utilizing dilution home base technique, where Phosphate Buffer Saline ( PBS, 1X ) was used as saline solution. The bacteriums were so grown on alimentary media i.e.

Tryptic Soy Agar ( TSA ) in sterilized petri home bases and placed in brooder at 28 & A ; deg ; C for at least 48 hours. After bacterial growing single settlements were picked and streaked on home bases incorporating TSA media for purification under sterilized conditions in laminar air flow cabinet. Single settlements were repeatedly re-streaked till the purified civilizations were obtained ( Ahmed et al. , 2007 ) .

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3.1.2 Morphologic Characterization Indian potato rhizobacteria

The purified strains were cultured on solid media for the observation of settlement morphological features. Slides of the stray and purified strains were prepared to analyze cell morphology ( Murray et al.

, 1994 ) .

3.1.3 Biochemical word picture Indian potato rhizobacteria

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

3.1.4 Preparation of DNA Template

Deoxyribonucleic acid templet were prepared by picking single settlement with the aid of sterilized toothpick and dissolve in 0.

2 milliliters ependorf tubing incorporating 1X Tris EDTA ( Ethylenediamine tetra acetic acid ) . PCR was performed at 95 & A ; deg ; C to obtain templet ( Ahmed et al. , 2007 ) .

3.1.5 PCR Reaction

The stray strains were identified utilizing modern techniques of PCR and DNA sequence analysis in Plant Biotechnology Program, NIGAB, NARC. For this intent, about complete 16S rRNA cistron sequences of the strains were obtained after PCR elaboration of the cistrons as described by Katsivela et Al.

( 1999 ) utilizing cosmopolitan frontward and rearward primers: 9F ( 5′-GAGTTTGATCCTGGCTCAG-3 ‘ ) and 1510R ( 5’-GGCTACCTTGTTACGA-3 ‘ ) . Chemical reaction mixture ( 25 µL ) , prepared for full-length 16S rRNA cistron elaboration was ab initio denatured at 94 & A ; deg ; C for 2 min, followed by 30 rhythms dwelling of denaturation at 94 & A ; deg ; C for 2 min ; primer tempering at 55 & A ; deg ; C for 60 sec and primer extension at 72 & A ; deg ; C for 2 min and eventually extension at 72 & A ; deg ; C for 10 min in a thermo cycler ( Ahmed et al. , 2007 ) .

3.1.6 Gel Electrophoresis

Amplified PCR merchandises of 16S ribosomal cistron were separated on 1 % agarose gel in 0.5 ten TE ( Tris-EDTA ) buffer incorporating 2 µL ethidium bromide ( 20 mg/mL ) . ? Hind-III ladder marker was used as a size marker.

The gel was viewed under UV visible radiation and photographed utilizing gel certification system ( Ahmed et al. , 2007 ) .

3.1.7 Word picture of rhizobacteria for PGP traits


1 Production of Indole Acetic Acid ( IAA )

IAA production was detected by the modii¬?ed method as bacterial civilizations were grown for 48 hour -72 hour on their several media at 36±2 & A ; deg ; C. Fully adult civilizations were centrifuged at 3000 revolutions per minute for 30min. The supernatant ( 2ml ) was assorted with two beads of orthophosphoric acid and 4ml of the Salkowski reagent ( 50ml, 35 % of perchloric acid, 1ml 0.5M FeCl3 solution ) . Development of pink colour indicated IAA production ( Brick et al.

, 1991 ) . NH3 production

Bacterial isolates were tested for the production of ammonium hydroxide in peptone H2O.

Freshly adult civilizations will inoculated in 10ml peptone H2O in each tubing and incubated for 48-72 hour at 36 0C. Nessler ‘s reagent ( 0.5ml ) will added in each tubing.

Development of brown to yellow colour was the positive trial for ammonium hydroxide production ( Cappuccino and Sherman, 1992 ) .On the footing of word picture, for the works growing advancing traits five discolorations were selected for the pot experiment

3.1.8 Preparation and application of the inoculant

Inocula were prepared in the broth civilization and applied 1 milliliter to the pots after the sprouting of the harvest ( Sapatnekar et al. , 2001 ) .


A pot experiment was carried out on garbanzo ( curriculum vitae.

DASHT ) under cotrolled conditions at PMAS-AAUR during, 2009-10 to look into the good consequence of Indian potato rhizobacterial strains on garbanzo growing and N2 arrested development. Each pot was filled 8 kg dirt and 4 seeds were sown in each pot. Strains inocula were prepared in Soil Science lab PGPRs was selected on the footing of their morphological and physiological traits. Experiment was replicated three times in a Complete Randomized Design ( CRD ) .

Detail of Treatments

T1 ControlT2 MediaT3 NCCP-94T4 NCCP-98T5 NCCP-99T6 NCCP- 100T7 NCCP-101


The dirt used for pot experiment was analyzed for following physiochemical belongingss.

3.2.1 Electrical Conductivity ( ECe )

Soil infusion was taken from the saturated dirt paste and the electrical conduction of the infusion was recorded by utilizing conduction metre calibrated with 0.

01 N KCl solution ( Rhoades, 1996 ) .


Air- dried dirt ( 50 gms ) was taken into a glass beaker ; 100 milliliter of distilled H2O was added. The contents was assorted and allowed to stand for 1 hr. After this, the dirt pH was measured by utilizing calibrated pH metre with buffer solution of pH 7 and 9 ( Thomas, 1996 ) .

3.2.3 Entire Organic Carbon ( TOC )

Dirt sample ( 2 gms ) was taken in separate digestion tubings and 5 milliliter of K bichromate ( K2Cr2O7 ) solution was added and assorted for few seconds.

While blending, 10 milliliter of sulfuric acid ( H2SO4 ) was added to the tubing. After add-on of all the acid, farther commixture was continued for 30 seconds. Then the tubings were placed in preheated block digester. Precisely, after 30 proceedingss, tubings was removed, allowed to chill, H2O was added to half manner and assorted. After farther chilling, the tubings were filled to graduated grade with H2O and whirl 3-4 times for thorough commixture.

After the colony of the suspension, the same sum from each digest solution was decanted into extractor tubing for 15 proceedingss. Similarly, the criterions will besides be prepared. Finally, optical density for sample and standard solution was measured utilizing spectrophotometer at 610 nanometer ( Heans, 1984 ) .Soil entire organic C was calculated as under:milligrams C% C = — — — — — — — — — — — — i‚? 100Oven dry dirt wt


4 Nitrate Nitrogen ( NO3-N )

Salicylic acid method was used for finding of NO3-N. A 10 gm dirt sample was taken into a 250 milliliter bottle and 20 milliliter of distilled H2O was added. The contents was shaken for an hr and filtered through Whatman No. 42 filter paper.

A sample of 0.5 milliliter was taken in trial tubing and 1 milliliter of 5 % salicylic acerb reagent was added, removed exhaustively, and tubing was left for 30 proceedingss. After this, 10 milliliter of 4 M NaOH reagent was added to each tubing.

Contentss were assorted exhaustively and were left for an hr for full coloring material development. Reading was taken by utilizing Spectronic 20 at 410 nanometer ( Mulvaney, 1996 ) .

3.2.5 Available Phosphorus ( P )

Five gms dirt was taken into 250 milliliter of Erlenmeyer flask along with 100 milliliters of 0.5M of NaHCO3 solution. Flasks were shaken for 30 proceedingss and filtrate was taken. 10 milliliter of filtrate was added into 50 milliliters volumetric flask along with 1 milliliters of 5 N H2SO4.

Entire volume was made up to 40 milliliters by add-on of distilled H2O. After this 8-ml of reagent B ( Ascorbic acid ) was added in order to develop the colour. Transmittance was recorded after 10 proceedingss by utilizing Spectrophotometer ( Kuo, 1996 ) .

Table 3.1: Chemical belongingss of dirt before start of experiment



ECe ( dS Garand rifle )0.281

Soil pH

7.27Entire Organic Carbon ( g 100g-1 )0.

90Nitrate Nitrogen ( ug g-1 )5.3Available P ( ug g-1 )6.1Exchangeable K ( µg g-1 )121

Exchangeable Potassium ( K )

Five gms dirt was taken into 50 milliliters centrifuge tubing, 33 milliliter of ammonium ethanoate solution was added into it, and so it was shaked for 5 proceedingss. The solution was centrifuged until the supernatant liquid and so infusion was collected into 100 milliliters volumetric flask.

Then the solution was diluted to 100 milliliters with ammonium ethanoate and concentration of K was read by utilizing fire photometer ( Helmke and Sparks, 1996 ) .

3.3 Plant analysis

Plant samples of garbanzo were taken at blooming. The samples were dried in oven at 70 oC for 48 hour and will anchor in a Wiley factory and samples were stored in plastic containers and were analyzed for entire N.

Digestion for Entire Nitrogen ( TN )

Land works stuff ( 0.2 gm ) was taken in separate digestion tubings, 4.4 milliliter of digestion mixture incorporating Se pulverization, Li sulfate and H peroxide was added and it was digested for two hours at 360 0C till solution is colourless, 50 milliliter of H2O was added and assorted well. After chilling, it was made up to 100 milliliters and assorted. After the colony, the clearer solution was used for farther analysis of TN calorimetrically ( Anderson and Ingram, 1993 )


3.1.1 Colorimetric Determination of Total Nitrogen ( % )

For this purpose 0.1 milliliter of each criterion and sample, 5 milliliter of reagent incorporating Na salicylate, Na citrate, Na tartarate and Na nitroprusside was added. It was assorted good and left for 15 proceedingss. Then 5 milliliter of reagent incorporating a solution of NaOH, H2O and Na hypochlorite was added to each trial tubing and left for one hr for full colour development.

Optical density of samples was measured utilizing spectrophotometer at 665 nanometers.Plant TN was calculated by the undermentioned expression:TN % = C/W x 0.01Where C is correctd concentration ( ?g/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 land to ticket pulverization. A sub sample of one gm for both leguminous plant and mention non leguminous plant ( 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 leguminous plant and mention works ( wheat ) was determined in eventually land 10 milligram sample by dry Dumas burning, followed by isotope ratio mass spectroscopy ( People et al. , 1989 ) .?15N= 1000 ( R sample – Rair ) / RairWhere R is the ratio of mass 29 / mass 28.An estimation of N derived from atmosphere ( % Ndfa ) was obtained utilizing following equation.% Ndfa= 100 ( x- Y ) / ( x-z )Whereten is ?15N of shoot of wheat deducing all N from the dirtY is he ?15N of garbanzo shootomega is the ?15N of garbanzo having all N from N2 arrested development. ( Shah et al.

1997 ) .

3.4 Statistical Analysis

The informations collected for assorted parametric quantities was subjected to Analysis of Variance ( ANOVA ) and the agencies obtained was compared by LSD at 5 % degree of significance ( Steel et al.

, 1997 ) .


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