materials and methods - shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/32645/14/14_chapter...
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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
Amplification Sequencing
Temperature Time Temperature Time
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
Amplification Sequencing
Temperature Time Temperature Time
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
Amplification Sequencing
Temperature Time Temperature Time
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.
• Instant FAME Reagent 2
− Added 250 µl of Reagent 2 to the vial.
− Capped vial vortexed for 3 s.
• Instant FAME Reagent 2
− Added 250 µl of Reagent 3 to the vial.
− 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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37
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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38
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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39
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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40
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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41
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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43
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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44
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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45
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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46
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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47
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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48
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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49
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
P concentration (µg ml-1
) 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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50
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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51
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
P concentration (µg ml-1
)
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
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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
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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.