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MATERIALS & METHODS
CHAPTER – III
MATERIALS & METHODS
(I) SELECTION OF PLANT SPECIES -
(A) A field survey was made from July 2006 - February 2008 to collect root nodules of the
cultivated & wild leguminous crops Two site were chosen for the collection of plants
viz; Indira Gandhi Krishi Vishwavidyalaya, (IGKVV) Raipur (C.G.) & Government
Nagarjuna
P.G. (NPG) College of Science Campus, Raipur (C.G.). Altogether ten
cultivated & three wild leguminous were selected for the isolation of Rhizobium spp.
a) Cultivated Plants :
Phaseolus aureus (PA) , Arachis hypogaea (AH), Dolichos lablab (DL), Glycine
max (GM), Trigonella foenum graecum (TFg), Cicer arietinum (CA) Vigna ungiculata
(VU) , Pisum sativum (PS), Lathyrus sativa (LO), Phaseolus vulgaris (PV)
The nodules of selected cultivated crops were collected from IGKVV campus,
Raipur, during both kharif & rabi crop season.
b) Wild Plants:
Desmodium triflorum (DT) Mimosa pudica (MP), Tephrosia purpurea (TP).
The nodules from selected wild species were collected from Govt.NPG college of Science
during rainy season.
Collection of Nodules: Nodules from different leguminous plants, which were pink in
colour & situated on the tap root were collected in sterile polythene bags. The selected nodules
were brought to the laboratory for the isolation. The nodules were separated from the root
carefully so that the piece of root on the side of the nodule remained attached. The nodules were
then thoroughly washed with running tap water.
Morphological Categorization of Nodules – The collected nodules were studied
morphologically on the basis of their size, shape & colour. The morphological
diversity amongst the collected nodules were recorded.
Selection of Basal media –
Different culture media were tested for the growth of Rhizobium bacteria. The media selected
were viz; 1) Yeast extract mannitol agar 2) Peptone glucose agar 3) Nutrient agar
1) Yeast extract mannitol agar : (YEMA)
K2HPO4
– 0.5g
MgSO4 H2O
– 0.2g
NaCl
– 0.1g
Mannitol
– 10g
CaCO3
- 0.5 g
Yeast extract powder
– 0.5 g
Agar-Agar
- 15 g
Distilled water
–
pH
1000ml.
6.8
– 2.5 ml
Congo red 1(%)
Aqueous solution of congo red 1% w/v.
2) Peptone glucose agar. (PGA)
Glucose
– 5.00g
Peptone
– 10.00 g
Agar
– 15.00 g
– 1000 ml
Distilled water
Bromo cresol purple 1% in ethanol – 10ml.
3) Nutrient agar medium.(NAM)
Beef extract
-
3.0g
NaCl
-
5.0g
Peptone
-
5.0g
Agar-agar
-
15.0g
Distilled Water PH
-
1000 ml
7.0
The growth of all the isolates of Rhizobia was optimum in YEMA hence it was selected for the
further studies.
ISOLATION –
Isolation of Rhizobium spp from 10 cultivated & 03 wild legume plants were done by a
general procedure described by Vincent (1970). The nodules were immersed (10-12) in 95%
ethanol for 1 minute & then 0.1% acidified mercuric chloride for surface sterilization (HgCl2
1.0g conc. HCl 5ml, water 1 litre ) for 3 to 4 minutes. Later the nodules were washed five times
in double distilled water and in 70 % ethyl alcohol for 1 minute. The nodules were then washed
in double distilled water 4 to 5 times and transferred to sterile petriplates (Solger, 1969). A few
drops of water was left in the test tube containing sterilized washed nodule.The nodules were
crushed with a sterilized glass rod with flat end to form a turbid suspension under aseptic
conditions. A loopful of the suspension was streaked on yeast extract mannitol agar medium
(Fred et al,.1932). The inoculated plates were incubated at 28 ±1°C for 24 to 48 hours till small
round colourless colonies of Rhizobium developed. The Rhizobial colonies were picked off &
streaked on clean YEMA plates to obtain pure cultures. The purified cultures were maintained on
YEMA slants. (Subha Rao, 1988).
IDENTIFICATION –
The following tests were made to confirm identification of the isolated bacteria as Rhizobium
(Vincent 1970). Rhizobium identification was based on the Bergey’s manual of determinative
bacteriology.
Gram staining - Rhizobia are gram negative rod shaped bacteria. Tests were made to
identify Rhizobia on the basis of their staining characteristics.
Staining Procedure – The smear was fixed in a drop of normal saline & stained with
crystal violet solution for 1 min. It was rinsed with water & the excess water was drained off.
Iodine solution was added & allowed to act (iodine) for 1 min. The iodine solution was
drained off & decolorized with iodinated alcohol (5 min). After washing with water, safranin
was added & then rinsed with water.The slide was then observed under the microscope using
oil immersion objective.
Growth Characteristics – To study the growth characteristics of all the isolates of
Rhizobium following tests were performed.
Growth on YEMA with congored – YEMA with congored (10ml/1) was prepared &
inoculated with Rhizobium culture in each case, inoculated slant, were incubated at 28 +1°c.
Generally the rhizobium strains absorb the dye very weakly which is a distinguishing character
from other bacteria which absorb the dye strongly.
Bromo Thymol Blue (BTB) Test - The acid production was tested by means of plate culture
methods.1ml of 1% Bromothymol blue (BTB) was prepared & added to 10 ml of YEMA
medium in sterile petriplates,& allowed to solidify. One loopful different Rhizobia were
inoculated on the plates & incubated at 28 +1°C for 48 hours . This test help to segregate alkali
& acid producing strains. Alkaline range gives green colour & in acidic range it turns yellow.
Growth on peptone glucose Agar - Rhizobia were cultured in peptone glucose agar & plate
were incubated at for 28 +1°C 48 hours. Many of the Rhizobial strains do not grow on peptone
glucose agar medium & this cultural test is usually considered suitable to distinguish them.
Effect of temperature - The effect of temperature was performed in terms of growth.
Rhizobia were grown in YEMA medium on petriplates & incubated at different temperatures viz;
30,35, 40, 45, 50 & 55ºC for 3 days.
Effect of salt (NaCl) concentration - Effect of NaCl was studied on the growth of Rhizobia.
The concentration of NaCl was increased in YEMA medium viz; 0.1 %1.0% 1.5% 2.0% 2.5%
Rhizobia were grown in petriplates having different concentration of salt for 3 days at 28 +1°C
Growth pattern - Biological characterization of the isolated Rhizobium spp were made to
determine their growth patterns. This method can also be relatively concerned to the total amount
of cell substance & number of cells for a particular bacterium. Growth estimation of each isolate
of Rhizobium was measured in terms of turbidity using nephelo turbidity meter & has been
represented as NTU (Nephelo Turbidity Units). Rhizobia were cultured in 150 ml flask each
containing 30 ml Nutrient broth medium One loopful of Rhizobial cultures were inoculated &
incubated at 28 + 10c for 48 hrs. After sufficient growth 1 loopful these cultures were further
cultured in screw cap glass vials, each containing 10 ml nutrient broth medium & was incubated
at 28 + 1°C. The turbidity readings were taken at 0,1,2,3,4,5,6, hours of growth thereafter
readings were noted after 24, 48, 72, 96,120,144 &168 hrs of growth. Data of growth at each
period of determination was taken as NTU mean for 3 replicates. (Vincent, 1982).
Nodulation testNodulation efficiency of the isolated Rhizobia were estimated in vitro in terms of nodulation &
cross inoculation tests.
The identification of Rhizobium was done through nodulation tests using sterilized polythene
bags in triplicates. The Rhizobium isolated was tested against their respective hosts & nodulation
was observed confirming the isolates as Rhizobium. The crop was grown with the help of
sterilized sand using nitrogen free nutrient solution by sand culture technique.
Composition of McKnight’s seedling N - free nutrient solution.
Solution A
mg/100 ml D.D.W.
MnSO4. 4H2O (Manganese Sulphate)
-
2.86
ZnSO4. 7H2O (Zinc Sulphate)
-
1.54
CuSO4. 5H2O (Copper Sulphate)
-
0.08
H2 MoO4
-
0.09
(Molybdic acid)
Solution B
FeCl3.
EDTA
mg/100 ml D.D.W.
(Ferric chloride)
-
16.8
-
2.0
Solution C
CaSO4
mg/100 ml D.D.W.
(Calcium Sulphate)
MgSO4. 7H2O (Magnesium Sulphate)
KH2PO4
KCL
(Potassium Dihydrogen Phosphate)
(Potassium Chloride)
-
24.0
4.0
-
4.0
-
6.0
To the 960 ml of solution C, 20 ml each of solution A & B were added & the whole solution was
diluted 20 times. pH of the nutrient solution was adjusted at 7.0 for sterilization the nutrient
solution was autoclaved at 15 Ib/ inch pressure for 30 minutes (Katre et al., 1997).
Seed inoculation & Sowing Healthy seeds of each selected legume host were surface sterilized with 0.01% Hgcl2 &
inoculated with Rhizobial culture in sterilized polythene bags. After 24 hours of inoculation of
seed bags were irrigated by Mcknight’ seedling N free nutrient solution. Sowing of 5 seeds per
bag was done & after germination of plant 3 healthy plants were maintained per bag. Time to
time uniform irrigation to all bags by the N-free nutrient solution was given. The crop was
harvested after 45 days. Success or failure of the Rhizobial inoculums determined its effectivity
or ineffectivity. Formation of nodules on their respective hosts confirmed as specific Rhizobium
isolates, (Nambiar et.al., 1984 & Nambiar, 1985).
Artificial cross inoculation under sterilized soil & seed conditions.
Experimental investigations were made to determine infectivity (inf.) & effectivity (eff.) of the
isolates on artificial cross inoculation under sterilized soil & seed conditions.
Rhizobium Cultures used:
[R1-PA] Rhizobium strain isolated from host Phaseolus aureus
[R2-PV] Rhizobium strain isolated from host Phaseolus vulgaris
[R3-AH] Rhizobium strain isolated from host Arachis hypogaea.
[R4-DL] Rhizobium strain isolated from host Dolichas lablab.
[R5-GM] Rhizobium strain isolated from host Glycine max.
[R6-TFG] Rhizobium strain isolated from host Trigonella foenum gracum
[R7-CA] Rhizobium strain isolated from host Cicer arietinum.
[R8-VU] Rhizobium strain isolated from host Vigna ungiculata.
[R9-PS] Rhizobium strain isolated from host Pisum sativum.
[R10-LS] Rhizobium strain isolated from host Lathyrus sativus.
Host species – The genotypes of legume species used for infectivity test were (i)
R1-PA, (ii) R2-PV, (iii) R3-AH, (iv) R4-DL (v) R5-GM (vi) R6-TFG, (vii) R7-CA,
(viii) R8-VU, (ix) R9-PS (x) R10-LS
Nodulation tests were done in sterilized plastic bags containing sterilized sand
saturated with water.Seeds of the above crop plants were surface sterilized with acidic alcohol
(Concentrated H2SO4 : Ethanol, 7:3) for 5 minutes, later followed by several washings with
sterile distilled water. The sterilized seeds were then inoculated with 7 days old cultures grown
on yeast extract mannitol agar medium seeds were sown in autoclaved soil in 5 replicates for
each treatment. Uninoculated seeds were sown as control. The plants were watered whenever
needed. Observation for nodulation their size shape & number were taken after 45 days of plant
growth. The amount of nitrogen fixed by the legume host was estimated by Kjeldahl method.
B) Aritificial Cross inoculation for nodulation test
Experimental investigations were made to determine infectivity (inf) & effectivity
(eff) of the isolates on artificial cross inoculation under sterilized sand & seed condition.
Rhizobium cultures: The Rhizobium strains under for nodulation studies were
i)
[R – PA] Rhizobium strain isolated from host Phaseolus aureus.
ii)
(a) [R – DL] Rhizobium strain isolated from host Dolichos lablab.
(b) [R-AH] Rhizobium strain isolated from host Arachis hypogaea.
iii)
(a) [R – Tfg] Rhizobium strain isolated from host Trigonella foenum graecum.
(b) [R-LS] Rhizobium strain isolated from host Lathyrus sativous.
(c) [R-CA] Rhizobium strain isolated from host cicer arietinum.
Host species – The genotypes of legume species under for cross inoculation
Tests were
Set - I) (a)Vigna ungiuculata (VU)
(b)Glycine max (GM)
(c)Phaseolus vulgaris (PV)
(d)Phaseolus aureus (PA)
Set - II) (a) Dolichos lablab (DL)
(b) Arachis hypogaea (AH)
(c) Phaseolus aureus (PA)
(d) Glycine max (GM)
(e) Lathyrus sativus (LS)
Set - III) (a) Pisum sativum (PM)
(b) Cicer arietinum (CA)
(c) Trigonella foenum graceum (Tfg)
(d) Lathyrus sativus (LS)
Nodulation tests were done in sterilized polythene bags as per the procedure described
earlier.
BIOCHEMICAL AND PHYSIOLOGICAL CHARACTERISTICS DNA estimation – Estimation of the total DNA content was done by spectrophotometrically
method. The Rhizobial cultures were grown in yeast extract mannitol broth for 3 days. After
sufficient growth was attained the culture were centrifuged at 10,000 RPM for 15 minutes.
The pellets were then dissolved in 400µl TE buffer. Further 40µl of 10% SDS and 5µl
proteinease K (20mg/ml) were added. This mixture was incubated at 56o C for 45 minutes
and then it was centrifuged at 10,000 rpm for 10 minutes.The supernatant was taken and
200l of tris saturated phenol with 200l of chloroform isoamyl alcohol (24:1) were added.
The above process was repeated twice. The supernatant was taken & add 400l of
chloroform: isomyl alcohol (24:1). The supernatant was taken & add 0.1 volume of 3 M
sodium acetate & 2 volumes of chilled absolute ethanol & incubated at 20°C for 2 hours and
then centrifuged at 15,000 rpm for 30 mins. The supernatant was decanted off the pellet was
washed with 70% ethanol by centrifugation at 10,000 rpm for 15 minutes. The supernatant
was decanted & the pellets were dried at room temperature. Further the pellets were
suspended in 50l of TE. The cuvette was filled with double distilled water. The
spectrophotometer was adjusted to zero at 260nm. DNA was diluted with at TE buffer(1µl:
999 µl ) & read at OD 260 nm/OD 280 nm was an indicator of DNA purity.
Sugar Utilization – Sugar utilization test was done with Drywood’s Anthrone reagent (Morris,
1948).
The Rhizobia were cultured in yeast extract mannitol broth medium. After 48 hours of
growth 1 ml culture was taken in a centrifuge tube & centrifuged at 10,000 rpm. Hereafter 02ml
of supernatant was taken in a test tube & 08ml of the reagent (0.2% anthrone in conc. H2SO4)
was added to it on ice bath. The test tubes were then transferred to boiling water bath for 15
minutes. A golden green colour was developed and the same was read on systronics digital
spectrophotometer at 620 nm. The quantity of sugar consumed was calculated by the help of
standard calibration curve of glucose,
Protein test – Protein was estimated by the method of Lowry et.al., (1951).
Preparation of sample for protein measurement –
Rhizobia were cultured in YEMA (yeast extract mannitol broth) medium for 48 hrs. After
sufficient growth 1 ml culture was taken in a centrifuge tube & centrifuged at 10000 rpm. The
supernatant was discarded. This procedure was repeated twice. The pellet was dissolved in 10 ml
1 N NaCl (saline). This sample was used for protein estimation.
Reagent required-
i)
Preparation of alkaline CuSO4
Solution A: Alkaline sodium carbonate solution (2% Na2CO3 in 0.1 N NaOH).
Solution B: Copper sulphate – sodium potassium tartrate (0.5% CuSO4 in 1% Na, K tartrate).
50ml of solution A was mixed with 1 ml of solution B.
ii)
Folin – Ciocalteu’s Phenol reagent Procedure –
1ml of aliquot was taken in a test tube to which 5 ml of freshly prepared alkaline CuSO4
solution was added. After 5 minutes, 0.5ml of Folin ciocalteu’s phenol reagent was added & the
solution was immediately shaken. After 15 minutes optical density (OD) of the developed colour
was read on systronics digital spectrophotometer at 750nm against a blank of 1 ml 1 N NaCl.
Standard graph was prepared by using different concentrations of bovine serum albumin
& total protein content of the test sample was calculated with the help of standard graph.
Antibiosis & Antagonism Antibiotic Sensitivity test
It was tested according to the method adopted by Prasuna (1987) sensitivity test was
performed by using the paper disc diffusion assay.
Procedure – The antibiotic sensitivity was tested by means of plate culture method. Five
antibiotics were used viz: streptomycin, penicillin, erythromycin, terramycin & norfloxacin. The
biodiscs were prepared in three different concentration viz : 0.5%, 1.0%, & 1.5%. The bacterial
suspensions were prepared in nutrient broth medium. Each test tube containing 10ml medium &
one loopful Rhizobia was inoculated. One ml of bacterial suspension & 10 ml of nutrient agar
medium were inoculated in petriplate. Afterplating & solidification of the medium biodiscs were
placed upon the surface of solidified nutrient agar medium. The plates were incubated for 48 hrs.
at 28 + 0C. Data for antibiotic sensitivity was recorded by measuring the diameter of growth
inhibition zone around the biodiscs after 48 hours of incubation.
Antagonism Rhizobia were tested to observe their antagonistic properties against some fungal
pathogens viz – Aspergillus sp, Trichoderma sp, Alternaria sp, Cladosporium sp, Fusarium sp
(Buonassisi et al., 1986). These Rhizobial isolates were also tested for their inhibitory properties
against the mycelial growth of the above fungal species. (Omar & Abdalla, 1998).
Fungi were grown on PDA (Potato Dextrose Agar) media.
Potato
– 200 .0g
Dextrose
– 20.0 g
Agar
– 20.0 g
Distilled water – 1000 ml
pH
- 4.5
Isolated Rhizobia & fungi were inoculated in petriplates containing YEMA media. They
were incubated for 7 days at 28 + 10C . The zone formation was measured in c.m.
Chemical Analysis of soil:
Total estimation of NPK by slandered method
Available N: Available N was determined by alkaline permanganate (KMnO4) method of
Subbiah & Asija, (1956).
Twenty gram soil sample was taken in one litre boiling flask & 200 ml distilled water
was added. 100 ml of 0.32 percent KMnO4 & 100 ml of 2.5 percent NaOH were then added in
sequence. The flask was connected to the condenser immediately after adding NaOH & the 5ml
boric acid solution containing mixed indicator. Ammonium in distillate was determined by
titrating against H2SO4 (Bremner, 1965).
Available P: Soil phosphorus was extracted as described by Olsen et.al.,(1954) & phosphorus
in the extract was determined by ascorbic acid method of Waterable & Olsen (1965).
Available K – Potassium was estimated by flame photometer (Hanway & Heidle, 1952).
Procedure – Five g of soil was shaken with 25ml neutral 1 N ammonium acetate (Ph-7) for 5
minutes & filtered immediately through a dry filter paper (Whatman No. 1) determine
potassium concentration was determined by flame photometer.
Chemical analysis of plant -
Nitrogen – One gram of plant sample was weighed in 100 ml. Kjeldahl flask 20 ml of cons
H2SO4 & 7 g of salt mixture (K2SO4 & CuSO4 in ratio of 7:1) were then added & the materials
were then transferred to 100 ml volumetric flask & make up to volume. A 10 ml aliquot was
pipetted into a distillation apparatus & distilled with steam in presence of 10 ml of 10 N NaOH.
The distilled NH3 was absorbed in 5ml of 2% boric acid containing mixed indicator (Bramner,
1965).
Enzymological Studies –
Assay for uptake Hydrogenase (Hup) – The pure colonies of Rhizobium isolates on YEM
medium were transferred on yeast extract – mannitol – glutamate – malate agar (YEMGMA)
medium slants were incubated for seven days at 280 ± 10 C.
After sufficient growth of bacterial culture was attained the Hup enzyme system was
determined by the enhanced effect of 10% H2 atmosphere on acetylene (C2H2) reduction in the
medium after 48 hrs. The amount of hydrogen & acetylene reduction was measured by Gas –
Chromatography.
Chromatographic equipment filled with a thermal detector & operated with a molecular
sieve 5-A & 2 M long column & N2 gas was used as the carrier gas to measure hydrogen
Ethylene was measured on AIMIC Chromotographic equipment filled with thermal detector with
parapak N, 2m long column. Expression of nitrogenase activity, in this hydrogenated
atmosphere, by acetylene reduction test gave positive uptake hydrogenic (Hup+) activity also
where failure of nitrogenase expression is considered as hydrogenase negative (Hup-).
Amylase – Enzyme activity was measured on solid medium (yeast extract mannitol agar)
medium containing 0.2% starch (without congored) after 7 days growth by using Lugol’s Iodine
solution (1gm Iodine + 3gm Potassium Iodide +125ml distilled water). A yellow zone around the
colony in an otherwise blue medium indicated amylolytic activity of the organism. The diameter
(mm) of the zone represented the amount of the enzyme production by the organism.
Protease – This test was performed on solid medium (yeast extract mannitol Agar medium)
containing 2% gelatin (without congored) following the method of Hankin & Anagnostakis
(1975). Observations were taken after 7 days of incubation for zone formed in the plate due to
acidic mercuric chloride (12gm HgCl2 +16 ml conc. HCl + 84 ml distilled water) acid reaction
with the culture medium.
Cellulase & polygalcteuronase
Enzyme Preparation
The test organisms were cultured in YEM broth medium & 3,5,7,9 days of incubation for
the estimation of enzymes. 1 ml culture was taken in a centrufuge tube & centrifuged at 10,000
RPM. The supernatan was served as crude enzyme source.
Cellulase (CX): Cellulase activity was assayed by the method of Gascoigne & Gascoigne
(1960).
Reaction Mixture –
Enzyme preparation
-
1.0 ml
0.55% Carboxy Methyl
Cellulose solution (pH5.5)
-
3.5 ml
DNS reagent
-
1.0 ml
Method The reaction mixture contained 1 ml of the enzyme preparation & 3.5 ml of the enzyme
substrate (0.55% carboxyl methyl cellulose solution in sodium citrate buffer of pH 5.5) was
taken in a test tube & incubated at 30 ± 10c for one hour. 1 ml of the reaction mixture was added
to a test tube containing 1 ml DNS reagent (Dinitrosalicylic acid – 1.0gm + crystalline phenol –
200mg + 1% sodium hydroxide – 100 ml + sodium sulphate – 50mg) & heated over a boiling
water bath for 5 minutes. It was then cooled under tap water to room temperature, 8ml of
distilled water was added to make up the volume up to 10ml. The absorption of the sample was
measured on systronics digital spectrophotometer at 540 nm. The amount of reducing sugar
liberated was obtained by a standard curve prepared by using an aqueous solution of D- glucose.
One unit of cellulose enzyme activity is defined as the amount of enzyme to liberate reducing
sugar equivalent to 10µg of glucose.
Polygalacturonase -
Enzyme preparation was obtained from the cultural broth form according to the usual
method & procedures described (Hussain, 1958; Hancock et.al., 1964; Betaman, 1963).
Enzyme assay was made viscometrically. The reaction mixtures contained :
1.2% sodium polypectate solution
-
3.5 ml
(pH 5.5 ml ) distilled water
-
1.5 ml
Enzyme preparation
-
1.5 ml
Mciivaines buffer (4.5 pH)
-
1.5 ml
The reaction mixtures were placed in the viscometers & the time of efflux was recorded
immediately as at 0 hours at 300C in each case. Subsequently the readings of the efflux time were
noted after incubation of 30,60,90 & 120 minutes. Boiled culture filtrates served as control.
Percentage loss in viscosity & relative enzyme activity (REA) were calculated as given below :
% loss in viscosity =
ET0 – Ett X 100
ET0 – ETw
Where
ET0 = efflux time at 0 hours
Ett
= effluxe time at time t
ETw = efflux time of control
REA = 1000/tat V 50
Where,
T at V50 is the time in minutes required to reduce viscosity by 50%.
RESULTS