a S c i T e c h n o l j o u r n a lResearch Article

Leo Daniel et al., J Plant Physiol Pathol 2013, 1:2http://dx.doi.org/10.4172/jppp.1000105 Journal of Plant

Physiology & Pathology

All articles published in Journal of Plant Physiology & Pathology are the property of SciTechnol, and is protected by copyright laws. Copyright © 2013, SciTechnol, All Rights Reserved.International Publisher of Science,

Technology and Medicine

Effect of Polymeric Additives, Adjuvants, Surfactants on Survival, Stability and Plant Growth Promoting Ability of Liquid BioinoculantsLeo Daniel Amalraj E1*, Venkateswarlu B1, Suseelendra Desai1, Praveen Kumar G1, Mir Hassan Ahmed SK1, Meenakshi T1, Uzma Sultana1, Sravani Pinisetty1 and Lakshmi Narasu M2

AbstractThe present study was conducted to evaluate the effect of polymeric additives, adjuvant and surfactants for their ability to support growth, shelf-life stability and bio-efficacy of liquid bioinoculants (Bacillus megaterium var. phosphaticum, Azospirilum brasilense and Azotobacter chrococcum). Liquid inoculants formulated with 2% polyvinylpyrollidone (PVP 30 K), 0.1% carboxy methylcellulose (CMC-high density) and 0.025% Polysorbate 20 promoted long-term survival of Bacillus megaterium var. phosphaticum, Azospirillum and Azotobacter with 5.6 x 107, 1.9x108 and 3.5x107 cfu ml-1, respectively after 480 days of formulation when stored at 30oC. Maize seeds treated with consortium of PSB and Azospirillum brasilense enhanced plant growth positively by a multitude of synergistic mechanisms when compared to single inoculant application.

KeywordsLiquid inoculants; Cell protectants; Shelf life; Growth promotion

IntroductionBioinoculants contain beneficial bacteria that enhances plant

growth by the virtue of its nutrient solubilisation [1], nitrogen fixation [2], phytohormone production [3,4] and induction of defence mechanism [5-7] abilities. In India, Azospirillum, Azotobacter, Rhizobium, potash mobilizing bacteria, phosphate and zinc solubilising bacteria were included under Fertilizer control order, 1985 in view of nourishing the organic agriculture through the application of quality bioinoculants. Bhattacharyya and Kumar [8], stated that, bioinoculants manufactured in India are mostly solid carrier based and have a shelf life of only six months. These carrier-based inoculants are inherent with certain constraints like lower shelf-life, poor survival under adverse environmental conditions, high degree of contamination, and inconsistent field performances. There have been many attempts to find alternatives for carrier based inoculants and, also to enhance viability of microorganisms in the inoculants. Besides, it requires a significant amount of processing,

*Corresponding author: Leo Daniel Amalraj E, Division of Crops Sciences, Central Research Institute for Dryland Agriculture, Santoshnagar, Hyderabad, Andhra Pradesh, India-500059, E-mail: leodanielamalraj@hotmail.com

Received: March 02, 2013 Accepted: Apirl 29, 2013 Published: May 03, 2013

such as mining, drying, milling, neutralizing and also becomes expensive in sterilization, formulation and transportation processes.

Liquid inoculant formulations (LIF) could be a possible solution to the foresaid problems. It containing not only the desired microorganisms and their nutrients but also special cell protectants or additives that promote for longer shelf life and tolerance to adverse conditions [9]. Moreover, additives in the broth will improve inoculants quality, such as including better adhesion to seed [10], stabilizing the product, binding or inactivating soluble seed coat toxins. Many kinds of polymers have been used for inoculant production because of their ability to limit heat transfer, their good rheological properties and high water activities. These polymers, such as sodium alginate [11], gum Arabic [12], polyvinyl pyrrolidone (PVP) [13], polyvinyl alcohol (PVA) [14] and polyethylene glycol (PEG) [15] are normally used as adhesive when they are applied to seed. Polymers are also used to entrap bacteria in microbeads [16,17]. They are also soluble in water and make convenient for seed application a simple process for farmers. The selection of ingredients was based on their properties, such as solubility in water, non-toxicity and complex chemical nature, which prevents microorganisms in the soil from rapidly degradation. It is always a herculean task for a single inoculant to instigate significant impact on the plant growth. Co-inoculation can benefit plant growth by different mechanisms [11]. Hence, we evaluated the optimum concentration of polymers, surfactants and adjuvants for LIF that could sustain the bacterial shelf life and also studied its bioefficacy in pot culture using maize (Zea mays. L) as test crop.

Materials and MethodsMicroorganisms

Bacillus megaterium var. phosphaticum (BMPSB 01), Azospirillum brasilense (ASP 20) and Azotobacter chrococcum (AZB 29) were taken from microbial culture bank, Central Research Institute for Dryland Agriculture, Hyderabad, India. They were isolated from rhizospheric soil of maize plants in Hayathnagar Research Farm, Hyderabad, India.

Growth medium for liquid inoculants

A modified Pikovaskaya’s broth composed of (gL-1) yeast extract: 5.0, dextrose: 10.0, Ca3(PO4)2: 5.0, KCl: 0.5, MgSO4: 0.2, MnSO4: 0.1, FeSO4: traces, (NH4)2SO4: traces, glycerol: 10 ml was used to culture Bacillus megaterium var phosphaticum. Modified Jenson’s broth was used to culture Azotobacter which composed of sucrose: 20, K2HPO4: 1.0, MgSO4: 0.5, Nacl: 0.5, FeSO4: 0.1, Na2MoO4: 0.005, glycerol: 10 ml and for Azospirillum, modified nitrogen free bromothymol blue medium containing L-Malic acid: 5.0, K2HPO4: 0.5, MgSO4: 0.2, Nacl: 0.1, CaCl2: 0.02, Na2MoO4 : 0.002, MnSO4: 0.01, FeEDTA (1.64% w/v): 4.0 ml, glycerol: 10 ml, bromo thymol blue (0.5% w/v in ethanol): 3.0 ml, KOH: 4.5, biotin: 0.1 were used as a basal media to evaluate the effect of additives on cell growth.

Screening different concentration of additives on bacterial growth

All the three media were amended with different concentrations of additives as follows: PVP at 1.0, 2.0, 3.0 and 5.0% (w/v); PEG at

Citation: Leo Daniel AE, Venkateswarlu B, Suseelendra D, Praveen Kumar G, Mir Hassan Ahmed SK, et al. (2013) Effect of Polymeric Additives, Adjuvants, Surfactants on Survival, Stability and Plant Growth Promoting Ability of Liquid Bioinoculants. J Plant Physiol Pathol 1:2.

• Page 2 of 5 •Volume 1 • Issue 2 • 1000105

doi:http://dx.doi.org/10.4172/jppp.1000105

0.1, 0.5, 1.0 and 5.0% (w/v); PVA at 0.1, 0.5, 1.0, 3.0% (w/v); Gum Arabic at 0.1, 0.3, 0.5, 0.8% (w/v); Sodium alginate at 0.1, 0.2, 0.3 and 0.5% (w/v); and cassava starch at 0.1, 0.5, 1.0 and 3.0% (w/v). The experiments were carried out in 125 ml Erlenmeyer flasks containing 50 ml of amended medium. Late log phase cultures of the respective bacterium were inoculated into test media (0.1% v/v), and grown in an incubator shaker at 28°C and 200 rpm for 4 days. The viable cell populations were determined by plate counts on respective agar medium.

Screening different concentration of adjuvants and surfactants on bacterial growth

Four adjuvants viz., carboxyl methyl cellulose (CMC), xanthan gum, carageenum and guar gum were test in different concentration (0.1, 0.2, 0.3, 0.4 and 0.5g l-1) in liquid formulation containing 5 x 1010 cfu ml-1. Similarly, three non-ionic surfactants (Polysorbate 80, 40 and 20) were tested in different concentrations viz., 0.025, 0.05 and 0.1% for the uniform dispersion of liquid formulation. The viable cell populations after 10 days in all the treatments of additives and surfactants were determined by plate counts on respective agar medium.

Shelf life studies of liquid inoculants

Liquid inoculants (PSB, Azospirillum and Azotobacter) were prepared with the cell population adjusted to 5 x 1010 cfu ml-1 and made upto 250 ml and were packed in surface sterilized high density polyethylene (HDPE). The formulated products were stored in room temperature and assessed for their shelf life, pH and temperature at monthly interval upto 16 months.

Bioefficacy evaluation of liquid inoculants

Bio-efficacy of liquid inoculants was studied by pot culture

assay with maize as the test crop. Soil and sand was mixed in 3:1 proportion and sterilized at 121°C for 1 h consecutively for three days. Each plastic bag (10 × 12 inches) was filled with five kg soil. Maize (DHM117) seeds were surface sterilized and treated with bioinoculants in different combination (Table 1) @ 10 ml kg-1 seed in case of liquid inoculants or 5 ml of each inoculant for consortia treatments. In each pot seeds were sown at 2 cm depth and at equal distance between the seeds. Six replications were maintained for all the treatments. In each pot exactly 6 plantlets were maintained by thinning the unevenly grown plant at 15 DAS. The maize plants were uprooted carefully on 45th day of sowing and analysed for various agronomical parameters like root volume, shoot length, root and shoot drymass and total drymass.

Result and DiscussionEffect of additives

Among the five different additives screened, PVP at 2% concentration, showed excellent cell retention trait as compared to other treatments (Table 2). After 10 days of incubation, media

Treatments Liquid inoculants

T1 Control

T2 PSB

T3 Azospirillum

T4 Azotobacter

T5 PSB+ Azospirillum

T6 PSB+ Azotobacter

Table 1: List of treatments used to evaluate the plant growth promoting effect of bioinoculants.

CarrierPercentconcentration of additives

1010 cfu ml-1

Bacillus Azotobacter Azospirillum

Polyvinyl pyrrolidone

1 0.91c (±0.042) 1.81b (±0.402) 0.79c (±0.361)2 3.7a (±0.169) 4.64a (±0.212) 3.88a (±0.177)3 1.96b (±0.089) 0.921c (±0.042) 0.811b (±0.037)5 0.88c (±0.04) 0.56d (±0.026) 0.67c (±0.031)

Polyethylene glycol

0.1 0.086d (±0.004) 0.093e (±0.004) 0.078d (±0.004)0.5 0.083d (±0.004) 0.093e (±0.004) 0.079d (±0.004)1 0.09d (±0.004) 0.095e (±0.004) 0.079d (±0.004)5 0.089d (±0.004) 0.085e (±0.004) 0.078d (±0.004)

Polyvinyl alcohol

0.1 0.081d (±0.004) 0.093e (±0.004) 0.078d (±0.004)0.5 0.085d (±0.004) 0.09e (±0.004) 0.082d (±0.004)1 0.082d (±0.004) 0.092e (±0.004) 0.082d (±0.004)3 0.076d (±0.003) 0.091e (±0.004) 0.084d (±0.004)

Gum Arabic

0.1 0.081d (±0.004) 0.093e (±0.004) 0.095d (±0.004)0.3 0.092d (±0.004) 0.092e (±0.004) 0.094d (±0.004)0.5 0.091d (±0.004) 0.087e (±0.004) 0.093d (±0.004)0.8 0.092d (±0.004) 0.089e (±0.004) 0.094d (±0.004)

Sodium alginate

0.1 0.076d (±0.003) 0.085e (±0.004) 0.071d (±0.003)0.2 0.074d (±0.003) 0.083e (±0.004) 0.042d (±0.002)0.3 0.073d (±0.003) 0.081e (±0.004) 0.039d (±0.002)0.5 0.032d (±0.001) 0.03e (±0.001) 0.026d (±0.001)

Control - 0.047d (±0.002) 0.096e (±0.004) 0.098d (±0.004)

Table 2: Effect of polymeric additives on liquid bioinoculants.

Values are the mean of three determinants ±SD. Within a column different letters (a,b) after values indicate that there is a significant difference at P value of 0.05 as determined by Fischer’s least significant difference test.

Citation: Leo Daniel AE, Venkateswarlu B, Suseelendra D, Praveen Kumar G, Mir Hassan Ahmed SK, et al. (2013) Effect of Polymeric Additives, Adjuvants, Surfactants on Survival, Stability and Plant Growth Promoting Ability of Liquid Bioinoculants. J Plant Physiol Pathol 1:2.

• Page 3 of 5 •Volume 1 • Issue 2 • 1000105

doi:http://dx.doi.org/10.4172/jppp.1000105

amended with 2% PVP showed 3.7 × 1010, 3.88 × 1010 and 4.64 × 1010 cfu ml-1 of Bacillus, Azospirillum and Azotobacter, respectively as compared to other treatments. It could be due to their ability to limit heat transfer, their good rheological properties and high water activities. This is in agreement with Singleton et al. [13] who reported that PVP blended with medium for normal culturing conditions of bradyrhizobia showed no adverse affect on growth. The cells cannot use these polymers as an energy source, but the polymers have other properties supporting the growth and survival of cells. They have a sticky consistency, which may enhance cell adherence to seed, and their viscous nature may slow the drying process of the inoculant after application to seed [15]. PVP also has a high water binding capacity, which could maintain water around the cells for their metabolism [14]. Besides, it also facilitates in seed treatment, a simple process for adoption by farmers. The cell population was significantly reduced from 1010 to 108 cfu ml-1 in all other additives amended media.

Effect of adjuvants and surfactants

Among the four different adjuvants screened carboxy methyl cellulose (CMC) at 0.1% concentration, showed excellent cell retention property as compared to other treatments (Table 3). After 10 days of incubation, Bacillus showed 3.25 x108 cfu ml-1, Azotobacter (4.1 x 108 cfu ml-1) and Azospirillum (3.56 × 108 cfu ml-1) cell populations as compared to other treatments. Proportional sedimentation was observed with the increase in concentration of adjuvants. The present findings are similar to the results obtained by Fernandes and Junior [18] who reported that CMC has shown the ability to sustain a high rhizobial concentration up to six months of storage, and the inoculants produced with these carriers were on par to the performance of peat inoculants in greenhouse conditions [17]. Among three surfactants screened, polysorbate 20 commercially called “Tween 20” at 0.025%

concentration was found to be effective surfactant that showed better cell population as compared to other treatments (Table 4). After 10 days of incubation, Bacillus exhibited 3.3 ×108 cfu ml-1, Azotobacter showed 3.99 x 108 cfu ml-1 and Azospirillum showed 3.52 x 108 cfu ml-1 populations as compared to other treatments. Polysorbate is a non ionic surfactant whose stability and relative non-toxicity allows it to be used as a detergent and emulsifier in a number of domestic, scientific, and pharmacological applications.

Shelf life of liquid inoculants

In all the cases, the initial set population 5 × 1010 cfu ml-1 significantly dropped with lapse of time. However, the degree of declination varied with the type of bacterial inoculant. The survival of three LIF was recorded upto 480 days under room temperature (Figure 1). The liquid formulation of PSB, Azospirillum and Azotobacter showed 5.6 × 107, 1.9 × 108 and 3.5 × 107 cfu ml-1 at 480 days. The obtained results were in agreement with Mahdi et al. [19] who reported that the shelf-life of common solid carrier based biofertilizer is around six months; however, it could be as high as two years for a liquid formulation. Unlike solid carrier based biofertilizers, liquid formulations allow the manufacturer to include sufficient amount of nutrients, cell protectant, and inducers responsible for spore/cyst formation to ensure prolonged shelf-life. The enhanced shelf life in individual formulation could be attributed to monoculture conditions. In addition, as concentrations of salts increase in the cell environment with the drying liquid inoculant, stabilizing polymers such as PVP may be useful in reducing the extent of protein precipitation or coagulation of cells. Maintenance of macromolecular structure may improve biological integrity, thus leading to improved survival [14]. Further, solid carrier based biofertilizers are less thermo-tolerant whereas liquid formulations

Table 3: Effect of adjuvant in liquid inoculant formulation.

Adjuvant g-L

108 cfu ml-1

PSB Azotobacter Azospirillum

Carboxyl methyl cellulose

0.1 3.25a (±0.148) 4.1a (±0.187) 3.56a (±0.162)0.2 3.21a (±0.147) 3.85b (±0.176) 3.11c (±0.142)0.3 2.1b (±0.096) 3.17d (±0.145) 3.33b (±0.152)0.4 1.31c (±0.06) 1.8g (±0.082) 1.75d (±0.08)0.5 0.7e (±0.032) 0.42f (±0.019) 0.32h (±0.015)

Xanthan gum

0.1 1.1d (±0.05) 1.22e (±0.056) 1.7d (±0.078)0.2 1.11d (±0.051) 1.43e (±0.065) 1.55e (±0.071)0.3 1.34c (±0.061) 1.44e (±0.066) 1.23f (±0.056)0.4 1.31c (±0.06) 1.41g (±0.064) 1.43e (±0.065)0.5 0.32g (±0.005) 0.33g (±0.015) 0.45g (±0.021)

Carageenum

0.1 0.54f (±0.025) 0.43g (±0.02) 0.62g (±0.028)0.2 0.41g (±0.019) 0.48g (±0.022) 0.6g (±0.027)0.3 0.33g (±0.015) 0.41g (±0.019) 0.55g (±0.025)0.4 0.31g (±0.014) 0.24g (±0.011) 0.11h (±0.005)0.5 0.21g (±0.01) 0.11g (±0.005) 0.07h (±0.003)

Guar gum

0.1 0.11g (±0.005) 0.3g (±0.014) 0.28h (±0.013)0.2 0.1g (±0.003) 0.21g (±0.01) 0.16h (±0.007)0.3 0.06g (±0.002) 0.04g (±0.002) 0.08h (±0.004)0.4 0.051g (±0.002) 0.04g (±0.002) 0.06h (±0.003)0.5 0.04g (±0.001) 0.03g (±0.001) 0.04h (±0.002)

Control - 3.23a (±0.147) 3.88b (±0.177) 3.15c (±0.144)Values are the mean of three determinants ±SD. Within a column different letters (a,b) after values indicate that there is a significant difference at P value of 0.05 as determined by Fischer’s least significant difference test.

Citation: Leo Daniel AE, Venkateswarlu B, Suseelendra D, Praveen Kumar G, Mir Hassan Ahmed SK, et al. (2013) Effect of Polymeric Additives, Adjuvants, Surfactants on Survival, Stability and Plant Growth Promoting Ability of Liquid Bioinoculants. J Plant Physiol Pathol 1:2.

• Page 4 of 5 •Volume 1 • Issue 2 • 1000105

doi:http://dx.doi.org/10.4172/jppp.1000105

Surfactant Percentconcentration

108 cfu ml-1

PSB Azotobacter Azospirillum

Polysorbate 800.025 1.3e (±1.19) 1.4d (±0.63) 1.1f (±0.5)0.05 0.2g (±0.18) 0.6f (±0.27) 0.1e (±0.05)0.1 0.02h (±0.02) 0.07g (±0.03) 0.01e (±0.04)

Polysorbate 400.025 2.1d (±1.92) 2.9b (±1.32) 2.5b (±1.14)0.05 1.0f (±0.91) 1.5d (±0.68) 1.4c (±0.64)0.1 0.25g (±0.23) 0.09g (±0.04) 0.12e (±0.05)

Polysorbate 200.025 3.3a (±3.01) 3.99a (±1.78) 3.52a (±1.55)0.05 3.1b (±2.83) 2.1c (±0.95) 1.1d (±0.5)0.1 2.9c (±2.65) 0.8e (±0.36) 0.2e (±0.09)

Control - 3.4a (±3.15) 4.12a (±1.88) 3.61a (±1.65)

Values are the mean of three determinants ±SD. Within a column different letters (a,b) after values indicate that there is a significant difference at P value of 0.05 as determined by Fischer’s least significant difference test.

Table 4: Effect of surfactants in liquid inoculants formulation.

PSB- LI Azo spirillum- LI Azotobacter- LI11.0

10.5

10.0

9.5

9.0

8.5

8.0

7.5

7.0

30 60 90 120

150

180

210

240

370

300

330

360

390

410

450

480

Days interval

cfu

ml-1

(log

10)

Figure 1: Shelf life of liquid formulated PSB, Azospirillum and Azotobacter.

Figure 2: Growth promotion by bioinoculants in maize 45 DAS.

can tolerate the temperature as high as 55°C. Hence, improved shelf-life could be achieved by the application of a liquid biofertilizer formulation. The mean temperature and pH (data not shown) of all the formulations were recorded at monthly interval. Significant drop in cell population was observed during March (35.6°C), April (38.5°C) and May (39.5°C), 2009. This observation correlates with the fact that the growth rate of bioinoculants at higher temperature (45°C) was low [20]. Under these stress conditions, maintenance of high number of viable cells in liquid inoculants could be attributed to the presence of PVP. The pH of the liquid formulation of PSB ranged from 7.0 to 6.46, Azospirillum (7.01 to 6.71) and Azotobacter (7.1 to 6.6) between 30 and 480 days, respectively.

Pot culture assay

Among five different treatments, maize seeds treated with consortium of PSB and Azospirillum showed significantly growth promotion by the virtue of increased root volume (3.38 cm3), shoot length (68.46 cm), shoot drymass (5.46 g), root drymass (6.47 g) and total drymass (11.9 g) as compared to other treatments (Table 5 and Figure 2). The growth promotion could be attributed to consortium of Bacillus for P solubilisation and Azospirillum for N2 assimilation. This is substantiate by finding of Sumner [21], who reported that inoculation with asymbiotic dinitrogen fixers like Azospirillum may improve plant growth and yield by supplementing the growing plants with fixed nitrogen and growth promoting substances

[21] whereas Rodríguez and Fraga [22], reported that phosphate-solubilizing bacteria (PSB) solubilize insoluble phosphorus and helps in assimilation. Besides, both the bacterium adopt different strategies for plant growth like production of phytohormone, hydrolytic enzymes and solubilisation of other macro and/or micronutrient. Lucangeli and Bottini [23] reported the involvement of GA3 produced by Azospirillum in promoting growth of maize. Co-inoculation of Pseudomonas striata, Bacillus polymyxa and Azospirillum brasilense resulted significant improvement of grain and dry matter yields, with a concomitant increase in N and P uptake, compared with separate inoculations with each strain [24]. Further studies are necessary to explore the possible mechanism of plant growth promotion and nutrient mobilization of consortia microbes. More importantly, on-farm field efficacy studies are essential to strengthen the obtained results.References

1. Leo Daniel Amalraj E, Maiyappan S, John Peter A (2012) In vivo and in vitro studies of Bacillus megaterium var. phosphaticum on nutrient mobilization, antagonism and plant growth promoting traits. Journal of Ecobiotechnology 4: 35-42.

TreatmentsMaize

RV (cm3) SL (cm) RDM (gm)

SDM (gm)

TDM (gm)

Control 1.8f (±0.08)

58.3d (±2.66)

3.23e (±0.148)

3.44e (±0.157)

6.67d (±0.305)

PSB 3.06c (±0.14)

65.3b (±2.98)

4.19c (±0.192)

3.71d (±0.17)

7.91c (±0.361)

Azospirillum 3.18b (±0.15)

66.2b (±3.02)

3.84d (±0.175)

4.23c (±0.193)

8.07c (±0.369)

Azotobacter 2.82e (±0.13)

62.4c (±2.85)

2.73f (±0.125)

3.35f (±0.153)

6.09e (±0.278)

PSB+ Azospirillum 3.38a (±0.15)

68.4a (±3.12)

6.47a (±0.296)

5.46a (±0.249)

11.9a (±0.545)

PSB + Azotobacter 2.91d (±0.13)

64.43bc (±2.94)

4.441b (±0.203)

4.54b (±0.207)

8.986b (±0.41)

CV% 19.42 5.45 31.24 19.39 24.99LSD 0.063 1.797 0.147 0.091 0.234

Table 5: Increase in various plant growth parameters in maize (45 DAS) on seed treatment with liquid bioinoculants in different combinations.

Values are the mean of three determinants ±SD. Within a column different letters (a,b) after values indicate that there is a significant difference at P value of 0.05 as determined by Fischer’s least significant difference test. RV: root volume; SL: shoot length; RDM: root dry mass; SDM: shoot dry mass; TDM: total dry mass

Citation: Leo Daniel AE, Venkateswarlu B, Suseelendra D, Praveen Kumar G, Mir Hassan Ahmed SK, et al. (2013) Effect of Polymeric Additives, Adjuvants, Surfactants on Survival, Stability and Plant Growth Promoting Ability of Liquid Bioinoculants. J Plant Physiol Pathol 1:2.

• Page 5 of 5 •Volume 1 • Issue 2 • 1000105

doi:http://dx.doi.org/10.4172/jppp.1000105

2. Hubbell DH, Kidder G (2009) Biological Nitrogen Fixation. University of Florida IFAS Extension Publication SL16: 1-4.

3. Bottini R, F Cassan, P Picolli (2004) Gibberellin production by bacteria and its involvement in plant growth promotion. Appl Microbiol Biotechnol 65: 497-503.

4. Bloemberg GV, Lugtenberg BJ (2001) Molecular basis of plant growth promotion and biocontrol by rhizobacteria. Curr Opin Plant Biol 4: 343-350.

5. Richardson AE, Barea JM, McNeill AM, Prigent-Combaret C (2009) Acquisition of phosphorus and nitrogen in the rhizosphere and plant growth promotion by microorganisms. Plant Soil 321: 305-339.

6. Idris EE, Iglesias DJ, Talon M, Borriss R (2007) Tryptophan-dependent production of Indole-3- Acetic Acid (IAA) affects level of plant growth promotion by Bacillus amyloliquefaciens FZB42. Mol Plant Microbe Interact 20: 619-626.

7. Whipps JM (2001) Microbial interactions and biocontrol in the rhizosphere. J Exp Bot 52: 487-512.

8. Bhattacharyya P, Kumar R (2000) Liquid biofertilizer-current Knowledge and Future prospect. National seminar on development and use of biofertilizers, biopesticides and organic manures. Bidhan Krishi Viswavidyalaya, Kalyani, West Bengal: 10- 12.

9. Hegde SV (2008) Liquid bio-fertilizers in Indian agriculture. Bio-fertilizer news letter: 17-22.

10. Smith RS (1995) Inoculant formulations and applications to meet changing needs. Current Plant Science and Biotechnology in Agriculture 27: 653-657.

11. Bashan Y, Holguin G, de-Bashan LE (2004) Azospirillum-plant relationships: physiological, molecular, agricultural and environmental advances (1997-2003). Can J Microbiol 50: 521-577.

12. Mugnier J and Jung G (1985) Survival of bacteria and fungi in relation to water activity and the solvent properties of water in biopolymer. Appl Environ Microbiol 50: 108-114.

13. Singleton P, H Keyser, E Sande (2002) Development and evaluation of

liquid inoculants. Australian Centre for International Agricultural Research, Canberra: 52-66.

14. Deaker R, Roughley RJ, Kennedy IR (2004) Legume seed inoculation technology-a review. Soil Biol Biochem 36: 1275-1288.

15. Temprano FJ, Albareda M, Camacho M, Daza A, Santamaría C, et al. (2002) Survival of several Rhizobium/Bradyrhizobium strains on different inoculant formulations and inoculated seeds. Int Microbiol 5: 81-86.

16. Dommergues YR, Diem HG, Divies C (1979) Polyacrylamide-entrapped Rhizobium as an inoculant for legumes. Appl Environ Microbiol 37: 779-781.

17. Bashan Y, Hernandez JP0, Leyva LA, Bacilio M (2002) Alginate microbeads as inoculant carriers for plant growth promoting bacteria. Biol Fertil Soils 35: 359-368.

18. Fernandes Junior PI (2006) A base polymeric composition of carboxymethylcellulose (CMC) and starch as vehicles of Rhizobium inoculation on legume. Dissertation. Federal Rural University of Rio de Janeiro-UFRRJ. 43.

19. Mahdi S, Hassan GI, Samoon SA, Rather HA, Dar SA, et al. (2010) Biofertilizers in organic agriculture. Journal of Phytology 2: 42-54.

20. Gaind S, Gaur AC (1990) Shelf life of phosphate-solubilizing inoculants as influenced by type of carrier, high temperature and low moisture. Can J Microbiol 36: 846-849.

21. Sumner ME (1990) Crop response to Azospirillum inoculation. Adv Soil Sci 12: 53-123.

22. Rodríguez H, Fraga F (1999) Phosphate solubilizing bacteria and their role in plant growth promotion. Biotechnol Adv 17: 319-339.

23. Lucangeli C, R Bottini (1997) Effects of Azospirillum spp. on endogenous gibberellin content and growth of maize (Zea mays L.) treated with uniconazole. Symbiosis 23: 63-72.

24. Alagawadi AR, Gaur AC (1992) Inoculation of Azospirillum brasilense and phosphate-solubilizing bacteria on yield of sorghum [Sorghum bicolour (L.) Moench] in dry land. Trop Agric 69: 347-350.

Submit your next manuscript and get advantages of SciTechnol submissions

� 50 Journals � 21 Day rapid review process � 1000 Editorial team � 2 Million readers � More than 5000 � Publication immediately after acceptance � Quality and quick editorial, review processing

Submit your next manuscript at ● www.scitechnol.com/submission

Author Affiliations Top

1Division of Crops Sciences, Central Research Institute for Dryland Agriculture, Santoshnagar, Hyderabad, Andhra Pradesh, India-5000592Centre for Biotechnology, IST, JNT University, Hyderabad, Andhra Pradesh, India