International Journal of Microbiological Research 4 (3): 275-287, 2013
ISSN 2079-2093
© IDOSI Publications, 2013
DOI: 10.5829/idosi.ijmr.2013.4.3.825
Azospirillum and its Formulations: A Review
P. Sivasakthivelan and P. Saranraj
Department of Microbiology, Annamalai University,
Annamalai Nagar-608 002, Tamil Nadu, India
Abstract: Biofertilizers manufactured in India are mostly carrier based, using lignite or charcoal as carriers.
Inspite of nearly three decades of existence of biofertilizers in India supported by central and state government,
the adoption level of biofertilizers by farmers remained low. There are many reasons for such a low adoption
of this technology, which are related to technological, developmental, social issues. Biofertilizers manufactured
in India presently are carrier based, in general and suffer from short shelf life, poor quality, high contamination
and low and unpredictable field performances. Death of the organisms in the inoculated seeds is one of the
important factors contributing the failure of inoculation response in field condition. Research conducted on the
inoculant production and formulation technologies is limited. A break through is needed in the inoculation
technology to improve the shelf life and field efficacy of biofertilizer in India to make them commercially viable
and acceptable to farmers. The present review was focused on different formulations of Azospirillum.
Key words: Azospirillum
Biofertilizer
Nitrogen Fixation and Formulations
INTRODUCTION
So studies to increase the shelf life of inoculants or
finding alternate formulations for carrier based inoculants
are important.
Liquid biofertilizers are special liquid formulation
containing not only the desired microorganisms and their
nutrients, but also special cell protectants or substances
that encourage formation of resting spores or cysts for
longer shelf life and tolerance to adverse conditions.
Azospirillum is one of the important biofertilizer, which is
found to fix nitrogen in association with world’s most
staple food crops like rice, maize, sorghum, wheat and
millets. Members of the genus Azospirillum are
widespread in soils and its inoculation of cereal and
forage crops resulted in yield increases in many field
experiments [4], not only due to the nitrogen fixation but
also through the production of plant growth promoting
substances [5].
Biofertilizers are carrier based preparations containing
beneficial microorganisms in a viable state intended for
seed or soil application and designed to improve soil
fertility and to help plant growth by increasing the number
and biological activity of desired microorganisms in the
root environment. Biofertilizers are products of selected
beneficial and live microorganisms, which help to improve
plant growth and productivity mainly through supply of
plant nutrients. Biofertilizers are also known as microbial
inoculants or bio inoculants. Biofertilizers have come to
stay in Indian agriculture since last three decades in
view of their cost effectiveness, contribution to crop
productivity, soil sustainability and ecofriendly
characters. Biofertilizers form an integral part of Integrated
Plant Nutrient Supply system (IPNS) and organic farming
which constitutes the present as well as future mandate of
Indian agriculture. One of the main problem in inoculant
technology is the survival of microorganisms during
storage and several parameters such as the culture
medium, physiological state of the microorganisms
when harvested [1] the process of dehydrates, rate of
drying [2] the temperature of storage and water activity
of the inoculum [3] have an influence on their shelf life.
Corresponding Author:
The Genus Azospirillum: Azospirillum spp. is of
ubiquitous distribution in many parts of the world
consisting of tropical, sub-tropical and temperate climatic
conditions and also with various standing crops grown
in a variety of soil types. Azospirillum spp. fixes
atmospheric nitrogen and has been isolated from the
rhizosphere of a variety of tropical and sub tropical non
P. Saranraj, Department of Microbiology,Annamalai University,
Annamalai Nagar - 608 002, Tamil Nadu, India.
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leguminous plants [6]. Lakshmikumari et al. [7] reported
the occurrence of nitrogen fixing Spirillum in the roots of
rice, sorghum and maize. Various cereals have responded
differently to field inoculation with this organism [8].
Okon and Labandera Gonzalez [9] evaluated world wide
data accumulated over the past 20 years on field
inoculation with Azospirillum and concluded that these
bacteria are capable of promoting the yield of
agriculturally important crops in different soil and
climatic regions. The genomes of five of the six
Azospirillum spp. were analyzed by pulsed-field gel
electrophoresis strains DNA hybridization indicated
multiple chromosomes in genomes ranging in size from
4.8 to 9.7Mbp. The nif HDK operon was identified in the
largest replicon [10].
Azospirillum mostly lives associated with the
plants, particularly in their rhizosphere, on the root
surface and to a lesser extent, inside the root [23],
Bashan and Holguin [24] reported that the ability of
Azospirillum to colonize atleast 64 plant species of which
18 are weed species. The association of Azospirillum with
cashew and its possible role in improving crop growth
and yield was reported by Purushothaman [25].
Nitrogen Fixation by Azospirillum: Azospirillum spp is
one of the most efficient N2 fixers in the field when all the
required conditions for biological nitrogen fixation are
present. Most studies on the Azospirillum inoculation
have suggested that nitrogen fixation was the major
mechanism of plant growth [26]. Lima et al. [27] noted that
up to 50 per cent of the N content of crops such as
sugarcane, Panicum maximum and Paspalum notatum
could be supplied by associated nitrogen fixers mainly
Azospirillum.
Hartmann et al. [28] reported the influence of amino
acids on nitrogen fixation ability and growth of
Azospirillum spp. The utilization of amino acids for
growth and their effects on nitrogen fixation differ
greatly among the several strains of each species of
Azospirillum spp. The different utilization of various
amino acids by Azospirillum spp may be important for
their establishment in the rhizosphere and for their
associative nitrogen fixation with plants. Kucey et al. [29]
indicated that upto 18 per cent of the plant nitrogen
was derived from nitrogen fixation. All wild type
Azospirillum strains were found to fix N2 efficiently either
as free-living or in association with plants [30].
Heulin et al. [31] reported that association with rice,
Azospirillum lipoferum N-4 contributed about 66% of
the total N in plants as was demonstrated with 15N isotope
studies. The mesophilic lac-z-marked Azospirillum
lipoferum ALP 3 did not grow and fix nitrogen at 45°C out
of 40 thermo tolerant marked mutants, developed from
ALP 3 and screened for N-fixing ability at 30°C and 45°C,
only 14 mutants could grow and fix nitrogen at 45°C [32].
Gadagi et al. [33] examined the effect of combined
Azospirillum inoculation and nitrogen fertilizer on plant
growth promotion and yield response of the blanket
flower Gaillardia pulchella. Azospirillum strain OAD-2
inoculation significantly increased plant height, number
of leaves per plant, branches per plant and total dry mass
accumulation in G. pulchella than other inoculations or
un inoculated controls. Azospirillum strains OAD-2 and
OAD-11 can play an important role in the N nutrition of
G pulchella.
Isolation of Azospirillum Species: The soil bacterium
Azospirillum was first isolated and originally named as
Spirillum lipoferum. This bacterium was later isolated
from soil and from dried seaweed in Indonesia [11]
and as a phyllosphere bacterium in tropical plants [12].
Breed et al. [13] classified the genus Spirillum in the
order Pseudomonodales. Krieg [14] noted that
S. lipoferum might belong to the genus Azospirillum.
De Polli et al. [15] identified A. lipoferum as more
homologous group, whereas A. brasilense recorded three
sub groups with respect to fluorescent antibody reaction.
Magalhaes et al. [16] reported a new acid tolerant
nitrogen fixing species, Azospirillum amazonanse which
grows at pH 6.0 temperature 35°C and with G + C percent
as 67. Khammas et al. [17] isolated a new strain of
Azospirillum from the rice rhizosphere of Diwaniya,
Irag and classified into a new species as Azospirillum
irakense. Fani et al. [18] investigated phylogenetic
relationship of Azospirillum based on 16S rRNA at the
inner and sub generic level. The phylogenetic analysis
confirmed that Azospirillum genus into separate entity
with in sub class of proteobacteria with five species
properly defined.
The occurrence of Azospirillum in the rhizosphere
varied from 1 to 10 per cent to the total rhizosphere
population [19]. The rhizosphere soil samples contained
100 times more number of Azospirillum than the
non-rhizosphere soil [20]. Azospirilla have been
isolated from a wide variety of plants from all over the
world from tropical, temperate, salt affected soils, desert
soils to water flooded conditions [21]. The occurrence of
Azospirillum sp. was reported in the rhizosphere of
various field grown crops such as rice, wheat, maize,
sorghum and pearl millet of different locations [22].
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Omay et al. [34]
reported
Azospirillum sp
inoculation in wheat, barley and oats
seeds in
greenhouse experiments. For wheat significant
increases were obtained when the inoculation was
associated to 100% of the recommended nitrogen. For
barley the presence of the inoculants substituted 20%
of the recommended nitrogen fertilization. For oats the
inoculation with Azospirillum sp RAM-7 did not provide
a significant increase in grain yields.
Gadagi Ravi et al. [35] reported the effect of
Azospirillum amazonense inoculation on growth, yield
and N2 fixation of rice. Bacteria of the genus Azospirillum
stimulate plant growth directly either by synthesizing
phyto-hormones or by promoting nutrition by the
process of biological nitrogen fixation. All A. amazonense
strains tested produced indoles, but only 10% of them
showed high production. The nitrogenase activity also
was variable and only 9% of isolates showed high
nitrogenase activity and the majority (54%) exhibited a
low potential.
Saikia et al. [36] described that dinitrogen fixation
activity of Azospirillum brasilense in maize. The presence
of nitrogenase activity was noticed in plants treated with
Azospirillum. Plant root- rhizosphere N2 fixing bacterial
interactions is required before any consistent, significant
and beneficial N2 - fixing association can be developed.
Plant Growth Substances Production by Azospirillum:
Hartmann et al. [37] revealed that certain mutants of
A. brasilense exerted higher quantities of IAA in the
culture upto 16 µg ml 1. Inoculation of the plants with
IAA over producing mutants of A. brasilense resulted in
increased root numbers, length and root hairs in wheat
[38]. Horemans et al. [39] reported that the in vitro
production of cytokinin by Azospirillum. Okon and Hadar
[40] observed in sorghum, an increase in root length and
root hairs due to Azospirillum inoculation. The higher
amount of IAA, IBA in the maize roots inoculated with
A. brasilense than in non inoculated roots were reported
by Fallik et al. [41]. The amounts of gibberellin and
cytokinins produced by the mixed culture of A. brasilense
and Arthrobacter giacomelloi were higher than those of
single cultures, suggesting that the interaction between
the rhizosphere inhabitants may affect their secondary
metabolism and indirectly the plants [42].
Zimmer et al. [43] screened the tryptophan dependent
IAA production of different Azospirillum spp and
revealed that A. irakense KA3 released 10 times less IAA
into the medium than A. brasilense SP 7. The culture of
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A. brasilense and the barley straw degrading fungus
Trichoderma harzianum produced a substantial increase
of GA was reported by Janzen et al. [44].
Barberi and Galli [45] noted that inoculated
A. brasilense SPM 7918 promoted the root system
development on wheat seedlings. In liquid culture of
A. brasilense Cd, the concentration of IAA increased
rapidly at the beginning of the stationary phase when the
sole carbon source (DL-Malic acid) was a limiting factor.
This suggested that the higher increase in IAA in the
stationary phase in the expression of an overall change in
cell metabolism when the carbon source is exhausted [46]
Gibberellin GA3 had effects similar to Azospirillum
lipoferum inoculation in increasing root hair density in
corn seedlings [47]. Gibberellin GA3 production by
stationary phase pure cultures of Azospirillum lipoferum
appeared to be correlated with culture viability and
polysaccharide excretion that is favoured in turn by a
specific N concentration and initial pH of the culture [48].
Production of major quantities of IAA and Gibberellin
at relatively older stages of the bacterial growth may imply
that this might be relevant to the field rhizosphere
interaction of Azospirillum with plants where the bacteria
are seldom at the logarithmic phase yet affect plant
development. This may also explain why very old
aggregated cultures (flocs) induced positive plant
responses after inoculation [49]. For a better evaluation
of the role of hormones in general and IAA in particular
in plant promotion, there is a need for a mutant
totally deficient in IAA production. This mutant is
still unavailable [50]. The same is true for other
phytohormones. The gene ipd C encoding, an indole1-pyruvate decarboxylase involved in the synthesis of
IAA showed growth phase dependent expression.
External IAA and synthate auxins could be involved in
the up regulation of expression of A.brasilense IAA
biosynthetic gene ipd [51].
Fabricio Cassan et al. [52] reported that Azospirillum
brasilense and Azospirillum lipoferum hydrolyze
conjugates of GA20 and metabolize the resultant
aglycones yo GA1in seedlings of rice dwarf mutants.
A. brasilense strain cd and A. lipoferum strain USA
5b promoted sheath elongation growth of two single gene
GA-deficient dwarf rice mutants, dy and dx when the
inoculated seedlings were supplied with GA20 – glucosyl
ether.
Kolb and Martin [53] described the isolation and
selection of indigenous Azospirillum spp. and the IAA of
superior strains effects on wheat roots. IAA produced by
Intl. J. Microbiol. Res., 4 (3): 275-287, 2013
bacteria of the genus Azospirillum spp. can promote
plant growth by stimulating root formation. Native
Azospirillum spp. isolated from Iranian soils had been
evaluated. The roots of wheat seedlings responded
positively to the several bacteria inoculations by an
increase in root length, dry weight and by the lateral root
hairs. Perrig et al. [54] reported that plant growth
promoting compounds produced by two strains of
Azospirillum phytohormones Indole-3 acetic acid (IAA),
Gibberillic acid (GA3) Abscisic acid (ABA) and growth
regulators putrescine, spermine, spermidine and
cadaverine were found in culture supernatant of both
strains. This is the first report on the evaluation of
important bioactive molecules in strains of A. brasilense
as potentially capable of direct plant growth production
or agronomic yield increase. This fact has important
technological implications for inoculant formulations as
different concentrations of growth regulators produced
by different strains.
successful inoculation is maximized. Since crop response
to inoculation, in most instances, is the result of
appropriate strain selection and target organism
population [60]. Okon [61] reported Azospirillum as a
commercial inoculant for improving crop yields. The
development of an effective seed or soil applied legume
inoculant requires the integration of physical, chemical
and biological processes was stated by Stephenes
et al.[62].
Different Carrier Used for Microbial Inoculant
Production: Peat is generally as most dependable carrier
because of its high content of organic matter and water
holding capacity [63]. In India, Peat like material is
available in Nilgris valley to the extent of 5.5. Million
tones and an unknown quality is also known to occur in
Kashmir valley [64]. Tilak [65] observed the survival of
Azospirillum upto 31 weeks in carriers like farm yard
manure (FYM), FYM + soil, FYM + charcoal and soil,
FYM + charcoal + soil gave higher viable counts of
Azospirillum than others.
Farm yard manure, compost, coconut shell powder,
vermiculite clay, teak leaf powder was also used as
carriers for inoculant production [66]. Sparrow and Han
[67] studied the survival of Azospirillum in peat,
powdered peanut shell, corn cobs, vermiculite and
polyacrylamide gel and reported that vermiculite
supported 107 cells of rhizobia g 1 of carrier even after
30 weeks of period. The survival of Azospirillum in
number of locally available material such as lignite,
pressmud, charcoal, soil and coffee waste and found to be
better than other carriers including peat for the survival of
Azospirillum upto 200 days the rate of decline in
Azospirillum population was least in pressmud [68].
Several experimental Azospirillum inoculant
formulations have been proposed based on the
biopolymer like Alginate and xanthan gum which were
shown to be good carriers. These carriers protect the
organisms against stress conditions [69].
Singaravadivel and Anthoni Raj [70] reported that
black ash, paddy husk, black ash plus husk mixture,
husk powder and pressmud were effective and
satisfactory carriers for Rhizobium and were on par with
lignite and peat. These carriers maintained the viable cell
load of 107 cells g 1 even after 6 months. Gaind and Gaur
[71] examined the charcoal-soil mixture as the best carrier
for phosphate solubilizing microorganisms. Fages [72]
reported that the survival of Azospirillum in various
carriers such as peat, vermiculite, Alginates and liquid
Biofertilizer Inoculant Technology: Tarrand et al. [55]
reported that some of the most promising organism,
capable of colonizing roots in large numbers and exerting
beneficial effects on plants belong to the genus
Azospirillum. Azospirillum helps in efficient use of
applied fertilizers and enriching the soil with nitrogen
fixed in association with the roots. The Azospirillum
inoculant is efficiently used so far as carrier based
inoculant and this is high adsorptive, easy to process and
non toxic to Azospirillum [56].
Inoculant preparation and inoculation technologies
must be improved. In addition, the inoculated
Azospirillum should reach the root even if the root
system is widely spread and the bacterial inoculation
should be at the precise time needed by the plant [57].
The inoculation techniques should be practical,
economical and easy to accomplish for the farmer.
The formulated products should deliver sufficient
inoculum to the plant must be competitive with existing
commercial standards and must possess a long shelf life
[58]. The development of adequate formulations, which
would ensure survival and protection of the strain and the
application technology, which would allow timely, easy
and precise delivery in the field could be a major step
towards this goal [59].
The use of a microbial inoculant is primarily
concerned with crop productivity, not bacterial
physiology nor ecology. Inoculants manufactures must
accept this reality and ensure that the probability of
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Aeration: Canadian and European workers observed
rapid death of rhizobia in sealed containers, but with
access to air their numbers remained high until the
carrier became desiccated [82]. In contrast, other workers
found that rhizobia were able to multiply 10 to 100 fold
and survive satisfactorily in either screw-capped jars or
sealed cans. Some of this confusion is possibly caused
by the fact that the demand for gas exchange in peat
cultures though definite is not high [83]. Most reports
compared only “unrestricted exchange” containers with
sealed containers and where the lacter were not sealed
under vacuum, the higher proportion of air trapped to
carrier material may have allowed better survival of the
organisms [84].
Roughley [85] examined the growth and survival of
clover and cowpea-type rhizobia in sterilized peat in
cotton wool stopper tubes, sealed cans and plastic film
packets with various gas exchange properties. The
practice of putting small holes in bags is both
unnecessary and harmful as in that moisture loss is
increased. Also, they allow entry of contaminants in
sterilized peat cultures [86].
formulation and reported that better results have been
obtained with cells in dried microgranulated Alginate
formulation.
Narendranath [73] studied the suitability of different
carrier materials for inoculant preparation and reported
that incorporation of amendments like soymeal (1%) and
molasses (1%) in the carrier enhanced the survival of
Rhizobium and also suggested that pressmud amended
with soymeal, as an alternative carrier to peat in inoculant
preparation. Abd-Alla et al. [74] explained different
carriers for biofertilizers.
Factors Affecting the Shelf Life of Microbial Inoculants
Moisture Content: Roughley [75] showed that clover and
cowpea type rhizobia are adversely affected by moisture
content of 30 per cent. Other organisms occurring
naturally in peat were less affected. Levels in the range of
40 to 50 per cent were optimal for survival of three strains
of Rhizobium [76]. In a sterilized peat, all strains are more
tolerant to higher levels of moisture and growth is optimal
in the range of 40 to 60% moisture. An interesting adverse
effect of a high moisture level is that numbers of certain
Rhizobia are highest at 60 to 65% and reduced carriers to
absorb different amounts of moisture may explain the
different optima for growth and survival [77].
Sterile and Non-Sterile Carrier: The inoculants prepared
with non-sterilized peat might contain 100-told fewer
rhizobia than sterilized peat because the death rate is
higher and this difference increases during the storage
period [87].
Sterilization has a great influence on the growth and
survival of rhizobia and other sterile inoculants and
their ability to achieve consistently high cell densities in
excess of 109 g 1 [88, 89, 90]. The choice of a method
of inoculating sterilized peat without introducing
contaminants depends on the type of packaging.
In Holland, an assembly of glass and rubber tubing
with a hallow needle is used to connect the sampling part
of a fermentor [91].
Santhanakrishnan and Thangaraju [92] revealed that
the polymer and amendments addition improved the
survival of Azospirillum. The hydrophilic polymers,
Jalsakthi and terra cotton both at 2% and 4% levels
and amendments, polyvinyl pyrrolidone and skim milk at
1% and 2% levels were observed for retaining moisture
and favoured the survival of Azospirillum cells in both
sterilized and un-sterilized carrier based inoculant.
Temperature: Roughley [78] reported that effect of
storage on growth and survival of organism is influenced
by both the purity of the culture and the amount of
moisture lost during storage. It cultures are prepared in
sterilized carriers, incubation at 26°C immediately after
inoculation promotes initial rapid growth of organisms
and has little or no effect on long term survival if moisture
content is maintained. In experiment on long term storage
of unsterilized peat cultures the weekly log death rate
of clover rhizobia increased from 0.04 at 5°C to 0.094 at
25°C [79]. The moisture content of cultures in cotton wool
stoppered bottles wrapped in cellophane may be kept
at a constant 50% by storage at 2°C [80], but storage at
such temperatures immediately after inoculant restricts
initial multiplication and maximum numbers are not
reached until 26 weeks. This period can be reduced to two
weeks if cultures are incubated for one week at 26°C prior
to storage at 4°C.
Saha et al. [81] studied the survival of Rhizobium
in the carriers at different temperatures using green
fluorescent protein marker and reported that survival rate
of Rhizobium transform at was minimum at higher
temperature above 28°C, in the paddy husk.
Storage Materials for Inoculants: Roughly [93] confirmed
that Rhizobium needed a definite gas exchange through
packing material. Better survival of sealed bags than in
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open aerated bags was observed by Iswaran [94].
Strijdom and Deschodt [95] studied the survival of
four Azospirillum spp in steam sterilized peat sealed in
high density polypropylene bag of 0.31-0.32 mm gauge.
They observed high survival (3.5 x 108 g 1 to 62 x 108 g 1)
in inoculants packed in high density polythene bags.
Subba Rao [96] studied the effect of packing
materials on the survival of Azospirillum. Double
polythene bag (600 gauge thickness) supported higher
survival of Azospirillum compared to single polythene
bad (300 guage) at room temperature. Packing the peat
based inoculant Azospirillum in two layered polythene
bag increased the shelf life when compared to packing in
single polythene bag or polythene container [97].
molassess (1%) in the carrier enhanced the survival of
Rhizobium. The soymeal (2%) or polyvinyl pyrrolidone
(2%) for soymeal (2%) or polyvinyl pyrrolidone (2%) for
BradyRhizobium japonicum (TAL 102) were found to be
the suitable amendments for enhancing the survival of
Rhizobium inoculants [108]. Daza et al. [109] have
demonstrated the usage of perlite as the carrier for
biofertilizers formulation and it could maintain a higher
population of organisms than peat inoculants at room
temperature for six months.
The hydrophilic polymers are synthetic water
absorbing monomers of high molecular weight and
they have been used for the past 30 years to increase the
water holding capacity, increase the pore size/number,
increase in nutrient reserves and reduction of compaction
of soil or artificial medium [110]. Polymers absorb water at
irrigation and release it as the soil surrounding the
polymer dries and has no effect on the physical
characteristics of water [111]. Dhumal [112] observed the
significant increase in the yield of tomato (58.8%) and
chillies (31.6%) by applying jalsakthi during water stress
condition.
The application of jalsakthi and Rhizobium
treatment has increased the yield and water use efficiency
of groundnut crop during summer [113]. Subbaraj et al.
[114] used Qemisorb, an agricultural polymers, which
hydrates about 100 times its initial weight with water and
increased the moisture content of soil by 67 per cent.
The hydrogels such as polyacrylamide or polyacrylic
acid are capable of absorbing in excess of 300 times their
dry weight and they are mixed with the growing medium
of tomato plants to retain the moisture in arid regions
[115]. Grula et al. [116] revealed that the polymers applied
for trapping water are used polymers applied for trapping
water are used as the carbon sources of soil micro
organisms and stimulated their growth. Terracotton, a
hydrophilic polymer can store water 150 times its own
weight and increased the yield of tomato by 150 per cent,
the germination per cent and plant height of papaya by
60 per cent and 20 percent respectively and soybean crop
by 10 per cent [117].
Amendments and Polymers Addition into Inoculant
Carrier:Fages [98] reported that the skim milk
addition into dehydrated Alginate beads helps to
contain 10 billion cells g 1 of Azospirillum in the carrier.
Kandasamy and Prasad [99] suggested that amending
lignite with one per cent soybean powder could be used
as carrier material for the Rhizobium spp than soil.
The cellulose powder appeared to be more satisfactory as
a carrier in as much as it gave the same case of handling
and low moisture loss as peat based inoculants and fairly
high survival of rhizobia [100]. Iswaran [101] used coir
dust and soy meal with soil to study the survival of an
efficient strain of R. trifolii and reported that coir dust
which is a carrier material along with soil for Rhizobium
was superior to soy meal.
Carriers consisting of 40 per cent coal, 40 per cent
bentonite and 20 per cent bone meal have been studied in
South Africa by Strijidom and Deschodt [102] and the
rhizobial survival in that carrier was comprehensive to
that in peat. Tilak and Subba Rao [103] evaluated 11
carrier materials and used amendments like charcoal and
coconut shell powder to some carrier materials (1: 1 ratio)
for rhizobia and they concluded that lignite, pressmud
and FYM amended with coconut shell powder or charcoal
could serve as good substitute for peat under Indian
conditions. Kumar Rao et al. [104] reported that the rate
of decline of Rhizobium population was least when
pressmud is used as a carrier.
Sparrow and Ham [105] and Graham-Weiss et al. [106]
have evaluated vermiculture as the carrier material and
suggested vermiculite supplemented with nutrients will
support the bacterial survival and could be used as an
inoculant carrier. Narendranath [107] reported that
incorporation of amendments like soymeal (1%) and
Liquid Formulation of Microbial Inoculants: Generally,
the inoculation procedures with carrier-based inoculants
are time consuming, messy and impractical when
sowing large quantities of seed. So, as an alternate,
liquid inoculants formulation was developed with
specific constituents of medium to grow Rhizobial cells.
As historical background, liquid inoculants were used in
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inoculating Azospirillum into the cyst inducing minimal
salts medium (MSM). One hundred percent conversion
of vegetative cells into cyst cells was noticed in 96h.
The population level of 108 was maintained till the final
observation [130].
the form of broth or suspensions of cells growing on agar
slants, but it was not common in market [118]. First report
on liquid inoculants for seed inoculation of Rhizobium
was reported from Holland [119]. Burton et al. [120]
reported that the liquid inoculum was as effective as
peat-based inoculant when the number of rhizobia per
seed was increased by 2.5 times.
Schiffmann and Aples [121] observed that liquid
inoculants were particularly suited to large seeded grain
legumes, which, because of their bulk, make seed
inoculation a formidable task. The liquid inoculation of
rhizobia provides higher cell load in the rhizosphere. Wani
and Rai [122] reported that foliar spray application of
Azotobacter increased the yield of paddy, wheat and
sorghum. The nodule numbers are increasing with
increasing rates of applied liquid rhizobia from log
4.59 up to log 9.59 viable cells g 1 [123]. The spray
method of liquid Rhizobium inoculant directly on to the
soil was also reported.
Generally, all manufactures of liquid inoculants
claim that their liquid products contain high titers value.
Lipha Tecj (France) product claimed (cfu of 17.21 x 1011 in
their liquid product. The soybean nodule numbers in a
tropical soil increased at increased rates of rhizobia
applied in a liquid form Smith [124]. Hynes et al. [125]
reported that the liquid rhizobial inoculants for pea and
lentil resulted in yield equal to or better than those
observed with peat inoculant.
Niftal has already claimed the cell number of 1 x
14
10 cells ml 1 in gliquid inoculant. The National chemical
laboratory of pine has reported Cfu of 108 - 109 in case of
Azotobacter (liquid) inoculants [126]. Inamadar et al. [127]
standardized cyst and spore based liquid inoculants for
Azotobacter and P-solubilizing Bacillus megaterium and
recorded higher population and comparatively better yield
of crops in inoculated area. Singleton et al. [128] reported
the development and evaluation of liquid inoculants and
nitrogen fixation of legumes in Vietnam.
Vendan and Thangaraju [129] reported that the carrier
based inoculants generally suffer from shortage shelf
life, poor quality, high contamination and low field
performance. The liquid formulations of Azospirillum
with the amendments viz., Trehalose, Polyvinylpyrollidine
and Glycerol enhanced and maintained the population
level at 108 upto 10 months of storage. The liquid
formulation shows better adherence and survival on
seeds, roots of seedlings and in the rhizosphere soil
than the solid carrier based Azospirillum inoculants.
Cyst based liquid formulation was developed by
Gel Based Microbial Inoculants: During last decade,
there has been an increased interest in synthetic beads
of various materials and dimensions for immobilization.
The main purpose of these beads was to immobilize or
entrap the target organisms for a longer period and slow
release of microorganism under favourable condition.
Poly acrylamide gel was observed as a suitable carrier for
Bradyrhizobium japonicum. Survival of wet gel entrapped
cells at high temperature was better than peat based cells.
Jung et al. [131] suggested the replacement of poly
acrylamide with Alginate (AER) which supported higher
survival. The Alginate beads (dried AER) accounted for
more than 88% survival even after 150 days of storage
and they proposed the use of Alginate beads with skim
milk. The dried beads containing Azospirillum could be
stored at ambient temperate over a period of twelve weeks
without any loss of Azospirillum cell content.
Fages [132] reported that highest survival of
Azosprillum could be obtained by addition of skim
milk and controlled dehydration of the Alginate beads.
The powdered product contained more than 1010 viable
cells per gram dry weight after several months of storage
at ambient temperature. Hegde and Brahmaprakash [133]
developed a free flow granular inoculant of Rhizobium
from sodium Alginate and perlite. They observed high
number of survival of two groundnut Rhizobium strains
in dry granules beyond 180 days than in peat.
Fages [134] reported better survival rate of
Azospirillum in dried micro granulated Alginate
formulation. Okon [135] studied the survival of
A. lipoferum with Alginate encapsulate, dehydrated
at room temperature. The viable cell count was 10 10 per
gram even after one year. Narendranath [136] reported
the new formulation like Alginate beads, clay balls and
liquid inoculation for the survival of Rhizobium. Alginate
beads supported higher survival of Rhizobium followed
by clay was reported. Long- term survival of inoculants
is a matter of commercial secrecy.
Bashan [137] stated that, the main advantages of
Alginate inoculants are their nontoxic nature, their
degradation in the soil and above all their slow release of
the entrapped microorganisms in the soil. Bashan and
Gonzalez [138] studied and evaluated the capacity of
281
Intl. J. Microbiol. Res., 4 (3): 275-287, 2013
A. brasilense and P. fluorescens for long term survival,
viability and growth promoting capacity after storage in
dry Alginate beads for 14 years. The two bacteria
survived for 14 years in dry Alginate beads at ambient
temperature upon recovery, both species retained the
capacity to enhance plant growth. Fillinger et al. [139]
reported that trehalose is required for the acquisition of
tolerance to a variety of stresses in the filamentous
fungus. Suresh Babu et al. [140] stated that shelf life of
Azospirillum inoculants was improved by the addition of
polymers, chemicals and amendments in the lignite carrier
[141, 142].
5.
6.
7.
8.
CONCLUSION
Formulation of biofertilizer plays a vital role in
helping to solve many problems in agricultural field and in
making an organism effective in the field. However this
must be achieved in a cost effective manner so that
product has to survive commercially. Formulation
comprises aids to preserving organisms and to
delivering them to their target fields and once-there to
improve their activities. A technical concentrate of an
organism that has been achieved by a particular
process is called as formulation. There are varieties of
formulation both liquid and solid. The carrier based
bioinoculants generally suffer from shorter shelf life,
poor quality, high contamination and low field
performance. Therefore, it is desirable that new inoculant
formulations being developed where liquid inoculants
play a significant role.
9.
10.
11.
12.
13.
REFERENCES
14.
1.
2.
3.
4.
Chen, M. and M. Alexander, 1973. Survival of soil
bacteria during prolonged desiccation. Soil Biol.
Biochem., 5: 213-221.
Mary, P., D. Ochin and R. Tailliez, 1985. Rates of
drying and survival of Rhizobium meliloti strains
during storage at different relative humidities.
Appl. Environ. Microbiol., 50: 207-211.
Hahn-Hagerdal, B., 1986. Water activity: a possible
external regulator in biotechnical process. Enzyme
Microb. Technol., 8: 322-327.
Boddey, R.M. and J. Dobereiner, 1988. Nitrogen
fixation associated with grasses and cereals: Recent
results and perspectives for future research. Plant
Soil, 108 : 53-65.
15.
16.
17.
282
Tien, T.M., M.H. Gaskin and D.H. Hubbel, 1979.
Plant growth substances produced by Azospirillum
brasilense and their effect on the growth of pearl
millet (Pennisetum americanum L.). Appl. Environ.
Microbiol., 37(5): 1016-1024.
Neyra, C.A. and J. Dobereiner, 1977. Nitrogen fixation
in greases. Adv. Agron., 29: 1-38.
Lakhsmi kumari, M.,
S.K. Kavimandan and
N.S. Subba Rao, 1976. Occurrence of nitrogen
fixing Spirillum in roots of rice, sorghum, maize and
other plants. Indian J. Exptl. Biol., 14: 638-639.
Barber, L.E., J.D. Tjepkema, S.A. Russel and
H.J. Evans, 1976. Acetylene reduction (N2 fixation)
associated with corn inoculated with Spirillum.
Appl. Environ. Microbiol., 32: 108-113.
Okon, Y. and C.A. Labandera-Gonzalez, 1994.
Agronomic applications of Azospirillum: An
evaluation of 20 years worldwide field inoculation.
Soil. Biol. Biochem., 26: 1591-1601.
Claudia, C. Martin and Didonet, 2000. Genome
structure of the genus Azospirillum. J Bacteriol.,
182 (14): 4113-4116.
Schroder, M., 1932. Die assimilation des Lufstick
stoffs durch einige Bacterian Lentrall. Bakteriol.
Parasitendkd. Infektion Skr. Hyg. Abt., 2(85): 177-212.
Becking, J.H., 1985. Pleomorphism in Azospirillum
spp. pp: 243-262. In: Azospirillum III Genetics,
Physiology,
Ecology, N. Klingmuller (Ed.)
Springer-Verlag, Berlin.
Breed, R.S., E.G.D. Murrary and N.R. Smith, 1957.
Bergey’s manual of determinative bacteriology,
pp: 254-257. Williams and Wilkins, Baltimore,
Maryland.
Krieg, N.R., 1976. The taxonomy of the
Chemoheterotrophic
spirilla.
Annu.
Rev.
Microbiol., 30: 303-325.
De-Polli, H., B.B. Bohlool and J. Dobereinder, 1980.
Serological differentiation of Azospirillum species
belonging to different host plant specificity groups.
Arch. Microbiol., 126: 211-222.
Magalhaes, F.M.M., J.I. Baldani, S.M. Souto,
J.R. Kuykendall and J. Dobereiner, 1983. A new
arid tolerant Azospirillum species. Ann. Acad.
Bras. Cienc., 55: 417-430.
Khammas, K.M., E. Ageron, P.A.D. Grimontand
P. Kaiser, 1989. Azospirillum irakense sp. nov. a
nitrogen-fixing bacterium associated with rice roots
and rhizosphere soil. Res. Microbiol., 140: 679-693.
Intl. J. Microbiol. Res., 4 (3): 275-287, 2013
18. Fani, R.C.,
C. Bandi,
M. Bazzicalupo,
M.T. Ceccherini, S. Fancelli, E. Gallori, L. Gerace,
A. Grifoni, N. Miclaus and G. Damiani, 1995.
Phylogeny of the genus Azospirillum based on
16 srRNA sequesnce. FEMS Microbiol. Lett.,
129: 195-200.
19. Okon, Y., 1985. Azospirillum as a potential
inoculant for agriculture. Trends Biotechnol.,
3: 223-228.
20. DeConincek, K., S. Horemans, S. Randombage and
K.Vlassak, 1988. Occurrence and survival of
Azospirillum spp. in temperate regions. Plant Soil,
110: 213.
21. Michiels, K., J. Vanderleyden and A.Van Gool, 1989.
Azospirillum-Plant root associations. A review,
Biol. Fertil. Soils., 8: 356-368.
22. Sumner, M.E. 1990. Crop response to Azospirillum
inoculation. Ad. Soil. Science, 12: 53-123.
23. Smith, R.L.,
J.H. Boiton,
S.C. Schank,
K.H. Quesenberry, M.E.
Tyler, J.R. Milam,
M.H. Gaskins and R.C. Little, 1976. Nitrogen fixation
in grasses inoculated with Spirillum lipoferum.
Science, 193: 1003-1005.
24. Bashan, Y. and G. Holguin, 1995. Survival of
Azospirillum brasilense in the bulk soil and
rhizosphere
of
23soil types. Appl Environ
Microbiol., 61: 1938-1935.
25. Purushothaman, D., 2002. Biology of Azospirillum
association with cashew (Anacardium occidentale
L.) pp: 1. In: National symposium and XII
Southern Regional Conference on ‘Microbial
inoculants’. Annamalai Univ., India, March 2002
(Abstr.)
26. Rennie, R.J., J.R. Defrietas, A.P. Ruschel and
P.V. Vose, 1983. 15N isotope dilution to quantify
dinitrogen fixation associated with Canadian and
Brazilian wheat. Can. J. Bot., 61: 1667-1671.
27. Lima, E., R.M. Boddey and J. Dobereiner, 1987.
Quantification of biological nitrogen fixation
associated with sugarcane using 15N aided nitrogen
balance. Soil Biol. Biochem., 19: 165-170.
28. Hartmann, A., H.A. Fu and R.H. Burris, 1988.
Influence of amino acids on nitrogen fixation ability
and growth of Azospirillum spp. Appl. Environ
Microbiol., 54(1): 87-93.
29. Kucey, E., Z. Liu and K. Kloppstech, 1993.
Expression of heat shock proteins during
development of barley. Plant Mol. Biol., 23: 111-122.
30. Hurek, T., B. Reinhold, G. Niemann and Fendrik,
1988. Nitrogen dependent growth of Azospirillum
spp. in batch culture at low concentration of
oxygen, pp: 115-121. In: Azospirillum IV. Genetics,
Physiology and Ecology. W. Klingmuller (Ed.),
Springer-Verlag, Berlin.
31. Heulin, T., M. Rahman, A.M.N. Omar, Z. Rafidison,
J.C. Pierrat and J. Balandreau, 1989. Experimental and
mathematical procedures for comparing N2-fixing
efficiencies of rhizosphere diazotrophs. J. Microbiol.
Methods, 9: 163-173.
32. Anand, R.C., R. Ger, M. Hazija and B.S. Kundu,
1999. Development of thermotolerant mutants of
Laz-z-marked
Azospirillum lipoferum, Indian
J. Microbiol., 39(2): 121-123.
33. Gadagi , S., P. Ravi, U. Krishnaraj, J.H. Kulkarni
and S.A. Tongmin, 2004.The Effect of combined
Azospirillum inoculation and nitrogen fertilizer on
plant growth promotion and yield response of the
blanket flower Gaillardia pulchella. Scientia
Horticulturae., 100(1-4): 323-332.
34. Omay, S.H., W.A. Schmidt,
P. Martin and
Bangertti, 1993. Indole acetic acid production by
the rhizosphere bacterium Azospirillum brasilense
cd under in vitro conditions Can. J. Microbiol.,
24: 734-742.
35. Gadagi, S., P. Ravi, U. Krishnaraj, J.H. Kulkarni
and S.A.Tongmin, 2003.The Effect of combined
Azospirillum inoculation and nitrogen fertilizer on
plant growth promotion and yield response of the
blanket flower Gaillardia pulchella. Scientia
Horticulturae., 100(1-4): 323-332.
36. Saikia, S.P., Vanita Jain, Sageeta Khetarpal and
Samitha Aravind, 2007. Dinitrogen fixation activity
of Azospirillum brasilense in maize (Zea mays).
Curr. Sci., 93(9):1296-1299).
37. Hartmann, A., A. Fussader and W. Klingmuller, 1983.
Mutants of Azospirillum affected in N2 Fixation and
Auxin Production, pp: 78-88.
38. Jain, D.K. and D.G. Patriquin, 1984. Root hair
deformation, bacterial attachment and plant growth in
wheat Azospirillum associations. Appl. Environ.
Microbiol., 48: 1208-1213.
39. Horemans, S., K. De Koninck, J. Neuray,
R. Hermans and K. Vlassak, 1986. Production of
plant growth substances by Azospirillum sp.
and other
rhizosphere
bacteria. Symbiosis,
2: 341-346.
283
Intl. J. Microbiol. Res., 4 (3): 275-287, 2013
40. Okon, Y. and Y. Hadar, 1987. Microbial inoculant as
crop-yield enhancers of CRC. Crit. Rev. Biotechnol.,
6: 61-85.
41. Fallik, E., Y. Okon, E. Epstein, A. Goldman and
M. Fischer, 1989. Identification and quantification of
IAA and IBA in Azospirillum brasilense inoculated
maize roots. Soil Biol. Biochem., 21: 147-153.
42. Cacciari, L.,
D. Lippi,
T. Pietrosanti and
W. Pietrosanti, 1989. Phytohormones like substances
produced by single and mixed diazotrophic
cultures of Azospirillum and Arthrobacter. Plant
Soil, 115: 151-153.
43. Zimmer, W., C. Aparicio and C. Elmerich, 1991.
Relationship between tryptophan biosynthesis and
undole-3 acetic acid production in Azospirillum:
identification and sequencing of a to GDC cluster.
Mol. Gen. Genet., 229: 41-51.
44. Janzen, R.A.,
S.B. Rood, J.F. Dormaar and
W.B.
McGill, 1992. Azospirillum brasilense
produces Gibberellin in pure culture on chemically
defined medium in co-culture on straw. Soil Biol.
Biochem., 24: 1061-1064.
45. Barberi, P. and E. Galli, 1993. Effect on wheat
root
development
of
inoculation with an
Azospirillum brasilense mutant with altered
indole-3 acetic acid production. Res. Microbiol.,
144: 69-75.
46. Omay, S.H., W.A. Schmidt, P. Martin
and
Bangertti, 1993 . Indole acetic acid production by
the rhizosphere bacterium Azospirillum brasilense
cd under in vitro conditions Can. J. Microbiol.,
24: 734-742.
47. Fulchieri, M. and L. Frioni, 1994. Azospirillum
inoculation on maize (Zea mays) : effect of yield in a
field experiment in Central Agentina. Soil Biol.
Biochem., 26: 921-924.
48. Piccoli, P. and R. Bottini, 1994. Effects of C/N ratio
N content, pH and inoculation time on growth and
gibberellin production by Azospirillum lipoferum.
49. Neyra, C.A, A. Alkinson and O. Olubayi 1995.
Coaggregation of Azospirillum with other bacteria:
basis for functional diversity. NATO, ASI, Ser. G.,
37: 429-439.
50. Vande Brock, A., M. Lambrecht, K. Eggermont
and J. Vanderleyden, 1995 . Auxins upregulate
expression of the indole-3-pyruvate decarboxylase
gene in Azospirillum brasilense. J. Bacteriol.,
181(4): 1338-1342.
51. Vande Brock, A., M. Lambrecht, K. Eggermont
and J. Vanderleyden, 1999. Auxins upregulate
expression of the indole-3-pyruvate decarboxylase
gene in Azospirillum brasilense. J. Bacteriol.,
181(4): 1338-1342.
52. Fabricio Cassan Ruben Bottini Gernot Schneider
and Patricia Piccoli, 2001. Azospirillum brasilense
and Azospirillum lipoferum Hydrolyze Conjugates
of GA20 and metabolize the resultant Aglycones to
GA1 in Seedlings of Rice Dwarf Mutants. Plant
Physiol., 125(4): 2053-2058.
53. Kolb, W. and P. Martin, 1985. Response of plant
roots to inoculation with Azospirillum brasilense
and to Application of Indole Acetic Acid,
pp: 215-221.
In: Azospirillum III. Genetics,
Physiology, Ecology, W. Klingmuller (Ed.) SpringerVerlag, Berlin.
54. Perrig, D., M. Boiero, O.L.A. Masciarelli, C. Penna,
O.A. Ruiz, F. Cassan and M. Dand Luna, 2007. Appl.
Microbiol., 75: 1143-1150.
55. Tarrand, J.J., N.R. Kreig and J. Dobereiner, 1978.
A taxonic study of the Spirillum lipoferum
group, with descriptions of a new genus,
Azospirillum gen. Nov. and two species,
Azospirillum brasilense (Beijerinck) comb. Nov. and
Azospirillum brasilense sp. nov. Can. J. Microbiol.,
24: 967-980.
56. Okon, Y., 1985. Azospirillum as a potential
inoculant for agriculture. Trends Biotechnol.,
3: 223-228.
57. Bashan, Y., 1986. Significance of timing and level of
inoculation with rhizosphere bacteria on wheat
plants. Soil Biol. Biochem., 18: 297-301.
58. Bashan, Y. and H. Levanony, 1990. Current status
of
Azospirillum
inoculation
technology.
Azospirillum as a challenge for agriculture. Can.
J. Microbiol., 35: 591-608.
59. Fages J., 1994. Azospirillum inoculants and field
Experiments, pp: 87-110. In: Azospirillum-Plant
Association. Y. Okon, (Ed.) CRC Press, Boca Raton,
Fla.
60. Hume, D.J. and D.H. Blair, 1992.Effects of members
of Bradyrhizobium japonicum applied in
commercial inoculants in soya bean seed fields in
Ontario. Can. J. Microbiol., 38: 582-593.
61. Okon, Y., 1985. Azospirillum as a potential
inoculant for agriculture. Trends Biotechnol.,
3: 223-228.
284
Intl. J. Microbiol. Res., 4 (3): 275-287, 2013
62. Omay, S.H., W.A. Schmidt, P. Martin and Bangertti,
1993. Indole acetic acid production by the
rhizosphere bacterium Azospirillum brasilense cd
under in vitro conditions Can. J. Microbiol.,
24: 734-742.
63. Iswaran, V., 1969. Growth and survival of R. trifoli
in coir dust and soybean meal compost. Madras
Agric. J., 59: 52.
64. Tilak, K.V.B.R. and N.S. Subba Rao, 1978. Carries for
legume inoculants. Fert. News., 23: 25-28.
65. Tilak, K.V.B.R., 1979. Survival of Azospirillum
brasilense in different carriers. Curr. Sci., 48: 412.
66. Tilak, K.V.B.R. and N.S. Subba Rao, 1978. Carries for
legume inoculants. Fert. News., 23: 25-28.
67. Sparrow, S.D. and G.E. Han, 1981. Survival of
Rhizobium phaseoli in six carrier materials. Agron.
J., 75: 181-184.
68. Kumar Rao, J.V.D., K.C. Mohan Kumar and
R.B. Patil, 1982. Alternate carrier materials for
Rhizobium inoculant production. Mysore J. Agric.
Sci., 16: 2520255.
69. Somasegaran, P., 1985. Inoculant production with
diluted liquid cultures of Rhizobium spp and
autoclaved
peat.
Evaluation
of
dilutents.
Rhizobium spp peats, sterility requirements, storage
and plant effectiveness. Appl. Environ. Microbiol.,
50(2): 398-405.
70. Singaravadivel, K. and S. Anthoni Raj, 1988. Rice mill
by products as carrier for Rhizobium. Legume Res.,
11: 143-145.
71. Gaind, S. and A.C. Gaur, 1990. Shelf life of phosphate
solubilizing inoculants as influenced by type of
carrier, high temperature and low moisture. Can. J.
Microbiol., 36: 846-849.
72. Fages, J., 1992. An industrial view on Azospirillum
inoculants: formulation and application technology.
Symbiosis, 13: 15.
73. Narendranath, N.V., 1995. Shelf life of Rhizobium
inoculant as influenced by carries and storage
conditions. M.Sc. (Ag.) Thesis, Tamil Nadu Agrl.
Univ., Coimbatore, India.
74. Abd-Alla, M.H. and S.A. Omar 2001.Carriers for
biofertilizer. J. Plant. Nutr., 24(2): 261-272.
75. Roughley, R.J., 1968. Some factors influencing the
growth and survival of root nodule bacteria in peat
culture. J. Appl. Bacteriol., 31: 259-265.
76. Date, R.A. and R.J. Roughley, 1977. Legume inoculant
production. Indian Natl. Sci. Acad Bull., 51: 67-686.
77. Kannaiyan, S., 2000. Biofertilizer technology and
quality control. Publication directorate, TNAU,
Coimbatore, India, pp: 256.
78. Roughley, R.J., 1968. Some factors influencing the
growth and survival of root nodule bacteria in peat
culture. J. Appl. Bacteriol., 31: 259-265.
79. Vincent, J.M., 1958. Nutrition of the legumes.
E.G. Hallsworth (Ed.) Butterwoetho. London. U.K.,
pp: 108-123.
80. Van Schreven, D.A., 1970. Some factors influencing
growth and survival of Rhizosphere spp. in soul peat
cultures. Plant Soil, 32: 113-130.
81. Saha, A.K., M.V. Deshpande and B.P. Kapadnis,
2001. Studies on survival of Rhizobium in the
carriers at different temperature using green
fluorescent protein marker. Curr. Sci., 80(5): 69-671.
82. Hedlin, R.A. and J.W. Newton, 1948. Some
factors influencing the growth and survival of
rhizobia in humus and soil culture. Can. J. Res.
Sect. C., 26: 174-187.
83. Roughley, R.J. and J.M. Vincent, 1967. Growth and
survival of Rhizobium spp. in peat culture. J. Appl.
Bacteriol., 30: 362-376.
84. Date, R.A. and R.J. Roughley, 1977. Legume inoculant
production. Indian Natl. Sci. Acad Bull., 51: 67-686.
85. Roughley, R.J., 1968. Some factors influencing the
growth and survival of root nodule bacteria in peat
culture. J. Appl. Bacteriol., 31: 259-265.
86. Smith, R.S., 1987. Production and quality control of
inoculants. In: Symbiotic nitrogen fixation
technology. G.H. Elkan, (Ed.) Marcel Dekker,
New York, pp. 391-411.
87. Burton, J.C., R.L. Curley and P.S. Nutman, 1965.
Comparative efficiency of liquid and peat based
inoculants of field grown soybean (Glycine max).
Agron. J., 57: 379-381.
88. Roughley, R.J., 1968. Some factors influencing the
growth and survival of root nodule bacteria in peat
culture. J. Appl. Bacteriol., 31: 259-265.
89. Strijdom, B.W. and C.C. Deschodt, 1976. Carriers of
rhizobia and the effect of prior treatment on the
survival of rhizobia. In: Symbiotic Nitrogen
Fixation in Plants (ed.) P.s. Nutman. International
Biological Programme 7. Cambridge University press,
Cambridge, pp: 151-168.
90. Somasegaran, P. and J. Halliday, 1982. Dilution of
liquid Rhizobium cultures to increase production
capacity of inoculant plants. Appl. Environ.
Microbiol., 44: 330-333.
285
Intl. J. Microbiol. Res., 4 (3): 275-287, 2013
91. Van Schreven, D.A., 1958. Some factors affecting
uptake of nitrogen by legumes. In: Nutrition of the
legumes. Butterwirth & Co., London, UK, pp: 137-163.
92. Santhanakrishnan, P. and M. Thangaraju, 2002.
Enhancement of shelf life of Azospirillum through
addition of polymers and chemicals is carrier
based inoculant. Biofertilizer Newsletter, Dec. 3-10.
93. Roughley, R.J., 1968. Some factors influencing the
growth and survival of root nodule bacteria in peat
culture. J. Appl. Bacteriol., 31: 259-265.
94. Iswaran, V., 1972. Growth and survival o f R.trifoli
in coir dust and soybean meal compost. Madras
Agric. J., 59: 52.
95. Strijdom, B.W. and C.C. Deschodt, 1976. Carriers of
rhizobia and the effect of prior treatment on the
survival of rhizobia. In: Symbiotic Nitrogen Fixation
in Plants (ed.) P.s. Nutman. International Biological
Programme 7. Cambridge University press,
Cambridge, pp: 151-168.
96. Subba Rao, N.S., 1982. Rhizobium inoculant,
pp: 15-69. In: Biofertilizers in Agriculture. Oxford
and IBH Pub. Co. New Delhi.
97. Govindarajan, K,. 1996. Shelf life of Azospirillum
inoculant as influenced by carrier material and type
of packing. Paper presented in National Seminar on
Biofertilizer production problems and constraints.
TNAU, Coimbatore, Jan. 24-25 (Abs.) pp: 38.
98. Fages, J., 1990. An optimized process for
manufacturing Azospirillum inoculant for crops.
Appl. Microbiol. Biotechnol., 32: 473-478.
99. Kandasamy, R. and N.N. Prasad, 1971. Lignite as a
carrier of rhizobia. Curr. Sci., 40: 496.
100. Pugashetti, B.K., H.S. Gopal Gowda and R.B. Patil,
1971. Cellulose powder as a legume inoculant base.
Curr. Sci., 40: 494.
101. Iswaran, V., 1972. Growth and survival of R. trifoli
in coir dust and soybean meal compost. Madras
Agric. J., 59: 52.
102. Strijdom, B.W. and C.C. Deschodt, 1976. Carriers of
rhizobia and the effect of prior treatment on the
survival of rhizobia. In: Symbiotic Nitrogen
Fixation in Plants (ed.) P.s. Nutman. International
Biological Programme 7. Cambridge University
press, Cambridge, pp: 151-168.
103. Tilak, K.V.B.R. and N.S. Subba Rao, 1978. Carries for
legume inoculants. Fert. News., 23: 25-28.
104. Kumar Rao, J.V.D., K.C. Mohan Kumar and
R.B. Patil, 1982. Alternate carrier materials for
Rhizobium inoculant production. Mysore J. Agric.
Sci., 16: 2520255.
105. Sparrow, Jr., S.D. and G.E. Ham, 1983. Survival of
Rhizobium phaseoli in six carrier materials.
Agron. J., 75: 181-184.
106. Graham-Weiss, L., M.L. Bennett and A.S. Paau,
1987. Production of bacterial inoculants by direct
fermentation on nutrient supplemented vermiculite.
Appl. Environ. Microbiol., 53: 2138-140.
107. Narendranath, N.V., 1995. Shelf life of Rhizobium
inoculant as influenced by carries and storage
conditions. M.Sc. (Ag.) Thesis, Tamil Nadu Agrl.
Univ., Coimbatore, India.
108. Senthil Kumar, M., 1999. Improved methodologies
for legume inoculant production, M.Sc. (Ag.)
Thesis, Tamil Nadu Agric. Univ., Coimbatore,
India, pp: 163.
109. Daza, A., D.N. Santamaria, P.N. Rodriguez-Navarro,
M. Camacho, R. Orive and F. Temprano, 2000.
Perlite as a carrier for bacterial inoculants. Soil Biol.
Biochem., 32: 567-572.
110. Orzolck, M.D., 1993. Use of hydrophilic polymers in
horticulture. Hort. Technology, 3(1): 41-44.
111. Blodgett, A.M., D.J. Beattie, J.W. White and
G.C. Elliott, 1993. Hydrophilic polymers and wetting
agents affect absorption and evaporative water
loss. Hort Science, 28(6): 633-635.
112. Dhumal, K.N., 1991. Effects of jalsakthi on growth
and yield of some vegetables under water stress
condition. J. Maharashtra Agric. Univ., 18(2): 307.
113. Bharambe, P.R., R.P. Rodge, P.R. Amhegamokar
and S.R. Oza, 1993. Effects of jalskathi and
Rhizobium application on yield and wat Burton,
J.C., R.L. Curley
and P.S. Nutman, 1965.
Comparative efficiency of liquid and peat based
inoculants of field grown soybean (Glycine max).
Agron. J., 57: 379-381.
114. Subbaraj, D., P.P. Ramaswamy and G. Arunachalam,
1993. Efficiency of Qemisorb agricultural polymers
for water retention in tree cropped their lands.
South Indian Horticulture, 416: 379-380.
115. Szmidt, D., 1994. Making the desert bloom.
The Horticulturist, 3: 10-25.
116. Grula, M.M., M.L. Huang and G. Sewell, 1994.
Interactions of certain polyacrylamides with soil
bacteria. Soil Sci., 158: 291-300.
117. Tomar, S.P., 1997a. Terracotton to save water and
increase net returns. Kisan World, Sep., pp: 43-44.
118. Nethery, A.A. 1991. Inoculant production with
nonsterile carrier. In: Expert consultation on legume
inoculant production and quality control. FAO,
Rome, pp: 43-50.
286
Intl. J. Microbiol. Res., 4 (3): 275-287, 2013
119. Van
Schreven,
D.A,
G.W. Harmsen,
D.T. Lindenbergh and D. Otzen, 1953. Experiments
on the cultivation of Rhizobium in liquid media
for use on zindderzee polders. Anton van
Leeuwenhoek, 19: 300-308.
120. Burton, J.C., R.L. Curley and P.S. Nutman, 1965.
Comparative efficiency of liquid and peat based
inoculants of field grown soybean (Glycine max).
Agron. J., 57: 379-381.
121. Schiffman, J. and Y. Apler, 1968. Inoculation of
peanuts by application of Rhizobium suspension
into planting furrows. Expt. Agric., 4: 219-226.
122. Wani, S.P. and P.V. Rai, 1980. Response of
sorghum to foliar sprays and seed inoculation
with Azotobacter. Indian J. Microbiol., 20: 319-320.
123. Smith, R.S., M.A. Ellis and R.E. Smith, 1981. Effect of
R. japonicum inoculant rates on soybean nodulation
of a tropical soil. Agron. J., 73: 505-508.
124. Smith, R.S., 1992. Legume inoculant formulation and
application. Can. J. Microbiol., 38: 485-492.
125. Hynes, R.K., K.A. Craig, D. Covart, R.S. Smith
and R.J. Rennie, 1995. Liquid rhizobial inoculants
for lentil and field pea. J. Prod. Agric., 8(4): 547-552.
126. Bhattacharya, P. and R. Kumar, 2000. Liquid
biofertilizer-Current
knowledge
and future
prospect. Paper presented in National Seminar on
Development & Use of Biofertilizers, Biopesticides
and Organic manures, Kalyani, West Bengal, India.
127. Inamdar, S., K. Raviu and W.G. Millind, 2000.
Longevity of Azotobacter cysts and a model for
optimization of cyst density in liquid bioinoculants.
Curr. Sci., 78(6): 719-722.
128. Singlenton, P.W., H.H. Keyser and E.S. Sande, 2002.
Development and evaluation of liquid inoculants.
In Inoculants and nitrogen fixation of legumes
in Vietnam.(ed.)D Herridge, AICAR Proceedings,
109e: 52-66.
129. Vendan, R.T. and M. Thangaraju, 2007. Acta
Microbiol. Immunol Hung., 54(2): 167-167.
130. Vendan R.T. and M. Thangaraju, 2006. Development
and standardization of liquid formulation for
Azospirillum bioinoculant Indian J. Microbiol.,
46(4): 379-387.
131. Jung, G., J. Mugnier, H.G. Dim, Y.R. Diem and
Y.R. Dommergues, 1982.
Polymer entrapped
Rhizobium. Curr. Sci., 38: 468-469.
132. Fages, J., 1990. An optimized process for
manufacturing Azospirillum inoculant for crops.
Appl. Microbiol. Biotechnol., 32: 473-478.
133. Hegde, S.V. and G.P. Brahmaprakash, 1992. A dry
granular inoculant
of
Rhizobium for soil
application. Plant Soil, 144: 309-311.
134. Fages, J., 1992. An industrial view on Azospirillum
inoculants: formulation and application technology.
Symbiosis, 13: 15.
135. Okon, Y., 1994. Azospirillum as a potential inoculant
for agriculture. Trends Biotechnol., 3: 223-228.
136. Narendranath, N.V., 1995. Shelf life of Rhizobium
inoculant as influenced by carries and storage
conditions. M.Sc. (Ag.) Thesis, Tamil Nadu Agrl.
Univ., Coimbatore, India.
137. Bashan, Y., 1998. Inoculants for plant growth promoting bacteria in agriculture. Biotechnol. Adv.,
16: 729-770.
138. Bashan, Y. and L.E. Gonzalez, 1999. Long-term
survival of the plant-growth-promoting bacteria
Azospirillum
brasilense
and
Pseudomonas
fluorescens in dry alginate inoculants. Appl.
Microbiol Biotechnol., 51: 262-266.
139. Fillinger, S., M. Chaeroche, P. Van Dijck and
C. De Enfert, 2001. Trehalose is required for the
acquisition of tolerance to a variety of stresses in
the filamentous fungus Aspergillus nidulans.
Microbiol., 147: 1851-1862.
140. Suresh Babu,
S.,
M.
Thangaraju and
P. Santhanakrishnan, 2002. Shelf life improvement of
Azospirillum inoculants by addition of polymers,
chemicals and amendments in the lignite carrier.
J. Microb. World, 4: 51-58.
141. Saranraj, P., P. Sivasakthivelan and S. Siva Sakthi,
2013. Prevalence and production of plant growth
promoting substance by Pseudomonas fluorescens
isolated from paddy rhizosphere soil of Cuddalore
district, Tamil Nadu, India. African Journal of Basic
and Applied Sciences, 5(2): 95-101.
142. Sivasakthi, S.,
D. Kanchana, G. Usharani and
P. Saranraj, 2013. Production of plant growth
promoting substance by Pseudomonas fluorescens
and Bacillus subtilis isolated from paddy
rhizosphere soil of Cuddalore district, Tamil Nadu,
India. International Journal of Microbiology
Research, 4(3): 227-233.
287