NEW MAN INTERNATIONAL JOURNAL OF MULTIDISCIPLINARY STUDIES (ISSN: ) Kolkar M.V., Bhosle P.K., Deo M.S., Dr. Bhutada S.A.

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1 6 PHOSPHATE SOLUBILIZING BACTERIA AND THEIR ROLE IN PLANTS Kolkar M.V., Bhosle P.K., Deo M.S., Dr. Bhutada S.A. Abstract: - Phosphorous is major plant growth limiting nutrient, though P is abundant in soils in both organic and inorganic forms, its availability is restricted as it occurs mostly in insoluble forms. Poor availability or deficiency of P markedly reduces plant size and growth. To satisfy crop nutritional requirement P is usually added to soil as chemical P fertilizer, however synthesis of chemical fertilizer is highly impact on the environment in terms of eutrophication, soil fertility depletion. In this regards phosphate solubilising microorganisms have been seen as best eco-friendly means for P nutrition of crops. Phosphate solubilising microorganisms are ubiquitous in soil and it provides P in soluble forms to plants, so it increases P uptake. The main purpose in managing soil phosphorous is to optimize crop production and minimize P loss from soil. This review focus on mechanism of P solubilisation, their role and application in managing sustainable environmental system. Keywords:- Soil phosphorous, phosphate solubilising organisms, phosphate solubilisation, Increase fertility of soil, Environmental balance. Introduction:- Phosphorous is second important key element after nitrogen as a mineral nutrient in terms of quantitative plant requirement, though Phosphorous is abundant in soils in both organic and inorganic forms. Low level of availability of phosphorous is due to high reactivity of soluble P with Ca, Fe and Al. Inorganic phosphate in acidic soil is associated with Fe and Al compounds. The ability to solubilise the mineral-phosphate complexes has been attributed to the ability of PSM to reduce ph of surroundings by releasing organic acids. These organic acids can either dissolve the mineral phosphate as a result of anion exchange can chelate Fe or Al ions associated with the phosphate. However, acidification does not seem to be the only mechanism of P solubilisation as a ability to reduce ph in some cases. The phosphorous content in average soil is about 0.5% but only 0.1% of the total phosphorous is available to plant because of poor solubility and its fixation in soil. 39

2 Phosphorous is one of the major essential macronutrients for plants and is applied to soil in the form of phosphate fertilizers. However, a large portion of soluble inorganic phosphate which is applied to soil as chemical fertilizer is immobilized rapidly and becomes unavailable to plants. It also affects the agricultural production as well as impacts on overall soil health. Thus increased phosphate levels responsible for eutrophication of surface water that may lead to algal blooms. Microbial phosphorous solubilisation trait was analysed by determining the phosphate solubilisation efficiency E (E-diameter of bacterial growth/ diameter of clearing zone *100) Phosphorous also plays an important role in plants in many physiological activities such as cell division, photosynthesis and development of good root system. Phosphorous deficiency results in leaves turning brown accompanied by small leaves, weak stem, and slow development. Occurrence of Phosphate Solubilising bacteria :- High proportion of phosphate solubilising microorganisms is concentrated in rhizosphere, and they are metabolically more active than other sources. It Is also occurs in rhizoplanic soils as well. Soil bacteria are in cocci, bacilli or spiral shapes. Bacilli are common in soil, where as spirilli are very rare in natural environments. Population of phosphate solubilising bacteria depends on different soil properties (physical and chemical properties, organic matter, and P content) and cultural activities. Large population of PSB are found in agricultural land. Some other role of PSM:- Nitrogen Fixation Production of nanoparticals Ability of N fixation: - Phosphate solubilizing bacteria as plant growth promoter. It is direct mechanism nitrogen is the most vital nutrient for plant for plant growth and productivity. Although there is about 78 % nitrogen in the atmosphere, it is unavailable to the growing plants. The atmospheric nitrogen converted into plant utilizable forms by biological nitrogen fixation, which changes nitrogen to ammonia by nitrogen fixing microorganisms using a 40

3 complex enzyme system known as nitrogenase. In fact bacterial nitrogen fixation accounts for approximately two third of the nitrogen fixed globally. Biological nitrogen fixation occurs generally at mild temperature which is fixed by nitrogen fixation microorganisms. In this way phosphate solubilising organisms also have ability to fix the nitrogen in the soil to promote the growth of plants. They are nitrogen fixing organisms. They acts as symbiotic or non symbiotic nitrogen fixing bacteria, it includes members of family rhizobiaceae.plant growth promoting bacteria carried out the process of nitrogen fixation by a complex enzyme the nitrogenise complex. Nitrogen fixing system varies among different bacterial genera. Methods and Material Collection of soil samples:- Soil samples were collected from neighbouring places like Spinach, black gram, bajara, soybeans, coriander, garden soil, tomato, lemon etc. Samples were collected from the selected sites rhizoplenic and rhizoplenic soils. Soil samples were also collected from the rhizosphere of different types of plants. Samples was then taken for the study. The samples were then air dried, powered and mixed well to represent a single sample. Sr.No. Source /place of soil Sample Type 1 Coriander Rhizoplenic 2 Wheat Rhizospheric 3 Spinach Rhizoplenic 4 Garden soil Rhizospheric 5 Fenugreek Rhizoplenic 41

4 Preparation Of Medium:- Ingredients of Pikovskay s agar medium (PVK)- Composition Grams/ml Glucose 10 Ca3 (Po4)2 5.0 (NH4)2So4 0.5 MgSo4 0.2 MnSo FeSo Yeast extract Agar 15.0 Water 1000 Pikovskay s Agar Medium was found to be as selective media for the isolation of Phosphate solubilising organisms. Adjust the ph at 7. Sterilize tricalcium separately; Cool at about 50 C and then mix both the solution. Use:- To test Po4 solubilising activity of microorganisms. Nitrogen free Medium:- Composition Grams/ml K2HPo4 1.0 MgSo4.7H2O 0.2 FeSO4.2H2O 0.5 CaCl2. 2H2O 0.1 NaMoO.2H2O Glucose 10.0 Molybdenum is included to activate nitrogenase for N fixation. 42

5 Isolation of PSM:- PSM were isolated from each sample by serial dilution and spread plate method. One gram of soil sample was dispersed in 9 ml of autoclaved distilled water and was thoroughly shaken. 1ml of above solution was again transferred to 9 ml of sterile distilled water to form 10 ² dilution. Similarly 10 ³, 10 ⁴, 10 ⁵, 10 ⁶, 10 ⁷, and 10 ⁸ serials were made for each sample. 0.1 ml of each dilution was spread on pikovskayas agar medium containing insoluble Tricalcium phosphate and incubated at C for 7 days. Colonies showing halo zones were picked and purified it on Pikovskayas agar medium for studying colony morphology. Study of morphology:- Colony characteristics were studied that is Size, shape, colour, opacity, elevation, consistency, margin, morphology, grams nature were observed and characterize it. Gram staining:- The isolates was characterized for its gram staining characteristics as per the standard procedure and identify it. Isolation of Phosphate Solubilising bacteria:- Out Of these all samples, only 27 isolate exhibiting halozone was found showing the capability of P solubilisation. This isolate had the morphological features like colourless colonies. The PKVK medium was used in present study because it act as specific isolation medium for PSM isolation due to presence of calcium triphosphate which is known for halozone formation. Inoculum preparation for pot assay:- Single colonies of each strain were transferred to 500ml flask containing nutrient broth; colonies were then grows aerobically in flasks on rotating shaker (150rpm) for 48 hours. The bacterial suspension was then diluted in sterile distilled water to a final concentration of 10CFU/ml and resulting suspension were used for treatment. Pot Assay This active culture was inoculated in N free mannitol broth, and incubated at 37 C for 5-6 days. Wheat seeds were soaked separately in distilled water as control.then seeds were soaked separately in different test cultures. The soaked seeds were put in sterile soil and the effect of growth of that seeds after germination was observed.the results were compared with control set. Production of ammonia by phosphate solubilizing bacteria:- 43

6 Bacterial isolates were tested for the production of ammonia in peptone water. Freshly grown cultures were inoculated in 10ml peptone water in each tube and incubated for hours at 37C.Nesslers reagent 0.5ml was added in each tube. Development of brown to yellow colour was positive test for ammonia production. Discussion: Microorganisms with phosphate solubilizing potential increase the availability of soluble phosphate and enhance the plant growth by improving biological nitrogen fixation (Kucey et al )Single and dual inoculation along with P fertilizer was % better than P fertilizer alone for improving grain yield of wheat, and dual inoculation without P fertilizer improved grain yield up to 20 % against sole P fertilization (Afzal and Bano, 2008).. Plants absorb fewer amounts of applied phosphatic fertilizers and the rest is rapidly converted into insoluble complexes in the soil (Mckenzie and Roberts, 1990). But regular application of phosphate fertilizers is not only costly but is also environmentally undesirable. This has led to search for an ecologically safe and economically reasonable option for improving crop production in low P soils. Observations and Results:- Pot assay : Sr. no. Weight of plants in grams Shoot length in cm Root length in cm Total length in cm Control

7 Test for Ammonia production:- Culture number Test Control - 1 Negative 2 Positive 3 Positive 4 Negative 5 Negative 6 Negative 7 Negative 8 Negative 9 Positive 10 Negative 11 Positive 12 Positive 13 Positive 14 Negative 15 Positive 16 Positive 17 Negative 18 Positive 45

8 19 Positive 20 Positive 21 Positive 22 Positive 23 Negative 24 Positive 25 Positive 26 Positive 27 Positive Conclusions:- Pot assay:- By comparing the physical characteristics of control and test plants it was observed that plant growth with test sample in sterile soil shows better results than control one. Ammonia production:- From the all above of phosphate solubilizing isolates some have the ability to produce ammonia which is essential for the plant growth promotion. Culture no.2,3,9,11,12,13,15,16,18,19,20,21,22,24,25,26 and 27 have ability to produce ammonia. Future prospectus:- Phosphate-solubilizing bacteria play an important role in plant nutrition through the increase in P uptake by the plant and their use as PGPR is an important contribution to biofertilization of agricultural crops. Accordingly, further investigation is needed to improve the performance and use of phosphate-solubilizing bacteria as bacterial inoculants. 46

9 References:- Mechanisms and applications of plant growth promoting rhizobacteria: Current perspective Munees Ahemad a,b,*, Mulugeta Kibret b. Phosphorus Solubilizing Bacteria: Occurrence, Mechanisms and their Role in Crop Production Ahmad Ali Khan 1, Ghulam Jilani 1,*, Mohammad Saleem Akhtar 1, Syed Muhammad Saqlan Naqvi 2, Mohammad Rasheed 3 Isolation of Phosphate Solubilizing Microorganism (PSMs) From Soil Sonam Sharma*,Vijay Kumar and Ram Babu Tripathi Phosphate solubilizing bacteria and their role in plant growth promotion Hilda Rodríguez *, Reynaldo Fraga Phosphate solubilizing microbes: Sustainable approach for managing phosphorous deficiency in agricultural soil ; Seema B Sharma, Riyaz Z Sayyad, Mrugesh H Trived, Thivakaran A Gobi. Influence of phosphate dissolving Bacilli on yield and phosphate uptake of wheat crop. Indian J Exp Biol. Gaur AC, Ostwal KP. Soil Microbiology and Biochemistry. San Diego, CA: Academic Press Paul EA, Clark FE. Phosphate solubilizing organisms in improving fertility status. In: Sen SP, Palit P, editors. Biofertilizers: Potentialities and Problems. Calcutta: Plant Physiology Forum, Naya Prokash, Dey KB. 47