Indian Patents. 226577:"A PROCESS FOR PREPARATION OF A SYNERGISTIC COMPOSITION USEFUL AS MICROBIOLOGICAL GROWTH MEDIUM FOR SCREENING OF PHOSPHATE SOLUBILISING MICRO-ORGANISM"

Title of Invention	"A PROCESS FOR PREPARATION OF A SYNERGISTIC COMPOSITION USEFUL AS MICROBIOLOGICAL GROWTH MEDIUM FOR SCREENING OF PHOSPHATE SOLUBILISING MICRO-ORGANISM"
Abstract	A synergistic composition useful as microbiological growth medium for screening of phosphate solubilising micro-organism: The present invention provides a synergistic composition useful as a microbiological growth media, to quickly evaluate the level of phosphate solubilisation.

Full Text	The present invention relates to a synergistic composition useful as microbiological growth medium for screening of phosphate solubilising microorganism. More particularly the present invention provides composition of microbiological growth medium for qualitative screening of phospnate solubilising microorganisms, based upon visual observation. A larce portion of inorganic phosphates applied to soil as fertiliser is rapidly immobilised after application and becomes unavailable to plants [Yadav KS. Dadarwal KR (1997) Phosphate solubilisation and mobilisation through soil microorganisms. In: Dadarwal KR (ed): Biotechnological approaches in soil microorganisms for sustainable crop production. Jodhpur. India: Scientific Publishers, pp. 293-308]. Thus, the release of insoluble and fixed forms of phosphorus is an important aspect of increasing soil phosphorus availability. Seed or soil inoculation with phosphate solubilising bacteria is known to improve solubilisation of fixed soil phosphorus and applied phosphates resulting in higher crop yields [Abd-Alla MH (1994) Phosphatases and the utilisation of organic phosphorus by Rhizobium leguminosarum biovar viceae. Lett Appl Microbiol 18:294-296: Jones DL. Darrah PR (1994) Role of root derived organic acids in the mobilisation of nutrients from the rhizosphere. Plant Soil 166:247-257; Leyval C, Barthelin J (1989) Interactions between Laccaria laccata. Agrobacierium radiobacier and beech roots: Influence on P. K. Mg and Fe mobilisation irom mineral and plant growth. Plant Soil 17:103-110: Yadav KS. Dadarwal KR (1997) Phosphate solubilisation and mobilisation through soil microorganisms. In: Dadarwal KR (ed). Biotechnological approaches in soil microorganisms for sustainable crop production. Jodhpur, India: Scientific Publishers, pp. 293-308]. Several authors attribute the solubilisation of inorganic insoluble phosphate by microorganisms to the production of organic acids and chelating oxo acids from sugars [Leyval C. Barthelin J (1989) Interactions between Laccaria iaccaia. Agrobacierium radiobacier and beech roots: Influence on P, K. Mg and Fe mobilisation from mineral and plant growth. Plant Soil 17:103-110; Yad2v KS. Dadarwal KR (1997) Phosphate solubilisation and mobilisation through soil microorganisms. In: Dadarwal KR (ed): Biotechnological approaches :r. soil microorganisms for sustainable crop production. Jodhpur. India: Scientific Publishers, pp. 293-308]. Therefore, most of the quantitative tests to assay the relative efficiency of the phosphate solubilising bacteria are based on the lowering of pH. due to production of organic acids into the surrounding medium [Bajpai PD. Sundara Rao WVB (1971) Phosphate solubilising bacteria II. Extracellular production of organic acids by selected bacteria solubilising insoluble phosphates. Soil Sci Plant Nutr 17:44-45; Gaind S. Gaur AC (1989) Effect of pH on phosphate solubilisation by microbes. Curr Sci 58:1208-1211: Johnston HW (1952) The solubilisation of phosphate : the action of various organic compounds on dicalcium and tricalcium phosphate. NZJ Sci Technol 33:436-444; Rose RE (1957) Techniques of determining the effect of microorganisms on insoluble inorganic phosphates. N.ZJ Sci Technol 38:773-780; Sethi RP, Subba Rao NS (1968) Solubilisation of tricalcium phosphate and calcium phytase by soil fungi. J Gen Appl Microbiol 14:329-331]. The initial isolation of phosphate solubilisers is usually made by using a medium suspended with insoluble-phosphates such as tricalcium phosphates [Tilak KVBR (1993) Bacterial Fertilisers. New Delhi. India: Indian Council of Agricultural Research]. The production of clearing zones around the colonies of the organism is an indication of the presence of phosphate solubilising organisms. Such cultures are isolated and extent of phosphate solubilisation determined quantitatively, using biochemical methods [Tilak KVBR (1993) Bacterial Fertilisers. New Delhi. India: Indian Council of Agricultural Research; Subba Rao NS (1993) Biofertilizers in agriculture and forestry. Oxford & IBH Publishing Company Pvt. Ltd.. New Delhi: Oxford & IBH Publishing Company Pvt. Ltd.]. Phosphate solibilising microorganisms are routinely screened by a plate assay method using Pikovskaya (PVK containing (gm / Lt) : glucose. 10; Ca3 (PO4)2, 5; (NH4)2SO4, 0.5; NaCI. 0.2; MgSO4.7H20. 0.1; KCI, 0.2; yeast extract. 0.5; MnSO4.H20. 0.002 and FeSO4.7H20. 0.002) agar [Pikovskaya RI (1948) Mobilisation of phosphorus in soil in connection with the vital activity of some microbial species. Mikrobiologiya 17:362-370]. Other phosphate solibilising media known in the prior art besides PVK are as follows containing (gm / Lt) : 1. Glucose. 10: CaHPO4. 5; yeast extract. 0.5; KCl. 0.2; MgSO4.7H20. 0.1; MnSO4. trace: and FeSO4.7H20, trace [Sundara Rao. WVM. Sinha MK (1963) Phosphate dissolving organisms in soil and rhizosphere. Ind. J. Agri. Sci. 33. 272-278]. 2. Glucose. 10: Ca3 (PO4)2, 5; (NH4)2SO4, 1: MgSO4.7H20, 0.5: KCI. 0.2; yeast extract. 0.2: MnSO4.H20. trace and FeCl3, trace [Haider AK. Mishra AK, Chakarbartty PK (1991) Solubilisation of inorganic phosphates by Bradyrhizobium Ind. J. Exp. Biol. 29.28-31]. 3. Sucrose. 5: CaHPO4, 5; MgSO4.7H20, 0.5; KNO3, l; KCI, 0.5; and FeSO4.7H20, 0.011 [Wenzel, C.L., Ashford, A.E. and Summerell, B.A. (1994) Phosphate solubilising bacteria associated with protoid roots of seedlings of waratoh (Telopea speciosissama,) New Phytol. 128. 487-496]. 4. Mannitol. 10; Hydroxyapetite. 2; NH4C12, 1: MgSO4.7H20, 0.5: KCI. 0.2; biotin. 0.001: calcium pantothenate. 0.001; MnSO4.H20. 0.002 and FeCh. 0.002 [Abd-Alla MH (1994) Phosphatases and the utilisation of organic phosphorus b> Rhizobium leguminosarum biovar viceae. Lett Appl Microbiol 18:294-296]. The test of the relative efficiency of isolated strains is carried out by selecting the microorganisms which are capable of producing a halo/clear zone on plate due to the production of organic acids into the surrounding medium [Pikovskaya RI (1948) Mobilisation of phosphorus in soil in connection with the vital activity of some microbial species. Mikrobiologiya 17:362-370]. However, as the reliability of this halo-based technique is questioned as many isolates which did not produce any visible halo/zone on agar plates could solubilise various types of insoluble inorganic phosphates in liquid medium a modified; PVK^} medium using bromophenoj_blueJlBPB), to improve the clarity and visibility of the yellow-coloured halo has not necessarily improved the plate assay [Gupta R, Singal R, Shankar A, Kuhad RC Saxena RK (1994) A modified plate assay for screening phosphate solubilising microorganisms. J Gen Appl Microbiol 40:255-260]. Thus, the existing plate assay fails where the halo is inconspicuous or absent. Contrary to indirect measurement of phosphate solubilisation by plate assay, the direct measurement of phosphate solubilisation in broth assay always resulted into reliable results [Nautiyal CS (1999) An efficient microbiological growth medium for screening phosphate solubilising microorganisms. FEMS Microbiol Lett 170:265-270]. It was suggested that microbes from soil may be screened in National Botanical Research Institute's phosphate growth medium (NBRIP) broth assay for the identification of most efficient phosphate solubilisers [Johri JK, Surange S Nautiyal CS (1999) Occurrence of salt, pH and temperature-tolerant, phosphate-solubilising bacteria in alkaline soils. Curr Microbiol 39:89-93; Nautiyal CS, Bhadauria S, Kumar P, Lai H, Mondal R, Verma D (2000) Stress induced phosphate solubilisation in bacteria isolated from alkaline soils. FEMS Microbiol Lett 182:291-296]. NBRIP contained (per liter): glucose, 10 g; Ca3 (P04)2, 5 g; MgCI2.6H20, 5 g; MgSO4.7H20, 0.25 g; KCI, 0.2 g, and (NH4)2SO4, 0.1 g [Nautiyal CS (1999) An efficient microbiological growth medium for screening phosphate solubilising microorganisms. FEMS Microbiol Lett 170:265-270]. However, screening large number of isolates for phosphate solubilisation by quantitative methods requires investment of time, labour and chemicals. Therefore, it was envisaged to a modify NBRIP medium by using BPB a blue coloured dye which decolorises due to drop in pH of the medium, as an indicator to quickly evaluate the level of phosphate solubilisation based upon visual observations. Therefore, it is desirable to formulate a defined media for screening phosphate solubilising microorganisms, based upon visual observations, to quickly evaluate the level of phosphate solubilisation. The main object of the present invention is to provide a composition useful as a microbiological growth media, which is based upon visual observations, and not on quantitative analysis involving investment of time, labour and chemicals, to quickly evaluate the level of phosphate solubilisation. Accordingly the present invention provides a process for preparation of a which comprises mixing for coventional manner for following ingrediengts : synergistic composition useful as microbiological to set the desired segnergistic composition growth medium for screening of phosphate solubilising micro-organism: Glucose 5 to 30 gm / Lt; Ca3(P04)2, 5 g 1 to 20 gm / Lt; MgCI2.6H20 0.5 to 20 gm / Lt; MgS04.7H20 0.02 to 5 gm / Lt; KCI. 0.2 g 0.025 to 5 gm / Lt; (NH4)2S04 0.01 to 2 gm / Lt; and BPB ((bromophenol blue) 0.01 to 0.1 gm/Lt, In an embodiment of the present invention the ingredients used in the composition of the present invention may be of commercial grade; However, the use of purer ingredient such as that of laboratory grade will result in better reproducible results. The composition of the present invention is not a mere admixture of the ingredients used resulting in aggregation of their properties but a synergistic mixture having enhanced properties resulting in identifying phosphate solubilising microorganisms. The composition of the present invention may be prepared by any one of the known methods for preparation of compositions useful in identifying phosphate solubilising microorganisms. The invention is described in detail with reference to following examples which are provided by way of illustration only and these examples should not be construed to limit the scope of the present invention. In our attempt to reduce the time required to perform quantitative assay we modified NBRIP medium by using BPB as an indicator dye for visual observations, to quickly evaluate the level of phosphate solubilisation. The results are shown in fig. 1 of the drawing accompanying this specification. Qualitative («)and quantitative (n) analysis of bacterial strain ASLUI3 (A,C,E,G) and N3 (B,D,F,G) grown in NBRIP medium, containing 0.1 (A,B); 0.025 (C,D); 0.05 (E, F) and 0.1 (G,H) gm/ Lt of bromophhenol blue, at 30°C, pH 7 for 3 days. It was of interest to compare the influence of BPB on qualitative and quantitative analysis, using bacterial strains ASLU13 ( an efficient phosphate solubilizer) and N3 ( a moderately efficient phosphate solubilizer) grown in NBRIP liquid medium containing 0.01, 0.025, 0.05 and 0.1 gm / Lt BPB, for 3 days (Fig. 1). Highest limit of decolorisation (-1.999 O.D. at 600 nm) in the presence of 0.01 (Fig. 1A) and 0.025 (Fig. 1C) gm/Lt BPB by ASLU13 was achieved by day 3. For N3 the highest limit of decolorisation of BPB was achieved for O.Oi gm / Lt BPB (Fig. IB), while it declorised 0.025 gm / Lt BPB to -0.25 O.D. by day 3 (Figure ID). Our data show that it is possible to distinguish among ASLU13 and N3 based on quantitative analysis, using 0.025 gm / Lt BPB (Fig. 1). It was observed that. NBRIP liquid medium containing 0.01. 0.025. 0.05 and 0.1 gm / Lt BPB. had no appreciable effect on quantitative analysis of phosphate solubilisation. by ASLU13 and N3 (Fig. 1). However, based on qualitative analysis increasing the concentration of BPB to more than 0.025 gm / Lt, unlike quantitative analysis, it was not possible to distinguish among ASLU13 and N3 (Fig. 1). Therefore, concentration of 0.025 gm / Lt BPB was used to formulate the new medium. This National Botanical Research Institute's phosphate growth medium containing 0.025 gm / Lt BPB. was designated as NBRI-BPB. Based on the observations obtained as above the novel composition of the present invention useful as a new medium NBRI-BPB was defined, which comprises : glucose : lOgm/Lt; Ca3 (P04)2,5 g : 5 gm / Lt; MgCI2.6H20 : 5 gm / Lt; MgS04.7H20 : 0.25 gm / Lt; KCI. 0.2 g 0.2 gm / Lt; (NH4)2S04 : 0.1gm/Lt;and BPB : 0.025 gm / Lt. The invention is described with reference to the examples which are provided by way of illustration only and these should not be be construed to limit the scope of the present invention. Example 1 Potential of NBRI-BPB to evaluate large number of phosphate solubilisers was tested by initial screening of 2015 bacterial strains. Based upon the quantitative assay ten bacterial strains. ASLU13. 12.33, AS7.8. 2661. 3246, 4003, N, P, SN15, and SN16 were selected for further work. The results are shown in Fig. 2 of the drawing accompanying this specification. Qualitative (■) and quantitative (D) analysis often bacterial strains. ASLU13 (A), 12.33 (B), AS7.8 (C), 2661 (D). 3246 (E). 4003 (F). N (G). P (H). SN15 (I), and SN16 (J) grown in NBRI-BPB medium, at 30°C. pH 7 for 3 days. Qualitative and quantitative analysis was carried out using ten bacterial strains. ASLU13 (Fig. 2A). 12.33 (Fig. 2B). AS7.8 (Fig. 2C), 2661 (Fig. 2D). 3246 (Fig. 2E). 4003 (Fig. 2F). N (Fig. 2G). P (Fig. 2H). SN15 (Fig. 21). and SN16 (Fig. 2J) grown on NBRI-BPB, for 3 days. The strains selected could be placed into two distinct groups based upon the level of phosphate solubilisation. First group of five strains ASLU13 (Fig. 2A). 12.33 (Fig. 2B). AS7.8 (Fig. 2C). 2661 (Fig. 2D), and 3246 (Fig. 2E) solubilised phosphate was atleast 3-fold more efficient than that of second group of five strains 4003 (Fig. 2F). N (Fig. 2G). P (Fig. 2H), SN15 (Fig. 21). and SN16 (Fig. 2J). Comparative studies on the strains both in qualitative and quantitative assay showed similar results, as the strains could easily be divided into two groups. These findings indicate that there is a correlation between qualitative and quantitative assay. However, in qualitative assay using NBRI-BPB it was possible to quickly distinguish the two groups of bacteria, without any need for time consuming biochemical methods usually involved in the quantitative assay of phosphate solubilisers. The results suggest that NBRI-BPB should serve as an excellent composition for the initial screening of large number of phosphate solubilisers. Example 2 An investigation was carried out to assess the probability of an early detection of the effect of various physiological parameters on qualitative and quantitative analysis, using NBRI-BPB. The results are shown in Fig. 3 of the drawing accompanying this specification. Qualitative (■) and quantitative (□) analysis of the effect of various carbon, nitrogen, and salts. To check the effect of carbon sources, glucose (A) in the NBRIP-BPB was replaced by the carbon source, arabinose (B), glycerol (C), xylose (D), fructose (E), as indicated. To check the effect of nitrogen sources, (NPL^SCu (A) in the NBRIP-BPB was replaced by the nitrogen source. (NH4)2Cr207 (F), C4Hi2N206 (G), NH4HCO3 (H), C24H2oBi4028.6NH3.10H20 (I), as indicated. To check the effect of salts. MnCl2.4H20: 2.5 gm / Lt (J), NaN03; 2.5 gm / Lt (K), CaCl2.2H20; 0.25 gm / Lt (L). M11SO4.H2O: 0.25 gm / Lt (M) was added in the NBRIP-BPB. as indicated. Tn5 mutants. ASLU13.T035 (N). ASLU13.T168 (O). ASLU13.T268 (P). and ASLU13.T483 (Q) were grown in NBRI-BPB medium, at 30°C, pH 7 for 3 days. Phosphate solubilisation activity of ASLU13 was monitored in the presence of various carbon, nitrogen, and salts. ASLU13 as compared with control NBRI-BPB (Fig. 3A). demonstrated diverse level of phosphate solubilisation activity in the presence of various carbon [arabinose (Fig. 3B), glycerol (Fig. 3C), xylose (Fig. 3D), fructose (Fig. 3E)]; nitrogen [(NH^CnOv (Fig. 3F). C4Hi2N206 (Fig. 3G), NH4HCO3 (Fig. 3H), C24H2oBi402s.6NH3.10H20 (Fig. 31)]; and salts [MnCl2.4H20 (Fig. 3J). NaN03 (Fig. 3K), CaCl2.2H20 (Fig. 3L). MnS04.H20 (Fig. 3M)]. The pattern of phosphate solubilisation by ASLU13 in qualitative assay using NBRI-BPB. correlated well with quantitative assay. This observation further augments well for the use of NBRI-BPB for qualitative analysis to detect the effect of various physiological factors on phosphate solubilisers. based upon visual observation. Pure culture evaluation using NBRI-BPB may be a useful tool in search of phosphate solubilising strains better suited for soil environments where physiological factors may constitute a limitation for phosphate solubilisation. Example 3 An experiment was conducted to screen five hundred phosphate solubilisation defective Tn5 mutants of ASLU13. The results are shown in Fig. 3 of the drawing accompanying this specification. Qualitative (■) and quantitative (O) analysis of the phosphate solubilisation defective transposon5 (Tn5 ) mutants. ASLU13 (A) and Tn5 mutants. ASLU13.T035 (N). ASLU13.T168 (O), ASLU13.T268 (P). and ASLU13.T483 (Q) were grown in NBRI-BPB medium, at 30°C. pH 7 for 3 days. Based upon visual observation, due to their incapability to decolorize BPB efficiently, as compared with ASLU13 (Fig. 3A), four mutants ASLU13.T035 (Fig. 3N). ASLU13.T168 (Fig. 30), ASLU13.T268 (Fig. 3P), and ASLU13.T483 (Fig. 3Q) were easily distinguishable by 3 day. Quantitative analysis further confirmed the diverse levels of phosphate solubilisation ability of the mutants (Fig. 3). These findings further demonstrate that there is a correlation between the pattern of phosphate solubilisation by the mutants in qualitative assay and quantitative assay. The data thus show that using our simple protocol, it is indeed possible to screen large number of phosphate solubilising defective mutants. Example 4 Phosphate solubilising bacteria have been used in the commercial preparation of phosphate-dissolving cultures to improve the growth of plants [Tilak KVBR (1993) Bacterial Fertilisers. New Delhi. India: Indian Council of Agricultural Research: Subba Rao NS (1993) Biofertilizers in agriculture and forestry. Oxford & IBH Publishing Company Pvt. Ltd.. New Delhi: Oxford & IBH Publishing Company Pvt. Ltd.]. Based on the observations obtained as above, a study was conducted to evaluate the possibility of using NBRI-BPB for quickly assessing the quality of commercial bioinoculant preparations, based on phosphate solubilsers. The results are shown in Fig. 3 of the drawing accompanying this specification. Qualitative (■) and quantitative (n) analysis of the ASLU13 (A. B. C) and various commercial bioinoculants. based on phosphate solubilsers. CPB1 (D. E. F). CPB2 (G. H. I). CPB3 (J, K. L). CPB4 (M. N. O), and CPB5 (P. Q. R), grown in NBRI-BPB medium, at 30oC. pH 7 for upto 1 day (A, D, G, J. M, P), 2 day (B, E. H. K. N, Q). and 3 day (C, F, I, L, O, R). In a comparative study, using our ASLU13 based bioinoculant preparation (4A, 4B. 4C). along with five commercial products CPB1 (4D. 4E.4 F). CPB2 (4G, 4H. 41). CPB3 (4J. 4K. 4L), CPB4 (4M. 4N. 40). and CPB5 (4P. 4Q. 4R), grown in NBRI-BPB medium, at 30oC. pH 7 for upto 1 day (4A. 4D. 4G, 4J. 4M. 4P), 2 day (4B. 4E. 4H. 4K. 4N. 4Q). and 3 day (4C. 4F. 41. 4L. 40. 4R), was subjected to testing by the new composition (Fig. 4). Decolorisation of BPB using our ASLU13 based bioinoculant preparation was achieved by 2 day (Fig. 4B). Among the five products tested, based upon visual observation the highest limit of decolorisation of BPB in products CPB1 (Fig. 4F) and CPB3 (Fig. 4L) was achieved by 3 day, while decolorisation of BPB in CPB2 (Fig. 4B), CPB4 (Fig. 40), and CPB5 (Fig. 4R) was under -0.180 by 3 day. Thus the commercial bioinoculant product CPB1 was easily distinguishable from other products in its ability to solubilise phosphate. Furthermore, the present work indicates that the composition can also be used as a quality control test for expeditious screen::;.: ::" the commercial bioinocuiant preparations, based on phosphate solubilsers Our results indicate that using our compos:;:on containing BPB. it is possible to quicklx screen on quahtame basis, the phosrnate soiubiiising bacteria. Qualitative anahsis of the phosphate solubilised by : anous groups, correlated well with grouping based upon quantitame analysis of bacteria isolated from soil, effect of various carbon, nitrogen, salts, and phosphate soiubilisation defective transposon mutants. However, unlike quantitative analysis methods which involve time consuming biochemical procedures, the time for screening phosphate solubilising bacteria is significantly reduced by using our simple protocol. Therefore, it is hereby suggested that this composition basea upon qualitative analysis should be used for the quick screening of phosphate solubilising bacteria. Our results indicate that the composition can also be used as a qualm control test for expeditious!) screening the commercial bioinocuiant preparations, based on phosphate solubilsers. The main advantages of the composition of the present invention are : 1. It can be used for quick screening of phosphate solubilising microorganisms, because our protocol is based upon visual observation. 2. The novel medium composition of the present invention NBRIP-BPB broth assa>. can be used for the qualitative analysis of bacteria isolated from soil, effect of various carbon, nitrogen, salts, and phosDhate soiubilisation defective transposon mutants. carbon, nitrogen, salts, and phosphate soiubilisation defective mutants. 3. The composition can also be used as a quaht;. control test for expeditious screening of the commercial bioinocuiant preparations, based on phosphate solubilsers. We Claim: 1. A synergistic composition useful as microbiological growth medium for screening of phosphate solubilising micro-organism: Glucose 5 to 30 gm / Lt; Ca3(PO4)2, 5 g 1 to 20 gm / Lt; MgCI2.6H2O 0.5 to 20 gm / Lt; MgSO4.7H2O 0.02 to 5 gm / Lt; KCI. 0.2 g 0.025 to 5 gm / Lt; (NH4)2SO4 0.01 to 2 gm / Lt; and BPB (bromophenol blue) : 0.01 to 0.1 gm / Lt. 2. A synergistic composition useful as microbiological growth medium for screening of phosphate solubilising micro-organism substantially as herein described with reference to the examples and drawing accompanying this specification.

Full Text

The present invention relates to a synergistic composition useful as microbiological growth medium for screening of phosphate solubilising microorganism.
More particularly the present invention provides composition of
microbiological growth medium for qualitative screening of phospnate
solubilising microorganisms, based upon visual observation.
A larce portion of inorganic phosphates applied to soil as fertiliser is rapidly immobilised after application and becomes unavailable to plants [Yadav KS. Dadarwal KR (1997) Phosphate solubilisation and mobilisation through soil microorganisms. In: Dadarwal KR (ed): Biotechnological approaches in soil microorganisms for sustainable crop production. Jodhpur. India: Scientific Publishers, pp. 293-308]. Thus, the release of insoluble and fixed forms of phosphorus is an important aspect of increasing soil phosphorus availability. Seed or soil inoculation with phosphate solubilising bacteria is known to improve solubilisation of fixed soil phosphorus and applied phosphates resulting in higher crop yields [Abd-Alla MH (1994) Phosphatases and the utilisation of organic phosphorus by Rhizobium leguminosarum biovar viceae. Lett Appl Microbiol 18:294-296: Jones DL. Darrah PR (1994) Role of root derived organic acids in the mobilisation of nutrients from the rhizosphere. Plant Soil 166:247-257; Leyval C, Barthelin J (1989) Interactions between Laccaria laccata. Agrobacierium radiobacier and beech roots: Influence on P. K. Mg and Fe mobilisation irom mineral and plant growth. Plant Soil 17:103-110: Yadav KS. Dadarwal KR (1997) Phosphate solubilisation and mobilisation through soil microorganisms. In: Dadarwal KR (ed). Biotechnological approaches in soil microorganisms for sustainable crop production. Jodhpur, India: Scientific Publishers, pp. 293-308]. Several authors attribute the solubilisation of inorganic insoluble phosphate by microorganisms to the production of organic acids and chelating oxo acids from sugars [Leyval C. Barthelin J (1989) Interactions between Laccaria iaccaia. Agrobacierium radiobacier and beech roots: Influence on P, K. Mg and Fe mobilisation from mineral and plant growth. Plant Soil 17:103-110; Yad2v KS. Dadarwal KR (1997) Phosphate solubilisation and mobilisation through soil microorganisms. In: Dadarwal KR (ed): Biotechnological approaches :r. soil
microorganisms for sustainable crop production. Jodhpur. India: Scientific Publishers, pp. 293-308]. Therefore, most of the quantitative tests to assay the relative efficiency of the phosphate solubilising bacteria are based on the lowering of pH. due to production of organic acids into the surrounding medium [Bajpai PD. Sundara Rao WVB (1971) Phosphate solubilising bacteria II. Extracellular production of organic acids by selected bacteria solubilising insoluble phosphates. Soil Sci Plant Nutr 17:44-45; Gaind S. Gaur AC (1989) Effect of pH on phosphate solubilisation by microbes. Curr Sci 58:1208-1211: Johnston HW (1952) The solubilisation of phosphate : the action of various organic compounds on dicalcium and tricalcium phosphate. NZJ Sci Technol 33:436-444; Rose RE (1957) Techniques of determining the effect of microorganisms on insoluble inorganic phosphates. N.ZJ Sci Technol 38:773-780; Sethi RP, Subba Rao NS (1968) Solubilisation of tricalcium phosphate and calcium phytase by soil fungi. J Gen Appl Microbiol 14:329-331]. The initial isolation of phosphate solubilisers is usually made by using a medium suspended with insoluble-phosphates such as tricalcium phosphates [Tilak KVBR (1993) Bacterial Fertilisers. New Delhi. India: Indian Council of Agricultural Research]. The production of clearing zones around the colonies of the organism is an indication of the presence of phosphate solubilising organisms. Such cultures are isolated and extent of phosphate solubilisation determined quantitatively, using biochemical methods [Tilak KVBR (1993) Bacterial Fertilisers. New Delhi. India: Indian Council of Agricultural Research; Subba Rao NS (1993) Biofertilizers in agriculture and forestry. Oxford & IBH Publishing Company Pvt. Ltd.. New Delhi: Oxford & IBH Publishing Company Pvt. Ltd.].
Phosphate solibilising microorganisms are routinely screened by a plate assay method using Pikovskaya (PVK containing (gm / Lt) : glucose. 10; Ca3 (PO4)2, 5; (NH4)2SO4, 0.5; NaCI. 0.2; MgSO4.7H20. 0.1; KCI, 0.2; yeast extract. 0.5; MnSO4.H20. 0.002 and FeSO4.7H20. 0.002) agar [Pikovskaya RI (1948) Mobilisation of phosphorus in soil in connection with the vital activity of some microbial species. Mikrobiologiya 17:362-370]. Other phosphate solibilising media known in the prior art besides PVK are as follows containing (gm / Lt) : 1.
Glucose. 10: CaHPO4. 5; yeast extract. 0.5; KCl. 0.2; MgSO4.7H20. 0.1; MnSO4. trace: and FeSO4.7H20, trace [Sundara Rao. WVM. Sinha MK (1963) Phosphate dissolving organisms in soil and rhizosphere. Ind. J. Agri. Sci. 33. 272-278].
2. Glucose. 10: Ca3 (PO4)2, 5; (NH4)2SO4, 1: MgSO4.7H20, 0.5: KCI. 0.2; yeast
extract. 0.2: MnSO4.H20. trace and FeCl3, trace [Haider AK. Mishra AK,
Chakarbartty PK (1991) Solubilisation of inorganic phosphates by Bradyrhizobium
Ind. J. Exp. Biol. 29.28-31].
3. Sucrose. 5: CaHPO4, 5; MgSO4.7H20, 0.5; KNO3, l; KCI, 0.5; and FeSO4.7H20, 0.011 [Wenzel, C.L., Ashford, A.E. and Summerell, B.A. (1994) Phosphate solubilising bacteria associated with protoid roots of seedlings of waratoh (Telopea speciosissama,) New Phytol. 128. 487-496].
4. Mannitol. 10; Hydroxyapetite. 2; NH4C12, 1: MgSO4.7H20, 0.5: KCI. 0.2; biotin. 0.001: calcium pantothenate. 0.001; MnSO4.H20. 0.002 and FeCh. 0.002 [Abd-Alla MH (1994) Phosphatases and the utilisation of organic phosphorus b> Rhizobium leguminosarum biovar viceae. Lett Appl Microbiol 18:294-296].
The test of the relative efficiency of isolated strains is carried out by selecting the microorganisms which are capable of producing a halo/clear zone on plate due to the production of organic acids into the surrounding medium [Pikovskaya RI (1948) Mobilisation of phosphorus in soil in connection with the vital activity of some microbial species. Mikrobiologiya 17:362-370].
However, as the reliability of this halo-based technique is questioned as many isolates which did not produce any visible halo/zone on agar plates could solubilise various types of insoluble inorganic phosphates in liquid medium a modified; PVK^} medium using bromophenoj_blueJlBPB), to improve the clarity and visibility of the yellow-coloured halo has not necessarily improved the plate assay [Gupta R, Singal R, Shankar A, Kuhad RC Saxena RK (1994) A modified plate assay for screening phosphate solubilising microorganisms. J Gen Appl Microbiol 40:255-260]. Thus, the existing plate assay fails where the halo is inconspicuous or absent. Contrary to indirect measurement of phosphate solubilisation by plate assay, the direct measurement of phosphate solubilisation in broth assay always resulted into reliable results [Nautiyal CS (1999) An efficient microbiological growth medium
for screening phosphate solubilising microorganisms. FEMS Microbiol Lett 170:265-270]. It was suggested that microbes from soil may be screened in National Botanical Research Institute's phosphate growth medium (NBRIP) broth assay for the identification of most efficient phosphate solubilisers [Johri JK, Surange S Nautiyal CS (1999) Occurrence of salt, pH and temperature-tolerant, phosphate-solubilising bacteria in alkaline soils. Curr Microbiol 39:89-93; Nautiyal CS, Bhadauria S, Kumar P, Lai H, Mondal R, Verma D (2000) Stress induced phosphate solubilisation in bacteria isolated from alkaline soils. FEMS Microbiol Lett 182:291-296]. NBRIP contained (per liter): glucose, 10 g; Ca3 (P04)2, 5 g; MgCI2.6H20, 5 g; MgSO4.7H20, 0.25 g; KCI, 0.2 g, and (NH4)2SO4, 0.1 g [Nautiyal CS (1999) An efficient microbiological growth medium for screening phosphate solubilising microorganisms. FEMS Microbiol Lett 170:265-270].
However, screening large number of isolates for phosphate solubilisation by quantitative methods requires investment of time, labour and chemicals. Therefore, it was envisaged to a modify NBRIP medium by using BPB a blue coloured dye which decolorises due to drop in pH of the medium, as an indicator to quickly evaluate the level of phosphate solubilisation based upon visual observations. Therefore, it is desirable to formulate a defined media for screening phosphate solubilising microorganisms, based upon visual observations, to quickly evaluate the level of phosphate solubilisation.
The main object of the present invention is to provide a composition useful as a microbiological growth media, which is based upon visual observations, and not on quantitative analysis involving investment of time, labour and chemicals, to quickly evaluate the level of phosphate solubilisation.
Accordingly the present invention provides a process for preparation of a which comprises mixing for coventional
manner for following ingrediengts :
synergistic composition useful as microbiological
to set the desired segnergistic composition
growth medium for screening of phosphate solubilising micro-organism:
Glucose 5 to 30 gm / Lt;
Ca3(P04)2, 5 g 1 to 20 gm / Lt;
MgCI2.6H20 0.5 to 20 gm / Lt;
MgS04.7H20 0.02 to 5 gm / Lt;
KCI. 0.2 g 0.025 to 5 gm / Lt;
(NH4)2S04 0.01 to 2 gm / Lt; and
BPB ((bromophenol blue) 0.01 to 0.1 gm/Lt,
In an embodiment of the present invention the ingredients used in the composition of the present
invention may be of commercial grade; However, the use of purer ingredient such as that of
laboratory grade will result in better reproducible results.
The composition of the present invention is not a mere admixture of the ingredients used resulting
in aggregation of their properties but a synergistic mixture having enhanced properties resulting in
identifying phosphate solubilising microorganisms.
The composition of the present invention may be prepared by any one of the known methods for
preparation of compositions useful in identifying phosphate solubilising microorganisms.
The invention is described in detail with reference to following examples which are provided by
way of illustration only and these examples should not be construed to limit the scope of the
present invention.
In our attempt to reduce the time required to perform quantitative assay we modified NBRIP
medium by using BPB as an indicator dye for visual observations, to quickly evaluate the level of
phosphate solubilisation. The results are shown in fig. 1 of the drawing accompanying this
specification. Qualitative («)and quantitative (n) analysis of bacterial strain ASLUI3 (A,C,E,G) and
N3 (B,D,F,G) grown in NBRIP medium, containing 0.1 (A,B); 0.025 (C,D); 0.05 (E, F) and 0.1
(G,H) gm/ Lt of bromophhenol blue, at 30°C, pH 7 for 3 days.
It was of interest to compare the influence of BPB on qualitative and quantitative analysis, using
bacterial strains ASLU13 ( an efficient phosphate solubilizer) and N3 ( a moderately efficient
phosphate solubilizer) grown in NBRIP liquid medium containing 0.01, 0.025, 0.05 and 0.1 gm / Lt
BPB, for 3 days (Fig. 1). Highest limit of decolorisation (-1.999 O.D. at 600 nm) in the presence of
0.01 (Fig. 1A) and 0.025 (Fig. 1C) gm/Lt BPB by ASLU13 was achieved by day 3. For N3 the

highest limit of decolorisation of BPB was achieved for O.Oi gm / Lt BPB (Fig. IB), while it declorised 0.025 gm / Lt BPB to -0.25 O.D. by day 3 (Figure ID). Our data show that it is possible to distinguish among ASLU13 and N3 based on quantitative analysis, using 0.025 gm / Lt BPB (Fig. 1). It was observed that. NBRIP liquid medium containing 0.01. 0.025. 0.05 and 0.1 gm / Lt BPB. had no appreciable effect on quantitative analysis of phosphate solubilisation. by ASLU13 and N3 (Fig. 1). However, based on qualitative analysis increasing the concentration of BPB to more than 0.025 gm / Lt, unlike quantitative analysis, it was not possible to distinguish among ASLU13 and N3 (Fig. 1). Therefore, concentration of 0.025 gm / Lt BPB was used to formulate the new medium. This National Botanical Research Institute's phosphate growth medium containing 0.025 gm / Lt BPB. was designated as NBRI-BPB.
Based on the observations obtained as above the novel composition of the present
invention useful as a new medium NBRI-BPB was defined, which comprises :
glucose : lOgm/Lt;
Ca3 (P04)2,5 g : 5 gm / Lt;
MgCI2.6H20 : 5 gm / Lt;
MgS04.7H20 : 0.25 gm / Lt;
KCI. 0.2 g 0.2 gm / Lt;
(NH4)2S04 : 0.1gm/Lt;and
BPB : 0.025 gm / Lt.
The invention is described with reference to the examples which are provided by way of illustration only and these should not be be construed to limit the scope of the present invention.
Example 1 Potential of NBRI-BPB to evaluate large number of phosphate solubilisers was tested by initial screening of 2015 bacterial strains. Based upon the quantitative assay ten bacterial strains. ASLU13. 12.33, AS7.8. 2661. 3246, 4003, N, P, SN15, and SN16 were selected for further work. The results are shown in Fig. 2 of the drawing accompanying this specification. Qualitative (■) and quantitative (D) analysis often bacterial strains. ASLU13 (A), 12.33 (B), AS7.8 (C), 2661 (D).
3246 (E). 4003 (F). N (G). P (H). SN15 (I), and SN16 (J) grown in NBRI-BPB medium, at 30°C. pH 7 for 3 days.
Qualitative and quantitative analysis was carried out using ten bacterial strains. ASLU13 (Fig. 2A). 12.33 (Fig. 2B). AS7.8 (Fig. 2C), 2661 (Fig. 2D). 3246 (Fig. 2E). 4003 (Fig. 2F). N (Fig. 2G). P (Fig. 2H). SN15 (Fig. 21). and SN16 (Fig. 2J) grown on NBRI-BPB, for 3 days. The strains selected could be placed into two distinct groups based upon the level of phosphate solubilisation. First group of five strains ASLU13 (Fig. 2A). 12.33 (Fig. 2B). AS7.8 (Fig. 2C). 2661 (Fig. 2D), and 3246 (Fig. 2E) solubilised phosphate was atleast 3-fold more efficient than that of second group of five strains 4003 (Fig. 2F). N (Fig. 2G). P (Fig. 2H), SN15 (Fig. 21). and SN16 (Fig. 2J). Comparative studies on the strains both in qualitative and quantitative assay showed similar results, as the strains could easily be divided into two groups. These findings indicate that there is a correlation between qualitative and quantitative assay. However, in qualitative assay using NBRI-BPB it was possible to quickly distinguish the two groups of bacteria, without any need for time consuming biochemical methods usually involved in the quantitative assay of phosphate solubilisers. The results suggest that NBRI-BPB should serve as an excellent composition for the initial screening of large number of phosphate solubilisers.
Example 2 An investigation was carried out to assess the probability of an early detection of the effect of various physiological parameters on qualitative and quantitative analysis, using NBRI-BPB. The results are shown in Fig. 3 of the drawing accompanying this specification. Qualitative (■) and quantitative (□) analysis of the effect of various carbon, nitrogen, and salts. To check the effect of carbon sources, glucose (A) in the NBRIP-BPB was replaced by the carbon source, arabinose (B), glycerol (C), xylose (D), fructose (E), as indicated. To check the effect of nitrogen sources, (NPL^SCu (A) in the NBRIP-BPB was replaced by the nitrogen source. (NH4)2Cr207 (F), C4Hi2N206 (G), NH4HCO3 (H), C24H2oBi4028.6NH3.10H20 (I), as indicated. To check the effect of salts. MnCl2.4H20: 2.5 gm / Lt (J), NaN03; 2.5 gm / Lt (K), CaCl2.2H20; 0.25 gm / Lt
(L). M11SO4.H2O: 0.25 gm / Lt (M) was added in the NBRIP-BPB. as indicated. Tn5 mutants. ASLU13.T035 (N). ASLU13.T168 (O). ASLU13.T268 (P). and ASLU13.T483 (Q) were grown in NBRI-BPB medium, at 30°C, pH 7 for 3 days. Phosphate solubilisation activity of ASLU13 was monitored in the presence of various carbon, nitrogen, and salts. ASLU13 as compared with control NBRI-BPB (Fig. 3A). demonstrated diverse level of phosphate solubilisation activity in the presence of various carbon [arabinose (Fig. 3B), glycerol (Fig. 3C), xylose (Fig. 3D), fructose (Fig. 3E)]; nitrogen [(NH^CnOv (Fig. 3F). C4Hi2N206 (Fig. 3G), NH4HCO3 (Fig. 3H), C24H2oBi402s.6NH3.10H20 (Fig. 31)]; and salts [MnCl2.4H20 (Fig. 3J). NaN03 (Fig. 3K), CaCl2.2H20 (Fig. 3L). MnS04.H20 (Fig. 3M)]. The pattern of phosphate solubilisation by ASLU13 in qualitative assay using NBRI-BPB. correlated well with quantitative assay. This observation further augments well for the use of NBRI-BPB for qualitative analysis to detect the effect of various physiological factors on phosphate solubilisers. based upon visual observation. Pure culture evaluation using NBRI-BPB may be a useful tool in search of phosphate solubilising strains better suited for soil environments where physiological factors may constitute a limitation for phosphate solubilisation.
Example 3 An experiment was conducted to screen five hundred phosphate solubilisation defective Tn5 mutants of ASLU13. The results are shown in Fig. 3 of the drawing accompanying this specification. Qualitative (■) and quantitative (O) analysis of the phosphate solubilisation defective transposon5 (Tn5 ) mutants. ASLU13 (A) and Tn5 mutants. ASLU13.T035 (N). ASLU13.T168 (O), ASLU13.T268 (P). and ASLU13.T483 (Q) were grown in NBRI-BPB medium, at 30°C. pH 7 for 3 days. Based upon visual observation, due to their incapability to decolorize BPB efficiently, as compared with ASLU13 (Fig. 3A), four mutants ASLU13.T035 (Fig. 3N). ASLU13.T168 (Fig. 30), ASLU13.T268 (Fig. 3P), and ASLU13.T483 (Fig. 3Q) were easily distinguishable by 3 day. Quantitative analysis further confirmed the diverse levels of phosphate solubilisation ability of the mutants (Fig. 3). These findings further demonstrate that there is a correlation between the pattern of phosphate solubilisation by the mutants in qualitative assay and
quantitative assay. The data thus show that using our simple protocol, it is indeed possible to screen large number of phosphate solubilising defective mutants.
Example 4 Phosphate solubilising bacteria have been used in the commercial preparation of phosphate-dissolving cultures to improve the growth of plants [Tilak KVBR (1993) Bacterial Fertilisers. New Delhi. India: Indian Council of Agricultural Research: Subba Rao NS (1993) Biofertilizers in agriculture and forestry. Oxford & IBH Publishing Company Pvt. Ltd.. New Delhi: Oxford & IBH Publishing Company Pvt. Ltd.]. Based on the observations obtained as above, a study was conducted to evaluate the possibility of using NBRI-BPB for quickly assessing the quality of commercial bioinoculant preparations, based on phosphate solubilsers. The results are shown in Fig. 3 of the drawing accompanying this specification. Qualitative (■) and quantitative (n) analysis of the ASLU13 (A. B. C) and various commercial bioinoculants. based on phosphate solubilsers. CPB1 (D. E. F). CPB2 (G. H. I). CPB3 (J, K. L). CPB4 (M. N. O), and CPB5 (P. Q. R), grown in NBRI-BPB medium, at 30oC. pH 7 for upto 1 day (A, D, G, J. M, P), 2 day (B, E. H. K. N, Q). and 3 day (C, F, I, L, O, R).
In a comparative study, using our ASLU13 based bioinoculant preparation (4A, 4B. 4C). along with five commercial products CPB1 (4D. 4E.4 F). CPB2 (4G, 4H. 41). CPB3 (4J. 4K. 4L), CPB4 (4M. 4N. 40). and CPB5 (4P. 4Q. 4R), grown in NBRI-BPB medium, at 30oC. pH 7 for upto 1 day (4A. 4D. 4G, 4J. 4M. 4P), 2 day (4B. 4E. 4H. 4K. 4N. 4Q). and 3 day (4C. 4F. 41. 4L. 40. 4R), was subjected to testing by the new composition (Fig. 4). Decolorisation of BPB using our ASLU13 based bioinoculant preparation was achieved by 2 day (Fig. 4B). Among the five products tested, based upon visual observation the highest limit of decolorisation of BPB in products CPB1 (Fig. 4F) and CPB3 (Fig. 4L) was achieved by 3 day, while decolorisation of BPB in CPB2 (Fig. 4B), CPB4 (Fig. 40), and CPB5 (Fig. 4R) was under -0.180 by 3 day. Thus the commercial bioinoculant product CPB1 was easily distinguishable from other products in its ability to solubilise phosphate. Furthermore, the present work indicates that the composition can also be used as a
quality control test for expeditious screen::;.: ::" the commercial bioinocuiant preparations, based on phosphate solubilsers
Our results indicate that using our compos:;:on containing BPB. it is possible to quicklx screen on quahtame basis, the phosrnate soiubiiising bacteria. Qualitative anahsis of the phosphate solubilised by : anous groups, correlated well with grouping based upon quantitame analysis of bacteria isolated from soil, effect of various carbon, nitrogen, salts, and phosphate soiubilisation defective transposon mutants. However, unlike quantitative analysis methods which involve time consuming biochemical procedures, the time for screening phosphate solubilising bacteria is significantly reduced by using our simple protocol. Therefore, it is hereby suggested that this composition basea upon qualitative analysis should be used for the quick screening of phosphate solubilising bacteria. Our results indicate that the composition can also be used as a qualm control test for expeditious!) screening the commercial bioinocuiant preparations, based on phosphate solubilsers. The main advantages of the composition of the present invention are :
1. It can be used for quick screening of phosphate solubilising microorganisms, because our protocol is based upon visual observation.
2. The novel medium composition of the present invention NBRIP-BPB broth assa>. can be used for the qualitative analysis of bacteria isolated from soil, effect of various carbon, nitrogen, salts, and phosDhate soiubilisation defective transposon mutants.
carbon, nitrogen, salts, and phosphate soiubilisation defective mutants.
3. The composition can also be used as a quaht;. control test for expeditious
screening of the commercial bioinocuiant preparations, based on phosphate
solubilsers.

We Claim:
1. A synergistic composition useful as microbiological growth medium for
screening of phosphate solubilising micro-organism:
Glucose 5 to 30 gm / Lt;
Ca3(PO4)2, 5 g 1 to 20 gm / Lt;
MgCI2.6H2O 0.5 to 20 gm / Lt;
MgSO4.7H2O 0.02 to 5 gm / Lt;
KCI. 0.2 g 0.025 to 5 gm / Lt;
(NH4)2SO4 0.01 to 2 gm / Lt; and
BPB (bromophenol blue) : 0.01 to 0.1 gm / Lt.
2. A synergistic composition useful as microbiological growth medium for
screening of phosphate solubilising micro-organism substantially as
herein described with reference to the examples and drawing
accompanying this specification.

Documents:

1157-del-2000-abstract.pdf

1157-del-2000-claims.pdf

1157-del-2000-correspondence-others.pdf

1157-del-2000-correspondence-po.pdf

1157-del-2000-description (complete).pdf

1157-del-2000-drawings.pdf

1157-del-2000-form-1.pdf

1157-del-2000-form-19.pdf

1157-del-2000-form-2.pdf

1157-del-2000-form-3.pdf

1157-del-2000-petition-137.pdf

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Patent Number

226577

Indian Patent Application Number

1157/DEL/2000

PG Journal Number

01/2009

Publication Date

02-Jan-2009

Grant Date

19-Dec-2008

Date of Filing

15-Dec-2000

Name of Patentee

COUNCIL OF SCIENTFIC AND INDUSTRIAL RESEARCH

Applicant Address

RAFI MARG, NEW DELHI-110 001, INDIA.

Inventors:

#	Inventor's Name	Inventor's Address
1	SANGEETA MEHTA	NATIONAL BOTANICAL RESEARCH INSTITUTE, P.B. NO. 436, LUCKHNOW 226001 (U.P.), INDIA.
2	CHANDRA SHEKHAR NAUTIYAL	NATIONAL BOTANICAL RESEARCH INSTITUTE, P.B. NO. 436, LUCKHNOW 226001 (U.P.), INDIA.
3	PALPU PUSHPANGADAN	NATIONAL BOTANICAL RESEARCH INSTITUTE, P.B. NO. 436, LUCKHNOW 226001 (U.P.), INDIA.

PCT International Classification Number

C12N 1/02

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA