Open Access
M.E Shilpa and G.P. Brahmaprakash
Department of Agricultural Microbiology, UAS, GKVK, Bengaluru, Karnataka, India.
J Pure Appl Microbiol. 2016;10(4):2835-2842
https://doi.org/10.22207/JPAM.10.4.44 | © The Author(s). 2016
Received: 13/06/2016 | Accepted: 19/09/2016 | Published: 31/12/2016
Abstract

The present investigation was conducted to find out the effect of vermicompost and urban compost as amendment with talc in maintaining the shelf life of microbial inoculants such as Bradyrhizobium japonicum, Bacillus megaterium and Pseudomonas fluorescens as single, dual and triple inoculants. Talc amended with organic materials maintained better shelf life of microbial inoculants than talc. Maximum viable cells and per cent survival of microbial inoculants were recorded in triple inoculants in all the carrier based formulations followed by dual and single inoculant formulations.

Keywords

Vermicompost, Urban compost, Talc and Microbial consortium.

Introduction

Population of introduced microbial inoculants starts to decline gradually (Roy et al., 2010), it is due to unpredictable and heterogeneous environment of soil. Survival of introduced microbial inoculants in soil depends on various factors like temperature, moisture and carrier material. Carrier material acts as a medium to transfer live microorganisms from laboratory to plant rhizosphere. It provides an environment to microbial inoculants which is conducive to their growth and development. Ideal properties of carrier materials are, should contain high organic matter content, high water holding capacity, it should have neutral pH, amenable to nutrient supplement, available in powder or granular form in abundant quantities at a reasonable price, manageable in mixing, curing and packaging operation, nontoxic to the microbial inoculants and to the plant itself, supports growth and survival of microbial inoculants (Smith, 1992).

Various organic materials such as FYM, vermicompost and compost have been used as carrier materials for formulation of beneficial microbial inoculants (Mohammadi and Sohrabi, 2012). The amendment of carrier with organic materials during the preparation of microbial inoculants will improve inoculants quality such as better adherence to seed, stabilizing the product, and enhances the survivability of microorganisms during storage, exhibit better field performance and tolerance to extreme environmental conditions. Many kinds of additives have been used for inoculant production because of their good rheological properties and high water holding capacity (Mugnier and Jung, 1985).

Many investigations have suggested compost as carrier material for biofertilizers, but the role of compost in maintaining microbial population has not been studied much. In the present study, vermicompost and urban compost are used as amendment with talc to develop carrier based formulation of microbial consortium and their effect on survival of microbial inoculants.

Materials and Methods

The experiment was carried out in the Department of Agricultural Microbiology, University of Agricultural Sciences, Bengaluru. Bradyrhizobium japonicum as nitrogen fixer, Pseudomonas fluorescens as PGPR and Bacillus megaterium as phosphorus solubilizer were used to develop single, dual and triple inoculant formulation in three carrier materials.

Preparation of carrier material
Vermicompost and urban compost were collected sieved through 2 mm sieve and shade dried for 4-5 days, to bring the moisture level up to 5 per cent, powdered and sieved through 90 ìm sieve. The processed samples were mixed separately with talc in the ratio of 1:10, autoclaved and used to develop single, dual and triple inoculant formulation.

Preparation of microbial consortium
Three single inoculant formulations of Bradyrhizobium japonicum, Pseudomonas fluorescens and Bacillus megaterium were prepared. These microorganisms were grown in yeast extract mannitol broth, King’s B broth and Pikovaskaya’s broth respectively and incubated on shaker for 3-4 days. After incubation period, 25 ml of each single microorganism broth was mixed separately with 0.5 per cent carboxy methyl cellulose (CMC) + sterilized talc powder and stored in polythene covers for further use. Same procedure was followed to develop single inoculant formulations for sterilized talc + 10 per cent vermicompost and sterilized talc + 10 per cent urban compost.

Three dual inoculants formulations were prepared with combinations of Bradyrhizobium japonicum + Pseudomonas fluorescens, Bradyrhizobium japonicum + Bacillus megaterium and Pseudomonas fluorescens + Bacillus megaterium. These inoculants broth were mixed in 1:1 ratio. 25 ml of broth containing Bradyrhizobium japonicum and Pseudomonas fluorescens were added to sterilized talc powder and 0.5 per cent CMC, mixed throughly and stored for further study. In the similar way dual inoculants formulations were prepared for talc + 10 per cent vermicompost and talc + 10 per cent urban compost formulations. Similarly dual inoculants of Bradyrhizobium japonicum + Bacillus megaterium and Pseudomonas fluorescens + Bacillus megaterium were prepared.

Triple inoculant formulation was prepared by mixing the broth containing Bradyrhizobium japonicum, Pseudomonas fluorescens and Bacillus megaterium in 1:1:1 ratio. This broth culture was used for the development of talc, talc + 10 per cent vermicompost and talc + 10 per cent urban compost formulations.

Storage and survival study
Inoculant formulations of talc, talc + 10 per cent vermicompost and talc + 10 per cent urban compost formulations were packed in sterilized polythene covers under aseptic conditions and stored at ambient temperature. Survival of developed inoculant formulations was monitored upto 360 days at intervals of 0, 10, 20, 30, 60, 90, 120, 150, 180 and 360 days. Viable counts were enumerated by standard plate count method and number of colony forming units (cfu) were recorded at 10 6 dilutions and converted to log10 cfu per gram.

Statistical analyses of population data was analyzed by using complete randomized design and means were compared by the Duncan’s Multiple Range Test (DMRT) (Little and Hills, 1978).

RESULTS AND DISCUSSION

Survival of single, dual and triple inoculants in talc + 10 % vermicompost formulation
Among single, dual and triple inoculants of talc + 10 % vermicompost formulation, survival of microbial inoculants was found better in triple inoculants compared to single and dual inoculants. Initial 90 days of storage, population of viable cells increased in single, dual and triple inoculants. At the end of 360 days, population of Bradyrhizobium japonicum (log10 6.98 cells/ g), Pseudomonas fluorescens (log10 6.97 cells/ g) and Bacillus megaterium (log10 6.98 cells/ g) were recorded in single inoculant formulation. Dual inoculants containing Bradyrhizobium japonicum + Pseudomonas fluorescens recorded (log10 6.96 and 7.04 cells/ g), Bradyrhizobium japonicum + Bacillus megaterium (log10 6.78 and 6.69 cells/ g) and Pseudomonas fluorescens + Bacillus megaterium (log10 6.84 and 6.98 cells/ g) respectively. Population of Bradyrhizobium japonicum, Pseudomonas fluorescens and Bacillus megaterium was log10 6.30, 6.45 and 6.31 cells/ g respectively in triple inoculants at the end of 360 days (Fig 1). The per cent reduction of cells on log values was found to be 80.60, 82.56 and 80.10 per cent in triple inoculants of B. japonicum, P. fluorescens and B. megaterium respectively (Table 1).

Fig. 1: Survival of microbial inoculants in talc + 10 per cent vermicompost based formulation upto 180 days

Table (1):
Per cent survival of microbial inoculants in talc + 10 % vermicompost based formulations.

Inoculants Population ( Per cent)
Duration of storage ( days)
0 10 20 30 60 90 120 150 180 360
Inoculant 1 Bj 100.00 100.86 102.52a 104.19a 103.97a 103.16a 102.95a 103.08a 100.00ab 89.49a
Inoculant 2 Pf 100.00 100.25 100.81bc 101.36bcd 103.31ab 102.80ab 101.65abc 101.40bc 100.21ab 88.57ab
Inoculant 3 Bm 100.00 100.43 101.02b 101.95bc 101.44cd 102.12ab 101.61abc 101.61b 100.64a 88.92a
Inoculant 4 Bj 100.00 99.83 100.55bcd 101.58bcd 102.81abc 102.13ab 100.77bcd 101.02bcd 100.47a 88.85a
Pf 100.00 99.79 100.55bcd 101.65bcd 100.98d 101.53bcd 99.75d 99.62ef 98.09e 89.50a
Inoculant 5 Bj 100.00 100.34 100.68bc 101.45bcd 102.52abcd 103.16a 102.18ab 100.09cdef 99.36bc 86.92c
Bm 100.00 100.17 99.58cd 100.85d 101.81bcd 100.59cd 99.75d 99.29f 98.82cde 84.57d
Inoculant 6 Pf 100.00 100.98 100.89bc 102.34b 101.66cd 100.30d 100.26cd 100.00def 99.24bcd 87.29bc
Bm 100.00 100.21 100.68bc 100.85cd 101.23cd 101.91abc 101.15bcd 100.00def 98.56cde 88.92a
Inoculant 7 Bj 100.00 99.62 100.34bcd 101.11cd 102.43abcd 102.22ab 101.58abc 100.90bcdf 100.17ab 80.60f
Pf 100.00 99.79 99.19d 101.11cd 101.54cd 101.71bc 100.94bcd 99.15f 98.25de 82.56e
Bm 100.00 100.17 100.47bcd 101.69bcd 101.61cd 100.25d 100.13cd 99.54f 98.60cde 80.10f
LSD at 1% NS NS 1.36 1.19 1.60 1.36 1.59 1.31 0.99 1.29

Note: Means with same superscript are statistically on par at P £ 0.01 by DMRT
Bj: Bradyrhizobium japonicum, Pf: Pseudomonas fluorescens, Bm: Bacillus megaterium

Survival of single, dual and triple inoculants in talc formulation
Talc formulation maintained population density of single inoculants of Bradyrhizobium japonicum, Pseudomonas fluorescens and Bacillus megaterium (log10 6.94, 6.82 and 6.64 cells/ g respectively) at the end of storage period. Consortium containing Bradyrhizobium japonicum + Pseudomonas fluorescens, Bradyrhizobium japonicum + Bacillus megaterium and Pseudomonas fluorescens + Bacillus megaterium maintained population of (log10 6.91 and 6.98 cells/ g), (log10 6.45 and 6.49 cells/ g) and (log10 6.78 and 6.81 cells/ g) respectively and triple inoculants maintained log10 5.95, 6.13 and 6.03 cells/ g of Bradyrhizobium japonicum, Pseudomonas fluorescens and Bacillus megaterium respectively at the end of 360 days (Fig 2). Maximum per cent survival was observed to be 75.45, 76.73 and 76.94 in B. japonicum, P. fluorescens and B. megaterium in triple inoculants respectively at 360 days (Table 2).

Fig. 2: Survival of microbial inoculants in talc based formulation upto 180 days

Table (2):
Per cent survival of microbial inoculants in talc based formulation.

Inoculants Population ( Per cent)
Duration of storage ( days)
0 10 20 30 60 90 120 150 180 360
Inoculant 1 Bj 100.00 99.62ab 100.90a 101.88ab 102.39 101.88a 101.02a 100.34bc 99.53a 88.75a
Inoculant 2 Pf 100.00 100.34a 100.81a 99.87de 101.36 102.04a 100.76a 99.96bc 98.68ab 86.84ab
Inoculant 3 Bm 100.00 99.92ab 100.13abc 99.92d 101.20 101.23ab 100.93a 100.38b 99.02ab 84.48bc
Inoculant 4 Bj 100.00 99.70ab 100.13abc 99.66de 99.41 98.30gh 97.92cd 97.45ef 97.11c 87.99a
Pf 100.00 100.17ab 99.92abc 100.34cd 99.98 99.75cde 98.96bc 98.08def 96.82cd 87.80a
Inoculant 5 Bj 100.00 99.79ab 100.30ab 101.07bc 100.77 100.68bc 100.04ab 99.02cd 98.50ab 82.77c
Bm 100.00 99.58ab 99.54bc 99.62de 100.60 98.61fgh 98.15cd 97.01fg 95.91de 82.06c
Inoculant 6 Pf 100.00 100.00ab 100.98a 102.04a 101.62 101.06ab 100.72a 98.25def 98.00bc 86.60ab
Bm 100.00 99.83ab 100.09abc 99.92d 101.11 99.45def 98.99bc 97.89def 96.91cd 86.43ab
Inoculant 7 Bj 100.00 99.92ab 98.90c 98.69f 100.69 100.30bcd 100.00ab 98.61de 95.52e 75.45d
Pf 100.00 99.04b 99.25bc 98.99ef 99.81 99.04efg 98.42cd 97.12a 93.66f 76.73d
Bm 100.00 100.13ab 100.04abc 99.45def 100.94 97.66h 97.70d 95.87g 94.17f 76.94d
LSD at 1% NS 1.22 1.22 0.88 NS 1.09 1.08 1.31 1.17 2.88

Note: Means with same superscript are statistically on par at P £ 0.01 by DMRT
Bj: Bradyrhizobium japonicum, Pf: Pseudomonas fluorescens, Bm: Bacillus megaterium

Survival of single, dual and triple inoculants in talc + 10 % urban compost formulation
Viable cells of Bradyrhizobium japonicum (log10 6.15 cells/ g), Pseudomonas fluorescens (log10 6.25 cells/ g) and Bacillus megaterium (log10 6.17 cells/ g) were noticed in triple inoculants of talc + 10 % urban compost formulation where as single inoculants of  Bradyrhizobium japonicum (log10 6.96 cells/ g), Pseudomonas fluorescens (log10 6.93 cells/ g) and Bacillus megaterium (log10 6.91 cells/ g) at the end of 360 days. Consortium of Bradyrhizobium japonicum + Pseudomonas fluorescens (log10 6.98 and 7.00 cells/ g), Bradyrhizobium japonicum + Bacillus megaterium (log10 6.68 and 6.56 cells/ g) and Pseudomonas fluorescens + Bacillus megaterium (log10 6.76 and 6.85 cells/ g) were maintained at the end of storage period (Fig 3). 78.39, 79.46 and 78.04 per cent cells were recorded at the end of 360 days in triple inoculants of B. japonicum, P. fluorescens and B. megaterium respectively (Table 3).

Fig. 3: Survival of microbial inoculants in talc + 10 per cent urban compost based formulation upto 180 days
Bj: Bradyrhizobium japonicum, Pf: Pseudomonas fluorescens, Bm: Bacillus megaterium, I: Inoculant

Table (3):
Per cent survival of microbial inoculants in talc + 10 % urbancompost based formulation.

Inoculants Population ( Per cent)
Duration of storage ( days)
0 10 20 30 60 90 120 150 180 360
Inoculant 1 Bj 100.00 100.90a 102.34a 102.94a 102.60a 102.04ab 101.53ab 100.73abc 100.30a 88.89ab
Inoculant 2 Pf 100.00 100.34abc 100.00b 100.68cd 101.40ab 101.65ab 101.27abc 101.10ab 98.73ab 87.83abc
Inoculant 3 Bm 100.00 99.66bcde 100.72b 101.36bc 101.83ab 102.34a 101.62a 101.11ab 99.71a 88.06ab
Inoculant 4 Bj 100.00 100.64ab 100.34b 99.74d 101.24ab 101.50ab 101.20abc 99.36cde 95.47d 89.49a
Pf 100.00 99.54cde 99.92b 100.46cd 101.73ab 101.18abc 99.92cd 99.88abcde 97.31bc 88.46ab
Inoculant 5 Bj 100.00 100.08abcde 99.54b 100.72cd 101.35ab 101.56ab 100.51abc 100.25abcd 97.55bc 84.53cd
Bm 100.00 100.85a 100.51b 101.27bc 102.03ab 101.48ab 100.05bcd 99.79bcde 98.86ab 83.18d
Inoculant 6 Pf 100.00 99.07e 99.92b 101.27bc 101.73ab 100.84bc 100.55abc 98.78e 97.68bc 85.57bcd
Bm 100.00 100.13abcd 101.02ab 102.04ab 101.45ab 101.82ab 100.47abc 98.90de 97.25bc 87.08abc
Inoculant 7 Bj 100.00 100.30abc 99.79b 101.32bc 101.91ab 102.25a 101.49ab 101.23a 99.49a 78.39e
Pf 100.00 100.34abc 100.93ab 101.61bc 101.06ab 100.00cd 100.80abc 98.94de 96.62cd 79.46e
Bm 100.00 99.24de 99.87b 100.55cd 100.38a 99.20d 98.82d 98.69e 97.43bc 78.04e
LSD at 1% NS 1.00 1.50 1.23 1.83 1.22 1.52 1.37 1.59 3.18

Note: Means with same superscript are statistically on par at P £ 0.01 by DMRT
Bj: Bradyrhizobium japonicum, Pf: Pseudomonas fluorescens, Bm: Bacillus megaterium.

The investigation aimed on two different aspects, one is carrier based formulation and another was consortium development. The survival of any microbial inoculants mainly depends on the carrier material. Carrier materials used in the study satisfies the essential criteria of ideal carrier such as high water holding capacity, rich organic matter content, neutral pH, easy availability and low cost (Arangarasan et al., 1998). The maximum viable cells of Bradyrhizobium japonicum, Pseudomonas fluorescens and Bacillus megaterium in single, dual and triple inoculants were maintained in talc amended with vemicompost and urbancompost formulation compared to talc formulation. This may be due to high nutrient content present in organic materials supported the survival of microbial inoculants during storage period. Similar results were obtained by Rajasekar and Karmegam (2010) reported more than 1 X 107 viable cells/ g (Azotobacter chroococcun, Bacillus megaterium and Rhizobium leguminosarum) observed in different combination of vermicast with lignite upto 10 months. Survival of Pseudomonas fluorescens, Bacillus subtilis, Azospirillum brasilense were reported in different organic carriers like vermicompost, spent mushroom, farm yard manure, pressmud, rice bran (Karunya and Reetha, 2014;  Gade et al., 2014 and Singh et al., 2014).

Highest survival rate of microbial inoculants were found in triple inoculants in all the developed carrier based formulations. The significant difference in survival was mainly due to synergistic effect of the inoculants on each other. The factors that affect the longevity of the cells of microbial inoculants include temperature, moisture and carrier materials. The result of the study is on par with the findings of Sahu et al., 2013 and Lavanya et al., 2015, reported triple inoculant formulation maintained maximum population of Acinetobacter sp., Pseudomonas fluorescens, Bacillus megaterium and Azotobacter chroococcum in consortium.

Commonly used carrier for microbial inoculants are peat, lignite, talc etc., which support the growth of microbial inoculants. Use of organic materials like vermicompost, urban compost as carrier increases the survival rate of microbial inoculants in biofertilizer formulation by providing nutrients for growth and metabolic activities of microbial inoculants during storage. The study indicated that the use of organic materials as amendment with talc was found to be successful in maintaining the viable cells of microbial inoculants in consortium for longer period of time which could meet the Bureau of Indian Standards and also possible delivery of microbial consortium in a single carrier. Hence organic materials can be used as amendment with talc which can be an efficient carrier for development of microbial inoculant formulation.

References
  1. Arangarasan, V., Palaniappan, S.P., Chelliah, S. Inoculation effects of diazotrophs and phosphobacteria on rice. Indian J. Microbiol., 1998; 38(2): 111-112.
  2. Gade, R.M., Chaithanya, B.H., Khurade, K.C. A comparative study of different carriers for shelf life of Pseudomonas fluorescens. The Bioscan, 2014; 9(1): 287-290.
  3. Karunya, S.K., Reetha, D. Survival of saline tolerant PGPR in different carriers and liquid formulations. Int. J. Adv. Res. Biol. Sci., 2014; 1: 179-183.
  4. Lavanya, G., Sahu, P.K., Manikanta, D.S., Brahmaprakash, G.P. Effect of fluid bed dried formulation in comparison with lignite formulation of microbial consortium on finger millet (Eleusine coracona Gaertn.). J. Pure Appl. Microbiol., 2015; 9(2): 193-199.
  5. Little, T.M., Hills, J.F. Agricultural experimentation. John Wiley and sons, New York, USA. 1978.
  6. Mohammadi, K., Sohrabi, Y. Bacterial biofertilizer for sustainable crop production, A review. J. Agril. Biol. Sci., 2012; 7: 2.
  7. Mugnier, J., Jung. G. Survival of bacteria and fungi in relation to water activity and the solvent properties of water in biopolymer. Appl. Environ. Microbiol., 1985; 50: 108-114.
  8. Rajasekar, K., Karmegam, N. Earthworm casts as an alternate carrier material for biofertilizers: Assessment of endurance and viability of Azotobacter chroococcum, Bacillus megaterium and Rhizobium leguminosarum. Scientia Horticulturae., 2010; 124: 286–289.
  9. Roy, B.D., Deb, B., Sharma, G.D. Evaluation of carrier based inoculants of Azotobacter Chroococcun strain SDSA-112/2 in improving growth and yield of summer rice. IR-36. Biofrent, 2010; 1: 36-40.
  10. Sahu, P.K., Lavanya, G., Brahmaprakash, G.P. Fluid bed dried microbial inoculants formulation with improved survival and reduced contamination level. J. Soil Biol. Ecol., 2013; 33(1-2): 81-94.
  11. Singh, S., Govind Gupta, Ekta Khare, Behal, K.K., Naveen, K.A. Effect of enrichment material on the shelf life and field efficiency of bioformulation of Rhizobium sp. and P-solubilizing Pseudomonas fluorescens. Sci. Res. Reporter, 2014; 4(1): 44-50.
  12. Smith, R.S. Legume inoculant formulation and application. Can. J. Microbiol., 1992; 38: 485-492.

Article Metrics

Article View: 2619

Share This Article

© The Author(s) 2016. Open Access. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License which permits unrestricted use, sharing, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.