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International Journal of Research in Pure and Applied Microbiology
Universal Research Publications. All rights reserved
ISSN 2277–3843
Original Article
Isolation and Identification of Root Nodule Bacteria of Mung Bean
(Vigna radiata L.) for Biofertilizer Production
Shraddha Bhatt*1, Dr.R.V.Vyas1, H. N. Shelat1 and Sneha J. Mistry
Address: 1 Anand Agricultural University, Department of Microbiology, Anand -388110, Gujarat, India, +91-09737674033
*Corresponding author: [email protected]
Received 20 October 2013; accepted 05 November 2013
Abstract
Nine different isolates of Rhizobium were isolated from Mung bean root nodules, cultivated in different Mung bean
agricultural fields in Gujarat. Background: Present investigation – nodules bearing a smooth surface of varying sizes were
well established on top as well as lateral portions of roots. Some of the nodules were found to be have rough surface and
the colour of the nodules ranged from pink to brown. Good growth of all the isolates was observed on congored yeast
extract mannitol agar (YEMA) medium but was unable to produce chromo genesis on congored medium. All the isolated
isolates of Rhizobium failed to show a growth on glucose peptone – agar medium as well as Hofer‟s alkaline broth. All test
isolates gave a negative test on ketolactose medium. The isolated isolates were found to be Gram negative in nature. In
case of casein hydrolysis and starch hydrolysis all the isolates gave negative results. All test isolates reduced nitrate to
nitrite and none test isolates produced H2S. Isolate AAU-6 produced a maximum gum of 122 mg while isolate AAU-9
produced a minimum of 90 mg. All isolates showed varied on growth characteristic in 2% NaCl while glucose
consumption and utilization of nitrogenous compound varied from poor to very good. Conclusion: On the basis of result
obtained in the present observation it can be concluded that isolate AAU-6 and AAU-7 were found to be the unsurpassed
isolate of Rhizobium and can be exploited as biofertilizers for better yield of Mung bean.
© 2013 Universal Research Publications. All rights reserved
Keywords: Mung bean, Rhizobium, root nodules, biochemical characterization
INTRODUCTION:
Chemical nitrogen fertilizers will continue to serve for
increasing grain production until a predictable future, but
efforts should also be oriented towards augmenting
biological nitrogen fixation which will mediated by
microorganisms. In developing countries like India the
construction of new nitrogen fertilizers plant is not only
expensive but time consuming. Therefore it is essential for
us to evolve and adopt a strategy of integrated nutrient
supply by using a judicious combination of chemical
fertilizer, organic manures and biofertilizers.
Biologically active product more appropriately called as
“microbial inoculants” contains active strength of selective
microorganisms like bacteria, algae, fungi; alone or in
combination helps in increasing crop productivity by
biological nitrogen fixation. The rhizobia are a group of
Gram-negative bacteria that form species-specific
symbioses with legume plant, traditionally thought to
include only members of the α-subdivision of
proteobacteria and more recently found to include members
of the β-proteobacteria. Nitrogen fixation, the reduction of
atmospheric dinitrogen (N2) to ammonia (NH3), by rhizobia
only occurs during symbiosis and provides a significant
127
proportion of available nitrogen in the biosphere (Figure 1).
Symbiotic nitrogen fixation is therefore of great ecological
and socio-economic importance. Sustainable agricultural
methods successfully exploit the rhizobia-legume
symbiosis as a natural fertiliser by cultivating legume crops
in rotation with non-symbiotic crops.
In many soils across India the nodule‟s producing bacteria
are absent or are present in non-adequate number or quality
to meet the nitrogen requirement of legumes. To meet the
requirement of nitrogen fixation it is necessary to inoculate
the seeds with highly effective rhizobia (bacteria) cultures.
Different legumes require different rhizobia in order to
produce an effective symbiosis. Inoculants are prepared in
laboratory by culturing nodule producing bacteria and are
generally mixed with a suitable carrier material such as
peat or lignite to make inoculants. The process of adding
these inoculants to seed is called inoculation.
Rhizobia are soil bacteria which can infect the roots of
legumes to form effective nitrogen fixing nodules. In many
soils rhizobia which are already present are either not in
sufficient number or quality, or are not compatible with the
farmer‟s legume crops to form effective symbiosis. In those
soils, it is necessary to inoculate legume with rhizobia to
International Journal of Research in Pure and Applied Microbiology 2013; 3(4): 127-133
increase the amount of nitrogen fixed by the crop and
increase the farmer‟s yield. By proper inoculation with
rhizobia, the farmer can introduce large number of effective
rhizobia with superior quality in producing nodules.
Legume inoculants are either liquid or solid substances
which contain live rhizobia and are inoculated by merely
bringing in contact with the seed or legume root. The
success of a technology depends on its techno economic
feasibility. Production of inoculants first commence with
the selection of individual rhizobial strains, those are
effective at biological nitrogen fixation with particular
legumes. These strains are grown to high population in
liquid broth culture (Yeast Extract Mannitol Agar medium)
in flasks and are subsequently transferred to larger
fermentation vessel at laboratory level. Like all pure
bacterial culture rhizobia must be grown under sterile
condition and do require minimum aeration provided by
shaking or sterilized air by bubbling and grow superlative
at 30 ± 2 0C. After an incubation period of 4-6 days the
broth culture is ready to be mixed into a suitable carrier i.e.
charcoal/lignite powder.
The farmer coats his seed with inoculants (Biofertilizer)
before a planting so that a high number of superior rhizobia
are present when the legume root emerges. These
inoculants, rhizobia can then quickly infect the root and
start the process of nodulation. The best type of seed
inoculants is very fine powder lignite or peat, which will
adhere to the seed. A sticker is recommended to find the
rhizobia to the seed. A sticker increase the amount of
inoculants that will adhere to seed Gum Arabic is a
common sticker.
MATERIAL AND METHOD:
The isolates were checked for contamination and
particularly to distinguish the Rhizobium from
Agrobacterium using the following biochemical tests:
(1) Congo red Yeast Extract Mannitol Agar medium
Incorporated medium (2.5 ml of 1% aqueous solution of the
dye per one litre of YEMA) colonies of rhizobia in congo
red are of white, translucent, glistening, elevated in nature
and comparatively smaller with entire margin in contrast to
the stained Agrobacterium colonies (Hahn 1966). The
composition of the medium was (Fred et al. 1931) as
Mannitol (10 g), K2HPO4 (0.5 g), MgSO4.7H2O (0.2 g),
NaCl (0.1 g), Yeast Extract (1 g), CaCO3 (1 g), Agar –
Agar (20 g), and distilled water (1000 ml) and a final pH 7.0. The medium was sterilized at 15 PSI at 121 0C for 1520 min in an autoclave. Three plates were streaked for
isolation from each nodule. Upon incubation up to 10 days,
the colonies of bacteria emerging were picked up and
transferred to YEMA slants.
(2) Hofer’s alkaline medium
Agrobacteria can grow at higher pH levels than rhizobia
and therefore their growth in YEM broth with elevated pH
of 11.0 is considered as a useful means to distinguish
between the two allied genera (Hofer 1935).
(3) Glucose Peptone Agar medium
Rhizobia cannot utilize peptone whereas Agrobacterium
can utilize and grow very fast on this medium. The
composition of the medium used during the
experimentation contained Glucose-10g, Peptone-20g,
128
NaCl-5 g, Agar–Agar- 20 g, distilled water-1000 ml and
pH is maintained at pH-7.2. In this medium 1 ml of 1.6 %
Bromocresol purple per liter was added and autoclaved for
three consecutive days. Rhizobia showed poor or no growth
after 24 hrs with neutral or alkaline reaction Presence of
Agrobacterium was acknowledged on the basis of
maximum growth and acid reaction. Colour change to
yellow due to production of acid by Agrobacteria and other
contaminants of the medium (Kleczkowska and Nutman
1968).
(4) Ketolactose test
Ketolactose medium was prepared by replacing mannitol
with lactose in YEMA medium. Sterilization was achieved
by steaming for a period of 30 min for two successive days.
After sterilization the medium was allowed to cool at room
temperature (30±2 0C) and stored at the same temperature
for 3 to 4 days to check the contamination and confirm its
sterility. A loopful of the inoculum from a fully grown
culture slant (7 days old culture) was transferred to a petri
plate containing the ketolactose agar medium. After
incubation for 5 to 7 days at 27 – 30 0C the plates were
flooded with a shallow layer of Benedict‟s solution. The
composition of Benedict‟s solution consisted of two
solutions: Solution A (Sodium citrate-173 g; Anhydrous
sodium carbonate-100 g; Distilled water-600 ml) and
Solution B (Copper sulphate-17.3 g; Distilled water-100
ml). The solutions A and B were prepared separately and
later solution B was mixed with A and then filtered. The
resultant solution was clear and having transparent blue
colour. After pouring the Benedict‟s solution into above
mentioned plates the excess of solution was drained off and
the plates were incubated for one hour at 30±1 0C without
any disturbance. The yellow colouration around bacterial
colonies confirms the presence of Agrobacterium
(Bernaerts and Delay 1963).
(5) Growth on Yeast Extract Mannitol Agar (YEMA)
slant
The Yeast Extract Mannitol Agar medium was prepared
and 3 ml of this medium was dispensed in culture tubes (15
x 150 mm) autoclaved at 15 PSI at 121 0C for 15 – 20
minutes. Slanted at 450C and kept for 24 hrs to check for
contamination. For inoculation 7 days old culture of
different strains of Rhizobium were used. After seven days
of incubation, the growth characteristics of each strain were
recorded and categorized into three groups fast, medium
and poor growing strains.
(6) Gram staining
For gram staining the following reagents were prepared;
Crystal violet solution (Crystal violet-10 g, Ethyl alcohol100 ml, Ammonium oxalate-4 g, Distilled water-400 ml);
Iodine solution (Iodine-1 g, Potassium iodide-2 g, Ethyl
alcohol-25 ml, Distilled water-100 ml); Iodinated alcohol
(Iodine solution (b)- 5ml, Ethyl alcohol-95 ml)
Counterstain (2.5% safranin in ethyl alcohol-10 ml,
Distilled water-100 ml). Gram stained smear were prepared
with a loop full of a selected bacterium and spread over on
a slide in a drop of water and allowed to dry in air (Graham
and Parker 1964). The slide is dried in the vicinity of the
flame and allowed to cool and then stained with crystal
violet solution as follows: for 1 min followed by rinsing
International Journal of Research in Pure and Applied Microbiology 2013; 3(4): 127-133
with water and removal of excess water, the slide is then
flooded with iodine solution followed by decolourized with
iodinated alcohol for one minute, for 5 min the slide is
washed in water, drained and counterstained with safranin.
Finally the slide is washed in water, drained and air dried
and observed under oil immersion (Vincent 1970).
(7) Growth in 2% sodium chloride concentration
To check the adaptability of Rhizobium at 2% NaCl
concentration slants of Yeast Extract Mannitol Agar
(YEMA) medium containing 2% salt were inoculated with
each isolate and observation of growth was taken after 5
day of incubation (Graham and Parker 1964).
(8) Gum Production
Gum production by Rhizobium was studied by the method
described by Anderson (1938). Flasks containing 100 ml of
the Yeast Extract Mannitol (YEM) broth were sterilized at
15 PSI at 121 0C for 15-20 min and inoculated in triplicate
with 1 ml of 5 days old culture broth of each isolate and
incubated for 15 days on a rotatary shaker. After
incubation, with the help of boiling the volume of culture
fluid was reduced to 30 ml. A mixture of ethanol and
acetone (3 volumes) was added to the flasks for
precipitation of the gum and were left undisturbed
overnight to ensure complete precipitation. The contents
from the flasks were filtered and the retentate (i.e. gum) on
the filter paper was dried in oven for 24 hrs at 78 0C after
complete drying the amount of gum produced by the
individual isolate was recorded.
(9)Reduction of 2, 3, 5 triphenyl tetrazolium chloride
(TTC)
The method of Herrigan et al. (1966) was used for this test.
TTC is redox indicator and it indicates the capability of the
isolates to produce dehydrogenase enzyme, by adding this
into culture. Tubes containing 5 ml of YEM broth were
inoculated with different isolates and after 7 days of
incubation, 1 ml of 2, 3, 5 TTC (1% solution) was added
and further incubated at 28 0C for 30 min. Appearance of
pink colour in the incubated tubes, indicated the reduction
of TTC.
(10) Sugar Fermentation Test
Andrade's Fermentation Broths produce acid as a metabolic
waste when inoculated with bacteria that is capable of
metabolizing the constituent substrate. Acid production
causes a decrease in pH which results in a colour shift from
pale pink to red in the medium. Change in color is the
indicator for fermentation of carbohydrates by the isolates.
(11) Starch Hydrolysis
Nutrient agar medium containing 0.2% starch powder was
used to determine the starch hydrolysis by the isolates.
Positive test indicates that isolates have the competence to
solubilise starch by producing amylase enzyme. After
incubation period, plates were flooded with Gram‟s iodine
and presence or absence of halos around the bacterial
colonies was recorded.
(12) Liquefaction of gelatin
Nutrient medium without agar was supplemented with 12%
of gelatin and used to determine the ability of the isolates to
liquefy the gelatine. The plates were prepared in triplicate
and incubated at 28±2 0C for 3-4 days and then flooded
with 0.2 % mercuric chloride in 20% HCl. The presence of
129
clear halos around the colonies due to liquefaction of
gelation indicated that the isolates are capable of liquefying
the gelatine.
(13) Action on Litmus milk
Litmus milk is a milk-based medium which is used to
distinguish between different species of bacteria. The
lactose (milk sugar), litmus (pH indicator), and casein (milk
protein) contained within the medium can all be
metabolized by different types of bacteria. Since milk is
usually the first substrate used to maintain bacteria, this test
allows for accurate depiction of bacterial types. The
addition of litmus, other than explaining the pH type, acts
as an oxidation-reduction indicator. The test itself tells
whether the bacterium can ferment lactose, reduce litmus,
form clots, form gas, or start peptonization. Bromocresol
purple solution was added to the skimmed milk to get a
grey – blue colour. The milk was distributed in test tube
and sterilized for 30 min for consecutive days. The tubes
were then inoculated in duplicate with respective culture
and incubated at 28±2 0C. The appearance and change in
colour of the milk was particularly examined after 24 hrs of
inoculation.
(14) Casein hydrolysis
The test is conducted to determine if an organism can
produce the exoenzyme casesase. Casease is an exoenzyme
that is produced by some bacteria in order to degrade
casein. Casein is a large protein that is responsible for the
white color of milk. This test is conducted on milk agar
which is a complex media containing casien, peptone and
beef extract. If an organism can produce casein, then there
will be a zone of clearing around the bacterial growth.
Skimmed milk agar (containing milk 1000 ml, agar-agar
15-20 g) was prepared, sterilized and poured on to sterile
petriplates. The plates were inoculated in the center using
short strokes of the culture loop with and incubated at
37 0C for 48 hrs. After 48 hrs of incubation, the plates were
observed against black background and presence or
absence of clear halos around the centre of the plate was
recorded.
(15) Methyl Red and Voges Proskauer test
This test is used to decide the capacity of the bacteria to
meatbolize pyruvic acid. A negative result of MR and VP
test indicates that isolates are using butylenes glycol
pathway to metabolize pyruvic acid to neutral end products.
Negative results of VP suggest that isolates are not capable
to form acetoin by using glucose. Glucose phosphate broth
containing glucose-5 g, peptone-5 g, K2HPO4-5 g, and
distilled water-1000 ml was prepared and dispersed (5 ml
in each test tube) followed by steam sterilized for
conducting Methyl Red and Voges Proskauer test. The
tubes were inoculated and incubated for 72 hrs. For methyl
red (MR) test 5 drops of the indicator (0.1 g methyl red in
300 ml of ethanol + 200 ml distilled water) was added to
each tube containing 2 ml of liquid culture. Development of
colour indicated positive reaction. For the voges-proskauer
test (0.6 ml of 5% Alfa nepthol in absolute alcohol and 0.2
ml of 40 % potassium hydroxide were added to one ml
culture.
(16) Production of ammonia from peptone
Production of ammonia from urea and by deamination of
International Journal of Research in Pure and Applied Microbiology 2013; 3(4): 127-133
peptone by bacteria can be examined by this test. Peptone
broth containing peptone-1 g, NaCl-0.5 g, potassium
nitrate-0.5 g and distilled water – 1000 ml was prepared
and 5 ml volume of the broth was dispensed in each test
tubes followed by sterilization. After inoculation test tubes
were incubated at 37 0C for 48 hrs. After incubation 1 ml of
Nessler‟s reagent was added to culture tube. The
development of orange to brown colour indicates the
presence of ammonia.
(17) Reduction of nitrate to nitrite
The nitrate broth containing peptone-5 g, yeast extract-3 g,
potassium nitrate-1 g and distilled water-1000 ml was
prepared, distributed (5 ml in each test tube) and sterilized.
After inoculation culture tubes were kept for seven days
incubation period and then the tubes were tested for the
nitrate reduction with the sulphanilic acid napthylamine
reagent.
(18) Production of hydrogen sulphide
Peptone containing 0.01% cysteine was prepared and
dispersed in 5 ml lot in each test tube. Dry filter paper
strips saturated with lead acetate solution was put in each
test tube. The test tube was then sterilized, incubated at
28±2 0C for 48 to 72 hrs. Blackening of lead acetate paper
indicated production of hydrogen sulphite.
(19)Triple sugar iron agar test
The test was performed to determine the capability of
isolates to use various carbohydrate sources e.g. sucrose,
glucose, lactose, etc as media for growth. Triple sugar iron
test is designed to differentiate among the different group
which are capable of fermenting glucose with the
production of acid and hydrogen sulphide production.
Triple Sugar Iron Agar media consisted of beef extract-3
g/l, yeast extract-3 g/l, peptone-15 g/l, NaCl-5 g/l, lactose
10 g/l, sucrose 10 g/l, dextrose-1 g/l, ferrous sulphate-0.2
g/l, sodium thiosulfate-0.3 g/l, phenol red-0.24 g/l, agar-15
g/l and the final pH is adjusted to pH 7.0 (Kligler, 1918;
Hajna, 1945). After inoculation and incubation, color on
the butt and the slant was observed.
(20) Simmon’s Citrate Agar Slant
Organisms growing on Simmons Citrate Agar are capable
of using citrate as the sole carbon source and they can
metabolize the ammonium salt in the medium. Use of
citrate increases the pH of the medium which causes colour
change in the bromothymol blue indicator, turning it blue.
This colour change is useful because growth on Simmons
Citrate Agar is often limited and would be hard to observe
if it were not for the colour change.
(21) Hugh and Leifson medium
A carbohydrate is added to the culture medium,
degradation of the carbohydrate to acid is indicated by the
pH indicator bromothymol blue which changes its colour to
yellow. The degradation is allowed to take place while the
medium is exposed to air (degradation may be oxidative or
fermentative) or under exclusion of air (degradation by
fermentation only). Composition (g/liter) of Hugh and
Leifson medium: Peptone from casein-2.0, yeast extract-1,
sodium chloride-5, di-potassium hydrogen phosphate-0.2,
bromothymol blue-0.08, agar-agar- 2.5 and carbohydrate10g/l.
(22) Urease production
Urease broth is a differential medium that tests the ability
of an organism to produce an exoenzyme, called urease,
which ydrolyzes urea to ammonia and carbon dioxide. The
broth contains two pH buffers, urea, a very small amount of
nutrients for the bacteria, and the pH indicator phenol red.
Phenol red turns yellow in an acidic environment and
fuchsia in an alkaline environment. If the urea in the broth
is degraded and ammonia is produced, an alkaline
environment is created, and the media turns pink.
RESULTS AND DISCUSSION
Nine different isolates of Rhizobium were isolated from
Mung bean root nodules, cultivated in different Mung bean
agricultural fields of Anand Agricultural University,
Gujarat. Identification and cultural characteristics of
Rhizobium species isolates was carried out. The nine
species were studied for their morphological, cultural,
physiological and biochemical characteristics.
Morphology of root nodule bacteria
The morphology features of root nodules bacteria are
presented in Table –1. The results indicate that all the
Table 1. Morphological characteristics of root nodules bacteria.
Growth on glucose –
Growth on “Hofer’s”
Isolates
Alkaline medium
peptone agar medium
3-Keto
Lactose test
Gram staining
AAU 1
-ve
-ve
-ve
Gram negative
AAU 2
-ve
-ve
-ve
Gram negative
AAU 3
-ve
-ve
-ve
Gram negative
AAU 4
-ve
-ve
-ve
Gram negative
AAU 5
-ve
-ve
-ve
Gram negative
AAU 6
-ve
-ve
-ve
Gram negative
AAU 7
-ve
-ve
-ve
Gram negative
AAU 8
-ve
-ve
-ve
Gram negative
AAU 9
-ve
-ve
-ve
Gram negative
130
International Journal of Research in Pure and Applied Microbiology 2013; 3(4): 127-133
Table 2. Growth on Congo red YEMA at incubation temperature of 28±2 ºC
Size of colonies (mm.) on Congo red YEMA at incubation temperature of 28±2 ºC
Isolates
After 24 hrs
After 40 hrs
After 72 hrs
After 96 hrs
After 112 hrs
After 136 hrs
AAU 1
0
0
1.1±0.2
3.1±0.5
5.0±0.4
7.2±0.3
AAU 2
0
1.0±0.3
1.0±0.5
3.2±0.4
5.2±0.3
7.1±0.4
AAU 3
0
0
1.2±0.5
3.0±0.4
4.8±0.2
5.8±0.2
AAU 4
0
0
1.0±0.3
3.2 ±0.5
4.7±0.1
5.5±0.1
AAU 5
0
1.2±0.4
2.2±0.4
3.2 ±0.2
5.1±0.3
6.2±0.2
AAU 6
0
1.1±0.5
2.0±0.2
5.0 ±0.2
7.3±0.2
11.1±0.1
AAU 7
0
0
1.0±0.1
2.0±0.1
4.8±0.2
6.9±0.2
AAU 8
0
0
1.0±0.4
2.4±0.5
4.6±0.1
5.8±0.1
AAU 9
0
1.0±0.2
2.0±0.3
4.1±0.5
5.9±0.3
7.9±0.3
Table 3. Biochemical characteristics of different isolates
Sr.
No.
1.
2.
3.
4.
5.
1% Peptone Broth
2% Peptone Broth
Liquefaction of gelatin
Urease test
Triple sugar iron agar test
AAU1
AC/Al
6.
Action on Litmus milk
curdling
7.
8.
5.
6.
Methyl Red
Vogues Proskauer‟s
Citrate utilization
+
+
AAU 2
AC/Al
curdlin
g
+
+
Glucose
Xylose
Mannitol
Starch Agar Plate
Indole
Phenylalanine Deamination
Nitrate reduction
H2S production
Organic Acid Production
Casein Hydrolysis
Gum Production on YEMA
broth (mg/100ml)
Growth in 2% sodium
chloride concentration
Reduction of 2,3,5 triphenyl
tetrazolium chloride (TTC)
Melanin Production
Amylase Production
±
±
±
+
+
++++
-
±
±
±
+
+
++++
-
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
Test
120
Good
Moderate
++++
++++
AAU 3
AC/Al
AAU 4
AC/Al
Isolates
AAU 5
AC/Al
AAU 6
AC/Al
AAU 7
AC/Al
AAU 8
AC/Al
AAU 9
AC/Al
curdling
curdling
curdling
curdling
curdling
curdling
curdling
+
+
+
+
+
+
+
+
-
+
+
+
±
±
±
+
+
-
±
±
±
+
++
-
±
±
±
+
+
+
-
±
±
±
+
+
++
-
±
±
±
+
+
++
-
100
90
90
98
96
Good
Poor
growth
Poor
growth
+
+
Sugar Utilization
±
±
±
±
±
±
+
+
+
+++
++
-
122
106
103
Very
Good
Moder
ate
++++
++++
Very
Good
Poor
growth
Good
Moderate
Poor
Poor
Moderate
Moderate
Moderate
++++
+++
+++
-
++
-
++
-
+++
++
Good
Modera
te
++
+
Table 4. Physiological characteristics of different isolates (incubation temp. 28±2ºC)
pH
Temperature
Isolates
28oC
32 oC
37 oC
40 oC
4
5
7
9
AAU 1
AAU 2
AAU 3
AAU 4
AAU 5
AAU 6
AAU 7
AAU 8
++++
++++
++++
++++
++++
++++
++++
++++
++
++
++
++
++
++
++
++
+
+
+
+
+
+
+
+
-
-
+
+
-
+
+
+
+
+
+
+
+
-
AAU 9
++++
++
+
-
-
-
+
-
131
International Journal of Research in Pure and Applied Microbiology 2013; 3(4): 127-133
isolates showed a negative growth on glucose-peptone agar
and on Hofer‟s Alkaline medium. Similarly all the nine
isolates did not produce yellow coloration around their
colonies indicating that Agrobacterium was absent and all
of them were Rhizobium. All the nine isolates were
reported to be Gram negative in nature.
Cultural characteristics
The cells of different isolates were small to medium sized
rods and were Gram negative. All the isolates grew well on
yeast extract mannitol agar slants and congored yeast
extract mannitol agar plates but didn‟t show chromo
genesis. All isolates failed to grow on glucose peptone agar
medium and Hofer‟s alkaline broth (pH 11.0). All isolates
showed the negative test of rhizobia for the production of
3-ketolactose. None of the nine isolates grew on congored
yeast extract mannitol agar medium on 24 hrs (Table 2). By
48 hrs, 3 isolates developed into puntiform (less than 1 to
2mm dia) colonies size indicating their ability to grow fast.
After 72 hrs all isolates showed moderate rate of growth.
By 4th day the colonies of most of the fast growing strain
showing 3-4 mm. dia and slow growing isolates AAU-7
and AAU-8 showed 2 and 2.4 mm dia respectively by this
time and were the slowest growing among the nine isolates.
The colonies of most of the fast growing strain took 7 days
to attain 8 to11 mm. dia whereas the colonies of slow or
moderate growing strains showing 5-6 mm dia in the same
incubation period. Colonies of both fast and slow growing
isolates were rounded with entire margin and smooth
surface and none of them showed chromogenesis.
Biochemical and physiological characteristics
In the present study, the 9 isolated from Vigna radiata to
check their growth in 2% (w/v) NaCl. Out of nine isolates 2
isolates grew best, 4 isolates grew good and 3 isolates grew
poorly in 2% NaCl. (Table 3). As suggested by previous
research Kucuk and Kivanc (2008), we found that fast
growing isolates were generally more tolerant to high NaCl
concentrations than slow growing isolates. Furthermore, the
majority of our isolates had similar demands for
carbohydrates i.e. Glucose, xylose and mannitol as several
fast growing rhizobia (Kucuk and Kivanc 2008). Among
the isolates we studied all were able to at grow 37 and 40
ºC, whereas 2 isolates (AAU 7 and AAU 9) showed only
minimal growth at 40 and 45 ºC (Table 4). All isolates were
grown in YEM medium with pH values of 4, 5, 7 and 9, but
differences were detected at pH 5 (Table 4). Of all the
isolates, 3(AAU2, 6, 9) isolates even grew at a acidic
condition as low as pH 5.
None of the test isolates gave positive results for casein,
hydrolysis, gelatin liquefaction but gave positive tests of
methyl red and voges-proskauer test. Milk was curdled by
all test isolate and the colour of Bromocresol purple (BCP)
was not changed. All test isolates reduced nitrate to nitrite
but didn‟t produced H2S from peptone.
The biochemical characteristics showed similarity of nine
test isolates. Isolates AAU1 produced maximum gum and
AAU6 lowest gum produced by isolates. The isolates were
identical morphologically but exhibited variations in
cultural and physiological characters (Wright 1925) studies
made at the isolates differed in their morphological,
physiological and biochemical characters (Graham and
132
Parker 1964; Nakul 1990; Tiwari 2003).
CONCLUSION:
Nine different isolates were isolated from Mung bean root
nodules, cultivated in different Mung bean agricultural
fields in Gujarat. All the test isolates were strictly Gram –
ve in nature. During the identification a notable feature was
recorded with them as they produced more gum and were
able to utilize nitrogenous compounds. All the isolates were
able to grow on 2% salt concentration. Similar findings
were also reported by Keneni et al.(2010); Nakul (1990);
Sindhu and Dadarwal (2000) and a final conclusion is that
all the isolates were Rhizobium. In future the same isolates
may be checked for ARDRA (Amplified rDNA (Ribosomal
DNA) Restriction Analysis), nif gene, nod gene etc. to
access the capacity of the isolates to produce nodules.
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Source of support: Nil; Conflict of interest: None declared
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