biosullllry

International Journal of Microbiological Research 4 (3): 275-287, 2013ISSN 2079-2093© IDOSI Publications, 2013DOI: 10.5829/idosi.ijmr.2013.4.3.825

Corresponding Author: P. Saranraj, Department of Microbiology,Annamalai University, Annamalai Nagar - 608 002, Tamil Nadu, India.

275

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 adoptionof this technology, which are related to technological, developmental, social issues. Biofertilizers manufacturedin India presently are carrier based, in general and suffer from short shelf life, poor quality, high contaminationand low and unpredictable field performances. Death of the organisms in the inoculated seeds is one of theimportant factors contributing the failure of inoculation response in field condition. Research conducted on theinoculant production and formulation technologies is limited. A break through is needed in the inoculationtechnology to improve the shelf life and field efficacy of biofertilizer in India to make them commercially viableand 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

Biofertilizers are carrier based preparations containing are important.beneficial microorganisms in a viable state intended for Liquid biofertilizers are special liquid formulationseed or soil application and designed to improve soil containing not only the desired microorganisms and theirfertility and to help plant growth by increasing the number nutrients, but also special cell protectants or substancesand biological activity of desired microorganisms in the that encourage formation of resting spores or cysts forroot environment. Biofertilizers are products of selected longer shelf life and tolerance to adverse conditions.beneficial and live microorganisms, which help to improve Azospirillum is one of the important biofertilizer, which isplant growth and productivity mainly through supply of found to fix nitrogen in association with world’s mostplant nutrients. Biofertilizers are also known as microbial staple food crops like rice, maize, sorghum, wheat andinoculants or bio inoculants. Biofertilizers have come to millets. Members of the genus Azospirillum arestay in Indian agriculture since last three decades in widespread in soils and its inoculation of cereal andview of their cost effectiveness, contribution to crop forage crops resulted in yield increases in many fieldproductivity, soil sustainability and ecofriendly experiments [4], not only due to the nitrogen fixation butcharacters. Biofertilizers form an integral part of Integrated also through the production of plant growth promotingPlant Nutrient Supply system (IPNS) and organic farming substances [5].which constitutes the present as well as future mandate ofIndian agriculture. One of the main problem in inoculant The Genus Azospirillum: Azospirillum spp. is oftechnology is the survival of microorganisms duringstorage and several parameters such as the culturemedium, physiological state of the microorganismswhen harvested [1] the process of dehydrates, rate ofdrying [2] the temperature of storage and water activityof the inoculum [3] have an influence on their shelf life.

finding alternate formulations for carrier based inoculants

ubiquitous distribution in many parts of the worldconsisting of tropical, sub-tropical and temperate climaticconditions and also with various standing crops grownin a variety of soil types. Azospirillum spp. fixesatmospheric nitrogen and has been isolated from therhizosphere of a variety of tropical and sub tropical non

Intl. J. Microbiol. Res., 4 (3): 275-287, 2013

276

leguminous plants [6]. Lakshmikumari et al. [7] reported Azospirillum mostly lives associated with thethe occurrence of nitrogen fixing Spirillum in the roots of plants, particularly in their rhizosphere, on the rootrice, sorghum and maize. Various cereals have responded surface and to a lesser extent, inside the root [23],differently to field inoculation with this organism [8]. Bashan and Holguin [24] reported that the ability ofOkon and Labandera Gonzalez [9] evaluated world wide Azospirillum to colonize atleast 64 plant species of whichdata accumulated over the past 20 years on field 18 are weed species. The association of Azospirillum withinoculation with Azospirillum and concluded that these cashew and its possible role in improving crop growthbacteria are capable of promoting the yield of and yield was reported by Purushothaman [25].agriculturally important crops in different soil andclimatic regions. The genomes of five of the six Nitrogen Fixation by Azospirillum: Azospirillum spp isAzospirillum spp. were analyzed by pulsed-field gelelectrophoresis strains DNA hybridization indicatedmultiple chromosomes in genomes ranging in size from4.8 to 9.7Mbp. The nif HDK operon was identified in thelargest replicon [10].

Isolation of Azospirillum Species: The soil bacteriumAzospirillum was first isolated and originally named as could be supplied by associated nitrogen fixers mainlySpirillum lipoferum. This bacterium was later isolated Azospirillum.from soil and from dried seaweed in Indonesia [11] Hartmann et al. [28] reported the influence of aminoand as a phyllosphere bacterium in tropical plants [12]. acids on nitrogen fixation ability and growth ofBreed et al. [13] classified the genus Spirillum in the Azospirillum spp. The utilization of amino acids fororder Pseudomonodales. Krieg [14] noted that growth and their effects on nitrogen fixation differS. lipoferum might belong to the genus Azospirillum. greatly among the several strains of each species ofDe Polli et al. [15] identified A. lipoferum as more Azospirillum spp. The different utilization of varioushomologous group, whereas A. brasilense recorded three amino acids by Azospirillum spp may be important forsub groups with respect to fluorescent antibody reaction. their establishment in the rhizosphere and for their

Magalhaes et al. [16] reported a new acid tolerant associative nitrogen fixation with plants. Kucey et al. [29]nitrogen fixing species, Azospirillum amazonanse which indicated that upto 18 per cent of the plant nitrogengrows at pH 6.0 temperature 35°C and with G + C percent was derived from nitrogen fixation. All wild typeas 67. Khammas et al. [17] isolated a new strain of Azospirillum strains were found to fix N efficiently eitherAzospirillum from the rice rhizosphere of Diwaniya, as free-living or in association with plants [30].Irag and classified into a new species as Azospirillum Heulin et al. [31] reported that association with rice,irakense. Fani et al. [18] investigated phylogenetic Azospirillum lipoferum N-4 contributed about 66% ofrelationship of Azospirillum based on 16S rRNA at the the total N in plants as was demonstrated with N isotopeinner and sub generic level. The phylogenetic analysis studies. The mesophilic lac-z-marked Azospirillumconfirmed that Azospirillum genus into separate entity lipoferum ALP 3 did not grow and fix nitrogen at 45°C outwith in sub class of proteobacteria with five species of 40 thermo tolerant marked mutants, developed fromproperly defined. ALP 3 and screened for N-fixing ability at 30°C and 45°C,

The occurrence of Azospirillum in the rhizosphere only 14 mutants could grow and fix nitrogen at 45°C [32].varied from 1 to 10 per cent to the total rhizosphere Gadagi et al. [33] examined the effect of combinedpopulation [19]. The rhizosphere soil samples contained Azospirillum inoculation and nitrogen fertilizer on plant100 times more number of Azospirillum than the growth promotion and yield response of the blanketnon-rhizosphere soil [20]. Azospirilla have been flower Gaillardia pulchella. Azospirillum strain OAD-2isolated from a wide variety of plants from all over the inoculation significantly increased plant height, numberworld from tropical, temperate, salt affected soils, desert of leaves per plant, branches per plant and total dry masssoils to water flooded conditions [21]. The occurrence of accumulation in G. pulchella than other inoculations orAzospirillum sp. was reported in the rhizosphere of un inoculated controls. Azospirillum strains OAD-2 andvarious field grown crops such as rice, wheat, maize, OAD-11 can play an important role in the N nutrition ofsorghum and pearl millet of different locations [22]. G pulchella.

one of the most efficient N fixers in the field when all the2

required conditions for biological nitrogen fixation arepresent. Most studies on the Azospirillum inoculationhave suggested that nitrogen fixation was the majormechanism of plant growth [26]. Lima et al. [27] noted thatup to 50 per cent of the N content of crops such assugarcane, Panicum maximum and Paspalum notatum

2

15


277

Omay et al. [34] reported Azospirillum sp A. brasilense and the barley straw degrading fungusinoculation in wheat, barley and oats seeds in Trichoderma harzianum produced a substantial increasegreenhouse experiments. For wheat significant of GA was reported by Janzen et al. [44].increases were obtained when the inoculation was Barberi and Galli [45] noted that inoculatedassociated to 100% of the recommended nitrogen. For A. brasilense SPM 7918 promoted the root systembarley the presence of the inoculants substituted 20% development on wheat seedlings. In liquid culture ofof the recommended nitrogen fertilization. For oats the A. brasilense Cd, the concentration of IAA increasedinoculation with Azospirillum sp RAM-7 did not provide rapidly at the beginning of the stationary phase when thea significant increase in grain yields. sole carbon source (DL-Malic acid) was a limiting factor.

Gadagi Ravi et al. [35] reported the effect of This suggested that the higher increase in IAA in theAzospirillum amazonense inoculation on growth, yield stationary phase in the expression of an overall change inand N fixation of rice. Bacteria of the genus Azospirillum cell metabolism when the carbon source is exhausted [46]2

stimulate plant growth directly either by synthesizing Gibberellin GA had effects similar to Azospirillumphyto-hormones or by promoting nutrition by the lipoferum inoculation in increasing root hair density inprocess of biological nitrogen fixation. All A. amazonense corn seedlings [47]. Gibberellin GA production bystrains tested produced indoles, but only 10% of them stationary phase pure cultures of Azospirillum lipoferumshowed high production. The nitrogenase activity also appeared to be correlated with culture viability andwas variable and only 9% of isolates showed high polysaccharide excretion that is favoured in turn by anitrogenase activity and the majority (54%) exhibited a specific N concentration and initial pH of the culture [48].low potential. Production of major quantities of IAA and Gibberellin

Saikia et al. [36] described that dinitrogen fixation at relatively older stages of the bacterial growth may implyactivity of Azospirillum brasilense in maize. The presence that this might be relevant to the field rhizosphereof nitrogenase activity was noticed in plants treated with interaction of Azospirillum with plants where the bacteriaAzospirillum. Plant root- rhizosphere N fixing bacterial are seldom at the logarithmic phase yet affect plant2

interactions is required before any consistent, significant development. This may also explain why very oldand beneficial N - fixing association can be developed. aggregated cultures (flocs) induced positive plant2

Plant Growth Substances Production by Azospirillum: of the role of hormones in general and IAA in particularHartmann et al. [37] revealed that certain mutants ofA. brasilense exerted higher quantities of IAA in theculture upto 16 µg ml . Inoculation of the plants with1

IAA over producing mutants of A. brasilense resulted inincreased root numbers, length and root hairs in wheat[38]. Horemans et al. [39] reported that the in vitroproduction of cytokinin by Azospirillum. Okon and Hadar[40] observed in sorghum, an increase in root length androot hairs due to Azospirillum inoculation. The higheramount of IAA, IBA in the maize roots inoculated withA. brasilense than in non inoculated roots were reportedby Fallik et al. [41]. The amounts of gibberellin andcytokinins produced by the mixed culture of A. brasilenseand Arthrobacter giacomelloi were higher than those ofsingle cultures, suggesting that the interaction betweenthe rhizosphere inhabitants may affect their secondarymetabolism and indirectly the plants [42].

Zimmer et al. [43] screened the tryptophan dependentIAA production of different Azospirillum spp andrevealed that A. irakense KA released 10 times less IAA3

into the medium than A. brasilense SP 7. The culture of

3

3

responses after inoculation [49]. For a better evaluation

in plant promotion, there is a need for a mutanttotally deficient in IAA production. This mutant isstill unavailable [50]. The same is true for otherphytohormones. The gene ipd C encoding, an indole-1-pyruvate decarboxylase involved in the synthesis ofIAA showed growth phase dependent expression.External IAA and synthate auxins could be involved inthe up regulation of expression of A.brasilense IAAbiosynthetic gene ipd [51].

Fabricio Cassan et al. [52] reported that Azospirillumbrasilense and Azospirillum lipoferum hydrolyzeconjugates of GA and metabolize the resultant20

aglycones yo GA in seedlings of rice dwarf mutants.1

A. brasilense strain cd and A. lipoferum strain USA5b promoted sheath elongation growth of two single geneGA-deficient dwarf rice mutants, dy and dx when theinoculated seedlings were supplied with GA – glucosyl20

ether.Kolb and Martin [53] described the isolation and

selection of indigenous Azospirillum spp. and the IAA ofsuperior strains effects on wheat roots. IAA produced by


278

bacteria of the genus Azospirillum spp. can promote successful inoculation is maximized. Since crop responseplant growth by stimulating root formation. Native to inoculation, in most instances, is the result ofAzospirillum spp. isolated from Iranian soils had been appropriate strain selection and target organismevaluated. The roots of wheat seedlings responded population [60]. Okon [61] reported Azospirillum as apositively to the several bacteria inoculations by an commercial inoculant for improving crop yields. Theincrease in root length, dry weight and by the lateral root development of an effective seed or soil applied legumehairs. Perrig et al. [54] reported that plant growth inoculant requires the integration of physical, chemicalpromoting compounds produced by two strains of and biological processes was stated by StephenesAzospirillum phytohormones Indole-3 acetic acid (IAA), et al.[62].Gibberillic acid (GA ) Abscisic acid (ABA) and growth3

regulators putrescine, spermine, spermidine and Different Carrier Used for Microbial Inoculantcadaverine were found in culture supernatant of both Production: Peat is generally as most dependable carrierstrains. This is the first report on the evaluation of because of its high content of organic matter and waterimportant bioactive molecules in strains of A. brasilense holding capacity [63]. In India, Peat like material isas potentially capable of direct plant growth production available in Nilgris valley to the extent of 5.5. Millionor agronomic yield increase. This fact has important tones and an unknown quality is also known to occur intechnological implications for inoculant formulations as Kashmir valley [64]. Tilak [65] observed the survival ofdifferent concentrations of growth regulators produced Azospirillum upto 31 weeks in carriers like farm yardby different strains. manure (FYM), FYM + soil, FYM + charcoal and soil,

Biofertilizer Inoculant Technology: Tarrand et al. [55] Azospirillum than others.reported that some of the most promising organism, Farm yard manure, compost, coconut shell powder,capable of colonizing roots in large numbers and exerting vermiculite clay, teak leaf powder was also used asbeneficial effects on plants belong to the genus carriers for inoculant production [66]. Sparrow and HanAzospirillum. Azospirillum helps in efficient use of [67] studied the survival of Azospirillum in peat,applied fertilizers and enriching the soil with nitrogen powdered peanut shell, corn cobs, vermiculite andfixed in association with the roots. The Azospirillum polyacrylamide gel and reported that vermiculiteinoculant is efficiently used so far as carrier based supported 10 cells of rhizobia g of carrier even afterinoculant and this is high adsorptive, easy to process and 30 weeks of period. The survival of Azospirillum innon toxic to Azospirillum [56]. number of locally available material such as lignite,

Inoculant preparation and inoculation technologies pressmud, charcoal, soil and coffee waste and found to bemust be improved. In addition, the inoculated better than other carriers including peat for the survival ofAzospirillum should reach the root even if the root Azospirillum upto 200 days the rate of decline insystem is widely spread and the bacterial inoculation Azospirillum population was least in pressmud [68].should be at the precise time needed by the plant [57]. Several experimental Azospirillum inoculantThe inoculation techniques should be practical, formulations have been proposed based on theeconomical and easy to accomplish for the farmer. biopolymer like Alginate and xanthan gum which wereThe formulated products should deliver sufficient shown to be good carriers. These carriers protect theinoculum to the plant must be competitive with existing organisms against stress conditions [69].commercial standards and must possess a long shelf life Singaravadivel and Anthoni Raj [70] reported that[58]. The development of adequate formulations, which black ash, paddy husk, black ash plus husk mixture,would ensure survival and protection of the strain and the husk powder and pressmud were effective andapplication technology, which would allow timely, easy satisfactory carriers for Rhizobium and were on par withand precise delivery in the field could be a major step lignite and peat. These carriers maintained the viable celltowards this goal [59]. load of 10 cells g even after 6 months. Gaind and Gaur

The use of a microbial inoculant is primarily [71] examined the charcoal-soil mixture as the best carrierconcerned with crop productivity, not bacterial for phosphate solubilizing microorganisms. Fages [72]physiology nor ecology. Inoculants manufactures must reported that the survival of Azospirillum in variousaccept this reality and ensure that the probability of carriers such as peat, vermiculite, Alginates and liquid

FYM + charcoal + soil gave higher viable counts of

7 1

7 1


279

formulation and reported that better results have been Aeration: Canadian and European workers observedobtained with cells in dried microgranulated Alginate rapid death of rhizobia in sealed containers, but withformulation. access to air their numbers remained high until the

Narendranath [73] studied the suitability of different carrier became desiccated [82]. In contrast, other workerscarrier materials for inoculant preparation and reported found that rhizobia were able to multiply 10 to 100 foldthat incorporation of amendments like soymeal (1%) and and survive satisfactorily in either screw-capped jars ormolasses (1%) in the carrier enhanced the survival of sealed cans. Some of this confusion is possibly causedRhizobium and also suggested that pressmud amended by the fact that the demand for gas exchange in peatwith soymeal, as an alternative carrier to peat in inoculant cultures though definite is not high [83]. Most reportspreparation. Abd-Alla et al. [74] explained different compared only “unrestricted exchange” containers withcarriers for biofertilizers. sealed containers and where the lacter were not sealed

Factors Affecting the Shelf Life of Microbial Inoculants carrier material may have allowed better survival of theMoisture Content: Roughley [75] showed that clover and organisms [84].cowpea type rhizobia are adversely affected by moisture Roughley [85] examined the growth and survival ofcontent of 30 per cent. Other organisms occurring clover and cowpea-type rhizobia in sterilized peat innaturally in peat were less affected. Levels in the range of cotton wool stopper tubes, sealed cans and plastic film40 to 50 per cent were optimal for survival of three strains packets with various gas exchange properties. Theof Rhizobium [76]. In a sterilized peat, all strains are more practice of putting small holes in bags is bothtolerant to higher levels of moisture and growth is optimal unnecessary and harmful as in that moisture loss isin the range of 40 to 60% moisture. An interesting adverse increased. Also, they allow entry of contaminants ineffect of a high moisture level is that numbers of certain sterilized peat cultures [86].Rhizobia are highest at 60 to 65% and reduced carriers toabsorb different amounts of moisture may explain the Sterile and Non-Sterile Carrier: The inoculants prepareddifferent optima for growth and survival [77]. with non-sterilized peat might contain 100-told fewer

Temperature: Roughley [78] reported that effect of higher and this difference increases during the storagestorage on growth and survival of organism is influenced period [87].by both the purity of the culture and the amount of Sterilization has a great influence on the growth andmoisture lost during storage. It cultures are prepared in survival of rhizobia and other sterile inoculants andsterilized carriers, incubation at 26°C immediately after their ability to achieve consistently high cell densities ininoculation promotes initial rapid growth of organisms excess of 10 g [88, 89, 90]. The choice of a methodand has little or no effect on long term survival if moisture of inoculating sterilized peat without introducingcontent is maintained. In experiment on long term storage contaminants depends on the type of packaging.of unsterilized peat cultures the weekly log death rate In Holland, an assembly of glass and rubber tubingof clover rhizobia increased from 0.04 at 5°C to 0.094 at with a hallow needle is used to connect the sampling part25°C [79]. The moisture content of cultures in cotton wool of a fermentor [91].stoppered bottles wrapped in cellophane may be kept Santhanakrishnan and Thangaraju [92] revealed thatat a constant 50% by storage at 2°C [80], but storage at the polymer and amendments addition improved thesuch temperatures immediately after inoculant restricts survival of Azospirillum. The hydrophilic polymers,initial multiplication and maximum numbers are not Jalsakthi and terra cotton both at 2% and 4% levelsreached until 26 weeks. This period can be reduced to two and amendments, polyvinyl pyrrolidone and skim milk atweeks if cultures are incubated for one week at 26°C prior 1% and 2% levels were observed for retaining moistureto storage at 4°C. and favoured the survival of Azospirillum cells in both

Saha et al. [81] studied the survival of Rhizobium sterilized and un-sterilized carrier based inoculant.in the carriers at different temperatures using greenfluorescent protein marker and reported that survival rate Storage Materials for Inoculants: Roughly [93] confirmedof Rhizobium transform at was minimum at higher that Rhizobium needed a definite gas exchange throughtemperature above 28°C, in the paddy husk. packing material. Better survival of sealed bags than in

under vacuum, the higher proportion of air trapped to

rhizobia than sterilized peat because the death rate is

9 1


280

open aerated bags was observed by Iswaran [94]. molassess (1%) in the carrier enhanced the survival ofStrijdom and Deschodt [95] studied the survival of Rhizobium. The soymeal (2%) or polyvinyl pyrrolidonefour Azospirillum spp in steam sterilized peat sealed in (2%) for soymeal (2%) or polyvinyl pyrrolidone (2%) forhigh density polypropylene bag of 0.31-0.32 mm gauge. BradyRhizobium japonicum (TAL 102) were found to beThey observed high survival (3.5 x 10 g to 62 x 10 g ) the suitable amendments for enhancing the survival of8 1 8 1

in inoculants packed in high density polythene bags. Rhizobium inoculants [108]. Daza et al. [109] haveSubba Rao [96] studied the effect of packing demonstrated the usage of perlite as the carrier formaterials on the survival of Azospirillum. Double biofertilizers formulation and it could maintain a higherpolythene bag (600 gauge thickness) supported higher population of organisms than peat inoculants at roomsurvival of Azospirillum compared to single polythene temperature for six months.bad (300 guage) at room temperature. Packing the peat The hydrophilic polymers are synthetic waterbased inoculant Azospirillum in two layered polythene absorbing monomers of high molecular weight andbag increased the shelf life when compared to packing in they have been used for the past 30 years to increase thesingle polythene bag or polythene container [97]. water holding capacity, increase the pore size/number,

Amendments and Polymers Addition into Inoculant of soil or artificial medium [110]. Polymers absorb water atCarrier:Fages [98] reported that the skim milk irrigation and release it as the soil surrounding theaddition into dehydrated Alginate beads helps to polymer dries and has no effect on the physicalcontain 10 billion cells g of Azospirillum in the carrier. characteristics of water [111]. Dhumal [112] observed the1

Kandasamy and Prasad [99] suggested that amending significant increase in the yield of tomato (58.8%) andlignite with one per cent soybean powder could be used chillies (31.6%) by applying jalsakthi during water stressas carrier material for the Rhizobium spp than soil. condition.The cellulose powder appeared to be more satisfactory as The application of jalsakthi and Rhizobiuma carrier in as much as it gave the same case of handling treatment has increased the yield and water use efficiencyand low moisture loss as peat based inoculants and fairly of groundnut crop during summer [113]. Subbaraj et al.high survival of rhizobia [100]. Iswaran [101] used coir [114] used Qemisorb, an agricultural polymers, whichdust and soy meal with soil to study the survival of an hydrates about 100 times its initial weight with water andefficient strain of R. trifolii and reported that coir dust increased the moisture content of soil by 67 per cent.which is a carrier material along with soil for Rhizobium The hydrogels such as polyacrylamide or polyacrylicwas superior to soy meal. acid are capable of absorbing in excess of 300 times their

Carriers consisting of 40 per cent coal, 40 per cent dry weight and they are mixed with the growing mediumbentonite and 20 per cent bone meal have been studied in of tomato plants to retain the moisture in arid regionsSouth Africa by Strijidom and Deschodt [102] and the [115]. Grula et al. [116] revealed that the polymers appliedrhizobial survival in that carrier was comprehensive to for trapping water are used polymers applied for trappingthat in peat. Tilak and Subba Rao [103] evaluated 11 water are used as the carbon sources of soil microcarrier materials and used amendments like charcoal and organisms and stimulated their growth. Terracotton, acoconut shell powder to some carrier materials (1: 1 ratio) hydrophilic polymer can store water 150 times its ownfor rhizobia and they concluded that lignite, pressmud weight and increased the yield of tomato by 150 per cent,and FYM amended with coconut shell powder or charcoal the germination per cent and plant height of papaya bycould serve as good substitute for peat under Indian 60 per cent and 20 percent respectively and soybean cropconditions. Kumar Rao et al. [104] reported that the rate by 10 per cent [117].of decline of Rhizobium population was least whenpressmud is used as a carrier. Liquid Formulation of Microbial Inoculants: Generally,

Sparrow and Ham [105] and Graham-Weiss et al. [106] the inoculation procedures with carrier-based inoculantshave evaluated vermiculture as the carrier material and are time consuming, messy and impractical whensuggested vermiculite supplemented with nutrients will sowing large quantities of seed. So, as an alternate,support the bacterial survival and could be used as an liquid inoculants formulation was developed withinoculant carrier. Narendranath [107] reported that specific constituents of medium to grow Rhizobial cells.incorporation of amendments like soymeal (1%) and As historical background, liquid inoculants were used in

increase in nutrient reserves and reduction of compaction


281

the form of broth or suspensions of cells growing on agar inoculating Azospirillum into the cyst inducing minimalslants, but it was not common in market [118]. First report salts medium (MSM). One hundred percent conversionon liquid inoculants for seed inoculation of Rhizobium of vegetative cells into cyst cells was noticed in 96h.was reported from Holland [119]. Burton et al. [120] The population level of 10 was maintained till the finalreported that the liquid inoculum was as effective as observation [130].peat-based inoculant when the number of rhizobia perseed was increased by 2.5 times. Gel Based Microbial Inoculants: During last decade,

Schiffmann and Aples [121] observed that liquid there has been an increased interest in synthetic beadsinoculants were particularly suited to large seeded grain of various materials and dimensions for immobilization.legumes, which, because of their bulk, make seed The main purpose of these beads was to immobilize orinoculation a formidable task. The liquid inoculation of entrap the target organisms for a longer period and slowrhizobia provides higher cell load in the rhizosphere. Wani release of microorganism under favourable condition.and Rai [122] reported that foliar spray application of Poly acrylamide gel was observed as a suitable carrier forAzotobacter increased the yield of paddy, wheat and Bradyrhizobium japonicum. Survival of wet gel entrappedsorghum. The nodule numbers are increasing with cells at high temperature was better than peat based cells.increasing rates of applied liquid rhizobia from log Jung et al. [131] suggested the replacement of poly4.59 up to log 9.59 viable cells g [123]. The spray acrylamide with Alginate (AER) which supported higher1

method of liquid Rhizobium inoculant directly on to the survival. The Alginate beads (dried AER) accounted forsoil was also reported. more than 88% survival even after 150 days of storage

Generally, all manufactures of liquid inoculants and they proposed the use of Alginate beads with skimclaim that their liquid products contain high titers value. milk. The dried beads containing Azospirillum could beLipha Tecj (France) product claimed (cfu of 17.21 x 10 in stored at ambient temperate over a period of twelve weeks11

their liquid product. The soybean nodule numbers in a without any loss of Azospirillum cell content.tropical soil increased at increased rates of rhizobia Fages [132] reported that highest survival ofapplied in a liquid form Smith [124]. Hynes et al. [125] Azosprillum could be obtained by addition of skimreported that the liquid rhizobial inoculants for pea and milk and controlled dehydration of the Alginate beads.lentil resulted in yield equal to or better than those The powdered product contained more than 10 viableobserved with peat inoculant. cells per gram dry weight after several months of storage

Niftal has already claimed the cell number of 1 x at ambient temperature. Hegde and Brahmaprakash [133]10 cells ml in gliquid inoculant. The National chemical developed a free flow granular inoculant of Rhizobium14 1

laboratory of pine has reported Cfu of 10 - 10 in case of from sodium Alginate and perlite. They observed high8 9

Azotobacter (liquid) inoculants [126]. Inamadar et al. [127] number of survival of two groundnut Rhizobium strainsstandardized cyst and spore based liquid inoculants for in dry granules beyond 180 days than in peat.Azotobacter and P-solubilizing Bacillus megaterium and Fages [134] reported better survival rate ofrecorded higher population and comparatively better yield Azospirillum in dried micro granulated Alginateof crops in inoculated area. Singleton et al. [128] reported formulation. Okon [135] studied the survival ofthe development and evaluation of liquid inoculants and A. lipoferum with Alginate encapsulate, dehydratednitrogen fixation of legumes in Vietnam. at room temperature. The viable cell count was 10 per

Vendan and Thangaraju [129] reported that the carrier gram even after one year. Narendranath [136] reportedbased inoculants generally suffer from shortage shelf the new formulation like Alginate beads, clay balls andlife, poor quality, high contamination and low field liquid inoculation for the survival of Rhizobium. Alginateperformance. The liquid formulations of Azospirillum beads supported higher survival of Rhizobium followedwith the amendments viz., Trehalose, Polyvinylpyrollidine by clay was reported. Long- term survival of inoculantsand Glycerol enhanced and maintained the population is a matter of commercial secrecy.level at 10 upto 10 months of storage. The liquid Bashan [137] stated that, the main advantages of8

formulation shows better adherence and survival onseeds, roots of seedlings and in the rhizosphere soilthan the solid carrier based Azospirillum inoculants.Cyst based liquid formulation was developed by

8

10

10

Alginate inoculants are their nontoxic nature, theirdegradation in the soil and above all their slow release ofthe entrapped microorganisms in the soil. Bashan andGonzalez [138] studied and evaluated the capacity of


282

A. brasilense and P. fluorescens for long term survival, 5. Tien, T.M., M.H. Gaskin and D.H. Hubbel, 1979.viability and growth promoting capacity after storage in Plant growth substances produced by Azospirillumdry Alginate beads for 14 years. The two bacteria brasilense and their effect on the growth of pearlsurvived for 14 years in dry Alginate beads at ambient millet (Pennisetum americanum L.). Appl. Environ.temperature upon recovery, both species retained the Microbiol., 37(5): 1016-1024.capacity to enhance plant growth. Fillinger et al. [139] 6. Neyra, C.A. and J. Dobereiner, 1977. Nitrogen fixationreported that trehalose is required for the acquisition of in greases. Adv. Agron., 29: 1-38.tolerance to a variety of stresses in the filamentous 7. Lakhsmi kumari, M., S.K. Kavimandan andfungus. Suresh Babu et al. [140] stated that shelf life of N.S. Subba Rao, 1976. Occurrence of nitrogenAzospirillum inoculants was improved by the addition of fixing Spirillum in roots of rice, sorghum, maize andpolymers, chemicals and amendments in the lignite carrier other plants. Indian J. Exptl. Biol., 14: 638-639.[141, 142]. 8. Barber, L.E., J.D. Tjepkema, S.A. Russel and

CONCLUSION associated with corn inoculated with Spirillum.

Formulation of biofertilizer plays a vital role in 9. Okon, Y. and C.A. Labandera-Gonzalez, 1994.helping to solve many problems in agricultural field and in Agronomic applications of Azospirillum: Anmaking an organism effective in the field. However this evaluation of 20 years worldwide field inoculation.must be achieved in a cost effective manner so that Soil. Biol. Biochem., 26: 1591-1601.product has to survive commercially. Formulation 10. Claudia, C. Martin and Didonet, 2000. Genomecomprises aids to preserving organisms and to structure of the genus Azospirillum. J Bacteriol.,delivering them to their target fields and once-there to 182 (14): 4113-4116.improve their activities. A technical concentrate of an 11. Schroder, M., 1932. Die assimilation des Lufstickorganism that has been achieved by a particular stoffs durch einige Bacterian Lentrall. Bakteriol.process is called as formulation. There are varieties of Parasitendkd. Infektion Skr. Hyg. Abt., 2(85): 177-212.formulation both liquid and solid. The carrier based 12. Becking, J.H., 1985. Pleomorphism in Azospirillumbioinoculants generally suffer from shorter shelf life, spp. pp: 243-262. In: Azospirillum III Genetics,poor quality, high contamination and low field Physiology, Ecology, N. Klingmuller (Ed.)performance. Therefore, it is desirable that new inoculant Springer-Verlag, Berlin.formulations being developed where liquid inoculants 13. Breed, R.S., E.G.D. Murrary and N.R. Smith, 1957.play a significant role. Bergey’s manual of determinative bacteriology,

REFERENCES Maryland.

1. Chen, M. and M. Alexander, 1973. Survival of soil Chemoheterotrophic spirilla. Annu. Rev.bacteria during prolonged desiccation. Soil Biol. Microbiol., 30: 303-325.Biochem., 5: 213-221. 15. De-Polli, H., B.B. Bohlool and J. Dobereinder, 1980.

2. Mary, P., D. Ochin and R. Tailliez, 1985. Rates of Serological differentiation of Azospirillum speciesdrying and survival of Rhizobium meliloti strains belonging to different host plant specificity groups.during storage at different relative humidities. Arch. Microbiol., 126: 211-222.Appl. Environ. Microbiol., 50: 207-211. 16. Magalhaes, F.M.M., J.I. Baldani, S.M. Souto,

3. Hahn-Hagerdal, B., 1986. Water activity: a possible J.R. Kuykendall and J. Dobereiner, 1983. A newexternal regulator in biotechnical process. Enzyme arid tolerant Azospirillum species. Ann. Acad.Microb. Technol., 8: 322-327. Bras. Cienc., 55: 417-430.

4. Boddey, R.M. and J. Dobereiner, 1988. Nitrogen 17. Khammas, K.M., E. Ageron, P.A.D. Grimontandfixation associated with grasses and cereals: Recent P. Kaiser, 1989. Azospirillum irakense sp. nov. aresults and perspectives for future research. Plant nitrogen-fixing bacterium associated with rice rootsSoil, 108 : 53-65. and rhizosphere soil. Res. Microbiol., 140: 679-693.

H.J. Evans, 1976. Acetylene reduction (N fixation)2

Appl. Environ. Microbiol., 32: 108-113.

pp: 254-257. Williams and Wilkins, Baltimore,

14. Krieg, N.R., 1976. The taxonomy of the


283

18. Fani, R.C., C. Bandi, M. Bazzicalupo, 30. Hurek, T., B. Reinhold, G. Niemann and Fendrik,M.T. Ceccherini, S. Fancelli, E. Gallori, L. Gerace, 1988. Nitrogen dependent growth of AzospirillumA. Grifoni, N. Miclaus and G. Damiani, 1995. spp. in batch culture at low concentration ofPhylogeny of the genus Azospirillum based on oxygen, pp: 115-121. In: Azospirillum IV. Genetics,16 srRNA sequesnce. FEMS Microbiol. Lett., Physiology and Ecology. W. Klingmuller (Ed.),129: 195-200. Springer-Verlag, Berlin.

19. Okon, Y., 1985. Azospirillum as a potential 31. Heulin, T., M. Rahman, A.M.N. Omar, Z. Rafidison,inoculant for agriculture. Trends Biotechnol., J.C. Pierrat and J. Balandreau, 1989. Experimental and3: 223-228. mathematical procedures for comparing N -fixing

20. DeConincek, K., S. Horemans, S. Randombage and efficiencies of rhizosphere diazotrophs. J. Microbiol.K.Vlassak, 1988. Occurrence and survival of Methods, 9: 163-173.Azospirillum spp. in temperate regions. Plant Soil, 32. Anand, R.C., R. Ger, M. Hazija and B.S. Kundu,110: 213. 1999. Development of thermotolerant mutants of

21. Michiels, K., J. Vanderleyden and A.Van Gool, 1989. Laz-z-marked Azospirillum lipoferum, IndianAzospirillum-Plant root associations. A review, J. Microbiol., 39(2): 121-123.Biol. Fertil. Soils., 8: 356-368. 33. Gadagi , S., P. Ravi, U. Krishnaraj, J.H. Kulkarni

22. Sumner, M.E. 1990. Crop response to Azospirillum and S.A. Tongmin, 2004.The Effect of combinedinoculation. Ad. Soil. Science, 12: 53-123. Azospirillum inoculation and nitrogen fertilizer on

23. Smith, R.L., J.H. Boiton, S.C. Schank, plant growth promotion and yield response of theK.H. Quesenberry, M.E. Tyler, J.R. Milam, blanket flower Gaillardia pulchella. ScientiaM.H. Gaskins and R.C. Little, 1976. Nitrogen fixation Horticulturae., 100(1-4): 323-332.in grasses inoculated with Spirillum lipoferum. 34. Omay, S.H., W.A. Schmidt, P. Martin andScience, 193: 1003-1005. Bangertti, 1993. Indole acetic acid production by

24. Bashan, Y. and G. Holguin, 1995. Survival of the rhizosphere bacterium Azospirillum brasilenseAzospirillum brasilense in the bulk soil and cd under in vitro conditions Can. J. Microbiol.,rhizosphere of 23soil types. Appl Environ 24: 734-742.Microbiol., 61: 1938-1935. 35. Gadagi, S., P. Ravi, U. Krishnaraj, J.H. Kulkarni

25. Purushothaman, D., 2002. Biology of Azospirillum and S.A.Tongmin, 2003.The Effect of combinedassociation with cashew (Anacardium occidentale Azospirillum inoculation and nitrogen fertilizer onL.) pp: 1. In: National symposium and XII plant growth promotion and yield response of theSouthern Regional Conference on ‘Microbial blanket flower Gaillardia pulchella. Scientiainoculants’. Annamalai Univ., India, March 2002 Horticulturae., 100(1-4): 323-332.(Abstr.) 36. Saikia, S.P., Vanita Jain, Sageeta Khetarpal and

26. Rennie, R.J., J.R. Defrietas, A.P. Ruschel and Samitha Aravind, 2007. Dinitrogen fixation activityP.V. Vose, 1983. 15N isotope dilution to quantify of Azospirillum brasilense in maize (Zea mays).dinitrogen fixation associated with Canadian and Curr. Sci., 93(9):1296-1299).Brazilian wheat. Can. J. Bot., 61: 1667-1671. 37. Hartmann, A., A. Fussader and W. Klingmuller, 1983.

27. Lima, E., R.M. Boddey and J. Dobereiner, 1987. Mutants of Azospirillum affected in N Fixation andQuantification of biological nitrogen fixation Auxin Production, pp: 78-88. associated with sugarcane using 15N aided nitrogen 38. Jain, D.K. and D.G. Patriquin, 1984. Root hairbalance. Soil Biol. Biochem., 19: 165-170. deformation, bacterial attachment and plant growth in

28. Hartmann, A., H.A. Fu and R.H. Burris, 1988. wheat Azospirillum associations. Appl. Environ.Influence of amino acids on nitrogen fixation ability Microbiol., 48: 1208-1213.and growth of Azospirillum spp. Appl. Environ 39. Horemans, S., K. De Koninck, J. Neuray,Microbiol., 54(1): 87-93. R. Hermans and K. Vlassak, 1986. Production of

29. Kucey, E., Z. Liu and K. Kloppstech, 1993. plant growth substances by Azospirillum sp.Expression of heat shock proteins during and other rhizosphere bacteria. Symbiosis,development of barley. Plant Mol. Biol., 23: 111-122. 2: 341-346.

2

2


284

40. Okon, Y. and Y. Hadar, 1987. Microbial inoculant as 51. Vande Brock, A., M. Lambrecht, K. Eggermontcrop-yield enhancers of CRC. Crit. Rev. Biotechnol., and J. Vanderleyden, 1999. Auxins upregulate6: 61-85. expression of the indole-3-pyruvate decarboxylase

41. Fallik, E., Y. Okon, E. Epstein, A. Goldman and gene in Azospirillum brasilense. J. Bacteriol.,M. Fischer, 1989. Identification and quantification of 181(4): 1338-1342.IAA and IBA in Azospirillum brasilense inoculated 52. Fabricio Cassan Ruben Bottini Gernot Schneidermaize roots. Soil Biol. Biochem., 21: 147-153. and Patricia Piccoli, 2001. Azospirillum brasilense

42. Cacciari, L., D. Lippi, T. Pietrosanti and and Azospirillum lipoferum Hydrolyze ConjugatesW. Pietrosanti, 1989. Phytohormones like substances of GA and metabolize the resultant Aglycones toproduced by single and mixed diazotrophic GA in Seedlings of Rice Dwarf Mutants. Plantcultures of Azospirillum and Arthrobacter. Plant Physiol., 125(4): 2053-2058.Soil, 115: 151-153. 53. Kolb, W. and P. Martin, 1985. Response of plant

43. Zimmer, W., C. Aparicio and C. Elmerich, 1991. roots to inoculation with Azospirillum brasilenseRelationship between tryptophan biosynthesis and and to Application of Indole Acetic Acid,undole-3 acetic acid production in Azospirillum: pp: 215-221. In: Azospirillum III. Genetics,identification and sequencing of a to GDC cluster. Physiology, Ecology, W. Klingmuller (Ed.) Springer-Mol. Gen. Genet., 229: 41-51. Verlag, Berlin.

44. Janzen, R.A., S.B. Rood, J.F. Dormaar and 54. Perrig, D., M. Boiero, O.L.A. Masciarelli, C. Penna,W.B. McGill, 1992. Azospirillum brasilense O.A. Ruiz, F. Cassan and M. Dand Luna, 2007. Appl.produces Gibberellin in pure culture on chemically Microbiol., 75: 1143-1150.defined medium in co-culture on straw. Soil Biol. 55. Tarrand, J.J., N.R. Kreig and J. Dobereiner, 1978.Biochem., 24: 1061-1064. A taxonic study of the Spirillum lipoferum

45. Barberi, P. and E. Galli, 1993. Effect on wheat group, with descriptions of a new genus,root development of inoculation with an Azospirillum gen. Nov. and two species,Azospirillum brasilense mutant with altered Azospirillum brasilense (Beijerinck) comb. Nov. andindole-3 acetic acid production. Res. Microbiol., Azospirillum brasilense sp. nov. Can. J. Microbiol.,144: 69-75. 24: 967-980.

46. Omay, S.H., W.A. Schmidt, P. Martin and 56. Okon, Y., 1985. Azospirillum as a potentialBangertti, 1993 . Indole acetic acid production by inoculant for agriculture. Trends Biotechnol.,the rhizosphere bacterium Azospirillum brasilense 3: 223-228.cd under in vitro conditions Can. J. Microbiol., 57. Bashan, Y., 1986. Significance of timing and level of24: 734-742. inoculation with rhizosphere bacteria on wheat

47. Fulchieri, M. and L. Frioni, 1994. Azospirillum plants. Soil Biol. Biochem., 18: 297-301.inoculation on maize (Zea mays) : effect of yield in a 58. Bashan, Y. and H. Levanony, 1990. Current statusfield experiment in Central Agentina. Soil Biol. of Azospirillum inoculation technology.Biochem., 26: 921-924. Azospirillum as a challenge for agriculture. Can.

48. Piccoli, P. and R. Bottini, 1994. Effects of C/N ratio J. Microbiol., 35: 591-608.N content, pH and inoculation time on growth and 59. Fages J., 1994. Azospirillum inoculants and fieldgibberellin production by Azospirillum lipoferum. Experiments, pp: 87-110. In: Azospirillum-Plant

49. Neyra, C.A, A. Alkinson and O. Olubayi 1995. Association. Y. Okon, (Ed.) CRC Press, Boca Raton,Coaggregation of Azospirillum with other bacteria: Fla.basis for functional diversity. NATO, ASI, Ser. G., 60. Hume, D.J. and D.H. Blair, 1992.Effects of members37: 429-439. of Bradyrhizobium japonicum applied in

50. Vande Brock, A., M. Lambrecht, K. Eggermont commercial inoculants in soya bean seed fields inand J. Vanderleyden, 1995 . Auxins upregulate Ontario. Can. J. Microbiol., 38: 582-593.expression of the indole-3-pyruvate decarboxylase 61. Okon, Y., 1985. Azospirillum as a potentialgene in Azospirillum brasilense. J. Bacteriol., inoculant for agriculture. Trends Biotechnol.,181(4): 1338-1342. 3: 223-228.

20

1


285

62. Omay, S.H., W.A. Schmidt, P. Martin and Bangertti, 77. Kannaiyan, S., 2000. Biofertilizer technology and1993. Indole acetic acid production by therhizosphere bacterium Azospirillum brasilense cdunder in vitro conditions Can. J. Microbiol.,24: 734-742.

63. Iswaran, V., 1969. Growth and survival of R. trifoliin coir dust and soybean meal compost. MadrasAgric. J., 59: 52.

64. Tilak, K.V.B.R. and N.S. Subba Rao, 1978. Carries forlegume inoculants. Fert. News., 23: 25-28.

65. Tilak, K.V.B.R., 1979. Survival of Azospirillumbrasilense in different carriers. Curr. Sci., 48: 412.

66. Tilak, K.V.B.R. and N.S. Subba Rao, 1978. Carries forlegume inoculants. Fert. News., 23: 25-28.

67. Sparrow, S.D. and G.E. Han, 1981. Survival ofRhizobium phaseoli in six carrier materials. Agron.J., 75: 181-184.

68. Kumar Rao, J.V.D., K.C. Mohan Kumar andR.B. Patil, 1982. Alternate carrier materials forRhizobium inoculant production. Mysore J. Agric.Sci., 16: 2520255.

69. Somasegaran, P., 1985. Inoculant production withdiluted liquid cultures of Rhizobium spp andautoclaved peat. Evaluation of dilutents.Rhizobium spp peats, sterility requirements, storageand plant effectiveness. Appl. Environ. Microbiol.,50(2): 398-405.

70. Singaravadivel, K. and S. Anthoni Raj, 1988. Rice millby products as carrier for Rhizobium. Legume Res.,11: 143-145.

71. Gaind, S. and A.C. Gaur, 1990. Shelf life of phosphatesolubilizing inoculants as influenced by type ofcarrier, high temperature and low moisture. Can. J.Microbiol., 36: 846-849.

72. Fages, J., 1992. An industrial view on Azospirilluminoculants: formulation and application technology.Symbiosis, 13: 15.

73. Narendranath, N.V., 1995. Shelf life of Rhizobiuminoculant as influenced by carries and storageconditions. M.Sc. (Ag.) Thesis, Tamil Nadu Agrl.Univ., Coimbatore, India.

74. Abd-Alla, M.H. and S.A. Omar 2001.Carriers forbiofertilizer. J. Plant. Nutr., 24(2): 261-272.

75. Roughley, R.J., 1968. Some factors influencing thegrowth and survival of root nodule bacteria in peatculture. J. Appl. Bacteriol., 31: 259-265.

76. Date, R.A. and R.J. Roughley, 1977. Legume inoculantproduction. Indian Natl. Sci. Acad Bull., 51: 67-686.

quality control. Publication directorate, TNAU,Coimbatore, India, pp: 256.


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 influencinggrowth and survival of Rhizosphere spp. in soul peatcultures. Plant Soil, 32: 113-130.

81. Saha, A.K., M.V. Deshpande and B.P. Kapadnis,2001. Studies on survival of Rhizobium in thecarriers at different temperature using greenfluorescent protein marker. Curr. Sci., 80(5): 69-671.

82. Hedlin, R.A. and J.W. Newton, 1948. Somefactors influencing the growth and survival ofrhizobia in humus and soil culture. Can. J. Res.Sect. C., 26: 174-187.

83. Roughley, R.J. and J.M. Vincent, 1967. Growth andsurvival of Rhizobium spp. in peat culture. J. Appl.Bacteriol., 30: 362-376.

84. Date, R.A. and R.J. Roughley, 1977. Legume inoculantproduction. Indian Natl. Sci. Acad Bull., 51: 67-686.


86. Smith, R.S., 1987. Production and quality control ofinoculants. In: Symbiotic nitrogen fixationtechnology. 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 basedinoculants of field grown soybean (Glycine max).Agron. J., 57: 379-381.


89. Strijdom, B.W. and C.C. Deschodt, 1976. Carriers ofrhizobia and the effect of prior treatment on thesurvival of rhizobia. In: Symbiotic NitrogenFixation in Plants (ed.) P.s. Nutman. InternationalBiological Programme 7. Cambridge University press,Cambridge, pp: 151-168.

90. Somasegaran, P. and J. Halliday, 1982. Dilution ofliquid Rhizobium cultures to increase productioncapacity of inoculant plants. Appl. Environ.Microbiol., 44: 330-333.


286

91. Van Schreven, D.A., 1958. Some factors affecting 105. Sparrow, Jr., S.D. and G.E. Ham, 1983. Survival ofuptake of nitrogen by legumes. In: Nutrition of the Rhizobium phaseoli in six carrier materials.legumes. Butterwirth & Co., London, UK, pp: 137-163. Agron. J., 75: 181-184.

92. Santhanakrishnan, P. and M. Thangaraju, 2002. 106. Graham-Weiss, L., M.L. Bennett and A.S. Paau,Enhancement of shelf life of Azospirillum through 1987. Production of bacterial inoculants by directaddition of polymers and chemicals is carrier fermentation on nutrient supplemented vermiculite.based inoculant. Biofertilizer Newsletter, Dec. 3-10. Appl. Environ. Microbiol., 53: 2138-140.

93. Roughley, R.J., 1968. Some factors influencing the 107. Narendranath, N.V., 1995. Shelf life of Rhizobiumgrowth and survival of root nodule bacteria in peat inoculant as influenced by carries and storageculture. J. Appl. Bacteriol., 31: 259-265. conditions. M.Sc. (Ag.) Thesis, Tamil Nadu Agrl.

94. Iswaran, V., 1972. Growth and survival o f R.trifoli Univ., Coimbatore, India.in coir dust and soybean meal compost. Madras 108. Senthil Kumar, M., 1999. Improved methodologiesAgric. J., 59: 52. for legume inoculant production, M.Sc. (Ag.)

95. Strijdom, B.W. and C.C. Deschodt, 1976. Carriers of Thesis, Tamil Nadu Agric. Univ., Coimbatore,rhizobia and the effect of prior treatment on the India, pp: 163.survival of rhizobia. In: Symbiotic Nitrogen Fixation 109. Daza, A., D.N. Santamaria, P.N. Rodriguez-Navarro,in Plants (ed.) P.s. Nutman. International Biological M. Camacho, R. Orive and F. Temprano, 2000.Programme 7. Cambridge University press, Perlite as a carrier for bacterial inoculants. Soil Biol.Cambridge, pp: 151-168. Biochem., 32: 567-572.

96. Subba Rao, N.S., 1982. Rhizobium inoculant, 110. Orzolck, M.D., 1993. Use of hydrophilic polymers inpp: 15-69. In: Biofertilizers in Agriculture. Oxford horticulture. Hort. Technology, 3(1): 41-44.and IBH Pub. Co. New Delhi. 111. Blodgett, A.M., D.J. Beattie, J.W. White and

97. Govindarajan, K,. 1996. Shelf life of Azospirillum G.C. Elliott, 1993. Hydrophilic polymers and wettinginoculant as influenced by carrier material and type agents affect absorption and evaporative waterof packing. Paper presented in National Seminar on loss. Hort Science, 28(6): 633-635.Biofertilizer production problems and constraints. 112. Dhumal, K.N., 1991. Effects of jalsakthi on growthTNAU, Coimbatore, Jan. 24-25 (Abs.) pp: 38. and yield of some vegetables under water stress

98. Fages, J., 1990. An optimized process for condition. J. Maharashtra Agric. Univ., 18(2): 307.manufacturing Azospirillum inoculant for crops. 113. Bharambe, P.R., R.P. Rodge, P.R. AmhegamokarAppl. Microbiol. Biotechnol., 32: 473-478. and S.R. Oza, 1993. Effects of jalskathi and

99. Kandasamy, R. and N.N. Prasad, 1971. Lignite as a Rhizobium application on yield and wat Burton,carrier of rhizobia. Curr. Sci., 40: 496. J.C., R.L. Curley and P.S. Nutman, 1965.

100. Pugashetti, B.K., H.S. Gopal Gowda and R.B. Patil, Comparative efficiency of liquid and peat based1971. Cellulose powder as a legume inoculant base. inoculants of field grown soybean (Glycine max).Curr. Sci., 40: 494. Agron. J., 57: 379-381.

101. Iswaran, V., 1972. Growth and survival of R. trifoli 114. Subbaraj, D., P.P. Ramaswamy and G. Arunachalam,in coir dust and soybean meal compost. Madras 1993. Efficiency of Qemisorb agricultural polymersAgric. J., 59: 52. for water retention in tree cropped their lands.

102. Strijdom, B.W. and C.C. Deschodt, 1976. Carriers of South Indian Horticulture, 416: 379-380.rhizobia and the effect of prior treatment on the 115. Szmidt, D., 1994. Making the desert bloom.survival of rhizobia. In: Symbiotic Nitrogen The Horticulturist, 3: 10-25. Fixation in Plants (ed.) P.s. Nutman. International 116. Grula, M.M., M.L. Huang and G. Sewell, 1994.Biological Programme 7. Cambridge University Interactions of certain polyacrylamides with soilpress, Cambridge, pp: 151-168. bacteria. Soil Sci., 158: 291-300.

103. Tilak, K.V.B.R. and N.S. Subba Rao, 1978. Carries for 117. Tomar, S.P., 1997a. Terracotton to save water andlegume inoculants. Fert. News., 23: 25-28. increase net returns. Kisan World, Sep., pp: 43-44.

104. Kumar Rao, J.V.D., K.C. Mohan Kumar and 118. Nethery, A.A. 1991. Inoculant production withR.B. Patil, 1982. Alternate carrier materials for nonsterile carrier. In: Expert consultation on legumeRhizobium inoculant production. Mysore J. Agric. inoculant production and quality control. FAO,Sci., 16: 2520255. Rome, pp: 43-50.


287

119. Van Schreven, D.A, G.W. Harmsen, 132. Fages, J., 1990. An optimized process forD.T. Lindenbergh and D. Otzen, 1953. Experiments manufacturing Azospirillum inoculant for crops.on the cultivation of Rhizobium in liquid media Appl. Microbiol. Biotechnol., 32: 473-478.for use on zindderzee polders. Anton van 133. Hegde, S.V. and G.P. Brahmaprakash, 1992. A dryLeeuwenhoek, 19: 300-308. granular inoculant of Rhizobium for soil

120. Burton, J.C., R.L. Curley and P.S. Nutman, 1965. application. Plant Soil, 144: 309-311. Comparative efficiency of liquid and peat based 134. Fages, J., 1992. An industrial view on Azospirilluminoculants of field grown soybean (Glycine max). inoculants: formulation and application technology.Agron. J., 57: 379-381. Symbiosis, 13: 15.

121. Schiffman, J. and Y. Apler, 1968. Inoculation of 135. Okon, Y., 1994. Azospirillum as a potential inoculantpeanuts by application of Rhizobium suspension for agriculture. Trends Biotechnol., 3: 223-228.into planting furrows. Expt. Agric., 4: 219-226. 136. Narendranath, N.V., 1995. Shelf life of Rhizobium

122. Wani, S.P. and P.V. Rai, 1980. Response of inoculant as influenced by carries and storagesorghum to foliar sprays and seed inoculation conditions. M.Sc. (Ag.) Thesis, Tamil Nadu Agrl.with Azotobacter. Indian J. Microbiol., 20: 319-320. Univ., Coimbatore, India.

123. Smith, R.S., M.A. Ellis and R.E. Smith, 1981. Effect of 137. Bashan, Y., 1998. Inoculants for plant growth -R. japonicum inoculant rates on soybean nodulation promoting bacteria in agriculture. Biotechnol. Adv.,of a tropical soil. Agron. J., 73: 505-508. 16: 729-770.

124. Smith, R.S., 1992. Legume inoculant formulation and 138. Bashan, Y. and L.E. Gonzalez, 1999. Long-termapplication. Can. J. Microbiol., 38: 485-492. survival of the plant-growth-promoting bacteria

125. Hynes, R.K., K.A. Craig, D. Covart, R.S. Smith Azospirillum brasilense and Pseudomonasand R.J. Rennie, 1995. Liquid rhizobial inoculants fluorescens in dry alginate inoculants. Appl.for lentil and field pea. J. Prod. Agric., 8(4): 547-552. Microbiol Biotechnol., 51: 262-266.

126. Bhattacharya, P. and R. Kumar, 2000. Liquid 139. Fillinger, S., M. Chaeroche, P. Van Dijck andbiofertilizer-Current knowledge and future C. De Enfert, 2001. Trehalose is required for theprospect. Paper presented in National Seminar on acquisition of tolerance to a variety of stresses inDevelopment & Use of Biofertilizers, Biopesticides the filamentous fungus Aspergillus nidulans.and Organic manures, Kalyani, West Bengal, India. Microbiol., 147: 1851-1862.

127. Inamdar, S., K. Raviu and W.G. Millind, 2000. 140. Suresh Babu, S., M. Thangaraju andLongevity of Azotobacter cysts and a model for P. Santhanakrishnan, 2002. Shelf life improvement ofoptimization of cyst density in liquid bioinoculants. Azospirillum inoculants by addition of polymers,Curr. Sci., 78(6): 719-722. chemicals and amendments in the lignite carrier.

128. Singlenton, P.W., H.H. Keyser and E.S. Sande, 2002. J. Microb. World, 4: 51-58.Development and evaluation of liquid inoculants. 141. Saranraj, P., P. Sivasakthivelan and S. Siva Sakthi,In Inoculants and nitrogen fixation of legumes 2013. Prevalence and production of plant growthin Vietnam.(ed.)D Herridge, AICAR Proceedings, promoting substance by Pseudomonas fluorescens109e: 52-66. isolated from paddy rhizosphere soil of Cuddalore

129. Vendan, R.T. and M. Thangaraju, 2007. Acta district, Tamil Nadu, India. African Journal of BasicMicrobiol. Immunol Hung., 54(2): 167-167. and Applied Sciences, 5(2): 95-101.

130. Vendan R.T. and M. Thangaraju, 2006. Development 142. Sivasakthi, S., D. Kanchana, G. Usharani andand standardization of liquid formulation for P. Saranraj, 2013. Production of plant growthAzospirillum bioinoculant Indian J. Microbiol., promoting substance by Pseudomonas fluorescens46(4): 379-387. and Bacillus subtilis isolated from paddy

131. Jung, G., J. Mugnier, H.G. Dim, Y.R. Diem and rhizosphere soil of Cuddalore district, Tamil Nadu,Y.R. Dommergues, 1982. Polymer entrapped India. International Journal of MicrobiologyRhizobium. Curr. Sci., 38: 468-469. Research, 4(3): 227-233.

biosullllry

Documents

liquid biofertilizers

short shelf life

annamalai nagar

annamalai university

beneficial microorganisms

important biofertilizer

live microorganisms

genus azospirillum arestay