Biochem. Cell. Arch. Vol. 14, No. 1, pp.

00-00, 2014 ISSN 0972-5075

OCCURRENCE AND MICROBIOLOGICAL CHARACTERISTICS OF

AZOSPIRILLUM STRAINS ASSOCIATED WITH WHEAT RHIZOPLANE AND
ENDORHIZOSPHERE
S. Shubha, V. P. Savalagi1, A. Anantha Rama and G. B. Santoshagowda
Department of Agril. Microbiology, University of Agricultural Sciences, Raichur - 584 102, India.
1
Department of Agril. Microbiology, University of Agricultural Sciences, Dharwad - 580 005, India.
e-mal: shubhad2003@yahoo.co.in
(Accepted 21 March 2014 )

ABSTRACT : Azosprillum spp. were isolated from the endorhizosphere of 27 wheat genotypes and 32 Azospirillum strains
were obtained. Among these six were Azospirillum lipoferum, 13 were Azosprillum brasilense and 13 isolates could not be
grouped into either of the two categories. And all the isolates were subjected to various biochemical characterization. All the
isolates were tested for siderophore production by growing them on M-9 medium. Out of 32 isolates, 18 strains showed good
growth on the medium, where as five others showed moderate growth and rest others totally failed to grow on the medium. The
variations in the isolates might be due to the genetic variations in the plant genotypes and their properties.

Key words : Azosprillum isolates, biochemical characterization, wheat genotypes.

INTRODUCTION Azospririllum is also known to produce growth

Azospirillum has been isolated from the roots of
numerous wild and cultivated grasses, cereals, legumes
and from tropical, sub-tropical and temperate soils world
wide. Thus, it appears that Azospirillum is a universal
bacterium found almost everywhere. They are nitrogen
fixers, exhibiting nitrogen dependent growth under
microaerophilic conditions. It is the widely exploited
bioinoculant for non-leguminous crops. A free-living
bacterium, it is diazotrophic under microaerophilic nitrogen
deficient condition, a situation that is present in the
endorhizosphere and pockets of rhizosphere. In various
studies Azospirillum inoculations have been reported to
reduce the use of chemical fertilizers in particular to
nitrogen by 20-50 per cent. The interactions showed better
results especially when organic fertilizers were
incorporated with the associative N2 fixers, Azospirillum
spp. in many grasses and cereals (Day and Doberiner,
1976). Based on physiological and morphological
difference between various strains and on DNA homology
experiments, (Tarrand et al., 1978) proposed the genus
Azospirillum and distinguished two species: A. brasilense
and A. lipoferum. Studies on the taxonomy, biochemistry,
physiology and genetics of Azospirillum have revealed a
very interesting microorganism (Patriquin et al, 1983;
Hartmann et al, 2000; Stoffels et al, 2001) which serve
as a model for elucidating the mode of action of beneficial
plant-rhizobacterial interaction (Okon et al, 1988; Bashan
and Holguin, 1997).
promoting substances which augment the growth of the
plants (Tien et al, 1979). There is a need to search for
location specific high nitrogen fixing Azospirilla and
enhance their performance in local soils. To obtain location
specific high nitrogen fixing Azospirilla, isolation and
screening for efficient Azospirilla from that location is
necessary. Enhancement of their Nitrogen fixing ability
has been done by various methods including mutagenesis
and recombinant DNA technology (Weniger and Van
Veen, 1991). One approach that has been followed was
to obtain NH4 excreting mutants. These mutants improved
nitrogen availability in rhizosphere (Machado et al, 1991).
MATERIALS AND METHODS
Isolation of Azospirillum
Azospirillum was isolated form wheat roots. The
cultivars of wheat were used for isolation are given in
Table 1. These were obtained from, Senior Scientist.,
MARS. Dharwad. Azospirillum cultures were isolated
from the roots of wheat by following the enrichment
culture techniques as described by Day and Dobereiner
(1976). Six root bits were taken from a single cultivar of
wheat and inoculated in different tubes. Tubes with white
dense pellicle formation 1-2 mm below the surface were
selected for further purification. The seedlings were
uprooted and washed thoroughly in running water. Root
bits were initially surface sterilized using 0.1 per cent
mercuric chloride for 30 seconds and with 70 per cent
alcohol for 30 seconds to remove all the surface microflora
 S. Shubha et al
present on the root bits. The root bits were thoroughly
washed with sterile distilled water to remove the traces
of mercuric chloride and alcohol and were aseptically
placed into 5 ml of the semisolid malate medium (NFb)
(Baladani and Dobereiner, 1980) supplemented with 50
mg per litre of yeast extract to enhance the growth of
Azospirillum. After 48 h of incubation at 29º±1ºC,
characteristic white subsurface pellicles of Azospirillum
were observed. The cells with good N2-fixing capacity in
N2 deficient conditions will only form the subsurface
pellicle. The above mentioned procedure was repeated
several times to purify the culture. From these tubes
loopful of culture was taken and streaked on NFb agar
plates supplemented with 50 mg yeast extract per litre to
obtain single colonies. These single colonies were further
transferred to semisolid NFb medium. Here the pellicle
formation in this medium was taken as an indication that
the isolate is Azospirillum. For final purification these
cultures were streaked on to BMS potato agar (Baldani
and Dobereiner, 1980) Congo red medium (Rodriguez-
Caceres, 1982) and on Luria agar and the colony
morphology was observed.
Identification of the isolates
The tentatively identified Azospirillum isolates were
subjected to morphological and biochemical activities. For
all the tests along with isolates a reference strain A.
brasilense was used for comparison.
Cultural and Morphological Description
Putative Azospirillum colonies were selected on the
basis of the culture plate morphology characteristics
namely: opacity, pale to deep pink pigmentation, no slimy
and wrinkled. Selected colonies were picked up and
cultured on NA slants.
Morphological Description of the Vegetative cells
Azospirillum was examined for cell-shape, Gram
reaction, inclusions and motility in the semisolid malate
medium after 1-3 days. Polymorphism was recorded after
2, 7 and 15 days of incubation.
Physiological and Biochemical tests
For species determination, utilization of different
carbon- sources was performed either in aerobic or
anaerobic conditions. The bacterial strains were grown
in semisolid malate medium containing carbohydrate
together with a pH indicator (Bromothymol blue) (Hugh
and Leifson, 1953). The development of a yellow colour
during 96 h incubation at 30ºC indicated acidification. The
tests involved catalase activity, determination test, growth
in the presence of 3 % NaCl and growth on the amino
acid L-Histidine as a sole C and N source (Hartmann et
al, 1988). Production of siderophore (growth on M-9
medium) (Bachhawat and Gosh, 1987) as well as starch
and gelatine hydrolysis were also tested (Martinez -Drets,
1984).
Biochemical test
Acid from glucose (Tarrand et al., 1978)
The acidified peptone medium was adjusted to 7.0-
7.1pH with KOH. The medium was dispensed in 5ml
quantities in test tubes and sterilized. Filter sterilized glucose
was added to a final concentration of one per cent. 48 h
old cultures from test tube slants were taken and
suspended in sterile phosphate buffer saline and 0.1 ml of
culture was inoculated into the medium. After a period of
four days of incubation at 29ºC, the appearance of yellow
colour indicated acid production.
Catalase activity
To test the isolates for the presence of catalase, 3%
H‚ O‚ was added to the single colony and release of
bubbles was recorded as catalase positive.
Urease activity
The urease activity was determined by using the
method of (Philpot, 1967). 24 h old culture was streaked
on the Christensens medium (Weniger, 1946) slants.
Development of deep red colour throughout the medium
when incubated at 29ºC for four days showed the cultures
were urease positive.
Phosphatase activity
The presence or absence of phosphatases in dense
suspension of resting cell was determined according to
method of (Burger, 1967). The substrate used for the test
of enzyme activity was P-nitrophenol which is yellow in
alkaline solution, 0.3 ml suspension of resting cells were
mixed with 0.3 ml of P-nitrophenol phosphate solution
and was incubated for 6 h at 37ºC. The presence of acid
phophatase was confirmed when a yellow colour was
developed on addition of 0.3 ml of glycine NaOH buffer.
Other biochemical tests
Further, the strains were checked for the growth on
triple sugar iron slants, eosine methylene blue agar,
MacConkey agar, MRVP broth, sellers slants and for the
growth in presence of 1 per cent bile and 1 per cent
glycine. Further, the strains were also checked for starch
hydrolysis, growth at pH 6.0, salt tolerance at 3 per cent
and 4.5 per cent NaCl. All these experiments were done
to characterize Azospirillum according to Bergey’s
manual of Determinative Bacteriology (VIII edition). The
ability of isolate to use different carbon sources as sole
carbon was tried. The malate free NFb was prepared
 Microbiological characteristics of Azospirillum strains
Table 1 : Different genotypes of wheat and their Azospirillum isolates.
Isolate No. Wheat genotype Isolate No. Source
WAS- 1 DWR-185 WAS-17 C-306
WAS-2 MSS- 3063 WAS-18 JWS-35
WAS-3 DDK-1009 WAS-19 NW-1085
WAS-4 DWR-241 WAS-20 MP-1090
WAS-5 DWR-240 WAS-21 PBW-175
WAS-6 C-306 WAS-22 MP-5010
WAS-7 C-306 WAS-23 NIAW-381
WAS-8 DT-115 WAS-24 PBN-3287
WAS-9 HW-2028 WAS-25 K-9744
WAS-10 WH-717 WAS-26 HO-2765
WAS-11 HI-1471 WAS-27 NI-5439
WAS-12 K-80627 WAS-28 HI-1474
WAS-13 HI-1471 WAS-29 UP-2472
WAS-14 NIAW-296 WAS-30 Filler-3
WAS-15 UP-2472 WAS-31 Filler-4
WAS-16 K-9741 WAS-32 Up-2472

with respective 1 per cent carbon sources. The different
carbon sources used were citrate, aconitate, isocitrate,
á-ketoglutarate, succinate, fumarate, L- malate,
oxaloacetate, pyruvate, L-lactate, malonate, acetate, â-
hydroxybutyrate, n-propanol, L-histidine, L-tyrosine, L-
phenylalanine, L-alanine, L-glutamate, L-aspartate, L-
asparagine, L-proline, L-hydroxyproline, L-serine, glycine,
putrescine, D-gluconate, D-mannitol, D-fructose, glycerol.
The growth from the Master plate Luria agar was
replicated on different plates containing medium with
different carbon sources and plates were incubated at
29ºC and readings were observed after 2 h of incubation.
RESULTS AND DISCUSSION
Investigations were carried out to isolate native
isolates of Azospirillum from wheat rhizoplane and
endorhizosphere. The isolates were further identified
based on their physiological characteristics. All the isolates
formed subsurface pellicle in the semi solid medium. The
isolates were urease positive, could grow in anaerobic
condition in presence of nitrate phosphatase positive, could
grow in anaerobic condition in presence of nitrate
phosphatase positive and most of them were catalase
positive, however but some of the isolates showed weak
catalase activity (WAS-3, WAS-5, WAS-19, WAS-27,
WAS-29 and WAS-31). None of the isolates could
produce H2S from cystine or could utilize gelatine. All the
isolates did not grow on EMB agar, one per cent bile,
MacConkey agar, MRVP broth and sellers slants. None
of the strains could hydrolyse the starch. Very few strains
WAS-6, WAS-11, WAS-29, WAS-30 could utilize
succinate as sole carbon source. WAS-31 could utilize
succinate as sole carbon source and WAS-20, WAS-26,
WAS-28 however, showed weak growth. Only WAS-10
strain could utilize glutamate as sole carbon source. Eleven
isolates could grow in the presence of 1 per cent bile
concentration and 13 strains showed good response in
triple sugar iron slants. Out of 32 isolates, 15 isolates
required biotin for its growth. Growth was observed at
pH 6.0, in only 5 isolates. Azospirillum is a general root
colonizer and is not a plant-specific bacterium (Bashan
and Holguin, 1997; Hartmann and Baldani, 2006). In the
present investigation 32 spirilla were isolated from the
endorhizosphere of different wheat genotypes. After
several transfers, 15 strains were identified as bacteria
belonging to the genus Azospirillum based on to their
common cultural and cell morphological characteristics.
The characteristics were the formation of a veil like pellicle
or ballon often 10mm below the surface of semi solid N-
free media. The formation of this pellicle is due to an
aerotactic response of the motile bacteria towards low
levels of PO4 that permit N2 fixation (Okon, 1985). The
dissolved O2 concentration in the media was just enough
for optimal respiration rates without inhibiting N2 fixation
(Day and Dobereiner, 1976), as a result of nitrogenase
inhibition.
Utilization of carbon compounds
The ability of isolates to use several carbonaceous
compounds as sole carbon source were checked using
31 different carbon sources. The observations were taken
with respect to growth in comparison with malate as
control. The results obtained are presented in Table 2. All
the isolated strains could utilize citrate, L- malate,
oxaloacetate, pyruvate, L-lactate, â- hydroxybutyrate, D-
gluconate, D-mannitol and glycerol. Screening of different
Table 2 : Utilization of different compounds as the sole carbon sources by Azospirillum isolates.
Isolate Citrate Aconitate Isocitrate α –ketoglu Succinate Fumarate L-malate Oxaloac- Pyruvate L-lactate Malonate Acetate β–Hydroxy- n-
tarate etate butyrate propanol
WAS-1 + - - - - + + + + + - - + -
WAS-2 + - - - - + + + + + - - + -
WAS-3 + - - - + + + + + + - - + -
WAS-4 + - - - - + + + + + - - + -
WAS-5 + - - - - + + + + + - - + -
WAS-6 + - - - +/- + + + + + - + + -
WAS-7 + - - - - + + + + + + - + +
WAS-8 + - - + - +/- + + + + + - + -
WAS-9 + - - - - +/- + + + + + - + -
WAS-10 + - - - - + + + + + - - + -
WAS-11 + + - + +/- + + + + + - - + -
WAS-12 + - - - - + + + + + + - + -
WAS-13 + - - - - + + + + + - - + -
WAS-14 + - - - - + + + + + - - + -

S. Shubha et al
WAS-15 + - - - - +/- + + + + - - + -
WAS-16 + - - - - + + + + + - - + -
WAS-17 + - - - - + + + + + - - + -
WAS-18 + - - - - +/- + + + +/- + - + -
WAS-19 + - - - - + + + + + - - + -
WAS-20 + + - + +/- + + + + + - - + -
WAS-21 + - - - - + + + + + - - + -
WAS-22 + - - - - + + + + + - - + -
WAS-23 + - - - - + + + + + - - + -
WAS-24 + - - - - + + + + + - - + -
WAS-25 + - - - - + + + + + - - + -
WAS-26 + - - + + + + + + + - - + -
WAS-27 + - - - - + + + + + + - + -
WAS-28 + - - + + + + + + + - - + -
WAS-29 + - - + + +/- + + + + - - + -
WAS-30 + - - + + + + + + + - - + -
WAS-31 + - - - + + + + + + - - + -
WAS-32 + - - - - + + + + + - - + -
A.brasilence - - - - + + + + + + - - + -
A.lipoferum + - - - + + + + + + - - + -

Table 2 contd...
Table 2 : Utilization of different compounds as the sole carbon sources by Azospirillum isolates (Contd....).
Isolate L- L- L-Phenyl- L- L-Glu- L-Aspar- L- L-Hydroxl L- Glycine Putres- D-Gl- D-Glu- D- D- Glyce- L-aspp-
histidine Tyrosine alanine Alanine tomate agine Proline proline serine cine ucose conate Mannitol fructose rol artate

WAS-1 + - - + - + + + - - - - + + + + -
WAS-2 - - - - + - - - - - - + + + +
WAS-3 - - - - + - - - - - ++ + + + -
WAS-4 - - - - - - - - - - - + + + +
WAS-5 + - - - - - + + - - - +/- + + + + +
WAS-6 - - + - - - - - - - +/- + + + -
WAS-7 - - - - + - - - - - +/- + + + -
WAS-8 - - - - - - - - - - - + + + -

Microbiological characteristics of Azospirillum strains

WAS-9 - - - - - - - - - - - + + + -
WAS-10 - - + - - - - - - - - + + + -
WAS-11 - - - - - - - - - - ++ + + + -
WAS-12 + - - - - + + + - - - +/- + + + + +
WAS-13 - - - - - - - - - - +/- + + + -
WAS-14 - - + - - - - - - - +/- + + + -
WAS-15 - - - - - - - - - - +/- + + + -
WAS-16 - - - - + + + - - - +/- + + + +
WAS-17 + - - - - - + + - - - +/- + + + + -
WAS-18 - - - - + + + - - - +/- + +/- +/- +
WAS-19 - - - - - - - - - - +/- + + + -
WAS-20 - - - - - - - - - - +/- + + + -
WAS-21 - - - - - - - - - - +/- + + + -
WAS-22 - - + - + + + - - - +/- + + + +
WAS-23 - - - - + - - - - - +/- + + + -
WAS-24 + - - - + + + + - - - +/- + + + + -
WAS-25 - - + - - - - - - - +/- + + + -
WAS-26 - - - - - - - - - - +/- + + + -
WAS-27 - - + - - - - - - - ++ + + + -
WAS-28 - - - - - - - - - - + + + + -
WAS-29 + - - + - + + - - - - - + + + + -
WAS-30 - - - - - - + - - - ++ + + + -
WAS-31 - - - - - - - - - - - + + + -
WAS-32 - - - - - - - - - - - + + + -
A.brasilence - - - - - - - - - - - - + + - + -
A.lipoferum + - - + - + + + - - - + + + +/- + -
+ Normal growth +/- Weak growth - No growth.
 S. Shubha et al
Table 3 : Morphological characteristics of Azospirillum isolates on different growth medium.
Tests Cell Motility Gram Polymor- Growth in Growth in Growth in Catalase Gelatin Starch
organism shape stain phism 3% NaCl M9 medium JNFB medium activity hydrolysis hydrolysis
WAS-1 Ovoid + - + + ++ - + - -
WAS-2 Curved + - - +/- + - + - -
WAS-3 Curved + - - +/- - - +/- - -
WAS-4 Curved + - - +/- ++ - + - -
WAS-5 Ovoid + - + + + - +/- - -
WAS-6 Curved + - - +/- ++ - + - -
WAS-7 Curved + - - +/- - - + - -
WAS-8 Rod + - - +/- - - + - -
WAS-9 Curved + - - + - - + - -
WAS-10 Curved + - - + + - + - -
WAS-11 Curved + - - +/- - - + - -
WAS-12 Ovoid + - + + ++ - + - -
WAS-13 Curved + - - + - - + - -
WAS-14 Rod + - - + - - + - -
WAS-15 Curved + - - +/- ++ - + - -
WAS-16 Curved + - - +/- - - + - -
WAS-17 Ovoid + - + + - - + - -
WAS-18 Curved + - - + ++ - + - -
WAS-19 Curved + - - +/- - - +/- - -
WAS-20 Curved + - - +/- - - + - -
WAS-21 Rod + - - + - - + - -
WAS-22 Curved + - - +/- - - + - -
WAS-23 Curved + - - + ++ - + - -
WAS-24 Ovoid + - + + + - + - -
WAS-25 Curved + - - +/- + - + - -
WAS-26 Rod + - - +/- + - + - -
WAS-27 Curved + - - +/- + - +/- - -
WAS-28 Curved + - - + + - + - -
WAS-29 Ovoid + - + + + - + - -
WAS-30 Curved + - - +/- - - + - -
WAS-31 Rod + - - +/- - - +/- - -
WAS-32 Rod + - - +/- + - + - -
A.brasilence Curved + - - +/- + - + - -
A.lipoferum Ovoid + - + + + - + - -

strains for carbohydrate utilization differed markedly with
respect to the Azospirillum species and to the carbon
source. However, all the 32 strains effectively oxidized
the tested organic acids (Succinate, Malate and Pyruvate)
when used as a sole carbon source auzanotrophically [in
presence of (NH2SO4)]. The preference of the organic
acids by different Azospirillum species was reported
earlier by (Reinhold et al, 1985). This can be explained
on the basis that organic acids were the major source of
nutrients for the microflora in the rhizosphere (Curl and
Truelove, 1986).
On aconitate, growth was observed in only WAS -11
and WAS-20 strains. Only seven isolates could utilize á-
ketoglutarate as sole carbon source. None of the isolates
could utilize isocitrate. Six strains could utilize succinate
and three of them however showed only weak growth.
All the isolates except 5 strains WAS-8, WAS-9, WAS-
15, WAS-18 and WAS-29 showed good growth on
fumarate medium. On L-lactate as sole carbon source,
all strains except WAS-18 showed good growth. On
malonate as sole carbon source, growth was observed in
only 6 strains. In acetate as carbon source, only WAS-6
strain could show good growth. Only WAS-7 isolate could
utilize n-propanol as sole carbon source. None of the
isolates could utilize L-histidine, L-tyrosine, L-
phenylalanine, L-serine, glycine and putrescine. Only 7
among the 32 isolates could utilize L-alanine as a sole
 Microbiological characteristics of Azospirillum strains
carbon source. On L- aspargaine as a sole carbon source
8 isolates could grow well. On L-proline, only few isolates
could grow well. 4 isolates grew well on L-hydroxy
proline. In D-glucose as a sole carbon source, only 6
isolates could grew very well. All others showed weak
growth. Only 7 strains could utilize L- aspirate as sole
carbon source. The additions of certain carbon source
encourage the growth of certain group of bacteria. For
instance, addition of manitol or glucose in N2- free medium
leads to the frequent isolation of Azotobacteraceae
(Thompson et al, 1979), whereas malate leads to the
isolation of Azospirillum (Okon et al, 1977) In the present
study, Azospirillum was isolated and enriched from the
rhizosphere, rhizoplane or bulk soil using the nitrogen-free
biotin medium (NFb) in which L-malic was the sole C-
source. Although reports on the isolation of Azospirillum
from graminaceous plants are common, other reports
showed that the bacterium is a natural inhabitant of many
nongraminaceous plants. Azospirillum was isolated from
roots of coconut palms grown under diverse agronomic
practices (George, 1990) and within the stem nodules,
root nodules and stem of Aeshynamene indica (Singh,
1992). The results of the present study also indicated that
A. lipoferum related strains were able to utilize large
group of carbohydrate, while A. brasilense strain was
more restricted in its use of carbon sources including
glucose which was not used by A. brasilense. Glucose
catabolised by Azospirillum spp. By the action of NAD
(P)- glucose- 6- p- dehydrogenase, is required for 6-
phosphoglucanate dehydrogenase synthesis which is a key
enzyme of the ED pathway for glucose catabolism. It
was the first enzyme produced in high level by A.
lipoferum but was undetectable in A. brasilense (Goebel
and Krieg, 1984). However, A. amazonense has
remarkable ability to grow and fix N2 in media containing
disaccharides (Martinez -Drets, 1984), which is a
characteristic of this species. The isolated Azospirillum
spp. In the present study were not able to utilize sucrose
(Table 2).
Siderophores are low molecular weight compounds
produced by the microorganisms which are able to bind
iron from the environment. The binding to the siderophore
allow transfer of iron to the cell. Enabling bacteria to
compete for this otherwise unavailable element
(Horemans et al., 1988). Azospirillum spp. Produces
siderophores that represent an important factor for their
competition and survival in the rhizosphere (Hartmann,
1989) The siderophore spirilobactin produced by A.
brasilense strain RG, was also reported by (Bacchwat
and Gosh, 1987) The siderophore iron uptake of A.
brasilense SP6 was studied by using molecular genetic
approach and the Lon gene was found to be involved in
the iron uptake of A. brasilense (Mori et al, 1992). Our
Azospirillum strains were examined for siderophore
production by testing their capability to grow in iron limiting
(M-9) medium (Hugh and Leifson, 1953). Table 3 indicates
that seven strains are able to grow well in the later medium,
whereas three other strains exhibited moderate growth
and the remaining strains cannot grow in that special
medium. More studies are needed to examine the
efficiency of such indigenous Azospirillum strains as it is
well proved that native strains perform better in all the
plant nutrient management rather than new strain isolated
from elsewhere in plant microbial interactions studies
which could be better utilized for plant nutrient
management aspects.
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