CHAPTER-III
ISOLATION AND
CHARACTERIZATION OF RHIZOBIA
FROM THE ROOT NODULES OF
PIGEON PEA (CAJANUS CAJAN)
Pigeon pea (Cajanus cajan) is a monotypic genus belonging to the family
Leguminosae, subfamily Papilionoideae, tribe, Phaseoleae and subtribe
Cajaninae. it ranks among the most important legume crops of the world (1). It
is cultivated as an annual plant and its height varies from 50 cm to more than
3.5 m with branches spreading up to 2 m. Pigeon pea is noted for its
adaptability to diverse ciimates and soil types. However, it is sensitive to water
logging and cannot tolerate frost (2).
Pigeon pea rbizobia belong to ‘cowpea miscellany' that nodulate legumes
(cowpea group) of mostly tropical and subtropical origin (3). In general, pigeon
pea is nodulated by slow-growing RNzobium species although fast growing
rhizobia have also been isolated from their nodules. Nodulation in pigeon pea
is rapid with about 25 nodules per plant formed in about 15 days of sowing (1).
Most nodules are formed on the secondary roots, the majority are located in the
top 30 cm of the soil profile (1).
There are two benefits that can arise from using legumes as a crop or in
pastures. The first is the plant's independence of the soil nitrogen and secondly
the potentially improved nitrogen states of the soil consequent to the use of the
legume. The nitrogen fixation of legumes could be improved by:
1.
Inoculation with effective Rhlzobium strains,
2.
A better understanding of the rhizospheric factors that affect legume-
Rhizoblum symbiosis and adoption of suitable management to
overcome the constraints and,
3.
Breeding and selection of legumes with increased nodulation and
nitrogen fixing abilities.
Therefore with an aim of developing a good rhizobial strain effective on the
agronomically important local crop, pigeon pea, rhizobia were isolated from its
nodule. For this, surface sterilised pigeon pea root nodules were crushed onto
AMA plates containing Congo red. Growth was observed after 24 h. Three
different isolates were selected based on the mucoid nature of their colonies.
The strains Rhizobium sp. T1 and Rhizobium sp. G1 had large mucoid
colonies while Rhizobium sp. G2 had comparatively less mucoid colonies.
Preliminary biochemical analyses were earned out according to Bergey’s
manual (4). Results (Table 1) show that all the three isolates are Gram negative
capsulated bacilli, it was observed that Rhizobium sp. T1 and Rhizobium sp.
G1 could not utilize citrate while Rhizobium sp. G2 could grow when citrate
was supplied as the sole source of carbon. The ability to utilize citrate has been
cited to be one of the properties segregating fast growers and slow growers (5).
Some of the other properties that differentiate fast and slow growers are (5) -
1) Generation time : Fast growers are found to have a generation time (GT) of
less than 6 h. Our results showed that Rhizobium sp. T1 and Rhizobium
sp. G1 have a GT of 1 and 3 h respectively whereas Rhizobium sp. G2 has
a GT more than 6 h (Table 2).
2) Fast growers are reported to utilize a wide variety of sugars - such as
hexoses, pentoses, mono- and di-saccharides in comparison with slow
49
1 :
Strains
Table
tH
?
?
i©
+ve
-ve
-ve
Nitrogenase
activity
+
Polysaccharide
production
from sucrose
+
+
+
+
+
+
+
i
5
+
f
t
?
?
CM
0
+ve and -ve indicates the positive or negative property and the number of plus denotes the increasing order of
function.
+ve
5
+
Glucose
Citrate
utilization
+
+
+
bacilli
+ve
5
+
Gram negative
Sucrose
L-------------
|
Carbohydrate utilization
+ve
Capsule
staining
c
bacilli
Gram negative
bacilli
;
I
Gram negative
Gram staining
Biochemical characterization of Rhizobium isolates from pigeon pea root nodule.
+
+
+•
Table 2 : Generation time of the strains Rhizobium sp.T1.G1 and G2 :
Strain
Generation Time (h)
T1
1
G1
3
G2
6
Table 3 : Antibiotic sensitivity of the strains Rhizobium sp. T1, G1 and G2 :
Antibiotics
Rhizobium Strains
Concentration
(^)
Tetracyclin
30
T1
S
G1
S
G2
S
Kanamycin
30
S
S
S
Cephaloridine
30
S
R
R
Streptomycin
10
S
R
S
Gentamycin
10
S
S
S
Ampicillin
20
R
R
R
300
R
R
R
Bactrim
25
R
R
R
Carbenicillin
50
R
R
R
Chloramphenicol
30
R
R
R
Colistin
10
R
R
R
Suiphatriad
R : Resistant; S : Sensitive.
growers which chiefly utilize pentoses
and
hexoses.
Studies were
conducted using carbon sources at 1% (w/v) concentration in Ashby’s
Mannitol Broth (AMB) where mannitol was replaced by other carbon
sources. It was observed that Rhizobium sp. T1 and Rhlzobium sp. G1
could utilize a wider spectrum of carbon sources than Rhizobium sp. G2
(Table 4). The pH of the spent medium was found to be slightly towards the
acidic range in the isolates, Rhizobium sp. T1 and Rhizobium sp. G1
except when TCA intermediates and mannitol were used as carbon sources
(Table 4). The production of alkaline metabolites when TCA intermediates
and mannitol were supplied as carbon sources is a property attributed to
slow growers (6).
3) Flagellation : Fast growing rhizobia are known to exhibit peritrichous
flagellation whereas slow growers show polar flagellation (5). In our study all
the isolates showed peritrichous flagellation.
4) Intrinsic antibiotic resistance : Fast growing rhizobia are reported to have a
low intrinsic antibiotic resistance (5). In our study the isolates Rhizobium sp.
G1 and Rhizobium sp. G2 showed a high intrinsic antibiotic resistance and
Rhizobium sp. T1 was found to be resistant to five of the eleven antibiotics
tested (Table 3).
5) The ability to express ex pianta nitrogenase activity has been described as
a characteristic of slow growing rhizobia (7). All the three strains under
study showed ex pianta nitrogenase activity. A direct correlation between
50
Carbon Source
CM
CP
ffl
Jif
?
c
Citrate
;
8.5
7.0
6.5
118.25
268.6
107.5
107.5
7.5
8.5
53.75
7.0
»n
N-'
- indicates absence of growth.
225
Gluconate
S
u.
172
1
CM
Malate
8.0
8.0
Succinate
1
172
o
oo'
8.0
270.2
Mannitol
l
6.0
l
o
oo
129
l
6.0
182.8
215
Lactose
I
6.5
161.3
107.5
li
I
5.5
236.5
l
150.5
5.5
i
Arabinose
7.0
6.5
193.5
5.0
00 in
in 8.
CM
OS
1i
OS
I
Xylose
96.8
w <o
G oo
l
107.5
|
S
l
129
00
in
Sucrose
G
6.0
p
161.25
pH of spent medium
5
Glucose
Growth (pg/ml protein)
Table 4: Carbohydrate nutritional characteristics of the isolates and pH change when grown
on
different carbon sources:
CM o o
G N-' N-"
o
<o
00
v."
rt
the high levels of EPS production and the ability to express ex pla
nitrogenase activity was also observed here. Rhizobium sp. Tt /and v
Rhizobium sp. G1 which showed good ex planta nitrogenase activity were
also good EPS producers (Table 5) whereas Rhizobium sp. G2 which
produces less EPS shewed comparatively a lower ex planta nitrogenase
activity. Reports drawing a positive correlation of EPS with nitrogenase
activity are available (8). It has been speculated that the EPS produced by
the rhizobia aid nitrogenase activity by reducing the oxygen diffusion into
the cells.
6) Another characteristic difference between the fast and slow growing rhizobia
is the ability of the formerto grow on 2% NaCI (1). The isolates Rhizobium
sp. T1 and Rhizobium sp. G1 were found to grow on 2% NaCI indicating
that they could be fast growers.
The results presented so far suggest that the pigeon pea isolates used in this
study were unique in comparison with cowpea rhizobia. Cowpea rhizobia are
generally believed to be slow growers. The isolates Rhizobium sp. T1 and
Rhizobium sp. G1 appear to exhibit several characteristics of fast growers such
as presence of peritrichous flagellation, ability to grow on 2% NaCI, a
generation time of less than 6 h, and the ability to utilize a wide spectrum of
carbon sources. However, they also showed properties of slow growers such as
inability to utilize citrate; production of alkaline metabolites when mannitol and
TCA intermediates were supplied as carbon sources; high antibiotic resistance;
expression of ex planta nitrogenase activity and the ability to nodulate pigeon
51
pea which is normally nodulated by slow growing rhizobia. Apart from
exhibiting peritrichous flagellation, Rhizobium sp. G2 showed all the properties
of slow growers. Rhizobium sp. T1 and Rhizobium sp. G1 therefore were found
to be closer to the intermediate group of rhizobia in the six group classification
proposed by Stowers (6). Reports suggesting the existence of such an
intermediate group of
rhizobia showing properties of both slow
growers and fast are also available (6,9).
In the legume nodule, though bacteroids can reduce nitrogen to ammonia, they
appear unable to assimilate it into amino acids. Instead the ammonia is
exported into the plant cytosol where it is assimilated into nitrogen compounds
(10, 11). ft has also been reported that at least some free living rhizobia are
poor at assimilating ammonia and have a preference for L-giutamate (12). In R.
ieguminosarum and ft. trifbiii ammonia was the most preferred nitrogen source
whereas glutamate and histidine were preferred by the broad host range
rhizobia, Rhizobium sp. NGR234 (13). Our studies showed that glutamate and
serine were the preferred nitrogen sources for Rhizobium sp. T1, while
Rhizobium sp. G1 grew best when histidine was supplied as tire sole source of
nitrogen in the AMB medium. RMzobium sp. G2 however showed a marked
preference for the inorganic nitrogen source, potassium nitrate (Table 6).
Soil acidity affects many areas of the world and limits legume productivity. Most
leguminous plants require a neutral or slightly acidic soil for growth especially
when depending on nitrogen fixation (14). Reports on the differences in the
levels of acid tolerance by legumes are available. Medicago sativa has been
52
Table 5: Ex planta nitrogenase activity and EPS production of the isolates T1, G1
and G2 :
Isolates
Ex planta Nitrogenase Activity
T1
(pi of C2H2 reduced/mg
protein)
4.045
EPS Production
(pg reducing sugar/mg
protein)
279.5
G1
3.088
172
G2
1.728
86
Table 6: Effect of different N-sources on the growth of the strains:
Nitrogen Sources
[0.2% (w/v) in AMB]
Strains
T1
Glutamate
268.5
G1
Growth (protein pg/ml)
258
Aspargine
172
172
86
Glycine
172
—
—
Threonine
236.5
43
86
Serine
279.5
301
86
--
64.5
—
225.8
--
—
129
408.5
129
53.8
—
_
—
258
172
KNO3
215
86
247.3
NI-UCI
139.8
64.5
86
Urea
193.5
75.25
75.25
Leucine
Lysine
Histidine
Methionine
Tyrosine
- indicates absence of growth.
G2
129
observed to be highly acid sensitive
whereas Lotus tenuis tolerates
relatively low pH. Similarly the nodule bacteria too vary in their response to
acidity when grown in liquid culture (15). R. melihti is reported to be acidsensitive whereas RMzobium loti is acid tolerant (15). The strains used in our
study were tolerant to a wide range of pH (Table 7). Earlier reports on rhizobia
indicate that these microsymbionts cannot tolerate very low pH (16). However,
recent reports on Rhizobium loti indicates that they can tolerate up to pH 4.0.
Low rhizobia! count and poor colonization of acidic soils have been shown to
restrict the growth and nodulation of host plants as well (17). Therefore, it has
been suggested that the selection of Rhizobium strains tolerant to low pH may
improve the acid tolerance of the legume (16).
One of the most important nutrients provided by the rhizosphere to the host
plant and its microsymbiont is iron. Importance of iron to rhizobia and its
legume host is of particular interest because of the prominent role of iron
- enzymes in the nitrogen fixation and assimilation process. The iron enzymes
and proteins involved include hydrogenase, nitrogenase, ferridoxin and
leghemoglobin, with nitrogenase (containing at least 30 iron atoms) and
leghemoglobin constituting about 12 to 30% of the total protein in the bacterial
and infected plant cells, respectively (18). Although iron is abundant in soil it is
often unavailable to plants and microorganisms because of very low solubility
of ferric hydroxides (18). It has been reported that iron-deficiency limits nodule
development in soybean - Bredyrhizobium symbiosis and the possession of
siderophore may significantly increase the ability of the differentiated bacterium
to fix nitrogen (19). Apart from fixing nitrogen in the nodules, rhizobia in the
53
Table 7 : pH tolerance of the Rhizobium isolates :
pH of the Medium
Strains
(AMB)
T1
G1
G2
Growth (protein pg/ml)
2.0
—
—
—
3.0
—
139.7
—
4.0
86.0
215.0
75.2
5.0
118.2
215.0
75.2
6.0
118.2
268.7
75.2
7.0
161.2
290.2
107.5
8.0
150.5
247.2
96.8
9.0
125.7
215.0
96.8
10.0
193.5
172.0
86.0
- indicates absence of growth.
rhizosphere may also promote the growth of its legume host by producing
growth hormones or enhancing nutrient uptake. Siderophore production and
iron transport may also contribute to the plant growth promoting activities by
Rhizobium species (20). Based on these facts it was of interest to check
whether the isolates have the ability to sequester iron from the rhizosphere by
producing siderophores. For tills the isolates Rhizobium sp. T1, Rhizobium sp.
G1 and Rhizobium sp. G2 were grown under iron-starved conditions and their
culture supernatants were treated with Chrome Azurol-S reagent (21). The
supernatant of Rhizobium sp. T1 produced a
yellow colour immediately
indicating the presence of a siderophore. The other two isolates tested
negative. Effect of iron on the growth of Rhizobium sp. T1 under iron starved
and iron supplemented conditions are shown in Fig. 1. An enhancement of the
lag phase in the iron-starved culture was clearly observed. Similar results have
been reported earlier with Azospirilium lipoferum M and cowpea Rhizobium
(groundnut isolate) (22, 23). Addition of iron has been shown to repress the
synthesis of siderophores in Azotobacter vineiandii and A. Upoferum M (25,
24). To determine the minimum iron concentration repressing siderophore
production, Rhizobium sp. T1 was grown in iron-starved conditions as well as
under conditions where different concentrations of iron were supplemented.
Siderophore production was found to
decrease with
increase
in
iron
concentration in the medium and was completely repressed on the addition of
100 pM of iron. Growth on the other hand increased proportionately with iron
concentration (Table 8). Siderophore extracted from the culture supernatant of
iron-starved Rhizobium sp. T1 showed the presence of 2,3-DHBA when
analyzed on thin
layer chromatography (TLC) (Table 9). Siderophore
54
Protein (pg/ml)
Fig.1 : Growth of Rhizobium sp. T1 under Fe-starved and Fe-supplemented conditions.
Time (h)
Table 8 : Effect of iron concentration on siderophore production and growth :
Iron Concentration
iiM)
0
Siderophore Production
(jig/ml)
9.8
Growth
Protein (pg/ml)]
19.78
1
6.2
24.08
5
5.6
30.53
10
5.2
41.71
25
4.3
43.86
50
4.03
66.65
100
--
73.05
- indicates absence of siderophore production.
Table 9: Preliminary characterization of siderophore by TLC :
Standards
Rf value
2,3 DHBA
0.36
3,5 DHBA
0.25
3,4 DHBA
0.32
Salicylate
Sample
indicates absence of the compound.
0.38
preparations
purified
by
preparative
TLC
were
subjected
to
UV-
spectrophotometric scan in the range 200 to 400 nm wavelength (Fig. 2). The
absorption spectra of the principal constituent matched that of authentic 2,3DHBA.
Rhizobium sp. G1, a non-producer of siderophore showed very low nitrogenase
activity under iron-starved conditions. The ability of Rhizobium sp. T1 to
express ex planta nitrogenase by virtue of its siderophore under similar
conditions highlights the importance of siderophore production to the enzyme
nitrogenase (Table 10). Iron is an important constituent of the nitrogenase
enzyme (26). Iron deficiency is reported to decrease nitrogenase activity.
Earlier reports have shown a 5 to 6 fold increase in ex pianta nitrogenase
activity in the presence of siderophore under iron-limited conditions, suggesting
a positive correlation between siderophore-mediated iron transport and the
nitrogen fixing property (24).
Rhizobia are bacteria that must adapt to two different lifestyles. Firstly, they
have to live in the soil with other soil bacteria and secondly they have to thrive
in the root nodule (in the bacteroid form) where they are supplied by the host
plant with photosyrrtoetate in exchange for the generation of NH3 from N2. The
success of the bacteroid form of a particular rhizobial strain depends partly on
the ability of the free living, motile form to compete effectively with other
rhizobial strains and with other microorganisms in the soil (27,28). Motility has
been described as a significant factor In the microbial competition for nutrients
and in the distribution of rhizobia in toe rhizosphere. The three isolates
53
.000
Figure 2 :
Spectrophotometric analysis of the siderophore from Rhizobium sp. T1.
Standard 2,3-DHBA (1); Siderophore sample (2).
Table 10: Nitrogenase activity of the strains under iron-starved and iron
supplemented conditions:
Strain
Nitrogenase Activity
(jil C2H2/mg protein)
Iron-supplemented
iron-starved
T1
G1
G2
2.09
3.7
—
2.17
—
—
- indicates absence of nitrogenase activity.
Table 11 : Chemotactlc efficiency of the isolates :
Strain
Migration rate (mm/h)
T1
0.125
G1
0.104
G2
0.083
Rhizobium sp. T1, G1 and G2 studied for this property were found to be motile
possessing peritrichous flagella. The migration rates (calculated as given in
Materials and Methods) of the isolates were as in Table 11. A motile parental
strain of Rhizobium meiiioti was shown to have a competitive advantage in
nodulation over a non-motile mutant strain (29). The access of inoculant strains
erf Rhizobium strain in the nodulation of their legume hosts may depend on a
variety of factors, but the key component needed to outstrip competition from
other strains would be the motility of toe inoculant strain and toe rapidity and
efficiency with which it responds chemotactically to the plant signals and
triggers toe initial pre-infection events.
Awareness of the benefits of inoculation has increased in the recent years and
hence it seems necessary to select efficient strains of rhizobia which when
used make significant contributions to nodulation and crop yield. The selection
criteria for a strain to be effective lies in its ability to express ex plants
nitrogenase
activity
effectively,
produce
good
amounts
of
surface
polysaccharides and above all to form effective nitrogen fixing nodules on
homologous and heterologus hosts under a wide range of field conditions. On
this basis, the isolate Rhizobium sp. T1
which was observed to be a better
producer of EPS compared to Rhizobium sp. G1 and Rhizobium sp. G2 was
selected for further studies.
The importance of seed inoculation for nitrogen fixation particularly in legumes
is now universally recognized. This is done to provide sufficient rhizobia to
nodulate the host effectively and to boost crop yield. Experiments were
56
conducted to assess the practical utility of the isolate Rhizobium sp. T1 as an
inoculant
for pigeon pea under greenhouse and field conditions. These
experiments were focused mainly on studying the effect of Rhizobium sp. T1
inoculation on pigeon pea and other homologous hosts like Vigna mungo
(black bean), Vigna radiate (mung bean) and Vigna sinensis (cowpea) and
heterologus hosts like Pisum sativum (pea), Clear aritenum (chick pea) and
Vicia faba (kidney bean).
Rhizobium sp. T1 inoculation on pigeon pea resulted in higher percentage of
germination and enhanced root and shoot growth (Table 12). The lush green
colour of the leaves are also one of tee observations made in pots inoculated
with Rhizobium sp. T1 (Plate 1A). Similar results were obtained not only in
Rhizobium sp. T1 inoculated homologous hosts but also in heterologous hosts
(pea, bean and chick pea) inoculated with tee same strain (Table 12, Plate 1
B,C,D). The association of rhizobia with tee roots of leguminous plants leading
to beneficial effects on plant growth is well known (19, 20). Though Rhizobium
sp. T1 exerted growth promoting effects on both homologous hosts and hosts
belonging to tee cross inoculation groups, noduiation was observed only in
black bean, mung bean and cowpea apart from pigeon pea. However, these
nodules were found to be smaller in size compared to the nodules initiated on
pigeon pea (Table 13; Plate 2 A.B.C.D). Earlier reports on pigeon pea rhizobia
have shown teat they can nodulate cowpea and soybean but not legumes
belonging to cross inoculation groups (1). However a majority of rhizobia
isolated from Glycine max, Arachis hypogea and Sesbania sp. are able. to
nodulate pigeon pea (1).
57
Table 12 : Effect of RMzobium sp. T1 on pigeon pea, pea, bean and chick pea :
Germination (%)
Average Shoot Length
(cm)
Uninoculated
50
12
Inoculated
100
17
Uninoculated
25
4
Inoculated
100
16
Uninoculated
25
7
Inoculated
75
14
Uninoculated
25
7
Inoculated
100
13
Plant Under Study
Piaeon Dea:
Pea:
Bean:
Chick Dea:
Plate 1 :
Effect of Rhizobium sp. T1 inoculation on (A) Pigeon pea;
(B) Pea; (C) Bean; (D) Chick pea.
Control - uninoculated; ino - inoculated with Rhizobium sp. T1.
c
3
Table 13 : Effect of Rhizobium sp. T1 inoculation on pigeon pea, black bean,
mung bean and cowpea under field conditions :
Description
Piaeon oea
Average no. of
nodules
1.0
Uninoculated
control
Rhizobium sp. T1
Nodule weight
Nodule size (mm)
(mg)
<0.5
-
3.0
15
3.0
Inoculated
Black bean
—
—
Uninoculated
control
Rhizobium sp. T1
3.0
2.5
0.5
Inoculated
*
Muna bean
Uninoculated
control
Rhizobium sp. T1
6.0
0.6
0.5
Inoculated
Cowoea
—
_
Uninoculated
control
Rhizobium sp. T1
10.0
inoculated
- indicates absence of nodules.
0.69
1.0
Plate 2 :
Effect of Rhizobium sp. T1 inoculation on (A) Pigeon pea; (B) Black
bean; (C) Mung bean; (D) Cowpea.
(Arrows point to the nodules).
A small scale field experiment was conducted to assess the practical utility of
Rhizobium sp. T1 as an inoculant for pigeon pea. The experiment conducted
was focussed mainly on the effect of Rhizobium inoculation on nodulation, in
plants nitrogenase activity and the effectiveness of the strain in noduiating
other hosts such as black bean, mung bean and cowpea (Table 14). A
significant difference was observed in the number of nodules and root-shoot
development of the pigeon pea plant inoculated with Rhizobium sp. T1 (Table
15; Plate 3 A,B). The effective nodules were surface sterilized and crushed
onto AMA plates as described earlier. The cultural characteristics and antibiotic
sensitivity pattern of the isolate thus obtained proved to be that of Rhizobium
sp. T1 thus indicating that the nodules were induced by RNzobium sp. T1 and
not the native rhizobia. Field trials have been described to be an ultimate test in
assessing a strain’s ability as the nodules initiated by it is an end resuit of
several challenging factors. Ability to persist in the soil and surviving the
competition from the native rhizobia! strains being some of them (1).
38
Table 14: Effect of Rhizobium sp. T1 inoculation on pigeon pea, black bean,
mung bean and cowpea:
Modulation (nod)
N2 fixation (fix)
Infection (inf)
Pigeon pea
nod*
fix*
inf
Black bean
nod+
fix'
inf
Mung bean
nod*
fix*
inf
Cowpea
nod*
fix*
inf
Host
+
ability
absence
Table 15 : Effect of Rhizobium sp. T1 inoculation on pigeon pea (under field
conditions):
Experimental Condition
Shoot Length (inch)
Average no. of Nodules
Uninoculated control
23.9
1.0
Rhizobium sp. T1
inoculated
31.3
3.0
Plate 3 :
Effect of
Rbtzobium sp. T1 inoculation on (A) Growth and (B)
(Modulation of pigeon pea under field conditions.
(control-)
REFERENCES
1. Subba Rao N.S. (1988). Modulation and nitrogen fixation. In: Biological
Nitrogen Fixation - Recent Developments (Subba Rao N.S. ed.), Oxford and
JBH Publishing Co. Pvt. Ltd. New Delhi, pp.21-52.
2. Pathak G.N. (1970). Redgram In: Pulse Crops of India (Kaehroo P. ed.),
ICAR New Delhi, pp.14-53.
3. van Rhijn P. and Vanderieyden J. (1995). The Rhizobium-plant symbiosis.
Microbiol. Rev. 59:124-142.
4. Jordan D.C. (1984). Family III Rhizobiaceae Conn. 1938. In: Bergey's
manual of systematic bacteriology. Vol. I (Krieg N.R. and Holt J.Q. eds.),
Williams and Wilkins, Baltimore, Md. pp. 234-254.
5. Eifcan G.H. (1992). Taxonomy of the rhlzobia. Can. J. Microbiol. 38 : 446450.
6. Stowers M.D. (1985). Carbon metabolism in
Rev. Microbiol. 39:89-108.
Rhizobium species. Ann.
7. Agarwal A.K. and Keister D.L. (1983). Physiology of ex planta nitrogenase
activity in R. japonhum. Appl. Environ. Microbiol. 45:1592-1601.
8. Mody B.R. and Modi V.V. (1987). Peanut agglutinin induced alterations in
capsular and extracellular polysaccharide synthesis and ex planta
nitrogenase activity of cowpea rhizobia. J. Biosci. 12:289-296.
9. Stowers M.D. and Eaglesham A.R.J. (1983). A stem nodulating Rhizobium
with the physiological characteristics of both slow and fast growers. J. Gen.
Microbiol. 129:3651-3655.
10. Brown C.M. and Diiworth M.J. (1975). Ammonium assimilation
Rhizobium cultures and bacteroids. J. Bacteriol. 135:114-123.
by
11. Kurz W.G.W., Rokosh DA and La Rue TA (1975). Enzymes of ammonia
assimilation in R. leguminosarum bacteroids. Can. J. Microbiol. 21 : 1009-
1012/
59
12. Ludwig R.A. (1978). Control of ammonium assimilation in Rhizobium 32H1.
J. Bacteriol. 135:114-123.
13. Poole P.S., Dilworth M.J. and Glenn A.R. (1987). Ammonia is preferred
nitrogen source in several rhizobia. J. Gen. Microbiol. 133:1707-1712.
14. Bordeleau L.M. and Prevost D. (1994). Nodulation and nitrogen fixation in
extreme environments. Plant and Soil, 161:115-124.
15. Correa O.S. and Barneix A.J. (1997). Cellular mechanisms of pH tolerance
in Rhizobium loti. World J. Microbiol. Biotechnol. 13:153-157.
16. Richardson A.E., Simpson R.J., Djordjevic MA and Rolfe B.G. (1988).
Expression of nodulation genes in R. leguminosarum biovar trifblii is
affected by low pH and by Ca and Al ions. Appl. Environ. Microbiol. 54 :
2541-2548.
17.Glenn A.R. and Dilworth M.J. (1994). The life of root nodule bacteria in the
acidic underground. FEMS Microbiol. Lett. 123:1-10.
18. Messenger A.J.M. and Ratledge C. (1985).
Siderophores. In:
Comprehensive Biotechnology (Young M.M. et al. eds.), Vol.3, Oxford :
Pergamon Press, pp.275-295.
19. Guerlnot M.L. (1991). Iron uptake and metabolism in the rhizobia-legume
symbioses. Plant and Soil, 130:199-209.
20. Jadhav R.S., Thaker N.V. and Desai A.J. (1994). Involvement of cowpea
Rhizobium in iron nutrition by peanut. World J. Microbiol. Biotechnol. 10 :
360-361.
21.Schwyn B. and Neiiands J.B. (1987). Universal chemical assay for the
detection of siderophores. Anal. Biochem. 160:47-56.
22.Shah S., Karkhanis V. and Desai A.J. (1992). Isolation and characterisation
of siderophore with antimicrobial activity from Azospirillum ttpolerum M.
Curr. Microbiol. 25:347-351.
23.Jadhav R.S. and Desai A.J. (1992). Isolation and characterization of
siderophore from cowpea Rhizobium (peanut isolate). Curr. Microbiol. 24 :
137-141.
60
24.Shah S., Rao K.K. and Desai A.J. (1993). Production of catecholate type
siderophores by Azospirillum iipoferum M. Ind. J. Expt. Biol. 31:41-44.
25. Corbin J.L. and Bulen WA (1969). The isolation and identification of 2,3dihydroxybenzoic acid and 2N 6N di(2,3-hydroxybenzoyl)-L-lysine formed
by iron deficient Azotobactervinefandii. Biochemistry, 8 :757-762.
26. Reeves M., Pine L., Neilands J.B. and Bullows' A. (1983). Absence of
siderophore activity in Legionella sp. grown in iron deficient media. J.
Bacterioi. 54:324-329.
27. Bergman K., Gulash-Hoffee, Hovestadt R.E., Larosiliere R.C., Ronco P.G.II
and Su L. (1988). Physiology of behavioural mutants of R. meiiioti :
Evidence for a dual chemotaxis pathways. J. Bacterioi. 170:3249-3254.
28. Parke D., Rivelli M. and Ornston L.N. (1985). Chemotaxis to aromatic and
hydroaromatic acids : Comparison of B. japonicum and R. trifoliL J.
Bacterioi. 163:417-422.
29. Ames P. and Bergman K. (1981). Competitive advantages provided by
bacterioi mobility in the formation of nodules by Rhizobium mefiloti. J.
Bacterioi. 148:728-729.
61