MATERIALS AND METHODS - Shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/32645/14/14_chapter 3.p… · TGA TCC TGG CTC AG-3’) and 1492r (3’-ACG GCT ACC TTG TTA CGA CTT-5’)

MMAATTEERRIIAALLSS AANNDD MMEETTHHOODDSS

3.1 Sampling

Samples were collected from cultivated pea fields from 22 locations in Lahaul and Spiti

valleys in the India trans-Himalayas (Figs. 3.1 and 3.2, Table 3.1). Samples for isolation of

plant growth-promoting bacteria were collected in pre-sterilized 50 ml conical FalconTM

tubes. Storage and transportation of samples was done under liquid nitrogen for root-

nodulating bacteria and at 4 °C for rhizosphere soil samples under refrigeration using

Mobile Laboratory Van. Bulk soil samples for chemical properties were collected in pre-

sterilized plastic sampling bags, air-dried at room temperature, and stored at 4 °C under

refrigeration.

3.1.1 Nodulated Pea Roots

Nodulated roots were collected by excavating soil up to 30 cm around the root system to dig

out the pea plants. Nodulated roots were rinsed with sterilized water were put in 50 ml pre-

sterilized conical FalconTM

tubes filled with 30% (v/v) sterilized glycerol. The sampling

locations included some large and some small agriculture areas. Accordingly, sampling was

done from 2 fields in small areas and 10-15 fields in large areas.

3.1.2 Rhizosphere Soil

Soil adhering to the roots of pea plants collected as above was brushed off into 50 ml

sterilized conical FalconTM

tube. Composite samples were made by pooling rhizosphere soil

of 9 randomly selected pea plants from each location.

3.1.3 Bulk Soil

Soil was dug out up to 30 cm depth at three spots around a plant from each sampling

location. Nine soil samples collected around three plants were pooled together for preparing

the composite sample. The soil samples were air-dried overnight at room temperature.

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Fig. 3.1 Map of sampling locations in Lahaul and Spiti valleys in the Indian trans-Himalayas.

L1-L10= Lahaul valley, S1-S12= Spiti valley. Latitude, longitude and altitude are given in

Table 3.1.

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Table 3.1 Sampling locations in Lahaul valley and Spiti valley in the Indian trans-

Himalayas

Sampling location Altitude MSL (m) Latitude Longitude

Dorni (L1) 3305 32° 21’ 30’’ 77° 18’ 42’’

Gramphoo (L2) 3264 32° 22’ 22’’ 77° 17’ 18’’

Khoksar (L3) 3134 32° 24’ 52” 77° 14’ 05’’

Kuthvihar (L4) 3135 32° 26’ 01’’ 77° 11’ 06’’

Teling (L5) 3254 32° 27’ 31’’ 77° 09’ 18’’

Sishu (L6) 3074 32° 28’ 49’’ 77° 07’ 25’’

Gompthang (L7) 3091 32° 29’ 00’’ 77° 06’ 50’’

Khangsar (L8) 3087 32° 30’ 11’’ 77° 02’ 29’’

Gondla (L9) 3166 32° 30’ 49’’ 77° 00’ 50’’

Tandi (L10) 2923 32° 32’ 58’’ 76° 58’ 28’’

Lahaul

valley

Lossar (S1) 4015 32° 26’ 07’’ 77° 45’ 42’’

Hansa (S2) 3990 32° 27’ 03’’ 77° 51’ 45’’

Kiato (S3) 3966 32° 26’ 44’’ 77° 53’ 48’’

Pangmoo (S4) 3863 32° 21’ 26’’ 77° 55’ 09’’

Hal (S5) 3840 32° 20’ 02’’ 77° 55’ 32’’

Morang (S6) 3785 32° 18’ 44’’ 77° 57’ 20’’

Sumling (S7) 3745 32° 17’ 52’’ 77° 58’ 35’’

Khurik (S8) 3705 32° 16’ 52’’ 77° 59’ 55’’

Rangrik (S9) 3685 32° 15’ 32’’ 78° 01’ 28’’

Kee (S10) 3747 32° 17’ 27’’ 78° 00’ 44’’

Kibber (S11) 4143 32° 19’ 23’’ 78° 00’ 30’’

Spiti

valley

Gete(S12) 4408 32° 18’ 21’’ 78° 01’ 33’’

L1-L10= Lahaul valley, S1-S12= Spiti valley.

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Fig. 3.2 Pea cultivation in Lahaul and Spiti valleys in the Indian trans-Himalayas.

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3.2 Isolation of Plant Growth-Promoting Bacteria

3.2.1 Root-Nodulating Bacteria

Root-nodulating bacteria were isolated from pink, unbroken, and firm pea root nodules on

yeast mannitol agar (YMA, Appendix I) supplemented with Congo Red (Himedia, Mumbai,

India) (Vincent, 1970). The nodules were surface sterilized with 20% (v/v) sodium

hypochlorite (4% available chlorine) (Himedia, Mumbai, India) for 5 min and washed

repeatedly in sterile distilled water. The nodules were dipped in 70% ethyl alcohol for 3 min

and washed 3 times with sterile distilled water. Surface sterilized nodules were crushed with

help of sterilized forceps and root sap streaked on YMA. The plates were incubated at

28±0.1 °C on the stationary shelf in Innova 4320 refrigerated incubator shaker (New

Brunswick Scientific, NJ, USA). Isolated white, translucent, glistening and elevated colonies

with entire margins were streaked on YMA plates to obtain pure colonies. Purified colonies

raised from nodules from different locations were preserved at -80 °C under 30% (v/v)

glycerol.

3.2.2 Phosphate-Solubilizing Rhizobacteria

Phosphate-solubilizing bacteria were isolated by plating in triplicate serial soil dilutions up

to 10-4

dilution of rhizosphere soil samples on modified Pikovskaya (PVK) agar containing

tricalcium phosphate (TCP) as the sole source of phosphate (Appendix I) (Gupta et al.,

1994). The colonies forming phosphate-solubilization zones were purified on modified PVK

and trypticase soya agar (TSA, Appendix I) (Himedia, Mumbai, India). The isolates were

maintained at -80 °C in equal volumes of nutrient broth (Appendix I) and 30% (v/v)

glycerol.

3.3 Selection of Widespread Genotypes

One hundred and twenty isolates of pea root-nodulating bacteria and 103 phosphate-

solubilizing rhizobacteria were subjected to diversity analysis for selection of widespread

genotypes.

3.3.1 Genomic DNA Isolation

Genomic DNA was extracted using GenEluteTM

Bacterial Genomic DNA Kit (Sigma, MO,

USA) according to the manufacturer’s instructions. The pea root-nodulating bacteria and

phosphate-solubilizing bacteria were grown overnight in 10 ml of yeast mannitol (YM) broth

and trypticase soya (TS) broth, respectively. The cultures pelleted at 10,000 rpm for 10 min

at 4 °C using Sigma 3K30 refrigerated centrifuge (Sigma, CA, USA) were suspended in 180

µl lysis solution T and added 20 µl RNase A solution, vortexed for 2-3 s, incubated at room

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temperature for 2 min. Added 20 µl proteinase K solution to the lysate, mixed and incubated

at 55 °C for 30 min. Added 200 µl of lysis solution C, vortexed for 15 s, incubated at 55 °C

for 10 min. Added 500 µl of column preparation solution to preassembled GenElute

Miniprep binding column seated in 2 ml collection tube, centrifuged at 14,000 rpm for 1 min

and the elute was discarded. Added 200 µl of ethanol (95-100%) to the lysate and mixed

thoroughly by vortexing for 5-10 s. Transferred the lysate to the binding column placed in 2

ml collection tube, centrifuged at 8,000 rpm for 2 min, discarded the collection tube

containing the elute and placed the column in a new 2 ml collection tube. Added 500 µl

wash solution 1 to the binding column and centrifuged for 1 min at 8,000 rpm. Discarded the

flow-through and added 500 µl wash solution to the binding column and centrifuged for 3

min at 14,000 rpm to dry the column. Placed the binding column into 1.5 ml microcentrifuge

tube and pipetted 100 µl elution-solution directly onto the column centre. Incubated the

column for 5 min at room temperature and centrifuged for 1 min at 8,000 rpm to elute the

genomic DNA. DNA quality was checked by obtaining a single and sharp band in

comparison to DNA ladder �DNA HindIII digested (Fermentas, Vilnius, Lithuania) on

0.75% agarose gel (Sigma-Aldrich, MO, USA) prepared in 1× TAE buffer (Appendix III)

and stained with 0.5 µg ml-1

ethidium bromide.

3.3.2 Amplified Ribosomal DNA Restriction Analysis

The amplification of 16S rRNA gene was performed using the primers 27f (5’-AGA GTT

TGA TCC TGG CTC AG-3’) and 1492r (3’-ACG GCT ACC TTG TTA CGA CTT-5’)

(Weisburg et al., 1991). PCR reaction mixture was comprised of 200 µM dNTPs (Fermentas,

Vilnius, Lithuania), 50 pmol each primer, 1× PCR buffer, 1 U Taq DNA polymerase

(Promega, WI, USA), and 100 ng genomic DNA. The thermocycling procedure involved an

initial denaturation at 94 °C for 4 min, followed by 35 cycles of 94 °C for 1 min, 52 °C for 1

min, and 72 °C for 2 min, and final extension at 72 °C for 8 min. The PCR products were

analyzed on 1.2% agarose gel and their size estimated using 1 kb DNA ladder (Fermentas,

Vilnius, Lithuania). The amplified products were digested using the restriction enzymes

AluI, HpaII, MaeII, MboI, RsaI and TaqI (Fermentas, Vilnius, Lithuania). Restriction

reaction consisted of 1.5 µl of 10× restriction enzyme buffer, 1U restriction enzyme, 10 µl

PCR product and nuclease-free water (18.2 �) to make final volume 15 µl. The reaction

mixture was incubated for 2 h at 37 ºC for AluI, HpaII, MaeII, MboI, and RsaI and at 65 º C

for Taq I. Reaction products were resolved on 2% (w/v) agarose gel prepared in 1× TAE

buffer stained with 0.5 µg ml-1

ethidium bromide. The restriction patterns were visualized

and photographed with Alpha Digidoc (Alpha Innotech, CA, USA) under UV light. The

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molecular weight of bands was estimated by comparing with 100 bp DNA ladder

(Fermentas, Vilinius, Lithuania).

3.3.3 ERIC Fingerprinting

ERIC-PCR fingerprint analysis was carried out using the primers ERICIR 3’-CAC TTA

GGG GTC CTC GAA TGT A-5’ and ERIC2 5’-AAG TAA GTG ACT GGG GTG AGC G-

3’ (Versalovic et al. 1994). The reaction mixture of 25 µl containing 50 ng genomic DNA,

1× PCR buffer, 2.5 mM MgCl2, 2.5 mM dNTPs, 50 pmol primers, and 1U Taq DNA

polymerase. The thermocycling conditions consisted of first denaturation cycle at 95 °C for

7 min followed by 30 cycles, including denaturation at 94 °C for 1 min, annealing for 1 min

at 52 °C for ERIC- PCR, extension at 65 °C for 8 min, one final extension cycle at 65 °C for

16 min, and hold at 4 °C. PCR products were separated on 2% agarose gel stained with 0.5

µg ml-1

ethidium bromide. The molecular weight of bands was estimated by comparing with

1 kbp DNA ladder.

3.4 Polyphasic Characterization

3.4.1 16S rRNA Gene Sequencing

16S rRNA gene amplification was performed as described in Section 3.3.2. The amplified

band corresponding to ~1500 bp was excised using a sharp blade and eluted employing

PureLinkTM

Quick Gel Extraction Kit according to the manufacturer’s instructions

(Invitrogen, CA, USA). Gel slice of ~400 mg containing DNA band was placed into a 1.5 ml

polypropylene tube, added 1.2 ml Gel Solubilization (GS1) buffer, and incubated at 50 °C

for 15 min and mixed every 3 min to ensure gel dissolution. After the gel dissolution

incubated for an additional 5 min. Placed Quick Gel Extraction column into 2 ml wash tube

and loaded the dissolved gel piece onto the column, centrifuged at 12,000 g for 1 min,

discarded the flow-through and positioned the column back into the wash tube. Added 500

µl GS1 buffer to the column, incubated at room temperature for 1 min, centrifuged at 12,000

g for 1 min. The flow-through was discarded and placed the column back into the wash tube.

Added 700 µl W9 wash buffer with ethanol to the column, incubated at room temperature

for 5 min, centrifuged at 12,000 g for 1 min and discarded the flow-through. Centrifuged at

12,000 g for 1 min to remove the residual W9 wash buffer and discarded the wash tube.

Placed the column into a 1.5 ml recovery tube, added 50 µl warm (60-70 °C) TE buffer to

the column center, incubated for 1 min at room temperature, centrifuged at 12,000 g for 2

min, discarded the column, and stored the purified 16S rRNA gene product at -20 °C.

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Sequencing of the purified 16S rRNA gene was done using four sequencing primers

(Table 3.2) and Big-Dye Terminator Cycle Sequencing Kit (Applied Biosystems, CA, USA).

The PCR reaction of 5 µl included 1 µl 5× sequencing buffer, 1 µl Big-Dye Terminator

premix, 1 µl primer (5 pmol) and 2 µl purified PCR product (~30 ng µl-1

). Thermal cycling

conditions consisted of an initial denaturation at 96 °C for 3 min, followed by 30 cycles of

94 °C for 10 s, 50 °C for 40 s and 60 °C for 4 min. The unincorporated dye terminators were

removed using Montage SEQ96 Sequencing Reaction Cleanup Kit (Mllipore, MA, USA).

The purified PCR products were transferred to 96 well injection plates for sequencing on

3130x Genetic Analyzer (Applied Biosystems, CA, USA).

Table 3.2 Primers for amplification and sequencing

Target gene*

Primer Sequence**

Reference

27f AGA GTT TGA TCC TGG CTC AG

685r TCT ACG CAT TTC ACC GCT AC

926f AAA CTC AAA GGA ATT GAC GG 16S rRNA

1492r TAC GGY TAC CTT GTT ACG ACT

Weisberg et

al., 1991

atpD-273F SCT GGG SCG YAT CMT GAA CGT

atpD-771R GCC GAC ACT TCC GAA CCN GCC TG

atpD

atpD-294F ATC GGC GAG CCG GTC GAC GA

Gaunt et

al., 2001

recA-6F CGK CTS GTA GAG GAY AAA TCG GTG GA

recA-555R CGR ATC TGG TTG ATG AAG ATC ACC AT

recA-63F ATC GAG CGG TCG TTC GGC AAG GG recA

recA-504R TTG CGC AGC GCC TGG CTC AT

Gaunt et

al., 2001

nodC-f AYG THG TYG AYG ACG GTT C nodC

nodC-i CYG GAC AGC CAN TCK CTA TTG

Laguerre et

al., 2003

nifH nifH-f

nifH-r

TAC GGN AAR GGS GGN ATC GGC AA AGC

ATG TCY TCS AGY TCN TCC A Laguerre et

al., 2003 *atpD = ATP synthase beta-subunit, recA = DNA recombinase A, nodC = N-

acetylglucosaminyl transferase, nifH = nitrogenase reductase. **

Symbols A, C, G, T = standard nucleotides; M = A, C; R = A, G; Y = C, T; S = G, C; K =

G, T; H = A, T, C; N= A, T, G, C.

3.4.2 Sequencing Housekeeping Genes

3.4.2.1 atpD Gene

atpD gene was amplified using 2× Promega GoTaq® Green master mix (Promega, WI,

USA) and 25 pmol primers atpD-273 and atpD-771R (Table 3.2), following the

thermocycling procedure given in Table 3.3. Molecular weight of PCR products resolved on

1.2% agarose gel (Sigma-Aldrich, MO, USA) was estimated by comparison with 100 bp

DNA ladder. The PCR products were gel purified as described in Section 3.4.1 and

sequenced using atpD-294F and atpD-771R primers (Table 3.2) and PCR conditions given in

Table 3.3.

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Table 3.3 PCR conditions for atpD gene

Amplification Sequencing

Temperature Time Temperature Time

95 °C 5 mins 1 Hold 95 °C 3 mins

95 °C

60-50 °C

72 °C

30 s

1 min

2 mins

10 Cycles

95 °C

50 °C

60 °C

20 s

40 s

4 mins

25 cycles

95 °C

50 °C

72 °C

45 s

1 min

2 mins

25 Cycles

4°C � Hold

72 °C 7 mins 1 Hold

4°C � Hold

3.4.2.2 recA Gene

recA gene was amplified using 2× Promega GoTaq® Green master mix and 25 pmol primers

recA-6F and recA-555R (Table 3.2) following the thermocycling procedure given in Table

3.4. Molecular weight of PCR products resolved on 1.2% agarose gel was estimated by

comparison with 100 bp DNA ladder. The PCR products were gel purified as described in

Section 3.4.1 and sequenced using recA-63F and recA-504R primers (Table 3.2), and PCR

conditions given in Table 3.4.

Table 3.4 PCR conditions for recA gene



95 °C 3 mins 1 Hold 95 °C 3 mins 1 Hold

95 °C

55 °C

72 °C

30 s

20 s

40 s

35 Cycles

95 °C

55 °C

60 °C

20 s

40 s

4 mins

25 cycles

72 °C 7 mins 1 Hold 4°C � Hold

4°C � Hold

3.4.3 Symbiotic Gene Sequencing

3.4.3.1 nodC Gene

nodC gene was amplified using 2× Promega GoTaq® Green master mix and 25 pmol

primers nodC-f and nodC-i (Table 3.2) following the thermocycling procedure given in

Table 3.5. Molecular weight of PCR products resolved on 1.2% agarose gel was estimated

by comparison with 100 bp DNA ladder. The PCR products were gel purified as described in

Section 3.4.1 and sequenced using the same primers employed for amplification (Table 3.2)

and PCR conditions given in Table 3.5.

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Table 3.5 PCR conditions for nodC gene



95 °C 5 mins 1 Hold

95 °C

62-55 °C

72 °C

30 s

1 min

2 mins

5 Cycles

95 °C

3 mins

95 °C

55 °C

72 °C

45 s

1 min

2 mins

25 Cycles

95 °C

55 °C

60 °C

20 s

40 s

4 mins

25 cycles

72 °C 7 mins 1 Hold 4 °C � Hold

4 °C � Hold

3.4.3.2 nifH Gene

nifH gene was amplified using 2× Promega GoTaq® Green master mix and 25 pmol primers

nifH-f and nifH-r (Table. 3.2), and following the thermocycling procedure given in Table

3.6. Molecular weight of PCR products resolved on 1.2% agarose gel was estimated by

comparison with 100 bp DNA ladder. The PCR products were gel purified as described in

Section 3.4.1 and sequenced using the same primers employed for amplification (Table 3.2)

and PCR conditions given in Table 3.6.

Table 3.6 PCR conditions for nifH gene



95 °C 3 mins 1 Hold 95 °C 3 mins 1 Hold

95 °C

55 °C

72 °C

30 s

40 s

2 mins

35 Cycles

95 °C

55 °C

60 °C

20 s

40 s

4 mins

25 cycles

72 °C 7 mins 1 Hold 4°C � Hold

4°C � Hold

3.4.4 Sequence Assembling and Phylogenetic Analysis

The sequence data obtained were assembled and analyzed using DNA sequence assembling

software SEQUENCHER™ 4.10.1 (Gene Codes Corporation, MI, USA). Sequence

polymorphism percentage and number of sequence types for each gene were calculated by

DNASP v5 (Librado and Rozas, 2009). The sequences were searched as nucleotide query in

nucleotide database of NCBI (http://www.ncbi.nlm.nib.gov/) to find the related sequences.

Phylogenetic tree was constructed after aligning all acquired and related sequences with

ClustalW software (Thompson et al., 1997), using the neighbour-joining method in MEGA 4

and the Kimura 2 parameter model, and bootstrapped with 1000 replications (Tamura et al.,

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2007). The sequences of atpD and recA genes were concatenated and combined

phylogenetic tree was constructed.

3.4.5 Carbon Source Utilization Pattern

Carbon source utilization pattern of the pea root-nodulating bacteria for 95 different carbon

sources was studied using BIOLOG MicrostationTM

system (BIOLOG, CA, USA). Cultures

grown on YMA for 48 h at 28±0.1 °C were re-suspended in 20 ml inoculation fluid with the

help of sterile cotton swab and their inoculum density adjusted to 52% transmittance using

BIOLOG turbidimeter. The BIOLOG GN2 microplates were inoculated with 150 µl of the

adjusted bacterial suspension per well and incubated at 30±0.1 °C for 24 h. The development

of color was read at 595 nm filter in the MicroStation reader between 16-24 h of incubation.

The substrate utilization profiles were compared with MicroLogTM

version 4.2 database

software and the identification was acknowledged when the similarity index used by

BIOLOG was 0.5 or more. The reactions were scored as negative (0), positive (1), or

borderline (0.5).

3.4.6 Whole-Cell Fatty Acid Methyl Ester (FAME) Analysis

Whole-cell fatty acid methyl esters composition of isolates was studied using Instant

FAMETM

protocol of Sherlock®

Microbial Identification System (MIDI Inc., DE, USA). The

isolates were grown on YMA at 28±0.1 °C for 24 h and 2.5-3.0 mg of cells were harvested

from the third quadrant of the quadrant streaked plates using a sterile inoculation loop in 2

ml GC vials. The extraction procedure was:

• Instant FAME Reagent 1

− Added 250 µl of Reagent 1 to the vial.

− Capped vial vortexed for 10 s.



− Capped vial vortexed for 3 s.



− Phase separation was observed, with clear the top layer and red bottom layer.

Using the Eppendrof Pipette 50 µl of the top layer was transferred into an insert and repeated

this step to obtain 100 µl of the top layer. The samples were loaded in the GC autosampler

(Agilent Technologies, CA, USA) along with the calibration standard and blank. The sample

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information was entered into the Sample Processor table and analysis was started by clicking

Start Batch tool in the Sherlock Sample Processor.

Gas chromatography: The samples were subjected to GC analysis on GC 6890 (Agilent

Technologies, CA, USA) equipped with Ultra 2 phenyl methyl silicone fused silica capillary

column 25 m × 0.2 mm (Agilent Technologies, CA, USA). Hydrogen was used as the fuel

gas and nitrogen as the carrier gas. Fatty acids were identified and quantified by comparison

to retention time and peak area obtained for the authentic standards. Qualitative and

quantitative differences in the fatty acid profiles were used to compute the distance for each

isolate relative to the strains in the Sherlock bacterial fatty acid reference ITSA library

version 1.1.

3.5 Screening for Plant Growth-Promoting Attributes

3.5.1 Nodulation and Plant Growth Promotion

The root-nodualting bacteria grown in YM broth for 54 h at 28 ºC were adjusted to 1.0 OD

at 600 nm with sterile YM broth for application as inoculum for pot studies (Pillai and

Swarup, 2002). Pisum sativum var. Palam Priya seeds were surface sterilized with 20% (v/v)

sodium hypochlorite (4% available chlorine) for 3 min and washed 5 times with sterile

distilled water. The seeds were germinated at 25±2 °C in moist sterile vermiculite. The

uniformly germinated seeds treated with inoculum by dipping for 30 min were sown at a

depth of 2.5 cm with 3 seeds per pot in 15 cm diameter pots filled with sterilized vermiculite.

The inoculated pots were placed in environment control chambers under 550 �M photon m-2

s-1

mixed incandescent and fluorescent illumination with 16 h photoperiod at 25±1 ºC and

50-60% RH. Jensen’s solution diluted ten times was given at 10 d interval to the plants

(Appendix II). The pre-germinated seeds treated with sterilized uninoculated YM broth

served as control.

Plants were carefully retrieved after 4 weeks of sowing for nodule count and plant

growth promotion. Data was recorded on nodule number per plant, root length and shoot

length. The plants were dried at 70 ºC for 3 d to determine the total plant dry weight.

3.5.2 Nitrogenase Activity

The nitrogenase activity of root-nodulating bacteria in pea plants inoculated and grown

under controlled environment as described in Section 3.5.1 was determined by acetylene

reduction assay (Hardy et al., 1973). Nodules were separated from the roots of a plant using

forceps, weighed and transferred to 50 ml air-tight serum bottles (Labconco, MO, USA).

Ten percent of air (v/v) from each bottle was replaced with pure acetylene using an air-tight

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hypodermic syringe (Sigma Gases, N. Delhi, India). The bottles were incubated at room

temperature for 1 h and 1 ml of gas mixture was assayed for ethylene concentration using

GC 6890 (Agilent Technologies, CA, USA), equipped with FID detector and CarboxenTM

fused silica capillary column 30 m × 0.53 mm (Supelco, PA, USA). Nitrogen was used as

the carrier gas at 5 ml min-1

flow rate. The GC oven temperature was programmed to

increase from 70 to 200 ºC at 20 ºC min-1

with 2 min hold at 70 ºC and 5 min hold at 200 ºC.

The standard curve was prepared using different concentrations of pure ethylene (Sigma

Gases, New Delhi, India) (Fig. 3.3). The acetylene reduction into ethylene was calculated as

nmol C2H4 produced mg nodule fresh weight

-1h

-1.

3.5.3 Phosphate Solubilization

3.5.3.1 Qualitative Analysis

Root-nodulating bacteria and rhizobacteria were screened for phosphate solubilization on

modified PVK agar containing TCP as the sole source of phosphate. A loop full of culture

placed on modified PVK agar plates was incubated at 28±0.1 °C for 7 d. Appearance of a

clear zone around the colony indicated phosphate-solubilization. The solubilization zone was

determined by subtracting colony diameter from total zone diameter inclusive of the colony.

3.5.3.2 Quantitative Estimation

Quantitative estimation of inorganic phosphate solubilization was done by growing the

bacterial isolates in National Botanical Research Institute’s Phosphate (NBRIP) broth

(Appendix I), containing 0.5% TCP as the sole source of phosphate (Nautiyal, 1999). The

flasks containing 50 ml medium inoculated with 500 µl culture (inoculum adjusted ~ 5 × 108

CFU ml-1

) in triplicate were incubated at 28±0.1 °C at 180 rpm in Innova 4230 refrigerated

incubator shaker. The cultures were harvested by centrifugation at 10,000 rpm for 10 min

Fig. 3.3 Standard curve for ethylene

y = 84.884x

R2 = 0.9983

0

5000

10000

15000

20000

25000

30000

35000

0 50 100 150 200 250 300 350 400

Ethylene concentration (mM)

Pea

k A

rea

y = 84.884x

R2 = 0.9983

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after 7 d incubation for root-nodulating bacteria and after 5 d incubation for rhizobacteria.

The uninoculated autoclaved NBRIP broth incubated under similar conditions was

employed as control.

Phosphorus content in culture supernatants was estimated by vanado-molybdate-

yellow colour method (Jackson, 1973). To a 0.5 ml aliquot of the supernatant, 2.5 ml

Barton’s reagent (Appendix II) was added and the volume made to 50 ml with de-ionized

water in volumetric flak. The absorbance of the reaction mixture was read after 10 min at

430 nm in Ultraspec 3000 UV/Visible spectrophotometer. Total soluble phosphate was

calculated from the regression equation of the standard curve. The values of soluble

phosphate were expressed as µg ml-1

over control. The pH of culture supernatants was

measured using CyberScan 510 pH meter (Eutech Instruments, Singapore).

The standard curve was prepared by dissolving 0.2195 g KH2PO4 in de-ionized water

to 1 l. The stock solution was diluted by adding 100 ml de-ionized water to 150 ml solution

to obtain a strength of 30 µg P ml-1

. Added 2.5 ml Barton’s reagent to 1, 2, 3, 4, 5, 6, 8, 10,

15, 20 and 25 ml aliquots of the diluted stock taken in 50 ml volumetric flasks and volume

made to 50 ml with de-ionized water. The blank included 2.5 ml Barton’s reagent made to

50 ml with de-ionized water. The absorbance of the resultant yellow color was read at 430

nm after 10 min. Values of concentration were plotted versus optical density and regression

equation used to calculate the liberated phosphorus (Fig. 3.4).

3.5.3.3 Detection of Organic Acids Produced during Phosphate Solubilization

The culture supernatants of phosphate solubilization experiments were filtered through 0.22

µm nylon filter. Detection and quantification of organic acids produced during phosphate

OD

at

43

0 n

m

y = 0.0008x + 0.0032

R2 = 0.9998

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0 200 400 600 800

P concentration (µg ml-1

)

Fig. 3.4 Standard curve for soluble phosphate

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solubilization were done on Waters 996 HPLC equipped with PDA detector, Waters 717

plus autosampler, Waters 600 controller, WatersTM

pump, Waters inline degasser AF

(Waters Corporation, CA USA), and Lichrosphere RP-18 column 250 mm × 4.6 mm and 5

�m particle size (Merck, Darmstadt, Germany). The mobile phase was 0.1% ortho-

phosphoric acid (Merck, Darmstadt, Germany) in a gradient programme of flow rate starting

at 0.4 ml/min at 0 min, increasing to 0.6 ml min-1

at 9.5 min, decreasing from 0.6 to 0.4

ml/min from 9.5 to 18 min, isocratic at 0.4 ml min-1

from 18 to 25 min, again increasing

from 0.4 to 0.6 ml min-1

from 25 to 30 min and equilibrating to 0.4 ml min-1

from 30 to 35

min. The eluates were detected at � 210 nm and identified by retention time and co-

chromatography of the samples spiked with the authentic standards of organic acids viz.

gluconic acid (Sigma-Aldrich, MO, USA), 2-keto gluconic acid (Sigma, CA, USA), formic

acid, isocitric acid, lactic acid, oxalic acid, succinic acid, malic acid, citric acid, and fumaric

acid (Supelco, PA, USA) (Fig. 3.5; Table 3.7). Each replicate was analyzed in a single run

on HPLC and the values were presented as the mean of three replicates.

Table 3.7 Regression equations for authentic organic acid standards

Standard Regression equation R2

value

Formic acid y = 0.0067x - 0.0237 0.999

Fumaric acid y = 0.0324x - 0.245 0.992

Gluconic acid y = 0.0009x + 0.024 0.994

�-keto gluconic acid y = 0.0011x - 0.0074 0.997

Lactic acid y = 0.0003x - 0.002 0.999

Malic acid y = 0.1186x - 0.0009 0.998

Oxalic acid y = 0.0041x - 0.048 0.994

Succinic acid y = 0.0734x + 0.0021 0.999

y is the peak area and x is the concentration; R2

= regression co-efficient

y = 0.1186x - 0.0009

R 2 = 0.9983

0

0.01

0.02

0.03

0.04

0.05

0 0.1 0.2 0.3 0.4 0.5

Fig. 3.5 Standard curve for malic acid

MA concentration (mg ml-1

)

Pea

k A

rea

y = 0.1186x - 0.0009

R2 = 0.9983

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3.5.4 IAA Production

3.5.4.1 Culture Growth Conditions

IAA production by root-nodulating bacteria was studied by inoculating 10 ml YM broth

supplemented with 0.1% DL-tryptophan taken in 30 ml screw-capped glass vials wrapped

with carbon paper and tin-foil with 500 µl of 24 h old cultures grown in YM broth. The

cultures were incubated for 72 h at 28±0.1 °C and 180 rpm in Innova 4230 refrigerated

incubator shaker. The cultures were centrifuged at 10,000 rpm for 10 min at 4 °C. IAA

production by rhizobacteria was estimated by growing the cultures for 48 h in nutrient broth

supplemented with 0.1% DL-tryptophan.

3.5.4.2 Colorimetric Estimation

Estimation of IAA in the culture supernatants was done by colorimetric assay (Loper and

Schroth, 1986). One millilitre supernatant was mixed with 4 ml Salkowski reagent

(Appendix II) and the absorbance read after 30 min at 535 nm in Ultraspec 3000 UV/Visible

spectrophotometer (Amersham Pharmacia, Buckinghamshire, England). The blank consisted

of 4 ml Salkowski reagent and 1 ml de-ionized water. A standard curve was prepared using

different concentrations of IAA (Sigma Aldrich, MO, USA). Values of concentration were

plotted versus optical density and the regression equation used to calculate IAA

concentration (Fig. 3.6). IAA content was expressed as µg ml-1

over control.

3.5.4.3 HPLC Analysis

IAA and its intermediates were quantified in the culture supernatants by HPLC (Chung et

al., 2003). The culture supernatants were acidified to pH 2.5 with 1 N HCl (Appendix II) and

indole derivatives extracted three times with equal volume of ethyl acetate. Ethyl acetate

extracts were pooled, evaporated to dryness under vacuum on R 210 rotatory evaporator

(Büchi, Essen, Germany), and the residue dissolved in 3 ml methanol. Sample analysis was

IAA concentration (µg ml-1

)

y = 0.0161x - 0.0024

R2 = 0.9991

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

0 10 20 30 40 50 60

Fig. 3.6 Standard curve for indole-3-acetic acid

OD

at

53

5 n

m y = 0.0161x -0.0024

R2 = 0.9991

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done on Waters 996 HPLC equipped with PDA detector, Waters 717 plus autosampler,

Waters 600 controller (Waters Corporation, CA, USA), and Lichrosphere RP-18 column 250

mm × 4.6 mm and 5 �m particle size (Merck, Darmstadt, Germany). A gradient of mobile

phase consisting of 0.5% acetic acid (A) and 100% acetonitrile (B) at a flow rate of 1 ml

min-1

was employed:

Time (min) A (%) B (%)

0 60 40

6.5 55 45

8.0 60 40

The sample injection volume was 20 µl. The eluates were detected using an isocratic

system and identified by retention time and co-chromatography by spiking the sample with

authentic indole derivatives. IAA and its intermediates were quantified by reference to the

peak areas obtained for authentic standards for tryptophan (Try), IAA, indole-3-pyruvic acid

(IPA), indole-3-acetaldehyde (IAAld), indole-3-acetamide (IAM), and indole-3-acetonitrile

(IAN) (Fig. 3.7, Table 3.8). Samples were analyzed in triplicates.

Table 3.8 Regression equations for authentic indole derivatives standards

Standard Regression equation R2

value

Indole-3-acetic acid y = 4.9429x - 0.002 0.999

Indole-3-acetaldehyde y = 1.3553x + 0.0101 0.994

Indole-3-acetamide y = 4.8571x + 2E-16 0.997

Indole-3-acetonitrile y = 4.3421x + 0.122 0.998

Indole-3-lactic acid y = 4.9029x – 0.002 0.999

Indole-3-pyruvic acid y = 3.1114x - 0.0577 0.998

Tryptophan y = 3.4257x + 0.0025 0.999

y is the peak area and x is the concentration; R2

= regression co-efficient

Fig. 3.7 Standard curve for indole-3-lactic acid

y = 4.9029x - 0.002

R 2 = 0.9999

0

0.1

0.2

0.3

0.4

0.5

0.6

0 0.02 0.04 0.06 0.08 0.1 0.12

ILA concentration (mg ml-1

)

Pea

k A

rea

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42

3.5.5 ACC-deaminase Activity

ACC-deaminase activity of bacterial isolates was determined by measuring the production of

�-ketobutyrate generated by ACC cleavage by ACC deaminase using 2, 4-

dinitrophenylhydrazine reagent (Penrose and Glick, 2003). Fifteen millilitre YM broth

inoculated with 100 µl of 48 h old cultures was incubated at 28±0.1 °C in Innova 4230

refrigerated incubator shaker for root-nodulating bacteria and TS broth for rhizobacteria.

Cultures after 48 h incubation were centrifuged at 4,000 rpm and 4 °C for 10 min. The

pellets were resuspended in 5 ml DF salts minimal medium and centrifuged at 3,000 rpm at 4

°C for 5 min. The supernatants were discarded and the pellets resuspended in 7.5 ml DF

minimal medium supplemented with 3.0 mM final concentration of ACC and incubated at

28±0.1 °C. After 24 h the cultures were centrifuged at 8,000 rpm for 10 min at 4 °C and the

pellets washed twice with 0.1 M Tris HCl, pH 7.5 and resuspended in 600 µl 0.1 M Tris

HCl, pH 8.5 (Appendix II). The bacterial cells made labile by adding 30 µl toluene were

vortexed at high speed for 30 s. A 100 µl aliquot of toluenized cells was stored at 4±0.1 °C

for protein estimation. The remaining toluenized cell suspension was assayed for ACC-

deaminase activity. To 200 µl aliquot of the cell suspension, 20 µl 0.5 M ACC (Appendix II)

was added, vortexed for 30 s and incubated at 30 °C for 30 min. The contents were

transferred to 10 ml glass vial, added 1 ml 0.56 N HCl (Appendix II) and centrifuged for 5

min at 13,000 rpm at 4 °C. One millilitre supernatant was vortexed with 800 µl 0.56 N HCl

and thereafter added 300 µl 2, 4-dinitrophenylhydrazine reagent (Appendix II). The contents

were vortexed and incubated at 30±0.1 °C for 30 min. Following addition of 2 ml 2 N NaOH

(Appendix II), absorbance was measured at 540 nm in Ultraspec 3000 UV/Visible

spectrophotometer. The negative control for the assay included 200 µl labilized cell

suspension without ACC and the blank included 200 µl 0.1 M Tris HCl (pH 8.5) with 20 µl

0.5 M ACC. ACC-deaminase activity was expressed as nM �-ketobutyrate mg protein-1

h-1

.

The concentration of �-ketobutyrate was determined by comparison with the standard

curve generated by mixing 1 ml aliquots of 0, 0.01, 0.05, 0.1, 0.2, 0.5, 0.75 and 1.0 mM �-

ketobutyrate with 800 µl 0.56 N HCl and 300 µl 2,4-dinitrophenylhydrazine reagent. The

reaction mixture was incubated at 30±0.1 °C for 30 min, added 2 ml 2 N NaOH, and

absorbance read at 540 nm. Values of concentration versus optical density were plotted and

regression equation used to calculate �-ketobutyrate concentration (Fig. 3.8).

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The protein concentration of toluenized cells was determined using Bicinchoninic

Acid (BCA) Protein Assay Kit (Novagen, Merck, Darmstadt, Germany) following

manufacturer’s instructions. A 26.5 µl aliquot of the toluene-labilized bacterial cell sample

was diluted with 173.5 µl 0.1 M Tris HCl, pH 8.0 and boiled with 200 µl 0.1 N NaOH for 10

min. After cooling the cell sample to room temperature, 50 µl cell lysate was taken out of it,

diluted to 150 µl with 0.1 M Tris HCl, pH 8.5 (Appendix II), and 50 µl pipetted out in a 10

ml glass tube. Following the addition of 1 ml BCA reagent, the mixture was mixed

thoroughly by vortexing, incubated at 60 °C for 15 min, and absorbance read at 562 nm in

Ultraspec 3000 UV/Visible spectrophotometer. Protein concentration was determined by

preparing the standard curve of bovine serum albumin (BSA). To 50 µl de-ionized water

with BSA dilutions of 5, 25, 50, 125 and 250 µg ml-1

was added 1 ml BCA reagent

incubated at 60 °C for 15 min and the absorbance read at 562 nm. Values of BSA

concentration were plotted versus optical density, and the regression equation used to

calculate protein content (Fig. 3.9).

y = 0.0024x + 0.0007

R2 = 0.9996

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0 50 100 150 200 250 300

y = 0.0024x + 0.0007

R2

= 0.9996

BSA concentration (µg ml-1

)

OD

at

56

2 n

m

Fig. 3.9 Standard curve for bovine serum albumin

Fig. 3.8 Standard curve for �-ketobutyrate

y = 1.9741x + 0.0159

R2 = 0.9997

0

0.5

1

1.5

2

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 1.1

OD

at

54

0 n

m

�-ketobutyrate concentration (mM)

y = 1.9741x + 0.0159

R2

= 0.9997

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3.5.6 Siderophore Production

3.5.6.1 Initial Screening

Siderophore production by the bacterial isolates was detected on Chrome Azurol Sulphonate

(CAS) agar (Schwyn and Neilands, 1987). CAS assay is based on the high affinity of

siderophores for ferric iron, whereby ferric iron bound to the dye is complexed and released:

Fe3+

-dye (blue) + siderophore = Fe3+

-siderophore + dye (orange)

CAS agar (Appendix I) plates were spot inoculated with 24 h old bacterial cultures and

incubated at 28±0.1 °C. Yellow-orange halos around colonies indicated siderophore

production. A siderophore zone was determined by subtracting colony diameter from total

zone diameter inclusive of the colony.

Siderophore production was recorded for root-nodulating bacteria after 7 d incubation

and rhizobacteria after 5 d incubation. The glassware used for siderophore detection and

quantification studies were cleaned in 20% HCl to remove iron and washed with de-ionized

water.

3.5.6.2 Culture Growth Conditions

Ten millilitre succinate broth (Appendix I) was inoculated with 100 µl of cultures of root-

nodulating bacteria grown in YM broth for 48 h and rhizobacteria in TS broth for 24 h at

28±0.1 °C. The vials in triplicates were incubated at 180 rpm at 28±0.1 °C in Innova 4230

refrigerated incubator shaker. The cultures of were centrifuged after 7 d for root-nodulating

bacteria and after 5 d for rhizobacteria at 13,000 rpm for 10 min at 4 °C, and the

supernatants used for further studies.

3.5.6.3 Quantitative Estimation

Quantitative estimation of siderophores was done by CAS-shuttle assay (Payne, 1994). To

0.5 ml aliquot of the culture supernatant was added 0.5 ml of CAS reagent (Appendix II) and

the absorbance measured at 630 nm against the reference consisting of 0.5 ml uninoculated

succinate broth and 0.5 ml of CAS reagent. The blank consisted of uninoculated succinate

broth. Siderophore content in the aliquot was calculated using the formula:

% siderophore units = (Ar-As/Ar) × 100

where Ar = absorbance of the reference and As = absorbance of the sample

3.5.6.4 Chemical Nature of Siderophores

Hydroxamate, catecholate and carboxylate nature of siderophores was determined by

examining absorption maxima (�max) of cell-free supernatant in UV-2450 UV/Visible

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spectrophotometer (Shimadzu, Kyoto, Japan). The �max reported for various siderophores is

between 400-410 nm for hydroxamate type, 320, 250 and 210 nm for catecholate type and

190-280 for copper carboxylate type (Neilands, 1981).

3.5.7 In vitro Antagonistic Activity against Phytopathogens

Rhizobacteria were evaluated for fungal antagonistic activity on yeast malt extract (YME)

agar by dual culture plate assay (Henis and Inbar, 1968). The pathogenic isolates of

Fusarium oxysporum f sp. pisi and F. solani f sp. pisi from pea were grown on potato

dextrose agar for 7 d at 28±0.1 °C. The bacterial isolates were grown on TSA for 48 h at 28

°C. The bacterial isolates were streaked in a file near the periphery in YME agar plates. The

agar discs nearly 6 mm diameter with full mycelium growth for each pathogen were seeded

perpendicular to the bacterial streak on the opposite side near the plate periphery. The Petri

plates without bacterial inoculation served as control. The plates in triplicates were incubated

at 28±0.1 °C for 7 d. The reduction in fungal growth was calculated using the formula:

Reduction in mycelium growth (%) = [(control-treatment)/control] × 100

Treatment = mycelial growth of fungus in plates with bacterium

Control = mycelial growth of fungus in plates without bacterium

3.5.8 Effect on Plant Growth in Controlled Environment

Surface sterilized seeds of Pisum sativum var. Palam Priya and Zea mays var. Girija were

germinated at 25±2 °C in moist sterile vermiculite. Rhizobacteria grown in TS broth for 30 h

at 28 ºC were adjusted to 1.0 OD at 600 nm with sterile TS broth for application under

controlled environment as inoculum for pot studies as described in Section 3.5.1. The pre-

germinated seeds treated with sterilized uninoculated TS broth served as control. The pots

were placed in complete Randomized Block Design under 550 �M photon m-2

s-1

mixed

incandescent and fluorescent illumination, 16/8 h light/dark cycle and 50-60% RH at 25±2

°C in environment control chamber. Hoagland nutrient solution (Hoagland and Arnon, 1938;

Appendix II) diluted ten times was given at 10 d interval to the plants. Data were recorded

on root length, shoot length and total dry weight after 30 d of sowing pre-germinated seeds.

The plants were oven-dried to a constant weight at 70 °C for 3 d to determine the total dry

weight.

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3.6 Microplot Evaluation

3.6.1 Inoculum Preparation

Root-nodulating bacteria grown in 250 ml Erlenmeyer flasks containing 100 ml YM broth

inoculated with 500 µl cultures grown for 24 h at 28±0.1 °C in YM broth. The cultures were

incubated at 28±0.1 °C with shaking at 180 rpm for 72 h in Innova 4320 refrigerated

incubator shaker and centrifuged at 10,000 rpm for 10 min at 4 °C. The bacterial pellets

were suspended in 0.85% NaCl, OD600 adjusted to 1.0 and 50 ml suspension added to 50 g

sterilized activated charcoal to obtain about 108

CFU per gram carrier-based inoculum.

Activated charcoal (RANKEM, N. Delhi, India) was sterilized in plastic bags by autoclaving

at 121 °C for 1 h on two alternate days.

Rhizobacteria were grown in 100 ml TS broth by transferring 500 µl of 24 h old

cultures grown in TS broth at 28±0.1 °C. The cultures were incubated at 28±0.1 °C for 48 h

at 180 rpm and processed for preparation of charcoal based inoculum.

3.6.2 Evaluation for Plant Growth and Yield

Plant growth-promoting bacteria were evaluated in comparison to untreated control on

Pisum sativum var. Palam Priya in a Randomized Block Design in 1 sqm microplots at

CSIR-IHBT Chandpur Experimental Farms (32° 6' 0" N and 76° 31' 0" E,�1300 m amsl).

The treated seeds were sown at 3 cm inter-plant distance and 25 cm inter-row distance. Data

were recorded on nodule number, plant height, and dry weight after 45 days of sowing, and

yield on harvest after 110 d of sowing on randomly selected 9 plants from the central rows of

each plot.

3.7 Small-Scale Field Trials

3.7.1 Plant Growth-Promoting Bacteria

Field evaluation of potential root-nodulating bacteria and phosphate-solubilizing

rhizobacteria was under taken singly and in combination on Pisum sativum var. Palam Priya

at CSIR-IHBT Chandpur Experimental Farms in comparison to Rhizobium leguminosarum

sv viciae USDA 3841 (courtesy Prof. JPW Young, University of York, UK) and strain

Pseudomonas putida UW4 (courtesy Prof. BR Glick, University of Waterloo, Canada).

3.7.2 Site Description

The mean monthly meteorological data of the location during the crop period from

December to March 2009-10 obtained from the Department of Agronomy, Forages and

Grassland Management, CSK HPKV, Palampur are presented in Table 3.9. Soil analysis of

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the surface soils upto 30 cm depth showed pH 5.61, organic matter 1.83%, available N 134.8

kg ha-1

, available P 45.2 ppm, and available K 118.6 ppm.

Table 3.9 Monthly meteorological data of Palampur for pea cropping season for

2009-10

Temperature (ºC) RH (%)

Month Max Min Max Min

Rainfall

(mm)

Rainy

days

November 2009 20.8 7.6 81 54 69.4 5

December 18.0 5.2 75 54 0.2 0

January 2010 18.3 4.9 76 49 25.2 3

February 18.3 6.2 82 64 120.6 6

March 25.6 12.4 61 40 26.0 3

April 30.3 15.7 48 30 27.9 5

RH = relative humidity, Min = minimum, Max = maximum

Source: Department of Agronomy, Forages and Grassland Management, CSK HPKV,

Palampur

3.7.3 Field Evaluations

The isolates were evaluated in comparison to untreated control and 100% NPK dosage in

2.25 × 1 m2

plots. The recommended NPK dosage was comprised of 108 kg N ha-1

as urea,

375 kg P ha-1

as SSP and 100 kg K ha-1

in the form of muriate of potash. The field trial was

conducted in a Randomized Block Design with 9 treatments of single inoculations, 20 of

combined inoculations of root-nodulating bacteria and phosphate-solubilizing bacteria, and

uninoculated controls with and without NPK dosage. The farmyard manure @ 20 tons ha-1

was applied to all the treatments.

The slurry of charcoal-based inoculum prepared by adding 10 g inoculum in 50-60

ml of 10% molasses was applied to the seeds @ 25 g kg-1

seeds. In co-inoculations, equal

quantities of charcoal-based preparations mixed were applied to the seeds. NPK was applied

before sowing to plots marked for the treatment without inoculations. Seeds treated with

sterilized activated charcoal in 10% molasses were employed for the control plots. The seeds

were sown at 3 cm inter-plant distance and 30 cm inter-row distance @ 125 kg ha-1

. Nine

plants from central rows of each plot were selected randomly for data recording on

nodulation, plant height and dry weight after 45 days of sowing, and yield after 110 d of

sowing.

3.8 Soil and Plant Analysis

Soil samples were air dried, ground with pestle and mortar, and passed through 2 mm sieve

for determining EC, pH, available N, P and K contents, and total organic matter. Soil EC

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and pH was determined in 1:2.5 soil:water suspension using CC 601 electrical conductivity

meter (Century Instruments, Chandigarh, India) and CyberScan 510 pH meter (Eutech

Instruments, Singapore), respectively (Jackson, 1973).

Plant root and shoot samples oven-dried at 70 °C for 3 d and powdered for estimation

of total N, P and K. Nine replicates of each treatment were analyzed as independent samples.

3.8.1 Available N

Available N was estimated by alkaline permanganate method (Subbiah and Asija, 1956).

Placed 20 g soil in a dry Kjeldahl flask, swirled with 20 ml de-ionized water, and added 1 ml

liquid-paraffin and 100 ml each of 0.32% potassium permanganate and 2.5% sodium

hydroxide solutions (Appendix II). The contents were distilled in a Kjeldahl assembly and

collected the ammonia in 250 ml Erlenmeyer flask containing 20 ml boric acid solution with

mixed indicator (Appendix II). The pink colour of boric acid solution turned green with

ammonia absorption. The contents were titrated with 0.02 N H2SO4 to the original pink

colour. Titration with the blank was carried out to the same end point as that for the sample.

Available N (kg ha-1

) = 31.36 × actual volume of H2SO4 used in titration

Available N (%) = (2.24 × value in kg ha-1

)/10000

3.8.2 Available P

Estimation of available P was done by sodium bicarbonate method (Olsen et al., 1954). To

2.5 g air-dried soil taken in a 150 ml Erlenmeyer flask was added 1 g activated charcoal and

50 ml Olsen’s reagent (Appendix II). The suspension was shaken for 30 min on

reciprocating shaker and filtered immediately through Whatman No. 40 filter paper. Placed 5

ml aliquot of the extract in a 25 ml volumetric flask, acidified with 2.5 M H2SO4 to pH 5.0,

added 4 ml Reagent B (Appendix II), and volume made 25 ml with de-ionized water. The

absorbance of blue colour was read at 882 nm after 10 min in T90+ UV/Visible

spectrophotometer (PG Instruments Ltd., Leicestershire, England).

The standard curve was prepared by taking 1, 2, 3, 4, 5 and 10 ml of 2 ppm P solution

(Appendix II) in 25 ml volumetric flasks. Added 5 ml Olsen’s reagent acidified with 2.5 M

H2SO4 to pH 5.0 and 4 ml Reagent B and volume made to 25 ml with de-ionized water. A

blank was prepared with 5 ml Olsen’s reagent, de-ionized water and 4 ml Reagent B. The

absorbance of blue colour was read at 882 nm after 10 min in T90+ UV/Visible

spectrophotometer. Values of concentration were plotted versus optical density and the

regression equation used to calculate the available P (Fig. 3.10).

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Available P (ppm) = y × 100 (dilution factor)

= z

Available P (%) = z/10000

3.8.3 Available K

Available K was estimated by flame photometric method (Jackson, 1973). To 5 g air-dried

soil sample taken in a 150 ml Erlenmeyer flask was added 25 ml ammonium acetate, pH 7.0

(Appendix II). Placed the flask on a reciprocating shaker for 5 min and filtered the contents

through Whatman No. 1 filter paper. K content in the extract was determined in BWB-XP

microprocessor flame photometer (BWB Technologies Ltd., Essex, UK) using K filter.

The standard curve for K was prepared by taking 5, 10, 15, 20 and 30 ml of 100 ppm

KCl standard solution (Appendix II) into 100 ml volumetric flasks and volume made with

ammonium acetate, pH 7.0 (Appendix II). Values of concentration were plotted versus

optical density and the regression equation used to calculate the available K (Fig. 3.11).

Available K (ppm) = (x × volume of extract)/weight of soil taken

= x × 5

Available K (%) = x × 5/10000

where x = ppm of K in the extract

Fig. 3.11 Standard curve for potassium

O

D

K concentration (µg ml-1

)

y = 1.0663x - 0.1671

R2 = 0.9988

0

5

10

15

20

25

30

35

0 5 10 15 20 25 30 35

y = 1.0663x –

0.1671


) O

D a

t 882

nm

y = 0.7235x - 0.0049

R2 = 0.9995

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0 0.2 0.4 0.6 0.8 1

Fig. 3.10 Standard curve for soluble phosphate

y = 0.7235x – 0.0049

R2

= 0.9995

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3.8.4 Total N

Estimation of total N was done by modified Kjeldahl’s method (Jackson, 1973). To 0.5 g

dried plant material taken in a 100 ml Kjeldahl flask was added 20 ml sulphuric-salicylic

acid mixture (Appendix II) and 5 g sulphate mixture (Appendix II). The flask was swirled

gently and allowed to stand overnight. The contents were heated gently for 5 min and then at

elevated temperature till the material became colourless. The contents were cooled and the

volume made to 100 ml with de-ionized water. 5 ml aliquot was taken in micro-Kjeldahl

apparatus and 10 ml 40% NaOH was slowly added. The contents were distilled till 100 ml

distillate was collected in 10 ml boric acid solution with mixed indicator (Appendix II). The

colour changed from wine-red to blue. The contents were titrated against 0.02 N H2SO4 to

the original wine-red colour. Titration with blank was carried out to the same end point as

that for the sample. Total nitrogen was calculated as follows:

N (%) = (TV × 0.00028 × 100 × 100)/(0.5 × 5)

where,

Weight of sample = 0.5 g

Normality of H2SO4 = 0.02

Volume of digestion = 100 ml

Aliquot taken = 5 ml

Titration value (TV) = Sample titration value - Blank titration value

1 ml 0.02 N H2SO4 = 0.00028 g nitrogen

3.8.5 Sample Digestion for Total P and K

Total phosphorus and total potassium in the plant material were estimated after digestion of

samples using mixed acid method (Jackson, 1973). To 1 g ground plant material taken in

100 ml volumetric flask was added 10 ml HNO3:H2SO4:HClO4 (9:4:1) mixture. The

contents were mixed by swirling and heated in a digestion chamber over a hot plate set at

low temperature initially and high temperature afterwards until the production of red fumes

ceased. The contents were further evaporated to 3-5 ml volume and completion of digestion

confirmed with colourless appearance of the contents. The volume after cooling was made to

100 ml with de-ionized water and the contents filtered through Whatman No. 1 filter paper.

Aliquots of the solution were used for the estimation of P and K.

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3.8.6 Total P

Total phosphorus was estimated by vanado-molybdate yellow colour method (Jackson,

1973).To 1 ml aliquot of the digested sample taken in 50 ml volumetric flask was added 10

ml Barton’s reagent (Appendix II) and volume made to 50 ml with de-ionized water. The

intensity of the yellow colour was read at 430 nm after 30 min in UV/Visible

spectrophotometer. The standard curve was prepared by taking 1, 2, 3, 4 and 5 ml of 50 ppm

standard stock P solution (Appendix II) in 50 ml volumetric flasks. 10 ml Barton’s reagent

was added and volume made to 50 ml with de-ionized water. The blank included 10 ml

Barton’s reagent and 40 ml de-ionized water, and absorbance was read at 430 nm after 30

min. Values of P concentration were plotted versus optical density and regression equation

used to calculate total P (Fig. 3.12).

P concentration (%) = (x × dilution factor)/10000

where x = value of P concentration (µg ml-1

) obtained from the standard curve

3.8.7 Total K

Total K was estimated by flame photometric method (Jackson, 1973). The plant material

was digested as described in the section 3.8.5 and total K was determined in BWB-XP

microprocessor flame photometer using K filter. The standard curve was prepared as

described for available K estimation (Fig. 3.10). K concentration in the samples was

calculated as follows:

K concentration (%) = (x × dilution factor)/10000

where x = value of K concentration (µg ml-1

) obtained from the standard curve


)

OD

at

430 n

m

y = 1.1829x - 0.0571

R2 = 0.9986

0

1

2

3

4

5

6

7

0 1 2 3 4 5

Fig. 3.12 Standard curve for total phosphate

y = 1.1829x – 0.0571

R2 = 0.9986

��

��

52

3.8.8 Total Organic Carbon

Total organic matter was estimated by potassium dichromate method. To 1 g air-dried soil

sample taken in a 250 ml Erlenmeyer flask was added 10 ml of 1N potassium dichromate

and swirled after adding 20 ml of conc. sulphuric acid and kept for 30 minutes on an

asbestos sheet. Added 100 ml of de-ionized water to flask and absorbance was read at 610

nm in UV/Visible spectrophotometer.

The standard curve was prepared by taking 1-25 mg of anhydrous sucrose in 250 ml

Erlenmeyer flask and added 10 ml of 1N potassium dichromate and 20 ml of conc. sulphuric

acid. After 10 min 100 ml of de-ionized water was added to each flask. Blank included 10

ml 1N potassium dichromate, 20 ml of conc. sulphuric acid and 100 ml of de-ionized water,

and the absorbance read at 610 nm. Values of concentration were plotted versus optical

density and the regression equation used to calculate total carbon (Fig. 3.13).

where,

Percent carbon Sucrose (mg)

0.00 0

0.042 1

0.084 2

0.168 4

0.262 6

0.336 8

0.42 10

0.504 12

0.588 14

0.672 16

0.756 18

0.840 20

1.05 25

Fig. 3.13 Standard curve for organic carbon

y = 0.1357x

R2 = 0.9923

0

0.02

0.04

0.06

0.08

0.1

0.12

0.14

0 0.2 0.4 0.6 0.8 1

OC concentration (%)

OD

at

61

0 n

m

y = 0.1357x

R2 = 0.9923

��

��

53

3.9 Experimental Design and Statistical Analysis

Randomized Block Design with two factor factorial arrangement was adopted for conducting

the experiments unless stated otherwise. The data were checked for normality and found to

be normally distributed. All the data were subjected to one way analysis of variance

(ANOVA) using the STATISTICA data analysis software system version 7 (StatSoft®

Inc.

Tulsa, USA, 2004) unless stated otherwise. All values are means of three replicates and

experiments repeated thrice unless stated otherwise. The mean of treatments of “screening

for plant growth-promoting attributes and plant growth promotion in controlled

environment” were compared by Tukey’s Honestly Significant Difference (HSD) at p =

0.01. The mean of treatments of “microplot and small-scale field studies” were compared

using Tukey’s HSD test at p = 0.05.

The presence (1) and absence (0) of bands produced by ARDRA gene and ERIC-

PCR were scored manually. The cluster analysis was performed using the hierarchic

Unweighted Pair Group Method with Arithmetic Mean (UPGMA) employing TREECON

software version 1.3b (Yves Van de Peer, University of Anterwerp, Belgium). The data on

carbon source utilization and whole-cell fatty acid methyl esters analysis were subjected to

Principal Component Analysis using PAST software (Hammer et al., 2001).

3.10 Sequence Submission

Original sequences of 16S rRNA, atpD, recA, nodC and nifH genes of Rlv strains have been

submitted with the NCBI GenBank under accession numbers: EF437220, EU730590-

EU730591, EU730594-EU730598, EU730600-EU730602, JF759685-JF759823. Sequences

of 16S rRNA gene of rhizobacteria have also been deposited with NCBI Genbank under

accession numbers: JF766686-JF766702.

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