Asian Journal of Agricultural Sciences 1(2): 62-65, 2009
ISSN: 2041-3890
© M axwell Scientific Organization, 2009
Submitted Date: August 09, 2009
Accepted Date: August 28, 2009
Published Date: October 05, 2009
Efficient Nitrogen Fixing Rhizobial Isolate Infecting Vigna radiata l.
1
Neeraj, 1 S.S. Gaurav, 2 S.C. Chatterjee ,4 Sachin and 3 Mahesh Chandra
Departm ent of Biotechnolo gy, C.C.S. University, M eerut, (U.P), India
2
Division of Plant Pathology IAR I, New D elhi, India
3
Division-FLOF , Central Institute of Medicinal an d Aromatic Plants, L ucknow , India
4
Sard ar Vallabh Bhai Patel (SV BP), University of Agriculture & Techno logy ,
Meerut- 250 110 (U.P) India
1
Abstract: Among 972 rhizobia, isolated from nodules o f different verities of Vigna radiata, 67 isolates we re
found to be potential ones on the basis o f plant growth promotion/ increased plan t biomass. These isolates w ere
further screened, where, only six potential isolates, showed higher plant biomass, maximum nodule numbe rs
and higher nitrogenase activity, were selected for further studies. Maximum range of fresh weights of nod ule
per plant w as sho wn again by isolate R0132 and noticed to be 26 1.37 mg n odule/plant. Nitrogenase activity
of plant nodule inoculated with d ifferent rhizobial isolates varied from 2.56 to 3.21 :g/mg/nodule. W hen
grow th abilities of these isolates studied, it had been observed that with different nitrogen sources, isolate
R0132 was found to be the best candidate as it utilized a wide range of nitrogen and carbon sources and was
able to grow at slightly higher temperature i.e. 32ºC and slightly acidic (pH 6.0) and alkaline (pH 8.0)
conditions. F urth ermore, th is iso late demo nstra te d the a bility to grow in pre se nc e 50mM concentration of N aCl.
Key w ords: Vigna rad iata, nitrogen fixation, nitrogenase activity and rhizobial isolates
INTRODUCTION
Deficiency in mineral nitrogen often limits plant
growth, and so sym biotic relationships have evolved
between plants and a variety of nitrogen-fixing organisms
(Estrada-De los, 2001). For more than 100 years,
biological nitrogen fixation (BNF) has commanded the
attention of scientists concerned with plant mineral
nutrition, and it has been exploited ex tensively in
agricultural practice (Burris, 1994). It is an essential step
in the global nitrogen cycle and this reaction is necessary
to sustain life on ea rth (See feldt, et al., 2004). Ho wev er,
international emphasis on environm entally sustainable
development with the use of renew able resourc es is likely
to focus attention on the poten tial role of B NF in
supp lying N for agriculture (P eoples, et al., 1995 ).
The establishment and maintenance of an effective
symbiosis depe nds o n several factors of w hich a favora ble
environm ent, that will allow maximum N 2 fixation, is
extrem ely important (Sin gleton , et al., 1982). Several
environmental factors such as soil pH, soil fertility,
temperature extremes impose limitations on the symbiotic
association between the host plant and microsymbiont
(Van Wyk, 2003). Furthermore, the amount of nitrogen
fixed by symbionts is variable; depending on the host
legume, cultivar, presence of saturated or near-saturated
soil water for movement, soil texture and composition,
bacterial species and g rowing co nditions - espe cially pH
and the presence of soil nitrog en etc (Gardner, et al.,
1985). In the present investigation different rhizobia w ere
isolated from nodules o f Vigna radiata for their ability to
grow in various environmental conditions like pH,
temperature, salt and other factors and further assessed for
their efficiency to fix nitrogen in in-vitro cond ition. Th is
study is not only useful in exploring the potential rhizob ia
but also in understanding of relation of natural rhizobial
isolates to their en vironmen t.
MATERIALS AND METHODS
Plant varities and chemicals: Varities of Vigna rad iata
viz., PM -2, PDM -54, K-851 and PM-5 were taken from
Indian Aagricultural Research Institute (IARI), New
Delhi. All the chem ical for m edia Y EM A were procured
from Himedia India Pvt Ltd and for gas chromatography
from Sigm a Ch emicals co. U SA .
Isolation and identification of rhizobial isolates:
Isolation of Rhizobium made by using followed usual
techniques (3, 19). Yeast extract mannitol agar (YEMA)
is the special medium used to grow Rhizobium. Colonies
grown on a me dium m ay not be o nly of rhizobium they
can be even of Agrobacterium. For definite conclusion,
one has to inocu late seeds of particular sequence and w ait
for formation of nodule on such plants. In order to
confirm whether isolated colonies a re of Rhizobium for
this purpose one has to see that inoculated plants form
effective nodule on roots. For further confirmation,
isolates were grown on Y EM A-congo red agar, glucose
peptone broth, bromocreserole purple agar medium and
plant infection assay .
Corresponding Author: T. Neeraj, Department of Biotechnology, C.C.S. University, Meerut-250110 (U.P) India
62
Asian J. Agric. Sci., 1(2): 62-65, 2009
period ically after 18 hours for ethylene formation on
sigma gas chromatography equipped with a Flame
Ionization Detector and D ual Po rpak N co lumn of 2.0 M
length.
The amou nt of ethylene produced through reduction of
acetylene calculated from calibration curve. Nitrogenase
activity expressed as the formation of n mol of
ethylene/ho ur/mg protein.
pH, temperature, N- source, C - source an d salt
tolerance: Effect of pH on gro wth of selec ted isolates
was measured in liquid culture using YEM broth at
differen t pH, rangin g form 5.5 to 9.0. For temperature
effect isolates were grown on YEM A agar media and
incubated at differen t (22, 24, 26, 28, 30, 32, 34, 36 and
38 ºC ) temperatures.
Ability of isolates utilizing different nitrogen sources
like yeast extract, casein hydrolysate, ammonium
chloride, and potassium nitrate were determined by
measuring the growth in liquid medium with mannitol as
carbon source. Four concentrations of each N source were
taken and growth was determined by CFU counts after 72
hr of in incubator shaker at 200 rpm. Different carbon
sources, viz., mannitol, sucrose, fructose and glucose w ere
selected to observe the effect of C-source on growth.
Growth profiles of rhizobial isolates were observed using
yeast extract mannitol broth medium with these sugar’s
supplements.
To study the effect of different salt concentrations on
the grow th of selected isolates of Rhizobium the isolates
were incubated at 28ºC for 6 days on surface agar and in
liquid culture med ium w ith YEM broth at 200 rpm for 72
h containing different concentrations of NaCl (Sodium
chloride) ra nged fro m 1 0 m M , 20 mM, 50 mM, 100 mM
a nd 15 0 m M to 50 0 mM .
RESULTS AND DISCUSSION
A total 972 isolates were collected from the nodules
of different verities of Vigna radiata viz., PM-2, PDM -54,
K-851 and PM-5. These isolates were designated as
rhizobia on the basis of their colony characteristics and
cell morphology and culture characteristics. Among these,
only 67 isolates were short listed on the basis of plant
grow th promotion/ increased plant biomass (data not
shown). These isolates were then assessed in pot
experiments for their ability to increase plant biomass,
maximum nodule numbers and higher nitrogenase
activity, where only six potential isolates (Table 1) which
were selec ted for further stud ies.
Although all the isolates show ed op timum grow th at
28 ºC but the isolate, designated as R0132, showed
slightly highe r temperature i.e. 32ºC for its optimum
growth (Fig. 1). Increased temperature optima of this
isolate may be beneficial for its application in temp erature
stress condition as symbiotic performance of different
rhizobial strains under temperature stress has been
correlated with their ability to grow in pure culture at
elevated tem peratu res (H ungria, 2000).
All the isolates showed their optimum growth at pH
7.2 but isolate, R0132 was able to tolerate slightly higher
pH that is 8.0 (Fig. 2). L arge v ariation in tolerance to pH
of the culture medium has been found within Rhizob ium
species (Graham and Parker, 1964). Isolates to isolates
differences are reported with regard to the growth in
relation to pH of the culture medium observed by
(Cooper, et al., 1988; Diat Loff, 1970).
No effect on grow th of any isolate was observed in
media containing upto 20 mM of sodium chloride.
Excessive inhibitory effect of presence of NaCl on the
grow th of all the iso lates was noticed by increasing the
concentration above 50 mM except isolate R0132 which
demonstrated good growth at this con centration of N aCl
(Fig. 3). Salinity, a com posite stress having both an ionic
as well as osm otic compo nent, can be extremely
detrimental for the growth of soil-inhabiting bacteria like
Rhizobium (Saxena, et al., 1996). With regard to the
salinity stress it has been observed that a number of
rhizobial isolates exhib it a large range of sensitivene ss to
salinity. Singleton et al. (Singleton, et al., 1982) showed
that some isolates from the different rhizobial types failed
to grow even at 0.6 percent NaC l while other show 3.7
fold range for insensitivity in term of fractional reduction
in growth. These results are in accordance w ith Man sari
et al. (2007) where Phaseolus vulgaris was able to grow
at high salt concentration.
Nodulation assay: For vetch assays, seeds of Vigna
radiata L. obtained, and sterilized in the same manner.
Seeds then placed on sterile water-aga r plates and TY
plates; water-agar plates placed in the dark 24-36 hours
at room temperature while TY plates were placed at 28ºC
to check for sterility. After germination on plates, seeds
placed in modified jars containing sterile carrier material
and 200 ml of plant medium. Three seeds placed in a pot.
Overnight cultures of Rhizobium isolates used for
inoculation. 1.0 ml of culture was added to 9.0 ml sterile
H 2 O mixed gen tly, and the mixture ad ded to one pot.
Control pots had 10.0 ml of water added instead of
inoculums.
Estimation of nitrogenase activity: Rhizobium cells
cultured in YEM medium and harvested at exponential
phase of grow th. These cells were suspen ded in sma ll
amount of medium and again inoculated into fresh culture
medium (10 ml of culture medium into a 100 ml bottles
with serum rubber stopper) of (Keister, 1975) with some
modification containing NaPO 4 7.4 mM, MgSO 4 , 0.8 mM,
FeSO 4 48 :m, NaMoO 4 :m, mannitol 0.5 percent and
sodium succinate 0.5 percent sodium glutamate 0.1
percent). The amount of other trace metals and vitamins
were same as used in defined medium. The pH of medium
adjusted to 6.8. After 96 hours of growth, the aero bic
phase of bottles replaced by flushing several times with
nitrogen. In final stages, 10 ml of acetylene as injected
into the bottles at partial pressure. The bottles were then
incubated upto 72 hours at 28 ºC on a rotary shaker at 200
rpm. The samples of gas phase 1.0 ml analyzed
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Asian J. Agric. Sci., 1(2): 62-65, 2009
Table 1: Effects of inoculation of selected rhizobial isolates on nodu le numb ers, weight and texture
SN.
Isolate Designation
Nu mb er of N odu le/plant
Fresh weight of Nodule (mg)
.
Contro
l2
118 1.2
2.
R0034
32
226 .3
3.
R0129
31
232 .4
4.
R0132
37
261 .3
5.
R0564
30
206 .8
6.
R0789
32
212 .3
7.
R1203
33
220 .6
Table 2: Nodulation efficiency parameter of selected rhizob ial Isolates
S.N
Isolate Designation
Fresh weight of Nodule (mg)
1
Control
181 .2
2
R0034
226 .3
3
R0129
232 .4
4
R0132
261 .3
5
R0564
206 .8
6
R0789
212 .3
7
R1203
220 .6
Dry weight of Nodule (mg)
22.6
31.9
32.0
35.4
29.3
30.6
31.6
Dry weight of Nodule (mg)
22.6
31.9
32.0
35.4
29.3
30.6
31.6
Nod ule texture
Pink color
Pink color
Pink color
Pink color
Pink color
Pink color
Pink color
AR A (µ mo l hG 1 mg G 1 FW of nodules)
2.73
2.56
2.90
3.21
2.73
2.94
2.81
Fig 2: Effect of Salt concentration on growth of rhizobial
isolated from Vigna radiate
Fig 1: Effect of temperature on growth of selected rhizobial
isolated in broth culture
Sucrose and fructose could not prove to be an
appropriate carbo n sou rce for growth of isolates. In case
of all the isolates maximum grow th was observed in
R0132 on m annitol containing med ium In present
investigation majority of the isolates showed their good
grow th on mannitol when was used as C-source (data not
show n). It has been reported that slow grow ing isolates
grow well on monosaccharides in comparison of other
carbo n sou rces.
The isolate were further assessed for the ability to
produce nodule and N-fixation ability. The data presented
in the Ta ble 1, revealed that the ability to produce no dule
by different isolates varied from 30-37. Higher number of
nodule (37) w ere ob served on the plant inoculated with
R0132. Nodule fresh weight of plant inoculated by
different rhizobial isolates also varied from 20 6.8 to 261.3
mg/p lant. The lowest range was observed in R0564 (206
mg nodule per plant). Maximum range of fresh w eights
of nodule per plant was shown by isolate R0132 wh ich
was notice d 261.37 mg n odule/plant.
The measurement of nitrogenase activity was based
on the reduction of acetylene to ethylene as quantities by
gas chrom atogra phy. Nitrog enase activity of plant nodu le
inoculated with d ifferent rhizobial isolates varied from
2.56 to 3.21 :g/mg/nodule (Table 2). The maximum
nitrogenase activity has been observed in isolates
designated as R0132 while minimum nitrogenase activity
has been recorded in iso lates R 0034.
Fig 3: Effect of temperature on growth of selected rhizobial
isolated in broth culture
It has been observed that growth of these isolates
varied on different nitrogen sources. Yeast extract proved
itself as a potential growth promoter at the concentration
of 1.0 to 0.5 g/l. Growths of isolates on ammonium
chloride were slightly low comparison of observed in case
of yeast extract while casein hydrolysate and potassium
nitrate were not supported (data not shown). W hen growth
comparison of these isolates studied it has been observed
that under different nitrogen sources isolates R0132 was
found to be the best performer as it utilized a wide range
of nitrog en sources.
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Asian J. Agric. Sci., 1(2): 62-65, 2009
How ever, Rhizobial isolates in present investigation
with regard to their physical environm ent for example
temperature; pH and salinity showed that some isolates
may have significant value of tolerance in diverse
condition. Efficiency of rhizobial isolates in pure culture
at different temperature, pH and salinity has far reaching
ecological significance in varied places (Du zan, et al.,
2004; Howieson and B allard, 2004).
Several environm ental conditions are limiting factors
to the growth and activity of the N 2 -fixing plan ts. A
principle of limiting factors states that "the level of crop
production can be no higher than that allowed by the
maximum limiting factor" (Bro ckw ell, 1980). In the
Rhizobium-legume symbiosis, w hich is a N 2 -fixing
system, the process o f N 2 fixation is strongly related to the
physiological state of the host plant. Therefo re, a
competitive and p ersistent rhizobial strain is not expected
to expre ss its full capacity for nitrogen fixation if limiting
factors (e.g., salinity, unfavorable soil pH, nutrient
deficiency and temperature extremes) impose limitations
on the vigor of the host leg ume (Thies, et al., 1995). From
the present investigation it may be concluded that the
isolate R0132 which showed broad range of pH and
temperature with minimum grow th requ irements, can fix
high level of N-fixation in in-vitro condition as observed
by ARA test. It is need less to state that such a candidate
could be potential one, which can address the problem of
nitrogen depletion.
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ACKNOWLEDGEMENT
The authors are grateful to the Director, Indian
Agriculture Research Institutes Pusa New Delhi, for
providing facilities and enco urageme nt.
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