ISSN: 2395-6887

Contents lists available at www.iosi.in

ISOI Journal of Microbiology, Biotechnology and Food Science

Volume 1 Issue 1 October 2015; Page No. 23-28

Pag

e23

ISOLATION AND CHARACTERIZATION OF POTENT PHOSPHATE SOLUBLIZING BACTERIA

1MAHANTESH S. P.,

2PATIL C S. AND

3HIMANSHU.

1,3Department of Biotechnology, OPJS University, Churu, Rajasthan

2Department of Biotechnology, B.V.Bhoomaraddi College of UG and PG Bidar, KARNATAKA

Author Email: mahanteshpanchal@gmail.com

ABSTRACT: Soil Microorganisms play an vital role in maintaining the ecological balance by active participation in Carbon, Nitrogen, Sulphur and Phosphorous cycles in nature. Phosphate Solubilizing Microbes plays an important role in plant nutrition through increase in phosphate uptake by plants and used as biofertilizers of agricultural crops. Phosphate is one of the most vital macronutrient required for the growth and development of plants. A large number of microorganisms present in the rhizosphere are known to solubilize and make available the insoluble phosphorus in the available form to the plants. The present investigation helps for identification, isolation and characterization of phosphate solublizing microbes which are more efficient in utilization of phosphorous present in soil. In the study 10 different samples were collected from root rhizosphere of different crop plants of Bidar and Kalaburagi, Karnataka and isolated 29 different phosphate solublizing bacteria’s isolated, in these two potent efficiently phosphate solublizing bacteria’s were isolated and they are characterized under invitro condition. Keywords: Phosphorous, Phosphate solublizing bacteria, Rhizosphere etc.

INTRODUCTION Phosphorus (P) is one of the essential macronutrients for plant growth and reproduction. However, it is a limiting factor in many soils, because an important part of this element is insoluble (Del Campillo et al., 1999). Mineral P solubilization is a common phenotype in several rhizobacteria, hence the term "phosphate solubilizing bacteria" PSB (Pérez et al., 2007). The application of these bacteria in the soil can increase plant productivity by improving P nutrition (Hameeda et al., 2008). These PSB also can stimulate plant growth by other mechanisms such as the production of phytohormones, nitrogen fixation, inhibition of phytopathogenic microorganisms, production of siderophores and ACC deaminase (Bhattacharyya & Jha, 2012).

Phosphate solubilizing microorganisms (PSM) play a significant role in making phosphorus available to plants by bringing about favourable changes in soil reaction in the soil microenvironment leading to solubilization of inorganic phosphate sources. Some microorganisms associated with different plant rhizosphere are able to solubilise inorganic insoluble P salts. Pseudomonas and Bacillus are two important genera of soil bacteria with promising activity of phosphate solubilisation (Reyes et al.,1999; Yadav and Tarafdar, 2011).

The majority of the isolated organisms are bacterial organisms, although several fungi are also known to solubilize phosphates. These bacteria and fungi have the potential to be used as biofertilizers. Their role in

increasing the soil nutrient value is of utmost importance. Their application to crop fields has resulted in an increased yield of several crops, such as cereals, legumes, fibers, vegetables, oils, and other crop plants (Silini-Cherif, 2012; Viruel et al., 2011; Khalimi et al., 2012).

The objective of this research work is to isolate the PSB strains from the rhizosphere soil of different crop plants such as bajra, red gram, maize, and vegetables as bean and tomato. Further, the isolated strains were identified and characterized

MATERIALS AND METHODS

Collection of Soil samples: The soil samples were collected from the depth of 10-15cm from the rhizosphere of different crop plants like Bajra, Redgram, maize and vegetables like bean and tomato etc. Around the agricultural land of Bidar and Kalaburagi, Karnataka. Collected soil samples were carried aseptically in polythene bags and maintained at the laboratory for the further study.

Isolation of PSM Isolation of Phosphate Solublizing microbes done with the suspension of different rhizospere soil samples(1g) in 100ml of distilled water. Aliquots of 100microlitre were inoculated of Pikovskaya medium by pour plate technique and also with swab spread plate technique and plates were incubated at 30°C. Colonies showing in plates considers as PSM. Single PSM colony were picked

MAHANTESH S. P., et al, ISOI Journal of Microbiology, Biotechnology and Food Science

Pag

e24

and transferred in broth and slants of Pikovskaya medium for further study.

IDENTIFICATION AND CHARACTERIZATION: Identification of the microbes were done by morphological and biochemical analysis. The bacterial isolates were identified by morphological characteristics in which gram staining, shapes, motility test, spore forming test and biochemical analysis as follows.

INDOLE TEST The organisms were grown in tryptone broth for 48 hr at 370C. Development of a cherry red colour at the upper layer by adding five drops of Kovac’s reagent (prepared by adding 10 g of p-dimethyl aminobenzaldehyde in 150 ml of isoamylalcohol and then added 50 ml of HCl.) indicated a positive test

METHYL RED (MR) TEST To 5 ml culture in MR-VP broth added 5 drops of methyl red indicator solution (0.2% in 50% alcohol). Appearance of red colour indicated a positive test.

VOGES PROSKAUER (VP) TEST Barritt’s reagent and MR-VP medium were used. In this test, production of acetoin (acetyl methyl carbinol) is detected by the addition of few drops of 5% alpha naphthol in 0.2 ml of 40% aqueous solution of KOH. The mixture in the tubes were shaken vigorously for few minutes and allowed to stand for two hours. Positive reaction was shown by development of crimson ruby colour.

CITRATE UTILIZATION TEST Tubes containing Simmon’s citrate agar media were inoculated by each organism. Following incubation citrate positive cultures were identified by the presence of growth on the surface of slant accompanied by blue colouration.

H2S PRODUCTION TEST Filter paper strips were saturated with 5% led acetate solution, air dried and autoclaved at 10 lb pressure for 15 min. Inoculated the nutrient broth tubes with the desired culture and the led acetate strips were inserted above the liquid between the plug and inner wall of tube. Incubated and examined the blackening of paper, indicating H2S production.

CASEIN HYDROLYSIS TEST Skim milk agar plates were prepared. Plates were incubated at 300C for 4 days after spot inoculation and observations were taken regularly. Formation of clear zone was taken as positive.

GELATIN HYDROLYSIS TEST Gelatin agar medium was prepared by adding 12% gelatine in nutrient broth, dispensed in tubes and autoclaved at 1210C for 12 minutes. Gelatin tubes were inoculated by stabbing to the bottom and incubated at

370C along with uninoculated controls for 30 days. Liquefaction at weekly intervals was detected after placing the tubes in refrigerator for 30 minutes.

STARCH HYDROLYSIS TEST Starch agar (prepared by adding 20 ml of 10% starch solution, steamed for 1 hr in 100 ml of melted nutrient agar) poured in petriplates was inoculated with test organisms and incubated for 3-5 days. The plates were flooded with Gram’s iodine solution. Blue colour of medium with colourless areas around growth indicated a positive test.

UREA HYDROLYSIS TEST Christensen’s urease agar medium containing the indicator phenol red were inoculated with test cultures and incubated at 370C for 18-24 hrs. A rose red pink colour is an indication of positive test for urease.

NITRATE REDUCTION TEST The cultures were grown in nitrate peptone broth containing potassium nitrate – 2 g, peptone- 5 g, distilled water- 1000 ml for five days and five drops of solution A and B each were added.

Solution A: Sulfanilic acid -8 g; Acetic acid 5 N – 1 litre Solution B: α- Napthylamine- 5 g; Acetic acid 5 N – 1 litre The tubes showing red colour within two minutes were taken as positive test for nitrate reduction test.

CATALASE TEST A small portion of microbial growth from solid medium was taken and emulsified with a drop of H2O2 (3-6%). Immediate effervescence indicated a positive catalase reaction.

OXIDASE TEST A strip of filter paper moistened with 1% solution of tetra methyl para phenyline diamine dihydrochloride was placed on the lid of a Petri dish, using a sterile platinum wire loop small portion of growth was taken and applied on the filter paper. Immediate development of deep purple colour indicated the test organism as oxidase positive.

TWEEN HYDROLYSIS Medium was prepared by adding 1 ml of Tween, 80 (Sigma) to molten nutrient agar cooled to 550C. mixed gently several times to disperse the tween and poured into Pertidish. Spot inoculated the culture under test on to the plate. Known positive and negative bacteria were also included. The plates were placed in a plastic bag to keep them moist and incubated at 370C up to 5 days. Read as Tween positive those colonies that develop a clearly visible halo of precipitated fatty acids when viewed against a dark background.

MAHANTESH S. P., et al, ISOI Journal of Microbiology, Biotechnology and Food Science

Pag

e25

Optimization of Physiological conditions

(pH and Temperature): The Phosphate solublizing microbes isolates were analyzed using various pH(6-7.5) and Temperature(25-35°C) and the results were recorded.

pH Tricalcium phosphate solubilizing efficiency of isolates were tested at various pH values (6, 6.5, 7, 7.25, and 7.5) after fourteen days of incubation period

Temperature Solubilization efficiency was examined at different temperatures (25, 27.5, 30, 32.5 and 35°C).

RESULT AND DISCUSSION

Isolation of PSM Ten soil samples were collected from different crop plants of Bidar and Kalaburagi (Table 5). All samples showed acidic pH. Twenty nine potential PSBs were isolated from different rhizosphere soil samples. All the isolates were subjected to in vitro tricalcium phosphate solubilization. The isolates showed variations in their P solubilizing ability. Their extents of P solubilization ranged between 11.38 – 73.52 mg/100 ml of soluble P in liquid medium. In general, P solubilizations of all the isolates were accompanied by reduction in pH of the culture filtrate. Maximum solubilization efficiency (SE%) was detected in PSB 13.

Among the isolates, 05 PSBs showed promising results and were re-evaluated for their phosphate solubilizing activity.

Quantitative evaluation of soluble P in the liquid medium showed that PSB 12 and 29 were the most efficient P solubilizing isolates. These two bacteria along with standard phosphate solubilizing Pseudomonas striata were utilized for further studies. The selected isolates were subjected to morphological, cultural physiological and biochemical analysis for identification.

Both isolates were aerobic, Gram negative, non-sporulating and motile rods arranged in single and pairs. PSB 29 found to possess diffusible brownish pigment. These isolates were able to grow over a wide range of temperature (25-35°C) and pH (6-7.5), PSB 29 found to be metabolically more active compared to PSB 12.

Table 1: Morphological Tests

Test PSB 12 PSB 29

Colony Morphology

Configuration Circular Circular

Margin Entire Lobate

Surface Smooth and shiny Pale brownish

Pigment - Pale brown

Grams Negative Negative

reaction

Cell Shape Rod Rod

Arrangement Single and pair Single and pair

Spore - -

Motility + +

Table 2: Physiological Tests

Tests(Growth) PSB 12 PSB29

Temperature

10°C - -

15°C - -

20°C + +

25°C + +

30°C + +

37°C + +

42°C + +

52°C - -

pH

pH 4.0 + +

pH 5.0 + +

pH 6.0 + +

pH 7.0 + +

pH 8.0 NG NG

pH 9.0 NG NG

NaCl

2.0 NG NG

4.0 NG NG

5.0 NG NG

7.0 NG NG

10.0 NG NG

Anaerobic - -

Table 3: Biochemical tests

Tests PSB12 PSB 29

Growth on MacConkey agar

NG NG

Inole test - -

Voge Proskauer test - -

Citrate utilization NG NG

H2S Production - -

Casein hydrolysis - -

Esculin Hydrolysis - -

Gelatin Hydrolysis NG -

Starch hydrolysis NG -

Urea hydrolysis NG NG

Nitrate reduction - -

Catalase test - -

Oxidase test - -

Lysine decarboxylase + -

Arginine dihydrolase - +

Ornithine decarboxylase - +

Tween 80 hydrolysis - +

Tyrosine degradation NG -

Hippurate hydrolysis - -

NG: No growth

MAHANTESH S. P., et al, ISOI Journal of Microbiology, Biotechnology and Food Science

Pag

e26

Out of 95 substrates utilized in BIOLOG GN2 plate PSB 12 was able to utilize 19 different substrates while PSB 29 assimilated 40 distinct substrates as their sole source of carbon. Both the isolates were able to utilize glucose, mannitol, mannose and arabinose as their C source. But assimilation of inositol and galactose was detected only in PSB 29.

The isolate PSB 12 was from Maize rhizosphere soil collected from Bidar, Karnataka. On the other hand PSB 29 was isolated from rhizosphere soil of Bajra collected from Kalaburagi disctrict, Karnataka (Plate I). Both the isolates showed considerably greater P solubilizing activity compared to standard P solubilizing Pseudomonas striata (50.12 mg/100 ml of soluble P). On the basis of above tests, the organism(s) has been identified as follows:

Fig 1: Growth of Phosphate solublizing microbes on Pikovaskaya’s medium

CONCLUSION: All the isolated bacterial strains were found to exhibit phosphate solubilizing capacity in varying degrees of culture filtrate. The strains isolated were gram negative rod shaped, non-spore forming, motiles arranged in single and pairs. The isolates able to grow over a wide range of temperature (25-35°C) and pH (6-7.5), the isolate PSB29 found to be more metabolically active compared to PSB 12. The PSB 29 solubilised significantly higher amount of

inorganic phosphate and demonstrating their higher potential to be used as soil ino-inoculants to enhance soil fertility and plant growth.

REFERENCES

1. Abdol Amir Yousefi, Kazem Khavazi, Abdol Amir Moezi, Farhad Rejali and HabibAllahNadian.2011. hosphate Solubilizing Bacteria and Arbuscular Mycorrhiza Fungi Impacts on Inorganic Phosphorus Fractions and Wheat Growth. World Applied Sciences Journal. 15 9: 1310-1318.

2. Aftab Afzal and Asghari Bano.2008. Rhizhobium and Phosphate Solubilizing Bacteria improve the yield and phosphours uptake in Wheat Triticum aestivum. International Journal of Agriculture and Biology.101:85-88.

3. Aftab Afzal, M, Ashraf, Saeed, A Asad and Farooq, M .2005.Effect of phosphate solubilizing microorganism on phosphorus uptake, yield traits of wheat Triticum aestivum L..International Journal of Agriculture and Biology.72:207-209

4. Ahmad Ali Khan, Ghulam Jilani, Mohammad Saleem Akhtar, Syed Muhammad Saqlan Naqvi,Mohammad Rasheed.2009. Phosphorus Solubilizing Bacteria: Occurrence, Mechanisms and their Role in Crop Production. Journal of Agricultural Biological science. 11:48-58.

5. Amardip Singh, Poonam and Ghosh, A.K.2012. Screening and Assessment of Phosphate Solubilising Microbes as Potential Biofertilizer, isolated from

MAHANTESH S. P., et al, ISOI Journal of Microbiology, Biotechnology and Food Science

Pag

e27

Ecosystem of Bihar. Asian Journal of Experimental Biological Science .32:397-406.

6. Amit Sagervanshi, Priyanka Kumari, Anju Nagee and Ashwani Kumar.2012. Isolation and Characterization Of Phosphate Solublizing Bacteria from Anand Agriculture Soil.International Journal of Life Sciences and Pharma Research. 23:256-266.

7. Barea, J., M. J. Pozo, R. Azcon and C. Azcon-Aguilar.2005. Microbial cooperation in the rhizosphere J. Exp. Bot. 56: 1761 - 1778.

8. Bottini, R., F. Cassán and P. Piccoli. 2004. Gibberellin production by bacteria and its involvement in plant growth promotion and yield increase. Appl. Microbiol. Biotechnol. 65:497-503.

9. Buddhi Charana Walpola and Min-Ho Yoon.2012. Prospectus of phosphate solubilizing microorganisms and phosphorus availability in agricultural soils: A review.African Journal of Microbiology Research. 637: 6600- 6605.

10. Federica Tamburini, Verena Pfahler, Else K. nemann, Kathi Guelland, Stefano M. Bernasconi and Emmanuel Frossard.2012.Oxygen Isotopes Unravel the Role of Microorganisms in Phosphate Cycling in Soils. Environmental Science and Technology.46:5956-5962.

11. Fengling Zhu, Lingyun Qu, Xuguang Hong, and Xiuqin Sun. 2011.Isolation and characterization of a phosphatesolubilizing Halophilic Bacterium Kushneria sp.YCWA18 from Daqiao Saltern on the Coast of Yellow Sea of China. Evidence-based Complementary Alternative Medicine.1-6.

12. Glick, B.R., 1995. The enhancement of plant growth by free living bacteria. Can.J. Microbiol. 41:109 117.

13. Gupta,A., J.M.Meyer and R.Goel.2002.Development of Heavy metal- resistant Mutants of phosphate Solubilizing Pseudomonas sp. NBRI 4014 and their characterization. Current Microbiology.45:323-327.

14. Gyaneshwar,P., G. Naresh Kumar, L. J. Parekh and P. S. Poole.2002.Role of soil microorganisms in improving P nutrition of plants. Plant and Soil. 245: 83 93.

15. Hafeez, F. Y., M. E. Safdar, A. U. Chaudhry and K. A. Malik 2004. Rhizobial inoculation improves seedling emergence, nutrient uptake and growth of cotton. Australian Journal of Experimental Agriculture .446: 617 622.

16. Hassan Shokri Vahed, Parisa Shahinrokhsar and Fatemeh Heydarnezhad.2012. improving growth and yield of rice Oryza Sativa L. in the presence of phosphorus fertilizer .International Journal of Agriculture and Crop Sciences. 4 17:1228-1232.

17. Hilda Rodríguez and Reynaldo Fraga.1999. Phosphate solubilizing bacteria and their role in plant growth promotion. Biotechnology Advances. 17 1999:319 339.

18. Humaria Yasmin and Asghari Bano.2011.Isolation and Characterization of Phosphate Solubilizing Bacteria from Rhizospheric soil of weeds of Khewra Salt Range and Attock. Pakistan .J .Bot., 433:1663- 1668.

19. Ivanova, R Bojinova,D Nedialkova, K.2006. Rock Phosphate Solubilization by Soil Bacteria. Journal of the University of Chemical Technology and Metallurgy. 413: 297-302

20. Jinhee Park, Nanthi Bolan, Mallavarapu Megharaj, and Ravi Naidu . 2010. Isolation of Phosphate-Solubilizing Bacteria and their Effects Characterization of on Lead Immobilization. Pedologist. 67-75.

21. Katiyar V and R. Goel, 2003. Solubilization of inorganic phosphate and plant growth promotion by cold tolerant mutants of Pseudomonas fluorescens. Microbiol Res .158:163 168.

22. Kannapiran, E and Sri Ramkumar, V. 2011. Isolation of phosphate Solubilizing bacteria from sediments of Thondi coast, Palk Strait, Southeast coast of India. Annals of Biological Research.25:157-163.

23. Khosro Mohammadi and Yousef Sohrabi2012. Bacterial Biofertilizers For Susstainable Crop production: A Review.ARPN Journal of Agricultural and biological Science.7:307- 316.

24. Kucey, R.M.N. 1983.Phosphate-Solubilizing bacteria and fungi in various cultivated and virgin Alberta soils. Canadian Journal of Soil Science.63:671- 678.

25. Panhwar,Q.A.,Radziah,O,Zaharah, A.R., Sariah,M.,Mohd.Razi,L.2010.Role of phosphate solubilizing bacteria on rock phosphate solubility and growth of aerobic rice. Journal of Environmental Biology.32:607-612.

26. Patten, C. L. and B. R. Glick 2002. Role of Pseudomonas putida indoleacetic acid in development of the host plant root system. Appl. Environ. Microbiol. 68:3795-3801.

27. Ponmurugan, P and Gopi, C.2006. In vitro production of growth regulators and phosphatase activity by phosphate solubilizing bacteria.African Journal of Biotechnology. 5 4: 348-350.

28. Pradhan, N and Sukla, L B.2005 .Solublization of inorganic phosphates by fungi isolated from agriculture soil. African Journal of Biotechnology. 510:850-854.

29. Sadia Alam, Samina Khalil, Najma Ayub and Maliha Rashid. 2002. In vitro solubilisation of inorganic phosphate by phosphate solubilizing microorganisms PSM from maize rhizosphere. International Journal of Agriculture and Biology.4:454-458.

30. Sanjotha,P., Mahantesh ,P.,and Patil,C.S.2011. Isolation and Screening of efficiency of Phosphate Solubilizing Microbes. International Journal of Microbiology Research. 31:56-58.

MAHANTESH S. P., et al, ISOI Journal of Microbiology, Biotechnology and Food Science

Pag

e28

31. Shruti Agrawal and R. K. Pathak.2012.Phosphate Solubilization by Alcaligenes faecalis over Pseudomonas fluorescens. Agricultural Science Research Journals. 22: 92-94.

32. Shekhar Nautiyal,C.1999. An efficient microbiological growth medium for screening phosphate solubilising microorganisms. FEMS Microbiology Letters. 170 . 265-270.

33. Sung-Man Woo, Min-Kyoung Lee, In-Soo Hong, S. Poonguzhali and Tong- Min Sa.2010. Isolation and characterization of phosphate solubilizing bacteria

from Chinese cabbage.19th World Congress of Soil Science, Soil Solutions for a Changing World.56-59.

34. Tripti, Vipin Kumar and Anshumali.2012.Phosphate Solubilizing Activity of Some Bacterial Strains Isolated from Chemical Pesticide Exposed Agriculture Soil. International Journal of Engineering Research and Development. 39:01-06.

35. Uma Maheswar ,N., and G. Sathiyavani.2012. Solubilization of phosphate by Bacillus Sps, from groundnut rhizosphere Arachis hypogaea L. Journal of Chemical and Pharmaceutical Research. 48:4007- 4011.